Hormonal Signaling in Biology and Medicine: Comprehensive Modern Endocrinology [Comprehensive ed.] 0128138149, 9780128138144

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Hormonal Signaling in Biology and Medicine: Comprehensive Modern Endocrinology [Comprehensive ed.]
 0128138149, 9780128138144

Table of contents :
Cover
Hormonal Signaling in Biology and Medicine: Comprehensive Modern Endocrinology
Copyright
Contributors
Preface
1. Hormones and Perinatal Development
1. Introduction
2. Development of Hepatic Metabolic Processes
2.1 Neonatal Development of Hepatic Gluconeogenesis
2.2 Neonatal Development of Hepatic Amino Acid Metabolism
2.3 Applying Neonatal Regulation of Hepatic Enzymes to Generate Functional Hepatocytes for Cell Therapy
3. Development of the Respiratory System
4. Postnatal Development of Intestinal Digestion
5. Conclusions and Future Directions
Acknowledgments
References
2. Hormones of Programmed Cell Death
1. Introduction
2. Role of Hormones in PCD
3. Steroids
3.1 Glucocorticoids
3.2 Androgens
3.3 Estrogens
3.4 Progestogens
4. Thyroids
5. Retinoids
6. Vitamin D3 Derivatives
7. Tumor Necrosis Factor Superfamily: Death Receptors and Ligands
8. Conclusions and Future Directions
References
3. Hypothalamic Releasing Hormones
1. Corticotropin Releasing Hormone
1.1 CRH Protein and Gene Structure
1.2 CRH Receptors
1.3 CRH–CRH Receptor Interaction and Signaling Pathway
1.4 Diseases
2. Thyrotropin-Releasing Hormone
3. Prolactin-Releasing Factors
4. Luteinizing Hormone-Releasing Hormone/Gonadotropin-Releasing Hormone
4.1 Receptors and Signaling
4.2 Therapeutic Use
5. Somatostatin (Somatotrophin Release-Inhibiting Factor)
5.1 Receptors and Signaling
5.2 Therapeutic Use
6. Growth Hormone-Releasing Hormone
6.1 The Gene
6.2 GH-RH Receptor and GHRH Receptor Gene
6.3 Signal Transduction
6.4 Disease and Therapy
Acknowledgements
References
4. Neurosteroids: Biosynthesis, Molecular Mechanisms, and Neurophysiological Functions in the Human Brain
1. Introduction
2. Biosynthesis of Neurosteroids
3. Molecular Mechanisms of Neurosteroids
3.1 Neurosteroid Modulation of GABA-A Receptors
3.2 Sulfated Neurosteroid Interactions With GABA and Glutamate Receptors
4. Neurophysiological Functions of Neurosteroids
4.1 Epilepsy and Neuronal Excitability Disorders
4.2 Status Epilepticus and Acute Seizures
4.3 Catamenial Epilepsy and Neuroendocrine Conditions
4.4 Infantile Spasms and Developmental Conditions
4.5 Menstrual Mood Disorders
4.6 Fragile X Syndrome and Genetic Abnormalities
4.7 Neuronal Injury and Neurotoxicity Conditions
4.8 Anxiety and Other Psychiatric Conditions
5. Conclusions and Future Directions
Acknowledgments
References
5. Neurotrophins and Neurotrophin Receptors
1. Introduction
2. Structures of Neurotrophins and Neurotrophin Receptors
2.1 Neurotrophins
2.2 Complex of Neurotrophin With Neurotrophin Receptor
2.2.1 p75NTR
2.2.2 TrkA
2.2.3 p75NTR Interactions With TrkA
3. Evolution of the Neurotrophin-Signaling System
4. Biochemical Reactions (Interaction With Receptors, Receptor Activation and Signaling Pathways)
4.1 p75NTR Signaling
4.1.1 p75 in Cell Death and Apoptosis: Mature Neurotrophins
4.1.2 p75 in Cell Death and Apoptosis: Proneurotrophins
4.1.3 p75 in Cell Survival and Activation of NF-κB
4.1.4 p75 in Cell Survival and Antioxidative Stress
4.2 Trk Signaling
4.2.1 PLC-γ
4.2.2 PI3K-Akt
4.2.3 MAPK
5. Physiologic Functions of the Neurotrophins
5.1 The Nervous System
5.1.1 TrkA/NGF Signaling
5.1.2 TrkB/BDNF/NT4 Signaling
5.1.3 TrkC-NT3
5.1.4 Truncated Trks
5.1.4.1 TrkB-T1
5.1.4.2 Truncated TrkC
5.2 Nonnervous System
5.2.1 Cardiovascular Functions
5.2.2 Regulation of Energy Balance and Body Weight
6. Disease and Aging Effects of Neurotrophins
6.1 Alzheimer Disease
6.2 Huntington Disease
6.3 Parkinson Disease
6.4 Amyotrophic Lateral Sclerosis
6.5 BDNF Polymorphism and Nervous System Disorders
6.6 Cancer and Tumor Formation
6.7 Inflammation, Allergy, and Pain
7. Conclusions and Future Directions
Acknowledgments
References
6. The Pineal as a Gland and Melatonin as a Hormone
1. Introduction: History of the Hypothesis that the Mammalian Pineal is a Gland and Secretes the Hormone Melatonin
2. The Pineal Gland as a Neuroendocrine Transducer: Converting a Neural Input to an Endocrine Output
3. Melatonin: Distribution, Biosynthesis, Metabolism
4. Melatonin Rhythms: Circadian and Annual
5. Melatonin as a “Nutritional Supplement”
6. Aging, Melatonin and Sleep
References
Further Reading
7. Anterior Pituitary: Glycoprotein Hormones From Gonadotrope (FSH and LH) and Thyrotrope (TSH) Cells
1. Introduction
2. Gene and Protein Structures
2.1 Ligand Structures
2.2 Ligand Glycosylation
2.3 Receptor Structures
3. Evolution
4. Regulation of Synthesis and Secretion
4.1 Gonadotropins
4.1.1 Regulation by GnRH
4.1.2 Regulation by Activins and Inhibins
4.1.3 Regulation by Sex Steroids
4.2 Thyrotropin
4.2.1 Regulation by TRH
4.2.2 Regulation by Thyroid Hormones
4.2.3 Regulation by Other Factors
5. Biochemical Reactions
5.1 Receptor Activation
5.2 Receptor Oligomerization
6. Physiologic Functions
6.1 Gonadotropins
6.2 Thyrotropin
7. Hormone Inactivation
8. Disease and Aging Effects
8.1 Gonadotropins
8.2 Thyrotropin
9. Conclusions and Future Directions
Funding
References
8. Anterior Pituitary and Pars Intermedia Space: Corticotrophs (ACTH) and Melanotrophs (α-MSH)
1. Introduction
2. POMC-Derived Peptides and Their Receptors: Structure and Processing
2.1 POMC—Synthesis and Processing
2.2 Transcriptional Control of the POMC Gene
2.3 Posttranslational Control of POMC Production
3. Pituitary Cells Expressing POMC: Corticotrophs and Melanotrophs
3.1 Corticotrophs and ACTH
3.1.1 ACTH Release from Corticotrophs: A Key Mediator of the Neuroendocrine Response to Stress
3.1.2 Corticotroph Development and Anatomy in the Anterior Pituitary Gland
3.1.3 CRH and Glucocorticoid Control of POMC Expression in Corticotrophs
3.1.4 Regulation of ACTH Secretion by CRH, AVP, and Glucocorticoids
3.1.5 CRH and AVP Activate Distinct G Protein–Coupled Receptor Signaling Pathways in Corticotrophs
3.1.6 Synergy of CRH- and AVP-Stimulated ACTH Secretion
3.1.7 Electrical Excitability, Calcium Signaling, and the Control of ACTH Secretion
3.1.8 Glucocorticoid Feedback Inhibition of ACTH Release
4. Melanotrophs and α-MSH
4.1 Anatomic Considerations
4.2 Control of Melanotroph Activity
5. The Melanocortin Receptors (MCRs), Accessory Proteins (MRAP), and Their Actions
5.1 MC2R
5.2 MC1R
5.3 MC5R
5.4 MC3R and MC4R
6. Hormone Inactivation
7. Disease and Aging Effects
7.1 Addison Disease—Adrenal Insufficiency
7.2 Cushing Disease
7.3 Familial Glucocorticoid Deficiency
7.4 Changes with Aging
8. Conclusions and Future Directions
References
9. Anterior Pituitary: Somatotrophs (GH) and Lactotrophs (PRL)
1. Introduction
2. Structure and Regulation
2.1 The Structure and Regulation of GH and PRL
2.2 The Structure and Regulation of GH and PRL Receptors
2.3 GH- and PRL-Binding Proteins
3. Evolution
3.1 Evolution of GH and PRL
3.2 Evolution of GHR and PRLR
4. Biochemical Reactions
4.1 Formation of Receptor Complexes
4.2 Receptor Signaling
5. The Pituitary Cells Producing GH and PRL
5.1 Regulation of GH and PRL Secretion: The Importance of the Hypothalamic–Pituitary Relationship
5.2 Regulation of GH Secretion
5.2.1 Hypothalamic Regulation of Pituitary GH Secretion
5.2.2 Regulation of GH Release by Nutritional Status and Metabolic Factors
5.2.3 Feedback Regulation of GH Secretion
5.3 Regulation of PRL Secretion
5.3.1 Dopamine Is the Primary Inhibitor of PRL Secretion
5.3.2 Other Factors Regulating PRL Secretion
5.3.3 Short Loop Feedback Regulation of PRL
5.4 Regulation of GH and PRL Secretion at the Level of the Pituitary
6. Physiologic Functions of GH and PRL
6.1 The Pattern of GH Secretion and Its Physiologic Consequences
6.2 Changes in GH Secretion Across the Lifespan
6.3 Physiologic Modifications of GH Secretion
6.3.1 Gonadal Steroids
6.3.2 Sleep
6.3.3 Stress
6.3.4 Pregnancy and Lactation
6.4 Changes in PRL Secretion Across the Lifespan
6.5 Physiologic Modifications of PRL Secretion
6.5.1 PRL secretion in Pregnant and Lactating Rodents
6.5.2 PRL Secretion in Pregnant and Lactating Women
6.6 GH and PRL in Circulation
6.7 The Physiologic Functions of GH
6.7.1 Skeletal Growth and Bone Metabolism
6.7.2 Metabolism
6.7.3 Protein Metabolism
6.7.4 Fat Metabolism
6.7.5 Carbohydrate Metabolism
6.7.6 Interaction With Insulin and Secretion
6.7.7 Electrolyte/Fluid Homeostasis
6.8 The Physiologic Functions of PRL
6.8.1 Mammary Gland Regulation
6.8.2 Pregnancy
6.8.3 Reproductive Behavior
6.8.4 Regulation of Hypothalamic Neurones
6.8.5 Effects on Metabolism
7. Disorders of the GH and PRL Axes
7.1 Hormone Deficiency
7.2 Congenital Causes of GH and PRL Deficiency
7.3 Acquired GH and PRL Deficiency
7.4 Consequences of GH and PRL Deficiency
7.5 Hormone Excess
7.6 Consequences of GH Excess
7.7 Consequences of PRL Excess
8. Conclusions
References
10. Posterior Pituitary Hormones
1. Introduction
2. Evolution of Neurohypophyseal Hormones
3. The Structure and Synthesis of Neurohypophyseal Hormones
3.1 Peptide Structure
3.2 Gene Structure
3.3 Synthesis of Vasopressin
3.4 Synthesis of Oxytocin
3.5 Distribution of Oxytocin- and Vasopressin-Synthesizing Neurons in the Brain
4. The Oxytocin/Vasopressin Receptor Family
4.1 Oxytocin Receptor
4.2 Vasopressin Receptors
5. Physiologic Functions and Behavior
5.1 Oxytocin
5.1.1 Oxytocin in Reproduction
5.1.2 Oxytocin in Cardiac Function
5.1.3 Oxytocin in Fluid Balance
5.1.4 Oxytocin Neurocircuitry and Behavior
5.1.4.1 Reproductive Behavior
5.1.4.2 Parental Care
5.1.4.3 Social Behavior
5.1.4.4 Stress Responsivity and Anxiety-Like Behavior
5.1.4.5 Osmoregulatory Behavior
5.1.4.6 Feeding Behavior
5.2 Vasopressin
5.2.1 Vasopressin in Water Balance
5.2.2 Vasopressin in Vasoregulation
5.2.3 Vasopressin Neurocircuitry and Behavior
5.2.3.1 Reproductive Behavior
5.2.3.2 Parental Care
5.2.3.3 Social Behavior
5.2.3.4 Stress Reactivity
6. Disease and Aging
6.1 Neurohypophyseal Hormones and Disease
6.2 Metabolic Disorders
6.3 Cardiovascular Disease
6.4 Neurodevelopmental Disorders
6.5 Neuropsychiatric Disorders
6.6 Neurodegenerative Disorders
6.7 Neurohypophyseal Hormones and Aging
7. Conclusions and Future Directions
Acknowledgments
References
11. Hormones and the Regulation of Neuronal Voltage-Sensing Ion Channels
1. Introduction
2. Ion Channels
3. Hormones and Neuronal Ion Channel Regulation
3.1 Peripheral Hormones
3.1.1 Leptin
3.1.2 Insulin
3.1.3 Ghrelin
3.1.4 Prolactin
3.2 Neurohormones
3.2.1 TRH
3.2.2 Oxytocin and Vasopressin
4. Concluding Remarks
References
12. Hormonal Regulation of Epithelial Sodium Channel (ENaC) and Other Nonneuronal Epithelial Ion Channels
1. The Epithelium
2. Structure of ENaC Channel
2.1 Amiloride Inhibitor and ENaC Current
2.2 Regulation of ENaC Mainly by Aldosterone and Other Hormones
2.3 Bradykinin
2.4 Endothelin
3. Renal Outer Medullary K+ (ROMK or Kir1) Channel
4. Potassium Channels Activated by Calcium, BK Channel (Maxi-K Channel)
4.1 Magnesium Channels (TRPM6 and TRPM7)
5. Epithelial Basolateral Na+/K+ ATPase Pump
6. Apical Brush Border Renal Phosphate Channels (SLC34 Transporters, NPT2 and NPT3)
6.1 Parathyroid Hormone
6.2 Fibroblast Growth Factor 23
7. Aquaporins
8. TRPV Epithelial Calcium Transport Channels (TRPV5 and TRPV6)
9. Chloride Transport, Cystic Fibrosis Transmembrane Conductance Regulator
10. Pendrin, Apical Iodide Transporting Channel in Thyroid Follicular Cell (Thyrocyte)
10.1 Aldosterone
11. Sodium/Iodide Transporter
12. MDR, Multidrug Resistance Channel, P-glycoprotein
References
Further Reading
13. Thymosins
1. Structures of Thymosin Proteins
1.1 Structures of Uncomplexed Thymosin Proteins
1.1.1 Prothymosin-α (ProTα), Parathymosin, and α-Thymosin-1 (Tα1)
1.1.2 Beta Thymosin Proteins
1.1.2.1 Thymosin Beta 4
1.1.2.2 Thymosin Beta 9 and Beta 10
1.2 Structures of Thymosin Proteins in Complexes and Chimeras
1.2.1 Structures of Alpha Thymosins in Complexes
1.2.2 Structures of Beta Thymosins in Complexes and Chimeras
2. Evolution of Thymosin Proteins
3. Biochemical Reactions
3.1 Alpha Thymosins
3.2 Beta Thymosins
4. Thymosins and Disease
4.1 Thymosins and Fibrosis
4.2 Thymosin and Viruses
4.2.1 HIV
4.2.2 Chronic Hepatitis
4.3 Thymosins and Vaccines
4.4 Thymosins and Stroke
4.5 Thymosins and Cancer
4.6 Thymosin and Liver
4.7 Cardiology
4.8 Sepsis
5. Conclusions and Future
References
Further Reading
14. Heart Hormones
1. Introduction
2. Natriuretic Peptides
2.1 Cardiac NPs
2.2 Evolution of NPs
2.3 NP Receptors
2.4 Structure of NPs
3. Physiologic Functions of NPs
3.1 Secretion of ANP
3.2 Clearance and Degradation of NPs
3.3 NPs Inhibit Cardiac Hypertrophy
3.4 NPs Regulate Cardiomyocyte Proliferation
3.5 NPs Inhibit Cardiac Fibrosis
3.6 NPs Decrease Blood Pressure
3.7 NPs Regulate Metabolism
3.8 NPs Control Satiety
4. Pathologic Functions of NPs
4.1 NP Secretion in Pathologic Conditions
4.2 NPs Suppress Inflammation
4.3 NPs Suppress Cardiovascular Diseases
4.4 NPs Affect Metabolic Diseases
4.5 NPs as Biomarkers
4.6 NPs as Therapeutic Targets
5. Other Cardiomyokines
5.1 Follistatin-Like 1
5.2 Secreted Phospholipase A2
5.3 Fibroblast Growth Factors
5.4 Others
6. Conclusions and Future Directions
References
15. Stomach Hormones
1. Gastric Cells Producing Signal Substances
2. Methods for Identifying Cells Producing Signal Substances
3. Types of “Endocrine” Cells in the Gastric Mucosa, Their Localization and Distribution
4. The EC Cell and 5-Hydroxytrypatamine (5-HT) (Serotonin)
5. The D Cell and Somatostatin
6. The G Cell and Its Regulation
7. The ECL Cell
8. Gastrin
8.1 History
8.2 Structure
8.3 Physiology
8.4 Role in Disease
9. A-Like Cells and Ghrelin/Obestatin
9.1 History
9.2 Structure
9.3 Physiology
9.4 Obestatin
10. Neural Influence on Gastric Secretory and Signal-Producing Cells
11. Conclusion
References
16. Intestinal Hormones
1. Overview of the Intestinal Hormones
2. The Intestinal Enteroendocrine Cells and the Enteroendocrine System
3. The Main Intestinal Hormones
3.1 Secretin
3.2 Cholecystokinin
3.3 Neurotensin
3.4 Peptide YY3−36
3.5 Incretins
3.5.1 Glucose-Dependent Insulinotropic Peptide
3.5.2 Glucagon-Like Peptide 1
3.6 Glucagon-Like Peptide 2
3.7 Oxyntomodulin
4. The Paradigm of Coevolution of Glucagon-Like Receptors and Their Ligands
5. The Glucagon-Like Peptide-1 Receptor
6. The Glucagon-Like Peptide-1 Receptor Ligands
7. Intestinal Hormones in the Pathogenesis of T2D
8. Intestinal Hormones in the Treatment of T2D
9. Intestinal Hormones in the Pathogenesis of Obesity
10. Intestinal Hormones in the Treatment of Obesity
11. Conclusions and Future Directions
References
Further Reading
17. Pancreatic Hormones
1. The Endocrine Pancreas
1.1 History
1.2 Evolution of the Endocrine Pancreas
1.3 Developmental Aspects
1.4 Cellular Architecture of the Islets of Langerhans
2. Insulin
2.1 History
2.2 Structure of Insulin
2.3 Biosynthesis, Processing, and Secretion
2.4 Insulin Clearance and Inactivation
2.5 The Insulin Receptor and Its Apo Structure
2.6 Mechanism of Insulin Receptor Binding
2.7 Structure of the Site 1 Insulin Receptor Complex
2.8 Structure of the Insulin Receptor Kinase and Mechanism of Activation
2.9 Insulin Intracellular Signaling Pathways
2.10 Pathophysiologic and Clinical Implications of Insulin Signaling
3. Glucagon
3.1 History
3.2 Amino Acid Sequence, Evolution, and Structure
3.3 Biosynthesis and Processing
3.4 The Glucagon Receptor
3.5 Glucagon Receptor Inactivation and Mutations
3.6 Glucagon Receptor Signaling Pathways and Endpoint Metabolic Effects
3.7 Control of Secretion
3.8 Physiologic Functions
3.9 Pathophysiologic and Therapeutic Implications for Diabetes Mellitus and Obesity
4. Other Pancreatic Hormones
4.1 Somatostatin
4.1.1 History
4.1.2 Amino Acid Sequence, Evolution, and Structure (Ando, 2016)
4.1.3 Biosynthesis and Processing (Ando, 2016)
4.1.4 Somatostatin Receptors and Signaling Pathways
4.1.5 Physiologic Actions (Huang, 1997; Ando, 2016)
4.1.6 Pathophysiologic and Therapeutic Implications (Ando, 2016)
4.2 Pancreatic Polypeptide (Williams, 2014)
4.2.1 History
4.2.2 Amino Acid Sequence, Evolution, and Structure
4.2.3 Biosynthesis, Processing, and Secretion
4.2.4 PP Receptors and Signaling Pathways
4.2.5 PP Receptors Inactivation
4.2.6 Physiologic Actions
4.2.7 Pathophysiologic and Therapeutic Implications
4.3 Amylin
4.3.1 History
4.3.2 Amino Acid Sequence and Structure
4.3.3 Biosynthesis, Processing, and Secretion (Ogoshi, 2016)
4.3.4 The Amylin Receptor and Signaling Pathways (Ogoshi, 2016)
4.3.5 Physiologic Functions
4.4 Ghrelin
4.5 Urocortin III
4.6 Stanniocalcin 2
4.7 Serotonin-Producing Enterochromaffin Cells and Gastrin-Producing G Cells
4.8 Gamma-Aminobutyric Acid (GABA) Production by β cells (Braun et al., 2010)
5. Conclusions and Perspectives
Acknowledgments
References
18. Liver Hormones
1. Angiotensinogen
1.1 Structure
1.2 Evolution
1.3 Biochemical Reactions
1.4 Physiological Functions
1.5 Mouse Models with Altered AGT Expression
1.6 AGT in Human Disease
1.7 Conclusions and Clinical Applications
2. Hepcidin
2.1 Structure
2.2 Evolution
2.3 Biochemical Reactions
2.4 Physiological Functions
2.5 Hormone Inactivation
2.5.1 Hereditary Hemochromatosis
2.5.2 Iron-Loading Anemias
2.5.3 Chronic Liver Diseases
2.6 Hormone Hyperactivation
2.6.1 Anemias with Iron-Restricted Erythropoiesis
2.6.2 Chronic Liver Diseases
2.7 Conclusions and Clinical Applications
3. Insulin-Like Growth Factors
3.1 Structure
3.2 Evolution
3.3 Biochemical Reactions
3.4 Physiological Functions
3.5 Hormone Inactivation
3.5.1 Growth Disorders
3.5.2 Metabolic Disorders
3.6 Hormone Hyperactivation
3.6.1 Acromegaly
3.6.2 Cancer
3.7 Conclusions and Clinical Applications
4. Thrombopoietin
4.1 Structure
4.2 Evolution
4.3 Biochemical Reactions
4.4 Physiological Functions
4.5 Hormone Inactivation
4.5.1 Congenital Amegakaryocytic Thrombocytopenia
4.5.2 Thrombocytopenia in Liver Disease
4.5.3 Exogenous TPO-Induced Immune Thrombocytopenia
4.6 Hormone Hyperactivation
4.6.1 Congenital Thrombocythemia
4.6.2 Myeloproliferative Neoplasms
4.7 Conclusions and Clinical Applications
References
19. The Endocrine Kidney: Local and Systemic Actions of Renal Hormones
1. Introduction
2. Renin-Angiotensin System
2.1 Angiotensin II-Activated Cellular Signaling
2.2 Adverse Effects of AngII in Chronic Kidney Disease
3. Vasoconstrictors: Endothelin-1 and Urotensin II
3.1 Endothelin-1
3.1.1 Endothelin Receptors and Signaling Mechanisms
3.1.2 Modulators of ET-1
3.1.3 Actions of ET-1
3.1.4 Endothelin-Enhanced H+ excretion
3.1.5 Endothelin-I in Chronic Kidney Diseases
3.2 Urotensin II
3.2.1 Ca2+-Mediated UII Signaling
3.2.2 Urotensin II in Chronic Diseases
4. The Natriuretic Peptides: ANP, BNP, and Urodilatin
4.1 Natriuretic Peptide Signaling
4.2 Renal Actions of Natriuretic Peptides
4.3 Natriuretic Peptides: Potential Interventions for Heart Failure Management
5. Calcitonin Gene-Related Peptides: Adrenomedullin and Intermedin
5.1 Adrenomedullin
5.1.1 Adrenomedullin Signaling Mechanisms
5.1.2 Physiologic Actions of Adrenomedullin
5.1.3 Renoprotective Mechanisms of Adrenomedullin
5.1.4 Antihypertensive Actions of Adrenomedullin
5.2 Intermedin
5.2.1 Physiologic Effects of Intermedin
5.2.2 Intermedin's Actions Against Renal and Cardiovascular Disease
6. Calcitriol and Ca2+ Metabolism
6.1 Regulation of Calcitriol Activity
6.2 FGF23-Klotho
6.3 Calcitriol in Renal Disease
6.4 Estrogen Replacement and Calcitriol
7. Erythropoietin
7.1 O2 regulation of Renal Erythropoietin Production
7.2 Erythropoietin: Renoprotective Hormone?
8. Summary
Acknowledgments
References
20. Adipocyte-Derived Hormones
1. Introduction
2. Leptin
2.1 Leptin Structure
2.2 Leptin Signaling and Metabolic Function
2.3 Other Functions of Leptin
2.4 Leptin and Cancer
2.5 Future of Leptin
3. Adiponectin
3.1 Adiponectin Structure
3.2 Adiponectin Signaling
3.3 Adiponectin Functions
4. Resistin
4.1 Background on Resistin/FIZZ3/ADSF
4.2 Resistin Structure
4.3 Complexities of Resistin Expression
4.4 Resistin Signaling
4.5 Physiological and Pathologic Roles of Resistin
4.6 Conclusions and Future Directions of Resistin Research
5. Future Outlook for Adipokines and Adipose Tissue Secreted Factors
Acknowledgments
References
21. Thyroid Hormones
1. Introduction
1.1 Structure and Synthesis of Thyroid Hormone
1.2 Evolution of Thyroid Hormone
1.3 Thyroid Hormone Transport
1.4 Thyroid Hormone Receptors
1.5 TH/THR Signaling Pathways and Mechanisms
1.6 Physiological Actions Development and Tissue-Specific Expression of THR
1.7 Regulation of Thyroid Hormone Metabolism
1.8 Functional Thyroid Diseases, Clinical Manifestations, and Pathophysiology
1.9 Changes in the Thyroid and Thyroid Function with Age
1.10 Conclusions and Future Directions
Acknowledgments
References
22. Parathyroid Hormones
1. Introduction
2. Structures
2.1 Parathyroid Hormone
2.1.1 PTH Synthesis and Secretion from Parathyroid Glands
2.1.2 PTH Regulation by Serum Calcium and Phosphate Homeostasis
2.1.3 PTH Regulation by Magnesium Homeostasis
2.1.4 PTH Regulation by Fluoride
2.1.5 PTH Regulation by Vitamin D
2.1.6 PTH Regulation by FGF23
2.1.7 Other PTH Fragments
2.1.8 PTH Mutations
2.1.8.1 Hyperparathyroidism
2.1.8.2 Hypoparathyroidism
2.2 PTH Receptors
2.2.1 PTH/PTHrP Receptor 1
2.2.1.1 PTH/PTHrP Receptor 1 Expression
2.2.1.2 PTH/PTHrP Receptor 1 Mutations
2.2.1.2.1 Loss of Function Mutations
2.2.1.2.2 Gain of Function Mutations
2.2.2 PTH Receptor 2
3. Biochemical Reactions
3.1 Interaction of the Hormones/Receptor
3.2 Signaling Pathways Activated by PTHR1
3.2.1 Canonical G Protein–Dependent Pathways
3.2.2 PTHR1 and Epigenetic Regulation
3.2.3 Β-Arrestin Pathway
3.3.1 Wnt Pathway
4. Physiologic Function of PTH
4.1 PTH in Calcium/Phosphate/Vitamin D Homeostasis
4.2 PTH and FGF23
4.3 PTH and Bone Remodeling
4.3.1 Anabolic Action of PTH
4.3.2 Catabolic Action of PTH
4.3.2.1 OPG/RANKL Balance
4.3.2.2 Monocyte Chemoattractant Protein-1 (MCP1)
5. Conclusions and Future Directions
References
23. Hormones and Hormone Precursors of the Skin
1. Introduction
2. Skin Function and Anatomy
3. Hormones Produced in the Skin
3.1 Vitamin D
3.1.1 Evolution and Vitamin D
3.1.2 Structure and Synthesis of Vitamin D
3.1.3 Structure of VDR
3.1.4 Biochemical Reactions (VDR Activation and Signaling)
3.1.4.1 Genomic Actions of VDR
3.1.4.2 Nongenomic Actions of VDR
3.1.5 Vitamin D Signaling in Physiology and Pathophysiology
3.1.5.1 VDR Signaling in Skin
3.1.5.2 Skeletal Effects of Vitamin D
3.1.5.3 Nonskeletal Effects of Vitamin D
3.1.6 Hormone Inactivation
3.1.7 Vitamin D and Aging
3.2 HPA Axis Equivalent of the Skin
3.2.1 Expression of HPA Axis Hormones in the Skin
3.2.2 Skin HPA Axis and UV
3.3 Steroid Regulation of the Skin
3.3.1 Sex Steroids
3.3.1.1 Androgens in the Skin
3.3.1.2 Estrogens in the Skin
3.3.2 Corticosteroids in the Skin
4. Skin Hormones in Dermatology
4.1 Acne
4.2 Rosacea
4.3 Atopic Dermatitis
4.4 Alopecia
4.5 Psoriasis
4.6 Skin Cancer
5. Future Directions
References
24. Hormones of the Testes
1. Steroid Hormones
2. Testicular Steroidogenesis
3. Secretion and Transport of Testicular Steroids
3.1 Associating Proteins
4. Testosterone is a Direct Precursor for Other Bioactive Steroids: DHT and Estrogen
5. Metabolism of Testicular Steroids
6. Function: Testosterone and DHT Receptors: Crosstalk of Genomic and Nongenomic Mechanisms
7. Testicular Protein and Peptide Hormones: Inhibin, Activin, and Follistatin
References
25. Ovarian Hormones
1. Introduction
1.1 Age-Related Changes in Ovarian Hormones
2. Estradiol
2.1 Structure
2.2 Synthesis
2.2.1 Aromatase
2.2.2 Estradiol Is Produced in Incomplete Synthesis Pathways
2.2.3 Regulation of Estradiol Synthesis
2.3 Estradiol Receptor and Signaling
2.3.1 Membrane Estrogen Receptors
2.4 Physiological Effects of Ovarian Estrogens
2.4.1 Ovarian Effects
2.4.2 Estradiol Guards the Embryo in the Oviduct
2.4.3 Estradiol Effects on the Uterus
2.4.4 Estradiol Regulation of the Mammary Gland
2.4.5 Effects of Estradiol on LH Secretion
2.4.6 Nonreproductive Effects of Estradiol
3. Progesterone
3.1 Structure and Synthesis
3.1.1 Hormonal Regulation
3.2 Progesterone Receptor and Signaling
3.2.1 Membrane Progesterone Receptors
3.3 Physiological Effects of Progesterone
3.3.1 Autoregulation of Ovarian Function by Progesterone
3.3.2 The Fallopian Tube
3.3.3 Progesterone Effects in the Uterus
3.3.4 Progesterone Regulation of the Mammary Gland
3.3.5 Effects of Progesterone on LH Secretion
3.3.6 Nonreproductive Effects of Progesterone in the Brain
4. Conclusions
Acknowledgments
References
26. Muscle Hormones
1. Introduction
2. Interleukin-6
2.1 Structure of IL-6 and Its Receptor Complex
2.2 Biochemical Reactions of IL-6: Signaling and Secretion
2.2.1 Mechanisms of Signal Transduction Induced by IL-6
2.2.2 IL-6 Signaling in Skeletal Muscle
2.2.3 IL-6 Production and Secretion by Muscle Cells
2.3 Physiological Functions of IL-6: Muscle IL-6 in Health and Disease
2.3.1 Muscle IL6 Physiology and Effects in Skeletal Muscle
2.3.2 Effects of Muscle IL-6 in Adipose Tissue
2.3.3 Effects of Muscle IL-6 in Brain
2.3.4 Effects of Muscle IL-6 on Liver Glucose Homeostasis
2.3.5 Effects of Muscle IL-6 in Pancreas: More About Glucose Homeostasis
2.3.6 IL-6 as an Antiinflammatory Myokine
2.3.7 Muscle IL-6 in Health and Disease, Cancer, and Beyond
3. Irisin
3.1 Structure of Irisin
3.2 Biochemical Reactions of Irisin: Signaling and Secretion
3.3 Physiological Functions of Irisin and Their Relation With Health and Disease
3.3.1 Effects of Irisin in Skeletal Muscle
3.3.2 Effects of Irisin in Adipose Tissues
3.3.3 Effects of Irisin in Bone
3.3.4 Effects of Irisin in Brain and Cognitive Function
3.3.5 Effects of Irisin in Liver
3.3.6 Effects of Irisin in Pancreas
3.3.7 Antiinflammatory Effects of Irisin
3.3.8 Irisin Effects on Disease and Aging
4. Other Myokines
5. Conclusions and Future Directions
References
27. Hormones From Bone
1. Confirmed Bone-Derived Endocrine Hormones
1.1 Fibroblast Growth Factor-23
1.1.1 Structures
1.1.2 Evolution
1.1.3 Biochemical Reactions
1.1.4 Physiologic Functions
1.1.5 Hormone Inactivation
1.1.6 Disease and Aging Effects
1.1.6.1 Inherited Loss-of-Function Disorders
1.1.6.2 Inherited Gain-of-Function Disorders
1.1.6.3 Tumor-Induced Osteomalacia
1.1.6.4 Renal Failure
1.1.6.5 Cardiovascular Disease
1.1.6.6 Aging
1.2 Osteocalcin
1.2.1 Structures
1.2.2 Evolution
1.2.3 Biochemical Reactions
1.2.4 Physiologic Functions
1.2.5 Hormone Inactivation
1.2.6 Disease and Aging Effects
2. Bone-Derived Paracrine Factors That Reach the Circulation
2.1 Sclerostin
2.2 RANK-L
3. Bone-Derived Paracrine Factors That Are Hormones When Produced Elsewhere
3.1 IGF-I and II
3.2 Parathyroid Hormone-Related Protein
3.3 Prostaglandins
3.4 Calcitriol
4. Conclusions and Future Directions
References
28. Adrenal Cortex Hormones
1. Adrenal Glands: Anatomy and Histology
2. Biosynthesis of Adrenal Cortex Hormones
3. Evolution of Adrenal Cortex Hormone Receptors
4. Glucocorticoids
4.1 Regulation of Glucocorticoid Secretion
4.2 Glucocorticoid Receptor
4.3 Glucocorticoid Signaling
5. Mineralocorticoids
5.1 Regulation of Mineralocorticoid Secretion
5.2 Mineralocorticoid Receptor
5.3 Mineralocorticoid Signaling
6. Adrenal Androgens
7. Clinical Implications
7.1 Adrenal Insufficiency
7.2 Congenital Adrenal Hyperplasia
7.3 Primary Generalized Glucocorticoid Resistance (Chrousos Syndrome)
7.4 Mineralocorticoid Resistance
8. Conclusions and Future Directions
References
Further Reading
29. Adrenal Medulla Hormones
1. Anatomy and Functional Organization of the Adrenal Medulla
1.1 General Organization of the Adrenal Medulla
1.2 Blood Supply to the Adrenal Medulla
1.3 Innervation of the Adrenal Medulla
2. Biosynthesis and Metabolism of Adrenal Catecholamines
2.1 Biosynthetic Pathway for Norepinephrine and Epinephrine
2.2 Catabolism of Norepinephrine and Epinephrine
3. Epinephrine and Norepinephrine Act Through G Protein–Coupled Adrenoceptors
3.1 GPCR Signaling Through β-Arrestin
4. Release of Catecholamines From Adrenal Chromaffin Cells (Stimulus-Secretion Coupling)
4.1 Autocrine/Paracrine Control of Adrenal Catecholamine Secretion
5. Physiologic Effects of Adrenal Catecholamines
5.1 The Sympathoadrenal Stress Response
5.2 Cardiovascular System Effects of Adrenal Catecholamines
5.3 Metabolic Effects of Adrenal Catecholamines
5.4 Effects of Adrenal Catecholamines on Pulmonary and Gastrointestinal Smooth Muscle
5.5 Other Effects of Adrenal Catecholamines
6. Adrenal Catecholamines and Pathophysiology
6.1 Pheochromocytoma
6.2 Cardiovascular Pathologies Associated With Adrenal Catecholamines
6.3 Metabolic Pathologies Associated With Adrenal Catecholamines
6.4 Other Pathologies Associated With Adrenal Catecholamines
7. Summary
References
30. Overproduction of Hormones by Pituitary Tumors
1. Introduction
2. Pituitary Adenomas
3. Classification of Pituitary Adenomas
4. Overproduction of Growth Hormone
5. Overproduction of Adrenocorticotropic Hormone
6. Overproduction of Prolactin
7. Overproduction of Thyroid-Stimulating Hormone
8. Overproduction of Follicle-Stimulating Hormone and Luteinizing Hormone
9. Conclusion
References
31. Hormone Effects on Tumors
1. Introduction
2. Corticosteroids
2.1 Tumors Affected by Corticosteroids
2.2 Mechanisms of Tumor Promotion
2.3 Agents, Approaches, and Mechanisms of Tumor Inhibition and Interference
2.4 Corticosteroid Resistance
3. Future Directions in Corticosteroid Therapeutic Approaches
3.1 Synergistic Actions of Corticosteroid Hormones With Novel Therapeutic Agents
3.2 Corticosteroid Regulation of Micro-RNAs
3.3 Antiinflammatory Action of Corticosteroids
4. Androgens
4.1 Perspective on Androgen Metabolism in Prostate Cancer
4.2 Androgen Deprivation Therapy
4.3 Targeting Androgen Synthesis and Androgen Receptor Blockade
5. Estrogens and Progestogens
5.1 Tumors Affected by Estrogens and Progestogens
5.2 Mechanisms of Tumor Promotion
5.3 Agents, Approaches, and Mechanisms of Tumor Inhibition and Interference
5.4 Breast Cancer Resistance to Endocrine Therapy
5.5 Future Directions
6. Insulin and Insulin-Like Peptides
6.1 Insulin, IGFs, and Insulin Receptors
6.2 Insulin-Promoted Tumors
6.3 Insulin Resistance and Hyperinsulinemia
6.4 Insulin Signaling as a Therapeutic Target
6.5 Oral Antidiabetic Agents in Cancer Therapy
7. Insulin-Like Growth Factors
7.1 IGFs and Cancer-Related Syndromes
7.2 IGFs and IGF Receptors
8. Growth Hormone
9. Adipocytokines
9.1 Leptin Effects on Tumors
9.2 Adiponectin Effects on Tumors
9.3 Visfatin Effects on Tumors
9.4 Retinol Binding Protein 4
Acknowledgments
References
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
R
S
T
U
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Back Cover

Citation preview

HORMONAL SIGNALING IN BIOLOGY AND MEDICINE COMPREHENSIVE MODERN ENDOCRINOLOGY Edited by

Gerald Litwack Emeritus Professor and Chair of Basic Sciences, Geisinger Commonwealth School of Medicine, Scranton, PA, United States Formerly Professor of Molecular and Cellular Medicine and Associate Director, Institute for Regenerative Medicine, Texas A&M Health Science Center, Temple, TX, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-813814-4 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisition Editor: Tari Broderick Editorial Project Manager: Timothy Bennett Production Project Manager: Sreejith Viswanathan Cover Designer: Miles Hitchen Typeset by TNQ Technologies

Contributors Marah Armouti Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA

Giulia Cantini Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy

John A. Arnott Geisinger Commonwealth School of Medicine, Scranton, PA, United States

Ayano Chiba Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Suita, Japan

Kushal Bakshi Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, United States

George P. Chrousos Division of Endocrinology, Metabolism and Diabetes, First Department of Pediatrics, National and Kapodistrian University of Athens Medical School, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece; Division of Endocrinology and Metabolism, Center of Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

Natalie J. Bales Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, United States Mary Beth Bauer Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, NJ, United States

Sila Cocciolillo Royal Victoria Hospital, McGill University Health Center, and Department of Medicine, McGill University, Montreal, QC, Canada

Nathan A. Berger Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, United States; Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, United States; Department of Medicine, Case Western Reserve University, Cleveland, OH, United States; Department of Biochemistry, Case Western Reserve University, Cleveland, OH, United States

R. Comaposada-Baro´ Molecular Basis of Neurodegeneration Unit, Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain Kevin P.M. Currie Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, NJ, United States

Daniel J. Bernard Departments of Pharmacology and Therapeutics, and Anatomy and Cell Biology, McGill University, Montreal, QC, Canada

Pierre De Meyts Department of Cell Signalling, de Duve Institute, Brussels, Belgium; Department of Stem Cell Research, Novo Nordisk A/S, Ma˚løv, Denmark

Mark Blostein Lady Davis Institute for Medical Research, Jewish General Hospital and Department of Medicine, McGill University, Montreal, QC, Canada

Ilaria Dicembrini Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy; Careggi University Hospital (AOUC), Florence, Italy

M. Luisa Bonet Laboratory of Molecular Biology, Nutrition and Biotechnology (Nutrigenomics), University of the Balearic Islands (UIB), CIBER Fisiopatologı´a de la Obesidad y Nutricio´n (CIBEROBN), and Institut d’Investigacio´ Sanita`ria Illes Balears (IdISBa), Palma de Mallorca, Spain

Clark W. Distelhorst Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, United States; Department of Medicine, Case Western Reserve University, Cleveland, OH, United States Nikoletta Dobos Department of Biopharmacy, Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary

Amanda P. Borrow Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, United States

David E. Fisher Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, United States

Gregory A. Brent Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Physiology David Geffen School of Medicine at UCLA Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, California, US Emilie Bruˆle´ Departments of Pharmacology and Therapeutics, and Anatomy and Cell Biology, McGill University, Montreal, QC, Canada

M.L. Franco Molecular Basis of Neurodegeneration Unit, Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain Gabor Halmos Department of Biopharmacy, Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary; Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL, United States

Max H. Cake School of Veterinary and Life Sciences, Murdoch University, Perth, WA, Australia

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CONTRIBUTORS

Robert J. Handa Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, United States Elie Hobeika Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA Eva Juhasz Institute of Pediatrics, Clinical Center, University of Debrecen, Debrecen, Hungary Hamsini Sudheer Kala Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA Lajos V. Kemeny Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, United States Ruth A. Keri Department of Pharmacology, Case Western Reserve University, Cleveland, OH, United States; Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, United States; Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, United States

David J. Lyons School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom Rong Ma Department of Physiology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX, United States Robert T. Mallet Department of Physiology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX, United States Edoardo Mannucci Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy; Careggi University Hospital (AOUC), Florence, Italy Michael J. Shipston Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, UK Anna Milanesi Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Physiology David Geffen School of Medicine at UCLA Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, California, US

Haruka Kobayashi Institute of Molecular Medicine, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Tokushima University Faculty of Medicine, Tokushima, Tokushima, Japan

Naoki Mochizuki Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Suita, Japan; AMED-CREST

Christopher S. Kovacs Faculty of Medicine, Memorial University of Newfoundland, Health Sciences Centre, St. John’s, NL, Canada Kalman Kovacs Department of Laboratory Medicine, Division of Pathology, University of Toronto, Toronto, ON, Canada; The Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada

Nicolas C. Nicolaides Division of Endocrinology, Metabolism and Diabetes, First Department of Pediatrics, National and Kapodistrian University of Athens Medical School, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece; Division of Endocrinology and Metabolism, Center of Clinical, Experimental Surgery and Translational Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

Pierre J. Lefe`bvre Division of Diabetes, Nutrition and Metabolic Diseases, Department of Medicine, CHU, Lie`ge, Belgium

Kostas Pantopoulos Lady Davis Institute for Medical Research, Jewish General Hospital and Department of Medicine, McGill University, Montreal, QC, Canada

Carole Le Henaff Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, NY, United States

Nicola C. Partridge Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, NY, United States

P.R. Le Tissier Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom

Doodipala Samba Reddy Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, United States

Gerald Litwack Formerly Institute for Regenerative Medicine, Texas A&M Health Science Center, Temple, TX, United States Yan-Yun Liu Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Physiology David Geffen School of Medicine at UCLA Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, California, US Michaela Luconi Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy; Careggi University Hospital (AOUC), Florence, Italy

J.F. Murray Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom

Lina M. Restrepo Department of Endocrinology, Clinica Medellin - Grupo Quiro´nsalud, Medellin, Colombia Joan Ribot Laboratory of Molecular Biology, Nutrition and Biotechnology (Nutrigenomics), University of the Balearic Islands (UIB), CIBER Fisiopatologı´a de la Obesidad y Nutricio´n (CIBEROBN), and Institut d’Investigacio´ Sanita`ria Illes Balears (IdISBa), Palma de Mallorca, Spain Allison J. Richard Pennington Biomedical Research Center, Baton Rouge, LA, United States

CONTRIBUTORS

Ana M. Rodrı´guez Laboratory of Molecular Biology, Nutrition and Biotechnology (Nutrigenomics), University of the Balearic Islands (UIB), CIBER Fisiopatologı´a de la Obesidad y Nutricio´n (CIBEROBN), and Institut d’Investigacio´ Sanita`ria Illes Balears (IdISBa), Palma de Mallorca, Spain Nicola Romano` Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, UK Fabio Rotondo Department of Laboratory Medicine, Division of Pathology, University of Toronto, Toronto, ON, Canada; The Keenan Research Centre for Biomedical Science at the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada Andrew V. Schally Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL, United States; Department of Pathology, Divisions of HematologyeOncology and Endocrinology, Miller School of Medicine, University of Miami, Miami, FL, United States; Department of Medicine, Divisions of Hematology eOncology and Endocrinology, Miller School of Medicine, University of Miami, Miami, FL, United States; Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, United States Giada Sebastiani Royal Victoria Hospital, McGill University Health Center, and Department of Medicine, McGill University, Montreal, QC, Canada Carlos A. Serna Laboratorio de Patologia y Citologia Rodrigo Restrepo, Department of Pathology, Clinica Las Ame´ricas, Universidad CES, Medellin, Colombia Nima Sharifi Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, United States; Genitourinary Malignancies Research Center, Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, United States Sonia Lobo Geisinger Commonwealth School of Medicine, Scranton, PA, United States Jacqueline M. Stephens Pennington Biomedical Research Center, Baton Rouge, LA, United States; Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, United States

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Carlos Stocco Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA Sally A. Stover Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, United States Luis V. Syro Department of Neurosurgery, Hospital Pablo Tobon Uribe, and Clinica Medellin - Grupo Quiro´nsalud, Medellin, Colombia Zsuzsanna Szabo Department of Biopharmacy, Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary Martina Trabucco Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy M. Vilar Molecular Basis of Neurodegeneration Unit, Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain David E. Volk Institute of Molecular Medicine, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States Helge Waldum Faculty of Medicine, Norwegian University of Science and Technology and St. Olav’s University Hospital, Trondheim, Norway Richard J. Wurtman Massachusetts Institute of Technology, Cambridge, MA, United States George C.T. Yeoh Harry Perkins Institute of Medical Research, QEII Medical Centre, Perth, WA, Australia Yue Yu Institute of Molecular Medicine, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Shanghai Jiao Tong University School of Medicine, Shanghai, China Eleonora Zakharian Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL, United States

Preface

Hormones are the messengers from specialized tissues to other tissues, near and far, of the body, so the complex organism can function efficiently. Modern research not only informs of the specific receptors for hormones, located distally, in the neighborhood, or even in the cell producing the hormone, but of the pathways that are stimulated or depressed inside the target cell as a result of receptor activation by the hormonal ligand. Emphasis in this volume is placed upon the physiologic effects of hormones, their interactions with specific receptors, and the consequent metabolic pathways within the target cells that are affected. In short, much is now known about the exact mechanisms by which hormones function, either upon local or upon distant cells within the organism. In addition, there is emphasis on the molecular structures of hormones and their receptors, so many chapters will illustrate X-ray crystal structures of the hormone under discussion, its receptor, and in some cases the complex of the receptor with its ligand. In brief, such an approach fulfills the understanding of comprehensive modern endocrinology. In this book, the classification of hormones is organized by the tissue cells that synthesize them. Nowadays, it is recognized that, occasionally, different types of cells may be the sites of synthesis of the same hormone, and this diversity is dealt with. The organization extends from the upper part of the body to the lower, the brain, and then downward. However, there are exceptions. First, there are two introductory chapters that survey hormones and perinatal development, followed by a chapter on hormones of programmed cell death, a phenomenon critical to the homeostasis of cell number. The effective organization of topics follows, first with the brain, as exemplified by successive chapters on hypothalamic releasing hormones, neurosteroids, neurotrophins, the pineal gland, and the anterior and posterior pituitary. The hormonal regulation of ion channels is then considered in two chapters, the first dealing with neural ion channels and the second dealing with epithelial nonneural ion channels. The thymosins are considered next. Originally, thymosin was considered to be a hormone of the thymus, and while one of the key thymosins is synthesized in the thymus, other key thymosins are synthesized in other tissues, however, not detracting from their potential importance. The sequence of the subsequent chapters returns to the original classification. The next chapter focuses on hormones produced in the heart and their actions. Similar approaches are illuminated in the following chapters, starting with the stomach, the intestine, pancreas, liver, kidney, adipose tissue (now certainly considered to be a hormone-producing gland), thyroid, parathyroid, skin, testes, ovaries, muscle, and adrenal gland (consisting of a chapter on the adrenal cortex and another chapter on the adrenal medulla). Finally, there are two chapters that emphasize clinical observations, the first dealing with pituitary tumors producing (ectopic) hormones and the second relating the effects of hormones upon tumors. Selecting 30 corresponding authors and approaching them to prepare manuscripts that emphasized the aspects envisioned was a hefty proposition. Fortunately, I had some help. Initially, I thought that the anterior pituitary hormones could be covered by a single author. That proved to be unrealistic. Here, I must recognize the fact that Dr. Daniel Bernard of McGill University suggested that the topic be divided into three portions, one of which he would undertake, and then he recommended two other authors for the remaining sections. Thankfully, they both agreed. I am most grateful to Dan for his help. Dr. Nathan Berger helped with the title, so it would be suitably representative of clinical interest as well as that of the basic sciences. Nate is an old friend and I was very open to his suggestion. Narmatha Mohan of Elsevier agreed to take on the awesome responsibility for obtaining permissions to reproduce figures and other materials existing in the literature for all of the authors. If required of the authors, themselves, this could have been a draining task. I am most grateful to Narmatha for shouldering this responsibility. As with my earlier experiences spanning more than 30 years, Tari Broderick of Elsevier was always available to smooth out any wrinkles. It has ever been a great pleasure to collaborate with her. Finally, perhaps the greatest burden for mounting manuscripts on the electronic submission system (EMSS) and for keeping myself and all of the information we have gathered on the straight and narrow, Timothy Bennett of Elsevier took up the reins and, obviously, was a key player in the production of this book. I extend my thanks for his efforts.

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PREFACE

The book cover is, in my view, a critical item. Searching for an impressive figure from the literature to grace the cover presented a number of problems. First, one would have to know whether such a figure had ever been used on the cover of some other book. To evade this problem, we decided to use a figure from one of the chapters in this book, and I had to make the selection. With this accomplished, the book cover designer at Elsevier would render three versions of a cover, after which I could switch elements of any of the three around to come up with a cover that, to me, would be striking and attractive. Fig. 15 of Chapter 18 was selected as a suitable figure. This is a crystal structure of the receptorbinding domainof thrombopoietin complexed to a neutralizing antibody (TnFab), a structure from the Protein Data Bank (PDB ID: 1V7N) that is in the public domain. Chapter 18 is entitled “Liver Hormones” by S. Cocciodillo, G. Sebastiani, M. Blostein, and K. Pantopoulos. Considering the coverage of virtually all of the known hormones, their receptors, the mechanisms of signaling from the activated receptor onward within the target cell together with structural aspects represented the best of what we know about modern endocrinology. In addition, virtually every chapter considers diseases evolving from hormone insufficiency or hormone excess and also the roles of participants in a given hormonal pathway that lead to disease. Therefore, it came to mind that the subtitle “Comprehensive Modern Endocrinology” would be beneficial. I have to thank Tari for agreeing with this proposition. As a result of this considerable effort, my hope is that this book will be instrumental in teaching, where, as a source, an instructor could mine the material needed for a specific lecture and, as well, make use of the extensive illustrative materials. In addition to its potential value for instructors who are responsible for various courses or specific lectures, I anticipate that both researchers and clinicians will find this to be a vital resource for reference. Gerald Litwack North Hollywood, CA June 10, 2019

C H A P T E R

1 Hormones and Perinatal Development Max H. Cake1, George C.T. Yeoh2 1

School of Veterinary and Life Sciences, Murdoch University, Perth, WA, Australia; 2Harry Perkins Institute of Medical Research, QEII Medical Centre, Perth, WA, Australia

1. INTRODUCTION

regulatory proteins with transcriptional response sequences. This causes activation or inhibition of expression of specific genes, resulting in an altered level of the protein product. Although there are numerous examples that could be chosen to highlight the importance of hormones in activating specific developmental events, this chapter will focus on the postnatal development of key processes in the liver (gluconeogenesis, amino acid catabolism, and urea cycle function), the lung (surfactant production), and the intestine (digestion) of eukaryotes and especially address the impact of glucocorticoids, glucagon, epinephrine, and the inhibitory effects of insulin and androgens. The processes discussed are vital to postnatal survival, and if they are impaired, at a minimum, the health status of the postnatal organism is compromised.

Cell division is one of the most fundamental activities of multicellular existence. It is obviously important during embryological development, during which the single, fertilized ovum undergoes a complex, programmed sequence of cell divisions to generate a mature organism consisting of many millions of cells. Even after a higher animal has reached maturity, cell division is required to replenish those tissues that have suffered cell losses due to wear and tear. For instance, based on a blood volume of 4.7 L, human erythrocytes having a lifespan of 120 days and being present in blood at a concentration of 5  1012 cells/L (Alberts et al., 2002), it was calculated that, just to maintain the erythrocyte population, an adult human must produce >2.3 million new erythrocytes per second, which confirms the value previously published (Sackmann, 1995). In this context, erythropoietin is the principal mediator that regulates the erythron (Adamson, 1996). There are a host of tissue-specific growth factors and hormones that determine the size of different organs of the body (Hafen and Stocker, 2003). Numerous studies have shown, however, that hormones are essential for many eukaryotic developmental processes as well as for cellular homeostasis. Work from the laboratories of O’Malley (Tsai et al., 1978) and Chambon (Wasylyk et al., 1980; Chambon, 2004) showed that, through an interaction with specific receptor proteins, the hormone estrogen regulates the rate of expression of the genes for the egg white proteins, ovalbumin, conalbumin, ovomucoid, and lysozyme by interacting with and activating its cognate receptor so it binds to specific, upstream enhancer sequences. Subsequently, it has been shown that the majority of developmental events orchestrated by hormones are the result of transcriptional events initiated by interaction of specific nuclear

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00001-8

2. DEVELOPMENT OF HEPATIC METABOLIC PROCESSES 2.1 Neonatal Development of Hepatic Gluconeogenesis The process of birth, upon completion of mammalian gestation, interrupts the continuous maternal supply of fuels that are provided via transplacental transfer, leading to an altered metabolic environment in the newborn. Although suckling provides an alternative fuel supply, there is a delay between birth and the onset of suckling. During this period, there is a reduction in the blood glucose concentration (hypoglycemia), which is quickly returned to normal levels as a result of phosphorylase activation (Dawkins, 1963; Cake and Oliver, 1969) that initiates glycogenolysis and the development of hepatic gluconeogenesis (Dawkins, 1963; Cake et al., 1971; Girard et al., 1973; Marsac et al., 1976).

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1. HORMONES AND PERINATAL DEVELOPMENT

In vertebrates, gluconeogenesis, which can be defined as the synthesis of glucose from noncarbohydrate precursors such as pyruvate, lactate, glycerol, and glycogenic amino acids, takes place mainly in the liver and in the cortex of the kidney (Gerich et al., 2001). Hepatic gluconeogenesis is virtually absent in the fetus and only becomes evident postnatally (Ballard and Oliver, 1963, 1965; Warnes et al., 1977; Platt and Deshpande, 2005). In rats, the inability to synthesize glucose from noncarbohydrate precursors prior to birth is due to the absence of the enzyme phosphoenolpyruvate carboxykinase (EC 4.1.1.32; PEP carboxykinase), despite the presence of the other three enzymes essential for gluconeogenesis, namely pyruvate carboxylase (EC 6.4.1.1), fructose 1,6-bisphosphatase (EC 3.1.3.11), and glucose 6-phosphatase (EC 3.1.3.9) (Ballard and Hanson, 1967; Yeung et al., 1967; Fig. 1.1). Natural birth or premature delivery by uterine section of fetal rats results in the appearance of PEP carboxykinase, which is linearly correlated with postnatal gluconeogenic activity (Yeung and Oliver, 1967). The appearance of the enzyme at this time is the result of a transcriptional event, which is apparent from the rapid accumulation of PEP carboxykinase mRNA from an undetectable level in the liver of the fetal rat late in gestation to a peak level 24 h after birth (Lyonnet et al., 1988). The administration of the synthetic glucocorticoid, triamcinolone, does not precociously induce hepatic PEP carboxykinase activity in the fetal rat (Yeung et al., 1967; Mencher and Reshef, 1979). In contrast, in

postnatal rats, which possess PEP carboxykinase activity, triamcinolone administration elicits a two- to threefold increase in enzyme activity. This increase was shown to be the result of increased synthesis of the enzyme, accompanied by enhanced PEP carboxykinase mRNA (Mencher and Reshef, 1979). Renal PEP carboxykinase, unlike that of the liver, was detectable prior to birth and was elevated in both fetal and postnatal animals when exposed to triamcinolone. Conflicting reports on the effect of glucocorticoids on PEP carboxykinase activity (Gunn et al., 1975; Mencher and Reshef, 1979) question the role played by glucocorticoids in the developmental regulation of this enzyme. The postnatal accumulation of PEP carboxykinase mRNA, and as a consequence the enhanced enzymatic activity, is now recognized as being promoted by the dramatic change in the circulating level of the pancreatic hormones that occurs at birth (Girard et al., 1973; Pegorier et al., 1992) together with a rise in epinephrine, released from the adrenal medulla as a consequence of the stress associated with birth. Within an hour of the birth of rats, the plasma glucagon concentration rose by more than threefold, whereas the insulin concentration, which was high in the fetal circulation, fell to less than 30% of that recorded for late fetal rats and remained at this low level over the first 2 postnatal weeks (Girard et al., 1972, 1973; Fig. 1.2). As a result, there was a greater than 10-fold decline in the insulin/glucagon molar ratio, implying that, soon after birth, there would be a dramatic increase in the hepatic concentration of cyclic AMP (cAMP). The in utero administration of either glucagon or dibutyryl cAMP precociously induced both PEP

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FIGURE 1.1 Developmental changes in the activities of the gluconeogenic enzymes pyruvate carboxylase (black), PEP carboxykinase (red), fructose 1,6-bisphosphatase (green), and glucose 6-phosphatase (blue) in the rat. Data was taken from Yeung, D., Stanley, R.S., Oliver, I.T., 1967. Development of gluconeogenesis in neonatal rat liver. Effect of triamcinolone. Biochem. J. 105, 1219e1227.

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Postnatal changes in the circulatory levels of the pancreatic hormones insulin (black) and glucagon (red) in the rat. Data was taken from Girard, J.R., Cuendet, G.S., Marliss, E.B., Kervran, A., Rieutort, M., Assan, R., 1973. Fuels, hormones and liver metabolism at term and during the early postnatal period in the rat. J. Clin. Investig. 52, 3190e3200.

2. DEVELOPMENT OF HEPATIC METABOLIC PROCESSES

carboxykinase mRNA levels (Garcia Ruiz et al., 1978; Cimbala et al., 1982; Mencher et al., 1984) and enzymatic activity (Yeung and Oliver, 1968; Girard et al., 1973). In contrast, the injection of insulin led to the opposite effect (Yeung and Oliver, 1968; Cimbala et al., 1982; Mencher et al., 1984). Hanson et al. (1973) concluded that the combination of an elevated liver cAMP concentration and a decreasing level of insulin during the perinatal period was the primary effector of the rapid accumulation of PEP carboxykinase in liver cytosol. Although it has been suggested that investigations of metabolic processes in the newborn rat may not be extrapolated to other mammals (Hanson et al., 1975), it has been shown that newborn humans experience postnatal hypoglycemia, which induces an increase in immunoreactive glucagon (Bloom and Johnston, 1972). Numerous regulatory elements have been shown to impinge on transcription of the PEP carboxykinase gene (Short et al., 1986; Magnuson et al., 1987; Croniger et al., 1998; Cassuto et al., 2005; Yang et al., 2009; Fig. 1.3). Early investigations of the sequence of the PEP carboxykinase promoter identified both basal and hormonal regulatory elements, including a cAMP- and two glucocorticoid-response elements (Short et al., 1986; Magnuson et al., 1987; Quinn et al., 1988). Elevated cAMP leads to the phosphorylation and activation of the cAMP-response element-binding protein (CREB) (Mayr and Montminy, 2001). The activated CREB binds to the cAMP-response element (CRE), located in the early upstream region of the promoter (see Fig. 1.3), and it is then bound by the CREB-binding protein that leads to transcription of the PEP carboxykinase gene. This interaction with the CRE is essential for both basal and cAMP-regulated expression of this gene and is influenced by binding of both C/EBPa and NF1 to this region (Croniger et al., 1998). A second region contains a hepatic nuclear factor 1 (HNF1) and a thyroid

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hormone-response element (TRE). It too binds C/EBPa, which is required for maximal response to cAMP. A third region contains two glucocorticoidresponse elements (GREs) that together constitute a glucocorticoid-response unit, which also contains an insulin-response element (IRE). This latter response element accounts for the inhibitory effect of insulin on the transcription of the PEP carboxykinase gene. Although glucocorticoids and glucagon strongly promote hepatic gluconeogenesis, as discussed, insulin suppresses this metabolic pathway in the liver (O’Brien et al., 1990; Hanson and Reshef, 1997; Hall and Granner, 1999). Two components known to have important physiologic roles in this process are the forkhead transcription factor FOXO1 and peroxisome proliferative activated receptor-g coactivator 1a (PGC-1a), a transcriptional coactivator. The latter binds and activates FOXO1, which is required for activation of PEP carboxykinase gene expression in hepatocytes (Puigserver et al., 2003). A fourth region of the promoter contains a PPARg-response element, which is essential for PEP carboxykinase expression in adipose tissue (Croniger et al., 1998; see Fig. 1.3). It has recently been shown that the insulin receptor itself translocates to the nucleus where it forms complexes with RNA polymerase II and various transcription factors that bind to a range of gene promoters thereby directly regulating the expression of those genes (Hancock et al., 2019). Thus, the mode of inhibition by insulin of PEP carboxykinase gene expression, as proposed above, is now brought into question. It is well established that the relative activities of the glycolytic and gluconeogenic pathways are reciprocally regulated by allosteric mechanisms. However, transcription of the genes coding for L-type pyruvate kinase (EC 2.7.1.40; L-PK) and PEP carboxykinase, which are key enzymes in these pathways, is also reciprocally controlled by the pancreatic hormones, glucagon and

FIGURE 1.3 Details of the promoter region of the PEP carboxykinase gene showing the various transcriptional regulatory elements that, when appropriately bound, are able to regulate the expression of this gene. C/EBP, CAAT/enhancer-binding protein; COUP, chicken ovalbumin upstream promoter; CRE, cAMP response element; CREB, cAMP response element-binding protein; GR, glucocorticoid receptor; GR1 and 2, GREs; IRS, IRE; NF1, nuclear factor 1; RAR/RXR, retinoic acid receptor/retinoid X receptor; T3R, thyroid hormone receptor; TRE, thyroid hormone regulatory element. P1eP6 are protein-binding sites identified by DNase I footprinting (Roesler et al., 1989). Reproduced from Yang, J., Reshef, L., Cassuto, H., Aleman, G., Hanson, R.W., 2009. Aspects of the control of phosphoenolpyruvate carboxykinase gene transcription. J. Biol. Chem. 284, 27031e27035.

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1. HORMONES AND PERINATAL DEVELOPMENT

insulin, and by carbohydrate availability (Lyonnet et al., 1988). Glucagon diminishes transcription of the L-PK gene, reduces the half-life of its mRNA, and inactivates the mature enzyme through phosphorylation by cAMPdependent protein kinase (Ljungstrom et al., 1976; Vaulont et al., 1986). In contrast, it enhances transcription of the PEP carboxykinase gene, thus stimulating hepatic glucose production (Lamers et al., 1982). Furthermore, insulin stimulates expression of the L-PK gene and blocks the transcription of the PEP carboxykinase gene (Cimbala et al., 1982; Noguchi et al., 1985). Kawaguchi et al. (2001) have shown that a carbohydrate response element-binding protein (ChREBP), which is activated by high glucose and inhibited by cAMP, binds to the regulatory domain of the L-PK gene and is essential for L-PK gene transcription. Whereas elevated glucose activates cAMP nuclear uptake and DNA-binding activity by enhancing dephosphorylation of the binding protein, cAMP produces the opposite effects (Kawaguchi et al., 2001). Thus, by modifying the activity of ChREBP, high carbohydrate and cAMP are able to regulate transcription of the L-PK gene.

2.2 Neonatal Development of Hepatic Amino Acid Metabolism The liver performs a myriad of functions that include producing the majority of plasma proteins and urea, as well as metabolizing and eliminating xenobiotics and integrating lipid, protein, and carbohydrate metabolic processes. Consequently, if its function is compromised, the health of the organism will be adversely affected. Many of the so-called “inborn errors of metabolism,” identified and characterized as genetic diseases, are manifest as liver diseases. The most common are hereditary hemochromatosis, Wilson disease, alpha1-antitrypsin deficiency, and phenylketonuria. The liver appears as a distinct entity early in the development of chicks and rodents, which, coupled with its metabolic versatility, makes it the organ of choice for elucidating molecular mechanisms that initiate the gene expression patterns responsible for cell differentiation and also those that regulate gene expression that ensure optimal functionality. Less common liver diseases include urea cycle disorders, where specific enzymes of the pathway such as argininosuccinate lyase (EC 4.3.2.1) or ornithine transcarbamylase (EC 2.1.3.3) are deficient, and the tyrosinemias, where fumarylacetoacetate hydrolase (EC 3.7.1.2) or tyrosine aminotransferase (EC 2.6.1.5) are rendered nonfunctional through mutations. Deficiencies in the ability to synthesize urea become evident when an enzyme in the pathway becomes rate limiting through either a lack of expression or expression of a functionally deficient enzyme. This then manifests as a postnatal

pathology. Dietary management to reduce ammonia production is generally effective (Haberle et al., 2012); however a liver transplant is necessary if this fails, which is often related to a deficiency of carbamoylphosphate synthetase (EC 6.3.5.5) or ornithine transcarbamylase (Saudubray et al., 1999). The limited availability of suitable liver for transplant and quality of life issues related to immune suppression following transplant have driven efforts to establish alternative therapies, such as gene and/or cell therapy. Both of these therapies must be based on a thorough understanding of mechanisms that regulate the six enzymes that constitute the urea cycle. Much of our understanding of mechanisms underlying the postnatal regulation of liver enzymes, especially those that increase substantially following birth, stems from the cloning of their respective genes. Good examples are PEP carboxykinase (Sasaki et al., 1984; Beale et al., 1985) (as discussed earlier) and tyrosine aminotransferase (Shinomiya et al., 1984), where the identification of transcription factor motifs and enhancers in their respective promoters have identified regulators of their expression (see Fig. 1.3) and also explained the coordinated appearance of the enzymes following birth. Subsequent studies have shown de novo transcription is the primary mechanism for producing enzymes that substantially improve the metabolic capacity and versatility of the postnatal liver (Beale et al., 1981; Reik et al., 1994). Although there has been a focus on the role of transcription, it is important to note that posttranscriptional mechanisms can also operate in unison. Panduro et al. (1987) provided compelling evidence that increased efficiency of translation of some liver-specific genes occurs following birth. It is also worth noting that although many genes are upregulated by increasing the abundance of positive regulators, there are instances whereby a diminution of negative regulators is just as important. This is illustrated by the suppressive effects of insulin on the expression of tyrosine aminotransferase (Cake et al., 1980, 1989; Ho et al., 1981; Panduro et al., 1987).

2.3 Applying Neonatal Regulation of Hepatic Enzymes to Generate Functional Hepatocytes for Cell Therapy Protein degradative pathways have provided valuable information on gene regulation that is applicable to current strategies that utilize liver stem/progenitor cells, embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs) in the treatment of liver disease using cell therapy. The transition from stem cell to functional liver cell requires two different levels of gene regulation. The first alters the gene and its regulatory elements so that it can be transcribed, while the second facilitates the developmental regulation of expression.

3. DEVELOPMENT OF THE RESPIRATORY SYSTEM

It is obvious that muscle or brain cells exposed to glucagon, cAMP, or glucocorticoids would not synthesize PEP carboxykinase nor tyrosine aminotransferase. The outcome would be the same if we treated ESCs or iPSCs, whereas adult hepatocytes or cell lines of the hepatic lineage can respond through enhanced transcription of the respective genes leading to more of the corresponding mRNA and thus production of more of each enzyme. In contrast, liver progenitor cells and even hepatocytes derived from 15-day gestation fetal rats will not transcribe the tyrosine aminotransferase gene when treated with glucagon, cAMP, or glucocorticoids (Yeoh et al., 1979; Yeoh and Oliver, 1980; Shelly and Yeoh, 1991; Yeoh and Fisher, 1993). However, in vivo or in vitro, these cells can acquire the ability to respond, which is markedly different from muscle or brain cells. Recent advances in our understanding of processes that direct the development of the three germ layers in early embryos (Wandzioch and Zaret, 2009) have facilitated the generation of liver progenitor cells and hepatocytes from ESCs and iPSCs (Si-Tayeb et al., 2010). Once generated using such approaches, the role of transcriptional regulators such as glucagon, cAMP, glucocorticoids, and insulin are important in ensuring the derived hepatocytes are fully functional. It should be emphasized that many of the component enzymes of metabolic pathways such as glycolysis, gluconeogenesis, amino acid catabolism, and the urea cycle are coordinately regulated by virtue of possessing common motifs such as those depicted in Fig. 1.3. Hence it is important to maintain in differentiated cells the appropriate combination of regulators to ensure all components of the various pathways are expressed at appropriate levels. Hence, protocols for differentiating and maintaining hepatocytes in 2D culture or as organoids contain these transcriptional regulators (Broutier et al., 2016) so that the cells acquire and retain the ability to synthesize glucose, glycogen, and urea and to metabolize drugs, i.e., the full repertoire of liver functions.

3. DEVELOPMENT OF THE RESPIRATORY SYSTEM The lung appears at a very early stage of embryological development as a consequence of an outpouching of the endodermal cells of the anterior foregut (Mendelson, 2000; Metzger et al., 2011). Branching morphogenesis of this diverticulum, which is dependent upon bidirectional epithelialemesenchyme interactions, contributes to the formation of the various structural features of the lung. This includes the appearance of the trachea, bronchi, bronchioles, alveoli, and later, the development of the saccular and alveolar capillary network required for gas exchange and the appearance of specialized cell populations through cellular differentiation of the

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epithelial cells (Mendelson, 2000). The process of branching morphogenesis has been shown to be enhanced by keratinocyte growth factor (KGF) (Post et al., 1996) and bombesin-like peptides, such as gastrin-releasing peptide (King et al., 1995; Shan et al., 2004), as well as dihydrotestosterone. This steroid, which induces its effect partially through an elevation in Hoxb5 expression (Volpe et al., 2013), is responsible for a faster rate of lung development in male embryos in the early stages of gestation. However, as detailed subsequently, androgens interfere with the production of surfactant at a later stage of lung development, thereby leading to a higher incidence of neonatal respiratory distress syndrome (RDS) in males (Nielsen and Torday, 1981). The advent of birth terminates the placental supply of oxygen to the fetus and dictates that the newborn has to rely on oxygen provision through ventilation of its own lungs. This necessitates that the lung has already undergone development and maturation prior to birth, which requires an interplay between transcriptional factors (such as hormones), growth factors, components of the matrix, and physical forces (Torday and Rehan, 2017). The pioneering work of Liggins (1969) demonstrated that exogenous glucocorticoids stimulate maturation of the lung, including the appearance of surfactant, which is essential for postnatal survival. Liggins subsequently showed that the offspring of mothers undergoing premature labor had dramatically reduced incidence of neonatal mortality if their mothers were subjected to antepartum glucocorticoid treatment (Liggins and Howie, 1972). For decades, this treatment approach was adopted in developed countries to reduce both infant mortality and the incidence of RDS that would otherwise occur in premature infants (N.I.H. Consensus Development Panel, 1995; Garbrecht et al., 2006; Vogel et al., 2017). A recently convened WHO expert group recommended that efficacy trials for this procedure are justified in low-resource countries where the majority of the world’s preterm births occur (Vogel et al., 2017). In those countries where health resources are more readily available, the incidence of this disease due to low levels of surfactant is markedly reduced in premature infants, if they are postnatally administered an artificial surfactant (Wigglesworth, 1980; Gitlin et al., 1987). Although both lung fibroblasts and type II alveolar epithelial cells contain glucocorticoid receptors, the effect of glucocorticoids on surfactant synthesis by type II cells was determined by Smith and colleagues (Smith, 1978; Post and Smith, 1984; Post et al., 1986) to be an indirect effect initiated by the lung mesenchymal cells (fibroblasts). Exposure of these latter cells to glucocorticoids results in the production of a peptide, termed fibroblast-pneumocyte factor (FPF) (Floros et al., 1985), a partially purified preparation of which accelerates lung maturation when injected into fetal rats (Smith, 1979). Moreover, monoclonal antibodies raised against

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1. HORMONES AND PERINATAL DEVELOPMENT

rat lung FPF diminished the glucocorticoid induction of surfactant synthesis in fetal rat lung cells and embryonic chicks (Post et al., 1984). The stimulatory effect of glucocorticoids and FPF on surfactant synthesis has been shown to be the result of elevated activity of the ratelimiting step in surfactant phospholipid synthesis, the reaction catalyzed by choline-phosphate cytidylyltransferase (EC 2.7.7.15) (Post et al., 1986; Rooney et al., 1986). Over the last 30 years or more, there have been numerous attempts to identify the chemical nature of FPF. In a recent review, King et al. (2016) investigated the possibility that KGF (otherwise known as fibroblast growth factor 7 (FGF-7)), leptin, or neuregulin-1b (NRG-1b) acted as FPF or a component of it. Each of these factors has been shown to be induced in lung fibroblasts by glucocorticoids, and they enhance the synthesis of surfactant after binding to their cognate receptors on type II epithelial cells (Chelly et al., 1999, 2001; Bergen et al., 2002; Torday et al., 2002; Dammann et al., 2003; Gesche et al., 2011; King et al., 2014b, 2016). King et al. (2016) concluded that, even if one or other of these factors is shown to have all of the attributes of the previously described FPF, they are all likely to contribute to lung development and/or maturation. As well as stimulating surfactant synthesis, some of them have additional activities that are unique. For instance, KGF also reduces lung injury by stimulating lung epithelial proliferation and repair (Deterding et al., 1997; Danan et al., 2002; Baba et al., 2007; Sakamoto et al., 2011), and NRG-1b directly stimulates surfactant secretion and enhances b-adrenergic receptor (b-AR) activity in type II cells (King et al., 2014a, 2016). In this context, it should be noted that glucocorticoids act directly on type II pneumocytes to elevate the concentrations of b-ARs and of b-AR mRNA, whereas NRG-1b does not directly activate b-AR gene expression but enhances the transcriptional response to glucocorticoids (King et al., 2014a). These observations are consistent with previous findings that glucocorticoids enhance the rate of transcription of the b-AR gene in both rat and human lung (Mak et al., 1995; McGraw et al., 1995; Aksoy et al., 2002) and that the b-AR gene contains a glucocorticoid-response element (Cornett et al., 1998). By elevating the level of b-AR, both glucocorticoids and NRG-1b enhance the rate of surfactant phospholipid secretion from type II cells in response to b-adrenergic agonists (King et al., 2014a). It is well established that premature male infants have a higher incidence of neonatal RDS than female infants (Nielsen and Torday, 1981; Torday et al., 1981; Torday, 1990; Carey et al., 2007; Ito et al., 2017). This has been attributed to a 5a-dihydrotestosterone (DHT) inhibition of glucocorticoid-enhanced surfactant synthesis in the later stages of gestation in male infants, resulting in a maturational delay in the enhancement of surfactant

synthesis (Nielsen et al., 1982). This effect is the result of diminished production of FPF by lung fibroblasts (Torday, 1985), which is a consequence of a pretranslational inhibition of FPF synthesis by androgens (Floros et al., 1987). In his commentary of the aforementioned review (King et al., 2016), Torday (2017) stated that, of the three agents being examined in that review (namely, KGF, leptin, and NRG-1b), only leptin has been shown to be reduced by DHT administration (Wabitsch et al., 1997). In this context, it should be emphasized that this latter study was conducted on cultured human adipocytes. It is also relevant that androgens have been shown to inhibit other components that have effects on lung maturation. For example, DHT treatment reduces the density of epidermal growth factor receptor (EGF-R or ErbB1) in fetal lung (Klein and Nielsen, 1993), which would lead to delayed lung maturation, as EGF has been shown to enhance surfactant synthesis (Gross et al., 1986; Sen and Cake, 1991) via elevated levels of FPF (Nielsen, 1989; Sen and Cake, 1991). It remains to be seen whether DHT also inhibits the activity of either ErbB3 or ErbB4, in which case the response to NRG-1b would be diminished. The higher incidence of neonatal RDS in premature male infants could also arise from defective lung fluid clearance as a result of immature expression of epithelial Naþ channels (ENaC) (Helve et al., 2009). Recent studies by Haase et al. (2017) have shown that, although DHT had a minor effect on decreasing lung epithelial transport of Naþ, exposure to testosterone had no effect, and the androgen receptor antagonist, flutamide, failed to abolish the gender difference in Naþ transport. In contrast, the female sex steroids, estradiol and progesterone, stimulate both Naþ transport and ENaC expression, particularly in female-derived epithelial cells. This is likely to result in more efficient fluid clearance from the lung of females and may account for the lower incidence of neonatal RDS in premature female infants. The incidence of RDS is also more prevalent in infants of diabetic mothers, which is likely to be due to the fetal hyperinsulinemia associated with maternal diabetes exerting a detrimental effect on lung development (Mendelson, 2000). In this instance, it appears that insulin has a profound inhibitory effect on the production of the surfactant-associated proteins SP-A and SP-B rather than on the synthesis of surfactant phospholipids (Dekowski and Snyder, 1992).

4. POSTNATAL DEVELOPMENT OF INTESTINAL DIGESTION In the suckling period immediately after birth and prior to weaning, the intestine is capable of hydrolyzing lactose but not the carbohydrates typically found in

5. CONCLUSIONS AND FUTURE DIRECTIONS

solid food. This is a consequence of the presence of small intestinal lactase (EC 3.2.1.108) but the near absence of salivary and pancreatic amylase as well as undetectable levels of the small intestinal disaccharidases maltaseglucoamylase (MGA) (EC 3.2.1.3), trehalase (EC 3.2.1.28), and sucrose-isomaltase (EC 3.2.1.10) (Agbemafle et al., 2005). The transition from a predominantly milk diet to one containing solid food, as occurs at weaning, is associated with a decline in lactase activity and a dramatic increase in the activities of salivary and pancreatic amylase and the aforementioned disaccharidases (Agbemafle et al., 2005). Although sucrose-isomaltase (SI) activity is induced in enterocytes during the sucklingeweaning transition (Henning et al., 1975), it is considered that the process of induction is under hormonal rather than dietary control (Henning, 1978; Fig. 1.4). As adrenalectomy interferes with the normal developmental process (Henning and Sims, 1979), glucocorticoids have been strongly implicated, and their administration during the first 2 weeks of life causes precocious appearance of SI as well as rapid disappearance of lactase (Moog et al., 1973; Yeh et al., 1989). Leeper and Henning (1990) concluded that, with respect to intestinal distribution and to both normal and precocious glucocorticoidinduced development, the SI activity is determined primarily by the level of its cognate mRNA. Keller et al. (1992), however, argue that, although the biosynthesis

FIGURE 1.4 Developmental patterns for lactase and sucrose activities in jejunal mucosa compared with the concentration of free corticosterone in rat plasma at the indicated postnatal ages. Reproduced from Henning, S.J., 1978. Plasma concentrations of total and free corticosterone during development in the rat. Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 4, E451eE456, with permission.

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of pro-SI correlates with its mRNA level, the SI activity along the small intestine is regulated both by mRNA levels and by posttranslational factors. One of the complications in elucidating the mode of action of glucocorticoids on SI gene regulation is that the precocious induction of SI mRNA by glucocorticoids is characterized by a 12-h lag, suggesting that this response is a secondary effect (Leeper and Henning, 1990). Using supershift assays, Oesterreicher and Henning (2004) showed the presence of the transcription factors GATA-4 and GATA-6 but not Cdx-2 or C/EBP in jejunal extracts from mice previously exposed to dexamethasone for 4 h. Moreover, Western blot analysis demonstrated GATA-4 and GATA-6 proteins were found to be present in jejunal tissue extracts from dexamethasone-treated but not control animals (Oesterreicher and Henning, 2004). Their proposal that GATA proteins are key regulatory components in the development of small intestinal epithelium is consistent with the research of others (Boudreau et al., 2002; Benoit et al., 2010; Aronson et al., 2014), which has demonstrated that these proteins are involved in stimulation of the evolutionarily conserved SI promoter.

5. CONCLUSIONS AND FUTURE DIRECTIONS A variety of hormones, including glucocorticoids, glucagon, epinephrine, insulin, androgens, and estrogens, have been shown to result in either the stimulation or inhibition of a range of events associated with the development of key metabolic processes, which are critical for postnatal survival. These include the acquisition of gluconeogenesis, amino acid metabolism, and urea cycle function in the neonatal liver, the development of the surfactant system by the perinatal lung, and modification of jejunal digestive enzyme capabilities upon weaning. Many of these are dependent on crucial genes, the expression of which is influenced by a range of regulatory elements. Many of these regulatory elements have become established at the time of a critical evolutionary event (Torday and Rehan, 2011). Diseases of the liver, including urea cycle disorders and the tyrosinemias, lead to postnatal hepatic pathology. In severe cases, this is currently overcome by liver transplantation. However, the limitations of this procedure have driven a desire to establish alternatives, such as gene or cell therapy. However, to successfully treat liver disease using one of a variety of stem cells available requires both a complete understanding of the regulation of key enzymes and the ability to convert the stem cells to fully functional, hormone-responsive hepatocytes. While it is well established that glucocorticoids stimulate (Liggins, 1969) and androgens inhibit (Nielsen and

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Torday, 1981; Nielsen et al., 1982) the production of surfactant in the developing lung, further research is required to elucidate the exact molecular mechanism by which these two responses are effected (Post et al., 1986; Nielsen, 1989; Torday, 1990; King et al., 2016). Such research would also aid in ascertaining, at a molecular level, why male infants are more susceptible to neonatal respiratory distress and provide an insight into how this circumstance might be overcome. Although the decline in lactase and rise in SI activity in the intestine of rats are concomitant with a rise in free plasma corticosterone levels (Henning, 1978), there is an even greater increase in free corticosterone just prior to birth (Martin et al., 1977). This raises the question as to why the elevated corticosterone during embryonic development does not induce the digestive changes that are associated with weaning. Given that GATA-4 and GATA-6 proteins are likely to be involved in the changes in digestive capabilities (Oesterreicher and Henning, 2004), it is possible that the genes coding for these two proteins only become sensitive to glucocorticoids after birth, perhaps through an age-dependent maturational event.

Acknowledgments Both authors wish to acknowledge the pivotal mentoring role that the late Professor Ivan T Oliver played in the development of their careers. The authors are grateful to Professor Heber Nielsen for his helpful comments on early drafts of the manuscript.

References Adamson, J.W., 1996. Regulation of red blood cell production. Am. J. Med. 101, 4Se6S. Agbemafle, B.M., Oesterreicher, T.J., Shaw, C.A., Henning, S.J., 2005. Immediate early genes of glucocorticoid action on the developing intestine. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G897eG906. Aksoy, M.O., Mardini, I.A., Yang, Y., Bin, W., Zhou, S., Kelsen, S.G., 2002. Glucocorticoid effects on the b-adrenergicdadenyl cyclase system of human airway epithelium. J. Allergy Clin. Immunol. 109, 491e497. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., 2002. Molecular Biology of the Cell, fourth ed. Garland Science, New York, NY. Aronson, B.E., Stapleton, K.A., Krasinski, S.D., 2014. Role of GATA factors in development, differentiation, and homeostasis of the small intestinal epithelium. Am. J. Physiol. Gastrointest. Liver Physiol. 306, G474eG490. Baba, Y., Yazawa, T., Kanegae, Y., Sakamoto, S., Saito, I., Morimura, N., Goto, T., Yamada, Y., Kurahashi, K., 2007. Keratinocyte growth factor gene transduction ameliorates acute lung injury and mortality in mice. Hum. Gene Ther. 18, 130e141. Ballard, F.J., Hanson, R.W., 1967. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver. Biochem. J. 104, 866e871. Ballard, F.J., Oliver, I.T., 1963. Glycogen metabolism in embryonic chick and neonatal rat liver. Biochim. Biophys. Acta 71, 578e588.

Ballard, F.J., Oliver, I.T., 1965. Carbohydrate metabolism in liver from foetal and neonatal sheep. Biochem. J. 95, 191e200. Beale, E.G., Katzen, C.S., Granner, D.K., 1981. Regulation of rat liver phosphoenolpyruvate carboxykinase (GTP) messenger ribonucleic acid activity by N6, O2’-dibutyryladenosine 3’,5’-phosphate. Biochemistry 20, 4878e4883. Beale, E.G., Chrapkiewicz, N.B., Scoble, H.A., Metz, R.J., Quick, D.P., Noble, R.L., Donelson, J.E., Biemann, K., Granner, D.K., 1985. Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene. J. Biol. Chem. 260, 10748e10760. Benoit, Y.D., Pare´, F., Francoeur, C., Jean, D., Tremblay, E., Boudreau, F., Escaffit, F., Beaulieu, J.-F., 2010. Cooperation between HNF-1a, Cdx2 and GATA-4 in initiating an enterocytic differentiation program in a normal human intestinal epithelial progenitor cell line. Am. J. Physiol. Gastrointest. Liver Physiol. 298, G504eG517. Bergen, H.T., Cherlet, T.C., Manuel, P., Scott, J.E., 2002. Identification of leptin receptors in lung and isolated fetal type II cells. Am. J. Respir. Cell Mol. Biol. 27, 71e77. Bloom, S.R., Johnston, D.I., 1972. Failure of glucagon release in infants of diabetic mothers. Br. Med. J. 4, 453e454. Boudreau, F., Rings, E.H.H.M., Van Wering, H.M., Kim, R.K., Swain, G.P., Krasinski, S.D., Moffett, J., Grand, R.J., Suh, E.R., Traber, P.G., 2002. Hepatocyte nuclear factor-a, GATA-4, and caudal related homeodomain protein Cdx2 interact functionally to modulate intestinal gene transcription. Implications for the developmental regulation of the sucrase-isomaltase gene. J. Biol. Chem. 277, 31909e31917. Broutier, L., Andersson-Rolf, A., Hindley, C.J., Boj, S.F., Clevers, H., Koo, B.-K., Huch, M., 2016. Culture and establishment of selfrenewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat. Protoc. 11, 1724e1743. Cake, M.H., Oliver, I.T., 1969. The activation of phosphorylase in neonatal rat liver. Eur. J. Biochem. 11, 576e581. Cake, M.H., Yeung, D., Oliver, I.T., 1971. The control of postnatal hypoglycaemia. Suggestions based on experimental observations in neonatal rats. Biol. Neonate 18, 183e192. Cake, M.H., Yeoh, G., Oliver, I.T., Litwack, G., 1980. Developmental changes in the glucocorticoid induction of hepatic tyrosine aminotransferase. Adv. Biosci. 25, 263e272. Cake, M.H., Ho, K.K.W., Shelly, L., Milward, E., Yeoh, G.C.T., 1989. Insulin antagonism of dexamethasone induction of tyrosine aminotransferase in cultured fetal hepatocytes. A correlation between enzyme activity, synthesis, level of messenger RNA and transcription. Eur. J. Biochem. 182, 429e435. Carey, M.A., Card, J.W., Voltz, J.W., Arbes Jr., S.J., Germolec, D.R., Korach, K.S., Zeldin, D.C., 2007. It’s all about sex: gender, lung development and lung disease. Trends Endocrinol. Metabol. 18, 308e313. Cassuto, H., Kochan, K., Chakravarty, K., Cohen, H., Blum, B., Olswang, Y., Hakimi, P., Xu, C., Massillon, D., Hanson, R.W., Reshef, L., 2005. Glucocorticoids regulate transcription of the gene for phosphoenolpyruvate carboxykinase in the liver via an extended glucocorticoid regulatory unit. J. Biol. Chem. 280, 33873e33884. Chambon, P., 2004. How I became one of the fathers of a superfamily. Nat. Med. 10, 1027e1031. Chelly, N., Mouhieddine-Gueddiche, O.-B., Barlier-Mur, A.-M., Chailley-Heu, B., Bourbon, J.R., 1999. Keratinocyte growth factor enhances maturation of fetal rat lung type II cells. Am. J. Respir. Cell Mol. Biol. 20, 423e432. Chelly, N., Henrion, A., Pinteur, C., Chailley-Heu, B., Bourbon, J.R., 2001. Role of keratinocyte growth factor in the control of surfactant synthesis by fetal lung mesenchyme. Endocrinology 142, 1814e1819. Cimbala, M.A., Lamers, W.H., Nelson, K., Monahan, J.E., YooWarren, H., Hanson, R.W., 1982. Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP. J. Biol. Chem. 257, 7629e7636.

REFERENCES

N.I.H. Consensus Development Panel, 1995. The effect of corticosteroids for fetal maturation on perinatal outcomes. J. Am. Med. Assoc. 273, 413e418. Cornett, L.E., Hiller, F.C., Jacobi, S.E., Cao, W., McGraw, D.W., 1998. Identification of a glucocorticoid response element in the rat b2adrenergic receptor gene. Mol. Pharmacol. 54, 1016e1023. Croniger, C., Leahy, P., Reshef, L., Hanson, R.W., 1998. C/EBP and the control of phosphoenolpyruvate carboxykinase gene transcription in the liver. J. Biol. Chem. 273, 31629e31632. Dammann, C.E.L., Nielsen, H.C., Carraway III, K.L., 2003. Role of neuregulin-1b in the developing lung. Am. J. Respir. Crit. Care Med. 167, 1711e1716. Danan, C., Franco, M.-L., Jarreau, P.-H., Dassieu, G., Chailley-Heu, B., Bourbon, J., Delacourt, C., 2002. High concentrations of keratinocyte growth factor in airways of premature infants predicted absence of bronchopulmonary dysplasia. Am. J. Respir. Crit. Care Med. 165, 1384e1387. Dawkins, M.J.R., 1963. Glycogen synthesis and breakdown in fetal and newborn rat liver. Annals N. Y. Acad. Sci. 111, 203e211. Dekowski, S.A., Snyder, J.M., 1992. Insulin regulation of messenger ribonucleic acid for the surfactant-associated proteins in human fetal lung in vitro. Endocrinology 131, 669e676. Deterding, R.R., Havill, A.M., Yano, T., Middleton, S.C., Jacoby, C.R., Shannon, J.M., Simonet, W.S., Mason, R.J., 1997. Prevention of bleomycin-induced injury in rats by keratinocyte growth factor. Proc. Assoc. Am. Phys. 109, 254e268. Floros, J., Post, M., Smith, B.T., 1985. Glucocorticoids affect the synthesis of pulmonary fibroblast-pneumonocyte factor at a pretranslational level. J. Biol. Chem. 260, 2265e2267. Floros, J., Nielsen, H.C., Torday, J.S., 1987. Dihydrotestosterone blocks fetal lung fibroblast-pneumonocyte factor at a pretranslational level. J. Biol. Chem. 262, 13592e13598. Garbrecht, M.R., Klein, J.M., Schmidt, T.J., Snyder, J.M., 2006. Glucocorticoid metabolism in the human fetal lung: implications for lung development and the pulmonary surfactant system. Biol. Neonate 89, 109e119. Garcia Ruiz, J.P., Ingram, R., Hanson, R.W., 1978. Changes in hepatic messenger RNA for phosphoenolpyruvate carboxykinase (GTP) during development. Proc. Natl. Acad. Sci. U. S. A. 75, 4189e4193. Gerich, J.E., Meyer, C., Woerle, H.J., Stumvoll, M., 2001. Renal gluconeogenesis. Its importance in human glucose homeostasis. Diabetes Care 24, 382e391. Gesche, J., Fehrenbach, H., Koslowski, R., Ohler, F.M., Pynn, C.J., Griese, M., Poets, C.F., Bernhard, W., 2011. rhKGF stimulates lung surfactant production in neonatal rats in vivo. Pediatr. Pulmonol. 46, 882e895. Girard, J., Bal, D., Assan, R., 1972. Glucagon secretion during the early postnatal period in the rat. Horm. Metab. Res. 4, 168e170. Girard, J.R., Cuendet, G.S., Marliss, E.B., Kervran, A., Rieutort, M., Assan, R., 1973. Fuels, hormones and liver metabolism at term and during the early postnatal period in the rat. J. Clin. Investig. 52, 3190e3200. Gitlin, J.D., Soll, R.F., Parad, R.B., Horbar, J.D., Feldman, H.A., Lucey, J.F., Taeusch, H.W., 1987. Randomized controlled trial of exogenous surfactant for the treatment of hyaline membrane disease. Pediatrics 79, 31e37. Gross, I., Dynia, D.W., Rooney, S.A., Smart, D.A., Warshaw, J.B., Sissom, J.F., Hoath, S.B., 1986. Influence of epidermal growth factor on fetal rat lung development in vitro. Pediatr. Res. 20, 473e477. Gunn, J.M., Hanson, R.W., Meyuhas, O., Reshef, L., Ballard, F.J., 1975. Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat. Biochem. J. 150, 195e203. Haase, M., Laube, M., Thome, U.H., 2017. Sex-specific effects of sex steroids on alveolar epithelial Naþ transport. Am. J. Physiol. Lung Cell Mol. Physiol. 312, L405eL414.

9

Haberle, J., Boddaert, N., Burlina, A., Chakrapani, A., Dixon, M., Huemer, M., Karall, D., Martinelli, D., Crespo, P.S., Santer, R., Servais, A., Valayannopoulos, V., Lindner, M., Rubio, V., DionisiVici, C., 2012. Suggested guidelines for the diagnosis and management of urea cycle disorders. Orphanet J. Rare Dis. 7, 32. Hafen, E., Stocker, H., 2003. How are the sizes of cells, organs and bodies controlled? PLoS Biol. 1, 319e323. Hall, R.K., Granner, D.K., 1999. Insulin regulates expression of metabolic genes through divergent signaling pathways. J. Basic Clin. Physiol. Pharmacol. 10, 119e133. Hancock, M.L., Meyer, R.C., Mistry, M., Khetani, R.S., Wagschal, A., Shin, T., Ho Sui, S.J., Na¨a¨r, A.M., Flanagan, J.G., 2019. Insulin receptor associates with promoters genome-wide and regulates gene expression. Cell 177, 722e736. Hanson, R.W., Reshef, L., 1997. Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression. Annu. Rev. Biochem. 66, 581e611. Hanson, R., Fisher, L., Ballard, F., Reshef, L., 1973. The regulation of phosphoenolpyruvate carboxykinase in fetal rat liver. Enzyme 15, 97e110. Hanson, R., Reshef, L., Ballard, F., 1975. Hormonal regulation of hepatic P-enolpyruvate carboxykinase (GTP) during development. Fed. Proc. 34, 166e171. Helve, O., Pitkanen, O., Jane´r, C., Andersson, S., 2009. Pulmonary fluid balance in the human newborn infant. Neonatology 95, 347e352. Henning, S.J., 1978. Plasma concentrations of total and free corticosterone during development in the rat. Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 4, E451eE456. Henning, S.J., Sims, J.M., 1979. Delineation of the glucocorticoidsensitive period of intestinal development in the rat. Endocrinology 104, 1158e1163. Henning, S.J., Ballard, P.L., Kretchmer, N., 1975. A study of the cytoplasmic receptors for glucocorticoids in intestine of pre- and postweanling rats. J. Biol. Chem. 250, 2073e2079. Ho, K.K.W., Cake, M.H., Yeoh, G.C.T., Oliver, I.T., 1981. Insulin antagonism of glucocorticoid induction of tyrosine aminotransferase in cultured foetal hepatocytes. Eur. J. Biochem. 118, 137e142. Ito, M., Tamura, M., Namba, F., Neonatal Research Network of Japan, 2017. Role of sex in morbidity and mortality of very premature neonates. Pediatr. Int. 59, 898e905. Kawaguchi, T., Takenoshita, M., Kabashima, T., Uyeda, K., 2001. Glucose and cAMP regulate the L-type pyruvate kinase gene by phoshorylation/dephosphorylation of the carbohydrate response element binding protein. Proc. Natl. Acad. Sci. U. S. A. 98, 13710e13715. Keller, P., Zwicker, E., Mantai, N., Semenza, G., 1992. The levels of lactase and of sucrase-isomaltase along the rabbit small intestine are regulated both at the mRNA level and post-translationally. FEBS (Fed. Eur. Biochem. Soc.) Lett. 313, 265e269. King, K.A., Torday, J.S., Sunday, M.E., 1995. Bombesin and [Leu8]phyllolitorin promote fetal mouse lung branching morphogenesis via a receptor-mediated mechanism. Proc. Natl. Acad. Sci. U. S. A. 92, 4357e4361. King, G., Damas, J.E., Cake, M.H., Berryman, D., Maker, G.L., 2014a. Influence of glucocorticoids, neuregulin-1b, and sex on surfactant phospholipid secretion from type II cells. Am. J. Physiol. Lung Cell Mol. Physiol. 306, L292eL298. King, G., Maker, G.L., Berryman, D., Trengove, R.D., Cake, M.H., 2014b. Role of neuregulin-1b in dexamethasone-enhanced surfactant synthesis in fetal type II cells. FEBS (Fed. Eur. Biochem. Soc.) Lett. 588, 975e980. King, G., Smith, M.E., Cake, M.H., Nielsen, H.C., 2016. What is the identity of fibroblast-pneumocyte factor? Pediatr. Res. 80, 768e776. Klein, J.M., Nielsen, H.C., 1993. Androgen regulation of epidermal growth factor receptor binding activity during fetal rabbit lung development. J. Clin. Investig. 91, 425e431.

10

1. HORMONES AND PERINATAL DEVELOPMENT

Lamers, W.H., Hanson, R.W., Meisner, H.M., 1982. cAMP stimulates transcription of the gene for cytosolic phosphoenolpyruvate carboxykinase in rat liver nuclei. Proc. Natl. Acad. Sci. U. S. A. 79, 5137e5141. Leeper, L.L., Henning, S.J., 1990. Development and tissue distribution of sucrase-isomaltase mRNA in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 258, G52eG58. Liggins, G.C., 1969. Premature delivery of foetal lambs infused with glucocorticoids. J. Endocrinol. 45, 515e523. Liggins, G.C., Howie, R.N., 1972. A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants. Pediatrics 50, 515e525. Ljungstrom, O., Berglund, L., Engstrom, L., 1976. Studies on the kinetic effects of adenosine-3’:5’-monophosphate-dependant phosphorylation of purified pig-liver pyruvate kinase type L. Eur. J. Biochem. 68, 497e506. Lyonnet, S., Coupe´, C., Girard, J.R., Kahn, A., Munnich, A., 1988. In vivo regulation of glycolytic and gluconeogenic enzyme gene expression in newborn rat liver. J. Clin. Investig. 81, 1682e1689. Magnuson, M., Quinn, P., Granner, D., 1987. Multihormone regulation of phosphoenolpyruvate carboxykinase-chloramphenicol acetyltransferase fusion genes. Insulin’s effects oppose those of cAMP and dexamethasone. J. Biol. Chem. 262, 14917e14920. Mak, J.C.W., Nishikawa, M., Barnes, P.J., 1995. Glucocorticosteroids increase b2-adrenergic receptor transcription in human lung. Am. J. Physiol. Lung Cell Mol. Physiol. 268, L41eL46. Marsac, C., Saudubray, J., Moncion, A., Leroux, J., 1976. Development of gluconeogenic enzymes in the liver of human newborns. Biol. Neonate 28, 317e325. Martin, C.E., Cake, M.H., Hartmann, P.E., Cook, I.F., 1977. Relationship between foetal corticosteroids, maternal progesterone and parturition in the rat. Acta Endocrinol. 84, 167e176. Mayr, B., Montminy, M., 2001. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat. Rev. 2, 599e609. McGraw, D.W., Chai, S.E., Hiller, F.C., Cornett, L.E., 1995. Regulation of the b2-adrenergic receptor and its mRNA in the rat lung by dexamethasone. Exp. Lung Res. 21, 535e546. Mencher, D., Reshef, L., 1979. Effect of triamcinolone on renal and hepatic phosphoenolpyruvate carboxykinase in the newborn rat. Changes in the rate of synthesis of the enzyme and in the activity of its translatable messenger RNA. Eur. J. Biochem. 94, 581e589. Mencher, D., Cohen, H., Benvenisty, N., Meyuhas, O., Reshef, L., 1984. Primary activation of cytosolic phosphoenolpyruvate carboxykinase gene in fetal rat liver and the biogenesis of its mRNA. Eur. J. Biochem. 141, 199e203. Mendelson, C.R., 2000. Role of transcription factors in fetal lung development and surfactant protein gene expression. Annu. Rev. Physiol. 62, 875e915. Metzger, R., Wachowiak, R., Kluth, D., 2011. Embryology of the early foregut. Semin. Pediatr. Surg. 20, 136e144. Moog, F., Denes, A.E., Powell, P.M., 1973. Disaccharidases in the small intestine of the mouse: normal development and influence of cortisone, actinomycin D, and cycloheximide. Dev. Biol. 35, 143e159. Nielsen, H.C., 1989. Epidermal growth factor influences the developmental clock regulating maturation of the fetal lung fibroblast. Biochim. Biophys. Acta 1012, 201e206. Nielsen, H.C., Torday, J.S., 1981. Sex differences in fetal rabbit pulmonary surfactant production. Pediatr. Res. 15, 1245e1247. Nielsen, H.C., Zinman, H.M., Torday, J.S., 1982. Dihydrotestosterone inhibits fetal rabbit pulmonary surfactant production. J. Clin. Investig. 69, 611e616. Noguchi, T., Inoue, H., Tanaka, T., 1985. Transcriptional and posttranscriptional regulation of L-type pyruvate kinase in diabetic rat liver by insulin and dietary fructose. J. Biol. Chem. 260, 14393e14397.

O’Brien, R.M., Lucas, P.C., Forest, C.D., Magnuson, M.A., Granner, D.K., 1990. Identification of a sequence in the PEPCK gene that mediates a negative effect of insulin on transcription. Science 249, 533e537. Oesterreicher, T.J., Henning, S.J., 2004. Rapid induction of GATA transcription factors in developing mouse intestine following glucocorticoid administration. Am. J. Physiol. Gastrointest. Liver Physiol. 286, G947eG953. Panduro, A., Shalaby, F., Shafritz, D.A., 1987. Changing patterns of transcriptional and post-transcriptional control of liver-specific gene expression during rat development. Genes Dev. 1, 1172e1182. Pegorier, J.-P., Salvado, J., Forestier, M., Girard, J., 1992. Dominant role of glucagon in the initial induction of phosphoenolpyruvate carboxykinase mRNA in cultured hepatocytes from fetal rats. Eur. J. Biochem. 210, 1053e1059. Platt, M.W., Deshpande, S., 2005. Metabolic adaptation at birth. Semin. Fetal Neonatal Med. 10, 341e350. Post, M., Smith, B.T., 1984. Effect of fibroblast-pneumonocyte factor on the synthesis of surfactant phospholipids in type II cells from fetal rat lung. Biochim. Biophys. Acta 793, 297e299. Post, M., Floros, J., Smith, B.T., 1984. Inhibition of lung maturation by monoclonal antibodies against fibroblast-pneumonocyte factor. Nature 308, 284e286. Post, M., Barsoumian, A., Smith, B.T., 1986. The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblastpneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity. J. Biol. Chem. 261, 2179e2184. Post, M., Souza, P., Liu, J., Tseu, I., Wang, J., Kuliszewski, M., Tanswell, A.K., 1996. Keratinocyte growth factor and its receptor are involved in regulating early lung branching. Development 122, 3107e3115. Puigserver, P., Rhee, J., Donovan, J., Walkey, C.J., Yoon, J.C., Oriente, F., Kitamura, Y., Altomonte, J., Dong, H., Accili, D., Spiegelman, B.M., 2003. Insulin-regulated hepatic gluconeogenesis through FOX01e PGC-1alpha interaction. Nature 423, 550e555. Quinn, P.G., Wong, T.W., Magnuson, M.A., Shabb, J.B., Granner, D.K., 1988. Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. Mol. Cell Biol. 8, 3467e3475. Reik, A., Stewart, A.F., Schutz, G., 1994. Cross-talk modulation of signal transduction pathways: two mechanisms are involved in the control of tyrosine aminotransferase gene expression by phorbol esters. Mol. Endocrinol. 8, 490e497. Roesler, W.J., Vandenbark, G.R., Hanson, R.W., 1989. Identification of multiple protein binding domains in the promoter-regulatory region of the phosphoenolpyruvate carboxykinase (GTP) gene. J. Biol. Chem. 264, 9657e9664. Rooney, S.A., Dynia, D.W., Smart, D.A., Chu, A.J., Ingleson, L.D., Wilson, C.M., Gross, I., 1986. Glucocorticoid stimulation of cholinephosphate cytidylyltransferase activity in fetal rat lung: receptorresponse relationships. Biochim. Biophys. Acta 888, 208e216. Sackmann, E., 1995. Biological membranes architecture and function. In: Lipowski, R., Sackmann, E. (Eds.), Structure and Dynamics of Membranes. Elsevier Science, Amsterdam. Sakamoto, S., Yazawa, T., Baba, Y., Sato, H., Kanegae, Y., Hirai, T., Saito, I., Goto, T., Kurahashi, K., 2011. Keratinocyte growth factor gene transduction ameliorates pulmonary fibrosis induced by bleomycin in mice. Am. J. Respir. Cell Mol. Biol. 45, 489e497. Sasaki, K., Cripe, T.P., Koch, S.R., Andreone, T.L., Petersen, D.D., Beale, E.G., Granner, D.K., 1984. Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription. The dominant role of insulin. J. Biol. Chem. 259, 15242e15251. Saudubray, J.M., Touati, G., Delonlay, P., Jouvet, P., Narcy, C., Laurent, J., Rabier, D., Kamoun, P., Jan, D., Revillon, Y., 1999. Liver transplantation in urea cycle disorders. Eur. J. Pediatr. 158, S55eS59.

REFERENCES

Sen, N., Cake, M.H., 1991. Enhancement of disaturated phosphatidylcholine synthesis by epidermal growth factor in cultured fetal lung cells involves a fibroblast-epithelial cell interaction. Am. J. Respir. Cell Mol. Biol. 5, 337e343. Shan, L., Emanuel, R.L., Dewald, D., Torday, J.S., Asokananthan, N., Wada, K., Wada, E., Sunday, M.E., 2004. Bombesin-like peptide receptor gene expression, regulation and function in fetal murine lung. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L165eL173. Shelly, L.L., Yeoh, G.C., 1991. Effects of dexamethasone and cAMP on tyrosine aminotransferase expression in cultured fetal rat hepatocytes. Eur. J. Biochem. 199, 475e481. Shinomiya, T., Scherer, G., Schmid, W., Zentgraf, H., Schutz, G., 1984. Isolation and characterization of the rat tyrosine aminotransferase gene. Proc. Natl. Acad. Sci. U. S. A. 81, 1346e1350. Short, J.M., Wynshaw-Boris, A., Short, H.P., Hanson, R.W., 1986. Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. J. Biol. Chem. 21, 9721e9726. Si-Tayeb, K., Noto, F.K., Nagaoka, M., Li, J., Battle, M.A., Duris, C., North, P.E., Dalton, S., Duncan, S.A., 2010. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51, 297e305. Smith, B.T., 1978. Fibroblast-pneumonocyte factor: intercellular mediator of glucocorticoid effect on fetal lung. In: Stern, L., Oh, W., Friis-Hansen, B. (Eds.), Intensive Care in the Newborn II. Masson, New York. Smith, B., 1979. Lung maturation in the fetal rat: acceleration by injection of fibroblast-pneumocyte factor. Science 204, 1094e1095. Torday, J.S., 1985. Dihydrotestosterone inhibits fibroblastpneumonocyte factor-mediated synthesis of saturated phosphatidylcholine by fetal rat lung cells. Biochim. Biophys. Acta 835, 23e28. Torday, J.S., 1990. Androgens delay human fetal lung maturation in vitro. Endocrinology 126, 3240e3244. Torday, J.S., 2017. Commentary on “What is the identity of fibroblastpneumocyte factor (FPF)?”. Pediatr. Pulmonol. 81, 391. Torday, J.S., Rehan, V.K., 2011. A cell-molecular approach predicts vertebrate evolution. Mol. Biol. Evol. 28, 2973e2981. Torday, J.S., Rehan, V.K., 2017. Evolution, the Logic of Biology. John Wiley & Sons, Inc, Hoboken, NJ. Torday, J., Nielsen, H., Fencl, M., Avery, M., 1981. Sex differences in fetal lung maturation. Am. Rev. Respir. Dis. 123, 205e208. Torday, J.S., Sun, H., Wang, L., Torres, E., Sunday, M.E., Rubin, L.P., 2002. Leptin mediates the parathyroid hormone-related protein (PTHrP) paracrine stimulation of fetal lung maturation. Am. J. Physiol. Lung Cell Mol. Physiol. 282, L405eL410. Tsai, S., Roop, D., Tsai, M.-J., Stein, J., Means, A., O’Malley, B., 1978. Effect of estrogen on gene expression in the chick oviduct. Regulation of the ovomucoid gene. Biochemistry 17, 5773e5780. Vaulont, S., Munnich, A., Decaux, J.-F., Kahn, A., 1986. Transcriptional and post-transcriptional regulation of L-type pyruvate kinase gene expression in rat liver. J. Biol. Chem. 261, 7621e7625. Vogel, J., Oladapo, O., Pilleggi-Castro, C., Adejuyigbe, E., Althabe, F., Ariff, S., Ayede, A., Baqui, A., Costello, A., Chikamata, D.,

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Crowther, C., Fawole, B., Gibbons, L., Jobe, A., Kapasa, M., Kinuthia, J., Kriplani, A., Kuti, O., Neilson, J., Patterson, J., Piaggio, G., Qureshi, R., Qureshi, Z., Sanka, M., Stringer, J., Temmerman, M., Yunis, K., Bahl, R., Gulmezoglu, A., 2017. Antenatal corticosteroids for women at risk of imminent preterm birth in low-resource countries: the case for equipoise and the need for efficiency trials. BMJ Glob. Health 2, 1e7. Volpe, M.V., Ramadurai, S.M., Mujahid, S., Vong, T., Brandao, M., Wang, K.T., Pham, L.D., Nielsen, H.C., 2013. Regulatory interactions between androgens, Hoxb5, and TGFb signaling in murine lung development. BioMed Res. Int. 2013, 320249. Wabitsch, M., Blum, W.F., Muche, R., Braun, M., Hube, F., Rascher, W., Heinze, E., Teller, W., Hauner, H., 1997. Contribution of androgens to the gender difference in leptin production in obese children and adolescents. J. Clin. Investig. 100, 808e813. Wandzioch, E., Zaret, K.S., 2009. Dynamic signaling network for the specification of embryonic pancreas and liver progenitors. Science 324, 1707e1710. Warnes, D., Seamark, R., Ballard, F., 1977. The appearance of gluconeogenesis at birth in sheep. Biochem. J. 162, 627e634. Wasylyk, B., Kedinger, C., Corden, J., Brison, O., Chambon, P., 1980. Specific in vitro initiation of transcription on conalbumin and ovalbumin genes and comparison with adenovirus-2 early and late genes. Nat. Med. 285, 367e373. Wigglesworth, J., 1980. Artificial surfactant therapy for hyaline membrane disease. Arch. Dis. Child. 55, 753e754. Yang, J., Reshef, L., Cassuto, H., Aleman, G., Hanson, R.W., 2009. Aspects of the control of phosphoenolpyruvate carboxykinase gene transcription. J. Biol. Chem. 284, 27031e27035. Yeh, K.-Y., Yeh, M., Holt, P.R., 1989. Differential effects of thyroxine and cortisone on jejunal sucrase expression in suckling rats. Am. J. Physiol. Gastrointest. Liver Physiol. 256, G604eG612. Yeoh, G.C., Fisher, C.J., 1993. Transformation-induced alterations in the regulation of tyrosine aminotransferase expression in fetal rat hepatocytes: changes in hormone inducibility and the DNasehypersensitive site. Cancer Res. 53, 515e522. Yeoh, G.C., Oliver, I.T., 1980. The effect of cytosine arabinoside and bromodeoxyuridine on the appearance of tyrosine aminotransferase in cultured foetal hepatocytes of the rat. Biochem. J. 188, 929e932. Yeoh, G.C., Bennett, F.A., Oliver, I.T., 1979. Hepatocyte differentiation in culture. Appearance of tyrosine aminotransferase. Biochem. J. 180, 153e160. Yeung, D., Oliver, I.T., 1967. Development of gluconeogenesis in neonatal rat liver. Effect of premature delivery. Biochem. J. 105, 1229e1233. Yeung, D., Oliver, I.T., 1968. Factors affecting the premature induction of phosphopyruvate carboxylase in neonatal rat liver. Biochem. J. 108, 325e331. Yeung, D., Stanley, R.S., Oliver, I.T., 1967. Development of gluconeogenesis in neonatal rat liver. Effect of triamcinolone. Biochem. J. 105, 1219e1227.

C H A P T E R

2 Hormones of Programmed Cell Death John A. Arnott, Sonia Lobo Geisinger Commonwealth School of Medicine, Scranton, PA, United States

1. INTRODUCTION

caspases orchestrate the execution phase of apoptosis by proteolytically cleaving hundreds of different protein substrates within the cell. These substrates include structural proteins, DNA repair enzymes, protein kinases, and other cellular proteins that promote the apoptotic phenotype (Kidd et al., 2000). Caspases are aspartate-specific proteases with a catalytic cysteine in the active site (Alnemri et al., 1996; Cerretti et al., 1992; Nicholson et al., 1995). They are expressed as inactive, monomeric procaspases and activated through heteroand autoproteolytic processing and chainechain interaction (Salvesen and Dixit, 1997; Thornberry and Lazebnik, 1998; Fig. 2.1A). Initiator caspases (caspase-8 and -9) become activated when their two identical chains are brought together by adapter proteins to produce an active enzyme (Boatright et al., 2003; Donepudi et al., 2003); they in turn activate executioner caspases (caspase-3, -6, and -7) by cleaving between the large and small subunits of the procaspase dimer, bringing the two active sites together (Chai et al., 2001; Riedl et al., 2001). Once activated, a single executioner caspase can cleave other executioner caspases, amplifying the death signal through an accelerated feedback loop of caspase activation (McIlwain et al., 2013; Fig. 2.1B). Two distinct initiating pathways converge on the same set of executioner caspases to mediate the final phase of apoptosis (Fig. 2.2). The extrinsic pathway is triggered by extracellular ligands that bind and activate membrane-bound death receptors of the tumor necrosis factor (TNF) superfamily. Binding of death receptor ligands to death receptors leads to the recruitment, dimerization, and activation of caspase-8 with the help of the adapter proteins Fas-associated death domain (FADD)/TRADD (Ashkenazi and Dixit, 1998); these complexes are referred to as death-induced signaling complexes or death-inducing signaling complexes (DISCs) (Kischkel et al., 1995). Activated caspase-8 initiates apoptosis directly by cleaving and activating executioner caspases or via the intrinsic

Apoptosis is a type of programmed cell death (PCD) that occurs in embryonic development and during involution of organs. It is characterized by biochemical and morphologic changes that are distinct from other types of cell death like necrosis or autophagy (Green, 2011; Berg et al., 2002). Apoptosis does not generally evoke an inflammatory response, and affected cells undergo shrinkage and separation from surrounding cells, membrane blebbing, DNA fragmentation, nuclear membrane breakdown, and cytolysis into membrane-bound apoptotic bodies. By contrast, necrosis is characterized by rupture of the cell membrane, leakage of the cell plasma, and triggering of an acute inflammatory response (Wyllie et al., 1980). Autophagic cell death is characterized by the appearance of vacuoles in the cell and does not include extensive nuclear condensation. PCD is an essential process for turnover of cells necessary for proper maintenance of the healthy organism and adaptation to environmental changes. In the course of normal development, specific molecules can signal a cell to undergo apoptosis. Chemically or physically induced stress as well as stress caused by infection can also trigger an active cell death process. When cell death occurs aberrantly, excessively, or not at all, the consequences can be devastating. Defects in the apoptotic machinery have been implicated in cancer (Wong, 2011) and autoimmune disease (Eguchi, 2001) and excessive apoptosis with myocardial infarction (Krijnen et al., 2002), stroke (Radak et al., 2017; Broughton et al., 2009), and neurodegenerative disorders such as Alzheimer disease (Obulesu and Lakshmi, 2014). These examples highlight the significance of apoptosis and cell turnover for homeostatic regulation. Apoptosis exhibits recognizable phases: the reversible induction phase, the irreversible execution phase, and the degradation phase in which the remains are digested and recycled. A family of proteins known as Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00002-X

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FIGURE 2.1 Caspases involved in apoptotic pathways. (A) Mammalian caspases involved in apoptosis can be subdivided into two functional groups: initiator caspases (caspase-2, -8, -9, and -10) and executioner caspases (caspase-3, -6, and -7). Initiator caspases initiate the apoptosis signal, and executioner caspases carry out the mass proteolysis that leads to apoptosis. Caspases are initially produced as inactive monomeric procaspases that require dimerization and cleavage for activation. Dimer assembly is facilitated by various adapter proteins that bind to specific regions in the prodomain of the procaspase, and caspase contains a conserved pentapeptide motif, QACXG, which includes the active-site cystine residues. Activation requires proteolytic processing between aspartic acid residues (DX). (B) Caspases become rapidly cleaved and activated in response to death receptors (DISC complex) and apoptosome stimuli. Caspases will then cleave a range of substrates, including downstream caspases, nuclear proteins, plasma membrane proteins, and mitochondrial proteins, ultimately leading to cell death. The prodomain of the intrinsic initiator caspases and the inflammatory caspases contains a single death fold known as caspase recruitment domain (CARD), while the prodomain of the extrinsic initiator caspases contains two death folds known as death effector domains (DED). The activation of initiator caspases is initiated by dimerization, which is facilitated by binding to adaptor proteins via protein interaction motifs (DED), while effector caspases use the CARD domain for this function. Once appropriately dimerized, caspases cleave at interdomain linker regions (DX), forming a large and small subunit. This cleavage allows the active-site loops to take up a conformation favorable for enzymatic activity. Initiator caspases autoproteolytically cleave, whereas effector caspases are cleaved by initiator caspases. Adapted from Mcilwain, D.R., Berger, T., Mak, T.W., 2013. Caspase functions in cell death and disease. Cold Spring Harb. Perspect. Biol. 5, a008656.

apoptotic pathway through cleavage of BH3 interacting-domain (Bid). The intrinsic or mitochondrial apoptosis pathway is regulated by pro- and antiapoptotic members of the Bcl-2 protein family (Fig. 2.3). Proapoptotic factors of the BH3-only family such as Bim, Bid, Bad, Puma, and Noxa induce apoptosis by activating the multidomain family members Bax and Bak or by stimulating other apoptosispromoting factors. This process is counterbalanced by antiapoptotic Bcl-2 family members such as Bcl-2 and

Bcl-xL, which bind and neutralize their proapoptotic counterparts. Various cellular stresses lead to an increase in proapoptotic Bax and Bak, forming a pore in the outer mitochondrial membrane (Green and Kroemer, 2004). Subsequent release of cytochrome c from mitochondria and formation of the apoptosome results in the activation of caspase-9 via the adapter protein, apoptotic protease-activating factor-1 (Apaf1). Physical binding of Apaf1 to caspase-9 is mediated by their caspase recruitment domains (CARDs). Active

2. ROLE OF HORMONES IN PCD

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FIGURE 2.2 The extrinsic and intrinsic apoptotic pathways. Activation of the extrinsic pathway requires binding of extracellular ligands (e.g., TNFa, Fas, and TRAIL) to cognate death receptors localized at the surface of cells. Ligand binding leads to recruitment of adaptor proteins (FADD/TRADD) that form DISC complexes that bind and activate effectors such as procaspase-8 to induce downstream apoptotic signaling. Death receptors also can activate other TFs (c-Jun, N-terminal kinase (JNK), and NF-kB) that can regulate apoptosis. Regulatory signaling proteins like cFLIPL/S isoforms can also modulate cytotoxic signaling from death receptors. Extrinsic apoptotic signaling may be amplified and activate the intrinsic pathway by caspase-mediated cleavage of the proapoptotic Bcl-2 protein Bid and release of mitochondrial proapoptotic factors, and intrinsic death signaling can be triggered by DNA damage, leading to activation of the TF p53. The intrinsic pathway involves activation of initiator caspases-2, -8, -9, and -10, and subsequent activation of effector/executioner caspases-3, -6, and -7, which catalyze downstream steps of apoptosis. Intrinsic apoptotic pathways are propagated upon permeabilization of the mitochondrial outer membrane. This process is regulated by members of the Bcl-2 family of proteins and results in release of mitochondrial factors involved in apoptosis and in loss of mitochondrial functions. The Bcl-2 family includes antiapoptotic proteins, which promote cell survival and can inhibit cell death by sequestering proapoptotic Bcl-2 proteins, and two groups of proapoptotic members: the multi-BH domain Bax, Bak, and Bok, and the BH3-only proteins, such as Bid, Bad, and Bim. Prosurvival Bcl-2 proteins are localized in the mitochondria, endoplasmic reticulum, and perinuclear membranes, where they are involved in maintaining the status of the mitochondrial membrane. The sensitivity of a cell to apoptosis is thus largely regulated by the relative expression levels and activities of anti- and proapoptotic Bcl-2 proteins, which in turn, are controlled at the level of transcription by posttranslational modifications, by modulation of subcellular localization, and by direct interactions with other proteins (Noy, 2010).

caspase-9 then initiates apoptosis by cleaving and thereby activating executioner caspases, such as procaspase-3 (Shiozaki et al., 2002; Cain et al., 2002). In many cases, PCD is under the control of extracellular ligands including physical stimuli, cytotoxic drugs, cytokines, growth factors, and hormones, depending on the biochemical and morphologic events during the process. In particular, hormones are known to induce or enhance apoptotic processes under physiologic or pathologic conditions and can act generally and/or in a tissue-specific manner to prevent the onset of PCD. In this chapter, we will summarize and discuss the role of hormones in PCD with a particular emphasis on steroid hormones and death-inducing ligands of the TNF superfamily and the mechanisms that link them to the execution phase of apoptosis.

2. ROLE OF HORMONES IN PCD Many hormonal factors are capable of inducing or facilitating PCD. In hormone-dependent tissues such as the mammary gland, ovary, testis, and prostate, PCD plays a key role in ensuring that tissue homeostasis and proper disposal of cells that are no longer needed occur at the appropriate time; for example, in milkproducing epithelial cells of the mammary gland, systemic hormonal changes after lactation induce apoptosis as part of involution (Hennighausen et al., 1997). In the ovary, luteal cells of the corpus luteum undergo apoptosis postpartum, and apoptosis of germ cells occurs before birth to limit the number of follicles that can develop (Tilly et al., 1997). Following surgical castration, secretory cells of the prostate undergo massive

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FIGURE 2.3 Bcl-2 core family proteins and apoptosis. The Bcl-2 family of proteins are gatekeepers to the apoptotic response. They contain both proapoptotic and antiapoptotic members that interact with each other. Family members contain short amino acid sequences known as Bcl-2 homology (BH1, BH2, BH3, and BH) motifs. At least one BH motif is contained in each of the Bcl-2 family members, and these motifs contribute to the function of each member. Antiapoptotic Bcl-2 proteins conserve all four BH domains. All antiapoptotic proteins contain BH1 and BH2 domains; some of them contain an additional BH4 domain. All proapoptotic proteins contain a BH3 domain necessary for dimerization with other Bcl-2 family proteins and crucial for cytotoxic activity. Adapted from Plas, D.R., Thompson, C.B., 2002. Cell metabolism in the regulation of programmed cell death. Trends Endocrinol. Metabol. 13, 75e78.

apoptosis within 2e3 weeks (Buttyan et al., 1999). Induction of apoptosis in these tissues is almost entirely due to the depletion or fall in systemic levels of steroid hormones; however, the specific pathways and mechanisms by which these hormones offer protection from apoptosis is not well understood. Several key molecular players are now known to be involved in the regulation and execution of cell death, including caspases, the Bcl-2 family of proteins, and mitochondrial elements. Understanding how hormones regulate apoptosis in responsive tissues is critical to designing strategies to prevent and treat diseases that affect these tissues, such as cancer, autoimmune, and degenerative disorders.

3. STEROIDS Steroid hormones are potent regulators of apoptosis, and their addition or ablation may induce cell death in steroid-dependent cell types and target tissues like those already mentioned. These hormones include

glucocorticoids (GCs), androgens, estrogens, and progestogens. Most studies suggest that steroids serve as survival factors and that hormone withdrawal results in the activation of PCD. For example, androgens protect the prostate from involution (Buttyan et al., 1999), GCs and progesterone exert a similar antiapoptotic effect in the postweaning mammary gland (Feng et al., 1995; Berg et al., 2002), and in the ovarian follicles, estrogens promote survival, while androgens potentiate apoptosis (Chun et al., 1996; Haanen and Vermes, 1996). In the uterine epithelium, estrogen stimulates epithelial growth and progesterone removal triggers apoptosis. Similarly, in the prostate, estrogens exert direct and indirect opposing effects on cell death. Thus, steroids use a variety of mechanisms to regulate PCD, and apoptotic events can be triggered directly by steroid action on target cells or indirectly by altering expression of paracrine effectors in the affected or in supporting stromal cells (Thompson, 1994). Among the steroid hormones, GC regulation of lymphocyte PCD has served as a paradigm for steroid activation of apoptosis.

3. STEROIDS

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FIGURE 2.4

Steroid hormone receptor domain structures. (A) Steroid hormones have a domain structure consisting of a variable N-terminal amino acid sequence (NTD), which is unique to each receptor, has variable sequence and length, and contains an autonomous transcriptional activation function known as AF-1. The AF1 shows weak conservation and may mediate differential promoter regulation in vivo. A1 functions as a ligand-independent transcriptional activator and can also functionally synergize with AF2. The highly conserved (DBD) region harbors the DNAbinding domain that confers sequence-specific DNA recognition. LR is a linker region and contains the nuclear localization signal (NLS). The LBD is responsible for the binding of cognate ligand or hormone. This domain also contains a ligand-dependent transcriptional activation function (AF2) necessary for recruiting transcriptional coactivators, which interact with chromatin remodeling proteins and the general transcriptional activation machinery. Nuclear receptors may or may not contain a final domain in the C-terminus, the VR region, whose sequence is extremely variable and whose structure and function are unknown (Aranda and Pascual, 2001). (B) The human GR gene comprises 9 exons. Alternative splicing in exon 9 generates two receptor isoforms, termed GRa and GRb, that are identical through to amino acid 727 but then diverge at their carboxyl-termini. GRa contains an additional 50 amino acids, whereas GRb has an additional, nonhomologous 15 amino acids. GRb does not bind glucocorticoids and resides constitutively in the nucleus of cells. GRb has been shown to function as a dominant negative inhibitor and repress the transcriptional activity of GRa.

3.1 Glucocorticoids GCs are steroid hormones produced in the adrenal cortex and regulated by the hypothalamic-pituitary adrenal axis. Their secretion follows a circadian rhythm but is also regulated by stress stimuli (Chrousos, 1995; Uchoa et al., 2014). GCs regulate most physiologic functions essential for life, including reproduction, growth, cognition, and behavior. They also ensure proper functioning of metabolic organs, cardiovascular and central nervous systems, stress-related homeostasis, and the immune/inflammatory response (Charmandari et al., 2005; Chrousos et al., 2004; Rhen and Cidlowski, 2005; Nicolaides et al., 2010). GCs exert most of their effects by altering transcription of various steroid-responsive genes through binding to the intracellular glucocorticoid receptor (GR), a ligand-activated TF belonging to nuclear hormone receptor family. Like other steroid receptors, GR consists of a ligand-binding domain (LBD) at the C-terminus, a central DNA-binding domain (DBD) harboring two zinc finger motifs, and an N-terminal domain (NTD) containing the activation function 1 (AF1), which activates target genes in a ligandindependent fashion. A second activation function

domain AF2 is located within the LBD as well as sequences important for heat shock protein (HSP) and coregulator interaction (Fig. 2.4A). Each of these domains is important for mediating GR’s transcriptional function and response to GCs. The human GR gene is located on chromosome 5q3132 and consists of nine exons, with exons 2 to 9 coding for the GR protein and exon 1 forming the 50 untranslated region. Alternative splicing of exon 9 produces two GR isoforms, GRa and GRb, which are nearly identical but vary beyond amino acid 727 (Oakley and Cidlowski, 2011; Fig. 2.4B). The GRb isoform consists of 742 amino acids, with 15 nonhomologous amino acids (encoded by exon 9b) replacing the 50 carboxy terminal amino acids that comprise GRa (777 amino acids total). GRa functions as a TF to trigger genomic effects; however, there is also evidence of rapid, “nongenomic” effects, mediated via membrane-associated receptors and their signaling cascades (Revollo and Cidlowski, 2009). In contrast, GRb cannot bind GCs but exerts dominant negative effects on GRa through heterodimerization (Bamberger et al., 1995). The possibility that GRb has intrinsic, GRa-independent transcriptional activity remains to be established, but recent data suggest it may

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modulate gene expression by altering the activity of transcriptional intermediate molecules or other TFs through physical proteineprotein interactions (Gougat et al., 2002; Kino et al., 2009). In the absence of GCs, GRa resides in the cytoplasm bound in a complex with HSPs, chaperones, and immunophilins that maintain the receptor in a conformation suitable for ligand binding. Lipophilic GCs diffuse freely across the cell membrane and into the cytoplasm, where binding to GRa induces a conformation change in the receptor, which leads to dissociation of the complex and translocation of GRa into the nucleus. The “activated” receptor binds as a homodimer to specific DNA sequences called glucocorticoid response elements (GREs) in the promoter of target genes or as a monomer or dimer with other TFs via proteineprotein interactions, resulting in target gene upregulation or downregulation (Fig. 2.5). Once bound to the GRE, GRa can recruit coactivators (i.e., histone acetyltransferases (HATs)) or corepressors (histone deacetylases) that open or close the chromatin structure to alter transcription (McKenna et al., 1999a). These so called “genomic effects” of GC action result in increased expression of antiinflammatory proteins and decreased expression of proinflammatory cytokines.

GRa can also enhance or repress gene activity using mechanisms other than classical GREs. GRa can bind to other DNA-bound TFs and modulate their activity through a process called tethering, thereby affecting the transcription rates of their respective genes. This mechanism is especially important in the suppression of immune function and inflammation by GCs and can be explained in part by the interaction between GRa and nuclear factor-kB (NF-kB), activator protein-1 (AP1) and the signal transducers and activators of transcription (STATs) (Barnes and Karin, 1997; De Bosscher and Haegeman, 2009; Karin and Chang, 2001). Competition with transcriptional activators for DNA-binding sites is another mechanism that GRa uses to repress target genes, such as is the case with repression of Fas ligand (FasL) expression by NF-kB (Novac et al., 2006). GC-induced apoptosis of thymocytes is one of the earliest recognized forms of PCD (Wyllie et al., 1980); GCs have also been reported to induce cell death in some nonlymphoid cells and tissues such as eosinophils, fibroblasts, bone, hippocampus, and certain cancer cells (Druilhe et al., 2003; Hammer et al., 2004; Pufall, 2015; Weinstein, 2012). GC-induced apoptosis is initiated by the interaction of GC with GRa; however, our understanding of GC-induced cell death is limited in part by

FIGURE 2.5 GR signaling and PCD. GRa resides in the cytoplasm bound in a complex with HSPs, chaperones, and immunophilins that maintain the receptor in ligand binding confirmation. GCs diffuse and bind to GRa, inducing a conformation change in the receptor, which leads to dissociation of the complex and translocation of GRa into the nucleus. The activated receptor binds as a homodimer to specific DNA sequences called glucocorticoid response elements (GREs) in the promoter of target genes or as a monomer or dimer with other TFs via proteineprotein interactions, and this involves coactivators or corepressors. GRa suppresses the transcription of survival gene leading to apoptosis. It also may induce apoptosis via gene transcription of proapoptotic proteins and via nongenomic mechanisms. GRa may be sequestered in the cytoplasm by p53 inducing apoptosis. Adapted from Greenstein, S., Ghias, K., Krett, N.L., Rosen, S.T., 2002. Mechanisms of glucocorticoid-mediated apoptosis in hematological malignancies. Clin. Cancer Res. 8, 1681e1694.

3. STEROIDS

the lack of knowledge surrounding changes in gene expression and whether the transactivation or transrepression functions of GRa trigger the process. There is substantial evidence supporting the involvement of both GRa functions for mediating GC-induced apoptosis as well as data demonstrating that GCs act via the intrinsic pathway (Fig. 2.2). Further, the maintenance and level of GR expression is also a critical determinant for GC sensitivity, as in vivo studies indicate that a decrease in steroid titer regulates thymocyte apoptosis, while an increase enhances resistance to stress (Pazirandeh et al., 2002; Reichardt et al., 2000). Studies involving mutant GRs defective in their transactivation function but able to interfere with AP-1 TF activity have supported the idea that GC-induced apoptosis is mediated by the transrepression function of GRa (Helmberg et al., 1995). Since AP-1 regulates genes involved in cell survival, its repression may contribute to GC-induced apoptosis. Repression of other prosurvival TFs such as nuclear factor of activated T lymphocytes (NFAT) (Wisniewska et al., 1997), NF-kB (De Bosscher et al., 2000), and c-Myc have also been shown to accompany the induction of apoptosis by GCs, while c-Myc overexpression inhibits GC-induced apoptosis (Thompson, 1994; Wisniewska et al., 1997). In contrast, mice harboring a dimerization-deficient GR, thus inactivating its transactivation function, were deficient in GC-induced thymocyte apoptosis, suggesting that the transactivation function is required (Reichardt et al., 1998). A similar conclusion was derived from experiments using RAP46 to inhibit DNA binding and transactivation by the receptor or expression of N-terminal-deleted GR constructs, which inhibited GC-induced apoptosis of S49 mouse lymphoma cells (Dieken and Miesfeld, 1992; Kullmann et al., 1998). In addition, experiments using VP16 and E1A transactivation domains in place of the GR N-terminus enhanced activity of lymphoid cells to GC-induced apoptosis, supporting a role for transactivation in inducing apoptosis (Chapman et al., 1996). Indeed, neither transrepression nor transactivation has been conclusively ruled out as a mechanism for GC-induced apoptosis, and it is likely that a combination of both may contribute depending on the cell origin and other molecular factors. The target genes whose transactivation of transrepression initiates the cell death pathway remain uncertain; however, microarray-based expression profiling has revealed candidate genes and patterns of gene expression associated with GC-induced apoptosis. In an analysis of eight profiling studies, Schmidt noted that not a single gene was found to be regulated in all eight biologic systems examined, and only a few genes such as NF-kB inhibitor a (IkB-a) and FK506-binding protein 5 (FKBP 51) appeared in five or more systems; further, of the 900 GC-regulated genes reported, only

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approximately 70 appeared in more than one data set, suggesting that distinct genes may be regulated in a cell typeedependent context to bring about GCinduced cell death (Schmidt et al., 2004). Other approaches to identify GC-induced genes have revealed c-myc, Bim, and Puma as promising candidates for mediating apoptosis; for example, GC-induced apoptosis is partially inhibited in Bim-deficient mice and in pre-B acute lymphoblastic leukemia (ALL) cell lines treated with Bim RNAi (Abrams et al., 2004; Wang et al., 2003). Using patient-derived xenografts, Jing et al. demonstrated that GR increases Bim expression by binding to a GR promoter within the Bim intronic region. Bim binds existing Bcl-2, preventing Bax binding and resulting in Bax-mediated apoptosis (Jing et al., 2015). Puma knockout mice also display partial resistance of thymocytes to GC-induced apoptosis (Jeffers et al., 2003; Villunger et al., 2003), suggesting that induction of proapoptotic BH3-only proteins may be implicated in GC-induced apoptosis. In most cell types examined, GC-induced cell death proceeds via the intrinsic pathway, although GCs can activate components of the extrinsic pathway. In human ALL cell lines (Geley et al., 1997; Planey et al., 2003) and transgenic mice (Smith et al., 1996), inhibition of caspase 8 with crmA did not prevent the cells from undergoing apoptosis, indicating that the extrinsic pathway is not necessary for GC-mediated apoptosis. However, overexpression of antiapoptotic Bcl-2 proteins were able to diminish GC-mediated apoptosis. GCs can activate cell death through induction of proapoptotic members of the Bcl-2 family, such as Bim, Bid, and Bad (Han et al., 2001; Lu et al., 2007; Wang et al., 2003; Jing et al., 2015) and/or repression of antiapoptotic members, such as Bcl-2, Mcl-1, and Bcl-xL (Casale et al., 2003; Chauhan et al., 2003; Rogatsky et al., 1999; Fig. 2.5); moreover, thymocytes from mice deficient in the proapoptotic proteins Apaf1 (Cecconi et al., 1998; Yoshida et al., 1998) and caspase-9 (Hakem et al., 1998; Kuida et al., 1998) are partially resistant to apoptosis, as are thymocytes from double knockout mice lacking Bak and Bax (Rathmell et al., 2002). p53 is known to regulate Bcl-2 and Bax, so it could play a role in regulating the bcl-2:bax rheostat that determines cell fate upon GC exposure; however, while thymocytes from p53 knockout mice are resistant to ionizing radiation, they are not susceptible to GCinduced cell death, suggesting that p53 is not required (Clarke et al., 1993; Lowe et al., 1993). In vitro studies showing that the GR and p53 interact and that GR transcriptional activity is repressed by p53 suggest that crosstalk may exist between the two pathways. Evidence for this negative crosstalk has been demonstrated in HSC-2 head and neck carcinoma cells and IMR32 primary neuroblastoma cells, where the GR and p53 form a strong complex in the presence of dexamethasone to

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inhibit each other’s transactivation by cytoplasmic sequestration (Sengupta et al., 2000). Other cell typeespecific mediators of GC-induced apoptosis that have been identified include the proteasome complex, which prevents disruption of the mitochondrial membrane potential and nuclear fragmentation in thymocytes upon its pharmacological inhibition (Tonomura et al., 2003) and changes in calcium homeostasis (Lam et al., 1993; Squier and Cohen, 1997). Lastly, there is evidence to suggest that GR can induce apoptosis through nongenomic mechanisms such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt), which in turn activates eNOS (Limbourg and Liao, 2003) and via membrane-bound GR (mGR) (Gametchu and Watson, 2002). GCs are among the most commonly used drugs worldwide due to their profound antiinflammatory and immunosuppressive properties. Additionally, because of their ability to induce cell death in lymphoma, leukemia, and myeloid cells, GCs are widely prescribed for treatment of hematological malignancies including Hodgkin lymphoma, ALL, and multiple myelomas (Frankfurt and Rosen, 2004). However, long-term GC administration is not without side effects and often results in tolerance and development of GC resistance. At the molecular level, resistance could be attributed to GR mutations that alter affinity for the ligand, insufficient GR expression or increased expression of GR variants, a reduced ability to bind DNA, or increased expression of TFs like AP-1 that compete for DNA binding. Downstream mechanisms involving mutations in GC-regulated genes crucial for apoptosis induction or

for activating survival signals may also contribute to resistance (for a review, see Ramamoorthy and Cidlowski, 2013). Indeed, elucidating the mechanisms of resistance to GC-induced apoptosis will be critical for optimizing the therapeutic benefit of GCs.

3.2 Androgens Androgens are synthesized from cholesterol and are produced primarily in the testis, ovaries, and the adrenal glands. Testosterone is the major physiologic androgen synthesized in the testis and secreted into the blood. The hypothalamic-pituitary testicular axis is tightly regulated to ensure that circulating testosterone levels in the blood are maintained within a defined physiologic range (Isaacs et al., 1992). In addition to testosterone, other androgens include dehydroepiandrosterone (DHEA), androstenedione (A4), androstenediol (A5), androsterone, and 5a-dihydrotestosterone (DHT). Androgens function as paracrine hormones required by Sertoli cells to support sperm production. They are also required for masculinization of the developing male fetus, including sexual and prostate development. In addition, androgens mediate cell differentiation, proliferation, apoptosis, and metabolism in many tissues and thus are important for maintenance and homeostasis (Nelson et al., 2002). Androgen is the major systemic hormone for the prostate. Testosterone enters basal epithelial cells within the prostate from the blood, where it is converted to DHT by 5a-reductase enzyme (Fig. 2.6; Wilson and Gloyna, 1970). More than 95% of the androgenic steroid present within prostatic cell nuclei is DHT, and the

FIGURE 2.6 Mechanisms of androgen receptor signaling. AR, androgen receptor; ARA-70, androgen receptor-associated protein-70; ARE, androgen response element; DHT, 5a-Dihydrotestosterone; HSP, heat shock protein.

3. STEROIDS

affinity of the androgen receptor for DHT is higher than for other endogenously occurring steroids; hence, DHT is believed to be the major intracellular effector of androgen action within the prostate (Lamb et al., 1992). DHT diffuses into the cytoplasm of epithelial cells, where it binds and activates the androgen receptor (AR) (Radmayr et al., 2008). Upon binding, AR undergoes a conformational change that promotes dissociation of HSPs and association of importin-a and androgen receptor-associated protein-70 (ARA70), stabilizing the receptor and promoting its nuclear translocation. AR dimerizes in the nucleus and binds to DNA at androgen response elements (ARE), which promotes recruitment of coactivators with HAT activity resulting in chromatin remodeling. Like GR, AR regulates gene transcription through ARE binding and interaction or crosstalk with TFs, but also utilizes nonclassical, nongenomic mechanisms of signal transduction (Bennett et al., 2010). DHT inhibits PCD in the prostate gland. Early studies conducted in adult male rats demonstrated that the supply of androgen regulates the balance between cell death and proliferation, so neither overgrowth nor involution of the gland occurs (Kyprianou and Isaacs, 1988). After castration, only the androgendependent, glandular epithelial cells cease proliferating and undergo apoptosis but not the androgen-independent basal or stromal cells. The rapid decrease in serum testosterone levels following castration correlates with a corresponding rapid decrease in DHT concentration; the reduction in prostatic DHT initiates a process that leads to the death of the androgendependent cells of the ventral prostate, resulting in involution. Hence, this involution demonstrates that normal prostatic glandular epithelial cells continuously require a physiologic level of androgen for maintenance, in addition to their growth phase in early development. Apoptosis in the normal prostate gland occurs in two phases: first columnar cell secretion stops, followed by altered gene transcription and translation within the first 24 h, initiating atrophy. During the second phase, characteristic apoptotic changes such as DNA fragmentation and apoptotic body formation are cytologically evident, and by 7e10 days after castration, the ventral prostate gland of the rat is reduced to 80% in mass (Gosden and Spears, 1997). The drop in prostatic DHT that follows castration leads to changes in nuclear AR function. Within 12 h, ARs are no longer retained in biochemically isolated, ventral prostatic nuclei, resulting in the synthesis of proteins like testosterone repressed prostate message-2 (TRMP-2), c-myc, calmodulin, TGF-b1, glutathione S-transferase, and a-prothymosin, which are normally not present in the intact prostate (Denmeade et al., 1996).

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In addition to normal prostate cells, androgen is known to suppress apoptosis in androgen-dependent prostate cancer cells via AR-mediated repression of apoptotic genes (Denmeade et al., 1996). Androgen deprivation therapies typically induce a regression of mature prostate tissue that is accompanied by the extensive loss of prostate cells through apoptosis. Importantly, human androgen-dependent prostatic cancer cells undergo apoptosis following androgen ablation. In a study by Kyprianou et al. nude mice harboring PC-82 human prostatic xenografts showed rapid tumor involution following castration, with the tumor reaching approximately half of its starting size within 3 weeks of castration. TGF-b1 and TRPM-2 mRNA levels and DNA fragmentation also increased within the first day of castration (Kyprianou et al., 1990). After 2 days, the number of PC-82 cells entering S phase declined to below 1%, indicating that the cells die in G0 phase. Additional studies have demonstrated that androgen ablation does not induce this apoptotic process in androgenindependent prostatic cancer cells perhaps due to nonsustained elevation in intracellular free calcium levels (Sells et al., 1994; Martikainen et al., 1991) or increased expression of antiapoptotic proteins (Levine et al., 1993). In both human and rat prostate cancer, increased frequency of Bcl-2 expression is associated with progression of localized cancer to the metastatic, androgenindependent phenotype (Furuya et al., 1996; McDonnell et al., 1992). Metastatic prostatic cancer within an individual patient is heterogeneously composed of clones of both androgen-dependent and -independent cancer cells. Regardless of the mechanism, once androgenindependent cancer cells are present within individual prostatic cancer patients, the patient is no longer curable by androgen withdrawal therapy, as these cells do not activate apoptosis. To affect all the heterogenous prostatic cancer cell populations within a patient, effective chemotherapy, specifically targeted against the androgen-independent cancer cell, must be simultaneously combined with androgen ablation to affect the androgen-dependent cells. Misregulated AR activity due to gene amplification, altered expression or mutations, and altered ligand-binding specificity can also promote cancer growth after androgen ablation, making prostate cancer difficult to treat. Recent studies indicate that androgen and AR promote stress-mediated apoptosis in prostate cancer cells by increasing Bax translocation to mitochondria. Further, introduction of AR into AR-negative prostate cancer cells upregulated expression of the BH3-only protein Noxa, while inhibition of Noxa expression blocked UV-induced apoptosis by AR, revealing crosstalk between the androgen/AR hormonal signaling pathway and the intrinsic apoptotic pathway that determines sensitivity of stress-induced

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apoptosis (Lin et al., 2006). Thus, therapeutic interventions via Bax-mediated apoptosis may be more suitable for AR-positive, but not AR-negative prostate cancer.

3.3 Estrogens Estrogens are synthesized from cholesterol and are produced primarily by the ovaries, with secondary contributions from the testes, placenta, adipose tissue, and adrenal glands. Estrogens are essential to the development, maintenance, and function of the female reproductive system, including the proliferation and differentiation of breast epithelium (Muramatsu and Inoue, 2000; Lumachi et al., 2011; Nilsson and Gustafsson, 2011). 17b-estradiol (E2), the predominant intracellular estrogen, promotes cell proliferation in both normal and transformed epithelial cells by modifying the expression of hormone-responsive genes involved in the cell cycle and/or PCD. These dynamic actions are mediated via two estrogen receptors (ERs): ERa and ERb. Estrogens and their receptors contribute to pathologic states in multiple tissues, including cancer (e.g., breast, endometrial, prostate, and colorectal), osteoporosis, and cardiovascular, metabolic, and cognitive diseases (Jaffe et al., 1994; Resnick et al., 1998). ERa is a 66 kDa protein encoded by 8 exons located on chromosome 6q34-27 (Ponglikitmongkol et al., 1988), while ERb is 55 kDa and encoded within the ESR2 gene located on chromosome 14q (Ichikawa et al., 2005). Both receptors contain an NTD, DBD, and an LBD, mirroring the structure of other steroid hormone receptors, and signal using the classical, ligandinduced mechanism of action (Fig. 2.7; Truss and Beato, 1993). ERa and ERb (including its various isoforms b1, b2, b4, and b5) comprise a large and flexible binding pocket that allows for biding of multiple steroids, xenobiotics, and phytoestrogens (Anstead et al., 1997). Unlike the DBD and LBD whose sequence homology appears to be conserved between ERa and ERb (Kuiper et al., 1996; Mosselman et al., 1996), the NTD of ERb is significantly shorter and displays different sequence homology that may alter ERb0 s downstream effects (Kumar et al., 2011). Within the NTD and LBD domains are the AF1 and AF2 domains (Beato, 1989; Tora et al., 1989), whose synergistic activity allows for full transcriptional activation (Planey et al., 2013); however, AF1 functions in a hormone-independent manner, and AF2 requires the presence of a ligand that could explain the difference in ligand-induced transcriptional activation of target genes by ERa and ERb (Berry et al., 1990). Following either independent or simultaneous stimulation of AF1 and AF2, transcriptional coregulator proteins are recruited and able to interact with the liganded or unliganded ER to alter gene transcription (Arnal et al., 2013; Beato et al., 1995).

FIGURE 2.7 Estrogen receptor signaling. Estrogens are able to induce changes in gene expression through genomic and nongenomic mechanisms. The “classical” or genomic mechanism of estrogen action relies on its lipophilic nature, which allows for direct diffusion into the cell nucleus where the hormone can then bind to estrogen receptors (ER) located within the nucleus. Genomic control of estrogen-mediated gene expression is a complex process that includes ligandereceptor interactions, receptoreDNA interactions, and receptorecofactor interactions. After estrogen binds its receptor, the ligand-bound ERs undergo a conformational change in their hormone-binding domain that allows for receptor dimerization and subsequent interaction with the estrogen response element (ERE) sequence, usually located within the promoter region of target genes. Interaction with the ERE can be direct or indirect. The indirect interaction is mediated via proteine protein interactions with activator protein 1 (AP1) or specificity protein 1 (SP1) sites in the promoter region of estrogen-responsive genes, which results in the recruitment of coregulator proteins (coactivators or corepressors) to the promoter and ultimately affects the quantity of mRNA and protein production levels that regulate physiologic responses. Following free diffusion of estrogen into the nucleus and binding to ERs, the receptors can dimerize with other ERs. The high degree of sequence homology within the DBD of ERa and ERb results in the ability for both receptors to bind the ERE, a 13-base-pair inverted-repeat DNA sequence (GGTCAnnnTGACC), with high affinity, specificity, and any combination of ERa/ERb dimer. Each composition (ERa/ERa, ERb/ERb, ERa/ERb) of dimer is believed to result in a unique response, and therefore the relative amounts of each receptor present in a given tissue influence the response.

Estrogens induce changes in gene expression through genomic and nongenomic mechanisms (Mauvais-Jarvis et al., 2013). Genomic control of estrogen-mediated gene expression is a complex process that includes ligandereceptor interactions, receptoreDNA interactions, and receptorecofactor interactions. After estrogen binds ER, the receptor undergoes a conformational

3. STEROIDS

change, dimerization, and subsequent interaction with DNA at EREs located within the promoter region of target genes (Beato and Sanchez-Pacheco, 1996; Beato et al., 1995). Interaction with the ERE can be direct or indirect. The indirect interaction is mediated via proteine protein interactions with AP-1 or specificity protein 1 (Sp-1) sites in the promoter region of estrogenresponsive genes, which results in the recruitment of coregulators to the promoter and transcription of genes that regulate physiologic responses. The nongenomic actions of estrogen occur over seconds or minutes and can proceed without direct ERegene interaction but rather by acting on membrane-bound ERs (mERs) (Deroo and Korach, 2006). mERs are transcribed in the same fashion as the classic nuclear ER but are transported to the plasma membrane where they associate via interactions with other membrane proteins such as caveolin, growth factor receptor-binding proteins like Shc, or guanosine (G) nucleotide-binding proteins (Levin and Pietras, 2008). mERs may undergo posttranslational modifications, such as palmitoylation, and/or associate with adaptor proteins like Shc, caveolins, flotillins, modulator of nongenomic activity of ER (MNAR), or lipid raft proteins (Acconcia et al., 2005; Marquez et al., 2006). mERs can bind estrogens and trigger downstream signaling pathways through direct action with either mERa or mERb at the plasma membrane (Vasudevan and Pfaff, 2007). Despite having only subtle differences in structure, ER receptors have varied tissue distribution and function resulting in tissue-dependent effects. The stimulation of ERa and ERb by estrogen results in a differential set of effects on growth, differentiation, and maintenance in a tissue-dependent fashion. For example, ERa is generally believed to have a proliferative role, whereas ERb may have an apoptotic role in transfected breast cancer cells (Sotoca et al., 2008). A possible explanation for their differential effects is the structural variation within the AF1/AF2 domains (Arnal et al., 2013). In mammary glands, E2 stimulates growth or inhibits apoptosis in both normal and transformed epithelial cells by regulating the expression of hormone-responsive genes involved in the cell cycle or PCD. In ER-positive MCF-7 breast cancer cells, E2 stimulates growth through the induction of G1-to S-phase transition and activation of cyclin E-CDK2 complexes. This induction is associated with c-myc upregulation, which controls cyclin D1 expression, activation of cyclin-dependent kinase, and phosphorylation of retinoblastoma protein (Rb) (Altucci et al., 1996; Foster and Wimalasena, 1996). Within minutes of E2 administration, mERs also mediate nongenomic effects through association with extranuclear sites, caveolae, or lipid raft domains in the plasma membrane. There, they interact with transmembrane growth factor receptors such as

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epidermal growth factor receptor (EGFR), HER2, and insulin-like growth factor receptor 1 (IGFR1) as well as other signaling molecules, including components of the MAPK and PI3K/AKT pathways, Shc, Src kinases, JAK/STAT, NOS, and G-proteins (Pedram et al., 2007; Pietras and Marquez-Garban, 2007). E2 is also a potent inhibitor of apoptosis, and it regulates the expression of several apoptotic proteins, including Bcl-2 in breast cancer cells. E2 is also capable of inducing apoptosis in numerous other cell types (i.e., prostate, neuronal, bone, thymocytes) that have been long-term estrogen-deprived (LTED) or treated extensively with antiestrogens. The development of E2 sensitivity in these cells is associated with upregulation of ERa and the MAPK, PI3K, and mammalian target of rapamycin (mTOR) growth factor pathways and the involvement of both the extrinsic and intrinsic apoptotic pathways (Santen et al., 2008). Specifically, activation of the CD95/FasL complex (Song et al., 2001), cytochrome c release from the mitochondria and alterations in Bcl-2 (Lewis et al., 2005a; Song et al., 2005), and downregulation of NF-kB are observed (Lewis et al., 2005b). Interestingly, a putative ERE has been identified in the promoter of the FasL gene, suggesting that estrogen may have direct transcriptional effects on FasL expression (Mor et al., 2000). Indeed, LTED MCF-7 cells expressed high levels of Fas compared to parental cells, with a marked increase in FasL following E2 treatment (Osipo et al., 2003; Song et al., 2001). Sp-1 and AP-1 are known regulators of FasL gene expression, and functional studies have demonstrated that Sp-1 is critical for E2-induced FasL gene expression; thus, it is likely that an apoptotic signal is initiated by FasL upregulation in breast cancer cells (Porter et al., 1997). In addition to the role of the extrinsic pathway in E2induced apoptosis, several proapoptotic Bcl-2 proteins have also been shown to be upregulated in response to E2 treatment in LTED breast cancer cells, including Bax, Bak, Bim, Noxa, and Puma. Bim and Bax short interfering RNAs (siRNAs) could reverse the apoptotic effect of E2 in these cells; moreover, loss of mitochondrial potential, release of cytochrome c, and activation of caspases-7 and -9 were observed, suggesting that the intrinsic pathway also plays a role in E2-induced apoptosis (Lewis et al., 2005a). In two other estrogendeprived breast cancer cell lines, MCF-7:2A and E8CASS, suppression of Bcl-2 expression greatly enhanced the apoptotic effect of E2, so there is great interest in developing small-molecule inhibitors of Bcl-2 as anticancer agents (Kang and Reynolds, 2009; LewisWambi et al., 2008). Akt is another rational target for breast cancer therapy, and studies have shown that E2 can induce apoptosis of breast cancer cells by inhibiting the P13K/Akt signaling pathway. E2 reduces Akt

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phosphorylation, which is associated with decreased cell proliferation (Zhang et al., 2009). Similarly, E2 has been shown to inhibit the activity of the prosurvival/ antiapoptotic factor NF-kB, inducing apoptosis (Lewis et al., 2005b; Osipo et al., 2003). Thus, development of inhibitors that block these survival pathways may enhance the apoptotic and antiproliferative effects of E2 in metastatic breast cancer that has become refractory to long-term, antihormone therapy.

3.4 Progestogens Progesterone (P4) belongs to the group of endogenous steroid hormones called progestogens that bind and activate progesterone receptor (PR). P4 is the most well studied of these family members and plays a predominant role in tissue physiology. P4 is synthesized by the placenta, ovaries, and adrenal glands and is key for coordinating normal female reproductive function and development (Lydon et al., 1995). Its major target organs include the uterus, ovary, mammary gland, bone, and brain (Kim et al., 2013). It is also involved in a myriad of functions in these tissues, including release and implantation of oocytes, maintenance of pregnancy, uterine growth, milk secretion in the mammary gland, and regulation of sexually responsive behavior in the brain (Graham and Clarke, 1997). Additionally, P4 functions as a metabolic intermediate in the production of endogenous steroids and corticosteroids and plays an important role in brain function as a neurosteroid (Baulieu and Schumacher, 2000). P4 signaling is transduced through a classical signaling mechanism via binding to PR. PR is a member of the steroid hormone receptor family of liganddependent TFs that function by binding progesterone response elements or by tethering to other DNA-bound TFs to activate specific target genes. PR also interacts with coregulatory proteins to alter chromatin structure and rates of gene transcription coactivation (Bulynko and O’Malley, 2011; Kastner et al., 1990; Li and O’Malley, 2003; Mangelsdorf et al., 1995). In humans, PR exists in three isoforms: PR-A, PR-B, and PR-C. PR-A and PR-B are both derived from a single gene with two distinct promoter regions encoding for each of the two isoforms (Mulac-Jericevic and Conneely, 2004). PR receptors are expressed in a variety of tissues, most notably smooth muscle, fallopian tube, breast, uterus, and the ovary; however, the ratios of the individual isoforms vary in tissues as a consequence of developmental (Schott et al., 1991) and hormonal (Duffy et al., 1997) status and during carcinogenesis (Graham et al., 1996), greatly impacting P4 effects. For example, studies in aggressive tumors have shown that the ratio between PR-A and PR-B is altered, with a predominance of PR-A

and loss of PR-B (Hopp et al., 2004). The third isoform, PR-C does not appear to have transcriptional activity, and to date, its expression is limited to breast cancer cell lines (Wei et al., 1997). Structurally, PRs have modules consisting of distinct, functional domains capable of (1) binding steroidal ligand, (2) dimerizing liganded receptors, (3) interacting with hormone-responsive DNA elements, and (4) interacting with coregulator proteins required for bridging receptors to the transcriptional apparatus (GuiochonMantel et al., 1989; Tsai and O’Malley, 1994; Giangrande and McDonnell, 1999; McKenna et al., 1999b; Conneely et al., 2002). While PR-A and PR-B have identical LBDs and DBDs, they differ in that PR-A has a 164 amino acid truncation of the NTD (Obr and Edwards, 2012; Nikaidou et al., 1992; Li and O’malley, 2003; Mangelsdorf et al., 1995; Mulac-Jericevic et al., 2000; Shyamala et al., 1998). PR-B contains an additional sequence of amino acids at its amino terminus encoding a third transactivation function (AF3) that is absent from PR-A (Sartorius et al., 1994; Wen et al., 1994). This additional AF3 domain allows binding of a subset of coactivators to PR-B that do not interact with PR-A, allowing PR-A and PR-B to act as two distinct TFs in vivo, thus affecting the physiologic response of certain tissues to P4 (Mc Cormack et al., 2007; Giangrande et al., 2000). Hence, PR-A and PR-B display different transactivation properties that are specific in both cell type and target gene promoter context (Tora et al., 1988; Meyer et al., 1992; Vegeto et al., 1993; Hovland et al., 1998) and allow PR-A and PRB to recruit specific coregulator proteins (Giangrande et al., 2000). In addition to this classical ligand-dependent transcription activation pathway, PR can function outside of the nucleus to mediate rapid P4-induced activation of protein phosphorylation signaling cascades (Edwards, 2005; Boonyaratanakornkit et al., 2001). These “nonclassical” pathways can be dependent or independent of transcriptional or genomic regulation (Vicent et al., 2013). Additionally, various second messengers and signal transduction pathways transduce these rapid hormonal effects mediated by nonclassical signaling (Mani et al., 2012; Schneider et al., 2014), including activation of cell membrane receptors (PGRMC1 and GABA-A), cytoplasmic PR, or receptor-independent intracellular signaling cascades like Src kinase, MAPK, intracellular cAMP, and CaMKII (Frye et al., 2006; Dressing et al., 2011; Garg et al., 2017). P4 can modulate both the intrinsic and extrinsic apoptotic pathway and also act in an apoptotic and antiapoptotic fashion depending on context and disease state (Fig. 2.8). P4 also modulates apoptosis via nongenomic mechanisms. Generally, P4 has been shown to induce apoptosis in cancer cell lines and can either enhance or prevent apoptosis in normal cells. With

3. STEROIDS

FIGURE 2.8 Summary of progesterone signaling on apoptosis. P4 can modulate both the intrinsic and extrinsic apoptotic pathways and also act in an apoptotic and antiapoptotic fashion depending on context and disease state. Numerous survival and cytotoxic genes are upregulated, downregulated, activated, or impaired.

respect to the intrinsic pathway, P4 can alter caspase expression and has been shown to modulate expression of Bcl-2 family proteins. In the extrinsic pathway, it has been shown to block apoptosis by downregulating Fas, Fas-L, and impairing TNF-a-induced apoptosis. Additionally, it can upregulate proapoptotic p53 expression in some cells. For example, P4 can cause apoptotic death of normal rat islet cells and enhance generation of reactive species (ROS), markers of oxidative stress. Incubation of the RINm5F rat pancreatic islet b-cell line with P4 increased the number of cells with loss of membrane integrity and DNA fragmentation, induced generation of reactive species, and increased upregulation of the proapoptotic TFs CREB2 (cAMP response element binding protein 2) and CHOP (CCAAT-enhancer-binding protein homologous protein) (Nunes et al., 2014). In ovarian and endometrial cancer cell lines, P4 caused a dose-dependent decrease in cell viability and activation of caspase-3. Levels of ROS and antioxidant proteins were elevated in endometrial cancer cells compared to normal cells, and a marked decrease in their expression was seen following P4 treatment. In these cells, P4 exposure resulted in increased proapoptotic p53 and BaX and decreased antiapoptotic Bcl-2 expression (Nguyen and Syed, 2011). Additionally, in a separate study, both endogenous P4 and synthetic progestin directly inhibited cellular proliferation and induced apoptosis and caspase 3/7 activity in human adenomyotic stromal cells (Yamanaka et al., 2014).

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In the brain, P4 has been shown to provide a neuroprotective effect that is linked to its ability to inhibit apoptosis. In a neonatal rat model of hypoxic ischemic brain damage (HIBD), P4 treatment prior to injury was demonstrated to increase the expression levels of phosphorylated Akt and antiapoptotic Bcl-2 but decreased that of NF-kB. It also decreased inflammation in HIBD, inhibited apoptosis, and protected the brain, suggesting that P4 activates the PI3K/Akt survival signaling pathway (Li et al., 2015). In another experimental model of traumatic brain injury (TBI), P4 treatment reduced Bax and Bad mRNA levels in the ipsilateral cerebral cortex of TBI rats, and decreased Bax and Bad protein levels. In addition, Bcl-2 and Bcl-xL mRNA and protein levels were increased by P4 in TBI injured cortex, indicating that one of the neuroprotective mechanisms of P4 may be related to its differential regulation of apoptotic signals (Yao et al., 2005). P4 can also mediate the extrinsic apoptotic pathway in numerous cell types. In a study examining the effects of P4 in the human trophoblast-derived HTR-8/SV neo cell line compared with untreated cultures, P4 treatment decreased the TUNEL-positive rate and the expression of Fas, FasL, caspase-8, caspase-3, and apoptosisassociated poly (ADP-ribose) polymerase (PARP). Bcl-2 expression was increased, suggesting that PR inhibits apoptosis by regulating a gene expression in HTR-8/ SV neo cells (Liu et al., 2007). P4 has also been implicated as a protective factor for epithelial ovarian cancers. When P4-induced effects on PCD were examined in two immortalized normal (HOSE 642, HOSE 12-12) and two malignant (OVCA 429, OVCA 432) HOSE cell lines, P4 was shown to induce apoptosis via activation of a caspase-8-initiated Fas/FasL signaling pathway. This study also demonstrated differential P4-regulation of FasL expression in HOSE and OVCA cells (Syed and Ho, 2003). P4 can also modulate apoptotic signaling in a nongenomic fashion via p53. P4 is known to inhibit the proliferation of normal breast epithelial cells in vivo, as well as breast cancer cells in vitro. In PR-positive T47D breast cancer cells, P4 treatment induced apoptosis, and expression of Bcl-2 was downregulated, while p53 was upregulated (Formby and Wiley, 1998). In the ovarian cancer cell line, SNU-840, P4 caused apoptosis, chromosomal DNA fragmentation, and upregulated p53 transiently (Yu et al., 2001). P4 administration also induced p53 gene expression and apoptosis in T47D breast cancer cells, induced apoptosis and suppressed proliferation in 211H mesothelioma cells (Horita et al., 2001), and induced apoptosis in HUVEC cells through a p38 and Jun, N-terminal kinase (JNK)-mediated pathway (Powazniak et al., 2009). In general, P4 appears to be apoptotic in nature with the exception of its antiapoptotic actions in neural tissue.

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However, there are other examples of antiapoptotic P4 actions impairing the intrinsic and extrinsic cytotoxic signaling pathways. For example, P4 has been suggested as a bone-trophic hormone, and studies have shown that P4 promotes bone formation by stimulating the proliferation and differentiation of osteoblasts (Seifert-Klauss and Prior, 2010). P4 suppresses apoptosis in the osteoblastic cell line, MC3T3-E1, induced by serum deprivation, and a PR antagonist RU486 blocked this effect. Furthermore, the suppressive effects of P4 on cytochrome c release, caspase-9 and caspase-3 activation in serum-deprived MC3T3-E1 cells were also reversed by RU486, demonstrating that P4 protects osteoblasts from apoptosis through PR and the downstream mitochondrial pathway (Wang et al., 2009). In the MCF-10A breast epithelial cell line that lacks nuclear PR expression, treatment with P4 or the progestin, R5020, increased mitochondrial activity, while a specific PR antagonist inhibited this reaction. Progestin treatment inhibited FasL-induced apoptosis and decreased caspase 3/7 levels. This suggests that progesterone treatment may stimulate the release of paracrine growth factors from nuclear receptor-positive cells to modulate apoptotic signaling (Behera et al., 2009). P4 was also found to inhibit basal caspase-3 activity and TNFa-induced apoptosis in term, fetal membranes (Luo et al., 2010) and to promote chemoresistance of A549 lung cancer cells in vitro by attenuating cisplatininduced apoptosis via nonclassical sex hormone signaling pathways (Grott et al., 2013).

4. THYROIDS Thyroid hormone (TH) is produced by the thyroid gland in response to actions of thyroid stimulating hormone (TSH) on the TSH receptor (TSH-R) expressed on the thyroid follicular cell basolateral membrane (Chiamolera and Wondisford, 2009). The thyroid hormone Lthyroxine (T4) is the major secretory product of the normal thyroid gland, and although T4 is known to play physiologic roles in regulating the actin cytoskeleton (Farwell et al., 2005) and angiogenesis (Chen et al., 2012; Luidens et al., 2010), it is primarily converted into 3,5,30 -triiodo-L-thyronine (T3) by deiodination to effect genomic hormonal actions that utilize nuclear receptors for thyroid hormone (TRs) (Cheng et al., 2010). TH is essential for normal development, metabolic regulation, and neural differentiation in mammals (Cheng et al., 2010; Williams, 2008; Tata, 2013), and TH deficiency during development or untreated congenital hypothyroidism result in neurologic deficits and growth retardation (Zimmermann, 2009). TH regulates a wide range of cellular activities, including tissue

differentiation/growth and maintenance of a cell’s metabolic balance (Yen et al., 2006). There are two types of TRsdTRa and TRbdand each displays different patterns of expression during development and adulthood (Cheng et al., 2010; Oetting and Yen, 2007). In addition, TRa and TRb have splice variants that are selectively expressed at different levels depending on the tissue type. TRa1 is a TRa splice variant that can bind T3 and is predominantly expressed in brain, cardiac, and skeletal muscle. TRa has two additional splice variants, TRa2 and TRa3, with several additional truncated forms that are widely expressed, but do not bind T3. TRb has three spliced variants, TRb1, TRb2 and TRb3, and while TRb1 is expressed widely in various tissues, TRb2 has more limited expression primarily in the brain, retina, and inner ear. TRb3 is expressed in kidney, liver, and lung (Brent, 2012; Cheng et al., 2010). The unliganded TR heterodimerizes with the retinoic acid receptor (RXR) and binds to the thyroid response element (TRE) in target genes with a corepressor to repress gene expression. Genomic TH signaling requires (1) transport of T3 into the target cell via TH transporters, (2) binding of T3 to a nuclear receptor, and (3) recruitment of coactivators that facilitate target gene transcription. This signaling pathway is highly regulated by tissue-specific TH transporters, expression and distribution of receptor isoforms, and the presence of corepressors/coactivators and the sequence and location of the TRE (Gereben et al., 2008; Oetting and Yen, 2007; Visser et al., 2011; Astapova et al., 2008; Shibusawa et al., 2003; Fig. 2.9). TH can also signal via av/b3 integrin receptors in a nongenomic fashion that is distinct from TRs; however, importantly, the hormone-binding site on avb3 may support nuclear uptake of cytoplasmic TH and may influence genomic hormonal actions (Davis et al., 2013a,b). Signaling through avb3 integrin can occur via T3 and T4 activation and generally induces proangiogenic effects, including transcription of vascular growth factor genes (Luidens et al., 2010; Cayrol et al., 2015; Liu et al., 2014; Yoshida et al., 2012), modulation of the function of vascular growth factor receptors (Mousa et al., 2008), and enhanced endothelial cell motility (Mousa et al., 2014). However, avb3 integrin expression is limited mainly to dividing endothelial cells, tumor cells, and certain other cell types; further, it is expressed or activated only limitedly in nonmalignant and nondividing endothelial cells (Bergh et al., 2005; Davis et al., 2011). T3 and T4 activation of avb3 causes activation (phosphorylation) of PI3K that ultimately activates the oncogenic ERK1/2 signal transduction pathway (Lin et al., 2009), leading to cell proliferation and inhibition of apoptosis (Lin et al., 2009, 2013). Additionally, this pathway allows TH to aid in rapid tumor growth through activation of fibroblast growth factor 2

4. THYROIDS

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FIGURE 2.9 Thyroid hormone apoptotic pathways. TH can modulate both the intrinsic and extrinsic apoptotic pathways and, in general, promote antiapoptotic gene expression and suppress apoptotic actions in both pathways, thus promoting survival.

expression (Davis et al., 2004) and the a subunit of the TF, hypoxia-inducible factor 1 (HIF-1), which plays a role in activating genes associated with angiogenesis, adaptation to hypoxia, invasion, and metastasis (Lin et al., 2009; Luidens et al., 2010; Moeller and BroeckerPreuss, 2011). TH can modulate both the intrinsic and extrinsic apoptotic pathways and, in general, promote antiapoptotic gene expression and suppress apoptotic actions in both pathways, thus promoting survival. Concerning modulation of the intrinsic apoptotic pathway, it is known that TH promotes expression of antiapoptotic Bcl-2 family genes (Genovese et al., 2013; Pietrzak and Puzianowska-Kuznicka, 2008). For example, T3 downregulates transcription of proapoptotic Bad (Bcl-2-associated death promoter) and upregulates antiapoptotic Bcl-2 in nonmalignant cells, without affecting proapoptotic Bax (Bcl-2-associated X protein) or apoptosis-inducing factor (Davis et al., 2014). It can also decrease expression of caspase-3 and Bax and increase the expression of antiapoptotic XIAP (X-linked inhibitor of apoptosis) and HIF-1a (Lin et al., 2009, 2013). In vivo studies have also demonstrated that T3 administration rescues hypothyroid rat liver cells from apoptosis induced by oxidative stress (Genovese et al., 2013). Additionally, TH activates expression of Mcl1 (Pietrzak and Puzianowska-Kuznicka, 2008)da Bcl-2-related protein that is critical for binding Bak and Bax proteinsdthus, leading to stabilization of the mitochondrial membrane preventing cytochrome c release and induction of apoptosis. In addition, T4 can downregulate expression

of proapoptotic Bax (Davis et al., 2014) and upregulate the HIF1a gene in cancer cells (Liao et al., 2014; Zhang et al., 2012). As mentioned before, TH can also modulate the extrinsic apoptotic pathway. TH can suppress apoptosis in trophoblast cells by decreasing expression of the death ligands TNF-a, Fas, and FasL (Laoag-Fernandez et al., 2004). It also decreases the activity of caspase-3 in these cells. Additionally, in mouse hepatocytes, T3 interfered with TNF-a and Fas signaling (Sukocheva and Carpenter, 2006) and prevented caspase activation and DNA fragmentation. The antiapoptotic effect of T3 could also be blocked by pharmacologic inhibition of the T3-regulated Naþ/Hþ exchanger (NHE1) pump, demonstrating that T3 may modulate essential proapoptotic TNF-induced cell acidification (D’Arezzo et al., 2004). In human hepatoma cells that were found to overexpress TRa, T3 induced apoptotic resistance (Chi et al., 2012). This effect was attributed in part to Bcl-xL overexpression, but interestingly, increased TRAIL expression in these cells was associated with increased metastatic potential (Lin et al., 2015). TH can also modulate cytotoxic signaling mechanisms through its interaction with av/b3 integrin receptors. One mechanism is through interfering with p53 mediated apoptotic pathways. Resveratrol (3,5,40 -trihydroxy-trans-stilbene) is an agent that is known to induce apoptosis in tumor cells (Chin et al., 2014). Resveratrol receptor is on a distinct domain on the av/b3 integrin, and it can induce p53-dependent nuclear accumulation of cyclooxygenase-2 (COX-2), which forms complexes

28

2. HORMONES OF PROGRAMMED CELL DEATH

with pERK1/2, p53, and SUMO-1 to act as a coactivator for p53-responsive genes (Lin et al., 2011; Chin et al., 2014). T4 treatment of cells exposed to resveratrol prevents phosphorylation (activation) of p53, inhibits nuclear accumulation of COX-2, and prevents apoptosis. Thus, it has been speculated that T4 actions may account, at least in part, for the limited clinical success of resveratrol as a chemotherapeutic agent (Davis et al., 2013a,b). The second example can be seen using the agent tetrac (tetraiodothyroacetic acid). Tetrac is a deaminated analogue of T4 that blocks the proangiogenesis and survival actions of T4 and T3, as well as other growth factors at the cell surface receptor for thyroid hormone on avb3 integrin (Yalcin et al., 2009). It leads to suppression of XIAP and Mcl1 gene expression and induced transcription of CASP2 and BCL-2L14 and other proapoptotic genes (Davis et al., 2011, D’arezzo et al., 2004). These results lend additional evidence toward understanding the antiapoptotic signaling mechanisms employed by T3 and T4.

5. RETINOIDS Vitamin A and its metabolites are collectively known as retinoids. Vitamin A refers to retinyl esters, retinol, retinal, retinoic acid, and oxidated and conjugated forms of both retinol and retinal. Retinoic acid (RA) is an active derivative of vitamin A and can be metabolically converted into isomers that subsequently exist in numerous oxidative states. RA constitutes three metabolically active metabolites that include all-trans-retinoic acid (tRA), 9-cis retinoic acid (9cRA), and 11-cis-retinal (11cRA), with tRA being the predominant physiologic form due to its stability. These metabolites are all composed of three distinct structural domains that contain a hydrophobic b-ionone ring, a linker polyunsaturated chain, and a polar end terminal group that can be oxidized to form the various biologically active metabolites. 11-cis-retinal is the visual chromophore essential for phototransduction, and 9cRA and tRA are transcriptionally active and have been shown to play a role in PCD. RA signaling in general involves ligand-activated TFs that belong to the steroid receptor family, and RA receptors contain two classes: retinoic acid receptors (RAR) and retinoid X receptors (RXR). Both RAR and RXR have three subtypes a, b, and g, each with different isoforms. RARa and RARg both have two isoforms 1 (RARa1, RARg1) and 2 (RARa2, RARg2), whereas RARb has five isoforms (RARb1e4, and 10 ) (Rhinn and Dolle, 2012). Additionally, these receptors have numerous coactivators and corepressors that modulate receptor activity. RAR can be activated by both tRA and 9cRA, while RXR is only activated by 9cRA. Most

retinol signaling in cells is thought to be mediated by tRA binding RAR in RAR/RXR heterodimers (Mic et al., 2003). Heterodimerization of retinoid receptors is essential for the biologic activity. RARs form heterodimers with RXRs, and these heterodimers and RXR homodimers function as TFs, activating retinoic acid response elements in the promoter regions of target genes. When ligand is not present, the RAR/RXR actively represses transcription by recruiting corepressors. Additionally, RXRs can heterodimerize with numerous other non-RA-associated nuclear receptors to mediate alternative signaling pathways. For example, peroxisome proliferator-activated receptors (PPARs), a, b/d, and g, are nuclear hormone receptors that can form heterodimers with RXR. These PPAR/RXR heterodimers can function as TFs, activating specific response elements of target genes (Mangelsdorf and Evans, 1995; Bocher et al., 2002; Willson et al., 2000). tRA has also been shown to be a ligand for PPAR b/d (Berry and Noy, 2007; Fig. 2.10). RA is known to be essential for numerous biologic functions and pathologic conditions including vision, embryonic development, reproduction, immune function, adipogenesis, homeostasis, metabolic syndrome, cancer, and aging (Rhinn and Dolle, 2012; Noy, 2010). RA has also been shown to regulate various cellular functions like differentiation, cell cycle arrest, and PCD; however, these occur in a cell-specific and context-dependent fashion. Concerning PCD, the two most well-studied RA metabolites include tRA and 9cRA. tRA has been studied for the treatment of various types of cancers, including kidney cancer, cervical cancer, lymphoma, leukemia, lung cancer, neuroblastoma, and glioblastoma (Das et al., 2014), while 9cRA is approved by the US Food Drug Administration for the topical treatment of cutaneous lesions of Kaposi sarcoma (Baumann et al., 2005) and has also been studied for the prevention of mammary and prostate cancer (Wu et al., 2000; Christov et al., 2002). The exact mechanisms and target genes involved in tRA- and 9cRAmediated cytotoxic signaling are a matter of investigation. It has been demonstrated that tRA can modulate both the intrinsic and extrinsic apoptotic pathways; yet to date, most studies indicate that 9cRA functions along the intrinsic route. For example, treatment of MCF-7 breast cancer cells with tRA can induce apoptosis and lead to direct activation of the RAR in these cells controlling upregulation of caspase-9 expression (Donato and Noy, 2005). In keratinocytes, tRA can sensitize them to apoptosis from UVB irradiation (Mrass et al., 2004) and can induce the expression of caspase-7 through de novo protein synthesis (Donato and Noy, 2005; Mrass et al., 2004). tRA has also been shown to induce the expression of caspase-3, -6, -7, and -9 in keratinocytes

5. RETINOIDS

FIGURE 2.10 RA hormone, metabolites, and apoptotic pathways. (A) RA constitutes three metabolically active metabolites that have been intensely studied and include all-trans-retinoic acid (tRA), 9-cisretinoic acid (9cRA), and 11-cis-retinal (11cRA), with tRA being the predominant physiologic form due to its stability. (B) RAR can be activated by both tRA and 9cRA, while RXR is only activated by 9cRA. Most retinol signaling in cells is thought to be mediated by tRA binding RAR in RAR/RXR heterodimers. Adapted from Noy, N., 2010. Between death and survival: retinoic acid in regulation of apoptosis. Annu. Rev. Nutr. 30, 201e217.

29

(Mrass et al., 2004), possibly increasing susceptibility of these cells to apoptosis-inducing genes (Shinoura et al., 1999, 2000a, 2000b, 2002; Yasui et al., 2005). Additionally, tRA can modulate the expression of both pro- and antiapoptotic Bcl-2 proteins to initiate mitochondrial-based cytotoxic signaling pathways in a number of cancer cell lines (Noy, 2010). Treatment of MCF-7 cells with tRA leads to Bax activation and cytochrome c release (Niu et al., 2001), and this tRA-induced apoptosis is also associated with downregulation of Bcl-2 and survivin (a protein that inhibits caspases) (Pratt et al., 2003; Raffo et al., 2000). In neuroblastoma cells, apoptosis induced by tRA is accompanied by downregulation of Bcl-2, activation of caspase-9, activation of caspase-3, and cytoplasmic release of cytochrome c (Noy, 2010; Niizuma et al., 2006). In metastatic melanoma (Zhang and Rosdahl, 2004) and myeloblastic leukemia cells (Zheng et al., 2000), tRA has also been shown to downregulate Bcl-2 during apoptosis. tRA triggers esophageal tumor cell apoptosis in mice with accompanied downregulation of survivin and upregulation of caspase-3 (Lu et al., 2010). Similarly, in gastrointestinal stromal tumor GIST-T1 and GIST-882 cells, tRA activates caspase-3, which is accompanied by downregulation of survivin and upregulation of Bax (Hoang et al., 2010). The 9cRA metabolite can also induce PCD through modifying expression of Bcl-2 proteins and activation of caspases. 9cRA is associated with reduced Bcl-2 and Bcl-XL expression, activation of procaspase-6, -7, -8, and -9, and release of cytochrome c (Gianni et al., 2000). When MCF-7 breast cancer cells were treated with 9cRA in combination with PPAR ligand rosiglitazone (BRL), cell viability decreased with a concomitant increase in the levels of both p53 and p21. Apoptosis induced by BRL and 9-RA was determined to activate the intrinsic apoptotic pathway (Bonofiglio et al., 2009). Interestingly, tRA has also been shown to upregulate the tumor suppressor p53 in numerous cancer cell lines; however the mechanism is not well understood and may involve the chromatin-modifying protein Chmp1A (Li et al., 2009) and the basic helix-loop-helix TF stimulated by RA 13 (Stra13) (Thin et al., 2007). tRA also interacts with death ligands and death receptors via the extrinsic apoptotic pathway. tRA has been shown to upregulate the death ligand TRAIL via RAR upregulation of interferon regulatory factorda known regulator of TRAIL expression in acute promyelocytic leukemia cells (Altucci et al., 2001)dand has been suggested to enhance Fas-mediated apoptosis in T cells by reversal of PKC-induced recruitment of caspase-8 to the DISC complex (Engedal et al., 2009). Finally, tRA can also induce expression of TNFa receptors, resulting in enhanced TNFa-induced apoptosis in both lung cancer and leukemia cells (Manna and Aggarwal, 2000; Witcher et al., 2003).

30

2. HORMONES OF PROGRAMMED CELL DEATH

It is important to note that in addition to the cytotoxic actions of RA, in the context of some normal as well as carcinoma cells, RA promotes rather than inhibits survival and can even promote cell proliferation. The ability of RA to enhance proliferation and suppress apoptosis is unlikely to be mediated by nuclear RA receptors, whose target genes are usually involved in inhibition of cell growth (Noy, 2010). Indeed, RA can exert effects using a transcription-independent, nonclassical pathway where the nuclear receptor is dispensable. For example, RA rapidly activates CREB in human bronchial epithelial cells via a RAR nuclear receptor-independent pathway (Aggarwal et al., 2006). Additionally, there are other proteins that can mediate apoptotic activities of the RAR in addition to RA, including CCAAT/ enhancer-binding protein, the RAR target gene Rig-I(a protein that initiates apoptosis upon sensing viral RNA in infected cells (Besch et al., 2009)), p38 MAP kinase (Hormi-Carver et al., 2007), and the programmed cell death-4 (PDCD4) tumor suppressor protein (Altucci and Gronemeyer, 2001; Ozpolat et al., 2007). Thus, determining the precise mechanistic explanation for and the cellular effects associated with RA and/or the receptors isoforms is an area that requires more investigation.

6. VITAMIN D3 DERIVATIVES Vitamin D is a secosteroid hormone that regulates many biologic and cellular functions. Classical vitamin D target tissues include the small intestine, kidney, and bone, where it participates in calcium homeostasis. But, epidemiological evidence has associated vitamin D deficiency with autoimmune disease, cancer, hypertension, and diabetes, thus indicating that vitamin D participates in diverse biologic actions. Indeed, many studies have demonstrated that vitamin D plays a role in bone metabolism, immunomodulation, cell cycling, cell proliferation, differentiation, control of other hormones, and PCD. Vitamin D has protective effects against numerous diseases inducing diabetes, metabolic syndrome, hypertension, cancer, and multiple sclerosis (Samuel and Sitrin, 2008; Dusso et al., 2005). Vitamin D starts out as a prohormone that is obtained through diet or synthesized from sunlight exposure that is metabolically converted to the active metabolite 1,25dihydroxyvitamin D (calcitriol; 1,25(OH)2D3) from sequential processing in the liver and kidneys. Most 1,25(OH)2D3 signaling requires interaction with its high-affinity receptor (VDR), which is a member of the steroid receptor family and acts as a ligand-activated TF, which alters the transcription rates of target genes responsible for the biologic responses. VDR-initiated gene expression requires (1) ligand binding, (2) heterodimerization with RXR, (3) vitamin D response element

interaction in target gene promoters, and (4) recruitment of coregulators/corepressors that can enhance or suppress subsequent gene transcription (Dusso et al., 2005). Various studies have demonstrated the anticancer activities of 1,25(OH)2D3 and its derivatives, and it has been shown to reduce tumor incidence and inhibit tumor progression (Eisman et al., 1989; Studzinski and Moore, 1995; Guyton et al., 2001; Colston and Hansen, 2002). This anticancer activity has been attributed to their direct ability to prevent cell proliferation and to induce cytotoxic effects on cancer cells, as they have been shown to cause cell cycle arrest followed by the induction of PCD in many tumor cells in vitro (Colston et al., 1992; Danielsson et al., 1997; Evans et al., 1999; Park et al., 2000; Mathiasen et al., 2001). Importantly, it has also been recognized that these anticancer activities are achieved through interaction with other signaling pathways. Vitamin D compounds have been shown to exert synergistic effects when used in combination with different agents used in anticancer therapies. For example, 1,25(OH)2D3 potentiates the cytotoxic action of different immune mediators, including TNFa (Mathiasen et al., 2001; Yacobi et al., 1996; Rocker et al., 1994) and interleukin-1 and -6 (Koren et al., 2000). TNFa is considered a major mediator of the anticancer activity of macrophages, which often infiltrate tumors (Steele et al., 1985; Mantovani et al., 1992), and may also produce calcitriol from its precursor 25-hydroxyvitamin D3 (Reichel et al., 1987, 1991; Adams et al., 1989; Weitsman et al., 2003). With respect to PCD, vitamin D signaling plays a role in both proapoptotic and antiapoptotic signaling, and this is dependent on the cell type or disease state. For example, 1,25(OH)2D3 can induce apoptosis in numerous cancers mainly through the intrinsic pathway but has also shown a protective effect with regard to PCD in normal tissues. 1,25(OH)2D3 can induce caspase-dependent apoptosis in MCF-7 breast cancer cells by reciprocal modulation of Bcl-2 and Bax (Wagner et al., 2003) or through induction of increases in intracellular calcium (Sergeev, 2004), which activates calciumdependent, proapoptotic proteases m-calpain and caspase-12 (Mathiasen et al., 2002). 1,25(OH)2D3 has also been shown to enhance apoptotic signaling as a result of TNFa stimulation or exposure to ionizing radiation in MCF-7 cells (Diker-Cohen et al., 2003). Additionally 1,25(OH)2D3 promotes cytotoxic signaling in hyperproliferative disorders and cancers including glioma (Elias et al., 2003), melanoma, breast cancer (Valrance and Welsh, 2004), colorectal adenoma and carcinoma (Diaz et al., 2000), prostate cancer (Guzey et al., 2002), epithelial caner (Sergeev, 2014), and ovarian cancer (Jiang et al., 2004). However, in normal cells, 1,25(OH)2D3 generally does not induce cytotoxic

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7. TUMOR NECROSIS FACTOR SUPERFAMILY: DEATH RECEPTORS AND LIGANDS

signaling and has even been shown to exert a protective effect in cases of UV-B-induced apoptosis in keratinocytes (De Haes et al., 2003). It has also been reported to enhance Bcl-2 expression to enhance survival in normal thyrocytes (Wang et al., 1999), to protect pancreatic islet cells from TNF/IL-1b/IFN-g-induced apoptosis by suppressing Fas expression (Riachy et al., 2002), and can increase the Bcl-2/Bax ratio in osteoblasts to inhibit caspase-8 activation (Christakos and Liu, 2004). Importantly, induction of cytotoxic signals by 1,25(OH)2D3 is not limited to hyperproliferative diseases and cancer. For example, it can also induce apoptotic signaling via activation of a calcium-dependent activation of proapoptotic proteases m-calpain and caspase-12 in normal mature adipocytes. Taken together, vitamin D demonstrates both cytotoxic and survival signaling in normal tissue development and plays an important role in growth arrest in both noncancerous hyperproliferative disorders and cancer (Dusso et al., 2005).

7. TUMOR NECROSIS FACTOR SUPERFAMILY: DEATH RECEPTORS AND LIGANDS The TNF superfamily comprises a diverse number of type I transmembrane protein cytokine receptors and ligands that regulate a diverse number of physiologic and pathologic processes including immune responses, hematopoiesis, morphogenesis, tumorigenesis, transplant rejection, septic shock, viral replication, bone resorption, rheumatoid arthritis, diabetes, and PCD (Aggarwal, 2003; Table 2.1). Structurally, these receptors contain a C-terminal intracellular tail, a membrane-spanning region, and a cysteine-rich extracellular domain that defines ligand specificity, which is characterized by the presence of up to six cysteine-rich domains (Guicciardi and Gores, 2009). One subset of receptors in this family are classified as the death receptors due to the fact they

can trigger a rapid and direct route to apoptosis. The death receptors are distinguished from other receptors in the TNF superfamily due to the presence of an w80 amino acid, homologous cytoplasmic sequence termed the death domain (DD), which is essential for induction of PCD (Brakebusch et al., 1992; Itoh and Nagata, 1993). Historically death receptors were thought to primarily induce PCD; however, other specific noncytotoxic functions have been identified for these receptors including effects on cell proliferation, differentiation, inflammation, and tumorigenesis. Thus, given the role of these receptors in controlling survival and other critical cellular functions and their potential therapeutic application in diseases such as cancer, they have become a target for intense clinical investigation. The cytotoxic signal transduction cascade initiated by death receptors utilizes both the extrinsic and intrinsic PCD signaling pathways. It proceeds generally through several steps, including (1) binding to the cognate ligand, (2) recruitment of specific adaptor/docking proteins, (3) initiator caspase activation, and finally, (4) specific signaling pathways that are adaptor protein and cell type dependent (Fig. 2.11; Guicciardi and Gores, 2009). The cognate ligands (death ligands) are a group of complementary cytokines that belong to the TNF protein family, and they can function in an autocrine and paracrine manner inducing trimerization of their respective cell surface receptors to initiate the apoptotic signal. Structurally, these ligands are type II transmembrane proteins containing an intracellular NTD, a transmembrane region, and a C-terminal extracellular tail. These ligands can also be in a free soluble form, as is the case with lymphocyte-derived cytokine LTa, or can be released from the membrane by proteolytic cleavage; however, the capacity of the soluble forms to induce apoptosis is generally significantly lower when compared to corresponding membrane-bound forms (Schneider et al., 1998; Shudo et al., 2001; Wajant et al., 2001; Grell et al., 1998). Once receptor activation has occurred, intracellular recruitment of specific adaptor proteins occurs through

TABLE 2.1 Death Receptors, Ligands and Decay Receptors. Receptor

Alternate Name

Ligand

TNF-R1

(DR1,CD120a, p55, p60)

TNF (LTa)

CD95

(DR2, Fas, Apo-1)

FasL (CD95L)

TRAMP

(DR3,APO-3, WSL1, LARD)

TWEAK(Apo3L)

TRAIL-R1

(DR4, APO-2)

TRAIL(Apo2L)

TRAIL-R2

(DR5, KILLER, TRICK2)

TRAIL (Apo2L)

DR6

(TNFRSF21)

N-APP?

EDAR (Ectodysplasin-A Receptor)

Ectodysplasin A

NGFR (Nerve Growth Factor Receptor)

Neurotrophins

Decoy Receptor

DcR3

DcR1, DcR2, OPG

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2. HORMONES OF PROGRAMMED CELL DEATH

FIGURE 2.11 Extrinsic apoptotic pathways mediated by death receptors. The cytotoxic signal transduction cascade initiated by death receptors utilizes both the extrinsic and intrinsic PCD signaling pathways. It proceeds generally through several steps, including (1) binding to the cognate ligand; (2) recruitment of specific adaptor/docking proteins; (3) initiator caspase activation; (4) and specific signaling pathways that are adaptor protein and cell type dependent. There are two general types of death receptor signaling strategies that form unique protein complexes. Adapted from Lavrik, I., Golks, A., Krammer, P.H., 2005. Death receptor signaling. J. Cell Sci. 118, 265e267.

hemophilic interactions, and the adaptor proteins involved are death receptor dependent. There are two general types of death receptor signaling strategies that form unique protein complexes. CD95, TRAIL-R1, and TRAIL-R2 form DISCs, which all contain similar signaling proteins that subsequently function to activate caspase-8 and induce apoptotic signaling. The second type involving TNFR1, DR3, DR6, EDAR, and NGFR are capable of inducing both apoptotic and survival signals through the formation of two subsequent signaling complexes where the initial complex (complex I) promotes survival and the latter complex (complex II) promotes apoptosis through a caspase-8-mediated mechanism. As far as specific signaling mechanism, CD95, TRAIL-R1, and TRAIL-R2 receptor activation leads to DISC formation through interactions of DDs found on both the receptor and its associated adaptor protein, FADD. FADD couples the receptor to downstream death effectors including initiator caspases (procaspases-8a/b, procaspase-10), variants of the cellular FLICE-inhibitory protein (cFLIPL and cFLIPS) (Lavrik et al., 2005; Peter and Krammer, 2003), and other less characterized proteins through homotypic contacts on death effector domains (DEDs) found on these death effectors. This leads to autoproteolytic activation of caspase-8 and subsequent propagation of the apoptotic signal and activation of effector caspases (caspase-3/6/7). In the case of the second type of signaling strategy involving TNFR1, DR3, DR6, EDAR, and NGFR,

TNFR1 is the most understood. Cytotoxic TNFR1 signaling involves two distinct signaling complexes. The first, complex I, is formed at the membrane by interactions with the receptor through its DD with receptorinteracting protein, adaptor protein TNFR-associated DD protein (TRADD), and TRAF-1/2 (TNFRassociated factor). This complex purportedly activates NF-kB signaling by recruitment of the IKK complex and additionally activates JNK through a TRAF-2 dependent mechanism. Activation of NF-kB leads to expression of survival genes, and while prolonged JNK activation can lead to caspase-8 activation, a balance between NF-kB and JNK signaling can promote cell survival or cell death (Tang et al., 2001; Chang et al., 2006). Complex I can translocate to the cytosol and recruit FADD, procaspase-8/10, and cFLIPL/S forming complex II and activate death signaling through caspase-8 activation (Micheau and Tschopp, 2003). The exact molecular mechanism through which adaptor and other downstream signaling proteins mediate PCD is a matter of investigation, and presumably numerous proteins and signaling cascades involved in this regulation are as of now unidentified. That being said, there are many known mechanisms that play a role in modulation of death receptor signaling, and this modulation can occur at many levels including cell contextual differences, regulatory proteins, and decoy receptors. Control of receptor and ligand expression is one way in which the cell context

33

REFERENCES

determines the sensitivity to PCD signals. For example, while some receptors like TNFR1, TRAIL-R1, TRAIL-R2, and CD95 are expressed in numerous tissue types, ligands like TNF or FasL are expressed in a more cellrestricted fashion (Kumar et al., 2005). In addition to controlling expression levels, cell context also determines the specific signaling pathway that is activated and ultimately leads to PCD. An example of this can be seen in the case of CD95 signaling, where two signaling mechanisms have been identified that occur in a cell-dependent fashion. Certain cell types contain high levels of DISC formation and active caspase-8. In these type I cells, activation of caspase-8 leads to direct activation of downstream caspases. However, some cells have lower levels of CD95-activated DISC formation and thus lower caspase-8 activation. In these type II cells, the Bcl-2family protein Bid is cleaved by procaspase-8 and translocates to the mitochondria where it mediates release of cytochrome c to activate the intrinsic pathway, which is essential for PCD in Type II cells (Scaffidi et al., 1998; Korsmeyer et al., 2000). Although the concept of Type I and II cells was originally related only to CD95 signaling, recent evidence suggests that this signaling paradigm may apply to other death receptors as well. Regulatory signaling proteins like cFLIPL/S isoforms can also modulate cytotoxic signaling from death receptors. As we have mentioned, cFLIPL/S plays an essential role in formation of the DISC complex and as part of complex II signaling and activation of caspase-8 and -10 at the DISC can be regulated by cFLIP (Tschopp et al., 1998). cFLIP has three isoforms, cFLIP long (cFLIPL), cFLIP short (cFLIPS), and a short variant (cFLIPR), all of which have DED domains and can be recruited to the DISC complex (Golks et al., 2005). The role of cFLIPS in inhibiting death receptor-mediated apoptosis is well established, as it can block caspase-8 processing and activation at the DISC. cFLIPR structurally resembles cFLIPS and likely works through a similar mechanism. However, cFLIPL has been described as antiapoptotic by impairing caspase-8 activation and participating in activation of NF-kB survival signaling (Hu et al., 2000). However, cFLIPL may also act in a cytotoxic fashion by promoting caspase-8 activation, suggesting that it has a dual function in both cytotoxic and survival signaling (Chang et al., 2002; Micheau et al., 2002; Dohrman et al., 2005). In addition to death receptors, the TNF superfamily comprises decoy receptors that inhibit death signaling through the sequestration of ligand. Decoy receptors include DcR1, DcR2, and osteoprotegerin (OPG), which bind to TRAIL R1 and R2, and DcR3, which binds CD95 ligand (Ashkenazi and Dixit, 1999). Decoy receptors can be cell surface molecules (DcR1 and DcR2) or can be secreted and function as soluble proteins (OPG and

DcR3) (Ashkenazi and Dixit, 1999). Structurally, DcR1 lacks a cytoplasmic region and appears to be attached to the cell surface through a glycophospholipid anchor, while DcR2 has a cytoplasmic DD, albeit shorter than a typical DD, which does not transmit the apoptotic signal. The extracellular domain of DcR1 and DcR2 competes with DR3 and DR4 for binding of TRAIL (Apo2L). DcR3 is structurally related to OPG and can bind FasL with an affinity equal to that of CD95 and also prevents the apoptotic signal.

8. CONCLUSIONS AND FUTURE DIRECTIONS The process of PCD is essential for maintenance of the healthy organism and adaptation to environmental changes. Hormones play a critical role in this process. Defects in the apoptotic machinery have been implicated in cancer (Wong, 2011) and autoimmune disease (Eguchi, 2001) and excessive apoptosis with myocardial infarction (Krijnen et al., 2002), stroke (Radak et al., 2017; Broughton et al., 2009), and neurodegenerative disorders such as Alzheimer disease (Obulesu and Lakshmi, 2014). These examples highlight the significance of apoptosis and cell turnover for homeostatic regulation. Hormones are known to induce or enhance apoptotic processes under physiologic or pathologic conditions and can act generally and/or in a tissue-specific manner to prevent the onset of PCD. Understanding how hormones regulate apoptosis in responsive tissues is critical to designing strategies to prevent and treat diseases that affect these tissues, such as cancer, autoimmune, and degenerative disorders.

References Abrams, M.T., Robertson, N.M., Yoon, K., Wickstrom, E., 2004. Inhibition of glucocorticoid-induced apoptosis by targeting the major splice variants of BIM mRNA with small interfering RNA and short hairpin RNA. J. Biol. Chem. 279, 55809e55817. Acconcia, F., Ascenzi, P., Bocedi, A., Spisni, E., Tomasi, V., Trentalance, A., Visca, P., Marino, M., 2005. Palmitoylationdependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol. Mol. Biol. Cell 16, 231e237. Adams, J.S., Modlin, R.L., Diz, M.M., Barnes, P.F., 1989. Potentiation of the macrophage 25-hydroxyvitamin D-1-hydroxylation reaction by human tuberculous pleural effusion fluid. J. Clin. Endocrinol. Metab. 69, 457e460. Aggarwal, B.B., 2003. Signalling pathways of the TNF superfamily: a double-edged sword. Nat. Rev. Immunol. 3, 745e756. Aggarwal, S., Kim, S.W., Cheon, K., Tabassam, F.H., Yoon, J.H., Koo, J.S., 2006. Nonclassical action of retinoic acid on the activation of the cAMP response element-binding protein in normal human bronchial epithelial cells. Mol. Biol. Cell 17, 566e575. Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G., Thornberry, N.A., Wong, W.W., Yuan, J., 1996. Human ICE/CED3 protease nomenclature. Cell 87, 171.

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2. HORMONES OF PROGRAMMED CELL DEATH

Altucci, L., Addeo, R., Cicatiello, L., Dauvois, S., Parker, M.G., Truss, M., Beato, M., Sica, V., Bresciani, F., Weisz, A., 1996. 17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells. Oncogene 12, 2315e2324. Altucci, L., Gronemeyer, H., 2001. The promise of retinoids to fight against cancer. Nat. Rev. Canc. 1, 181e193. Altucci, L., Rossin, A., Raffelsberger, W., Reitmair, A., Chomienne, C., Gronemeyer, H., 2001. Retinoic acid-induced apoptosis in leukemia cells is mediated by paracrine action of tumor-selective death ligand TRAIL. Nat. Med. 7, 680e686. Anstead, G.M., Carlson, K.E., Katzenellenbogen, J.A., 1997. The estradiol pharmacophore: ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site. Steroids 62, 268e303. Aranda, A., Pascual, A., 2001. Nuclear hormone receptors and gene expression. Physiol. Rev. 81, 1269e1304. Arnal, J.F., Fontaine, C., Abot, A., Valera, M.C., Laurell, H., Gourdy, P., Lenfant, F., 2013. Lessons from the dissection of the activation functions (AF-1 and AF-2) of the estrogen receptor alpha in vivo. Steroids 78, 576e582. Ashkenazi, A., Dixit, V.M., 1998. Death receptors: signaling and modulation. Science 281, 1305e1308. Ashkenazi, A., Dixit, V.M., 1999. Apoptosis control by death and decoy receptors. Curr. Opin. Cell Biol. 11, 255e260. Astapova, I., Lee, L.J., Morales, C., Tauber, S., Bilban, M., Hollenberg, A.N., 2008. The nuclear corepressor, NCor, regulates thyroid hormone action in vivo. Proc. Natl. Acad. Sci. U. S. A. 105, 19544e19549. Bamberger, C.M., Bamberger, A.M., DE Castro, M., Chrousos, G.P., 1995. Glucocorticoid receptor beta, a potential endogenous inhibitor of glucocorticoid action in humans. J. Clin. Investig. 95, 2435e2441. Barnes, P.J., Karin, M., 1997. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336, 1066e1071. Baulieu, E., Schumacher, M., 2000. Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination. Steroids 65, 605e612. Baumann, L., Vujevich, J., Halem, M., Martin, L.K., Kerdel, F., Lazarus, M., Pacheco, H., Black, L., Bryde, J., 2005. Open-label pilot study of alitretinoin gel 0.1% in the treatment of photoaging. Cutis 76, 69e73. Beato, M., 1989. Gene regulation by steroid hormones. Cell 56, 335e344. Beato, M., Herrlich, P., Schutz, G., 1995. Steroid hormone receptors: many actors in search of a plot. Cell 83, 851e857. Beato, M., Sanchez-Pacheco, A., 1996. Interaction of steroid hormone receptors with the transcription initiation complex. Endocr. Rev. 17, 587e609. Behera, M.A., Dai, Q., Garde, R., Saner, C., Jungheim, E., Price, T.M., 2009. Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis in MCF-10A benign breast epithelial cells. Am. J. Physiol. Endocrinol. Metab. 297, E1089eE1096. Bennett, N.C., Gardiner, R.A., Hooper, J.D., Johnson, D.W., Gobe, G.C., 2010. Molecular cell biology of androgen receptor signalling. Int. J. Biochem. Cell Biol. 42, 813e827. Berg, M.N., Dharmarajan, A.M., Waddell, B.J., 2002. Glucocorticoids and progesterone prevent apoptosis in the lactating rat mammary gland. Endocrinology 143, 222e227. Bergh, J.J., Lin, H.Y., Lansing, L., Mohamed, S.N., Davis, F.B., Mousa, S., Davis, P.J., 2005. Integrin alphaVbeta3 contains a cell surface receptor site for thyroid hormone that is linked to activation of mitogen-activated protein kinase and induction of angiogenesis. Endocrinology 146, 2864e2871. Berry, D.C., Noy, N., 2007. Is PPARbeta/delta a retinoid receptor? PPAR Res. 2007, 73256.

Berry, M., Metzger, D., Chambon, P., 1990. Role of the two activating domains of the oestrogen receptor in the cell-type and promotercontext dependent agonistic activity of the anti-oestrogen 4hydroxytamoxifen. EMBO J. 9, 2811e2818. Besch, R., Poeck, H., Hohenauer, T., Senft, D., Hacker, G., Berking, C., Hornung, V., Endres, S., Ruzicka, T., Rothenfusser, S., Hartmann, G., 2009. Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells. J. Clin. Investig. 119, 2399e2411. Boatright, K.M., Renatus, M., Scott, F.L., Sperandio, S., Shin, H., Pedersen, I.M., Ricci, J.E., Edris, W.A., Sutherlin, D.P., Green, D.R., Salvesen, G.S., 2003. A unified model for apical caspase activation. Mol. Cell 11, 529e541. Bocher, V., Pineda-Torra, I., Fruchart, J.C., Staels, B., 2002. PPARs: transcription factors controlling lipid and lipoprotein metabolism. Ann. N. Y. Acad. Sci. 967, 7e18. Bonofiglio, D., Cione, E., Qi, H., Pingitore, A., Perri, M., Catalano, S., Vizza, D., Panno, M.L., Genchi, G., Fuqua, S.A., Ando, S., 2009. Combined low doses of PPARgamma and RXR ligands trigger an intrinsic apoptotic pathway in human breast cancer cells. Am. J. Pathol. 175, 1270e1280. Boonyaratanakornkit, V., Scott, M.P., Ribon, V., Sherman, L., Anderson, S.M., Maller, J.L., Miller, W.T., Edwards, D.P., 2001. Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases. Mol. Cell 8, 269e280. Brakebusch, C., Nophar, Y., Kemper, O., Engelmann, H., Wallach, D., 1992. Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor. EMBO J. 11, 943e950. Brent, G.A., 2012. Mechanisms of thyroid hormone action. J. Clin. Investig. 122, 3035e3043. Broughton, B.R., Reutens, D.C., Sobey, C.G., 2009. Apoptotic mechanisms after cerebral ischemia. Stroke 40, e331ee339. Bulynko, Y.A., O’malley, B.W., 2011. Nuclear receptor coactivators: structural and functional biochemistry. Biochemistry 50, 313e328. Buttyan, R., Shabsigh, A., Perlman, H., Colombel, M., 1999. Regulation of apoptosis in the prostate gland by androgenic steroids. Trends Endocrinol. Metabol. 10, 47e54. Cain, K., Bratton, S.B., Cohen, G.M., 2002. The Apaf-1 apoptosome: a large caspase-activating complex. Biochimie 84, 203e214. Casale, F., Addeo, R., D’angelo, V., Indolfi, P., Poggi, V., Morgera, C., Crisci, S., DI Tullio, M.T., 2003. Determination of the in vivo effects of prednisone on Bcl-2 family protein expression in childhood acute lymphoblastic leukemia. Int. J. Oncol. 22, 123e128. Cayrol, F., Diaz Flaque, M.C., Fernando, T., Yang, S.N., Sterle, H.A., Bolontrade, M., Amoros, M., Isse, B., Farias, R.N., Ahn, H., Tian, Y.F., Tabbo, F., Singh, A., Inghirami, G., Cerchietti, L., Cremaschi, G.A., 2015. Integrin alphavbeta3 acting as membrane receptor for thyroid hormones mediates angiogenesis in malignant T cells. Blood 125, 841e851. Cecconi, F., Alvarez-Bolado, G., Meyer, B.I., Roth, K.A., Gruss, P., 1998. Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727e737. Cerretti, D.P., Kozlosky, C.J., Mosley, B., Nelson, N., Van Ness, K., Greenstreet, T.A., March, C.J., Kronheim, S.R., Druck, T., Cannizzaro, L.A., et al., 1992. Molecular cloning of the interleukin-1 beta converting enzyme. Science 256, 97e100. Chai, J., Wu, Q., Shiozaki, E., Srinivasula, S.M., Alnemri, E.S., Shi, Y., 2001. Crystal structure of a procaspase-7 zymogen: mechanisms of activation and substrate binding. Cell 107, 399e407. Chang, D.W., Xing, Z., Pan, Y., Algeciras-Schimnich, A., Barnhart, B.C., Yaish-Ohad, S., Peter, M.E., Yang, X., 2002. c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. EMBO J. 21, 3704e3714.

REFERENCES

Chang, L., Kamata, H., Solinas, G., Luo, J.L., Maeda, S., Venuprasad, K., Liu, Y.C., Karin, M., 2006. The E3 ubiquitin ligase itch couples JNK activation to TNFalpha-induced cell death by inducing c-FLIP(L) turnover. Cell 124, 601e613. Chapman, M.S., Askew, D.J., Kuscuoglu, U., Miesfeld, R.L., 1996. Transcriptional control of steroid-regulated apoptosis in murine thymoma cells. Mol. Endocrinol. 10, 967e978. Charmandari, E., Tsigos, C., Chrousos, G., 2005. Endocrinology of the stress response. Annu. Rev. Physiol. 67, 259e284. Chauhan, S., Leach, C.H., Kunz, S., Bloom, J.W., Miesfeld, R.L., 2003. Glucocorticoid regulation of human eosinophil gene expression. J. Steroid Biochem. Mol. Biol. 84, 441e452. Chen, J., Ortmeier, S.B., Savinova, O.V., Nareddy, V.B., Beyer, A.J., Wang, D., Gerdes, A.M., 2012. Thyroid hormone induces sprouting angiogenesis in adult heart of hypothyroid mice through the PDGFAkt pathway. J. Cell Mol. Med. 16, 2726e2735. Cheng, S.Y., Leonard, J.L., Davis, P.J., 2010. Molecular aspects of thyroid hormone actions. Endocr. Rev. 31, 139e170. Chi, H.C., Chen, S.L., Liao, C.J., Liao, C.H., Tsai, M.M., Lin, Y.H., Huang, Y.H., Yeh, C.T., Wu, S.M., Tseng, Y.H., Chen, C.Y., Tsai, C.Y., Chung, I.H., Chen, W.J., Lin, K.H., 2012. Thyroid hormone receptors promote metastasis of human hepatoma cells via regulation of TRAIL. Cell Death Differ. 19, 1802e1814. Chiamolera, M.I., Wondisford, F.E., 2009. Minireview: thyrotropinreleasing hormone and the thyroid hormone feedback mechanism. Endocrinology 150, 1091e1096. Chin, Y.T., Hsieh, M.T., Yang, S.H., Tsai, P.W., Wang, S.H., Wang, C.C., Lee, Y.S., Cheng, G.Y., Huangfu, W.C., London, D., Tang, H.Y., Fu, E., Yen, Y., Liu, L.F., Lin, H.Y., Davis, P.J., 2014. Antiproliferative and gene expression actions of resveratrol in breast cancer cells in vitro. Oncotarget 5, 12891e12907. Christakos, S., Liu, Y., 2004. Biological actions and mechanism of action of calbindin in the process of apoptosis. J. Steroid Biochem. Mol. Biol. 89e90, 401e404. Christov, K.T., Moon, R.C., Lantvit, D.D., Boone, C.W., Steele, V.E., Lubet, R.A., Kelloff, G.J., Pezzuto, J.M., 2002. 9-cis-retinoic acid but not 4-(hydroxyphenyl)retinamide inhibits prostate intraepithelial neoplasia in Noble rats. Cancer Res. 62, 5178e5182. Chrousos, G.P., 1995. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N. Engl. J. Med. 332, 1351e1362. Chrousos, G.P., Charmandari, E., Kino, T., 2004. Glucocorticoid action networksdan introduction to systems biology. J. Clin. Endocrinol. Metab. 89, 563e564. Chun, S.Y., Eisenhauer, K.M., Minami, S., Billig, H., Perlas, E., Hsueh, A.J., 1996. Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor. Endocrinology 137, 1447e1456. Clarke, A.R., Purdie, C.A., Harrison, D.J., Morris, R.G., Bird, C.C., Hooper, M.L., Wyllie, A.H., 1993. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 362, 849e852. Colston, K.W., Chander, S.K., Mackay, A.G., Coombes, R.C., 1992. Effects of synthetic vitamin D analogues on breast cancer cell proliferation in vivo and in vitro. Biochem. Pharmacol. 44, 693e702. Colston, K.W., Hansen, C.M., 2002. Mechanisms implicated in the growth regulatory effects of vitamin D in breast cancer. Endocr. Relat. Cancer 9, 45e59. Conneely, O.M., Mulac-Jericevic, B., Demayo, F., Lydon, J.P., O’malley, B.W., 2002. Reproductive functions of progesterone receptors. Recent Prog. Horm. Res. 57, 339e355. D’arezzo, S., Incerpi, S., Davis, F.B., Acconcia, F., Marino, M., Farias, R.N., Davis, P.J., 2004. Rapid nongenomic effects of 3,5,3’triiodo-L-thyronine on the intracellular pH of L-6 myoblasts are mediated by intracellular calcium mobilization and kinase pathways. Endocrinology 145, 5694e5703.

35

Danielsson, C., Mathiasen, I.S., James, S.Y., Nayeri, S., Bretting, C., Hansen, C.M., Colston, K.W., Carlberg, C., 1997. Sensitive induction of apoptosis in breast cancer cells by a novel 1,25-dihydroxyvitamin D3 analogue shows relation to promoter selectivity. J. Cell. Biochem. 66, 552e562. Das, B.C., Thapa, P., Karki, R., Das, S., Mahapatra, S., Liu, T.C., Torregroza, I., Wallace, D.P., Kambhampati, S., Van Veldhuizen, P., Verma, A., Ray, S.K., Evans, T., 2014. Retinoic acid signaling pathways in development and diseases. Bioorg. Med. Chem. 22, 673e683. Davis, F.B., Mousa, S.A., O’connor, L., Mohamed, S., Lin, H.Y., Cao, H.J., Davis, P.J., 2004. Proangiogenic action of thyroid hormone is fibroblast growth factor-dependent and is initiated at the cell surface. Circ. Res. 94, 1500e1506. Davis, P.J., Davis, F.B., Mousa, S.A., Luidens, M.K., Lin, H.Y., 2011. Membrane receptor for thyroid hormone: physiologic and pharmacologic implications. Annu. Rev. Pharmacol. Toxicol. 51, 99e115. Davis, P.J., Glinsky, G.V., Lin, H.Y., Leith, J.T., Hercbergs, A., Tang, H.Y., Ashur-Fabian, O., Incerpi, S., Mousa, S.A., 2014. Cancer cell gene expression modulated from plasma membrane integrin alphavbeta3 by thyroid hormone and nanoparticulate tetrac. Front. Endocrinol. 5, 240. Davis, P.J., Lin, H.Y., Tang, H.Y., Davis, F.B., Mousa, S.A., 2013a. Adjunctive input to the nuclear thyroid hormone receptor from the cell surface receptor for the hormone. Thyroid 23, 1503e1509. Davis, P.J., Yalcin, M., Lin, H.Y., Tang, H.Y., Hercbergs, A., Leith, J.T., Davis, F.B., Luidens, M.K., Mousa, S.A., 2013b. Incomplete success of angioinhibitor therapy in cancer: estimation of contribution of pro-angiogenic activity of patient thyroid hormone. J. Cancer Sci. Ther. 5, 441e445. De Bosscher, K., Haegeman, G., 2009. Minireview: latest perspectives on antiinflammatory actions of glucocorticoids. Mol. Endocrinol. 23, 281e291. De Bosscher, K., Vanden Berghe, W., Vermeulen, L., Plaisance, S., Boone, E., Haegeman, G., 2000. Glucocorticoids repress NFkappaB-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell. Proc. Natl. Acad. Sci. U. S. A. 97, 3919e3924. De Haes, P., Garmyn, M., Degreef, H., Vantieghem, K., Bouillon, R., Segaert, S., 2003. 1,25-Dihydroxyvitamin D3 inhibits ultraviolet Binduced apoptosis, Jun kinase activation, and interleukin-6 production in primary human keratinocytes. J. Cell. Biochem. 89, 663e673. Denmeade, S.R., Lin, X.S., Isaacs, J.T., 1996. Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer. Prostate 28, 251e265. Deroo, B.J., Korach, K.S., 2006. Estrogen receptors and human disease. J. Clin. Investig. 116, 561e570. Diaz, G.D., Paraskeva, C., Thomas, M.G., Binderup, L., Hague, A., 2000. Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy. Cancer Res. 60, 2304e2312. Dieken, E.S., Miesfeld, R.L., 1992. Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis. Mol. Cell Biol. 12, 589e597. Diker-Cohen, T., Koren, R., Liberman, U.A., Ravid, A., 2003. Vitamin D protects keratinocytes from apoptosis induced by osmotic shock, oxidative stress, and tumor necrosis factor. Ann. N. Y. Acad. Sci. 1010, 350e353. Dohrman, A., Russell, J.Q., Cuenin, S., Fortner, K., Tschopp, J., Budd, R.C., 2005. Cellular FLIP long form augments caspase activity and death of T cells through heterodimerization with and activation of caspase-8. J. Immunol. 175, 311e318. Donato, L.J., Noy, N., 2005. Suppression of mammary carcinoma growth by retinoic acid: proapoptotic genes are targets for retinoic

36

2. HORMONES OF PROGRAMMED CELL DEATH

acid receptor and cellular retinoic acid-binding protein II signaling. Cancer Res. 65, 8193e8199. Donepudi, M., MAC Sweeney, A., Briand, C., Grutter, M.G., 2003. Insights into the regulatory mechanism for caspase-8 activation. Mol. Cell 11, 543e549. Dressing, G.E., Goldberg, J.E., Charles, N.J., Schwertfeger, K.L., Lange, C.A., 2011. Membrane progesterone receptor expression in mammalian tissues: a review of regulation and physiological implications. Steroids 76, 11e17. Druilhe, A., Letuve, S., Pretolani, M., 2003. Glucocorticoid-induced apoptosis in human eosinophils: mechanisms of action. Apoptosis 8, 481e495. Duffy, D.M., Wells, T.R., Haluska, G.J., Stouffer, R.L., 1997. The ratio of progesterone receptor isoforms changes in the monkey corpus luteum during the luteal phase of the menstrual cycle. Biol. Reprod. 57, 693e699. Dusso, A.S., Brown, A.J., Slatopolsky, E., 2005. Vitamin D. Am. J. Physiol. Renal. Physiol. 289, F8eF28. Edwards, D.P., 2005. Regulation of signal transduction pathways by estrogen and progesterone. Annu. Rev. Physiol. 67, 335e376. Eguchi, K., 2001. Apoptosis in autoimmune diseases. Intern. Med. 40, 275e284. Eisman, J.A., Koga, M., Sutherland, R.L., Barkla, D.H., Tutton, P.J., 1989. 1,25-Dihydroxyvitamin D3 and the regulation of human cancer cell replication. Proc. Soc. Exp. Biol. Med. 191, 221e226. Elias, J., Marian, B., Edling, C., Lachmann, B., Noe, C.R., Rolf, S.H., Schuster, I., 2003. Induction of apoptosis by vitamin D metabolites and analogs in a glioma cell line. Recent Results Canc. Res. 164, 319e332. Engedal, N., Auberger, P., Blomhoff, H.K., 2009. Retinoic acid regulates Fas-induced apoptosis in Jurkat T cells: reversal of mitogenmediated repression of Fas DISC assembly. J. Leukoc. Biol. 85, 469e480. Evans, S.R., Soldatenkov, V., Shchepotin, E.B., Bogrash, E., Shchepotin, I.B., 1999. Novel 19-nor-hexafluoride vitamin D3 analog (Ro 25-6760) inhibits human colon cancer in vitro via apoptosis. Int. J. Oncol. 14, 979e985. Farwell, A.P., Dubord-Tomasetti, S.A., Pietrzykowski, A.Z., Stachelek, S.J., Leonard, J.L., 2005. Regulation of cerebellar neuronal migration and neurite outgrowth by thyroxine and 3,3’,5’triiodothyronine. Brain Res. Dev. Brain Res. 154, 121e135. Feng, Z., Marti, A., Jehn, B., Altermatt, H.J., Chicaiza, G., Jaggi, R., 1995. Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland. J. Cell Biol. 131, 1095e1103. Formby, B., Wiley, T.S., 1998. Progesterone inhibits growth and induces apoptosis in breast cancer cells: inverse effects on Bcl-2 and p53. Ann. Clin. Lab. Sci. 28, 360e369. Foster, J.S., Wimalasena, J., 1996. Estrogen regulates activity of cyclindependent kinases and retinoblastoma protein phosphorylation in breast cancer cells. Mol. Endocrinol. 10, 488e498. Frankfurt, O., Rosen, S.T., 2004. Mechanisms of glucocorticoid-induced apoptosis in hematologic malignancies: updates. Curr. Opin. Oncol. 16, 553e563. Frye, C.A., Sumida, K., Lydon, J.P., O’malley, B.W., Pfaff, D.W., 2006. Mid-aged and aged wild-type and progestin receptor knockout (PRKO) mice demonstrate rapid progesterone and 3alpha,5alphaTHP-facilitated lordosis. Psychopharmacology (Berlin) 185, 423e432. Furuya, Y., Krajewski, S., Epstein, J.I., Reed, J.C., Isaacs, J.T., 1996. Expression of bcl-2 and the progression of human and rodent prostatic cancers. Clin. Cancer Res. 2, 389e398. Gametchu, B., Watson, C.S., 2002. Correlation of membrane glucocorticoid receptor levels with glucocorticoid-induced apoptotic competence using mutant leukemic and lymphoma cells lines. J. Cell. Biochem. 87, 133e146.

Garg, D., Ng, S.S.M., Baig, K.M., Driggers, P., Segars, J., 2017. Progesterone-mediated non-classical signaling. Trends Endocrinol. Metabol. 28, 656e668. Geley, S., Hartmann, B.L., Kapelari, K., Egle, A., Villunger, A., Heidacher, D., Greil, R., Auer, B., Kofler, R., 1997. The interleukin 1beta-converting enzyme inhibitor CrmA prevents Apo1/Fas- but not glucocorticoid-induced poly(ADP-ribose) polymerase cleavage and apoptosis in lymphoblastic leukemia cells. FEBS Lett. 402, 36e40. Genovese, T., Impellizzeri, D., Ahmad, A., Cornelius, C., Campolo, M., Cuzzocrea, S., Esposito, E., 2013. Post-ischaemic thyroid hormone treatment in a rat model of acute stroke. Brain Res. 1513, 92e102. Gereben, B., Zavacki, A.M., Ribich, S., Kim, B.W., Huang, S.A., Simonides, W.S., Zeold, A., Bianco, A.C., 2008. Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling. Endocr. Rev. 29, 898e938. Giangrande, P.H., Kimbrel, E.A., Edwards, D.P., Mcdonnell, D.P., 2000. The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding. Mol. Cell Biol. 20, 3102e3115. Giangrande, P.H., Mcdonnell, D.P., 1999. The A and B isoforms of the human progesterone receptor: two functionally different transcription factors encoded by a single gene. Recent Prog. Horm. Res. 54, 291e313 discussion 313-4. Gianni, M., Ponzanelli, I., Mologni, L., Reichert, U., Rambaldi, A., Terao, M., Garattini, E., 2000. Retinoid-dependent growth inhibition, differentiation and apoptosis in acute promyelocytic leukemia cells. Expression and activation of caspases. Cell Death Differ. 7, 447e460. Golks, A., Brenner, D., Fritsch, C., Krammer, P.H., Lavrik, I.N., 2005. cFlipr, a new regulator of death receptor-induced apoptosis. J. Biol. Chem. 280, 14507e14513. Gosden, R., Spears, N., 1997. Programmed cell death in the reproductive system. Br. Med. Bull. 53, 644e661. Gougat, C., Jaffuel, D., Gagliardo, R., Henriquet, C., Bousquet, J., Demoly, P., Mathieu, M., 2002. Overexpression of the human glucocorticoid receptor alpha and beta isoforms inhibits AP-1 and NFkappaB activities hormone independently. J. Mol. Med. (Berl.) 80, 309e318. Graham, J.D., Clarke, C.L., 1997. Physiological action of progesterone in target tissues. Endocr. Rev. 18, 502e519. Graham, J.D., Yeates, C., Balleine, R.L., Harvey, S.S., Milliken, J.S., Bilous, A.M., Clarke, C.L., 1996. Progesterone receptor A and B protein expression in human breast cancer. J. Steroid Biochem. Mol. Biol. 56, 93e98. Green, D.R., 2011. Means to an End Apoptosis and Other Cell Death Mechanisms. Cold Spring Harbor Laboratory Press, New York. Green, D.R., Kroemer, G., 2004. The pathophysiology of mitochondrial cell death. Science 305, 626e629. Greenstein, S., Ghias, K., Krett, N.L., Rosen, S.T., 2002. Mechanisms of glucocorticoid-mediated apoptosis in hematological malignancies. Clin. Cancer Res. 8, 1681e1694. Grell, M., Wajant, H., Zimmermann, G., Scheurich, P., 1998. The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor. Proc. Natl. Acad. Sci. U. S. A. 95, 570e575. Grott, M., Karakaya, S., Mayer, F., Baertling, F., Beyer, C., Kipp, M., Kopp, H.G., 2013. Progesterone and estrogen prevent cisplatininduced apoptosis of lung cancer cells. Anticancer Res. 33, 791e800. Guicciardi, M.E., Gores, G.J., 2009. Life and death by death receptors. FASEB J. 23, 1625e1637. Guiochon-Mantel, A., Loosfelt, H., Lescop, P., Sar, S., Atger, M., PerrotApplanat, M., Milgrom, E., 1989. Mechanisms of nuclear localization of the progesterone receptor: evidence for interaction between monomers. Cell 57, 1147e1154. Guyton, K.Z., Kensler, T.W., Posner, G.H., 2001. Cancer chemoprevention using natural vitamin D and synthetic analogs. Annu. Rev. Pharmacol. Toxicol. 41, 421e442.

REFERENCES

Guzey, M., Kitada, S., Reed, J.C., 2002. Apoptosis induction by 1alpha,25-dihydroxyvitamin D3 in prostate cancer. Mol. Canc. Therapeut. 1, 667e677. Haanen, C., Vermes, I., 1996. Apoptosis: programmed cell death in fetal development. Eur. J. Obstet. Gynecol. Reprod. Biol. 64, 129e133. Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M., Soengas, M.S., Elia, A., DE LA Pompa, J.L., Kagi, D., Khoo, W., Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S.W., Penninger, J.M., Mak, T.W., 1998. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339e352. Hammer, S., Sauer, B., Spika, I., Schraut, C., Kleuser, B., SchaferKorting, M., 2004. Glucocorticoids mediate differential anti-apoptotic effects in human fibroblasts and keratinocytes via sphingosine-1-phosphate formation. J. Cell. Biochem. 91, 840e851. Han, J., Flemington, C., Houghton, A.B., Gu, Z., Zambetti, G.P., Lutz, R.J., Zhu, L., Chittenden, T., 2001. Expression of bbc3, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals. Proc. Natl. Acad. Sci. U. S. A. 98, 11318e11323. Helmberg, A., Auphan, N., Caelles, C., Karin, M., 1995. Glucocorticoidinduced apoptosis of human leukemic cells is caused by the repressive function of the glucocorticoid receptor. EMBO J. 14, 452e460. Hennighausen, L., Robinson, G.W., Wagner, K.U., Liu, W., 1997. Prolactin signaling in mammary gland development. J. Biol. Chem. 272, 7567e7569. Hoang, T.C., Bui, T.K., Taguchi, T., Watanabe, T., Sato, Y., 2010. All-trans retinoic acid inhibits KIT activity and induces apoptosis in gastrointestinal stromal tumor GIST-T1 cell line by affecting on the expression of survivin and Bax protein. J. Exp. Clin. Cancer Res. 29, 165. Hopp, T.A., Weiss, H.L., Hilsenbeck, S.G., Cui, Y., Allred, D.C., Horwitz, K.B., Fuqua, S.A., 2004. Breast cancer patients with progesterone receptor PR-A-rich tumors have poorer disease-free survival rates. Clin. Cancer Res. 10, 2751e2760. Horita, K., Inase, N., Miyake, S., Formby, B., Toyoda, H., Yoshizawa, Y., 2001. Progesterone induces apoptosis in malignant mesothelioma cells. Anticancer Res. 21, 3871e3874. Hormi-Carver, K., Feagins, L.A., Spechler, S.J., Souza, R.F., 2007. All trans-retinoic acid induces apoptosis via p38 and caspase pathways in metaplastic Barrett’s cells. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G18eG27. Hovland, A.R., Powell, R.L., Takimoto, G.S., Tung, L., Horwitz, K.B., 1998. An N-terminal inhibitory function, If, suppresses transcription by the A-isoform but not the B-isoform of human progesterone receptors. J. Biol. Chem. 273, 5455e5460. Hu, W.H., Johnson, H., Shu, H.B., 2000. Activation of NF-kappaB by Fadd, casper, and caspase-8. J. Biol. Chem. 275, 10838e10844. Ichikawa, S., Koller, D.L., Peacock, M., Johnson, M.L., Lai, D., Hui, S.L., Johnston, C.C., Foroud, T.M., Econs, M.J., 2005. Polymorphisms in the estrogen receptor beta (ESR2) gene are associated with bone mineral density in Caucasian men and women. J. Clin. Endocrinol. Metab. 90, 5921e5927. Isaacs, J.T., Lundmo, P.I., Berges, R., Martikainen, P., Kyprianou, N., English, H.F., 1992. Androgen regulation of programmed death of normal and malignant prostatic cells. J. Androl. 13, 457e464. Itoh, N., Nagata, S., 1993. A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J. Biol. Chem. 268, 10932e10937. Jaffe, A.B., Toran-Allerand, C.D., Greengard, P., Gandy, S.E., 1994. Estrogen regulates metabolism of Alzheimer amyloid beta precursor protein. J. Biol. Chem. 269, 13065e13068. Jeffers, J.R., Parganas, E., Lee, Y., Yang, C., Wang, J., Brennan, J., Maclean, K.H., Han, J., Chittenden, T., Ihle, J.N., Mckinnon, P.J., Cleveland, J.L., Zambetti, G.P., 2003. Puma is an essential mediator of p53-dependent and -independent apoptotic pathways. Cancer Cell 4, 321e328.

37

Jiang, F., Bao, J., Li, P., Nicosia, S.V., Bai, W., 2004. Induction of ovarian cancer cell apoptosis by 1,25-dihydroxyvitamin D3 through the down-regulation of telomerase. J. Biol. Chem. 279, 53213e53221. Jing, D., Bhadri, V.A., Beck, D., Thoms, J.A., Yakob, N.A., Wong, J.W., Knezevic, K., Pimanda, J.E., Lock, R.B., 2015. Opposing regulation of BIM and BCL2 controls glucocorticoid-induced apoptosis of pediatric acute lymphoblastic leukemia cells. Blood 125, 273e283. Kang, M.H., Reynolds, C.P., 2009. Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin. Cancer Res. 15, 1126e1132. Karin, M., Chang, L., 2001. AP-1dglucocorticoid receptor crosstalk taken to a higher level. J. Endocrinol. 169, 447e451. Kastner, P., Krust, A., Turcotte, B., Stropp, U., Tora, L., Gronemeyer, H., Chambon, P., 1990. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J. 9, 1603e1614. Kidd, V.J., Lahti, J.M., Teitz, T., 2000. Proteolytic regulation of apoptosis. Semin. Cell Dev. Biol. 11, 191e201. Kim, J.J., Kurita, T., Bulun, S.E., 2013. Progesterone action in endometrial cancer, endometriosis, uterine fibroids, and breast cancer. Endocr. Rev. 34, 130e162. Kino, T., Su, Y.A., Chrousos, G.P., 2009. Human glucocorticoid receptor isoform beta: recent understanding of its potential implications in physiology and pathophysiology. Cell. Mol. Life Sci. 66, 3435e3448. Kischkel, F.C., Hellbardt, S., Behrmann, I., Germer, M., Pawlita, M., Krammer, P.H., Peter, M.E., 1995. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14, 5579e5588. Koren, R., Rocker, D., Kotestiano, O., Liberman, U.A., Ravid, A., 2000. Synergistic anticancer activity of 1,25-dihydroxyvitamin D(3) and immune cytokines: the involvement of reactive oxygen species. J. Steroid Biochem. Mol. Biol. 73, 105e112. Korsmeyer, S.J., Wei, M.C., Saito, M., Weiler, S., Oh, K.J., Schlesinger, P.H., 2000. Pro-apoptotic cascade activates Bid, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ. 7, 1166e1173. Krijnen, P.A., Nijmeijer, R., Meijer, C.J., Visser, C.A., Hack, C.E., Niessen, H.W., 2002. Apoptosis in myocardial ischaemia and infarction. J. Clin. Pathol. 55, 801e811. Kuida, K., Haydar, T.F., Kuan, C.Y., Gu, Y., Taya, C., Karasuyama, H., Su, M.S., Rakic, P., Flavell, R.A., 1998. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325e337. Kuiper, G.G., Enmark, E., Pelto-Huikko, M., Nilsson, S., Gustafsson, J.A., 1996. Cloning of a novel receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. U. S. A. 93, 5925e5930. Kullmann, M., Schneikert, J., Moll, J., Heck, S., Zeiner, M., Gehring, U., Cato, A.C., 1998. RAP46 is a negative regulator of glucocorticoid receptor action and hormone-induced apoptosis. J. Biol. Chem. 273, 14620e14625. Kumar, R., Herbert, P.E., Warrens, A.N., 2005. An introduction to death receptors in apoptosis. Int. J. Surg. 3, 268e277. Kumar, R., Zakharov, M.N., Khan, S.H., Miki, R., Jang, H., Toraldo, G., Singh, R., Bhasin, S., Jasuja, R., 2011. The dynamic structure of the estrogen receptor. J. Amino Acids 2011, 812540. Kyprianou, N., English, H.F., Isaacs, J.T., 1990. Programmed cell death during regression of PC-82 human prostate cancer following androgen ablation. Cancer Res. 50, 3748e3753. Kyprianou, N., Isaacs, J.T., 1988. Activation of programmed cell death in the rat ventral prostate after castration. Endocrinology 122, 552e562. Lam, M., Dubyak, G., Distelhorst, C.W., 1993. Effect of glucocorticosteroid treatment on intracellular calcium homeostasis in mouse lymphoma cells. Mol. Endocrinol. 7, 686e693. Lamb, J.C., English, H., Levandoski, P.L., Rhodes, G.R., Johnson, R.K., Isaacs, J.T., 1992. Prostatic involution in rats induced by a novel 5

38

2. HORMONES OF PROGRAMMED CELL DEATH

alpha-reductase inhibitor, SK&F 105657: role for testosterone in the androgenic response. Endocrinology 130, 685e694. Laoag-Fernandez, J.B., Matsuo, H., Murakoshi, H., Hamada, A.L., Tsang, B.K., Maruo, T., 2004. 3,5,3’-Triiodothyronine downregulates Fas and Fas ligand expression and suppresses caspase-3 and poly (adenosine 5’-diphosphate-ribose) polymerase cleavage and apoptosis in early placental extravillous trophoblasts in vitro. J. Clin. Endocrinol. Metab. 89, 4069e4077. Lavrik, I., Golks, A., Krammer, P.H., 2005. Death receptor signaling. J. Cell Sci. 118, 265e267. Levin, E.R., Pietras, R.J., 2008. Estrogen receptors outside the nucleus in breast cancer. Breast Canc. Res. Treat. 108, 351e361. Levine, B., Huang, Q., Isaacs, J.T., Reed, J.C., Griffin, D.E., Hardwick, J.M., 1993. Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene. Nature 361, 739e742. Lewis, J.S., Meeke, K., Osipo, C., Ross, E.A., Kidawi, N., Li, T., Bell, E., Chandel, N.S., Jordan, V.C., 2005a. Intrinsic mechanism of estradiolinduced apoptosis in breast cancer cells resistant to estrogen deprivation. J. Natl. Cancer Inst. 97, 1746e1759. Lewis, J.S., Osipo, C., Meeke, K., Jordan, V.C., 2005b. Estrogen-induced apoptosis in a breast cancer model resistant to long-term estrogen withdrawal. J. Steroid Biochem. Mol. Biol. 94, 131e141. Lewis-Wambi, J.S., Kim, H.R., Wambi, C., Patel, R., Pyle, J.R., KleinSzanto, A.J., Jordan, V.C., 2008. Buthionine sulfoximine sensitizes antihormone-resistant human breast cancer cells to estrogeninduced apoptosis. Breast Cancer Res. 10, R104. Li, J., Orr, B., White, K., Belogortseva, N., Niles, R., Boskovic, G., Nguyen, H., Dykes, A., Park, M., 2009. Chmp 1A is a mediator of the anti-proliferative effects of all-trans retinoic acid in human pancreatic cancer cells. Mol. Canc. 8, 7. Li, X., O’malley, B.W., 2003. Unfolding the action of progesterone receptors. J. Biol. Chem. 278, 39261e39264. Li, X., Zhang, J., Zhu, X., Wang, P., Wang, X., Li, D., 2015. Progesterone reduces inflammation and apoptosis in neonatal rats with hypoxic ischemic brain damage through the PI3K/Akt pathway. Int. J. Clin. Exp. Med. 8, 8197e8203. Liao, H.Y., Wang, G.P., Huang, S.H., Li, Y., Cai, S.W., Zhang, J., Chen, H.G., Wu, W.B., 2014. HIF-1alpha silencing suppresses growth of lung adenocarcinoma A549 cells through induction of apoptosis. Mol. Med. Rep. 9, 911e915. Limbourg, F.P., Liao, J.K., 2003. Nontranscriptional actions of the glucocorticoid receptor. J. Mol. Med. (Berl.) 81, 168e174. Lin, H.Y., Glinsky, G.V., Mousa, S.A., Davis, P.J., 2015. Thyroid hormone and anti-apoptosis in tumor cells. Oncotarget 6, 14735e14743. Lin, H.Y., Su, Y.F., Hsieh, M.T., Lin, S., Meng, R., London, D., Lin, C., Tang, H.Y., Hwang, J., Davis, F.B., Mousa, S.A., Davis, P.J., 2013. Nuclear monomeric integrin alphav in cancer cells is a coactivator regulated by thyroid hormone. FASEB J. 27, 3209e3216. Lin, H.Y., Sun, M., Tang, H.Y., Lin, C., Luidens, M.K., Mousa, S.A., Incerpi, S., Drusano, G.L., Davis, F.B., Davis, P.J., 2009. L-Thyroxine vs. 3,5,3’-triiodo-L-thyronine and cell proliferation: activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Am. J. Physiol. Cell Physiol. 296, C980eC991. Lin, H.Y., Tang, H.Y., Davis, F.B., Davis, P.J., 2011. Resveratrol and apoptosis. Ann. N. Y. Acad. Sci. 1215, 79e88. Lin, Y., Kokontis, J., Tang, F., Godfrey, B., Liao, S., Lin, A., Chen, Y., Xiang, J., 2006. Androgen and its receptor promote Bax-mediated apoptosis. Mol. Cell Biol. 26, 1908e1916. Liu, J., Matsuo, H., Laoag-Fernandez, J.B., Xu, Q., Maruo, T., 2007. The effects of progesterone on apoptosis in the human trophoblastderived HTR-8/SV neo cells. Mol. Hum. Reprod. 13, 869e874. Liu, X., Zheng, N., Shi, Y.N., Yuan, J., Li, L., 2014. Thyroid hormone induced angiogenesis through the integrin alphavbeta3/protein kinase D/histone deacetylase 5 signaling pathway. J. Mol. Endocrinol. 52, 245e254.

Lowe, S.W., Schmitt, E.M., Smith, S.W., Osborne, B.A., Jacks, T., 1993. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362, 847e849. Lu, N.Z., Collins, J.B., Grissom, S.F., Cidlowski, J.A., 2007. Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor. Mol. Cell Biol. 27, 7143e7160. Lu, T.Y., Li, W.B., Wu, X.A., Wang, L.X., Wang, R.L., Gao, D.L., Lu, S.X., Fan, Q.X., 2010. [Mechanism of apoptosis of esophageal cancer EC9706 in nude mice induced by all-trans retinoic acid]. Zhonghua Zhongliu Zazhi 32, 892e896. Luidens, M.K., Mousa, S.A., Davis, F.B., Lin, H.Y., Davis, P.J., 2010. Thyroid hormone and angiogenesis. Vasc. Pharmacol. 52, 142e145. Lumachi, F., Luisetto, G., Basso, S.M., Basso, U., Brunello, A., Camozzi, V., 2011. Endocrine therapy of breast cancer. Curr. Med. Chem. 18, 513e522. Luo, G., Abrahams, V.M., Tadesse, S., Funai, E.F., Hodgson, E.J., Gao, J., Norwitz, E.R., 2010. Progesterone inhibits basal and TNF-alphainduced apoptosis in fetal membranes: a novel mechanism to explain progesterone-mediated prevention of preterm birth. Reprod. Sci. 17, 532e539. Lydon, J.P., Demayo, F.J., Funk, C.R., Mani, S.K., Hughes, A.R., Montgomery Jr., C.A., Shyamala, G., Conneely, O.M., O’malley, B.W., 1995. Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev. 9, 2266e2278. Mangelsdorf, D.J., Evans, R.M., 1995. The RXR heterodimers and orphan receptors. Cell 83, 841e850. Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., Evans, R.M., 1995. The nuclear receptor superfamily: the second decade. Cell 83, 835e839. Mani, S.K., Mermelstein, P.G., Tetel, M.J., Anesetti, G., 2012. Convergence of multiple mechanisms of steroid hormone action. Horm. Metab. Res. 44, 569e576. Manna, S.K., Aggarwal, B.B., 2000. All-trans-retinoic acid upregulates TNF receptors and potentiates TNF-induced activation of nuclear factors-kappab, activated protein-1 and apoptosis in human lung cancer cells. Oncogene 19, 2110e2119. Mantovani, A., Bottazzi, B., Colotta, F., Sozzani, S., Ruco, L., 1992. The origin and function of tumor-associated macrophages. Immunol. Today 13, 265e270. Marquez, D.C., Chen, H.W., Curran, E.M., Welshons, W.V., Pietras, R.J., 2006. Estrogen receptors in membrane lipid rafts and signal transduction in breast cancer. Mol. Cell. Endocrinol. 246, 91e100. Martikainen, P., Kyprianou, N., Tucker, R.W., Isaacs, J.T., 1991. Programmed death of nonproliferating androgen-independent prostatic cancer cells. Cancer Res. 51, 4693e4700. Mathiasen, I.S., Hansen, C.M., Foghsgaard, L., Jaattela, M., 2001. Sensitization to TNF-induced apoptosis by 1,25-dihydroxy vitamin D(3) involves up-regulation of the TNF receptor 1 and cathepsin B. Int. J. Cancer 93, 224e231. Mathiasen, I.S., Sergeev, I.N., Bastholm, L., Elling, F., Norman, A.W., Jaattela, M., 2002. Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells. J. Biol. Chem. 277, 30738e30745. Mauvais-Jarvis, F., Clegg, D.J., Hevener, A.L., 2013. The role of estrogens in control of energy balance and glucose homeostasis. Endocr. Rev. 34, 309e338. Mc Cormack, O., Harrison, M., Kerin, M.J., Mccann, A., 2007. Role of the progesterone receptor (PR) and the PR isoforms in breast cancer. Crit. Rev. Oncog. 13, 283e301. Mcdonnell, T.J., Troncoso, P., Brisbay, S.M., Logothetis, C., Chung, L.W., Hsieh, J.T., Tu, S.M., Campbell, M.L., 1992. Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res. 52, 6940e6944. Mcilwain, D.R., Berger, T., Mak, T.W., 2013. Caspase functions in cell death and disease. Cold Spring Harb. Perspect. Biol. 5, a008656.

REFERENCES

Mckenna, N.J., Lanz, R.B., O’malley, B.W., 1999a. Nuclear receptor coregulators: cellular and molecular biology. Endocr. Rev. 20, 321e344. Mckenna, N.J., Xu, J., Nawaz, Z., Tsai, S.Y., Tsai, M.J., O’malley, B.W., 1999b. Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple functions. J. Steroid Biochem. Mol. Biol. 69, 3e12. Meyer, M.E., Quirin-Stricker, C., Lerouge, T., Bocquel, M.T., Gronemeyer, H., 1992. A limiting factor mediates the differential activation of promoters by the human progesterone receptor isoforms. J. Biol. Chem. 267, 10882e10887. Mic, F.A., Molotkov, A., Benbrook, D.M., Duester, G., 2003. Retinoid activation of retinoic acid receptor but not retinoid X receptor is sufficient to rescue lethal defect in retinoic acid synthesis. Proc. Natl. Acad. Sci. U. S. A. 100, 7135e7140. Micheau, O., Thome, M., Schneider, P., Holler, N., Tschopp, J., Nicholson, D.W., Briand, C., Grutter, M.G., 2002. The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex. J. Biol. Chem. 277, 45162e45171. Micheau, O., Tschopp, J., 2003. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, 181e190. Moeller, L.C., Broecker-Preuss, M., 2011. Transcriptional regulation by nonclassical action of thyroid hormone. Thyroid Res. 4 (Suppl. 1), S6. Mor, G., Kohen, F., Garcia-Velasco, J., Nilsen, J., Brown, W., Song, J., Naftolin, F., 2000. Regulation of fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen. J. Steroid Biochem. Mol. Biol. 73, 185e194. Mosselman, S., Polman, J., Dijkema, R., 1996. ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett. 392, 49e53. Mousa, S.A., Bergh, J.J., Dier, E., Rebbaa, A., O’connor, L.J., Yalcin, M., Aljada, A., Dyskin, E., Davis, F.B., Lin, H.Y., Davis, P.J., 2008. Tetraiodothyroacetic acid, a small molecule integrin ligand, blocks angiogenesis induced by vascular endothelial growth factor and basic fibroblast growth factor. Angiogenesis 11, 183e190. Mousa, S.A., Lin, H.Y., Tang, H.Y., Hercbergs, A., Luidens, M.K., Davis, P.J., 2014. Modulation of angiogenesis by thyroid hormone and hormone analogues: implications for cancer management. Angiogenesis 17, 463e469. Mrass, P., Rendl, M., Mildner, M., Gruber, F., Lengauer, B., Ballaun, C., Eckhart, L., Tschachler, E., 2004. Retinoic acid increases the expression of p53 and proapoptotic caspases and sensitizes keratinocytes to apoptosis: a possible explanation for tumor preventive action of retinoids. Cancer Res. 64, 6542e6548. Mulac-Jericevic, B., Conneely, O.M., 2004. Reproductive tissue selective actions of progesterone receptors. Reproduction 128, 139e146. Mulac-Jericevic, B., Mullinax, R.A., Demayo, F.J., Lydon, J.P., Conneely, O.M., 2000. Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science 289, 1751e1754. Muramatsu, M., Inoue, S., 2000. Estrogen receptors: how do they control reproductive and nonreproductive functions? Biochem. Biophys. Res. Commun. 270, 1e10. Nelson, P.S., Clegg, N., Arnold, H., Ferguson, C., Bonham, M., White, J., Hood, L., Lin, B., 2002. The program of androgen-responsive genes in neoplastic prostate epithelium. Proc. Natl. Acad. Sci. U. S. A. 99, 11890e11895. Nguyen, H., Syed, V., 2011. Progesterone inhibits growth and induces apoptosis in cancer cells through modulation of reactive oxygen species. Gynecol. Endocrinol. 27, 830e836. Nicholson, D.W., Ali, A., Thornberry, N.A., Vaillancourt, J.P., Ding, C.K., Gallant, M., Gareau, Y., Griffin, P.R., Labelle, M., Lazebnik, Y.A., et al., 1995. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376, 37e43.

39

Nicolaides, N.C., Galata, Z., Kino, T., Chrousos, G.P., Charmandari, E., 2010. The human glucocorticoid receptor: molecular basis of biologic function. Steroids 75, 1e12. Niizuma, H., Nakamura, Y., Ozaki, T., Nakanishi, H., Ohira, M., Isogai, E., Kageyama, H., Imaizumi, M., Nakagawara, A., 2006. Bcl-2 is a key regulator for the retinoic acid-induced apoptotic cell death in neuroblastoma. Oncogene 25, 5046e5055. Nikaidou, N., Kamio, Y., Izaki, K., 1992. Molecular cloning and nucleotide sequence of a pectin lyase gene from Pseudomonas marginalis N6301. Biochem. Biophys. Res. Commun. 182, 14e19. Nilsson, S., Gustafsson, J.A., 2011. Estrogen receptors: therapies targeted to receptor subtypes. Clin. Pharmacol. Ther. 89, 44e55. Niu, M.Y., Menard, M., Reed, J.C., Krajewski, S., Pratt, M.A., 2001. Ectopic expression of cyclin D1 amplifies a retinoic acid-induced mitochondrial death pathway in breast cancer cells. Oncogene 20, 3506e3518. Novac, N., Baus, D., Dostert, A., Heinzel, T., 2006. Competition between glucocorticoid receptor and NFkappaB for control of the human FasL promoter. FASEB J. 20, 1074e1081. Noy, N., 2010. Between death and survival: retinoic acid in regulation of apoptosis. Annu. Rev. Nutr. 30, 201e217. Nunes, V.A., Portioli-Sanches, E.P., Rosim, M.P., Araujo, M.S., Praxedes-Garcia, P., Valle, M.M., Roma, L.P., Hahn, C., GurgulConvey, E., Lenzen, S., Azevedo-Martins, A.K., 2014. Progesterone induces apoptosis of insulin-secreting cells: insights into the molecular mechanism. J. Endocrinol. 221, 273e284. Oakley, R.H., Cidlowski, J.A., 2011. Cellular processing of the glucocorticoid receptor gene and protein: new mechanisms for generating tissue-specific actions of glucocorticoids. J. Biol. Chem. 286, 3177e3184. Obr, A.E., Edwards, D.P., 2012. The biology of progesterone receptor in the normal mammary gland and in breast cancer. Mol. Cell. Endocrinol. 357, 4e17. Obulesu, M., Lakshmi, M.J., 2014. Apoptosis in Alzheimer’s disease: an understanding of the physiology, pathology and therapeutic avenues. Neurochem. Res. 39, 2301e2312. Oetting, A., Yen, P.M., 2007. New insights into thyroid hormone action. Best Pract. Res. Clin. Endocrinol. Metabol. 21, 193e208. Osipo, C., Gajdos, C., Liu, H., Chen, B., Jordan, V.C., 2003. Paradoxical action of fulvestrant in estradiol-induced regression of tamoxifenstimulated breast cancer. J. Natl. Cancer Inst. 95, 1597e1608. Ozpolat, B., Akar, U., Steiner, M., Zorrilla-Calancha, I., TiradoGomez, M., Colburn, N., Danilenko, M., Kornblau, S., Berestein, G.L., 2007. Programmed cell death-4 tumor suppressor protein contributes to retinoic acid-induced terminal granulocytic differentiation of human myeloid leukemia cells. Mol. Canc. Res. 5, 95e108. Park, W.H., Seol, J.G., Kim, E.S., Hyun, J.M., Jung, C.W., Lee, C.C., Binderup, L., Koeffler, H.P., Kim, B.K., Lee, Y.Y., 2000. Induction of apoptosis by vitamin D3 analogue EB1089 in NCI-H929 myeloma cells via activation of caspase 3 and p38 MAP kinase. Br. J. Haematol. 109, 576e583. Pazirandeh, A., Xue, Y., Prestegaard, T., Jondal, M., Okret, S., 2002. Effects of altered glucocorticoid sensitivity in the T cell lineage on thymocyte and T cell homeostasis. FASEB J. 16, 727e729. Pedram, A., Razandi, M., Sainson, R.C., Kim, J.K., Hughes, C.C., Levin, E.R., 2007. A conserved mechanism for steroid receptor translocation to the plasma membrane. J. Biol. Chem. 282, 22278e22288. Peter, M.E., Krammer, P.H., 2003. The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ. 10, 26e35. Pietras, R.J., Marquez-Garban, D.C., 2007. Membrane-associated estrogen receptor signaling pathways in human cancers. Clin. Cancer Res. 13, 4672e4676. Pietrzak, M., Puzianowska-Kuznicka, M., 2008. Triiodothyronine utilizes phosphatidylinositol 3-kinase pathway to activate antiapoptotic myeloid cell leukemia-1. J. Mol. Endocrinol. 41, 177e186.

40

2. HORMONES OF PROGRAMMED CELL DEATH

Planey, S.L., Abrams, M.T., Robertson, N.M., Litwack, G., 2003. Role of apical caspases and glucocorticoid-regulated genes in glucocorticoid-induced apoptosis of pre-B leukemic cells. Cancer Res. 63, 172e178. Planey, S.L., Kumar, R., Arnott, J.A., 2013. Post-translational modification of transcription factors: mechanisms and potential therapeutic interventions. Curr. Mol. Pharmacol. 6, 173e182. Plas, D.R., Thompson, C.B., 2002. Cell metabolism in the regulation of programmed cell death. Trends Endocrinol. Metabol. 13, 75e78. Ponglikitmongkol, M., Green, S., Chambon, P., 1988. Genomic organization of the human oestrogen receptor gene. EMBO J. 7, 3385e3388. Porter, W., Saville, B., Hoivik, D., Safe, S., 1997. Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol. Endocrinol. 11, 1569e1580. Powazniak, Y., Kempfer, A.C., De La Paz Dominguez, M., Farias, C., Keller, L., Calderazzo, J.C., Lazzari, M.A., 2009. Effect of estradiol, progesterone and testosterone on apoptosis- and proliferationinduced MAPK signaling in human umbilical vein endothelial cells. Mol. Med. Rep. 2, 441e447. Pratt, M.A., Niu, M., White, D., 2003. Differential regulation of protein expression, growth and apoptosis by natural and synthetic retinoids. J. Cell. Biochem. 90, 692e708. Pufall, M.A., 2015. Glucocorticoids and cancer. Adv. Exp. Med. Biol. 872, 315e333. Radak, D., Katsiki, N., Resanovic, I., Jovanovic, A., SudarMilovanovic, E., Zafirovic, S., Mousad, S.A., Isenovic, E.R., 2017. Apoptosis and acute brain ischemia in ischemic stroke. Curr. Vasc. Pharmacol. 15, 115e122. Radmayr, C., Lunacek, A., Schwentner, C., Oswald, J., Klocker, H., Bartsch, G., 2008. 5-alpha-reductase and the development of the human prostate. Indian J. Urol. 24, 309e312. Raffo, P., Emionite, L., Colucci, L., Belmondo, F., Moro, M.G., Bollag, W., Toma, S., 2000. Retinoid receptors: pathways of proliferation inhibition and apoptosis induction in breast cancer cell lines. Anticancer Res. 20, 1535e1543. Ramamoorthy, S., Cidlowski, J.A., 2013. Exploring the molecular mechanisms of glucocorticoid receptor action from sensitivity to resistance. Endocr. Dev. 24, 41e56. Rathmell, J.C., Lindsten, T., Zong, W.X., Cinalli, R.M., Thompson, C.B., 2002. Deficiency in Bak and Bax perturbs thymic selection and lymphoid homeostasis. Nat. Immunol. 3, 932e939. Reichardt, H.M., Kaestner, K.H., Tuckermann, J., Kretz, O., Wessely, O., Bock, R., Gass, P., Schmid, W., Herrlich, P., Angel, P., Schutz, G., 1998. DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93, 531e541. Reichardt, H.M., Umland, T., Bauer, A., Kretz, O., Schutz, G., 2000. Mice with an increased glucocorticoid receptor gene dosage show enhanced resistance to stress and endotoxic shock. Mol. Cell Biol. 20, 9009e9017. Reichel, H., Bishop, J.E., Koeffler, H.P., Norman, A.W., 1991. Evidence for 1,25-dihydroxyvitamin D3 production by cultured porcine alveolar macrophages. Mol. Cell. Endocrinol. 75, 163e167. Reichel, H., Koeffler, H.P., Barbers, R., Norman, A.W., 1987. Regulation of 1,25-dihydroxyvitamin D3 production by cultured alveolar macrophages from normal human donors and from patients with pulmonary sarcoidosis. J. Clin. Endocrinol. Metab. 65, 1201e1209. Resnick, S.M., Maki, P.M., Golski, S., Kraut, M.A., Zonderman, A.B., 1998. Effects of estrogen replacement therapy on PET cerebral blood flow and neuropsychological performance. Horm. Behav. 34, 171e182. Revollo, J.R., Cidlowski, J.A., 2009. Mechanisms generating diversity in glucocorticoid receptor signaling. Ann. N. Y. Acad. Sci. 1179, 167e178. Rhen, T., Cidlowski, J.A., 2005. Antiinflammatory action of glucocorticoidsdnew mechanisms for old drugs. N. Engl. J. Med. 353, 1711e1723.

Rhinn, M., Dolle, P., 2012. Retinoic acid signalling during development. Development 139, 843e858. Riachy, R., Vandewalle, B., Kerr Conte, J., Moerman, E., Sacchetti, P., Lukowiak, B., Gmyr, V., Bouckenooghe, T., Dubois, M., Pattou, F., 2002. 1,25-dihydroxyvitamin D3 protects RINm5F and human islet cells against cytokine-induced apoptosis: implication of the antiapoptotic protein A20. Endocrinology 143, 4809e4819. Riedl, S.J., Fuentes-Prior, P., Renatus, M., Kairies, N., Krapp, S., Huber, R., Salvesen, G.S., Bode, W., 2001. Structural basis for the activation of human procaspase-7. Proc. Natl. Acad. Sci. U. S. A. 98, 14790e14795. Rocker, D., Ravid, A., Liberman, U.A., Garach-Jehoshua, O., Koren, R., 1994. 1,25-Dihydroxyvitamin D3 potentiates the cytotoxic effect of TNF on human breast cancer cells. Mol. Cell. Endocrinol. 106, 157e162. Rogatsky, I., Hittelman, A.B., Pearce, D., Garabedian, M.J., 1999. Distinct glucocorticoid receptor transcriptional regulatory surfaces mediate the cytotoxic and cytostatic effects of glucocorticoids. Mol. Cell Biol. 19, 5036e5049. Salvesen, G.S., Dixit, V.M., 1997. Caspases: intracellular signaling by proteolysis. Cell 91, 443e446. Samuel, S., Sitrin, M.D., 2008. Vitamin D’s role in cell proliferation and differentiation. Nutr. Rev. 66, S116eS124. Santen, R.J., Song, R.X., Masamura, S., Yue, W., Fan, P., Sogon, T., Hayashi, S., Nakachi, K., Eguchi, H., 2008. Adaptation to estradiol deprivation causes up-regulation of growth factor pathways and hypersensitivity to estradiol in breast cancer cells. Adv. Exp. Med. Biol. 630, 19e34. Sartorius, C.A., Melville, M.Y., Hovland, A.R., Tung, L., Takimoto, G.S., Horwitz, K.B., 1994. A third transactivation function (AF3) of human progesterone receptors located in the unique N-terminal segment of the B-isoform. Mol. Endocrinol. 8, 1347e1360. Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K.J., Debatin, K.M., Krammer, P.H., Peter, M.E., 1998. Two CD95 (APO1/Fas) signaling pathways. EMBO J. 17, 1675e1687. Schmidt, S., Rainer, J., Ploner, C., Presul, E., Riml, S., Kofler, R., 2004. Glucocorticoid-induced apoptosis and glucocorticoid resistance: molecular mechanisms and clinical relevance. Cell Death Differ. 11 (Suppl. 1), S45eS55. Schneider, A.E., Karpati, E., Schuszter, K., Toth, E.A., Kiss, E., Kulcsar, M., Laszlo, G., Matko, J., 2014. A dynamic network of estrogen receptors in murine lymphocytes: fine-tuning the immune response. J. Leukoc. Biol. 96, 857e872. Schneider, P., Holler, N., Bodmer, J.L., Hahne, M., Frei, K., Fontana, A., Tschopp, J., 1998. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J. Exp. Med. 187, 1205e1213. Schott, D.R., Shyamala, G., Schneider, W., Parry, G., 1991. Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor. Biochemistry 30, 7014e7020. Seifert-Klauss, V., Prior, J.C., 2010. Progesterone and bone: actions promoting bone health in women. J. Osteoporos. 2010, 845180. Sells, S.F., Wood Jr., D.P., Joshi-Barve, S.S., Muthukumar, S., Jacob, R.J., Crist, S.A., Humphreys, S., Rangnekar, V.M., 1994. Commonality of the gene programs induced by effectors of apoptosis in androgendependent and -independent prostate cells. Cell Growth Differ. 5, 457e466. Sengupta, S., Vonesch, J.L., Waltzinger, C., Zheng, H., Wasylyk, B., 2000. Negative cross-talk between p53 and the glucocorticoid receptor and its role in neuroblastoma cells. EMBO J. 19, 6051e6064. Sergeev, I.N., 2004. Calcium as a mediator of 1,25-dihydroxyvitamin D3-induced apoptosis. J. Steroid Biochem. Mol. Biol. 89e90, 419e425. Sergeev, I.N., 2014. Vitamin D-mediated apoptosis in cancer and obesity. Horm. Mol. Biol. Clin. Investig. 20, 43e49.

REFERENCES

Shibusawa, N., Hollenberg, A.N., Wondisford, F.E., 2003. Thyroid hormone receptor DNA binding is required for both positive and negative gene regulation. J. Biol. Chem. 278, 732e738. Shinoura, N., Muramatsu, Y., Nishimura, M., Yoshida, Y., Saito, A., Yokoyama, T., Furukawa, T., Horii, A., Hashimoto, M., Asai, A., Kirino, T., Hamada, H., 1999. Adenovirus-mediated transfer of p33ING1 with p53 drastically augments apoptosis in gliomas. Cancer Res. 59, 5521e5528. Shinoura, N., Muramatsu, Y., Yoshida, Y., Asai, A., Kirino, T., Hamada, H., 2000a. Adenovirus-mediated transfer of caspase-3 with Fas ligand induces drastic apoptosis in U-373MG glioma cells. Exp. Cell Res. 256, 423e433. Shinoura, N., Sakurai, S., Shibasaki, F., Asai, A., Kirino, T., Hamada, H., 2002. Co-transduction of Apaf-1 and caspase-9 highly enhances p53-mediated apoptosis in gliomas. Br. J. Canc. 86, 587e595. Shinoura, N., Yamamoto, N., Yoshida, Y., Fujita, T., Saito, N., Asai, A., Kirino, T., Hamada, H., 2000b. Adenovirus-mediated gene transduction of IkappaB or IkappaB plus Bax gene drastically enhances tumor necrosis factor (TNF)-induced apoptosis in human gliomas. Jpn. J. Canc. Res. 91, 41e51. Shiozaki, E.N., Chai, J., Shi, Y., 2002. Oligomerization and activation of caspase-9, induced by Apaf-1 CARD. Proc. Natl. Acad. Sci. U. S. A. 99, 4197e4202. Shudo, K., Kinoshita, K., Imamura, R., Fan, H., Hasumoto, K., Tanaka, M., Nagata, S., Suda, T., 2001. The membrane-bound but not the soluble form of human Fas ligand is responsible for its inflammatory activity. Eur. J. Immunol. 31, 2504e2511. Shyamala, G., Yang, X., Silberstein, G., Barcellos-Hoff, M.H., Dale, E., 1998. Transgenic mice carrying an imbalance in the native ratio of A to B forms of progesterone receptor exhibit developmental abnormalities in mammary glands. Proc. Natl. Acad. Sci. U. S. A. 95, 696e701. Smith, K.G., Strasser, A., Vaux, D.L., 1996. CrmA expression in T lymphocytes of transgenic mice inhibits CD95 (Fas/APO-1)-transduced apoptosis, but does not cause lymphadenopathy or autoimmune disease. EMBO J. 15, 5167e5176. Song, R.X., Mor, G., Naftolin, F., Mcpherson, R.A., Song, J., Zhang, Z., Yue, W., Wang, J., Santen, R.J., 2001. Effect of long-term estrogen deprivation on apoptotic responses of breast cancer cells to 17beta-estradiol. J. Natl. Cancer Inst. 93, 1714e1723. Song, R.X., Zhang, Z., Mor, G., Santen, R.J., 2005. Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradioldepleted ER(þ) breast cancer cells. Apoptosis 10, 667e678. Sotoca, A.M., Van Den Berg, H., Vervoort, J., Van Der Saag, P., Strom, A., Gustafsson, J.A., Rietjens, I., Murk, A.J., 2008. Influence of cellular ERalpha/ERbeta ratio on the ERalpha-agonist induced proliferation of human T47D breast cancer cells. Toxicol. Sci. 105, 303e311. Squier, M.K., Cohen, J.J., 1997. Calpain, an upstream regulator of thymocyte apoptosis. J. Immunol. 158, 3690e3697. Steele, R.J., Brown, M., Eremin, O., 1985. Characterisation of macrophages infiltrating human mammary carcinomas. Br. J. Canc. 51, 135e138. Studzinski, G.P., Moore, D.C., 1995. Sunlightdcan it prevent as well as cause cancer? Cancer Res. 55, 4014e4022. Sukocheva, O.A., Carpenter, D.O., 2006. Anti-apoptotic effects of 3,5,3’tri-iodothyronine in mouse hepatocytes. J. Endocrinol. 191, 447e458. Syed, V., Ho, S.M., 2003. Progesterone-induced apoptosis in immortalized normal and malignant human ovarian surface epithelial cells involves enhanced expression of FasL. Oncogene 22, 6883e6890. Tang, G., Minemoto, Y., Dibling, B., Purcell, N.H., Li, Z., Karin, M., Lin, A., 2001. Inhibition of JNK activation through NF-kappaB target genes. Nature 414, 313e317. Tata, J.R., 2013. The road to nuclear receptors of thyroid hormone. Biochim. Biophys. Acta 1830, 3860e3866.

41

Thin, T.H., Li, L., Chung, T.K., Sun, H., Taneja, R., 2007. Stra13 is induced by genotoxic stress and regulates ionizing-radiationinduced apoptosis. EMBO Rep. 8, 401e407. Thompson, E.B., 1994. Apoptosis and steroid hormones. Mol. Endocrinol. 8, 665e673. Thornberry, N.A., Lazebnik, Y., 1998. Caspases: enemies within. Science 281, 1312e1316. Tilly, J.L., Tilly, K.I., Perez, G.I., 1997. The genes of cell death and cellular susceptibility to apoptosis in the ovary: a hypothesis. Cell Death Differ. 4, 180e187. Tonomura, N., Mclaughlin, K., Grimm, L., Goldsby, R.A., Osborne, B.A., 2003. Glucocorticoid-induced apoptosis of thymocytes: requirement of proteasome-dependent mitochondrial activity. J. Immunol. 170, 2469e2478. Tora, L., Gronemeyer, H., Turcotte, B., Gaub, M.P., Chambon, P., 1988. The N-terminal region of the chicken progesterone receptor specifies target gene activation. Nature 333, 185e188. Tora, L., White, J., Brou, C., Tasset, D., Webster, N., Scheer, E., Chambon, P., 1989. The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59, 477e487. Truss, M., Beato, M., 1993. Steroid hormone receptors: interaction with deoxyribonucleic acid and transcription factors. Endocr. Rev. 14, 459e479. Tsai, M.J., O’malley, B.W., 1994. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63, 451e486. Tschopp, J., Irmler, M., Thome, M., 1998. Inhibition of fas death signals by FLIPs. Curr. Opin. Immunol. 10, 552e558. Uchoa, E.T., Aguilera, G., Herman, J.P., Fiedler, J.L., Deak, T., DE Sousa, M.B., 2014. Novel aspects of glucocorticoid actions. J. Neuroendocrinol. 26, 557e572. Valrance, M.E., Welsh, J., 2004. Breast cancer cell regulation by highdose Vitamin D compounds in the absence of nuclear vitamin D receptor. J. Steroid Biochem. Mol. Biol. 89e90, 221e225. Vasudevan, N., Pfaff, D.W., 2007. Membrane-initiated actions of estrogens in neuroendocrinology: emerging principles. Endocr. Rev. 28, 1e19. Vegeto, E., Shahbaz, M.M., Wen, D.X., Goldman, M.E., O’malley, B.W., Mcdonnell, D.P., 1993. Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol. Endocrinol. 7, 1244e1255. Vicent, G.P., Nacht, A.S., Zaurin, R., Font-Mateu, J., Soronellas, D., Le Dily, F., Reyes, D., Beato, M., 2013. Unliganded progesterone receptor-mediated targeting of an RNA-containing repressive complex silences a subset of hormone-inducible genes. Genes Dev. 27, 1179e1197. Villunger, A., Michalak, E.M., Coultas, L., Mullauer, F., Bock, G., Ausserlechner, M.J., Adams, J.M., Strasser, A., 2003. p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science 302, 1036e1038. Visser, W.E., Friesema, E.C., Visser, T.J., 2011. Minireview: thyroid hormone transporters: the knowns and the unknowns. Mol. Endocrinol. 25, 1e14. Wagner, N., Wagner, K.D., Schley, G., Badiali, L., Theres, H., Scholz, H., 2003. 1,25-dihydroxyvitamin D3-induced apoptosis of retinoblastoma cells is associated with reciprocal changes of Bcl-2 and bax. Exp. Eye Res. 77, 1e9. Wajant, H., Moosmayer, D., Wuest, T., Bartke, T., Gerlach, E., Schonherr, U., Peters, N., Scheurich, P., Pfizenmaier, K., 2001. Differential activation of TRAIL-R1 and -2 by soluble and membrane TRAIL allows selective surface antigen-directed activation of TRAIL-R2 by a soluble TRAIL derivative. Oncogene 20, 4101e4106. Wang, Q.P., Xie, H., Yuan, L.Q., Luo, X.H., Li, H., Wang, D., Meng, P., Liao, E.Y., 2009. Effect of progesterone on apoptosis of murine MC3T3-E1 osteoblastic cells. Amino Acids 36, 57e63.

42

2. HORMONES OF PROGRAMMED CELL DEATH

Wang, S.H., Koenig, R.J., Giordano, T.J., Myc, A., Thompson, N.W., Baker Jr., J.R., 1999. 1Alpha,25-dihydroxyvitamin D3 up-regulates Bcl-2 expression and protects normal human thyrocytes from programmed cell death. Endocrinology 140, 1649e1656. Wang, Z., Malone, M.H., He, H., Mccoll, K.S., Distelhorst, C.W., 2003. Microarray analysis uncovers the induction of the proapoptotic BH3-only protein Bim in multiple models of glucocorticoidinduced apoptosis. J. Biol. Chem. 278, 23861e23867. Wei, L.L., Norris, B.M., Baker, C.J., 1997. An N-terminally truncated third progesterone receptor protein, PR(C), forms heterodimers with PR(B) but interferes in PR(B)-DNA binding. J. Steroid Biochem. Mol. Biol. 62, 287e297. Weinstein, R.S., 2012. Glucocorticoid-induced osteoporosis and osteonecrosis. Endocrinol Metab. Clin. N. Am. 41, 595e611. Weitsman, G.E., Ravid, A., Liberman, U.A., Koren, R., 2003. Vitamin D enhances caspase-dependent and independent TNF-induced breast cancer cell death: the role of reactive oxygen species. Ann. N. Y. Acad. Sci. 1010, 437e440. Wen, D.X., Xu, Y.F., Mais, D.E., Goldman, M.E., Mcdonnell, D.P., 1994. The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells. Mol. Cell Biol. 14, 8356e8364. Williams, G.R., 2008. Neurodevelopmental and neurophysiological actions of thyroid hormone. J. Neuroendocrinol. 20, 784e794. Willson, T.M., Brown, P.J., Sternbach, D.D., Henke, B.R., 2000. The PPARs: from orphan receptors to drug discovery. J. Med. Chem. 43, 527e550. Wilson, J.D., Gloyna, R.E., 1970. The intranuclear metabolism of testosterone in the accessory organs of reproduction. Recent Prog. Horm. Res. 26, 309e336. Wisniewska, M., Stanczyk, M., Grzelakowska-Sztabert, B., Kaminska, B., 1997. Nuclear factor of activated T cells (NFAT) is a possible target for dexamethasone in thymocyte apoptosis. Cell Biol. Int. 21, 127e132. Witcher, M., Ross, D.T., Rousseau, C., Deluca, L., Miller Jr., W.H., 2003. Synergy between all-trans retinoic acid and tumor necrosis factor pathways in acute leukemia cells. Blood 102, 237e245. Wong, R.S., 2011. Apoptosis in cancer: from pathogenesis to treatment. J. Exp. Clin. Cancer Res. 30, 87. Wu, K., Kim, H.T., Rodriquez, J.L., Munoz-Medellin, D., Mohsin, S.K., Hilsenbeck, S.G., Lamph, W.W., Gottardis, M.M., Shirley, M.A., Kuhn, J.G., Green, J.E., Brown, P.H., 2000. 9-cis-Retinoic acid suppresses mammary tumorigenesis in C3(1)-simian virus 40 T antigen-transgenic mice. Clin. Cancer Res. 6, 3696e3704. Wyllie, A.H., Kerr, J.F., Currie, A.R., 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251e306. Yacobi, R., Koren, R., Liberman, U.A., Rotem, C., Wasserman, L., Ravid, A., 1996. 1,25-dihydroxyvitamin D3 increases the sensitivity of human renal carcinoma cells to tumor necrosis factor alpha but

not to interferon alpha or lymphokine-activated killer cells. J. Endocrinol. 149, 327e333. Yalcin, M., Bharali, D.J., Lansing, L., Dyskin, E., Mousa, S.S., Hercbergs, A., Davis, F.B., Davis, P.J., Mousa, S.A., 2009. Tetraidothyroacetic acid (tetrac) and tetrac nanoparticles inhibit growth of human renal cell carcinoma xenografts. Anticancer Res. 29, 3825e3831. Yamanaka, A., Kimura, F., Kishi, Y., Takahashi, K., Suginami, H., Shimizu, Y., Murakami, T., 2014. Progesterone and synthetic progestin, dienogest, induce apoptosis of human primary cultures of adenomyotic stromal cells. Eur. J. Obstet. Gynecol. Reprod. Biol. 179, 170e174. Yao, X.L., Liu, J., Lee, E., Ling, G.S., Mccabe, J.T., 2005. Progesterone differentially regulates pro- and anti-apoptotic gene expression in cerebral cortex following traumatic brain injury in rats. J. Neurotrauma 22, 656e668. Yasui, H., Adachi, M., Hamada, H., Imai, K., 2005. Adenovirusmediated gene transfer of caspase-8 sensitizes human adenocarcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand. Int. J. Oncol. 26, 537e544. Yen, P.M., Ando, S., Feng, X., Liu, Y., Maruvada, P., Xia, X., 2006. Thyroid hormone action at the cellular, genomic and target gene levels. Mol. Cell. Endocrinol. 246, 121e127. Yoshida, H., Kong, Y.Y., Yoshida, R., Elia, A.J., Hakem, A., Hakem, R., Penninger, J.M., Mak, T.W., 1998. Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739e750. Yoshida, T., Gong, J., Xu, Z., Wei, Y., Duh, E.J., 2012. Inhibition of pathological retinal angiogenesis by the integrin alphavbeta3 antagonist tetraiodothyroacetic acid (tetrac). Exp. Eye Res. 94, 41e48. Yu, S., Lee, M., Shin, S., Park, J., 2001. Apoptosis induced by progesterone in human ovarian cancer cell line SNU-840. J. Cell. Biochem. 82, 445e451. Zhang, H., Chen, G.G., Zhang, Z., Chun, S., Leung, B.C., Lai, P.B., 2012. Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK. Apoptosis 17, 325e334. Zhang, H., Rosdahl, I., 2004. Expression profiles of p53, p21, bax and bcl-2 proteins in all-trans-retinoic acid treated primary and metastatic melanoma cells. Int. J. Oncol. 25, 303e308. Zhang, Y., Zhao, H., Asztalos, S., Chisamore, M., Sitabkhan, Y., Tonetti, D.A., 2009. Estradiol-induced regression in T47D:A18/ PKCalpha tumors requires the estrogen receptor and interaction with the extracellular matrix. Mol. Canc. Res. 7, 498e510. Zheng, A., Mantymaa, P., Saily, M., Savolainen, E., Vahakangas, K., Koistinen, P., 2000. p53 pathway in apoptosis induced by alltrans-retinoic acid in acute myeloblastic leukaemia cells. Acta Haematol. 103, 135e143. Zimmermann, M.B., 2009. Iodine deficiency. Endocr. Rev. 30, 376e408.

C H A P T E R

3 Hypothalamic Releasing Hormones Gabor Halmos1,2, Nikoletta Dobos1, Eva Juhasz3, Zsuzsanna Szabo1, Andrew V. Schally2,4,5,6 1

Department of Biopharmacy, Faculty of Pharmacy, University of Debrecen, Debrecen, Hungary; 2Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL, United States; 3Institute of Pediatrics, Clinical Center, University of Debrecen, Debrecen, Hungary; 4Department of Pathology, Divisions of HematologyeOncology and Endocrinology, Miller School of Medicine, University of Miami, Miami, FL, United States; 5Department of Medicine, Divisions of HematologyeOncology and Endocrinology, Miller School of Medicine, University of Miami, Miami, FL, United States; 6Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, United States

1. CORTICOTROPIN RELEASING HORMONE

Although CRH was initially described for its function as an HPA regulator, CRH is widely expressed in central circuits. CRH is also synthesized in peripheral tissues, including the heart, blood vessels, skin, lung, spleen, pancreas, kidney, liver, adipose tissue, digestive tract, testes, ovaries, placenta, and even T-lymphocytes (Contoreggi, 2015; Inda et al., 2017; Keller-Wood, 2015). A nuclear magnetic resonance (NMR) study of the conformation of the human CRF (hCRF) was determined in an aqueous trifluoro-ethanol solution (TFE). This method identified that in hCRF, the N-terminal tetrapeptide is connected to an a-helix between residues 6 and 36 and a poorly resolved C-terminal region. It was proven that the side chains containing residues 5e19 are very important for receptor binding and activation. The amino acid side chains of the second half of the molecule are more conserved and have less functions, while C-terminal region, at least up to residue 36, is a hydrophilic a-helix (Romier et al., 1993). This a-helix is amphiphilic and exists in other peptide hormones as well (Kaiser and Kezdy, 1984). This confirmation plays a role in the initiation of the formation of the amphipathic helix in TFE or in the neighborhood in the membrane. The activating region of CRF includes the two neighboring proline residues (residues 4e8) and the first turn of the a-helix (Romier et al., 1993).

Corticotropin releasing hormone or CRH is a highly conserved neuropeptide hormone. It is also known as corticotropin-releasing factor (CRF) or corticoliberin. In keeping with the historical nomenclature, the original name CRF is still CRF in much of the literature, rather than CRH, but both terms are used (Maclean and Jackson, 1988; Reichlin et al., 1976; Vale et al., 1981). In the 1950s, Hans Selye called attention to the importance of “the first mediator” of the ACTH response upon exposure to stress. In 1955, Saffran and Schally demonstrated the presence of CRF in hypothalamic tissue. This was the first experimental proof that hypothalamic hormones regulate pituitary function, as postulated by the great English physiologist G. W. Harris (Saffran and Schally, 1955). CRF was characterized as a 41-residue peptide 30 years later by the group led by W. Vale. This peptide hormone is derived from a 196amino acid preprohormone (Majzoub, 2006; Vale et al., 1981). Some earlier reviews describe CRF, its variants, multiple receptors, mechanism of action, multiple physiologic functions, and clinical implications (Vale et al., 1981; Yanaihara et al., 1986). Immunohistochemical localization of CRF has demonstrated that the hormone has a broad extrahypothalamic distribution in the central nervous system (CNS) (De Souza, 1995). CRH is an important activator of the hypothalamicepituitaryeadrenal (HPA) axis (Fig. 3.1; De Souza, 1995).

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00003-1

1.1 CRH Protein and Gene Structure CRH is chemically classified as a neuropeptide hormone, a protein-like molecule made up of a short chain

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Copyright © 2020 Elsevier Inc. All rights reserved.

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FIGURE 3.1 The hypothalamusepituitaryeadrenal axis (HPA) (Sherin and Nemeroff, 2011).

of amino acids that is produced in the brain and functions as a hormone. It was first purified from sheep hypothalamus (Thomson, 2013). The chemical status of CRF was summarized by Blackwell and Guillemin and Schally et al. in 1973 (Schally et al., 1973). Human CRH is a product of the preproCRH molecule, which is 196 amino acids in length, at its amino terminal end containing a hydrophobic signal sequence, required for secretion. There are 124 amino acids with unknown function in the middle of the molecule. The carboxyl terminal end of preproCRH contains a 41 amino acid sequence of the mature peptide hormone. Amidation of the carboxyl end of CRH is essential for biologic activity (Majzoub, 2006). The CRH gene is highly conserved among vertebrate species. In humans, CRH protein is encoded by the CRH gene. CRH gene is composed of two exons and an 800bp intron. The entire protein-coding region is contained in the second exon (Fig. 3.2). The gene for CRH is assigned to 8q13 chromosome (Arbiser et al., 1988).

FIGURE 3.2 CRH gene structure. http://genatlas.medecine.univparis5.fr/fiche.php?symbol¼CRH.

It is known that in addition to the 41 amino acid peptide CRF, there are three other CRF related peptide forms belonging to the CRF signaling family proteins. These are urocortin I (urotensin I, Ucn I), which has 45% homology to CRH, urocortin II (a stresscopinrelated peptide, Ucn II) with 55% homology to CRH, and urocortin III (stresscopin, Ucn III), which has 32% homology to CRH (Stengel and Tache, 2010; Thomson, 2013). CRF has a well-conserved primary structure among mammalian species including humans, primates, dogs, and rodents (Lovejoy and Jahan, 2006; Stengel and Tache, 2010). As observed for CRF, the primary structure of Ucn I is highly conserved across mammalian species, including rat, mouse, and sheep. Mouse Ucn II (mUcn II) is a 38 amino acid peptide that shares 34% homology with rat/human CRF and 42% with rat/mouse UcnI, whereas human Ucn III has only a little overlap with the structure of rat/human CRF, with 18% and 21% homology, respectively (Cardoso et al., 2016; Stengel and Tache, 2010).

1.2 CRH Receptors The two major forms of CRF receptor are named CRHR1 and CRHR2. Both proteins are seventransmembrane G proteinecoupled receptors (GPCRs; Figs. 3.3 and 3.4). Structurally, the types 1 and 2 CRHR

FIGURE 3.3 The CRF system signaling (Ducarouge and JacquierSarlin, 2011).

1. CORTICOTROPIN RELEASING HORMONE

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factor in binding specificity. A residue at position 11 is found only in CRHR2, which makes it selective to ligands Ucn2 and Ucn3 (Grammatopoulos and Ourailidou, 2017; Perrin and Vale, 1999; Fig. 3.3). CRHR1 binds CRH as well as Ucn1, but not Ucn2 or Ucn3, with equivalent high affinity to CRH, suggesting that these peptides may be its natural ligands (Grammatopoulos and Ourailidou, 2017; Fig. 3.3). CRF and Ucn1 can bind to the CRF1 and CRF2 receptors with a lower affinity for CRF. Ucn2 and Ucn3 bind exclusively the CRF2 receptor. These ligand/receptor interactions are challenged by a competition with the CRF binding protein (CRF-BP), or soluble truncated forms of the receptors. The activated receptors are endocytosed and either further degraded or recycled to the cell membrane.

1.3 CRHeCRH Receptor Interaction and Signaling Pathway

FIGURE 3.4

The CRF receptor signaling (Ducarouge and Jacquier-

Sarlin, 2011).

are approximately 70% identical in the amino acids, but there is a significant divergence at the N-terminal extracellular domain (ECD) (approximately 47%), consequent with their distinct pharmacological properties and agonist selectivity. CRH-R1b, the largest variant, has 444 amino acids, and in its sequence, a structural difference produces a higher affinity for CRH. This different characteristic is important for their unique physiologic roles, when natively coexpressed in specific tissues. In humans, there are three described splice variants of CRHR2: a, b, and g. CRHR2 a and CRHR2b are synthesized in the placenta. Splice variant CRHR2g is specifically expressed in the limbic system of the brain. Different splice variants of CRHR1 and CRHR2 have diverse biologic roles (Grammatopoulos and Ourailidou, 2017). Combinations of different isoforms of CRH receptors are found in various tissues (including the immune system, integument, the blood brain barrier, gastrointestinal tract, and parasympathetic ganglions) Nevertheless, it is proposed that CRH family peptides can stimulate different intracellular systems (Contoreggi, 2015; De Souza, 1995; Liapakis et al., 2011; Thomson, 2013). Within the amino acid sequence of CRHR ligands, a number of residues have been identified as an important

Actions of CRH are mediated through the activation of two types of GPCRs: CRH Receptor 1 (CRHR1) and CRH Receptor 2 (CRHR2). The ubiquitous distribution of CRHR makes it capable of activating diverse signaling mechanisms in different tissues. In most cells of peripheral tissues, the physiologic actions of CRF and urocortins involve coupling of both types of CRHR to Ga(s) and to GbY proteins. In human tissues, stimulation of any CRHR and its coupling to Ga(s) leads to the activation of adenyl cyclase (AC), stimulating cAMP-mediated signaling cascades. However, in some tissues (i.e., testes, placenta), CRH stimulates alternative signaling cascades, such as phosphoinositol hydrolysis. In a tissue-specific manner, CRHRs modulate a number of intracellular enzymes (PKA, PKC, Akt, ERKs, p38 MAPKs) and other important signaling intermediates, such as Ca2þ, NOS, GC, prostaglandins, and FasL. These proteins in turn stimulate various transcription factors like c-Jun, c-Fos, JunD, Elk1, MEF2, and cAMP response element binding (CREB), which act directly on the POMC promoter and stimulate ACTH biosynthesis along with the expression of several other genes (Majzoub, 2006). The activation of cellular G protein influences the activities of the neuronal, endothelial, endocrine, smooth muscle, epithelial and immune cells. Different effects depend on a number of environmental factors and also on tissues where the activation occurs (Inda et al., 2017; Larauche et al., 2009). For example, the stimulation of MAPKs cascade involves the activation of the PKC pathway and leads to the effect of CRH on Leydig, myometrial, and hippocampus cells. The CRHR-MAPK cascade mediates the neuroprotective effects of the CRH and CRH-like peptides, and activation of FasL by CRHR-activated p38 MAPK

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culminates in apoptosis. In the heart and vasculature, CRHRs mediate the ionotropic, vasodilatory, and antiedema effects of CRH. In immune cells, CRH both inhibits and stimulates production of the proinflammatory cytokines (IL-1 and IL-6) by mononuclear cells in peripheral blood. CRH has proinflammatory effects in mast cells. It induces VEGF release in mast cells through selective activation of the signaling pathway cAMPPKA-p38 MAPK (Grammatopoulos and Ourailidou, 2017; Inda et al., 2017; Larauche et al., 2009). Downstream signaling of the CRF receptors is connected to the GPCR Gabg activity and the Src kinase, which interacts with the receptors’ cytoplasmic domains. Numerous activated intracellular proteins could regulate cell processes such as survival, proliferation, apoptosis, or migration. When physiologic and psychological stress is processed in the cerebral hemispheres, the hypothalamus is activated. As a consequence, it releases CRH into the hypophyseal portal system that transports blood to the anterior pituitary partly to synthesize and release ACTH and b-endorphin into the blood stream (Majzoub, 2006; Thomson, 2013). ACTH acts on the adrenal cortex to release glucocorticoids and other steroid hormones including androgens and aldosterone. The HPA system is regulated by negative feedback (Hillhouse and Grammatopoulos, 2006; Majzoub, 2006; Stengel and Tache, 2010; Thomson, 2013). The negative regulation of glucocorticoids on CRH and the ability of ACTH from the pituitary to inhibit CRH release represent a short feedback loop. In the adrenal cortex, ACTH stimulates the synthesis and release of steroid hormones including cortisol. Cortisol exerts a range of physiologic functions including the stimulation of gluconeogenesis and glycogen catabolism, release of fatty acids from adipose tissue, and immune response modulations. Released cortisol exerts negative feedback on the pituitary, where it suppresses POMC gene expression and ACTH secretion. In the hypothalamus, cortisol decreases pro-CRH gene expression and the release of CRH. As cortisol has profound effects on the immune system, the HPA axis and the immune system are closely coupled. Moreover, cytokines, particularly IL-1, IL-2, and IL-6, stimulate the HPA axis. ACTH release represents a long feedback loop (Fig. 3.5). CRH is required for the stimulation of pituitary ACTH gene expression, which occurs with loss of negative feedback during adrenal insufficiency (Majzoub, 2006). Vasopressin, cosynthesized with CRH in hypothalamic PVN neurons and altogether with CRH, stimulates ACTH secretion. CRF plays a major role in the coordination of endocrine, autonomic, behavioral, and immune responses to stress through

the actions in the brain and the periphery (Contoreggi, 2015; De Souza, 1995; Fig. 3.5).

1.4 Diseases As important integrators of the stress response in the amygdala, CRH and urocortin III peptides have been involved in the mediation of behavioral responses to stress (Contoreggi, 2015). Excessive CRH within the CNS may induce depressive symptoms consisting of loss of appetite, insomnia, and intense fear and anxiety (De Souza, 1995; Stengel and Tache´, 2010). CRF-induced dysregulation of neuronal activity within the CNS may also be strongly involved in the pathology of depressive disorders (Contoreggi, 2015; Kasckow et al., 2001). Neuroendocrine findings in posttraumatic stress disorder (PTSD) revealed that PTSD patients are characterized with low adrenocortical activity and high levels of CRH in the CNS (Kasckow et al., 2001; Thomson, 2013). Dual action of IL-1 and CRH mediates both pro- and antiinflammatory responses. IL-1 is produced by stimulated macrophages and directly via the stimulation of the HPA axis, and it increases the secretion of CRH and ACTH, leading to elevated levels of antiinflammatory glucocorticoids (Majzoub, 2006; Stengel and Tache, 2010). CRF receptors are also involved in stress-related motility of the small intestine and the colon. Studies in rodents showed that the administration of CRH antagonists induces bowel emptying by increased colonic motility and inhibits gastric acid secretion and gastric emptying (De Souza, 1995; Stengel and Tache, 2010). CRH also inhibits feeding behavior even in fooddeprived experimental animals. It is possible that one of the peptides related to CRH, such as urocortin I or III, mediates these gastrointestinal actions (Gilligan and Li, 2004; Majzoub, 2006; Stengel and Tache, 2010). CRH is also a major inhibitor of the reproductive functions in both sexes. For example, intense or prolonged stress has been shown to inhibit gonadotropin secretion. Additional central effects of CRF include the attenuation of sexual behavior (Majzoub, 2006; Thomson, 2013). Tumors, including prostate cancers, bronchial carcinoids, nephroblastomas, medullary carcinomas of the thyroid, and islet cell tumors are able to secrete CRH ectopically. Such tumors usually secrete ACTH as well, and therefore, it is often difficult to establish whether any associated Cushing syndrome is due to the hypersecretion of CRH or ACTH. Nevertheless, under some circumstances, CRH can serve as a tumor marker to assess the course of the disease and its response to therapy (Ur and Grossman, 1992).

2. THYROTROPIN-RELEASING HORMONE

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FIGURE 3.5 Hypothalamusepituitaryeadrenal (HPA) axis, illustrating factors regulating the secretion of corticotropin releasing hormone (CRH). Pathway diagram showing the HPA axis, stressor activating the HPA axis, and the feedback regulation of glucocorticoid hormone secretion by the HPA axis. ACTH, adrenocorticotropic hormone; AP, anterior pituitary; CRH, corticotropin releasing hormone; PP, posterior pituitary (Porterfield and White, 2007) http://blp6.atw.hu/BLP6/HTML/C0409780323045827.htm.

2. THYROTROPIN-RELEASING HORMONE Thyrotropin-releasing hormone (TRH) was the first hypothalamic releasing hormone to be isolated and synthesized (Boler et al., 1969). In 1969, the groups of Guillemin and Schally independently purified and determined the amino acid sequence of TRH from ovine and porcine hypothalamic extracts, respectively (Boler et al., 1969; Burgus et al., 1969; Ladram et al., 1994). TRH of both species was a tripeptide (pyro)Glu-His-Pro-NH2. The synthetic TRH was tested biologically in animals and clinically (Anderson et al., 1971; Bowers et al., 1970). Anatomically and physiologically, TRH, previously called thyrotropin-releasing factor (TRF) or thyroliberin, is one of the releasing hormones produced by the hypothalamus, and it stimulates the release of thyrotropin (also known as thyroid-stimulating hormone or TSH) and prolactin from the anterior pituitary. TRH is synthesized in various tissues and organs and is present in

different parts of the CNS. TRH is produced mainly in the hypothalamus, particularly in the parvocellular neurosecretory neurons, suprachiasmatic preoptic nucleus, dorsomedial nucleus, and basolateral hypothalamus (Fig. 3.6). TRH forms the topmost component of the hypothalamicepituitaryethyroid (HPT) axis, and it plays a crucial role in the regulation of this axis (Nillni, 2010). Interestingly, the hypothalamus contains only 30%e32% of the total brain TRH, the other 70% is located and produced in other areas, e.g., in the forebrain, posterior diencephalon, hindbrain, cranial nerves nuclei, neuropituitary, and pineal gland (Ghamari-Langroudi et al., 2010). Furthermore, TRH was also shown to be present in other tissues and organs, e.g., in the placenta, in the gastrointestinal tract, as well as in tumors. TRH has a very short half-life, approximately 4 min in circulation due to its rapid enzymatic inactivation in blood (Golubeva, 2013). TRH is synthesized as a large, 242 amino acid precursor TRH protein mainly in the hypothalamus but also in

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FIGURE 3.7

FIGURE 3.6 Distribution of TRH-synthesizing neurons in the PVN. Low-power micrographs (AeC) illustrate the TRH neurons at three rostrocaudal levels of the PVN. Schematic drawings (DeF) illustrate the subdivisions of the PVN where hypophysiotropic TRH neurons are localized (gray). AP, anterior parvocellular subdivision; DP, dorsal parvocellular subdivision; LP, lateral parvocellular subdivision; MN, magnocellular part of PVN; MP, medial parvocellular subdivision; PV, periventricular parvocellular subdivision; III, third ventricle (Mariotti and Beck-Peccoz, 2000; Fekete and Lechan, 2007).

other tissues outside the brain, such as pancreatic b cells, the C cells of the thyroid gland, the myocardium, reproductive organs (prostate and testis), the spinal cord, and in the anterior pituitary (Bruhn et al., 1994, 1998; Martino et al., 1978; Mariotti and Beck-Peccoz, 2000; Jeffcoate et al., 1976; Jackson, 1982; Gkonos et al., 1989; Bilek, 2000). As indicated already, the mature human TRH is a tripeptide pGlu-His-Pro-NH2 with a molecular weight of 362.4 Da (Fig. 3.7), originating from a precursor protein (preproTRH) with multiple copies of five progenitor sequences for TRH: (Kastin, 2013; Mariotti and BeckPeccoz, 2000). Since TRH is first synthesized as a large prohormone, it is posttranslationally modified to generate a bioactive peptide. Thus, TRH is synthesized from a larger inactive preproTRH molecule by a series of posttranslational modifications while being transported through the secretory pathway (RSP) (Nillni, 2010) These posttranslational modifications require various enzymes to produce the mature form of TRH. Nillni et al. have described two members of the family of prohormone convertases (PCs), PC1/3 and secondarily by PC2, implicated in TRH maturation (Nillni, 2010). After a protease cleaves the C-terminal side of the flanking Lys-Arg or Arg-Arg, carboxyl peptidase E and D remove the

Chemical structure of TRH (EPA.gov).

C-terminal basic amino acids. TRH-glycine, the immediate precursor to TRH, is amidated by peptidylglycine a-amidating monooxygenase enzyme (PAM) (Eipper et al., 1992; Nillni et al., 2002; Perello and Nillni, 2007) The N-terminal glutamine is then converted into pyroglutamate (Nillni, 2010). In vertebrates, TRH is a highly conserved neuropeptide that is distributed throughout the animal kingdom, and it can even occur in species that lack the pituitary (Kastin, 2013). In 2017, Sinay et al. documented a functional TRH neuropeptide-receptor pathway in the nematode Caenorhabditis elegans, which suggests that TRH signaling had evolved in a bilaterian ancestor more than 700 million years ago (Van Sinay et al., 2017). The human TRH gene is located on chromosome 3 (3q13.3a`q21) (Yamada et al., 1999). Potential glucocorticoid and cyclic AMP response elements (GRE and CRE) are detected on the 50 flanking sequence of the TRH gene (Lee et al., 1988; Fig. 3.8). The preproTRH molecule serves as a precursor for proTRH peptides as well as non-TRH peptides with likely physiologic function, e.g., stimulation of TSH b gene expression, and enhancement of TRH-induced release of TSH and prolactin (PRL) from the pituitary (Bulant et al., 1990). The expression of hypophysiotropic preproTRH gene is regulated in the medial and periventricular regions of the hypothalamus (Nillni, 2010). Parvocellular neurosecretory neurons originating in the paraventricular nucleus of the hypothalamus (PVN) have projections to the median eminence (ME), where their axons end close to the capillaries of the hypophysial-portal system’s. TRH peptide is transported through axon terminals to these capillaries and activates the synthesis and secretion of pituitary TSH, which in turn stimulates the thyroid and induces the biosynthesis and secretion of thyroid hormones (Nillni,

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FIGURE 3.8 Genomic and promoter structure of TRH. The murine and human TRH genes are composed of three exons and two introns (A). Exon 1 encodes the 50 untranslated region, while exon 2 and 3 contain the sequence for preproTRH. The two intron sequences are poorly conserved among the species. The promoter region of the preproTRH gene is located immediately 50 to exon 1, and this is likely the major site where the regulation of the gene occurs (Nillni, 2010). As depicted, the TRH promoter region precedes the transcription start site in exon 1. The proximal 250bp sequences of the human, mouse, and rat promoters are similar and share the transcription factor binding sites. The location of the CREB binding site (Site 4) and sequences in human (H), mouse (M), and rat (R) are shown. (B, C) Hypothesized schematic representation of the interaction between PCREB and the thyroid hormone receptor at Site 4. (B) Illustrates that in the presence of abundant PCREB, there may be less availability for binding of the thyroid hormone receptor/T3 complex, hence, an increase in TRH gene transcription. When PCREB concentrations fall, as shown in (C), increased binding of the thyroid hormone receptor/T3 complex reduces TRH gene transcription (Fekete and Lechan, 2007; Mariotti and Beck-Peccoz, 2000).

2010; Ortiga-Carvalho et al., 2016). The thyroid hormones, thyroxine (T4) and triiodothyronine (T3), regulate the secretion of TRH and TSH secretion by negative feedback (Fig. 3.9). The regulation of TSH by TRH and negative feedback mechanisms of thyroid hormones are essential for the maintenance of euthyroidism. TRH secreted from the hypothalamus is responsible for the secretion of TSH from the anterior pituitary. TSH induces the secretion of T4 and T3 (Nillni, 2010). T4 regulates the secretion of TSH by negative feedback and decreases the responsiveness of the anterior pituitary to TRH stimulation. Unlike hypothalamic TRH that is controlled by negative feedback, extrahypothalamic biosynthesis and release of TRH is not responsive to thyroid hormones (Kastin, 2013). TRH exerts its biologic effects through stimulation of its cell-surface receptors (TRH-R), which belong to the superfamily of GPCRs with seven highly conserved transmembrane domains (Mariotti and Beck-Peccoz, 2000; Fig. 3.10). TRH binds to its receptors activating adenylate cyclase to produce cAMP or stimulating the phosphoinositol system. This leads to phosphorylation of protein kinases. Upon the binding of TRH to TRHR, the Gq/11 dependent pathway is activated, producing the mobilization of intracellular calcium and activation of protein

FIGURE 3.9 Normal control path and negative feedback for thyroid hormones. Adapted from Slominski, A.T., Zmijewski, M.A., Skobowiat, C., Zbytek, B., Slominski, R.M., Steketee, J.D., 2012. Sensing the environment: regulation of local and global homeostasis by the skin’s neuroendocrine system. Adv. Anat. Embryol. Cell Biol. 212, v, vii, 1e115.

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FIGURE 3.10 Crystallographic modeling of TRHR showing the positions (red spheres) of the two mutations associated with central hypothyroidism: R17X truncating the protein in the extracellular domain and an in-frame deletion of 3 amino acids (Ser115eThr117) plus a missense change (Ala118 for Thr118; p.S115-T117delCT118) located at the cytoplasmic end of the third transmembrane domain of the receptor. Structural model of the TRHR was generated by homology modeling using the PHYRE server and Pymol. The N-terminal start codon and C-terminal end codon are highlighted in green. Adapted from Schoenmakers, N., Alatzoglou, K.S., Chatterjee, V.K., Dattani, M.T., 2015. Recent advances in central congenital hypothyroidism. J. Endocrinol. 227, R51eR71.

kinase C, which is responsible for the synthesis and secretion of TSH. Subsequent activation of pyroglutamyl peptidase II (PPII) produces degradation of extracellular TRH (Hinkle et al., 2012; Fekete and Lechan, 2007; Schoenmakers et al., 2015). Despite interaction with its receptors, TRH can maintain its cell function by nonspecific inclusion into the membrane (Golubeva, 2013). Because of its small size, TRH is transported easily through the cell membrane. It was demonstrated that the plasma membrane, mitochondria, and nucleus contain intracellular TRH receptors (Golubeva, 2013; Miranda et al., 2005). Perlman et al. provided evidence that only the unprotonated form of TRH can bind to TRH-R1. Under physiologic conditions, the His residue of TRH is present in a form of charged and uncharged species, and the state of ionization of TRH is sensitive to pH changes within the physiologic range. The biologic significance of this event is not clear, but it might affect the function of TRH (Engel and Gershengorn, 2007; Perlman et al., 1992). Activation of receptors for TRH starts with binding of TRH to TRHR in a sequential process (Fig. 3.11). First,

FIGURE 3.11 The activation model of mouse TRH-R1. The upper panel shows a representative structure of the inactive, unoccupied TRH-R1, while the lower panel shows that of the activated, TRHoccupied TRH-R1. These are viewed from the intracellular aspect. TRH, located in the transmembrane binding pocket, is marked in yellow. The lipid bilayer and water molecules are not shown. The TRHinduced conformational changes at the cytoplasmic ends of TMH5 and TMH6 are indicated with yellow arrows. Adapted from Engel, S., Gershengorn, M.C., 2007. Thyrotropin-releasing hormone and its receptorsea hypothesis for binding and receptor activation. Pharmacol. Ther. 113, 410e419; Huang, W., Osman, R., Gershengorn, M.C., 2005. Agonistinduced conformational changes in thyrotropin-releasing hormone receptor type I: disulfide cross-linking and molecular modeling approaches. Biochemistry 44, 2419e2431.

TRH interacts with an extracellular binding pocket that serves as an entry channel of TRHR formed by the residues in the extracellular loop. This complex undergoes conformational changes creating a highaffinity TRH-TRHR complex. Subsequently, TRH moves then further toward the second TRH binding pocket situated in the transmembrane domain of the receptor, leading to changes in conformation and coupling of G protein (Engel and Gershengorn, 2007). As mentioned before, TRH has a very short half-life. A cell-surface peptidase, called TRH-degrading ectoenzyme (TRH-DE), rapidly inactivates TRH within the CNS. This enzyme is very specific to TRH, and there are no other known enzymes degrading TRH (Heuer et al., 1998; Engel and Gershengorn, 2007).

3. PROLACTIN-RELEASING FACTORS

TRH functions as an agent responsible for the stimulation and release of TSH from the anterior pituitary. The density of the receptors in the pituitary gland is crucial, and it is negatively regulated by circulating thyroid hormones. TSH has essential effects on the functions of the thyroid to produce T3 and T4. In turn, the thyroid hormones control iodine metabolism, protein synthesis, catabolism, and the developmental processes. TRH is indirectly involved in growth and development through the regulation of the synthesis of T3 and T4 as well (Golubeva, 2013). Although the primary function of TRH is the stimulation of TSH from the anterior pituitary. TRH can also stimulate the secretion of other pituitary hormones, prolactin, and under certain pathologic conditions, growth hormone as well. Many other functions and effects are ascribed to TRH. TRH has important roles as a neurotransmitter, neuromodulator, and neuroprotective agent in the central and peripheral nervous systems. TRH is a natural opioid agonist; however, opioid effects, such as suppression of breath, decreased heart rate, and catalepsy, do not develop (Kastin, 2013, Golubeva, 2013). In addition, TRH can inhibit the expression of glycogen synthase kinase-3b (GSK-3b), and thus, it could be of interest for novel therapies of Alzheimer disease, major depression, bipolar disorder, and diabetes. TRH is also an important regulator of lean body mass (Kastin, 2013). TRH may also increase blood pressure and spontaneous locomotor activity (Golubeva, 2013). This peptide can similarly potentiate the excitatory effect of acetylcholine on cortical neurons, provoke anorexia, stimulate norepinephrine and dopamine release from the synaptosomes, and accelerate norepinephrine exchange (Horita, 1998). Thyroid hormones may modulate lymphocyte activity, and it has been shown that TRH has an indirect effect on lymphocyte proliferation (Klecha et al., 2006). The HPT axis plays a critical role in mediating changes in metabolism and thermogenesis (Nillni, 2010). Thyroid hormones as well as TRH are responsible for thermoregulation through an augmented intracellular lipid oxidation, tonus, and muscle tremor triggering (Golubeva, 2013). TRH is present in the pancreas and has been shown to decrease glucose and xylose absorbance from the intestine (Golubeva, 2013). TRH is also able to reduce the microviscosity of the lipid component of biomembranes, and it can change the temperature of the structural transition in the membrane. The effect of TRH on membranes is concentration dependent (Golubeva, 2013). TRH (maternal or embryonic) is not required for the normal development of the fetal pituitary thyrotrophs,

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and mice lacking TRH do not develop hypothyroidism at birth. However, TRH is required later for the postnatal maintenance of the normal function of the pituitary thyrotrophs (Mariotti and Beck-Peccoz, 2000; Shibusawa et al., 2000). In conclusion, TRH has a broad spectrum of activity and potential beneficial effects on different tissues and organs. However, the clinical use of TRH other than diagnostic is limited, first by its main hormonal activity, and secondly, by its poor metabolic stability. Therefore, to overcome these limitations, metabolically stable TRH analogs with similar potency and activity to natural TRH might be interesting.

3. PROLACTIN-RELEASING FACTORS Prolactin (PRL) is a multipurpose hormone of the mammalian body, exerting more than 300 biologic activities including reproductive, sexual, metabolic, and immune functions (Bole-Feysot et al., 1998). Thus, it is not surprising that the secretion of prolactin is highly complex, involving the coordinated actions of several hypothalamic nuclei (Egli et al., 2010). Moreover, it has been shown that the synthesis and secretion of prolactin is not restricted to the anterior pituitary gland; other organs, e.g., the hypothalamus, placenta, amnion, decidua, uterus, mammary gland, and even the immune system, particularly lymphocytes, have this capability as well (Freeman et al., 2000). The major form of prolactin is a 23-kDa protein; however, various forms of prolactin have been characterized mainly as a result of alternative splicing of the primary transcript, proteolytic cleavage, and other posttranslational modifications (Freeman et al., 2000). Several potent PRFs have been proposed; however, regarding the stimulation of prolactin release by a hypothalamic factor(s), none of them appears to be a unique prolactin-releasing hormone. Among other substances, TRH, vasoactive intestinal polypeptide (VIP), oxytocin, and prolactin-releasing peptide (PrRP) can stimulate prolactin release (Squire et al., 2014a). The neurotransmitter serotonin can also stimulate prolactin synthesis and secretion; however, the primary control mechanism does not include the serotonergic pathway (Van de Kar et al., 1996). Neurohormones and neurotransmitters playing a role in the regulation of prolactin secretion are well summarized in the review of Freeman et al. (2000). In 1971, Tashjian et al. described TRH as a potent prolactin-releasing factor (PRF), and since then, TRH has become one of the best-studied PRFs (Fig. 3.12). However, it is still not clear whether TRH is a physiologic regulator of prolactin. Most likely, TRH is one of

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FIGURE 3.12

3. HYPOTHALAMIC RELEASING HORMONES

Regulation of prolactin (Hilal-Dandan and Brunton,

2014b).

the regulators, but not the primary releasing factor (Tashjian et al., 1971). The release and synthesis of prolactin is regulated by different TRH-induced signaling pathways, as shown in GH3 cells (GH- and prolactin-synthesizing and releasing cells established from rat pituitary adenoma; Fig. 3.13). The activation of ERK signaling induced by TRH is strongly involved in prolactin gene expression. On the other hand, an MEK inhibitor, which is an activator of ERK, cannot inhibit TRH-induced prolactin

release, indicating that prolactin release is regulated differently. It has been demonstrated that intracellular Ca2þ from intracellular or extracellular Ca2þ storage sites can stimulate Ca2þ-dependent protein kinases, e.g., calcium/calmodulin-dependent kinase II (CaMK II) or myosin light chain kinase, inducing prolactin release (Kanasaki et al., 2015). In 1999, Hinuma et al. discovered another novel peptide with prolactin-releasing properties named PrRP (Hinuma et al., 1998). Although PrRP has been reported as a potent and specific PRF, the mechanism of action of PrRP remained unclear, until Jarry et al. showed that PrRP cannot directly stimulate PRL secretion, indicating that PrRP most likely exerts neuromodulatory functions in the CNS and thus should not be classified as a classical hypophysiotrophic factor (Jarry et al., 2000). Correspondingly, in 2007, Spuch and Navarro demonstrated that pituitary PrRP is a more important modulator of prolactin release mediated by TRH than PRF itself (Spuch and Navarro, 2011). PrRP displays prolactin-releasing capacity at nanomolar doses (Swinnen et al., 2005); nevertheless, PRL release induced by PrRP is several times lower than that brought about by TRH (Spuch et al., 2007) Thus, PrRP has been proposed as an important modulator of TRH as PRF in the pituitary. Interestingly, increasing PRL response to TRH suggested a paracrine action of PrRP (Spuch et al., 2006, 2007).

FIGURE 3.13 The release and synthesis of prolactin is regulated by different TRH-induced signaling pathways. Adapted from Kanasaki, H., Oride, A., Mijiddorj, T., Kyo, S., 2015. Role of thyrotropin-releasing hormone in prolactin-producing cell models. Neuropeptides 54, 73e77.

4. LUTEINIZING HORMONE-RELEASING HORMONE/GONADOTROPIN-RELEASING HORMONE

In 2007, Christian et al. published their results on the regulation of PRL, suggesting that TRH, PrRP, or VIP regulates selectively three different subtypes of lactotroph cells, whereas dopamine is an inhibitor of all cells (Christian et al., 2007). As cited earlier, it is widely accepted that VIP also directly stimulates lactotroph cells to induce PRL release. Although VIP and TRH cause fundamentally different secretion profiles of PRL, their effects on PRL release are additive (Bjoro et al., 1990). In summary, different putative PRFs have been postulated to induce prolactin release. Nonetheless, compared to other adenohypophysial hormones and their hypothalamic releasing factor counterparts, these stimulating factors do not seem to have the one-to-one relationship with prolactin. Thus, the search for a unique hypothalamic releasing hormone for prolactin is still ongoing (Squire et al., 2014a).

4. LUTEINIZING HORMONE-RELEASING HORMONE/GONADOTROPINRELEASING HORMONE Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone (LHRH), is the primary link between the brain and the pituitary. It plays a key role in the neuroendocrine control of reproduction in vertebrates and in the regulation of gonadal functions (Schally et al., 2001a). In 1971, Andrew Schally and his associates were the first to accomplish the isolation and elucidation of the chemical structure of LHRH from pig hypothalami and its synthesis, winning the race with other groups (Schally et al., 1971; Schally et al., 2001b; Schally and Halmos, 2012). They also showed that natural LHRH and synthetic decapeptide corresponding to its structure possess follicle-stimulating hormone (FSH)-releasing activity (FSHRH) as well as luteinizing hormone (LH)releasing activity (LHRH). The concept formulated by one of us (A.V.S.), that one hypothalamic hormone regulates the secretion of both gonadotropins from the pituitary gland, is now supported by much experimental and clinical evidence (Schally et al., 2001a; Schally and Halmos, 2012). It was originally suggested by Schally et al. (1971) that the abbreviation LHRH/FSH-RH be changed to GnRH for gonadotropin-releasing hormone. However, this leads to confusion with the abbreviation GHRH for growth hormone-releasing hormone, for which many agonistic and antagonistic analogs already exist. Consequently, we now prefer to use the original name and abbreviation, LHRH, especially for its analogs. LHRH was then isolated from the ovine hypothalami by the group of Guillemin. In 1977, Andrew Schally was awarded the Nobel Prize in physiology/medicine

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for the isolation, elucidation of structure, and synthesis of hypothalamic LHRH, which he shared with Roger Guillemin (Kochman, 2012; Schally et al., 2001a; Fig. 3.14). The structure of LHRH in all mammals including humans is the same with LHRH, playing a basic physiologic role in the regulation of the synthesis and release of LH and FSH. Moreover, to date, 30 structurally different isoforms of LHRH peptide have been reported (Kochman, 2012). Interestingly, each isoform of the LHRH decapeptide has well-conserved N-terminal (Glp-His-Trp-Ser) and C-terminal (Pro-Gly-NH2) amino acid sequences (Maggi et al., 2016). Particularly, in addition to mammalian GnRH, GnRH-II (commonly referred to as “chicken GnRHII”), and GnRH-III (also known as “salmon GnRH”) are the most studied isoforms of GnRH (Kochman, 2012). The structure of GnRH-II is uniquely conserved from fish to mammals, and it shows 70% similarity to the mammalian GnRH. GnRH-III was first isolated from sea lamprey (Petromyzon marinus), showing 60% homology with GnRH (Maggi et al., 2016). Since various LHRH peptide forms are present in a single species, it is not surprising that multiple LHRH gene variations exist. A single copy of LHRH gene is

FIGURE 3.14

The structure of preepro-gonadotrophin-releasing hormone (GnRH). Highlighted is the decapeptide of the active molecule GnRH (molecular weight 1181). Sites of cleavage from the gonadotrophin-associated peptide are shown, as well as the main sites of enzymatic degradation of GnRH by endo- and carboxamide peptidases in the pituitary. Adapted from Control of Hypothalamice PituitaryeOvarian Function with permission.

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3. HYPOTHALAMIC RELEASING HORMONES

FIGURE 3.15 Schematic diagram showing organization of GnRH genes. The genes encoding GnRH in the cichlid fish, Haplochromis burtoni, are shown. Within each gene, a given exon encodes a corresponding preprohormone region. Single hatched bars, signal sequence coding region; cross-hatched bars, GnRH-coding region; open bars, GnRH-associated peptide (GAP) coding region. Horizontal lines adjacent to exons represent introns. Modified from Fernald, R.D., White, R.B., 1999. Gonadotropin-releasing hormone genes: phylogeny, structure, and functions. Front. Neuroendocrinol. 20, 224e240; White, R.B., Fernald, R.D., 1998. Genomic structure and expression sites of three gonadotropin-releasing hormone genes in one species. Gen. Comp. Endocrinol. 112, 17e25 with permission.

located on the short arm of chromosome 8 (8p21-p11.2). It contains four exons and three introns (Fig. 3.15). A 92 amino acid prohormone is encoded in the LHRH gene. This prohormone first undergoes a specific enzymatic cleavage and is further modified into the secretory granules (Fig. 3.16). The LHRH prohormone consists of the following: (1) a signal peptide (23 amino acids) being responsible for intracellular packaging and secretion, (2) the LHRH decapeptide, (3) a proteolytic processing

FIGURE 3.16 Biosynthetic pathway for the synthesis of GnRH decapeptide. Adapted from Gore, A.C., Roberts, J.L. 1997. Regulation of gonadotropin-releasing hormone gene expression in vivo and in vitro. Front. Neuroendocrinol. 18, 209e245 with permission.

site that is three amino acids (Gly-Lys-Arg) long, and (4) an LHRH-associated protein (GAP) with sequence that is 56 amino acids long with so far unknown functions (Maggi et al., 2016; Fernald and White, 1999) In the adult brain, the various GnRH isoforms show different expression pattern: GnRH-I is expressed in the hypothalamus, GnRH-II in the midbraintegmentum, and GnRH-III in the forebrain telencephalon (Fernald and White, 1999). Nevertheless, the expression of LHRHs is not restricted to the brain. Outside the brain, LHRH-I expression has been also found in splenocytes and peripheral lymphocytes, in the liver, heart, skeletal muscle, kidney, placenta, and in the testis, ovary, and prostate. The expression of GnRH-II and GnRH-III was much less investigated. In addition to its expression in the brain, GnRH-II is also found in the prostate, bone marrow, kidneys, testis and ovary, while GnRH-III was reported in the testis and ovary (Fernald and White, 1999) The regulation of the LHRH genes is not yet fully understood. Little is known about the regulation of GnRH-II and GnRH-III, while transcriptional, posttranscriptional, and posttranslational regulatory mechanisms of LHRH-I were widely investigated. For example, Gore and Roberts provided an outstanding review of GnRH1 gene regulation in vitro and in vivo (Gore and Roberts, 1997). LHRH secretion is silenced in childhood, but it becomes pronounced in puberty, when sexual development requires higher production of gonadotropins and gonadal steroids (Flanagan and Manilall, 2017). As mentioned earlier, LHRH-I is a tropic peptide hormone

55

4. LUTEINIZING HORMONE-RELEASING HORMONE/GONADOTROPIN-RELEASING HORMONE

FIGURE 3.17

Structure of the GnRH receptor. Adapted from Millar, R.P., 2005. GnRHs and GnRH receptors. Anim. Reprod. Sci. 88, 5e28 with

permission.

synthesized in specialized LHRH-secreting neurons of the hypothalamus and released into the hypophyseal portal circulation in a pulsatile manner. In the anterior pituitary gland, LHRH-I interacts with membranous receptors on gonadotropes (Grosse et al., 2000; Hotchkiss and Knobil, 1994), inducing the synthesis and release of LH and FSH (Naor et al., 1998).

The binding of LHRH to LHRH receptor results in intracellular responses, leading to secretion and synthesis of LH and FSH. Activation of LHRH receptor signal is shown in Fig. 3.18. Upon binding of LHRH, the LHRH receptors on the pituitary gonadotrophs are activated

GnRh

4.1 Receptors and Signaling LHRH receptor is a member of the rhodopsin-like GPCR superfamily, having a characteristic seventransmembrane (TM) domain structure (Fig. 3.17). LHRH receptors of various mammalian species have a well-conserved structure with 85% similarity in amino acid sequence and nearly identical TM domains (Kochman, 2012). The human LHRH receptor molecule consists of 328 amino acids. Its TM domain is connected by extracellular coils and intracellular coils. Important structural elements include the residues of the binding site and binding pocket formation containing disulphide bonds and glycosylation sites (Fig. 3.17). Certain residues involved in receptor activation are highly conserved in the GPCR superfamily. Residues involved in coupling to G proteins, protein kinase C (PKC), and protein kinase A (PKA) phosphorylation sites have also been reported (Kochman, 2012). The presence of LHRH isoforms suggests that three different LHRH receptor subtypes also exist. The extracellular coil domain 3 (EC3) in the receptor is primarily responsible for receptor selectivity for LHRH variants (Schally et al., 2001a).

GnRH receptor GTP binding proteins

Diacylglycerol

PLC

Protein kinase C

Inosityltriphosphate Intracellular stores Ca2+

Adenyl cyclase complex

AMP cAMP

Ca2+

P Proteins

P

P

Proteins P

P

P

P

FIGURE 3.18 Schematic representation of signal activation of gonadotrophin-releasing hormone (GnRH)-receptor. AMP, adenosine monophosphate; cAMP, cyclic AMP; GTP, guanosine triphosphate; P, phosphate group; PLC, phospholipase-C enzyme. Adapted from Shaw, R.W., 2015. Control of HypothalamicePituitaryeOvarian Function with permission.

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3. HYPOTHALAMIC RELEASING HORMONES

within seconds. As a consequence of the membranebound phospholipase-C enzyme, inositol triphosphate is released and induces Ca2þ mobilization, initially from intracellular storage sites (e.g., endoplasmic reticulum). Then, extracellular Ca2þ also enters the gonadotroph to increase concentration of free Ca2þ to maintain continuous release of LH. Due to the action of phospholipase-C, diacylglycerol is also released, leading to the activation of the phosphorylating enzyme protein kinase C. Furthermore, the stimulation of AC complex results in the generation of cyclic adenosine monophosphate (cAMP). Altogether, the interaction of Ca2þ, protein kinase C, and cAMP stimulates the secretion of stored LH and FSH and subsequent biosynthesis (Schally et al., 2001a; Clayton, 1989; Schally and Halmos, 2012).

4.2 Therapeutic Use Over the past 30 years, more than 3000 analogs of LHRH have been synthesized. Both agonists and antagonists of LHRH can serve as potential therapeutic agents, acting directly on the target cells. Many agonistic analogs are 50e100 times more potent than LHRH itself (Schally et al., 2001a; Schally and Halmos, 2012). Agonistic analogs, such as Decapeptyl, Leuprolide, Zoladex, and Buserelin, have various important clinical applications in gynecology and oncology (Schally et al., 2001a; Schally and Halmos, 2012). Continuous exposure to LHRH agonists leads to inhibition of the gonadotropin secretion. Thus, sustained stimulation of the pituitary by chronic administration of LHRH or its superagonists, which have a higher affinity to LHRH receptors, produces inhibition of the hypophyseale gonadal axis through a process of “downregulation” of pituitary receptors for LHRH, desensitization of the pituitary gonadotrophs, and suppression of circulating levels of LH and sex steroids (Schally et al., 2001a; Schally and Halmos, 2012). Downregulation of LHRH receptors, produced by repeated administration or depot preparation of LHRH agonists, provides the basis for clinical applications in gynecology and oncology of this class of compounds. Treatment of central precocious puberty, polycystic ovarian syndrome, and some aspects of the use for in vitro fertilization and embryo transfer (IVF-ET) programs and controlled ovarian stimulationassisted reproductive technology (COS-ART) are based on the suppression of gonadotropin secretion (selective medical hypophysectomy) (Schally et al., 2001a). Treatment of sex hormone-dependent malignant neoplasms, represented by prostate and breast cancer, endometrial carcinoma, and other diseases or medical conditions,

such as benign prostate hyperplasia, preoperative treatment of large uterine leiomyomas, and endometriosis, is based on the reversible medical castration and the creation of a state of sex steroid deprivation (Schally et al., 2001a; Schally et al., 2001a; Schally and Halmos, 2012). In contrast, antagonists of LHRH produce a competitive blockade of LHRH receptors, preventing a stimulation by endogenous LHRH and leading to an immediate cessation of the secretion of gonadotropins and sex steroid. The LHRH antagonists reduce the time of the onset of therapeutic effects compared to the agonists (Schally et al., 2001a; Engel and Schally, 2007; Schally and Halmos, 2012). Potent antagonists of LHRH, such as Cetrorelix, Ganirelix, Abarelix, and Degarelix, have been developed and are now available for clinical use in gynecology, especially in COS-ART and oncology (Schally et al., 2001a; Engel and Schally, 2007; Schally and Halmos, 2012). The receptors for LHRH on human tumors can also serve as targets for LHRH analogs linked to cytotoxic agents. The cytotoxic analog AN-152 (AEZS-108, zoptarelin doxorubicin), consisting of [D-Lys6]-LHRH linked through a glutaric acid spacer to doxorubicin (DOX) was designed for receptor-mediated chemotherapy aimed at the inhibition of the growth of tumors expressing LHRH receptors (Nagy et al., 1996). This analog has been extensively investigated in a large number of experimental studies (Schally et al., 2001a; Schally and Halmos, 2012) and also tested in Phase I/II clinical trials in castration-resistant prostate cancer and in Phase II and III trials in endometrial and ovarian cancers (Emons et al., 2010; Schally and Halmos, 2012). Regrettably, clinical trials with zoptarelin doxorubicin in various human cancers were terminated because these studies did not achieve their primary endpoint.

5. SOMATOSTATIN (SOMATOTROPHIN RELEASE-INHIBITING FACTOR) Somatostatin (SST) is a hormonal neuropeptide that was isolated from ovine and later from porcine hypothalami (Brazeau et al., 1973; Schally, 1978). This tetradecapeptide, discovered in 1973, was named originally hypothalamic growth hormone (GH) inhibiting factor (Brazeau et al., 1973; Schally, 1978). Brazeau et al. proved that somatostatin inhibits secretion of immunoreactive rat or human GHs in vitro at 109 M concentration and has similar activity in vivo in rats (Brazeau et al., 1973). This peptide discovered as a GH releaseinhibitory substance in the hypothalamus was subsequently characterized as a cyclic peptide consisting of 14 amino acids. Its structure in ovine and porcine

5. SOMATOSTATIN (SOMATOTROPHIN RELEASE-INHIBITING FACTOR)

FIGURE 3.19 Somatostatin structure. The two somatostatin isoforms in humans are depicted on the left side. Somatostatin (SST) is a cyclic peptide of 14 (SST-14) or 28 (SST-28) amino acids. Essential functional groups of the SST peptide with high-binding affinity to SSTRs were detected using the alanine scanning technology. SRL, somatostatin receptor ligand; SST, somatostatin; SSTR, somatostatin receptor (Cuevas-Ramos and Fleseriu, 2014).

hypothalami is H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-TrpLys-Thr-Phe-Thr-Ser-Cys-OH (Brazeau et al., 1973; Schally, 1978). Somatostatin was named for its ability to inhibit pituitary GH release, and it is also called growth hormone-inhibiting hormone or somatotropin release-inhibiting factor (SRIF). Biologically active somatostatin exits in two molecular forms: somatostatin14 (SST-14) (Brazeau et al., 1973) and an amino terminally extended version consisting of 28 amino acids, somatostatin-28 (SST-28) (Schally, 1978; Schally et al., 2001a; Schally and Halmos, 2012; Fig. 3.19). A disulfide bond between cysteine residues maintains the cyclic structure. Somatostatin-14 consists of carboxyl terminal 14 amino acids of somatostatin-28. Both SST-14 and SST-28 exhibit very similar biologic effect on a wide variety of cells and appear to be endogenous growth inhibitors. The two forms are produced from a precursor encoded by the somatostatin 11 gene (Antonio Blanco, 2017). SST-14 is the predominant form in the brain, hypothalamus, and the pancreas, while SS-28 is mainly produced by intestinal enteroendocrine cells (Antonio Blanco, 2017). Six SST genes have been identified and reported in vertebrates, and two SST-related peptide analogs, cortistatin and neuronostatin, are derived from SST family genes. Somatostatin exhibits neuroendocrine, neuromodulatory, and gastrointestinal actions through binding to SST receptors that belong to the family of GPCRs (Yoshio Takei and Tsutsui, 2015). The biologic activity of

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SST-14 and SST-28 resides in the cyclic region of the mature peptide. The Phe-Trp-Lys-Thr amino acid sequence of the ring structure is required for receptor binding and occupancy. Somatostatin is produced by paracrine cells that are scattered throughout the gastrointestinal tract (GI) and inhibits gastrointestinal endocrine secretion. Somatostatin is also found in various locations in the central and peripheral nervous system and exerts neural control over many physiologic functions. Besides being produced in the hypothalamus, somatostatin is also produced by the d cells of the pancreas islets, enterocytes, and thyroid parafollicular C cells (Antonio Blanco, 2017). Both forms of somatostatin are present in the GI tract and inhibit the secretion of many hormones including GH, thyrotropin, corticotropin, insulin, glucagon, gastrin, secretin, cholecystokinin (CCK), and renal renin under physiologic and pathologic conditions. Somatostatin is also present in the gastric mucosa, pancreas, and duodenum, where it suppresses exocrine secretions including gastric acid, pancreatic enzymes, and bicarbonate, respectively (Schally et al., 2001a, 2004; Schally and Halmos, 2012). The action of SST is often targeted at the same tissue in which the peptide is located. Thus, in addition to its endocrine effects, SST can also serve as an autocrine/paracrine regulator, and SST present in discrete cells of the pancreas, gastric mucosa, and duodenum may, through paracrine control, regulate the endocrine pancrease and the GI tract (Schally et al., 2001a, 2004; Schally and Halmos, 2012). Somatostatin secretion occurs in response to a variety of stimuli. Meal ingestion and gastric acid secretion increase the output of somatostatin from gastric D cells. Production of gut somatostatin is regulated by the autonomic nervous system with catecholamines inhibiting and cholinergic mediators stimulating peptide release (Yamada et al., 1984). Somatostatin is also a paracrine transmitter that is released from somatostatincontaining D cells and acts on adjacent cells. However, somatostatin is also released into the blood after a meal and considerable work has been done to characterize the role of the circulating somatostatin levels. Administration of exogenous somatostatin inhibits secretion of pancreatic bicarbonate stimulated by a meal, intestinal perfusion of amino acids, sodium oleate, HCl, or infusion of secretin and CCK (Said, 2018). Somatostatin has only a short, 2-min half-life in tissue and in blood. Its concentration in the serum is low, generally in subpicomolar amounts. After intravenous administration, 50% of the peptide is removed from the circulation in less than 3 min. Because of the short half-life of somatostatin-14, as a result of intense research, several more stable and more potent synthetic

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FIGURE 3.20

3. HYPOTHALAMIC RELEASING HORMONES

Therapeutic uses of somatostatin and its analogs: Current view and potential applications (Adapted from Rai et al., 2015).

somatostatin analogs have been developed, including octreotide (Sandostatin), vapreotide (RC-160, Octastatin), and lanreotide (Somatulin) (Schally et al., 2001a, 2004; Schally and Halmos, 2012; Fig. 3.20). These analogs have a longer plasma half-life of about 120 min and are about 50 times more potent than somatostatin in inhibiting GH release from the pituitary. Recombinant SST is a recombinant peptide chemically identical or similar to endogenous somatostatin. Somatostatin and its octapeptide analogs exert their effects through specific GPCRs that are widely distributed in normal and cancerous cells (Schally et al., 2001a, 2004; Schally and Halmos, 2012).

5.1 Receptors and Signaling Somatostatin and its octapeptide analogs exert their effects through specific GPCRs that are widely distributed in normal and cancerous cells. At least five distinct somatostatin receptor subtypes, designated (sst1e5; Fig. 3.21), have been cloned and characterized (Patel, 1997; Reisine and Bell, 1995). They share a common molecular topology including a hydrophobic core of seven transmembrane-spanning a-helices, three intracellular loops, three extracellular loops, an amino-terminus outside the cell, and a carboxyl-terminus inside the cell. For most of the GPCRs, intracytosolic sequences, and more particularly the C-terminus, are believed to interact with proteins. It was reported that these proteins are mandatory for either exporting neosynthesized receptor, anchoring receptor at the plasma membrane, internalization, recycling, or degradation after ligand binding (Csaba et al., 2012). These five subtypes of somatostatin receptors are encoded by five genes that were mapped to chromosomes 14, 17, 22, 20, and 16, respectively (Patel, 1997; Reisine and Bell, 1995; Guillermet-Guibert et al., 2005). The sst2 gene generates two splice variants, sst2A and sst2B, which

have a shorter cytoplasmic domain. Most of the SRIF receptors can traffic not only in vitro within different cell types but also in vivo. While native somatostatin shows similar high affinity to sst1e5, the synthetic octapeptides such as octreotide or vapreotide bind preferentially to sst2 and sst5, display moderate affinity to sst3, and a low affinity to sst1 and sst4 (Schally et al., 2001a, 2004; Reisine and Bell, 1995). These SST receptors are widely distributed in normal and malignant tumor tissues, with cells very often expressing more than one subtype of SST receptors. All somatostatin receptor subtypes have been found in the brain. In contrast, peripheral tissues vary in the subtype expressed. All subtypes of the somatostatin receptor are coupled to adenylate cyclase through an inhibitory G protein. Activation of receptor results in a reduction of cAMP accumulation. In addition, occupancy of some receptor subtypes by agonists couples to activation of ion channels (e.g., potassium channels) and mobilization of intracellular calcium. It was also reported that vapreotide (RC-160) stimulates tyrosine phosphatase activity and inhibits the proliferation of the cells expressing the sst2 gene. Thus, tyrosine phosphatase might be a transducer of the growth inhibition signal (Buscail et al., 1994). Previous studies also show that in sst5-expressing cells, the phosphatase pathway is not involved in the mechanism of action of vapreotide on cell growth, and the phosphoinositide/calcium pathway could be implicated in the inhibitory effect (Buscail et al., 1994; Schally et al., 2001a). Certain agonists exhibit functional selectivity at individual somatostatin receptors and activate only a portion of the receptor’s potential effects. This property is known as selective agonism and has been described with other GPC receptors. This may make it possible to develop agonists with various somatostatin-like activities. Somatostatin receptors can interact with each other and form dimers. Agonist-induced receptor dimerization changes ligand

5. SOMATOSTATIN (SOMATOTROPHIN RELEASE-INHIBITING FACTOR)

59

FIGURE 3.21 Schematic depiction of the seven-transmembrane topology of the human sst1e5 receptors. CHO are the potential sites for Nlinked glycosylation within the amino terminal segment and second extracellular loops (ECL); PO4 are the putative sites for phosphorylation by protein kinase A, protein kinase C, and casein kinase. The cysteine residue 12 amino acids downstream from the VIIth TM is conserved in sst1,2,4,5 and may be the site of a potential palmitoyl membrane anchor: The YANSCAN PI/VLY sequence in the VIIth TM is highly conserved in all members of the sst family. Residues Asp122, Asn276, and Phe294 in TMs III, VI, and VII, respectively, of sst2A have been proposed to form part of a ligand-binding pocket for octreotide and are shown by the closed circles. From Patel, Y.C., Srikant, C.B., 1997. Somatostatin receptors. With permission of Trends Endocrinol. Metab. 8, 398e405.

binding affinity and receptor internalization. Different receptor types can interact with the somatostatin receptor (receptor heterodimerization), thus providing a novel pathway for cellular communication. Cross-talk between somatostatin receptors and growth factor receptors of the receptor tyrosine kinase family may be important in cancer (Kumar, 2011). Pasireotide is a new SST analog developed for the treatment of acromegaly, neoroendocrine tumors, and Cushing disease (Fig. 3.22). It is a small, stable cyclohexapeptide with high affinity for sst5 > sst2 > sst3 > sst1 and no affinity

FIGURE 3.22 Chemical structure of pasireotide (Cuevas-Ramos and Fleseriu, 2014).

for sst4. This receptor binding affinity profile is closer to that of endogenous SST, which suggests that pasireotide could be a better therapeutic analog than either ocreotide or lanreotide, which selectively bind sst2. Pasireotide has a 39-, 30-, and 5-fold higher binding affinity for sst5, sst1, and sst3 respectively, and 2.6 times lower affinity for sst2 compared with ocreotide (Bruns et al., 2002; Schmid, 2008; Lewis et al., 2003; Golor et al., 2012).

5.2 Therapeutic Use The development of synthetic analogs of SST such as octreotide, lanreotide, vapreotide, and pasireotide has led to treatment of clinical disorders such as acromegaly, hormone-secreting neuroendocrine tumors of the gastrointestinal tract (VIPoma, carcinoid tumors, insulinomas, gastrinomas, glucagonomas), thyrotroph adenoma, and portal hypertensive bleeding. Consequently, attempts have been made to use modern SST analogs for the therapy of human cancers, such as prostatic, breast, pancreatic, and lung. However, clinical trials in prostatic, breast, and lung cancers were not successful, and relevant palliative benefits have been obtained only in experimental hepatocellular carcinoma (Schally et al., 2004; Schally and Halmos, 2012). Pediatric and neonatal use of octreotide is also documented for the treatment of refractory hyperinsulinemic hypoglycemia

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and also adjunctive treatment of congenital and postoperative chylothorax (Yin et al., 2017). Synthetic analogs are also used for diagnostic imaging, as the neuroendocrine tumors express somatostatin receptors on their cell surface. Radiolabeled analogs of SST, such as [111InDTPA-D-Phe1]-octreotide (OctreoScan), are most commonly used clinically for the localization of tumors expressing receptors for SST. It was reported that various primary tumors and their metastases, both neuroendocrine or nonneuroendocrine, can be localized in vivo using SST receptor scintigraphy (Krenning et al., 1993). Various neuroendocrine tumors that could be localized with OctreoScan include pituitary tumors, gastrinomas, insulinomas, glucagonomas, medullary thyroid carcinoma, neuroblastomas, carcinoids, and small cell lung carcinomas (SCLC). Nonneuroendocrine tumors that could be visualized by tumor scintigraphy included non-SCLC, meningiomas, breast cancer, and astrocytomas. A positive scintigram may also predict a good response to treatment with octreotide (Krenning et al., 1993). Targeted radiotherapy, in which somatostatin analogs are linked to various radionuclides such as 68gallium or 90yttrium, is also being developed (Schally et al., 2004; Schally and Halmos, 2012). These SST analogs linked to radionuclides appear to have a great therapeutic potential. In the past 2 decades, the molecular mechanism of the antitumor activity of somatostatin has been extensively investigated. These antitumor actions of somatostatin and its analogs appear to be exerted through both direct and indirect mechanisms (Schally et al., 2004; Schally and Halmos, 2012). The presence of receptors for SST on various neuroendocrine malignancies and many other solid tumors also serves as a rationale to use SST octapeptides as carriers to deliver cytotoxic agents specifically to cancerous cells. Thus, in an endeavor to develop chemotherapy targeted to somatostatin receptors, a series of novel cytotoxic analogs of somatostatin have been synthesized, developed, and tested (Nagy et al., 1998). These cytotoxic hybrids of somatostatin-containing DOX or its superactive derivative, 2-pyrrolino-DOX (AN-201) were conjugated to octapeptide analogs RC-121 or RC-160 (Nagy et al., 1998). The oncologic use of cytotoxic SST analogs were reported in several experimental models in vitro and in vivo including carcinoma of the prostate, breast, endometrial, and epithelial ovarian cancers, renal cell carcinoma, bladder cancer, brain tumors, melanomas, SCLC and non-SCLC, hepatocellular carcinoma, nonHodgkin lymphoma, pancreatic and gastric cancers (Schally et al., 2004; Schally and Halmos, 2012). These new classes of SST analogs could lead to a more effective treatment for various human cancers.

6. GROWTH HORMONE-RELEASING HORMONE GHRH is a hypothalamic peptide secreted by the hypothalamus and stimulates the synthesis and the release of GH in the pituitary. GHRH also stimulates the synthesis and proliferation of pituitary somatotroph cells. GHRH is initially synthesized as a larger preprohormone precursor of 108 amino acids whose N-terminal signal sequence is then enzymatically cleaved to generate the “mature” 44-amino acid form of GHRH and a C-terminal GHRH-related peptide (GHRH-RP) (Alba and Salvatori, 2004). GHRH is a member of a superfamily of brain-gut peptides, which also includes, among others, pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon, secretin, and VIP. GHRH-containing neurosecretory cells originate primarily from the arcuate nucleus of the hypothalamus. The axons of these GHRH neurons project to the ME and terminate on the capillaries of the hypophyseal portal system to stimulate GH release. GHRH was also found in the limbic system structures, cerebral cortex, and hindbrain region, as well as in the peripheral nervous system, gastrointestinal (GI) tract, pituitary, gonads, adrenal, thyroid, lung, and kidney (Fridlyand et al., 2016). GHRH, GHRH receptors, and the expression of GHRH gene are also present in diverse extrahypothalamic tissues including the gastrointestinal tract, placenta, ovary, testis, and many tumors (Schally et al., 2001a, 2008; Schally and A, 2006; Barabutis and Schally, 2010; Schally and Halmos, 2012). After the discovery of an ectopic production of GHRH in pancreatic cancers by Frohman and Szabo (Frohman and Szabo, 1981), the 44- and 40-amino acid forms of GHRH were isolated and identified from human carcinoid and pancreatic tumors (Guillemin et al., 1982; Schally et al., 2008; Rivier et al., 1982) and only subsequently purified from hypothalamic tissue (reviewed in Schally and A, 2006; Schally et al., 2001a, 2008; Barabutis and Schally, 2010; Schally and Halmos, 2012). Subsequently, the cDNA and/or gene of GHRH was isolated and characterized in rat, mouse, and other mammals, as well as in birds, amphibians, and fish (reviewed in Schally et al., 2001a, 2008; Schally and Halmos, 2012). The full biologic activity is contained in the N-terminal 29 amino acid sequence of [GHRH(1-29)NH2] (Schally et al., 2001a, 2008; Barabutis and Schally, 2010; Schally and Halmos, 2012). GHRH is secreted in a pulsatile manner about every 3 h and more frequently during sleep. The pulsatile release of GHRH is age dependent, and there is also a sexually dimorphic pattern, particularly at puberty, when GHRH function becomes less responsive to testosterone, and the feedback of GH on GHRH neurons also declines

6. GROWTH HORMONE-RELEASING HORMONE

considerably with aging. GHRH also can regulate sleep; specifically, it can increase the duration of slow wave sleep while inhibiting the secretion of ACTH and cortisol (Mayo et al., 1995; Lin-Su and Wajnrajch, 2002). GHRH undergoes rapid enzymatic degradation in blood and can be inactivated by dipeptidylpeptidase IV with the removal of the N-terminal peptide (Try-Ala) and cleavage of GHRH at positions 2 and 3, making inactive the GHRH fragment (3-44)-NH2 (Frohman et al., 1989). Initially, the role of GHRH has been thought to be the regulation of physiologic levels of growth hormone and insulin-like growth factor 1 (IGF-1) through the pituitary GH/hepatic IGF-1 axis. Accumulating evidence also suggests that, in addition to the neuroendocrine action of GHRH, extrahypothalamic GHRH has been implicated in many peripheral actions through autocrine/paracrine mechanisms (Fig. 3.23). GHRH and its agonists can regulate cell proliferation and survival, wound healing, cardiac repair, heart function, diabetic retinopathy, type 1 diabetes, apoptosis, and differentiation in several tissues and cell types, as reported in various studies (Fridlyand et al., 2016; Cui et al., 2016; Kiaris et al., 2011; Gesmundo et al., 2017; Schally and Halmos, 2012; Kanashiro-Takeuchi et al., 2012; Zhang et al., 2015). The expression of mRNA for GHRH and the presence of biologically or immunologically active GHRH has also been demonstrated in various human tumors, including those of the breast, endometrium, ovary, prostate, lung, pancreas, stomach, colon, and uveal melanoma (Schally et al., 2001a, 2008; Barabutis and Schally, 2010; Schally and Halmos, 2012).

6.1 The Gene Mayo et al. (1985) isolated and characterized overlapping clones from phage lambda and cosmid human genomic libraries that predict the entire structure of the gene encoding GHRH (Mayo et al., 1985). Dot blot analysis of DNA from high-resolution dual-laser-sorter human chromosomes also indicated that the GHRH gene is located on chromosome 20 and spans over 10 kilobases (Mayo et al., 1985). The gene has five exons and four introns. The GHRH sequence has an arginine (Arg) residue at the terminal end that serves as a signal for proteolytic processing (Mayo et al., 1985; Schally and Halmos, 2012).

6.2 GH-RH Receptor and GHRH Receptor Gene Pituitary type GHRH receptor is a seventransmembrane GPCR that, in rat and human, is 423 amino acids in length. The GHRH receptor gene is

61

FIGURE 3.23 Hypothalamic-pituitary regulation of growth hormone (GH). GH is produced and secreted by the somatotroph cells of the AP under the influence of two hypothalamic hormones. GHRH stimulates GH and SS inhibits GH secretion. Ghrelin also stimulates GH secretion from the AP via its own receptor. GH exerts direct metabolic effects on target tissues and exerts its growth effects through IGF-I, which is produced primarily by the liver. Additional regulation of GH secretion is achieved through feedback control by IGF-I and GH at the pituitary and at the hypothalamus. AP, anterior pituitary; GHRH, growth hormone-releasing hormone; IGF-I, insulin-like growth factor type I; SS, somatostatin. With permission from Lin-Su, K., Wajnrajch, M.P., 2002. Growth Hormone Releasing Hormone (GHRH) and the GHRH receptor. Rev. Endocr. Metab. Disord. https://doi.org/10.1023/A: 1020949507265.

localized to human chromosome 7p 14e15 and rat chromosome 4p24 and a member of the class II B of GPCRs, which includes the secretin/glucagon/VIP/PACAP peptide receptors. Alternative splicing in GPCRs is one of many emerging mechanisms by which this class of receptors diversifies its activities (Fridlyand et al., 2016; Lin-Su and Wajnrajch, 2002; Gaylinn, 2006). Characterization of the genomic sequence has revealed that the human GHRH receptor gene consists of 13 exons and spans 15 kilobases (Lin-Su and Wajnrajch, 2002; B., 2006; Fig. 3.24).

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FIGURE 3.24 Structure and translation of human GHRH. With permission from Lin-Su, K., Wajnrajch, M.P., 2002. Growth Hormone Releasing Hormone (GHRH) and the GHRH receptor. Rev. Endocr. Metab. Disord. https://doi.org/10.1023/A:1020949507265.

GHRH receptors are primarily expressed in the pituitary gland and, using in situ hybridization, have been localized to the anterior pituitary. Levels of GHRH receptor-expression in the pituitary vary with different developmental stages. GHRH receptors diminish after aging. GHRH receptors are also present in several other human tissues, such as myocardium, lymphocytes, testes, ovaries, skin, and pancreas and are involved in a variety of physiologic and pathophysiological effects and biologic processes (Kiaris et al., 2011; Schally and Halmos, 2012). GHRH receptor mRNA has also been detected in the placenta and the kidney (Lin-Su and Wajnrajch, 2002; Schally and Halmos, 2012). Hormonal activities of GHRH and its analogs are mediated by pituitary type GHRH receptor (pGHRHR) (Lin-Su and Wajnrajch, 2002; Schally et al., 2008; Schally and Halmos, 2012). However, our group has also identified a splice variant of GHRH receptors in human tumors and other extrapituitary tissues that can mediate the effects of GHRH and its agonistic and antagonistic analogs (Barabutis and Schally, 2010; Schally and Halmos, 2012). Isolation and sequencing of cDNAs encoding tumoral GHRH receptors revealed several splicing variants of the pGHRH-R (Rekasi et al., 2000). The major part of the cDNA sequence for the splicing variant 1 (SV1) is identical with the corresponding cDNA sequence of the pGHRH-R, but the first 334 nucleotides of SV1 and SV2 genes are different from those of the pGHRH-R gene. The deduced protein sequence of SV1 differs from that of pGHRH-R only in the N-terminal extracellular domain, the first 89 amino acids of pGHRH-R being replaced by a 25-amino acid sequence. It was also reported that SV1 encodes a

functional GPCR with seven-transmembrane domains, with the third intracellular loop critical for interaction with G protein. SV2 might encode a GHRH-R isoform truncated after the second transmembrane domain (Rekasi et al., 2000; Schally and Halmos, 2012). Based on these studies, SV1 appears to be the major isoform of GHRH receptors. Subsequent studies showed that SV1 is a functional receptor. Both pGHRH-R and SV1 have been identified in normal and neoplastic human tissues, where they mediate GHRH signaling. Thus, the expression of GHRH receptors has been found in primary human prostatic, breast, endometrial, lung, and adrenal carcinomas and uveal melanomas, as well as in experimental human cell lines of virtually all major types of malignancies, including prostatic, ovarian, breast, endometrial, lung (SCLC and non-SCLC), colorectal, gastric, pancreatic, renal, glioblastomas, osteogenetic and Ewing sarcomas, lymphomas, and uveal melanomas (Schally and Halmos, 2012; Schally et al., 2008; Barabutis and Schally, 2010; Kiaris et al., 2011). Collectively, these findings suggest that in various human tumors, GHRH and its tumoral receptors might form an autocrine/paracrine mitogenic loop involved in the control of malignant growth.

6.3 Signal Transduction After binding of GHRH to the GHRH receptors, the occupied GHRH receptor activates a G protein by catalyzing the binding of guanosine 50 -triphosphate (GTP) to the a-subunit on the intracellular side. Activation of these receptors results in increased GH production, mainly by intracellular cyclic 30 ,50 - adenosine

6. GROWTH HORMONE-RELEASING HORMONE

63

FIGURE 3.25 Mechanism of action of GHRH on the pituitary somatotrope to increase synthesis and secretion of GH. Binding of GHRH to its receptor activates a stimulatory G protein, which arouses adenylyl cyclase to increase cAMP. Increased cAMP levels augment intracellular calcium, which induces GH release. Increased cAMP also stimulates protein kinase A (PKA) to activate cAMP response element binding protein (CREB), which raises GH1 and GHRHR gene transcription. With permission from Lin-Su, K., Wajnrajch, M.P., 2002. Growth Hormone Releasing Hormone (GHRH) and the GHRH receptor. Rev. Endocr. Metab. Disord. https://doi.org/10.1023/A:1020949507265.

monophosphate (cAMP) dependent pathway, and also by the phospholipase C pathway (IP3/DAG pathway), and some other minor pathways. This causes stimulation of membrane-bound adenylyl cyclase and increased cAMP. The ensuing process produces the opening of a sodium channel, leading to its depolarization. In turn the resultant change in the intracellular voltage opens a voltage-gated Ca2þ, allowing the influx of calcium, which directly causes the release of GH, stored in secretory granules (Lin-Su and Wajnrajch, 2002; B., 2006; Fridlyand et al., 2016; Cui et al., 2016; Fig. 3.25). cAMP also binds to and activates the regulatory subunits of protein kinase A (PKA), allowing the free catalytic subunits to translocate to the nucleus and to phosphorylate and activate the transcription factor CREB protein. Phosphorylated CREB, together with its coactivators, p300 and CREB binding protein, enhances the transcription of GH by binding to CREs cAMP response elements in the promoter region of the GH gene. CREB also stimulates GHRH receptor gene transcription. In the phospholipase C pathway, GHRH stimulates phospholipase C (PLC) through the bg-complex of heterotrimeric G proteins. Activation of PLC produces both diacylglycerol (DAG) and inositol triphosphate (IP3), IP3 induces the

release of intracellular Ca2þ from the endoplasmic reticulum, increases cytosolic Ca2þ concentration, resulting in fusion of vesicles and the release of secretory vesicles containing premade GH. Some Ca2þ influx is also produced by direct action of cAMP, which is distinct from the cAMP-dependent pathway for activating protein kinase A. Upon binding of the GHRH ligand to the GHRH receptor, the activated second messengers include not only the adenylate cyclaseecAMP-PKA and Ca2þcalmodulin but also inositol phosphatee diacylglyceroleprotein kinase C (PKC), L-type calcium channels, and arachidonic acideeicosanoic pathways as well. The final result is the stimulation of the production and secretion of GH. The activation of MAP kinase and ERK phosphorylation has also been reported in the pituitary in a cAMP/PKA/PKC-dependent manner. GHRH can stimulate the Ras/MAPK through bg-subunits, to promote cell growth. Some studies also show that in the myocardium inhibition of apoptosis, mediated by GHRH receptor, involves modulation of ERK1 and ERK2 and PI3KAkt signaling. This became apparent because ERK1/2- and PI3K/Akt-specific inhibitors abolish these effects (Lin-Su and Wajnrajch, 2002; Fridlyand et al., 2016).

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6.4 Disease and Therapy

Acknowledgements

In the endeavor to explore novel methods for treatment of cancer and other malignancies, a large number of potent analogs of GHRH have been designed, synthetized, and developed. Over the past 2 decades, many advanced and powerful agonists and antagonists of GHRH with high receptor affinity have been developed (Schally et al., 2001a, 2008; Barabutis and Schally, 2010; Schally and Halmos, 2012; Cai et al., 2014; Zarandi et al., 2017). GHRHR antagonists strongly augment apoptosis and decrease proliferation of multiple types of cancer cells in vitro and in vivo. The tumor inhibitory effects of GHRH antagonists in 16 types of human cancers, represented by nearly 50 human cancer lines including prostatic, breast, ovarian, renal, gastric, pancreatic, lung, and recently, acute myeloid leukemia appear to be based in part on the interference with the local stimulatory GHRH system. Thus, GHRH antagonists can directly block the tumoral receptors for GHRH and prevent the activation of the autocrine/paracrine GHRH in cancers. In addition, the production of tumoral IGF-I and IGF-II also appears to be inhibited directly by GHRH antagonists. The antitumor action of GHRH antagonists can be also indirect and exerted through the inhibition of GH secretion from the pituitary. This mechanism causes the suppression of the pituitary GH/hepatic IGF-1 axis (Schally et al., 2001a, 2008; Barabutis and Schally, 2010; Schally and Halmos, 2012). GHRH antagonists also have beneficial effects on cognition, reduction of amyloid plaque and Tau filaments and inflammation in the most advanced transgenic mouse model of Alzheimer disease (Jaszberenyi et al., 2012). In addition, we have reported reduction of dyslipidemia in rats with MIA-602 class of GHRH antagonists (Romero et al., 2016). Promising and remarkable results have also been reported on the agonists of GHRH. Therapeutic effects of new GHRH agonists include cardioprotective action, regeneration of the heart after an infarct, protection from pneumolysin-induced pulmonary permeability edema, reduction in vascular calcification, and wound healing (Barabutis and Schally, 2010; Kiaris et al., 2011; Kanashiro-Takeuchi et al., 2015; Cui et al., 2016; Gesmundo et al., 2017; Cai et al., 2014). Recently, it was also reported that GHRH and its agonistic analogs can attenuate cardiac hypertrophy, and they are able to improve heart function in pressure overload-induced heart failure (Gesmundo et al., 2017). Beneficial effects of GHRH agonists in diabetes mellitus include the stimulation of pancreatic islets and insulin production and protective action in early experimental diabetic retinopathy (Fridlyand et al., 2016; Zhang et al., 2015). Both the agonists and antagonists of GHRH appear to be devoid of detectable side effects.

Some early experimental studies on TRH, LHRH, and somatostatin originating in one of our laboratories (A.V.S.) and cited in this chapter were supported by various US Public Health Services Grants. Since 1962, this work of the A.V.S. group was mainly supported by the Medical Research Services of the Department of Veterans Affairs. The chapter is also supported by the GINOP-2.3.2-15-2016-00043 project (G.H.) and by the Higher Education Institutional Excellence Program of the Ministry of Human Capacities in Hungary, within the framework of the Biotechnology Thematic Program of the University of Debrecen (20428-3/2018/FEKUTSTRAT) (G.H.). These projects are cofinanced by the European Union and the European Regional Development Fund.

References Alba, M., Salvatori, R., 2004. A mouse with targeted ablation of the growth hormone-releasing hormone gene: a new model of isolated growth hormone deficiency. Endocrinology 145, 4134e4143. Anderson, M.S., Bowers, C.Y., Kastin, A.J., Schalch, D.S., Schally, A.V., Snyder, P.J., Utiger, R.D., Wilber, J.F., Wise, A.J., 1971. Synthetic thyrotropin-releasing hormone. A potent stimulator of thyrotropin secretion in man. N. Engl. J. Med. 285, 1279e1283. Antonio Blanco, G.B., 2017. Medical Biochemistry. Academic Press. Arbiser, J.L., Morton, C.C., Bruns, G.A., Majzoub, J.A., 1988. Human corticotropin releasing hormone gene is located on the long arm of chromosome 8. Cytogenet. Cell Genet. 47, 113e116. Barabutis, N., Schally, A.V., 2010. Growth hormone-releasing hormone: extrapituitary effects in physiology and pathology. Cell Cycle 9, 4110e4116. Bilek, R., 2000. TRH-like peptides in prostate gland and other tissues. Physiol. Res. 49 (Suppl. 1), S19eS26. Bjoro, T., Sand, O., Ostberg, B.C., Gordeladze, J.O., Torjesen, P., Gautvik, K.M., Haug, E., 1990. The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells. Biosci. Rep. 10, 189e199. Bole-Feysot, C., Goffin, V., Edery, M., Binart, N., Kelly, P.A., 1998. Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr. Rev. 19, 225e268. Boler, J., Enzmann, F., Folkers, K., Bowers, C.Y., Schally, A.V., 1969. The identity of chemical and hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl-proline amide. Biochem. Biophys. Res. Commun. 37, 705e710. Bowers, C.Y., Schally, A.V., Enzmann, F., Boler, J., Folkers, K., 1970. Porcine thyrotropin releasing hormone is (pyro)glu-his-pro(NH2). Endocrinology 86, 1143e1153. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., Guillemin, R., 1973. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179, 77e79. Bruhn, T.O., Rondeel, J.M., Bolduc, T.G., Jackson, I.M., 1994. Thyrotropin-releasing hormone (TRH) gene expression in the anterior pituitary. I. Presence of pro-TRH messenger ribonucleic acid and pro-TRH-derived peptide in a subpopulation of somatotrophs. Endocrinology 134, 815e820. Bruhn, T.O., Huang, S.S., Vaslet, C., Nillni, E.A., 1998. Glucocorticoids modulate the biosynthesis and processing of prothyrotropin releasing-hormone (proTRH). Endocrine 9, 143e152. Bruns, C., Lewis, I., Briner, U., Meno-Tetang, G., Weckbecker, G., 2002. SOM230: a novel somatostatin peptidomimetic with broad somatotropin release inhibiting factor (SRIF) receptor binding and a unique antisecretory profile. Eur. J. Endocrinol. 146, 707e716.

REFERENCES

Bulant, M., Roussel, J.P., Astier, H., Nicolas, P., Vaudry, H., 1990. Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates a biologically active peptide, prepro-TRH-(160-169), which regulates TRH-induced thyrotropin secretion. Proc. Natl. Acad. Sci. U. S. A. 87, 4439e4443. Burgus, R., Dunn, T.F., Desiderio, D., Guillemin, R., 1969. [Molecular structure of the hypothalamic hypophysiotropic TRF factor of ovine origin: mass spectrometry demonstration of the PCA-His-Pro-NH2 sequence]. C R Acad. Sci. Hebd Seances Acad. Sci. D 269, 1870e1873. Buscail, L., Delesque, N., Esteve, J.P., Saint-Laurent, N., Prats, H., Clerc, P., Robberecht, P., Bell, G.I., Liebow, C., Schally, A.V., et al., 1994. Stimulation of tyrosine phosphatase and inhibition of cell proliferation by somatostatin analogues: mediation by human somatostatin receptor subtypes SSTR1 and SSTR2. Proc. Natl. Acad. Sci. U. S. A. 91, 2315e2319. Cai, R., Schally, A.V., Cui, T., Szalontay, L., Halmos, G., Sha, W., Kovacs, M., Jaszberenyi, M., He, J., Rick, F.G., Popovics, P., Kanashiro-Takeuchi, R., Hare, J.M., Block, N.L., Zarandi, M., 2014. Synthesis of new potent agonistic analogs of growth hormonereleasing hormone (GHRH) and evaluation of their endocrine and cardiac activities. Peptides 52, 104e112. Cardoso, J.C., Bergqvist, C.A., Felix, R.C., Larhammar, D., 2016. Corticotropin-releasing hormone family evolution: five ancestral genes remain in some lineages. J. Mol. Endocrinol. 57, 73e86. Christian, H.C., Chapman, L.P., Morris, J.F., 2007. Thyrotrophinreleasing hormone, vasoactive intestinal peptide, prolactinreleasing peptide and dopamine regulation of prolactin secretion by different lactotroph morphological subtypes in the rat. J. Neuroendocrinol. 19, 605e613. Clayton, R.N., 1989. Gonadotrophin-releasing hormone: its actions and receptors. J. Endocrinol. 120, 11e19. Contoreggi, C., 2015. Corticotropin releasing hormone and imaging, rethinking the stress axis. Nucl. Med. Biol. 42, 323e339. Csaba, Z., Peineau, S., Dournaud, P., 2012. Molecular mechanisms of somatostatin receptor trafficking. J. Mol. Endocrinol. 48, R1eR12. Cuevas-Ramos, D., Fleseriu, M., 2014 Jun. Somatostatin receptor ligands and resistance to treatment in pituitary adenomas. J. Mol. Endocrinol. 52 (3), R223eR240. https://doi.org/10.1530/JME-140011. Epub 2014 Mar 19. Cui, T., Jimenez, J.J., Block, N.L., Badiavas, E.V., RodriguezMenocal, L., Vila Granda, A., Cai, R., Sha, W., Zarandi, M., Perez, R., Schally, A.V., 2016. Agonistic analogs of growth hormone releasing hormone (GHRH) promote wound healing by stimulating the proliferation and survival of human dermal fibroblasts through ERK and AKT pathways. Oncotarget 7, 52661e52672. de Souza, E.B., 1995. Corticotropin-releasing factor receptors: physiology, pharmacology, biochemistry and role in central nervous system and immune disorders. Psychoneuroendocrinology 20, 789e819. Ducarouge, B., Jacquier-Sarlin, M., 2011. Stress neuromediators are key regulators of the intestinal barrier: link to inflammation and cancer. Trends Cell Mol. Biol. 6, 59e88. Egli, M., Leeners, B., Kruger, T.H., 2010. Prolactin secretion patterns: basic mechanisms and clinical implications for reproduction. Reproduction 140, 643e654. Eipper, B.A., Stoffers, D.A., Mains, R.E., 1992. The biosynthesis of neuropeptides: peptide alpha-amidation. Annu. Rev. Neurosci. 15, 57e85. Emons, G., Kaufmann, M., Gorchev, G., Tsekova, V., Grundker, C., Gunthert, A.R., Hanker, L.C., Velikova, M., Sindermann, H., Engel, J., Schally, A.V., 2010. Dose escalation and pharmacokinetic study of AEZS-108 (AN-152), an LHRH agonist linked to

65

doxorubicin, in women with LHRH receptor-positive tumors. Gynecol. Oncol. 119, 457e461. Engel, S., Gershengorn, M.C., 2007. Thyrotropin-releasing hormone and its receptorsea hypothesis for binding and receptor activation. Pharmacol. Ther. 113, 410e419. Engel, J.B., Schally, A.V., 2007. Drug Insight: clinical use of agonists and antagonists of luteinizing-hormone-releasing hormone. Nat. Clin. Pract. Endocrinol. Metabol. 3, 157e167. EPA.GOV. Available: https://comptox.epa.gov/dashboard/dsstoxdb/ results?search¼Thyrotropin-releasingþhormone%2Cþbeta-%28pyrazolyl-1%29-ala%282%29. Fekete, C., Lechan, R.M., 2007. Negative feedback regulation of hypophysiotropic thyrotropin-releasing hormone (TRH) synthesizing neurons: role of neuronal afferents and type 2 deiodinase. Front. Neuroendocrinol. 28, 97e114. Fernald, R.D., White, R.B., 1999. Gonadotropin-releasing hormone genes: phylogeny, structure, and functions. Front. Neuroendocrinol. 20, 224e240. Flanagan, C.A., Manilall, A., 2017. Gonadotropin-releasing hormone (GnRH) receptor structure and GnRH binding. Front. Endocrinol. 8, 274. Freeman, M.E., Kanyicska, B., Lerant, A., Nagy, G., 2000. Prolactin: structure, function, and regulation of secretion. Physiol. Rev. 80, 1523e1631. Fridlyand, L.E., Tamarina, N.A., Schally, A.V., Philipson, L.H., 2016. Growth hormone-releasing hormone in diabetes. Front. Endocrinol. 7, 129. Frohman, L.A., Szabo, M., 1981. Ectopic production of growth hormone-releasing factor by carcinoid and pancreatic islet tumors associated with acromegaly. Prog. Clin. Biol. Res. 74, 259e271. Frohman, L.A., Downs, T.R., Heimer, E.P., Felix, A.M., 1989. Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma. J. Clin. Investig. 83, 1533e1540. Gaylinn, B., 2006. Current research on the structure and function of the growth hormone releasing hormone receptor. J. Korean Endocr. Soc. 21, 173e183. Gesmundo, I., Miragoli, M., Carullo, P., Trovato, L., Larcher, V., di Pasquale, E., Brancaccio, M., Mazzola, M., Villanova, T., Sorge, M., Taliano, M., Gallo, M.P., Alloatti, G., Penna, C., Hare, J.M., Ghigo, E., Schally, A.V., Condorelli, G., Granata, R., 2017. Growth hormone-releasing hormone attenuates cardiac hypertrophy and improves heart function in pressure overload-induced heart failure. Proc. Natl. Acad. Sci. U. S. A. 114, 12033e12038. Ghamari-Langroudi, M., Vella, K.R., Srisai, D., Sugrue, M.L., Hollenberg, A.N., Cone, R.D., 2010. Regulation of thyrotropinreleasing hormone-expressing neurons in paraventricular nucleus of the hypothalamus by signals of adiposity. Mol. Endocrinol. 24, 2366e2381. Gilligan, P.J., Li, Y.W., 2004. Corticotropin-releasing factor antagonists: recent advances and exciting prospects for the treatment of human diseases. Curr. Opin. Drug Discov. Dev. 7, 487e497. Gkonos, P.J., Tavianini, M.A., Liu, C.C., Roos, B.A., 1989. Thyrotropinreleasing hormone gene expression in normal thyroid parafollicular cells. Mol. Endocrinol. 3, 2101e2109. Golor, G., Hu, K., Ruffin, M., Buchelt, A., Bouillaud, E., Wang, Y., Maldonado, M., 2012. A first-in-man study to evaluate the safety, tolerability, and pharmacokinetics of pasireotide (SOM230), a multireceptor-targeted somatostatin analog, in healthy volunteers. Drug Des. Dev. Ther. 6, 71e79. Golubeva, M.G., 2013. Thyrotropin-releasing hormone: structure, synthesis, receptors, and basic effects. Neurochem. J. 7, 98e102. Gore, A.C., Roberts, J.L., 1997. Regulation of gonadotropin-releasing hormone gene expression in vivo and in vitro. Front. Neuroendocrinol. 18, 209e245.

66

3. HYPOTHALAMIC RELEASING HORMONES

Grammatopoulos, D.K., Ourailidou, S., 2017. CRH receptor signalling: potential roles in pathophysiology. Curr. Mol. Pharmacol. 10, 296e310. Grosse, R., Schmid, A., Schoneberg, T., Herrlich, A., Muhn, P., Schultz, G., Gudermann, T., 2000. Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins. J. Biol. Chem. 275, 9193e9200. Guillemin, R., Brazeau, P., Bohlen, P., Esch, F., Ling, N., Wehrenberg, W.B., 1982. Growth hormone-releasing factor from a human pancreatic tumor that caused acromegaly. Science 218, 585e587. Guillermet-Guibert, J., Lahlou, H., Cordelier, P., Bousquet, C., Pyronnet, S., Susini, C., 2005. Physiology of somatostatin receptors. J. Endocrinol. Investig. 28, 5e9. Heuer, H., Schafer, M.K., Bauer, K., 1998. The thyrotropin-releasing hormone-degrading ectoenzyme: the third element of the thyrotropin-releasing hormone-signaling system. Thyroid 8, 915e920. Hilal-Dandan, R., Brunton, L.L., 2014b. Goodman and Gilman’s Manual of Pharmacology and Therapeutics. McGraw-Hill Companies. Hillhouse, E.W., Grammatopoulos, D.K., 2006. The molecular mechanisms underlying the regulation of the biological activity of corticotropin-releasing hormone receptors: implications for physiology and pathophysiology. Endocr. Rev. 27, 260e286. Hinkle, P.M., Gehret, A.U., Jones, B.W., 2012. Desensitization, trafficking, and resensitization of the pituitary thyrotropin-releasing hormone receptor. Front. Neurosci. 6, 180. Hinuma, S., Habata, Y., Fujii, R., Kawamata, Y., Hosoya, M., Fukusumi, S., Kitada, C., Masuo, Y., Asano, T., Matsumoto, H., Sekiguchi, M., Kurokawa, T., Nishimura, O., Onda, H., Fujino, M., 1998. A prolactin-releasing peptide in the brain. Nature 393, 272e276. Horita, A., 1998. An update on the CNS actions of TRH and its analogs. Life Sci. 62, 1443e1448. Hotchkiss, J., Knobil, E., 1994. The menstrual cycle and its neuroendocrine control. In: Knobil, E., Neill, J.D. (Eds.), The Physiology of Reproduction. Raven Press, New York. Huang, W., Osman, R., Gershengorn, M.C., 2005. Agonist-induced conformational changes in thyrotropin-releasing hormone receptor type I: disulfide cross-linking and molecular modeling approaches. Biochemistry 44, 2419e2431. Inda, C., Armando, N.G., Dos Santos Claro, P.A., Silberstein, S., 2017. Endocrinology and the brain: corticotropin-releasing hormone signaling. Endocr. Connect. 6, R99eR120. Jackson, I.M., 1982. Thyrotropin-releasing hormone. N. Engl. J. Med. 306, 145e155. Jarry, H., Heuer, H., Schomburg, L., Bauer, K., 2000. Prolactin-releasing peptides do not stimulate prolactin release in vivo. Neuroendocrinology 71, 262e267. Jaszberenyi, M., Rick, F.G., Szalontay, L., Block, N.L., Zarandi, M., Cai, R.Z., Schally, A.V., 2012. Beneficial effects of novel antagonists of GHRH in different models of Alzheimer’s disease. Aging (Albany NY) 4, 755e767. Jeffcoate, S.L., White, N., Hokfelt, T., Fuxe, K., Johansson, O., 1976. Proceedings: localization of thyrotrophin releasing hormone in the spinal cord of the rat by immunohisto-chemistry and radioimmunoassay. J. Endocrinol. 69, 9Pe10P. Kaiser, E.T., Kezdy, F.J., 1984. Amphiphilic secondary structure: design of peptide hormones. Science 223, 249e255. Kanasaki, H., Oride, A., Mijiddorj, T., Kyo, S., 2015. Role of thyrotropinreleasing hormone in prolactin-producing cell models. Neuropeptides 54, 73e77. Kanashiro-Takeuchi, R.M., Takeuchi, L.M., Rick, F.G., Dulce, R., Treuer, A.V., Florea, V., Rodrigues, C.O., Paulino, E.C., Hatzistergos, K.E., Selem, S.M., Gonzalez, D.R., Block, N.L.,

Schally, A.V., Hare, J.M., 2012. Activation of growth hormone releasing hormone (GHRH) receptor stimulates cardiac reverse remodeling after myocardial infarction (MI). Proc. Natl. Acad. Sci. U. S. A. 109, 559e563. Kastin, A.J. (Ed.), 2013. Handbook of Biologically Active Peptides, second ed. Elsevier/Academic Press, San Diego, CA, p. 2032. Kanashiro-Takeuchi, R.M., Szalontay, L., Schally, A.V., Takeuchi, L.M., Popovics, P., Jaszberenyi, M., Vidaurre, I., Zarandi, M., Cai, R.Z., Block, N.L., Hare, J.M., Rick, F.G., 2015. New therapeutic approach to heart failure due to myocardial infarction based on targeting growth hormone-releasing hormone receptor. Oncotarget 6, 9728e9739. Kasckow, J.W., Baker, D., Geracioti Jr., T.D., 2001. Corticotropinreleasing hormone in depression and post-traumatic stress disorder. Peptides 22, 845e851. Keller-Wood, M., 2015. Hypothalamic-pituitaryeadrenal axis-feedback control. Comp. Physiol. 5, 1161e1182. Kiaris, H., Chatzistamou, I., Papavassiliou, A.G., Schally, A.V., 2011. Growth hormone-releasing hormone: not only a neurohormone. Trends Endocrinol. Metabol. 22, 311e317. Klecha, A.J., Genaro, A.M., Gorelik, G., Barreiro Arcos, M.L., Silberman, D.M., Schuman, M., Garcia, S.I., Pirola, C., Cremaschi, G.A., 2006. Integrative study of hypothalamuspituitary-thyroid-immune system interaction: thyroid hormonemediated modulation of lymphocyte activity through the protein kinase C signaling pathway. J. Endocrinol. 189, 45e55. Kochman, K., 2012. Evolution of gonadotropin-releasing hormone (GnRH) structure and its receptor. J. Anim. Feed Sci. 21, 3e30. Krenning, E.P., Kwekkeboom, D.J., Bakker, W.H., Breeman, W.A., Kooij, P.P., Oei, H.Y., van hagen, M., Postema, P.T., De Jong, M., Reubi, J.,C., et al., 1993. Somatostatin receptor scintigraphy with [111In-DTPA-D-Phe1]- and [123I-Tyr3]-octreotide: the Rotterdam experience with more than 1000 patients. Eur. J. Nucl. Med. 20, 716e731. Kumar, U., 2011. Cross-talk and modulation of signaling between somatostatin and growth factor receptors. Endocrine 40, 168e180. Ladram, A., Bulant, M., Delfour, A., Montagne, J.J., Vaudry, H., Nicolas, P., 1994. Modulation of the biological activity of thyrotropin-releasing hormone by alternate processing of proTRH. Biochimie 76, 320e328. Larauche, M., Kiank, C., Tache, Y., 2009. Corticotropin releasing factor signaling in colon and ileum: regulation by stress and pathophysiological implications. J. Physiol. Pharmacol. 60 (Suppl. 7), 33e46. Lee, S.L., Stewart, K., Goodman, R.H., 1988. Structure of the gene encoding rat thyrotropin releasing hormone. J. Biol. Chem. 263, 16604e16609. Lewis, I., Bauer, W., Albert, R., Chandramouli, N., Pless, J., Weckbecker, G., Bruns, C., 2003. A novel somatostatin mimic with broad somatotropin release inhibitory factor receptor binding and superior therapeutic potential. J. Med. Chem. 46, 2334e2344. Liapakis, G., Venihaki, M., Margioris, A., Grigoriadis, D., Gkountelias, K., 2011. Members of CRF family and their receptors: from past to future. Curr. Med. Chem. 18, 2583e2600. Lin-Su, K., Wajnrajch, M.P., 2002. Growth hormone releasing hormone (GHRH) and the GHRH. Receptor Rev. Endocr. Metab. Disord. 3, 313e323. Lovejoy, D.A., Jahan, S., 2006. Phylogeny of the corticotropin-releasing factor family of peptides in the metazoa. Gen. Comp. Endocrinol. 146, 1e8. Maclean, D.B., Jackson, I.M., 1988. Molecular biology and regulation of the hypothalamic hormones. Baillieres Clin. Endocrinol. Metab. 2, 835e868. Maggi, R., Cariboni, A.M., Marelli, M.M., Moretti, R.M., Andre, V., Marzagalli, M., Limonta, P., 2016. GnRH and GnRH receptors in the pathophysiology of the human female reproductive system. Hum. Reprod. Update 22, 358e381.

REFERENCES

Majzoub, J.A., 2006. Corticotropin-releasing hormone physiology. Eur. J. Endocrinol. 71e76. Mariotti, S., Beck-Peccoz, P., 2000. Physiology of the HypothalamicPituitary-Thyroid Axis. Martino, E., Lernmark, A., Seo, H., Steiner, D.F., Refetoff, S., 1978. High concentration of thyrotropin-releasing hormone in pancreatic islets. Proc. Natl. Acad. Sci. U. S. A. 75, 4265e4267. Mayo, K.E., Cerelli, G.M., Lebo, R.V., Bruce, B.D., Rosenfeld, M.G., Evans, R.M., 1985. Gene encoding human growth hormonereleasing factor precursor: structure, sequence, and chromosomal assignment. Proc. Natl. Acad. Sci. U. S. A. 82, 63e67. Mayo, K.E., Godfrey, P.A., Suhr, S.T., Kulik, D.J., Rahal, J.O., 1995. Growth hormone-releasing hormone: synthesis and signaling. Recent Prog. Horm. Res. 50, 35e73. Millar, R.P., 2005. GnRHs and GnRH receptors. Anim. Reprod. Sci. 88, 5e28. Miranda, P., Giraldez, T., de la Pena, P., Manso, D.G., Alonso-Ron, C., Gomez-Varela, D., Dominguez, P., Barros, F., 2005. Specificity of TRH receptor coupling to G-proteins for regulation of ERG Kþ channels in GH3 rat anterior pituitary cells. J. Physiol. 566, 717e736. Nagy, A., Schally, A.V., Armatis, P., Szepeshazi, K., Halmos, G., Kovacs, M., Zarandi, M., Groot, K., Miyazaki, M., Jungwirth, A., Horvath, J., 1996. Cytotoxic analogs of luteinizing hormonereleasing hormone containing doxorubicin or 2pyrrolinodoxorubicin, a derivative 500-1000 times more potent. Proc. Natl. Acad. Sci. U. S. A. 93, 7269e7273. Nagy, A., Schally, A.V., Halmos, G., Armatis, P., Cai, R.Z., Csernus, V., Kovacs, M., Koppan, M., Szepeshazi, K., Kahan, Z., 1998. Synthesis and biological evaluation of cytotoxic analogs of somatostatin containing doxorubicin or its intensely potent derivative, 2pyrrolinodoxorubicin. Proc. Natl. Acad. Sci. U. S. A. 95, 1794e1799. Naor, Z., Harris, D., Shacham, S., 1998. Mechanism of GnRH receptor signaling: combinatorial cross-talk of Ca2þ and protein kinase C. Front. Neuroendocrinol. 19, 1e19. Nillni, E.A., 2010. Regulation of the hypothalamic thyrotropin releasing hormone (TRH) neuron by neuronal and peripheral inputs. Front. Neuroendocrinol. 31, 134e156. Nillni, E.A., Xie, W., Mulcahy, L., Sanchez, V.C., Wetsel, W.C., 2002. Deficiencies in pro-thyrotropin-releasing hormone processing and abnormalities in thermoregulation in Cpefat/fat mice. J. Biol. Chem. 277, 48587e48595. Ortiga-Carvalho, T.M., Chiamolera, M.I., Pazos-Moura, C.C., Wondisford, F.E., 2016. Hypothalamus-pituitary-thyroid Axis. Comp. Physiol. 6, 1387e1428. Patel, Y.C., 1997. Molecular pharmacology of somatostatin receptor subtypes. J. Endocrinol. Investig. 20, 348e367. Perello, M., Nillni, E.A., 2007. The biosynthesis and processing of neuropeptides: lessons from prothyrotropin releasing hormone (proTRH). Front. Biosci. 12, 3554e3565. Perlman, J.H., Nussenzveig, D.R., Osman, R., Gershengorn, M.C., 1992. Thyrotropin-releasing hormone binding to the mouse pituitary receptor does not involve ionic interactions. A model for neutral peptide binding to G protein-coupled receptors. J. Biol. Chem. 267, 24413e24417. Perrin, M.H., Vale, W.W., 1999. Corticotropin releasing factor receptors and their ligand family. Ann. N. Y. Acad. Sci. 885, 312e328. Porterfield, S.P., White, B.A., 2007. Endocrine Physiology, third ed. Rai, U., Thrimawithana, T.R., Valery, C., Young, S.A., 2015 Aug. Therapeutic uses of somatostatin and its analogues: Current view and potential applications. Pharmacol Ther. 152, 98e110. https:// doi.org/10.1016/j.pharmthera.2015.05.007. Epub 2015 May 5. Reichlin, S., Saperstein, R., Jackson, I.M., Boyd III, A.E., Patel, Y., 1976. Hypothalamic hormones. Annu. Rev. Physiol. 38, 389e424. Reisine, T., Bell, G.I., 1995. Molecular biology of somatostatin receptors. Endocr. Rev. 16, 427e442.

67

Rekasi, Z., Czompoly, T., Schally, A.V., Halmos, G., 2000. Isolation and sequencing of cDNAs for splice variants of growth hormonereleasing hormone receptors from human cancers. Proc. Natl. Acad. Sci. U. S. A. 97, 10561e10566. Rivier, J., Spiess, J., Thorner, M., Vale, W., 1982. Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 300, 276e278. Romero, M.J., Lucas, R., Dou, H., Sridhar, S., Czikora, I., Mosieri, E.M., Rick, F.G., Block, N.L., Sridhar, S., Fulton, D., Weintraub, N.L., Bagi, Z., Schally, A.V., 2016. Role of growth hormone-releasing hormone in dyslipidemia associated with experimental type 1 diabetes. Proc. Natl. Acad. Sci. U. S. A. 113, 1895e1900. Romier, C., Bernassau, J.M., Cambillau, C., Darbon, H., 1993. Solution structure of human corticotropin releasing factor by 1H NMR and distance geometry with restrained molecular dynamics. Protein Eng. 6, 149e156. Saffran, M., Schally, A.V., 1955. The release of corticotrophin by anterior pituitary tissue in vitro. Can. J. Biochem. Physiol. 33, 408e415. Said, H.M., 2018. Physiology of the Gastrointestinal Tract. Academic Press. Schally, A.V., 1978. Aspects of hypothalamic regulation of the pituitary gland. Science 202, 18e28. Schally, A.V., Comaru-Schally, A.M., 2003. Hypothalamic and Other Peptide Hormones. In: Kufe, D.W., Pollock, R.E., Weichselbaum, R.R., et al. (Eds.), Holland-Frei Cancer Medicine, 6th edition. BC Decker, Hamilton (ON). Chapter 61. Available from: https://www.ncbi.nlm.nih.gov/books/NBK14041/. Schally, A.V., Halmos, G., 2012. Targeting to Peptide Receptors. Drug Delivery in Oncology. Wiley-VCH Verlag GmbH & Co. KGaA. Schally, A.V., Arimura, A., Kastin, A.J., Matsuo, H., Baba, Y., Redding, T.W., Nair, R.M., Debeljuk, L., White, W.F., 1971. Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones. Science 173, 1036e1038. Schally, A.V., Arimura, A., Kastin, A.J., 1973. Hypothalamic regulatory hormones. Science 179, 341e350. Schally, A.V., Comaru-Schally, A.M., Nagy, A., Kovacs, M., Szepeshazi, K., Plonowski, A., Varga, J.L., Halmos, G., 2001a. Hypothalamic hormones and cancer. Front. Neuroendocrinol. 22, 248e291. Schally, A.V., Halmos, G., Rekasi, A., Arencibia, J.M., 2001b. The actions of LH-RH agonists, antagonists, and cytotoxic analogs on the LHRH receptors on the pituitary and tumors, in infertility and reproductive medicine. In: Devroey, P. (Ed.), Clinics of North America: GnRH Analogs. Saunders, Pennsylvania. Schally, A.V., Szepeshazi, K., Nagy, A., Comaru-Schally, A.M., Halmos, G., 2004. New approaches to therapy of cancers of the stomach, colon and pancreas based on peptide analogs. Cell. Mol. Life Sci. 61, 1042e1068. Schally, A.V., Varga, J.L., Engel, J.B., 2008. Antagonists of growthhormone-releasing hormone: an emerging new therapy for cancer. Nat. Clin. Pract. Endocrinol. Metabol. 4, 33e43. Schmid, H.A., 2008. Pasireotide (SOM230): development, mechanism of action and potential applications. Mol. Cell. Endocrinol. 286, 69e74. Schoenmakers, N., Alatzoglou, K.S., Chatterjee, V.K., Dattani, M.T., 2015. Recent advances in central congenital hypothyroidism. J. Endocrinol. 227, R51eR71. Shaw, R.W., 2015. Control of HypothalamicePituitaryeOvarian Function ([Online]). Sherin, J.E., Nemeroff, C.B., 2011. Post-traumatic stress disorder: the neurobiological impact of psychological trauma. Dialogues Clin. Neurosci. 13, 263e278. Shibusawa, N., Yamada, M., Hirato, J., Monden, T., Satoh, T., Mori, M., 2000. Requirement of thyrotropin-releasing hormone for the

68

3. HYPOTHALAMIC RELEASING HORMONES

postnatal functions of pituitary thyrotrophs: ontogeny study of congenital tertiary hypothyroidism in mice. Mol. Endocrinol. 14, 137e146. Slominski, A.T., Zmijewski, M.A., Skobowiat, C., Zbytek, B., Slominski, R.M., Steketee, J.D., 2012. Sensing the environment: regulation of local and global homeostasis by the skin’s neuroendocrine system. Adv. Anat. Embryol. Cell Biol. 212 v, vii, 1e115. Spuch, C., Navarro, C., 2011. New insights in prolactin releasing peptide (PrRP) in the brain. Immunol. Endocr. Metab. Agents Med. Chem. 11, 228e233. Spuch, C., diz-Chaves, Y., Perez-Tilve, D., Mallo, F., 2006. Fibroblast growth factor-2 and epidermal growth factor modulate prolactin responses to TRH and dopamine in primary cultures. Endocrine 29, 317e324. Spuch, C., diz-Chaves, Y., Perez-Tilve, D., Alvarez-Crespo, M., Mallo, F., 2007. Prolactin-releasing Peptide (PrRP) increases prolactin responses to TRH in vitro and in vivo. Endocrine 31, 119e124. Squire, L., Berg, D., Bloom, F.E., du Lac, S., Ghosh, A., Spitzer, N.C. (Eds.), 2014a. Fundamental Neuroscience, 4th Edition. Academic Press, p. 1152. Stengel, A., Tache, Y., 2010. Corticotropin-releasing factor signaling and visceral response to stress. Exp. Biol. Med. 235, 1168e1178. Swinnen, E., Boussemaere, M., Denef, C., 2005. Stimulation and inhibition of prolactin release by prolactin-releasing Peptide in rat anterior pituitary cell aggregates. J. Neuroendocrinol. 17, 379e386. Tashjian Jr., A.H., Barowsky, N.J., Jensen, D.K., 1971. Thyrotropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture. Biochem. Biophys. Res. Commun. 43, 516e523. Thomson, M., 2013. The physiological roles of placental corticotropin releasing hormone in pregnancy and childbirth. J. Physiol. Biochem. 69, 559e573. Ur, E., Grossman, A., 1992. Corticotropin-releasing hormone in health and disease: an update. Acta Endocrinol. 127, 193e199. Vale, W., Spiess, J., Rivier, C., Rivier, J., 1981. Characterization of a 41residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213, 1394e1397.

van de Kar, L.D., Rittenhouse, P.A., Li, Q., Levy, A.D., 1996. Serotonergic regulation of renin and prolactin secretion. Behav. Brain Res. 73, 203e208. van Sinay, E., Mirabeau, O., Depuydt, G., Van Hiel, M.B., Peymen, K., Watteyne, J., Zels, S., Schoofs, L., Beets, I., 2017. Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A. 114, E4065eE4074. White, R.B., Fernald, R.D., 1998. Genomic structure and expression sites of three gonadotropin-releasing hormone genes in one species. Gen. Comp. Endocrinol. 112, 17e25. Yamada, T., Soll, A.H., Park, J., Elashoff, J., 1984. Autonomic regulation of somatostatin release: studies with primary cultures of canine fundic mucosal cells. Am. J. Physiol. 247, G567eG573. Yamada, M., Satoh, T., Monden, T., Mori, M., 1999. Assignment of the thyrotropin-releasing hormone gene (TRH) to human chromosome 3q13.3–>q21 by in situ hybridization. Cytogenet. Cell Genet. 87, 275. Yanaihara, N., Yanaihara, C., Mochizuki, T., Iguchi, K., 1986. [Chemical structure, biosynthesis and body distribution of the corticotropin releasing factor]. Nihon Rinsho 44, 486e491. Yin, R., Zhang, R., Wang, J., Yuan, L., Hu, L., Jiang, S., Chen, C., Cao, Y., 2017. Effects of somatostatin/octreotide treatment in neonates with congenital chylothorax. Medicine (Baltim.) 96, e7594. Yoshio Takei, H.A., Tsutsui, K., 2015. Handbook of Hormonese Comparative Endocrinology for Basic and Clinical Research. Academic Press. Zarandi, M., Cai, R., Kovacs, M., Popovics, P., Szalontay, L., Cui, T., Sha, W., Jaszberenyi, M., Varga, J., Zhang, X., Block, N.L., Rick, F.G., Halmos, G., Schally, A.V., 2017. Synthesis and structure-activity studies on novel analogs of human growth hormone releasing hormone (GHRH) with enhanced inhibitory activities on tumor growth. Peptides 89, 60e70. Zhang, X., Cui, T., He, J., Wang, H., Cai, R., Popovics, P., Vidaurre, I., Sha, W., Schmid, J., Ludwig, B., Block, N.L., Bornstein, S.R., Schally, A.V., 2015. Beneficial effects of growth hormone-releasing hormone agonists on rat INS-1 cells and on streptozotocininduced NOD/SCID mice. Proc. Natl. Acad. Sci. U. S. A. 112, 13651e13656.

C H A P T E R

4 Neurosteroids: Biosynthesis, Molecular Mechanisms, and Neurophysiological Functions in the Human Brain Doodipala Samba Reddy, Kushal Bakshi Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M University Health Science Center, Bryan, TX, United States

1. INTRODUCTION

neurosteroids are positive allosteric modulators of g-aminobutyric acid type A (GABA-A) receptors, the primary mediators of inhibitory neurotransmission. Neurosteroid sulfates are known to interact with a wider variety of receptors including N-methyl-D-aspartate (NMDA) receptors and sigma receptors (Bowlby, 1993; Reddy, 2010). This chapter describes current knowledge of neurosteroids, with an emphasis on their biosynthesis, molecular mechanisms of action, and their neurophysiological functions in brain conditions such as epilepsy, anxiety, and psychiatric disorders.

Neurosteroids are endogenous steroids synthesized within the central nervous system that rapidly alter neuronal excitability through interactions with receptors at the neuronal membrane. Neurosteroids are also called neuroactive steroids and are synthesized from cholesterol, independently of the peripheral steroidogenic endocrine glands. Cholesterol and circulating steroid hormones serve as precursors for the synthesis of neurosteroids, which are produced locally in the hippocampus and other brain structures (Baulieu and Robel, 1990). Allopregnanolone (AP), allotetrahydrodeoxycorticosterone (THDOC), and androstanediol (AD) are examples of some of the most widely studied neurosteroids (Table 4.1). These neurosteroids do not directly interact with steroid hormone receptors, but their metabolites can bind to intracellular steroid receptors. Neurosteroids and neurosteroid-based compounds offer a vast therapeutic potential for treatment of conditions such as epilepsy, anxiety, and psychiatric disorders. Neurosteroids are classified into three classes based on their structure (Fig. 4.1): (1) pregnane neurosteroids, such as AP and THDOC; (2) androstane neurosteroids, such as androstanediol and etiocholanone; and (3) sulfated neurosteroids, such as pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS) (Aird and Gordan, 1951; Gyermek, 1967; Green et al., 1978; Reddy, 2003, 2010). Neurosteroids and neurosteroid sulfates interact with a wide variety of neurotransmitter receptors in the brain. Pregnane and androstane

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00004-3

2. BIOSYNTHESIS OF NEUROSTEROIDS Neurosteroids can be synthesized in the brain or the periphery. Due to their lipophilic structures, neurosteroids synthesized in the periphery can readily cross the bloodebrain barrier. In the brain, neurosteroids are synthesized from cholesterol or steroid precursors, such as progesterone or deoxycorticosterone, via progressive A-ring reductions (Reddy, 2010). The steroid precursors of neurosteroids are mainly synthesized in the gonads, adrenal gland, and fetoplacental unit. The conversion steps require two reducing enzymes that are found in reproductive endocrine tissues, liver, and skin, and recent studies have shown they are also present in the brain (Akk et al., 2004, 2007). Pregnane neurosteroids are derived from progesterone. The initial step in steroidogenesis is the

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TABLE 4.1

An Overview of Endogenous Neurosteroids.

Neurosteroid

Mechanism of Action

Neurophysiological Role

Allopregnanolone (AP) (Brexanolone)

GABA-A receptor (þ) modulator

Sedative Anxiolytic anticonvulsant Antistress Neurogenesis Neuroprotection

Allotetrahydrodeoxycorticosterone (THDOC)

GABA-A receptor (þ) modulator

Sedative Anxiolytic anticonvulsant Antistress Neuroprotection

Androstanediol (AD)

GABA-A receptor (þ) modulator

Anxiolytic anticonvulsant Neuroprotection

Pregnenolone sulfate (PS)

GABA-A receptor () modulator NMDA receptor (þ) modulator

Anxiogenic Proconvulsant Memory enhancer neuroprotection

Dehydroepiandrosterone sulfate (DHEAS)

GABA-A receptor () modulator NMDA receptor (þ) modulator

Anxiogenic Proconvulsant Memory enhancer Neurogenesis Neuroprotection

FIGURE 4.1

Classification and chemical structures of neurosteroids. DHEAS, dehydroepiandrosterone sulfate; PS, pregnenolone sulfate; THDOC, allotetrahydrodeoxycorticosterone.

3. MOLECULAR MECHANISMS OF NEUROSTEROIDS

conversion of cholesterol to pregnenolone by the mitochondrial enzyme P450scc. Progesterone is converted to AP by sequential A-ring reductions. Deoxycorticosterone is reduced to yield THDOC. Androstane neurosteroids are derived from testosterone by similar, sequential A-ring reductions to yield androstanediol. The two key enzymes that enable these A-ring reductions are expressed in the brain where synthesis occurs in glia and neurons across several regions, including the hippocampus and neocortex. The two key enzymes implicated in neurosteroid synthesis are 5a-reductase (NADPH-D4-3-oxosteroid-5a-oxidoreductase; EC 1.3.99.5) and 3a-hydroxysteroid oxidoreductase (3a-HSOR). 5a-reductase is an NADPH-dependent enzyme that was originally characterized to catalyze the reduction of testosterone to the more potent androgen dihydrotestosterone. Progesterone and deoxycorticosterone and other 3-keto-pregnane steroids were later discovered to be substrates for the 5a-reductase enzyme. Since the activity of the 3a-HSOR is far greater than 5a-reductase, the 5a-reduction is the rate-limiting step in the biosynthesis of neurosteroids. Mechanism-based, irreversible inhibition of 5a-reductase by finasteride and dutasteride can shut down the neurosteroid biosynthesis, thereby affecting neuronal excitability and behavior (Ellsworth et al., 1998). Neurosteroid sulfates are derived from sulfation reactions with their parent compound. Pregnenolone is derived from cholesterol by the P450scc enzyme. PS is synthesized when pregnenolone undergoes sulfation. The cytosolic sulfotransferase enzymes (SULT) are the primary mediators of neurosteroid sulfate synthesis. SULT2A1, SULT2B1a, and SULT2B1b have been implicated in PS biosynthesis (Her et al., 1998; Strott, 2002). Dehydroepiandrosterone sulfate (DHEAS) is synthesized from pregnenolone, which forms DHEA through a series of reactions catalyzed by hydroxylase enzymes. The SULT2A1 enzyme allows DHEA sulfation to form DHEAS (Le Goascogne et al., 1987; Li et al., 1996). Cholesterol transport mechanisms play a vital role in steroid and neurosteroid biosynthesis (Papadopoulos et al., 2015). In fact, our currently changing perception of the role of steroids in the brain has generated controversies, such as the significance of the mitochondrial translocator protein (TSPO) in their biosynthesis, or the importance of intracellular receptor mediated versus membrane actions of steroids. Molecular and gene knockout studies are providing new insights on neurosteroid biosynthesis and signaling mechanisms, in particular the role of TSPO in the biosynthesis of neurosteroids (Fan et al., 2015; Venugopal et al., 2016).

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3. MOLECULAR MECHANISMS OF NEUROSTEROIDS 3.1 Neurosteroid Modulation of GABA-A Receptors GABA-A receptors are ligand-gated chloride ion channels and the primary receptor mediators of inhibitory neurotransmission in the brain. When bound by the ligand GABA, GABA-A receptors conduct chloride ions into the neuron and hyperpolarize the membrane, decreasing the likelihood of that neuron generating action potentials. Structurally, GABA-A receptors are pentameric channels made from a combination of various subunits (a1e6, b1e4, g1e3, d, ε, q, r1e3), usually consisting of two a and two b subunits with a fifth subunit that varies based on receptor type and location. The subunit composition of GABA-A receptors determines its pharmacological sensitivity to drugs and other endogenous compounds. Neurosteroids, for example, preferentially bind to d-containing receptors. Based on their location on neuronal sites, GABA-A receptors are classified into synaptic (primarily g-containing) and extrasynaptic (primarily d-containing) receptors (Akk et al., 2005, 2007; Chuang and Reddy, 2018a). Synaptic and extrasynaptic receptors differ in their GABA affinity (Mortensen et al., 2011), desensitization rate, agonist efficacy (Bianchi and Macdonald, 2002, 2003), and neurosteroid sensitivity (Brown et al., 2002; Wohlfarth et al., 2002). Patch-clamp electrophysiology studies reveal key differences between synaptic and extrasynaptic GABA-A receptors (Carver and Reddy, 2013). Synaptic receptors, located at neuronal synapses ubiquitously within the brain, produce phasic currents in response to vesicular release of GABA. Extrasynaptic receptors, on the other hand, are located outside the neuronal synapse and are expressed in specific brain regions including hippocampus, thalamus, and cerebellum (Whissell et al., 2015). Extrasynaptic receptors exhibit high affinity for GABA and generate nondesensitizing tonic currents that are continuously activated by ambient GABA. Tonic current sets the baseline inhibitory tone and provides shunting inhibition via continuous chloride conductance in specific neurons. All GABA-A receptors have allosteric binding sites where endogenous and exogenous compounds bind and affect channel function. These allosteric sites bind compounds such as neurosteroids, benzodiazepines, and other anesthetics. Neurosteroids are positive allosteric agonists of synaptic and extrasynaptic GABA-A receptors (Reddy and Rogawski, 2000a,b; Carver and Reddy, 2013; Reddy and Estes, 2016). At high concentrations (>1 mM), neurosteroids directly activate receptors,

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whereas at low concentrations (200 in the FMR1 gene considered diagnostic for disease. Individuals with 55e200 repeats are considered to have an FXS premutation, which is frequently clinically silent during childhood but can develop in late adulthood into fragile X-associated tremor/ataxia syndrome. FXS affects about 100,000 people in the United States. Patients with FXS show autism-like symptoms including cognitive impairment, anxiety and mood swings, and behavioral and learning challenges. Many patients with FXS have seizures, and mouse models have indicated an imbalance in excitatory/inhibitory signaling (Cao et al., 2012b). Because this imbalance was theorized to stem at least in part from deficits in GABAergic function, positive allosteric modulation of GABA-A receptors using AP has been explored. AP treatment was found to reversibly mitigate the functional impairments identified in this model, restoring WT firing behavior. Conversely, GABA-A receptor blockade of WT neurons using picrotoxin phenocopied the clustered burst firing pattern observed in the premutation model.

4.7 Neuronal Injury and Neurotoxicity Conditions Neurosteroids have strong neuroprotectant properties in a variety of experimental models of neuronal injury and chemical neurotoxicity (Reddy, 2016b; Stein DG & Sayeed, 2018). Progesterone plays an important role in the resistance of the brain to ischemic and traumatic brain injury by signaling via its intracellular progesterone receptors (PR) (Schumacher et al., 2014). Consequently, it is evident that treatment with progesterone protects the brain of wild-type, but not of PR knockout, male and female mice against damage after middle cerebral artery occlusion. Importantly, the brain of both sexes contains significant amounts of endogenous progesterone, but it was not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Recently, Schumacher and colleagues found that in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5a-dihydroprogesterone are rapidly

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REFERENCES

upregulated to pregnancy-like levels in the male but surprisingly not in the female brain (Frechou et al., 2015; Bielecki et al., 2016). Deletion of PR in the brain reduced its resistance to ischemic damage, resulting in increased infarct volumes and neurological deficits in both sexes. Overall, these reports uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. In addition, there is recent evidence that AP attenuates neurotoxicity associated with the with the HIV-1 regulatory protein, trans-activator of transcription (Paris et al., 2014, 2015; 2016), indicating the neuroprotectant potential of this endogenous neurosteroid in the brain.

4.8 Anxiety and Other Psychiatric Conditions Downregulation of neurosteroid biosynthesis has been linked to be a possible contributor to the development of anxiety and behavior disorders (Schu¨le et al., 2014). Reduced levels of AP in the peripheral blood or cerebrospinal fluid were found to be associated with depression, anxiety disorders, and negative symptoms in schizophrenia, or impulsive aggression. The importance of AP for the regulation of emotion and its clinical use in depression and anxiety may not only involve GABAergic mechanisms, but probably also includes enhancement of neurogenesis, myelination, neuroprotection, and regulatory effects on HPA axis function. While benzodiazepines and SSRIs continue to be the most popular treatments for behavioral and anxietyrelated disorders, some neurosteroids may provide beneficial effects (Westenberg, 2009). Neurosteroids that act as positive allosteric modulators of GABA-A receptors have been demonstrated to have an anxiolytic, sedative, and anticonvulsive effect (Smith, 2002). On the other hand, systemically administered neurosteroid sulfates exhibit a biphasic effect on anxiety responses depending on the dosage applied in different behavioral tests but may provide therapeutic benefit in these disorders in the correct dosages (Melchior and Ritzmann, 1994; Reddy and Kulkarni, 1997). Behavioral studies have shown that the increase of progesterone or AP in proestrus female rats increases the anxiolytic profile in anxiety tests such as the elevated plus-maze or the open field test (Frye et al., 2000). In addition, systemically injected 3a, 5a-THP showed an anxiolytic profile similar to benzodiazepines or barbiturates in the mirrored chamber test in mice (Reddy and Kulkarni, 1997). Glutamatergic neurotransmission involved in the biologic mechanisms underlying stress response and anxiety-related disorders could be targeted by PS or DHEAS (Rupprecht and Holsboer, 1999). Both PS and DHEAS have been described as both positive NMDA receptor modulators (Kussius et al., 2009) and negative GABA-A receptor modulators

(Mtchedlishvili and Kapur, 2003). The broad spectrum of activity and interactions of neurosteroid sulfates may prove to be beneficial in alleviating anxiety and behavior symptoms. Specific TSPO agonists, which increases endogenous synthesis of neurosteroids, are being investigated as clinical anxiolytics (Nothdurfter et al., 2012).

5. CONCLUSIONS AND FUTURE DIRECTIONS The endogenous neurosteroids AP, THDOC, and AD interact with many neurotransmitter receptors and play a key role in the balance of excitation and inhibition in the human brain. Pregnane and androstane neurosteroids positively modulate GABA-A receptormediated inhibitory neurotransmission, whereas neurosteroid sulfates positively modulate NMDA receptor-mediated excitatory neurotransmission through intricate mechanisms. While the exact physiologic function of these compounds continue to be elucidated, their role in neuronal excitability disorders such as epilepsy and behavior conditions such as anxiety is becoming better understood. Through interactions with extrasynaptic GABA-A receptors, neurosteroids control basal excitability state in brain regions, could provide therapeutic benefit to many forms of idiopathic epilepsies, and may even offer treatment options for conditions such as FXS or PMDD. Neurosteroid sulfates, through modulation of presynaptic and postsynaptic neurotransmission, could affect mood and memory conditions. Further investigation of these compounds may uncover additional therapeutic options for specific conditions linked to impaired inhibition in the brain.

Acknowledgments This work was partly supported by National Institutes of Health Grant R01NS051398 (to D.S.R.). This work was also partly supported by the CounterACT Program, National Institutes of Health, Office of the Director and the National Institute of Neurologic Disorders and Stroke [Grant U01 NS083460] (to D.S.R.). The authors declare no competing financial interests.

References Aird, R.B., Gordan, G.S., 1951. Anticonvulsive properties of desoxycorticosterone. J. Am. Med. Assoc. 145, 715e719. Akk, G., Bracamontes, J., Steinbach, J.H., 2004. Activation of GABA(A) receptors containing the alpha4 subunit by GABA and pentobarbital. J. Physiol. 556, 387e399. Akk, G., Covey, D.F., Evers, A.S., Steinbach, J.H., Zorumski, C.F., Mennerick, S., 2007. Mechanisms of neurosteroid interactions with GABA(A) receptors. Pharmacol. Ther. 116, 35e57.

78

4. NEUROSTEROIDS: BIOSYNTHESIS, MOLECULAR MECHANISMS, AND NEUROPHYSIOLOGICAL FUNCTIONS IN THE HUMAN BRAIN

Akk, G., Shu, H.J., Wang, C., Steinbach, J.H., Zorumski, C.F., Covey, D.F., Mennerick, S., 2005. Neurosteroid access to the GABAA receptor. J. Neurosci. 25, 11605e11613. Baulieu, E.E., Robel, P., 1990. Neurosteroids: a new brain function? J. Steroid Biochem. Mol. Biol. 37, 395e403. Bianchi, M.T., Macdonald, R.L., 2002. Slow phases of GABA(A) receptor desensitization: structural determinants and possible relevance for synaptic function. J. Physiol. 544, 3e18. Bianchi, M.T., Macdonald, R.L., 2003. Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns. J. Neurosci. 23, 10934e10943. Bielecki, B., Mattern, C., Ghoumari, A.M., Javaid, S., Smietanka, K., Abi Ghanem, C., Mhaouty-Kodja, S., Ghandour, M.S., Baulieu, E.E., Franklin, R.J., Schumacher, M., Traiffort, E., 2016. Unexpected central role of the androgen receptor in the spontaneous regeneration of myelin. Proc. Natl. Acad. Sci. U. S. A. 113, 14829e14834. Bowlby, M.R., 1993. Pregnenolone sulfate potentiation of N-methyl-Daspartate receptor channels in hippocampal neurons. Mol. Pharmacol. 43, 813e819. Brown, N., Kerby, J., Bonnert, T.P., Whiting, P.J., Wafford, K.A., 2002. Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA(A) receptors. Br. J. Pharmacol. 136, 965e974. Cao, Y., Pahlberg, J., Sarria, I., Kamasawa, N., Sampath, A.P., Martemyanov, K.A., 2012a. Regulators of G protein signaling RGS7 and RGS11 determine the onset of the light response in ON bipolar neurons. Proc. Natl. Acad. Sci. U. S. A. 109, 7905e7910. Cao, Z., Hulsizer, S., Tassone, F., Tang, H.T., Hagerman, R.J., Rogawski, M.A., Hagerman, P.J., Pessah, I.N., 2012b. Clustered burst firing in FMR1 premutation hippocampal neurons: amelioration with allopregnanolone. Hum. Mol. Genet. 21, 2923e2935. Carver, C.M., Reddy, D.S., 2013. Neurosteroid interactions with synaptic and extrasynaptic GABA(A) receptors: regulation of subunit plasticity, phasic and tonic inhibition, and neuronal network excitability. Psychopharmacology (Berl) 230, 151e188. Carver, C.M., Wu, X., Gangisetty, O., Reddy, D.S., 2014. Perimenstruallike hormonal regulation of extrasynaptic delta-containing GABAA receptors mediating tonic inhibition and neurosteroid sensitivity. J. Neurosci. 34, 14181e14197. Carver, C.M., Reddy, D.S., 2016. Neurosteroid structure-activity relationships for functional activation of extrasynaptic deltaGABA(A) receptors. J. Pharmacol. Exp. Ther. 357, 188e204. Carver, C.M., Chuang, S.-H., Reddy, D.S., 2016. Zinc selectively inhibits the neurosteroid-sensitive, extrasynaptic d-GABA-A receptors in the hippocampus. J. Neurosci. 36 (31), 8070e8077. Ceccon, M., Rumbaugh, G., Vicini, S., 2001. Distinct effect of pregnenolone sulfate on NMDA receptor subtypes. Neuropharmacology 40, 491e500. Charalampopoulos, I., Dermitzaki, E., Vardouli, L., Tsatsanis, C., Stournaras, C., Margioris, A.N., Gravanis, A., 2005. Dehydroepiandrosterone sulfate and allopregnanolone directly stimulate catecholamine production via induction of tyrosine hydroxylase and secretion by affecting actin polymerization. Endocrinology 146, 3309e3318. Chen, L., Dai, X.N., Sokabe, M., 2006a. Chronic administration of dehydroepiandrosterone sulfate (DHEAS) primes for facilitated induction of long-term potentiation via sigma 1 (sigma1) receptor: optical imaging study in rat hippocampal slices. Neuropharmacology 50, 380e392. Chen, L., Miyamoto, Y., Furuya, K., Dai, X.N., Mori, N., Sokabe, M., 2006b. Chronic DHEAS administration facilitates hippocampal long-term potentiation via an amplification of Src-dependent NMDA receptor signaling. Neuropharmacology 51, 659e670.

Chisari, M., Eisenman, L.N., Krishnan, K., Bandyopadhyaya, A.K., Wang, C., Taylor, A., Benz, A., Covey, D.F., Zorumski, C.F., Mennerick, S., 2009. The influence of neuroactive steroid lipophilicity on GABA-A receptor modulation: evidence for a low-affinity interaction. J. Neurophysiol. 102, 1254e1264. Chuang, S.-H., Reddy, D.S., 2018a. Genetic and molecular regulation of extrasynaptic GABA-A receptors in the brain: therapeutic insights for epilepsy. J. Pharmacol. Exp. Ther. 364, 180e197. Chuang, S.-H., Reddy, D.S., 2018b. 3b-Methyl-neurosteroid analogs are preferential positive allosteric modulators and direct activators of extrasynaptic dGABA-A receptors in the hippocampus dentate gyrus subfield. J. Pharmacol. Exp. Ther. 365 (3), 583e601. Covey, D.F., Nathan, D., Kalkbrenner, M., Nilsson, K.R., Hu, Y., Zorumski, C.F., Evers, A.S., 2000. Enantioselectivity of pregnanolone-induced gamma-aminobutyric acid(A) receptor modulation and anesthesia. J. Pharmacol. Exp. Ther. 293, 1009e1016. Clossen, B.L., Reddy, D.S., 2017. Catamenial-like seizure exacerbation in mice lacking extrasynaptic dGABA-A receptors. J. Neurosci. Res. 95, 1906e1916. Demirgoren, S., Majewska, M.D., Spivak, C.E., London, E.D., 1991. Receptor binding and electrophysiological effects of dehydroepiandrosterone sulfate, an antagonist of the GABA-A receptor. Neuroscience 45, 127e135. Devaud, L.L., Purdy, R.H., Finn, D.A., Morrow, A.L., 1996. Sensitization of gamma-aminobutyric acidA receptors to neuroactive steroids in rats during ethanol withdrawal. J. Pharmacol. Exp. Ther. 278, 510e517. Dong, L., Zhu, Y., Dong, Y., Yang, J., Zhao, Y., Qi, Y., Wu, P., Zhu, Y., Zheng, P., 2009. Neuroactive steroid dehydroepiandrosterone sulfate inhibits 5-hydroxytryptamine (5-HT)-evoked glutamate release via activation of sigma-1 receptors and then inhibition of 5-HT3 receptors in rat prelimbic cortex. J. Pharmacol. Exp. Ther. 330, 494e501. Dong, L.Y., Cheng, Z.X., Fu, Y.M., Wang, Z.M., Zhu, Y.H., Sun, J.L., Dong, Y., Zheng, P., 2007. Neurosteroid dehydroepiandrosterone sulfate enhances spontaneous glutamate release in rat prelimbic cortex through activation of dopamine D1 and sigma-1 receptor. Neuropharmacology 52, 966e974. Duncan, L.M., Deeds, J., Hunter, J., Shao, J., Holmgren, L.M., Woolf, E.A., Tepper, R.I., Shyjan, A.W., 1998. Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis. Cancer Res. 58, 1515e1520. Ellsworth, K.P., Azzolina, B.A., Cimis, G., Bull, H.G., Harris, G.S., 1998. Cloning, expression and characterization of rhesus macaque types 1 and 2 5alpha-reductase: evidence for mechanism-based inhibition by finasteride. J. Steroid Biochem. Mol. Biol. 66, 271e279. Endicott, J., Nee, J., Cohen, J., Halbreich, U., 1986. Premenstrual changes: patterns and correlates of daily ratings. J. Affect. Disord. 10, 127e135. Fan, J., Campioli, E., Midzak, A., Culty, M., Papadopoulos, V., 2015. Conditional steroidogenic cell-targeted deletion of TSPO unveils a crucial role in viability and hormone-dependent steroid formation. Proc. Natl. Acad. Sci. U. S. A. 112, 7261e7266. Ffrench-Mullen, J.M., Spence, K.T., 1991. Neurosteroids block Ca2þ channel current in freshly isolated hippocampal CA1 neurons. Eur. J. Pharmacol. 202, 269e272. Frechou, M., Zhang, S., Liere, P., Delespierre, B., Soyed, N., Pianos, A., Schumacher, M., Mattern, C., Guennoun, R., 2015. Intranasal delivery of progesterone after transient ischemic stroke decreases mortality and provides neuroprotection. Neuropharmacology 97, 394e403.

REFERENCES

Frye, C.A., Petralia, S.M., Rhodes, M.E., 2000. Estrous cycle and sex differences in performance on anxiety tasks coincide with increases in hippocampal progesterone and 3alpha,5alpha-THP. Pharmacol. Biochem. Behav. 67, 587e596. Gangisetty, O., Reddy, D.S., 2010. Neurosteroid withdrawal regulates GABA-A receptor alpha4-subunit expression and seizure susceptibility by activation of progesterone receptor-independent early growth response factor-3 pathway. Neuroscience 170, 865e880. Giller, E., Tsai, J., Shaw, K., Pieribone, V., Soufflet, C., Dulac, O., 2006. Inpatient flexible dose, open-label add-on ganaxolone in treatment-refractory pediatric epilepsy. In: American Epilepsy Society Annual Meeting. Green, C.J., Halsey, M.J., Precious, S., Wardley-Smith, B., 1978. Alphaxolone-alphadolone anaesthesia in laboratory animals. Lab. Anim. 12, 85e89. Griffin, L.D., Mellon, S.H., 1999. Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes. Proc. Natl. Acad. Sci. U. S. A. 96, 13512e13517. Grimm, C., Kraft, R., Sauerbruch, S., Schultz, G., Harteneck, C., 2003. Molecular and functional characterization of the melastatinrelated cation channel TRPM3. J. Biol. Chem. 278, 21493e21501. Grimm, C., Kraft, R., Schultz, G., Harteneck, C., 2005. Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine [corrected]. Mol. Pharmacol. 67, 798e805. Gunn, B.G., Cunningham, L., Mitchell, S.G., Swinny, J.D., Lambert, J.J., Belelli, D., 2015. GABA-A receptor-acting neurosteroids: a role in the development and regulation of the stress response. Front. Neuroendocrinol. 36, 28e48. Gyermek, L., 1967. Pregnanolone: a highly potent, naturally occurring hypnotic-anesthetic agent. Proc. Soc. Exp. Biol. Med. 125, 1058e1062. Harrison, N.L., Majewska, M.D., Harrington, J.W., Barker, J.L., 1987. Structure-activity relationships for steroid interaction with the gamma-aminobutyric acidA receptor complex. J. Pharmacol. Exp. Ther. 241, 346e353. Harrison, N.L., Simmonds, M.A., 1984. Modulation of the GABA receptor complex by a steroid anaesthetic. Brain Res. 323, 287e292. Her, C., Wood, T.C., Eichler, E.E., Mohrenweiser, H.W., Ramagli, L.S., Siciliano, M.J., Weinshilboum, R.M., 1998. Human hydroxysteroid sulfotransferase SULT2B1: two enzymes encoded by a single chromosome 19 gene. Genomics 53, 284e295. Hesdorffer, D.C., Beck, V., et al., 2013. Research implications of the Institute of medicine report, epilepsy across the spectrum: promoting health and understanding. Epilepsia 54, 207e216. Herzog, A.G., 2015. Catamenial epilepsy: update on prevalence, pathophysiology and treatment from the findings of the NIH Progesterone Treatment Trial. Seizure 28, 18e25. Herzog, A.G., Fowler, K.M., Smithson, S.D., Kalayjian, L.A., Heck, C.N., Sperling, M.R., Liporace, J.D., Harden, C.L., Dworetzky, B.A., Pennell, P.B., Massaro, J.M., Progesterone Trial Study, G., 2012. Progesterone vs placebo therapy for women with epilepsy: a randomized clinical trial. Neurology 78, 1959e1966. Herzog, A.G., Klein, P., Ransil, B.J., 1997. Three patterns of catamenial epilepsy. Epilepsia 38, 1082e1088. Horak, M., Vlcek, K., Petrovic, M., Chodounska, H., Vyklicky Jr., L., 2004. Molecular mechanism of pregnenolone sulfate action at NR1/NR2B receptors. J. Neurosci. 24, 10318e10325. Hosie, A.M., Clarke, L., da Silva, H., Smart, T.G., 2009. Conserved site for neurosteroid modulation of GABA A receptors. Neuropharmacology 56, 149e154. Hosie, A.M., Wilkins, M.E., da Silva, H.M., Smart, T.G., 2006. Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature 444, 486e489. Hosie, A.M., Wilkins, M.E., Smart, T.G., 2007. Neurosteroid binding sites on GABA-A receptors. Pharmacol. Ther. 116, 7e19.

79

Irwin, R.P., Lin, S.Z., Rogawski, M.A., Purdy, R.H., Paul, S.M., 1994. Steroid potentiation and inhibition of N-methyl-D-aspartate receptor-mediated intracellular Caþþ responses: structureactivity studies. J. Pharmacol. Exp. Ther. 271, 677e682. Jacobs, M.P., Leblanc, G.G., et al., 2009. Curing epilepsy: progress and future directions. Epilepsy Behav. 14, 438e445. Jacob, T.C., Moss, S.J., Jurd, R., 2008. GABA-A receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nat. Rev. Neurosci. 9, 331e343. Jacquemont, S., Hagerman, R.J., Hagerman, P.J., Leehey, M.A., 2007. Fragile-X syndrome and fragile X-associated tremor/ataxia syndrome: two faces of FMR1. Lancet Neurol. 6, 45e55. Jensen, M.L., Wafford, K.A., Brown, A.R., Belelli, D., Lambert, J.J., Mirza, N.R., 2013. A study of subunit selectivity, mechanism and site of action of the delta selective compound 2 (DS2) at human recombinant and rodent native GABA-A receptors. Br. J. Pharmacol. 168, 1118e1132. Kaminski, R.M., Livingood, M.R., Rogawski, M.A., 2004. Allopregnanolone analogs that positively modulate GABA receptors protect against partial seizures induced by 6-Hz electrical stimulation in mice. Epilepsia 45, 864e867. Kaminski, R.M., Marini, H., Kim, W.J., Rogawski, M.A., 2005. Anticonvulsant activity of androsterone and etiocholanolone. Epilepsia 46, 819e827. Kerrigan, J.F., Shields, W.D., Nelson, T.Y., Bluestone, D.L., Dodson, W.E., Bourgeois, B.F., Pellock, J.M., Morton, L.D., Monaghan, E.P., 2000. Ganaxolone for treating intractable infantile spasms: a multicenter, open-label, add-on trial. Epilepsy Res. 42, 133e139. Koike, C., Obara, T., Uriu, Y., Numata, T., Sanuki, R., Miyata, K., Koyasu, T., Ueno, S., Funabiki, K., Tani, A., Ueda, H., Kondo, M., Mori, Y., Tachibana, M., Furukawa, T., 2010. TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade. Proc. Natl. Acad. Sci. U. S. A. 107, 332e337. Kokate, T.G., Svensson, B.E., Rogawski, M.A., 1994. Anticonvulsant activity of neurosteroids: correlation with gamma-aminobutyric acidevoked chloride current potentiation. J. Pharmacol. Exp. Ther. 270, 1223e1229. Kokate, T.G., Yamaguchi, S., Pannell, L.K., Rajamani, U., Carroll, D.M., Grossman, A.B., Rogawski, M.A., 1998. Lack of anticonvulsant tolerance to the neuroactive steroid pregnanolone in mice. J. Pharmacol. Exp. Ther. 287, 553e558. Kussius, C.L., Kaur, N., Popescu, G.K., 2009. Pregnanolone sulfate promotes desensitization of activated NMDA receptors. J. Neurosci. 29, 6819e6827. Lambert, J.J., Belelli, D., Peden, D.R., Vardy, A.W., Peters, J.A., 2003. Neurosteroid modulation of GABA-A receptors. Prog. Neurobiol. 71, 67e80. Laurie, D.J., Bartke, I., Schoepfer, R., Naujoks, K., Seeburg, P.H., 1997. Regional, developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies. Brain Res. Mol. Brain Res. 51, 23e32. Le Goascogne, C., Robel, P., Gouezou, M., Sananes, N., Baulieu, E.E., Waterman, M., 1987. Neurosteroids: cytochrome P-450scc in rat brain. Science 237, 1212e1215. Li, P.K., Milano, S., Kluth, L., Rhodes, M.E., 1996. Synthesis and sulfatase inhibitory activities of non-steroidal estrone sulfatase inhibitors. J. Steroid Biochem. Mol. Biol. 59, 41e48. Li, Z., Zhou, R., Cui, S., Xie, G., Cai, W., Sokabe, M., Chen, L., 2006. Dehydroepiandrosterone sulfate prevents ischemia-induced impairment of long-term potentiation in rat hippocampal CA1 by up-regulating tyrosine phosphorylation of NMDA receptor. Neuropharmacology 51, 958e966. Majeed, Y., Agarwal, A.K., Naylor, J., Seymour, V.A., Jiang, S., Muraki, K., Fishwick, C.W., Beech, D.J., 2010. Cis-isomerism and other chemical requirements of steroidal agonists and partial agonists acting at TRPM3 channels. Br. J. Pharmacol. 161, 430e441.

80

4. NEUROSTEROIDS: BIOSYNTHESIS, MOLECULAR MECHANISMS, AND NEUROPHYSIOLOGICAL FUNCTIONS IN THE HUMAN BRAIN

Majewska, M.D., Schwartz, R.D., 1987. Pregnenolone-sulfate: an endogenous antagonist of the gamma-aminobutyric acid receptor complex in brain? Brain Res. 404, 355e360. Malayev, A., Gibbs, T.T., Farb, D.H., 2002. Inhibition of the NMDA response by pregnenolone sulphate reveals subtype selective modulation of NMDA receptors by sulphated steroids. Br. J. Pharmacol. 135, 901e909. Mameli, M., Carta, M., Partridge, L.D., Valenzuela, C.F., 2005. Neurosteroid-induced plasticity of immature synapses via retrograde modulation of presynaptic NMDA receptors. J. Neurosci. 25, 2285e2294. Martinez, P.E., Rubinow, D.R., Nieman, L.K., Koziol, D.E., Morrow, A.L., Schiller, C.E., Cintron, D., Thompson, K.D., Khine, K.K., Schmidt, P.J., 2016. 5alpha-Reductase inhibition prevents the luteal phase increase in plasma allopregnanolone levels and mitigates symptoms in women with premenstrual dysphoric disorder. Neuropsychopharmacology 41, 1093e1102. Maurice, T., Lockhart, B.P., 1997. Neuroprotective and anti-amnesic potentials of sigma (sigma) receptor ligands. Prog. Neuropsychopharmacol. Biol. Psychiatry 21, 69e102. Meera, P., Wallner, M., Otis, T.S., 2011. Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABA-A receptors. J. Neurophysiol. 106, 2057e2064. Melchior, C.L., Ritzmann, R.F., 1994. Pregnenolone and pregnenolone sulfate, alone and with ethanol, in mice on the plus-maze. Pharmacol. Biochem. Behav. 48, 893e897. Meyer, D.A., Carta, M., Partridge, L.D., Covey, D.F., Valenzuela, C.F., 2002. Neurosteroids enhance spontaneous glutamate release in hippocampal neurons. Possible role of metabotropic sigma1-like receptors. J. Biol. Chem. 277, 28725e28732. Mienville, J.M., Vicini, S., 1989. Pregnenolone sulfate antagonizes GABA-A receptor-mediated currents via a reduction of channel opening frequency. Brain Res. 489, 190e194. Mihalek, R.M., Banerjee, P.K., Korpi, E.R., Quinlan, J.J., Firestone, L.L., Mi, Z.P., Lagenaur, C., Tretter, V., Sieghart, W., Anagnostaras, S.G., Sage, J.R., Fanselow, M.S., Guidotti, A., Spigelman, I., Li, Z., DeLorey, T.M., Olsen, R.W., Homanics, G.E., 1999. Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice. Proc. Natl. Acad. Sci. U. S. A. 96, 12905e12910. Miquel, M.C., Emerit, M.B., Nosjean, A., Simon, A., Rumajogee, P., Brisorgueil, M.J., Doucet, E., Hamon, M., Verge, D., 2002. Differential subcellular localization of the 5-HT3-As receptor subunit in the rat central nervous system. Eur. J. Neurosci. 15, 449e457. Mitchell, E.A., Herd, M.B., Gunn, B.G., Lambert, J.J., Belelli, D., 2008. Neurosteroid modulation of GABA-A receptors: molecular determinants and significance in health and disease. Neurochem. Int. 52, 588e595. Monaghan, E.P., McAuley, J.W., Data, J.L., 1999. Ganaxolone: a novel positive allosteric modulator of the GABA-A receptor complex for the treatment of epilepsy. Expert Opin. Investig. Drugs 8, 1663e1671. Montell, C., Birnbaumer, L., Flockerzi, V., Bindels, R.J., Bruford, E.A., Caterina, M.J., Clapham, D.E., Harteneck, C., Heller, S., Julius, D., Kojima, I., Mori, Y., Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Shimizu, N., Zhu, M.X., 2002. A unified nomenclature for the superfamily of TRP cation channels. Mol. Cell 9, 229e231. Monyer, H., Burnashev, N., Laurie, D.J., Sakmann, B., Seeburg, P.H., 1994. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12, 529e540. Morgans, C.W., Zhang, J., Jeffrey, B.G., Nelson, S.M., Burke, N.S., Duvoisin, R.M., Brown, R.L., 2009. TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells. Proc. Natl. Acad. Sci. U. S. A. 106, 19174e19178. Mortensen, M., Patel, B., Smart, T.G., 2011. GABA potency at GABA-A receptors found in synaptic and extrasynaptic zones. Front. Cell. Neurosci. 6, 1.

Mtchedlishvili, Z., Kapur, J., 2003. A presynaptic action of the neurosteroid pregnenolone sulfate on GABAergic synaptic transmission. Mol. Pharmacol. 64, 857e864. Murray, H.E., Gillies, G.E., 1997. Differential effects of neuroactive steroids on somatostatin and dopamine secretion from primary hypothalamic cell cultures. J. Neuroendocrinol. 9, 287e295. Naylor, D.E., Liu, H., Wasterlain, C.G., 2005. Trafficking of GABA-A receptors, loss of inhibition, and a mechanism for pharmacoresistance in status epilepticus. J. Neurosci. 25, 7724e7733. Nohria, V., Giller, E., 2007. Ganaxolone. Neurotherapeutics 4, 102e105. Nothdurfter, C., Baghai, T.C., Schu¨le, C., Rupprecht, R., 2012. Translocator protein (18 kDa) (TSPO) as a therapeutic target for anxiety and neurologic disorders. Eur. Arch. Psychiatry Clin. Neurosci. 262 (Suppl. 2), S107eS112. Oberwinkler, J., Lis, A., Giehl, K.M., Flockerzi, V., Philipp, S.E., 2005. Alternative splicing switches the divalent cation selectivity of TRPM3 channels. J. Biol. Chem. 280, 22540e22548. Paris, J.J., Zou, S., Hahn, Y.K., Knapp, P.E., Hauser, K.F., 2016. 5alphareduced progestogens ameliorate mood-related behavioral pathology, neurotoxicity, and microgliosis associated with exposure to HIV-1 Tat. Brain Behav. Immun. 55, 202e214. Paris, J.J., Singh, H.D., Carey, A.N., McLaughlin, J.P., 2015. Exposure to HIV-1 Tat in brain impairs sensorimotor gating and activates microglia in limbic and extralimbic brain regions of male mice. Behav. Brain Res. 291, 209e218. Paris, J.J., Fenwick, J., McLaughlin, J.P., 2014. Progesterone protects normative anxiety-like responding among ovariectomized female mice that conditionally express the HIV-1 regulatory protein, Tat, in the CNS. Horm. Behav. 65, 445e453. Papadopoulos, V., Aghazadeh, Y., Fan, J., Campioli, E., Zirkin, B., Midzak, A., 2015. Translocator protein-mediated pharmacology of cholesterol transport and steroidogenesis. Mol. Cell. Endocrinol. 408, 90e98. Park-Chung, M., Wu, F.S., Purdy, R.H., Malayev, A.A., Gibbs, T.T., Farb, D.H., 1997. Distinct sites for inverse modulation of Nmethyl-D-aspartate receptors by sulfated steroids. Mol. Pharmacol. 52, 1113e1123. Peng, Z., Hauer, B., Mihalek, R.M., Homanics, G.E., Sieghart, W., Olsen, R.W., Houser, C.R., 2002. GABA-A receptor changes in delta subunit-deficient mice: altered expression of alpha4 and gamma2 subunits in the forebrain. J. Comp. Neurol. 446, 179e197. Pieribone, V.A., Tsai, J., Soufflet, C., Rey, E., Shaw, K., Giller, E., Dulac, O., 2007. Clinical evaluation of ganaxolone in pediatric and adolescent patients with refractory epilepsy. Epilepsia 48, 1870e1874. Pinna, G., Costa, E., Guidotti, A., 2006. Fluoxetine and norfluoxetine stereospecifically and selectively increase brain neurosteroid content at doses that are inactive on 5-HT reuptake. Psychopharmacology (Berl) 186, 362e372. Puia, G., Mienville, J.M., Matsumoto, K., Takahata, H., Watanabe, H., Costa, E., Guidotti, A., 2003. On the putative physiological role of allopregnanolone on GABA-A receptor function. Neuropharmacology 44, 49e55. Puia, G., Santi, M.R., Vicini, S., Pritchett, D.B., Purdy, R.H., Paul, S.M., Seeburg, P.H., Costa, E., 1990. Neurosteroids act on recombinant human GABA-A receptors. Neuron 4, 759e765. Qian, M., Krishnan, K., Kudova, E., Li, P., Manion, B.D., Taylor, A., Elias, G., Akk, G., Evers, A.S., Zorumski, C.F., Mennerick, S., Covey, D.F., 2014. Neurosteroid analogues. 18. Structure-activity studies of ent-steroid potentiators of gamma-aminobutyric acid type A receptors and comparison of their activities with those of alphaxalone and allopregnanolone. J. Med. Chem. 57, 171e190. Rapkin, A.J., Lewis, E.I., 2013. Treatment of premenstrual dysphoric disorder. Women’s Health 9, 537e556. Reddy, D.S., 2002. The clinical potentials of endogenous neurosteroids. Drugs Today 38, 465e485.

REFERENCES

Reddy, D.S., 2003. Pharmacology of endogenous neuroactive steroids. Crit. Rev. Neurobiol. 15, 197e234. Reddy, D.S., 2004a. Pharmacology of catamenial epilepsy. Methods Find Exp. Clin. Pharmacol. 26, 547e561. Reddy, D.S., 2004b. Role of neurosteroids in catamenial epilepsy. Epilepsy Res. 62, 99e118. Reddy, D.S., 2008. Mass spectrometric assay and physiologicalpharmacological activity of androgenic neurosteroids. Neurochem. Int. 52, 541e553. Reddy, D.S., 2009. The role of neurosteroids in the pathophysiology and treatment of catamenial epilepsy. Epilepsy Res. 85, 1e30. Reddy, D.S., 2010. Neurosteroids: endogenous role in the human brain and therapeutic potentials. Prog. Brain Res. 186, 113e137. Reddy, D.S., 2011. Role of anticonvulsant and antiepileptogenic neurosteroids in the pathophysiology and treatment of epilepsy. Front. Endocrinol. 2, 38. Reddy, D.S., 2013. Neuroendocrine aspects of catamenial epilepsy. Horm. Behav. 63, 254e266. Reddy, D.S., 2014. Neurosteroids and their role in sex-specific epilepsies. Neurobiol. Dis. 72 (Pt B), 198e209. Reddy, D.S., 2016a. Catamenial epilepsy: discovery of an extrasynaptic molecular mechanism for targeted therapy. Front. Cell. Neurosci. 10 (101), 1e14. Reddy, D.S., 2016b. Neurosteroids for the potential protection of humans against organophosphate toxicity. Ann. N. Y. Acad. Sci. 1378 (1), 25e32. Reddy, D.S., 2017a. Clinical pharmacology of modern antiepileptic drugs. Int. J. Pharm. Sci. Nanotech. 10, 3875e3890. Reddy, D.S., 2017b. Sex differences in the anticonvulsant neurosteroids. J. Neurosci. Res. 95, 661e670. Reddy, D.S., Estes, W.A., 2016. Clinical potential of neurosteroids for CNS disorders. Trends Pharmacol. Sci. 37, 543e561. Reddy, D.S., Gangisetty, O., Briyal, S., 2010. Disease-modifying activity of progesterone in the hippocampus kindling model of epileptogenesis. Neuropharmacology 59, 573e581. Reddy, D.S., Gould, J., Gangisetty, O., 2012. A mouse kindling model of perimenstrual catamenial epilepsy. J. Pharmacol. Exp. Ther. 341, 784e793. Reddy, D.S., Jian, K., 2010. The testosterone-derived neurosteroid androstanediol is a positive allosteric modulator of GABAA receptors. J. Pharmacol. Exp. Ther. 334, 1031e1041. Reddy, D.S., Kim, H.Y., Rogawski, M.A., 2001. Neurosteroid withdrawal model of perimenstrual catamenial epilepsy. Epilepsia 42, 328e336. Reddy, D.S., Kulkarni, S.K., 1997. Differential anxiolytic effects of neurosteroids in the mirrored chamber behavior test in mice. Brain Res. 752, 61e71. Reddy, D.S., Kulkarni, S.K., 2000. Development of neurosteroid-based novel psychotropic drugs. Prog. Med. Chem. 37, 135e175. Reddy, D.S., Mohan, A., 2011. Development and persistence of limbic epileptogenesis are impaired in mice lacking progesterone receptors. J. Neurosci. 31, 650e658. Reddy, D.S., O’Malley, B.W., Rogawski, M.A., 2005. Anxiolytic activity of progesterone in progesterone receptor knockout mice. Neuropharmacology 48, 14e24. Reddy, D.S., Rogawski, M.A., 2000a. Chronic treatment with the neuroactive steroid ganaxolone in the rat induces anticonvulsant tolerance to diazepam but not to itself. J. Pharmacol. Exp. Ther. 295, 1241e1248. Reddy, D.S., Rogawski, M.A., 2000b. Enhanced anticonvulsant activity of ganaxolone after neurosteroid withdrawal in a rat model of catamenial epilepsy. J. Pharmacol. Exp. Ther. 294, 909e915. Reddy, D.S., Rogawski, M.A., 2001. Enhanced anticonvulsant activity of neuroactive steroids in a rat model of catamenial epilepsy. Epilepsia 42, 337e344. Reddy, D.S., Rogawski, M.A., 2002. Stress-induced deoxycorticosteronederived neurosteroids modulate GABA-A receptor function and seizure susceptibility. J. Neurosci. 22, 3795e3805.

81

Reddy, D.S., Rogawski, M.A., 2009. Neurosteroid replacement therapy for catamenial epilepsy. Neurotherapeutics 6, 392e401. Reddy, D.S., Woodward, R., 2004c. Ganaxolone: a prospective overview. Drugs Future 29, 227e242. Reddy, S.D., Younus, I., Clossen, B.L., Reddy, D.S., 2015. Antiseizure activity of midazolam in mice lacking delta-subunit extrasynaptic GABA-A receptors. J. Pharmacol. Exp. Ther. 353, 517e528. Riikonen, R., Rener-Primec, Z., Carmant, L., Dorofeeva, M., Hollody, K., Szabo, I., Krajnc, B.S., Wohlrab, G., Sorri, I., 2015. Does vigabatrin treatment for infantile spasms cause visual field defects? An international multicentre study. Dev. Med. Child Neurol. 57, 60e67. Rogawski, M.A., Loya, C.M., Reddy, K., Zolkowska, D., Lossin, C., 2013. Neuroactive steroids for the treatment of status epilepticus. Epilepsia 54 (Suppl. 6), 93e98. Rogawski, M.A., Reddy, D.S., 2002. Neurosteroids and infantile spasms: the deoxycorticosterone hypothesis. Int. Rev. Neurobiol. 49, 199e219. Romeo, E., Strohle, A., Spalletta, G., di Michele, F., Hermann, B., Holsboer, F., Pasini, A., Rupprecht, R., 1998. Effects of antidepressant treatment on neuroactive steroids in major depression. Am. J. Psychiatry 155, 910e913. Rupprecht, R., Holsboer, F., 1999. Neuroactive steroids: mechanisms of action and neuropsychopharmacological perspectives. Trends Neurosci. 22, 410e416. Sanna, E., Mostallino, M.C., Murru, L., Carta, M., Talani, G., Zucca, S., Mura, M.L., Maciocco, E., Biggio, G., 2009. Changes in expression and function of extrasynaptic GABAA receptors in the rat hippocampus during pregnancy and after delivery. J. Neurosci. 29, 1755e1765. Schally, A.V., Halmos, G., Rekasi, A., Arencibia, J.M., 2001. The actions of LH-RH agonists, antagonists, and cytotoxic analogs on the LHRH receptors on the pituitary and tumors, in infertility and reproductive medicine. In: Devroey, P. (Ed.), Clinics of North America: GnRH Analogs. Saunders, Pennsylvania. Schiller, C.E., Schmidt, P.J., Rubinow, D.R., 2014. Allopregnanolone as a mediator of affective switching in reproductive mood disorders. Psychopharmacology (Berl) 231, 3557e3567. Schmidt, C.L., de Ziegler, D., Gagliardi, C.L., Mellon, R.W., Taney, F.H., Kuhar, M.J., Colon, J.M., Weiss, G., 1989. Transfer of cryopreservedthawed embryos: the natural cycle versus controlled preparation of the endometrium with gonadotropin-releasing hormone agonist and exogenous estradiol and progesterone (GEEP). Fertil. Steril. 52, 609e616. Schu¨le, C., Nothdurfter, C., Rupprecht, R., 2014. The role of allopregnanolone in depression and anxiety. Prog. Neurobiol. 113, 79e87. Schumacher, M., Mattern, C., Ghoumari, A., Oudinet, J.P., Liere, P., Labombarda, F., Sitruk-Ware, R., De Nicola, A.F., Guennoun, R., 2014. Revisiting the roles of progesterone and allopregnanolone in the nervous system: resurgence of the progesterone receptors. Prog. Neurobiol. 113, 6e39. Shen, H., Gong, Q.H., Yuan, M., Smith, S.S., 2005. Short-term steroid treatment increases delta GABAA receptor subunit expression in rat CA1 hippocampus: pharmacological and behavioral effects. Neuropharmacology 49, 573e586. Shen, Y., Rampino, M.A., Carroll, R.C., Nawy, S., 2012. G-protein-mediated inhibition of the Trp channel TRPM1 requires the Gbetagamma dimer. Proc. Natl. Acad. Sci. U. S. A. 109, 8752e8757. Shim, H., Wang, C.T., Chen, Y.L., Chau, V.Q., Fu, K.G., Yang, J., McQuiston, A.R., Fisher, R.A., Chen, C.K., 2012. Defective retinal depolarizing bipolar cells in regulators of G protein signaling (RGS) 7 and 11 double null mice. J. Biol. Chem. 287, 14873e14879. Shirakawa, H., Katsuki, H., Kume, T., Kaneko, S., Akaike, A., 2005. Pregnenolone sulphate attenuates AMPA cytotoxicity on rat cortical neurons. Eur. J. Neurosci. 21, 2329e2335. Smith, S.S., 2002. Withdrawal properties of a neuroactive steroid: implications for GABA-A receptor gene regulation in the brain and anxiety behavior. Steroids 67, 519e528.

82

4. NEUROSTEROIDS: BIOSYNTHESIS, MOLECULAR MECHANISMS, AND NEUROPHYSIOLOGICAL FUNCTIONS IN THE HUMAN BRAIN

Snyder, S.H., Largent, B.L., 1989. Receptor mechanisms in antipsychotic drug action: focus on sigma receptors. J. Neuropsychiatry Clin. Neurosci. 1, 7e15. Sousa, A., Ticku, M.K., 1997. Interactions of the neurosteroid dehydroepiandrosterone sulfate with the GABA(A) receptor complex reveals that it may act via the picrotoxin site. J. Pharmacol. Exp. Ther. 282, 827e833. Spence, K.T., Plata-Salaman, C.R., ffrench-Mullen, J.M., 1991. The neurosteroids pregnenolone and pregnenolone-sulfate but not progesterone, block Ca2þ currents in acutely isolated hippocampal CA1 neurons. Life Sci. 49, PL235e239. Spigelman, I., Li, Z., Banerjee, P.K., Mihalek, R.M., Homanics, G.E., Olsen, R.W., 2002. Behavior and physiology of mice lacking the GABAA-receptor delta subunit. Epilepsia 43 (Suppl. 5), 3e8. Spivak, C.E., 1994. Desensitization and noncompetitive blockade of GABAA receptors in ventral midbrain neurons by a neurosteroid dehydroepiandrosterone sulfate. Synapse 16, 113e122. Spivak, V., Lin, A., Beebe, P., Stoll, L., Gentile, L., 2004. Identification of a neurosteroid binding site contained within the GluR2-S1S2 domain. Lipids 39, 811e819. Stein, D.G., Sayeed, I., 2018. Repurposing and repositioning neurosteroids in the treatment of traumatic brain injury: a report from the trenches. Neuropharmacology 147, 66e73. Steiner, M., Steinberg, S., Stewart, D., Carter, D., Berger, C., Reid, R., Grover, D., Streiner, D., 1995. Fluoxetine in the treatment of premenstrual dysphoria. Canadian fluoxetine/premenstrual dysphoria collaborative study group. N. Engl. J. Med. 332, 1529e1534. Stell, B.M., Brickley, S.G., Tang, C.Y., Farrant, M., Mody, I., 2003. Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit-containing GABAA receptors. Proc. Natl. Acad. Sci. U. S. A. 100, 14439e14444. Strott, C.A., 2002. Sulfonation and molecular action. Endocr. Rev. 23, 703e732. Su, T.P., Schmidt, P.J., Danaceau, M.A., Tobin, M.B., Rosenstein, D.L., Murphy, D.L., Rubinow, D.R., 1997. Fluoxetine in the treatment of premenstrual dysphoria. Neuropsychopharmacology 16, 346e356. Tokuyama, S., Hirata, K., Yoshida, A., Maruo, J., Matsuno, K., Mita, S., Ueda, H., 1999. Selective coupling of mouse brain metabotropic sigma receptor with recombinant Gi1. Neurosci. Lett. 268, 85e88. Tsai, J., Farfel, G.M., Nohria, V., 2011. Ganaxolone is safe and welltolerated as add-on therapy in adults in uncontrolled partial seizures. In: American Epilepsy Society Annual Meeting. Tsai, J., Giller, E., Shaw, K., Pieribone, V., Soufflet, C., Dulac, O., 2006. Outpatient flexible dose, open-label add-on ganaxolone in treatment-refractory pediatric epilepsy. In: American Epilepsy Society Annual Meeting. Tsuda, M., Suzuki, T., Misawa, M., 1997. Modulation of the decrease in the seizure threshold of pentylenetetrazole in diazepam withdrawn mice by the neurosteroid 5alphapregnan-3alpha,21-diol-20-one (alloTHDOC). Addict. Biol. 2, 455e460.

Ueda, H., Yoshida, A., Tokuyama, S., Mizuno, K., Maruo, J., Matsuno, K., Mita, S., 2001. Neurosteroids stimulate G proteincoupled sigma receptors in mouse brain synaptic membrane. Neurosci. Res. 41, 33e40. Upasani, R.B., Yang, K.C., Acosta-Burruel, M., Konkoy, C.S., McLellan, J.A., Woodward, R.M., Lan, N.C., Carter, R.B., Hawkinson, J.E., 1997. 3alpha-Hydroxy-3beta-(phenylethynyl)5beta-pregnan-20-ones: synthesis and pharmacological activity of neuroactive steroids with high affinity for GABA-A receptors. J. Med. Chem. 40, 73e84. Uzunov, D.P., Cooper, T.B., Costa, E., Guidotti, A., 1996. Fluoxetineelicited changes in brain neurosteroid content measured by negative ion mass fragmentography. Proc. Natl. Acad. Sci. U. S. A. 93, 12599e12604. Uzunova, V., Sheline, Y., Davis, J.M., Rasmusson, A., Uzunov, D.P., Costa, E., Guidotti, A., 1998. Increase in the cerebrospinal fluid content of neurosteroids in patients with unipolar major depression who are receiving fluoxetine or fluvoxamine. Proc. Natl. Acad. Sci. U. S. A. 95, 3239e3244. Venugopal, S., Martinez-Arguelles, D.B., Chebbi, S., HullinMatsuda, F., Kobayashi, T., Papadopoulos, V., 2016. Plasma membrane origin of the steroidogenic pool of cholesterol used in hormone-induced acute steroid formation in Leydig cells. J. Biol. Chem. 291, 26109e26125. Vriens, J., Owsianik, G., Hofmann, T., Philipp, S.E., Stab, J., Chen, X., Benoit, M., Xue, F., Janssens, A., Kerselaers, S., Oberwinkler, J., Vennekens, R., Gudermann, T., Nilius, B., Voets, T., 2011. TRPM3 is a nociceptor channel involved in the detection of noxious heat. Neuron 70, 482e494. Westenberg, H.G., 2009. Recent advances in understanding and treating social anxiety disorder. CNS Spectr. 14, 24e33. Whissell, P.D., Lecker, I., Wang, D.S., Yu, J., Orser, B.A., 2015. Altered expression of deltaGABA-A receptors in health and disease. Neuropharmacology 88, 24e35. Wohlfarth, K.M., Bianchi, M.T., Macdonald, R.L., 2002. Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit. J. Neurosci. 22, 1541e1549. Wu, F.S., Chen, S.C., 1997. Mechanism underlying the effect of pregnenolone sulfate on the kainate-induced current in cultured chick spinal cord neurons. Neurosci. Lett. 222, 79e82. Wu, F.S., Gibbs, T.T., Farb, D.H., 1991. Pregnenolone sulfate: a positive allosteric modulator at the N-methyl-D-aspartate receptor. Mol. Pharmacol. 40, 333e336. Wu, X., Gangisetty, O., Carver, C.M., Reddy, D.S., 2013. Estrous cycle regulation of extrasynaptic delta-containing GABA-A receptormediated tonic inhibition and limbic epileptogenesis. J. Pharmacol. Exp. Ther. 346, 146e160. Xu, Y., Dhingra, A., Fina, M.E., Koike, C., Furukawa, T., Vardi, N., 2012. mGluR6 deletion renders the TRPM1 channel in retina inactive. J. Neurophysiol. 107, 948e957. Yaghoubi, N., Malayev, A., Russek, S.J., Gibbs, T.T., Farb, D.H., 1998. Neurosteroid modulation of recombinant ionotropic glutamate receptors. Brain Res. 803, 153e160.

C H A P T E R

5 Neurotrophins and Neurotrophin Receptors M.L. Franco, R. Comaposada-Baro´, M. Vilar Molecular Basis of Neurodegeneration Unit, Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain

1. INTRODUCTION

deleting the NTs or their receptors in mice and the implication of the NTs in several human diseases.

The discovery of nerve growth factor (NGF) by Rita Levi-Montalcini and Stanley Cohen in the 1950s represents an important milestone in the processes that led to modern cell biology and molecular neuroscience (Cohen et al., 1954; Levi-Montalcini et al., 1954; LeviMontalcini and Hamburger, 1951). NGF was the first growth factor to be identified, paving the way to the identification of new soluble growth factors. NGF was discovered due to its trophic effects on sensory and sympathetic neurons. In 1982, brain-derived neurotrophic factor (BDNF), the second member of the “neurotrophic” family of neurotrophic factors, was shown to promote survival of a subpopulation of dorsal root ganglion neurons, and it subsequently was purified from pig brain (Barde et al., 1982). Since then, other members of the neurotrophin family such as neurotrophin-3 (NT3) (Maisonpierre et al., 1990b) and neurotrophin-4/5 (NT-4/5) (Hallbook et al., 1991) have been described, each with a distinct profile of trophic effects on subpopulations of neurons in the peripheral and central nervous systems. The neurotrophin (NT) protein family is implicated in the maintenance and survival of the peripheral and central nervous systems (Ceni et al., 2014; Chao, 2003; Hempstead, 2014; Lu et al., 2014). NTs interact with two distinct receptors, a cognate member of the Trk receptor tyrosine kinase family and the common p75 neurotrophin receptor, which belongs to the tumor necrosis factor receptor superfamily of death receptors (Friedman and Greene, 1999; Huang and Reichardt, 2003). The interaction of mature neurotrophins with their receptors induces cell survival, cell death, differentiation, and synaptic plasticity activities. In this chapter we will describe the structural determinants of NTs binding to its receptors, the signalling pathways triggered, the functional consequences of

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00005-5

2. STRUCTURES OF NEUROTROPHINS AND NEUROTROPHIN RECEPTORS 2.1 Neurotrophins Neurotrophins are initially synthesized as precursors or proneurotrophins consisting of an N-terminal prodomain and a C-terminal mature domain. Following translation, proneurotrophins form noncovalent dimers via interactions of the mature domain. Mature neurotrophin proteins are noncovalent homodimers that contain a special three-dimensional structure, known as the cysteine knot (McDonald et al., 1991). The first reported structure of the cystine-knot family of growth factors was the one from NGF (McDonald et al., 1991). This structure is shared by other growth factors like transforming growth factor-b, platelet-derived growth factor, and others, forming a large superfamily of growth factors with a similar tertiary fold (McDonald and Hendrickson, 1993; Sun and Davies, 1995). The cysteine knot consists of three disulfide bonds that form a true knot of the polypeptide chain (Fig. 5.1A). The NGF protomer contains three pairs of antiparallel beta strands connected by three P-hairpin loop structures (Fig. 5.1A). Based on sequence alignments, neurotrophin residues are generally divided into two categories, conserved or variable. The dimer interface is composed of b-strands that maintain the conformation; these hydrophobic core residues are highly conserved. In addition, conserved residues are implicated in neurotrophin binding to their receptors (Fig. 5.1B). Variable residues represent elements of specificity to the different receptors and are usually located in the b-loops.

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5. NEUROTROPHINS AND NEUROTROPHIN RECEPTORS

(A)

(B)

NGF dimer

cysteine-knot cysteine-knot NGF cys 15- cys 80 cys 58- cys 108 cys 68- cys 110 90º NGF protomer

(D)

(C) NT-3

p75-ECD p75-ECD

TrkA-ECD TrkA-ECD NGF

FIGURE 5.1 Structure of neurotrophins and neurotrophin binding domains. (A) The three-dimensional structure of one protomer of NGF showing the location of the representative cysteine-knot (inside the box). The cysteine residues forming the cysteine-knot are highlighted on the right. (B) The three-dimensional structure of NGF non-covalent dimer from two different angles, showing the two protomers (red and green) (PDB: 1BET (McDonald et al., 1991)). (C) The three-dimensional structure of p75 extracellular domain (ECD) bound to NT3 (PDB: 3BUK (Gong et al., 2008)). (D) The three-dimensional structure of TrkA extracellular domain (ECD) bound to NGF (PDB: 2IFG (Wehrman et al., 2007)).

2.2 Complex of Neurotrophin With Neurotrophin Receptor 2.2.1 p75NTR p75 is a type I membrane protein (N-terminal outside of the cell) with an extracellular region very rich in cysteines residues, and an intracellular domain without a catalytic activity and containing a Death Domain (DD). The extracellular domain (ECD) of p75NTR, is characterized by four cysteine-rich domains (CRDs) termed CRD1 through CRD4 in an elongated and kinked structure (Fig. 5.1C). Twelve pairs of disulfide bonds are evenly spaced along p75ECD simulating a ladder. Experimental data from several laboratories demonstrated that p75NTR interacts with the neurotrophins through the four CRDs in its extracellular domain (Baldwin et al., 1992; Yan and Chao, 1991) and does it by forming homodimers (Grob and Bothwell, 1983; Grob et al., 1983, 1985; Fig. 5.1C). In 2004, He and Garcia reported the structure of the complex of p75ECD with NGF (He and Garcia, 2004) and found that it contains only one p75NTR protomer for each NGF dimer, forming a 2:1 stoichiometry (p75:NT) (He and Garcia, 2004). Later, however, experiments of the interaction of the

isolated p75ECD and NGF in solution (Aurikko et al., 2005) and the reported crystal structure of p75ECD with NT-3 (Gong et al., 2008) confirmed a 2:2 stoichiometry for p75ECD:NT3 (Fig. 5.1C). p75 forms constitutive dimers through a conserved cysteine residue close to the transmembrane domain (Nadezhdin et al., 2016; Vilar et al., 2009). Mutation of this cysteine residue impairs the activity of p75 in several cell-death paradigms in vitro and in vivo (Tanaka et al., 2016; Vilar et al., 2009). p75 forms constitutive dimers through a conserved cysteine residue close to the transmembrane domain (Nadezhdin et al., 2016; Vilar et al., 2009). Mutation of this cysteine residue impairs the activity of p75 in several cell-death paradigms in vitro and in vivo (Tanaka et al., 2016; Vilar et al., 2009). The intracellular domain of p75 presents two different regions. A flexible intrinsically disordered region of 60-70 residues called the juxtamembrane domain (JTM) and a well folded region that constitutes the death domain (DD). DDs are found in other death receptors such as Fas and TNF-R1 and death receptorinteracting adapter proteins. A region in the JTM called Chopper has been involved in mediating the cell death (Coulson et al., 2008; Coulson et al., 2000; Underwood et al., 2008). The intracellular domain of p75 presents

3. EVOLUTION OF THE NEUROTROPHIN-SIGNALING SYSTEM

two different regions. A flexible intrinsically disordered region of 60-70 residues called the juxtamembrane domain (JTM) and a well folded region that constitutes the death domain (DD). p75NTR contains a death domain (DD) in the intracellular region (Liepinsh et al., 1997). DDs are found in other death receptors such as Fas and TNF-R1 and death receptor-interacting adapter proteins such as FADD and TRADD. DDs are protein-protein interaction domains and they function as modules to bring closer different proteins involved in several signaling cascades (Park et al., 2007). The DD of p75 interacts with several protein interactors to mediate different functional roles as it is shown below. 2.2.2 TrkA Trks belong to the receptor tyrosine kinase (RTK) family. They consist of an extracellular regions, and single transmembrane domain and an intracellular tyrosine kinase domain. The first three domains of Trk consist of a leucine-rich region (Trk-d1) flanked by two CRDs (Trkd2 and Trk-d3). The fourth and fifth domains (Trk-d4 and Trk-d5) are immunoglobulin (Ig)-like domains, which are followed by a 30-residue-long linker connecting the extracellular portion of the receptor to the single transmembrane region and to the intracellular region that contains the kinase domain. The NT binding domain is located in the Trk-d5 Ig domain (Wiesmann et al., 1999), although other domains also seem to participate in activation by neurotrophins (Zaccaro et al., 2001; Arevalo et al., 2001). In 2007 the laboratory of Dr. Christopher Garcia reported the three-dimensional structure of full-length TrkA-ECD in complex with NGF (Wehrman et al., 2007). The overall structure of the NGF/ TrkA complex was described by Garcia’s laboratory as a “crab with extended pinchers” (Wehrman et al., 2007; Fig. 5.1D). The crab body is composed of the NGF homodimer and is flanked by the pinchers, comprised of the TrkA d1ed5 domains (Wehrman et al., 2007). The C-terminal Ig-C2 domain of TrkA engages the sides of the NGF homodimer, as previously shown. One copy of a TrkA ECD binds to each side of NGF homodimer, confirming the originally determined 2:2 stoichiometry of the NGF/Ig-C2 complex (Wiesmann et al., 1999; Ultsch et al., 1999). The long axis of TrkA is roughly parallel to the long axis of NGF. Of the five TrkA domains, only the C-terminal Ig-C2 domain interacts with NGF in the complex structure, as originally seen in the truncated complex structure (Wiesmann et al., 1999; Ultsch et al., 1999). The interaction between Ig-C2 and NGF is rather striking in that the top loops of the Ig domain penetrate into the saddle-like depression along the tops of the NGF central b sheets, in an almost orthogonal fashion (Wehrman et al., 2007; Wiesmann et al., 1999; Ultsch et al., 1999).

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2.2.3 p75NTR Interactions With TrkA p75NTR interacts with TrkA to form a high-affinity binding complex (Hempstead et al., 1991). While TrkA alone was found to bind NGF with nanomolar affinity, coexpression with p75NTR was discovered to increase this interaction by 100-fold (Esposito et al., 2001; Hempstead et al., 1991). When p75 is coexpressed with TrkA, mature NGF binds to the complex with higher affinity than is observed when TrkA is expressed in the absence of p75 and facilitates cell survival (Lee et al., 1994; Davies et al., 1993). The mechanism of this function of p75 is not yet clear, as some authors suggests that the binding of NGF to p75 is required to pass the NGF to TrkA (Barker, 2009) and others suggests that p75 induces a conformational change on TrkA independent of the binding of the neurotrophin (Esposito et al., 2001). Thus, p75NTR can augment TrkA-mediated survival by increasing its interaction with neurotrophins. This may explain the sensory neuron loss in the animals lacking p75 and that higher doses of NGF are needed to promote survival of sensory and sympathetic neurons from p75NTR KO mice (Lee et al., 1994; Davies et al., 1993). Although functional interaction between p75NTR and Trk receptors is clear, the molecular details are not fully understood. The existence of this complex has been demonstrated by crosslinking (Ross et al., 1998), coimmunoprecipitation (Bibel et al., 1999; Huber and Chao, 1995; Jung et al., 2003), copatching (Ross et al., 1996), fluorescence recovery after photobleaching (Wolf et al., 1995), and FRET studies (Sykes et al., 2012; Iacaruso et al., 2011). Biochemical studies suggest that the transmembrane and intracellular domains of p75NTR, but not the neurotrophin-binding portion of the extracellular domain, are required for the high-affinity complex (Esposito et al., 2001).

3. EVOLUTION OF THE NEUROTROPHIN-SIGNALING SYSTEM Although initially it was thought that the neurotrophin system was a recent invention of the vertebrate lineage, whole genome sequencing efforts showed that the neurotrophin-signaling system, including neurotrophinlike ligands, p75NTR, and Trk-like receptors, evolved before the evolution of vertebrates (see review in Bothwell, 2006). Bilaterian organisms fall within two major branches: (1) protostomes including arthropods and (2) deuterostomes, from which vertebrates evolved (Fig. 5.2). As clear orthologs of neurotrophin, p75NTR, and Trk genes were not identified in model protostomes species such as Drosophila melanogaster or Caenorhabditis elegans (Bothwell, 2006), it was then assumed that the neurotrophin system was a recent invention. However, it has been recently described that the genome of the

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Neurotrophins, p75NTR, Trk

ra

Porife

ria Cnida

lida Anne

ata ?) sca ematoda (rthropoda chinoderm hordata N C Mollu E A

PROTOSTOMES

DEUTEROSTOMES

BILATERIA

Choanoflagellate ancestor

FIGURE 5.2 Phylogenetic tree of the animal kingdom. The presence of at least one member of a neurotrophin (NT), p75NTR, and a Trk in each phylum inside the green box suggests that the neurotrophin system appeared at least in the common ancestor of the Bilateria. The question mark indicates that a bona fide neurotrophin system has not been described yet in the Nematoda. The analysis of the genomes of the non-Bilateria animals will reveal if the neurotrophins were already present in the last common animal ancestor.

arthropod Daphnia pulex encodes a full neurotrophin system formed by a neurotrophin, Trk, and p75NTR orthologs (Wilson, 2009). A similar system has been found recently in the protostome marine annelid Platynereis dumerilii (Lauri et al., 2016). And the genomes of prechordate deuterostomes Strongylocentrotus purpuratus (sea urchin) and Saccoglossus kowalevskii (acorn worm) also encode neurotrophin, p75NTR, and Trk orthologs (Bothwell, 2006) together with the presence of Trk proteins in the Lymnaea mollusc (Beck et al., 2004). Thus, the shared ancestor of protostomes and deuterostomes must have possessed neurotrophins, p75NTR, and Trk orthologs (Fig. 5.2). Interestingly the neurotrophin-signaling system has apparently been lost during evolution of several bilaterian classes, like in C. elegans and in Drosophila. In Drosophila a family of neurotrophic factors has been named as neurotrophins (Ulian-Benitez et al., 2017; Zhu et al., 2008), although they do not share a high homology with the neurotrophins. The chordate ancestor of modern vertebrates probably had only one neurotrophin and one Trk gene. The multiple neurotrophin and Trk paralogs in modern vertebrates (four neurotrophins in mammals, three neurotrophins in birds, three Trks in both mammals and birds) apparently arose as a result of the two genome duplications that occurred as an early event in vertebrate evolution (Hallbook et al., 2006; Hallbook, 1999). Genome-wide sequencing of early species like cnidarians or sponges with primitive nervous systems will reveal how far the neurotrophin system could be tracked.

4. BIOCHEMICAL REACTIONS (INTERACTION WITH RECEPTORS, RECEPTOR ACTIVATION AND SIGNALING PATHWAYS) The four mammalian neurotrophins interact with four receptors: p75NTR, TrkA, TrkB, and TrkC (Fig. 5.3). All four neurotrophins, both as proneurotrophins and as mature neurotrophins, can bind and activate signaling by p75NTR, whereas Trks bind only to the mature neurotrophins. p75NTR binds with similar affinity to all four neurotrophins, NGF, BDNF, NT3, and NT4, and proneurotorphins (Rodriguez-Tebar et al., 1990, 1991; Rodriguez-Tebar et al., 1992; Hempstead, 2014), although with somewhat different kinetics. NGF binds and activates TrkA, NT3 preferentially binds and activates TrkC, and BDNF and NT4 preferentially bind and activate TrkB. NT3 is the most promiscuous of the neurotrophins, as alternative splicing of the TrkA transcript can be activated by NT3 (Clary and Reichardt, 1994). However, NGF and NT3 influence TrkA signaling differently and are not functionally equivalent (Harrington et al., 2011). Neurotrophin receptors are expressed in a wide variety of types of neurons and glia in both the central and peripheral nervous system and also in a variety of nonneural cell types. This explains why neurotrophins have an extraordinary range of biologic functions. The function of these multiple receptors is complex, as p75NTR and Trk receptors can

4. BIOCHEMICAL REACTIONS

NGF NT-3

BDNF NT-4

NT-3

NGF BDNF NT-3 pro-NGF pro-BDNF pro-NT3

TrkA

TrkB

TrkC

p75NTR

FIGURE 5.3 Specificity of neurotrophins and neurotrophin receptors. Trks bind only to the mature neurotrophins as indicated, whereas p75NTR binds with similar affinity to all four neurotrophins NGF, BDNF, NT3, and NT4 and to proneurotrophins.

function independently, but in neurons that express both p75NTR and Trk receptors, the receptors interact physically in ways that may alter the signaling properties of each other.

4.1 p75NTR Signaling The overall effect of p75NTR signaling is highly dependent upon the presence or absence of the Trk receptor or the cell type. In general, though not always, simultaneous activation of both Trk receptors and p75NTR by mature neurotrophins results in cell survival, and selective activation of p75NTR in the absence of Trk receptor activation or by proneurotrophins often promotes cell death rather than survival. 4.1.1 p75 in Cell Death and Apoptosis: Mature Neurotrophins During development, p75NTR promotes the naturally occurring elimination of neurons within the developing basal forebrain (Naumann et al., 2002), trigeminal ganglia (Agerman et al., 2000), retina (Frade et al., 1996), superior cervical ganglion (Bamji et al., 1998), and spinal cord (Frade and Barde, 1999). The ability of p75NTR to induce programmed cell death in response to ligand binding has been observed in a wide variety of neuronal and nonneuronal cell types, including retinal neurons in the chick (Frade et al., 1996), oligodendrocytes in rat (Casaccia-Bonnefil et al., 1996), sympathetic (Bamji et al., 1998; Linggi et al., 2005), motor (Sedel et al., 1999), and hippocampal neurons (Volosin et al., 2008).

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During embryonic development the role of p75NTR has been particularly well characterized in sympathetic neurons. These neurons express TrkA and p75NTR, which together mediate a survival signal in response to NGF. However, stimulation of these neurons with BDNF led to apoptosis by activation of p75 promoting p75NTR-dependent death of neighboring neurons receiving insufficient NGF signal (Deppmann et al., 2008; Bamji et al., 1998). Although the mechanism of cell death is still under scrutiny an accumulation of evidence indicates that p75NTR-induced apoptosis occurs via the mitochondrial pathway cascade. Activation of JNK by p75NTR has been demonstrated in several cases (Casaccia-Bonnefil et al., 1996; Bamji et al., 1998; Friedman, 2000), and inhibition of JNK prevented the cell death (Friedman, 2000; Kenchappa et al., 2010). Induction of apoptosis by p75NTR has also been linked to phosphorylation of Bim (Becker et al., 2004) and Bad (Bhakar et al., 2003), cytochrome c release (Bhakar et al., 2003), and cleavage of procaspase-3, -6, -7, or -9 (Bhakar et al., 2003; Tabassum et al., 2003). In some cases, cell death is associated with upregulation of p53 (Aloyz et al., 1998; Linggi et al., 2005). The mechanism of activation of JNK by p75NTR is not clear. One mechanism could be mediated by the participation of protein adaptors. TRAF1e6 have been reported to associate with p75NTR (Khursigara et al., 1999). TRAF6 associates with p75NTR in a liganddependent manner and mediates signaling from the receptor to both JNK and NF-k B (Khursigara et al., 1999). In addition, sympathetic neurons from traf6_/_ mice fail to undergo apoptosis (Yeiser et al., 2004), indicating that TRAF6 is essential for p75NTR-mediated apoptotic signaling in vivo. TRAF6 also associates with the neurotrophin receptor-interacting factor (NRIF) to promote JNK activation (Gentry et al., 2004), and interaction of NRIF with TRAF6 and p75NTR appears to be critical for p75NTR-mediated JNK activation and apoptosis. A recent report suggest that TRAF6 and RIP2 compete with the DD of p75 to sort cell death/survival pathways (Kisiswa et al. 2018). Beside the mitochondrial pathway, p75 induces cell death by the generation of the lipid molecule ceramide (Chao, 1995; Dobrowsky et al., 1994). Multiple reports showed the ability of p75NTR to stimulate ceramide production in oligodendrocytes (Casaccia-Bonnefil et al., 1996), hippocampal neurons (Brann et al., 2002), and Schwann cells (Hirata et al., 2001). An increase in the levels of ceramide leads to the activation of JNK (Westwick et al., 1995), so ceramide may couple p75NTR to JNK phosphorylation. Inhibition of sphingomyelinase, the enzyme that produces ceramide from sphingomyelin, prevented JNK activation and apoptosis (Westwick et al., 1995). However, the increase of ceramide levels does not always result in cell death. In

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fact, p75NTR-mediated ceramide production has also been linked to the promotion of cell survival (DeFreitas et al., 2001; McCollum and Estus, 2004). Further studies are needed to elucidate the mechanisms of ceramide production mediated by p75NTR activation and how ceramide elicits its effects in various cellular contexts. 4.1.2 p75 in Cell Death and Apoptosis: Proneurotrophins Unprocessed proneurotrophins interact with high affinity to a complex consisting of p75 and the protein sortilin, wherein the mature domain binds to p75, and the prodomain binds to sortilin (Nykjaer et al., 2004; Teng et al., 2005; Jansen et al., 2007; Yano et al., 2009). Proneurotrophins do not activate Trk receptors (Boutilier et al., 2008). Sortilin is a member of the Vps10p-domain receptor family (Nykjaer et al., 2004; Teng et al., 2005), which include SorLA, and SorCS-1, -2, and -3; they are type I transmembrane receptors with multifunctional roles like modulation of protein sorting and trafficking, as well as regulation of signal transduction (Willnow et al., 2008). In vivo, sortilin is required for developmental p75NTRmediated cell death of retinal ganglion cells (Jansen et al., 2007). Sortilin is not required for all p75NTR-mediated cell death, as sortilin KO mice did not have defects in the apoptosis of sympathetic neurons (Jansen et al., 2007). However, sortilin seems to play a role in the impairment of age-related neurodegeneration of these neurons (Jansen et al., 2007). Pro-BDNF has been also implicated in cell death by forming a complex with p75 and sortilin (Deinhardt et al., 2011, Teng et al., 2005). The mechanism is expected to be similar to the case of pro-NGF. However the case of the pro-domain of pro-BDNF requires a special mention. It has been recently reported that isolated from the mature form, the prodomain is a new synaptic modulator in the central nervous system and enhanced hippocampal LTD (Mizui et al., 2015). The effect of the prodomain requires the expression of p75NTR, although the mechanism is not yet elucidated. 4.1.3 p75 in Cell Survival and Activation of NF-kB Apart from cell death, there is evidence that p75NTR can activate an independent prosurvival signal. The molecular mechanisms by which p75NTR promotes survival independent of the Trk receptors are not fully understood; however, one downstream pathway that has been identified involves the transcription factor nuclear factor kappa B (NF-kB) (Kraemer et al., 2014). The activation of NF-kB by p75NTR has been reported in Schwann cells (Carter et al., 1996; Khursigara et al., 1999, 2001), Schwannoma cells (Gentry et al., 2000), trigeminal neurons (Hamanoue et al., 1999), and hippocampal neurons (Culmsee et al., 2002). Since TRAF6 promotes both NF-kB and JNK activation, it was recognized as a potential control point for determining

survival versus apoptotic signaling (Kraemer et al., 2014). How TRAF6 selectively promotes one pathway over the other remains to be fully elucidated; however, the finding that the adaptor protein receptorinteracting protein 2 (RIP2) directly associates with the death domain of p75NTR provided an important clue (Khursigara et al., 2001; Lin et al., 2015). Expression of RIP2 in Schwann cells conferred NGF-dependent activation of NF-kB through interaction with TRAF6 (Khursigara et al., 2001). Thus, RIP2 expression may serve as the key toggle, switching p75 signaling from JNK to NF-kB. Actually this could be the case. Mice lacking RIP2 has increased cerebellar granule neurons (CGN) apoptosis and this is mediated by p75NTR. Mechanistically, this resulted from increased association of TRAF6 with p75NTR, leading to enhanced JNK activity in CGNs lacking RIP2 (Kisisva et al. 2018). 4.1.4 p75 in Cell Survival and Antioxidative Stress The p75NTR has been shown to mediate antioxidant function in PC12 rat pheochromocytoma cells, and this requires the g-secretase cleavage on p75 and release of its intracellular domain (Tyurina et al., 2005). However the mechanism of this antioxidative and protective role of p75 is not known, although tyrosine phosphorylation of p75-ICD plays a role (Zhang et al., 2009). In addition, p75NTR was recently shown to regulate the stability of HIF1a (Le Moan et al., 2011), a transcription factor induced by oxidative stress that controls the expression of a wide variety of genes involved in protection from reactive oxygen species and, importantly, promoting cell survival. p75-ICD binds to Siah2, an E3 ubiquitin ligase, which targets HIF1a for degradation. The interaction between p75-ICD and Siah2 leads to upregulation of HIF1a and increased expression of vascular endothelial growth factor, which promoted angiogenesis after retinal hypoxia (Le Moan et al., 2011). As HIF1a induce genes involved in cell survival, it will be interesting to determine whether this pathway has a role in promoting neuronal survival in response to activation of the receptor.

4.2 Trk Signaling The signaling pathways activated by Trk receptors impact many diverse neuronal functions, including cell survival and differentiation, axonal and dendritic growth and arborization, synapse formation, and synaptic plasticity (Fig. 5.4; Huang and Reichardt, 2003; Deinhardt and Chao, 2014). The mechanism of Trk signaling involves phosphorylation of specific tyrosine residues upon neurotrophin binding (Huang and Reichardt, 2003). These phosphorylated tyrosine sites create docking sites to recruit proteins that initiate the activation of intracellular signaling pathways. The Y490 and Y785

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Trk p75NTR

C-C Frs2 SOS

RhoA RhoGDI

Gbr2

PLCγ GAB1

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IKK

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NFκB ERK

survival

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LTP Akt

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differentiation

actin

axonal growth

apoptosis

survival

FIGURE 5.4 Signaling pathways and main protein interactors of Trk and p75NTR. The signaling pathways of Trk (left) and p75NTR (right) upon NT binding are shown. Trk signaling induces cell survival, cell differentiation, and synaptic plasticity. P75NTR may induce cell survival or cell death depending on the cell type and ligand.

and their corresponding residues in TrkB and TrkC serve as the main docking sites to signaling pathways such as the Shc-extracellular signal-regulated kinase (ERK) or phospholipase C-g (PLC-g) pathways, respectively. The Y670, Y674, and Y675 residues located within the tyrosine kinase domain can also recruit adaptor proteins after phosphorylation, including the Grb2 and SH2B adaptor proteins. The three most studied pathways downstream of Trk receptor activation are described in the following sections. 4.2.1 PLC-g Phosphorylation of the most C-terminal tyrosine, Y785, leads to the recruitment and activation of PLC-g, which hydrolyzes phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) into diacylglycerol (DAG) and inositol tris-phosphate (IP3). IP3 leads to release of intracellular Ca2þ, which activates Ca2þ-dependent enzymes such as Ca2þ-calmodulin-regulated protein kinases (CaM kinases) and the phosphatase calcineurin. Additionally, the release of Ca2þ and the production of DAG activate protein kinase C (PKC), which stimulates ERK and the transient receptor potential channel, contributes to the BDNF-induced rise of Ca2þ at growth cones and

synapses. Other activities that are affected include the formation of TrkB-postsynaptic density 95 (PSD95) complexes at synapses and cAMP response element binding protein (CREB)-dependent transcription. In addition, PLC-g signaling in response to both NGF and BDNF has been implicated in chemoattraction of axonal growth cones. The physiologic functions of TrkBmediated PLCg signaling pathways have been tested in vivo by mutating the recruitment site, Y816, to phenylalanine (Minichiello et al., 2002). Mice homozygous for the Y816F mutation (trkBPLC-/PLC-) have a normal life span but are hyperactive compared with control littermates. Moreover, these mice have significant deficiencies in the induction of both the early and late phases of hippocampal CA1 long-term potentiation (LTP) (Minichiello et al., 2002). Taken together, these data indicate that signaling initiated at the PLCg1 docking site on TrkB is important for the initiation and maintenance of hippocampal LTP. 4.2.2 PI3K-Akt Phosphorylation of Y490 in TrkA or Y515 in TrkB creates a Shc binding site. This leads to activation of phosphatidylinositol 3-kinase (PI3K). As a consequence, Akt

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translocates to the plasma membrane and becomes activated. Akt activity results in increased protein translation, in enhancing axonal growth, and in mediating neuronal survival. Direct activation by Ras of PI3K is a major pathway through which Trk signaling promotes cell survival (Vaillant et al., 1999; Holgado-Madruga et al., 1997). After its own phosphorylation, Shc recruits the adaptor protein, Grb2, complexed with SOS, an exchange factor for Ras. Phosphorylated Grb2 recruits the adaptor proteins Gab1 and Gab2 that recruit and facilitate the activation of Class I PI3-kinases. In response to growth factor signaling, Gab proteins also nucleate formation of complexes that include the tyrosine phosphatase Shp2 (Liu and Rohrschneider, 2002), enhancing the activation of MAP kinase signaling. Akt also phosphorylates glycogen synthase kinase 3-b (GSK3b), downregulating its activity, (Hetman et al., 2000) and contributes to the prosurvival effects of Trk activation. Like p75, activation of Trk also promotes activation of NFkB (Foehr et al., 2000). IkB is a substrate of Akt, and Akt-mediated phosphorylation of IkB promotes its degradation, which results in NFkBpromoted gene transcription and neuronal survival (Foehr et al., 2000). The forkhead transcription factor, FKHRL1, is another substrate of Akt (Brunet et al., 2002). Phosphorylation of FKHRL1 induces its export from the nucleus, preventing the expression of proapoptotic genes. 4.2.3 MAPK In addition to PI3K-Akt signaling, the creation of a Shc binding site at Y490 initiates downstream RasERK signaling (Fig. 5.4). Activation of the Ras-MAPK/ ERK signaling cascade is essential for neurotrophindependent differentiation of PC12 cells and neurons. In PC12 cells, MAPK signaling is associated with cell proliferation and cell differentiation. Recruitment of the Grb2/SOS complex stimulates the activation of Ras and the transient activation of the c-Raf/MEK/ERK cascade but not prolonged activation of ERK signaling. In contrast, prolonged MAPK activation depends on a distinct signaling pathway involving the adapter protein Crk, the guanine nucleotide exchange factor C3G, the small G protein Rap1, the protein tyrosine phosphatase Shp2, and the serine threonine kinase B-Raf (Huang and Reichardt, 2003). Trk signaling appears to initiate activation of this pathway by recruitment of an adaptor fibroblast growth factor receptor substrate-2 (Frs2) to phosphorylated Y490. Frs2 provides binding sites for additional signaling proteins, including the adaptors Grb2 and Crk and the enzymes c-Src and Shp2. Association with Crk results in activation of the guanyl nucleotide exchange factor C3G for Rap1. Rap-1-GTP stimulates B-Raf, which initiates the ERK cascade. The various MAP kinases activated through Ras and Rap1

have different downstream targets that synergize with each other to mediate gene transcription and cell differentiation. Sustained ERK signaling lasting 3e4 h in response to NGF induces neuronal differentiation in PC12 cells, whereas more transient ERK signaling lasting 30e60 min in response to EGF promotes proliferation. It has been described a transcriptional program in PC12 cells whose activity is associated with sustained ERK signaling and neuronal differentiation. Mullenbrock et al. identified a set of 69 genes preferentially upregulated during sustained ERK signaling and provided evidence that transcription factors AP-1 and CREB cooperatively contribute to the preferential expression of some genes (Mullenbrock et al., 2011). In addition, recent data suggest that the transcription factor Egr1 also plays a cooperative role in the induction of some of these genes (Adams et al., 2017). MAPK signaling may lead to local axonal growth as well as to the initiation of CREB-mediated transcriptional events.

5. PHYSIOLOGIC FUNCTIONS OF THE NEUROTROPHINS The identification of trophic factors that promote neuronal survival began with the seminal work of Levi-Montalcini and colleagues, who demonstrated that specific populations of peripheral neurons during the period of target tissue innervation require the NGF supplied at the target (Levi-Montalcini and Cohen, 1960). The “neurotrophic hypothesis” predicts that growth factor concentration is limiting, resulting in selective neuronal survival. However, as NGF promotes survival of only a subset of neurons, subsequent searches led to the identification of the additional members of the NGF family (BDNF, NT3, NT4). A major contribution to understanding the in vivo functions of neurotrophins has come from the study of the distribution of neurotrophins and their receptors in embryonic and adult tissues of different animal species. Among them, sensory neurons are the most accessible neuronal populations for experimental studies during development both in vitro and in vivo. Experiments using different types of sensory neurons in cell culture showed that neurotrophins prevent or inhibit neuronal cell death. In addition, some neurons switch their requirement for particular neurotrophins during development. However, it was not until the availability of genetically engineered mouse models obtained by targeted deletion of neurotrophins or their receptors that it was possible to test the early hypotheses on neurotrophin functions and unravel their in vivo function during mouse development. Next, we will summarize their role in the nervous system, obtained using some of the most important neurotrophin and

5. PHYSIOLOGIC FUNCTIONS OF THE NEUROTROPHINS

neurotrophin receptor deletion mice (reviewed elsewhere like in Snider, 1994; Fritzsch et al., 2004; Kirstein and Farinas, 2002; Tessarollo and Hempstead, 1998; Tessarollo, 1998; Chalazonitis, 2004; Casaccia-Bonnefil et al., 1999; Conover and Yancopoulos, 1997).

5.1 The Nervous System 5.1.1 TrkA/NGF Signaling TrkA null animals have normal motor function, but display severe sensory and sympathetic neuropathies and die within 1 month of birth (Snider, 1994; Smeyne et al., 1994). They have extensive neuronal cell loss in trigeminal, sympathetic, and dorsal root ganglia, as well as a decrease in the cholinergic basal forebrain projections to the hippocampus and cortex. This was an almost identical phenotype as the NGF deletion mice (Crowley et al., 1994). Animals homozygous for NGF disruption failed to respond to noxious mechanical stimuli, and histological analysis revealed profound cell loss in both sensory and sympathetic ganglia (Crowley et al., 1994). Within dorsal root ganglia, effects of the mutation appeared to be restricted to small and medium peptidergic neurons. These observations confirm the critical dependence of sensory and sympathetic neurons on NGF and demonstrate that other neurotrophins are not able to compensate for the loss of NGF action on these cells. However the effect of the TrkA/NGF signaling in the basal forebrain cholinergic neurons (BFCN) was milder, and they continue to differentiate and express phenotypic markers for the life span of the null mutant mice (Crowley et al., 1994). Due of the importance of the BFCN in Alzheimer disease and the early death of TrkA and NGF knockout animals, the role of TrkA/NGF signaling in the development and survival of BCFN has been recently reanalyzed using conditional KO animals. Two independent groups generated a specific deletion of the TrkA gene in the BCFN of mice (Muller et al., 2012; Sanchez-Ortiz et al., 2012). In both animals the number of BFCN is reduced to respect the wild type but in few numbers. In addition the laboratory of Dr. Minichiello generated a specific deletion of the NGF gene in the BCFN, and a similar phenotype was described (Muller et al., 2012). This suggests that the TrkA/NGF signaling is only necessary for the development of a small population of BFCN in mice, and the expression of the cholinergic markers, ChAT, AChE, and p75 are not affected by TrkA/NGF signaling. Thus in general terms, deletion of TrkA or NGF is accompanied by a profound loss in superior cervical, dorsal root, and trigeminal ganglion neurons, but with limited defects within the central nervous system (Muller et al., 2012; Sanchez-Ortiz et al., 2012; Smeyne et al., 1994; Crowley et al., 1994).

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5.1.2 TrkB/BDNF/NT4 Signaling Animals deficient for TrkB fail to feed and die within hours after birth (Klein et al., 1993). These animals lack populations of dorsal root and trigeminal ganglia neurons, and they have loss of trigeminal ganglion cells that innervate whisker follicles and substantial neuron loss in the nodose petrosal ganglion (NPG) that contains visceral afferents. The NPG is comprised of neurons that relay sensory information critical for the regulation of autonomic functions like respiration, heart rate, and blood pressure to nuclei located in the brain stem. In mice, disruption of either the BDNF or NT4 gene results in a greater than 50% loss of neurons in the NPG implicating both BDNF and NT4 in the survival of specific populations of these neurons (Liu et al., 1995; Ernfors et al., 1994a; Jones et al., 1994). The 90% loss of NPG neurons found in TrkB(/) and BDNF(/)/NT4(/) mice indicates that the majority of nodose/petrosal neurons are either BDNF or NT4 dependent (Snider, 1994; Conover et al., 1995). A role for BDNF in mammalian inner ear development has been suggested based on mRNA expression patterns of both BDNF and TrkB, and on the ability of BDNF to support survival of vestibular and cochlear ganglia neurons in culture. TrkB is localized to neurons in both the vestibular and cochlear ganglia, and BDNF is expressed in the target tissues of these neurons. BDNF(/) mice have severe movement and balance abnormalities, consistent with the loss of a high number of vestibular ganglion neurons (Liu et al., 1995; Ernfors et al., 1994a; Jones et al., 1994). Mice with a mutation in the NT4 gene show no apparent loss of either vestibular or cochlear ganglia neurons, indicating that the TrkB-dependent neurons of the inner ear require exclusively BDNF, but not NT4 (Liu et al., 1995; Conover et al., 1995). Interestingly, despite the broad expression of TrkB throughout the central nervous system, there is no profound neuronal loss within the brain of TrkB null animals (Klein, 1994; Klein et al., 1993; Snider, 1994). Interfering with BDNFeTrkB signaling leads to axonal and dendritic outgrowth and arborization defects. BDNFeTrkB has a well-established role in dendritic spine formation and therefore synapse development. BDNFeTrkB enhance synaptic transmission in paradigms of LTP, a form of synaptic plasticity associated with learning and memory formation. Indeed, blocking BDNF-dependent TrkB activation leads to a decrease in hippocampal LTP (Bath et al., 2012), in both the early and late response. Decreased levels of BDNF has been linked to the hippocampal atrophy seen in depressed patients (Duman and Monteggia, 2006). Antidepressant treatments increased the expression of neurotrophic factors in the hippocampus to achieve their therapeutic effects. Because KO mice lacking either BDNF or its

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receptor TrkB do not survive past early postnatal periods, recent studies have used heterozygous knockout mice or mice with impaired BDNF/TrkB signaling (Berton et al., 2006; Monteggia et al., 2004; Saarelainen et al., 2003). Results from these studies indicate that impairment of BDNF/TrkB signaling does not lead to depression- or anxiety-like behavior but rather a blunted behavioral response to antidepressants . Therefore, these studies suggest that BDNF/TrkB signaling plays a pivotal role in the action of antidepressants, rather than in the development of depression (Berton et al., 2006; Monteggia et al., 2004; Saarelainen et al., 2003; Bjorkholm and Monteggia, 2016). Recent studies have implicated BDNF in the pathophysiology of psychiatric and neurodegenerative diseases with a potential mechanism likely due to a single nucleotide polymorphism caused by a valine (Val) to methionine (Met) base change at position 66 in the BDNF prodomain (Ventriglia et al., 2002; Sklar et al., 2002; Neves-Pereira et al., 2002). This modification results in a decrease in regulated BDNF secretion, leading to alterations in anxiety-related behavior, learning, and memory (Egan et al., 2003; Anastasia et al., 2013). 5.1.3 TrkC-NT3 The targeted disruption of the NT3 gene in mice results in death within several weeks of birth, with the mice showing severe sensory and sympathetic neuron loss (Snider, 1994; Ernfors et al., 1994b; Farinas et al., 1994). Mice deficient for TrkC show similar, but less dramatic, sensory neuron loss and also exhibit athetotic movements and die within several weeks of birth (Klein et al., 1994). The NT-3 and TrkC knockout animals have a phenotype of abnormal movements and postures (Ernfors et al., 1994b; Farinas et al., 1994; Klein et al., 1994) but can sense pain, suggesting that proprioception is specifically affected. DRG neurons that project to the primary endings of muscle spindles in the periphery extend a collateral branch to the motor pools in the spinal cord and mediate proprioception (sense of position of the limbs in space). These are the largest neurons within the DRGs, and their axons have the most rapid conduction velocities (Klein et al., 1994; Klein, 1994; Ernfors et al., 1994b). Other sensory neuron losses are also seen, most notably in the trigeminal, cochlear, and nodose/petrosal ganglia. Based on a phenotypic comparison of the NT3 and TrkC mutant mice, mice lacking NT3 show more dramatic neuronal loss in several regions than TrkC mutant mice. This suggests that NT3 may be capable of signaling via receptors other than TrkC, as has been previously suggested. In addition to DRGs, TrkC/NT-3 signaling has a physiologic role in the development and maintenance of a subset of enteric neurons (Chalazonitis, 2004). NT-3 acts directly on the precursor cells and in combination

with other neurotrophic factors, promoting the survival and differentiation of enteric neurons and glia. There is loss of neurons in both the myenteric and submucosal plexuses of mice lacking NT-3/TrkC signaling and selective hyperplasia in the myenteric plexus of mice overexpressing NT-3 (Chalazonitis, 2004). Altogether, NT-3 is required for the differentiation, maintenance, and physiologic function of late-developing enteric neurons that are important for the control of gut peristalsis. Most regions of the CNS express TrkC; however, most regions of the brain, brain stem, spinal cord, and cerebellum appear to be grossly normal (Klein et al., 1994). Outside the nervous system, TrkC expression was found in developing cardiomyocytes, and consequently TrkC null animals have severe cardiac deficiencies, such as atrial and ventricular septal defects and valvular defects (Tessarollo et al., 1997). 5.1.4 Truncated Trks 5.1.4.1 TrkB-T1 The TrkB and TrkC loci, in addition to the full-length kinase receptors, can generate, by alternative splicing, truncated receptors lacking the canonical intracellular tyrosine kinase domain (Tessarollo, 1998). These shorter isoforms are expressed at high levels throughout the mature nervous system (Fryer et al., 1996). However, compared to their full-length counterparts, relatively little is known about the biologic function of these truncated isoforms. Initially, it was thought that the truncated Trk receptors act as dominant negatives, which sequester free neurotrophin ligands away from the active full-length receptors (Eide et al., 1996). More recently, it has become clear that these truncated versions are not just neurotrophin sinks, but they are also actively signaling molecules (Michaelsen et al., 2010; Huang et al., 2009; Hartmann et al., 2004; Kryl and Barker, 2000; Gestwa et al., 1999; Venero and Hefti, 1998). Although full-length Trk kinase receptors have the strongest prosurvival activities, recently, it has become apparent that physiologic truncated Trk receptors could have multiple functions in mammalian development. For example, TrkB.T1 deletion in mouse causes increased anxiety and morphologic abnormalities in basolateral amygdala neurons consistent with an independent signaling function for this receptor (Carim-Todd et al., 2009). Moreover, physiologic levels of TrkB.T1 receptors are important regulators of fulllength TrkB (TrkB.FL) signaling in vivo since loss of TrkB.T1 can partially rescue BDNF haploinsufficiency (Carim-Todd et al., 2009). Truncated TrkB receptors can affect specific pathologies. For example, it has been reported that the frontal cortex of patients with Alzheimer disease has decreased BDNF and TrkB.FL expression

5. PHYSIOLOGIC FUNCTIONS OF THE NEUROTROPHINS

accompanied by increased truncated TrkB expression (Ferrer et al., 1999). Moreover, in amyotrophic lateral sclerosis (ALS) patients, BDNF mRNA and protein are dramatically upregulated in muscle, although phosphorylation of the TrkB receptor is reduced, suggesting that TrkB signaling impairments in ALS is caused by a mechanism affecting the TrkB response to BDNF (Mutoh et al., 2000). One possibility is the upregulation of TrkB.T1. Indeed, deletion of TrkB.T1 prevents the loss of motoneurons and muscle function in SOD1 mice, an ALS model, at the early disease stage without affecting the survival of the animals (Yanpallewar et al., 2012). In addition, deletion of TrkB.T1 in the SOD1 mouse rescues the number of parvalbumin expressing interneurons and normalizes the hippocampal LTP in this model (Quarta et al., 2018). Although the signaling of TrkB.T1 is still unknown, it seems that TrkB.T1/BDNF signaling modulates the actin cytoskeleton (Fenner, 2012). Overexpression of TrkB.T1 in hippocampal neurons induces the formation of dendritic filopodia independent of BDNF (Hartmann et al., 2004). One way to activate the formation of filopodia is through the inactivation of RhoA. Indeed, BDNF binding leads to the release of Rho-GDI and to the inhibition of RhoA (Ohira et al., 2005, 2006, 2007). Coexpression of a p75NTR lacking an intracellular domain inhibits the TrkB.T1-induced effect in a dominant negative manner (Michaelsen et al., 2010). These findings suggest that the TrkB.T1 and p75NTR receptor signaling systems might be cross-linked. As the ratio of expression levels of p75NTR and TrkB.T1 changes during development and in adulthood (Michaelsen et al., 2010), this mechanism may play an important role in modulating dendritic architecture and synaptic plasticity in the adult rodent hippocampus (Fenner, 2012). Recently the role of TrkB.T1 has been extended to nonneuronal tissues (Fulgenzi et al., 2015). TrkB.T1 receptor is expressed in cardiomyocytes. TrkB.T1 is activated by BDNF produced by cardiomyocytes, suggesting an autocrine/paracrine loop. Loss of TrkB.T1 in these cells impairs calcium signaling and causes cardiomyopathy (Fulgenzi et al., 2015). In the cardiovascular system, BDNF and its receptor TrkB have been described to have an early developmental role in cardiac endothelium formation (Anastasia et al., 2014), and in the adult heart only TrkB.T1 is expressed (Stoilov et al., 2002). It has been recently shown that TrkB.T1/ BDNF signaling mediates inotropic function by regulating Ca2þ signaling (Fulgenzi et al., 2015). TrkB.T1 was already found to mediate BDNF-induced calcium signaling in astrocytes (Rose et al., 2003), suggesting that this may be one important aspect of TrkB.T1 function. Specific deletion of TrkB.T1 or BDNF in cardiomyocytes causes cardiomyopathy (Fulgenzi et al., 2015). BDNF is secreted by cardiomyocytes, and its specific

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deletion in cardiomyocytes causes a cardiomyopathy resembling that caused by TrkB.T1 deficiency, indicating that BDNF is the ligand for TrkB.T1 (Fulgenzi et al., 2015). 5.1.4.2 Truncated TrkC TrkC receptors are also alternatively spliced to yield isoforms containing the catalytic tyrosine kinase domain (TKþ) and truncated isoforms that lack this domain (TK). Mice lacking all TrkC isoforms, including the truncated ones, have a more severe phenotype, including reduced viability and a more pronounced neuronal deficit, compared with mice lacking only the full-length tyrosine kinase receptors, suggesting a positive role for truncated TrkC receptors in vivo (Tessarollo et al., 1997). However, no signaling molecules downstream of truncated receptors have been identified to date. Ectopic expression of this truncated receptor results in severe deficiencies in the heart and the PNS (Palko et al., 1999). Ectopically, expression of TrkCTKþ and TrkC-TK in neural crest cells, the stem cell population that gives rise to the vast majority of the peripheral nervous system, showed that NT-3 activation promoted both proliferation and neuronal differentiation of TrkC-TKþ. Strikingly, the TrkC-TK isoform was significantly more effective at promoting neuronal differentiation, but it had no effect on proliferation. Furthermore, the TrkC-TK response was dependent on a conserved receptor cytoplasmic domain and required the participation of the p75NTR neurotrophin receptor (Hapner et al., 1998). Although the signaling downstream of TrkC-TK is still unknown, it has been demonstrated that TrkC-TK binds to the scaffold protein tamalin in a ligand-dependent manner (Esteban et al., 2006). NT-3 binding to TrkCT1etamalin induces Arf6 translocation to the membrane, which in turn causes membrane ruffling (Esteban et al., 2006). In addition, truncated TrkC has been involved in some diseases. TrkC.TK- isoform is significantly upregulated during the early phase of glaucoma and induces the production of TNF-a in activated retinal glia or Mu¨ller cells, leading to retinal ganglionar cells death over time (Bai et al., 2010).

5.2 Nonnervous System 5.2.1 Cardiovascular Functions Neurotrophins are required for the normal development of the heart and a critical regulator of vascular development (reviewed in Caporali and Emanueli, 2009). Neurotrophins are implicated in the development of the heart and the coronary vasculature (Bernd, 2008). In particular, BDNF deficiency reduces endothelial cellcell contact in the mouse embryonic heart, thus leading

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to intraventricular wall hemorrhage and depressed cardiac contractility (Donovan et al., 2000). Similarly, TrkB/ mice show a marked reduction of blood vessel density and increased number of TUNEL-positive apoptotic endothelial cells (EC) (Caporali and Emanueli, 2009). Genetic deficiency of either NT-3 or TrkC impaired cardiac morphogenesis in mice, and these developmental defects appeared as early as embryonic day 9.5, which is before the onset of cardiac innervation in mice, thus suggesting the existence of a direct control of NTs on cardiovascular development (Tessarollo, 1998). Postnatally, neurotrophins control the survival of EC, vascular smooth muscle cells, and cardiomyocytes and regulate angiogenesis and vasculogenesis (Risau and Flamme, 1995). All NTs are expressed by the mammalian heart, and their receptors are expressed by the developing heart and vessels (Bernd et al., 2004; Hohn et al., 1990; Maisonpierre et al., 1990a), and NGF was the first NT to be implicated in postnatal angiogenesis (Santos et al., 1991). TrkA activation by NGF is required for survival of the sympathetic (Crowley et al., 1994) and sensory neurons that innervate the heart (Ieda et al., 2006). The cardiac ventricles are innervated by TrkA-expressing sympathetic and sensory neurons whose production of neuropeptides can be regulated by NGF. TrkA activation promotes survival, proliferation, and migration of endothelial cells, while selective activation of p75NTR induces endothelial cell death (Caporali et al., 2008; Kim et al., 2004; Skoff and Adler, 2006). The activation of TrkB by BDNF is necessary for survival of endothelial cells and development of the cardiac vasculature, and it stimulates angiogenesis (Kermani and Hempstead, 2007; Kermani et al., 2005). NT-3, NT-4, and BDNF are all involved in the development of chemoafferent sensory neurons that innervate the carotid body (Conover et al., 1995; Erickson et al., 1996). TrkC activation is required for the development of atria, ventricles, and cardiac outflow tracts (Tessarollo et al., 1997; Donovan et al., 1996). The coexpression of NT-3 and TrkC in developing cardiomyocytes mediate their proliferation (Lin et al., 2000). 5.2.2 Regulation of Energy Balance and Body Weight Neurotrophins play significant roles in regulating energy metabolism in the body, most notably BDNF (Rios, 2013). A role of BDNF in metabolism came from animal models, wherein heterozygous BDNF-/þ animals show an age-related increase in body weight (Kernie et al., 2000; Lyons et al., 1999). Many studies showed that delivery of exogenous BDNF induced reduction in body weight gain (Lapchak and Hefti, 1992; Rios, 2013). It was also observed that BDNF mutant mice develop other aspects of metabolic syndrome including leptin and insulin resistance, dyslipidemia, and

hyperglycemia (Kernie et al., 2000; Rios, 2011). Central and systemic BDNF administration improved glucose metabolism in various models of obesity including leptin and leptin receptoredeficient mice (Nakagawa et al., 2000, 2002; Tonra et al., 1999). Later, it was shown that overexpression of BDNF in the rodent hypothalamus resulted in the activation of brown fat transcriptional programs in white fat cells. This white to brown cellular switch promotes energy expenditure and resistance to diet-induced obesity. Recently, p75 neurotrophin receptor (p75NTR) has been shown to control energy expenditure in obese mice on a high-fat diet (HFD) (Baeza-Raja et al., 2016). p75NTR-null mice are protected from HFD-induced obesity and remained lean as a result of increased energy expenditure without developing insulin resistance or liver steatosis. The authors found that p75NTR directly interacts with the catalytic subunit of protein kinase A (PKA) and regulates cAMP signaling in adipocytes, leading to decreased lipolysis and thermogenesis (Baeza-Raja et al., 2016). In addition p75NTR KO in a HFD are resistant to the increase of cholesterol levels. This suggests that p75NTR may be involved in the regulation of cholesterol levels or in its synthesis. This agrees with previous publications where cholesterol biosynthesis-related enzymes, including HMG CoA reductase, farnesyl-diphosphate synthase, and 7-dehydro-cholesterol reductase, are upregulated in p75NTR-positive cells as compared with p75NTR-negative cells (Korade et al., 2007; Yan et al., 2005). Not only p75NTR regulates the cholesterol synthesis but its uptake by the cells as well. Recently a novel pathway has been described by which the neurotrophins, such as NGF and pro-NGF, signaling via p75NTR, can activate the sterol regulatory elementbinding protein-2 (SREBP2) that regulates genes involved in lipid metabolism (Pham et al., 2016). SREBP2 increased the receptor for the low density lipoprotein (LDL) and the uptake of LDL by hepatocyte Huh7 cells. In this cascade, the SREBP2 is activated by the cleavage at an amino-terminal site subsequent to the activation of caspase-3 and p38 MAPK by p75NTR signaling. Surprisingly no cell death of Huh7 cells despite an increase in active caspase-3 by pro-NGF was observed. Instead, an activation of the NF-kB signaling pathways by p75NTR activation increased the viability of the hepatocytes.

6. DISEASE AND AGING EFFECTS OF NEUROTROPHINS 6.1 Alzheimer Disease It is well known that two of the neuromorphological changes of Alzheimer disease (AD) are the loss of

6. DISEASE AND AGING EFFECTS OF NEUROTROPHINS

BFCN and the widespread degeneration of cholinergic functions in the brain, which contribute to cognitive decline and dementia (Schliebs and Arendt, 2011; Niewiadomska et al., 2011; Mufson et al., 2003, 2008; Cuello et al., 2007). The role of NGF in BFCN survival has been demonstrated using different approaches (Cuello et al., 2007). For instance, knocking out NGF in adult mice by expressing transgenic anti-NGF antibodies resulted in basal forebrain and hippocampal cholinergic neuron reductions in adult mice (55% and 62% reductions, respectively) (Ruberti et al., 2000). In addition, adult mice showed deficits in spatial learning behavioral tasks (Ruberti et al., 2000). Increasing evidence supports that basal forebrain cholinergic metabolic dysfunction of NGF is involved in AD and that defective NGF processing, inducing an increase in the levels of pro-NGF rather than mature NGF, leads to an increased accumulation of Ab (Iulita and Cuello, 2016). The link between p75NTR and AD has been most studied (Coulson et al., 2009). Besides Purkinje neurons in the cerebellum, p75NTR is expressed at high levels in cholinergic neurons of the adult basal forebrain. Several in vitro studies have indicated that amyloid beta 1e42 (Ab), the main component of plaques commonly found within brains of AD patients, is a proapoptotic ligand for p75NTR (Costantini et al., 2005a, b, c; Tsukamoto et al., 2003; Yaar et al., 1997, 2002). These findings have led to the hypothesis that activation of p75NTR by Ab contributes to neurodegeneration caused by AD (Coulson et al., 2009). Deletion of p75NTR prevented the degeneration of cholinergic basal forebrain neurons in vivo following Ab injection into the hippocampus (Sotthibundhu et al., 2008). Furthermore, when p75NTR/ mice were crossed with the Thy1hAPPLond/Swe mouse model of AD, the degeneration of hippocampal and forebrain cholinergic fibers was dramatically rescued (Knowles et al., 2009). This has been confirmed recently when p75NTR was conditionally deleted in basal forebrain cholinergic neurons in the APP/PS1 familial AD mouse strain (Qian et al., 2018). Conditional loss of p75NTR slowed cognitive decline and reduced both Ab accumulation into plaques and gliosis in the APP/PS1 mice. In general removal of p75NTR in mice genetic models of AD, that produced high levels of Ab, leads to a general improvement in several behavioural tests (see Table in (Qian et al., 2018) ), suggesting that p75NTR is deleterious in such conditions and a good therapeutical target in dementia. However, these studies were challenged by a study by Wang et al., which indicated that p75NTR reduces the formation of fibrils in vivo and in vitro by competitive binding of p75-ECD to Ab peptides (Wang et al., 2011). In addition a reduction in the levels of p75-ECD is observed during aging and in AD. Normalizing the levels of p75-ECD by brain delivery of the gene

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encoding human p75ECD before or after Ab deposition in the brain of APP/PS1 mice reversed the behavioral deficits and AD-type pathologies, such as Ab deposit, apoptotic events, neuroinflammation, Tau phosphorylation, and loss of dendritic spine, neuronal structures, and synaptic proteins (Yao et al., 2015). This may suggest that p75NTR shedding is an important therapeutical target in AD (Chao, 2016). In general, this points to a prosurvival role of p75 in AD. This role of p75 has been also observed in other reports, indicating that expression of p75NTR is protective against Ab-induced toxicity (Bengoechea et al., 2009). Although the mechanism of cell survival is not clear, recently a model where Ab was cleared from the extracellular space by binding to p75NTR and retrogradely transported to the lysosome pathway was shown (Ovsepian et al., 2013, 2016; Ovsepian and Herms, 2013) supporting the pro-survival role of p75NTR in AD. In any case all these studies should be taken with caution as all the genetic mouse models of AD produce higher levels of human fibrillar Ab, a product that is never generated in the mice because mouse Ab-peptide do not form fibrils. Apart from Ab-induced apoptosis, studies have also implicated proNGF in AD pathology (Fahnestock et al., 2004). Increased amounts of proNGF have been detected in human brains affected by AD (Pedraza et al., 2005). ProNGF is converted to mature NGF by plasmin, a serine protease that derives from the zymogen plasminogen by the action of tissue plasminogen activator (tPA). Analysis of postmortem frontal cortex tissue from AD patients revealed a dramatic reduction in tPA, plasminogen, and plasmin protein levels, which explained the increase in proNGF, supporting the case that the proNGF cleavage loop is defective in AD brains. Moreover, analysis of the zymogenic activity of MMP-9, the main NGF-degrading protease, revealed a strong upregulation of MMP-9 activity that would increase the NGF degradation in AD. Thus, in addition to activation of p75NTR by Ab, alterations in the NGF metabolic pathway increasing the levels of proNGF contribute to neurodegeneration within the AD brain.

6.2 Huntington Disease Huntington disease (HD) is a dominant inherited neurodegenerative disorder that is caused by an unstable expansion of a CAG repeat within the huntingtin protein gene (Zuccato and Cattaneo, 2014). HD is characterized by massive loss of medium spiny neurons in the striatum. However, the mechanisms by which mutant huntingtin leads to this selective neuronal death remain incompletely understood. One of the physiologic functions of huntingtin is to regulate BDNF expression that contributes to maintain the BDNF pool in the brain

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(Zuccato et al., 2001, 2003). In light of this evidence a number of studies have tested the impact of reducing or augmenting the level of BDNF on disease onset and progression (Zuccato and Cattaneo, 2014). In addition to its role as a transcriptional regulator of BDNF expression, huntingtin has a role in the control of BDNF vesicle transport, presenting the evidence that BDNF vesicle transport is reduced in HD (Gauthier et al., 2004). These studies provided support to the idea that cortical BDNF depletion and dysfunction is one of the critical factors in the pathology of HD and that BDNF administration could be beneficial to HD patients (Zuccato and Cattaneo, 2014). In this sense, BDNF has been shown to be neuroprotective on HD striatal neurons both in vitro and in vivo. Not only BDNF levels are decreased in HD, but also the levels of its receptors are disturbed. There is a marked reduction in the number and activity of TrkB receptors levels in the striatum in mouse models of HD (Gines et al., 2006), whereas the mRNA levels of p75NTR are increased in the caudate (Brito and Gines, 2016). This indicates that, in addition to the reduction in BDNF mRNA and transport, there is also unbalanced neurotrophic receptor trafficking and signaling in HD. Actually a normalization of the levels of p75NTR has been suggested as a therapeutical intervention in HD (Brito and Gines, 2016; Brito et al., 2013, 2014).

6.3 Parkinson Disease Parkinson disease (PD) is the second most common age-related neurodegenerative disease after AD and the most common movement disorder. It affects about 1% of the population aged over 60 years and 4% of those over 80 in industrial countries. PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Degeneration of dopaminergic neurons in PD could also involve p75NTR. A study by Simon and colleagues demonstrated that loss of the engrailed transcription factors results in increased expression of p75NTR in the ventral midbrain (Alavian et al., 2009). This finding has implications for PD because mice deficient in Engrailed-1 and Engrailed-2 exhibit progressive loss of mesencephalic dopaminergic neurons and have PD-like motor deficiencies (Alavian et al., 2009). However, direct evidence for p75NTR expression in nigral dopaminergic neurons in PD and causal evidence linking expression of p75NTR to PD-associated nigral neurodegeneration in vivo is still missing.

6.4 Amyotrophic Lateral Sclerosis ALS is a fatal neurodegenerative disorder that involves the gradual loss of motoneurons in the cortex, brainstem, and spinal cord. Average mortality after

onset of ALS is 5 years. Approximately 10% of all cases of ALS concern a familial form; most cases of ALS, however, are considered to be sporadic. p75NTR may contribute to degeneration of motor neurons during the progression of ALS. Though p75NTR is downregulated in motor neurons of the spinal cord during the perinatal period, reexpression of the receptor was detected in spinal motor neurons of an ALS mouse model (Copray et al., 2003; Lowry et al., 2001), as well as in spinal cord samples from human patients with ALS (Lowry et al., 2001; Seeburger et al., 1993). The p75NTR receptor is reinduced postnatally when motor neurons are injured. Furthermore, the receptor was implicated in ALS-associated motor neuron death by a study in which knockdown of p75NTR delayed locomotor impairment and mortality in the SOD1G93A mouse model of ALS (Turner et al., 2003). However, when the SOD1G93A mice were crossed with the p75NTR knockout mice, prolonged survival was only detected in the female mice, and this improvement did not correlate with increased motor neuron survival, but with reduced astrocytosis (Kust et al., 2003). Nevertheless, the SOD mutation represents a very small fraction of ALS patients, so further study into the role of p75NTR in this disease is warranted.

6.5 BDNF Polymorphism and Nervous System Disorders In humans an SNP in the BDNF gene that causes a valine (Val) to methionine (Met) substitution at codon 66 (Val66Met, c.196G>A, dbSNP: rs6265) has been identified. In BDNF Val/Met heterozygotes and Met/Met homozygotes, the prodomain structure of the gene is altered. Though this alteration would not necessarily change the intrinsic biologic activity of the mature BNDF protein, the polymorphism can lead to improper protein folding, secretion, and a reduced binding of the mature BDNF to its receptor TrkB, causing impairments in hippocampal function. The Val66Met polymorphism frequency varies depending on both region and ethnicity. The Met carriers make up approximately 30%e50% of Caucasian populations, whereas among Asian populations, almost 70% of people carry the Met variant form of BDNF gene (Shen et al., 2018). The Met allele of BDNF impairs intracellular trafficking and synaptic localization of mature BDNF and significantly weakens the activity-dependent secretion of BDNF (up to 30%) while keeping the constitutive secretion of BDNF unaffected (Egan et al., 2003). A similar reduction of activity-dependent BDNF secretion was also observed in neurons derived from BDNF Val66Met knock-in mice (Chen et al., 2006). Although BDNF polymorphism has been implicated in several

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neurodegenerative disorders, like in AD and in glaucoma, or in some neuronal disorders like schizophrenia, those evidences are mixed, and further cohort studies with longitudinal assessments of the progression of neurodegenerative disorders are needed (Shen et al., 2018; Notaras et al., 2015).

6.6 Cancer and Tumor Formation Mutations or rearrangements in the Trk family have recently been described as a new mechanism of oncogenesis (Passiglia et al., 2016; Prasad et al., 2016; Sartore-Bianchi et al., 2016; Kim et al., 2014; Vaishnavi et al., 2013). Gene fusions lead to chimeric Trk proteins that either lead to the overexpression of the kinase domain or possess constitutive activity of the kinase function. Fusions have been identified in various solid tumors, including the lung, gastrointestinal tract, thyroid, primary brain tumors, and sarcomas. NTRK fusions were originally identified in 1986 in colon cancer when a TPM3-NTRK1 translocation was detected in a tumor biopsy (Martin-Zanca et al., 1986). Since this observation, gene fusions involving NTRK1, 2, and 3 genes have been documented in 11 specific tumor types, most notably NSCLC, papillary thyroid carcinoma, secretory breast cancer, and glioblastoma (Lange and Lo, 2018). As sequencing techniques continue to develop, it is expected that more NTRK gene fusions will be identified, renewing interest in Trk proteins as therapeutic targets. TrkC expression has been associated with a favorable outcome in two pediatric neoplasia medulloblastoma and neuroblastoma, supporting the view that expression of TrkC is rather a tumor suppressor. Two independent studies have demonstrated that TrkC is a dependence receptor (Ichim et al., 2013; Genevois et al., 2013). TrkC induces apoptosis in the absence of NT-3, is doublecleaved by caspase, and the released fragment is translocated to the mitochondria, which allows the activation of Bax, the subsequent release of cytochrome c, and the apoptosome activation (Ichim et al., 2013). As such, TrkC expression would represent a constraint for tumor escape by inducing apoptosis in settings of NT-3 limitation. Of interest, TrkC expression is downregulated in colon carcinoma tumors and when reexpressed induces apoptosis and inhibits tumor progression in vitro and in vivo (Genevois et al., 2013). The silencing of TrkC is due to the methylation and deacetylation of histones of its promoter, supporting the view that TrkC behaves as a tumor suppressor in colon carcinoma (Mehlen, 2010). p75NTR is a crucial determinant of tumorigenicity, stem-like properties, heterogeneity, and plasticity in melanoma cells. In the cancer field, p75NTR is best known as CD271. p75NTR/CD271 is a marker of neural crest cells from which melanocytes are derived and is

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considered a melanoma dedifferentiation marker, which is generally correlated with high aggressiveness. p75NTR-positive melanoma cells have a high tumorigenic potential compared with p75NTR-negative cells originating from the same tumor, when injected into immunodeficient mice (Boiko et al., 2010). Different groups demonstrated the exacerbated tumorigenic potential of p75NTR-positive melanoma cells that might be explained by an increased expression of stemness and mesenchymal markers. Silencing the expression of p75NTR prevents melanoma tumor development in a xenograft model (Redmer et al., 2014) and inhibits cell migration (Radke et al., 2017).

6.7 Inflammation, Allergy, and Pain The levels of NGF mRNA and protein are increased in several human pain disorders, especially in the context of inflammation (Minnone et al., 2017); this has been shown in both patient-derived samples and experimental pain models. Examples include bladder pain syndrome/interstitial cystitis (Chen et al., 2016), inflammatory bowel disease (di Mola et al., 2000), chronic pancreatitis (Friess et al., 1999), osteoarthritis (Iannone et al., 2002), and in rheumatoid arthritis and spondyloarthritis (Aloe et al., 1992; Barthel et al., 2009). In addition, enhanced NGF levels are found in the cerebrospinal fluid of multiple sclerosis patients and in systemic lupus erythematosus patients (Laudiero et al., 1992; Bracci-Laudiero et al., 1993). One of the effects of this enhanced production of NGF is the inflammatory pain associated with expression of transient receptor potential vanilloid 1 (TRPV1) channels and sodium channels and the alteration of peripheral innervation (Minnone et al., 2017). During the inflammatory process, there is a release of cytokines involved in inflammation, such as IL-1b, TNF-a, and IL-6, that have been shown to induce the NGF synthesis in a variety of cell types. However, in vivo the specific cell types and mediators responsible for the enhanced production of NGF during inflammatory response in patients and in animal models of inflammatory diseases are still mostly unknown. There is growing evidence that NTs are involved in the processes of allergic inflammation (for extensive reviews, see Barrios and Ai, 2018; Manti et al., 2017; Raap and Braunstahl, 2010; Freund-Michel and Frossard, 2008; Nassenstein et al., 2006; Frossard et al., 2004; Raychaudhuri and Raychaudhuri, 2004; Tabakman et al., 2004). To date, it is known that neurons and activated structural cells are able to influence the immune response through the release of cytokines and soluble mediators. Several studies indicate that neurotrophins mediate inflammatory signals between neurons and cells of immune and structural tissue and indicate that neurons and neuron-associated cells are the potential

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sources of neurotrophins in allergic inflammation (Thoenen, 1995) as well immune cells such as macrophages (Kerschensteiner et al., 1999), NK cells (Hammarberg et al., 2000), T cells, B cells, epithelial cells, eosinophils (Barouch et al., 2000; Kobayashi et al., 2002), and mast cells (Leon et al., 1994). Mast cells secrete NGF after IgE-mediated stimulation, but also depend on NGF as a maturation and survival factor. Neurotrophins act as inflammatory cytokines delivering signals of activation and survival to the effector cells of the allergic response. Inflammatory processes and responses to proinflammatory mediators are potent inducers of the release of neurotrophins by immune and nervous cells (Cho et al., 1997). In some allergic diseases, the data suggest that neurotrophins are produced locally after the provocation of allergens (Virchow et al., 1998). One of the first data that report the participation of neurotrophins in the pathology of allergic diseases comes from studies of patients with vernal kerato-conjunctivitis, a disease with local activation of mast cells (Lambiase et al., 1995). The expression of neurotrophins, such as NGF and BDNF, is highly regulated during allergic inflammation in atopic dermatitis and other variants of this chronic inflammatory skin disease (Raap et al., 2006). Elevated levels of serum NTs have been found in patients with other allergic diseases including urticaria, rhinitis, and allergic asthma (Bonini et al., 1996; Sanico et al., 2000). Elevated levels of BDNF and NT3 were found in patients with asthma (Lommatzsch et al., 2005; Noga et al., 2001), and this increase induces hyperreactivity and obstruction in the airway (Watanabe et al., 2015). The discovery of the potential role of neurotrophins within the immune system has led the concept that NTs not only react to local inflammatory stimuli, which results in various biologic effects that include cell survival, differentiation, and proliferation, but are also capable of promoting the activation and release of proinflammatory cytokines and mediators by autocrine and paracrine mechanisms (Brown et al., 2015).

7. CONCLUSIONS AND FUTURE DIRECTIONS This field has seen an explosion of new in vivo functions and physiologic roles of neurotrophins in the last 60 years of its discovery. However, still there are new discoveries to come. Although much of the work has been done in the nervous system, things are changing, and new avenues are being built to bring neurotrophin studies to new arenas, like immunology or cancer. A new field is emerging in the neuromodulation of the inflammatory response by neurotrophins. A better understanding of the physiologic role of NGF and the rest of the NTs and receptors in regulating neuroimmune

responses could open new therapeutic avenues for human diseases like asthma, arthritis, and other inflammatory diseases. The use of NGF to preserve the in vivo functions of BFCN in AD and of BDNF in other neurodegenerative diseases like HD still has some potential, probably with the new development of iPSC-derived neurons. In cancer, new discoveries are revealing the role of neurotrophins in perivascular innervation, tumor progression, and migration. New neurotrophin antagonists and agonists are being developed in the help to fight cancer metastasis and to decrease all general forms of cancer-derived pain, specifically inflammatory pain and bone pain in cancer patients.

Acknowledgments The authors thank to the Spanish Government Ministry of Science, Innovation and Universities for the funding (grant SAF2015-84096-R) for our research on neurotrophins.

References Adams, K.W., Kletsov, S., Lamm, R.J., Elman, J.S., Mullenbrock, S., Cooper, G.M., 2017. Role for egr1 in the transcriptional program associated with neuronal differentiation of PC12 cells. PLoS One 12, e0170076. Agerman, K., Baudet, C., Fundin, B., Willson, C., Ernfors, P., 2000. Attenuation of a caspase-3 dependent cell death in NT4- and p75-deficient embryonic sensory neurons. Mol. Cell. Neurosci. 16, 258e268. Alavian, K.N., Sgado, P., Alberi, L., Subramaniam, S., Simon, H.H., 2009. Elevated P75NTR expression causes death of engraileddeficient midbrain dopaminergic neurons by Erk1/2 suppression. Neural Dev. 4, 11. Aloe, L., Tuveri, M.A., Carcassi, U., Levi-Montalcini, R., 1992. Nerve growth factor in the synovial fluid of patients with chronic arthritis. Arthritis Rheum. 35, 351e355. Aloyz, R.S., Bamji, S.X., Pozniak, C.D., Toma, J.G., Atwal, J., Kaplan, D.R., Miller, F.D., 1998. p53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors. J. Cell Biol. 143, 1691e1703. Anastasia, A., Deinhardt, K., Chao, M.V., Will, N.E., Irmady, K., Lee, F.S., Hempstead, B.L., Bracken, C., 2013. Val66Met polymorphism of BDNF alters prodomain structure to induce neuronal growth cone retraction. Nat. Commun. 4, 2490. Anastasia, A., Deinhardt, K., Wang, S., Martin, L., Nichol, D., Irmady, K., Trinh, J., Parada, L., Rafii, S., Hempstead, B.L., Kermani, P., 2014. Trkb signaling in pericytes is required for cardiac microvessel stabilization. PLoS One 9, e87406. Arevalo, J.C., Conde, B., Hempstead, B.I., Chao, M.V., MartinZanca, D., Perez, P., 2001. A novel mutation within the extracellular domain of TrkA causes constitutive receptor activation. Oncogene 20, 1229e1234. Aurikko, J.P., Ruotolo, B.T., Grossmann, J.G., Moncrieffe, M.C., Stephens, E., Leppanen, V.M., Robinson, C.V., Saarma, M., Bradshaw, R.A., Blundell, T.L., 2005. Characterization of symmetric complexes of nerve growth factor and the ectodomain of the panneurotrophin receptor, p75NTR. J. Biol. Chem. 280, 33453e33460. Baeza-Raja, B., Sachs, B.D., Li, P., Christian, F., Vagena, E., Davalos, D., LE Moan, N., Ryu, J.K., Sikorski, S.L., Chan, J.P., Scadeng, M., Taylor, S.S., Houslay, M.D., Baillie, G.S., Saltiel, A.R., Olefsky, J.M., Akassoglou, K., 2016. p75 neurotrophin receptor regulates energy balance in obesity. Cell Rep. 14, 255e268.

REFERENCES

Bai, Y., Shi, Z., Zhuo, Y., Liu, J., Malakhov, A., Ko, E., Burgess, K., Schaefer, H., Esteban, P.F., Tessarollo, L., Saragovi, H.U., 2010. In glaucoma the upregulated truncated TrkC.T1 receptor isoform in glia causes increased TNF-alpha production, leading to retinal ganglion cell death. Invest. Ophthalmol. Vis. Sci. 51, 6639e6651. Baldwin, A.N., Bitler, C.M., Welcher, A.A., Shooter, E.M., 1992. Studies on the structure and binding properties of the cysteine-rich domain of rat low affinity nerve growth factor receptor (p75NGFR). J. Biol. Chem. 267, 8352e8359. Bamji, S.X., Majdan, M., Pozniak, C.D., Belliveau, D.J., Aloyz, R., Kohn, J., Causing, C.G., Miller, F.D., 1998. The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death. J. Cell Biol. 140, 911e923. Barde, Y.A., Edgar, D., Thoenen, H., 1982. Purification of a new neurotrophic factor from mammalian brain. EMBO J. 1, 549e553. Barker, P.A., 2009. A p75(NTR) pivoting paradigm propels perspicacity. Neuron 62, 3e5. Barouch, R., Appel, E., Kazimirsky, G., Braun, A., Renz, H., Brodie, C., 2000. Differential regulation of neurotrophin expression by mitogens and neurotransmitters in mouse lymphocytes. J. Neuroimmunol. 103, 112e121. Barrios, J., Ai, X., 2018. Neurotrophins in asthma. Curr. Allergy Asthma Rep. 18, 10. Barthel, C., Yeremenko, N., Jacobs, R., Schmidt, R.E., Bernateck, M., Zeidler, H., Tak, P.P., Baeten, D., Rihl, M., 2009. Nerve growth factor and receptor expression in rheumatoid arthritis and spondyloarthritis. Arthritis Res. Ther. 11, R82. Bath, K.G., Jing, D.Q., Dincheva, I., Neeb, C.C., Pattwell, S.S., Chao, M.V., Lee, F.S., Ninan, I., 2012. BDNF Val66Met impairs fluoxetine-induced enhancement of adult hippocampus plasticity. Neuropsychopharmacology 37, 1297e1304. Beck, G., Munno, D.W., Levy, Z., Dissel, H.M., VAN-Minnen, J., Syed, N.I., Fainzilber, M., 2004. Neurotrophic activities of trk receptors conserved over 600 million years of evolution. J. Neurobiol. 60, 12e20. Becker, E.B., Howell, J., Kodama, Y., Barker, P.A., Bonni, A., 2004. Characterization of the c-Jun N-terminal kinase-BimEL signaling pathway in neuronal apoptosis. J. Neurosci. 24, 8762e8770. Bengoechea, T.G., Chen, Z., O’leary, D.A., Masliah, E., Lee, K.F., 2009. p75 reduces beta-amyloid-induced sympathetic innervation deficits in an Alzheimer’s disease mouse model. Proc. Natl. Acad. Sci. U. S. A. 106, 7870e7875. Bernd, P., 2008. The role of neurotrophins during early development. Gene Expr. 14, 241e250. Bernd, P., Miles, K., Rozenberg, I., Borghjid, S., Kirby, M.L., 2004. Neurotrophin-3 and TrkC are expressed in the outflow tract of the developing chicken heart. Dev. Dynam. 230, 767e772. Berton, O., McClung, C.A., Dileone, R.J., Krishnan, V., Renthal, W., Russo, S.J., Graham, D., Tsankova, N.M., Bolanos, C.A., Rios, M., Monteggia, L.M., Self, D.W., Nestler, E.J., 2006. Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress. Science 311, 864e868. Bhakar, A.L., Howell, J.L., Paul, C.E., Salehi, A.H., Becker, E.B., Said, F., Bonni, A., Barker, P.A., 2003. Apoptosis induced by p75NTR overexpression requires Jun kinase-dependent phosphorylation of Bad. J. Neurosci. 23, 11373e11381. Bibel, M., Hoppe, E., Barde, Y.A., 1999. Biochemical and functional interactions between the neurotrophin receptors trk and p75NTR. EMBO J. 18, 616e622. Bjorkholm, R.S., Monteggia, L.M., 2006. A neurotrophic model for stress-related mood disorders. Biol Psychiatry 59, 1116e1127. Bjorkholm, C., Monteggia, L.M., 2016. BDNF - a key transducer of antidepressant effects. Neuropharmacology 102, 72e79. Boiko, A.D., Razorenova, O.V., Van de Rijn, M., Swetter, S.M., Johnson, D.L., Ly, D.P., Butler, P.D., Yang, G.P., Joshua, B., Kaplan, M.J., Longaker, M.T., Weissman, I.L., 2010. Human

99

melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature 466, 133e137. Bonini, S., Lambiase, A., Bonini, S., Angelucci, F., Magrini, L., Manni, L., Aloe, L., 1996. Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma. Proc. Natl. Acad. Sci. U. S. A. 93, 10955e10960. Bothwell, M., 2006. Evolution of the neurotrophin signaling system in invertebrates. Brain Behav. Evol. 68, 124e132. Boutilier, J., Ceni, C., Pagdala, P.C., Forgie, A., Neet, K.E., Barker, P.A., 2008. Proneurotrophins require endocytosis and intracellular proteolysis to induce TrkA activation. J. Biol. Chem. 283, 12709e12716. Bracci-Laudiero, L., Aloe, L., Levi-Montalcini, R., Galeazzi, M., Schilter, D., Scully, J.L., Otten, U., 1993. Increased levels of NGF in sera of systemic lupus erythematosus patients. Neuroreport 4, 563e565. Brann, A.B., Tcherpakov, M., Williams, I.M., Futerman, A.H., Fainzilber, M., 2002. Nerve growth factor-induced p75-mediated death of cultured hippocampal neurons is age-dependent and transduced through ceramide generated by neutral sphingomyelinase. J. Biol. Chem. 277, 9812e9818. Brito, V., Gines, S., 2016. p75NTR in Huntington’s disease: beyond the basal ganglia. Oncotarget 7, 1e2. Brito, V., Giralt, A., Enriquez-Barreto, L., Puigdellivol, M., Suelves, N., Zamora-Moratalla, A., Ballesteros, J.J., Martin, E.D., DominguezIturza, N., Morales, M., Alberch, J., Gines, S., 2014. Neurotrophin receptor p75(NTR) mediates Huntington’s disease-associated synaptic and memory dysfunction. J. Clin. Investig. 124, 4411e4428. Brito, V., Puigdellivol, M., Giralt, A., DEL Toro, D., Alberch, J., Gines, S., 2013. Imbalance of p75(NTR)/TrkB protein expression in Huntington’s disease: implication for neuroprotective therapies. Cell Death Dis. 4, e595. Brown, P.M., Schneeberger, D.L., Piedimonte, G., 2015. Biomarkers of respiratory syncytial virus (RSV) infection: specific neutrophil and cytokine levels provide increased accuracy in predicting disease severity. Paediatr. Respir. Rev. 16, 232e240. Brunet, A., Kanai, F., Stehn, J., Xu, J., Sarbassova, D., Frangioni, J.V., Dalal, S.N., Decaprio, J.A., Greenberg, M.E., Yaffe, M.B., 2002. 143-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. J. Cell Biol. 156, 817e828. Caporali, A., Emanueli, C., 2009. Cardiovascular actions of neurotrophins. Physiol. Rev. 89, 279e308. Caporali, A., Pani, E., Horrevoets, A.J., Kraenkel, N., Oikawa, A., SalaNewby, G.B., Meloni, M., Cristofaro, B., Graiani, G., Leroyer, A.S., Boulanger, C.M., Spinetti, G., Yoon, S.O., Madeddu, P., Emanueli, C., 2008. Neurotrophin p75 receptor (p75NTR) promotes endothelial cell apoptosis and inhibits angiogenesis: implications for diabetes-induced impaired neovascularization in ischemic limb muscles. Circ. Res. 103, e15e26. Carim-Todd, L., Bath, K.G., Fulgenzi, G., Yanpallewar, S., Jing, D., Barrick, C.A., Becker, J., Buckley, H., Dorsey, S.G., Lee, F.S., Tessarollo, L., 2009. Endogenous truncated TrkB.T1 receptor regulates neuronal complexity and TrkB kinase receptor function in vivo. J. Neurosci. 29, 678e685. Carter, B.D., Kaltschmidt, C., Kaltschmidt, B., Offenhauser, N., BohmMatthaei, R., Baeuerle, P.A., Barde, Y.A., 1996. Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75. Science 272, 542e545. Casaccia-Bonnefil, P., Carter, B.D., Dobrowsky, R.T., Chao, M.V., 1996. Death of oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature 383, 716e719. Casaccia-Bonnefil, P., Gu, C., Khursigara, G., Chao, M.V., 1999. p75 neurotrophin receptor as a modulator of survival and death decisions. Microsc. Res. Tech. 45, 217e224. Ceni, C., Unsain, N., Zeinieh, M.P., Barker, P.A., 2014. Neurotrophins in the regulation of cellular survival and death. Handb. Exp. Pharmacol. 220, 193e221.

100

5. NEUROTROPHINS AND NEUROTROPHIN RECEPTORS

Chalazonitis, A., 2004. Neurotrophin-3 in the development of the enteric nervous system. Prog. Brain Res. 146, 243e263. Chao, M.V., 1995. Ceramide: a potential second messenger in the nervous system. Mol. Cell. Neurosci. 6, 91e96. Chao, M.V., 2003. Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat. Rev. Neurosci. 4, 299e309. Chao, M.V., 2016. Cleavage of p75 neurotrophin receptor is linked to Alzheimer’s disease. Mol. Psychiatr. 21, 300e301. Chen, W., Ye, D.Y., Han, D.J., Fu, G.Q., Zeng, X., Lin, W., Liang, Y., 2016. Elevated level of nerve growth factor in the bladder pain syndrome/interstitial cystitis: a meta-analysis. SpringerPlus 5, 1072. Chen, Z.Y., Jing, D., Bath, K.G., Ieraci, A., Khan, T., Siao, C.J., Herrera, D.G., Toth, M., Yang, C., Mcewen, B.S., Hempstead, B.L., Lee, F.S., 2006. Genetic variant BDNF (Val66Met) polymorphism alters anxiety-related behavior. Science 314, 140e143. Cho, H.J., Kim, S.Y., Park, M.J., Kim, D.S., Kim, J.K., Chu, M.Y., 1997. Expression of mRNA for brain-derived neurotrophic factor in the dorsal root ganglion following peripheral inflammation. Brain Res. 749, 358e362. Clary, D.O., Reichardt, L.F., 1994. An alternatively spliced form of the nerve growth factor receptor TrkA confers an enhanced response to neurotrophin 3. Proc. Natl. Acad. Sci. U. S. A. 91, 11133e11137. Cohen, S., Levi-Montalcini, R., Hamburger, V., 1954. A nerve growthstimulating factor isolated from sarcom as 37 and 180. Proc. Natl. Acad. Sci. U. S. A. 40, 1014e1018. Conover, J.C., Erickson, J.T., Katz, D.M., Bianchi, L.M., Poueymirou, W.T., Mcclain, J., Pan, L., Helgren, M., Ip, N.Y., Boland, P., et al., 1995. Neuronal deficits, not involving motor neurons, in mice lacking BDNF and/or NT4. Nature 375, 235e238. Conover, J.C., Yancopoulos, G.D., 1997. Neurotrophin regulation of the developing nervous system: analyses of knockout mice. Rev. Neurosci. 8, 13e27. Copray, J.C., Jaarsma, D., Kust, B.M., Bruggeman, R.W., Mantingh, I., Brouwer, N., Boddeke, H.W., 2003. Expression of the low affinity neurotrophin receptor p75 in spinal motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis. Neuroscience 116, 685e694. Costantini, C., Della-Bianca, V., Formaggio, E., Chiamulera, C., Montresor, A., Rossi, F., 2005a. The expression of p75 neurotrophin receptor protects against the neurotoxicity of soluble oligomers of beta-amyloid. Exp. Cell Res. 311, 126e134. Costantini, C., Rossi, F., Formaggio, E., Bernardoni, R., Cecconi, D., Della-Bianca, V., 2005b. Characterization of the signaling pathway downstream p75 neurotrophin receptor involved in beta-amyloid peptide-dependent cell death. J. Mol. Neurosci. 25, 141e156. Costantini, C., Weindruch, R., Della Valle, G., Puglielli, L., 2005c. A TrkA-to-p75NTR molecular switch activates amyloid betapeptide generation during aging. Biochem. J. 391, 59e67. Coulson, E.J., Reid, K., Baca, M., Shipham, K.A., Hulett, S.M., Kilpatrick, T.J., Bartlett, P.F., Chopper, 2000. A new death domain of the p75 neurotrophin receptor that mediates rapid neuronal cell death. J. Biol. Chem. 275, 30537e30545. Coulson, E.J., May, L.M., Osborne, S.L., Reid, K., Underwood, C.K., Meunier, F.A., Bartlett, P.F., Sah, P., 2008. P75 neurotrophin receptor mediates neuronal cell death by activating GIRK channels through phosphatidylinositol 4,5-bisphosphate. J. Neurosci. 28, 315e324. Coulson, E.J., May, L.M., Sykes, A.M., Hamlin, A.S., 2009. The role of the p75 neurotrophin receptor in cholinergic dysfunction in Alzheimer’s disease. Neuroscientist 15, 317e323. Crowley, C., Spencer, S.D., Nishimura, M.C., Chen, K.S., Pitts-Meek, S., Armanini, M.P., Ling, L.H., Mcmahon, S.B., Shelton, D.L., Levinson, A.D., et al., 1994. Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 76, 1001e1011.

Cuello, A.C., Bruno, M.A., Bell, K.F., 2007. NGF-cholinergic dependency in brain aging, MCI and Alzheimer’s disease. Curr. Alzheimer Res. 4, 351e358. Culmsee, C., Gerling, N., Lehmann, M., Nikolova-Karakashian, M., Prehn, J.H., Mattson, M.P., Krieglstein, J., 2002. Nerve growth factor survival signaling in cultured hippocampal neurons is mediated through TrkA and requires the common neurotrophin receptor P75. Neuroscience 115, 1089e1108. Davies, A.M., Lee, K.F., Jaenisch, R., 1993. p75-deficient trigeminal sensory neurons have an altered response to NGF but not to other neurotrophins. Neuron 11, 565e574. Defreitas, M.F., Mcquillen, P.S., Shatz, C.J., 2001. A novel p75NTR signaling pathway promotes survival, not death, of immunopurified neocortical subplate neurons. J. Neurosci. 21, 5121e5129. Deinhardt, K., Chao, M.V., 2014. Trk receptors. Handb. Exp. Pharmacol. 220, 103e119. Deppmann, C.D., Mihalas, S., Sharma, N., Lonze, B.E., Niebur, E., Ginty, D.D., 2008. A model for neuronal competition during development. Science 320, 369e373. di Mola, F.F., Friess, H., Zhu, Z.W., Koliopanos, A., Bley, T., Di Sebastiano, P., Innocenti, P., Zimmermann, A., Buchler, M.W., 2000. Nerve growth factor and Trk high affinity receptor (TrkA) gene expression in inflammatory bowel disease. Gut 46, 670e679. Dobrowsky, R.T., Werner, M.H., Castellino, A.M., Chao, M.V., Hannun, Y.A., 1994. Activation of the sphingomyelin cycle through the low-affinity neurotrophin receptor. Science 265, 1596e1599. Donovan, M.J., Hahn, R., Tessarollo, L., Hempstead, B.L., 1996. Identification of an essential nonneuronal function of neurotrophin 3 in mammalian cardiac development. Nat. Genet. 14, 210e213. Donovan, M.J., Lin, M.I., Wiegn, P., Ringstedt, T., Kraemer, R., Hahn, R., Wang, S., Ibanez, C.F., Rafii, S., Hempstead, B.L., 2000. Brain derived neurotrophic factor is an endothelial cell survival factor required for intramyocardial vessel stabilization. Development 127, 4531e4540. Egan, M.F., Kojima, M., Callicott, J.H., Goldberg, T.E., Kolachana, B.S., Bertolino, A., Zaitsev, E., Gold, B., Goldman, D., Dean, M., Lu, B., Weinberger, D.R., 2003. The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 112, 257e269. Eide, F.F., Vining, E.R., Eide, B.L., Zang, K., Wang, X.Y., Reichardt, L.F., 1996. Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling. J. Neurosci. 16, 3123e3129. Erickson, J.T., Conover, J.C., Borday, V., Champagnat, J., Barbacid, M., Yancopoulos, G., Katz, D.M., 1996. Mice lacking brain-derived neurotrophic factor exhibit visceral sensory neuron losses distinct from mice lacking NT4 and display a severe developmental deficit in control of breathing. J. Neurosci. 16, 5361e5371. Ernfors, P., Lee, K.F., Jaenisch, R., 1994a. Mice lacking brain-derived neurotrophic factor develop with sensory deficits. Nature 368, 147e150. Ernfors, P., Lee, K.F., Kucera, J., Jaenisch, R., 1994b. Lack of neurotrophin-3 leads to deficiencies in the peripheral nervous system and loss of limb proprioceptive afferents. Cell 77, 503e512. Esposito, D., Patel, P., Stephens, R.M., Perez, P., Chao, M.V., Kaplan, D.R., Hempstead, B.L., 2001. The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor. J. Biol. Chem. 276, 32687e32695. Esteban, P.F., Yoon, H.Y., Becker, J., Dorsey, S.G., Caprari, P., Palko, M.E., Coppola, V., Saragovi, H.U., Randazzo, P.A., Tessarollo, L., 2006. A kinase-deficient TrkC receptor isoform activates Arf6-Rac1 signaling through the scaffold protein tamalin. J. Cell Biol. 173, 291e299. Fahnestock, M., Yu, G., Coughlin, M.D., 2004. ProNGF: a neurotrophic or an apoptotic molecule? Prog. Brain Res. 146, 101e110.

REFERENCES

Farinas, I., Jones, K.R., Backus, C., Wang, X.Y., Reichardt, L.F., 1994. Severe sensory and sympathetic deficits in mice lacking neurotrophin-3. Nature 369, 658e661. Fenner, B.M., 2012. Truncated TrkB: beyond a dominant negative receptor. Cytokine Growth Factor Rev. 23, 15e24. Ferrer, I., Marin, C., Rey, M.J., Ribalta, T., Goutan, E., Blanco, R., Tolosa, E., Marti, E., 1999. BDNF and full-length and truncated TrkB expression in Alzheimer disease. Implications in therapeutic strategies. J. Neuropathol. Exp. Neurol. 58, 729e739. Foehr, E.D., Lin, X., O’mahony, A., Geleziunas, R., Bradshaw, R.A., Greene, W.C., 2000. NF-kappa B signaling promotes both cell survival and neurite process formation in nerve growth factorstimulated PC12 cells. J. Neurosci. 20, 7556e7563. Frade, J.M., Barde, Y.A., 1999. Genetic evidence for cell death mediated by nerve growth factor and the neurotrophin receptor p75 in the developing mouse retina and spinal cord. Development 126, 683e690. Frade, J.M., Rodriguez-Tebar, A., Barde, Y.A., 1996. Induction of cell death by endogenous nerve growth factor through its p75 receptor. Nature 383, 166e168. Freund-Michel, V., Frossard, N., 2008. The nerve growth factor and its receptors in airway inflammatory diseases. Pharmacol. Ther. 117, 52e76. Friedman, W.J., 2000. Neurotrophins induce death of hippocampal neurons via the p75 receptor. J. Neurosci. 20, 6340e6346. Friedman, W.J., Greene, L.A., 1999. Neurotrophin signaling via Trks and p75. Exp. Cell Res. 253, 131e142. Friess, H., Zhu, Z.W., Di Mola, F.F., Kulli, C., Graber, H.U., AndrenSandberg, A., Zimmermann, A., Korc, M., Reinshagen, M., Buchler, M.W., 1999. Nerve growth factor and its high-affinity receptor in chronic pancreatitis. Ann. Surg. 230, 615e624. Fritzsch, B., Tessarollo, L., Coppola, E., Reichardt, L.F., 2004. Neurotrophins in the ear: their roles in sensory neuron survival and fiber guidance. Prog. Brain Res. 146, 265e278. Frossard, N., Freund, V., Advenier, C., 2004. Nerve growth factor and its receptors in asthma and inflammation. Eur. J. Pharmacol. 500, 453e465. Fryer, R.H., Kaplan, D.R., Feinstein, S.C., Radeke, M.J., Grayson, D.R., Kromer, L.F., 1996. Developmental and mature expression of fulllength and truncated TrkB receptors in the rat forebrain. J. Comp. Neurol. 374, 21e40. Fulgenzi, G., Tomassoni-Ardori, F., Babini, L., Becker, J., Barrick, C., Puverel, S., Tessarollo, L., 2015. BDNF modulates heart contraction force and long-term homeostasis through truncated TrkB.T1 receptor activation. J. Cell Biol. 210, 1003e1012. Gauthier, L.R., Charrin, B.C., Borrell-Pages, M., Dompierre, J.P., Rangone, H., Cordelieres, F.P., DE Mey, J., Macdonald, M.E., Lessmann, V., Humbert, S., Saudou, F., 2004. Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules. Cell 118, 127e138. Genevois, A.L., Ichim, G., Coissieux, M.M., Lambert, M.P., Lavial, F., Goldschneider, D., Jarrosson-Wuilleme, L., Lepinasse, F., Gouysse, G., Herceg, Z., Scoazec, J.Y., Tauszig-Delamasure, S., Mehlen, P., 2013. Dependence receptor TrkC is a putative colon cancer tumor suppressor. Proc. Natl. Acad. Sci. U. S. A. 110, 3017e3022. Gentry, J.J., Casaccia-Bonnefil, P., Carter, B.D., 2000. Nerve growth factor activation of nuclear factor kappaB through its p75 receptor is an anti-apoptotic signal in RN22 schwannoma cells. J. Biol. Chem. 275, 7558e7565. Gentry, J.J., Rutkoski, N.J., Burke, T.L., Carter, B.D., 2004. A functional interaction between the p75 neurotrophin receptor interacting factors, TRAF6 and NRIF. J. Biol. Chem. 279, 16646e16656. Gestwa, G., Wiechers, B., Zimmermann, U., Praetorius, M., Rohbock, K., Kopschall, I., Zenner, H.P., Knipper, M., 1999. Differential expression of trkB.T1 and trkB.T2, truncated trkC, and

101

p75(NGFR) in the cochlea prior to hearing function. J. Comp. Neurol. 414, 33e49. Gines, S., Bosch, M., Marco, S., Gavalda, N., Diaz-Hernandez, M., Lucas, J.J., Canals, J.M., Alberch, J., 2006. Reduced expression of the TrkB receptor in Huntington’s disease mouse models and in human brain. Eur. J. Neurosci. 23, 649e658. Gong, Y., Cao, P., Yu, H.J., Jiang, T., 2008. Crystal structure of the neurotrophin-3 and p75NTR symmetrical complex. Nature 454, 789e793. Grob, P.M., Berlot, C.H., Bothwell, M.A., 1983. Affinity labeling and partial purification of nerve growth factor receptors from rat pheochromocytoma and human melanoma cells. Proc. Natl. Acad. Sci. U. S. A. 80, 6819e6823. Grob, P.M., Bothwell, M.A., 1983. Modification of nerve growth factor receptor properties by wheat germ agglutinin. J. Biol. Chem. 258, 14136e14143. Grob, P.M., Ross, A.H., Koprowski, H., Bothwell, M., 1985. Characterization of the human melanoma nerve growth factor receptor. J. Biol. Chem. 260, 8044e8049. Hallbook, F., 1999. Evolution of the vertebrate neurotrophin and Trk receptor gene families. Curr. Opin. Neurobiol. 9, 616e621. Hallbook, F., Ibanez, C.F., Persson, H., 1991. Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary. Neuron 6, 845e858. Hallbook, F., Wilson, K., Thorndyke, M., Olinski, R.P., 2006. Formation and evolution of the chordate neurotrophin and Trk receptor genes. Brain Behav. Evol. 68, 133e144. Hamanoue, M., Middleton, G., Wyatt, S., Jaffray, E., Hay, R.T., Davies, A.M., 1999. p75-mediated NF-kappaB activation enhances the survival response of developing sensory neurons to nerve growth factor. Mol. Cell. Neurosci. 14, 28e40. Hammarberg, H., Lidman, O., Lundberg, C., Eltayeb, S.Y., Gielen, A.W., Muhallab, S., Svenningsson, A., Linda, H., van der Meide, P.H., Cullheim, S., Olsson, T., Piehl, F., 2000. Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells. J. Neurosci. 20, 5283e5291. Hapner, S.J., Boeshore, K.L., Large, T.H., Lefcort, F., 1998. Neural differentiation promoted by truncated trkC receptors in collaboration with p75(NTR). Dev. Biol. 201, 90e100. Harrington, A.W., St Hillaire, C., Zweifel, L.S., Glebova, N.O., Philippidou, P., Halegoua, S., Ginty, D.D., 2011. Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival. Cell 146, 421e434. Hartmann, M., Brigadski, T., Erdmann, K.S., Holtmann, B., Sendtner, M., Narz, F., Lessmann, V., 2004. Truncated TrkB receptor-induced outgrowth of dendritic filopodia involves the p75 neurotrophin receptor. J. Cell Sci. 117, 5803e5814. He, X.L., Garcia, K.C., 2004. Structure of nerve growth factor complexed with the shared neurotrophin receptor p75. Science 304, 870e875. Hempstead, B.L., 2014. Deciphering proneurotrophin actions. Handb. Exp. Pharmacol. 220, 17e32. Hempstead, B.L., Martin-Zanca, D., Kaplan, D.R., Parada, L.F., Chao, M.V., 1991. High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor. Nature 350, 678e683. Hetman, M., Cavanaugh, J.E., Kimelman, D., Xia, Z., 2000. Role of glycogen synthase kinase-3beta in neuronal apoptosis induced by trophic withdrawal. J. Neurosci. 20, 2567e2574. Hirata, H., Hibasami, H., Yoshida, T., Ogawa, M., Matsumoto, M., Morita, A., Uchida, A., 2001. Nerve growth factor signaling of p75 induces differentiation and ceramide-mediated apoptosis in Schwann cells cultured from degenerating nerves. Glia 36, 245e258.

102

5. NEUROTROPHINS AND NEUROTROPHIN RECEPTORS

Hohn, A., Leibrock, J., Bailey, K., Barde, Y.A., 1990. Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature 344, 339e341. Holgado-Madruga, M., Moscatello, D.K., Emlet, D.R., Dieterich, R., Wong, A.J., 1997. Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. Proc. Natl. Acad. Sci. U. S. A. 94, 12419e12424. Huang, E.J., Reichardt, L.F., 2003. Trk receptors: roles in neuronal signal transduction. Annu. Rev. Biochem. 72, 609e642. Huang, S.H., Zhao, L., Sun, Z.P., Li, X.Z., Geng, Z., Zhang, K.D., Chao, M.V., Chen, Z.Y., 2009. Essential role of Hrs in endocytic recycling of full-length TrkB receptor but not its isoform TrkB.T1. J. Biol. Chem. 284, 15126e15136. Huber, L.J., Chao, M.V., 1995. A potential interaction of p75 and trkA NGF receptors revealed by affinity crosslinking and immunoprecipitation. J. Neurosci. Res. 40, 557e563. Iacaruso, M.F., Galli, S., Marti, M., Villalta, J.I., Estrin, D.A., JaresErijman, E.A., Pietrasanta, L.I., 2011. Structural model for p75(NTR)-TrkA intracellular domain interaction: a combined FRET and bioinformatics study. J. Mol. Biol. 414, 681e698. Iannone, F., de Bari, C., Dell’accio, F., Covelli, M., Patella, V., Lo Bianco, G., Lapadula, G., 2002. Increased expression of nerve growth factor (NGF) and high affinity NGF receptor (p140 TrkA) in human osteoarthritic chondrocytes. Rheumatology 41, 1413e1418. Ichim, G., Genevois, A.L., Menard, M., Yu, L.Y., Coelho-Aguiar, J.M., Llambi, F., Jarrosson-Wuilleme, L., Lefebvre, J., Tulasne, D., Dupin, E., le Douarin, N., Arumae, U., Tauszig-Delamasure, S., Mehlen, P., 2013. The dependence receptor TrkC triggers mitochondria-dependent apoptosis upon cobra-1 recruitment. Mol. Cell 51, 632e646. Ieda, M., Kanazawa, H., Ieda, Y., Kimura, K., Matsumura, K., Tomita, Y., Yagi, T., Onizuka, T., Shimoji, K., Ogawa, S., Makino, S., Sano, M., Fukuda, K., 2006. Nerve growth factor is critical for cardiac sensory innervation and rescues neuropathy in diabetic hearts. Circ. 114, 2351e2363. Iulita, M.F., Cuello, A.C., 2016. The NGF metabolic pathway in the CNS and its dysregulation in down syndrome and Alzheimer’s disease. Curr. Alzheimer Res. 13, 53e67. Jansen, P., Giehl, K., Nyengaard, J.R., Teng, K., Lioubinski, O., Sjoegaard, S.S., Breiderhoff, T., Gotthardt, M., Lin, F., Eilers, A., Petersen, C.M., Lewin, G.R., Hempstead, B.L., Willnow, T.E., Nykjaer, A., 2007. Roles for the pro-neurotrophin receptor sortilin in neuronal development, aging and brain injury. Nat. Neurosci. 10, 1449e1457. Jones, K.R., Farinas, I., Backus, C., Reichardt, L.F., 1994. Targeted disruption of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development. Cell 76, 989e999. Jung, K.M., Tan, S., Landman, N., Petrova, K., Murray, S., Lewis, R., Kim, P.K., Kim, D.S., Ryu, S.H., Chao, M.V., Kim, T.W., 2003. Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor. J. Biol. Chem. 278, 42161e42169. Kenchappa, R.S., Tep, C., Korade, Z., Urra, S., Bronfman, F.C., Yoon, S.O., Carter, B.D., 2010. p75 neurotrophin receptor-mediated apoptosis in sympathetic neurons involves a biphasic activation of JNK and upregulation of tumor necrosis factor-alpha-converting enzyme/ ADAM17. J. Biol. Chem. 285, 20358e20368. Kermani, P., Hempstead, B., 2007. Brain-derived neurotrophic factor: a newly described mediator of angiogenesis. Trends Cardiovasc. Med. 17, 140e143. Kermani, P., Rafii, D., Jin, D.K., Whitlock, P., Schaffer, W., Chiang, A., Vincent, L., Friedrich, M., Shido, K., Hackett, N.R., Crystal, R.G., Rafii, S., Hempstead, B.L., 2005. Neurotrophins promote

revascularization by local recruitment of TrkBþ endothelial cells and systemic mobilization of hematopoietic progenitors. J. Clin. Investig. 115, 653e663. Kernie, S.G., Liebl, D.J., Parada, L.F., 2000. BDNF regulates eating behavior and locomotor activity in mice. EMBO J. 19, 1290e1300. Kerschensteiner, M., Gallmeier, E., Behrens, L., Leal, V.V., Misgeld, T., Klinkert, W.E., Kolbeck, R., Hoppe, E., Oropeza-Wekerle, R.L., Bartke, I., Stadelmann, C., Lassmann, H., Wekerle, H., Hohlfeld, R., 1999. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J. Exp. Med. 189, 865e870. Khursigara, G., Bertin, J., Yano, H., Moffett, H., Distefano, P.S., Chao, M.V., 2001. A prosurvival function for the p75 receptor death domain mediated via the caspase recruitment domain receptorinteracting protein 2. J. Neurosci. 21, 5854e5863. Khursigara, G., Orlinick, J.R., Chao, M.V., 1999. Association of the p75 neurotrophin receptor with TRAF6. J. Biol. Chem. 274, 2597e2600. Kim, H., Li, Q., Hempstead, B.L., Madri, J.A., 2004. Paracrine and autocrine functions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brain-derived endothelial cells. J. Biol. Chem. 279, 33538e33546. Kim, J., Lee, Y., Cho, H.J., Lee, Y.E., An, J., Cho, G.H., Ko, Y.H., Joo, K.M., Nam, D.H., 2014. NTRK1 fusion in glioblastoma multiforme. PLoS One 9, e91940. Kirstein, M., Farinas, I., 2002. Sensing life: regulation of sensory neuron survival by neurotrophins. Cell. Mol. Life Sci. 59, 1787e1802. Kisiswa, L., Fernandez-Suarez, D., Sergaki, M.C., Ibanez, C.F., 2018. RIP2 Gates TRAF6 Interaction with Death Receptor p75(NTR) to Regulate Cerebellar Granule Neuron Survival. Cell reports 24, 1013e1024. Klein, R., 1994. Role of neurotrophins in mouse neuronal development. FASEB J. 8, 738e744. Klein, R., Silos-Santiago, I., Smeyne, R.J., Lira, S.A., Brambilla, R., Bryant, S., Zhang, L., Snider, W.D., Barbacid, M., 1994. Disruption of the neurotrophin-3 receptor gene trkC eliminates la muscle afferents and results in abnormal movements. Nature 368, 249e251. Klein, R., Smeyne, R.J., Wurst, W., Long, L.K., Auerbach, B.A., Joyner, A.L., Barbacid, M., 1993. Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death. Cell 75, 113e122. Knowles, J.K., Rajadas, J., Nguyen, T.V., Yang, T., Lemieux, M.C., Vander Griend, L., Ishikawa, C., Massa, S.M., Wyss-Coray, T., Longo, F.M., 2009. The p75 neurotrophin receptor promotes amyloid-beta(1-42)-induced neuritic dystrophy in vitro and in vivo. J. Neurosci. 29, 10627e10637. Kobayashi, H., Gleich, G.J., Butterfield, J.H., Kita, H., 2002. Human eosinophils produce neurotrophins and secrete nerve growth factor on immunologic stimuli. Blood 99, 2214e2220. Korade, Z., Mi, Z., Portugal, C., Schor, N.F., 2007. Expression and p75 neurotrophin receptor dependence of cholesterol synthetic enzymes in adult mouse brain. Neurobiol Aging 28, 1522e1531. Kraemer, B.R., Yoon, S.O., Carter, B.D., 2014. The biological functions and signaling mechanisms of the p75 neurotrophin receptor. Handb. Exp. Pharmacol. 220, 121e164. Kryl, D., Barker, P.A., 2000. TTIP is a novel protein that interacts with the truncated T1 TrkB neurotrophin receptor. Biochem. Biophys. Res. Commun. 279, 925e930. Kust, B.M., Brouwer, N., Mantingh, I.J., Boddeke, H.W., Copray, J.C., 2003. Reduced p75NTR expression delays disease onset only in female mice of a transgenic model of familial amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 4, 100e105. Lambiase, A., Bonini, S., Bonini, S., Micera, A., Magrini, L., BracciLaudiero, L., Aloe, L., 1995. Increased plasma levels of nerve

REFERENCES

growth factor in vernal keratoconjunctivitis and relationship to conjunctival mast cells. Invest. Ophthalmol. Vis. Sci. 36, 2127e2132. Lange, A.M., Lo, H.W., 2018. Inhibiting TRK proteins in clinical cancer therapy. Cancers 10. Lapchak, P.A., Hefti, F., 1992. BDNF and NGF treatment in lesioned rats: effects on cholinergic function and weight gain. Neuroreport 3, 405e408. Laudiero, L.B., Aloe, L., Levi-Montalcini, R., Buttinelli, C., Schilter, D., Gillessen, S., Otten, U., 1992. Multiple sclerosis patients express increased levels of beta-nerve growth factor in cerebrospinal fluid. Neurosci. Lett. 147, 9e12. Lauri, A., Bertucci, P., Arendt, D., 2016. Neurotrophin, p75, and trk signaling module in the developing nervous system of the marine annelid Platynereis dumerilii. BioMed Res. Int. 2016, 2456062. le Moan, N., Houslay, D.M., Christian, F., Houslay, M.D., Akassoglou, K., 2011. Oxygen-dependent cleavage of the p75 neurotrophin receptor triggers stabilization of HIF-1alpha. Mol. Cell 44, 476e490. Lee, K.F., Davies, A.M., Jaenisch, R., 1994. p75-deficient embryonic dorsal root sensory and neonatal sympathetic neurons display a decreased sensitivity to NGF. Development 120, 1027e1033. Leon, A., Buriani, A., dal Toso, R., Fabris, M., Romanello, S., Aloe, L., Levi-Montalcini, R., 1994. Mast cells synthesize, store, and release nerve growth factor. Proc. Natl. Acad. Sci. U. S. A. 91, 3739e3743. Levi-Montalcini, R., Cohen, S., 1960. Effects of the extract of the mouse submaxillary salivary glands on the sympathetic system of mammals. Ann. N. Y. Acad. Sci. 85, 324e341. Levi-Montalcini, R., Hamburger, V., 1951. Selective growth stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo. J. Exp. Zool. 116, 321e361. Levi-Montalcini, R., Meyer, H., Hamburger, V., 1954. In vitro experiments on the effects of mouse sarcomas 180 and 37 on the spinal and sympathetic ganglia of the chick embryo. Cancer Res. 14, 49e57. Liepinsh, E., Ilag, L.L., Otting, G., Ibanez, C.F., 1997. NMR structure of the death domain of the p75 neurotrophin receptor. EMBO J 16, 4999e5005. Lin, M.I., Das, I., Schwartz, G.M., Tsoulfas, P., Mikawa, T., Hempstead, B.L., 2000. Trk C receptor signaling regulates cardiac myocyte proliferation during early heart development in vivo. Dev. Biol. 226, 180e191. Lin, Z., Tann, J.Y., Goh, E.T., Kelly, C., Lim, K.B., Gao, J.F., Ibanez, C.F., 2015. Structural basis of death domain signaling in the p75 neurotrophin receptor. Elife 4, e11692. Linggi, M.S., Burke, T.L., Williams, B.B., Harrington, A., Kraemer, R., Hempstead, B.L., Yoon, S.O., Carter, B.D., 2005. Neurotrophin receptor interacting factor (NRIF) is an essential mediator of apoptotic signaling by the p75 neurotrophin receptor. J. Biol. Chem. 280, 13801e13808. Liu, X., Ernfors, P., Wu, H., Jaenisch, R., 1995. Sensory but not motor neuron deficits in mice lacking NT4 and BDNF. Nature 375, 238e241. Liu, Y., Rohrschneider, L.R., 2002. The gift of Gab. FEBS Lett. 515, 1e7. Lommatzsch, M., Schloetcke, K., Klotz, J., Schuhbaeck, K., Zingler, D., Zingler, C., Schulte-Herbruggen, O., Gill, H., Schuff-Werner, P., Virchow, J.C., 2005. Brain-derived neurotrophic factor in platelets and airflow limitation in asthma. Am. J. Respir. Crit. Care Med. 171, 115e120. Lowry, K.S., Murray, S.S., Mclean, C.A., Talman, P., Mathers, S., Lopes, E.C., Cheema, S.S., 2001. A potential role for the p75 lowaffinity neurotrophin receptor in spinal motor neuron degeneration in murine and human amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 2, 127e134. Lu, B., Nagappan, G., Lu, Y., 2014. BDNF and synaptic plasticity, cognitive function, and dysfunction. Handb. Exp. Pharmacol. 220, 223e250.

103

Lyons, W.E., Mamounas, L.A., Ricaurte, G.A., Coppola, V., Reid, S.W., Bora, S.H., Wihler, C., Koliatsos, V.E., Tessarollo, L., 1999. Brainderived neurotrophic factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain serotonergic abnormalities. Proc. Natl. Acad. Sci. U. S. A. 96, 15239e15244. Maisonpierre, P.C., Belluscio, L., Friedman, B., Alderson, R.F., Wiegand, S.J., Furth, M.E., Lindsay, R.M., Yancopoulos, G.D., 1990a. NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 5, 501e509. Maisonpierre, P.C., Belluscio, L., Squinto, S., Ip, N.Y., Furth, M.E., Lindsay, R.M., Yancopoulos, G.D., 1990b. Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Science 247, 1446e1451. Manti, S., Brown, P., Perez, M.K., Piedimonte, G., 2017. The role of neurotrophins in inflammation and allergy. Vitam. Horm. 104, 313e341. Martin-Zanca, D., Hughes, S.H., Barbacid, M., 1986. A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences. Nature 319, 743e748. Mccollum, A.T., Estus, S., 2004. NGF acts via p75 low-affinity neurotrophin receptor and calpain inhibition to reduce UV neurotoxicity. J. Neurosci. Res. 77, 552e564. Mcdonald, N.Q., Hendrickson, W.A., 1993. A structural superfamily of growth factors containing a cystine knot motif. Cell 73, 421e424. Mcdonald, N.Q., Lapatto, R., Murray-Rust, J., Gunning, J., Wlodawer, A., Blundell, T.L., 1991. New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor. Nature 354, 411e414. Mehlen, P., 2010. Dependence receptors: the trophic theory revisited. Sci. Signal. 3, pe47. Michaelsen, K., Zagrebelsky, M., Berndt-Huch, J., Polack, M., Buschler, A., Sendtner, M., Korte, M., 2010. Neurotrophin receptors TrkB.T1 and p75NTR cooperate in modulating both functional and structural plasticity in mature hippocampal neurons. Eur. J. Neurosci. 32, 1854e1865. Minichiello, L., Calella, A.M., Medina, D.L., Bonhoeffer, T., Klein, R., Korte, M., 2002. Mechanism of TrkB-mediated hippocampal longterm potentiation. Neuron 36, 121e137. Minnone, G., de Benedetti, F., Bracci-Laudiero, L., 2017. NGF and its receptors in the regulation of inflammatory response. Int. J. Mol. Sci. 18. Monteggia, L.M., Barrot, M., Powell, C.M., Berton, O., Galanis, V., Gemelli, T., Meuth, S., Nagy, A., Greene, R.W., Nestler, E.J., 2004. Essential role of brain-derived neurotrophic factor in adult hippocampal function. Proc Natl Acad Sci U S A 101, 10827e10832. Mufson, E.J., Counts, S.E., Perez, S.E., Ginsberg, S.D., 2008. Cholinergic system during the progression of Alzheimer’s disease: therapeutic implications. Expert Rev. Neurother. 8, 1703e1718. Mufson, E.J., Ginsberg, S.D., Ikonomovic, M.D., Dekosky, S.T., 2003. Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction. J. Chem. Neuroanat. 26, 233e242. Mullenbrock, S., Shah, J., Cooper, G.M., 2011. Global expression analysis identified a preferentially nerve growth factor-induced transcriptional program regulated by sustained mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and AP-1 protein activation during PC12 cell differentiation. J. Biol. Chem. 286, 45131e45145. Muller, M., Triaca, V., Besusso, D., Costanzi, M., Horn, J.M., Koudelka, J., Geibel, M., Cestari, V., Minichiello, L., 2012. Loss of NGF-TrkA signaling from the CNS is not sufficient to induce cognitive impairments in young adult or intermediate-aged mice. J. Neurosci. 32, 14885e14898. Mutoh, T., Sobue, G., Hamano, T., Kuriyama, M., Hirayama, M., Yamamoto, M., Mitsuma, T., 2000. Decreased phosphorylation levels of TrkB neurotrophin receptor in the spinal cords from

104

5. NEUROTROPHINS AND NEUROTROPHIN RECEPTORS

patients with amyotrophic lateral sclerosis. Neurochem. Res. 25, 239e245. Nadezhdin, K.D., Garcia-Carpio, I., Goncharuk, S.A., Mineev, K.S., Arseniev, A.S., Vilar, M., 2016. Structural basis of p75 transmembrane domain dimerization. J. Biol. Chem. 291, 12346e12357. Nakagawa, T., Ono-Kishino, M., Sugaru, E., Yamanaka, M., Taiji, M., Noguchi, H., 2002. Brain-derived neurotrophic factor (BDNF) regulates glucose and energy metabolism in diabetic mice. Diabetes Metab. Res. Rev. 18, 185e191. Nakagawa, T., Tsuchida, A., Itakura, Y., Nonomura, T., Ono, M., Hirota, F., Inoue, T., Nakayama, C., Taiji, M., Noguchi, H., 2000. Brain-derived neurotrophic factor regulates glucose metabolism by modulating energy balance in diabetic mice. Diabetes 49, 436e444. Nassenstein, C., Schulte-Herbruggen, O., Renz, H., Braun, A., 2006. Nerve growth factor: the central hub in the development of allergic asthma? Eur. J. Pharmacol. 533, 195e206. Naumann, T., Casademunt, E., Hollerbach, E., Hofmann, J., Dechant, G., Frotscher, M., Barde, Y.A., 2002. Complete deletion of the neurotrophin receptor p75NTR leads to long-lasting increases in the number of basal forebrain cholinergic neurons. J. Neurosci. 22, 2409e2418. Neves-Pereira, M., Mundo, E., Muglia, P., King, N., Macciardi, F., Kennedy, J.L., 2002. The brain-derived neurotrophic factor gene confers susceptibility to bipolar disorder: evidence from a familybased association study. Am. J. Hum. Genet. 71, 651e655. Niewiadomska, G., Mietelska-Porowska, A., Mazurkiewicz, M., 2011. The cholinergic system, nerve growth factor and the cytoskeleton. Behav. Brain Res. 221, 515e526. Noga, O., Hanf, G., Schaper, C., O’connor, A., Kunkel, G., 2001. The influence of inhalative corticosteroids on circulating nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in allergic asthmatics. Clin. Exp. Allergy 31, 1906e1912. Notaras, M., Hill, R., van den Buuse, M., 2015. A role for the BDNF gene Val66Met polymorphism in schizophrenia? A comprehensive review. Neurosci. Biobehav. Rev. 51, 15e30. Nykjaer, A., Lee, R., Teng, K.K., Jansen, P., Madsen, P., Nielsen, M.S., Jacobsen, C., Kliemannel, M., Schwarz, E., Willnow, T.E., Hempstead, B.L., Petersen, C.M., 2004. Sortilin is essential for proNGF-induced neuronal cell death. Nature 427, 843e848. Ohira, K., Funatsu, N., Homma, K.J., Sahara, Y., Hayashi, M., Kaneko, T., Nakamura, S., 2007. Truncated TrkB-T1 regulates the morphology of neocortical layer I astrocytes in adult rat brain slices. Eur. J. Neurosci. 25, 406e416. Ohira, K., Homma, K.J., Hirai, H., Nakamura, S., Hayashi, M., 2006. TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells. Biochem. Biophys. Res. Commun. 342, 867e874. Ohira, K., Shimizu, K., Yamashita, A., Hayashi, M., 2005. Differential expression of the truncated TrkB receptor, T1, in the primary motor and prefrontal cortices of the adult macaque monkey. Neurosci. Lett. 385, 105e109. Ovsepian, S.V., Antyborzec, I., O’leary, V.B., Zaborszky, L., Herms, J., Oliver Dolly, J., 2013. Neurotrophin receptor p75 mediates the uptake of the amyloid beta (Abeta) peptide, guiding it to lysosomes for degradation in basal forebrain cholinergic neurons. Brain Struct. Funct. 219 (5), 1527e1541. Ovsepian, S.V., Herms, J., 2013. Drain of the brain: low-affinity p75 neurotrophin receptor affords a molecular sink for clearance of cortical amyloid beta by the cholinergic modulator system. Neurobiol. Aging 34, 2517e2524. Ovsepian, S.V., O’leary, V.B., Zaborszky, L., 2016. Cholinergic mechanisms in the cerebral cortex: beyond synaptic transmission. Neuroscientist 22, 238e251. Palko, M.E., Coppola, V., Tessarollo, L., 1999. Evidence for a role of truncated trkC receptor isoforms in mouse development. J. Neurosci. 19, 775e782.

Park, H.H., Lo, Y.C., Lin, S.C., Wang, L., Yang, J.K., Wu, H., 2007. The death domain superfamily in intracellular signaling of apoptosis and inflammation. Annu Rev Immunol 25, 561e586. Passiglia, F., Caparica, R., Giovannetti, E., Giallombardo, M., Listi, A., Diana, P., Cirrincione, G., Caglevic, C., Raez, L.E., Russo, A., Rolfo, C., 2016. The potential of neurotrophic tyrosine kinase (NTRK) inhibitors for treating lung cancer. Expert Opin. Investig. Drugs 25, 385e392. Pedraza, C.E., Podlesniy, P., Vidal, N., Arevalo, J.C., Lee, R., Hempstead, B., Ferrer, I., Iglesias, M., Espinet, C., 2005. Pro-NGF isolated from the human brain affected by Alzheimer’s disease induces neuronal apoptosis mediated by p75NTR. Am. J. Pathol. 166, 533e543. Prasad, M.L., Vyas, M., Horne, M.J., Virk, R.K., Morotti, R., Liu, Z., Tallini, G., Nikiforova, M.N., Christison-Lagay, E.R., Udelsman, R., Dinauer, C.A., Nikiforov, Y.E., 2016. NTRK fusion oncogenes in pediatric papillary thyroid carcinoma in northeast United States. Cancer 122, 1097e1107. Qian, L., Milne, M.R., Shepheard, S., Rogers, M.L., Medeiros, R., Coulson, E.J., 2018. Removal of p75 Neurotrophin Receptor Expression from Cholinergic Basal Forebrain Neurons Reduces Amyloidbeta Plaque Deposition and Cognitive Impairment in Aged APP/ PS1 Mice. Mol Neurobiol. Quarta, E., Fulgenzi, G., Bravi, R., Cohen, E.J., Yanpallewar, S., Tessarollo, L., Minciacchi, D., 2018. Deletion of the endogenous TrkB.T1 receptor isoform restores the number of hippocampal CA1 parvalbumin-positive neurons and rescues long-term potentiation in pre-symptomatic mSOD1(G93A) ALS mice. Mol. Cell. Neurosci. 89, 33e41. Raap, U., Braunstahl, G.J., 2010. The role of neurotrophins in the pathophysiology of allergic rhinitis. Curr. Opin. Allergy Clin. Immunol. 10, 8e13. Raap, U., Werfel, T., Goltz, C., Deneka, N., Langer, K., Bruder, M., Kapp, A., Schmid-Ott, G., Wedi, B., 2006. Circulating levels of brain-derived neurotrophic factor correlate with disease severity in the intrinsic type of atopic dermatitis. Allergy 61, 1416e1418. Radke, J., Rossner, F., Redmer, T., 2017. CD271 determines migratory properties of melanoma cells. Sci. Rep. 7, 9834. Raychaudhuri, S.P., Raychaudhuri, S.K., 2004. Role of NGF and neurogenic inflammation in the pathogenesis of psoriasis. Prog. Brain Res. 146, 433e437. Redmer, T., Welte, Y., Behrens, D., Fichtner, I., Przybilla, D., Wruck, W., Yaspo, M.L., Lehrach, H., Schafer, R., Regenbrecht, C.R., 2014. The nerve growth factor receptor CD271 is crucial to maintain tumorigenicity and stem-like properties of melanoma cells. PLoS One 9, e92596. Rios, M., 2011. New insights into the mechanisms underlying the effects of BDNF on eating behavior. Neuropsychopharmacology 36, 368e369. Rios, M., 2013. BDNF and the central control of feeding: accidental bystander or essential player? Trends Neurosci. 36, 83e90. Risau, W., Flamme, I., 1995. Vasculogenesis. Annu. Rev. Cell Dev. Biol. 11, 73e91. Rodriguez-Tebar, A., Dechant, G., Barde, Y.A., 1990. Binding of brainderived neurotrophic factor to the nerve growth factor receptor. Neuron 4, 487e492. Rodriguez-Tebar, A., Dechant, G., Barde, Y.A., 1991. Neurotrophins: structural relatedness and receptor interactions. Philos. Trans. R. Soc. Lond. B Biol. Sci. 331, 255e258. Rodriguez-Tebar, A., Dechant, G., Gotz, R., Barde, Y.A., 1992. Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor. EMBO J. 11, 917e922. Rose, C.R., Blum, R., Pichler, B., Lepier, A., Kafitz, K.W., Konnerth, A., 2003. Truncated TrkB-T1 mediates neurotrophin-evoked calcium signalling in glia cells. Nature 426, 74e78.

REFERENCES

Ross, A.H., Daou, M.C., Mckinnon, C.A., Condon, P.J., Lachyankar, M.B., Stephens, R.M., Kaplan, D.R., Wolf, D.E., 1996. The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA. J. Cell Biol. 132, 945e953. Ross, G.M., Shamovsky, I.L., Lawrance, G., Solc, M., Dostaler, S.M., Weaver, D.F., Riopelle, R.J., 1998. Reciprocal modulation of TrkA and p75NTR affinity states is mediated by direct receptor interactions. Eur. J. Neurosci. 10, 890e898. Ruberti, F., Capsoni, S., Comparini, A., di Daniel, E., Franzot, J., Gonfloni, S., Rossi, G., Berardi, N., Cattaneo, A., 2000. Phenotypic knockout of nerve growth factor in adult transgenic mice reveals severe deficits in basal forebrain cholinergic neurons, cell death in the spleen, and skeletal muscle dystrophy. J. Neurosci. 20, 2589e2601. Saarelainen, T., Hendolin, P., Lucas, G., Koponen, E., Sairanen, M., MacDonald, E., Agerman, K., Haapasalo, A., Nawa, H., Aloyz, R., Ernfors, P., Castren, E., 2003. Activation of the TrkB neurotrophin receptor is induced by antidepressant drugs and is required for antidepressant-induced behavioral effects. J Neurosci 23, 349e357. Sanchez-Ortiz, E., Yui, D., Song, D., Li, Y., Rubenstein, J.L., Reichardt, L.F., Parada, L.F., 2012. TrkA gene ablation in basal forebrain results in dysfunction of the cholinergic circuitry. J. Neurosci. 32, 4065e4079. Sanico, A.M., Stanisz, A.M., Gleeson, T.D., Bora, S., Proud, D., Bienenstock, J., Koliatsos, V.E., Togias, A., 2000. Nerve growth factor expression and release in allergic inflammatory disease of the upper airways. Am. J. Respir. Crit. Care Med. 161, 1631e1635. Santos, P.M., Winterowd, J.G., Allen, G.G., Bothwell, M.A., Rubel, E.W., 1991. Nerve growth factor: increased angiogenesis without improved nerve regeneration. Otolaryngol. Head Neck Surg. 105, 12e25. Sartore-Bianchi, A., Ardini, E., Bosotti, R., Amatu, A., Valtorta, E., Somaschini, A., Raddrizzani, L., Palmeri, L., Banfi, P., Bonazzina, E., Misale, S., Marrapese, G., Leone, A., Alzani, R., Luo, D., Hornby, Z., Lim, J., Veronese, S., Vanzulli, A., Bardelli, A., Martignoni, M., Davite, C., Galvani, A., Isacchi, A., Siena, S., 2016. Sensitivity to entrectinib associated with a novel LMNA-NTRK1 gene fusion in metastatic colorectal cancer. J. Natl. Cancer Inst. 108. Schliebs, R., Arendt, T., 2011. The cholinergic system in aging and neuronal degeneration. Behav. Brain Res. 221, 555e563. Sedel, F., Bechade, C., Triller, A., 1999. Nerve growth factor (NGF) induces motoneuron apoptosis in rat embryonic spinal cord in vitro. Eur. J. Neurosci. 11, 3904e3912. Seeburger, J.L., Tarras, S., Natter, H., Springer, J.E., 1993. Spinal cord motoneurons express p75NGFR and p145trkB mRNA in amyotrophic lateral sclerosis. Brain Res. 621, 111e115. Shen, T., You, Y., Joseph, C., Mirzaei, M., Klistorner, A., Graham, S.L., Gupta, V., 2018. BDNF polymorphism: a review of its diagnostic and clinical relevance in neurodegenerative disorders. Aging Dis. 9, 523e536. Sklar, P., Gabriel, S.B., Mcinnis, M.G., Bennett, P., Lim, Y., Tsan, G., Schaffner, S., Kirov, G., Jones, I., Owen, M., Craddock, N., Depaulo, J.R., Lander, E.S., 2002. Family-based association study of 76 candidate genes in bipolar disorder: BDNF is a potential risk locus. Brain-derived neutrophic factor. Mol. Psychiatr. 7, 579e593. Skoff, A.M., Adler, J.E., 2006. Nerve growth factor regulates substance P in adult sensory neurons through both TrkA and p75 receptors. Exp. Neurol. 197, 430e436. Smeyne, R.J., Klein, R., Schnapp, A., Long, L.K., Bryant, S., Lewin, A., Lira, S.A., Barbacid, M., 1994. Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 368, 246e249. Snider, W.D., 1994. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell 77, 627e638.

105

Sotthibundhu, A., Sykes, A.M., Fox, B., Underwood, C.K., Thangnipon, W., Coulson, E.J., 2008. Beta-amyloid(1-42) induces neuronal death through the p75 neurotrophin receptor. J. Neurosci. 28, 3941e3946. Stoilov, P., Castren, E., Stamm, S., 2002. Analysis of the human TrkB gene genomic organization reveals novel TrkB isoforms, unusual gene length, and splicing mechanism. Biochem. Biophys. Res. Commun. 290, 1054e1065. Sun, P.D., Davies, D.R., 1995. The cystine-knot growth-factor superfamily. Annu. Rev. Biophys. Biomol. Struct. 24, 269e291. Sykes, A.M., Palstra, N., Abankwa, D., Hill, J.M., Skeldal, S., Matusica, D., Venkatraman, P., Hancock, J.F., Coulson, E.J., 2012. The effects of transmembrane sequence and dimerization on cleavage of the p75 neurotrophin receptor by gamma-secretase. J. Biol. Chem. 287, 43810e43824. Tabakman, R., Lecht, S., Sephanova, S., Arien-Zakay, H., Lazarovici, P., 2004. Interactions between the cells of the immune and nervous system: neurotrophins as neuroprotection mediators in CNS injury. Prog. Brain Res. 146, 387e401. Tabassum, A., Khwaja, F., Djakiew, D., 2003. The p75(NTR) tumor suppressor induces caspase-mediated apoptosis in bladder tumor cells. Int. J. Cancer 105, 47e52. Teng, H.K., Teng, K.K., Lee, R., Wright, S., Tevar, S., Almeida, R.D., Kermani, P., Torkin, R., Chen, Z.Y., Lee, F.S., Kraemer, R.T., Nykjaer, A., Hempstead, B.L., 2005. ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. J. Neurosci. 25, 5455e5463. Tessarollo, L., 1998. Pleiotropic functions of neurotrophins in development. Cytokine Growth Factor Rev. 9, 125e137. Tessarollo, L., Hempstead, B.L., 1998. Regulation of cardiac development by receptor tyrosine kinases. Trends Cardiovasc. Med. 8, 34e40. Tessarollo, L., Tsoulfas, P., Donovan, M.J., Palko, M.E., Blair-Flynn, J., Hempstead, B.L., Parada, L.F., 1997. Targeted deletion of all isoforms of the trkC gene suggests the use of alternate receptors by its ligand neurotrophin-3 in neuronal development and implicates trkC in normal cardiogenesis. Proc. Natl. Acad. Sci. U. S. A. 94, 14776e14781. Tanaka, K., Kelly, C.E., Goh, K.Y., Lim, K.B., Ibanez, C.F., 2016. Death domain signaling by disulfide-linked dimers of the p75 neurotrophin receptor mediates neuronal death in the CNS. J. Neurosci. 36, 5587e5595. Thoenen, H., 1995. Neurotrophins and neuronal plasticity. Science 270, 593e598. Tonra, J.R., Ono, M., Liu, X., Garcia, K., Jackson, C., Yancopoulos, G.D., Wiegand, S.J., Wong, V., 1999. Brain-derived neurotrophic factor improves blood glucose control and alleviates fasting hyperglycemia in C57BLKS-Lepr(db)/lepr(db) mice. Diabetes 48, 588e594. Tsukamoto, E., Hashimoto, Y., Kanekura, K., Niikura, T., Aiso, S., Nishimoto, I., 2003. Characterization of the toxic mechanism triggered by Alzheimer’s amyloid-beta peptides via p75 neurotrophin receptor in neuronal hybrid cells. J. Neurosci. Res. 73, 627e636. Turner, B.J., Cheah, I.K., Macfarlane, K.J., Lopes, E.C., Petratos, S., Langford, S.J., Cheema, S.S., 2003. Antisense peptide nucleic acidmediated knockdown of the p75 neurotrophin receptor delays motor neuron disease in mutant SOD1 transgenic mice. J. Neurochem. 87, 752e763. Tyurina, Y.Y., Nylander, K.D., Mirnics, Z.K., Portugal, C., Yan, C., Zaccaro, C., Saragovi, H.U., Kagan, V.E., Schor, N.F., 2005. The intracellular domain of p75NTR as a determinant of cellular reducing potential and response to oxidant stress. Aging Cell 4, 187e196. Ulian-Benitez, S., Bishop, S., Foldi, I., Wentzell, J., Okenwa, C., Forero, M.G., Zhu, B., Moreira, M., Phizacklea, M., Mcilroy, G., Li, G., Gay, N.J., Hidalgo, A., 2017. Kek-6: a truncated-Trk-like receptor for Drosophila neurotrophin 2 regulates structural synaptic plasticity. PLoS Genet. 13, e1006968.

106

5. NEUROTROPHINS AND NEUROTROPHIN RECEPTORS

Ultsch, M.H., Wiesmann, C., Simmons, L.C., Henrich, J., Yang, M., Reilly, D., Bass, S.H., De Vos, A.M., 1999. Crystal structures of the neurotrophin-binding domain of trka, TrkB and TrkC. J. Mol. Biol. 290, 149e159. Underwood, C.K., Reid, K., May, L.M., Bartlett, P.F., Coulson, E.J., 2008. Palmitoylation of the C-terminal fragment of p75 (NTR) regulates death signaling and is required for subsequent cleavage by gamma-secretase. Mol. Cell. Neurosci. 37, 346e358. Vaillant, A.R., Mazzoni, I., Tudan, C., Boudreau, M., Kaplan, D.R., Miller, F.D., 1999. Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival. J. Cell Biol. 146, 955e966. Vaishnavi, A., Capelletti, M., Le, A.T., Kako, S., Butaney, M., Ercan, D., Mahale, S., Davies, K.D., Aisner, D.L., Pilling, A.B., Berge, E.M., Kim, J., Sasaki, H., Park, S.I., Kryukov, G., Garraway, L.A., Hammerman, P.S., Haas, J., Andrews, S.W., Lipson, D., Stephens, P.J., Miller, V.A., Varella-Garcia, M., Janne, P.A., Doebele, R.C., 2013. Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer. Nat. Med. 19, 1469e1472. Venero, J.L., Hefti, F., 1998. Regionally specific induction of BDNF and truncated trkB.T1 receptors in the hippocampal formation after intraseptal injection of kainic acid. Brain Res. 790, 270e277. Ventriglia, M., Bocchio Chiavetto, L., Benussi, L., Binetti, G., Zanetti, O., Riva, M.A., Gennarelli, M., 2002. Association between the BDNF 196 A/G polymorphism and sporadic Alzheimer’s disease. Mol. Psychiatr. 7, 136e137. Vilar, M., Charalampopoulos, I., Kenchappa, R.S., Simi, A., Karaca, E., Reversi, A., Choi, S., Bothwell, M., Mingarro, I., Friedman, W.J., et al., 2009. Activation of the p75 neurotrophin receptor through conformational rearrangement of disulphide-linked receptor dimers. Neuron 62, 72e83. Virchow, J.C., Julius, P., Lommatzsch, M., Luttmann, W., Renz, H., Braun, A., 1998. Neurotrophins are increased in bronchoalveolar lavage fluid after segmental allergen provocation. Am. J. Respir. Crit. Care Med. 158, 2002e2005. Volosin, M., Trotter, C., Cragnolini, A., Kenchappa, R.S., Light, M., Hempstead, B.L., Carter, B.D., Friedman, W.J., 2008. Induction of proneurotrophins and activation of p75NTR-mediated apoptosis via neurotrophin receptor-interacting factor in hippocampal neurons after seizures. J. Neurosci. 28, 9870e9879. Wang, Y.J., Wang, X., Lu, J.J., Li, Q.X., Gao, C.Y., Liu, X.H., Sun, Y., Yang, M., Lim, Y., Evin, G., Zhong, J.H., Masters, C., Zhou, X.F., 2011. p75NTR regulates Abeta deposition by increasing Abeta production but inhibiting Abeta aggregation with its extracellular domain. J. Neurosci. 31, 2292e2304. Watanabe, T., Fajt, M.L., Trudeau, J.B., Voraphani, N., Hu, H., Zhou, X., Holguin, F., Wenzel, S.E., 2015. Brain-derived neurotrophic factor expression in asthma. Association with severity and type 2 inflammatory processes. Am. J. Respir. Cell Mol. Biol. 53, 844e852. Wehrman, T., He, X., Raab, B., Dukipatti, A., Blau, H., Garcia, K.C., 2007. Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors. Neuron 53, 25e38. Westwick, J.K., Bielawska, A.E., Dbaibo, G., Hannun, Y.A., Brenner, D.A., 1995. Ceramide activates the stress-activated protein kinases. J. Biol. Chem. 270, 22689e22692. Wiesmann, C., Ultsch, M.H., Bass, S.H., De Vos, A.M., 1999. Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor. Nature 401, 184e188. Willnow, T.E., Petersen, C.M., Nykjaer, A., 2008. VPS10P-domain receptorseregulators of neuronal viability and function. Nat. Rev. Neurosci. 9, 899e909.

Wilson, K.H., 2009. The genome sequence of the protostome Daphnia pulex encodes respective orthologues of a neurotrophin, a Trk and a p75NTR: evolution of neurotrophin signaling components and related proteins in the bilateria. BMC Evol. Biol. 9, 243. Wolf, D.E., Mckinnon, C.A., Daou, M.C., Stephens, R.M., Kaplan, D.R., Ross, A.H., 1995. Interaction with TrkA immobilizes gp75 in the high affinity nerve growth factor receptor complex. J. Biol. Chem. 270, 2133e2138. Yaar, M., Zhai, S., Fine, R.E., Eisenhauer, P.B., Arble, B.L., Stewart, K.B., Gilchrest, B.A., 2002. Amyloid beta binds trimers as well as monomers of the 75-kDa neurotrophin receptor and activates receptor signaling. J. Biol. Chem. 277, 7720e7725. Yaar, M., Zhai, S., Pilch, P.F., Doyle, S.M., Eisenhauer, P.B., Fine, R.E., Gilchrest, B.A., 1997. Binding of beta-amyloid to the p75 neurotrophin receptor induces apoptosis. A possible mechanism for Alzheimer’s disease. J. Clin. Investig. 100, 2333e2340. Yan, H., Chao, M.V., 1991. Disruption of cysteine-rich repeats of the p75 nerve growth factor receptor leads to loss of ligand binding. J. Biol. Chem. 266, 12099e12104. Yan, C., Mirnics, Z.K., Portugal, C.F., Liang, Y., Nylander, K.D., Rudzinski, M., Zaccaro, C., Saragovi, H.U., Schor, N.F., 2005. Cholesterol biosynthesis and the pro-apoptotic effects of the p75 nerve growth factor receptor in PC12 pheochromocytoma cells. Brain Res Mol Brain Res 139, 225e234. Yano, H., Torkin, R., Martin, L.A., Chao, M.V., Teng, K.K., 2009. Proneurotrophin-3 is a neuronal apoptotic ligand: evidence for retrograde-directed cell killing. J. Neurosci. 29, 14790e14802. Yanpallewar, S.U., Barrick, C.A., Buckley, H., Becker, J., Tessarollo, L., 2012. Deletion of the BDNF truncated receptor TrkB.T1 delays disease onset in a mouse model of amyotrophic lateral sclerosis. PLoS One 7, e39946. Yao, X.Q., Jiao, S.S., Saadipour, K., Zeng, F., Wang, Q.H., Zhu, C., Shen, L.L., Zeng, G.H., Liang, C.R., Wang, J., Liu, Y.H., Hou, H.Y., Xu, X., Su, Y.P., Fan, X.T., Xiao, H.L., Lue, L.F., Zeng, Y.Q., Giunta, B., Zhong, J.H., Walker, D.G., Zhou, H.D., Tan, J., Zhou, X.F., Wang, Y.J., 2015. p75NTR ectodomain is a physiological neuroprotective molecule against amyloid-beta toxicity in the brain of Alzheimer’s disease. Mol. Psychiatr. 20, 1301e1310. Yeiser, E.C., Rutkoski, N.J., Naito, A., Inoue, J., Carter, B.D., 2004. Neurotrophin signaling through the p75 receptor is deficient in traf6-/mice. J. Neurosci. 24, 10521e10529. Zaccaro, M.C., Ivanisevic, L., Perez, P., Meakin, S.O., Saragovi, H.U., 2001. p75 Co-receptors regulate ligand-dependent and ligandindependent Trk receptor activation, in part by altering Trk docking subdomains. J. Biol. Chem. 276, 31023e31029. Zhang, T., Mi, Z., Schor, N.F., 2009. Role of tyrosine phosphorylation in the antioxidant effects of the p75 neurotrophin receptor. Oxid. Med. Cell. Longev. 2, 238e246. Zhu, B., Pennack, J.A., Mcquilton, P., Forero, M.G., Mizuguchi, K., Sutcliffe, B., Gu, C.J., Fenton, J.C., Hidalgo, A., 2008. Drosophila neurotrophins reveal a common mechanism for nervous system formation. PLoS Biol. 6, e284. Zuccato, C., Cattaneo, E., 2014. Huntington’s disease. Handb. Exp. Pharmacol. 220, 357e409. Zuccato, C., Ciammola, A., Rigamonti, D., Leavitt, B.R., Goffredo, D., Conti, L., Macdonald, M.E., Friedlander, R.M., Silani, V., Hayden, M.R., Timmusk, T., Sipione, S., Cattaneo, E., 2001. Loss of huntingtin-mediated BDNF gene transcription in Huntington’s disease. Science 293, 493e498. Zuccato, C., Tartari, M., Crotti, A., Goffredo, D., Valenza, M., Conti, L., Cataudella, T., Leavitt, B.R., Hayden, M.R., Timmusk, T., Rigamonti, D., Cattaneo, E., 2003. Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes. Nat. Genet. 35, 76e83.

C H A P T E R

6 The Pineal as a Gland and Melatonin as a Hormone Richard J. Wurtman Massachusetts Institute of Technology, Cambridge, MA, United States

1. INTRODUCTION: HISTORY OF THE HYPOTHESIS THAT THE MAMMALIAN PINEAL IS A GLAND AND SECRETES THE HORMONE MELATONIN Melatonin (5-methoxy N-acetyltryptamine) is the majordand possibly exclusivedhormone produced by and secreted from the human pineal gland (Lerner et al., 1958). It is synthesized from the essential amino acid tryptophan, which is first converted to serotonin (by 5-hydroxylation and then decarboxylation), then N-acetylated, and finally, O-methylated (Fig. 6.5) (Axelrod and Weissbach, 1960). Melatonin’s synthesis and its blood levels in humans and other mammals exhibit marked circadian rhythms (Lynch et al., 1975), so its concentrations at midnight may be tenfold or greater than those observed at the middle of the day. This rhythm enables melatonin to serve as a “time giver” (Zeitgeber) for other circadian rhythms (Lewy et al., 1992; Arendt, 1999) and as an important signal in promoting sleep onset and maintenance (Zhdanova et al., 1996, 2001; Haimov et al., 1995; Zhdanova et al., 1996; Garfinkel et al., 1995). Melatonin is presently used both to promote sleep onset, e.g., during eastbound flights, when the user wishes to sleep during part of the daily light period, or when anxiety or obsessive thinking interfere with sleep onset at its usual time of day, as well as “hormone replacement therapy” for people unable to sustain the normally elevated nocturnal plasma melatonin levels because of age-related pineal calcification. Its use has become widespread: Between 2007 and 2101 (the last year for which data are available through NIHeNCCIH (National Center for Complementary and Integrative Health)), its use by American adults more than doubled,

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00006-7

from 1,296,000 to 3,065,000 (nccih.nih.gov/research/statistics/NHIS; 2015). Most of the information now available about melatonin’s actions in humans and other mammals concerns the effects of administering small, physiologic doses (i.e., under 1 mg) that elevate its blood levels within their normal nocturnal ranges (Figs. 6.6 and 6.7) (Lewy et al., 1992; Arendt, 1999; Zhdanova et al., 2001). Data from some recent studies employing pharmacologic doses suggest, however, that the hormone could also have additional actions and possibly uses in higher doses (Wurtman, 2017), much as exogenous glucocorticoids given in one dosage range provide hormone replacement therapy for Addisonian patients and, at higher doses, drugs for treating poison ivy. Recognition that the mammalian pineal is a gland and that melatonin, its product, is a hormone came to endocrinology relatively late (Wurtman et al., 1963a). For much of the 20th century the mammalian pineal was generally dismissed by scientists as a “vestige,” a functioning third eye in certain lower vertebrates but only a calcified remnant in humans (Kitay and Altschule, 1954). Tumors of the pineal were known sometimes to be associated with precocious puberty, especially in boys, and some scientists attributed this to the tumor’s destruction of functioning pineal tissue that would otherwise have secreted a putative gonad inhibitor. However, the prevailing belief was that the accelerated gonadal maturation was simply a nonspecific consequence of increased intracranial pressure. The modern history of the mammalian pineal began in 1917, with the discovery that extracts of bovine pineals contained a compound that could lighten the skins of tadpoles or frogs (McCord and Allen, 1917). The physiologic significance of this finding was obscure, inasmuch as

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bovine pineal extracts had no discernible effects on pigmentation in bovines (nor humans), and frog pineals were not found to be involved in physiologic skin lightening in the frogs. However the finding did demonstrate that mammalian pineals contained a compound with at least some biologic activity, and probably most important, it suggested a successful strategy for isolating and identifying this compound. This strategy was based on using a novel skin-lightening assay that could follow the ability of sequentially purified pineal extracts to lighten frog skin (by causing the melanin granules in the frog’s pigment cells, the melanophores, to aggregate). In 1958, Aaron Lerner, a professor of dermatology who hoped to identify the skin-lightening agent so that he could use it to lighten human skin (e.g., in treating vitiligo) developed such an assay and used it to identify the structure of this agent. Once he succeeded in doing so, he named the compound melatonin (Lerner et al., 1958). A few years later, we showed that, besides affecting pigmentation in amphibians, melatonin was also a true hormone in mammals, secreted into the circulation and then suppressing gonadal maturation in laboratory rodents (Wurtman et al., 1963a). Two years later, Hoffman and Reiter (1965) showed that the hamster’s pinealepossibly also acting via a secretionemediated the gonadal atrophy that occurred when the animals were maintained in a mostly dark environment. In the decade prior to the isolation of melatonin, scientists had made four seemingly unrelated discoveries about the mammalian pineal that became coherent once melatonin was identified and its effects could be tested. These included the following: 1. The demonstration that pinealectomy accelerated the growth of the rat’s ovaries, while administration of bovine pineal extracts had the opposite effect (Wurtman et al., 1961). 2. The observation that housing rats in a continuously lighted environment decreased the weights of their pineals (Fiske et al., 1960). (As discussed subsequently, the lighting was found not to be acting directly on the pineals, as would have been the case if they were still photoreceptive in mammals as they are in some amphibians and reptiles, but indirectly, via the eyes.) 3. The discovery that, though the mammalian pineal originates embryologically as part of the brain, it loses most of its CNS connections by the time of birth, and instead receives its innervation via its postganglionic sympathetic nerves (Ariens-Kappers, 1960). 4. The demonstration that, in rats, pinealectomy or exposing the animals to prolonged light exposure, or giving both treatments, all accelerate the growth of the rat’s ovaries to the same extent, and that the effects of either treatment can be blocked by giving

the animals bovine pineal extracts (Wurtman et al., 1961). This suggested that the rat pineal secretes or at least contains a gonad-inhibiting substance, perhaps a hormone, which could be depleted by removing the pineal or by exposing the animal to supplemental lighting. (As discussed subsequently, though that substance turned out to be melatonin in rats and other laboratory rodents, melatonin has taken on entirely different functionsdrelating to circadian rhythms and to sleepdin humans.) Between 1963 and 1964, it was shown that melatonin, in rats, satisfied the formal criteria for classification as a hormone (Wurtman et al., 1963a), that it was the gonadinhibiting substance previously found in pineal extracts (Wurtman et al., 1963b), and that its synthesis in the pineal is controlled by circadian and seasonal variations in the environmental lightedark cycle. When rats are exposed continuously to light, the light acts not directly, as in the “third eye” of some amphibians and reptiles, but indirectly, via the animal’s lateral eyes and sympathetic nerves (Wurtman et al., 1963b, 1964) to suppress melatonin formation. Subsequently, the neurotransmitter that mediated the sympathetic nervous signals controlling the pineal was shown to be norepinephrine (Wurtman et al., 1981), which acted by enhancing cyclic-AMP production (Shein and Wurtman, 1969). The rates at which the rat’s pineal synthesizes serotonin and melatonin were shown to vary with circadian rhythms, coupled to the 24-h lightedark cycle, but dependent for their continuation not on that cycle (Quay, 1963; Lynch, 1971) but on a neural signal emanating from the suprachiasmatic nucleus (Moore and Klein, 1974). Finally, in 1975, it was discovered that melatonin secretion from the human pineal exhibits the pronounced circadian rhythm described earlier, with nocturnal plasma levels an order of magnitude or greater than those found in daytime (Lynch et al., 1975). This rhythm also was truly circadian: it persisted even if normal subjects underwent a sudden 12-hour phase shift in their lighting environment, and it required 5e7 days to reentrain to the new light cycle (Lynch et al., 1978).

2. THE PINEAL GLAND AS A NEUROENDOCRINE TRANSDUCER: CONVERTING A NEURAL INPUT TO AN ENDOCRINE OUTPUT As described before, the amount of melatonin that the pineal contains (Lynch, 1971) and secretes and its levels in the plasma normally vary from hour to hour, depending on, synchronously, an endogenous signal from the suprachiasmatic nucleus and an exogenous one

3. MELATONIN: DISTRIBUTION, BIOSYNTHESIS, METABOLISM

provided by the photic input to the eyes. Both signals affect the neural input to the gland, the firing frequency of the pineal’s sympathetic nerves and their release of norepinephrine (Axelrod et al., 1969; Wurtman et al., 1981). The pineal then transduces this input into what will be a circulating outputdthe release of melatonin into the blood streamdwhich in turn affects the brain and, probably, other tissues. Temporal variations in the rate at which melatonin is secreted generate two major biologic rhythms: a circadian rhythm, as noted before, and an annual rhythm in the number of light (or darkness) hours during each 24-h period. There are more hours of darkness in fall/winterdpeaking on the first day of winterdthan in spring/summer, so integrated plasma melatonin levels in fall/winter are also higher than those observed during spring/summer. Similarly, hours of light per 24-h day peak on the first day of summer, and integrated plasma melatonin levels per 24 h are also lower in summer than the annual average. These variations impact the brain, with biologic consequences, as discussed subsequently. Different species utilize the rhythms differentially (Brzezinski, 1997): among diurnally active mammals like humans, the nocturnal rise in plasma melatonin promotes sleepiness, and it facilitates sleep maintenance

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through the night (Wurtman and Zhdanova, 1995; Zhdanova et al., 2001). A relative deficiency in nighttime plasma melatonin levels, common among older people (Fig. 6.6) and related to the age-dependent calcification of the pineal, often impairs the ability to maintain sleep throughout the night. This deficiency is often treatable using low oral doses of melatonin, which are just sufficient to restore nighttime plasma levels to those characteristic of younger people (Zhdanova et al., 2001) (Figs. 6.7 and 6.1). Among animals that breed seasonally, the annual rhythm in day lengthdwhich causes plasma melatonin levels to be higher during winter months than during the summerdaffects the choice of breeding season, i.e., whether it coincides with increasing (i.e., spring) or decreasing (i.e., fall) day length (Hoffman and Reitman, 1965).

3. MELATONIN: DISTRIBUTION, BIOSYNTHESIS, METABOLISM All, or almost all, of the melatonin in mammalian tissues and body fluids is synthesized within the pineal gland. So not surprisingly, plasma melatonin and its

FIGURE 6.1 Metabolism of tryptophan to melatonin in the pineal gland. Reproduced from Zhdanova, I.V., Wurtman, R.J., 1997. In: Conn, P.M., Melmed, S. (Eds.), Endocrinology: Basic and Clinical Principles. Humana, Totowa, NJ, p. 281. Copyright 1997, Humana Press.

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FIGURE 6.2 Nighttime peak serum melatonin levels in subjects of different ages, years. Reproduced with permission from Zhdanova, I.V., Wurtman, R.J., 1997. In: Conn, P.M., Melmed, S. (Eds.), Endocrinology: Basic and Clinical Principles. Humana, Totowa, NJ, p. 281. Copyright © 1997 Humana Press.

circadian rhythm largely disappear following pinealectomy, as shown in animal studies (McConnell and Hinds, 1985) and in human subjects (Neuwelt and Lewy, 1983). Small quantities of melatonin and the key melatoninforming enzymes hydroxyindole-O-methyltransferase (HIOMT) and serotonin-N-acetyltransferase (SNAT)

have sometimes been described in the retina, Harderian gland, and gastrointestinal organs. However, most such claims have not been supported by studies that used the highly specific bioassay or GCMS (gas chromatography/mass spectroscopy, the chemical “gold standard”) techniques to confirm the authenticity of the melatonin.

FIGURE 6.3 Mean serum melatonin profiles. Mean serum melatonin profiles of 20 subjects sampled at intervals after ingesting 0.1, 0.3, 1.0, and 10 mg of melatonin or placebo at 11:45 a.m. Reproduced from Dollins, A.B., Zhdanova, I.V., Wurtman, R.J., Lynch, H.J., et al., 1994. Effect of inducing nocturnal serum melatonin concentrations in daytime on sleep, mood, body temperature, and performance. PNAS 91, 1824. Copyright 1994 National Academy of Sciences, USA.

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FIGURE 6.4 Mean group (n ¼ 30) plasma melatonin profiles after melatonin or placebo treatment 30 min before bedtime. Reproduced from Zhdanova, I.V., Wurtman, R.J., Regan, M.M., et al., 2001. Melatonin treatment for age-related insomnia. J. Clin. Endocrinol. Metab. 86, 4727.

Assays based on showing that a compound obtained from a tissue or body fluid and thought to be melatonin binds to antimelatonin antibodies or migrates with authentic melatonin on chromatography or HPLC are simply too nonspecific to distinguish melatonin from the large number of other compounds that might share these properties. One study by Ozaki and Lynch (1976) did confirmd using a bioassay methoddthat very small quantities of melatonin do persist in rat blood and urine following successful pinealectomy; however, circadian rhythms

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in the melatonin do not. This extrapineal melatonin could have been produced in nonpineal tissue or, given melatonin’s high lipid-solubility, could have been stored premortem in a fatty tissue and gradually released from that tissue or perhaps ingested with a fermented food product. Whether the melatonin attains sufficient concentrations to be physiologically active is problematic. Generally, readers are advised to maintain a healthy skepticism when asked to believe publications that claimdwithout supporting bioassay or GCMS datad that melatonin has now been discovered in another tissue or cellular structure. The same warning applies to articles that claim the presence of melatonin in foods. Several reports (e.g., Dubbels et al., 1995; Hattori et al., 1995; Van Tassel et al., 2001) have described a compound they believed to be melatonin in dietary fruits or vegetables (e.g., tomato), but in only one such article (Van Tassel et al., 2001) was the compound’s identity unambiguously confirmed by an adequately described GCMS method, and in that study the melatonin concentrations were very low (less than 20 ng per kilogram of fruit) and were thought to reflect contamination. Of greatest significance, it has never been reported that consumption of any nonfermented food by humans can raise plasma melatonin concentrations. (A Japanese fermentation product, a beer, was described by one laboratory as raising plasma melatonin levels.) Perhaps similarly, over the years the literature has abounded with largely unconfirmed reports that the pineal produces and secretes other biologically active compounds besides melatonin, most often peptides or other indoles. Surely, it is possible that the pineal could secrete multiple compounds; however, proof that it does

FIGURE 6.5 Structures useful for the study of MT1 receptor structureefunction relationships. Docking of melatonin in the solvent accessible cavity was achieved by energy relaxation by 300-ns molecular dynamics simulations. Jockers, R., Delagrange, P., Bubocovich, M.L., et al., 2016. Update on melatonin receptors: IUPHAR Review 20. Br. J. Pharmacol. 173, 2702e2725.

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FIGURE 6.6 Distribution of nonsynonymous MT1 (A) and MT2 (B) receptor variants identified in various human populations. Positions of variants are highlighted in light brown. Typical signatures of MT receptors, such as the 3.49NRY3.51 motif and the 7.49NAXXY7.53 motif are highlighted in red. Residues suspected to be directly involved in melatonin binding (S3.35 and S3.39 in MT1 and H5.46 in both MT1 and MT2 receptors) are highlighted in a blue circle. The putative palmitoylation site ar C314 is indicated in MT1 receptors. Jockers, R., Delagrange, P., Bubocovich, M.L., et al., 2016. Update on melatonin receptors: IUPHAR Review 20. Br. J. Pharmacol. 173, 2702e2725.

so, and that any such compounds actually function as hormones, is missing. Like all mammalian cells, pinealocytes take up circulating tryptophan and use it, along with the other amino acids, for synthesizing proteins. And like enterochromaffin cells and particular brain neurons, they also use some of their tryptophan to form serotonin (Fig. 6.5). As described before, the conversion of tryptophan to serotonin requires two enzymes: tryptophan hydroxylase, which forms 5-hydroxytryptophan (5-HTP), and aromatic-L-amino acid decarboxylase, which decarboxylates the 5-HTP to form the serotonin (5hydroxytrytamine). The hydroxylation step is rate limiting in this sequence because the hydroxylase enzyme has a very low affinity for tryptophan, its substrate, and pineal tryptophan levels are insufficient to saturate the enzyme. The conversion of serotonin to melatonin requires two additional enzymatic steps, as mentioned earlier, the acetylation of the amine nitrogen

to form N-acetylserotonin, followed by that intermediate’s O-methylation to form the melatonin (Axelrod and Weissbach, 1960). It should be noted that blood serotonindfree or in plateletsdor the serotonin in brain neurons have never been shown to be intermediates in pineal melatonin synthesis. During daylight hours the serotonin formed in pinealocytes tends to be stored in vesicles, so it is unavailable for further metabolism by the two intracellular serotonin-metabolizing enzymes, the N-acetylating enzyme (SNAT) and monoamine oxidase (MAO). With the onset of darkness, postganglionic sympathetic outflow to the pineal increases, and the consequent release of norepinephrine onto pinealocytes causes stored serotonin to become accessible for intracellular metabolism. At the same time the norepinephrine activates the enzymes that convert serotonin to melatonin, especially serotonin-N-acetyltransferase (SNAT) but also hydroxyindole-O-methyl transferase (HIOMT).

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FIGURE 6.7 Melatonin receptor signaling. (A) MT1 or MT2 monomeric receptor signaling. Both MT1 and MT2 melatonin receptors couple to pertussis toxin-sensitive GaI, b, g, and insensitive Gq, b, and g proteins inhibiting forskolin-stimulated cAMP, protein kinase A signaling and CREB phosphorylation. MT1/MT2 heterodimer receptor signaling. Human MT1 and MT2 receptors form homo- and heterodimer receptor signaling. Human MT1 and MT2 receptors form homo- and heterooligomers between themselves, altering the pharmacological properties of the individual receptors. Formation densities of the MT1/MT2 heterodimer and the MT2/MT2 homodimer are similar and three- to fourfold lower than the MT1/ Mt1 homodimer. Native functional MT1/MT2 heterodimers have been characterized in mouse rod photoreceptors, where they mediate the enhancement of scotopic light sensitivity by melatonin through a heterodimer-specific PLC and PKC pathway. Abbreviations: AC, adenylyl cyclase; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol-4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C. Liu, J., Shannon, J., Clough, A.J., et al., 2016. Melatonin 1 and melatonin 2 receptors: a therapeutic perspective. Annu. Rev. Pharmacol. Toxicol. 56, 361e383.

Consequently, melatonin levels rise many-fold, and by virtue of its lipid solubility, it rapidly diffuses out of the pinealocytes into the blood and cerebrospinal fluid (Tricoire et al., 2002). Pineal levels of the corresponding deaminated and O-methylated metabolite of serotonin, 5-methoxytryptophol, also rise (Skene et al., 1990), even though this compound’s formation is independent of SNAT. This suggests that the daily rhythm in melatonin synthesis is not caused by serotonin’s acetylation, as has been proposed. Melatonin is highly lipid soluble because both of the ionizable groups in serotonin, the hydroxyl and the amine, have been blocked by its Omethylation and N-acetylation. Thus, it diffuses freely across cell membranes, including those of the bloode brain barrier, and it travels in the blood largely bound to albumin (Cardinali et al., 1972). Most of the melatonin in the circulation is inactivated in the liver, where it is first oxidized to 6-OHmelatonin by a P450-dependent microsomal oxidase

and then largely conjugated to sulfate or glucuronide before being excreted into the urine or feces (Kopin et al., 1961). Approximately 2e3% of the circulating melatonin is excreted unchanged into the urine or the saliva, enabling measurements of urinary or salivary melatonin to be used as rough estimates of plasma melatonin concentrations. Salivary melatonin levels apparently correspond to those of the 25% to 30% of blood melatonin that is not bound to albumin (Cardinali et al., 1972). Studies using radioactively labeled melatonin have identified three probable melatonin receptors, two of which have been cloned using human sources (Gerdin et al., 2004, 2005). The macromolecules, which interact with G-proteins (Brydon et al., 1999), are concentrated, respectively, within the suprachiasmatic nucleus (SCN) of the hypothalamus, the pars tuberalis of the pituitary, and cardiac blood vessels (MT1); the retina and hippocampus (MT2); and in kidney, brain, and various

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FIGURE 6.8 Core body temperature profiles following melatonin or placebo treatment. Reproduced from Zhdanova, I.V., Wurtman, R.J., Regan, M.M., et al., 2001. Melatonin treatment for age-related insomnia. J. Clin. Endocrinol. Metab. 86, 4727.

peripheral organs (MT3) (Ekmekcioglu, 2006; Witt-Enderby et al., 2003). The MT1 receptors in the SCN allow melatonin to inhibit the firing of SCN neurons during the nighttime, an action that might contribute to melatonin’s sleep-promoting effects. The SCN’s MT2 receptors apparently mediate melatonin’s

effects on the SCN’s own circadian rhythms. Melatonin’s MT1 and MT2 receptors are highly susceptible to “desensitization,” their activity decreasing markedly after exposure to supranormal concentrations of the hormone (Dubocovich and Markowska, 2005). This is highly relevant to the prolonged use of high melatonin

FIGURE 6.9 Sleep efficiency in insomniacs during three consecutive parts (I, II, and III) of the night, following placebo or melatonin (0.3 mg) treatment. Reproduced from Zhdanova, I.V., Wurtman, R.J., Regan, M.M., et al., 2001. Melatonin treatment for age-related insomnia. J. Clin. Endocrinol. Metab. 86, 4727.

4. MELATONIN RHYTHMS: CIRCADIAN AND ANNUAL

FIGURE 6.10 Sleep efficiency in subjects with normal sleep and age-related insomnia. Reproduced from Zhdanova, I.V., Wurtman, R.J., Regan, M.M., et al., 2001. Melatonin treatment for age-related insomnia. J. Clin. Endocrinol. Metab. 86, 4727.

doses to promote sleep, particularly among older adults with insomnia who might inadvertently purchase excessively large doses of the hormone. Understandably, such people often find that their usual daily melatonin dose stops working after several weeks. Structures of MT1 and MT2 receptors are shown in Figs. 6.5 and 6.6 (Jockers et al., 2016). Activated receptor signaling is diagrammed in Fig. 6.7 (Liu et al., 2016). Melatonin receptors are either monomeric, such as MT1/MT1 and MT2/MT@2, or heteromeric, such as MT1/MT2. Both MT1 and MT2 melatonin receptors couple to pertussis toxin-sensitive Ga, b, and g proteins, inhibiting forskolin-stimulated cAMP, protein kinase A signaling, and CREB phosphorylation. Human MT1 and MT2 receptors form homo- and heterodimer receptor signaling. MT1 and MT2 receptors form homo- and heterooligomers between themselves, altering the pharmacological properties of the individual receptors. Further information on the signaling of these receptors is illustrated in Fig. 6.7.

4. MELATONIN RHYTHMS: CIRCADIAN AND ANNUAL In all mammals examined thus far, melatonin secretion manifests a similar circadian rhythm, with plasma and urine concentrations low during daylight, ascending after the onset of darkness, peaking in the middle of the night

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between 11 p.m. and 3 a.m., and then falling sharply before the time of light onset (Lewy et al., 1980; Burgess et al., 2001). As stated earlier, high nocturnal plasma melatonin concentrations are characteristic of both diurnally active species (like humans), in which the high levels promote sleep onset and maintenance, and nocturnally active ones (like rats), in which melatonin has no obvious relationship to sleep. While this rhythm normally is tightly entrained to the environmental light cycle, it does persist when people are placed for a few days in a dark room and, as described before, does not immediately phase shift when the light schedule is altered (Lynch et al., 1978), indicating that it is not simply generated by the lightedark cycle but also by cyclic endogenous signals, possibly arising in the SCN. Signals originating in the retina or the SCN reach the pineal via a retinohypothalamic tract, the superior cervical ganglia, and postganglionic sympathetic fibers that reenter the cranial cavity (Wurtman et al., 1964; Moore and Klein, 1994). Light has no known direct effects on pineal melatonin synthesis in humans and other mammals. The ability of exogenous melatonin to synchronize and to shift the phases of various human circadian rhythms, including in blind people (Sack et al., 1991) and those with disturbed sleep rhythms Dahlitz et al. (1991), is generally accepted (Lewy et al., 1992; Brzezinski, 1997; Arendt, 1999). In studies of healthy volunteers, 0.5 mg of pure melatonin or 0.05 mg of melatonin in corn oil (which causes earlier peaks in, and the more rapid disappearance of, elevated plasma melatonin concentrations) was able to advance the onset of nocturnal melatonin secretion when administered at 5 p.m., and larger doses caused greater phase advances (Arendt, 1999; Brzezinski et al., 2005; Lewy et al., 2002). Supraphysiologic doses of melatonin are able to shift the core body temperature rhythm (Fig. 6.2); however, a statistically significant effect is found in human subjects, only with doses > 0.5 mg. These doses increase plasma melatonin concentrations well above its upper limits of normal (Arendt, 1999). A fall in body temperature is not required for the hormone to promote sleep. Melatonin secretion by the human pineal varies markedly with age. The hormone starts to be secreted during the third or fourth months of life, coincident with the consolidation of sleeping at nighttime. Its secretion then increases rapidly, causing nocturnal melatonin concentrations to peak at ages 1 to 3 years; they then decline slightly to a plateau that persists throughout early adulthood (Fig. 6.1) (Waldhauser et al., 1984; Brzezinski, 1997). Cyclic nocturnal melatonin secretion then exhibits a marked and continuing decline in most people, with peak nocturnal concentrations in 70 year olds being only a quarter or less of what they are in young adults (Fig. 6.6). As proposed before, this decline may reflect

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the progressive, age-related calcification of the pineal gland and its resulting loss of pineal secretory tissue. Obviously, one strategy in using supplemental melatonin to promote sleep is to administer it to older adults with age-associated insomnia in doses just sufficient to compensate for this age-related decline. Nocturnal melatonin concentrations can be affected by drugs that interfere with the transmission of neurotransmitter signals to pineal cells (like propranolol, a beta-blocking agent) or those that affect melatonin’s metabolism (e.g., caffeine or ethanol). Nocturnal melatonin secretion is also suppressed by a relatively dim 100e200 lux when the pupils have been dilated. The most potent wavelength for suppressing melatonin secretion appears to be 446e477 nm, which differs from the peak absorbance of the photopigments for vision. Prolonged use of portable light-emitting devices (laptops, tablets, and smartphones) before bedtime may have a negative impact on melatonin secretion, circadian rhythms, and sleep. One study compared the effects of reading an electronic book illuminated by a light-emitting device (LE-ebook) versus a printed book (by reflected light) for 4 hours prior to bedtime for five consecutive nights. Subjects in the LE-ebook group exhibited suppressed melatonin concentrations in the early part of the night, a delayed endogenous circadian melatonin phase, felt less sleepy before bedtime, took longer to fall asleep, and reported feeling sleepier the following morning. The observations suggest but do not yet prove that evening use of some light-emitting devices may contribute to phase delays in the circadian clock and difficulty initiating sleep.

5. MELATONIN AS A “NUTRITIONAL SUPPLEMENT” In the United States, the hormone melatonin is termed a “dietary supplement” by the US Food and Drug Administration’s Dietary Health and Education Act, even though melatonin is not normally obtained from, nor even present, in the diet. It may have been so classified to encourage its use, without a prescription, because of its unusual safety if compared to sleeping pills. However, this misnomer probably diminishes the utility of the term when used like vitamins. In the European Union, melatonin has been evaluated by an official regulatory agency, the European Food Safety Authority (EFSA, 2011), for use in reducing sleep latency, the time it takes for normal sleepers or people with insomnia to fall asleep (at bedtime or after a nocturnal awakening). Based on the evidence provided in all three of the statistically valid published metaanalyses (Brzezenski et al., 2005; Buscemi et al., 2005, 2006), the agency concluded that “a cause and effect

relationship exists between the consumption of melatonin and a reduction of sleep onset latency,” and that “in order to obtain the claimed effect, 1 mg of melatonin should be consumed close to bedtime.” (Too few data were available to enable evaluation of the efficacy of lower doses). Such doses have little or no effect on sleep efficiency among adults in whom it was normal (Fig. 6.3). However, doses as low as 0.1 mg partially restore diminished sleep efficiency in subjects with age-related insomnia (Fig. 6.3). In such subjects, sleep during the first third of the night tends to be normal (Fig. 6.4) but depressed in the second and third parts; a 0.3-mg melatonin dose largely restores sleep during these periods (Fig. 6.4). Occasional claims to the contrary notwithstanding, apparently all of the melatonin sold in the United States is of synthetic origin. Although melatonin is relatively nontoxic, some marketed doses (1e10 mg) can elevate plasma concentrations to 3 to 60 times their normal peak values (Fig. 6.7). Supraphysiologic concentrations of melatonin can produce various biologic effects, including daytime sleepiness, impaired mental and physical performance, hypothermia, and hyperprolactinemia. These effects are not observed with physiologic concentrations of melatonin. There is considerable person-to-person variability in the bioavailability of melatonin. In one study using 0.5-mg oral doses, peak plasma melatonin concentrations among subjects varied over a greater than 10-fold range. Melatonin’s bioavailability was relatively poor (10%e56%), which the authors attributed to person-toperson differences in first-pass hepatic extraction. Perhaps reflecting such differences in hepatic function, older adult subjects in another study given a 0.3-mg oral dose of melatonin were found to exhibit considerable greater increments in plasma melatonin concentrations, with correspondingly greater variability, than young adults receiving that dose. These findings all suggest that, while a 0.3-mg dose given to young subjects during the daytime or to older adult insomniacs at night can, on average, product normal nocturnal plasma melatonin concentrations, some individuals, particularly older people, may need slightly more or significantly less melatonin to attain the desired effects on plasma melatonin and sleep. A nonprescription melatonin preparation providing a total of 0.9 mg melatonin is available in the United States: an outer capsule contains both a liquid providing 0.3 mg and a second capsule which slowly releases 0.6 mg. At least two synthetic melatonin agonists, ramelteon and tasimelteon, are thought to activate MT1 and MT2 melatonin receptors selectively, and they have been available in the United States for the treatment of sleep and rhythm disorders. Their efficacy in accelerating sleep onset, or sustaining sleep, is uncertain. Melatonin

REFERENCES

and its analogs may be useful in sustaining circadian rhythms among blind people. Despite the fact that melatonin is an unregulated drug in many countries and is often used in excessive doses, it has not generated a clear pattern of side effects. In clinical studies, one report described a search for reports of adverse effects with melatonin in data going back over 35 years (Morera et al., 2001). Of the nine studies that reported such effects, pharmacologic doses were used (1e36 mg), and the numbers of patients were not described. However, headache, confusion, and fragmented sleep were among the side effects reported. Dizziness, somnolence, nausea, and headache have been reported with the melatonin agonist ramelteon.

6. AGING, MELATONIN AND SLEEP As indicated earlier, nocturnal melatonin levels in plasma decrease markedly with aging, probably a consequence of the pineal’s poorly understood, progressive calcification (which can be first noted on skull X-rays early in adult life) (Fig. 6.5). These decreases are temporally associated with impaired sleep regulation, so about 30% of people over age 50 complain of insomnia characterized by decreased total nighttime sleep time, frequent nocturnal awakenings with difficulty falling back asleep, and early morning awakening. These sleep impairments are often associated with daytime sleepiness, memory and attention deficits, and changes in mood. Daytime administration of low doses of melatonin (0.3e1.0 mg) that raise plasma levels to their normal nocturnal values can facilitate daytime sleep onset, for example, in healthy people taking eastbound flights (Arendt, 1999; Dollins et al., 1994; Wyatt et al., 1999). Low doses of exogenous melatonin (e.g., less than 1 mg) taken near bedtime also ameliorate the insomnia of aging (Zhdanova et al., 2001) and restore sleep efficiency (Figs. 6.2 and 6.3). Higher doses, which raise plasma levels well beyond their normal nocturnal range, are not initially more effective than physiologic doses, and if taken repeatedly become less effective because melatonin receptors in the brain desensitize (Gerdin et al., 2004; Dubocovich and Markowska, 2005) (Figs. 6.8e6.10).

References Arendt, J., 1999. Jet-lag and shift work: therapeutic use of melatonin. J. Royal Soc. Med. 92, 402e405. Axelrod, J., Shein, H.M., Wurtman, R.J., 1969. Stimulation of C14melatonin synthesis from C14-tryptophan by noradrenaline in rat pineals in organ culture. Proc. Natl. Acad. Sci. U. S. A. 62, 544. Axelrod, J., Weissbach, H., 1960. Enzymatic O-methylation of Nacetylserotonin to melatonin. Science 131, 1312e1313.

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Ariens-Kappers, J., 1960. Innervation of the epiphysis cerebri in the albino rat. Anat. Rec. 136, 220. Brydon, L., Roka, F., Petic, L., et al., 1999. Dual signaling of human mel1a melatonin receptors via Gi2, Gi3, and Gq/11 proteins. Mol. Endocrinol. 13 (12), 2025. Brzezinski, A., Vangel, M.G., Wurtman, R.J., et al., 2005. Effects of exogenous melatonin on sleep: a meta-analysis. Sleep Med. Rev. 9, 41. Brzezinski, A., 1997. Melatonin in humans. N. Engl. J. Med. 336, 186. Burgess, H.J., Sletten, T., Savic, N., et al., 2001. Effects of bright light and melatonin on sleep propensity, temperature and cardiac activity at night. J. Appl. Physiol. 91, 1214. Buscermi, N., Vandermeer, B., Hooton, N., et al., 2005. The efficacy and safety of exogenous melatonin for primary sleep disorders. A metaanalysis. J. Gen. Intern. Med. 20, 1151. Buscemi, N., Vandermeer, B., Hooton, N., et al., 2006. Efficacy and safety of exogenous melatonin for secondary sleep disorders and sleep disorders accompanying sleep restriction: meta-analysis. Br. Med. J. 332, 385. Cardinali, D.P., Lynch, H.J., Wurtman, R.J., 1972. Binding of melatonin to human and rat plasma proteins. Endocrinol. 91, 1213. Dahlitz, M., Alvarez, B., Fignau, J., et al., 1991. Delayed sleep phase syndrome response to melatonin. Lancet 337, 1121. Dollins, A., Zhdanova, I., Wurtman, R.J., et al., 1994. Effect of inducing nocturnal serum melatonin concentrations in daytime on sleep, mood, body temperature and performance. Proc. Nat. Acad. Sci. U. S. A. 91, 1824e1828. Dubbels, R., Reiter, R.J., Klenke, E., et al., 1995. Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry. J. Pineal Res. 18, 28. Dubocovich, M.L., Markowska, M., 2005. Function al MT1, and MT2 melatonin receptors in mammals. Endocrinol. 27 (2), 101e110. Ekmekcioglu, C., 2006. Melatonin receptors in humans: biological role and clinical relevance. Biomed. Pharmacother. 60, 97e108. European Food Safety Authority (EFSA), 2011. Scientific opinion on the substantiation of a health claim related to melatonin and reduction of sleep onset latency (ID 1968, 1780, 4080) pursuant to article 13(1) of regulation (EC) No 1924/2006. EFSA J. 9, 2241. http://www.efsa. eujropa.eu/en/efsajourn al/doc/2241.pdf. Fiske, V.M., Bryant, K., Putnam, J., 1960. Effect of light on the weight of pineal in the rat. J. Endocrinol. 66, 489. Garfinkel, D., Laudon, M., Nof, D., et al., 1995. Improvement of sleep quality in elderly people by controlled-release melatonin. Lancet 346, 541. Gerdin, M.J., Masana, M.I., Rivera-Bermύdez, M.A., et al., 2004. Melatonin desensitizes endogenous MT2 melatonin receptors in the rat suprachiasmatic nucleus: relevance for defining the periods of sensitivity of the mammalian circadian clock to melatonin. FASEB J. 18, 1646. Gerdin, M.J., Masana, M.I., Dubocovich, M.L., 2005. Melatoninmediated regulation of human MT (1) melatonin receptors expressed in mammalian cells. Biochem. Pharmacol. 67, 2023. Haimov, I., Lavie, P., Laudon, M., Herer, P., Vigder, C., Zisapel, C., 1995. Melatonin Replacement Therapy of Elderly Insomniacs. Sleep 18, 598e603. Hattori, A., Migitaka, H., Iigo, M., et al., 1995. Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates. Biochem. Mol. Biol. Int. 35, 627. Hoffman, A.A., Reiter, R.J., 1965. Pineal gland influence on gonads in male hamsters. Science 148, 1609. Jockers, R., Delagrange, P., Bubocovich, M.L., et al., 2016. Update on melatonin receptors: IUPHAR review 20. Br. J. Pharmacol. 173, 2702e2725. Kitay, J.I., Altschule, M.D., 1954. The Pineal Gland e A Review of the Physiologic Literature. Harvard Univ Press, Cambridge, MA.

118

6. GLAND AND MELATONIN AS A HORMONE

Kopin, I.J., Pare, C.M., Axelrod, J., et al., 1961. 6-Hydroxylation, the major metabolic pathway for melatonin. Biochem. Biophys. Acta 40, 377. Lerner, A.B., Case, J.D., Takahashi, Y., et al., 1958. Isolation of melatonin, the pineal gland factor that lightens melanocytes. J. Am. Chem. Soc. 80, 2587. Lewy, A.J., Wehr, T.A., Goodwin, F.K., et al., 1980. Light suppresses melatonin secretion in humans. Science 210, 1267. Lewy, A.J., Ahmed, S., Jackson, J.M., et al., 1992. Melatonin shifts human circadian rhythms according to a phase-response curve. Chronobiol. Int. 9, 380. Lewy, A.J., Emens, J.S., Lefler, B.J., et al., 2002. Melatonin entrains freerunning blind people according to a physiological dose-response curve. Chronobiol. Int. 22, 1093. Liu, J., Shannon, J., Clough, A.J., et al., 2016. Melatonin 1 and melatonin 2 receptors: a therapeutic perspective. Annu. Rev. Pharmacol. Toxicol. 56, 361e383. Lynch, H.J., 1971. Diurnal oscillations in pineal melatonin content. Life Sci. 10 (1), 791e795. Lynch, H.J., Jimerson, D.C., Ozaki, Y., et al., 1978. Entrainment of rhythmic melatonin secretion in man to a 12-hour phase sift in the light/ dark cycle. Life Sci. 23, 1557. Lynch, H.J., Wurtman, R.J., Moskowitz, M.A., et al., 1975. Daily rhythm in human urinary melatonin. Science 187, 169. McCord, C.P., Allen, F.P., 1917. Evidence associating pineal gland function with alterations in pigmentation. J. Exp. Zool. 23, 207. McConnell, S.J., Hinds, L.A., 1985. Effect of pinealectomy on plasma melatonin, prolactin and progesterone concentrations during seasonal reproductive quiescence in the tammar, Macropus eugenii. J. Reprod. Fertil. 75, 433e440. Moore, R.Y., Klein, D.C., 1974. Visual pathways and the central neural control of a circadian rhythm in pineal serotonin Nacetyltransferase activity. Brain Res. 71, 17. Morera, A.L., Henry, M., de La Varga, M., 2001. Safety in melatonin use. Actas Exp. Psiquiatr. 29, 334. Neuwelt, E.A., Lewy, A.J., 1983. Disappearance of plasma melatonin after removal of a neoplastic plasma gland. N. Engl. J. Med. 308, 1132. Ozaki, Y., Lynch, H.J., 1976. Presence of melatonin in plasma and urine of pinealectomized rats. Endocrinol. 641e644. Quay, W.B., 1963. Circadian rhythm in rat pineal serotonin and its modifications by estrous cycle and photoperiod. Gen. Comp. Endocrinol. 24, 473. Sack, R.L., Lewy, A.J., Blood, M.L., et al., 1991. Melatonin administration to blind people: phase advances and entrainment. J. Biol. Rhythm. 649. Shein, H.M., Wurtman, R.J., 1969. Cyclic adenosine monohosphate: stimulation of melatonin and serotonin synthesis in cultured rat pineals. Science 166, 519. Skene, D.J., Vivien-Roels, B., Sparks, D.L., et al., 1990. Daily variation in the concentration of melatonin and 5-methoxytryptophol in the human pineal gland: effect of age and Alzheimer’s disease. Brain Res. 528, 170.

Tricoire, H., Locatella, A., Chemineau, P., Malpaux, B., 2002. Melatonin enters the cerebrospinal fluid through the pineal recess. Endocrinol. 143, 84. Van Tassel, D.L., Roberts, N., Lewy, A., O’Neill, S.D., 2001. Melatonin in plant organs. J. Pineal Res. 31, 8. Waldhauser, F., Weiszenbacher, G., Frisch, H., et al., 1984. Fall in nocturnal serum melatonin during prepuberty and pubescence. Lancet 1, 362. Witt-Enderby, P.A., Bennett, J., Jarzynka, M.J., et al., 2003. Melatonin receptors and their regulation: biochemical and structural mechanisms. Life Sci. 72, 2183. Wurtman, R.J., 2017. Multiple sclerosis, melatonin, and neurobehavioral diseases. Front. Endocrinol. 8, 280, 1-3. Wurtman, R.J., Axelrod, J., Chu, E.W., 1963a. Melatonin, a pineal substance: effect on the rat ovary. Science 36, 317. Wurtman, R.J., Axelrod, J., Fischer, J.E., 1964. Melatonin synthesis in the pineal gland: effect of light mediated by the sympathetic nervous system. Science 143, 1328. Wurtman, R.J., Axelrod, J., Phillips, L.S., 1963b. Melatonin synthesis in the pineal gland: control by light. Science 142, 1071. Wurtman, R.J., Roth, W., Altschule, M.D., Wurtman, J.J., 1961. Interactions of the pineal and exposure to continuous light on organ weights of female rats. Acta Endocrinol. 36, 617. Wurtman, R.J., Shein, H.M., Larin, F., 1981. Mediation by eadrenergic receptors of effect of norepinephrine on pineal synthesis of (14C)serotonin and (14)melatonin. J. Neurochem. 18, 1683. Wyatt, J.K., Ritz-DeCecco, Czeisler, et al., 1999. Circadian temperature and melatonin rhythms, sleep, and neurobehavioral function in humans living on a 20h day. Am. J. Physiol. 277, R1152eR1163. Wurtman, R.J., Zhdanova, I., 1995. Improvement of sleep quality by melatonin. Lancet 346, 1491. Zhdanova, I., Wurtman, R.J., Morabito, C., et al., 1996. Effects of low oral doses of melatonin, given 2-4 hours before habitual bedtime, on sleep in normal young humans. Sleep 19, 423. Zhdanova, I.V., Wurtman, R.J., Regan, M.M., et al., 2001. Melatonin treatment for age-related insomnia. J. Clin. Endocrinol. Metab. 86 (10), 4727e4730.

Further Reading Hughes, R.J., Sack, R.L., Lewy, A.J., 1998. The role of melatonin and circadian phase in age-related sleep-maintenance insomnia: assessment in a clinical trial of melatonin replacement. Sleep 21, 52. James, S.P., Sack, D.A., Rosenthal, N.E., et al., 1990. Melatonin administration in insomnia. Neuropsychopharmacology 3, 19. Jonas, M., Garfinkel, D., Zisapel, N., et al., 2003. Impaired nocturnal melatonin secretion in non-dipper hypertensive e patients. Blood Press. 12, 19. Waldhauser, F., Dietzel, M., 1985. Daily and annual rhythms in human melatonin secretion: role in puberty control. Ann. N. Y. Acad. Sci. 205e214.

C H A P T E R

7 Anterior Pituitary: Glycoprotein Hormones From Gonadotrope (FSH and LH) and Thyrotrope (TSH) Cells Daniel J. Bernard, Emilie Bruˆle´ Departments of Pharmacology and Therapeutics, and Anatomy and Cell Biology, McGill University, Montreal, QC, Canada

1. INTRODUCTION The anterior pituitary gland produces and secretes three structurally related glycoprotein hormones: folliclestimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH). FSH and LH are produced by the same cells, gonadotropes, and play fundamental roles in the control of reproductive physiology. FSH and LH are collectively known as the gonadotropins as they are trophic hormones that principally regulate gonadal function, including sex steroid production and gametogenesis. TSH, also known as thyrotropin, is produced by pituitary thyrotrope cells. The principal function of TSH is to stimulate thyroid hormone production by the thyroid gland. Thyroid hormones have many and diverse roles throughout the body during development and in adulthood, such as regulation of brain development and basal metabolism. With a major focus on results from analyses of human and rodent physiology, this chapter reviews the basic biology of FSH, LH, and TSH, including their structures and evolution, how they are produced and regulated, how they signal in target cells and organs, and how they are altered in disease states. At the end, we briefly highlight some gaps in our current knowledge that need to be addressed with more research.

2. GENE AND PROTEIN STRUCTURES 2.1 Ligand Structures The glycoprotein hormones (GPHs) are heterodimers composed of a common a subunit (formerly

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00007-9

called the a gonadotropin subunit or aGSU, but now referred to as chronic gonadotropin a or CGA) noncovalently linked to hormone-specific b subunits (Pierce et al., 1976; Pierce and Parsons, 1981) (Fig. 7.1A). There are at least four members of this protein family in modern mammals: FSH, LH, TSH, and chorionic gonadotropin (CG). CG appears to be restricted to a few species, including humans and horses, and is produced mainly by early embryos and the placenta (Casarini et al., 2018a; Talmadge et al., 1984a, 1984b). The pituitary glycoproteins, particularly in humans and rodents, are the focus of this chapter. Those interested in CG are referred to the following reviews (Fournier et al., 2015; Cole, 2009). As FSH, LH, and TSH share an a subunit, their identities and biological functions are defined by their unique b subunits: FSHb, LHb, and TSHb. CGA, which encodes the common a subunit, is a fiveexon gene located on chromosome six in humans (Chr. 4 in mice) (Naylor et al., 1983). The mature CGA protein possesses 92 amino acids. CGA is expressed in gonadotrope and thyrotrope cells in the pituitary and in trophoblast cells of the placenta (Horn et al., 1992; Fiddes and Goodman, 1981). FSHb, LHb, and TSHb are encoded by the FSHB, LHB, and TSHB genes, respectively. All three genes contain three exons, reflecting their common ancestry. FSHB, located on chromosome 11 in humans (Chr. 2 in mice), encodes a mature protein of 111 amino acids. LHB maps to chromosome 19 in humans (Chr. 7 in mice) and encodes a 121-amino acid protein. FSHB and LHB are expressed predominantly, if not exclusively, by gonadotrope cells. TSHB is located on human

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7. ANTERIOR PITUITARY: GLYCOPROTEIN HORMONES FROM GONADOTROPE (FSH AND LH) AND THYROTROPE (TSH) CELLS

(A)

Monomers

LH β N-

-C 121

1

FSH β N-

-C 111

1

TSH β N-

-C

NN-

-C

NN-

-C -C

NN-

-C

LH

FSH

-C 118

1

CGA N-

-C

TSH

-C 92

1

(B)

Dimers

FSHβ FSHβ

of CG and FSH in their resolved structures (Fox et al., 2001). For dimerization, the a subunit establishes lowaffinity interactions with a common sequence motif, CAGYC, in the b subunit. A seatbelt region of the b subunit strengthens the interaction by looping around the L2 loop of the a subunit, “buckling” the C-terminus of the b subunit back to its core via an intramolecular disulfide bond (Jiang et al., 2014a; Xing et al., 2004). At the center of the seatbelt, there is a region of charged amino acids (net positive charge in LHb and CGb; net negative in TSHb and FSHb) that is thought to confer receptor binding specificity to the b subunits (Jiang et al., 2014a). The C-terminus of the a subunit and the L2 loop of the b subunit are important for receptor binding and activation (Jiang et al., 2012, 2014a; Fan and Hendrickson, 2005).

CGA ECD

2.2 Ligand Glycosylation

CGA ECD

FIGURE 7.1 Glycoprotein hormone structures. (A) Schematic representation of the luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH) b subunits and the common a subunit (CGA, chorionic gonadotropin subunit a). The left depicts the individual subunits, with the amino (N) and carboxy (C) termini at the left and right, respectively. Amino acid lengths are indicated. At the right, the dimeric LH, FSH, and TSH hormones are presented. Y, reflects the approximate sites of asparagine residues (N) to which glycans are attached. (B) X-ray crystal structure of FSH bound to the extracellular domain (ECD) of the FSH receptor. Two different orientations of the same structure (PDB: 4AY9) are shown. The FSHb subunit is in yellow, the a subunit in orange, and the receptor ECD is in green. Balls and sticks represent the locations of glycan attachment.

chromosome 1 (Chr. 3 in mice) and expressed by thyrotrope cells. TSHB encodes a 118-amino acid protein. The human CG (hCG) was crystallized (PDB: 1HCN, ˚ , respectively) in 1994 1HRP; resolutions: 2.6 and 3 A (Lapthorn et al., 1994; Wu et al., 1994), providing the first insight into the atomic structure of GPHs. In 2001, the structure of a partially glycosylated human ˚ ) (Fox FSH was solved (PDB: 1FL7; resolution: 3A et al., 2001). The atomic structures of TSH and LH have yet to be reported. Comparison of the CG and FSH crystal structures reveals substantial similarities. Each of the GPH subunits are folded into elongated structures with three b-hairpin loops extending from their cystine-knot cores, providing a high surface-tovolume ratio (Jiang et al., 2014a) (Fig. 7.1B). The b loops share similar folds; however, there are differences in the conformations of the L2 and L3 loops in the b subunits

All GPH subunits are glycosylated on asparagine residues (Bousfield et al., 2018; Wide and Eriksson, 2017). The a subunit contains two N-linked glycosylation sites, whereas the b subunits contain either one (LHb and TSHb) or two (FSHb) (Bousfield et al., 2018; Nunez Miguel et al., 2017; Choi and Smitz, 2014) (Fig. 7.1A). The two sites in the a subunit are constitutively glycosylated, whereas glycosylation of the b subunits is regulated. For example, FSHb can either be hypoglycosylated (only one site glycosylated) or hyperglycosylated (both sites glycosylated) (Bousfield et al., 2018; Wide and Eriksson, 2017). The extent and nature of glycosylation of the b subunits affect ligand stability (half-life) and activity (Wide and Eriksson, 2017; Nunez Miguel et al., 2017; Sarapura and Samuel, 2017). For example, hyperglycosylated forms of FSH have longer half-lives in circulation, but reduced activity in the ovary (Bousfield et al., 2018; Jiang et al., 2015). FSH glycosylation varies across the menstrual cycle and postmenopausally in humans (Anobile et al., 1998; Wide and Eriksson, 2018). In the later context, FSH is predominantly hyperglycosylated, which is clinically relevant (Wang et al., 2016a, 2016b). FSH purified from the urine of postmenopausal women is used in the context of ovarian stimulation in assisted reproductive technologies. Recombinant FSH, made principally in Chinese hamster ovary cells, is similarly hyperglycosylated (Wang et al., 2016c; Hakola et al., 1997). Therefore, therapeutic preparations of FSH are generally of lower activity than the hypoglycosylated FSH produced during the follicular phase of typical menstrual cycles (Jiang et al., 2015; Wide and Eriksson, 2018) (Fig. 7.2A). This is the stage of the cycle when FSH stimulates ovarian follicles to grow and, eventually, ovulate.

2. GENE AND PROTEIN STRUCTURES

(A) Human

Relave hormone level

FSH LH

P4 E2

InhA InhB

(B)

Follicular phase

Luteal phase 1°

Rat

Relave hormone level



FSH LH

P4 E2 InhB

InhA

Metestrus

Diestrus

Proestrus

Estrus

FIGURE 7.2 Reproductive cycles in female humans and rats. (A) Schematic representation of serum hormonal dynamics across the (A) human menstrual and (B) rat estrous cycles. (A) The human menstrual cycle is approximately 28 days in length and is divided into two general phases: follicular and luteal. During the follicular phase, ovarian follicles grow in response to FSH stimulation. The dominant (preovulatory follicle) secretes high levels of E2, which triggers a surge of LH and FSH in the middle of the cycle. This surge triggers ovulation of the dominant follicle (arrow indicates the approximate time of ovulation). The remnants of the follicle differentiate into the corpus luteum (CL), which produces high levels of P4 and E2 during the luteal phase. Inhibin B levels are elevated in the follicular phase, whereas inhibin A is secreted by the CL during the luteal phase. (B) Rats and mice have 4- to 5-day estrous cycles. Gonadotropins are low on metestrus and diestrus. On the afternoon of proestrus, high levels of E2 from preovulatory follicles stimulate the primary surges of LH and FSH, which trigger ovulation (arrow). Inhibin B is elevated on metestrus and diestrus, and only decreases after the LH/FSH surges. Inhibin A increases as preovulatory follicles grow and then, like inhibin B, decreases after the primary LH/FSH surge. The loss of inhibins on the morning of estrus enables intrapituitary activins to stimulate the secondary FSH surge. Inhibin B levels increase in response to this FSH stimulation of the ovary. E2, estradiol; InhA, inhibin A; InhB, inhibin B; P4, progesterone.

2.3 Receptor Structures FSH, LH, and TSH bind and signal via distinct receptors: FSHR, LHCGR, and TSHR, respectively (Casarini et al., 2018a). All three belong to the leucine-rich

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repeat-containing subfamily of class A (rhodopsin-like) G proteinecoupled receptors (GPCRs) (Smits et al., 2003; Kleinau and Krause, 2009). FSHR, a mature protein of 678 amino acids, is encoded by a 10-exon gene, FSHR, located on human chromosome 2 (Chr. 17 in mice) (Gromoll et al., 1994, 1996; Rousseau-Merck et al., 1993). Several mRNA splice variants of FSHR have been reported, some of which encode isoforms with either reduced or no signaling often due to protein misfolding (Karakaya et al., 2014; Gerasimova et al., 2010). FSHR is predominantly expressed by granulosa cells of ovarian follicles and Sertoli cells of the testicular seminiferous tubules (Kumar, 2018; Kumar et al., 1997) (Fig. 7.3). More recent studies have reported FSHR expression in extragonadal tissues such as osteoclasts in bone, monocytes, developing placenta, liver, endothelial cells of the umbilical vein, uterus, and in some tumors (Sun et al., 2006; Zaidi et al., 2018; Liu et al., 2017; Song et al., 2016; Stilley and Segaloff, 2018; Stelmaszewska et al., 2016; Stilley et al., 2014; Ghinea, 2018). Whether FSH plays physiologic or pathophysiologic roles in these tissues is unresolved. The LH receptor, which binds both LH and CG, is encoded by LHCGR, an 11-exon gene located on human chromosome 2 (Chr. 17 in mice). Mature LHCGR is 673 amino acids in length. In women, LHCGR is expressed by ovarian thecal and mural granulosa cells, and by the corpus luteum (Casarini et al., 2018b; Richards and Ascoli, 2018). In men, LHCGR is expressed in interstitial Leydig cells of the testis (Richards and Ascoli, 2018) (Fig. 7.3). Extragonadal LHCGR expression has also been reported (Sacchi et al., 2018; Kokk et al., 2011; Pakarainen et al., 2007; Toth, 2001). TSHR is a 12-exon gene located on human chromosome 14 (Chr. 12 in mice) (Libert et al., 1989, 1990; Rousseau-Merck et al., 1990). Mature TSHR is a 744amino acid protein predominantly expressed at the basolateral surface of thyroid follicle cells (Fig. 7.4), although the receptor has also been reported in other tissues such as astrocytes and neurons, folliculo-stellate cells in the pituitary, adipose tissue, bone, liver, ovary, lymphocytes, erythrocytes, and parafollicular cells in the thyroid (Briet et al., 2018; Williams, 2011; Naicker and Naidoo, 2018). The three GPH receptors (GPHRs) share sequence and structural homology. All possess a large N-terminal extracellular domain (ECD), seven transmembrane a-helical domains (TMDs) typical of GPCRs, three extracellular and intracellular loops, and an intracellular hydrophilic C-terminus. The ECDs have two distinct functional domains, a hormone-binding domain composed of multiple leucine-rich repeats (LRRs), followed by a hinge region N-terminal to the first TMD (Jiang et al., 2014a; Ulloa-Aguirre et al., 2018). Each LRR motif is composed of approximately 20e30

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(A)

LHR

(B)

Secondary follicle

Granulosa

Primordial Primary follicle follicle

Theca

Gonadotropin-independent follicle growth

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cAMP/PKA

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FSHR

Theca cells Mural granulosa cells Cumulus cells Oocyte

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(C)

(D)

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Sertoli

Leydig

Seminiferous tubule

LHR cAMP/PKA

Testosterone

ABP

AR Inhibin B

cAMP/PKA

Spermatogonia

FSHR Spermatozoa

FIGURE 7.3 Gonadotropin actions in the gonads. (A) Stages of ovarian follicle development. Growth of the follicle from the primordial stage (oocyte surrounded by immature granulosa cells) to the early antral stage (oocyte surrounded by multiple layers of granulosa cells) occurs independently of gonadotropins. Growth from the early antral to preovulatory stage is dependent on FSH. Ovulation of these follicles and their differentiation into the CL is controlled by LH. The different cell types of the follicle are labeled in the preovulatory follicle. (B) Two cell/two gonadotropin model of estrogen biosynthesis. LH binds the LH receptor (LHR) on theca cells, stimulating androgen production: testosterone (Test.) and androstenedione (A-dione). These androgens diffuse into adjacent granulosa cells where they are converted to estrogens: estradiol (E2) and estrone (E1). FSH binds to its receptor (FSHR) on granulosa cells and thereby stimulates expression of the aromatase (arom.) enzyme, which converts androgens to estrogens. FSH also stimulates granulosa cell proliferation and expression of the LHR in mural (but not cumulus) granulosa cells. (C) Schematic representation of cross-sections of the testis, showing seminiferous tubules and interstitial cells. Sertoli, Leydig, and germ cells are labeled (peritubular myoid cells are not shown). (D) Model of gonadotropin action in testicular cells. LH binds the LHR on Leydig cells, stimulating testosterone production. This testosterone diffuses into adjacent Sertoli cells where it binds the androgen receptor (AR) and regulates spermatogenesis. FSH binds the FSHR on Sertoli cells, stimulating the production of androgen binding protein (ABP), which helps concentrate testosterone in the testis.

residues folded into a sequential b-sheet and a-helix, with the b-sheets facing the concave surface of the ECD (Smits et al., 2003) (Fig. 7.1B). The number of LRRs varies between GPHRs, with 12 in FSHR, 9 in LHCGR, and 11 in TSHR. Thus far, there have been a total of four independent crystal structures solved of the ECDs of GPHRs, two each for FSHR and TSHR. The crystal structure of FSH in complex with the hormone binding domain of

FSHR lacking the hinge region (amino acids 1e268) ˚) was described in 2005 (PDB: 1XWD; resolution: 2.92 A (Fan and Hendrickson, 2005). This was the first published structure of any GPHR. The FSH-FSHR structure showed the hormone binding region of FSHR, composed of 10 of the 12 LRRs, folded into a slightly arched, horseshoe-like structure. FSH binds to the concave surface of the FSHR-ECD (see Fig. 7.1B and more subsequently).

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2. GENE AND PROTEIN STRUCTURES

(A)

(B)

Na+/I-

T4 T3

Thyroid follicular cells (thyrocytes)

TSHR

Thyrocyte

NIS

MCT8

cAMP

T3

T4

Colloid

Tg

Na+/I-

Tg

Nucleus

(MIT/DIT) Lysosome

Blood vessel

Tg Thyroid follicles

Pendrin TPO H2O2

Tg II0 Tg

(MIT/DIT)

Colloid

FIGURE 7.4 Thyroid gland anatomy and hormone synthesis. (A) The thyroid gland is composed of thyroid follicles. These follicles are spherical structures with follicular cells (thyrocytes) at the periphery and colloid in the lumen. (B) Schematic representation of thyroid hormone synthesis. Thyrocytes take up iodide (I) via the sodium/iodide symporter (NIS). Iodide is then pumped into the lumen via the transporter pendrin. Once in the lumen, iodide is converted to iodine (I0) via the actions of thyroid peroxidase (TPO) in the presence of hydrogen peroxide (H2O2). Thyrocytes produce TPO and the large, tyrosine-rich protein thyroglobulin (Tg). TPO is expressed at apical membrane of thyrocytes, its catalytic domain in the lumen. Tg is secreted into the lumen of the follicle. Iodine reacts with the tyrosines in Tg in the colloid-filled lumen creating mono- and diiodotyrosines (MIT and DIT). Iodinated Tg is endocytosed by thyrocytes and degraded inside lysosomes. This yields molecules of the thyroid hormones triiodothyronine (T3) and tetraiodothyronine (T4, thyroxine), which are secreted from the cells via transporters such as monocarboxylate transporter 8 (MCT8). TSH binds to its receptor on the membrane of thyrocytes, regulating many of the steps in the thyroid hormone synthesis pathway (see text for details).

Two years later, the crystal structure of the ECD of TSHR (amino acids 1e260) in complex with a TSHRstimulating autoantibody, M22, was reported (PDB: ˚ ) (Sanders et al., 2007). As previ3G04; resolution: 2.55 A ously predicted, the structure of the extracellular portion of TSHR bound to M22 was similar to that of FSHR bound to FSH. M22 (antibody) and FSH (ligand) bind to the concave surfaces of their respective receptors in similar positions. In 2011, the crystal structure of the extracellular domain of TSHR (amino acids 1e260) in complex with the TSHR blocking autoantibody, K1-70, ˚ ) (Sanders was solved (PDB: 2XWT; resolution: 1.9 A et al., 2011). Comparison of the TSHR ECD structure bound to either M22 or K1-70 reveals similar receptor conformations in both complexes. The higher resolution of the K1-70-bound structure provided details of the arrangement of intramolecular disulfide bonds between Cys24-Cys29 and Cys31-Cys41 at the N-terminus of TSHR. There are no interactions between the cysteines and autoantibodies, leading to the hypothesis that these residues do not contribute to hormone binding. However, it has been reported that the N-terminal cysteine cluster is

required for proper folding of recombinant LH receptor (LHR) (Fan and Hendrickson, 2005). In 2012, the structure of the entire ECD of human FSHR, including the hinge region, was solved in complex ˚ ) (Jiang et al., 2012) with FSH (PDB: 4AY9; resolution: 2.5 A (Fig. 7.1B). In contrast to previous predictions, the hormone binding and hinge regions interact with FSH as a single unit, rather than the hinge region functioning as a distinct structure only involved in receptor activation. To date, there have been no LHCGR structures published; however, the receptor shares approximately 70% sequence identity with FSHR and TSHR. Therefore, its structure has been modeled based on the structures of the other GPHRs (Troppmann et al., 2013; Grzesik et al., 2015). The structure of unsolved regions of GPHRs, such as the TMDs have been modeled using related GPCRs with known structures, such as rhodopsin (Baldwin, 1993; Kajava et al., 1995). A series of structure-function (mutagenesis) analyses have also identified residues mediating ligand binding and effector (G protein) coupling (Briet et al., 2018; Ulloa-Aguirre et al., 2018; Puett et al., 2010).

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3. EVOLUTION All glycoprotein hormones are thought to have evolved from a common ancestor over 500 million years ago (Kawauchi and Sower, 2006). There are two major hypotheses on how vertebrate evolution occurred. The longest standing hypothesis is that two large-scale genome duplications, coined as the two round (2R) hypothesis, occurred during early vertebrate evolution (Sower, 2015, 2018; Sower and Hausken, 2017; Holland et al., 1994). More recently, some groups have proposed that only a single large genome duplication occurred followed by many small duplications in the genome (Sower, 2018; Smith and Keinath, 2015). The former hypothesis is most generally accepted. The two rounds of genome duplication in the 2R hypothesis are thought to have permitted the specialization of the hypothalamicepituitary axes. During the first round (1R), the ancestral glycoprotein hormone b subunit gene (ancGphB) duplicated into two paralogous genes, pregonadotropin b subunit gene (preGthB) and the precursor of the Tshb subunit gene (preTshb) (Dos Santos et al., 2011; Maugars et al., 2014). In early vertebrates, Lhb and Fshb arose during 2R, concurrently with two Tshb genes (Dos Santos et al., 2011; Maugars et al., 2014). The second Tshb gene remains in some fish species; however, it was lost during mammalian evolution (Dos Santos et al., 2011; Maugars et al., 2014). A “new” GPH that activates the TSH receptor, thyrostimulin, was recently discovered (Hsu et al., 2002). Phylogenetic analyses suggest that the thyrostimulin subunits are ancestral to the better-known vertebrate glycoprotein subunits (Sower and Hausken, 2017), making this an old rather than new hormone. Like other GPHs, thyrostimulin is composed of both a (GPA2) and b subunits (GPB5), which share structural homology, including cystine knots and N-glycosylation sites, to the more recently evolved GPH subunits (Sower and Hausken, 2017; Dos Santos et al., 2009; Hausken et al., 2018; Nakabayashi et al., 2002). However, thyrostimulin’s b subunit does not have a seatbelt region, resulting in a less stable protein (Sower and Hausken, 2017; Dos Santos et al., 2009; Nakabayashi et al., 2002; Sudo et al., 2005). Thyrostimulin subunits are expressed in both invertebrates and vertebrates, including but not restricted to sea lamprey, fly, mouse, rat, and human (Sower and Hausken, 2017; Dos Santos et al., 2009; Hausken et al., 2018; Sudo et al., 2005). In humans, GPA2 is expressed in pituitary, ovary, testis, kidney, and pancreas, and GPB5 is expressed in brain and pituitary (Hsu et al., 2002). Although thyrostimulin activates TSHR, its physiologic function has not yet been established (Nakabayashi et al., 2002). Given its tissue distribution, it may act as a paracrine, rather than endocrine, factor (Hausken et al., 2018).

4. REGULATION OF SYNTHESIS AND SECRETION 4.1 Gonadotropins 4.1.1 Regulation by GnRH LH and FSH are regulated by hormones and paracrine factors from the brain, pituitary, and gonads (Fig. 7.5A). Gonadotropin-releasing hormone (GnRH) is a decapeptide produced by a specialized set of neurons in the preoptic area and/or mediobasal hypothalamus in the brain. These cells, which number around 1000 in the brain of most mammals, are relatively widely distributed rather than being clustered in a discrete hypothalamic nucleus. GnRH neurons send their axons to the median eminence at the base of the brain, where they release GnRH into the pituitary portal vessels. GnRH is secreted in pulses, with the frequency and amplitude of release varying across both the lifespan and female reproductive cycles. Pulse generation appears to be governed by a second group of neurons in the arcuate nucleus of the hypothalamus (Herbison, 2018). These cells produce kisspeptin, a potent GnRH secretagogue (Popa et al., 2008). According to the current model, arcuate kisspeptin neurons synapse near the terminals of GnRH neurons in the median eminence, enabling coordination of GnRH release from an otherwise disperse population of cells. GnRH pulses, which likely derive from kisspeptin pulses, occur every 1e8 h, depending on the physiologic state of the animal. Changes in pulse frequency have consequences for gonadotropin synthesis and secretion (Thompson and Kaiser, 2014; Wildt et al., 1981). At high pulse frequencies (e.g., one pulse per hour), the pituitary preferentially secretes LH relative to FSH (i.e., more LH than FSH, though both hormones are secreted). At lower pulse frequencies (e.g., one pulse every 2 h or longer), the ratio inverts, with more FSH secreted relative to LH. The mechanisms underlying pulse frequency decoding by pituitary gonadotropes are unresolved, at least in part because the mechanisms through which GnRH regulates FSH synthesis are incompletely described. GnRH binds to the GnRH receptor (GnRHR) on the membrane of gonadotropes (Fig. 7.6A). GnRHR is a GPCR, which preferentially activates the Gaq/11 pathway upon ligand binding (Millar et al., 2004). GTP-bound Gaq/11 activates phospholipase C, which in turn cleaves phosphatidylinositol bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5trisphosphate (IP3). DAG activates protein kinase C (PKC) isoforms, which promote mitogen-activated protein kinase (MAPK) signaling, including the extracellular regulated kinases (ERK1/2). ERK1/2 mediates the expression of the transcription factor early growth

4. REGULATION OF SYNTHESIS AND SECRETION

HPG axis Hypothalamus

(A)

(B)

HPT axis Hypothalamus

AVPV

E2/P4

ARC

Kiss

Kiss

GnRH Acvin B E2/P4

E2/P4/T

PVN

TRH GnRHR

Pituitary

FSH/LH

LHR FSHR

Gonads

E2 inhibins

TRHR1

T3/T4

T3/T4

Pituitary TSH TSHR

Thyroid

FIGURE 7.5 The hypothalamicepituitaryegonadal (HPG) and hypothalamicepituitaryethyroid (HPT) axes. (A) Hypothalamic kisspeptin neurons in the arcuate nucleus (ARC) form part of the gonadotropin-releasing hormone (GnRH) pulse generator. Kisspeptin (Kiss) secretion from these cells stimulates pulsatile GnRH release at the level of the GnRH neuron terminals in the median eminence. A GnRH neuron is depicted in black. GnRH travels via the pituitaryportal system to the anterior pituitary where it binds to the GnRH receptor (GnRHR) on the plasma membrane of gonadotrope cells. GnRH stimulates the synthesis of the gonadotropins, LH and FSH. LH and FSH travel via systemic circulation to the gonads where they bind to LH and FSH receptors (LHR/FSHR) and stimulate the production of sex steroids: estradiol (E2), progesterone (P4), and testosterone (T). These hormones negatively feed back (red lines) to the ARC kisspeptin neurons, decreasing kisspeptin expression and, in turn, GnRH and gonadotropin secretion. The gonads also produce inhibins, which feed back to the pituitary to suppress FSH. In females, E2 and P4 can also have positive feedback effects (green lines) on GnRH and gonadotropin secretion. At the level of the brain, E2 stimulates production and presumably release of Kiss from a second population of kisspeptin neurons in the anteroventral periventricular nucleus (AVPV). E2 and P4 also feedback at the pituitary level to potentiate GnRH stimulation of the gonadotropins. The pituitary produces activin B, which is thought to stimulate FSH in an autocrine or paracrine fashion. (B) Thyrotropinreleasing hormone (TRH) neurons (black neuron) in the paraventricular nucleus (PVN) secrete TRH into the pituitary portal system at the level of the median eminence. TRH travels to the pituitary gland and binds to the TRH receptor (TRHR or TRHR1) on the plasma membrane of thyrotrope cells. In response, thyrotropes synthesize and release TSH (thyrotropin). TSH travels to the thyroid gland and binds to the TSH receptor (TSHR) on the plasma membrane of thyrocytes, stimulating thyroid hormone (T3, triiodothyronine; T4, thyroxine) production and secretion. T3 and T4 negatively feed back (red lines) to the PVN to downregulate TRH synthesis and secretion, and to the pituitary to inhibit expression of the TSH subunits and TRHR.

response 1 (EGR1) (Duan et al., 2002). EGR1 binds the proximal Lhb promoter in concert with the nuclear receptor steroidogenic factor 1 (SF1 or NR5A1) and paired-like homeodomain transcription factor 1 (PITX1) (Jorgensen et al., 2004). EGR1-SF1-PITX1 complexes drive Lhb transcription, at least in vitro. This mechanism appears to be conserved across the mammalian species that have been investigated thus far, including humans (Fortin et al., 2009). How GnRH regulates transcription of the Fshb subunit gene is less clear. Based on in vitro observations,

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it appeared as though GnRH induction of activator protein 1 (AP-1) proteins, such as Fos and Jun family members, played an important role in Fshb transcription (Wang et al., 2008; Coss et al., 2004; Strahl et al., 1998). However, this model has not been supported by the majority of in vivo observations, in particular from conditional knockout and transgenic mice (Huang et al., 2001; Jonak et al., 2018; Xie et al., 2015). Other in vitro results implicated the Gas-cyclic AMP-protein kinase A (PKA)-CREB pathway in GnRH’s induction of Fshb transcription (Thompson et al., 2013, 2016). However, we did not observe alterations in FSH or LH synthesis in gonadintact mice lacking Gas selectively in gonadotrope cells (our unpublished observations). Nonetheless, consistent with the aforementioned model for Lhb regulation, mice lacking EGR1 globally or ERK1/2 specifically in gonadotropes display selective impairments in LH (relative to FSH) synthesis (Brown et al., 2018; Bliss et al., 2009; Lee et al., 1996). Thus, it is clear that GnRH uses distinct mechanisms to regulate the gonadotropin b subunits, providing a means for their differential regulation in response to high or low GnRH pulse frequencies. As indicated, GnRH stimulates increases in IP3 production. In turn, IP3 stimulates the release of calcium (Ca2þ) from the endoplasmic reticulum (ER) (Fig. 7.6A). This increase in intracellular Ca2þ promotes the acute release of LH stored in dense core granules (secretory vesicles) (Naor, 1990). Ca2þ also enters gonadotropes via L-type channels (Stutzin et al., 1989). Interestingly, GnRH is a relatively weak regulator of acute FSH release. This is, in part, because FSH is predominantly trafficked through the constitutive secretory pathway during its biosynthesis (McNeilly et al., 2003). In contrast, LH is processed through the regulated pathway and is therefore preferentially stored in secretory granules. Some FSH can also be found in these granules and a small amount of LH can be constitutively released. However, in general, GnRH is thought to first stimulate release of stored LH and then promote the synthesis of new LH to replenish the secreted hormone. In contrast, GnRH stimulates production of FSH, which is then released via the constitutive pathway. It is for this reason that GnRH-stimulated FSH release is delayed and more modest relative to GnRH-stimulated LH release. The differential sorting of LH and FSH in the secretory pathway is mediated by a heptapeptide sequence at the C-terminus of the LHb subunit, which directs LH to the regulated pathway (Jablonka-Shariff et al., 2008). This sequence is absent in FSHb. One exception to these general modes of GnRHstimulated gonadotropin release occurs prior to ovulation in females. At this stage of the reproductive cycle (Fig. 7.2), high levels of estrogens (see more subsequently) trigger surges of LH and FSH. These increases are an order of magnitude higher than typical LH

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(A) GnRHR PIP2

PIP2

PLCβ1

PLCβ1

DAG Raf? PKCδ/ε

IP3

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Egr1 SF1 EGR1 PITX1

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Gonadotrope cell

Cytosol

(B) TRHR PIP2

PLCβ

PLCβ

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MEK1

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Nr4a1 NR4A1 Pit1 GATA2

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Nucleus Cytosol

Thyrotrope cell

FIGURE 7.6 GnRH and TRH signaling. (A) Schematic representation of GnRH signaling in gonadotrope cells. The GnRHR couples to Gq/11 (only Gq is pictured). Upon ligand binding, Gq/11 binds GTP and dissociates from the Gbg subunits. GTP-bound Gq/11 activate phospholipase Cb1 (PLCb1), which cleaves PIP2 into IP3 and diacylglycerol (DAG). IP3 stimulates calcium mobilization from the ER and exocytosis of LH from secretory granules. DAG stimulates protein kinase C isoforms d and ε, which activate MAP kinase signaling. Extracellular regulated kinases 1 and 2 (ERK1/2) phosphorylate ELK1, stimulating the expression of early growth response 1 (EGR1). EGR1 partners with steroidogenic factor 1 (SF1; NR5A1) and paired-like homeodomain transcription factor 1 (PITX1). Complexes of these transcription factors bind to the proximal Lhb/LHB promoter, driving its transcription. (B) Schematic representation of TRH signaling in thyrotrope cells. Many of the features resemble those observed with GnRH in gonadotrope cells, though some details are missing. The most recent data suggest that TRH signaling stimulates the production of the nuclear receptor NR4A1, which then partners with Pit1 and GATA2 to drive Tshb transcription. Note that in both figures, PIP2 is cleaved into both IP3 and DAG, but these events are shown separately because of space limitations. PIP2, phosphatidyl inositol bisphosphate. IP3, inositol 1,4,5-trisphosphate; Raf, raf kinase; MEK1, mitogen-activated kinase kinase 1.

4. REGULATION OF SYNTHESIS AND SECRETION

pulses, lack a clear pulsatile nature, and are protracted in length (at least several hours). The surge of LH drives processes in the ovary that culminate in ovulation (see Fig. 7.3A and more subsequently). Though sex steroids, like estrogens, ordinarily inhibit gonadotropin secretion through negative feedback, the high preovulatory levels of estrogens promote GnRH secretion from the brain and enhance pituitary sensitivity to GnRH (Fig. 7.5A). The estrogen positive feedback effect in the brain is mediated via a second population of kisspeptin neurons distinct from the pulse generator cells in the arcuate nucleus, at least in rodents (Wang et al., 2019; Smith, 2013). In female rats and mice, kisspeptin neurons are observed in the anteroventral periventricular nucleus (AVPV) of the preoptic area. In response to high estrogens, these cells increase their production (and presumably secretion) of kisspeptin, which stimulates GnRH release (Smith et al., 2005a). This population of cells is largely absent in males. Perinatal and/or neonatal androgens from the immature testes promote the loss of these neurons during development (Kauffman et al., 2007; Homma et al., 2009). As a result, male rodents do not exhibit estrogen positive feedback. At the same time that high estrogen levels stimulate the AVPV kisspeptin neurons (positive feedback), they inhibit the arcuate kisspeptin neurons (negative feedback on the pulse generator). In humans and nonhuman primates, estrogen positive feedback on LH release (i.e., the surge) involves a critical pituitary component. In fact, some observations suggest that it is principally increased pituitary sensitivity to GnRH, rather than enhanced GnRH secretion that drives the surge in these species. For example, lesions of the arcuate nuclei in the brain abolish GnRH release and gonadotropin synthesis and secretion in rhesus monkeys. Similarly, patients with Kallman syndrome have hypogonadotropic hypogonadism (underdeveloped gonads due to low LH and FSH levels) because GnRH neurons, which are born in or near the olfactory placode, fail to migrate into the brain during embryonic development (Schwanzel-Fukuda et al., 1992). Treatment of female monkeys with brain lesions or Kallman patients with exogenous pulsatile GnRH is sufficient to restore gonadotropin production and, indeed, menstrual cyclicity (Knobil et al., 1980; Crowley and McArthur, 1980). This includes the occurrence of LH surges, despite the invariant nature of GnRH administration in the treatment protocol. These observations led to the conclusion that high estrogen levels from the preovulatory follicle (Fig. 7.3A) enhance pituitary sensitivity to GnRH, leading to the LH (and FSH) surge. Conditional knockout studies in mice similarly implicate a role for estrogen action in the pituitary in the positive feedback mechanism (Singh et al., 2009). The mechanisms of enhanced pituitary responsiveness to GnRH

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at the time of the LH surge have not been fully resolved, though estrogens stimulate GnRHR expression and the number of receptors in gonadotropes peaks at this stage of the cycle in rodents (Naik et al., 1985; Park et al., 1976; Bauer-Dantoin et al., 1993). 4.1.2 Regulation by Activins and Inhibins In addition to GnRH, FSH synthesis is strongly and selectively regulated by proteins in the transforming growth factor b (TGFb) family, the activins and inhibins (Ying, 1988). These are structurally related disulfidelinked dimers composed of inhibin a and b subunits (Fig. 7.7A). The inhibin b (bA and bB) subunits homoor heterodimerize to form activin A (bA-bA), activin B (bB-bB), and activin AB (bA-bB). Dimerization of the inhibin a subunit with one of the inhibin b subunits forms inhibin A (a-bA) or inhibin B (a-bB). Activin A, activin AB, inhibin A, and inhibin B were initially purified from ovarian follicular fluid (Robertson et al., 1985; Ling et al., 1985, 1986a, 1986b; Mason et al., 1985; Vale et al., 1986). The gonads are the major source of circulating inhibins, whereas activins are produced in multiple tissues and cells (Woodruff et al., 1996). According to our current understanding, gonadotrope cells produce activin B, which acts in autocrine and/or paracrine fashion to stimulate FSH synthesis (Corrigan et al., 1991). Activins bind to complexes of type I and II receptor serine/threonine kinases (Fig. 7.7B). Both classes of receptors have a ligandbinding extracellular domain, a single transmembrane domain, and an intracellular kinase domain. Activins can bind three of the five type II receptors in the family: activin type II receptors A and B (ACVR2A and ACVR2B) and the bone morphogenetic protein type II receptor (BMPR2) (Rejon et al., 2013; Mathews and Vale, 1991; Attisano et al., 1992). Mice lacking a functional Acvr2a gene have reduced FSH levels (Matzuk et al., 1995a). According to our unpublished data, ACVR2B but not BMPR2 can partially compensate for the loss of ACVR2A in gonadotropes of mice (Schang et al., in preparation; Ongaro et al., in preparation). Two of seven type I receptors in the family have been proposed to mediate the actions of activins: activin receptor-like kinases (ALK) 4 (product of the Acvr1b gene) and 7 (product of the Acvr1c gene) (Bernard et al., 2006; Tsuchida et al., 2004). Pituitaries of Acvr1c knockout mice appear to make FSH normally (Sandoval-Guzman et al., 2012). Acvr1b mice die during embryonic development (Gu et al., 1998); therefore, the role of this receptor in FSH synthesis in vivo has not yet been reported. Upon ligand binding to the high affinity type II receptor, the type I receptor is recruited into the complex. The type II receptor then phosphorylates the type I receptor in a juxtamembrane domain called the GS box. Phosphorylation of the type I receptor

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Inhibin α

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FIGURE 7.7 Activins and inhibins. (A) Activins are disulfidelinked dimers of inhibin b subunits (bA or bB). Inhibins are heterodimers of the inhibin a subunit and one of the two inhibin b subunits. (B) Schematic representation of activin and inhibin regulation of FSH synthesis. Activin B, produced by gonadotrope cells, binds to complexes of type II (ACVR2A or ACVR2B) and type I (ALK4 or ALK7) serine/threonine kinase receptors. The type II receptors trans-phosphorylate the type I receptors, which then phosphorylate the signaling protein SMAD3. Phospho-SMAD3 binds SMAD4 and accumulates in the nucleus. SMAD3/4 partner with FOXL2 and bind to the proximal Fshb promoter to drive its transcription. Activin’s actions are antagonized in at least two ways. First, follistatin binds and bioneutralizes activins. Second, inhibins form high-affinity ternary complexes with activin type II receptors and the type III receptor betaglycan. This enables inhibins to outcompete activins for binding to type II receptors. ACVR2, activin type II receptor; ALK, activin receptor-like kinase (type I receptor).

activates its kinase activity. The type I receptor then phosphorylates signaling proteins in the homolog of Drosophila mothers against decapentaplegic (SMAD) family, in particular SMAD2 and SMAD3 (Shi and Massague, 2003). According to both in vitro and in vivo observations, SMAD3 appears most critical for FSH synthesis (Li

et al., 2017; Suszko et al., 2005; Bernard, 2004). Phosphorylated SMAD3 partners with the co-SMAD, SMAD4, in the cytoplasm. The SMAD complex then translocates to and accumulates in the nucleus. There, these SMADs partner with the DNA binding protein forkhead box L2 (FOXL2), and bind to the proximal Fshb promoter, driving its transcription (Lamba et al., 2010; Tran et al., 2011). This mechanism of action has been most firmly established in mice, though it is likely to be conserved across other mammalian species (Li et al., 2017, 2018a; Fortin et al., 2014; Tran et al., 2013). Interestingly, the available data in mice suggest that a ligand other than activin B may be the principal driver of FSH synthesis. That is, mice lacking activin B have elevated rather than reduced levels of FSH (Vassalli et al., 1994). It is possible that another TGFb family member that uses similar receptors and signal proteins may regulate FSH or, at least, can compensate in the absence of activin B. Activin B bioneutralization does reduce FSH production by rat pituitaries in culture, however (Corrigan et al., 1991). Therefore, the TGFb ligand(s) driving FSH synthesis may differ across species. The stimulatory effects of activins (or activin-like ligands) are antagonized in at least two ways, by the actions of inhibins and follistatins (Fig. 7.7B). In rodents, ovarian granulosa cells produce both inhibin A and B, whereas testicular Sertoli cells make inhibin B exclusively. In humans, inhibin B is a product of growing follicles in the follicular phase of the menstrual cycle, whereas inhibin A is principally produced by the corpus luteum during the luteal phase of the cycle (Woodruff and Mayo, 1990; O’Connor and De Kretser, 2004) (Fig. 7.2). Inhibins function as endocrine hormones that are released from the gonads and travel to the pituitary to suppress FSH production by competitively binding to activin type II receptors in gonadotrope cells (Xu et al., 1995; Lewis et al., 2000). However, inhibins do not recruit type I receptors and therefore cannot promote the formation of receptor complexes capable of signaling in cells. Inhibins bind activin type II receptors with at least 10fold lower affinity than do activins (Mathews and Vale, 1991; Bernard et al., 2002). Whereas, activins bind type II receptors in the high picomolar range, inhibins circulate in the mid-picomolar range. This would suggest that inhibins should be incapable of competing with activins for type II receptor binding. However, in the presence of a coreceptor, the TGFb type III receptor (also known as TGFBR3 or betaglycan), inhibins form high-affinity ternary complexes with the activin type II receptors that are resistant to disruption by activins (Lewis et al., 2000) (Fig. 7.7B). Activin A is unable to compete for inhibin A binding to betaglycan. This has led to the inference that inhibins bind betaglycan principally via their a subunit (which activins lack) and to type II

4. REGULATION OF SYNTHESIS AND SECRETION

receptors via their b subunits (which they share with activins). Inhibin A binds type II receptor-betaglycan complexes with higher affinity than does inhibin B, though the latter may more potently inhibit FSH synthesis (Makanji et al., 2009). This suggests that inhibin B may act via additional mechanisms. Consistent with this idea, gonadotrope cells deficient in betaglycan have reduced sensitivity to inhibin A, but not inhibin B (Li et al., 2018b). Whether inhibin B binds to a distinct receptor or coreceptor needs to be resolved. The second form of activin antagonism comes in the form of soluble proteins in the follistatin family (Ueno et al., 1987) (Fig. 7.7B). Two transcripts are derived via alternative splicing from the follistatin (Fst) gene. These mRNAs encode proteins of 288 and 315 amino acids, Fst288 and Fst-315 (Inouye et al., 1991). Both proteins bind to activins in a 2:1 stoichiometry (Thompson et al., 2005). This binding, which is reportedly irreversible, occludes receptor interaction surfaces on the ligand, inhibiting activin action. Follistatins also bind to inhibins, though with lower affinities, and can bioneutralize other members of the TGFb family, such as growth differentiation factors 8 and 11 (GDF8 and GDF11) (Schneyer et al., 2008). Activins in circulation are largely bound to Fst315 (McConnell et al., 1998). Fst-288, in contrast, has a high affinity for heparan sulfate proteoglycans, which localizes the protein at the plasma membrane (Sugino et al., 1993; Nakamura et al., 1991). Activins bound to Fst-288 are internalized by cells and degraded intracellularly (Hashimoto et al., 1997; Cash et al., 2009). A second molecule, follistatin-like 3 (Fstl3), similarly binds and bioneutralizes activins, GDF8, and GDF11 (Sidis et al., 2006). However, Fstl3 knockout mice are fertile, suggesting that the protein does not play essential roles in FSH synthesis (Mukherjee et al., 2007). Fst mice die shortly after birth, indicating that the protein plays pleiotropic roles, in addition to regulating FSH (Matzuk et al., 1995b). 4.1.3 Regulation by Sex Steroids Under most conditions, gonadal sex steroids negatively regulate the gonadotropins. Most of these effects are indirect, via inhibition of GnRH secretion from the brain (Fig. 7.5A). That said, steroids can also regulate FSH and LH at the pituitary level. This is perhaps most clearly demonstrated in mice lacking steroid receptors in their gonadotropes. Estrogens act via two nuclear receptors, ERa and ERb. Global knockout models demonstrate that feedback regulation of estrogens is mediated principally via ERa (Lindzey et al., 1998; Couse et al., 2003). In female mice lacking ERa specifically in gonadotropes, FSH appears to be unaffected, but LH levels are either increased or unaffected (Singh et al., 2009; Lindzey et al., 1998; Couse et al., 2003; Wersinger et al., 1999; Gieske et al., 2008). The basis for

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the incomplete penetrance of the phenotype is unclear at present. The LH surge (caused by estrogen positive feedback) may be impaired in these mice, but this was not investigated systematically. Gonadotropin production in males was not reported in these mice. In mice lacking the androgen receptor (AR) in gonadotropes, FSH levels are reduced in females (Wu et al., 2014). This is consistent with observations that androgens directly stimulate expression of the Fshb subunit. This effect may be species-specific, however, as androgens inhibit expression of the human FSHB subunit gene. In unpublished work, we investigated the effects of gonadotrope-specific ablation of the progesterone receptor (Pgr; Toufaily et al., submitted). Gonadotropin production and secretion were unaffected in both females and males. However, the amplitude of the LH surge was blunted in females. Estrogens upregulate Pgr expression in gonadotropes and other tissues. These data suggest that at least part of the estrogen positive feedback effect on LH secretion in mice is mediated via the progesterone receptor in the pituitary.

4.2 Thyrotropin 4.2.1 Regulation by TRH Analogous to the situation with GnRH and the gonadotropins (LH and FSH), a hypothalamic peptide, thyrotropin-releasing hormone (TRH), is the main driver of TSH secretion from thyrotrope cells of the anterior pituitary gland (Ortiga-Carvalho et al., 2016) (Fig. 7.5B). TRH, a tripeptide, is produced by neurons in the paraventricular nucleus (PVN), which project their axons to the median eminence and release TRH into the pituitary portal vessels. Like LH, TSH is released in pulses, approximately every 2e3 h, with variability between individuals. TRH affects the pulse amplitude of TSH; however, it does not appear to regulate TSH pulse frequency, as constant infusions of TRH do not change the pulse frequency (Nillni, 2010; Samuels et al., 1993). The nature of TSH pulse generator has yet to be described (Samuels et al., 1995). Like the GnRH receptor, TRH receptors (TRHR) are Gaq/11-coupled GPCRs in the rhodopsin family (Fig. 7.6B). There is one form of the receptor in humans and two in rodents. TRHR1 is the primary form of the receptor in the rodent pituitary, where it is expressed in thyrotropes, lactotropes (produce prolactin), and somatotropes (produce growth hormone) (Joseph-Bravo et al., 2015; Hinkle et al., 2012). Ligand binding promotes increases in intracellular Ca2þ from the ER and through L-type channels. The increases in Ca2þ promote TSH release from secretory vesicles. TRH also stimulates transcription of the TSH subunit genes and regulates TSH glycosylation. The mechanisms of TRH-induced

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Tshb expression have been studied in the greatest detail, though more details need to be worked out and validated in vivo. The available data suggest that TRH acts via PKC and MAPK pathways to upregulate the expression of the nuclear receptor NR4A1. NR4A1 then works in concert with the transcription factors Pit1 (POU1F1) and GATA2 to drive transcription of the Tshb subunit gene (Nakajima et al., 2012; Ohba et al., 2011; Gordon et al., 1997; Steinfelder et al., 1991, 1992). 4.2.2 Regulation by Thyroid Hormones Once released from the pituitary, TSH stimulates thyroid hormone, thyroxine (T4) and triiodothyronine (T3), production in the thyroid gland (see Figs. 7.4A and 7.5B, more subsequently). In addition to producing effects throughout the body, thyroid hormones negatively feed back to the brain and pituitary to regulate their own production. At the level of the brain, thyroid hormones suppress Trh expression (and presumably TRH release) in the PVN (Koller et al., 1987). In the pituitary, they inhibit expression of the TSH subunit genes (Gurr and Kourides, 1983). In thyrotrope cells, thyroid hormones act via the thyroid hormone receptor TRb2 to regulate expression of the TSH subunits. For example, serum TSH and pituitary TSH subunit expression are upregulated in mice lacking the TRb2 receptors (Forrest et al., 1996). The receptor resides in the nucleus where it is constitutively bound to thyroid hormone response elements in the promoter regions of TSHB and other genes. Upon binding of T3 to TRb2, TSHB transcription is inhibited (Nakano et al., 2004; Shibusawa et al., 2003; Bodenner et al., 1991). It should be noted that thyroid hormones are metabolized into more or less active forms by iodothyronine deiodinase enzymes in target tissues throughout the body, including the brain and pituitary gland (Schweizer and Steegborn, 2015). At the level of the pituitary, T4 is converted into the more active T3 via the type 2 iodothyronine deiodinase enzyme (Dio2). Thyrotropespecific ablation of Dio2 in mice leads to mild to modest increases in TSH secretion, suggesting that T4 is locally converted to T3 (Fonseca et al., 2013; Luongo et al., 2015). Thyroid hormones also require active transport into cells. Two transporters, MCT8 and OATP1C1, have proven critical for thyroid hormone transport into the brain; however the relevant transporters in the pituitary have not yet been resolved (Muller and Heuer, 2012). 4.2.3 Regulation by Other Factors There are many factors other than thyroid hormones that regulate TSH. For example, growth hormone deficiency is associated with an increase in TSH responsiveness to TRH. Conversely, an excess of growth hormone decreases basal, pulsatile, and TRH-stimulated TSH

release, possibly due to growth hormone stimulation of hypothalamic somatostatin (Lippe et al., 1975; Samuels et al., 1992). Somatostatin binds its receptors, subtypes 1 and 5, on thyrotropes to inhibit TSH secretion in humans and rats (James et al., 1997). Cortisol affects circadian TSH secretion. TSH has a circadian pattern of release, with nocturnal levels twice those observed during the day in humans (Roelfsema et al., 2014). Patients with adrenal insufficiency studied under glucocorticoid withdrawal have increased daytime TSH and abolished circadian rhythmicity in TSH release. Normal circadian rhythmicity of TSH secretion is reestablished following hydrocortisone treatment (Samuels, 2000). Dopamine inhibits basal and TRH-stimulated TSH secretion (Cooper et al., 1983). Dopamine, binding to D2 dopamine receptors on thyrotropes, inhibits transcription of both Cga and Tshb in rat primary culture (Shupnik et al., 1986). Dopamine infusions in humans suppress TSH pulse amplitude and abolish the nocturnal increase in TSH; however, they do not affect TSH pulse frequency (Samuels et al., 1992).

5. BIOCHEMICAL REACTIONS 5.1 Receptor Activation The current model of GPHR activation involves a two-step process, and it has been largely generated based on the structure of FSH and the ECD of FSHR (Jiang et al., 2012, 2014a; Fan and Hendrickson, 2005; Kleinau and Krause, 2009; Ulloa-Aguirre et al., 2018). First, FSH specifically binds to the b-strands of FSHR’s concave surface, forming electrostatic interactions and hydrogen bonds with both the a and b subunits of FSH (Fig. 7.1B). Each hormone contains a determinant loop in proximity to the seatbelt loop that is at the center of the ligand-receptor interface. The C-terminus of the a subunit and the L2 loop of the b subunit also participate in receptor binding. Second, following initial binding to its receptor, a hydrophobic sulfated tyrosine-binding pocket becomes accessible between the a and b subunits of the ligand, allowing for binding of a sulfated tyrosine residue, found in the hinge region of each GPHR (FSHR Y335, LHR Y331, TSHR Y385) (Bonomi et al., 2006; Bruysters et al., 2008). Binding of the sulfated tyrosine to the ligand leads to rotation of the a helix of the hinge region of the receptor. This sulfated tyrosine is indispensable for hormone recognition and signaling by the receptor. The hinge region of each GPHR functions as an inverse agonist, specifically through a highly conserved decapeptide sequence located N-terminal to the first

6. PHYSIOLOGIC FUNCTIONS

TMD (Bonomi et al., 2006; Bruysters et al., 2008; Agrawal and Dighe, 2009; Mueller et al., 2010). In the unliganded state, the decapeptide’s contacts with the TMD keeps the otherwise constitutively active receptor in an inactive state. Upon ligand binding, specifically the binding of the sulfated tyrosine, this inhibition is alleviated, and there is conformational change in the TMDs, allowing for effector binding intracellularly and activation of downstream signaling cascades. In fact, removal of the hinge region leads to an FSHR with higher basal activity and impaired ability to respond to FSH (Agrawal and Dighe, 2009). The hinge region in TSHR is unique compared to FSHR and LHCGR. The TSHR hinge region is cleaved at two sites, which leads to the release of a small peptide (C-peptide) of approximately 50 amino acids (Rapoport and McLachlan, 2016; Kaczur et al., 2007; Tanaka et al., 1999a; Hamidi et al., 2011). The exact cleavage sites and size of the C-peptide have yet to be determined. Cleavage and release of the C-peptide results in shedding of the extracellular domain (A-peptide) from the TMD (B-peptide). In vivo, there is a combination of both uncleaved and cleaved receptor at the cell surface (Tanaka et al., 1999b; Chen et al., 2006). The A- and Bpeptides of the cleaved receptor interact via disulfide bonds (Rapoport and McLachlan, 2016). The roles and consequences of this cleavage are not well understood (Vassart and Costagliola, 2004). However, when wildtype or uncleavable TSHRs are overexpressed in vitro, there is no difference in receptor constitutive activity in the absence of ligand or in downstream signaling upon TSH binding (Chazenbalk et al., 1999). Therefore, it does not appear that receptor cleavage influences receptor activity. Each GPHR can couple to multiple G proteins, often in a tissue-specific manner. However, all GPHRs predominantly couple to Gas, leading to activation of the adenylyl cyclase-cAMP-PKA signaling pathway (Figs. 7.3B, D, and 7.4B). For example, in the gonads, FSHR primarily couples to Gas; however, at high levels of FSH, the receptor can also engage Gaq/11, activating PLC and ultimately increasing intracellular Ca2þ (Ulloa-Aguirre et al., 2007; Quintana et al., 1994; Escamilla-Hernandez et al., 2008). A similar phenomenon has been described for the LHCGR, and Gaq/11mediated responses may be critical for LH-induced ovulation (Breen et al., 2013). In extragonadal tissues, such as osteoclasts and adipocytes, FSHR couples to Gai, inhibiting production of cAMP (Sun et al., 2006; Zaidi et al., 2018; Liu et al., 2017). At high concentrations of TSH, TSHR couples to Gaq/11, which is important for both thyroid growth and thyroid hormone synthesis (Biebermann et al., 1998; Claus et al., 2006; Neumann et al., 2005).

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5.2 Receptor Oligomerization GPHRs can form both homo- and heterodimers/oligomers; however, the role and physiologic importance of these higher order complexes remains unresolved and disputed (Thomas et al., 2007; Zoenen et al., 2012). It is generally thought that GPHRs interact through their TMDs; however, a crystal structure of the FSHR ECD bound to FSH showed formation of a trimer, suggesting that the receptors can interact extracellularly (Jiang et al., 2012, 2014b; Guan et al., 2010). Various studies have shown that TSHR oligomers form in the ER, a process that may be required for proper receptor expression. It was proposed that upon TSH binding, TSHR oligomers dissociate into monomers, and only monomers mediate G protein activation and signal transduction (Latif et al., 2002, 2015). For example, hyt/hyt mice, which have a spontaneous mutation in the fourth TMD of TSHR (P556L), have decreased cAMP activation, due to defects in TSH-induced dissociation of TSHR dimers into monomers, resulting in hypothyroidism (Endo and Kobayashi, 2012). However, in other contexts, dimerization TSHR and other GPHRs were not affected by hormone binding, and hormone binding to dimer/oligomers led to receptor transactivation (Rivero-Muller et al., 2010).

6. PHYSIOLOGIC FUNCTIONS 6.1 Gonadotropins Both LH and FSH are released into systemic circulation by gonadotrope cells. Their main targets are cells in the gonads (i.e., the testes and ovaries) (Fig. 7.3B, D). In males, LH binds its receptor (LHCGR) on interstitial Leydig cells to promote production of testosterone (O’Shaughnessy, 2014; Kaiser, 2017). LH stimulates the phosphorylation and activation of the steroidogenic acute regulatory (StAR) protein, which facilitates the transport of cholesterol from the outer to inner mitochondrial membrane (Kallen et al., 1998). Once inside the mitochondria, the P450 side chain cleavage enzyme converts cholesterol into pregnenolone, the ratelimiting step in steroid biosynthesis. Steroidogenic enzymes in the Leydig cells, which are also regulated by LH signaling, convert pregnenolone into testosterone, which is immediately secreted. Testosterone acts in a paracrine fashion on Sertoli cells to support latter stages of spermatogenesis (O’Shaughnessy, 2014; Kaiser, 2017; Smith and Walker, 2014). Sertoli cells directly support the developing spermatogonia and spermatocytes. Testosterone also negatively feeds back to the brain and pituitary to regulate its own production (Fig. 7.5A). Interestingly, many of the effects of

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testosterone in the brain are mediated via estrogens (Smith et al., 2005b). That is, testosterone is converted into estrogens by the aromatase enzyme in the brain and acts via ERa to suppress kisspeptin expression in the arcuate nucleus. FSH acts on Sertoli cells in the seminiferous tubules to stimulate production of androgen binding protein (ABP) and inhibin B (Hansson et al., 1973; Ramaswamy and Plant, 2001). ABP binds and concentrates testosterone in the testis, whereas inhibin B feeds back to the pituitary to inhibit FSH production (O’Shaughnessy, 2014; Kaiser, 2017). In females, LHCGR is expressed by the theca cells at the periphery of ovarian follicles and in mural granulosa cells of mature follicles (Richards and Ascoli, 2018). Postovulation, the remnants of the dominant follicles differentiate into the corpus luteum (CL). Luteal cells of the CL also express LHCGR and produce progesterone (and estradiol) in response to LH during the luteal phase of the menstrual cycle or to CG from the conceptus/placenta if pregnancy occurs. During the follicular phase of the menstrual cycle in women (Fig. 7.2A), LH stimulates androgen (androstenedione and testosterone) synthesis by theca cells in a manner analogous to LH-stimulated testosterone production in Leydig cells (Casarini et al., 2018a; Choi and Smitz, 2014) (Fig. 7.3B, D). Thecal androgens are converted into estrogens (e.g., estrone and 17b-estradiol) by the aromatase enzyme expressed in adjacent granulosa cells. Aromatase expression is stimulated by FSH actions in granulosa cells (Chan and Tan, 1987). During the late follicular phase of the cycle, elevated estradiol levels have positive feedback effects in the brain and pituitary. Estradiol promotes GnRH secretion from the brain, while enhancing pituitary sensitivity to GnRH. These two effects culminate in the generation of a massive discharge of LH (the LH surge) from the pituitary. The LH surge, via its actions in mural granulosa cells, triggers a cascade of events that culminates in ovulation (Casarini et al., 2018a; Choi and Smitz, 2014; Richards and Ascoli, 2018; Richards and Hedin, 1988). A surge of FSH also occurs alongside the LH surge, though its physiologic significance is less clear. Postovulation, during the luteal phase (Fig. 7.2A), LH pulse frequency diminishes greatly due principally to the negative feedback effects of luteal progesterone on GnRH secretion from the brain (Norman et al., 1984; Soules et al., 1984). These LH pulses promote CL survival and progesterone production for much of the luteal phase. In the absence of a pregnancy, the CL degenerates through a process called luteolysis. Progesterone (and estradiol) levels correspondingly decrease, removing negative feedback to the brain and pituitary, leading to increases in GnRH, LH, and FSH secretion,

driving the initiation of the next follicular phase. If fertilization occurs, the conceptus and, later, placenta produce CG, which acts via the LHCGR in luteal cells to maintain the CL. During the luteal phase, the CL also produces inhibin A, which inhibits FSH secretion from the pituitary at this stage of the cycle (McConnell et al., 1996; Yamoto et al., 1997). The loss of the CL also leads to reductions in inhibin A, which contributes to increases in FSH observed at the beginning of the follicular phase. FSH acts on granulosa cells of the follicle, leading to their proliferation, expression of LHCGR, and synthesis of aromatase (Richards and Pangas, 2010) (Fig. 7.3B). Aromatase converts thecal androgens to estrogens. The estrogens support follicular growth and oocyte maturation (Richards and Ascoli, 2018; Richards and Hedin, 1988). Gonadotropins play similar roles and are similarly regulated across rodent estrous cycles (Fig. 7.2B). These cycles are divided into four basic stages: metestrus, diestrus, proestrus, and estrus. On the early morning of estrus, there is a selective increase in FSH (known as the secondary FSH surge), which is analogous to the increase in FSH seen during the early follicular phase of the menstrual cycle. This increase in FSH selects the cohort of follicles that will ovulate in response to the LH surge 2e3 days later (Hoak and Schwartz, 1980). Though FSH and LH are generally low during the metestrous and diestrous stages that follow, there is sufficient gonadotropin support to drive continued follicle development and estradiol production. Once estradiol levels exceed a particular threshold, they promote, through positive feedback, surges of LH (and FSH). The LH surge triggers ovulation of the dominant follicles. During metestrus and diestrus, inhibin B from growing follicles maintains FSH at relatively low levels, preventing the recruitment of additional follicles (Rivier and Vale, 1989). The dominant follicles also start to produce inhibin A, which reaches a peak at the time of the LH surge (Woodruff et al., 1996). Following the surge, both inhibin A and B levels plummet. It is the loss of this inhibin negative feedback that enables activins to potently stimulate FSH production, leading to the secondary FSH surge. Though ovulated follicles form CLs, rodents do not have luteal stages in their cycles. Rather, on the late evening of proestrus, there is a transient increase in progesterone secretion, which likely contributes to the amplitude of the LH surge at both the hypothalamic and pituitary levels (Stephens et al., 2015; Gal et al., 2016). The critical importance of both LH and FSH to mammalian reproduction is clearly evident in individuals with loss or gain of function mutations in the gonadotropin subunits or their receptors (Themmen, 2005) (see more subsequently).

8. DISEASE AND AGING EFFECTS

6.2 Thyrotropin The main actions of TSH are on the thyroid gland (Fig. 7.4B). We have learned from TSH- and TSHRdeficient mice that TSH action is not required for embryonic development of the thyroid (Sarapura and Samuel, 2017; Marians et al., 2002; Postiglione et al., 2002). However, TSH is the main regulator of thyroid hormone synthesis during adulthood. The thyroid is composed of spherical follicles, with follicular cells (thyrocytes) at the periphery and colloid in the lumen (Fig. 7.4A). Soluble iodide (I) is acquired through our diet and is transported in the plasma to the thyroid, where it is transported into thyrocyte cells via the sodium/iodide symporter (NIS, encoded by SLC5A) located on the basolateral membrane of thyrocytes (Paire et al., 1997; Kogai et al., 1997) (Fig. 7.4B). NIS uses an electrochemical sodium gradient to transport anionic I across the membrane. Next, I is concentrated in the cytosol of thyrocytes before it is pumped through the apical membrane by pendrin (encoded by SLC26A4) into the follicular lumen of the colloid (Royaux et al., 2000; Yoshida et al., 2002; Scott et al., 1999). Thyroid peroxidase (TPO), a transmembrane glycoprotein located at the apical membrane of thyrocytes, with its catalytic domain facing in the colloid lumen, and hydrogen peroxide (H2O2) catalyze the oxidation of I to iodine (I0) (Krinsky and Alexander, 1971; Penel et al., 1998; Damante et al., 1989). I0 reacts with tyrosine residues of thyroglobulin (Tg) in the colloid to form mono- and diiodotyrosines (MIT and DIT) (Marians et al., 2002; Tosta et al., 1983; Virion et al., 1981). TPO catalyzes the coupling of neighboring iodotyrosine molecules to form thyroid hormones. MIT and DIT residues are coupled to form triiodothyronine (T3) or two DIT residues are coupled to form thyroxine (T4) (Cahnmann et al., 1977). T3 and T4 are commonly referred to as thyroid hormone. Iodinated Tg is endocytosed by thyrocytes and degraded in lysosomes, releasing the thyroid hormones (BernierValentin et al., 1990, 1991; Kostrouch et al., 1993). Thyroid hormones are secreted from thyrocytes into circulation by transporters such as monocarboxylate transporter 8 (MCT8, encoded by SLC16A2) (Di Cosmo et al., 2010). TSH regulates thyroid hormone production via 1) NIS, 2) TPO, and 3) Tg (Sarapura and Samuel, 2017; Postiglione et al., 2002). First, TSH promotes NIS biosynthesis, phosphorylation, and its proper trafficking to the plasma membrane (Paire et al., 1997; Kogai et al., 1997; Riedel et al., 2001). Second, TSH is required for TPO expression during embryogenesis. After birth, TSH regulates both TPO transcription and mRNA stability, as well as TPO translocation to the apical surface of thyrocytes (Sarapura and Samuel, 2017; Penel et al., 1998; Damante et al., 1989). Third, whereas basal Tg

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gene transcription is TSH-independent, TSH can regulate its rate of transcription and mRNA stability (Marians et al., 2002; Tosta et al., 1983; Bernier-Valentin et al., 1991). Additionally, TSH regulates expression and activation of the small GTPases, Rab5a and Rab7, which are the rate-limiting catalysts of Tg internalization and transfer to lysosomes (van den Hove et al., 2007). Finally, TSH acts on iodinated Tg stored in luminal colloid and stimulates its hydrolysis, resulting in the release of thyroid hormones (Sarapura and Samuel, 2017; Bernier-Valentin et al., 1991). The extrathyroidal actions of TSH remain unclear and understudied. TSHR expression has been found in astrocytes and neurons of the brain, where TSH stimulates Dio2 activity, as well as in folliculo-stellate cells of the pituitary, which may provide paracrine feedback and inhibit TSH secretion (Prummel et al., 2000, 2004; Crisanti et al., 2001). TSHR expression has been reported many other tissues (see earlier); however, their functions therein have yet to be fully characterized.

7. HORMONE INACTIVATION GPHs are eliminated via the kidneys and liver as intact proteins. Indeed, FSH and LH are purified from the urine of postmenopausal women and are used for ovarian stimulation and to treat hypogonadism (Deeks, 2018). LH has a serum half-life of 20e30 min, whereas FSH is longer-lived in circulation with a half-life of 2e4 h, depending on the nature and extent of glycosylation. This difference in elimination kinetics helps explain the pulsatile versus relatively constant levels of LH and FSH, respectively, in circulation. TSH has a serum halflife of about 1 h.

8. DISEASE AND AGING EFFECTS 8.1 Gonadotropins In humans, gonadotropins are elevated in the first few months after birth, during a period known as mini-puberty on infancy. They are then suppressed until the onset of puberty proper. In normal males, LH and FSH remain at adult levels throughout the rest of their lives, barring incident. In females, LH and FSH are cyclically released (as described before) from puberty to menopause (Fig. 7.2). During the menopausal transition, FSH levels begin to rise because of decreases in the remaining pool of follicles (follicular exhaustion) and the inhibin B that they secrete (Burger, 1999). Postmenopausally, both FSH and LH are elevated, and remain so because of the loss of both inhibin and steroid hormone negative feedback. Elevated gonadotropins are also

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observed in younger women experiencing premature ovarian failure in their 30s or 40s (normal menopause occurs around age 51). Here too, it is the loss of negative feedback regulators from the ovary that leads to this condition of hypergonadotropic hypogonadism. Gonadotropin dysregulation is also seen in polycystic ovary syndrome (PCOS). PCOS is the most common endocrinopathy in reproductive age women and is a common cause of anovulation and infertility. Diagnostic features include hyperandrogenemia, polycystic ovaries, and anovulation or oligo-ovulation (Azziz et al., 2016). In many women with PCOS, the ratio of LH to FSH is elevated in circulation. This has been attributed to enhanced GnRH pulse generator activity (Pastor et al., 1998). Recall from earlier that higher GnRH pulse frequencies favor LH relative to FSH secretion. The current model suggests that elevated androgens in PCOS impair progesterone negative feedback to the brain (Eagleson et al., 2000; Chhabra et al., 2005). As a result, GnRH pulse frequency remains inappropriately high. The skewed LH/FSH ratio contributes to increased androgen levels, as the LH-induced androgen production is not counterbalanced fully by FSH-induced aromatization into estrogens. Impairments in both gonadotropins (hypogonadotropism) are relatively rare but are observed under a variety of (mostly congenital) conditions. Kallman syndrome or any other defect in the development or function of the kisspeptin-GnRH system in the brain or pituitary leads to profound deficiencies in LH and FSH (Seminara et al., 2000; Balasubramanian et al., 2010). These defects are also observed in mice with naturally occurring or engineered mutations in the genes encoding GnRH, the GnRH receptor, kisspeptin, or the kisspeptin receptor (Mason et al., 1986; Seminara et al., 2003; Pask et al., 2005; Lapatto et al., 2007). Indeed, any condition blocking GnRH secretion or action will impair gonadotropin synthesis and secretion. Selective reductions in FSH are also observed in individuals with tumors (most often of granulosa cell origin) that hypersecrete inhibins (Hugon-Rodin et al., 2016; Healy et al., 1993). Isolated FSH or LH deficiency derives from mutations in the FSHB or LHB subunit genes that prevent production of dimeric FSH or LH (Layman and McDonough, 2000; Berger et al., 2005; Huhtaniemi et al., 1999). These mutations, which occur in homozygous or compound heterozygous form, are relatively rare, but they produce consistent and penetrant phenotypes. In males, LH deficiency is associated with hypoandrogenemia, incomplete sexual virilization during development, and azoospermia. Similar phenotypes are observed in male Lhb subunit knockout mice (Ma et al., 2004; Weiss et al., 1992). Women with LH deficiency are sterile, because of estrogen deficiency, and

anovulation (Lofrano-Porto et al., 2007). Again, these characteristics are phenocopied in female knockout mice. Women with FSHB subunit mutations fail to go through menarche because of arrested ovarian follicle development and estrogen deficiency (Misgar et al., 2019). Fshb knockout mice similarly display sterility due to a block in follicle development (Kumar et al., 1997). Men with FSH deficiency have external genitalia that are appropriately masculinized, although they have smaller than average sized testes. These men are, however, azoospermic and sterile (Phillip et al., 1998). In contrast, male Fshb-deficient mice are oligospermic (low sperm count) but fertile (Kumar et al., 1997). Similarly, men and mice with loss of function mutations in the FSH receptor gene (FSHR/Fshr) are oligospermic and fertile (Tapanainen et al., 1997; Krishnamurthy et al., 2000). These observations have raised important, but unresolved questions about the necessity for FSH in male reproduction. Thus far, only a single FSHR mutation has been investigated in men. Therefore, it is possible that not all receptor function is compromised by this particular amino acid substitution. At a minimum, the extant data indicate that FSH may be more important in spermatogenesis and fertility in men than in male mice.

8.2 Thyrotropin Thyroid disorders are common in humans, with conditions of thyroid hormone excess (hyperthyroidism) and deficiency (hypothyroidism) mainly caused by defects at the level of the thyroid gland. Graves disease, the most common form of autoimmune hyperthyroidism, is caused by antibodies binding to and activating the TSH receptor in a manner analogous to TSH (Smith and Hegedus, 2016). Hypothyroidism can similarly be caused by autoimmunity, as well as by iodine deficiency and congenital disorders that impair thyroid gland development or thyroid hormone biosynthesis (Chaker et al., 2017). Less frequently, hypothyroidism can be central in origin, arising from congenital or acquired defects at the level of the brain and/or pituitary (Beck-Peccoz et al., 2017). Inactivating mutations in the human TRH gene have not yet been described, but Trh knockout mice have reduced thyroid hormone levels (Yamada et al., 1997). Interestingly, pituitary TSH content is reduced in these mice, whereas serum TSH levels are increased. This suggests that most of the TSH produced by these mice is constitutively released and of reduced bioactivity. A similar phenotype is observed in humans and mice with inactivating mutations in the TRH receptor gene (TRHR/Trhr1) (Rabeler et al., 2004; Bonomi et al.,

9. CONCLUSIONS AND FUTURE DIRECTIONS

2009; Collu et al., 1997). Patients with inactivating mutations in the TSHB gene have also been described. These individuals present as children and must be treated with thyroid hormone replacement. Tshb-deficient mice are viable, but severely growth restricted and infertile (Tsujino et al., 2013). Collectively, these observations indicate that TSH is essential for thyroid hormone production but that TSH synthesis and secretion are not wholly dependent on TRH. This contrasts with the situation in the reproductive axis where FSH and LH synthesis depend on GnRH. TSH deficiency most frequently occurs in the context of what is referred to as combined pituitary hormone deficiency, which often arises from mutations in transcription factors that play essential roles in pituitary gland development and the specification of multiple cell lineages in the tissue (Fang et al., 2016). The most common cause of congenital central hypothyroidism appears to derive from mutations in the X-linked immunoglobulin superfamily member 1 gene (IGSF1), affecting an estimated 1: 100,000 live births (Joustra et al., 2013, 2016). IGSF1 is a transmembrane glycoprotein of unknown function that is highly expressed in thyrotrope cells (Joustra et al., 2015). In Igsf1-deficient mice, the TRH receptor mRNA is downregulated, and the animals show reduced TRHstimulated release of TSH (Sun et al., 2012; Turgeon et al., 2017). Similarly, humans with IGSF1 deficiency, especially affected children, show reduced TSH release in response to TRH challenge. How IGSF1 regulates TRHR expression is currently unknown.

9. CONCLUSIONS AND FUTURE DIRECTIONS We have learned a great deal about the evolution, structure, synthesis, and actions of the pituitary glycoprotein hormones. Given their common ancestry, it is perhaps not surprising how many parallels exist between FSH, LH, and TSH, but there are also some notable differences. Both the gonadotropins and thyrotropin are regulated by neuropeptides that act via Gaq/11-coupled GPCRs in the pituitary. The downstream pathways that regulate hormone secretion and b subunit expression are also conserved, at least for LH and TSH. GnRH and TRH stimulate LH and TSH release in a Ca2þ-dependent manner, and they regulate the Lhb and Tshb subunits via transcription factors induced downstream of ERK1/2 signaling. FSH is the "odd man out" among the three. Not only is GnRH a poor FSH secretagogue (with the exception of the primary gonadotropin surges), but it is potently regulated by TGFb superfamily ligands in a manner not observed for the other two hormones.

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All three hormones exert their actions via structurally related Gas-coupled GPCRs, and all promote the production of endocrine hormones, though in distinct ways. LH stimulates androgen synthesis in the testes and ovaries by regulating the first step in steroid biosynthesis. FSH acts to convert these androgens into estrogens (in females). TSH regulates many of the steps in the complex process of thyroid hormone synthesis. Steroid and thyroid hormones are all small molecules that work through nuclear receptors to regulate target gene transcription. However, where the hydrophobic nature of steroids enables them to diffuse across membranes, the zwitterionic thyroid hormones require facilitated transport. All have potent negative feedback on the glycoprotein hormones that promote their synthesis but achieve these effects in different ways. Thyroid hormones suppress both the neuropeptide that stimulates TSH synthesis and secretion (TRH) and have direct inhibitory effects on the expression of the TSH subunits in the pituitary. In contrast, the negative feedback effects of sex steroids on the gonadotropins are largely indirect. They principally inhibit a main driver of GnRH secretion (kisspeptin), without directly regulating (at least not in a significant manner) GnRH synthesis in the brain or gonadotropin subunit expression in the pituitary. Whereas gonadotropin synthesis depends on GnRH, TSH can still be produced in the absence of TRH. The preceding is not meant to be an exhaustive list, but rather it is designed to highlight some of the shared and unique aspects of glycoprotein hormone biology. Next, we briefly review some current gaps in knowledge, focusing on one or more major unanswered questions for each of the three hormones. Arguably, of the three pituitary glycoprotein hormones, we know most about the mechanisms of LH synthesis and action. As a result, there are probably fewer burning questions for LH than for FSH and TSH. Nonetheless, we still do not understand how gonadotropes decode GnRH pulse frequency. That is, how does high pulse frequency favor LH versus FSH synthesis and secretion? This remains an important question to answer so that we can better understand the mechanisms underlying elevated LH levels in women with PCOS. There are several "known unknowns" left to resolve for FSH. First, how does GnRH regulate FSH synthesis? Though there have been some tantalizing hints over the years from in vitro experiments, most have gone unsubstantiated in vivo. Second, how do GnRH and "activin" work together to regulate FSH? The data from a variety of mammalian species indicate that both factors are necessary for FSH synthesis, but neither is sufficient. Do the two work in series or in parallel to stimulate Fshb transcription? Third, data from mice suggest that activin B is not the primary TGFb ligand driving FSH synthesis in vivo. What is the ligand, where is it made,

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and how does it reach gonadotrope cells? Finally, it recently came to light that betaglycan functions as an obligate coreceptor for inhibin A, but not inhibin B in gonadotropes. How then does inhibin B suppress FSH with high affinity? Does it have its own unique coreceptor? Finally, we still have much to learn about TSH regulation. For example, how are TRH and T3 actions integrated to regulate Tshb expression? There is also relatively little known about transcriptional regulation of TSH synthesis in vivo. Though Pit1 and NR4A1 have been implicated in Tshb expression in vitro, the necessity for either protein in TSH production in vivo has not been resolved (e.g., with conditional knockout mice). What is the TSH pulse generator? TSH is released in a pulsatile fashion; however, TRH does not appear to influence TSH pulse frequency. Another important question from our perspective is how IGSF1 functions in thyrotropes to regulate TRH receptor expression. Loss of function mutations in the IGSF1 gene are the most common cause of central hypothyroidism, and this appears to derive from impaired TRH receptor expression and TRH action. However, the function of IGSF1 is unknown. Until this is resolved, it will be challenging to fully understand the disease process in affected patients. Research over the next decade should focus on these and related questions to provide a more complete picture of pituitary glycoprotein hormone synthesis and action in health and disease. The results of this work may identify novel therapeutic strategies to treat different forms of infertility and thyroid dysfunction, as well as to establish new contraceptive targets.

Funding DJB is supported by operating grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada (NSERC). EB holds a doctoral research scholarship from NSERC.

References Agrawal, G., Dighe, R.R., 2009. Critical involvement of the hinge region of the follicle-stimulating hormone receptor in the activation of the receptor. J. Biol. Chem. 284, 2636e2647. https://doi.org/10.1074/ jbc.M808199200. Anobile, C.J., Talbot, J.A., McCann, S.J., Padmanabhan, V., Robertson, W.R., 1998. Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the postmenopausal state. Mol. Hum. Reprod. 4, 631e639. https:// doi.org/10.1093/molehr/4.7.631. Attisano, L., Wrana, J.L., Cheifetz, S., Massague, J., 1992. Novel activin receptors: distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors. Cell 68, 97e108. Azziz, R., et al., 2016. Polycystic ovary syndrome. Nat. Rev. Dis. Primers 2, 16057. https://doi.org/10.1038/nrdp.2016.57.

Balasubramanian, R., et al., 2010. Human GnRH deficiency: a unique disease model to unravel the ontogeny of GnRH neurons. Neuroendocrinology 92, 81e99. https://doi.org/10.1159/000314193. Baldwin, J.M., 1993. The probable arrangement of the helices in G protein-coupled receptors. EMBO J. 12, 1693e1703. Bauer-Dantoin, A.C., Hollenberg, A.N., Jameson, J.L., 1993. Dynamic regulation of gonadotropin-releasing hormone receptor mRNA levels in the anterior pituitary gland during the rat estrous cycle. Endocrinology 133, 1911e1914. https://doi.org/10.1210/ endo.133.4.8404635. Beck-Peccoz, P., Rodari, G., Giavoli, C., Lania, A., 2017. Central hypothyroidism e a neglected thyroid disorder. Nat. Rev. Endocrinol. 13, 588e598. https://doi.org/10.1038/nrendo.2017.47. Berger, K., et al., 2005. Clinical and hormonal features of selective follicle-stimulating hormone (FSH) deficiency due to FSH betasubunit gene mutations in both sexes. Fertil. Steril. 83, 466e470. https://doi.org/10.1016/j.fertnstert.2004.06.069. Bernard, D.J., 2004. Both SMAD2 and SMAD3 mediate activinstimulated expression of the follicle-stimulating hormone beta subunit in mouse gonadotrope cells. Mol. Endocrinol. 18, 606e623. https://doi.org/10.1210/me.2003-0264. Bernard, D.J., Chapman, S.C., Woodruff, T.K., 2002. Inhibin binding protein (InhBP/p120), betaglycan, and the continuing search for the inhibin receptor. Mol. Endocrinol. 16, 207e212. https:// doi.org/10.1210/mend.16.2.0783. Bernard, D.J., Lee, K.B., Santos, M.M., 2006. Activin B can signal through both ALK4 and ALK7 in gonadotrope cells. Reprod. Biol. Endocrinol. 4, 52. https://doi.org/10.1186/1477-7827-4-52. Bernier-Valentin, F., Kostrouch, Z., Rabilloud, R., Munari-Silem, Y., Rousset, B., 1990. Coated vesicles from thyroid cells carry iodinated thyroglobulin molecules. First indication for an internalization of the thyroid prohormone via a mechanism of receptor-mediated endocytosis. J. Biol. Chem. 265, 17373e17380. Bernier-Valentin, F., Kostrouch, Z., Rabilloud, R., Rousset, B., 1991. Analysis of the thyroglobulin internalization process using in vitro reconstituted thyroid follicles: evidence for a coated vesicle-dependent endocytic pathway. Endocrinology 129, 2194e2201. https://doi.org/10.1210/endo-129-4-2194. Biebermann, H., et al., 1998. A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G-protein coupling. FASEB J. 12, 1461e1471. https://doi.org/10.1096/fasebj.12.14.1461. Bliss, S.P., et al., 2009. ERK signaling in the pituitary is required for female but not male fertility. Mol. Endocrinol. 23, 1092e1101. https:// doi.org/10.1210/me.2009-0030. Bodenner, D.L., Mroczynski, M.A., Weintraub, B.D., Radovick, S., Wondisford, F.E., 1991. A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene. J. Biol. Chem. 266, 21666e21673. Bonomi, M., Busnelli, M., Persani, L., Vassart, G., Costagliola, S., 2006. Structural differences in the hinge region of the glycoprotein hormone receptors: evidence from the sulfated tyrosine residues. Mol. Endocrinol. 20, 3351e3363. https://doi.org/10.1210/ me.2005-0521. Bonomi, M., et al., 2009. A family with complete resistance to thyrotropin-releasing hormone. N. Engl. J. Med. 360, 731e734. https://doi.org/10.1056/NEJMc0808557. Bousfield, G.R., May, J.V., Davis, J.S., Dias, J.A., Kumar, T.R., 2018. In vivo and in vitro impact of carbohydrate variation on human follicle-stimulating hormone function. Front. Endocrinol. 9, 216. https://doi.org/10.3389/fendo.2018.00216. Breen, S.M., et al., 2013. Ovulation involves the luteinizing hormonedependent activation of G(q/11) in granulosa cells. Mol. Endocrinol. 27, 1483e1491. https://doi.org/10.1210/me.2013-1130. Briet, C., Suteau-Courant, V., Munier, M., Rodien, P., 2018. Thyrotropin receptor, still much to be learned from the patients. Best Pract. Res.

REFERENCES

Clin. Endocrinol. Metabol. 32, 155e164. https://doi.org/10.1016/ j.beem.2018.03.002. Brown, J.L., et al., 2018. Sex- and age-specific impact of ERK loss within the pituitary gonadotrope in mice. Endocrinology 159, 1264e1276. https://doi.org/10.1210/en.2017-00653. Bruysters, M., Verhoef-Post, M., Themmen, A.P., 2008. Asp330 and Tyr331 in the C-terminal cysteine-rich region of the luteinizing hormone receptor are key residues in hormone-induced receptor activation. J. Biol. Chem. 283, 25821e25828. https://doi.org/ 10.1074/jbc.M804395200. Burger, H.G., 1999. The endocrinology of the menopause. J. Steroid Biochem. Mol. Biol. 69, 31e35. Cahnmann, H.J., Pommier, J., Nunez, J., 1977. Spatial requirement for coupling of iodotyrosine residues to form thyroid hormones. Proc. Natl. Acad. Sci. U.S.A. 74, 5333e5335. https://doi.org/ 10.1073/pnas.74.12.5333. Casarini, L., Santi, D., Brigante, G., Simoni, M., 2018. Two hormones for one receptor: evolution, biochemistry, actions and pathophysiology of LH and hCG. Endocr. Rev. https://doi.org/10.1210/er.201800065. Casarini, L., Santi, D., Simoni, M., Poti, F., 2018. ’Spare’ luteinizing hormone receptors: facts and fiction. Trends Endocrinol. Metab. 29, 208e217. https://doi.org/10.1016/j.tem.2018.01.007. Cash, J.N., Rejon, C.A., McPherron, A.C., Bernard, D.J., Thompson, T.B., 2009. The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding. EMBO J. 28, 2662e2676. https://doi.org/10.1038/emboj.2009.205. Chaker, L., Bianco, A.C., Jonklaas, J., Peeters, R.P., 2017. Hypothyroidism. Lancet 390, 1550e1562. https://doi.org/ 10.1016/S0140-6736(17)30703-1. Chan, W.K., Tan, C.H., 1987. Induction of aromatase activity in porcine granulosa cells by FSH and cyclic AMP. Endocr. Res. 13, 285e299. Chazenbalk, G.D., Tanaka, K., McLachlan, S.M., Rapoport, B., 1999. On the functional importance of thyrotropin receptor intramolecular cleavage. Endocrinology 140, 4516e4520. https://doi.org/ 10.1210/endo.140.10.7031. Chen, C.R., Chazenbalk, G.D., Wawrowsky, K.A., McLachlan, S.M., Rapoport, B., 2006. Evidence that human thyroid cells express uncleaved, single-chain thyrotropin receptors on their surface. Endocrinology 147, 3107e3113. https://doi.org/10.1210/en.20051514. Chhabra, S., et al., 2005. Progesterone inhibition of the hypothalamic gonadotropin-releasing hormone pulse generator: evidence for varied effects in hyperandrogenemic adolescent girls. J. Clin. Endocrinol. Metab. 90, 2810e2815. https://doi.org/10.1210/jc.2004-2359. Choi, J., Smitz, J., 2014. Luteinizing hormone and human chorionic gonadotropin: origins of difference. Mol. Cell. Endocrinol. 383, 203e213. https://doi.org/10.1016/j.mce.2013.12.009. Claus, M., Neumann, S., Kleinau, G., Krause, G., Paschke, R., 2006. Structural determinants for G-protein activation and specificity in the third intracellular loop of the thyroid-stimulating hormone receptor. J. Mol. Med. 84, 943e954. https://doi.org/10.1007/ s00109-006-0087-8. Cole, L.A., 2009. New discoveries on the biology and detection of human chorionic gonadotropin. Reprod. Biol. Endocrinol. 7, 8. https://doi.org/10.1186/1477-7827-7-8. Collu, R., et al., 1997. A novel mechanism for isolated central hypothyroidism: inactivating mutations in the thyrotropin-releasing hormone receptor gene. J. Clin. Endocrinol. Metab. 82, 1561e1565. https://doi.org/10.1210/jcem.82.5.3918. Cooper, D.S., Klibanski, A., Ridgway, E.C., 1983. Dopaminergic modulation of TSH and its subunits: in vivo and in vitro studies. Clin. Endocrinol. 18, 265e275. Corrigan, A.Z., et al., 1991. Evidence for an autocrine role of activin B within rat anterior pituitary cultures. Endocrinology 128, 1682e1684. https://doi.org/10.1210/endo-128-3-1682.

137

Coss, D., Jacobs, S.B., Bender, C.E., Mellon, P.L., 2004. A novel AP-1 site is critical for maximal induction of the follicle-stimulating hormone beta gene by gonadotropin-releasing hormone. J. Biol. Chem. 279, 152e162. https://doi.org/10.1074/jbc.M304697200. Couse, J.F., Yates, M.M., Walker, V.R., Korach, K.S., 2003. Characterization of the hypothalamic-pituitary-gonadal axis in estrogen receptor (ER) Null mice reveals hypergonadism and endocrine sex reversal in females lacking ERalpha but not ERbeta. Mol. Endocrinol. 17, 1039e1053. https://doi.org/10.1210/me.2002-0398. Crisanti, P., et al., 2001. The expression of thyrotropin receptor in the brain. Endocrinology 142, 812e822. https://doi.org/10.1210/ endo.142.2.7943. Crowley Jr., W.F., McArthur, J.W., 1980. Simulation of the normal menstrual cycle in Kallman’s syndrome by pulsatile administration of luteinizing hormone-releasing hormone (LHRH). J. Clin. Endocrinol. Metab. 51, 173e175. https://doi.org/10.1210/jcem-51-1-173. Damante, G., et al., 1989. Thyrotropin regulation of thyroid peroxidase messenger ribonucleic acid levels in cultured rat thyroid cells: evidence for the involvement of a nontranscriptional mechanism. Endocrinology 124, 2889e2894. https://doi.org/10.1210/endo124-6-2889. Deeks, E.D., 2018. Highly purified human menopausal gonadotropin (MenopurÒ): a profile of its use in infertility. Clin. Drug Investig. 38, 1077e1084. https://doi.org/10.1007/s40261-018-0703-8. Di Cosmo, C., et al., 2010. Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion. J. Clin. Investig. 120, 3377e3388. https://doi.org/10.1172/JCI42113. Dos Santos, S., et al., 2009. Distinct expression patterns of glycoprotein hormone-alpha2 and -beta5 in a basal chordate suggest independent developmental functions. Endocrinology 150, 3815e3822. https://doi.org/10.1210/en.2008-1743. Dos Santos, S., Mazan, S., Venkatesh, B., Cohen-Tannoudji, J., Querat, B., 2011. Emergence and evolution of the glycoprotein hormone and neurotrophin gene families in vertebrates. BMC Evol. Biol. 11, 332. https://doi.org/10.1186/1471-2148-11-332. Duan, W.R., et al., 2002. GnRH regulates early growth response protein 1 transcription through multiple promoter elements. Mol. Endocrinol. 16, 221e233. https://doi.org/10.1210/mend.16.2.0779. Eagleson, C.A., et al., 2000. Polycystic ovarian syndrome: evidence that flutamide restores sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and progesterone. J. Clin. Endocrinol. Metab. 85, 4047e4052. https://doi.org/ 10.1210/jcem.85.11.6992. Endo, T., Kobayashi, T., 2012. Dominant negative effect of mutated thyroid stimulating hormone receptor (P556L) causes hypothyroidism in C.RF-Tshr(hyt/wild) mice. PLoS One 7, e42358. https://doi.org/ 10.1371/journal.pone.0042358. Escamilla-Hernandez, R., Little-Ihrig, L., Zeleznik, A.J., 2008. Inhibition of rat granulosa cell differentiation by overexpression of Galphaq. Endocrine 33, 21e31. https://doi.org/10.1007/s12020008-9064-z. Fan, Q.R., Hendrickson, W.A., 2005. Structure of human folliclestimulating hormone in complex with its receptor. Nature 433, 269e277. https://doi.org/10.1038/nature03206. Fang, Q., et al., 2016. Genetics of combined pituitary hormone deficiency: roadmap into the genome era. Endocr. Rev. 37, 636e675. https://doi.org/10.1210/er.2016-1101. Fiddes, J.C., Goodman, H.M., 1981. The gene encoding the common alpha subunit of the four human glycoprotein hormones. J. Mol. Appl. Genet. 1, 3e18. Fonseca, T.L., et al., 2013. Coordination of hypothalamic and pituitary T3 production regulates TSH expression. J. Clin. Investig. 123, 1492e1500. https://doi.org/10.1172/JCI61231. Forrest, D., et al., 1996. Recessive resistance to thyroid hormone in mice lacking thyroid hormone receptor beta: evidence for tissue-specific modulation of receptor function. EMBO J. 15, 3006e3015.

138

7. ANTERIOR PITUITARY: GLYCOPROTEIN HORMONES FROM GONADOTROPE (FSH AND LH) AND THYROTROPE (TSH) CELLS

Fortin, J., Lamba, P., Wang, Y., Bernard, D.J., 2009. Conservation of mechanisms mediating gonadotrophin-releasing hormone 1 stimulation of human luteinizing hormone beta subunit transcription. Mol. Hum. Reprod. 15, 77e87. https://doi.org/10.1093/molehr/ gan079. Fortin, J., Boehm, U., Deng, C.X., Treier, M., Bernard, D.J., 2014. Folliclestimulating hormone synthesis and fertility depend on SMAD4 and FOXL2. FASEB J. 28, 3396e3410. https://doi.org/10.1096/fj.14249532. Fournier, T., Guibourdenche, J., Evain-Brion, D., 2015. Review: hCGs: different sources of production, different glycoforms and functions. Placenta 36 (Suppl. 1), S60eS65. https://doi.org/ 10.1016/j.placenta.2015.02.002. Fox, K.M., Dias, J.A., Van Roey, P., 2001. Three-dimensional structure of human follicle-stimulating hormone. Mol. Endocrinol. 15, 378e389. https://doi.org/10.1210/mend.15.3.0603. Gal, A., et al., 2016. Loss of fertility in the absence of progesterone receptor expression in kisspeptin neurons of female mice. PLoS One 11, e0159534. https://doi.org/10.1371/journal.pone.0159534. Gerasimova, T., et al., 2010. Identification and in vitro characterization of follicle stimulating hormone (FSH) receptor variants associated with abnormal ovarian response to FSH. J. Clin. Endocrinol. Metab. 95, 529e536. https://doi.org/10.1210/jc.2009-1304. Ghinea, N., 2018. Vascular endothelial FSH receptor, a target of interest for cancer therapy. Endocrinology 159, 3268e3274. https:// doi.org/10.1210/en.2018-00466. Gieske, M.C., et al., 2008. Pituitary gonadotroph estrogen receptoralpha is necessary for fertility in females. Endocrinology 149, 20e27. https://doi.org/10.1210/en.2007-1084. Gordon, D.F., et al., 1997. Pit-1 and GATA-2 interact and functionally cooperate to activate the thyrotropin beta-subunit promoter. J. Biol. Chem. 272, 24339e24347. https://doi.org/10.1074/ jbc.272.39.24339. Gromoll, J., Ried, T., Holtgreve-Grez, H., Nieschlag, E., Gudermann, T., 1994. Localization of the human FSH receptor to chromosome 2 p21 using a genomic probe comprising exon 10. J. Mol. Endocrinol. 12, 265e271. Gromoll, J., Pekel, E., Nieschlag, E., 1996. The structure and organization of the human follicle-stimulating hormone receptor (FSHR) gene. Genomics 35, 308e311. https://doi.org/10.1006/ geno.1996.0361. Grzesik, P., et al., 2015. Differences in signal activation by LH and hCG are mediated by the LH/CG receptor’s extracellular hinge region. Front. Endocrinol. 6, 140. https://doi.org/10.3389/ fendo.2015.00140. Gu, Z., et al., 1998. The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse. Genes Dev. 12, 844e857. https://doi.org/10.1101/gad.12.6.844. Guan, R., et al., 2010. Structural determinants underlying constitutive dimerization of unoccupied human follitropin receptors. Cell. Signal. 22, 247e256. https://doi.org/10.1016/j.cellsig.2009.09.023. Gurr, J.A., Kourides, I.A., 1983. Regulation of thyrotropin biosynthesis. Discordant effect of thyroid hormone on alpha and beta subunit mRNA levels. J. Biol. Chem. 258, 10208e10211. Hakola, K., et al., 1997. Recombinant rat follicle-stimulating hormone; production by Chinese hamster ovary cells, purification and functional characterization. Mol. Cell. Endocrinol. 127, 59e69. Hamidi, S., Chen, C.R., Mizutori-Sasai, Y., McLachlan, S.M., Rapoport, B., 2011. Relationship between thyrotropin receptor hinge region proteolytic posttranslational modification and receptor physiological function. Mol. Endocrinol. 25, 184e194. https:// doi.org/10.1210/me.2010-0401. Hansson, V., et al., 1973. FSH stimulation of testicular androgen binding protein. Nat. New Biol. 246, 56e58. Hashimoto, O., et al., 1997. A novel role of follistatin, an activinbinding protein, in the inhibition of activin action in rat pituitary

cells. Endocytotic degradation of activin and its acceleration by follistatin associated with cell-surface heparan sulfate. J. Biol. Chem. 272, 13835e13842. https://doi.org/10.1074/ jbc.272.21.13835. Hausken, K.N., et al., 2018. Cloning and characterization of a second lamprey pituitary glycoprotein hormone, thyrostimulin (GpA2/ GpB5). Gen. Comp. Endocrinol. 264, 16e27. https://doi.org/ 10.1016/j.ygcen.2018.04.010. Healy, D.L., et al., 1993. Elevated serum inhibin concentrations in postmenopausal women with ovarian tumors. N. Engl. J. Med. 329, 1539e1542. https://doi.org/10.1056/NEJM199311183292104. Herbison, A.E., 2018. The gonadotropin-releasing hormone pulse generator. Endocrinology 159, 3723e3736. https://doi.org/ 10.1210/en.2018-00653. Hinkle, P.M., Gehret, A.U., Jones, B.W., 2012. Desensitization, trafficking, and resensitization of the pituitary thyrotropin-releasing hormone receptor. Front. Neurosci. 6, 180. https://doi.org/ 10.3389/fnins.2012.00180. Hoak, D.C., Schwartz, N.B., 1980. Blockade of recruitment of ovarian follicles by suppression of the secondary surge of folliclestimulating hormone with porcine follicular field. Proc. Natl. Acad. Sci. U.S.A. 77, 4953e4956. https://doi.org/10.1073/ pnas.77.8.4953. Holland, P.W., Garcia-Fernandez, J., Williams, N.A., Sidow, A., 1994. Gene duplications and the origins of vertebrate development. Dev. Suppl. 125e133. Homma, T., et al., 2009. Significance of neonatal testicular sex steroids to defeminize anteroventral periventricular kisspeptin neurons and the GnRH/LH surge system in male rats. Biol. Reprod. 81, 1216e1225. https://doi.org/10.1095/biolreprod.109.078311. Horn, F., Windle, J.J., Barnhart, K.M., Mellon, P.L., 1992. Tissue-specific gene expression in the pituitary: the glycoprotein hormone alphasubunit gene is regulated by a gonadotrope-specific protein. Mol. Cell. Biol. 12, 2143e2153. https://doi.org/10.1128/mcb.12.5.2143. Hsu, S.Y., Nakabayashi, K., Bhalla, A., 2002. Evolution of glycoprotein hormone subunit genes in bilateral metazoa: identification of two novel human glycoprotein hormone subunit family genes, GPA2 and GPB5. Mol. Endocrinol. 16, 1538e1551. https://doi.org/ 10.1210/mend.16.7.0871. Huang, H.J., Sebastian, J., Strahl, B.D., Wu, J.C., Miller, W.L., 2001. Transcriptional regulation of the ovine follicle-stimulating hormonebeta gene by activin and gonadotropin-releasing hormone (GnRH): involvement of two proximal activator protein-1 sites for GnRH stimulation. Endocrinology 142, 2267e2274. https:// doi.org/10.1210/endo.142.6.8203. Hugon-Rodin, J., et al., 2016. Inhibin A and inhibin B producing ovarian fibrothecoma revealed by suppression of follicle stimulating hormone (FSH) in a post-menopausal woman: report of the first case. Gynecol. Endocrinol. 32, 872e874. https://doi.org/ 10.1080/09513590.2016.1222364. Huhtaniemi, I., Jiang, M., Nilsson, C., Pettersson, K., 1999. Mutations and polymorphisms in gonadotropin genes. Mol. Cell. Endocrinol. 151, 89e94. Inouye, S., et al., 1991. Recombinant expression of human follistatin with 315 and 288 amino acids: chemical and biological comparison with native porcine follistatin. Endocrinology 129, 815e822. https://doi.org/10.1210/endo-129-2-815. Jablonka-Shariff, A., Pearl, C.A., Comstock, A., Boime, I., 2008. A carboxyl-terminal sequence in the lutropin beta subunit contributes to the sorting of lutropin to the regulated pathway. J. Biol. Chem. 283, 11485e11492. https://doi.org/10.1074/ jbc.M800654200. James, R.A., et al., 1997. Thyroid hormone-induced expression of specific somatostatin receptor subtypes correlates with involution of the TtT-97 murine thyrotrope tumor. Endocrinology 138, 719e724. https://doi.org/10.1210/endo.138.2.4951.

REFERENCES

Jiang, X., et al., 2012. Structure of follicle-stimulating hormone in complex with the entire ectodomain of its receptor. Proc. Natl. Acad. Sci. U.S.A. 109, 12491e12496. https://doi.org/10.1073/ pnas.1206643109. Jiang, X., Dias, J.A., He, X., 2014. Structural biology of glycoprotein hormones and their receptors: insights to signaling. Mol. Cell. Endocrinol. 382, 424e451. https://doi.org/10.1016/ j.mce.2013.08.021. Jiang, X., et al., 2014. Evidence for follicle-stimulating hormone receptor as a functional trimer. J. Biol. Chem. 289, 14273e14282. https:// doi.org/10.1074/jbc.M114.549592. Jiang, C., et al., 2015. Hypoglycosylated hFSH has greater bioactivity than fully glycosylated recombinant hFSH in human granulosa cells. J. Clin. Endocrinol. Metab. 100, E852eE860. https:// doi.org/10.1210/jc.2015-1317. Jonak, C.R., Lainez, N.M., Boehm, U., Coss, D., 2018. GnRH receptor expression and reproductive function depend on JUN in GnRH receptor-expressing cells. Endocrinology 159, 1496e1510. https:// doi.org/10.1210/en.2017-00844. Jorgensen, J.S., Quirk, C.C., Nilson, J.H., 2004. Multiple and overlapping combinatorial codes orchestrate hormonal responsiveness and dictate cell-specific expression of the genes encoding luteinizing hormone. Endocr. Rev. 25, 521e542. https://doi.org/10.1210/ er.2003-0029. Joseph-Bravo, P., Jaimes-Hoy, L., Uribe, R.M., Charli, J.L., 2015. 60 years of neuroendocrinology: TRH, the first hypophysiotropic releasing hormone isolated: control of the pituitary-thyroid axis. J. Endocrinol. 226, T85eT100. https://doi.org/10.1530/JOE-150124. Joustra, S.D., et al., 2013. IGSF1 deficiency syndrome: a newly uncovered endocrinopathy. Rare Dis. 1, e24883. https://doi.org/ 10.4161/rdis.24883. Joustra, S.D., et al., 2015. Spatial and temporal expression of immunoglobulin superfamily member 1 in the rat. J. Endocrinol. 226, 181e191. https://doi.org/10.1530/JOE-15-0204. Joustra, S.D., et al., 2016. IGSF1 deficiency: lessons from an extensive case series and recommendations for clinical management. J. Clin. Endocrinol. Metab. 101, 1627e1636. https://doi.org/10.1210/ jc.2015-3880. Kaczur, V., et al., 2007. Cleavage of the human thyrotropin receptor by ADAM10 is regulated by thyrotropin. J. Mol. Recognit. 20, 392e404. https://doi.org/10.1002/jmr.851. Kaiser, U.B., 2017. In: Melmed, S. (Ed.), The Pituitary, fourth ed. Academic Press, pp. 203e250. Kajava, A.V., Vassart, G., Wodak, S.J., 1995. Modeling of the threedimensional structure of proteins with the typical leucine-rich repeats. Structure 3, 867e877. https://doi.org/10.1016/S09692126(01)00222-2. Kallen, C.B., et al., 1998. Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein. Mol. Cell. Endocrinol. 145, 39e45. Karakaya, C., et al., 2014. Follicle-stimulating hormone receptor (FSHR) alternative skipping of exon 2 or 3 affects ovarian response to FSH. Mol. Hum. Reprod. 20, 630e643. https://doi.org/10.1093/ molehr/gau024. Kauffman, A.S., et al., 2007. Sexual differentiation of Kiss1 gene expression in the brain of the rat. Endocrinology 148, 1774e1783. https:// doi.org/10.1210/en.2006-1540. Kawauchi, H., Sower, S.A., 2006. The dawn and evolution of hormones in the adenohypophysis. Gen. Comp. Endocrinol. 148, 3e14. https://doi.org/10.1016/j.ygcen.2005.10.011. Kleinau, G., Krause, G., 2009. Thyrotropin and homologous glycoprotein hormone receptors: structural and functional aspects of extracellular signaling mechanisms. Endocr. Rev. 30, 133e151. https:// doi.org/10.1210/er.2008-0044.

139

Knobil, E., Plant, T.M., Wildt, L., Belchetz, P.E., Marshall, G., 1980. Control of the rhesus monkey menstrual cycle: permissive role of hypothalamic gonadotropin-releasing hormone. Science 207, 1371e1373. Kogai, T., et al., 1997. Regulation by thyroid-stimulating hormone of sodium/iodide symporter gene expression and protein levels in FRTL-5 cells. Endocrinology 138, 2227e2232. https://doi.org/ 10.1210/endo.138.6.5189. Kokk, K., et al., 2011. Expression of luteinizing hormone receptors in the mouse penis. J. Androl. 32, 49e54. https://doi.org/10.2164/ jandrol.109.008623. Koller, K.J., Wolff, R.S., Warden, M.K., Zoeller, R.T., 1987. Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus. Proc. Natl. Acad. Sci. U.S.A. 84, 7329e7333. https://doi.org/10.1073/pnas.84.20.7329. Kostrouch, Z., et al., 1993. Thyroglobulin molecules internalized by thyrocytes are sorted in early endosomes and partially recycled back to the follicular lumen. Endocrinology 132, 2645e2653. https://doi.org/10.1210/endo.132.6.8504765. Krinsky, M.M., Alexander, N.M., 1971. Thyroid peroxidase. Nature of the heme binding to apoperoxidase. J. Biol. Chem. 246, 4755e4758. Krishnamurthy, H., Danilovich, N., Morales, C.R., Sairam, M.R., 2000. Qualitative and quantitative decline in spermatogenesis of the follicle-stimulating hormone receptor knockout (FORKO) mouse. Biol. Reprod. 62, 1146e1159. https://doi.org/10.1095/ biolreprod62.5.1146. Kumar, T.R., 2018. Fshb knockout mouse model, two decades later and into the future. Endocrinology 159, 1941e1949. https://doi.org/ 10.1210/en.2018-00072. Kumar, T.R., Wang, Y., Lu, N., Matzuk, M.M., 1997. Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat. Genet. 15, 201e204. https://doi.org/10.1038/ ng0297-201. Lamba, P., et al., 2010. Activin A regulates porcine follicle-stimulating hormone beta-subunit transcription via cooperative actions of SMADs and FOXL2. Endocrinology 151, 5456e5467. https:// doi.org/10.1210/en.2010-0605. Lapatto, R., et al., 2007. Kiss1-/- mice exhibit more variable hypogonadism than Gpr54-/- mice. Endocrinology 148, 4927e4936. https:// doi.org/10.1210/en.2007-0078. Lapthorn, A.J., et al., 1994. Crystal structure of human chorionic gonadotropin. Nature 369, 455e461. https://doi.org/10.1038/ 369455a0. Latif, R., Graves, P., Davies, T.F., 2002. Ligand-dependent inhibition of oligomerization at the human thyrotropin receptor. J. Biol. Chem. 277, 45059e45067. https://doi.org/10.1074/jbc.M206693200. Latif, R., Ali, M.R., Mezei, M., Davies, T.F., 2015. Transmembrane domains of attraction on the TSH receptor. Endocrinology 156, 488e498. https://doi.org/10.1210/en.2014-1509. Layman, L.C., McDonough, P.G., 2000. Mutations of follicle stimulating hormone-beta and its receptor in human and mouse: genotype/ phenotype. Mol. Cell. Endocrinol. 161, 9e17. Lee, S.L., et al., 1996. Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1). Science 273, 1219e1221. Lewis, K.A., et al., 2000. Betaglycan binds inhibin and can mediate functional antagonism of activin signalling. Nature 404, 411e414. https://doi.org/10.1038/35006129. Li, Y., et al., 2017. SMAD3 regulates follicle-stimulating hormone synthesis by pituitary gonadotrope cells in vivo. J. Biol. Chem. 292, 2301e2314. https://doi.org/10.1074/jbc.M116.759167. Li, Y., et al., 2018. Conditional deletion of FOXL2 and SMAD4 in gonadotropes of adult mice causes isolated FSH deficiency. Endocrinology 159, 2641e2655. https://doi.org/10.1210/en.2018-00100. Li, Y., et al., 2018. Betaglycan (TGFBR3) functions as an inhibin A, but not inhibin B, coreceptor in pituitary gonadotrope cells in mice.

140

7. ANTERIOR PITUITARY: GLYCOPROTEIN HORMONES FROM GONADOTROPE (FSH AND LH) AND THYROTROPE (TSH) CELLS

Endocrinology 159, 4077e4091. https://doi.org/10.1210/en.201800770. Libert, F., et al., 1989. Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem. Biophys. Res. Commun. 165, 1250e1255. Libert, F., Passage, E., Lefort, A., Vassart, G., Mattei, M.G., 1990. Localization of human thyrotropin receptor gene to chromosome region 14q3 by in situ hybridization. Cytogenet. Cell Genet. 54, 82e83. https://doi.org/10.1159/000132964. Lindzey, J., et al., 1998. Effects of castration and chronic steroid treatments on hypothalamic gonadotropin-releasing hormone content and pituitary gonadotropins in male wild-type and estrogen receptor-alpha knockout mice. Endocrinology 139, 4092e4101. https://doi.org/10.1210/endo.139.10.6253. Ling, N., et al., 1985. Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc. Natl. Acad. Sci. U.S.A. 82, 7217e7221. https://doi.org/ 10.1073/pnas.82.21.7217. Ling, N., et al., 1986. Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin. Nature 321, 779e782. https://doi.org/10.1038/321779a0. Ling, N., et al., 1986. A homodimer of the beta-subunits of inhibin A stimulates the secretion of pituitary follicle stimulating hormone. Biochem. Biophys. Res. Commun. 138, 1129e1137. Lippe, B.M., et al., 1975. Reversible hypothyroidism in growth hormone-deficient children treated with human growth hormone. J. Clin. Endocrinol. Metab. 40, 612e618. https://doi.org/10.1210/ jcem-40-4-612. Liu, P., et al., 2017. Blocking FSH induces thermogenic adipose tissue and reduces body fat. Nature 546, 107e112. https://doi.org/ 10.1038/nature22342. Lofrano-Porto, A., et al., 2007. Luteinizing hormone beta mutation and hypogonadism in men and women. N. Engl. J. Med. 357, 897e904. https://doi.org/10.1056/NEJMoa071999. Luongo, C., et al., 2015. The selective loss of the type 2 iodothyronine deiodinase in mouse thyrotrophs increases basal TSH but blunts the thyrotropin response to hypothyroidism. Endocrinology 156, 745e754. https://doi.org/10.1210/en.2014-1698. Ma, X., Dong, Y., Matzuk, M.M., Kumar, T.R., 2004. Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility. Proc. Natl. Acad. Sci. U.S.A. 101, 17294e17299. https://doi.org/10.1073/ pnas.0404743101. Makanji, Y., Temple-Smith, P.D., Walton, K.L., Harrison, C.A., Robertson, D.M., 2009. Inhibin B is a more potent suppressor of rat follicle-stimulating hormone release than inhibin a in vitro and in vivo. Endocrinology 150, 4784e4793. https://doi.org/ 10.1210/en.2008-1783. Marians, R.C., et al., 2002. Defining thyrotropin-dependent and -independent steps of thyroid hormone synthesis by using thyrotropin receptor-null mice. Proc. Natl. Acad. Sci. U.S.A. 99, 15776e15781. https://doi.org/10.1073/pnas.242322099. Mason, A.J., et al., 1985. Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-beta. Nature 318, 659e663. Mason, A.J., et al., 1986. A deletion truncating the gonadotropinreleasing hormone gene is responsible for hypogonadism in the hpg mouse. Science 234, 1366e1371. Mathews, L.S., Vale, W.W., 1991. Expression cloning of an activin receptor, a predicted transmembrane serine kinase. Cell 65, 973e982. Matzuk, M.M., Kumar, T.R., Bradley, A., 1995. Different phenotypes for mice deficient in either activins or activin receptor type II. Nature 374, 356e360. https://doi.org/10.1038/374356a0. Matzuk, M.M., et al., 1995. Multiple defects and perinatal death in mice deficient in follistatin. Nature 374, 360e363. https://doi.org/ 10.1038/374360a0.

Maugars, G., Dufour, S., Cohen-Tannoudji, J., Querat, B., 2014. Multiple thyrotropin beta-subunit and thyrotropin receptor-related genes arose during vertebrate evolution. PLoS One 9, e111361. https:// doi.org/10.1371/journal.pone.0111361. McConnell, D.S., et al., 1996. Development of a two-site solid-phase immunochemiluminescent assay for measurement of dimeric inhibin-A in human serum and other biological fluids. Clin. Chem. 42, 1159e1167. McConnell, D.S., et al., 1998. A two-site chemiluminescent assay for activin-free follistatin reveals that most follistatin circulating in men and normal cycling women is in an activin-bound state. J. Clin. Endocrinol. Metab. 83, 851e858. https://doi.org/10.1210/ jcem.83.3.4651. McNeilly, A.S., Crawford, J.L., Taragnat, C., Nicol, L., McNeilly, J.R., 2003. The differential secretion of FSH and LH: regulation through genes, feedback and packaging. Reprod. Suppl. 61, 463e476. Millar, R.P., et al., 2004. Gonadotropin-releasing hormone receptors. Endocr. Rev. 25, 235e275. https://doi.org/10.1210/er.2003-0002. Misgar, R.A., et al., 2019. FSH beta-subunit mutations in two sisters: the first report from the Indian sub-continent and review of previous cases. Gynecol. Endocrinol. 35, 290e293. https://doi.org/ 10.1080/09513590.2018.1529159. Mueller, S., Jaeschke, H., Gunther, R., Paschke, R., 2010. The hinge region: an important receptor component for GPHR function. Trends Endocrinol. Metab. 21, 111e122. https://doi.org/10.1016/ j.tem.2009.09.001. Mukherjee, A., et al., 2007. FSTL3 deletion reveals roles for TGF-beta family ligands in glucose and fat homeostasis in adults. Proc. Natl. Acad. Sci. U.S.A. 104, 1348e1353. https://doi.org/10.1073/ pnas.0607966104. Muller, J., Heuer, H., 2012. Understanding the hypothalamus-pituitarythyroid axis in mct8 deficiency. Eur. Thyroid J. 1, 72e79. https:// doi.org/10.1159/000339474. Naicker, M., Naidoo, S., 2018. Expression of thyroid-stimulating hormone receptors and thyroglobulin in limbic regions in the adult human brain. Metab. Brain Dis. 33, 481e489. https://doi.org/ 10.1007/s11011-017-0076-3. Naik, S.I., Young, L.S., Charlton, H.M., Clayton, R.N., 1985. Evidence for a pituitary site of gonadal steroid stimulation of GnRH receptors in female mice. J. Reprod. Fertil. 74, 615e624. Nakabayashi, K., et al., 2002. Thyrostimulin, a heterodimer of two new human glycoprotein hormone subunits, activates the thyroidstimulating hormone receptor. J. Clin. Investig. 109, 1445e1452. https://doi.org/10.1172/JCI14340. Nakajima, Y., et al., 2012. NR4A1 (Nur77) mediates thyrotropinreleasing hormone-induced stimulation of transcription of the thyrotropin beta gene: analysis of TRH knockout mice. PLoS One 7, e40437. https://doi.org/10.1371/journal.pone.0040437. Nakamura, T., Sugino, K., Titani, K., Sugino, H., 1991. Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells. J. Biol. Chem. 266, 19432e19437. Nakano, K., et al., 2004. Thyroid-hormone-dependent negative regulation of thyrotropin beta gene by thyroid hormone receptors: study with a new experimental system using CV1 cells. Biochem. J. 378, 549e557. https://doi.org/10.1042/BJ20031592. Naor, Z., 1990. Signal transduction mechanisms of Ca2þ mobilizing hormones: the case of gonadotropin-releasing hormone. Endocr. Rev. 11, 326e353. https://doi.org/10.1210/edrv-11-2-326. Naylor, S.L., et al., 1983. Chromosome assignment of genes encoding the alpha and beta subunits of glycoprotein hormones in man and mouse. Somat. Cell Genet. 9, 757e770. Neumann, S., Krause, G., Claus, M., Paschke, R., 2005. Structural determinants for g protein activation and selectivity in the second intracellular loop of the thyrotropin receptor. Endocrinology 146, 477e485. https://doi.org/10.1210/en.2004-1045.

REFERENCES

Nillni, E.A., 2010. Regulation of the hypothalamic thyrotropin releasing hormone (TRH) neuron by neuronal and peripheral inputs. Front. Neuroendocrinol. 31, 134e156. https://doi.org/ 10.1016/j.yfrne.2010.01.001. Norman, R.L., et al., 1984. Pulsatile secretion of luteinizing hormone during the menstrual cycle of rhesus macaques. Endocrinology 115, 261e266. https://doi.org/10.1210/endo-115-1-261. Nunez Miguel, R., Sanders, J., Furmaniak, J., Rees Smith, B., 2017. Glycosylation pattern analysis of glycoprotein hormones and their receptors. J. Mol. Endocrinol. 58, 25e41. https://doi.org/10.1530/ JME-16-0169. O’Connor, A.E., De Kretser, D.M., 2004. Inhibins in normal male physiology. Semin. Reprod. Med. 22, 177e185. https://doi.org/ 10.1055/s-2004-831893. O’Shaughnessy, P.J., 2014. Hormonal control of germ cell development and spermatogenesis. Semin. Cell Dev. Biol. 29, 55e65. https:// doi.org/10.1016/j.semcdb.2014.02.010. Ohba, K., et al., 2011. GATA2 mediates thyrotropin-releasing hormoneinduced transcriptional activation of the thyrotropin beta gene. PLoS One 6, e18667. https://doi.org/10.1371/ journal.pone.0018667. Ortiga-Carvalho, T.M., Chiamolera, M.I., Pazos-Moura, C.C., Wondisford, F.E., 2016. Hypothalamus-pituitary-thyroid Axis. Comp. Physiol. 6, 1387e1428. https://doi.org/10.1002/ cphy.c150027. Paire, A., Bernier-Valentin, F., Selmi-Ruby, S., Rousset, B., 1997. Characterization of the rat thyroid iodide transporter using anti-peptide antibodies. Relationship between its expression and activity. J. Biol. Chem. 272, 18245e18249. https://doi.org/10.1074/ jbc.272.29.18245. Pakarainen, T., et al., 2007. Extragonadal LH/hCG action e not yet time to rewrite textbooks. Mol. Cell. Endocrinol. 269, 9e16. https://doi.org/10.1016/j.mce.2006.10.019. Park, K.R., Saxena, B.B., Gandy, H.M., 1976. Specific binding of LH-RH to the anterior pituitary gland during the oestrous cycle in the rat. Acta Endocrinol. 82, 62e70. Pask, A.J., et al., 2005. A novel mouse model of hypogonadotrophic hypogonadism: N-ethyl-N-nitrosourea-induced gonadotropinreleasing hormone receptor gene mutation. Mol. Endocrinol. 19, 972e981. https://doi.org/10.1210/me.2004-0192. Pastor, C.L., Griffin-Korf, M.L., Aloi, J.A., Evans, W.S., Marshall, J.C., 1998. Polycystic ovary syndrome: evidence for reduced sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and progesterone. J. Clin. Endocrinol. Metab. 83, 582e590. https://doi.org/10.1210/jcem.83.2.4604. Penel, C., Gruffat, D., Alquier, C., Benoliel, A.M., Chabaud, O., 1998. Thyrotropin chronically regulates the pool of thyroperoxidase and its intracellular distribution: a quantitative confocal microscopic study. J. Cell. Physiol. 174, 160e169. https://doi.org/10.1002/ (SICI)1097-4652(199802)174:23.0.CO;2-M. Phillip, M., Arbelle, J.E., Segev, Y., Parvari, R., 1998. Male hypogonadism due to a mutation in the gene for the beta-subunit of folliclestimulating hormone. N. Engl. J. Med. 338, 1729e1732. https:// doi.org/10.1056/NEJM199806113382404. Pierce, J.G., Parsons, T.F., 1981. Glycoprotein hormones: structure and function. Annu. Rev. Biochem. 50, 465e495. https://doi.org/ 10.1146/annurev.bi.50.070181.002341. Pierce, J.G., Faith, M.R., Giudice, L.C., Reeve, J.R., 1976. Structure and structure-function relationships in glycoprotein hormones. Ciba Found. Symp. 41, 225e250. Popa, S.M., Clifton, D.K., Steiner, R.A., 2008. The role of kisspeptins and GPR54 in the neuroendocrine regulation of reproduction. Annu. Rev. Physiol. 70, 213e238. https://doi.org/10.1146/ annurev.physiol.70.113006.100540. Postiglione, M.P., et al., 2002. Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the

141

thyroid gland. Proc. Natl. Acad. Sci. U.S.A. 99, 15462e15467. https://doi.org/10.1073/pnas.242328999. Prummel, M.F., et al., 2000. Expression of the thyroid-stimulating hormone receptor in the folliculo-stellate cells of the human anterior pituitary. J. Clin. Endocrinol. Metab. 85, 4347e4353. https:// doi.org/10.1210/jcem.85.11.6991. Prummel, M.F., Brokken, L.J., Wiersinga, W.M., 2004. Ultra short-loop feedback control of thyrotropin secretion. Thyroid 14, 825e829. https://doi.org/10.1089/thy.2004.14.825. Puett, D., Angelova, K., da Costa, M.R., Warrenfeltz, S.W., Fanelli, F., 2010. The luteinizing hormone receptor: insights into structurefunction relationships and hormone-receptor-mediated changes in gene expression in ovarian cancer cells. Mol. Cell. Endocrinol. 329, 47e55. https://doi.org/10.1016/j.mce.2010.04.025. Quintana, J., Hipkin, R.W., Sanchez-Yague, J., Ascoli, M., 1994. Follitropin (FSH) and a phorbol ester stimulate the phosphorylation of the FSH receptor in intact cells. J. Biol. Chem. 269, 8772e8779. Rabeler, R., et al., 2004. Generation of thyrotropin-releasing hormone receptor 1-deficient mice as an animal model of central hypothyroidism. Mol. Endocrinol. 18, 1450e1460. https:// doi.org/10.1210/me.2004-0017. Ramaswamy, S., Plant, T.M., 2001. Operation of the follicle-stimulating hormone (FSH)-inhibin B feedback loop in the control of primate spermatogenesis. Mol. Cell. Endocrinol. 180, 93e101. Rapoport, B., McLachlan, S.M., 2016. TSH receptor cleavage into subunits and shedding of the A-subunit; a molecular and clinical perspective. Endocr. Rev. 37, 114e134. https://doi.org/10.1210/ er.2015-1098. Rejon, C.A., et al., 2013. Activins bind and signal via bone morphogenetic protein receptor type II (BMPR2) in immortalized gonadotrope-like cells. Cell. Signal. 25, 2717e2726. https:// doi.org/10.1016/j.cellsig.2013.09.002. Richards, J.S., Ascoli, M., 2018. Endocrine, paracrine, and autocrine signaling pathways that regulate ovulation. Trends Endocrinol. Metab. 29, 313e325. https://doi.org/10.1016/j.tem.2018.02.012. Richards, J.S., Hedin, L., 1988. Molecular aspects of hormone action in ovarian follicular development, ovulation, and luteinization. Annu. Rev. Physiol. 50, 441e463. https://doi.org/10.1146/ annurev.ph.50.030188.002301. Richards, J.S., Pangas, S.A., 2010. The ovary: basic biology and clinical implications. J. Clin. Investig. 120, 963e972. https://doi.org/ 10.1172/JCI41350. Riedel, C., Levy, O., Carrasco, N., 2001. Post-transcriptional regulation of the sodium/iodide symporter by thyrotropin. J. Biol. Chem. 276, 21458e21463. https://doi.org/10.1074/jbc.M100561200. Rivero-Muller, A., et al., 2010. Rescue of defective G protein-coupled receptor function in vivo by intermolecular cooperation. Proc. Natl. Acad. Sci. U.S.A. 107, 2319e2324. https://doi.org/10.1073/ pnas.0906695106. Rivier, C., Vale, W., 1989. Immunoneutralization of endogenous inhibin modifies hormone secretion and ovulation rate in the rat. Endocrinology 125, 152e157. https://doi.org/10.1210/endo-125-1-152. Robertson, D.M., et al., 1985. Isolation of inhibin from bovine follicular fluid. Biochem. Biophys. Res. Commun. 126, 220e226. Roelfsema, F., et al., 2014. Thyrotropin secretion in healthy subjects is robust and independent of age and gender, and only weakly dependent on body mass index. J. Clin. Endocrinol. Metab. 99, 570e578. https://doi.org/10.1210/jc.2013-2858. Rousseau-Merck, M.F., et al., 1990. Assignment of the human thyroid stimulating hormone receptor (TSHR) gene to chromosome 14q31. Genomics 8, 233e236. Rousseau-Merck, M.F., Atger, M., Loosfelt, H., Milgrom, E., Berger, R., 1993. The chromosomal localization of the human folliclestimulating hormone receptor gene (FSHR) on 2p21-p16 is similar to that of the luteinizing hormone receptor gene. Genomics 15, 222e224. https://doi.org/10.1006/geno.1993.1041.

142

7. ANTERIOR PITUITARY: GLYCOPROTEIN HORMONES FROM GONADOTROPE (FSH AND LH) AND THYROTROPE (TSH) CELLS

Royaux, I.E., et al., 2000. Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells. Endocrinology 141, 839e845. https://doi.org/10.1210/endo.141.2.7303. Sacchi, S., Sena, P., Degli Esposti, C., Lui, J., La Marca, A., 2018. Evidence for expression and functionality of FSH and LH/hCG receptors in human endometrium. J. Assist. Reprod. Genet. 35, 1703e1712. https://doi.org/10.1007/s10815-018-1248-8. Samuels, M.H., 2000. Effects of variations in physiological cortisol levels on thyrotropin secretion in subjects with adrenal insufficiency: a clinical research center study. J. Clin. Endocrinol. Metab. 85, 1388e1393. https://doi.org/10.1210/jcem.85.4.6540. Samuels, M.H., Henry, P., Ridgway, E.C., 1992. Effects of dopamine and somatostatin on pulsatile pituitary glycoprotein secretion. J. Clin. Endocrinol. Metab. 74, 217e222. https://doi.org/10.1210/ jcem.74.1.1345783. Samuels, M.H., Henry, P., Luther, M., Ridgway, E.C., 1993. Pulsatile TSH secretion during 48-hour continuous TRH infusions. Thyroid 3, 201e206. https://doi.org/10.1089/thy.1993.3.201. Samuels, M.H., Veldhuis, J., Ridgway, E.C., 1995. Copulsatile release of thyrotropin and prolactin in normal and hypothyroid subjects. Thyroid 5, 369e372. https://doi.org/10.1089/thy.1995.5.369. Sanders, J., et al., 2007. Crystal structure of the TSH receptor in complex with a thyroid-stimulating autoantibody. Thyroid 17, 395e410. https://doi.org/10.1089/thy.2007.0034. Sanders, P., et al., 2011. Crystal structure of the TSH receptor (TSHR) bound to a blocking-type TSHR autoantibody. J. Mol. Endocrinol. 46, 81e99. https://doi.org/10.1530/JME-10-0127. Sandoval-Guzman, T., et al., 2012. Neuroendocrine control of female reproductive function by the activin receptor ALK7. FASEB J. 26, 4966e4976. https://doi.org/10.1096/fj.11-199059. Sarapura, V.D., Samuel, M.H., 2017. In: Melmed, S. (Ed.), The Pituitary, fourth ed. Academic Press, pp. 163e201. Schneyer, A.L., et al., 2008. Differential antagonism of activin, myostatin and growth and differentiation factor 11 by wild-type and mutant follistatin. Endocrinology 149, 4589e4595. https:// doi.org/10.1210/en.2008-0259. Schwanzel-Fukuda, M., Jorgenson, K.L., Bergen, H.T., Weesner, G.D., Pfaff, D.W., 1992. Biology of normal luteinizing hormonereleasing hormone neurons during and after their migration from olfactory placode. Endocr. Rev. 13, 623e634. https://doi.org/ 10.1210/edrv-13-4-623. Schweizer, U., Steegborn, C., 2015. New insights into the structure and mechanism of iodothyronine deiodinases. J. Mol. Endocrinol. 55, R37eR52. https://doi.org/10.1530/JME-15-0156. Scott, D.A., Wang, R., Kreman, T.M., Sheffield, V.C., Karniski, L.P., 1999. The Pendred syndrome gene encodes a chloride-iodide transport protein. Nat. Genet. 21, 440e443. https://doi.org/10.1038/7783. Seminara, S.B., Oliveira, L.M., Beranova, M., Hayes, F.J., Crowley Jr., W.F., 2000. Genetics of hypogonadotropic hypogonadism. J. Endocrinol. Investig. 23, 560e565. https:// doi.org/10.1007/BF03343776. Seminara, S.B., et al., 2003. The GPR54 gene as a regulator of puberty. N. Engl. J. Med. 349, 1614e1627. https://doi.org/10.1056/ NEJMoa035322. Shi, Y., Massague, J., 2003. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685e700. Shibusawa, N., et al., 2003. Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo. J. Clin. Investig. 112, 588e597. https://doi.org/10.1172/JCI18377. Shupnik, M.A., Greenspan, S.L., Ridgway, E.C., 1986. Transcriptional regulation of thyrotropin subunit genes by thyrotropin-releasing hormone and dopamine in pituitary cell culture. J. Biol. Chem. 261, 12675e12679. Sidis, Y., et al., 2006. Biological activity of follistatin isoforms and follistatin-like-3 is dependent on differential cell surface binding

and specificity for activin, myostatin, and bone morphogenetic proteins. Endocrinology 147, 3586e3597. https://doi.org/ 10.1210/en.2006-0089. Singh, S.P., et al., 2009. Impaired estrogen feedback and infertility in female mice with pituitary-specific deletion of estrogen receptor alpha (ESR1). Biol. Reprod. 81, 488e496. https://doi.org/ 10.1095/biolreprod.108.075259. Smith, J.T., 2013. Sex steroid regulation of kisspeptin circuits. Adv. Exp. Med. Biol. 784, 275e295. https://doi.org/10.1007/978-1-4614-61999_13. Smith, T.J., Hegedus, L., 2016. Graves’ disease. N. Engl. J. Med. 375, 1552e1565. https://doi.org/10.1056/NEJMra1510030. Smith, J.J., Keinath, M.C., 2015. The sea lamprey meiotic map improves resolution of ancient vertebrate genome duplications. Genome Res. 25, 1081e1090. https://doi.org/10.1101/gr.184135.114. Smith, L.B., Walker, W.H., 2014. The regulation of spermatogenesis by androgens. Semin. Cell Dev. Biol. 30, 2e13. https://doi.org/ 10.1016/j.semcdb.2014.02.012. Smith, J.T., Cunningham, M.J., Rissman, E.F., Clifton, D.K., Steiner, R.A., 2005. Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 146, 3686e3692. https:// doi.org/10.1210/en.2005-0488. Smith, J.T., et al., 2005. Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology 146, 2976e2984. https://doi.org/10.1210/en.2005-0323. Smits, G., et al., 2003. Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity. EMBO J. 22, 2692e2703. https://doi.org/10.1093/emboj/cdg260. Song, Y., et al., 2016. Follicle-stimulating hormone induces postmenopausal dyslipidemia through inhibiting hepatic cholesterol metabolism. J. Clin. Endocrinol. Metab. 101, 254e263. https:// doi.org/10.1210/jc.2015-2724. Soules, M.R., et al., 1984. Progesterone modulation of pulsatile luteinizing hormone secretion in normal women. J. Clin. Endocrinol. Metab. 58, 378e383. https://doi.org/10.1210/jcem-58-2-378. Sower, S.A., 2015. Breaking dogma on the hypothalamic-pituitary anatomical relations in vertebrates. Endocrinology 156, 3882e3884. https://doi.org/10.1210/en.2015-1778. Sower, S.A., 2018. Landmark discoveries in elucidating the origins of the hypothalamic-pituitary system from the perspective of a basal vertebrate, sea lamprey. Gen. Comp. Endocrinol. 264, 3e15. https://doi.org/10.1016/j.ygcen.2017.10.016. Sower, S.A., Hausken, K.N., 2017. A lamprey view on the origins of neuroendocrine regulation of the thyroid axis. Mol. Cell. Endocrinol. 459, 21e27. https://doi.org/10.1016/j.mce.2017.04.012. Steinfelder, H.J., et al., 1991. Thyrotropin-releasing hormone regulation of human TSHB expression: role of a pituitary-specific transcription factor (Pit-1/GHF-1) and potential interaction with a thyroid hormone-inhibitory element. Proc. Natl. Acad. Sci. U.S.A. 88, 3130e3134. https://doi.org/10.1073/pnas.88.8.3130. Steinfelder, H.J., Radovick, S., Wondisford, F.E., 1992. Hormonal regulation of the thyrotropin beta-subunit gene by phosphorylation of the pituitary-specific transcription factor Pit-1. Proc. Natl. Acad. Sci. U.S.A. 89, 5942e5945. https://doi.org/10.1073/ pnas.89.13.5942. Stelmaszewska, J., et al., 2016. Revisiting the expression and function of follicle-stimulation hormone receptor in human umbilical vein endothelial cells. Sci. Rep. 6, 37095. https://doi.org/10.1038/ srep37095. Stephens, S.B., et al., 2015. Absent progesterone signaling in kisspeptin neurons disrupts the LH surge and impairs fertility in female mice. Endocrinology 156, 3091e3097. https://doi.org/10.1210/en.20151300. Stilley, J.A.W., Segaloff, D.L., 2018. FSH actions and pregnancy: looking beyond ovarian FSH receptors. Endocrinology 159, 4033e4042. https://doi.org/10.1210/en.2018-00497.

REFERENCES

Stilley, J.A., Guan, R., Duffy, D.M., Segaloff, D.L., 2014. Signaling through FSH receptors on human umbilical vein endothelial cells promotes angiogenesis. J. Clin. Endocrinol. Metab. 99, E813eE820. https://doi.org/10.1210/jc.2013-3186. Strahl, B.D., Huang, H.J., Sebastian, J., Ghosh, B.R., Miller, W.L., 1998. Transcriptional activation of the ovine follicle-stimulating hormone beta-subunit gene by gonadotropin-releasing hormone: involvement of two activating protein-1-binding sites and protein kinase C. Endocrinology 139, 4455e4465. https://doi.org/10.1210/ endo.139.11.6281. Stutzin, A., Stojilkovic, S.S., Catt, K.J., Rojas, E., 1989. Characteristics of two types of calcium channels in rat pituitary gonadotrophs. Am. J. Physiol. 257, C865eC874. https://doi.org/10.1152/ ajpcell.1989.257.5.C865. Sudo, S., Kuwabara, Y., Park, J.I., Hsu, S.Y., Hsueh, A.J., 2005. Heterodimeric fly glycoprotein hormone-alpha2 (GPA2) and glycoprotein hormone-beta5 (GPB5) activate fly leucine-rich repeat-containing G protein-coupled receptor-1 (DLGR1) and stimulation of human thyrotropin receptors by chimeric fly GPA2 and human GPB5. Endocrinology 146, 3596e3604. https://doi.org/10.1210/en.2005-0317. Sugino, K., et al., 1993. Molecular heterogeneity of follistatin, an activin-binding protein. Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell. J. Biol. Chem. 268, 15579e15587. Sun, L., et al., 2006. FSH directly regulates bone mass. Cell 125, 247e260. https://doi.org/10.1016/j.cell.2006.01.051. Sun, Y., et al., 2012. Loss-of-function mutations in IGSF1 cause an Xlinked syndrome of central hypothyroidism and testicular enlargement. Nat. Genet. 44, 1375e1381. https://doi.org/ 10.1038/ng.2453. Suszko, M.I., Balkin, D.M., Chen, Y., Woodruff, T.K., 2005. SMAD3 mediates activin-induced transcription of follicle-stimulating hormone beta-subunit gene. Mol. Endocrinol. 19, 1849e1858. https:// doi.org/10.1210/me.2004-0475. Talmadge, K., Boorstein, W.R., Vamvakopoulos, N.C., Gething, M.J., Fiddes, J.C., 1984. Only three of the seven human chorionic gonadotropin beta subunit genes can be expressed in the placenta. Nucleic Acids Res. 12, 8415e8436. https://doi.org/10.1093/nar/ 12.22.8415. Talmadge, K., Vamvakopoulos, N.C., Fiddes, J.C., 1984. Evolution of the genes for the beta subunits of human chorionic gonadotropin and luteinizing hormone. Nature 307, 37e40. Tanaka, K., Chazenbalk, G.D., McLachlan, S.M., Rapoport, B., 1999. Thyrotropin receptor cleavage at site 1 involves two discontinuous segments at each end of the unique 50-amino acid insertion. J. Biol. Chem. 274, 2093e2096. https://doi.org/10.1074/jbc.274.4.2093. Tanaka, K., Chazenbalk, G.D., McLachlan, S.M., Rapoport, B., 1999. Subunit structure of thyrotropin receptors expressed on the cell surface. J. Biol. Chem. 274, 33979e33984. https://doi.org/ 10.1074/jbc.274.48.33979. Tapanainen, J.S., Aittomaki, K., Min, J., Vaskivuo, T., Huhtaniemi, I.T., 1997. Men homozygous for an inactivating mutation of the folliclestimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nat. Genet. 15, 205e206. https://doi.org/10.1038/ng0297-205. Themmen, A.P., 2005. An update of the pathophysiology of human gonadotrophin subunit and receptor gene mutations and polymorphisms. Reproduction 130, 263e274. https://doi.org/ 10.1530/rep.1.00663. Thomas, R.M., et al., 2007. Follice-stimulating hormone receptor forms oligomers and shows evidence of carboxyl-terminal proteolytic processing. Endocrinology 148, 1987e1995. https://doi.org/ 10.1210/en.2006-1672. Thompson, I.R., Kaiser, U.B., 2014. GnRH pulse frequency-dependent differential regulation of LH and FSH gene expression. Mol. Cell.

143

Endocrinol. 385, 28e35. https://doi.org/10.1016/ j.mce.2013.09.012. Thompson, T.B., Lerch, T.F., Cook, R.W., Woodruff, T.K., Jardetzky, T.S., 2005. The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding. Dev. Cell 9, 535e543. https://doi.org/10.1016/j.devcel.2005.09.008. Thompson, I.R., et al., 2013. GnRH pulse frequency-dependent stimulation of FSHbeta transcription is mediated via activation of PKA and CREB. Mol. Endocrinol. 27, 606e618. https://doi.org/ 10.1210/me.2012-1281. Thompson, I.R., et al., 2016. GnRH pulse frequency control of Fshb gene expression is mediated via ERK1/2 regulation of ICER. Mol. Endocrinol. 30, 348e360. https://doi.org/10.1210/ me.2015-1222. Tosta, Z., Chabaud, O., Chebath, J., 1983. Identification of thyroglobulin mRNA sequences in the nucleus and the cytoplasm of cultured thyroid cells: a post-transcriptional effect of thyrotropin. Biochem. Biophys. Res. Commun. 116, 54e61. Toth, P., 2001. Clinical data supporting the importance of vascular LH/ hCG receptors of uterine blood vessels. Semin. Reprod. Med. 19, 55e61. Tran, S., Lamba, P., Wang, Y., Bernard, D.J., 2011. SMADs and FOXL2 synergistically regulate murine FSHbeta transcription via a conserved proximal promoter element. Mol. Endocrinol. 25, 1170e1183. https://doi.org/10.1210/me.2010-0480. Tran, S., et al., 2013. Impaired fertility and FSH synthesis in gonadotrope-specific FOXL2 knockout mice. Mol. Endocrinol. 27, 407e421. https://doi.org/10.1210/me.2012-1286. Troppmann, B., Kleinau, G., Krause, G., Gromoll, J., 2013. Structural and functional plasticity of the luteinizing hormone/choriogonadotrophin receptor. Hum. Reprod. Update 19, 583e602. https:// doi.org/10.1093/humupd/dmt023. Tsuchida, K., et al., 2004. Activin isoforms signal through type I receptor serine/threonine kinase ALK7. Mol. Cell. Endocrinol. 220, 59e65. https://doi.org/10.1016/j.mce.2004.03.009. Tsujino, K., et al., 2013. Establishment of TSH beta real-time monitoring system in mammalian photoperiodism. Genes Cells 18, 575e588. https://doi.org/10.1111/gtc.12063. Turgeon, M.O., et al., 2017. TRH action is impaired in pituitaries of male IGSF1-deficient mice. Endocrinology 158, 815e830. https:// doi.org/10.1210/en.2016-1788. Ueno, N., et al., 1987. Isolation and partial characterization of follistatin: a single-chain Mr 35,000 monomeric protein that inhibits the release of follicle-stimulating hormone. Proc. Natl. Acad. Sci. U.S.A. 84, 8282e8286. https://doi.org/10.1073/pnas.84.23.8282. Ulloa-Aguirre, A., et al., 2007. Role of the intracellular domains of the human FSH receptor in G(alphaS) protein coupling and receptor expression. Mol. Cell. Endocrinol. 260e262, 153e162. https:// doi.org/10.1016/j.mce.2005.11.050. Ulloa-Aguirre, A., Zarinan, T., Jardon-Valadez, E., Gutierrez-Sagal, R., Dias, J.A., 2018. Structure-function relationships of the folliclestimulating hormone receptor. Front. Endocrinol. 9, 707. https:// doi.org/10.3389/fendo.2018.00707. Vale, W., et al., 1986. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature 321, 776e779. https://doi.org/10.1038/321776a0. van den Hove, M.F., et al., 2007. Thyrotropin activates guanosine 5’diphosphate/guanosine 5’-triphosphate exchange on the ratelimiting endocytic catalyst, Rab5a, in human thyrocytes in vivo and in vitro. J. Clin. Endocrinol. Metab. 92, 2803e2810. https:// doi.org/10.1210/jc.2006-2351. Vassalli, A., Matzuk, M.M., Gardner, H.A., Lee, K.F., Jaenisch, R., 1994. Activin/inhibin beta B subunit gene disruption leads to defects in eyelid development and female reproduction. Genes Dev. 8, 414e427. https://doi.org/10.1101/gad.8.4.414.

144

7. ANTERIOR PITUITARY: GLYCOPROTEIN HORMONES FROM GONADOTROPE (FSH AND LH) AND THYROTROPE (TSH) CELLS

Vassart, G., Costagliola, S., 2004. A physiological role for the posttranslational cleavage of the thyrotropin receptor? Endocrinology 145, 1e3. https://doi.org/10.1210/en.2003-1225. Virion, A., Pommier, J., Deme, D., Nunez, J., 1981. Kinetics of thyroglobulin iodination and thyroid hormone synthesis catalyzed by peroxidases: the role of H2O2. Eur. J. Biochem. 117, 103e109. Wang, Y., et al., 2008. Activator protein-1 and smad proteins synergistically regulate human follicle-stimulating hormone beta-promoter activity. Endocrinology 149, 5577e5591. https://doi.org/10.1210/ en.2008-0220. Wang, H., Butnev, V., Bousfield, G.R., Kumar, T.R., 2016. A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7Delta) Asn(24Delta)) FSHbeta subunit fails to rescue Fshb null mice. Mol. Cell. Endocrinol. 426, 113e124. https://doi.org/ 10.1016/j.mce.2016.02.015. Wang, H., et al., 2016. Evaluation of in vivo bioactivities of recombinant hypo- (FSH(21/18)) and fully- (FSH(24)) glycosylated human FSH glycoforms in Fshb null mice. Mol. Cell. Endocrinol. 437, 224e236. https://doi.org/10.1016/j.mce.2016.08.031. Wang, H., et al., 2016. Comparative assessment of glycosylation of a recombinant human FSH and a highly purified FSH extracted from human urine. J. Proteome Res. 15, 923e932. https://doi.org/ 10.1021/acs.jproteome.5b00921. Wang, L., et al., 2019. Genetic dissection of the different roles of hypothalamic kisspeptin neurons in regulating female reproduction. Elife 8. https://doi.org/10.7554/eLife.43999. Weiss, J., et al., 1992. Hypogonadism caused by a single amino acid substitution in the beta subunit of luteinizing hormone. N. Engl. J. Med. 326, 179e183. https://doi.org/10.1056/ NEJM199201163260306. Wersinger, S.R., Haisenleder, D.J., Lubahn, D.B., Rissman, E.F., 1999. Steroid feedback on gonadotropin release and pituitary gonadotropin subunit mRNA in mice lacking a functional estrogen receptor alpha. Endocrine 11, 137e143. https://doi.org/10.1385/ENDO: 11:2:137. Wide, L., Eriksson, K., 2017. Molecular size and charge as dimensions to identify and characterize circulating glycoforms of human FSH, LH and TSH. Ups. J. Med. Sci. 122, 217e223. https://doi.org/ 10.1080/03009734.2017.1412373. Wide, L., Eriksson, K., 2018. Low-glycosylated forms of both FSH and LH play major roles in the natural ovarian stimulation. Ups. J. Med. Sci. 123, 100e108. https://doi.org/10.1080/03009734.2018.1467983. Wildt, L., et al., 1981. Frequency and amplitude of gonadotropinreleasing hormone stimulation and gonadotropin secretion in the rhesus monkey. Endocrinology 109, 376e385. https://doi.org/ 10.1210/endo-109-2-376. Williams, G.R., 2011. Extrathyroidal expression of TSH receptor. Ann. Endocrinol. 72, 68e73. https://doi.org/10.1016/j.ando.2011.03.006.

Woodruff, T.K., Mayo, K.E., 1990. Regulation of inhibin synthesis in the rat ovary. Annu. Rev. Physiol. 52, 807e821. https://doi.org/ 10.1146/annurev.ph.52.030190.004111. Woodruff, T.K., et al., 1996. Inhibin A and inhibin B are inversely correlated to follicle-stimulating hormone, yet are discordant during the follicular phase of the rat estrous cycle, and inhibin A is expressed in a sexually dimorphic manner. Endocrinology 137, 5463e5467. https://doi.org/10.1210/endo.137.12.8940372. Wu, H., Lustbader, J.W., Liu, Y., Canfield, R.E., Hendrickson, W.A., 1994. Structure of human chorionic gonadotropin at 2.6 A resolution from MAD analysis of the selenomethionyl protein. Structure 2, 545e558. Wu, S., et al., 2014. Conditional knockout of the androgen receptor in gonadotropes reveals crucial roles for androgen in gonadotropin synthesis and surge in female mice. Mol. Endocrinol. 28, 1670e1681. https://doi.org/10.1210/me.2014-1154. Xie, C., Jonak, C.R., Kauffman, A.S., Coss, D., 2015. Gonadotropin and kisspeptin gene expression, but not GnRH, are impaired in cFOS deficient mice. Mol. Cell. Endocrinol. 411, 223e231. https:// doi.org/10.1016/j.mce.2015.04.033. Xing, Y., et al., 2004. Glycoprotein hormone assembly in the endoplasmic reticulum: II. Multiple roles of a redox sensitive betasubunit disulfide switch. J. Biol. Chem. 279, 35437e35448. https://doi.org/10.1074/jbc.M403053200. Xu, J., McKeehan, K., Matsuzaki, K., McKeehan, W.L., 1995. Inhibin antagonizes inhibition of liver cell growth by activin by a dominantnegative mechanism. J. Biol. Chem. 270, 6308e6313. https:// doi.org/10.1074/jbc.270.11.6308. Yamada, M., et al., 1997. Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene. Proc. Natl. Acad. Sci. U.S.A. 94, 10862e10867. https://doi.org/10.1073/pnas.94.20.10862. Yamoto, M., Imai, M., Otani, H., Nakano, R., 1997. Serum levels of inhibin A and inhibin B in women with normal and abnormal luteal function. Obstet. Gynecol. 89, 773e776. Ying, S.Y., 1988. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr. Rev. 9, 267e293. https://doi.org/10.1210/edrv-9-2-267. Yoshida, A., et al., 2002. Pendrin is an iodide-specific apical porter responsible for iodide efflux from thyroid cells. J. Clin. Endocrinol. Metab. 87, 3356e3361. https://doi.org/10.1210/jcem.87.7.8679. Zaidi, M., et al., 2018. FSH, bone mass, body fat, and biological aging. Endocrinology 159, 3503e3514. https://doi.org/10.1210/en.201800601. Zoenen, M., Urizar, E., Swillens, S., Vassart, G., Costagliola, S., 2012. Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers. Nat. Commun. 3, 1007. https://doi.org/10.1038/ncomms1991.

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8 Anterior Pituitary and Pars Intermedia Space: Corticotrophs (ACTH) and Melanotrophs (a-MSH) Nicola Romano`, Michael J. Shipston Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, UK

1. INTRODUCTION Proopiomelanocortin (POMC) is the 241-amino-acidlong peptide whose posttranslational processing gives rise to several bioactive peptide hormones controlling a large array of different physiologic functions ranging from stress, feeding, and energy balance to pigmentation and analgesia. Two separate cell lineages in the pituitary gland express POMC: the corticotrophs of the anterior pituitary, central to the hormonal stress response, and the melanotrophs of the pars intermedia (PI), important for determination of skin and fur pigmentation. Differential posttranslational processing of POMC in these two populations results in production of adrenocorticotropic hormone (ACTH) in corticotrophs and a-melanocyte-stimulating hormone (a-MSH) in melanotrophs. This chapter will focus on the physiology of ACTH and a-MSH, although references to other POMC-derived hormones will be made where necessary.

2. POMC-DERIVED PEPTIDES AND THEIR RECEPTORS: STRUCTURE AND PROCESSING 2.1 POMCdSynthesis and Processing The synthesis and processing of POMC and its related peptides have been extensively studied since the 1940s, when a peptide with the ability of “repairing” the adrenal cortex of hypophysectomized rats, later named adrenocorticotropic hormone (ACTH), was initially isolated from the sheep pituitary gland (Li et al., 1955, 1954, Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00008-0

1942). It was not until several decades later with the cloning of the POMC gene that the existence of a precursor gene for ACTH and beta lipotropin (b-LPH) was confirmed (Nakanishi et al., 1979). Expression of POMC is highest in the pituitary gland and the hypothalamus; however, lower levels of POMC and its products have been detected in other tissues such as the male reproductive tract, which, in many species including humans, produces b-endorphin (Shu-Dong et al., 1982; Sharp and Pekary, 1981), and the skin, where keratinocytes and melanocytes produce and secrete POMC as well as ACTH, a-MSH, and b-endorphin (Rousseau et al., 2007; Wakamatsu et al., 1997). The production of POMC and its products is regulated by a complex interplay of transcriptional and posttranscriptional control.

2.2 Transcriptional Control of the POMC Gene The transcriptional control of the POMC gene allows for its cell-specific expression and has been the subject of a large amount of studies. Three main genomic regions are involved in the control of pituitary POMC expression; the promoter region, a 300 bp enhancer, and a 7 kb enhancer. Hypothalamic POMC neurons use two further enhancers at 10 kb and 12 kb (Drouin, 2016; de Souza et al., 2005). Along with binding sites for some widely expressed transcription factors (TFs), these regulatory elements also contain additional binding sites for TFs expressed in a cell-specific manner, which mediate the corresponding POMC cell type-specific expression pattern in corticotrophs and melanotrophs. Tpit (T-box TF) and Pitx1 (bicoid-type homeodomain TF pituitary homeobox 1) are important for both corticotrophs and

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melanotrophs; NeuroD1 (neurogenic differentiation 1), a member of the basic helix-loop-helix (bHLH) family, is key for corticotroph POMC expression; finally, Pax7 (Paired Box 7) is central for defining the specific transcriptional profile of melanotrophs (Drouin, 2016). Tpit and Pitx1 bind to a composite Tpit/Pitx1 responsive element (Poulin et al., 1997; Lamonerie et al., 1996), while NeuroD1 binds to an E-box sequence, named EboxNeuro, upstream of the Tpit/Pitx1 binding site (Philips et al., 1997). Pitx1 gives broad pituitary specificity, due to its role in transcription of many anterior pituitary hormones (Tremblay et al., 1998). TPIT expression, on the other hand, is restricted to corticotrophs and melanotrophs (Lamolet et al., 2001), limiting POMC transcription to these cell populations. Finally, NeuroD1 expression is high in corticotrophs during development, being required for their early differentiation, but it strongly declines in the adult (Lamolet et al., 2004); it is never expressed by melanotrophs. The binding of these TFs to the enhancer allows synergistic regulation of POMC transcription (Therrien and Drouin, 1991); for instance, NeuroD1 can synergistically interact with Pitx1, as shown by an approximately 10-fold increase in POMC transcription when both of these factors are present compared to either of them alone (Poulin et al., 2000). Furthermore, studies on the binding site for NeuroD1 have revealed that it acts as a heterodimer with another TF of the bHLH family, Pan1, through a NeuroD1-specific E-box, called E-boxNeuro. Upstream of

this element is E-boxUbi, which is activated by binding of ubiquitously present bHLH TFs. The two E-box elements are equally effective in activating POMC transcription; indeed, E-boxNeuro can be substituted by an E-boxUbi (that does not bind NeuroD1) without affecting transcription levels (Poulin et al., 2000). However, mutations or removal of E-boxNeuro severely affect the levels of POMC transcription both during development and in the adult (Lavoie et al., 2008; Poulin et al., 2000). Since NeuroD1 levels are low in adult corticotrophs, this has suggested that another bHLH TF, such as Ascl1, which is expressed in adult corticotrophs and melanotrophs, may bind to E-boxNeuro in adult life (Drouin, 2016). Melanotroph identity is instead defined by the Pax7 TF; this acts as a pioneer factor able to remodel chromatin to allow access to promoters for genes that are not normally accessible in corticotrophs (Budry et al., 2012). Indeed, exogenous expression of Pax7 in the AtT-20 corticotroph cell line is sufficient to change their epigenetic asset and confer them a melanotroph-like identity (Mayran et al., 2018).

2.3 Posttranslational Control of POMC Production A second layer of control in the production of POMCderived peptides happens at the level of posttranslational modifications (Fig. 8.1). POMC is processed through the regulated secretory pathway (RSP),

FIGURE 8.1 POMC posttranscriptional processing. Schematic of the posttranscriptional processing of POMC. The precursor peptide POMC is processed through a series of proteolytic cuts by the proteases PC1 and PC2. Corticotrophs express only PC1 and can therefore process POMC into ACTH and b-lipotropin, while in melanotrophs PC2 further processes these products to form a- and b-MSH and b-endorphin. Production of a-MSH also requires further cleavage by carboxypeptidase E (CPE), and amidation and acetylation through the subsequent actions of peptidylglycine a-amidating monooxygenase (PAM) and N-acetyltransferase (NAT). Finally the lysosomal enzyme prolylcarboxypeptidase (PRCP) is involved in degradation of a-MSH. CLIP, Corticotropin-like intermediate lobe peptide; EP, endorphin; JP, junction peptide; LPH, lipotropin; Sig, signal peptide.

3. PITUITARY CELLS EXPRESSING POMC: CORTICOTROPHS AND MELANOTROPHS

whereby the precursor peptide is transported through the rough endoplasmic reticulum (RER) to the Golgi apparatus, where it is cleaved and modified, for its products to be targeted in the secretory vesicles of the RSP. In the Golgi apparatus, a complex series of posttranslational cleavage and modification reactions of the POMC precursor results in the production of several peptide hormones including the melanocortins ACTH, a-, b-, and g-melanocyte-stimulating hormone (a-, b-, and g-MSH), and the opioids b-endorphin and b- and g-lipotropin (b- and g-LPH) (Roberts and Herbert, 1977; Mains et al., 1977) (Fig. 8.1). Cell type-specific expression of the two main endoproteases responsible for the processing of POMC, the proprotein convertases 1 and 2 (PC1 also called PC3, and PC2) (Benjannet et al., 1991), allows control of the production of specific hormones. PC1 and PC2 are endoproteases that recognize the [R/K]-Xn-[R/K]Y motif (where Y indicates the cutting site) (Seidah and Chre´tien, 1999). Studies in rodents and humans (Takumi et al., 1998; Seidah et al., 1991) have shown localization of PC1 in the anterior pituitary corticotrophs; this results in cleavage of POMC to produce ACTH and b-LPH. In the melanotrophs of the PI, on the other hand, expression of both PC1 and PC2 allows almost complete cleavage of b-LPH to b-endorphin and g-LPH, and it allows selective production of a-MSH from cleavage of ACTH specifically in these cells (Day et al., 1992; Seidah et al., 1991). Processing of POMC and sorting through the RSP happens in a sequential fashion. Translocation to the RER is allowed by the presence of two disulphide bridges at the N-terminal of POMC, forming a hairpin loop structure (Cool et al., 1995; Cool and Peng, 1994; Tam et al., 1993). This structure allows binding to the sorting receptor, which has been identified as carboxypeptidase E (CPE, also known as carboxypeptidase H or enkephalin convertase), a widely expressed exopeptidase present in many endocrine tissues and involved in the biosynthesis of several hormones (Fricker, 1988; Hook et al., 1982). Specifically, it is the membrane-bound form of CPE that functions as the sorting receptor for POMC in the trans-Golgi network (Cool et al., 1997). Electron microscopy studies have shown that processing of POMC is limited to the outermost cisternae of the Golgi in corticotrophs (Schnabel et al., 1989). Here, acidic pH allows the autocatalytic activation of PC1 (Seidah et al., 2008), which first cleaves POMC into b-LPH and pro-ACTH, then further cleaves the latter to produce ACTH, a junction peptide, and NPOMC. In parallel to the activity of PC1 and PC2, there is evidence for the role of the protease cathepsin L, which is located in secretory vesicles, in the maturation of POMC to ACTH, a-MSH, and b-endorphin, as evidenced by the fact that cathepsin L knockout mice show major reduction in the levels of these hormones (Hook et al., 2009; Funkelstein et al., 2008).

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In cells expressing PC2, such as the melanotrophs of the intermediate zone, the skin melanocytes, or the hypothalamic POMC neurons, further processing of ACTH to a-MSH and of b-LPH to g-LPH and b-endorphin happens at the level of secretory vesicles (Fig. 8.1). After cleavage of the full-length ACTH(1-39) molecule by PC2 to give ACTH(1-14), the C-terminal of the latter is cleaved by a soluble form of CPE (Ji et al., 2017). Finally, through the subsequent actions of peptidylglycine a-amidating monooxygenase (PAM) and an N-acetyltransferase (NAT), the active acetylated and amidated form of a-MSH is synthesized. Finally, these posttranslational events can also be modulated by changes in the level of the enzymes involved in POMC processing: for example, in rat melanotrophs, PC1, PC2, and PAM transcription and translation are under dopaminergic control, being increased by haloperidol treatment and decreased by bromocriptine (Oyarce et al., 1996; Day et al., 1992; Birch et al., 1991). Similarly, expression of PC1 in corticotrophs has been shown to depend on activation of the cAMP/PKA pathway, for instance through the inducible cAMP early repressor (ICER), an isoform of the cAMP response element modulator (Lamas et al., 1997); this is also in line with evidence showing that PC1 is regulated through CRE elements (Jansen et al., 1995).

3. PITUITARY CELLS EXPRESSING POMC: CORTICOTROPHS AND MELANOTROPHS 3.1 Corticotrophs and ACTH 3.1.1 ACTH Release from Corticotrophs: A Key Mediator of the Neuroendocrine Response to Stress The release of ACTH from corticotrophs of the anterior pituitary gland is a key component of the hypothalamicepituitaryeadrenal (HPA) axis that coordinates the neuroendocrine response to stress (Fig. 8.2). Activation of the HPA axis results in the release of ACTH from corticotrophs into the circulation. ACTH in turn stimulates the synthesis and release of glucocorticoid hormones from the adrenal gland (primarily cortisol in man, corticosterone in rodents) that have powerful and pleiotropic roles in a range of systems. Glucocorticoids, in turn, feed back at the pituitary corticotroph, as well as other levels of the HPA axis, to limit output of the axis as part of an adaptive response to restore homeostasis. Corticotrophs and ACTH release thus provide an important communication link between the brain neural circuitry and the peripheral release of glucocorticoids from the endocrine adrenal gland. Importantly, release of ACTH is pulsatile across multiple time domains with both ultradian and circadian

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FIGURE 8.2 Control of ACTH and a-MSH production. Schematic of the control of corticotroph and melanotroph activity. PC1 in anterior pituitary corticotrophs processes POMC to ACTH. This is released in response to release of corticotropin-releasing hormone (CRH) and/or arginine vasopressin (AVP) from hypothalamic neuroendocrine neurons in the portal vasculature as part of the hormonal response to stress. ACTH stimulates the synthesis and release of glucocorticoid hormones from fasciculata cells of the adrenal cortex to control a variety of physiologic functions. Glucocorticoid negative feedback controls the HPA axis at multiple levels and over different time scales. Melanotrophs of the pars intermedia are directly innervated from hypothalamic fibers. They are under tonic inhibitory control from dopaminergic and GABAergic inputs, as well as receiving other neuropeptidergic inputs that may control their function (see text for details). In these cells the combined actions of PC1 and PC2 allow the production of a-MSH, important for the control of skin pigmentation.

rhythmsdthis pulsatility is critically important for the physiologic response of target organs to ACTH and glucocorticoids and is modified in aging and disease (Spiga et al., 2014; Lightman and Conway-Campbell, 2010). The release of ACTH from corticotrophs is thus tightly controlled to allow ultradian/circadian rhythmicity while being able to respond appropriately to stressors throughout the day. Acute stress is beneficial by priming the organism for the demands of its immediate environment, for example, by increasing alertness and behavioral and cognitive performance. Indeed, glucocorticoids mediate powerful and pleiotropic physiologic and metabolic responses including energy mobilization, cardiovascular and antiinflammatory responses, as well as suppression of digestive and reproductive function to prepare the organism for a stressful situation (Sapolsky et al., 2000). However, our inability to adapt to persistent stress is now well recognized as increasing the likelihood of developing a range of debilitating disorders including cardiovascular disease, metabolic disruption such as obesity and diabetes, as well as neurological disorders such as depression, anxiety, and cognitive dysfunction. In general, we are not well equipped to cope with

chronic activation of stress pathways, and as recognized by Hans Selye, “it is not stress that kills us, it is our reaction to it.” While “activation” of the HPA axis is commonly associated with responses to aversive stimuli, it is important to remember that output is under both ultradian and circadian control (Spiga et al., 2014; Lightman and Conway-Campbell, 2010), as well stimulated by “pleasurable” behaviors including appetite reward and sexual encounter. 3.1.2 Corticotroph Development and Anatomy in the Anterior Pituitary Gland In the adult anterior pituitary gland, corticotrophs represent approximately 5%e10% of the endocrine secretory cell population. Until relatively recently, corticotrophs been considered largely “randomly” distributed throughout the anterior pituitary as single cells/ clusters intermingled with other pituitary cells. However, using whole tissue imaging and genetic labeling of corticotrophs reveals that corticotrophs, as for other pituitary cells, form homotypic networks that extend throughout the gland (Le Tissier et al., 2012; Budry et al., 2011). In mice, corticotrophs form columns and

3. PITUITARY CELLS EXPRESSING POMC: CORTICOTROPHS AND MELANOTROPHS

sheets of cells from the ventral surface toward the center of the anterior pituitary gland, and many cells have long cytoplasmic projections (cytonemes) that make contact with corticotrophs separated by other cell lineages (Le Tissier et al., 2012; Budry et al., 2011). Moreover, corticotrophs are generally further away from capillary blood vessels, so whether ACTH release into the blood stream is from cytonemes located adjacent to the vessels and/or is first released into the interstitial space is unclear (Le Tissier et al., 2017). However, whether this anatomic network in fact results in functional corticotroph networks, as has been demonstrated for somatotrophs and lactrotrophs (Hodson et al., 2012; Lafont et al., 2010; Bonnefont et al., 2005), is not known. Functional corticotroph networks and the relationship of corticotrophs to the vascular bed are both likely to play an important role in the dynamics (including amplitude, synchronization, and robustness) of pulsatile ACTH release into the peripheral vasculature, and perhaps for a form of corticotroph “memory” following a stressor, as for other pituitary hormones (Hodson et al., 2012). Corticotrophs are the first cells of the anterior pituitary to form anatomic networks and the first cells to reach terminal differentiation that is determined by a complex network of TFs (Drouin, 2016). Corticotroph terminal differentiation is dependent upon the expression of Tpit, which binds to the POMC promoter in corticotrophs, resulting in corticotrophs being detected by about e12.5a in mice (Lamolet et al., 2001). Tpit is expressed 0.5 days before detectable expression of POMC and as well as being critical for corticotroph terminal differentiation genetic deletion of the TPIT gene in mice reveals a role for Tpit in both corticotroph expansion and maintenance (Pulichino et al., 2003). In humans, multiple TPIT mutations are associated with the inherited recessive condition of isolated ACTH deficiency that results in a lack of pituitary ACTH and secondary adrenal glucocorticoid deficiency (Couture et al., 2012; Vallette-Kasic et al., 2005) while sparing other pituitary hormones. Corticotroph specificity of POMC expression is also dependent upon the neurogenic basic helix-loop-helix factor NeuroD1 (Poulin et al., 1997) and a complex network of protein interactions. In particular, NeuroD1 binds to a bipartite regulatory element that includes a Nur response element (NurRE) important for corticotrophin-releasing hormone (CRH) stimulation of POMC transcription (Philips et al., 1997). Together the Tpit/PitX and NeuroD1/Nur response elements act synergistically to stimulate POMC transcription (Drouin, 2016). Importantly, corticotroph specification is not dependent upon CRH or AVP from the hypothalamus. For example, corticotroph development is largely normal in CRH-deficient mice (Muglia et al., 1995), and in mice lacking mature CRH-

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and AVP-producing hypothalamic neurons, maturation of anterior pituitary cells was normal (Schonemann et al., 1995). 3.1.3 CRH and Glucocorticoid Control of POMC Expression in Corticotrophs CRH, but not AVP, is however a powerful stimulator of POMC transcription and thus ACTH synthesis and storage in corticotrophs. A major target for CRHmediated stimulation of POMC transcription is through an orphan nuclear receptor related to nerve growth factor 1-B (Nurr77) that binds the NurRE and is activated by the MAPK pathway (Philips et al., 1997). The effect of CRH via Nurr77 is enhanced by a number of coactivators including members of the steroid receptor coactivator family (Maira et al., 2003). In turn, glucocorticoids antagonize CRH-stimulated POMC transcription that appears to be mediated primarily through a transrepression mechanism resulting from proteineprotein interactions of the glucocorticoid receptor (GR) complex with Nur factors rather than the GR binding directly to glucocorticoid response elements in the POMC promoter (Martens et al., 2005; Philips et al., 1997). This glucocorticoid-mediated transrepression of POMC transcription requires recruitment of histone deacetylase (HDAC2) and the ATPase component of the SW1/SNF chromatin remodeling complex, BRG1. Indeed, loss of nuclear expression of either HDAC2 or BRG1 may account for loss of glucocorticoid repression of POMC in a significant proportion of patients with Cushing syndrome (Drouin, 2016). 3.1.4 Regulation of ACTH Secretion by CRH, AVP, and Glucocorticoids The release of ACTH from corticotrophs is controlled by two major hypothalamic neuropeptides, CRH, also known as corticotrophin releasing factor (CRF), and arginine vasopressin (AVP). Perception of a stressor by the brain is ultimately relayed to neuroendocrine neurons in the hypothalamus that release CRH and/or AVP into the hypophysial portal circulation (Fig. 8.2). At the level of the corticotroph, CRH and AVP can act synergistically through the activation of distinct G proteinecoupled receptor (GPCR) signaling pathways (Fig. 8.3). 3.1.5 CRH and AVP Activate Distinct G ProteineCoupled Receptor Signaling Pathways in Corticotrophs CRH, originally isolated and characterized from ovine hypothalamus by Wyllie Vale and colleagues in 1981 (Vale et al., 1981), belongs to a family of four related neuropeptidesdCRH, urocortin 1 (UCN1), UCN2, and UCN3dthat are widely expressed in the brain (Dedic et al., 2018). CRH is most closely related to UCN1 (43% sequence identity), and its biologic active, mature form

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FIGURE 8.3 Stimulus secretion-coupling in corticotrophs and melanotrophs. In corticotrophs (left), CRH and AVP stimulate ACTH secretion through activation of distinct G proteinecoupled receptors (CRHR1 and AVPR1, respectively). Activation of CRHR1 activates adenylate cyclase (AC) with a resulting increase in the second messenger cyclic adenosine monophosphate (cAMP) that activates protein kinase A (PKA). Activation of AVP1b results in stimulation of phospholipase C (PLC), resulting in liberation of diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG activates protein kinase C (PKC), whereas IP3 releases calcium from intracellular calcium stores. CRH and AVP control electrical excitability of corticotrophs through a variety of plasma membrane ions channels including voltage-gated calcium channels and potassiumselective channels. The exocytosis of ACTH is ultimately controlled by an increase in intracellular free calcium. Similar mechanisms control a-MSH secretion from melanotrophs where, as in corticotrophs, cAMP and intracellular calcium are central for hormonal secretion. In these cells the two main hypothalamic controllers are dopamine (DA) and GABA. Through its action on the D2 receptors, DA inhibits production of cAMP by AC. GABA acts through both the through the ionotropic GABAA receptor, which provides rapid depolarization, and through the metabotropic GABAB receptor, which provides sustained inhibition by blocking cAMP production.

is a 41 amino acid peptide generated from a 196 amino acid precursor by proteolytic cleavage. CRH is the major physiologic activator of the HPA axis in man and rodents, and its bioavailability is regulated by CRHbinding protein (Seasholtz et al., 2001). In mice, constitutive genetic deletion of CRH blunts basal and stress-evoked stress responses (Muglia et al., 1995). CRH mRNA and peptide is high in parvocellular neurons of the hypothalamus that project to the median eminence and release CRH into the hypophysial portal circulation. CRH signals through activation of two G proteinecoupled receptors (CRHR1 and CRHR2), which share w70% amino acid identity (Dedic et al., 2018). CRH shows a much higher affinity for CRHR1 than CRHR2; however, at the level of anterior pituitary corticotrophs, CRHR1 is the major CRH GPCR on corticotrophs, where it couples to adenylate cyclase via Gs proteins to stimulate the synthesis of cAMP. Thus, the majority, if not all, effects of CRH at the level of pituitary corticotrophs are mediated through cAMP and subsequent activation of cAMP-dependent protein kinase, although the role of other cAMP-mediated pathways has not been systematically explored. Genetic deletion of CRHR1 in mice abolishes CRH-induced ACTH secretion and cAMP accumulation in isolated pituitary cells and significantly blunts stress-induced ACTH and corticosterone secretion in vivo (Timpl et al., 1998; Smith et al., 1998).

A role for AVP in driving ACTH secretion in response to a variety of stressors has long been recognized, including from studies using the Brattleboro rat, which lacks endogenous AVP (Antoni, 1993). AVP, also known as antidiuretic hormone, is a nonapeptide, structurally related to the related neuropeptide oxytocin (OT) that is synthesized from a larger precursor pre-proAVP. AVP is expressed in both magnocellular and parvocellular neuroendocrine neurons of the paraventricular nucleus that project to the median eminence (Antoni, 1993). AVP mediates its physiologic actions through three distinct GPCRs the V1a, V1b, and V2 receptors that share 40%e55% amino acid identity (Koshimizu et al., 2012). However, genetic and pharmacologic evidence reveals that the V1b receptor, which is highly expressed in pituitary corticotrophs (Roper et al., 2011; Lolait et al., 1995; Antoni, 1993; Jard et al., 1986), mediates the actions of AVP to stimulate ACTH release from corticotrophs. Genetic deletion of V1b receptors in mice (V1bR-KO) leads to loss of AVP-stimulated but not CRH-stimulated ACTH release in isolated pituitary cells (Roper et al., 2010; Tanoue et al., 2004). Moreover, in vivo, V1bR-KO mice or wild-type animals injected with V1b receptor antagonists such as ORG52186 or SSR149415 display blunted ACTH responses to AVP stimulation (Roper et al., 2010; Spiga et al., 2009; Tanoue et al., 2004). In corticotrophs, the V1b receptor is coupled to Gq/11 proteins and stimulates phospholipase C

3. PITUITARY CELLS EXPRESSING POMC: CORTICOTROPHS AND MELANOTROPHS

(PLC) activity, resulting in the activation of protein kinase C signaling (via diacylglycerol) and release of calcium from intracellular inositol trisphosphate (IP3)dsensitive stores (Roper et al., 2011; Lolait et al., 1995; Antoni, 1993; Jard et al., 1986). 3.1.6 Synergy of CRH- and AVP-Stimulated ACTH Secretion Although CRH is the major ACTH secretagogue in most species, AVP plays an important role through its synergistic action on ACTH secretion in combination with CRH observed in vitro and in vivo (Lamberts et al., 1984; Gillies et al., 1982). Mechanistically, synergy is also observed at the level of CRH-stimulated cAMP production and is dependent upon activation of PKC by AVP in cell population assays. In isolated rat corticotrophs, CRH has been proposed to stimulate two distinct isoforms of adenylate cyclase (AC): the Ca2þ-dependent AC9 and Ca2þ-independent, but PKC-activated, AC7 (Antoni et al., 2003). AC9 is the major adenylate cyclase responsible for CRH-stimulated cAMP accumulation at low physiologic levels of CRH. As AC9 is inhibited by Ca2þ, cAMP accumulation at low CRH is subject to robust Ca2þ-dependent negative feedback. In this model, AVP, via PKC-dependent activation of AC7, is proposed to act as a switch, so CRH-dependent cAMP accumulation is now predominantly through activation of AC7, rather than AC9. This promotes cAMP synthesis that is now largely resistant to Ca2þ-feedback inhibition and likely also involves differential regulation of phosphodiesterases that break down cAMP (Antoni et al., 2003). Whether synergy occurs downstream of cAMP signaling is not well understood. As discussed subsequently, differential regulation of ion channels may lead to synergistic actions of CRH and AVP at the level of electrical excitability. However, at the population level, rat corticotrophs do not display synergy at the level of intracellular Ca2þ accumulation evoked by physiologic levels of CRH and AVP (Romano` et al., 2017). However, distinct intercellular heterogeneity in synergy is observed with approximately 40% of corticotrophs displaying synergy in response to repeated exposure to CRH, AVP, and CRH/AVP (Romano` et al., 2017). Thus, synergy at the corticotroph population level of ACTH secretion may also arise due to heterogeneity in ACTH secretion from single cells in response to CRH and AVP. Indeed, using the hemolytic plaque assay that allows secretion detection in response to several hours of secretagogue exposure has revealed corticotrophs that respond differentially to CRH and AVP. For example, ACTH secretion from single cells was dose dependently increased by CRH, but not AVP, whereas AVP recruited more cells to secrete ACTH (Canny et al., 1992). Finally, synergy may occur at the level of Ca2þ-dependent exocytosis of ACTH vesicles. In

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corticotrophs the Ca2þ dependence of fast exocytosis has a third-power relationship (Tse and Lee, 2000). Thus, as CRH and AVP together promote a stronger depolarization (Zemkova et al., 2016; Lee et al., 2015; Duncan et al., 2015), and hence increase in voltagedependent Ca2þ entry, due to the steep dependence of voltage-gated calcium channels (VGCC) at physiologic membrane potentials, this may provide a synergistic effect at the level of ACTH vesicle exocytosis. 3.1.7 Electrical Excitability, Calcium Signaling, and the Control of ACTH Secretion Metabolically intact corticotrophs are electrically excitable cells that display spontaneous (20- to 100-ms duration) action potentials, although there is considerable heterogeneity between cells (Fletcher et al., 2017; Zemkova et al., 2016; Liang et al., 2011; Kuryshev et al., 1996). Modeling studies suggest this heterogeneity can arise from small differences in functional ion channel expression rather than from distinct “subtypes” of corticotrophs, as individual cells can display multiple behaviors (Fletcher et al., 2017). The ion channel targets downstream of CRH and AVP receptor activation are still poorly understood; however, recent evidence reveals both secretagogues control distinct as well as common ion channels to ultimately control elevation of intracellular free calcium. Physiologic (subnanomolar) concentrations of CRH in murine corticotrophs result in cell depolarization and an increase in electrical excitability characterized by both an increase in action potential frequency as well a transition to a “pseudo plateau bursting” mode (Fletcher et al., 2017; Zemkova et al., 2016; Duncan et al., 2015). Modeling and experimental studies suggest that the initial membrane depolarization is dependent on activation of a background sodium conductance and inhibition of a background potassium conductance. In mouse, the background Kþ current may be mediated by the two-pore TREK-1 potassium channel that is also reported to be inhibited by AVP, providing a mechanism for additive effects of CRH and AVP on initial depolarization (Lee et al., 2015). This depolarization is important for activation of VGCC, with L-type (dihydropyridine sensitive) VGCCs, playing a critical role in both calcium entry and CRH-evoked ACTH secretion (Ritchie et al., 1996; Kuryshev et al., 1996). The transition to bursting is promoted by large conductance calcium-and voltage-activated potassium channels that play a paradoxical role in facilitating sustained membrane depolarization and voltage-gated Ca2þ entry (Fletcher et al., 2017; Zemkova et al., 2016; Duncan et al., 2015). This increase in excitability is maintained for several minutes following removal of CRH. In contrast, physiologic levels of AVP largely evoke a smaller depolarization and an increase in action

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potential frequency rather than a significant transition to bursting. Importantly, the response to AVP is maintained >10 min following AVP withdrawal. At supraphysiological (>2 nM) levels of AVP, the release of intracellular free calcium is predominant and reported to result in transient hyperpolarization of the membrane potential in both rat and mouse corticotrophs (Lee et al., 2015; Tse and Lee, 1998). Indeed, the relative importance of voltage-gated Ca2þ entry compared to release from intracellular IP3-sensitive stores for AVP-stimulated ACTH release is likely to be concentration dependent. The more potent effect of CRH, compared to AVP, in stimulating secretion does not appear to be a result of either cAMP or the CRH-induced spatial calcium gradient during VGCC Ca2þ entry, being more effective in promoting exocytosis of the readily releasable pool of ACTH vesicles in rat corticotrophs (Tse and Lee, 2000, 1998). However, whether CRH is more effective at sustaining recruitment and slower exocytosis of vesicles compared to AVP during sustained stimulation is not known. 3.1.8 Glucocorticoid Feedback Inhibition of ACTH Release Glucocorticoids feed back at multiple levels of the stress axis to suppress HPA activity in both a timeand concentration-dependent manner (Keller-Wood and Dallman, 1984). At the level of the anterior pituitary, corticotroph glucocorticoids inhibit ACTH secretion in three, mechanistically distinct, time domains: (1) fast inhibition (seconds to minutes) that is rapidly reversible and nongenomic; (2) early inhibition (10 mine2 h); and (3) slow inhibition (h/days). Fast glucocorticoid inhibition at the level of the pituitary is observed in vivo upon injection of corticosterone immediately prior to stimulation of ACTH secretion with CRH (Hinz and Hirschelmann, 2000). Recent in vitro studies, using perifused pituitary cells at physiologic CRH concentrations, have suggested that fast inhibition is mediated downstream of CRH-induced calcium signaling involving a membrane-bound form of the classical GR through a nongenomic mechanism (Deng et al., 2015). Importantly, this rapid inhibition was seen at concentrations of corticosterone that would typically be observed during stress (>1 mM), or near the peak of an ultradian pulse, in corticotrophs that had been previously exposed to nanomolar concentrations of glucocorticoid prior to stimulation. In corticotrophs exposed to glucocorticoid-free conditions for 6 h prior to stimulation, 10e100 nM corticosterone could now also evoke rapid and reversible inhibition of CRHstimulated ACTH release. Slow glucocorticoid inhibition results in both suppression of basal and evoked ACTH secretion involving multiple genomic mechanisms including suppression of

POMC transcription (see earlier) (Drouin, 2016) as well as suppression of CRH-R1 mRNA expression (Pozzoli et al., 1996). The long-term effects of glucocorticoids or stress on other parameters of corticotroph function such as Ca2þ signaling or electrical excitability have not been explored systematically. In contrast, early glucocorticoid inhibition involves multiple mechanisms that largely inhibit evoked, rather than basal ACTH secretion through control of both membrane excitability and suppression of CRH- and AVP- evoked elevations of intracellular free calcium. In perifused rat corticotrophs subjected to pulses of CRH and/or CRH þ AVP, glucocorticoid inhibition of evoked ACTH secretion is observed within 10 min of glucocorticoid exposure that lasts for w2 h following steroid withdrawal. Inhibition is blocked by classical type II glucocorticoid antagonists as well as inhibitors of transcription and translation and is thus a largely genomic effect of glucocorticoid (Shipston and Antoni, 1991; Dayanithi and Antoni, 1989). In the same time frame, glucocorticoids suppress both CRH- and AVP-evoked increases in intracellular calcium signaling at the single cell level in rat corticotrophs (Romano` et al., 2017) and suppress CRH-mediated Ca2þ signaling in mouse AtT20D16:16 cells (Antoni et al., 1992). Importantly, in native rat corticotrophs, suppression of calcium signaling involved an increase in the recruitment of individual cells to glucocorticoid inhibition, the time course also being dependent on the secretagogue combination (Romano` et al., 2017). Cells exposed to CRH and AVP in combination cells were also more resistant to feedback inhibition. Glucocorticoid feedback inhibition of Ca2þ signaling is, at least in part, due to suppression of CRH- and AVP-stimulated electrical excitability through multiple mechanisms. In native mouse corticotrophs, glucocorticoids prevent the transition to bursting in response to CRH. This inhibition of CRH-induced bursting involves control of BK channels, as corticotrophs that were pretreated with corticosterone could be induced to burst, in the presence of CRH, by reintroducing a BK-like fast current into cells using dynamic clamp (Duncan et al., 2016). Corticosterone also dampens excitability independently of BK channels as corticosterone inhibited both CRH- and AVP-induced increases in action potential frequency in the absence of BK channels. However, glucocorticoids had little effect on CRH/AVP-induced depolarization, suggesting that the mechanisms largely target channels involved in control spike frequency and bursting, rather than initial depolarization from rest (Duncan et al., 2016). Corticosterone had differential effects on basal activitydwith no effect on resting membrane potential following pretreatment for 4 or 150 min but with a significant hyperpolarization of RMP observed after 90 min of exposure. Thus, glucocorticoids

4. MELANOTROPHS AND a-MSH

control excitability through multiple ionic mechanisms in the early time domain although the molecular targets and mechanisms remain to be defined.

4. MELANOTROPHS AND a-MSH Production of a-MSH from melanotrophs is key for the control of skin and fur pigmentation in vertebrates. A large volume of studies on melanotrophs has been performed in fish, amphibians, and reptiles, where the role of a-MSH in controlling color changes and skin pigmentation is well established (Nilsson Sko¨ld et al., 2013; Vaudry et al., 2006; Tonon et al., 1993, 1988). The role of a-MSH in pigmentation in mammals has also been subject of many investigations, and although its role in human skin pigmentation was initially debated, it is now clear that a-MSH acting through its receptor MC1R plays a central role in the control of eumelanin production in humans as well (Nasti and Timares, 2015; Abdel-Malek et al., 2014, 1999).

4.1 Anatomic Considerations Melanotrophs are located in the PI of the pituitary gland, a band of cells located at the interface between the anterior and neural part of the hypophysis, where these cells receive direct innervation from hypothalamic neurons (Takeuchi, 2001). Melanotrophs are the main cell population of the PI; morphologic studies have revealed heterogeneity in this population, in terms of shape, amount, and size of secretory granules, as well as levels of POMC expression (Takeuchi, 2001; Ishii and Ishibashi, 1989; Chronwall et al., 1987; Murakami et al., 1968). Interestingly, it has been shown that some species, including humans and higher apes (Plaut, 1936), cetaceans (Geiling et al., 1940), elephants (Wislocki, 1940), and birds (Rahn and Painter, 1941), lack a well-defined PI or only present a rudimentary or vestigial one. The presence of a functional PI in humans has been a longstanding matter of debate; although a clear PI is found in the fetus (Murakami et al., 1968), this is thought to regress in the adult, leaving residual colloid-filled cysts (Larkin and Ansorge, 2000; Horvath et al., 1999; Rasmussen, 1930). Although in cetaceans there are reports of melanotrophs in the anterior pituitary (Panin et al., 2013), it is unknown whether this is the case in humans.

4.2 Control of Melanotroph Activity In most species, vascularization of the PI is fairly limited when compared to that of the anterior pituitary (Stojilkovic et al., 2010; Takeuchi, 2001), in agreement

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with the fact that cells in this region are controlled through direct innervation from dopaminergic, GABAergic, and neuropeptidergic hypothalamic neurons (Fig. 8.2), rather than receiving hormonal signals from the hypothalamus through the portal vasculature, as it is the case for hormone-producing cells in the anterior pituitary. Mammalian melanotrophs are under tonic GABAergic and dopaminergic inhibition (Stojilkovic et al., 2010); in amphibians, the role of several neuropeptides (such as TRH, CRH, and NPY) has also been investigated (Shibuya and Douglas, 1993; Leenders et al., 1993; de Rijk et al., 1992), while the role of these peptides in mammals remains less clear. When cultured (thus relieving their tonic hypothalamic inhibition), dissociated melanotrophs show a diversity of spontaneous activity; they have been reported to display low frequency (70% of cases) of endogenous Cushing syndrome results from ACTH-secreting pituitary adenomas (called Cushing disease) with ectopic (nonpituitary) ACTH secretion (such as ACTH-secreting lung tumors) being less common (Sbiera et al., 2015; Raff and Carroll, 2015). In both cases, excessive ACTH stimulates adrenal MC2R receptors, leading to excessive glucocorticoid release and adrenal hyperplasia. Transphenoidal surgery to remove the adenoma is the treatment of choice with pharmacological interventions to either inhibit pituitary ACTH release, glucocorticoid synthesis, or antagonize GRs in patients with inoperable tumors or following relapse. A major hallmark of Cushing disease is a relative resistance to glucocorticoid-negative feedback inhibition of ACTH release arising from a higher than normal set point for effective glucocorticoid inhibition. Two major cellular mechanisms are thus implicated in Cushing disease. Firstly, ACTH-secreting pituitary corticotroph adenomas result predominantly from monoclonal expansion of corticotrophs with genetic mutations (Zhou et al., 2014; Gicquel et al., 1992; Biller

8. CONCLUSIONS AND FUTURE DIRECTIONS

et al., 1992; Schulte et al., 1991). These adenomas are typically associated with increased expression of CRHR1 and AVPR1b as well as cell cycle regulators (such as epidermal growth factor receptor and cyclin E) and tumor suppressors, including bone morphogenic protein-4 (BMP-4) and suppressor of cytokine signaling 1 (SOCS1) as well as nuclear TFs such as testicular orphan receptor 4 (TR4) (Sbiera et al., 2015; Zhou et al., 2014). In approximately 60% of adenomas from patients with Cushing disease, whole-exome sequencing has identified somatic mutations in the deubiquitinase gene USP8 (Reincke et al., 2015; PerezRivas et al., 2015; Ma et al., 2015). An important target for USP8-dependent deubiquitination is the EGFR receptor, and thus USP8 protects the degradation of the EGFR receptor by the lysosomal pathway. The activity of USP8 is normally kept in check by binding to the adapter protein 14-3-3. Identified gain-of-function mutations in USP8 disrupt the interaction of USP8 with 14-3-3, resulting in an increase in constitutive USP8 deubiquitination activity. This, in turn, increases plasma membrane EGFR expression in adenomas, resulting in increased activation of the MAPK signaling pathway that enhances POMC transcription and ACTH synthesis. Thus, knockdown of USP8 or pharmacological inhibition of EGFR activity results in a suppression of ACTH release and tumor size, suggesting potential new therapeutic avenues (Sbiera et al., 2015; Reincke et al., 2015; Perez-Rivas et al., 2015; Ma et al., 2015; Fukuoka et al., 2011). Secondly, the levels of ACTH in the circulating plasma of patients with Cushing syndrome are inappropriate for the elevated levels of circulating glucocorticoid. Although not abolished, glucocorticoid negative feedback is significantly attenuated in Cushing’ disease. The mechanisms of reduced glucocorticoid feedback are poorly understood; however as discussed earlier, dysregulation of glucocorticoidmediated transrepression of POMC transcription due to a loss of nuclear HDAC2 and BRG1 is also likely involved (Drouin, 2016; Bilodeau et al., 2006).

7.3 Familial Glucocorticoid Deficiency Several mutations in MC2R have been associated with familial glucocorticoid deficiency (FGD), a rare autosomal recessive disorder (Clark et al., 1993) in which the cells of the zona fasciculata of the adrenal gland fail to produce cortisol in response to ACTH. This is characterized by hypocortisolism in the presence of high ACTH, which results in hypoglycemic symptoms in early life, accompanied by hyperpigmentation of the skin due to activation of MC1R in the skin (Tsigos, 1999; Weber and Clark, 1994). Furthermore, the

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high levels of ACTH have been suggested to stimulate bone growth, probably through activation of MC3R, explaining the tall stature often associated with FGD (Imamine et al., 2005; Elias et al., 2000). FGD, however, is a heterogeneous disease, and mutations in MC2R are not the only cause of FGD, accounting for approximately 25% of cases, designated as type I FGD (Ge´nin et al., 2002; Naville et al., 1996; Weber and Clark, 1994). Genetic mapping of FGD patients not showing MC2R mutations have identified two other loci linked to this disease, on chromosome 21q22.1 (Metherell et al., 2005) and 8q12.1 to 8q21.2 (Ge´nin et al., 2002). The first have shown to depend on mutations of MRAP2 (Metherell et al., 2005) and have been designated FGD type 2. Mutations in STAR (which have usually been associated with nonclassic lipoid congenital adrenal hyperplasia) have been shown to be the underlying cause of FGD in the second group of patients, identified as type 3 FGD (Metherell et al., 2009). Overall, these account for just over 50% of FGD cases (Dias et al., 2010), and other genetic causes of FGD are presently unknown.

7.4 Changes with Aging As for most pituitary functions, the activity of corticotrophs and melanotrophs is also changed by aging (Veldhuis, 2013). ACTH secretion following CRH injection is amplified in the elderly, in a gender-dependent manner (Born et al., 1995). However, ACTH secretion was higher in young compared to old men (but not women) in response to social stress (Kudielka et al., 2004). Furthermore, a decrease in the amplitude of circadian variations in ACTH and glucocorticoid secretion has been reported in older individuals (Sherman et al., 1985). Rats display an age-dependent increase in the size of the PI; however, they do not display major changes in plasma a-MSH levels. The precise molecular mechanisms underlying these changes, and the cellular and molecular properties of corticotrophs and melanotrophs during aging, however, still remain to be determined.

8. CONCLUSIONS AND FUTURE DIRECTIONS This chapter has highlighted the main features of the two different cell populations of the pituitary gland producing POMC and its proteolytic products. Corticotrophs in the anterior pituitary gland, central to the endocrine stress response, and melanotrophs of the PI, important for the control of pigmentation.

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The control of these cell populations has been widely studied and modeled both at the cell and system levels, in a variety of cellular and animal model systems, including humans. Still, many questions remain open. Several anatomic and functional observations over the years have supported the idea that both these cell types are highly heterogeneous, but the cause and functional role of this heterogeneity still remains to be clarified. Furthermore, much work on these systems has been performed in vitro on cell line or primary dissociated pituitary cells; while these studies have been instrumental for understanding the molecular function of corticotrophs and melanotrophs, an always growing literature supports the idea of a functional role for the 3D structure of pituitary cells. Exploration of the functionality of corticotrophs and melanotrophs at the single cell level and in their native anatomic context (e.g., in slices or in vivo) has not been attempted to date. This as well as studies targeted to get a better understanding of the interactions between the different cell types of the pituitary gland are much needed. Secondly, a better understanding of how timevarying hypothalamic signals (e.g., CRH and AVP) are interpreted by corticotrophs, how these inputs vary, and how they affect glucocorticoid output during the course of different type of stressors or different physiologic situations will help developing better systemwide models of the HPA axis. This is of particular clinical relevance, since glucocorticoids are one of the most widely prescribed drugs for a variety of clinical conditions, and approximately 1% of the general population receives long-term glucocorticoid treatment (Overman et al., 2013; Fardet et al., 2011; van Staa et al., 2000). Although some treatment regimens with glucocorticoids try to mimic the physiologic circadian variations in the level of these hormones (Oksnes et al., 2014; Johannsson et al., 2012; Verma et al., 2010), they fail to recapitulate their physiologic ultradian release, which has been shown to be important for their function, resulting in important undesired side effects. Proof-of-concept studies using a subcutaneous delivery system show that it is possible to replicate near-physiologic levels of glucocorticoids, to produce pulsatile administration that could improve clinical outcomes (Russell and Lightman, 2014; Russell et al., 2014). Thus, understanding the mechanisms that control ACTH and glucocorticoid pulsatility may in the future inform clinicians, with the hope of developing better treatment for stress-related pathologies as well as improving the use of glucocorticoids in the clinic.

References Abdel-Malek, Z., Suzuki, I., Tada, A., Im, S., Akcali, C., 1999. The melanocortin-1 receptor and human pigmentation. Ann. N. Y. Acad. Sci. 885, 117e133. Abdel-Malek, Z.A., Swope, V.B., Starner, R.J., Koikov, L., Cassidy, P., Leachman, S., 2014. Melanocortins and the melanocortin 1 receptor, moving translationally towards melanoma prevention. Arch. Biochem. Biophys. 563, 4e12. https://doi.org/10.1016/ j.abb.2014.07.002. Anderson, E.J.P., C ¸ akir, I., Carrington, S.J., Cone, R.D., GhamariLangroudi, M., Gillyard, T., Gimenez, L.E., Litt, M.J., 2016. 60 YEARS OF POMC: regulation of feeding and energy homeostasis by a-MSH. J. Mol. Endocrinol. 56, T157eT174. https://doi.org/ 10.1530/JME-16-0014. Andrioli, M., Pecori Giraldi, F., Cavagnini, F., 2006. Isolated corticotrophin deficiency. Pituitary 9, 289e295. https://doi.org/10.1007/ s11102-006-0408-5. Antakly, T., Sasaki, A., Liotta, A.S., Palkovits, M., Krieger, D.T., 1985. Induced expression of the glucocorticoid receptor in the rat intermediate pituitary lobe. Science 229, 277e279. Antoni, F.A., 1993. Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Front. Neuroendocrinol. 14, 76e122. https://doi.org/10.1006/frne.1993.1004. Antoni, F.A., Hoyland, J., Woods, M.D., Mason, W.T., 1992. Glucocorticoid inhibition of stimulus-evoked adrenocorticotrophin release caused by suppression of intracellular calcium signals. J. Endocrinol. 133, R13eR16. Antoni, F.A., Sosunov, A.A., Haunso, A., Paterson, J.M., Simpson, J., 2003. Short-term plasticity of cyclic adenosine 30 ,50 -monophosphate signaling in anterior pituitary corticotrope cells: the role of adenylyl cyclase isotypes. Mol. Endocrinol. 17, 692e703. https://doi.org/ 10.1210/me.2002-0369. Autelitano, D.J., Snyder, L., Sealfon, S.C., Roberts, J.L., 1989. Dopamine D2-receptor messenger RNA is differentially regulated by dopaminergic agents in rat anterior and neurointermediate pituitary. Mol. Cell. Endocrinol. 67, 101e105. Baker, J.R., Bennett, H.P., Hudson, A.M., McMartin, C., Purdon, G.E., 1976. On the metabolism of two adrenocorticotrophin analogues. Clin. Endocrinol. 5 (Suppl. l), 61Se72S. Barrett, P., MacDonald, A., Helliwell, R., Davidson, G., Morgan, P., 1994. Cloning and expression of a new member of the melanocytestimulating hormone receptor family. J. Mol. Endocrinol. 12, 203e213. Baumann, J.B., Eberle, A.N., Christen, E., Ruch, W., Girard, J., 1986. Steroidogenic activity of highly potent melanotropic peptides in the adrenal cortex of the rat. Acta Endocrinol. 113, 396e402. Beaumont, K.A., Newton, R.A., Smit, D.J., Leonard, J.H., Stow, J.L., Sturm, R.A., 2005. Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk. Hum. Mol. Genet. 14, 2145e2154. https://doi.org/ 10.1093/hmg/ddi219. Benjannet, S., Rondeau, N., Day, R., Chre´tien, M., Seidah, N.G., 1991. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc. Natl. Acad. Sci. U. S. A. 88, 3564e3568. Bertolotto, C., Abbe, P., Hemesath, T.J., Bille, K., Fisher, D.E., Ortonne, J.P., Ballotti, R., 1998. Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes. J. Cell Biol. 142, 827e835. Biller, B.M., Alexander, J.M., Zervas, N.T., Hedley-Whyte, E.T., Arnold, A., Klibanski, A., 1992. Clonal origins of adrenocorticotropin-secreting pituitary tissue in Cushing’s disease. J. Clin. Endocrinol. Metab. 75, 1303e1309. https:// doi.org/10.1210/jcem.75.5.1358909.

REFERENCES

Bilodeau, S., Vallette-Kasic, S., Gauthier, Y., Figarella-Branger, D., Brue, T., Berthelet, F., Lacroix, A., Batista, D., Stratakis, C., Hanson, J., Meij, B., Drouin, J., 2006. Role of Brg1 and HDAC2 in GR trans-repression of the pituitary POMC gene and misexpression in Cushing disease. Genes Dev. 20, 2871e2886. https://doi.org/ 10.1101/gad.1444606. Birch, N.P., Tracer, H.L., Hakes, D.J., Loh, Y.P., 1991. Coordinate regulation of mRNA levels of pro-opiomelanocortin and the candidate processing enzymes PC2 and PC3, but not furin, in rat pituitary intermediate lobe. Biochem. Biophys. Res. Commun. 179, 1311e1319. https://doi.org/10.1016/0006-291X(91)91716-P. Bonnefont, X., Lacampagne, A., Sanchez-Hormigo, A., Fino, E., Creff, A., Mathieu, M.-N., Smallwood, S., Carmignac, D., Fontanaud, P., Travo, P., Alonso, G., Courtois-Coutry, N., Pincus, S.M., Robinson, I.C.A.F., Mollard, P., 2005. Revealing the large-scale network organization of growth hormone-secreting cells. Proc. Natl. Acad. Sci. U. S. A. 102, 16880e16885. https:// doi.org/10.1073/pnas.0508202102. Born, J., Ditschuneit, I., Schreiber, M., Dodt, C., Fehm, H.L., 1995. Effects of age and gender on pituitary-adrenocortical responsiveness in humans. Eur. J. Endocrinol. 132, 705e711. Bornstein, S.R., 2009. Predisposing factors for adrenal insufficiency. N. Engl. J. Med. 360, 2328e2339. https://doi.org/10.1056/ NEJMra0804635. Boston, B.A., 2006. The role of melanocortins in adipocyte function. Ann. N. Y. Acad. Sci. 885, 75e84. https://doi.org/10.1111/j.17496632.1999.tb08666.x. Broersen, L.H.A., Pereira, A.M., Jørgensen, J.O.L., Dekkers, O.M., 2015. Adrenal insufficiency in corticosteroids use: systematic review and meta-analysis. J. Clin. Endocrinol. Metab. 100, 2171e2180. https:// doi.org/10.1210/jc.2015-1218. Budry, L., Balsalobre, A., Gauthier, Y., Khetchoumian, K., L’honore´, A., Vallette, S., Brue, T., Figarella-Branger, D., Meij, B., Drouin, J., 2012. The selector gene Pax7 dictates alternate pituitary cell fates through its pioneer action on chromatin remodeling. Genes Dev. 26, 2299e2310. https://doi.org/10.1101/gad.200436.112. Budry, L., Lafont, C., El Yandouzi, T., Chauvet, N., Cone´jero, G., Drouin, J., Mollard, P., 2011. Related pituitary cell lineages develop into interdigitated 3D cell networks. Proc. Natl. Acad. Sci. U. S. A. 108, 12515e12520. https://doi.org/10.1073/pnas.1105929108. Burlet, A., Tonon, M.C., Tankosic, P., Coy, D., Vaudry, H., 1983. Comparative immunocytochemical localization of corticotropin releasing factor (CRF-41) and neurohypophysial peptides in the brain of Brattleboro and Long-Evans rats. Neuroendocrinology 37, 64e72. https://doi.org/10.1159/000123517. Canny, B.J., Jia, L.G., Leong, D.A., 1992. Corticotropin-releasing factor, but not arginine vasopressin, stimulates concentration-dependent increases in ACTH secretion from a single corticotrope. Implications for intracellular signals in stimulus-secretion coupling. J. Biol. Chem. 267, 8325e8329. Cats, A., Kassenaar, A.A., 1957. Influence of the kidney on the disappearance rate of labelled corticotrophin from the blood stream. Acta Endocrinol. 24, 43e49. Challis, B.G., Pritchard, L.E., Creemers, J.W.M., Delplanque, J., Keogh, J.M., Luan, J., Wareham, N.J., Yeo, G.S.H., Bhattacharyya, S., Froguel, P., White, A., Farooqi, I.S., O’Rahilly, S., 2002. A missense mutation disrupting a dibasic prohormone processing site in pro-opiomelanocortin (POMC) increases susceptibility to early-onset obesity through a novel molecular mechanism. Hum. Mol. Genet. 11, 1997e2004. Chan, L.F., Webb, T.R., Chung, T.-T., Meimaridou, E., Cooray, S.N., Guasti, L., Chapple, J.P., Egertova´, M., Elphick, M.R., Cheetham, M.E., Metherell, L.A., Clark, A.J.L., 2009. MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family. Proc. Natl. Acad. Sci. U. S. A. 106, 6146e6151. https:// doi.org/10.1073/pnas.0809918106.

161

Charmandari, E., Nicolaides, N.C., Chrousos, G.P., 2014. Adrenal insufficiency. Lancet 383, 2152e2167. https://doi.org/10.1016/ S0140-6736(13)61684-0. Chen, W., Kelly, M.A., Opitz-Araya, X., Thomas, R.E., Low, M.J., Cone, R.D., 1997. Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides. Cell 91, 789e798. Chhajlani, V., Wikberg, J.E., 1992. Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Lett. 309, 417e420. Chhajlani, V., Xu, X., Blauw, J., Sudarshi, S., 1996. Identification of ligand binding residues in extracellular loops of the melanocortin 1 receptor. Biochem. Biophys. Res. Commun. 219, 521e525. https://doi.org/10.1006/bbrc.1996.0266. Chronwall, B.M., Millington, W.R., Griffin, W.S., Unnerstall, J.R., O’Donohue, T.L., 1987. 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 120, 1201e1211. https://doi.org/10.1210/endo-120-3-1201. Clark, A.J.L., Grossman, A., McLoughlin, L., 1993. Familial glucocorticoid deficiency associated with point mutation in the adrenocorticotropin receptor. Lancet 341 (8843), 461e462. https://doi.org/ 10.1016/0140-6736(93)90208-X. Originally published as vol. 1. Cool, D.R., Fenger, M., Snell, C.R., Loh, Y.P., 1995. Identification of the sorting signal motif within pro-opiomelanocortin for the regulated secretory pathway. J. Biol. Chem. 270, 8723e8729. https://doi.org/ 10.1074/jbc.270.15.8723. Cool, D.R., Normant, E., Shen, F., Chen, H.-C., Pannell, L., Zhang, Y., Loh, Y.P., 1997. Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpefat mice. Cell 88, 73e83. https://doi.org/ 10.1016/S0092-8674(00)81860-7. Cool, D.R., Peng, L., 1994. Identification of a sorting signal for the regulated secretory pathway at the N-terminus of proopiomelanocortin. Biochimie 76, 265e270. https://doi.org/ 10.1016/0300-9084(94)90156-2. Cooray, S.N., Chung, T.-T., Mazhar, K., Szidonya, L., Clark, A.J.L., 2011. Bioluminescence resonance energy transfer reveals the adrenocorticotropin (ACTH)-Induced conformational change of the activated ACTH receptor complex in living cells. Endocrinology 152, 495e502. https://doi.org/10.1210/en.2010-1053. Costa, J.L., Bui, S., Reed, P., Dores, R.M., Brennan, M.B., Hochgeschwender, U., 2004. Mutational analysis of evolutionarily conserved ACTH residues. Gen. Comp. Endocrinol. 136, 12e16. https://doi.org/10.1016/j.ygcen.2003.11.005. Cote, T.E., Felder, R., Kebabian, J.W., Sekura, R.D., Reisine, T., Affolter, H.U., 1986. D-2 dopamine receptor-mediated inhibition of pro-opiomelanocortin synthesis in rat intermediate lobe. Abolition by pertussis toxin or activators of adenylate cyclase. J. Biol. Chem. 261, 4555e4561. Couture, C., Saveanu, A., Barlier, A., Carel, J.C., Fassnacht, M., Flu¨ck, C.E., Houang, M., Maes, M., Phan-Hug, F., Enjalbert, A., Drouin, J., Brue, T., Vallette, S., 2012. Phenotypic homogeneity and genotypic variability in a large series of congenital isolated ACTHdeficiency patients with TPIT gene mutations. J. Clin. Endocrinol. Metab. 97, E486eE495. https://doi.org/10.1210/jc.2011-1659. Creemers, J.W.M., Lee, Y.S., Oliver, R.L., Bahceci, M., Tuzcu, A., Gokalp, D., Keogh, J., Herber, S., White, A., O’Rahilly, S., Farooqi, I.S., 2008. Mutations in the amino-terminal region of proopiomelanocortin (POMC) in patients with early-onset obesity impair POMC sorting to the regulated secretory pathway. J. Clin. Endocrinol. Metab. 93, 4494e4499. https://doi.org/10.1210/jc.2008-0954. Cripps, M.M., Bromberg, B.B., Patchen-Moor, K., Welch, M.H., 1987. Adrenocorticotropic hormone stimulation of lacrimal peroxidase secretion. Exp. Eye Res. 45, 673e682.

162

8. ANTERIOR PITUITARY AND PARS INTERMEDIA SPACE: CORTICOTROPHS (ACTH) AND MELANOTROPHS (a-MSH)

Day, R., Schafer, M.K., Watson, S.J., Chre´tien, M., Seidah, N.G., 1992. Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary. Mol. Endocrinol. 6, 485e497. https://doi.org/10.1210/mend.6.3.1316544. Dayanithi, G., Antoni, F.A., 1989. Rapid as well as delayed inhibitory effects of glucocorticoid hormones on pituitary adrenocorticotropic hormone release are mediated by type II glucocorticoid receptors and require newly synthesized messenger ribonucleic acid as well as protein. Endocrinology 125, 308e313. https://doi.org/ 10.1210/endo-125-1-308. de Rijk, E.P., van Strien, F.J., Roubos, E.W., 1992. Demonstration of coexisting catecholamine (dopamine), amino acid (GABA), and peptide (NPY) involved in inhibition of melanotrope cell activity in Xenopus laevis: a quantitative ultrastructural, freeze-substitution immunocytochemical study. J. Neurosci. 12, 864e871. De Souza, E.B., Perrin, M.H., Rivier, J., Vale, W.W., Kuhar, M.J., 1984. Corticotropin-releasing factor receptors in rat pituitary gland: autoradiographic localization. Brain Res. 296, 202e207. de Souza, F.S.J., Santangelo, A.M., Bumaschny, V., Avale, M.E., Smart, J.L., Low, M.J., Rubinstein, M., 2005. Identification of neuronal enhancers of the proopiomelanocortin gene by transgenic mouse analysis and phylogenetic footprinting. Mol. Cell. Biol. 25, 3076e3086. https://doi.org/10.1128/MCB.25.8.3076-3086.2005. Dedic, N., Chen, A., Deussing, J.M., 2018. The CRF family of neuropeptides and their receptorsemediators of the central stress response. Curr. Mol. Pharmacol. 11, 4e31. https://doi.org/10.2174/ 1874467210666170302104053. Demeneix, B.A., Desaulles, E., Feltz, P., Loeffler, J.P., 1984. Dual population of GABAA and GABAB receptors in rat pars intermedia demonstrated by release of alpha MSH caused by barium ions. Br. J. Pharmacol. 82, 183e190. Demeneix, B.A., Taleb, O., Loeffler, J.P., Feltz, P., 1986. GABAA and GABAB receptors on porcine pars intermedia cells in primary culture: functional role in modulating peptide release. Neuroscience 17, 1275e1285. Deng, Q., Riquelme, D., Trinh, L., Low, M.J., Tomic, M., Stojilkovic, S., Aguilera, G., 2015. Rapid glucocorticoid feedback inhibition of ACTH secretion involves ligand-dependent membrane association of glucocorticoid receptors. Endocrinology 156, 3215e3227. https://doi.org/10.1210/EN.2015-1265. Desrues, L., Castel, H., Malagon, M.M., Vaudry, H., Tonon, M.-C., 2005. The regulation of alpha-MSH release by GABA is mediated by a chloride-dependent [Ca2þ]c increase in frog melanotrope cells. Peptides 26, 1936e1943. https://doi.org/10.1016/ j.peptides.2004.11.022. Desrues, L., Vaudry, H., Lamacz, M., Tonon, M.C., 1995. Mechanism of action of gamma-aminobutyric acid on frog melanotrophs. J. Mol. Endocrinol. 14, 1e12. Dias, R.P., Chan, L.F., Metherell, L.A., Pearce, S.H.S., Clark, A.J.L., 2010. Isolated Addison’s disease is unlikely to be caused by mutations in MC2R, MRAP or STAR, three genes responsible for familial glucocorticoid deficiency. Eur. J. Endocrinol. 162, 357e359. https:// doi.org/10.1530/EJE-09-0720. Dores, R.M., Baron, A.J., 2011. Evolution of POMC: origin, phylogeny, posttranslational processing, and the melanocortins. Ann. N. Y. Acad. Sci. 1220, 34e48. https://doi.org/10.1111/j.17496632.2010.05928.x. Douglas, W.W., Taraskevich, P.S., 1978. Action potentials in gland cells of rat pituitary pars intermedia: inhibition by dopamine, an inhibitor of MSH secretion. J. Physiol. 285, 171e184. Drouin, J., 2016. 60 YEARS OF POMC: transcriptional and epigenetic regulation of POMC gene expression. J. Mol. Endocrinol. 56, T99eT112. https://doi.org/10.1530/JME-15-0289. Duncan, P.J., Sengu¨l, S., Tabak, J., Ruth, P., Bertram, R., Shipston, M.J., 2015. Large conductance Ca2þ-activated Kþ (BK) channels promote secretagogue-induced transition from spiking to bursting in murine

anterior pituitary corticotrophs. J. Physiol. 593, 1197e1211. https:// doi.org/10.1113/jphysiol.2015.284471. Duncan, P.J., Tabak, J., Ruth, P., Bertram, R., Shipston, M.J., 2016. Glucocorticoids inhibit CRH/AVP-evoked bursting activity of male murine anterior pituitary corticotrophs. Endocrinology 157, 3108e3121. https://doi.org/10.1210/en.2016-1115. Ebling, F.J., Ebling, E., Randall, V., Skinner, J., 1975. The synergistic action of alpha-melanocyte-stimulating hormone and testosterone of the sebaceous, prostate, preputial, Harderian and lachrymal glands, seminal vesicles and brown adipose tissue in the hypophysectomized-castrated rat. J. Endocrinol. 66, 407e412. Elias, L.L., Huebner, A., Metherell, L.A., Canas, A., Warne, G.L., Bitti, M.L., Cianfarani, S., Clayton, P.E., Savage, M.O., Clark, A.J., 2000. Tall stature in familial glucocorticoid deficiency. Clin. Endocrinol. 53, 423e430. Everson, R.A., Smith, P.D., Dobson, E.L., 1969. Kinetics of Adrenocorticotropic Hormone Inactivation in the Dog. Fardet, L., Petersen, I., Nazareth, I., 2011. Prevalence of long-term oral glucocorticoid prescriptions in the UK over the past 20 years. Rheumatology 50, 1982e1990. https://doi.org/10.1093/rheumatology/ ker017. Farooqi, I.S., Drop, S., Clements, A., Keogh, J.M., Biernacka, J., Lowenbein, S., Challis, B.G., O’Rahilly, S., 2006. Heterozygosity for a POMC-null mutation and increased obesity risk in humans. Diabetes 55, 2549e2553. https://doi.org/10.2337/db06-0214. Fletcher, P.A., Zemkova, H., Stojilkovic, S.S., Sherman, A., 2017. Modeling the diversity of spontaneous and agonist-induced electrical activity in anterior pituitary corticotrophs. J. Neurophysiol. https://doi.org/10.1152/jn.00948.2016 jn.00948.2016. Fricker, L.D., 1988. Carboxypeptidase E. Annu. Rev. Physiol. 50, 309e321. https://doi.org/10.1146/annurev.ph.50.030188.001521. Fridmanis, D., Petrovska, R., Kalnina, I., Slaidina, M., Peculis, R., Schio¨th, H.B., Klovins, J., 2010. Identification of domains responsible for specific membrane transport and ligand specificity of the ACTH receptor (MC2R). Mol. Cell. Endocrinol. 321, 175e183. https://doi.org/10.1016/j.mce.2010.02.032. Fukuoka, H., Cooper, O., Ben-Shlomo, A., Mamelak, A., Ren, S.-G., Bruyette, D., Melmed, S., 2011. EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas. J. Clin. Investig. 121, 4712e4721. https://doi.org/10.1172/ JCI60417. Funkelstein, L., Toneff, T., Mosier, C., Hwang, S.-R., Beuschlein, F., Lichtenauer, U.D., Reinheckel, T., Peters, C., Hook, V., 2008. Major role of cathepsin L for producing the peptide hormones ACTH, beta-endorphin, and alpha-MSH, illustrated by protease gene knockout and expression. J. Biol. Chem. 283, 35652e35659. https://doi.org/10.1074/jbc.M709010200. Fuse, N., Yasumoto, K., Suzuki, H., Takahashi, K., Shibahara, S., 1996. Identification of a melanocyte-type promoter of the microphthalmia-associated transcription factor gene. Biochem. Biophys. Res. Commun. 219, 702e707. https://doi.org/10.1006/ bbrc.1996.0298. Gagner, J.P., Drouin, J., 1987. Tissue-specific regulation of pituitary proopiomelanocortin gene transcription by corticotropin-releasing hormone, 30 ,50 -cyclic adenosine monophosphate, and glucocorticoids. Mol. Endocrinol. 1, 677e682. https://doi.org/ 10.1210/mend-1-10-677. Gantz, I., Konda, Y., Tashiro, T., Shimoto, Y., Miwa, H., Munzert, G., Watson, S.J., DelValle, J., Yamada, T., 1993. Molecular cloning of a novel melanocortin receptor. J. Biol. Chem. 268, 8246e8250. Gantz, I., Shimoto, Y., Konda, Y., Miwa, H., Dickinson, C.J., Yamada, T., 1994. Molecular cloning, expression, and characterization of a fifth melanocortin receptor. Biochem. Biophys. Res. Commun. 200, 1214e1220. https://doi.org/10.1006/bbrc.1994.1580. Gary, K.A., Chronwall, B.M., 1992. The onset of dopaminergic innervation during ontogeny decreases melanotrope proliferation in the

REFERENCES

intermediate lobe of the rat pituitary. Int. J. Dev. Neurosci. 10, 131e142. Geiling, E.M.K., Vos, B.J., Oldham, F.K., 1940. The pharmacology and anatomy of the hypophysis of the porpoise. Endocrinology 27, 309e316. https://doi.org/10.1210/endo-27-2-309. Ge´nin, E., Huebner, A., Jaillard, C., Faure, A., Halaby, G., Saka, N., Clark, A.J.L., Durand, P., Be´geot, M., Naville, D., 2002. Linkage of one gene for familial glucocorticoid deficiency type 2 (FGD2) to chromosome 8q and further evidence of heterogeneity. Hum. Genet. 111, 428e434. https://doi.org/10.1007/s00439-002-0806-3. Gicquel, C., Le Bouc, Y., Luton, J.P., Girard, F., Bertagna, X., 1992. Monoclonality of corticotroph macroadenomas in Cushing’s disease. J. Clin. Endocrinol. Metab. 75, 472e475. https://doi.org/ 10.1210/jcem.75.2.1322426. Gillies, G.E., Linton, E.A., Lowry, P.J., 1982. Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin. Nature 299, 355e357. https://doi.org/10.1038/299355a0. Gomez, M.T., Magiakou, M.A., Mastorakos, G., Chrousos, G.P., 1993. The pituitary corticotroph is not the rate limiting step in the postoperative recovery of the hypothalamic-pituitary-adrenal axis in patients with Cushing syndrome. J. Clin. Endocrinol. Metab. 77, 173e177. https://doi.org/10.1210/jcem.77.1.8392083. Griffon, N., Mignon, V., Facchinetti, P., Diaz, J., Schwartz, J.C., Sokoloff, P., 1994. Molecular cloning and characterization of the rat fifth melanocortin receptor. Biochem. Biophys. Res. Commun. 200, 1007e1014. Guo, Q., Lu, J., Mu, Y., Chen, K., Pan, C., 2013. Adult idiopathic isolated ACTH deficiency: a short series and literature review. Neuroendocrinol. Lett. 34, 693e700. Haskell-Luevano, C., Sawyer, T.K., Trumpp-Kallmeyer, S., Bikker, J.A., Humblet, C., Gantz, I., Hruby, V.J., 1996. Three-dimensional molecular models of the hMC1R melanocortin receptor: complexes with melanotropin peptide agonists. Drug Des. Discov. 14, 197e211. Hinz, B., Hirschelmann, R., 2000. Rapid non-genomic feedback effects of glucocorticoids on CRF-induced ACTH secretion in rats. Pharm. Res. 17, 1273e1277. Hodson, D.J., Schaeffer, M., Romano, N., Fontanaud, P., Lafont, C., Birkenstock, J., Molino, F., Christian, H., Lockey, J., Carmignac, D., Fernandez-Fuente, M., Le Tissier, P., Mollard, P., 2012. Existence of long-lasting experience-dependent plasticity in endocrine cell networks. Nat. Commun. 3, 605. https://doi.org/ 10.1038/ncomms1612. Ho¨llt, V., Przewłocki, R., Haarmann, I., Almeida, O.F., Kley, N., Millan, M.J., Herz, A., 1986. Stress-induced alterations in the levels of messenger RNA coding for proopiomelanocortin and prolactin in rat pituitary. Neuroendocrinology 43, 277e282. https:// doi.org/10.1159/000124541. Hook, V., Funkelstein, L., Toneff, T., Mosier, C., Hwang, S.-R., 2009. Human pituitary contains dual cathepsin L and prohormone convertase processing pathway components involved in converting POMC into the peptide hormones ACTH, a-MSH, and b-endorphin. Endocrine 35, 429e437. https://doi.org/10.1007/ s12020-009-9163-5. Hook, V.Y., Eiden, L.E., Brownstein, M.J., 1982. A carboxypeptidase processing enzyme for enkephalin precursors. Nature 295, 341e342. Horvath, E., Kovacs, K., Lloyd, R.V., 1999. Pars intermedia of the human pituitary revisited: morphologic aspects and frequency of hyperplasia of POMC-peptide immunoreactive cells. Endocr. Pathol. 10, 55e64. https://doi.org/10.1007/BF02738816. Huber, W.E., Price, E.R., Widlund, H.R., Du, J., Davis, I.J., Wegner, M., Fisher, D.E., 2003. A tissue-restricted cAMP transcriptional response: SOX10 modulates alpha-melanocyte-stimulating hormone-triggered expression of microphthalmia-associated transcription factor in melanocytes. J. Biol. Chem. 278, 45224e45230. https://doi.org/10.1074/jbc.M309036200.

163

Huszar, D., Lynch, C.A., Fairchild-Huntress, V., Dunmore, J.H., Fang, Q., Berkemeier, L.R., Gu, W., Kesterson, R.A., Boston, B.A., Cone, R.D., Smith, F.J., Campfield, L.A., Burn, P., Lee, F., 1997. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88, 131e141. Imamine, H., Mizuno, H., Sugiyama, Y., Ohro, Y., Sugiura, T., Togari, H., 2005. Possible relationship between elevated plasma ACTH and tall stature in familial glucocorticoid deficiency. Tohoku J. Exp. Med. 205, 123e131. Imura, H., Matsuyama, H., Matsukura, S., Miyake, T., Fukase, M., 1967. Stability of ACTH preparations in human plasma incubated in vitro. Endocrinology 80, 599e602. https://doi.org/10.1210/ endo-80-4-599. Isales, C.M., Zaidi, M., Blair, H.C., 2010. ACTH is a novel regulator of bone mass. Ann. N. Y. Acad. Sci. 1192, 110e116. https://doi.org/ 10.1111/j.1749-6632.2009.05231.x. Ishii, T., Ishibashi, T., 1989. The ultrastructure of the pars intermedia and the subdivision of its glandular cells in the young sheep pituitary. Arch. Histol. Cytol. 52, 123e133. Jackson, R.S., Creemers, J.W., Ohagi, S., Raffin-Sanson, M.L., Sanders, L., Montague, C.T., Hutton, J.C., O’Rahilly, S., 1997. Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene. Nat. Genet. 16, 303e306. https://doi.org/10.1038/ng0797-303. Jahn, R., Padel, U., Porsch, P.H., So¨ling, H.D., 1982. Adrenocorticotropic hormone and alpha-melanocyte-stimulating hormone induce secretion and protein phosphorylation in the rat lacrimal gland by activation of a cAMP-dependent pathway. Eur. J. Biochem. 126, 623e629. Jansen, E., Ayoubi, T.A., Meulemans, S.M., Van de Ven, W.J., 1995. Neuroendocrine-specific expression of the human prohormone convertase 1 gene. Hormonal regulation of transcription through distinct cAMP response elements. J. Biol. Chem. 270, 15391e15397. Jard, S., Gaillard, R.C., Guillon, G., Marie, J., Schoenenberg, P., Muller, A.F., Manning, M., Sawyer, W.H., 1986. Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol. Pharmacol. 30, 171e177. Ji, L., Wu, H.-T., Qin, X.-Y., Lan, R., 2017. Dissecting carboxypeptidase E: properties, functions and pathophysiological roles in disease. Endocr. Connect. 6, R18eR38. https://doi.org/10.1530/EC-17-0020. Johannsson, G., Nilsson, A.G., Bergthorsdottir, R., Burman, P., Dahlqvist, P., Ekman, B., Engstro¨m, B.E., Olsson, T., Ragnarsson, O., Ryberg, M., Wahlberg, J., Biller, B.M.K., Monson, J.P., Stewart, P.M., Lennerna¨s, H., Skrtic, S., 2012. Improved cortisol exposure-time profile and outcome in patients with adrenal insufficiency: a prospective randomized trial of a novel hydrocortisone dual-release formulation. J. Clin. Endocrinol. Metab. 97, 473e481. https://doi.org/10.1210/jc.2011-1926. Kehl, S.J., 1994. Voltage-clamp analysis of the voltage-gated sodium current of the rat pituitary melanotroph. Neurosci. Lett. 165, 67e70. Keller-Wood, M.E., Dallman, M.F., 1984. Corticosteroid inhibition of ACTH secretion. Endocr. Rev. 5, 1e24. https://doi.org/10.1210/ edrv-5-1-1. Klungland, H., Va˚ge, D.I., Gomez-Raya, L., Adalsteinsson, S., Lien, S., 1995. The role of melanocyte-stimulating hormone (MSH) receptor in bovine coat color determination. Mamm. Genome 6, 636e639. Koshimizu, T., Nakamura, K., Egashira, N., Hiroyama, M., Nonoguchi, H., Tanoue, A., 2012. Vasopressin V1a and V1b receptors: from molecules to physiological systems. Physiol. Rev. 92, 1813e1864. https://doi.org/10.1152/physrev.00035.2011. Krasner, A.S., 1999. Glucocorticoid-induced adrenal insufficiency. J. Am. Med. Assoc. 282, 671e676. Krude, H., Biebermann, H., Luck, W., Horn, R., Brabant, G., Gru¨ters, A., 1998. Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans. Nat. Genet. 19, 155e157. https://doi.org/10.1038/509.

164

8. ANTERIOR PITUITARY AND PARS INTERMEDIA SPACE: CORTICOTROPHS (ACTH) AND MELANOTROPHS (a-MSH)

Krude, H., Biebermann, H., Schnabel, D., Tansek, M.Z., Theunissen, P., Mullis, P.E., Gru¨ters, A., 2003. Obesity due to proopiomelanocortin deficiency: three new cases and treatment trials with thyroid hormone and ACTH4-10. J. Clin. Endocrinol. Metab. 88, 4633e4640. https://doi.org/10.1210/jc.2003-030502. Krueger, R.J., Orme-Johnson, N.R., 1983. Acute adrenocorticotropic hormone stimulation of adrenal corticosteroidogenesis. Discovery of a rapidly induced protein. J. Biol. Chem. 258, 10159e10167. Kudielka, B.M., Buske-Kirschbaum, A., Hellhammer, D.H., Kirschbaum, C., 2004. HPA axis responses to laboratory psychosocial stress in healthy elderly adults, younger adults, and children: impact of age and gender. Psychoneuroendocrinology 29, 83e98. Kuryshev, Y.A., Childs, G.V., Ritchie, A.K., 1996. Corticotropinreleasing hormone stimulates Ca2þ entry through L- and P-type Ca2þ channels in rat corticotropes. Endocrinology 137, 2269e2277. https://doi.org/10.1210/endo.137.6.8641175. Labbe´, O., Desarnaud, F., Eggerickx, D., Vassart, G., Parmentier, M., 1994. Molecular cloning of a mouse melanocortin 5 receptor gene widely expressed in peripheral tissues. Biochemistry 33, 4543e4549. Lafont, C., Desarme´nien, M.G., Cassou, M., Molino, F., Lecoq, J., Hodson, D., Lacampagne, A., Mennessier, G., El Yandouzi, T., Carmignac, D., Fontanaud, P., Christian, H., Coutry, N., Fernandez-Fuente, M., Charpak, S., Le Tissier, P., Robinson, I.C.A.F., Mollard, P., 2010. Cellular in vivo imaging reveals coordinated regulation of pituitary microcirculation and GH cell network function. Proc. Natl. Acad. Sci. U. S. A. 107, 4465e4470. https://doi.org/10.1073/pnas.0902599107. Lamas, M., Molina, C., Foulkes, N.S., Jansen, E., Sassone-Corsi, P., 1997. Ectopic ICER expression in pituitary corticotroph AtT20 cells: effects on morphology, cell cycle, and hormonal production. Mol. Endocrinol. 11, 1425e1434. https://doi.org/10.1210/ mend.11.10.9987. Lamberts, S.W., Verleun, T., Oosterom, R., de Jong, F., Hackeng, W.H., 1984. Corticotropin-releasing factor (ovine) and vasopressin exert a synergistic effect on adrenocorticotropin release in man. J. Clin. Endocrinol. Metab. 58, 298e303. https://doi.org/10.1210/jcem58-2-298. Lamolet, B., Poulin, G., Chu, K., Guillemot, F., Tsai, M.-J., Drouin, J., 2004. Tpit-independent function of NeuroD1(BETA2) in pituitary corticotroph differentiation. Mol. Endocrinol. 18, 995e1003. https://doi.org/10.1210/me.2003-0127. Lamolet, B., Pulichino, A.M., Lamonerie, T., Gauthier, Y., Brue, T., Enjalbert, A., Drouin, J., 2001. A pituitary cell-restricted T box factor, Tpit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 104, 849e859. Lamonerie, T., Tremblay, J.J., Lanctoˆt, C., Therrien, M., Gauthier, Y., Drouin, J., 1996. Ptx1, a bicoid-related homeo box transcription factor involved in transcription of the pro-opiomelanocortin gene. Genes Dev. 10, 1284e1295. Larkin, S., Ansorge, O., 2000. Development and microscopic anatomy of the pituitary gland. In: De Groot, L.J., Chrousos, G., Dungan, K., Feingold, K.R., Grossman, A., Hershman, J.M., Koch, C., Korbonits, M., McLachlan, R., New, M., Purnell, J., Rebar, R., Singer, F., Vinik, A. (Eds.), Endotext. MDText.com, Inc., South Dartmouth, MA. Lavoie, P.-L., Budry, L., Balsalobre, A., Drouin, J., 2008. Developmental dependence on NurRE and EboxNeuro for expression of pituitary proopiomelanocortin. Mol. Endocrinol. 22, 1647e1657. https:// doi.org/10.1210/me.2007-0567. Le Tissier, P., Campos, P., Lafont, C., Romano`, N., Hodson, D.J., Mollard, P., 2017. An updated view of hypothalamic-vascular-pituitary unit function and plasticity. Nat. Rev. Endocrinol. 13, 257e267. https://doi.org/10.1038/nrendo.2016.193.

Le Tissier, P.R., Hodson, D.J., Lafont, C., Fontanaud, P., Schaeffer, M., Mollard, P., 2012. Anterior pituitary cell networks. Front. Neuroendocrinol. 33, 252e266. https://doi.org/10.1016/ j.yfrne.2012.08.002. Lee, A.K., 1996. Dopamine (D2) receptor regulation of intracellular calcium and membrane capacitance changes in rat melanotrophs. J. Physiol. 495, 627e640. Lee, A.K., Tse, F.W., Tse, A., 2015. Arginine vasopressin potentiates the stimulatory action of CRH on pituitary corticotropes via a protein kinase Cedependent reduction of the background TREK-1 current. Endocrinology 156, 3661e3672. https://doi.org/10.1210/ en.2015-1293. Leenders, H.J., de Koning, H.P., Ponten, S.P., Jenks, B.G., Roubos, E.W., 1993. Differential effects of coexisting dopamine, GABA and NPY on alpha-MSH secretion from melanotrope cells of Xenopus laevis. Life Sci. 52, 1969e1975. Li, C.H., Geschwind, I.I., Cole, R.D., Raacke, I.D., Harris, J.I., Dixon, J.S., 1955. Amino-acid sequence of alpha-corticotropin. Nature 176, 687. https://doi.org/10.1038/176687a0. Li, C.H., Geschwind, I.I., Levy, A.L., Harris, J.I., Dixon, J.S., Pon, N.G., Porath, J.O., 1954. Isolation and properties of alpha-corticotrophin from sheep pituitary glands. Nature 173, 251e253. Li, C.H., Simpson, M.E., Evans, H.M., 1942. Isolation of adrenocorticotropic hormone from sheep pituitaries. Science 96, 450. https:// doi.org/10.1126/science.96.2498.450. Liang, Z., Chen, L., McClafferty, H., Lukowski, R., MacGregor, D., King, J.T., Rizzi, S., Sausbier, M., McCobb, D.P., Knaus, H.-G., Ruth, P., Shipston, M.J., 2011. Control of hypothalamic-pituitary-adrenal stress axis activity by the intermediate conductance calciumactivated potassium channel, SK4. J. Physiol. 589, 5965e5986. https://doi.org/10.1113/jphysiol.2011.219378. Lightman, S.L., Conway-Campbell, B.L., 2010. The crucial role of pulsatile activity of the HPA axis for continuous dynamic equilibration. Nat. Rev. Neurosci. 11, 710e718. https://doi.org/ 10.1038/nrn2914. Lolait, S.J., O’Carroll, A.M., Mahan, L.C., Felder, C.C., Button, D.C., Young, W.S., Mezey, E., Brownstein, M.J., 1995. Extrapituitary expression of the rat V1b vasopressin receptor gene. Proc. Natl. Acad. Sci. U. S. A. 92, 6783e6787. Louiset, E., Cazin, L., Lamacz, M., Tonon, M.C., Vaudry, H., 1988. Patch-clamp study of the ionic currents underlying action potentials in cultured frog pituitary melanotrophs. Neuroendocrinology 48, 507e515. https://doi.org/10.1159/000125057. Lu, D., Willard, D., Patel, I.R., Kadwell, S., Overton, L., Kost, T., Luther, M., Chen, W., Woychik, R.P., Wilkison, W.O., 1994. Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor. Nature 371, 799e802. https://doi.org/10.1038/ 371799a0. Lugo, D.I., Pintar, J.E., 1996. Ontogeny of basal and regulated secretion from POMC cells of the developing anterior lobe of the rat pituitary gland. Dev. Biol. 173, 95e109. https://doi.org/10.1006/ dbio.1996.0009. Ma, Z.-Y., Song, Z.-J., Chen, J.-H., Wang, Y.-F., Li, S.-Q., Zhou, L.-F., Mao, Y., Li, Y.-M., Hu, R.-G., Zhang, Z.-Y., Ye, H.-Y., Shen, M., Shou, X.-F., Li, Z.-Q., Peng, H., Wang, Q.-Z., Zhou, D.-Z., Qin, X.L., Ji, J., Zheng, J., Chen, H., Wang, Y., Geng, D.-Y., Tang, W.-J., Fu, C.-W., Shi, Z.-F., Zhang, Y.-C., Ye, Z., He, W.-Q., Zhang, Q.-L., Tang, Q.-S., Xie, R., Shen, J.-W., Wen, Z.-J., Zhou, J., Wang, T., Huang, S., Qiu, H.-J., Qiao, N.-D., Zhang, Y., Pan, L., Bao, W.-M., Liu, Y.-C., Huang, C.-X., Shi, Y.-Y., Zhao, Y., 2015. Recurrent gainof-function USP8 mutations in Cushing’s disease. Cell Res. 25, 306e317. https://doi.org/10.1038/cr.2015.20. Mains, R.E., Eipper, B.A., Ling, N., 1977. Common precursor to corticotropins and endorphins. Proc. Natl. Acad. Sci. U. S. A. 74, 3014e3018.

REFERENCES

Maira, M., Couture, C., Le Martelot, G., Pulichino, A.-M., Bilodeau, S., Drouin, J., 2003. The T-box factor Tpit recruits SRC/p160 coactivators and mediates hormone action. J. Biol. Chem. 278, 46523e46532. https://doi.org/10.1074/jbc.M305626200. Mano-Otagiri, A., Nemoto, T., Yamauchi, N., Kakinuma, Y., Shibasaki, T., 2016. Distribution of corticotrophin-releasing factor type 1 receptor-like immunoreactivity in the rat pituitary. J. Neuroendocrinol. 28 https://doi.org/10.1111/jne.12440. Marklund, L., Moller, M.J., Sandberg, K., Andersson, L., 1996. A missense mutation in the gene for melanocyte-stimulating hormone receptor (MC1R) is associated with the chestnut coat color in horses. Mamm. Genome 7, 895e899. Martens, C., Bilodeau, S., Maira, M., Gauthier, Y., Drouin, J., 2005. Protein-protein interactions and transcriptional antagonism between the subfamily of NGFI-B/Nur77 orphan nuclear receptors and glucocorticoid receptor. Mol. Endocrinol. 19, 885e897. https://doi.org/10.1210/me.2004-0333. Matsuyama, H., Mims, R.B., Ruhmann-Wennhold, A., Nelson, D.H., 1971. Bioassay and radioimmunoassay of plasma ACTH in adrenalectomized rats. Endocrinology 88, 696e701. https://doi.org/ 10.1210/endo-88-3-696. Matsuyama, H., Ruhmann-Wennhold, A., Johnson, L.R., Nelson, D.H., 1972. Disappearance rates of exogenous and endogenous ACTH from rat plasma measured by bioassay and radioimmunoassay. Metabolism 21, 30e35. Mayran, A., Khetchoumian, K., Hariri, F., Pastinen, T., Gauthier, Y., Balsalobre, A., Drouin, J., 2018. Pioneer factor Pax7 deploys a stable enhancer repertoire for specification of cell fate. Nat. Genet. 50, 259e269. https://doi.org/10.1038/s41588-017-0035-2. Mendiratta, M.S., Yang, Y., Balazs, A.E., Willis, A.S., Eng, C.M., Karaviti, L.P., Potocki, L., 2011. Early onset obesity and adrenal insufficiency associated with a homozygous POMC mutation. Int. J. Pediatr. Endocrinol. 2011, 5. https://doi.org/10.1186/1687-98562011-5. Metherell, L.A., Chapple, J.P., Cooray, S., David, A., Becker, C., Ru¨schendorf, F., Naville, D., Begeot, M., Khoo, B., Nu¨rnberg, P., Huebner, A., Cheetham, M.E., Clark, A.J.L., 2005. Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2. Nat. Genet. 37, 166e170. https://doi.org/10.1038/ng1501. Metherell, L.A., Naville, D., Halaby, G., Begeot, M., Huebner, A., Nu¨rnberg, G., Nu¨rnberg, P., Green, J., Tomlinson, J.W., Krone, N.P., Lin, L., Racine, M., Berney, D.M., Achermann, J.C., Arlt, W., Clark, A.J.L., 2009. Nonclassic lipoid congenital adrenal hyperplasia masquerading as familial glucocorticoid deficiency. J. Clin. Endocrinol. Metab. 94, 3865e3871. https://doi.org/ 10.1210/jc.2009-0467. Meunier, H., Labrie, F., 1982. beta-Adrenergic, CRF-ergic and dopaminergic mechanisms controlling alpha-MSH secretion in rat pars intermedia cells in primary culture. Prog. NeuroPsychopharmacol. Biol. Psychiatry 6, 411e415. ˚ ., Holst, B., CondeMøller, C.L., Raun, K., Jacobsen, M.L., Pedersen, T.A Frieboes, K.W., Wulff, B.S., 2011. Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved. Mol. Cell. Endocrinol. 341, 9e17. https://doi.org/10.1016/j.mce.2011.03.010. Morgan, C., Cone, R.D., 2006. Melanocortin-5 receptor deficiency in mice blocks a novel pathway influencing pheromone-induced aggression. Behav. Genet. 36, 291e300. https://doi.org/10.1007/ s10519-005-9024-9. Morgan, C., Thomas, R.E., Ma, W., Novotny, M.V., Cone, R.D., 2004. Melanocortin-5 receptor deficiency reduces a pheromonal signal for aggression in male mice. Chem. Senses 29, 111e115. Mountjoy, K.G., 2015. Pro-opiomelanocortin (POMC) neurones, POMC-derived peptides, melanocortin receptors and obesity: how understanding of this system has changed over the last

165

decade. J. Neuroendocrinol. 27, 406e418. https://doi.org/ 10.1111/jne.12285. Mountjoy, K.G., 2010. Functions for pro-opiomelanocortin-derived peptides in obesity and diabetes. Biochem. J. 428, 305e324. https://doi.org/10.1042/BJ20091957. Mountjoy, K.G., Mortrud, M.T., Low, M.J., Simerly, R.B., Cone, R.D., 1994. Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol. Endocrinol. 8, 1298e1308. https://doi.org/10.1210/mend.8.10.7854347. Mountjoy, K.G., Robbins, L.S., Mortrud, M.T., Cone, R.D., 1992. The cloning of a family of genes that encode the melanocortin receptors. Science 257, 1248e1251. Muglia, L., Jacobson, L., Dikkes, P., Majzoub, J.A., 1995. Corticotropinreleasing hormone deficiency reveals major fetal but not adult glucocorticoid need. Nature 373, 427e432. https://doi.org/ 10.1038/373427a0. Murakami, M., Yoshida, T., Nakayama, Y., Hashimoto, J., Hirata, S., 1968. The fine structure of the pars intermedia of the pituitary in human fetus. Arch. Histol. Jpn. Nihon Soshikigaku Kiroku 30, 61e73. Nakanishi, S., Inoue, A., Kita, T., Inoue, A., Nakamura, M., Chang, A.C.Y., Cohen, S.N., Numa, S., 1979. Nucleotide sequence of cloned cDNA for bovine corticotropin-b-lipotropin precursor. Nature 278, 423e427. https://doi.org/10.1038/278423a0. Nasti, T.H., Timares, L., 2015. Invited review MC1R, eumelanin and pheomelanin: their role in determining the susceptibility to skin cancer. Photochem. Photobiol. 91, 188e200. https://doi.org/ 10.1111/php.12335. Naville, D., Barjhoux, L., Jaillard, C., Faury, D., Despert, F., Esteva, B., Durand, P., Saez, J.M., Begeot, M., 1996. Demonstration by transfection studies that mutations in the adrenocorticotropin receptor gene are one cause of the hereditary syndrome of glucocorticoid deficiency. J. Clin. Endocrinol. Metab. 81, 1442e1448. https:// doi.org/10.1210/jcem.81.4.8636348. Newton, R.A., Roberts, D.W., Leonard, J.H., Sturm, R.A., 2007. Human melanocytes expressing MC1R variant alleles show impaired activation of multiple signaling pathways. Peptides 28, 2387e2396. https://doi.org/10.1016/j.peptides.2007.10.003. Newton, R.A., Smit, S.E., Barnes, C.C., Pedley, J., Parsons, P.G., Sturm, R.A., 2005. Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells. Peptides 26, 1818e1824. https://doi.org/10.1016/ j.peptides.2004.11.031. Nilsson Sko¨ld, H., Aspengren, S., Wallin, M., 2013. Rapid color change in fish and amphibians e function, regulation, and emerging applications. Pigm. Cell Melanoma Res. 26, 29e38. https:// doi.org/10.1111/pcmr.12040. Noon, L.A., Franklin, J.M., King, P.J., Goulding, N.J., Hunyady, L., Clark, A.J.L., 2002. Failed export of the adrenocorticotrophin receptor from the endoplasmic reticulum in non-adrenal cells: evidence in support of a requirement for a specific adrenal accessory factor. J. Endocrinol. 174, 17e25. Nussey, S.S., Soo, S.C., Gibson, S., Gout, I., White, A., Bain, M., Johnstone, A.P., 1993. Isolated congenital ACTH deficiency: a cleavage enzyme defect? Clin. Endocrinol. 39, 381e385. Oksnes, M., Bjo¨rnsdottir, S., Isaksson, M., Methlie, P., Carlsen, S., Nilsen, R.M., Broman, J.-E., Triebner, K., Ka¨mpe, O., Hulting, A.L., Bensing, S., Husebye, E.S., Løva˚s, K., 2014. Continuous subcutaneous hydrocortisone infusion versus oral hydrocortisone replacement for treatment of Addison’s disease: a randomized clinical trial. J. Clin. Endocrinol. Metab. 99, 1665e1674. https:// doi.org/10.1210/jc.2013-4253. Opdecamp, K., Nakayama, A., Nguyen, M.T., Hodgkinson, C.A., Pavan, W.J., Arnheiter, H., 1997. Melanocyte development in vivo and in neural crest cell cultures: crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor. Development 124, 2377e2386.

166

8. ANTERIOR PITUITARY AND PARS INTERMEDIA SPACE: CORTICOTROPHS (ACTH) AND MELANOTROPHS (a-MSH)

Ouleghzal, H., Rosales, C., Raffin-Sanson, M.-L., 2012. Treatment of corticotroph deficiency. Ann. Endocrinol. 73, 12e19. https:// doi.org/10.1016/j.ando.2012.01.001. Overman, R.A., Yeh, J.-Y., Deal, C.L., 2013. Prevalence of oral glucocorticoid usage in the United States: a general population perspective. Arthritis Care Res. 65, 294e298. https://doi.org/10.1002/ acr.21796. Oyarce, A.M., Hand, T.A., Mains, R.E., Eipper, B.A., 1996. Dopaminergic regulation of secretory granule-associated proteins in rat intermediate pituitary. J. Neurochem. 67, 229e241. Panin, M., Giurisato, M., Peruffo, A., Ballarin, C., Cozzi, B., 2013. Immunofluorescence evidence of melanotrophs in the pituitary of four odontocete species. An immunohistochemical study and a critical review of the literature. Ann. Anat. Anat. Anz. 195, 512e521. https://doi.org/10.1016/j.aanat.2013.06.004. Perez-Rivas, L.G., Theodoropoulou, M., Ferrau`, F., Nusser, C., Kawaguchi, K., Stratakis, C.A., Faucz, F.R., Wildemberg, L.E., Assie´, G., Beschorner, R., Dimopoulou, C., Buchfelder, M., Popovic, V., Berr, C.M., To´th, M., Ardisasmita, A.I., Honegger, J., Bertherat, J., Gadelha, M.R., Beuschlein, F., Stalla, G., Komada, M., Korbonits, M., Reincke, M., 2015. The gene of the ubiquitin-specific protease 8 is frequently mutated in adenomas causing Cushing’s disease. J. Clin. Endocrinol. Metab. 100, E997eE1004. https://doi.org/10.1210/jc.2015-1453. Philips, A., Lesage, S., Gingras, R., Maira, M.H., Gauthier, Y., Hugo, P., Drouin, J., 1997. Novel dimeric Nur77 signaling mechanism in endocrine and lymphoid cells. Mol. Cell. Biol. 17, 5946e5951. Plaut, A., 1936. Investigations on the pars intermedia of the hypophysis in anthropoid apes and man. J. Anat. 70, 242e249. Poulin, G., Lebel, M., Chamberland, M., Paradis, F.W., Drouin, J., 2000. Specific protein-protein interaction between basic helix-loop-helix transcription factors and homeoproteins of the Pitx family. Mol. Cell. Biol. 20, 4826e4837. Poulin, G., Turgeon, B., Drouin, J., 1997. NeuroD1/beta2 contributes to cell-specific transcription of the proopiomelanocortin gene. Mol. Cell. Biol. 17, 6673e6682. Pozzoli, G., Bilezikjian, L.M., Perrin, M.H., Blount, A.L., Vale, W.W., 1996. Corticotropin-releasing factor (CRF) and glucocorticoids modulate the expression of type 1 CRF receptor messenger ribonucleic acid in rat anterior pituitary cell cultures. Endocrinology 137, 65e71. https://doi.org/10.1210/endo.137.1.8536643. Price, E.R., Horstmann, M.A., Wells, A.G., Weilbaecher, K.N., Takemoto, C.M., Landis, M.W., Fisher, D.E., 1998. a-melanocytestimulating hormone signaling regulates expression of microphthalmia, a gene deficient in Waardenburg syndrome. J. Biol. Chem. 273, 33042e33047. https://doi.org/10.1074/jbc.273.49.33042. Proulx-Ferland, L., Labrie, F., Dumont, D., Coˆte´, J., Coy, D.H., Sveiraf, J., 1982. Corticotropin-releasing factor stimulates secretion of melanocyte-stimulating hormone from the rat pituitary. Science 217, 62e63. Prusis, P., Schio¨th, H.B., Muceniece, R., Herzyk, P., Afshar, M., Hubbard, R.E., Wikberg, J.E.S., 1997. Modeling of the threedimensional structure of the human melanocortin 1 receptor, using an automated method and docking of a rigid cyclic melanocytestimulating hormone core peptide. J. Mol. Graph. Model. 15, 307e317. https://doi.org/10.1016/S1093-3263(98)00004-7. Pulichino, A.-M., Vallette-Kasic, S., Tsai, J.P.-Y., Couture, C., Gauthier, Y., Drouin, J., 2003. Tpit determines alternate fates during pituitary cell differentiation. Genes Dev. 17, 738e747. https:// doi.org/10.1101/gad.1065703. Raff, H., Carroll, T., 2015. Cushing’s syndrome: from physiological principles to diagnosis and clinical care. J. Physiol. 593, 493e506. https://doi.org/10.1113/jphysiol.2014.282871. Rahn, H., Painter, B.T., 1941. A comparative histology of the bird pituitary. Anat. Rec. 79, 297e311. https://doi.org/10.1002/ ar.1090790304.

Ramachandran, J., Farmer, S.W., Liles, S., Li, C.H., 1976. Comparison of the steroidogenic and melanotropic activities of corticotropin, alpha-melanotropin and analogs with their lipolytic activities in rat and rabbit adipocytes. Biochim. Biophys. Acta 428, 347e354. Rasmussen, A.T., 1930. Origin of the basophilic cells in the posterior lobe of the human hypophysis. Am. J. Anat. 46, 461e475. https://doi.org/10.1002/aja.1000460305. Reincke, M., Sbiera, S., Hayakawa, A., Theodoropoulou, M., Osswald, A., Beuschlein, F., Meitinger, T., Mizuno-Yamasaki, E., Kawaguchi, K., Saeki, Y., Tanaka, K., Wieland, T., Graf, E., Saeger, W., Ronchi, C.L., Allolio, B., Buchfelder, M., Strom, T.M., Fassnacht, M., Komada, M., 2015. Mutations in the deubiquitinase gene USP8 cause Cushing’s disease. Nat. Genet. 47, 31e38. https://doi.org/10.1038/ng.3166. Ritchie, A.K., Kuryshev, Y.A., Childs, G.V., 1996. Corticotropinreleasing hormone and calcium signaling in corticotropes. Trends Endocrinol. Metab. 7, 365e369. Robbins, L.S., Nadeau, J.H., Johnson, K.R., Kelly, M.A., RoselliRehfuss, L., Baack, E., Mountjoy, K.G., Cone, R.D., 1993. Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72, 827e834. Roberts, J.L., Herbert, E., 1977. Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule. Proc. Natl. Acad. Sci. U. S. A. 74, 4826e4830. Romano`, N., McClafferty, H., Walker, J.J., Le Tissier, P., Shipston, M.J., 2017. Heterogeneity of calcium responses to secretagogues in corticotrophs from male rats. Endocrinology. https://doi.org/10.1210/ en.2017-00107. Roper, J., O’Carroll, A.-M., Young, W., Lolait, S., 2011. The vasopressin Avpr1b receptor: molecular and pharmacological studies. Stress 14, 98e115. https://doi.org/10.3109/10253890.2010.512376. Roper, J.A., Craighead, M., O’Carroll, A.-M., Lolait, S.J., 2010. Attenuated stress response to acute restraint and forced swimming stress in arginine vasopressin 1b receptor subtype (Avpr1b) receptor knockout mice and wild-type mice treated with a novel Avpr1b receptor antagonist. J. Neuroendocrinol. 22, 1173e1180. https:// doi.org/10.1111/j.1365-2826.2010.02070.x. Roselli-Rehfuss, L., Mountjoy, K.G., Robbins, L.S., Mortrud, M.T., Low, M.J., Tatro, J.B., Entwistle, M.L., Simerly, R.B., Cone, R.D., 1993. Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system. Proc. Natl. Acad. Sci. U. S. A. 90, 8856e8860. Rousseau, K., Kauser, S., Pritchard, L.E., Warhurst, A., Oliver, R.L., Slominski, A., Wei, E.T., Thody, A.J., Tobin, D.J., White, A., 2007. Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis. FASEB J. 21, 1844e1856. https:// doi.org/10.1096/fj.06-7398com. Roy, S., Roy, S.J., Pinard, S., Taillefer, L.-D., Rached, M., Parent, J.-L., Gallo-Payet, N., 2011. Mechanisms of melanocortin-2 receptor (MC2R) internalization and recycling in human embryonic kidney (HEK) cells: identification of Key Ser/Thr (S/T) amino acids. Mol. Endocrinol. 25, 1961e1977. https://doi.org/10.1210/me.2011-0018. Russell, G.M., Durant, C., Ataya, A., Papastathi, C., Bhake, R., Woltersdorf, W., Lightman, S., 2014. Subcutaneous pulsatile glucocorticoid replacement therapy. Clin. Endocrinol. 81, 289e293. https://doi.org/10.1111/cen.12470. Russell, G.M., Lightman, S.L., 2014. Can side effects of steroid treatments be minimized by the temporal aspects of delivery method? Expert Opin. Drug Saf. 13, 1501e1513. https://doi.org/10.1517/ 14740338.2014.965141. Sahm, U.G., Olivier, G.W.J., Branch, S.K., Moss, S.H., Pouton, C.W., 1994. Synthesis and biological evaluation of a-MSH analogues substituted with alanine. Peptides 15, 1297e1302. https:// doi.org/10.1016/0196-9781(94)90157-0.

REFERENCES

Saiardi, A., Bozzi, Y., Baik, J.H., Borrelli, E., 1997. Antiproliferative role of dopamine: loss of D2 receptors causes hormonal dysfunction and pituitary hyperplasia. Neuron 19, 115e126. Sa´nchez-Ma´s, J., Guillo, L.A., Zanna, P., Jime´nez-Cervantes, C., Garcı´aBorro´n, J.C., 2005. Role of G protein-coupled receptor kinases in the homologous desensitization of the human and mouse melanocortin 1 receptors. Mol. Endocrinol. 19, 1035e1048. https://doi.org/ 10.1210/me.2004-0227. Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev. 21, 55e89. https://doi.org/10.1210/edrv.21.1.0389. Sbiera, S., Deutschbein, T., Weigand, I., Reincke, M., Fassnacht, M., Allolio, B., 2015. The new molecular landscape of Cushing’s disease. Trends Endocrinol. Metab. 26, 573e583. https://doi.org/ 10.1016/j.tem.2015.08.003. Schio¨th, H.B., Haitina, T., Ling, M.K., Ringholm, A., Fredriksson, R., Cerda´-Reverter, J.M., Klovins, J., 2005. Evolutionary conservation of the structural, pharmacological, and genomic characteristics of the melanocortin receptor subtypes. Peptides 26, 1886e1900. https://doi.org/10.1016/j.peptides.2004.11.034. Schio¨th, H.B., Muceniece, R., Szardenings, M., Prusis, P., Lindeberg, G., Sharma, S.D., Hruby, V.J., Wikberg, J.E.S., 1997. Characterisation of D117A and H260A mutations in the melanocortin 1 receptor. Mol. Cell. Endocrinol. 126, 213e219. https://doi.org/10.1016/S03037207(96)03993-7. Schio¨th, H.B., Raudsepp, T., Ringholm, A., Fredriksson, R., Takeuchi, S., Larhammar, D., Chowdhary, B.P., 2003. Remarkable synteny conservation of melanocortin receptors in chicken, human, and other vertebrates. Genomics 81, 504e509. Schnabel, E., Mains, R.E., Farquhar, M.G., 1989. Proteolytic processing of pro-ACTH/endorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells. Mol. Endocrinol. 3, 1223e1235. https://doi.org/10.1210/mend-3-8-1223. Schonemann, M.D., Ryan, A.K., McEvilly, R.J., O’Connell, S.M., Arias, C.A., Kalla, K.A., Li, P., Sawchenko, P.E., Rosenfeld, M.G., 1995. Development and survival of the endocrine hypothalamus and posterior pituitary gland requires the neuronal POU domain factor Brn-2. Genes Dev. 9, 3122e3135. Schulte, H.M., Oldfield, E.H., Allolio, B., Katz, D.A., Berkman, R.A., Ali, I.U., 1991. Clonal composition of pituitary adenomas in patients with Cushing’s disease: determination by X-chromosome inactivation analysis. J. Clin. Endocrinol. Metab. 73, 1302e1308. https://doi.org/10.1210/jcem-73-6-1302. Seasholtz, A.F., Burrows, H.L., Karolyi, I.J., Camper, S.A., 2001. Mouse models of altered CRH-binding protein expression. Peptides 22, 743e751. Sebag, J.A., Hinkle, P.M., 2010. Regulation of G proteinecoupled receptor signaling: specific dominant-negative effects of melanocortin 2 receptor accessory protein 2. Sci. Signal. 3, ra28. https://doi.org/ 10.1126/scisignal.2000593. Sebag, J.A., Hinkle, P.M., 2009. Opposite effects of the melanocortin-2 (MC2) receptor accessory protein MRAP on MC2 and MC5 receptor dimerization and trafficking. J. Biol. Chem. 284, 22641e22648. https://doi.org/10.1074/jbc.M109.022400. Sebag, J.A., Hinkle, P.M., 2007. Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers. Proc. Natl. Acad. Sci. U. S. A. 104, 20244e20249. https://doi.org/10.1073/pnas.0708916105. Seidah, N.G., Chre´tien, M., 1999. Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides. Brain Res. 848, 45e62. https://doi.org/10.1016/ S0006-8993(99)01909-5. Published on the World Wide Web on 17 August 1999.

167

Seidah, N.G., Marcinkiewicz, M., Benjannet, S., Gaspar, L., Beaubien, G., Mattei, M.G., Lazure, C., Mbikay, M., Chre´tien, M., 1991. Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol. Endocrinol. 5, 111e122. https://doi.org/10.1210/mend-5-1-111. Seidah, N.G., Mayer, G., Zaid, A., Rousselet, E., Nassoury, N., Poirier, S., Essalmani, R., Prat, A., 2008. The activation and physiological functions of the proprotein convertases. Int. J. Biochem. Cell Biol. 40, 1111e1125. https://doi.org/10.1016/j.biocel.2008.01.030. Sharp, B., Pekary, A.E., 1981. b-endorphin 61e91 and other b-endorphin-immunoreactive peptides in human semen. J. Clin. Endocrinol. Metab. 52, 586e588. https://doi.org/10.1210/jcem-52-3-586. Sherman, B., Wysham, C., Pfohl, B., 1985. Age-related changes in the circadian rhythm of plasma cortisol in man. J. Clin. Endocrinol. Metab. 61, 439e443. https://doi.org/10.1210/jcem-61-3-439. Shibahara, S., Takeda, K., Yasumoto, K., Udono, T., Watanabe, K., Saito, H., Takahashi, K., 2001. Microphthalmia-associated transcription factor (MITF): multiplicity in structure, function, and regulation. J. Investig. Dermatol. Symp. Proc. 6, 99e104. https:// doi.org/10.1046/j.0022-202x.2001.00010.x. Shibuya, I., Douglas, W.W., 1993. Spontaneous cytosolic calcium pulsing detected in Xenopus melanotrophs: modulation by secretoinhibitory and stimulant ligands. Endocrinology 132, 2166e2175. https://doi.org/10.1210/endo.132.5.8386613. Shipston, M.J., Antoni, F.A., 1991. Early glucocorticoid feedback in anterior pituitary corticotrophs: differential inhibition of hormone release induced by vasopressin and corticotrophin-releasing factor in vitro. J. Endocrinol. 129, 261e268. Shu-Dong, T., Phillips, D.M., Halmi, N., Krieger, D., Bardin, C.W., 1982. Beta-endorphin is present in the male reproductive tract of five species. Biol. Reprod. 27, 755e764. Smith, G.W., Aubry, J.M., Dellu, F., Contarino, A., Bilezikjian, L.M., Gold, L.H., Chen, R., Marchuk, Y., Hauser, C., Bentley, C.A., Sawchenko, P.E., Koob, G.F., Vale, W., Lee, K.F., 1998. Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron 20, 1093e1102. Spencer, J.D., Schallreuter, K.U., 2009. Regulation of pigmentation in human epidermal melanocytes by functional high-affinity betamelanocyte-stimulating hormone/melanocortin-4 receptor signaling. Endocrinology 150, 1250e1258. https://doi.org/ 10.1210/en.2008-1212. Spiga, F., Harrison, L.R., Wood, S., Knight, D.M., MacSweeney, C.P., Thomson, F., Craighead, M., Lightman, S.L., 2009. Blockade of the V(1b) receptor reduces ACTH, but not corticosterone secretion induced by stress without affecting basal hypothalamic-pituitaryadrenal axis activity. J. Endocrinol. 200, 273e283. https:// doi.org/10.1677/JOE-08-0421. Spiga, F., Walker, J.J., Terry, J.R., Lightman, S.L., 2014. HPA axisrhythms. Compr. Physiol. 4, 1273e1298. https://doi.org/10.1002/ cphy.c140003. Spiga, F., Zavala, E., Walker, J.J., Zhao, Z., Terry, J.R., Lightman, S.L., 2017. Dynamic responses of the adrenal steroidogenic regulatory network. Proc. Natl. Acad. Sci. U. S. A. 114, E6466eE6474. https://doi.org/10.1073/pnas.1703779114. Stack, J., Surprenant, A., 1991. Dopamine actions on calcium currents, potassium currents and hormone release in rat melanotrophs. J. Physiol. 439, 37e58. Stojilkovic, S.S., Tabak, J., Bertram, R., 2010. Ion channels and signaling in the pituitary gland. Endocr. Rev. 31, 845e915. https://doi.org/ 10.1210/er.2010-0005.

168

8. ANTERIOR PITUITARY AND PARS INTERMEDIA SPACE: CORTICOTROPHS (ACTH) AND MELANOTROPHS (a-MSH)

Sundstro¨m, G., Dreborg, S., Larhammar, D., 2010. Concomitant duplications of opioid peptide and receptor genes before the origin of jawed vertebrates. PLoS One 5, e10512. https://doi.org/10.1371/ journal.pone.0010512. Swope, V., Jameson, J., McFarland, K., Supp, D., Miller, W., McGraw, D., Patel, M.A., Nix, M.A., Milhauser, G., Babcock, G., Abdel-Malek, Z.A., 2012. Defining MC1R regulation in human melanocytes by its agonist a-melanocortin and antagonists agouti signaling protein and b-defensin 3. J. Investig. Dermatol. 132, 2255e2262. https://doi.org/10.1038/jid.2012.135. Takeuchi, M., 2001. The mammalian pars intermedia e structure and function. Zool. Sci. 18, 133e144. Takumi, I., Steiner, D.F., Sanno, N., Teramoto, A., Osamura, R.Y., 1998. Localization of prohormone convertases 1/3 and 2 in the human pituitary gland and pituitary adenomas: analysis by immunohistochemistry, immunoelectron microscopy, and laser scanning microscopy. Mod. Pathol. 11, 232e238. Tam, W.W.H., Andreasson, K.I., Loh, Y.P., 1993. The amino-terminal sequence of pro-opiomelanocortin directs intracellular targeting to the regulated secretory pathway. Eur. J. Cell Biol. 62, 294e306. Tanoue, A., Ito, S., Honda, K., Oshikawa, S., Kitagawa, Y., Koshimizu, T.-A., Mori, T., Tsujimoto, G., 2004. The vasopressin V1b receptor critically regulates hypothalamic-pituitary-adrenal axis activity under both stress and resting conditions. J. Clin. Investig. 113, 302e309. https://doi.org/10.1172/JCI19656. Taraskevich, P.S., Douglas, W.W., 1990. Dopamine (D2) or gammaaminobutyric acid (GABAB) receptor activation hyperpolarizes rat melanotrophs and pertussis toxin blocks these responses and the accompanying fall in [Ca2þ]i. Neurosci. Lett. 112, 205e209. Taraskevich, P.S., Douglas, W.W., 1982. GABA directly affects electrophysiological properties of pituitary pars intermedia cells. Nature 299, 733e734. Tatro, J.B., Reichlin, S., 1987. Specific receptors for alpha-melanocytestimulating hormone are widely distributed in tissues of rodents. Endocrinology 121, 1900e1907. https://doi.org/10.1210/endo121-5-1900. Therrien, M., Drouin, J., 1991. Pituitary pro-opiomelanocortin gene expression requires synergistic interactions of several regulatory elements. Mol. Cell. Biol. 11, 3492e3503. Thiboutot, D., Sivarajah, A., Gilliland, K., Cong, Z., Clawson, G., 2000. The melanocortin 5 receptor is expressed in human sebaceous glands and rat preputial cells. J. Investig. Dermatol. 115, 614e619. https://doi.org/10.1046/j.1523-1747.2000.00094.x. Thody, A.J., Higgins, E.M., Wakamatsu, K., Ito, S., Burchill, S.A., Marks, J.M., 1991. Pheomelanin as well as eumelanin is present in human epidermis. J. Investig. Dermatol. 97, 340e344. Thody, A.J., Shuster, S., 1975. Control of sebaceous gland function in the rat by alpha-melanocyte-stimulating hormone. J. Endocrinol. 64, 503e510. Thody, A.J., Shuster, S., 1973. Possible role of MSH in the mammal. Nature 245, 207e209. https://doi.org/10.1038/245207a0. Timpl, P., Spanagel, R., Sillaber, I., Kresse, A., Reul, J.M., Stalla, G.K., Blanquet, V., Steckler, T., Holsboer, F., Wurst, W., 1998. Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1. Nat. Genet. 19, 162e166. https://doi.org/10.1038/520. Tomiko, S.A., Taraskevich, P.S., Douglas, W.W., 1983. GABA acts directly on cells of pituitary pars intermedia to alter hormone output. Nature 301, 706e707. Tonon, M.-C., Danger, J.-M., Lamacz, M., Leroux, P., Adjeroud, S., Andersen, A.C., Verburg-van, K.L., Jenks, B.G., Pelletier, G., Stoeckel, L., Burlet, A., Kupryszewski, G., Vaudry, H., 1988. Multihormonal control of melanotropin secretion in cold-blooded vertebrates. In: The Melanotropic Peptides. Taylor & Francis Group.

Tonon, M.C., Desrues, L., Lamacz, M., Chartrel, N., Jenks, B., Vaudry, H., 1993. Multihormonal regulation of pituitary melanotrophs. Ann. N. Y. Acad. Sci. 680, 175e187. Tremblay, J.J., Lanctoˆt, C., Drouin, J., 1998. The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Limhomeodomain gene Lim3/Lhx3. Mol. Endocrinol. 12, 428e441. https://doi.org/10.1210/mend.12.3.0073. Trouslard, J., Demeneix, B.A., Feltz, P., 1989. Spontaneous spiking activities of porcine pars intermedia cells: effects of thyrotropinreleasing hormone. Neuroendocrinology 50, 33e43. https:// doi.org/10.1159/000125199. Tse, A., Lee, A.K., 2000. Voltage-gated Ca2þ channels and intracellular Ca2þ release regulate exocytosis in identified rat corticotrophs. J. Physiol. 528 (Pt 1), 79e90. Tse, A., Lee, A.K., 1998. Arginine vasopressin triggers intracellular calcium release, a calcium-activated potassium current and exocytosis in identified rat corticotropes. Endocrinology 139, 2246e2252. https://doi.org/10.1210/endo.139.5.5999. Tsigos, C., 1999. Isolated glucocorticoid deficiency and ACTH receptor mutations. Arch. Med. Res. 30, 475e480. https://doi.org/10.1016/ S0188-0128(99)00057-3. Va˚ge, D.I., Lu, D., Klungland, H., Lien, S., Adalsteinsson, S., Cone, R.D., 1997. A non-epistatic interaction of agouti and extension in the fox, Vulpes vulpes. Nat. Genet. 15, 311e315. https://doi.org/10.1038/ ng0397-311. Vale, W., Spiess, J., Rivier, C., Rivier, J., 1981. Characterization of a 41residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213, 1394e1397. Vale, W., Vaughan, J., Smith, M., Yamamoto, G., Rivier, J., Rivier, C., 1983. Effects of synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines, neurohypophysial peptides, and other substances on cultured corticotropic cells. Endocrinology 113, 1121e1131. https://doi.org/10.1210/endo-113-3-1121. Valentijn, J.A., Louiset, E., Vaudry, H., Cazin, L., 1991a. Involvement of non-selective cationic channels in the generation of pacemaker depolarizations and firing behaviour in cultured frog melanotrophs. Brain Res. 560, 175e180. Valentijn, J.A., Louiset, E., Vaudry, H., Cazin, L., 1991b. Dopamine regulates the electrical activity of frog melanotrophs through a G protein-mediated mechanism. Neuroscience 44, 85e95. Vallette-Kasic, S., Brue, T., Pulichino, A.-M., Gueydan, M., Barlier, A., David, M., Nicolino, M., Malpuech, G., De´chelotte, P., Deal, C., Van Vliet, G., De Vroede, M., Riepe, F.G., Partsch, C.-J., Sippell, W.G., Berberoglu, M., Atasay, B., de Zegher, F., Beckers, D., Kyllo, J., Donohoue, P., Fassnacht, M., Hahner, S., Allolio, B., Noordam, C., Dunkel, L., Hero, M., Pigeon, B., Weill, J., Yigit, S., Brauner, R., Heinrich, J.J., Cummings, E., Riddell, C., Enjalbert, A., Drouin, J., 2005. Congenital isolated adrenocorticotropin deficiency: an underestimated cause of neonatal death, explained by TPIT gene mutations. J. Clin. Endocrinol. Metab. 90, 1323e1331. https://doi.org/10.1210/jc.2004-1300. Valverde, P., Healy, E., Jackson, I., Rees, J.L., Thody, A.J., 1995. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nat. Genet. 11, 328e330. https://doi.org/10.1038/ng1195-328. van Staa, T.P., Leufkens, H.G., Abenhaim, L., Begaud, B., Zhang, B., Cooper, C., 2000. Use of oral corticosteroids in the United Kingdom. QJM 93, 105e111. Vance, K.W., Goding, C.R., 2004. The transcription network regulating melanocyte development and melanoma. Pigm. Cell Res. 17, 318e325. https://doi.org/10.1111/j.1600-0749.2004.00164.x. Vaudry, H., Chartrel, N., Desrues, L., Galas, L., Kikuyama, S., Mor, A., Nicolas, P., Tonon, M.C., 2006. The pituitary-skin connection in amphibians: reciprocal regulation of melanotrope cells and dermal

REFERENCES

melanocytes. Ann. N. Y. Acad. Sci. 885, 41e56. https://doi.org/ 10.1111/j.1749-6632.1999.tb08664.x. Veldhuis, J.D., 2013. Changes in pituitary function with aging and implications for patient care. Nat. Rev. Endocrinol. 9, 205e215. https://doi.org/10.1038/nrendo.2013.38. Verma, S., Vanryzin, C., Sinaii, N., Kim, M.S., Nieman, L.K., Ravindran, S., Calis, K.A., Arlt, W., Ross, R.J., Merke, D.P., 2010. A pharmacokinetic and pharmacodynamic study of delayed- and extended-release hydrocortisone (Chronocort) vs. conventional hydrocortisone (Cortef) in the treatment of congenital adrenal hyperplasia. Clin. Endocrinol. 72, 441e447. https://doi.org/ 10.1111/j.1365-2265.2009.03636.x. Wakamatsu, K., Graham, A., Cook, D., Thody, A.J., 1997. Characterisation of ACTH peptides in human skin and their activation of the melanocortin-1 receptor. Pigm. Cell Res. 10, 288e297. Wallingford, N., Perroud, B., Gao, Q., Coppola, A., Gyengesi, E., Liu, Z.-W., Gao, X.-B., Diament, A., Haus, K.A., Shariat-Madar, Z., Mahdi, F., Wardlaw, S.L., Schmaier, A.H., Warden, C.H., Diano, S., 2009. Prolylcarboxypeptidase regulates food intake by inactivating alpha-MSH in rodents. J. Clin. Investig. 119, 2291e2303. https:// doi.org/10.1172/JCI37209. Weber, A., Clark, A.J.L., 1994. Mutations of the ACTH receptor gene are only one cause of familial glucocorticoid deficiency. Hum. Mol. Genet. 3, 585e588. https://doi.org/10.1093/hmg/3.4.585. Wislocki, G.B., 1940. The topography of the hypophysis in the elephant, manatee and hyrax. Anat. Rec. 77, 427e445.

169

Yang, Y. k, Dickinson, C., Haskell-Luevano, C., Gantz, I., 1997. Molecular basis for the interaction of [Nle4,D-Phe7]melanocyte stimulating hormone with the human melanocortin-1 receptor. J. Biol. Chem. 272, 23000e23010. Yaswen, L., Diehl, N., Brennan, M.B., Hochgeschwender, U., 1999. Obesity in the mouse model of pro-opiomelanocortin deficiency responds to peripheral melanocortin. Nat. Med. 5, 1066e1070. https://doi.org/10.1038/12506. Zaidi, M., Sun, L., Robinson, L.J., Tourkova, I.L., Liu, L., Wang, Y., Zhu, L.-L., Liu, X., Li, J., Peng, Y., Yang, G., Shi, X., Levine, A., Iqbal, J., Yaroslavskiy, B.B., Isales, C., Blair, H.C., 2010. ACTH protects against glucocorticoid-induced osteonecrosis of bone. Proc. Natl. Acad. Sci. U. S. A. 107, 8782e8787. https://doi.org/ 10.1073/pnas.0912176107. Zemkova, H., Tomic, M., Kucka, M., Aguilera, G., Stojilkovic, S.S., 2016. Spontaneous and CRH-induced excitability and calcium signaling in mice corticotrophs involves sodium, calcium, and cationconducting channels. Endocrinology 157, 1576e1589. https:// doi.org/10.1210/en.2015-1899. Zhong, Q., Sridhar, S., Ruan, L., Ding, K.-H., Xie, D., Insogna, K., Kang, B., Xu, J., Bollag, R.J., Isales, C.M., 2005. Multiple melanocortin receptors are expressed in bone cells. Bone 36, 820e831. https:// doi.org/10.1016/j.bone.2005.01.020. Zhou, Y., Zhang, X., Klibanski, A., 2014. Genetic and epigenetic mutations of tumor suppressive genes in sporadic pituitary adenoma. Mol. Cell. Endocrinol. 386, 16e33. https://doi.org/10.1016/ j.mce.2013.09.006.

C H A P T E R

9 Anterior Pituitary: Somatotrophs (GH) and Lactotrophs (PRL) J.F. Murray, P.R. Le Tissier Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom

1. INTRODUCTION Growth hormone (GH) and prolactin (PRL) share many common features that suggest that they should be considered in parallel. As will be described in this chapter, the hormones have a common evolutionary origin, as do their respective receptors, so there is potential cross-talk between these two hormonal axes. Given a common ancestral origin for the two hormones, it is not surprising that the cells of the anterior pituitary producing these hormones also have a common developmental origin and that there are some cells capable of secreting both hormones. Further common features are that whilst neither is essential for life and both are well-known for a specific function (statural growth and lactation for GH and PRL, respectively), both have an extensive array of pleiotropic actions. The evolution of the hormonal axes, together with variation in their pleiotropic actions between species, suggest that both hormones have critical roles in maximizing the successful survival of a species: in particular, as important mediators facilitating adaptation to a range of environments and survival strategies. Throughout this chapter, we will describe aspects of the two hormonal axes in parallel to emphasize the commonality and distinctions of their evolution, mode of actions, and regulation. We will focus on the roles of the two hormones in humans but draw on studies in rodents and other species where these are informative.

2. STRUCTURE AND REGULATION 2.1 The Structure and Regulation of GH and PRL Bovine GH was first isolated to purity in 1944 (Li and Evans, 1944), but it was not until 1971 that the consensus

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00009-2

amino acid sequence for human GH was published (Niall, 1971; Li and Dixon, 1971). The predominate pituitary GH isoform is a 191-amino acid single peptide chain with two disulphide bridges, with a mass of approximately 22 kDa (Niall et al., 1971) (Fig. 9.1A). The gene encoding GH has five exons, which can be alternatively spliced to produce two other known GH isoforms that are produced within the pituitary: a 20kDa isoform that represents about 5% of the total GH synthesized (Lewis et al., 1978) and a 17.5-kDa isoform (Lecomte et al., 1987) that appears to only be produced in a rare condition, congenital isolated GH deficiency type II (reviewed in Alatzoglou et al., 2015). Unless specifically stated, throughout this chapter, GH will refer to the 22-kDa isoform produced by the pituitary. The high level of GH gene expression in the pituitary gland is directed by the pituitary-specific POU-class homeodomain protein POU1F1, also known as PIT1 (Kelberman et al., 2009). Both humans and mice with mutations leading to loss of functional POU1F1 are GH deficient, although the extent that this is a result of reduced GH gene expression, as opposed to a failure of somatotroph proliferation, is unclear (Tuggle and Trenkle, 1996). Transfection of nonpituitary cells with POU1F1 has been shown to result in activation of GH promoter activity, suggesting that it may be the limiting factor for GH expression in these cells (Ingraham et al., 1988). GH gene expression is also increased by cAMP through phosphorylation of the protein cAMPresponsive element binding protein, and this is the pathway that mediates effects of growth hormone releasing hormone (GHRH) on GH gene expression (see Section 5.2; Mayo et al., 2000). Other factors that have been shown to increase GH gene expression are thyroid hormone, retinoic acid, and glucocorticoids (reviewed in Tuggle and Trenkle, 1996).

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FIGURE 9.1 Primary and tertiary protein structures of growth hormone (GH: A, B) and prolactin (PRL: C, D). GH is 191 amino acids long and has two disulphide bridges (A) compared with PRL, which is 199 amino acids long with three disulphide bridges (C). Within their tertiary structures, both hormones have four antiparallel a-helices arranged in a left-twisted helical bundle (B and D for GH and PRL, respectively). Image (C) taken from https://www.ncbi.nlm.nih.gov/Structure/pdb/1HGU and image (D) from https://www.ncbi.nlm.nih.gov/Structure/pdb/1RW5.

Growing wild-type GH crystals for structure determination studies has proven to be problematic, so mutated polypeptides have been used. The first structure determination of GH was from crystals of genetically engineered methionyl porcine GH (in which the first amino acid alanine was replaced with a methionine), with X-ray diffraction to generate an electron density ˚ (Abdel-Meguid et al., map with a resolution of 2.8 A 1987). Fifty-four percent of the amino acids are organized as four antiparallel a-helices arranged in a lefttwisted helical bundle (Fig. 9.1B). Two-thirds of the amino acids forming these a-helices are conserved between species, and the helical bundle is therefore thought to be important in the maintenance of the structural integrity of the hormone (Abdel-Meguid et al., 1987). GH was the first protein described with the four antiparallel a-helices, and it has since been shown to be present in several cytokines, including PRL. The primary structure of PRL from sheep was the first to be described (Li et al., 1970) and its structural similarity with both GH and human placental lactogen (hPL) noted (Niall et al., 1971). Although the amino acid sequence homology is not high the location of two of the disulphide bridges in PRL is the same as for both GH and hPL (Niall et al., 1971). Mature human PRL is a 199-amino acid single peptide chain with three disulphide bridges and a mass of approximately 23 kDa (Fig. 9.1C; Shome and Parlow, 1977; reviewed by Freeman et al., 2000). Descriptions of the tertiary structure of PRL lagged behind those for GH, and advances were made when nuclear magnetic resonance could be

employed to explore the structure of PRL in solution (Keeler et al., 2003; Teilum et al., 2005). Not surprisingly, the tertiary structure of PRL was confirmed as being similar to GH; that is, it too has four antiparallel a-helices arranged in a left-twisted helical bundle (Fig. 9.1D; Keeler et al., 2003; Teilum et al., 2005). Like GH, the PRL gene has five exons, but whilst there is evidence for alternative splicing, this is not considered to have an important role in the generation of PRL variants (Freeman et al., 2000). In humans and primates, there is an additional, first exon that is utilized for expression of PRL in a range of tissues other than the pituitary, but as this is untranslated, it does not lead to any change in the PRL protein. There are three variants of PRL that result from proteolytic cleavage of the 23-kDa mature PRL: 22, 16, and 14 kDa. The 16- and 14-kDa proteolytic cleavage isoforms of PRL, referred to as vasoinhibins, have the C-terminus removed so bind to the PRL receptor (PRLR) very weakly (Clapp et al., 2006). Vasoinhibins have recently been shown to bind to integrin a5b1 on endothelial cells (Morohoshi et al., 2018) and elicit a diverse range of antiangiogenic effects, hence their name (reviewed by Triebel et al., 2015). The remainder of the chapter will refer to only 23-kDa mature PRL as made by the lactotrophs of the anterior pituitary. In addition to this proteolytic cleavage, PRL can also be modified by glycosylation, phosphorylation, sulfation, and deamidation (Freeman et al., 2000). The major factor required for PRL gene expression is POU1F1 and, like GH, loss or mutation of this protein leads to hormone deficiency (Kelberman et al., 2009).

2. STRUCTURE AND REGULATION

A number of POU1F1 binding sites have been identified in the promoter of both the human and rodent PRL genes (reviewed in Gourdji and Laverriere, 1994; Quentien et al., 2006), and transfection of the transcription factor into nonpituitary cells results in increased expression of PRL (Ingraham et al., 1988). Cyclic AMP and intracellular calcium have been shown to be important regulators of PRL gene expression and mediate the inhibitory effects of dopamine and stimulatory effects of factors such as thyrotrophin-releasing hormone (reviewed in Gourdji and Laverriere, 1994). In rodents, estrogen is a major stimulator of Prl gene expression, although this may of less importance in humans (Featherstone et al., 2012).

2.2 The Structure and Regulation of GH and PRL Receptors GH receptor (GHR) protein was first isolated and purified from rabbit liver (Spencer et al., 1988). Using liver cDNA libraries, clones for both human and rabbit GHR were obtained, and each were shown to encode for a 620-amino acid protein (Leung et al., 1987). The GHR gene consists of nine coding exons and several noncoding exons in the 50 -untranslated region of the gene (Godowski et al., 1989). In humans but not other species studied to date, a genomic deletion of exon 3 (rather than alternative splicing) results in a short isoform of the GHR (d3GHR; Pantel et al., 2003). In an early study, 10% of individuals were homozygous for this genomic deletion and appeared normal, suggesting that the 22 amino acids that exon 3 encode for are not essential for

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the normal function of the receptor (Stallings-Mann et al., 1996). Later studies indicate that the frequency of the d3GHR allele is higher than the initial reports suggested with more than 50% of some sampled populations having one copy or more of the allele (Dos Santos et al., 2004; Palizban et al., 2014). Whilst there appears to be no difference in the binding affinities of the two receptor isoforms, the responsiveness of d3GHR is enhanced in transfected cells (Dos Santos et al., 2004). A single copy of either GHR or d3GHR has since been shown to be sufficient for normal growth (Pantel et al., 2003). Currently, the physiologic consequences of being hetero- or homozygous for d3GHR are not well understood and have been recently reviewed by Boguszewski et al. (2017). In some human tissues, a third isoform has been identified: truncated GHR (trGHR; Amit et al., 1997), which fails to activate intracellular signaling in response to GH binding, can inhibit activity of the full length receptor, and produces large amounts of GH binding protein (GHBP; see later; Ross et al., 1997; Fig. 9.2A). Regulation of transcription of GHR is complex. As stated earlier, there are several noncoding exons in the 50 -untranslated region of the gene: there is significant diversity in the usage of these exons between tissue and physiologic states as a result of alternative splicing (Schwartzbauer and Menon, 1998). The ontology and regulation of GHR expression has been reviewed by Schwartzbauer and Menon (1998). Briefly, there appears to be little GHR expression in the fetus, but expression steadily increases in the neonate. This transition is positively correlated with the activity of the thyroid axis, so

FIGURE 9.2 Variants of (A) GH receptor (GHR) and (B) PRL receptor (PRLR) in the human. The extracellular binding domain of both receptors comprises two subdomains, D1 and D2. Within D1 are two disulphide linked cysteines (CeC) that are involved in binding, whilst in the D2 subdomain, the YGeFS (YG) motif of the GHR and the WSxWS (WS) motif of the PRLR have roles in receptor expression and stability. Each receptor makes one pass through the membrane. The intracellular domains are crucial for signaling, so variants with truncated domains have reduced intracellular signaling capacity. Box 1 (red square) contains a well-conserved motif that is crucial for the binding of Jak2, whilst Box 2 (green square) represents a less well-conserved motif of unknown function.

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it would appear that GHR may be transcriptionally regulated by thyroid hormone/receptor complexes. GH itself may regulate GHR gene expression in the liver since hypophysectomy reduces the number of GHRs in the liver. In general, hepatic GHR expression is higher in females than males, with estrogen increasing and testosterone decreasing expression. Liver expression of GHR increases during pregnancy. Glucocorticoids and nutrition may also have direct effects on GHR expression, with a reduction possibly contributing to the GH resistance seen in undernutrition (Vijayakumar et al., 2011). Like GHR, the PRLR protein was first isolated from rat liver, which allowed isolation of cDNA clones of the receptor in both the rabbit and human (Kelly et al., 1989). Full-length human PRLR (hPRLR) protein is 598 amino acids. In both the human and mouse, alternative splicing of the PRLR gene results in several different isoforms of the receptor (reviewed by Bernard et al., 2015). The long form has full signaling capabilities, whilst the short form lacks the domains required for activation of many signaling pathways and has been proposed to act as a dominant-negative regulator of the long form (Hu et al., 2001; Qazi et al., 2006). However, recent studies have begun to identify specific roles for short isoforms (Sangeeta Devi and Halperin, 2014). In humans, there is also an intermediate form of the PRLR that may have different signaling characteristics (Kline et al., 1999; Fig. 9.2B). PRLR gene transcription is controlled by multiple alternative promoters linked with variable 50 exons, which direct tissue-specific expression (Hu et al., 2002). The expression of the receptor can be modified in specific tissues by physiologic status; for example, PRLR expression in the choroid plexus is increased in lactation (Hirai et al., 2013). Currently, the factors that are responsible for regulation of transcription have not been fully characterized, but it has been shown that estradiol (Dong et al., 2006), PRL itself (Kavarthapu et al., 2014), and GH (Baxter and Zaltsman, 1984) can increase PRLR gene expression. Both the GHR and the PRLR belong to class 1 of the cytokine receptor family, so they both have the following features characteristic of this receptor family (reviewed by Waters, 2016): the extracellular domain is formed of two fibronectin III-like constructs, each with a sevenspanning b-sandwich structure and two disulphide bridges that are important for ligand binding; within the fibronectin III-like construct closest to the cell membrane is a WSxWS (GHR has a variant: YGeFS) motif box, which has a role in both the expression and stability of the receptors (Dagil et al., 2012); the receptor makes a single pass through the membrane; within the intracellular domain and closest to the cell membrane, there is both a conserved proline-rich Box 1 motif, that binds Janus kinase 2 (Jak2), and further from the cell membrane, a less conserved Box 2 motif that contains both aromatic

and acidic residues. The receptors themselves lack intrinsic kinase activity, so the recruitment of Jak2 is essential for their function. A further description of the intracellular domains, activation of Jak2, and the mechanisms involved in eliciting the intracellular signaling cascade following the binding of their ligand is described in Section 4.2. Although there are similarities with the overall structure between the GHR and PRLR, there are important differences, and it is in the intracellular domain where the greatest differences occur. The transmembrane domain of PRLR does not appear to have the rigid structure of the GHR, which appears to be integral to GHR’s activation of signaling (Bugge et al., 2016; Waters, 2016). The intracellular domains are similar with respect to having both Box 1 and Box 2 motifs, as well as long, disorganized tails that are thought to be important in STAT recruitment; however, the PRLR also has several lipid interaction domains. The long, intrinsically disordered domains within the tail of PRLR produce very different transient structures to GHR, and this is thought to be important in their differing functions (Haxholm et al., 2015).

2.3 GH- and PRL-Binding Proteins A circulating protein that binds GH specifically and with high affinity was first described in pregnant mice in 1977 (Peeters and Friesen, 1977) and then largely ignored until a similar protein was identified in rabbits (Leung et al., 1987) and humans (Herington et al., 1986; Baumann et al., 1986). GHBP (246 amino acids in humans) is the extracellular domain of the GHR and reversibly binds a single molecule of GH. The origin of GHBP differs between species. In mice and rats, GHBP is an alternative splice product of the Ghr gene (partially exon 2 and exons 3e7) and consists of the extracellular domain plus a C-terminal “tail” of 27 and 17 amino acids in the mouse and rat, respectively (Rosenfeld, 1994). By contrast, in rabbits and humans, GHBP is the extracellular domain of the GHR and is cleaved from the plasma membrane by tumor necrosis factor-a-converting enzyme (TACE; also known as ADAM17) in a process called ectodomain shedding (Baumann, 2002). TACE may be negatively regulated by tissue inhibitors of metalloproteases-3 (TIMP3; Zhang et al., 2016). Curiously the Rhesus macaque appears to use both alternative splicing and ectodomain shedding to produce GHBP (Martini et al., 1997). Similar PRL-binding proteins (PRLBP), generated by alternative splicing (Trott et al., 2004) or proteolytic cleavage (Kline and Clevenger, 2001), have been shown to be present in human blood and milk and to affect PRL function (Fleming et al., 2013), but their physiologic relevance is currently unclear.

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3. EVOLUTION

3. EVOLUTION As described subsequently, duplication of single genes encoding hormone and receptor have generated GH, PRL, GHR, and PRLR genes. Subsequent divergence (GH and PRL have only 16% sequence identity) has resulted in the individual hormones and their receptors; however, there is a curious dichotomy: both the GH and PRL genes are hypothesized to be significantly more ancient than their respective receptors (Ocampo Daza and Larhammar, 2018a).

3.1 Evolution of GH and PRL The GH gene (GH1) is located on human chromosome 17q23.3 (George et al., 1981), whilst the gene for PRL is located on human chromosome 6q22.3 (Owerbach et al., 1981). Phylogenetic and synteny analyses of 28 vertebrate species has suggested that the GH and PRL genes arose from duplications and divergence of a single ancestral gene (Ocampo Daza and Larhammar, 2018a) that within tetrapods has led to differences in the number and organization of PRL-like genes (Fig. 9.3A). The human GH locus has subsequently

(A)

undergone expansion, whilst there has been no expansion in the PRL locus: conversely in rodents, it is the Prl locus that has undergone expansion, whilst there has been no expansion in the Gh locus (Ben-Jonathan et al., 2008). In humans, the expanded GH locus resulted in a gene cluster consisting of two GH and three chorionic somatomammotropin (CS) genes (Harper et al., 1982; Barsh et al., 1983). There is over 90% sequence homology between the five genes (Seeburg, 1982; Chen et al., 1989). The cluster is organized from 50 to 30 as GH1, CS hormone 1 (CSH1: also known as human placental growth hormone or GH-V, where V ¼ variant, but better known as human placental lactogen (hPL-A or hPL)), GH2, CSH2 (also known as hPL-B), and CSH3 (also known as hPLL and which produces no detectable protein product) (Barsh et al., 1983; Hirt et al., 1987). GH is predominately expressed in the anterior pituitary, although there is evidence of extrapituitary synthesis, and in these tissues, it is thought to exert autocrine/paracrine roles (reviewed by Perez-Ibave et al., 2014; Harvey et al., 2015). The remaining four GH-like genes are all expressed and synthesized by the syncytiotrophoblasts of the human placenta in a developmentally

(B)

1R

1R

or 2R

2R

jawless vertebrates jawed vertebrates extensive gene losses

local duplicaon

jawless vertebrates jawed vertebrates carlaginous fish ray-finned fish

carlaginous fish

lobe-finned fish

coelacanths

ray finned fish

duplicaon and translocaon of PRL amphibians

lobe-finned fish

coelacanths

tetrapods

tetrapods sauropsids

mammals

amniotes

sauropsids

mammals

FIGURE 9.3 Evolutionary pathways proposed by Ocampo Daza and Larhammar for (A) the GH and PRL genes and (B) the GHR and PRLR genes. 1R ¼ first tetraploidization event; 2R ¼ second tetraploidization event; number to left of gene indicates the chromosome the gene is found on in that exemplar species; * indicates that at this locus, there was local expansion of the gene. ** See Ocampo Daza and Larhammar (2018a,b) for information on the ray-finned fish side of vertebrate evolution. Adapted from Ocampo Daza, D., Larhammar, D., 2018a. Evolution of the growth hormone, prolactin, prolactin 2 and somatolactin family. Gen. Comp. Endocrinol., 264, 94e112, Ocampo Daza, D., Larhammar, D., 2018b. Evolution of the receptors for growth hormone, prolactin, erythropoietin and thrombopoietin in relation to the vertebrate tetraploidizations. Gen. Comp. Endocrinol., 257, 143e160.

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coordinated manner (MacLeod et al., 1992). Of these four, only GH2 binds to the GHR and the PRLR with strong affinity (Ray et al., 1990), whereas hPL binds weakly to the GHR and more strongly to the PRLR (Ben-Jonathan et al., 2008). The roles of GH2 in both normal and pathologic pregnancies have been reviewed recently (Liao et al., 2018). In rodents, it is the Prl locus that has been expanded, and compared to the expansion of the human GH locus the expansion is extensive. At least 26 paralogous mouse Prl genes have been identified within a 1-megabase segment of chromosome 13 (Wiemers et al., 2003). Mouse placental lactogen is the product of three different genes that are differentially expressed through gestation (Colosi et al., 1987; Wiemers et al., 2003). A similar expansion has been demonstrated within the rat genome on chromosome 17 (Alam et al., 2006) with many orthologs of the mouse genes being identified. In cows, an expansion has also occurred within the PRL locus on chromosome 23 (reviewed by Schuler and Kessler, 1992), but it is considered to be independent of the expansion that occurred in rodents since it involves fewer genes and the expanded genes are not orthologs of the rodent genes (reviewed by Soares et al., 1991; Southard and Talamantes, 1991). In all three species, the expanded genes are predominately expressed in the placenta and/or uterus and associated with pregnancy. In this chapter, only pituitary prolactin will be reviewed.

3.2 Evolution of GHR and PRLR Both the GHR and PRLR genes are found on the same chromosome and are the result of gene duplication in jawed vertebrates (Ocampo Daza and Larhammar, 2018b): in humans on chromosome 5p13.1-p12 and 5p13.2 (Arden et al., 1990), respectively. Both the GHR and PRLR genes belong to the superfamily of cytokine class I receptors that also includes receptors for somatolactin, erythropoietin, and thrombopoietin. As for their study on the evolution of the GH and PRL genes, Ocampo Daza and Larhammar used a similar analytical approach including sequence phylogeny of 21 different vertebrae species combined with comparative genomic synteny (Ocampo Daza and Larhammar, 2018b). A common ancestral gene for both GHR and PRLR (GHR/ PRLR) arose from the first basal tetraploidization (1R), and then following the second basal tetraploidization (2R) a duplication of this common ancestral gene in jawed vertebrates resulted in two independent genes: GHR and PRLR. Unlike either the GH or PRL loci, there have been no expansion events at the loci for GHR and PRLR (Fig. 9.3B).

4. BIOCHEMICAL REACTIONS 4.1 Formation of Receptor Complexes Both the GHR and the PRLR form homodimers: this was first shown by crystallization of the extracellular domain of GHR with GH, which showed a 2:1 ratio of receptor to hormone (de Vos et al., 1992). It was initially thought that the receptors existed in the cell membrane as monomers and activation of the receptor first required the hormone to bind to a receptor to form a 1: 1 complex. To initiate an intracellular signaling cascade the 1:1 complex must then recruit a second receptor to form a 1:2 complex. For GHR, this explanation has since been demonstrated to be incorrect. The evidence against this hypothesis and the evidence for the existence of preformed unbound homodimers of GHR is reviewed by Waters et al. (2014). It has also been demonstrated that not only is ligand not required for GHR activation but that dimer formation is ligand independent (reviewed by Waters and Brooks, 2015; Gadd and Clevenger, 2006). There are two binding sites on GH: one with high affinity (Site 1) and one with lower affinity (Site 2) (Cunningham et al., 1991). Binding of GH Site 1 to a single receptor first occurs; this then facilitates binding of GH Site 2 to a second receptor, and receptor activation occurs through movement of the two receptors within the transmembrane domain (Chen et al., 1997). Studies to date suggest that activation of PRLR shares many features with GHR: sequential binding of PRL to two different receptors results in homodimer activation and activation of cell signaling through a conformational change of the receptor. However, there may be distinct differences between GHR and PRLR activation, and the three dimensional model of the PRLR generated by Bugge et al. (2016) supports this suggestion. It is currently unclear whether PRLR forms homodimers in the absence of ligand (Gadd and Clevenger, 2006; Brooks, 2012), and there must be additional complexities in PRLR activation, since in humans the receptor binds and is activated by three different ligands: PRL, GH, and hPL. This contrasts with the human GHR, which only binds hGH. Each of these ligands has differences in key residues that interact with the receptor and are likely to differ in their mechanisms and dynamics of receptor activation (Brooks, 2012). The affinity of hGH for the hPRLR increases dramatically (8000-fold) in the presence of zinc ions (Cunningham et al., 1990): zinc ions are not required for hGH to bind to the hGHR or hPRL to bind to hPRLR. It is thought that some of the negative effects observed in normal GH function in zinc deficiency will be due to a reduced affinity of hGH for the hPRLR.

4. BIOCHEMICAL REACTIONS

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FIGURE 9.4 The pleiotropic effects of both GH and PRL are a result of the multiple signaling pathways initiated in response to their binding to two of their respective transmembrane receptors. Ligand binding to the receptor dimer activates receptor-associated Jak2 and causes phosphorylation of both the Jak2 as well as multiple tyrosyl phosphorylation sites (indicated by yellow circles) on the intracellular domains. Phosphorylation in turn activates three main signaling pathways: Jak-STAT, PI3K, and MAPK.

In addition to receptor homodimer, there is some evidence that heterodimers of GHR and PRLR may be formed. Heterodimerization between the ruminant GHR and ruminant PRLR for binding ovine placental lactogen has been shown (Herman et al., 2000; Biener et al., 2003), and in human tissue, particularly cancer cell lines, hGH has been shown to bind to hGHR and hPRLR heterodimers (Xu et al., 2011).

4.2 Receptor Signaling Activation of GHR and PRLR results in the interaction of the two intracellular regions of the receptor dimers. Since the PRLR has multiple isoforms of the intracellular region, there is greater complexity and hence diversity in the signaling pathways initiated. For both receptors for full signaling transduction the “full” length of the intracellular region is required, and the following description is with respect to this. The conformational change arising from the ligand binding to the two receptors results in a repositioning of the transmembrane regions of the two receptors relative to each other, which then brings structural realignment to the intracellular regions: the consequence is transphosphorylation of Janus kinase 2 (Jak2) proteins (GHR: Argetsinger et al.,1993; reviewed by Brooks et al., 2008; PRLR: Dagil et al., 2012). As a result of the Jak2 transphosphorylation,

at least three intracellular signaling pathways for GHR and PRLR may be activated and/or modulated: (1) the Jak-STAT pathway; (2) the PI3K pathway; and 3. the MAPK pathway (Fig. 9.4). 1. The Jak-STAT pathway. Jak2 transphosphorylation leads to receptor phosphorylation and establishment of docking sites of SH2 domain proteins; in particular, the signal transducer and activator of transcription (Stat) molecules, Stat5a and Stat5b (Bole-Feysot et al., 1998). This binding results in phosphorylation of Stat5 and the generation of transcriptionally active Stat5 dimers, which then enter the nucleus of the cell and bind to g-interferon activation sites (GAS) of target genes to induce transcription. The GHR is also able to activate in the same manner both Stat1 and Stat3 (Meyer et al., 1994; Campbell et al., 1995). 2. The PI3K pathway. Jak2 transphosphorylation can also lead to phosphorylation of insulin receptor substrate 1 and 2, providing docking sites for p85, the regulatory subunit of the p85/p110 cytosolic heterodimer (Yamauchi et al., 1998). This results in PI3K-activation of Akt, which has a vast array of diverse cellular functions including regulation of cell proliferation, survival, and metabolism. 3. The MAPK pathway. Activated Jak2 can also in turn activate SH2 domains on the receptor known to be associated with recruitment and activation of the

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adaptor protein, growth factor receptor-bound protein 2 (Grb2), which in turn activates the Ras/Raf/ Mek/Erk, that is, the MAPK, pathway. As for Akt, MAPK pathway acts to regulate cellular growth and proliferation. This pathway can also be activated independently of Jak2 activation by the GHR (Zhu et al., 2002). Although the “short” form of the PRLR lacks the SH2 domains required for Jak2 signaling, there is now evidence to suggest that the “short” form of the receptor has unique signaling transduction pathways and can be either activating or repressing (Devi et al., 2009a, 2009b). In both mouse ovary and decidua, the “short” form of the receptor is involved in the downregulation of a specific transcription factor (Sp1) and appears to involve calmodulin dependent protein kinase (CamK; Devi et al., 2009b). With respect to the MAPK pathway, the same group has identified a unique phosphatase (dual specific phosphatase DUPD1) in the same tissues, which is activated by PRL signaling through the “short” form of PRLR, leading to inhibition of the MAPK pathway (Devi et al., 2011). There are three classes of negative regulators of both the GHR and PRLR: the suppressors of cytokine signaling (SOCS) protein family; the protein tyrosine phosphatase; and protein inhibitor of activated STAT (PIAS) (GHR: reviewed by Wojcik et al., 2018; PRLR: Swaminathan et al., 2008). The SOCS protein family has eight members, cytokine inducible SH2 domain-containing protein (CISH) and SOCS1e7, and roles for CISH and SOCS 1e3 in regulation of GHR and PRLR signaling have been best characterized to date. CISH has been shown to be a specific negative regulator of Stat5, and ubiquitous overexpression in transgenic mice leads to a reduction in growth and a failure of lactation (Matsumoto et al., 1999), suggesting that it has important roles in regulating both GHR and PRLR signaling. SOCS1, which binds directly to Jak2 and appears to inhibit its catalytic activity, has been shown to inhibit GHR signaling in vitro (Greenhalgh and Alexander, 2004), and SOCS1 deficiency rescues lactational defects in mice with reduced PRLR expression (Lindeman et al., 2001). SOCS2 has been shown to inhibit signaling through interference of Jak2 interactions with the receptor (Ram and Waxman, 1999). By 6 weeks of age, SOCS2-deficient mice are 30% e40% greater in size then their wild-type littermates, illustrating the importance of SOCS2 in negative regulation of the GHR (Metcalf et al., 2000). Similarly, the importance of SOCS2 in the negative regulation of the PRLR was demonstrated by the restoration of lactation in mice by deletion of Socs2 in the mammary gland of heterozygous PRLR knockout mice (Harris et al., 2006). SOCS3, which has a similar mode of action to SOCS2 (Ram and Waxman, 1999), is a potent inhibitor of GHR

signaling in vitro (Greenhalgh and Alexander, 2004) and is induced in breast cancer lines in response to prolactin (Barclay et al., 2009).

5. THE PITUITARY CELLS PRODUCING GH AND PRL Both GH and PRL are produced by specialized cells of the anterior lobe of the pituitary gland, termed somatotrophs and lactotrophs, respectively. The gland contains three other hormonal cell types, which along with somatotrophs and lactotrophs have a common developmental origin. Studies in mice have defined a range of signaling pathways and transcription factors that specify each cell type: analysis of patients with inherited pituitary hormone deficiencies have confirmed that these differentiation processes are largely conserved between humans and mice (Kelberman et al., 2009). Expression of POU1F1 transcription factor is required for differentiation of both somatotrophs and lactotrophs, but the factors leading to terminal differentiation of the two cell types are currently unknown. In rodents, where the populations have been best characterized, a small number of somatotroph and lactotroph cells are present at birth, after which both populations increase rapidly until puberty and then remain stable (Taniguchi et al., 2002). In the adult, somatotrophs and lactotrophs are the most numerous populations of cells in the anterior pituitary, together making up 50%e65% of the hormonal cell population. Somatotrophs are more numerous in males and lactotrophs in females in rodents; however, this sexual dimorphism of cell number may be less apparent in humans (Asa et al., 1982). A population of cells producing both hormones, mammosomatotrophs, has also been described and in humans may be as abundant as the monohormonal somatotrophs and lactotrophs (Frawley and Boockfor, 1991). The relative stability of cell number is altered in pregnancy in humans, when there is an increase in the lactotroph population from early pregnancy that returns to prepregnant numbers after cessation of lactation (Scheithauer et al., 1990). GHRH, synthesized and released by neuroendocrine cells of the hypothalamus, is the major factor driving somatotroph cell proliferation: in mice with a loss of functional GHRH receptor (GHRHR), there is severe somatotroph hypoplasia (Lin et al., 1993), whilst transgenic animals with global overproduction of GHRH have somatotroph hyperplasia (Mayo et al., 2000). A relatively minor inhibitory role of somatostatin (SST), another hypothalamic hormone, is suggested by the normal number of somatotrophs in mice with the Sst gene knocked out and minor effects when knockout of SST is combined with transgenic excess expression of

5. THE PITUITARY CELLS PRODUCING GH AND PRL

GHRH (Luque et al., 2009). In humans, the importance of GHRH for somatotroph proliferation is clear from the pituitary hypoplasia that occurs in patients with a mutation leading to functional loss of GHRHR signaling (Murray et al., 2000) and hyperplasia in patients with ectopic overproduction of GHRH from a tumor (Borson-Chazot et al., 2012). The factors regulating lactotroph cell proliferation are less clear in comparison with those responsible for somatotrophs, although a number of mouse genetic models have identified potential regulators. IGF1 knockout mice have a reduced number of lactotrophs, which is apparent in animals before weaning, suggesting that this factor may be required for the early expansion of this cell population (Hikake et al., 2009). In the adult, estradiol modulates lactotroph proliferation and may account for the transient increase in mitotic activity of this cell type at estrus (Seilicovich, 2010); knocking out ERa in mice leads to lactotroph hypoplasia (Scully et al., 1997). It has been suggested that PRL itself may increase proliferation of lactotrophs, as PRL and PRLR knockout animals have lactotroph hyperplasia; however, a recent study in which Prlr was specifically deleted in lactotrophs has shown that this is not the case (Bernard et al., 2018). Other factors that may increase lactotroph proliferation include paracrine or autocrine pituitary factors, such as galanin (Cai et al., 1999), nerve growth factor (Borrelli et al., 1992), and transforming growth factor-a (McAndrew et al., 1995). In common with its role in inhibiting PRL secretion (to be described later), dopamine has an inhibitory effect on lactotroph proliferation, although this may only have an important role in late adulthood, as lactotroph hyperplasia in mice with the dopamine 2 receptor (D2R) knocked out only occurs after 3 months of age (Kelly et al., 1997).

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5.1 Regulation of GH and PRL Secretion: The Importance of the HypothalamicePituitary Relationship The seminal studies of Harris more than 60 years ago demonstrated that the secretion of anterior pituitary hormones is primary regulated by factors produced by hypothalamic neurones and secreted into the specialized capillary bed of the median eminence (Harris, 1955). Release into this structure has important implications for pituitary regulation as the capillaries of the median eminence drain into the pituitary portal vessel and are delivered directly to the capillary bed of the pituitary (Clarke, 2015). The consequences of this anatomic relationship are that there is a very rapid effect of hypothalamic factors on the pituitary at concentrations that are several orders of magnitude higher than in the periphery, but also that modification of blood flow dynamics may have an important modulatory role on the dynamics of pituitary regulation (Le Tissier et al., 2017).

5.2 Regulation of GH Secretion 5.2.1 Hypothalamic Regulation of Pituitary GH Secretion Two hypothalamic factors are primarily responsible for regulation of somatotroph function, including hormone secretion: GHRH and SST with stimulatory and inhibitory actions, respectively, on GH secretion (Steyn et al., 2016) (Fig. 9.5A). Loss of GHRH (Alba and Salvatori, 2004; Le Tissier et al., 2005) or its receptor (Maheshwari et al., 1998; Lin et al., 1993) results in dwarfism as a result of reduced hormone secretion. Ectopic GHRH

FIGURE 9.5 (A) Binding of hypothalamic GHRH to its GPCR, GHRHR, on the anterior pituitary somatotroph increases intracellular cAMP to

increase GH synthesis as well increasing IP3 and hence Ca2þ resulting in GH release. GH synthesis is inhibited by hypothalamic somatostatin binding to its GPCR, SST2R. Stomach-derived ghrelin binds to GHRS1a, another GPCR, to stimulate IP3, with Ca2þ resulting in increased GH release. (B) Hypothalamic dopamine binds to the GPCR, D2R, on the lactotroph to inhibit PRL synthesis and release.

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excess in humans (Rivier et al., 1982) and mice (Hammer et al., 1985) has the opposite effect: GH cell hyperplasia and chronically increased GH secretion results. Loss of SST signaling through immunoneutralization (Tannenbaum, 1988) or gene ablation (Low et al., 2001) leads to an increase in GH secretion. The effects of these two neurohormones, GHRH and SST, on somatotrophs is primarily mediated by alterations in intracellular cAMP concentrations resulting from activation of their respective G proteinecoupled receptors, with increased cAMP in response to GHRH resulting in higher levels of somatotroph proliferation, gene expression, and secretion (Mayo et al., 2000). Models of the interplay between GHRH and SST output from the hypothalamus suggest that interactions between the two neuronal populations result in rhythmic surges of release of both neuropeptides that are 180 degrees out of phase and hence drive pulsatile GH release (Tannenbaum and Ling, 1984; MacGregor and Leng, 2005). A number of neuromodulators (for example, serotonin) and neuropeptides (for example, neuropeptide Y (NPY), galanin, corticotrophin-releasing hormone and proopiomelanocortin-derived peptides) have been described as modulating the activities of GHRH and SST neurones (reviewed in Steyn et al. (2016)) and therefore may mediate central regulation of GH secretion. 5.2.2 Regulation of GH Release by Nutritional Status and Metabolic Factors Since GH has important roles in the regulation of metabolism and response to nutritional challenge, it is not surprising that its release is also modified by peripheral factors that also regulate food intake and metabolism. Ghrelin, principally secreted from specialized endocrine cells of the stomach, is a potent stimulator of GH secretion (Fig. 9.5A). Ghrelin concentrations increase in circulation before meals, following weight loss and food deprivation and decrease after feeding (Cummings, 2006). The receptor for ghrelin, the growth hormone secretagogue receptor (GHS-R), is expressed in somatotrophs and stimulates GH secretion by an independent pathway to GHRH (Bowers et al., 1990). GHS-R is also expressed in GHRH neurones, and in vivo ghrelin stimulation of GH secretion is mediated in part by increased GHRH release (Tannenbaum et al., 2003) and possibly by inhibiting SST release (Tolle et al., 2001). Studies of the potential actions of insulin on GH release are complicated by its effects on circulating glucose; however, loss of insulin receptors specifically in somatotrophs has been shown to result in increased GH synthesis and secretion (Gahete et al., 2013), suggesting that insulin may have a modulatory role on the GH axis at the level of the pituitary. It is currently unclear whether insulin has any effects at the level of the hypothalamus. Leptin, which is secreted from

adipocytes in proportion to fat mass, has also been shown to modify GH secretion, but there are conflicting reports of whether it is stimulatory or inhibitory (Steyn et al., 2016). Specific loss of leptin receptors in somatotrophs in genetically modified mice results in decreased pituitary GH content and secretion. In general, excess nutrients in circulation are associated with lower circulating concentrations of GH, suggesting that they inhibit GH release. In the case of glucose, it is currently unclear whether the suppressive effects of high plasma glucose concentrations on GH release are a direct effect on the GH axis or a result of altered plasma insulin (Steyn et al., 2016). Free fatty acids have an inhibitory effect on GH secretion by reducing somatotroph response to GHRH stimulation (Alvarez et al., 1991) as well as potential effects at the level of the hypothalamus (Briard et al., 1998). An exception to the suppressive effects of increased nutrients on GH secretion is the effects of high plasma concentrations of amino acids (in particular, lysine and arginine) that specifically stimulate GH secretion (van Vught et al., 2008). 5.2.3 Feedback Regulation of GH Secretion Feedback regulation of GH secretion occurs through multiple mechanisms at both the level of the pituitary and hypothalamus. There is some evidence for ultrashort feedback within the pituitary, as it has been shown that somatotrophs express GHR, although it is unclear if these are functional (Fraser et al., 1991). There is much more compelling evidence for a short feedback loop with GH altering the activities of hypothalamic GHRH and SST neurones, although only a small proportion of these neurones show a response to GH (Burton et al., 1995). It is possible that effects on this small proportion alter the activity of a much larger population or that other neuronal cell types, such as NPY neurones, respond to GH and in turn modify the activities of both GHRH and SST neurones (Minami et al., 1998). Whilst the precise mechanism is unclear, it is accepted that short-loop GH feedback results in activation of SST release (Chihara et al., 1981) and possibly reduced GHRH release (Conway et al., 1985). It is well established that feedback from peripheral, in particular liver, insulin-like growth factor-1 (IGF-1) leads to inhibition of GH secretion at the level of the pituitary (Steyn et al., 2016), resulting from both reduced GH gene transcription and secretory response to GHRH stimulation (Morita et al., 1987). Consistent with this, specific deletion of IGF-1 receptors (IGF1R) in somatotrophs results in increased GH gene expression and circulating concentrations of GH (Romero et al., 2010). The feedback effects of IGF-1 at the level of the hypothalamus are less clear: IGF1R is expressed throughout the hypothalamus (Werther et al., 1989),

5. THE PITUITARY CELLS PRODUCING GH AND PRL

and IGF1 has been shown to decrease GHRH and increase SST gene expression. There is some evidence to suggest that this is in response to IGF1 production from within the brain rather than from the periphery (Sato and Frohman, 1993).

5.3 Regulation of PRL Secretion There is a high level of basal PRL secretion from lactotrophs, driven by the influx of calcium through voltage-gated calcium channels (VGCCs), which in turn are stimulated by high levels of spontaneous electrical activity (Gregerson, 2006). Unusually for pituitary hormones, the primary mechanism of regulation of this spontaneous activity is inhibition, originally demonstrated by experiments severing the pituitary stalk or implanting fragments of the pituitary under the kidney capsule (reviewed by Grattan and Le Tissier, 2015). These experiments demonstrated that in the absence of hypothalamic input the resulting hyperprolactinemia would induce pseudopregnancy in nonpregnant rats or the maintenance of lactation: clearly demonstrating that PRL secretion is under inhibitory and not stimulatory control by the hypothalamus. 5.3.1 Dopamine Is the Primary Inhibitor of PRL Secretion The primary factor inhibiting PRL secretion is dopamine, a catecholamine neurotransmitter, produced by three populations of neurones in the hypothalamus that together are described as the neuroendocrine dopamine (NEDA) neurones. The NEDA neurones comprise the tuberoinfundibular dopaminergic neurones of the dorsomedial hypothalamus, which project to the median eminence; the tuberohypophysial dopaminergic neurones originating in the rostral arcuate nucleus with terminals in the intermediate and neural lobes of the pituitary; and the periventricular dopaminergic neurones that arise in the periventricular nucleus and terminate in the intermediate lobe of the pituitary (Grattan, 2015). Since PRL is under tonic inhibition, sustained release of dopamine is required, which requires coordination of neuronal activities: such coordination has recently been described in the mouse (Romano et al., 2017) and rat (Stagkourakis et al., 2018), although the mechanisms involved may differ between species. Dopamine is transported to the anterior pituitary either from the median eminence through the portal circulation or via short portal vessels from the posterior pituitary. Lactotroph cells express the D2R, and it is the stimulation of this G proteinecoupled receptor, which couples to a pertussis-sensitive G protein (Gi/o), that alters the activities of the VGCCs to inhibit the secretion of PRL (Stojilkovic et al., 2010) (Fig. 9.5B). The importance

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of the D2R is demonstrated in mice where this receptor has been knocked out: the animals are hyperprolactinemic, and this is attributed in part to an associated dramatic increase in lactotroph cell proliferation (Kelly et al., 1997; Saiardi et al., 1997). This tonic inhibition of a spontaneously secreting lactotroph also provides a mechanism for regulated stimulation of PRL output, for example, by inhibition of the activity of NEDA neurones by the circadian, steroid, and opioid inputs that are required for PRL surges both at proestrus and in early pregnancy (Freeman et al., 2000). 5.3.2 Other Factors Regulating PRL Secretion Estradiol has a stimulatory role in PRL secretion, but this is principally a result of increased PRL gene expression or modification of the response of lactotroph cells to other regulators with evidence for increased TRH receptors, decreased D2R, as well as changes in ion channel density and electrical excitability (Gregerson, 2006). A number of factors have been identified, principally through in vitro studies, as modifiers of pituitary PRL secretion, although their physiologic importance is yet to be established (Grattan and Le Tissier, 2015). Several of these are hypothalamic factors: somatostatin and calcitonin have been identified as inhibitors of secretion, whilst thyrotrophin-releasing hormone, oxytocin, vasointestinal peptide (VIP), and galanin have been identified as potential stimulators of PRL secretion. VIP and galanin are also produced in the pituitary gland itself and can be included in a large list of factors that may be autocrine or paracrine regulators of lactotroph secretion (Denef, 2008). 5.3.3 Short Loop Feedback Regulation of PRL As PRL does not regulate a specific target endocrine organ, it is not surprising that it is the hormone itself that provides the feedback for its own regulation (Grattan, 2015). The hypothalamic dopaminergic neurones regulating PRL secretion express the PRLR and increase their firing rate and dopamine secretion within seconds of exposure to PRL, which is mediated through alterations in ion channel activities and electrical activity. As well as these rapid actions, PRL also induces hypothalamic expression and phosphorylation of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, resulting in increased dopamine synthesis and release. This short-loop feedback is altered and underlies the sustained increase in PRL secretion in lactation, with a marked reduction in dopamine release (Grattan, 2015). This appears not to be a result of reduced sensitivity of the NEDA neurones to PRL but rather an alteration in the activity of tyrosine hydroxylase, a disconnect between neuronal firing and dopamine release, and a possible switch in neuronal phenotype to opioid secretion (Romano et al., 2013).

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Maintenance of neuronal response to PRL and reduced dopamine production through posttranslational modification of tyrosine hydroxylase may provide a mechanism for the rapid reactivation of NEDA-mediated inhibition of PRL secretion at weaning.

5.4 Regulation of GH and PRL Secretion at the Level of the Pituitary In addition to possible paracrine regulation of both GH and PRL secretion (see Denef, 2008 for an extensive review), recent studies have shown that the secretory activity of both somatotrophs and lactotrophs may be modified by pituitary cell network organization that allows enhanced responses to regulation through cellecell communication (Le Tissier et al., 2012). It is now clear that both somatotrophs and lactotrophs are organized into homotypic structural networks that are distinct for each cell type (Fig. 9.6). This topographic organization, with somatotrophs forming clusters and lactotrophs a honeycomb-like structure, is plastic, and changes are correlated with physiologic status where secretion is altered; for example, the organization of somatotrophs is more apparent in males than females and is altered

FIGURE 9.6 The organization of GH (A) and PRL (B) cells within the anterior pituitary into distinct network motifs is revealed by imaging 2-photon imaging of cells in pituitary slices. (A) Cells from two month-old male GH-GFP transgenic mice are organized as interconnected clusters. (B) In contrast, cells of adult PRL-DsRed transgenic female mice are organized in a honeycomb structure. Figure reproduced from Le Tissier, P.R., Hodson, D.J., Lafont, C., Fontanaud, P., Schaeffer, M., Mollard, P., 2012. Anterior pituitary cell networks. Front. Neuroendocrinol., 33, 252e266 with permission.

by sex steroids, with remodeling around puberty (when GH secretion peaks), which is then reversed in adulthood. This structural organization underlies celle cell communication through gap junctions and mediates coordination of cell activities and enhanced secretory output. Similarly, lactotroph organization in females is enhanced during lactation when there is a high demand for PRL, but remarkably in this case the remodeling persists for a long period following cessation of demand at weaning. This persistent change in organization allows anticipation that a second pregnancy will occur and increases secretory output on the second lactation.

6. PHYSIOLOGIC FUNCTIONS OF GH AND PRL 6.1 The Pattern of GH Secretion and Its Physiologic Consequences A key feature of GH secretion in virtually all species where it has been studied is episodic release (Robinson and Hindmarsh, 2010). Estimates of the number of pulses over a 24-hour period are highly dependent on sampling frequency but have been estimated in some studies with intensive sampling as approximately 7 pulses/day in men and 11 pulses/day in women (Hartman et al., 1990) (Fig. 9.7). Basal secretion, which has been estimated as between 8% and 12% of total daily GH production (Veldhuis et al., 2001), combines with this episodic release to generate the varying GH concentrations in the circulation, meaning that both pulse amplitude, frequency, and nadir concentrations must be considered to fully understand the importance of the pattern of GH secretion. In addition, a proportion of GH in the circulation will be bound by GHBP, which increases the half-life of the hormone but also reduces its bioavailability (Veldhuis et al., 1993). This will have important consequences for the dynamics of pulse stimulation of the GH receptor, since it is likely that high amplitude pulses saturate GHBP and thus increase the concentrations of unbound circulating hormone, which will be free to bind to receptors but will have a short half-life. The pattern of GH secretion has an important role in determining its downstream effects (Robinson and Hindmarsh, 2010). An elegant study in dwarf rats, which have minimal endogenous GH secretion, has addressed the relative effects of continuous (akin to basal) or pulsatile administration of the same total dose of GH (Gevers et al., 1996) (Fig. 9.8). Pulsatile GH was shown to be markedly more effective at stimulating growth than a similar dose administered continuously, with no effect of basal concentrations in addition to pulsatile stimulation. This demonstrates the importance of GH pulsatility and its amplitude for stimulation of

6. PHYSIOLOGIC FUNCTIONS OF GH AND PRL

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FIGURE 9.7 Mean serum GH concentration over 24 h in two normal males (left panels) and two normal female (right panels) subjects. At the top of each panel is a continuous line that indicates each GH pulse, indicated using cluster analysis. Figure reproduced from Hartman, M.L., Veldhuis, J.D., Vance, M.L., Faria, A.C., Furlanetto, R.W., Thorner, M.O., 1990. Somatotropin pulse frequency and basal concentrations are increased in acromegaly and are reduced by successful therapy. J. Clin. Endocrinol. Metab., 70, 1375e1384.

growth but also shows that low nadir concentrations are not required for effective growth stimulation. In contrast to growth, continuous administration of GH was more effective at stimulating liver production of both GHR and circulating concentrations of GHBP (Gevers et al., 1996). Many other studies have shown that a range of other liver proteins are sensitive to GH secretion pattern, so pulsatile and basal GH secretion allows differential encoding of hormone action on different tissues. In humans, pulsatile administration of exogenous hormone has been shown to be most effective at stimulating bone formation and resorption (Jaffe et al., 2002) as well as exerting its lipolytic effect (Cersosimo et al., 1996).The expression of specific liver enzyme activities are differentially affected by continuous and pulsatile GH secretion (Jaffe et al., 2002). Other studies in humans monitoring the effects of endogenous pulsatile secretion have shown that basal GH concentrations are the principal determinant of circulating IGF1 concentrations (Faje and Barkan, 2010).

6.2 Changes in GH Secretion Across the Lifespan GH secretion in the human fetus can be detected at 12 weeks of gestation, and secretion gradually increases

until around 20e24 weeks of gestation before it declines until birth (Robinson and Hindmarsh, 2010). At birth, there are relatively high circulating concentrations of GH secretion due to frequent, high-amplitude pulsatile release, but both the frequency and amplitude of the pulses decline within the first 4 days of birth (Miller et al., 1993). This fetal and neonatal GH does not have an important role in statural growth since GH-deficient offspring of GH-deficient mothers have a relatively normal birth weight and length (Rimoin et al., 1966). From 2 to 3 months of age, however, GH is necessary for normal statural growth (Robinson and Hindmarsh, 2010). GH secretion gradually increases throughout childhood, principally through increased pulse amplitude, until puberty. At puberty, altered sex steroids drive a dramatic increase in GH secretion: there is a two- to threefold increase in the amplitude of GH pulses (Robinson and Hindmarsh, 2010). This increased secretion continues until early adulthood (18e25 years of age), after which there is an exponential decrease in GH secretion, with output halving approximately every 7 years (Giustina and Veldhuis, 1998). This progressive decrease in circulating GH concentrations in adults may be related to changes in pituitary function in adulthood with age, and this is probably caused by changes in hypothalamic regulation since reduced GHRH and

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FIGURE 9.8 The importance of pulsatile GH secretion is demonstrated by treatment of GH-deficient dwarf (dw/dw) rats by intravenous infusion of the same total amount of GH but with varying amounts of GH delivered either in pulses (P), continuously (C), or in a mixture of both patterns. Top panel: the pattern of GH infusion. Bottom panel: the resulting weight gain of animals above those of controls, showing that pulsatile delivery is more effective than continuous. *, P ¼ .095 versus 144C; **, P < .01 versus 144C; þ, P < .05 versus 200C. Redrawn from Gevers, E.F., Wit, J.M., Robinson, I.C., 1996. Growth, growth hormone (GH)-binding protein, and GH receptors are differentially regulated by peak and trough components of the GH secretory pattern in the rat. Endocrinology, 137, 1013e1018 with permission.

increased SST release have been reported in primate models (Nakamura et al., 2003).

6.3 Physiologic Modifications of GH Secretion 6.3.1 Gonadal Steroids At puberty in females the increase in GH secretion is driven by estrogen, and this steroid is also responsible for both the higher baseline and total GH secretion measured in women than men (Ho et al., 1987), a slower decline in GH secretion with age before menopause (Hudson et al., 2010), and the periovulatory increase in GH secretion during the female menstrual cycle (Birzniece and Ho, 2017). These regulatory effects occur at both the level of the pituitary and hypothalamus and may, in part at least, result from local estrogen production (Birzniece and Ho, 2017). Indeed, the effects of testosterone on GH secretion may result from its local metabolism by somatotroph aromatase, since aromatase knockout mice have hypoplastic pituitaries with reduced GH secretion (Yan et al., 2004).

6.3.2 Sleep An increase in GH secretion at night is well described in humans, and there is strong evidence that this is principally a result of increased output during slow-wave sleep, although this relationship may be bidirectional as patients with GH deficiency have disturbed sleep (Van Cauter et al., 2004). In experiments where sleep is delayed in healthy subjects by 5e6 h, an increase in GH output still occurs at the usual time of slow-wave sleep, but the amplitude of pulses is diminished, suggesting that there is both a circadian and sleep component to increased nocturnal GH secretion (Thorner et al., 1990). 6.3.3 Stress Psychosocial and physical stress, as well as that caused by conditions such as sepsis, initially cause an increase in GH secretion but in the longer term lead to its suppression (Robinson and Hindmarsh, 2010). This may be a result of an initial stimulation of GH synthesis and secretion by glucocorticoids but an increase in SST tone with prolonged chronic stress (Steyn et al., 2016).

6. PHYSIOLOGIC FUNCTIONS OF GH AND PRL

6.3.4 Pregnancy and Lactation In humans, increasing release of the placental form of GH from 8 to 10 weeks of gestation leads to a progressive suppression of pituitary GH secretion until parturition when pituitary GH secretion resumes (Steyn et al., 2016). In lactation, GH may have an important role in milk production, since inhibition of GH in lactating rats leads to a reduction in both the volume of milk produced and its fat content (Flint and Vernon, 1998). Suckling induces a rise in GH secretion in rats (Riskind et al., 1984), which may result from corelease with PRL from mammosomatotroph cells. Whether this is simply a consequence of coexpression of hormone from these cells or has a more important role in coordinating the actions of GH and PRL is currently unclear.

6.4 Changes in PRL Secretion Across the Lifespan Prl gene expression can first be detected in late pregnancy in rodents (embryonic day 17 in mice and 18 in rats (Ben-Jonathan et al., 2008)). In humans, fetal pituitary PRL production begins toward the end of the first trimester and continues to increase throughout the remainder of the pregnancy: this is reflected by increasing PRL concentrations in fetal blood until term (Ben-Jonathan et al., 2008). Newborn infants have relatively high circulating PRL concentrations that decline within the first day of life (de Zegher et al., 1993). These high concentrations of circulating PRL during fetal life and at birth may result from exposure of the fetus to maternal estradiol. Pituitary lactotroph cells continue to proliferate after birth until adulthood, and this is reflected in increased pituitary hormone content, although the limited available data does not suggest that this leads to increased secretion before puberty. In adulthood, basal PRL secretion is higher in females than males, with normal concentrations of 4e20 and 1e4 ng/mL in adult females and males, respectively. In both genders, these concentrations are modestly increased at orgasm, during rapid eye movement sleep, and during stress (Gregerson, 2006). In nonseasonal breeding animals, the most dramatic changes in secretion occur in females during the reproductive cycle, as documented subsequently.

6.5 Physiologic Modifications of PRL Secretion There are marked species differences in PRL secretion that largely reflect species differences in reproductive strategies. In seasonal breeding animals, for example, PRL is used as a circannual timer: with high secretion during the long days of summer regulating a diverse range of physiologic changes that affect reproductive

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success, such as horn growth in rams (Lincoln and Richardson, 1998). In all mammalian species, PRL secretion is increased by the suckling stimulus of offspring, and there are large changes in secretion dependent on the reproductive status of females (as detailed later.) Another important physiologic state leading to increased PRL secretion is stress, which may have important roles in both attenuating the effects of longterm stress (Torner, 2016) and modulating the immune system (Tang et al., 2017). 6.5.1 PRL secretion in Pregnant and Lactating Rodents The variations that occur in PRL secretion and their functional consequences have been best characterized in rats (Grattan and Le Tissier, 2015). During the estrous cycle, PRL secretion is maintained at low concentrations, with the exception of a surge that occurs on the afternoon of proestrus (Palm et al., 2001). This changes with the cervical stimulation that occurs at mating to twice-daily surges that continue for approximately 10e12 days. If mating is unsuccessful (pseudopregnancy), then secretion returns to low concentrations when estrous cyclicity resumes, whilst in pregnant animals the production of placental lactogens leads to termination of pituitary PRL secretion. Immediately before parturition, there is a resumption of PRL secretion from the pituitary, which is maintained at high levels throughout lactation, with hormone release now closely associated with the suckling stimulus of the young. At weaning, the high levels of PRL secretion are rapidly reduced and resume the same pattern as before pregnancy. Basal concentrations of PRL are lower following the first lactation compared to virgin animals (Byrnes and Bridges, 2005). 6.5.2 PRL Secretion in Pregnant and Lactating Women PRL secretion in women is maintained at low concentrations throughout the menstrual cycle, with conflicting evidence for a small increase at ovulation (Grattan and Le Tissier, 2015). In pregnancy, there is an increase in pituitary release from 6 to 8 weeks of gestation that continues to rise until term and is augmented by the release of decidual and fetal PRL (Ben-Jonathan et al., 2008). In lactation, as in the rodent and other species, PRL is released in response to suckling in proportion to the duration and intensity of the suckling episode (Grattan and Le Tissier, 2015).

6.6 GH and PRL in Circulation There is limited evidence that some GH may exist as multimeric complexes in circulation, but the biologic significance, if any, of these complexes is unknown.

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Similarly, in circulation, 23-kDa PRL can form complexes with IgG resulting in “big PRL” (an inactive dimer of 23-kDa PRL) and “big big PRL” (also referred to as macroprolactin: large complexes of PRL either with itself or else with immunoglobulin) (Rogol and Rosen, 1974; Suh and Frantz, 1974; von Werder and Clemm, 1974). Neither of these PRL complexes are thought to have biologic significance and are not associated with any known pathologies. An awareness of the presence of these complexes is important for those trying to measure biologically active GH or PRL concentrations in circulation as the complexes may interfere in some assays. There is little evidence that any of the GH in circulation is from extra-anterior pituitary sources, whereas in humans, PRL is also synthesized in extra-anterior pituitary tissues (reviewed by Marano and Ben-Jonathan, 2014). It should be noted that PRL from extra-anterior pituitary synthesis is identical to pituitary PRL and binds to the PRLR. A significant proportion of circulating GH is not biologic available as it is bound to GHBP (Baumann, 2002). It is possible that the same may be true for PRL; however there is a dearth of studies characterizing PRLBP in any species, although there is one report that measured no gender differences in the amount of PRLBP in circulation in adult humans (Kline and Clevenger, 2001). Again an awareness that in circulation a significant proportion of the hormone is not “free” is important when selecting an immunological-based assay to measure either GH or PRL; that is, clarification is required regarding whether total or free hormone is being measured. In all species, GH bound to GHBP is protected from degradation whilst in circulation, and therefore the half-life of the hormone is extended, with bound hormone acting as a reservoir. GHBP concentrations are undetectable to low in the fetus and neonate, steadily increase over childhood, and remain relatively constant over the life of the adult, with no marked gender differences (Schilbach and Bidlingmaier, 2015). Characterization of circulating concentrations in health and disease were reviewed by Baumann (2002): notably, GHBP is absent or nonfunctional in Laron syndrome (Daughaday and Trivedi, 1987). A low affinity GHBP was first described in 1990 (Baumann and Shaw, 1990), and it was later clarified that this isoform of GHBP is a product of the d3GHR gene (Urbanek et al., 1992): the genomic deletion results in a BP 22 amino acids shorter. Studies to determine the pathways involved in the metabolic clearance of both GH and PRL have determined that both hormones are rapidly removed from circulation predominately by specific liver uptake and renal clearance. For both hormones, the liver is a major target organ, and hepatocytes express both GHR and PRLR (Kelly et al., 1974; Posner, 1976). The GHR-GH-

GHR complex is endocytosed into clathrin-coated vesicles and trafficked to lysosomes for degradation: this process is dependent on the ubiquitin-dependent endocytosis (UbE)-motif on the cytoplasmic tail of the GHR (Govers et al., 1999; Sachse et al., 2001). Ubiquitindependent degradation is also believed to be involved in the turnover of PRLR-PRL-PRLR complexes, but the process is not yet as well described as it is for GHRGH-GHR (Li et al., 2004). In the kidney, both GH and PRL are freely filtered by the glomerulus of the nephron and then reabsorbed within the proximal tubule (Owens et al., 1973; Sonenberg et al., 1951; Turyn et al., 1997). Radiolabeled ovine GH infused into the tubule of the nephron was traced as it moved across the apical surface of the epithelial cells lining the proximal tubule in the rat kidney (Stacy et al., 1976). The GH is endocytosed into small then to large vacuoles before entering lysosomes, where it was rapidly degraded into its constituent amino acids, which are then transported over the basolateral cell surface via specific amino acid transporters, in the same manner as other proteins are degraded in the kidney. Very little intact GH and PRL is spared reabsorption at the proximal tubule: in humans, it is estimated that less than 0.01% of all secreted GH is excreted as intact GH in the urine (Baumann and Abramson, 1983). Curiously, more intact PRL was measured in male human urine than in females using radioimmunoassay, but as for GH, the amounts measured are a miniscule proportion of the total PRL secreted (Sinha et al., 1973). The measured half-life of endogenous GH, following release from the pituitary gland and not bound to GHBP, varies depending on the method using to calculate it. There are reports in the literature ranging from a mean of 8.9 min up to 50 min (discussed in Holl et al., 1993), although in a healthy adult using deconvolution analysis (2 compartments) the half-life appears to be about 20 min. The half-life of endogenous GH is shorter (by a couple of minutes) in the morning than in the evening (Holl et al., 1993); increased in the presence of increased free estradiol concentrations (Holl et al., 1993); decreased in obese prepubertal, pubertal, and adult males compared to age-matched lean males (Roemmich et al., 2005); and decreased with age in males (Iranmanesh et al., 1991). Complexed GH (that is, bound to GHBP) is retained within the vascular space, so it is more slowly degraded and has a metabolic clearance rate six times slower than uncomplexed GH, which is found mostly in extracellular compartments (Baumann et al., 1987). The overall capacity of the liver to uptake GH is substantial greater than for PRL, probably a reflection of the greater number of hepatic GHR relative to PRLR; hence the half-life of PRL has been found in several studies to be about 30% longer than the halflife of GH (Sievertsen et al., 1980; Roelfsema et al., 2012).

6. PHYSIOLOGIC FUNCTIONS OF GH AND PRL

6.7 The Physiologic Functions of GH Consistent with the wide range of tissues expressing GHR, GH has a diverse range of physiologic functions beyond those classically recognized in both skeletal growth and metabolism. There is strong evidence for a modulatory role of GH in the function of the immune system (Bodart et al., 2017), the cardiovascular system (Caicedo et al., 2018), and neural stem cells, as well as memory and cognition (Waters and Blackmore, 2011). Here, we will limit the description to the most classically recognized physiologic functions of GH. Understanding the mechanisms of GH action on tissues is complicated by its regulation of the production and secretion of growth factors and their receptors. The existence of an endocrine factor that mediated at least a proportion of the effects of GH was described more than 60 years ago (Daughaday and Reeder, 1966) and was termed somatomedin (Daughaday et al., 1972). Subsequently, the major somatomedin mediating the action of GH was identified as IGF-1 (Klapper et al., 1983), and it was proposed that the actions of GH were principally mediated by IGF-1 produced from the liver (the “somatomedin hypothesis”) (Le Roith et al., 2001). IGF-1 was subsequently found to be produced in a diverse range of tissues (Roberts et al., 1987); however, it also became clear that GH could act directly on some tissues, for example, the cartilage growth plate (Isaksson et al., 1982). It is now clear that GH may elicit its effects both indirectly and directly: indirectly, GH stimulates both circulating endocrine and locally produced paracrine IGF-1, which in turn mediates GH’s effects, and GH also has direct, IGF-1 independent actions (Le Roith et al., 2001). Dissecting the roles of direct GH versus the indirect endocrine and paracrine effects is clearly complex, in particular, as IGF-1 has a feedback effect on GH secretion (see preceding) and can be produced in the absence of GH signaling (Le Roith et al., 2001). The use of genetically modified mice (in particular, those with global loss of GHR (Ghr/ (Zhou et al., 1997)), IGF-1 (Igf-1/), or IGF1R (Igf1R/) (both Liu et al., 1993) and tissue-specific modification of the function of these proteins has furthered our understanding of the mechanisms of GH actions within different tissues (see later for examples). It should also be noted that GH modifies a range of other growth factors and their receptors in addition to IGF-1 (Le Roith et al., 2001), further complicating dissection of its modes of action. 6.7.1 Skeletal Growth and Bone Metabolism GH deficiency, resulting from either hypothalamic or pituitary dysfunction, leads to dwarfism and reduced skeletal acquisition in humans if the deficiency occurs in childhood (Alatzoglou et al., 2014) and in rodents

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(reviewed in Yakar and Isaksson, 2016). Conversely, excessive GH secretion in both human childhood and rodents leads to skeletal gigantism (Vilar et al., 2017; Yakar and Isaksson, 2016). Both conditions clearly demonstrate the essential regulatory role of GH in skeletal growth. The extent that this growth is regulated by direct GH actions on skeletal cells or through either circulating IGF-1 and/or by local IGF-1 secretion in response to GH has only recently become clear through the use of genetic models in mice (Yakar and Isaksson, 2016). Loss of GH activity in several mouse models, either through loss of the hormone itself or in Ghr/ mice, results in animals that are born with normal body weight but that only grow to w50% of adult body size (Borrelli et al., 1989), whilst loss of IGF-1 leads to w70% reduction (Liu et al., 1993). The increased severity of loss of IGF-1 signaling compared with GH alone suggests that there are also some IGF-1 GHindependent actions on bone growth. In mice with a loss of both GHR and IGF-1, body size is further reduced to w15% of wild-type animals (Lupu et al., 2001), demonstrating that GH has effects on bone that are direct and not mediated by IGF-1. Further studies have shown that both GH and locally produced IGF-1 are principally responsible for the increased chondrocyte proliferation and differentiation that underlies longitudinal bone growth, whilst GH, circulating and local IGF-1 have effects on the osteoblasts, osteoclasts, and osteocytes responsible for radial bone growth, modeling, and remodeling (Yakar and Isaksson, 2016). 6.7.2 Metabolism The role of GH in metabolism can be considered primarily as that of protein conservation, with effects that are dependent on nutritional status. As described in an excellent review (Moller et al., 2009), the metabolic actions of GH can be considered as having been selected during evolution to combine with insulin to increase protein stores during times of plentiful food whilst promoting fat utilization, thereby sparing both protein and glucose during famine and stress. 6.7.3 Protein Metabolism The anabolic effects of GH on protein metabolism are apparent from the loss of lean body mass in patients with hormone deficiency and increased lean body mass in acromegaly; however, these are most likely the result of subtle and minor increases over a prolonged period of time (Moller et al., 2009). Studies of the acute protein catabolic effects of GH are not consistent and are complicated by the use of prolonged exposure to relatively large doses of GH. It is clear that acute GH administration results in an increased partitioning of amino acids from oxidative to synthetic pathways, but this effect is attenuated when treatment is

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prolonged over periods of several months (reviewed in Chikani and Ho, 2014). The site of action is less clear than the whole-body effects. Whilst direct effects on increasing muscle protein synthesis have been described (Fryburg et al., 1991), others have either reported no effect or a decrease (Copeland and Nair, 1994), so it is likely that the majority of the protein anabolic effects of GH occur in tissue or organs other than skeletal muscle through a reduction in protein oxidation (Chikani and Ho, 2014). The relative contribution of direct actions of GH and those mediated by IGF-1 have again been studied in mouse models. Transgenic mice with targeted alterations of the IGF-1 axis specifically in muscle have provided evidence that the actions of GH are mediated by IGF-1: mice with muscle overexpression of IGF-1 have muscle hypertrophy (Coleman et al., 1995), whilst the effects of GH on muscle are attenuated in mice if IGF-1 signaling is blocked through expression of a dominantnegative form of the IGF1R (Kim et al., 2005). Together, these results suggest that the indirect actions of GH mediated by circulating and local IGF-1 mediate the protein anabolic actions. Study of the relative effects of direct GHR signaling and those mediated by IGF-1 may be complicated by their other effects on muscle cell biology other than protein accumulation per se (Sun and Bartke, 2014); for example, the effects of GH leading to increased muscle cell fusion are independent of IGF-1 (Sotiropoulos et al., 2006).

expression, as well as reducing lipid breakdown and/ or lipogenesis (Vijayakumar et al., 2010). In muscle, GH acts to increase triglyceride uptake, again through increased lipoprotein lipase activity, but here the fate of triglyceride can be storage or oxidation (Vijayakumar et al., 2010).

6.7.4 Fat Metabolism

6.7.6 Interaction With Insulin and Secretion

Although GH is generally considered an anabolic hormone, its effects on fat are catabolic. Following in vivo GH treatment, there is an increase in both fatty acids and glycerol as a result of lipolytic breakdown of triglycerides (Moller and Jorgensen, 2009). Patients with GH deficiency have an increased fat mass that is reduced to the normal range of non-GH-deficient patients by GH treatment (Chaves et al., 2011). In animal models, GH can mediate a resistance to diet-induced obesity: transgenic mice with increased circulating GH concentrations become less obese when fed a high-fat diet compared with GHR/ mice (Berryman et al., 2006). The effects of GH on fat metabolism are tissue dependent and in the case of adipose tissue vary between the type of fat (white vs. brown) and specific fat depots. The primary lipolytic effect occurs in visceral, and to a lesser extent in subcutaneous, adipose tissue (Freda et al., 2008) and is due to increased lipolysis mediated by enhanced hormone-sensitive lipase expression, rather than inhibition of triglyceride uptake and reduced lipogenesis (Vijayakumar et al., 2010). In contrast, GH increases uptake and hepatic storage of triglyceride through increased lipoprotein lipase

It is well recognized that GH induces insulin resistance; however, the underlying mechanism(s) are not clear. It is possible that the increased circulating free fatty acids resulting from the lipolytic actions of GH on adipose tissue results in an insensitivity of target tissues to insulin (Kovacs and Stumvoll, 2005); however, liver IGF-1-deficient mice with high circulating GH concentrations and normal amounts of free fatty acids have insulin resistance (Yakar et al., 2001), and inhibiting GH action in this model improves insulin sensitivity (Yakar et al., 2004). This suggests that other direct actions of GH may have a role, such as altered SOCS signaling or expression of the p85a subunit of PI3K in response to GH (Vijayakumar et al., 2010). An additional modifying effect of GH is its effects on the b-cells of the pancreas, which are the source of insulin. Mice with a global loss of GHR have a four- to fivefold reduction in b-cell mass, which is a result of decreased cell proliferation (Liu et al., 2004), and those with a specific loss of GHR in pancreatic b cells have reduced cell proliferation in response to a high-fat diet (Wu et al., 2011). An additional effect of GH on reducing b-cell apoptosis has also been shown, as well as an

6.7.5 Carbohydrate Metabolism An understanding of the effects of GH on carbohydrate metabolism is complicated by its interactions with insulin. Early classic studies of GH action showed that injection of high doses of GH led to hyperglycemia in patients, and it is clear that at physiologic levels, GH leads to an acute reduction in glucose uptake and a delayed reduction in glucose oxidation in muscle; however, the contribution of a direct action of GH or secondary effects of increased lipid utilization is currently unclear (Moller and Jorgensen, 2009). In the liver, GH stimulates glucose production, but again the mechanisms underlying this are unclear, although the current consensus is that glycogenolysis is the principal mechanism rather than gluconeogenesis (Vijayakumar et al., 2011). The kidney becomes an important source of glucose in starvation (Cahill, 2006), and there is strong evidence that the high concentrations of GH secreted in response to food deprivation act directly on renal proximal tubule cells to increase gluconeogenesis, an activity that is independent of local or circulating IGF-1 (Rogers et al., 1989).

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enhancement of insulin synthesis and secretion (Wang et al., 2017), suggesting that an enhancement of insulin secretion may ameliorate the impairment of its signaling by GH.

species for understanding the roles of PRL, but species differences mean that only some of these effects can be generalized amongst mammals and, in particular, extended to humans.

6.7.7 Electrolyte/Fluid Homeostasis

6.8.1 Mammary Gland Regulation Although there is no evidence for a requirement for PRL in embryonic and prepubertal development of the mammary gland, it does have important roles in all of the changes that occur in the adult, especially in lactation (Trott et al., 2012). Analyses of Prlr/ mice have provided the most conclusive evidence for the importance of PRL in the postpubertal development of ductal side branches and the formation of alveolar buds gland as well as the further development of the mammary gland in pregnancy with the elaboration of the lobuloalveoli and cytological structures that are required for milk secretion (Ormandy et al., 1997a). The requirement for PRL for the maintenance of lactation is well described, with important roles in the production of the constituents of milk (for example, b-casein) as well as prevention of mammary involution (Trott et al., 2012). The long form of the PRLR appears to mediate a majority of these effects, although there may be specific roles for the short form of the receptor (Grattan and Le Tissier, 2015).

A role for GH in sodium, water, calcium, and phosphate homeostasis has been recognized for more than 5 decades (Corvilain et al., 1962). GH-deficient patients have reduced extracellular volume, total body water, and sodium ion concentrations: the reverse is true for acromegalics, and treatment leads to a normalization of fluid and sodium homeostasis (Moller, 2003). The mechanisms underlying these effects of GH are not fully understood, but alterations in kidney function underlie some of the effects. Short-term treatment with GH leads to an increase in glomerular filtration rate resulting from IGF-1 mediated reduction in renal vascular resistance and increased glomerular perfusion (Kamenicky et al., 2014). Other, direct effects of GH on the kidney are also likely; indeed the kidneys of GHR/ mice are disproportionally small compared with controls, whilst those of IGF1/ animals are not altered in proportion to overall body size. A direct role for GH in podocyte function, leading to an increased permeability to albumin, has also been described (Mukhi et al., 2017). The role of other endocrine systems regulating electrolyte and fluid homeostasis remains controversial (reviewed in Kamenicky et al., 2014; Moller, 2003), although there is convincing evidence that calcitriol-driven increased intestinal absorption of calcium and phosphate mediates some of the effects of GH (Kamenicky et al., 2014). Mild increases in blood phosphate and calcium ion concentrations as well as increased urinary calcium excretion are found in some patients with acromegaly as well as in GH-deficient patients treated with GH (Kamenicky et al., 2014). These observations suggest that calcium and therefore phosphate homeostasis are modulated by GH, hardly surprising given the role of GH in increasing statural growth.

6.8 The Physiologic Functions of PRL Although PRL is principally recognized for its essential role in lactation, it can be considered to have a modulatory role in a very diverse range of physiologic functions (Grattan and Le Tissier, 2015). These are best characterized in female reproduction, where changes in the pattern of secretion are associated with physiologic adaptations that are required for, or optimize, successful reproduction. The experimental manipulations that are possible in rats and mice, in particular the generation of mouse models with deletion of the PRL (Prl/) and the PRLR (Prlr/) genes, have made these the model

6.8.2 Pregnancy It has long been recognized that PRL has an essential role in the maintenance of pregnancy in nonprimate mammals through maintenance of the corpora lutea and hence progesterone production consistent with this; both Prl/ and Prlr/ mice are infertile (Horseman et al., 1997; Ormandy et al., 1997b). In primates, PRL is not required as the corpus luteum is maintained by chorionic gonadotrophin (Devoto et al., 2009). In addition to the maintenance of pregnancy, PRL appears to be required for normal preimplantation embryo development and implantation in the mouse (Binart et al., 2000). In the nonpregnant rodent, PRL may also have a role in luteolysis of the corpora lutea from the previous ovulations (Gaytan et al., 2001). 6.8.3 Reproductive Behavior There is evidence that PRL has roles in receptivity to mating: lordosis is stimulated in female rats following acute administration of PRL into the midbrain (Harlan et al., 1983), and it has been suggested that the loss of sex drive in men immediately following ejaculation may result from the increase in circulating PRL associated with orgasm (Kruger et al., 2002). Perhaps the most intriguing reproductive behavior associated with PRL, however, is parental care, with evidence for stimulation of pup care in both mothers (Larsen and Grattan, 2012) and fathers (Bales and Saltzman, 2016). Recent studies have provided compelling evidence for a direct

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role of PRL in the induction of maternal care and have identified the PRL surges in the first few days of pregnancy, before implantation, as a critical window (Larsen and Grattan, 2010), with the medial preoptic area as the target area of the brain (Brown et al., 2017). 6.8.4 Regulation of Hypothalamic Neurones PRL has been shown to regulate several populations of hypothalamic neurones in addition to the dopamine neurones mediating short-loop feedback of PRL secretion, with modulatory effects of important physiologic systems that are altered in pregnancy and lactation. The following points provide a summary: • Suppression of fertility: In most mammals, fertility is reduced or completely suppressed in lactation, which may be important for ensuring appropriate spacing of pregnancies for the nutrition of offspring (Valeggia and Ellison, 2009). As reduced fertility is a known effect of hyperprolactinemia in both patients (Prabhakar and Davis, 2008) and rats (Cohen-Becker et al., 1986), a role for PRL in inducing this reduced fertility has been suggested. Indeed, recent studies have demonstrated that kisspeptin neurones are a target for PRL, resulting in decreased release of kisspeptin, which in turn results in a decrease in gonadotrophin-releasing hormone secretion and a loss of fertility (Brown et al., 2014). • Reduced anxiety and stress response: Hypothalamicepituitaryeadrenal responses to anxiety and/or stress are reduced in pregnancy and lactation, and this has an important role in protecting the offspring from adverse exposure to glucocorticoids (Brunton and Russell, 2015). • Activation of oxytocin neurones: Oxytocin has an important role in parturition, milk ejection, and maternal behavior, and there is evidence for a modulatory role of PRL to increase oxytocin secretion, although this may be restricted to pregnancy and lactation (Parker et al., 1991). • Appetite regulation: Intracerebroventricular administration of PRL in rats leads to an increase in food intake (Sauve and Woodside, 1996), suggesting that direct actions of the hormone in the hypothalamus increase appetite. This along with the PRL-induced steroid hormone changes that occur in early pregnancy likely contribute to increased food intake and appetite in pregnancy (Ladyman et al., 2010) and lactation (Woodside et al., 2012). This hyperphagia of pregnancy is maintained despite high circulating concentrations of leptin resulting from increased fat mass, which results from a reduced hypothalamic response to leptin that may be due to altered JAK-STAT signaling in response to chronic PRL stimulation (Ladyman et al., 2010).

6.8.5 Effects on Metabolism PRL modifies several different metabolic processes and is likely to have an important role in mediating the adaptations in metabolism that are required in pregnancy and lactation in addition to its effects on appetite regulation (see preceding). A summary follows: • Body weight and energy partitioning: Humans with high circulating concentrations of PRL resulting from pituitary tumors have been shown to have increased weight gain, and normalization of circulating PRL concentrations by surgery or pharmacologic treatment results in weight loss in a majority of these patients (Greenman et al., 1998). Consistent with this, high circulating PRL concentrations in rats lead to increased food intake and fat deposition (Moore et al., 1986), and although in mouse models with loss of PRL (LaPensee et al., 2006) there is little effect on body weight, loss of the receptor leads to a reduction in fat mass (Flint et al., 2006). This reduction in adipose tissue occurs despite normal food intake, indicating that PRL has a role in the partitioning of nutrients, which may involve a shift from brown to white fat (Auffret et al., 2012). • Nutrient supply: The expression of PRLRs in the digestive tract (Nagano et al., 1995) is consistent with it having a role in altering gastrointestinal function, leading to increased calcium (Teerapornpuntakit et al., 2012), lipid, and fat-soluble vitamin (Cao et al., 2001) transport. Also, it has effects on bone and calcium homeostasis, as there is a delay in bone formation in Prlr/ mice (Clement-Lacroix et al., 1999). • Glucose homeostasis: Women with hyperprolactinemia have increased basal and stimulated insulin secretion, similar to that found in pregnancy, which is reduced after treatment to normalize the PRL concentrations (Berinder et al., 2011). Enhanced insulin secretion in pregnancy is important in counteracting the maternal insulin resistance of late pregnancy, thereby ensuring an enhanced glucose supply to the developing fetus (Baeyens et al., 2016). PRL receptors are present in pancreatic b-cells, and their expression increases during pregnancy (Sorenson and Stout, 1995), and a 50% reduction in expression in Prlrþ/ mice leads to reduced b-cell expansion, glucose-stimulated insulin release, and glucose tolerance in pregnancy (Huang et al., 2009).

7. DISORDERS OF THE GH AND PRL AXES As may be expected from their important roles in multiple physiologic processes, both deficiency and excess of either GH or PRL can have serious

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consequences for human health. Throughout this chapter, we have used examples from humans or animal models where pathologic dysregulation of these hormones has informed their function, so we will limit the descriptions of the consequences of disorders to a brief description here. The common developmental origin of somatotrophs and lactotrophs means that dysregulation of both hormones can occur, but there are also a number of conditions where only one hormone may be affected.

expression have also been described with both recessive and dominant modes of inheritance, although the phenotype of these patients may be variable (Alatzoglou et al., 2014).

7.1 Hormone Deficiency

• Intrauterine insults: Perinatal injury has been reported in 80% of patients with hypopituitarism, suggesting that this may be an important cause of pituitary hormone deficiency, although it is possible that the associated trauma may only augment congenital defects or that pituitary dysfunction may increase the risk of birth trauma (Di Iorgi et al., 2016). An increased risk of pituitary stalk abnormalities is also associated with breech deliveries, suggesting that a disruption of the portal circulation may lead to abnormal postnatal pituitary development and regulation (Maghnie et al., 1996). • Pituitary tumors and their surgical removal: Macroadenomas or craniopharyngiomas can have a secondary effect on the secretion of both GH and PRL through a mass effect causing a compression of the pituitary portal blood supply (Arafah et al., 2000). Pituitary hormone deficiency is also a common consequence of surgery to remove pituitary tumors, with an increased likelihood if the tumor mass is large or has infiltrated the gland (Higham et al., 2016). • Radiation therapy: The pituitary can become damaged with a resulting loss of function if it is within the field irradiated in the treatment of a number of conditions, including tumors of the hypothalamic-pituitary area and other brain tumors requiring whole head irradiation (Darzy and Shalet, 2009). • Traumatic brain and vascular injury: Estimates of the incidence of pituitary hormone deficiency following traumatic brain injury (TBI) vary from 15% to 50% and may be caused by either a single trauma (such as a blast injury or motor vehicle accident) or by repeated trauma (for example, in sports such as rugby or kickboxing) (Tanriverdi et al., 2015). Infarction or hemorrhage into the pituitary, usually associated with a pituitary tumor, can lead to apoplexy of the gland and pituitary hormone deficiencies (Capatina et al., 2015). Pituitary infarction can also occur as a result of severe postpartum hemorrhage, with the ensuing loss of pituitary function named Sheehan syndrome (Higham et al., 2016). • Autoimmune disease and infection: Inflammation of the pituitary resulting from an autoimmune disorder

Pituitary hormone deficiency can be congenital or acquired and may present during childhood or adulthood: in all cases the most frequently affected hormone is GH (Alatzoglou et al., 2014). Estimates of the occurrence of all cases of pituitary hormone deficiency in the human population vary but had a prevalence of 29e45 per 100,000 in one large study in Spain (Regal et al., 2001). Estimates for the prevalence of GH deficiency range from 12 to 25 per 100,000 (Phillips and Cogan, 1994), whilst PRL deficiency is rare (Higham et al., 2016).

7.2 Congenital Causes of GH and PRL Deficiency Although a large proportion of cases of pituitary deficiency are idiopathic in origin, familial inheritance accounts for 5%e30% of all cases (Phillips and Cogan, 1994). Many of the genes responsible have not yet been identified, but a number that have a role in either hypothalamic or pituitary development have been shown to be found in a proportion of patients that most commonly present with multiple pituitary hormone deficiencies, including GH and PRL (reviewed in Fang et al., 2016). Isolated GH deficiency can also result from genes with roles in pituitary and hypothalamic development, but in a high proportion of cases, these patients go on to develop deficiencies in multiple pituitary hormones. Mutations in those factors with direct roles in GH regulation or action have also been identified in children with isolated GH deficiency (reviewed extensively in Alatzoglou et al., 2014). Mutations in the GH1 gene itself can either lead to GH deficiency with recessive or dominant modes of inheritance depending on whether a functional GH protein is produced and whether the mutated protein affects normal somatotroph secretory function and interacts with GHR. A number of GHRHR mutations have been described in patients with short stature, and 93 mutations have been described in GHRs that affect receptor interaction with GH, downstream signaling, or receptor expression (Lin et al., 2018). Mutations in the gene encoding the POU1F1 transcription factor required for both GH and PRL gene

7.3 Acquired GH and PRL Deficiency GH and PRL deficiency can result from a number of adverse events that may affect the pituitary gland or its regulation by the hypothalamus:

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is a rare condition that may be activated by immunotherapy drugs. This is described as hypophysitis and can variably lead to deficiency of both GH and PRL (Faje, 2016). GH deficiency may also be rarely a secondary consequence of diseases such as Langerhans cell histiocytosis, meningitis, or encephalitis (Di Iorgi et al., 2016).

7.4 Consequences of GH and PRL Deficiency As may be expected from its physiologic roles (see before), the consequences of GH deficiency are short stature in children, and in adults, increased fat and reduced muscle mass, low energy, and reduced quality of life. There is also evidence that GH deficiency may have impacts on cognitive function, bone architecture, and cardiovascular function, although the extent to which these are secondary to metabolic changes is unclear (Alatzoglou et al., 2014). PRL deficiency in humans primarily leads to failure of lactation (Ascoli and Cavagnini, 2006).

7.5 Hormone Excess Excess secretion of GH has been estimated to occur with a prevalence of six per 100,000, with males and females equally affected (Holdaway and Rajasoorya, 1999). The prevalence of excess PRL secretion is much higher in both men and women, with estimates of 20 and 90 per 100,000, respectively (Kars et al., 2009). The principal cause of both GH and PRL excess is the development of pituitary adenomas (see Chapters 30 and 31) but hyperprolactinemia occurs as a result of antipsychotic drug treatment, and increased use of this treatment is therefore causing a dramatic rise in the prevalence of hyperprolactinemia (Soto-Pedre et al., 2017). Additional causes of overproduction of GH are hypothalamic tumors leading to excess GHRH secretion, as well as, very rarely, ectopic GHRH or GH secretion from tumors in the periphery (Doga et al., 2001; Beuschlein et al., 2000). PRL excess can also occur following sellar and parasellar lesions that lead to a loss of dopaminergic control, which can also result from TBI (Agha et al., 2004).

7.6 Consequences of GH Excess Excess GH can cause a range of effects, but these vary greatly with age as result of closure of the epiphyseal plates at puberty in humans. Thus, GH excess in childhood leads to gigantism, with linear growth increased by more than 3 standard deviations above the mean for age and height (Vilar et al., 2017). In adults, the effects of GH excess are progressive and insidious,

meaning that diagnosis is often delayed for up to 10 years, and there are a range of systemic manifestations (Vilar et al., 2017). The main physical manifestations of GH excess in adults are excessive growth of hands and feet; facial coarsening (pronounced brow protrusion and increased interdental spacing); macroglossia, which may result in sleep apnoea; skin thickening and increased hair coarseness; and excessive sweating. Excess GH also leads to increased incidences of joint disease and musculoskeletal pain, hypertension, and a higher risk of certain tumors (especially colon cancer). There are several metabolic complications associated with GH excess, in particular, insulin resistance, decreased peripheral glucose uptake, increased gluconeogenesis and lipolysis, which together result in impaired glucose tolerance and an increased risk of diabetes mellitus and/or dyslipidemia.

7.7 Consequences of PRL Excess The principal effects of PRL excess are those that may be expected from its roles in milk production and modulation of fertility (Grattan and Le Tissier, 2015). In females, hyperprolactinemia leads to delayed puberty, galactorrhea, amenorrhea, infertility, reduced libido, and in some cases, recurrent miscarriage. Males with high levels of PRL can also experience galactorrhea, delayed puberty, infertility, and loss of libido.

8. CONCLUSIONS The pleiotropic actions of both GH and PRL mean that they are ideal hormones to coordinate physiologic responses to a number of challenges. This in turn also results in modulation of their secretion and activities by a wide range of both internal and external factors, some of which have been poorly characterized to date. Compared with PRL, the actions of GH, regulation of secretion, and the mechanisms underlying its actions have been the subject of intense study and are well understood. In contrast, many aspects of PRL biology have relied on its similarity to GH. It is clear, however, that there are considerable differences in the regulation and mechanisms of actions of the two hormones, and more intense study of PRL is warranted. In this regard, study of placental forms of GH and PRL may be informative, as these may have intermediate actions and define new roles, or novel aspects of known roles, for both hormones. The impetus for intense study of GH has been its importance in disease, both of deficiency and excess, as well as its roles in the regulation of metabolism. In terms of understanding its normal function and potential

REFERENCES

therapy to ameliorate its dysregulation, a clearer understanding of the importance of its patterned output, the modifying effects of GHBP, and how these may interact to result in tissue-specific effects is required. The importance of pulsatile output may provide a rationale for regenerating these patterns in therapy for GH deficiency, which may be possible using stem cells to regenerate a functional pituitary. The privileged relationship of the pituitary with the hypothalamus means this would most likely require cell replacement within the gland, but the relatively routine use of transphenoidal surgery suggest this may be feasible. The findings that cell networks have an important role in the output of the gland, however, suggest that this must be accounted for in any approaches utilizing stem cell therapy. The complex issue of direct GH effects, local and circulating IGF-1 also requires more study, although sophisticated mouse models are increasingly allowing dissection of their relative contributions to the modification of tissue function by GH. Important differences between humans and rodents in the biology of GH must be addressed, however, and the use of a broader range of model species (such as the rabbit) may allow this. The major disorder of the PRL axis in humans is that of excess. The relatively ease with which this is treated in most cases by dopamine analogs may explain, in part, the relative lack of research on this hormone in comparison to GH. The long-term use of dopamine analogs is not without side-effects, so more effective treatments, such as other therapies to suppress PRL secretion, inhibit lactotroph proliferation, or block the actions of the hormone, may be beneficial. The increasing prevalence of hyperprolactinemia as a consequence of the use of psychoactive drugs suggests that an increased understanding of PRL regulation, its modes of action, and physiologic functions should be addressed in more detail in future studies.

References Abdel-Meguid, S.S., Shieh, H.S., Smith, W.W., Dayringer, H.E., Violand, B.N., Bentle, L.A., 1987. Three-dimensional structure of a genetically engineered variant of porcine growth hormone. Proc. Natl. Acad. Sci. U.S.A. 84, 6434e6437. Agha, A., Rogers, B., Sherlock, M., O’kelly, P., Tormey, W., Phillips, J., Thompson, C.J., 2004. Anterior pituitary dysfunction in survivors of traumatic brain injury. J. Clin. Endocrinol. Metab. 89, 4929e4936. Alam, S.M., Ain, R., Konno, T., HO-Chen, J.K., Soares, M.J., 2006. The rat prolactin gene family locus: species-specific gene family expansion. Mamm. Genome 17, 858e877. Alatzoglou, K.S., Kular, D., Dattani, M.T., 2015. Autosomal dominant growth hormone deficiency (type II). Pediatr. Endocrinol. Rev. 12, 347e355. Alatzoglou, K.S., Webb, E.A., LE Tissier, P., Dattani, M.T., 2014. Isolated growth hormone deficiency (GHD) in childhood and adolescence: recent advances. Endocr. Rev. 35, 376e432.

193

Alba, M., Salvatori, R., 2004. A mouse with targeted ablation of the growth hormone-releasing hormone gene: a new model of isolated growth hormone deficiency. Endocrinology 145, 4134e4143. Alvarez, C.V., Mallo, F., Burguera, B., Cacicedo, L., Dieguez, C., Casanueva, F.F., 1991. Evidence for a direct pituitary inhibition by free fatty acids of in vivo growth hormone responses to growth hormone-releasing hormone in the rat. Neuroendocrinology 53, 185e189. Amit, T., Bergman, T., Dastot, F., Youdim, M.B., Amselem, S., Hochberg, Z., 1997. A membrane-fixed, truncated isoform of the human growth hormone receptor. J. Clin. Endocrinol. Metab. 82, 3813e3817. Arafah, B.M., Prunty, D., Ybarra, J., Hlavin, M.L., Selman, W.R., 2000. The dominant role of increased intrasellar pressure in the pathogenesis of hypopituitarism, hyperprolactinemia, and headaches in patients with pituitary adenomas. J. Clin. Endocrinol. Metab. 85, 1789e1793. Arden, K.C., Boutin, J.M., Djiane, J., Kelly, P.A., Cavenee, W.K., 1990. The receptors for prolactin and growth hormone are localized in the same region of human chromosome 5. Cytogenet. Cell Genet. 53, 161e165. Argetsinger, L.S., Campbell, G.S., Yang, X., Witthuhn, B.A., Silvennoinen, O., Ihle, J.N., Carter-Su, C., 1993. Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell 74, 237e244. Asa, S.L., Penz, G., Kovacs, K., Ezrin, C., 1982. Prolactin cells in the human pituitary. A quantitative immunocytochemical analysis. Arch. Pathol. Lab Med. 106, 360e363. Ascoli, P., Cavagnini, F., 2006. Hypopituitarism. Pituitary 9, 335e342. Auffret, J., Viengchareun, S., Carre, N., Denis, R.G., Magnan, C., Marie, P.Y., Muscat, A., Feve, B., Lombes, M., Binart, N., 2012. Beige differentiation of adipose depots in mice lacking prolactin receptor protects against high-fat-diet-induced obesity. FASEB J. 26, 3728e3737. Baeyens, L., Hindi, S., Sorenson, R.L., German, M.S., 2016. beta-Cell adaptation in pregnancy. Diabetes Obes. Metab. 18 (Suppl. 1), 63e70. Bales, K.L., Saltzman, W., 2016. Fathering in rodents: neurobiological substrates and consequences for offspring. Horm. Behav. 77, 249e259. Barclay, J.L., Anderson, S.T., Waters, M.J., Curlewis, J.D., 2009. SOCS3 as a tumor suppressor in breast cancer cells, and its regulation by PRL. Int. J. Cancer 124, 1756e1766. Barsh, G.S., Seeburg, P.H., Gelinas, R.E., 1983. The human growth hormone gene family: structure and evolution of the chromosomal locus. Nucleic Acids Res. 11, 3939e3958. Baumann, G., 2002. Growth hormone binding protein. The soluble growth hormone receptor. Minerva Endocrinol. 27, 265e276. Baumann, G., Abramson, E.C., 1983. Urinary growth hormone in man: evidence for multiple molecular forms. J. Clin. Endocrinol. Metab. 56, 305e311. Baumann, G., Amburn, K.D., Buchanan, T.A., 1987. The effect of circulating growth hormone-binding protein on metabolic clearance, distribution, and degradation of human growth hormone. J. Clin. Endocrinol. Metab. 64, 657e660. Baumann, G., Shaw, M.A., 1990. A second, lower affinity growth hormone-binding protein in human plasma. J. Clin. Endocrinol. Metab. 70, 680e686. Baumann, G., Stolar, M.W., Amburn, K., Barsano, C.P., Devries, B.C., 1986. A specific growth hormone-binding protein in human plasma: initial characterization. J. Clin. Endocrinol. Metab. 62, 134e141. Baxter, R.C., Zaltsman, Z., 1984. Induction of hepatic receptors for growth hormone (GH) and prolactin by GH infusion is sex independent. Endocrinology 115, 2009e2014.

194

9. ANTERIOR PITUITARY: SOMATOTROPHS (GH) AND LACTOTROPHS (PRL)

Ben-Jonathan, N., Lapensee, C.R., Lapensee, E.W., 2008. What can we learn from rodents about prolactin in humans? Endocr. Rev. 29, 1e41. Berinder, K., Nystrom, T., Hoybye, C., Hall, K., Hulting, A.L., 2011. Insulin sensitivity and lipid profile in prolactinoma patients before and after normalization of prolactin by dopamine agonist therapy. Pituitary 14, 199e207. Bernard, V., Lamothe, S., Beau, I., Guillou, A., Martin, A., le Tissier, P., Grattan, D., Young, J., Binart, N., 2018. Autocrine actions of prolactin contribute to the regulation of lactotroph function in vivo. FASEB J. 32, 4791e4797. Bernard, V., Young, J., Chanson, P., Binart, N., 2015. New insights in prolactin: pathological implications. Nat. Rev. Endocrinol. 11, 265e275. Berryman, D.E., List, E.O., Kohn, D.T., Coschigano, K.T., Seeley, R.J., Kopchick, J.J., 2006. Effect of growth hormone on susceptibility to diet-induced obesity. Endocrinology 147, 2801e2808. Beuschlein, F., Strasburger, C.J., Siegerstetter, V., Moradpour, D., Lichter, P., Bidlingmaier, M., Blum, H.E., Reincke, M., 2000. Acromegaly caused by secretion of growth hormone by a nonHodgkin’s lymphoma. N. Engl. J. Med. 342, 1871e1876. Biener, E., Martin, C., Daniel, N., Frank, S.J., Centonze, V.E., Herman, B., Djiane, J., Gertler, A., 2003. Ovine placental lactogeninduced heterodimerization of ovine growth hormone and prolactin receptors in living cells is demonstrated by fluorescence resonance energy transfer microscopy and leads to prolonged phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3. Endocrinology 144, 3532e3540. Binart, N., Helloco, C., Ormandy, C.J., Barra, J., Clement-Lacroix, P., Baran, N., Kelly, P.A., 2000. Rescue of preimplantatory egg development and embryo implantation in prolactin receptor-deficient mice after progesterone administration. Endocrinology 141, 2691e2697. Birzniece, V., Ho, K.K.Y., 2017. Sex steroids and the GH axis: implications for the management of hypopituitarism. Best Pract. Res. Clin. Endocrinol. Metabol. 31, 59e69. Bodart, G., Farhat, K., Charlet-Renard, C., Salvatori, R., Geenen, V., Martens, H., 2017. The somatotrope growth hormone-releasing hormone/growth hormone/insulin-like growth factor-1 axis in immunoregulation and immunosenescence. Front. Horm. Res. 48, 147e159. Boguszewski, C.L., Barbosa, E.J.L., Svensson, P.A., Johannsson, G., Glad, C.A.M., 2017. Mechanisms in endocrinology: clinical and pharmacogenetic aspects of the growth hormone receptor polymorphism. Eur. J. Endocrinol. 177, R309eR321. Bole-Feysot, C., Goffin, V., Edery, M., Binart, N., Kelly, P.A., 1998. Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr. Rev. 19, 225e268. Borrelli, E., Heyman, R.A., Arias, C., Sawchenko, P.E., Evans, R.M., 1989. Transgenic mice with inducible dwarfism. Nature 339, 538e541. Borrelli, E., Sawchenko, P.E., Evans, R.M., 1992. Pituitary hyperplasia induced by ectopic expression of nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 89, 2764e2768. Borson-Chazot, F., Garby, L., Raverot, G., Claustrat, F., Raverot, V., Sassolas, G., Group, G.T.E., 2012. Acromegaly induced by ectopic secretion of GHRH: a review 30 years after GHRH discovery. Ann. Endocrinol. 73, 497e502. Bowers, C.Y., Reynolds, G.A., Durham, D., Barrera, C.M., Pezzoli, S.S., Thorner, M.O., 1990. Growth hormone (GH)-releasing peptide stimulates GH release in normal men and acts synergistically with GHreleasing hormone. J. Clin. Endocrinol. Metab. 70, 975e982. Briard, N., Rico-Gomez, M., Guillaume, V., Sauze, N., Vuaroqueaux, V., Dadoun, F., Le Bouc, Y., Oliver, C., Dutour, A., 1998. Hypothalamic mediated action of free fatty acid on growth hormone secretion in sheep. Endocrinology 139, 4811e4819.

Brooks, A.J., Wooh, J.W., Tunny, K.A., Waters, M.J., 2008. Growth hormone receptor; mechanism of action. Int. J. Biochem. Cell Biol. 40, 1984e1989. Brooks, C.L., 2012. Molecular mechanisms of prolactin and its receptor. Endocr. Rev. 33, 504e525. Brown, R.S., Herbison, A.E., Grattan, D.R., 2014. Prolactin regulation of kisspeptin neurones in the mouse brain and its role in the lactationinduced suppression of kisspeptin expression. J. Neuroendocrinol. 26, 898e908. Brown, R.S.E., Aoki, M., Ladyman, S.R., Phillipps, H.R., Wyatt, A., Boehm, U., Grattan, D.R., 2017. Prolactin action in the medial preoptic area is necessary for postpartum maternal nursing behavior. Proc. Natl. Acad. Sci. U.S.A. 114, 10779e10784. Brunton, P.J., Russell, J.A., 2015. Chapter 44 e Maternal brain adaptations in pregnancy. In: Knobil and Neill’s Physiology of Reproduction, fourth ed. Academic Press, San Diego. Bugge, K., Papaleo, E., Haxholm, G.W., Hopper, J.T., Robinson, C.V., Olsen, J.G., Lindorff-Larsen, K., Kragelund, B.B., 2016. A combined computational and structural model of the fulllength human prolactin receptor. Nat. Commun. 7, 11578. Burton, K.A., Kabigting, E.B., Steiner, R.A., Clifton, D.K., 1995. Identification of target cells for growth hormone’s action in the arcuate nucleus. Am. J. Physiol. 269, E716eE722. Byrnes, E.M., Bridges, R.S., 2005. Lactation reduces prolactin levels in reproductively experienced female rats. Horm. Behav. 48, 278e282. Cahill Jr., G.F., 2006. Fuel metabolism in starvation. Annu. Rev. Nutr. 26, 1e22. Cai, A., Hayes, J.D., Patel, N., Hyde, J.F., 1999. Targeted overexpression of galanin in lactotrophs of transgenic mice induces hyperprolactinemia and pituitary hyperplasia. Endocrinology 140, 4955e4964. Caicedo, D., Diaz, O., Devesa, P., Devesa, J., 2018. Growth hormone (GH) and cardiovascular system. Int. J. Mol. Sci. 19. Campbell, G.S., Meyer, D.J., Raz, R., Levy, D.E., Schwartz, J., CarterSu, C., 1995. Activation of acute phase response factor (APRF)/ Stat3 transcription factor by growth hormone. J. Biol. Chem. 270, 3974e3979. Cao, J., Huang, L., Liu, Y., Hoffman, T., Stieger, B., Meier, P.J., Vore, M., 2001. Differential regulation of hepatic bile salt and organic anion transporters in pregnant and postpartum rats and the role of prolactin. Hepatology 33, 140e147. Capatina, C., Inder, W., Karavitaki, N., Wass, J.A., 2015. Management of endocrine disease: pituitary tumour apoplexy. Eur. J. Endocrinol. 172, R179eR190. Cersosimo, E., Danou, F., Persson, M., Miles, J.M., 1996. Effects of pulsatile delivery of basal growth hormone on lipolysis in humans. Am. J. Physiol. 271, E123eE126. Chaves, V.E., Frasson, D., Kawashita, N.H., 2011. Several agents and pathways regulate lipolysis in adipocytes. Biochimie 93, 1631e1640. Chen, C., Brinkworth, R., Waters, M.J., 1997. The role of receptor dimerization domain residues in growth hormone signaling. J. Biol. Chem. 272, 5133e5140. Chen, E.Y., Liao, Y.C., Smith, D.H., Barrera-Saldana, H.A., Gelinas, R.E., Seeburg, P.H., 1989. The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics 4, 479e497. Chihara, K., Minamitani, N., Kaji, H., Arimura, A., Fujita, T., 1981. Intraventricularly injected growth hormone stimulates somatostatin release into rat hypophysial portal blood. Endocrinology 109, 2279e2281. Chikani, V., Ho, K.K., 2014. Action of GH on skeletal muscle function: molecular and metabolic mechanisms. J. Mol. Endocrinol. 52, R107eR123. Clapp, C., Aranda, J., Gonzalez, C., Jeziorski, M.C., Martinez de la Escalera, G., 2006. Vasoinhibins: endogenous regulators of angiogenesis and vascular function. Trends Endocrinol. Metabol. 17, 301e307. Clarke, I.J., 2015. Hypothalamus as an endocrine organ. Comp. Physiol. 5, 217e253.

REFERENCES

Clement-Lacroix, P., Ormandy, C., Lepescheux, L., Ammann, P., Damotte, D., Goffin, V., Bouchard, B., Amling, M., GaillardKelly, M., Binart, N., Baron, R., Kelly, P.A., 1999. Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice. Endocrinology 140, 96e105. Cohen-Becker, I.R., Selmanoff, M., Wise, P.M., 1986. Hyperprolactinemia alters the frequency and amplitude of pulsatile luteinizing hormone secretion in the ovariectomized rat. Neuroendocrinology 42, 328e333. Coleman, M.E., Demayo, F., Yin, K.C., Lee, H.M., Geske, R., Montgomery, C., Schwartz, R.J., 1995. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J. Biol. Chem. 270, 12109e12116. Colosi, P., Talamantes, F., Linzer, D.I., 1987. Molecular cloning and expression of mouse placental lactogen I complementary deoxyribonucleic acid. Mol. Endocrinol. 1, 767e776. Conway, S., Mccann, S.M., Krulich, L., 1985. On the mechanism of growth hormone autofeedback regulation: possible role of somatostatin and growth hormone-releasing factor. Endocrinology 117, 2284e2292. Copeland, K.C., Nair, K.S., 1994. Acute growth hormone effects on amino acid and lipid metabolism. J. Clin. Endocrinol. Metab. 78, 1040e1047. Corvilain, J., Abramow, M., Bergans, A., 1962. Some effects of human growth hormone on renal hemodynamics and on tubular phosphate transport in man. J. Clin. Investig. 41, 1230e1235. Cummings, D.E., 2006. Ghrelin and the short- and long-term regulation of appetite and body weight. Physiol. Behav. 89, 71e84. Cunningham, B.C., Bass, S., Fuh, G., Wells, J.A., 1990. Zinc mediation of the binding of human growth hormone to the human prolactin receptor. Science 250, 1709e1712. Cunningham, B.C., Ultsch, M., De Vos, A.M., Mulkerrin, M.G., Clauser, K.R., Wells, J.A., 1991. Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. Science 254, 821e825. Dagil, R., Knudsen, M.J., Olsen, J.G., O’shea, C., Franzmann, M., Goffin, V., Teilum, K., Breinholt, J., Kragelund, B.B., 2012. The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation: insight from structures of the prolactin receptor. Structure 20, 270e282. Darzy, K.H., Shalet, S.M., 2009. Hypopituitarism following radiotherapy revisited. Endocr. Dev. 15, 1e24. Daughaday, W.H., Hall, K., Raben, M.S., Salmon Jr., W.D., van den Brande, J.L., van Wyk, J.J., 1972. Somatomedin: proposed designation for sulphation factor. Nature 235, 107. Daughaday, W.H., Reeder, C., 1966. Synchronous activation of DNA synthesis in hypophysectomized rat cartilage by growth hormone. J. Lab. Clin. Med. 68, 357e368. Daughaday, W.H., Trivedi, B., 1987. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism). Proc. Natl. Acad. Sci. U.S.A. 84, 4636e4640. de Vos, A.M., Ultsch, M., Kossiakoff, A.A., 1992. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255, 306e312. de Zegher, F., Devlieger, H., Veldhuis, J.D., 1993. Properties of growth hormone and prolactin hypersecretion by the human infant on the day of birth. J. Clin. Endocrinol. Metab. 76, 1177e1181. Denef, C., 2008. Paracrinicity: the story of 30 years of cellular pituitary crosstalk. J. Neuroendocrinol. 20, 1e70. Devi, Y.S., Seibold, A.M., Shehu, A., Maizels, E., Halperin, J., Le, J., Binart, N., Bao, L., Gibori, G., 2011. Inhibition of MAPK by prolactin signaling through the short form of its receptor in the ovary and decidua: involvement of a novel phosphatase. J. Biol. Chem. 286, 7609e7618. Devi, Y.S., Shehu, A., Halperin, J., Stocco, C., Le, J., Seibold, A.M., Gibori, G., 2009a. Prolactin signaling through the short isoform of

195

the mouse prolactin receptor regulates DNA binding of specific transcription factors, often with opposite effects in different reproductive issues. Reprod. Biol. Endocrinol. 7, 87. Devi, Y.S., Shehu, A., Stocco, C., Halperin, J., Le, J., Seibold, A.M., Lahav, M., Binart, N., Gibori, G., 2009b. Regulation of transcription factors and repression of Sp1 by prolactin signaling through the short isoform of its cognate receptor. Endocrinology 150, 3327e3335. Devoto, L., Fuentes, A., Kohen, P., Cespedes, P., Palomino, A., Pommer, R., Munoz, A., Strauss 3rd, J.F., 2009. The human corpus luteum: life cycle and function in natural cycles. Fertil. Steril. 92, 1067e1079. Di Iorgi, N., Morana, G., Allegri, A.E., Napoli, F., Gastaldi, R., Calcagno, A., Patti, G., Loche, S., Maghnie, M., 2016. Classical and non-classical causes of GH deficiency in the paediatric age. Best Pract. Res. Clin. Endocrinol. Metabol. 30, 705e736. Doga, M., Bonadonna, S., Burattin, A., Giustina, A., 2001. Ectopic secretion of growth hormone-releasing hormone (GHRH) in neuroendocrine tumors: relevant clinical aspects. Ann. Oncol. 12 (Suppl. 2), S89eS94. Dong, J., Tsai-Morris, C.H., Dufau, M.L., 2006. A novel estradiol/estrogen receptor alpha-dependent transcriptional mechanism controls expression of the human prolactin receptor. J. Biol. Chem. 281, 18825e18836. Dos Santos, C., Essioux, L., Teinturier, C., Tauber, M., Goffin, V., Bougneres, P., 2004. A common polymorphism of the growth hormone receptor is associated with increased responsiveness to growth hormone. Nat. Genet. 36, 720e724. Faje, A., 2016. Hypophysitis: evaluation and management. Clin. Diabetes. Endocrinol. 2, 15. Faje, A.T., Barkan, A.L., 2010. Basal, but not pulsatile, growth hormone secretion determines the ambient circulating levels of insulin-like growth factor-I. J. Clin. Endocrinol. Metab. 95, 2486e2491. Fang, Q., George, A.S., Brinkmeier, M.L., Mortensen, A.H., Gergics, P., Cheung, L.Y., Daly, A.Z., Ajmal, A., Perez Millan, M.I., Ozel, A.B., Kitzman, J.O., Mills, R.E., Li, J.Z., Camper, S.A., 2016. Genetics of combined pituitary hormone deficiency: roadmap into the genome era. Endocr. Rev. 37, 636e675. Featherstone, K., White, M.R., Davis, J.R., 2012. The prolactin gene: a paradigm of tissue-specific gene regulation with complex temporal transcription dynamics. J. Neuroendocrinol. 24, 977e990. Fleming, J.M., Ginsburg, E., Mcandrew, C.W., Heger, C.D., Cheston, L., Rodriguez-Canales, J., Vonderhaar, B.K., Goldsmith, P., 2013. Characterization of Delta7/11, a functional prolactin-binding protein. J. Mol. Endocrinol. 50, 79e90. Flint, D.J., Binart, N., Boumard, S., Kopchick, J.J., Kelly, P., 2006. Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity. J. Endocrinol. 191, 101e111. Flint, D.J., Vernon, R.G., 1998. Effects of food restriction on the responses of the mammary gland and adipose tissue to prolactin and growth hormone in the lactating rat. J. Endocrinol. 156, 299e305. Fraser, R.A., Siminoski, K., Harvey, S., 1991. Growth hormone receptor gene: novel expression in pituitary tissue. J. Endocrinol. 128, R9eR11. Frawley, L.S., Boockfor, F.R., 1991. Mammosomatotropes: presence and functions in normal and neoplastic pituitary tissue. Endocr. Rev. 12, 337e355. Freda, P.U., Shen, W., Heymsfield, S.B., Reyes-Vidal, C.M., Geer, E.B., Bruce, J.N., Gallagher, D., 2008. Lower visceral and subcutaneous but higher intermuscular adipose tissue depots in patients with growth hormone and insulin-like growth factor I excess due to acromegaly. J. Clin. Endocrinol. Metab. 93, 2334e2343.

196

9. ANTERIOR PITUITARY: SOMATOTROPHS (GH) AND LACTOTROPHS (PRL)

Freeman, M.E., Kanyicska, B., Lerant, A., Nagy, G., 2000. Prolactin: structure, function, and regulation of secretion. Physiol. Rev. 80, 1523e1631. Fryburg, D.A., Gelfand, R.A., Barrett, E.J., 1991. Growth hormone acutely stimulates forearm muscle protein synthesis in normal humans. Am. J. Physiol. 260, E499eE504. Gadd, S.L., Clevenger, C.V., 2006. Ligand-independent dimerization of the human prolactin receptor isoforms: functional implications. Mol. Endocrinol. 20, 2734e2746. Gahete, M.D., Cordoba-Chacon, J., Lin, Q., Bruning, J.C., Kahn, C.R., Castano, J.P., Christian, H., Luque, R.M., Kineman, R.D., 2013. Insulin and IGF-I inhibit GH synthesis and release in vitro and in vivo by separate mechanisms. Endocrinology 154, 2410e2420. Gaytan, F., Bellido, C., Morales, C., Sanchez-Criado, J.E., 2001. Luteolytic effect of prolactin is dependent on the degree of differentiation of luteal cells in the rat. Biol. Reprod. 65, 433e441. George, D.L., Phillips 3rd, J.A., Francke, U., Seeburg, P.H., 1981. The genes for growth hormone and chorionic somatomammotropin are on the long arm of human chromosome 17 in region q21 to qter. Hum. Genet. 57, 138e141. Gevers, E.F., Wit, J.M., Robinson, I.C., 1996. Growth, growth hormone (GH)-binding protein, and GH receptors are differentially regulated by peak and trough components of the GH secretory pattern in the rat. Endocrinology 137, 1013e1018. Giustina, A., Veldhuis, J.D., 1998. Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human. Endocr. Rev. 19, 717e797. Godowski, P.J., Leung, D.W., Meacham, L.R., Galgani, J.P., Hellmiss, R., Keret, R., Rotwein, P.S., Parks, J.S., Laron, Z., Wood, W.I., 1989. Characterization of the human growth hormone receptor gene and demonstration of a partial gene deletion in two patients with Laron-type dwarfism. Proc. Natl. Acad. Sci. U.S.A. 86, 8083e8087. Gourdji, D., Laverriere, J.N., 1994. The rat prolactin gene: a target for tissue-specific and hormone-dependent transcription factors. Mol. Cell. Endocrinol. 100, 133e142. Govers, R., ten Broeke, T., van Kerkhof, P., Schwartz, A.L., Strous, G.J., 1999. Identification of a novel ubiquitin conjugation motif, required for ligand-induced internalization of the growth hormone receptor. EMBO J. 18, 28e36. Grattan, D.R., 2015. 60 Years of neuroendocrinology: the hypothalamoprolactin axis. J. Endocrinol. 226, T101eT122. Grattan, D.R., Le Tissier, P., 2015. Chapter 12 e Hypothalamic control of prolactin secretion, and the multiple reproductive functions of prolactin, fourth ed. In: Knobil and Neill’s Physiology of Reproduction. Academic Press, San Diego. Greenhalgh, C.J., Alexander, W.S., 2004. Suppressors of cytokine signalling and regulation of growth hormone action. Growth Hormone IGF Res. 14, 200e206. Greenman, Y., Tordjman, K., Stern, N., 1998. Increased body weight associated with prolactin secreting pituitary adenomas: weight loss with normalization of prolactin levels. Clin. Endocrinol. 48, 547e553. Gregerson, K.A., 2006. Chapter 32 e Prolactin: structure, function, and regulation of secretion. In: Neill, J.D. (Ed.), Knobil and Neill’s Physiology of Reproduction, third ed. Academic Press, St Louis. Hammer, R.E., Brinster, R.L., Rosenfeld, M.G., Evans, R.M., Mayo, K.E., 1985. Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth. Nature 315, 413e416. Harlan, R.E., Shivers, B.D., Pfaff, D.W., 1983. Midbrain microinfusions of prolactin increase the estrogen-dependent behavior, lordosis. Science 219, 1451e1453. Harper, M.E., Barrera-Saldana, H.A., Saunders, G.F., 1982. Chromosomal localization of the human placental lactogen-growth hormone gene cluster to 17q22-24. Am. J. Hum. Genet. 34, 227e234.

Harris, G.W., 1955. Neural Control of the Pituitary Gland. Eward Arnold (Publishers) Ltd, London. Harris, J., Stanford, P.M., Sutherland, K., Oakes, S.R., Naylor, M.J., Robertson, F.G., Blazek, K.D., Kazlauskas, M., Hilton, H.N., Wittlin, S., Alexander, W.S., Lindeman, G.J., Visvader, J.E., Ormandy, C.J., 2006. Socs2 and elf5 mediate prolactin-induced mammary gland development. Mol. Endocrinol. 20, 1177e1187. Hartman, M.L., Veldhuis, J.D., Vance, M.L., Faria, A.C., Furlanetto, R.W., Thorner, M.O., 1990. Somatotropin pulse frequency and basal concentrations are increased in acromegaly and are reduced by successful therapy. J. Clin. Endocrinol. Metab. 70, 1375e1384. Harvey, S., Martinez-Moreno, C.G., Luna, M., Aramburo, C., 2015. Autocrine/paracrine roles of extrapituitary growth hormone and prolactin in health and disease: an overview. Gen. Comp. Endocrinol. 220, 103e111. Haxholm, G.W., Nikolajsen, L.F., Olsen, J.G., Fredsted, J., Larsen, F.H., Goffin, V., Pedersen, S.F., Brooks, A.J., Waters, M.J., Kragelund, B.B., 2015. Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes. Biochem. J. 468, 495e506. Herington, A.C., Ymer, S., Stevenson, J., 1986. Identification and characterization of specific binding proteins for growth hormone in normal human sera. J. Clin. Investig. 77, 1817e1823. Herman, A., Bignon, C., Daniel, N., Grosclaude, J., Gertler, A., Djiane, J., 2000. Functional heterodimerization of prolactin and growth hormone receptors by ovine placental lactogen. J. Biol. Chem. 275, 6295e6301. Higham, C.E., Johannsson, G., Shalet, S.M., 2016. Hypopituitarism. Lancet 388, 2403e2415. Hikake, T., Hayashi, S., Iguchi, T., Sato, T., 2009. The role of IGF1 on the differentiation of prolactin secreting cells in the mouse anterior pituitary. J. Endocrinol. 203, 231e240. Hirai, J., Nishita, M., Nakao, N., Saito, T.R., Tanaka, M., 2013. Regulation of prolactin receptor gene expression in the rat choroid plexus via transcriptional activation of multiple first exons during postnatal development and lactation. Exp. Anim. 62, 49e56. Hirt, H., Kimelman, J., Birnbaum, M.J., Chen, E.Y., Seeburg, P.H., Eberhardt, N.L., Barta, A., 1987. The human growth hormone gene locus: structure, evolution, and allelic variations. DNA 6, 59e70. Ho, K.Y., Evans, W.S., Blizzard, R.M., Veldhuis, J.D., Merriam, G.R., Samojlik, E., Furlanetto, R., Rogol, A.D., Kaiser, D.L., Thorner, M.O., 1987. Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J. Clin. Endocrinol. Metab. 64, 51e58. Holdaway, I.M., Rajasoorya, C., 1999. Epidemiology of acromegaly. Pituitary 2, 29e41. Holl, R.W., Schwarz, U., Schauwecker, P., Benz, R., Veldhuis, J.D., Heinze, E., 1993. Diurnal variation in the elimination rate of human growth hormone (GH): the half-life of serum GH is prolonged in the evening, and affected by the source of the hormone, as well as by body size and serum estradiol. J. Clin. Endocrinol. Metab. 77, 216e220. Horseman, N.D., Zhao, W., Montecino-Rodriguez, E., Tanaka, M., Nakashima, K., Engle, S.J., Smith, F., Markoff, E., Dorshkind, K., 1997. Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J. 16, 6926e6935. Hu, Z.Z., Meng, J., Dufau, M.L., 2001. Isolation and characterization of two novel forms of the human prolactin receptor generated by alternative splicing of a newly identified exon 11. J. Biol. Chem. 276, 41086e41094. Hu, Z.Z., Zhuang, L., Meng, J., Tsai-Morris, C.H., Dufau, M.L., 2002. Complex 5’ genomic structure of the human prolactin receptor:

REFERENCES

multiple alternative exons 1 and promoter utilization. Endocrinology 143, 2139e2142. Huang, C., Snider, F., Cross, J.C., 2009. Prolactin receptor is required for normal glucose homeostasis and modulation of beta-cell mass during pregnancy. Endocrinology 150, 1618e1626. Hudson, S.B., Schroeder, D.R., Bailey, J.N., Mielke, K.L., Erickson, D., Miles, J.M., Bowers, C.Y., Veldhuis, J.D., 2010. Pre-versus postmenopausal age, estradiol, and peptide-secretagogue type determine pulsatile growth hormone secretion in healthy women: studies using submaximal agonist drive and an estrogen clamp. J. Clin. Endocrinol. Metab. 95, 353e360. Ingraham, H.A., Chen, R.P., Mangalam, H.J., Elsholtz, H.P., Flynn, S.E., Lin, C.R., Simmons, D.M., Swanson, L., Rosenfeld, M.G., 1988. A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55, 519e529. Iranmanesh, A., Lizarralde, G., Veldhuis, J.D., 1991. Age and relative adiposity are specific negative determinants of the frequency and amplitude of growth hormone (GH) secretory bursts and the halflife of endogenous GH in healthy men. J. Clin. Endocrinol. Metab. 73, 1081e1088. Isaksson, O.G., Jansson, J.O., Gause, I.A., 1982. Growth hormone stimulates longitudinal bone growth directly. Science 216, 1237e1239. Jaffe, C.A., Turgeon, D.K., Lown, K., Demott-Friberg, R., Watkins, P.B., 2002. Growth hormone secretion pattern is an independent regulator of growth hormone actions in humans. Am. J. Physiol. Endocrinol. Metab. 283, E1008eE1015. Kamenicky, P., Mazziotti, G., Lombes, M., Giustina, A., Chanson, P., 2014. Growth hormone, insulin-like growth factor-1, and the kidney: pathophysiological and clinical implications. Endocr. Rev. 35, 234e281. Kars, M., Souverein, P.C., Herings, R.M., Romijn, J.A., Vandenbroucke, J.P., de Boer, A., Dekkers, O.M., 2009. Estimated age- and sex-specific incidence and prevalence of dopamine agonist-treated hyperprolactinemia. J. Clin. Endocrinol. Metab. 94, 2729e2734. Kavarthapu, R., Tsai Morris, C.H., Dufau, M.L., 2014. Prolactin induces up-regulation of its cognate receptor in breast cancer cells via transcriptional activation of its generic promoter by cross-talk between ERalpha and STAT5. Oncotarget 5, 9079e9091. Keeler, C., Dannies, P.S., Hodsdon, M.E., 2003. The tertiary structure and backbone dynamics of human prolactin. J. Mol. Biol. 328, 1105e1121. Kelberman, D., Rizzoti, K., Lovell-Badge, R., Robinson, I.C., Dattani, M.T., 2009. Genetic regulation of pituitary gland development in human and mouse. Endocr. Rev. 30, 790e829. Kelly, M.A., Rubinstein, M., Asa, S.L., Zhang, G., Saez, C., Bunzow, J.R., Allen, R.G., Hnasko, R., Ben-Jonathan, N., Grandy, D.K., Low, M.J., 1997. Pituitary lactotroph hyperplasia and chronic hyperprolactinemia in dopamine D2 receptor-deficient mice. Neuron 19, 103e113. Kelly, P.A., Boutin, J.M., Jolicoeur, C., Okamura, H., Shirota, M., Edery, M., Dusanter-Fourt, I., Djiane, J., 1989. Purification, cloning, and expression of the prolactin receptor. Biol. Reprod. 40, 27e32. Kelly, P.A., Posner, B.I., Tsushima, T., Friesen, H.G., 1974. Studies of insulin, growth hormone and prolactin binding: ontogenesis, effects of sex and pregnancy. Endocrinology 95, 532e539. Kim, H., Barton, E., Muja, N., Yakar, S., Pennisi, P., Leroith, D., 2005. Intact insulin and insulin-like growth factor-I receptor signaling is required for growth hormone effects on skeletal muscle growth and function in vivo. Endocrinology 146, 1772e1779. Klapper, D.G., Svoboda, M.E., van Wyk, J.J., 1983. Sequence analysis of somatomedin-C: confirmation of identity with insulin-like growth factor I. Endocrinology 112, 2215e2217. Kline, J.B., Clevenger, C.V., 2001. Identification and characterization of the prolactin-binding protein in human serum and milk. J. Biol. Chem. 276, 24760e24766.

197

Kline, J.B., Roehrs, H., Clevenger, C.V., 1999. Functional characterization of the intermediate isoform of the human prolactin receptor. J. Biol. Chem. 274, 35461e35468. Kovacs, P., Stumvoll, M., 2005. Fatty acids and insulin resistance in muscle and liver. Best Pract. Res. Clin. Endocrinol. Metabol. 19, 625e635. Kruger, T.H., Haake, P., Hartmann, U., Schedlowski, M., Exton, M.S., 2002. Orgasm-induced prolactin secretion: feedback control of sexual drive? Neurosci. Biobehav. Rev. 26, 31e44. Ladyman, S.R., Augustine, R.A., Grattan, D.R., 2010. Hormone interactions regulating energy balance during pregnancy. J. Neuroendocrinol. 22, 805e817. Lapensee, C.R., Horseman, N.D., Tso, P., Brandebourg, T.D., Hugo, E.R., Ben-Jonathan, N., 2006. The prolactin-deficient mouse has an unaltered metabolic phenotype. Endocrinology 147, 4638e4645. Larsen, C.M., Grattan, D.R., 2010. Prolactin-induced mitogenesis in the subventricular zone of the maternal brain during early pregnancy is essential for normal postpartum behavioral responses in the mother. Endocrinology 151, 3805e3814. Larsen, C.M., Grattan, D.R., 2012. Prolactin, neurogenesis, and maternal behaviors. Brain Behav. Immun. 26, 201e209. Le Roith, D., Bondy, C., Yakar, S., Liu, J.L., Butler, A., 2001. The somatomedin hypothesis: 2001. Endocr. Rev. 22, 53e74. Le Tissier, P., Campos, P., Lafont, C., Romano, N., Hodson, D.J., Mollard, P., 2017. An updated view of hypothalamic-vascular-pituitary unit function and plasticity. Nat. Rev. Endocrinol. 13, 257e267. Le Tissier, P.R., Carmignac, D.F., Lilley, S., Sesay, A.K., Phelps, C.J., Houston, P., Mathers, K., Magoulas, C., Ogden, D., Robinson, I.C., 2005. Hypothalamic growth hormone-releasing hormone (GHRH) deficiency: targeted ablation of GHRH neurons in mice using a viral ion channel transgene. Mol. Endocrinol. 19, 1251e1262. Le Tissier, P.R., Hodson, D.J., Lafont, C., Fontanaud, P., Schaeffer, M., Mollard, P., 2012. Anterior pituitary cell networks. Front. Neuroendocrinol. 33, 252e266. Lecomte, C.M., Renard, A., Martial, J.A., 1987. A new natural hGH variante17.5 kdeproduced by alternative splicing. An additional consensus sequence which might play a role in branchpoint selection. Nucleic Acids Res. 15, 6331e6348. Leung, D.W., Spencer, S.A., Cachianes, G., Hammonds, R.G., Collins, C., Henzel, W.J., Barnard, R., Waters, M.J., Wood, W.I., 1987. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature 330, 537e543. Lewis, U.J., Dunn, J.T., Bonewald, L.F., Seavey, B.K., Vanderlaan, W.P., 1978. A naturally occurring structural variant of human growth hormone. J. Biol. Chem. 253, 2679e2687. Li, C.H., Dixon, J.S., 1971. Human pituitary growth hormone. 32. The primary structure of the hormone: revision. Arch. Biochem. Biophys. 146, 233e236. Li, C.H., Dixon, J.S., Lo, T.B., Schmidt, K.D., Pankov, Y.A., 1970. Studies on pituitary lactogenic hormone. XXX. The primary structure of the sheep hormone. Arch. Biochem. Biophys. 141, 705e737. Li, C.H., Evans, H.M., 1944. The isolation of pituitary growth hormone. Science 99, 183e184. Li, Y., Kumar, K.G., Tang, W., Spiegelman, V.S., Fuchs, S.Y., 2004. Negative regulation of prolactin receptor stability and signaling mediated by SCF(beta-TrCP) E3 ubiquitin ligase. Mol. Cell. Biol. 24, 4038e4048. Liao, S., Vickers, M.H., Stanley, J.L., Baker, P.N., Perry, J.K., 2018. Human placental growth hormone variant in pathological pregnancies. Endocrinology 159, 2186e2198. Lin, S., Li, C., Li, C., Zhang, X., 2018. Growth hormone receptor mutations related to individual dwarfism. Int. J. Mol. Sci. 19. Lin, S.C., Lin, C.R., Gukovsky, I., Lusis, A.J., Sawchenko, P.E., Rosenfeld, M.G., 1993. Molecular basis of the little mouse

198

9. ANTERIOR PITUITARY: SOMATOTROPHS (GH) AND LACTOTROPHS (PRL)

phenotype and implications for cell type-specific growth. Nature 364, 208e213. Lincoln, G.A., Richardson, M., 1998. Photo-neuroendocrine control of seasonal cycles in body weight, pelage growth and reproduction: lessons from the HPD sheep model. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 119, 283e294. Lindeman, G.J., Wittlin, S., Lada, H., Naylor, M.J., Santamaria, M., Zhang, J.G., Starr, R., Hilton, D.J., Alexander, W.S., Ormandy, C.J., Visvader, J., 2001. SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice. Genes Dev. 15, 1631e1636. Liu, J.L., Coschigano, K.T., Robertson, K., Lipsett, M., Guo, Y., Kopchick, J.J., Kumar, U., Liu, Y.L., 2004. Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice. Am. J. Physiol. Endocrinol. Metab. 287, E405eE413. Liu, J.P., Baker, J., Perkins, A.S., Robertson, E.J., Efstratiadis, A., 1993. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75, 59e72. Low, M.J., Otero-Corchon, V., Parlow, A.F., Ramirez, J.L., Kumar, U., Patel, Y.C., Rubinstein, M., 2001. Somatostatin is required for masculinization of growth hormone-regulated hepatic gene expression but not of somatic growth. J. Clin. Investig. 107, 1571e1580. Lupu, F., Terwilliger, J.D., Lee, K., Segre, G.V., Efstratiadis, A., 2001. Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth. Dev. Biol. 229, 141e162. Luque, R.M., Soares, B.S., Peng, X.D., Krishnan, S., Cordoba-Chacon, J., Frohman, L.A., Kineman, R.D., 2009. Use of the metallothionein promoter-human growth hormone-releasing hormone (GHRH) mouse to identify regulatory pathways that suppress pituitary somatotrope hyperplasia and adenoma formation due to GHRHreceptor hyperactivation. Endocrinology 150, 3177e3185. Macgregor, D.J., Leng, G., 2005. Modelling the hypothalamic control of growth hormone secretion. J. Neuroendocrinol. 17, 788e803. Macleod, J.N., Lee, A.K., Liebhaber, S.A., Cooke, N.E., 1992. Developmental control and alternative splicing of the placentally expressed transcripts from the human growth hormone gene cluster. J. Biol. Chem. 267, 14219e14226. Maghnie, M., Genovese, E., Villa, A., Spagnolo, L., Campan, R., Severi, F., 1996. Dynamic MRI in the congenital agenesis of the neural pituitary stalk syndrome: the role of the vascular pituitary stalk in predicting residual anterior pituitary function. Clin. Endocrinol. 45, 281e290. Maheshwari, H.G., Silverman, B.L., Dupuis, J., Baumann, G., 1998. Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone-releasing hormone receptor: dwarfism of Sindh. J. Clin. Endocrinol. Metab. 83, 4065e4074. Marano, R.J., Ben-Jonathan, N., 2014. Minireview: extrapituitary prolactin: an update on the distribution, regulation, and functions. Mol. Endocrinol. 28, 622e633. Martini, J.F., Pezet, A., Guezennec, C.Y., Edery, M., Postel-Vinay, M.C., Kelly, P.A., 1997. Monkey growth hormone (GH) receptor gene expression. Evidence for two mechanisms for the generation of the GH binding protein. J. Biol. Chem. 272, 18951e18958. Matsumoto, A., Seki, Y., Kubo, M., Ohtsuka, S., Suzuki, A., Hayashi, I., Tsuji, K., Nakahata, T., Okabe, M., Yamada, S., Yoshimura, A., 1999. Suppression of STAT5 functions in liver, mammary glands, and T cells in cytokine-inducible SH2-containing protein 1 transgenic mice. Mol. Cell. Biol. 19, 6396e6407. Mayo, K.E., Miller, T., Dealmeida, V., Godfrey, P., Zheng, J., Cunha, S.R., 2000. Regulation of the pituitary somatotroph cell by GHRH and its receptor. Recent Prog. Horm. Res. 55, 237e266 discussion 266-7. Mcandrew, J., Paterson, A.J., Asa, S.L., Mccarthy, K.J., Kudlow, J.E., 1995. Targeting of transforming growth factor-alpha expression to

pituitary lactotrophs in transgenic mice results in selective lactotroph proliferation and adenomas. Endocrinology 136, 4479e4488. Metcalf, D., Greenhalgh, C.J., Viney, E., Willson, T.A., Starr, R., Nicola, N.A., Hilton, D.J., Alexander, W.S., 2000. Gigantism in mice lacking suppressor of cytokine signalling-2. Nature 405, 1069e1073. Meyer, D.J., Campbell, G.S., Cochran, B.H., Argetsinger, L.S., Larner, A.C., Finbloom, D.S., Carter-Su, C., Schwartz, J., 1994. Growth hormone induces a DNA binding factor related to the interferon-stimulated 91-kDa transcription factor. J. Biol. Chem. 269, 4701e4704. Miller, J.D., Esparza, A., Wright, N.M., Garimella, V., Lai, J., Lester, S.E., Mosier Jr., H.D., 1993. Spontaneous growth hormone release in term infants: changes during the first four days of life. J. Clin. Endocrinol. Metab. 76, 1058e1062. Minami, S., Kamegai, J., Sugihara, H., Suzuki, N., Wakabayashi, I., 1998. Growth hormone inhibits its own secretion by acting on the hypothalamus through its receptors on neuropeptide Y neurons in the arcuate nucleus and somatostatin neurons in the periventricular nucleus. Endocr. J. 45 (Suppl. l), S19eS26. Moller, J., 2003. Effects of growth hormone on fluid homeostasis. Clinical and experimental aspects. Growth Hormone IGF Res. 13, 55e74. Moller, N., Jorgensen, J.O., 2009. Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects. Endocr. Rev. 30, 152e177. Moller, N., Vendelbo, M.H., Kampmann, U., Christensen, B., Madsen, M., Norrelund, H., Jorgensen, J.O., 2009. Growth hormone and protein metabolism. Clin. Nutr. 28, 597e603. Moore, B.J., Gerardo-Gettens, T., Horwitz, B.A., Stern, J.S., 1986. Hyperprolactinemia stimulates food intake in the female rat. Brain Res. Bull. 17, 563e569. Morita, S., Yamashita, S., Melmed, S., 1987. Insulin-like growth factor I action on rat anterior pituitary cells: effects of intracellular messengers on growth hormone secretion and messenger ribonucleic acid levels. Endocrinology 121, 2000e2006. Morohoshi, K., Mochinaga, R., Watanabe, T., Nakajima, R., Harigaya, T., 2018. 16 kDa vasoinhibin binds to integrin alpha5 beta1 on endothelial cells to induce apoptosis. Endocr. Connect 7, 630e636. Mukhi, D., Nishad, R., Menon, R.K., Pasupulati, A.K., 2017. Novel actions of growth hormone in podocytes: implications for diabetic nephropathy. Front. Med. 4, 102. Murray, R.A., Maheshwari, H.G., Russell, E.J., Baumann, G., 2000. Pituitary hypoplasia in patients with a mutation in the growth hormonereleasing hormone receptor gene. Am. J. Neuroradiol. 21, 685e689. Nagano, M., Chastre, E., Choquet, A., Bara, J., Gespach, C., Kelly, P.A., 1995. Expression of prolactin and growth hormone receptor genes and their isoforms in the gastrointestinal tract. Am. J. Physiol. 268, G431eG442. Nakamura, S., Mizuno, M., Katakami, H., Gore, A.C., Terasawa, E., 2003. Aging-related changes in in vivo release of growth hormone-releasing hormone and somatostatin from the stalkmedian eminence in female rhesus monkeys (Macaca mulatta). J. Clin. Endocrinol. Metab. 88, 827e833. Niall, H.D., 1971. Revised primary structure for human growth hormone. Nat. New Biol. 230, 90e91. Niall, H.D., Hogan, M.L., Sauer, R., Rosenblum, I.Y., Greenwood, F.C., 1971. Sequences of pituitary and placental lactogenic and growth hormones: evolution from a primordial peptide by gene reduplication. Proc. Natl. Acad. Sci. U.S.A. 68, 866e870. Ocampo Daza, D., Larhammar, D., 2018a. Evolution of the growth hormone, prolactin, prolactin 2 and somatolactin family. Gen. Comp. Endocrinol. 264, 94e112. Ocampo Daza, D., Larhammar, D., 2018b. Evolution of the receptors for growth hormone, prolactin, erythropoietin and thrombopoietin in

REFERENCES

relation to the vertebrate tetraploidizations. Gen. Comp. Endocrinol. 257, 143e160. Ormandy, C.J., Binart, N., Kelly, P.A., 1997a. Mammary gland development in prolactin receptor knockout mice. J. Mammary Gland Biol. Neoplasia 2, 355e364. Ormandy, C.J., Camus, A., Barra, J., Damotte, D., Lucas, B., Buteau, H., Edery, M., Brousse, N., Babinet, C., Binart, N., Kelly, P.A., 1997b. Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev. 11, 167e178. Owens, D., Srivastava, M.C., Tompkins, C.V., Nabarro, J.D., Sonksen, P.H., 1973. Studies on the metabolic clearance rate, apparent distribution space and plasma half-disappearance time of unlabelled human growth hormone in normal subjects and in patients with liver disease, renal disease, thyroid disease and diabetes mellitus. Eur. J. Clin. Investig. 3, 284e294. Owerbach, D., Rutter, W.J., Cooke, N.E., Martial, J.A., Shows, T.B., 1981. The prolactin gene is located on chromosome 6 in humans. Science 212, 815e816. Palm, I.F., van der Beek, E.M., Swarts, H.J., van der Vliet, J., Wiegant, V.M., Buijs, R.M., Kalsbeek, A., 2001. Control of the estradiol-induced prolactin surge by the suprachiasmatic nucleus. Endocrinology 142, 2296e2302. Palizban, A.A., Radmansorry, M., Bozorgzad, M., 2014. Exon 3-deleted and full-length growth hormone receptor polymorphism frequencies in an Iranian population. Res. Pharm. Sci. 9, 489e494. Pantel, J., Grulich-Henn, J., Bettendorf, M., Strasburger, C.J., Heinrich, U., Amselem, S., 2003. Heterozygous nonsense mutation in exon 3 of the growth hormone receptor (GHR) in severe GH insensitivity (Laron syndrome) and the issue of the origin and function of the GHRd3 isoform. J. Clin. Endocrinol. Metab. 88, 1705e1710. Parker, S.L., Armstrong, W.E., Sladek, C.D., Grosvenor, C.E., Crowley, W.R., 1991. Prolactin stimulates the release of oxytocin in lactating rats: evidence for a physiological role via an action at the neural lobe. Neuroendocrinology 53, 503e510. Peeters, S., Friesen, H.G., 1977. A growth hormone binding factor in the serum of pregnant mice. Endocrinology 101, 1164e1183. Perez-Ibave, D.C., Rodriguez-Sanchez, I.P., Garza-Rodriguez Mde, L., Barrera-Saldana, H.A., 2014. Extrapituitary growth hormone synthesis in humans. Growth Hormone IGF Res. 24, 47e53. Phillips 3rd, J.A., Cogan, J.D., 1994. Genetic basis of endocrine disease. 6. Molecular basis of familial human growth hormone deficiency. J. Clin. Endocrinol. Metab. 78, 11e16. Posner, B.I., 1976. Characterization and modulation of growth hormone and prolactin binding in mouse liver. Endocrinology 98, 645e654. Prabhakar, V.K., Davis, J.R., 2008. Hyperprolactinaemia. Best Pract. Res. Clin. Obstet. Gynaecol. 22, 341e353. Qazi, A.M., Tsai-Morris, C.H., Dufau, M.L., 2006. Ligand-independent homo- and heterodimerization of human prolactin receptor variants: inhibitory action of the short forms by heterodimerization. Mol. Endocrinol. 20, 1912e1923. Quentien, M.H., Barlier, A., Franc, J.L., Pellegrini, I., Brue, T., Enjalbert, A., 2006. Pituitary transcription factors: from congenital deficiencies to gene therapy. J. Neuroendocrinol. 18, 633e642. Ram, P.A., Waxman, D.J., 1999. SOCS/CIS protein inhibition of growth hormone-stimulated STAT5 signaling by multiple mechanisms. J. Biol. Chem. 274, 35553e35561. Ray, J., Okamura, H., Kelly, P.A., Cooke, N.E., Liebhaber, S.A., 1990. Human growth hormone-variant demonstrates a receptor binding profile distinct from that of normal pituitary growth hormone. J. Biol. Chem. 265, 7939e7944. Regal, M., Paramo, C., Sierra, S.M., Garcia-Mayor, R.V., 2001. Prevalence and incidence of hypopituitarism in an adult Caucasian population in northwestern Spain. Clin. Endocrinol. 55, 735e740.

199

Rimoin, D.L., Merimee, T.J., mc Kusick, V.A., 1966. Growth-hormone deficiency in man: an isolated, recessively inherited defect. Science 152, 1635e1637. Riskind, P.N., Millard, W.J., Martin, J.B., 1984. Opiate modulation of the anterior pituitary hormone response during suckling in the rat. Endocrinology 114, 1232e1237. Rivier, J., Spiess, J., Thorner, M., Vale, W., 1982. Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature 300, 276e278. Roberts Jr., C.T., Lasky, S.R., Lowe Jr., W.L., Seaman, W.T., Leroith, D., 1987. Molecular cloning of rat insulin-like growth factor I complementary deoxyribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues. Mol. Endocrinol. 1, 243e248. Robinson, I.C.A.F., Hindmarsh, P.C., 2010. The Growth Hormone Secretory Pattern and Statural Growth. Comprehensive Physiology. John Wiley & Sons, Inc. Roelfsema, F., Pijl, H., Keenan, D.M., Veldhuis, J.D., 2012. Prolactin secretion in healthy adults is determined by gender, age and body mass index. PLoS One 7, e31305. Roemmich, J.N., Clark, P.A., Weltman, A., Veldhuis, J.D., Rogol, A.D., 2005. Pubertal alterations in growth and body composition: IX. Altered spontaneous secretion and metabolic clearance of growth hormone in overweight youth. Metabolism 54, 1374e1383. Rogers, S.A., Karl, I.E., Hammerman, M.R., 1989. Growth hormone directly stimulates gluconeogenesis in canine renal proximal tubule. Am. J. Physiol. 257, E751eE756. Rogol, A.D., Rosen, S.W., 1974. Prolactin of apparent large molecular size: the major immunoactive prolactin component in plasma of a patient with a pituitary tumor. J. Clin. Endocrinol. Metab. 38, 714e717. Romano, N., Guillou, A., Hodson, D.J., Martin, A.O., Mollard, P., 2017. Multiple-scale neuroendocrine signals connect brain and pituitary hormone rhythms. Proc. Natl. Acad. Sci. U.S.A. 114, 2379e2382. Romano, N., Yip, S.H., Hodson, D.J., Guillou, A., Parnaudeau, S., Kirk, S., Tronche, F., Bonnefont, X., LE Tissier, P., Bunn, S.J., Grattan, D.R., Mollard, P., Martin, A.O., 2013. Plasticity of hypothalamic dopamine neurons during lactation results in dissociation of electrical activity and release. J. Neurosci. 33, 4424e4433. Romero, C.J., Ng, Y., Luque, R.M., Kineman, R.D., Koch, L., Bruning, J.C., Radovick, S., 2010. Targeted deletion of somatotroph insulin-like growth factor-I signaling in a cell-specific knockout mouse model. Mol. Endocrinol. 24, 1077e1089. Rosenfeld, R.G., 1994. Circulating growth hormone binding proteins. Horm. Res. 42, 129e132. Ross, R.J., Esposito, N., Shen, X.Y., von Laue, S., Chew, S.L., Dobson, P.R., Postel-Vinay, M.C., Finidori, J., 1997. A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein. Mol. Endocrinol. 11, 265e273. Sachse, M., van Kerkhof, P., Strous, G.J., Klumperman, J., 2001. The ubiquitin-dependent endocytosis motif is required for efficient incorporation of growth hormone receptor in clathrin-coated pits, but not clathrin-coated lattices. J. Cell Sci. 114, 3943e3952. Saiardi, A., Bozzi, Y., Baik, J.H., Borrelli, E., 1997. Antiproliferative role of dopamine: loss of D2 receptors causes hormonal dysfunction and pituitary hyperplasia. Neuron 19, 115e126. Sangeeta Devi, Y., Halperin, J., 2014. Reproductive actions of prolactin mediated through short and long receptor isoforms. Mol. Cell. Endocrinol. 382, 400e410. Sato, M., Frohman, L.A., 1993. Differential effects of central and peripheral administration of growth hormone (GH) and insulin-like growth factor on hypothalamic GH-releasing hormone and somatostatin gene expression in GH-deficient dwarf rats. Endocrinology 133, 793e799.

200

9. ANTERIOR PITUITARY: SOMATOTROPHS (GH) AND LACTOTROPHS (PRL)

Sauve, D., Woodside, B., 1996. The effect of central administration of prolactin on food intake in virgin female rats is dose-dependent, occurs in the absence of ovarian hormones and the latency to onset varies with feeding regimen. Brain Res. 729, 75e81. Scheithauer, B.W., Sano, T., Kovacs, K.T., Young Jr., W.F., Ryan, N., Randall, R.V., 1990. The pituitary gland in pregnancy: a clinicopathologic and immunohistochemical study of 69 cases. Mayo Clin. Proc. 65, 461e474. Schilbach, K., Bidlingmaier, M., 2015. Growth hormone binding protein - physiological and analytical aspects. Best Pract. Res. Clin. Endocrinol. Metabol. 29, 671e683. Schuler, L.A., Kessler, M.A., 1992. Bovine placental prolactin-related hormones. Trends Endocrinol. Metabol. 3, 334e338. Schwartzbauer, G., Menon, R.K., 1998. Regulation of growth hormone receptor gene expression. Mol. Genet. Metab. 63, 243e253. Scully, K.M., Gleiberman, A.S., Lindzey, J., Lubahn, D.B., Korach, K.S., Rosenfeld, M.G., 1997. Role of estrogen receptor-alpha in the anterior pituitary gland. Mol. Endocrinol. 11, 674e681. Seeburg, P.H., 1982. The human growth hormone gene family: nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA 1, 239e249. Seilicovich, A., 2010. Cell life and death in the anterior pituitary gland: role of oestrogens. J. Neuroendocrinol. 22, 758e764. Shome, B., Parlow, A.F., 1977. Human pituitary prolactin (hPRL): the entire linear amino acid sequence. J. Clin. Endocrinol. Metab. 45, 1112e1115. Sievertsen, G.D., Lim, V.S., Nakawatase, C., Frohman, L.A., 1980. Metabolic clearance and secretion rates of human prolactin in normal subjects and in patients with chronic renal failure. J. Clin. Endocrinol. Metab. 50, 846e852. Sinha, Y.N., Selby, F.W., Vanderlaan, W.P., 1973. Radioimmunoassay of prolactin in the urine of mouse and man. J. Clin. Endocrinol. Metab. 36, 1039e1042. Soares, M.J., Faria, T.N., Roby, K.F., Deb, S., 1991. Pregnancy and the prolactin family of hormones: coordination of anterior pituitary, uterine, and placental expression. Endocr. Rev. 12, 402e423. Sonenberg, M., Money, W.L., Keston, A.S., Fitzgerald, P.J., Godwin, J.T., 1951. Localization of radioactivity after administration of labeled prolactin preparations to the female rat. Endocrinology 49, 709e719. Sorenson, R.L., Stout, L.E., 1995. Prolactin receptors and JAK2 in islets of Langerhans: an immunohistochemical analysis. Endocrinology 136, 4092e4098. Sotiropoulos, A., Ohanna, M., Kedzia, C., Menon, R.K., Kopchick, J.J., Kelly, P.A., Pende, M., 2006. Growth hormone promotes skeletal muscle cell fusion independent of insulin-like growth factor 1 upregulation. Proc. Natl. Acad. Sci. U.S.A. 103, 7315e7320. Soto-Pedre, E., Newey, P.J., Bevan, J.S., Greig, N., Leese, G.P., 2017. The epidemiology of hyperprolactinaemia over 20 years in the tayside region of Scotland: the prolactin epidemiology, audit and research study (PROLEARS). Clin. Endocrinol. 86, 60e67. Southard, J.N., Talamantes, F., 1991. Placental prolactin-like proteins in rodents: variations on a structural theme. Mol. Cell. Endocrinol. 79, C133eC140. Spencer, S.A., Hammonds, R.G., Henzel, W.J., Rodriguez, H., Waters, M.J., Wood, W.I., 1988. Rabbit liver growth hormone receptor and serum binding protein. Purification, characterization, and sequence. J. Biol. Chem. 263, 7862e7867. Stacy, B.D., Wallace, A.L., Gemmell, R.T., Wilson, B.W., 1976. Absorption of 125I-labelled sheep growth hormone in single proximal tubules of the rat kidney. J. Endocrinol. 68, 21e30. Stagkourakis, S., Perez, C.T., Hellysaz, A., Ammari, R., Broberger, C., 2018. Network oscillation rules imposed by species-specific electrical coupling. Elife 7.

Stallings-Mann, M.L., Ludwiczak, R.L., Klinger, K.W., Rottman, F., 1996. Alternative splicing of exon 3 of the human growth hormone receptor is the result of an unusual genetic polymorphism. Proc. Natl. Acad. Sci. U.S.A. 93, 12394e12399. Steyn, F.J., Tolle, V., Chen, C., Epelbaum, J., 2016. Neuroendocrine regulation of growth hormone secretion. Comp. Physiol. 6, 687e735. Stojilkovic, S.S., Tabak, J., Bertram, R., 2010. Ion channels and signaling in the pituitary gland. Endocr. Rev. 31, 845e915. Suh, H.K., Frantz, A.G., 1974. Size heterogeneity of human prolactin in plasma and pituitary extracts. J. Clin. Endocrinol. Metab. 39, 928e935. Sun, L.Y., Bartke, A., 2014. Tissue-specific GHR knockout mice: metabolic phenotypes. Front. Endocrinol. 5, 243. Swaminathan, G., Varghese, B., Fuchs, S.Y., 2008. Regulation of prolactin receptor levels and activity in breast cancer. J. Mammary Gland Biol. Neoplasia 13, 81e91. Tang, M.W., Garcia, S., Gerlag, D.M., Tak, P.P., Reedquist, K.A., 2017. Insight into the endocrine system and the immune system: a review of the inflammatory role of prolactin in rheumatoid arthritis and psoriatic arthritis. Front. Immunol. 8, 720. Taniguchi, Y., Yasutaka, S., Kominami, R., Shinohara, H., 2002. Proliferation and differentiation of rat anterior pituitary cells. Anat. Embryol. 206, 1e11. Tannenbaum, G.S., 1988. Somatostatin as a physiological regulator of pulsatile growth hormone secretion. Horm. Res. 29, 70e74. Tannenbaum, G.S., Epelbaum, J., Bowers, C.Y., 2003. Interrelationship between the novel peptide ghrelin and somatostatin/growth hormone-releasing hormone in regulation of pulsatile growth hormone secretion. Endocrinology 144, 967e974. Tannenbaum, G.S., Ling, N., 1984. The interrelationship of growth hormone (GH)-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion. Endocrinology 115, 1952e1957. Tanriverdi, F., Schneider, H.J., Aimaretti, G., Masel, B.E., Casanueva, F.F., Kelestimur, F., 2015. Pituitary dysfunction after traumatic brain injury: a clinical and pathophysiological approach. Endocr. Rev. 36, 305e342. Teerapornpuntakit, J., Wongdee, K., Thongbunchoo, J., Krishnamra, N., Charoenphandhu, N., 2012. Proliferation and mRNA expression of absorptive villous cell markers and mineral transporters in prolactin-exposed IEC-6 intestinal crypt cells. Cell Biochem. Funct. 30, 320e327. Teilum, K., Hoch, J.C., Goffin, V., Kinet, S., Martial, J.A., Kragelund, B.B., 2005. Solution structure of human prolactin. J. Mol. Biol. 351, 810e823. Thorner, M.O., Vance, M.L., Hartman, M.L., Holl, R.W., Evans, W.S., Veldhuis, J.D., van Cauter, E., Copinschi, G., Bowers, C.Y., 1990. Physiological role of somatostatin on growth hormone regulation in humans. Metabolism 39, 40e42. Tolle, V., Zizzari, P., Tomasetto, C., Rio, M.C., Epelbaum, J., BluetPajot, M.T., 2001. In vivo and in vitro effects of ghrelin/ motilin-related peptide on growth hormone secretion in the rat. Neuroendocrinology 73, 54e61. Torner, L., 2016. Actions of prolactin in the brain: from physiological adaptations to stress and neurogenesis to psychopathology. Front. Endocrinol. 7, 25. Triebel, J., Bertsch, T., Bollheimer, C., Rios-Barrera, D., Pearce, C.F., Hufner, M., Martinez de la Escalera, G., Clapp, C., 2015. Principles of the prolactin/vasoinhibin axis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 309, R1193eR1203. Trott, J.F., Hovey, R.C., Koduri, S., Vonderhaar, B.K., 2004. Multiple new isoforms of the human prolactin receptor gene. Adv. Exp. Med. Biol. 554, 495e499.

REFERENCES

Trott, J.F., Schennink, A., Petrie, W.K., Manjarin, R., Vanklompenberg, M.K., Hovey, R.C., 2012. Triennial lactation symposium: prolactin: the multifaceted potentiator of mammary growth and function. J. Anim. Sci. 90, 1674e1686. Tuggle, C.K., Trenkle, A., 1996. Control of growth hormone synthesis. Domest. Anim. Endocrinol. 13, 1e33. Turyn, D., Dominici, F.P., Sotelo, A.I., Bartke, A., 1997. Growth hormone-binding protein enhances growth hormone activity in vivo. Am. J. Physiol. 273, E549eE556. Urbanek, M., Macleod, J.N., Cooke, N.E., Liebhaber, S.A., 1992. Expression of a human growth hormone (hGH) receptor isoform is predicted by tissue-specific alternative splicing of exon 3 of the hGH receptor gene transcript. Mol. Endocrinol. 6, 279e287. Valeggia, C., Ellison, P.T., 2009. Interactions between metabolic and reproductive functions in the resumption of postpartum fecundity. Am. J. Hum. Biol. 21, 559e566. Van Cauter, E., Latta, F., Nedeltcheva, A., Spiegel, K., Leproult, R., Vandenbril, C., Weiss, R., Mockel, J., Legros, J.J., Copinschi, G., 2004. Reciprocal interactions between the GH axis and sleep. Growth Hormone IGF Res. 14 (Suppl. A), S10eS17. van Vught, A.J., Nieuwenhuizen, A.G., Brummer, R.J., WesterterpPlantenga, M.S., 2008. Effects of oral ingestion of amino acids and proteins on the somatotropic axis. J. Clin. Endocrinol. Metab. 93, 584e590. Veldhuis, J.D., Anderson, S.M., Shah, N., Bray, M., Vick, T., Gentili, A., Mulligan, T., Johnson, M.L., Weltman, A., Evans, W.S., Iranmanesh, A., 2001. Neurophysiological regulation and targettissue impact of the pulsatile mode of growth hormone secretion in the human. Growth Hormone IGF Res. 11 (Suppl. A), S25eS37. Veldhuis, J.D., Johnson, M.L., Faunt, L.M., Mercado, M., Baumann, G., 1993. Influence of the high-affinity growth hormone (GH)-binding protein on plasma profiles of free and bound GH and on the apparent half-life of GH. Modeling analysis and clinical applications. J. Clin. Investig. 91, 629e641. Vijayakumar, A., Novosyadlyy, R., Wu, Y., Yakar, S., Leroith, D., 2010. Biological effects of growth hormone on carbohydrate and lipid metabolism. Growth Hormone IGF Res. 20, 1e7. Vijayakumar, A., Yakar, S., Leroith, D., 2011. The intricate role of growth hormone in metabolism. Front. Endocrinol. 2. Vilar, L., Vilar, C.F., Lyra, R., Lyra, R., Naves, L.A., 2017. Acromegaly: clinical features at diagnosis. Pituitary 20, 22e32. von Werder, K., Clemm, C., 1974. Evidence for ’big’ and ’little’ components of circulating immunoreactive prolactin in humans. FEBS Lett. 47, 181e184. Wang, S., Wu, J., Wang, N., Zeng, L., Wu, Y., 2017. The role of growth hormone receptor in beta cell function. Growth Hormone IGF Res. 36, 30e35. Waters, M.J., 2016. The growth hormone receptor. Growth Hormone IGF Res. 28, 6e10. Waters, M.J., Blackmore, D.G., 2011. Growth hormone (GH), brain development and neural stem cells. Pediatr. Endocrinol. Rev. 9, 549e553. Waters, M.J., Brooks, A.J., 2015. JAK2 activation by growth hormone and other cytokines. Biochem. J. 466, 1e11. Waters, M.J., Brooks, A.J., Chhabra, Y., 2014. A new mechanism for growth hormone receptor activation of JAK2, and implications for related cytokine receptors. JAKSTAT 3, e29569.

201

Werther, G.A., Hogg, A., Oldfield, B.J., Mckinley, M.J., Figdor, R., Mendelsohn, F.A., 1989. Localization and characterization of insulin-like growth factor-I receptors in rat brain and pituitary gland using in vitro autoradiography and computerized densitometry* a distinct distribution from insulin receptors. J. Neuroendocrinol. 1, 369e377. Wiemers, D.O., Shao, L.J., Ain, R., Dai, G., Soares, M.J., 2003. The mouse prolactin gene family locus. Endocrinology 144, 313e325. Wojcik, M., Krawczynska, A., Antushevich, H., Herman, A.P., 2018. Post-receptor inhibitors of the GHR-JAK2-STAT pathway in the growth hormone signal transduction. Int. J. Mol. Sci. 19. Woodside, B., Budin, R., Wellman, M.K., Abizaid, A., 2012. Many mouths to feed: the control of food intake during lactation. Front. Neuroendocrinol. 33, 301e314. Wu, Y., Liu, C., Sun, H., Vijayakumar, A., Giglou, P.R., Qiao, R., Oppenheimer, J., Yakar, S., Leroith, D., 2011. Growth hormone receptor regulates beta cell hyperplasia and glucose-stimulated insulin secretion in obese mice. J. Clin. Investig. 121, 2422e2426. Xu, J., Zhang, Y., Berry, P.A., Jiang, J., Lobie, P.E., Langenheim, J.F., Chen, W.Y., Frank, S.J., 2011. Growth hormone signaling in human T47D breast cancer cells: potential role for a growth hormone receptor-prolactin receptor complex. Mol. Endocrinol. 25, 597e610. Yakar, S., Isaksson, O., 2016. Regulation of skeletal growth and mineral acquisition by the GH/IGF-1 axis: lessons from mouse models. Growth Hormone IGF Res. 28, 26e42. Yakar, S., Liu, J.L., Fernandez, A.M., Wu, Y., Schally, A.V., Frystyk, J., Chernausek, S.D., Mejia, W., le Roith, D., 2001. Liver-specific igf-1 gene deletion leads to muscle insulin insensitivity. Diabetes 50, 1110e1118. Yakar, S., Setser, J., Zhao, H., Stannard, B., Haluzik, M., Glatt, V., Bouxsein, M.L., Kopchick, J.J., Leroith, D., 2004. Inhibition of growth hormone action improves insulin sensitivity in liver IGF1-deficient mice. J. Clin. Investig. 113, 96e105. Yamauchi, T., Kaburagi, Y., Ueki, K., Tsuji, Y., Stark, G.R., Kerr, I.M., Tsushima, T., Akanuma, Y., Komuro, I., Tobe, K., Yazaki, Y., Kadowaki, T., 1998. Growth hormone and prolactin stimulate tyrosine phosphorylation of insulin receptor substrate-1, -2, and -3, their association with p85 phosphatidylinositol 3-kinase (PI3kinase), and concomitantly PI3-kinase activation via JAK2 kinase. J. Biol. Chem. 273, 15719e15726. Yan, M., Jones, M.E., Hernandez, M., Liu, D., Simpson, E.R., Chen, C., 2004. Functional modification of pituitary somatotrophs in the aromatase knockout mouse and the effect of estrogen replacement. Endocrinology 145, 604e612. Zhang, Y., Wang, X., Loesch, K., May, L.A., Davis, G.E., Jiang, J., Frank, S.J., 2016. TIMP3 modulates GHR abundance and GH sensitivity. Mol. Endocrinol. 30, 587e599. Zhou, Y., Xu, B.C., Maheshwari, H.G., He, L., Reed, M., Lozykowski, M., Okada, S., Cataldo, L., Coschigamo, K., Wagner, T.E., Baumann, G., Kopchick, J.J., 1997. A mammalian model for laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the laron mouse). Proc. Natl. Acad. Sci. U.S.A. 94, 13215e13220. Zhu, T., Ling, L., Lobie, P.E., 2002. Identification of a JAK2-independent pathway regulating growth hormone (GH)-stimulated p44/42 mitogen-activated protein kinase activity. GH activation of Ral and phospholipase D is Src-dependent. J. Biol. Chem. 277, 45592e45603.

C H A P T E R

10 Posterior Pituitary Hormones Amanda P. Borrow, Sally A. Stover, Natalie J. Bales, Robert J. Handa Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, United States S SON V1aR, V1bR and V2R V1R V2R VMH VTA

Abbreviations AC ACTH AH ANP AQP2 ASD ATP AVP AVT BNST cAMP ceA CRF1 CRH CVO DAG eCFP GPCR H1 H2 hnRNA HPA I ICV IP3 LG LS LV M MDD meA MEC MPOA NO NTS OB OT OTR OVLT PAG PIP2 PKA PKC PLC PP PVN

adenylyl cyclase adrenocorticotropic hormone anterior hypothalamus atrial natriuretic peptide aquaporin-2 autism spectrum disorder adenosine triphosphate arginine vasopressin arginine vasotocin bed nucleus of the stria terminalis cyclic adenosine monophosphate central nuclei of the amygdala corticotropin-releasing hormone receptor 1 corticotropin-releasing hormone circumventricular organ diacylglycerol enhanced cyan fluorescent protein G proteinecoupled receptor hydrin 1 hydrin 2 heterologous nuclear RNA hypothalamic-pituitary-adrenal isotocin intracerebroventricular inositol triphosphate licking and grooming lateral septum lysine vasopressin mesotocin major depressive disorder medial amygdala myoepithelial cell medial preoptic area nitric oxide nucleus of the solitary tract olfactory bulb oxytocin oxytocin receptor organum vasculosum of the lamina terminalis periaqueductal gray phosphatidylinositol biphosphate protein kinase A protein kinase C phospholipase C phenypressin paraventricular nucleus

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00010-9

seritocin supraoptic nucleus vasopressin receptors hepatic vasopressin receptor renal vasopressin receptor ventromedial hypothalamus ventral tegmental area

1. INTRODUCTION The posterior pituitary, or neurohypophysis, constitutes one lobe of the pituitary gland. The primary function of the posterior pituitary is the transmission of hormones originating from neurons located in hypothalamic brain regions such as the supraoptic nucleus (SON) and paraventricular nucleus (PVN) for secretion directly into peripheral circulation. Accordingly, the posterior pituitary is largely composed of axons and axon terminals. These terminals store oxytocin (OT), arginine vasopressin (AVP), and other neuropeptides within secretory granules (Gaddum, 1928; Silverman, 1976) (Fig. 10.1). While OT and AVP are the predominant hormones secreted by the posterior pituitary, others have also been identified, including somatostatin, which is received via projections from the PVN (Larsen et al., 1992), and endothelin, located within terminals of PVN and SON neurons (Yoshizawa et al., 1990). These hormones generally serve to regulate the synthesis, storage, and secretion of OT and AVP. The posterior pituitary includes two main structures: the posterior lobe, and the contiguous infundibular stalk and median eminence. In contrast to the anterior pituitary, which contains neuroendocrine epithelial cells, about 42% of the posterior pituitary is comprised of axons and axon terminals arising from hypothalamic neurons (Nordmann, 1977). These axon terminals are supported by glial cells known as pituicytes and contain neurosecretory granules storing OT and AVP. Unmyelinated axons originating from the hypothalamus project to

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Paraventricular nucleus Supraoptic nucleus

Hypothalamus

Optic chiasm

Oxytocin

Vasopressin

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Anterior pituitary

Oxytocin

Vasopressin

FIGURE 10.1 Diagrammatic representation of the neurohypophyseal control of oxytocin and vasopressin secretion into the general circulation. Oxytocin- and vasopressin-synthesizing neurons are located in the supraoptic and paraventricular nuclei of the hypothalamus. Axons from these neurons extend down the infundibular stalk to terminate along blood vessels within the posterior lobe of the pituitary. The neurosecretory products are stored in axon terminals of the posterior pituitary, where they are released into the circulation upon neural stimulation that activates soma in the hypothalamus.

the posterior lobe via the pars infundibularis of the infundibular stalk. The unmyelinated axons of the posterior pituitary contain Herring bodies, distinguishable axonal swellings housing neurosecretory granules (Yukitake et al., 1977). This chapter will focus on the principle components of these granules, the hormones OT and AVP.

2. EVOLUTION OF NEUROHYPOPHYSEAL HORMONES While OT and AVP are present in all placental mammals, related peptide analogs can be found throughout the animal kingdom. Furthermore, the precursor proteins for these hormones share an identical exon-intron organization across mammalian, fish, and invertebrate species. The existence of AVP- and OT-like neuropeptides in representatives of both Protostomia and Deuterostomia lineages has revealed a phylogenetically ancient origin for these hormones, which occurred sometime

before the split of these lineages some 640e760 million years ago (Stafflinger et al., 2008). Researchers have identified 16 nonapeptides to date in the vertebrates (Fig. 10.2), all sharing a high level of structural homology. These peptides can be categorized as members of either the basic or neutral nonapeptide family based on the amino acid at position 8. All vertebrates, with the exception of cyclostomes, have at least two of these peptides, with a minimum of one from each family. As cyclostomes possess a single nonapeptide, arginine vasotocin (AVT), AVT has been proposed to be the ancestral vertebrate nonapeptide through which all others have since been derived (Sawyer, 1977). AVT shares a ring structure with OT and a tail structure with AVP. The gene for AVT is believed to have duplicated at the base of the gnathostome lineage, resulting in two genes that formed the foundation of the basic and neutral families. The subsequent development of functionally distinct paralogs is thought to result from the occurrence of novel ligandereceptor interactions (Banerjee et al., 2017). The evolutionary history of AVP is fairly straightforward, as indicated by the presence of AVP in mammals and of AVT in cyclostomes and nonmammalian vertebrates. As AVT acts on both AVP and OT receptors, the existence of AVP in mammals can be explained as an increase in receptor selectivity (Acher, 1996). In contrast, the history of OT-like peptides is considerably more complex. This family includes OT, which is found in eutherian mammals, mesotocin, which is produced in birds, reptiles, marsupials, amphibians, and lungfishes, isotocin, which is found in bony fishes, and a variety of additional peptides within the family of cartilaginous fishes (Acher et al., 1997). OT/AVP homologs have also been identified in a number of invertebrate species (Banerjee et al., 2017). AVP-related peptides have been found in various insects, mollusks, and annelids, while OT-related peptides have also been observed in mollusks and annelids (Stafflinger et al., 2008; Oumi et al., 1994; Reich, 1992). Contrary to vertebrates, virtually all invertebrates possess a single nonapeptide, supporting the hypothesis that the presence of two nonapeptide families occurred early on in vertebrate evolution (Van Kesteren et al., 1995). However, two cephalopods, the cuttlefish and the octopus, contain two unique members of the OT/AVP superfamily, suggesting that a separate gene duplication event occurred independently for invertebrates. While AVP and OT share a similar origin, the same may not be said with certainty for their receptors. Four receptors attributed to the OT/AVP family have been reported in the mammals: three for AVP (V1aR, V1bR, and V2R) and one for OT (OTR). Nonapeptide receptors have been cloned and characterized in a variety of nonmammalian species. Two additional V2R subtypes,

2. EVOLUTION OF NEUROHYPOPHYSEAL HORMONES

Logomorphes Rodentia Primates Cetacea Artiodactyla Perissodactyla Didelphidae

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Peramelidae Macropodidae Phalangeridae Dasyuridae

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Crossopterygii Neopterygii Cyclostomata

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Marsupial Mammals

AT,H1,H2 Amphibia AVT AVT

Bony Fish

AVT

Cyclostomes

FIGURE 10.2 The evolutionary lineage of bony vertebrate nonapeptides. Diagram showing the phylogenetic relationship of different forms of arginine vasopressin (AVP) and oxytocin (OT) in vertebrates. AVP, arginine vasopressin; AVT, arginine vasotocin; H1, hydrin 1; H2, hydrin 2; LV, lysine vasopressin; I, isotocin; M, mesotocin; OT, oxytocin; PP, phenypressin; S, seritocin. Adapted from Acher, R., 1996. Molecular evolution of fish neurohypophysial hormones: neutral and selective evolutionary mechanisms. Gen. Comp. Endocrinol. 102 (2), 157e172.

named V2B, identified in teleost fishes and birds (Daza et al., 2012), and V2C, found in the sea lamprey (Mayasich and Clarke, 2016), have been reported, leading to the hypothesis that at least six receptor subtypes were present in the gnathostome ancestor. The absence of V2B in mammals and V2R (or V2A) in birds indicates reciprocal losses in multiple lineages. While receptor characterization in the invertebrate has received considerably less attention to date, current findings have been illuminating with respect to the origin of AVP and OT receptors. A G proteinecoupled receptor (GPCR) for the OT/AVP-like peptide inotocin has been cloned in the red flour beetle and the parasitic wasp Nasonia vitripennis (Stafflinger et al., 2008). This receptor is structurally similar to the insect adipokinetic hormone, crustacean cardioprotective, and corazonin receptors, suggesting a common origin. An overlap of hormonal systems resulting from gene duplications and subsequent mutations of GPCRs and their associated ligands may have led to the abandonment of OT/AVP-like hormonal systems in certain insect species. This hypothesis would explain the limited expression of these peptides in holometabolous insects (Stafflinger et al., 2008). In addition to conservation of hormone structure across species, conservation of function has also been described for OT- and AVP-like peptides. AVP and AVT are both antidiuretic hormones, with a

demonstrated role in osmoregulation identified in mammals, nonmammalian tetrapods, birds, amphibians, reptiles, and fishes (Acher, 1996; Balment et al., 2006). AVP and AVT are also implicated in male sexual behavior in species ranging from birds (Jurkevich et al., 1996) to mollusks (Van Kesteren et al., 1995). OT and OT-like peptides show a more diverse range of functions; while they are primarily associated with reproduction (Parry et al., 1996), they also appear to mediate smooth muscle contractions in processes including gut contractility (Oumi et al., 1994) and contribute to osmoregulation in the amphibian (Akhundova et al., 1996) and in annelids (Oumi et al., 1994). Finally, the functional similarities across phyla are further supported by similarities in the location of peptide synthesis and of associated receptors. The synthesis of nonapeptides within a preoptic brain area and subsequent axonal transport into a neurohypophysis appears across vertebral species (Banerjee et al., 2017). Research in organisms including the mollusk (Van Kesteren et al., 1995), the cricket (Musiol et al., 1990), and the earthworm (Takahama et al., 1998) has found that OT- and AVP-like peptides are neurohormones produced within even the most rudimentary of nervous systems. The location of nonapeptide receptors belies their roles in reproduction and fluid homeostasis, with receptors localized in many organs involved in these processes.

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In summary, OT and AVP share a well-described, phylogenetically ancient origin. These peptides and their associated receptors are ubiquitously expressed across the animal kingdom in both chordates and nonchordates. While many insects have abandoned the usage of OT- or AVP-like hormones, the persistence of nonapeptides across animal species underscores their critical importance, particularly with regard to their roles in fluid homeostasis and reproduction.

3. THE STRUCTURE AND SYNTHESIS OF NEUROHYPOPHYSEAL HORMONES In addition to their conserved evolutionary history, the neuropeptides OT and AVP share many similarities in peptide and gene structure, mechanisms of synthesis, and even the location of the neurons that produce these hormones. Nonetheless, critical differences in the structure of the two ligands and their selective interactions with receptors and coupling to second messenger pathways allow cellular and system specificity when considering the diverse array of functions controlled by OT and AVP.

(A)

3.1 Peptide Structure In mammals, the structure of mature AVP and OT is very similar, in that both are nonapeptides containing two cysteine residues in the 1 and 6 positions. The cysteine residues form a disulfide bridge resulting in a cyclic core of six amino acids with a flexible amidated tail consisting of the remaining amino acids (amino acids 7e9). The two peptides differ in their amino acid identity at the 3 and 8 positions, resulting in a molecular weight of 1007 for OT and 1084 for AVP (see Fig. 10.3A). A difference in polarity at amino acid 8, where OTrelated peptides contain a neutral residue and AVP peptides have a basic amino acid, confers selectivity for the molecule’s interaction with its receptor. However, because of their common structure, both can bind and act on multiple members of the receptor family (see below). The disulfide bond does not appear to be implicated in binding to the OTR, although reductions in the ring size through disulfide engineering can abolish OTR activity (Muttenthaler et al., 2010), and improved stability and selectivity can be achieved by substituting dibromo-xylene analogs (Beard et al., 2018). Differences in the amino acid sequence of OT and AVP in various species have been discussed in the previous section.

Oxytocin

Vasopressin Gly

Gly

Gln

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Cys

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Exon 2

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Neurophysin II

Signal

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Neurophysin I

Exon 3

Copeptin

Gly-Lys-Arg

FIGURE 10.3 Diagrammatic representation of the amino acid sequence and structure of mammalian oxytocin (OT) and arginine vasopressin (AVP) (panel A) and the overlapping structure of the OT and AVP genes (panel B) and preprohormones (panel C). The components of the OT gene are shown in blue whereas those of the AVP gene are shown in green. Panel B shows the tail-to-tail alignment of the OT and AVP genes. Panel C shows the protein product derived from the three exons. The gly-lys-arg site is used for processing and for amidation of the mature peptide. A glycosylated (shown as yellow) carboxy fragment termed copeptin is characteristic of the AVP preprohormone, but is not found in the OT preprohormone. AVP, vasopressin; OT, oxytocin.

3. THE STRUCTURE AND SYNTHESIS OF NEUROHYPOPHYSEAL HORMONES

3.2 Gene Structure Both OT and AVP are predominantly synthesized by magnocellular neurons found in the SON and PVN of the hypothalamus, although they can also be synthesized in several other brain regions, as well as in peripheral tissues. The OT and AVP genes are both located on chromosome 20 and are organized in a tail-to-tail manner, separated by a short intergenic region. The two genes are consequently transcribed in opposite directions (Fig. 10.3B) (Gainer, 2012). Both the AVP and OT genes are composed of three exons and two introns, with the first intron and second exon being extremely homologous. Both are synthesized as preprohormones with a carboxy-terminal neurophysin. The prohormone of vasopressin is glycosylated at the carboxy-terminus, which gives rise to a glycopeptide (copeptin) of little known function. The length of the intergenic region differs by species.

3.3 Synthesis of Vasopressin As discussed before, AVP molecules are found in all known vertebrates. Although almost all mammals synthesize AVP, some, such as the pig, express a lysine-vasopressin variant. Invertebrate OT- and AVPlike molecules have also been identified in arthropods, and it is believed that the OT/AVP signaling system dates back more than 600 million years, having evolved from an ancestral vasotocin molecule (Gruber, 2014). The AVP preprohormone consists of a 19 amino acid signal peptide followed by the sequence coding for mature AVP. Separating the AVP carboxy-terminus from the adjacent neurophysin II is a processing and amidation site (Gly-Lys-Arg) sequence. A 39 amino acid glycopeptide (copeptin) is found on the carboxyterminus of the gene and is separated from neurophysin II by an arginine residue (Brownstein, 1983) (Fig. 10.3C). An important enhancer region is found in the intergenic region downstream of the AVP gene, which is responsible for cell-specific expression of AVP and OT. Glucocorticoid response elements, cyclic AMP (cAMP) response elements and AP-1/2 regulatory elements are found in an upstream promoter region of the AVP gene (Hyodo, 2015). The greatest concentration of AVP is found in magnocellular neurons of the SON and PVN. Within magnocellular neurons, AVP is synthesized on ribosomes as part of the preprohormone precursor. Following cleavage of the signal sequence, the prohormone is glycosylated in the rough endoplasmic reticulum and disulfide bond formation in the AVP molecule, as well as between cysteine residues in neurophysin II. The glycosylation of proteins is thought to be an important step in the transfer of material from the rough endoplasmic

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reticulum to the Golgi complex. Further processing of the prohormone happens in the Golgi apparatus where enzymatic cleavage to AVP, neurophysin II, and copeptin occurs. This cleavage also takes place in neurosecretory vesicles as the peptides are transported to axon terminals residing in the posterior pituitary (Castel et al., 1984). A more complete description of the intracellular processing of AVP can be found in Morris et al. (1987).

3.4 Synthesis of Oxytocin Although the synthesis of OT is comparable to that of AVP, it is transcribed in the opposite direction. Similar enhancer elements exist in the intergenic region between the two genes, and a composite hormone response element is found in the promoter region upstream of the OT start site. This composite response element is comprised of many different motifs, allowing potential interactions with estrogen receptors, glucocorticoid receptors, thyroid hormone receptors, retinoic acid receptors, and orphan receptors such as COUP-TF1 (see Fig. 10.3B) (Gainer, 2012). Other regulatory regions containing an SP-1 element are also found in upstream regulatory regions (Hiroi et al., 2013). Unlike AVP, the OT preprohormone is not glycosylated (Tasso et al., 1977). After the signal peptide is cleaved off, the remaining prohormone is packaged into neurosecretory granules for transport to the posterior pituitary. Of importance, the OT prohormone does not contain copeptin, but rather a small nonglycosylated adduct is found at the carboxy-terminal end. The mature peptide, OT, and its carrier molecular, neurophysin I, are stored in axon terminals until release is elicited by neural inputs.

3.5 Distribution of Oxytocin- and VasopressinSynthesizing Neurons in the Brain OT and AVP are synthesized predominantly by two groups of magnocellular neurons found in the SON and PVN of the hypothalamus. The axons of these cells are unmyelinated and form the hypothalamohypophyseal tract that transports OT and AVP to be stored, along with their respective neurophysin, in terminals within the posterior pituitary. Magnocellular OT neurons have also been described in a series of accessory nuclei that are scattered between the PVN and SON in mammals (Knobloch and Grinevich, 2014). Magnocellular OT neurons, particularly those in the SON, provide the OT that is released into the general circulation by the posterior pituitary. Of importance, magnocellular OT neurons, particularly from the PVN, also innervate forebrain structures including the nucleus accumbens the medial (meA) and central (ceA) nuclei of the amygdala,

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bed nucleus of the stria terminalis (BNST), lateral septum (LS), and other limbic areas (Knobloch et al., 2012). These fibers are thought to account for the varied effects of OT on specific behaviors. Parvocellular OT neurons of the PVN are distinct from magnocellular neurons based on size, shape, location, and involvement in different OT-dependent circuitries. Parvocellular OT neurons have been shown to project to specific brainstem nuclei and spinal cord regions. There, OT release can regulate autonomic functions including cardiovascular, breathing, feeding behavior, and nociception (Petersson, 2002; Conde´sLara et al., 2003; Mack et al., 2007; Atasoy et al., 2012). To date, it is not known how parvocellular OT neurons interact with magnocellular OT neurons, although recent evidence suggests that this can occur through direct release of OT onto spinal cord neurons to inhibit their activity and through descending connections from PVN OT neurons to the magnocellular OT neurons of the SON to modify OT release (Eliava et al., 2016). Magnocellular neurons that synthesize AVP are also found in the PVN and SON. Although earlier studies suggested that the expression of AVP and OT occurred in a nonoverlapping fashion (Sokol and Valtin, 1967), more recent studies in the SON indicate that there is overlap in OT and AVP expression, with some cells preferentially expressing OT, others that preferentially express AVP, and a third type that express both in near equivalent amounts (Glasgow et al., 1999). In any case, it appears that SON AVP neurons extend axons to the posterior pituitary for secretion of their product to the general circulation following action potentials generated in magnocellular AVP neurons. AVP neurons are also found in many other brain areas including the BNST. These neurons exhibit a robust sex difference in their expression, where the number of AVP-expressing neurons in males is twofold greater than in females. These AVP neurons project, in a similar sexually dimorphic fashion, to behaviorally relevant brain areas such as the LS and lateral habenula (De Vries et al., 1994). In addition, the PVN also contains a group of parvocellular neurons that express AVP and secrete into the hypothalamo-hypophyseal portal blood where AVP synergizes with cosecreted corticotropin-releasing hormone (CRH) to augment the actions of CRH on adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary (Pei et al., 2014). Moreover, pharmacological activation of PVN AVP neurons acutely blunts food intake, suggesting that PVN AVP can have a number of different actions aside from those occurring following secretion by the posterior pituitary gland into peripheral circulation.

4. THE OXYTOCIN/VASOPRESSIN RECEPTOR FAMILY In vertebrates, receptors for OT and AVP are placed into a subfamily of GPCRs that mediate a vast array of functions. These include the regulation of blood pressure and water balance, parturition and lactation, social behaviors, neuroendocrine stress and autonomic responses, feeding, and mood and anxiety-like behaviors. Currently, four members of this family have been identified in mammals and include the OTR that is activated by OT and the V1aR, V1bR (also known as V3), and V2R that respond to AVP (Fig. 10.4).

4.1 Oxytocin Receptor The OTR interacts with the cyclic part of OT through interactions with transmembrane domains 3, 4, and 6 (Zingg, 2002), whereas the carboxy-terminal end of the OT molecule associates with transmembrane domains 2 and 3 and the first extracellular loop connecting these domains. The OTR is predominant in uterine myometrium, epithelium, and decidua (Arrowsmith and Wray, 2014), but it has also been shown to be expressed in mammary gland, testes, adrenal gland, and in select brain areas. Like all members of the AVP receptor subfamily, it is a GPCR linked to G-alpha(q) and upon activation leads to a rise in cytosolic calcium through release from intracellular stores and from extracellular influx. The OTR is also linked to Galpha(i) that inhibits increases in cAMP. Activation of the OTR has been shown to lead to stimulation of prostaglandin F2a. It has been proposed that phospholipase A2 can act as an intermediary to activate the mitogenactivated protein kinase pathway that is coupled to prostaglandin E2 synthesis in uterine decidual cells (Viero et al., 2010). AVP is a partial agonist of the OTR, suggesting that differences in OT and AVP responses are due to differences in amplitude of the functional response (Chini et al., 1996). Evidence for OTR function can be gleaned from studies of OTR knockout mice that show aberrant behaviors, obesity, and impaired thermoregulation (Nishimori et al., 2008).

4.2 Vasopressin Receptors The original nomenclature for AVP receptors was based on the finding that AVP triggered rises in cytosolic free calcium and increases in phosphatidylinositol breakdown leading to activation of protein kinase C (PKC) in hepatic tissues, whereas AVP activated adenylate cyclase and increased cAMP within the kidney. Hence, the

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4. THE OXYTOCIN/VASOPRESSIN RECEPTOR FAMILY

V1aR

PLC

αq

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αq

αq

β γ

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PKA AQP2 PAQP2

H 2O PAQP2

FIGURE 10.4 Diagrammatic representation of the actions of the different members of the AVP subfamily of GPCRs. The OTR, V1aR, and V1bR are G proteinecoupled receptors linked to G-alpha(q). These receptors regulate intracellular Ca2þ levels through the IP3 receptor located on the sarcoplasmic reticulum in smooth muscle cells or the endoplasmic reticulum of pituitary corticotrophs (V1bR) and other cells such as neurons. The right panel represents a kidney tubule cell where the V2R activation regulates cAMP levels through G-alpha(s). This results in the insertion of aquaporin-2 into the apical membrane of the cell, thereby allowing water to flow into the cell and out of the kidney tubule. AC, adenylyl cyclase; AQP2, aquaporin-2; ATP, adenosine triphosphate; AVP, arginine vasopressin; cAMP, cyclic adenosine monophosphate; DAG, diacylglycerol; IP3, inositol triphosphate; OT, oxytocin; PIP2, phosphatidylinositol biphosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C.

hepatic receptor was termed V1R, whereas the renal receptor was termed V2R (Michell et al., 1979). Subsequently, a novel receptor was identified in the anterior pituitary gland that possessed a different pharmacological profile than that of the V1 receptor and was labeled V1bR, whereas the original hepatic-like receptor was designated V1aR (Jard et al., 1986). The anterior pituitary V1bR was found exclusively in corticotrophs and additionally was shown to potentiate the actions of CRH to increase secretion of ACTH following costimulation of the CRF1 receptor (Antoni, 1993), which can occur whether the V1bR is activated by AVP (Tanoue et al., 2004) or OT (Schlosser et al., 1994). V1aR was subsequently cloned from rat liver (Morel et al., 1992) and from a human liver cDNA library (Thibonnier et al., 1994). The human and rat receptors share approximately 72% sequence identity. V1aR is a seven transmembrane GPCR coupled to G-alpha(q), whereby it increases cytosolic free calcium and the activity of PKC. It is widely distributed throughout the body (liver, smooth muscle, adrenal gland, testes, and bladder) and the brain (cortex, brainstem, hippocampus, hypothalamus, and striatum) (Ostrowski et al., 1992). OT can also bind the V1aR but with lower affinity and potency (Chini et al., 2008). V1bR was cloned from a human pituitary cDNA library (Sugimoto et al., 1994). It has been shown to be

expressed at highest levels in the anterior pituitary, and in pancreatic b-cells, but also in other areas besides the anterior pituitary (Saito et al., 1995) including the brain, heart, small intestine, lung, liver, and kidney. Similar to the V1aR subtype, V1bR is coupled to Galpha(q) to increase production of inositol triphosphate (IP3) and diacylglycerol, resulting in increases in cytosolic free calcium and PKC upon activation. V1bR has also been shown to activate adenylyl cyclase through G-alpha(s) activation, but with lower potency than that for G-alpha(q). Structural analogs of AVP have helped delineate the binding characteristics of V1bR, which differ from V1aR and V2R. The renal AVP receptor, or V2R, is predominantly expressed by the principal cells of the renal collecting duct and is responsible for the antidiuretic actions of AVP. It was originally cloned from rat kidney and human renal cDNA libraries (Lolait et al., 1992). V2R mutations are responsible for congenital diabetes insipidus, characterized by renal resistance to AVP and the failure to concentrate urine, leading to polyuria. The V2R is unique from the other members of the subfamily in that it is primarily coupled to G-alpha(s), resulting in activation of adenylyl cyclase and increases in cAMP. V2R can be secondarily coupled to G-alpha(q) which can, albeit with low potency, increase cytosolic free calcium.

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5. PHYSIOLOGIC FUNCTIONS AND BEHAVIOR 5.1 Oxytocin While OT is implicated in a diverse group of physiologic functions, it is primarily responsible for smooth muscle contraction, in regulating affiliative and reproductive behaviors, and in mediating stress responsivity via the hypothalamicepituitaryeadrenal (HPA) axis. The primary functions of OT in mammalian physiology are described below. 5.1.1 Oxytocin in Reproduction OT plays a critical role in both male and female reproduction and contributes to both sexual arousal and orgasm (Borrow and Cameron, 2012). OTRs are richly expressed in reproductive tissues. OTR activation facilitates uterine contractions during the follicular phase of the menstrual cycle and during orgasm in women, while epididymal OTRs facilitate contractions during ejaculation and contribute to the emission of semen in men. Plasma OT levels increase during sexual arousal in both men and women, with levels returning to baseline shortly after orgasm. Several reports have also suggested that intranasal OT may increase sexual arousal and sexual satisfaction. While the effects of intranasal OT treatment on sexual arousal may be limited to men (Kruger et al., 2018), intranasal OT did increase sexual contentment and the ability for healthy women to have an orgasm (Zhang et al., 2015), and male partners of women with hypoactive sexual disorder reported an increase in their partners’ sexual performance following treatment with intranasal OT (Muin et al., 2017). It is unclear whether intranasal OT influences sexual function by acting on peripheral OTRs in reproductive organs or if direct central activation of reproductive neurocircuitry occurs. OT has also been implicated in pregnancy. In the rodent, vaginocervical stimulation induces OT release, which in turn stimulates the secretion of prolactin, a hormone critical for the initiation and maintenance of pregnancy. OT may continue to regulate gestational prolactin pulses through suprachiasmatic nucleus input. Interestingly, while infusion of an OTR antagonist into the ventromedial nucleus of the hypothalamus inhibits the establishment of pregnancy in the rat (Northrop and Erskine, 2007), OT and OTR knockout mice are still capable of reproducing normally (Nishimori et al., 1996; Takayanagi et al., 2005). It is unclear whether findings from knockout models indicate that OT is not essential for the establishment and maintenance of pregnancy, or if these mutants develop compensatory mechanisms to ensure reproductive viability. Regardless, both OT and OTR knockout mice are incapable of lactation, and cross-fostering is required for offspring survival.

OT has historically been assumed to be the initiating factor of parturition; indeed, OT is named from the Greek words for “quick birth.” Elevated plasma OT and uterine OTR during parturition have been reported in all placental mammals studied to date, and synthetic OT reliably induces labor. In the rat, the concentration of OT within the posterior pituitary increases by approximately 50% during pregnancy, then decreases immediately postpartum. Cervical distention induces action potentials within the hypothalamus, resulting in a large pulse of OT coinciding with the expulsion of each pup, a phenomenon known as the Ferguson reflex. Interestingly, the hypothalamus is not the only source of OT during parturition. While transection of the neurohypophyseal stalk has prolonged labor in the mouse, women with posterior pituitary dysfunction are still capable of normal delivery. OT mRNA levels increase during parturition in the amnion, chorion, and decidua, purportedly via estrogenic mediation. The activation of OTR acts on the uterine myometrium to stimulate contractions and functions indirectly through prostaglandin formation in the decidua. The previously established view of OT as an essential component of parturition has recently been challenged by the demonstration that OT null mice retain the ability to deliver viable pups. However, infusion of OT at gestational day 15.5 affected the onset of labor in OTdeficient mice, suggesting that OT focuses the timing of labor onset (Imamura et al., 2000). OTR inhibition induces uterine quiescence in placental mammals and has been successfully used as a treatment for preterm labor, indicating that OTRs are likely essential for parturition (Blanks and Thornton, 2003). Although OT null mice are capable of delivering offspring, pups are not able to successfully suckle and die within 24 h without investigator intervention. As OT is essential for milk ejection in placental mammals, injection of OT or rescue with the rat OT gene is able to restore the milk ejection reflex in OT knockout dams in response to suckling (Young et al., 1998). During suckling, mechanoreceptors of sensory nerve terminals in the areolus send cholinergic afferents to the PVN and SON, stimulating the pulsatile secretion of OT. Interestingly, infant-related visual or auditory stimuli have a similar effect on OT secretion in lactating women. Circulating OT induces the contraction of myoepithelial cells (MECs) within the mammary gland. Milk is then expelled into the lactiferous sinuses and ducts that are shortened and widened by MEC contraction, ultimately leading to ejection of milk from the nipple (Truchet and Honvo-Houe´to, 2017). Finally, OT facilitates bonding in romantic and parentechild relationships. Extensive investigation of one of the few socially monogamous, biparental mammals, the prairie vole, has reinforced the critical role of

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OT in the formation and maintenance of pair bonding (Johnson and Young, 2015). OT is also essential for maternal behaviors in rats and mice. Far fewer studies have examined the influence of OT on human relationships. In humans, baseline levels and patterns of release of OT in plasma or saliva are associated with partner interactions and relationship survival in romantic relationships and with parentechild interactions and parental behaviors. A recent report found that lower maternal plasma OT during the perinatal period was a predictor of relationship dissolution by the time the child was a toddler (Sunahara et al., 2018). While studies investigating human bonding and peripheral levels of OT are largely correlative, they provide intriguing insight into the involvement of OT in human relationships. 5.1.2 Oxytocin in Cardiac Function OT is a cardioprotective agent with both direct and indirect effects on the cardiovascular system. It is highly active within the heart; in fact, OT content in the right atrium of the rat was reported to be comparable to levels found in the hypothalamus (Jankowski et al., 1998). The known cardiovascular actions of OT include regulating blood pressure and vasodilation, antioxidant and antiinflammatory activities, mediating cardiac glucose intake, inhibiting inotropic and chronotropic effects, and cardiomyogenesis (for review, see Gutkowska et al., 2014). OT’s effects on the cardiovascular system occur directly, as OTRs are found in both the heart and large vessels, as well as indirectly via autonomic nervous system actions. OT neurons project to brain stem autonomic regulatory centers of the cardiovascular system, and to the spinal cord, where OT acts to regulate sympathetic nerve activity (Pyner, 2009). Circulating OT binds to atrial OTRs, stimulating the secretion of atrial natriuretic peptide (ANP). In turn, ANP regulates vascular tone and electrolyte balance by inducing hypotension, natriuresis, and diuresis. OT also regulates nitric oxide (NO), an inhibitor of sympathetic nervous activity. NO augments the natriuretic effects of ANP and is implicated in several of OT’s known cardiovascular functions, including negative inotropic and chronotropic effects and regulation of cardiomyogenesis. 5.1.3 Oxytocin in Fluid Balance Plasma hypertonicity creates an osmotic gradient that draws fluid out of cells, causing an elevation in blood pressure and volume and subsequent activation of osmoreceptors. Alterations in fluid balance such as acute hypernatremia potentiate the secretion of OT into the periphery in humans and in the rodent (Steinwall et al., 1998). Research in the rodent has demonstrated that OT inhibits renin release and potentiates renal sodium excretion, ultimately restoring plasma

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sodium concentration (Bernal et al., 2015; Sjo¨quist et al., 1999). OT may also facilitate natriuresis indirectly by stimulating ANP secretion from atrial cardiomyocytes, resulting in sodium excretion through direct actions on the kidney and through indirect inhibitory effects on ACTH and corticosterone secretion (Chriguer et al., 2003). In addition to its effects on sodium excretion, OT also functions as an osmoregulator by influencing salt and water consumption in the rodent. OT’s influence on behaviors associated with fluid homeostasis is described in Section 5.6. 5.1.4 Oxytocin Neurocircuitry and Behavior To understand the means by which OT regulates mammalian behavior, it is essential to review the brain areas affected by OT. The neurocircuitry of OTmediated behaviors such as parental care and sexual behavior has been best described in the rodent; however, recent advances in neuroimaging coupled with experiments utilizing intranasal OT have highlighted parallels between humans and rodents. As discussed, OTRs are distributed throughout the brain, underscoring the wide range of functions mediated by OT. The OT system is fairly plastic, with changes reported in OTR expression and in firing patterns, cellular morphology, and afferent inputs of OT neurons following events such as pregnancy, lactation, and chronic stress. 5.1.4.1 Reproductive Behavior Studies using transgenic knockout models have shown that OT is not essential for mating but does influence sexual behaviors. Both OT knockout and OTR knockout mice are still capable of successfully reproducing (Nishimori et al., 1996; Takayanagi et al., 2005). While OT null males do not have any apparent functional or behavioral deficits during mating, sexual receptivity is decreased in females (Nishimori et al., 1996; Zimmermann-Peruzatto et al., 2017). Further support for OT’s role in rodent sexual behavior has been provided by pharmacological studies. Intracerebroventricular (ICV) injection of OT stimulates proceptive and receptive behavior in the female rat and increases erections and reduces ejaculation latency in the male rat. Conversely, ICV administration of OT antagonists inhibits female and male sexual behaviors and abolishes ejaculation. The effects of exogenous OT on female sexual behavior may be site-dependent, as infusion of OT into the lateral ventricle has been shown to inhibit sexual receptivity in the rat (Schulze and Gorzalka, 1992), while infusion into the third ventricle has been found to facilitate receptivity. In the female rodent, sexual receptivity is primarily defined by the lordosis reflex, a dorsiflexed posture generally accompanied by lateroflexion of the tail that

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permits intromission by the male during copulation. This hormone-dependent reflex is triggered by pressure applied to the female’s flanks by a mounting male or by an investigator’s hand. The female rodent’s pelvic organs contain OTR and receive OT fibers descending through the lumbosacral spinal cord. This OT-ergic regulation of pelvic organs has several copulationrelated functions, including the mediation of lubrication, pain suppression, and muscle contraction during mating. The neurocircuitry of the lordosis reflex is well characterized. Several of its neural componentsdthe PVN, the ventromedial hypothalamus (VMH), the medial preoptic area (MPOA), and the periaqueductal gray (PAG)dhave been found to be regulated by OT (for review, see Veening et al., 2015). Despite the apparent lack of effects of OT gene deletion on male copulatory behavior, OT is still a critical component of male sexual function. Sexual behavior or electrical stimulation of the glans penis or dorsal nerve of the penis activates OT neurons in the PVN and SON and increases OT release in the PVN and cerebrospinal fluid. It is thought that the activation of magnocellular OT neurons in the PVN leads to activation of nearby parvocellular OT neurons, which facilitate genital reflexes via the sacral parasympathetic neurons in the spinal cord. In addition, PVN OT neurons send collateral projections to the nucleus paragigantocellularis of the brainstem, which provides tonic inhibition of penile reflexes (Veening et al., 2015). 5.1.4.2 Parental Care Maternal behavior is impaired in mice lacking the OTR (Takayanagi et al., 2005). OTR expression in the rat increases during the peripartum period in regions associated with maternal care and maternal aggression, including the MPOA, LS, BNST, ceA, olfactory bulb (OB), and the ventral tegmental area (VTA) (Bosch and Neumann, 2012; Sabihi et al., 2014). Administration of OT to regions such as the MPOA, VTA, and OB can initiate maternal behavior in virgin females, while OTR antagonists applied to these regions inhibit maternal care in lactating dams. Research investigating maternal aggression in rodents has identified the ceA, BNST, and LS as sites through which OT mediates aggressive behaviors (Bosch and Neumann, 2012). Finally, OTR expression is believed to play an integral role in natural variations in maternal care in the rodent. Assessment of rat dams that are previously characterized as being Low or High providers of licking and grooming (LG) of offspring during the first week of life has revealed elevated OTR binding in the MPOA, BNST, LS, PVN, and ceA of High LG dams compared with Low LG females. Moreover, levels of LG received by female rodents during early life are predictive of the maternal care that

they themselves will provide their offspring in adulthood. Accordingly, OTR expression in regions associated with maternal care is programmed by the levels of LG received during early life (Bales and Perkeybile, 2012). 5.1.4.3 Social Behavior OT regulates human and rodent social behaviors, including juvenile play, social recognition, pair bonding, sociability, and aggression. ICV OT has been shown to reverse social defeat-induced social avoidance and also improve social recognition in male, but not female, rats. Furthermore, ICV administration of an OTR antagonist inhibited social recognition in both male and female mice. Research using transgenic mice has confirmed the importance of OT in social recognition, as OT and OTR knockout mice show impairments in this behavior (Dumais and Veenema, 2016). Conversely, OT administered to the LS induced juvenile play behavior in female rats but not males. While sex differences were not found in the quantity of OTimmunoreactive neurons in these regions in the rat brain (DiBenedictis et al., 2017), male rats have been found to have higher OTR binding densities in the LS, BNST, meA, and VMH, among other regions, when compared to female rats (Dumais et al., 2013). Collectively, these findings indicate that OT has sex-specific effects on the regulation of social behavior. 5.1.4.4 Stress Responsivity and Anxiety-Like Behavior OT is known to dampen stress reactivity and for associated decreases in anxiety-like behaviors. Central administration of OT decreases anxiety-like behavior in rats and mice, and OT knockout mice show more anxiety-like behavior and a greater physiologic response to stress than wildtype controls (Amico et al., 2004). OT regulates function of the HPA axis, the primary circuit for the mammalian stress response, in a site-specific manner. Acute stress stimulates the secretion of OT from both the PVN and SON (Neumann, 2007), likely via the activation of glucocorticoid receptors on or near OT neurons (Torner et al., 2017). Within the PVN, OT has been found to inhibit CRH, resulting in dampened HPA axis activity as indicated by attenuated ACTH and adrenal corticosterone release. In contrast, OT acts at the level of the pituitary to potentiate the actions of CRH (Schlosser et al., 1994), thereby increasing ACTH release. Finally, OT suppresses stress responsivity and decreases anxiety-like behavior through projections to limbic brain regions such as the meA, ceA, and LS (Neumann, 2007). 5.1.4.5 Osmoregulatory Behavior OT regulates consummatory behaviors associated with fluid homeostasis. Studies using OT knockout

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mice have reported an enhanced intake of sodium chloride solution under basal conditions or following dehydration, while central administration of OT inhibits salt intake (Amico et al., 2003). These findings suggest that OT typically suppresses salt consumption. OT administration has also been shown to enhance the consumption of water in rodents that were food deprived, fed a lowsodium diet, or administered polyethylene glycol (Bernal et al., 2015), indicating that OT selectively drives behaviors associated with the restoration of fluid homeostasis. Information about body fluid volume and electrolyte balance reaches the brain via the sensory circumventricular organs (CVOs) of the lamina terminalis, the area postrema, and the organum vasculosum of the lamina terminalis (OVLT). The OVLT has been shown to project information about changes in osmolality to OT and AVP neurons in the PVN and SON (Johnson and Gross, 1993). The dorsal raphe nucleus, the parabrachial nucleus, and the area postrema have all been implicated as regions regulating the response of OT neurons to osmotic challenge (Olszewski et al., 2010; Vivas et al., 2014). These neurons are activated in the mouse following isotonic blood volume expansion or the induction of acute hypernatremia (Ruginsk et al., 2007). In the rat, chronic salt loading increased OT mRNA and OTenhanced cyan fluorescent protein (eCFP) expression in the PVN and SON in a transgenic OT-eCFP reporter model (Katoh et al., 2010). The secretion of OT from the hypothalamus in response to osmotic stimuli has also been shown to be mediated by ANP, which colocalizes with PVN and SON OT neurons (Chriguer et al., 2003). While research has defined many of the key brain areas involved in osmoregulation, the downstream effects of OT secretion on consummatory behaviors related to the restoration of fluid balance have not been characterized. Interestingly, osmotic challenge also influences fear and anxiety behaviors, suggesting a link through OT. In the mouse, hypertonic saline injection has been shown to decrease anxiety-like behavior and stress responsivity (Smith et al., 2015). It is postulated that the murine behavioral response to hypertonia is due to an OT-mediated inhibition of CRH neurons within the PVN and the amygdala and from the axonal release of OT from PVN neurons into the BNST, thereby activating specific OT responsive populations of neurons within this brain area (Smith et al., 2015). The HPA axis also regulates the OT response to an osmotic stimulus, since pretreatment with the synthetic glucocorticoid, dexamethasone, reduces OT secretion and inhibits the activation of PVN and SON OT neurons (Ruginsk et al., 2007). This system may be dysregulated

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in patients with panic disorder and in a rat model of panic disorder, as injection of a hypertonic saline solution can increase symptoms of panic and anxietylike behavior, respectively (Jensen et al., 1991; Molosh et al., 2010). 5.1.4.6 Feeding Behavior Central OT has also been shown to mediate feeding behavior, with primarily anorexigenic actions. ICV injections of OT or OTR agonists suppress food intake in the rat, while OTR antagonists stimulate food consumption. Since only high-dose OT is capable of suppressing feeding when administered peripherally, OT’s hypophagic effects likely occur through central mechanisms. These findings are consistent with the observation that OT does not readily cross the bloodebrain barrier. The OT system is also believed to regulate homeostasis associated with feeding. OT responds to physiologic cues indicating fullness such as changes in gastric distention and alterations in plasma osmolarity (discussed in Section 5.1.4.5) resulting from food intake. OT also regulates feeding in a selective manner, as it is implicated in conditioned tasted aversion and in regulating carbohydrate consumption. Parvocellular OT neurons in the PVN have reciprocal projections to the area postrema, the nucleus of the solitary tract (NTS), and the dorsal motor nucleus of the vagus nerve, brainstem nuclei known to regulate feeding. OT has been found to facilitate the acquisition of conditioned taste aversion by activating the ceA (Olszewski et al., 2013) and suppressing sucrose intake via actions on the VTA (Mullis et al., 2013). Results from studies examining the distribution of OTR have led to hypothesized roles for OT neurotransmission in regulating food intake via reward, affect, and energy homeostasis neurocircuitry; however, the contribution of OT to feeding behavior mediated by these circuits has not been comprehensively evaluated (Olszewski et al., 2010).

5.2 Vasopressin AVP released from the posterior pituitary has two main sites of action. At the kidneys, it functions to regulate extracellular fluid volume, and on vascular smooth muscle, it causes vasoconstriction. 5.2.1 Vasopressin in Water Balance AVP maintains fluid homeostasis through its selective actions on the renal collecting duct. The AVP receptor V2R is found in the basolateral membrane of these cells and is activated upon AVP binding. Activation of V2R increases water permeability on the apical side of the

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kidney epithelial cells in the short term through increased trafficking of aquaporin-2 to the apical membrane and in the long term by increased aquaporin-2 gene expression (Fig. 10.4). Aquaporins are integral membrane proteins that play a critical role in regulating water content of cells by transferring water across the membrane. Aquaporin-2, the only aquaporin regulated by AVP, is found in the apical cell membrane of collecting ducts in the kidney. The primary function of AVP’s regulation of aquaporin-2 is to conserve the body’s water and to reduce the amount of water lost in urine. Under dehydrated conditions, reabsorption of water from the collecting duct rebalances fluid homeostasis by transporting solute-free water back into circulation, resulting in a decrease in plasma osmolarity and an increase in urine osmolarity. AVP restores homeostasis by decreasing the formation of urine, which in turn increases blood volume, arterial pressure, and cardiac output. 5.2.2 Vasopressin in Vasoregulation AVP plays a major role in blood pressure regulation through its actions as a potent vasoconstrictor. This occurs through the V1aR, which is found on vascular smooth muscle cells. Vasoconstriction is mediated by the IP3 and Rho-kinase signal transduction pathway and by the release of Ca2þ from the sarcoplasmic reticulum. AVP-mediated vasoconstriction is also dependent upon activation of PKC and L-type voltage-sensitive Ca2þ channels (Henderson and Byron, 2007). This ultimately increases arterial pressure as a result of increased systemic vascular resistance. Excess AVP can result in too much vascular constriction, which can lead to vasospasm and subarachnoid hemorrhage (Nishihashi et al., 2005). Both fluid resorption and blood vessel constriction are regulated by AVP originating from the hypothalamus and secreted by the posterior pituitary. In both cases, increased blood volume and vascular resistance cause elevations in arterial pressure. These two systems can work in tandem to maintain blood pressure and fluid balance. For example, dehydration or hemorrhage will cause a decrease in atrial pressure. In response, cardiopulmonary baroreceptors in the walls of the atrium decrease their firing rate under decreased pressure conditions. Normally, these atrial receptors project to and synapse within the NTS, causing increased ANP neurotransmission through efferent fibers that project to the SON to inhibit the release of AVP. During dehydration the baroreceptors decrease their firing rate, thereby decreasing the amount of ANP neurotransmission that usually inhibits AVP. This decrease in signaling results in increased AVP secretion until pressure returns to normal conditions.

An additional mechanism for blood pressure regulation has been identified that is specific to periods of dehydration. This mechanism utilizes hypothalamic osmoreceptors that detect the concentration of solutes in plasma. When the body is well hydrated, these osmoreceptors stay below threshold stimulation and AVP secretion is suppressed. With rising osmolarity, the osmoreceptors detect the increase and stimulate the secretion of AVP. AVP increases linearly with increasing plasma osmolarity, resulting in the retention of water. This process appears to be more sensitive than that of the baroreceptors. 5.2.3 Vasopressin Neurocircuitry and Behavior Several of the behaviors mediated by OT, including sexual behavior, social behavior, affective behavior, and parental care, have also been shown to be regulated by AVP. These hormones often assume oppositional functions and influence males and females differently. Many of the brain areas through which OT acts also contain AVP receptors and have been identified as key components of AVP behavioral neurocircuitry. AVP is involved in the processing of olfactory information from social contact, which, for the rodent, has important implications for several different types of behaviors. 5.2.3.1 Reproductive Behavior The effects of AVP on rodent reproductive behavior are particularly salient in males. Mating and matingrelated stimuli activate AVP neurons in brain areas such as the BNST and the meA of the male rat (Dass and Vyas, 2014; Ho et al., 2010), and administration of AVP reverses impairments in sexual behavior following castration. Conversely, ICV AVP has been shown to inhibit sexual behavior in the female rat (Zimmermann-Peruzatto et al., 2015). In the male prairie vole, 3 days of mating and social experience with a female will increase the number of AVP mRNA-labeled neurons in the BNST and has been shown to decrease the concentration of AVP-immunoreactive fibers in the LS. These changes were absent in female prairie voles. In the monogamous prairie vole, AVP appears to be critical for pair bonding behavior for males and, to a potentially lesser extent, females (Numan and Young, 2016). Thus, sex differences in the impact of AVP on reproductive behavior may be species- and behavior-specific. Moreover, this is consistent with the demonstration that there are profound sex differences in the number of AVP neurons in the brains of many rodents (Rood and De Vries, 2011; Wang et al., 1994; De Vries and AlShamma, 1990), with more AVP neurons in males relative to females.

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5.2.3.2 Parental Care AVP regulates both maternal and paternal care. The AVP system becomes activated during the perinatal period, with a reported increase in PVN AVP mRNA and elevated levels of AVP in the hippocampus and in plasma (Landgraf et al., 1991). AVP facilitates maternal behaviors in both rats and mice through its actions on the V1aR. Manipulation of V1aR in the MPOA has revealed an important role for these receptors in behaviors such as arched back nursing and pup retrieval that is specific to this brain area. AVP also regulates maternal aggression via V1aR in the ceA and BNST (Bosch and Neumann, 2012). While paternal behavior is not typically observed in the rodent, research conducted in the prairie vole, a species that displays biparental behavior, has provided insight into the influence of AVP on parental behavior in the male rodent. PVN and SON AVP mRNA increases in both male and female prairie voles during the postpartum period (Wang et al., 2000). Although pharmacological manipulation of V1aR in the LS has revealed that AVP acts on the LS to influence paternal behavior, the persistence of paternal care following castration, a manipulation that virtually abolishes AVP immunoreactivity within the LS, suggests that AVP signaling in this region is not essential for paternal behavior in the vole (Zimmermann-Peruzatto et al., 2015). 5.2.3.3 Social Behavior AVP and its receptors are found in many of the brain structures that comprise the social behavior neural network. The species-specific distribution of this hormone and its receptors is thought to contribute to the profound differences in social behavior between species (Albers, 2012). The formation of partner preference in both male and female prairie voles has been shown to occur through a V1aR-dependent mechanism, primarily via AVP signaling in the LS and the ventral pallidum. These two structures show differences in V1aR binding between monogamous and nonmonogamous vole species (Tickerhoof and Smith, 2017). AVP has been shown to regulate social recognition through its actions on the V1aR in the LS and OB in the rodent. AVP-mediated effects on social recognition have also been reported in the hippocampus and the meA. It has been proposed that AVP does not alter the salience of social stimuli, but instead facilitates the formation of memory, as social recognition can be affected by the administration of AVP or a V1aR antagonist after exposure to a social stimulus. AVP may be more critical for social recognition in male rodents, whereas it may only facilitate recognition in females (Gabor et al., 2012).

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AVP is also implicated in social aggression, although its effects may be mediated by prior social experience. In socially housed, unmanipulated rodents, AVP or a V1aR antagonist administered to the anterior hypothalamus (AH) facilitated and inhibited aggression, respectively. In addition, social isolation in hamsters and matinginduced pair bonding in prairie voles have been shown to increase aggressive behavior in males and concomitantly increase V1aR binding in the AH. Interestingly, voles with prior experience with aggressive interactions continued to show elevated levels of aggression following ICV administration of a V1aR antagonist (Winslow et al., 1993). Thus, activation of V1aR may not be required for the expression of aggressive behavior. A final AVP-mediated social behavior, communication, has been best described in the hamster. Flank marking is a form of scent marking through which social information, including social status, is transmitted. This behavior is mediated by V1a receptors in the MPOA/ AH (Albers, 2012), and it is also regulated by AVP signaling in extrahypothalamic regions such as the LS and the PAG (Terranova et al., 2017). While it was previously assumed that AVP regulates social behaviors solely through the V1aR, more recent research has indicated that V1bR also influence several social behaviors. V1bR knockout mice were shown to be less aggressive than wildtype controls, and to have mild deficits in social memory and in motivation to interact with social stimuli (Stevenson and Caldwell, 2012). Partial restoration of aggressive behavior has been shown following lentiviral-induced restoration of V1bR function within the hippocampus of knockout mice, suggesting that hippocampal V1bR are involved in the regulation of aggressive behavior (Pagani et al., 2015). 5.2.3.4 Stress Reactivity When considering AVP as a factor in regulating the neuroendocrine response to stressors, it is important to note that some parvocellular neurons in the PVN coexpress AVP and CRH. These neurons have axons that terminate in the external zone of the median eminence and release hormones into the hypophyseal portal vasculature. Significant evidence from rat studies have demonstrated that AVP and CRH are copackaged and coreleased by the same secretory granules and thereby work together to control the secretion of ACTH from the anterior pituitary corticotrophs (Whitnall et al., 1985). In this way, AVP has been shown to potentiate the actions of CRH through its binding to the V1bR. However, recent studies in mice indicate that the colocalization of AVP and CRH in PVN neurons could be

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much less than that reported for rats (40 C), dramatically increasing current amplitude and duration in response to both stimuli. In vivo, PRL administration enhances the nocifensive response to capsaicin in proestrous or ovariectomized females following E2 administration (Diogenes et al., 2006). A similar sensitizing effect of PRL has been observed in DRG cells, with the response including not only TRPV1 but also TRPA1 and TRPM8 channels, additional transducers of painful stimuli (Patil et al., 2013). Moreover, PRL has been shown to increase the excitability of DRG neurons, enhancing discharge frequency in response to depolarizing stimulus (Patil et al., 2014). Thus, given this wealth of information, PRL and its modulation of ion channel function appears to be a regulator of nociceptive perception, potentially linking the sensitization of key TRP channels to the numerous hyperalgesic conditions associated with estrogen and the estrous cycle. Given PRL’s functional promiscuity, the broad pattern of its receptor expression, and the now clear demonstration that it can regulate ion channel function, the more complete elucidation of its impact on neuronal network processing promises to be an exciting avenue of research.

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3.2 Neurohormones 3.2.1 TRH The tripeptide TRH was the first hypothalamic releasing factor to be identified. Signaling via two GPCRsdTRHR1 and TRHR2 (Sun et al., 2003)dthis hypophysiotropic hormone is the principal regulator of the hypothalamicepituitaryethyroid axis, stimulating the release of both thyroid-stimulating hormone and PRL from the anterior pituitary. However, complementary to its neuroendocrine functions, TRH has additional centrally mediated roles in various other processes, including energy balance, cognitive arousal and sleep, mood, and locomotion. Accordingly, in addition to the hypophysiotropic neurons of the PVH responsible for the delivery of TRH to the pituitary, both this peptide and its receptors are found in other brain regions, with particularly high levels of expression in structures associated with the preceding processes: these include the thalamus, hypothalamus, brainstem, and spinal cord. The electrophysiological impact of TRH in many of these systems has been well characterized, and this neurohormone has been shown to dramatically alter neuronal and network function through the activation or inhibition of numerous ionic conductances. The responses of TRH are predominantly excitatory in nature, with these effects achieved primarily via the activation of TRPC channels and/or the inhibition of various Kþ currents. The hypothalamus (TRPC): Having a prominent role in the setting of arousal and the promotion of wakefulness, TRH receptors are expressed in key thalamic and hypothalamic structures engaged in the regulation of sleep wake cycles and vigilance. For example, in the LHA, there are two anatomically adjacent neuronal populations that have opposing actions on these processes: neurons expressing the wakefulness-promoting peptide Hcrt/Ox and neurons expressing the sleep-promoting melanin-concentrating hormone (MCH) (Konadhode et al., 2013). Hypocretin/orexin neurons express TRH receptors and receive innervation from TRHexpressing neurons residing in multiple structures, including the DMH, a neighboring nucleus critical for the circadian organization of multiple physiologies (Chou et al., 2003). Application of TRH to Hcrt/Ox neurons results in a rapid depolarization and commencement of AP discharge. This postsynaptic, AP-independent, excitatory response was the result of a TRHR1-dependent activation of a mixed cationic TRPC-like current that was partially dependent upon intracellular Ca2þ release (Gonza´lez et al., 2009; Hara et al., 2009). In contrast, TRH application to MCH neurons results in a hyperpolarization and cessation of AP discharge. However, this effect was blocked by both TTX and the GABAA antagonist bicuculline, indicating an indirect, presynaptic enhancement of inhibitory

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tone. Indeed, electrophysiological recording of LHA GABAergic interneurons has revealed a proportion of these cells to be excited via a PLC-dependent activation of a TRPC channel in manner similar to that of Hcrt/Ox neurons. Importantly, this presynaptic mechanism proved sufficiently strong to attenuate the previously reported Hcrt/Ox-induced excitation of MCH neurons (van den Pol et al., 2004; Zhang and van den Pol, 2012). Thus, this multisite, circuit-wide effect of TRHd exciting Hcrt/Ox neurons, while concurrently diminishing the impact of their excitatory input to sleep promoting MCH neuronsdtilts the balance of this circuit toward an output conducive to the enhanced arousal observed with TRH administration (Nishino et al., 1997). In the TMN, another key hypothalamic center of arousal control (Yu et al., 2015), TRH excites histaminergic neurons by the activation of a TRPC-like mixed cationic conductance and the activation of the Naþ/ Ca2þ exchanger. This excitatory effectdfunctionally similar, though mechanistically distinct to that described for ghrelin beforedis thought to constitute a particularly prominent component of the arousal promoting effects of TRH, as the ability of the TRH receptor agonist montirelin to induce waking was dramatically reduced in histidine decarboxylase knockout mice, which lack histamine, and wild-type animals treated with an inhibitor of histamine biosynthesis (Parmentier et al., 2009). Elsewhere in the hypothalamus, TRH employs another TRPC-like dependent mechanism for the excitation of oscillating TIDA neurons (Lyons et al., 2010). The resultant switch from phasic to tonic discharge provides a possible explanation for the site-specific effects of TRH on PRL secretion, with centrally acting TRH behaving as a PRL-inhibiting factor, while conversely, TRH acting at the lactotroph promoting PRL release (Ohta et al., 1985; Lyons and Broberger, 2014). The thalamus (TRPC, GIRK, L-Type): This structure is a critical gateway system that polices the flow of sensory information to the cortex and as such plays a prominent role in the regulation arousal and sleep, undergoing tightly regulated state-dependent shifts in cellular and network activity that either faithfully transmit or impede the flow of information from the sensorium (McCormick and Bal, 1997). In brain slices of the geniculate complex spontaneously displaying rhythmic activity characteristic of slow-wave sleep, TRH application results in a transient disturbance in network activity, replacing spindle wave oscillations with discharge patterns associated with waking state. This dramatic shift in network performance was achieved via the simultaneous, TRH-dependent, depolarization of both dorsal lateral geniculate thalamocortical relay neurons (dLGN), which transmit visual information to the cortex, and neighboring GABAergic neurons of the reticular thalamus, which regulate local network function.

These responses were underpinned by the closure of a Kþ conductance, depolarizing both sets of neurons to membrane potentials where they were no longer capable of recruiting the LVA T-type Ca2þ channels required for rebound discharge, burst firing, and network rhythmogenesis. This depolarization of dLGN cells was also associated with the enhancement of the AP after depolarization, a response dependent upon the activation of a Ca2þ-dependent mixed cationic current (Broberger and McCormick, 2005). In the thalamic paraventricular nucleus, which receives arousal-related inputs from Hcrt/Ox neurons, TRH enhances neuronal excitability via a net depolarizing response with similar components to those reported in the geniculate formation. Here, TRH-induced excitation was driven by the concurrent inhibition of a tertiapin Q-sensitive GIRK-like Kþ conductance and the activation of a TRPC5 conductance that, in addition to depolarization, enhanced low-threshold spiking (Zhang et al., 2013) (Fig. 11.16). Further investigation revealed the GIRK inhibition alone to be dependent upon MAPK, while the TRPC conductance proved extremely sensitive to both extracellular and intracellular Ca2þ, requiring TRH activation of L-type Ca2þ channels and the subsequent release of Ca2þ from the endoplasmic reticulum, in a process of Ca2þ-activated Ca2þ release (Kolaj et al., 2016). The brainstem (TRPC, GIRK, K2P, KV, L-Type): TRH has numerous electrophysiological effects in the brainstem, from where it further influences arousal and has a prominent impact on parasympathetic outflow and the autonomic regulation of the gastrointestinal and respiratory tracts. The locus coeruleus (LC), located in the pontine and medullary brainstem, is a key part of the arousal-promoting ascending reticular activating system and is the principle central source of noradrenaline, sending noradrenergic projections throughout the brain. Application of TRH to noradrenergic neurons of the LC initiates a cascade of postsynaptic electrophysiological events that lead to AP discharge and depolarizing Ca2þ transients. Calcium spikes were blocked by the L-type channel antagonist nifedipine, while the underlying membrane potential depolarization was independent of TRP channel blockade and the result of an inward current with a reversal potential approaching that of Kþ. Pharmacological analysis of the TRHinhibited Kþ conductance revealed that it was not the consequence of the inhibition of a constitutively active GIRK channel, as in the thalamic paraventricular nucleus, but rather the result of a PLC-dependent inhibition of an pH-sensitive Kþ leak conductance (Ishibashi et al., 2009), with the electrophysiological and acidsensing properties of channels composed of K2P3.1 and K2P9.1, subunits prominently expressed in the LC (Talley et al., 2001) (Fig. 11.17).

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FIGURE 11.16

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Hormone modulation of the neuronal inwardly rectifying KD channels underpinning IGIRK and TRPC channels. Graphical representation of the TRH-induced excitation of PVT neurons. TRH is released within the PVT via wired transmission from TRH neurons, most likely residing in the hypothalamic parvocellular paraventricular and dorsomedial nuclei, and brainstem raphe nuclei. Activation of TRH receptors on PVT neurons leads to the inhibition of KIR3 channels and the activation of TRPC5 channels. This simultaneous reduction in constitutively active IGIRK and the activation of ITRPC results in a rapid and powerful depolarization and enhancement of AP discharge (Control, solid line; TRH, dashed line).

Hormone modulation of the neuronal two-pore KD channels underpinning leak IK2P and CaV1 channels. Graphical representation of the TRH-induced excitation of noradrenergic neurons of the locus coeruleus. TRH is released within the locus coeruleus via wired transmission from TRH neurons. Activation of TRH receptors on noradrenergic neurons leads to the inhibition of K2P channels and the activation of CaV1 channels. This simultaneous reduction in a constitutively active IK2P and activation of IL results in both a rapid and powerful depolarization and enhancement of AP discharge and the initiation of dihydropyridine (DHP)-sensitive Ca2þ spiking (Control, solid line; TRH, dashed line).

In the DMV, TRH induces an excitation, with the underlying depolarization driven by the activation of a mixed cationic current and the associated enhancement of AP discharge facilitated by an inhibition in the activation of the KV channels underpinning IA (Travagli et al., 1992). This excitation is thought to stimulate the gastric branch of the vagus nerve and to be a crucial component of the vagally mediated gut responses of the preparatory cephalic phase of digestion (Tache´ et al., 2014). Hypoglossal motor neurons, which have respiratory coupled motor patterns and contribute to the resistance of the upper respiratory tract, are also excited by TRH, which once again employs a Kþ-dependent mechanism in which the closure of K2P channels is explicitly

implicated (Bayliss et al., 1992; Talley et al., 2000). TRH further modulates brainstem respiratory control via its effects upon inspiratory activated and inspiratory inhibited vagal preganglionic neurons of the nucleus ambiguus. Application of TRH to these cellsdwhich have dominant roles in the regulation of the vasculature, musculature, and submucosal secretion of both the trachea and bronchiadresults in the activation of an unidentified postsynaptic inward current and an acceleration of inspiratory related inhibitory events (Hou et al., 2012), most likely driven by the TRHdependent excitation of pre-Bo¨tzinger complex neurons (Ruangkittisakul et al., 2006) via what is thought to be a K2P-dependent mechanism (Chen et al., 2006).

FIGURE 11.17

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3.2.2 Oxytocin and Vasopressin Oxytocin and AVP are closely related neuroendocrine factors, whose nonapeptide structures show remarkable similarity, differing only at the third and eighth amino acid. These evolutionary conserved neurohypophysial hormones are synthesized in predominantly exclusive, yet adjacent, neuronal populations, being expressed primarily in MSNs of the SON and PVH. In addition to MSNs, AVP and OT are also found in parvocellular populations of the PVH and a small number of discrete intra- and extrahypothalamic structures, though the distribution patterns of the latter displays marked interspecies variability. The physiological effects of AVP and OT are generated via interaction with their cognate receptors, all of which belong to the G proteinecoupled receptor superfamily. In rodents and humans a total of four receptors have been identified, with AVP interacting primarily with V1A, V1B, and V2, and OT displaying highest affinity for the oxytocin receptor (OTR). Oxytocin and AVP receptors appear to couple primarily to Gq (with the exception of the peripheral V2 receptor, which couples to Gs), though there are reports of additional interactions with Gi/o and Gs, with the OTR appearing the most promiscuous (Alberi et al., 1997; Birnbaumer, 2000; Gimpl and Fahrenholz, 2001; Gravati et al., 2010). It should be noted that receptor signaling by OT and AVP is further complicated by a degree of ligand cross-reactivity, with AVP binding to OTRs with only 10 times less affinity than that of OT itself (Kimura et al., 1994), while in a reciprocal fashion, OT is able to bind to AVP receptors, albeit with 100 times less affinity than AVP (Manning et al., 2012). These neurohypophysial hormones are released into the systemic circulation from MSN axon terminals located in the posterior pituitary, and their subsequent effects on peripheral physiology have been long studied and are well characterized, with peripheral AVP being involved primarily in the regulation of vascular function and fluid homeostasis, while peripheral OT has well-defined roles in milk ejection and the contraction of uterine smooth muscle during parturition. The effects of AVP and OT, however, are not restricted to the periphery, as both neurohypophysial hormones have emerging, complimentary, yet often antagonistic roles in the central regulation of numerous processes. These include social behaviors, autonomic and neuroendocrine control, feeding, nociception, learning and memory, and arousal. Accordingly, V1A and OTRsdthe receptor subtypes most abundantly expressed within the CNSdare widely distributed throughout the brain and spinal cord, being found in a variety of function-specific neuronal populations residing in structures such as the cortex, thalamus, olfactory system, VTA, brainstem, spinal cord, and in various sites within the limbic system, including the

hypothalamus, amygdala, hippocampus, and the lateral septum (Gimpl and Fahrenholz, 2001; Raggenbass, 2008). As the BBB is relatively impermeable to both OT and AVP, the contribution of circulating hormones to central receptor activation is thought to be negligible, and instead, OT and AVP are believed to arrive at central receptors via two principal modes of transmission: “wired” axonal release from fibers in close apposition to target neurons and “unwired” diffusional release from the dendrites and other extrasynaptic compartments (Ludwig et al., 2016). This latter mechanism, lacking the spatiotemporal precision of axonal release, is prominent in MSNs, and OT and AVP released via this process, in addition to local autocrine effects, is thought to travel via the extracellular and cerebrospinal fluids to activate receptors at distant sites in a process referred to as volume transfer (Ludwig et al., 2002). While this form of neurosecretion is perhaps most closely associated with MSNs, it appears not to be restricted to OT and AVP, with extrasynaptic release potentially constituting a relevant secretory mechanism for additional peptide and nonpeptide signaling factors, though there is ongoing debate as to the transmitter substances utilizing this form of release and the range of distances over which it may influence signal transduction (Leng and Ludwig, 2008; van den Pol, 2012; Leng, 2018). Irrespective of transmission mode, there is a growing body of literature detailing the ability of OT and AVP receptor activation to modulate neuronal excitability via the rapid activation and inhibition of ion channel activity. A selection of structures in which these responses, and the ion channels responsible, are best characterized shall be discussed subsequently and include the VTA, the septo-hippocampal complex, the spinal cord, brainstem, and the hypothalamus. The ventral tegmental area (TRPC): The VTA is a key regulatory node in the brain’s reward circuitry, and the delivery of DA from this structure to the striatum and the prefrontal cortex is important for the regulation of rewarding behavior and the processing of emotional and cognitive functions. Behavioral studies have identified DA-ergic neurons in the VTA as important sites for OT’s permissive effects upon the reward-like properties of social interactions that are critical for the establishment and maintenance of prosocial behaviors. Recent work has begun to unravel this complicated system, while firmly establishing a role for OT’s regulation of ion channel activity in its physiological functioning. Both OT fibers and OTRs are found in the VTA and the related midline interfascicular nucleus (IF) and application of OT to these structures results in the powerful excitation of almost 80% of IF neurons and an electrophysiologically distinct subset of VTA cells: putative DA-ergic neurons that possess a small IH and narrow AP half-width when

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contrasted with their nonresponsive counterparts. Electrophysiological and pharmacological analysis of these responses revealed the excitation in both structures to be the result of an OTR-dependent activation of a large, postsynaptic, mixed cationic current, most likely carried by a TRPC channel and dependent upon the action of PLC. Moreover, it was demonstrated that a smaller proportion of neurons in these structures were similarly excited by vasopressin via interaction with the V1A receptor (Tang et al., 2014). A subpopulation of OT-ergic parvocellular neurons within the PVH has been identified as an axonal source of OT to the VTA, and optogenetic stimulation of fibers originating from these cells selectively excites VTA DA-ergic neurons projecting to the striatum and hence likely involved in the processing of rewarding stimuli. This mechanism was shown to be OTR dependent and driven by the simultaneous activation of a postsynaptic current (presumably that described before) and enhancement of presynaptic inhibitory tone, which nevertheless proved insufficient to prevent a pronounced net excitatory effect. Parvocellular OT-ergic neurons of the PVH were also shown to send projections to the SNpc, the optogenetic stimulation of which resulted in a robust inhibition. This response, clearly diverging from that in the VTA, was the result of OTR and V1A receptor-dependent excitation of GABA-ergic interneurons that inhibited SNpc DA-ergic cells via both GABAA and GABA-B receptor activation (Xiao et al., 2017). Thus, it appears that OT engages in a biased modulation of midbrain DA-ergic signaling, whichdgiven the social reward (Song et al., 2016b) and locomotor (Angioni et al., 2016) effects of OTR agonists delivered to the VTA and SNpc, respectivelydhas been hypothesized to promote social interaction over exploratory locomotion. And indeed, subsequent optogenetic examination of the behavioral impact of this VTA circuit demonstrated the bidirectional gating of social reward, clearly identifying a network substrate for OT’s observed ability to promote the perception of social interactions as rewarding experience (Hung et al., 2017). Hippocampus and lateral septum (TRPC, K2P): Consistent with OT and AVP’s roles in the limbic processing of social memory and cognition, receptors for both of these hormones are found within the hippocampus and related structures, and accordingly, there are numerous examples of their activation inducing changes in membrane excitability. It was early demonstrated that application of both OT and AVP directly excites putative hippocampal interneurons, while simultaneously inhibiting CA1 pyramidal cells via an associated increase in presynaptic inhibitory tone (Mu¨hlethaler et al., 1984; Zaninetti and Raggenbass, 2000). Yet it remained unclear as to how this apparent OTR-mediated enhancement of inhibition tallied with OT’s permissive effects on learning and memory in

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general, and those associated with social and familial scenarios in particular (Tomizawa et al., 2003). This apparent paradox was resolved by Owen and colleagues, who demonstrated that OT-induced excitation of hippocampal interneurons was restricted exclusively to fast spiking (FS) cells of the stratum pyramidale, with OT having no effect on the regular spiking population. This FS-specific excitation was a consequence of a postsynaptic OTR-dependent activation of a TRPClike mixed cationic current that, in agreement with previous work, resulted in a dramatic increase in FS discharge and a concomitant inhibition of CA1 pyramidal neurons. However, despite the prevailing inhibition, in this network configuration, transmission fidelity and temporal precision of CA1 spiking in response to stimulation of Schaffer collateral (SC) excitatory inputs was greatly increased. This surprising improvement in circuit performance was the consequence of an enhancement of signal-to-noise ratio, with CA1 inhibition reducing the incidence of principal cell spiking independent of SC stimulation, i.e., noise, while the same enhanced GABAergic transmission, via a process of frequency-dependent depression, reduced the feedforward inhibition associated with SC excitatory inputs, leading to a dramatic increase in stimulus-spike coupling, i.e., signal (Owen et al., 2013). The observed enhancement of transmission fidelity and signal strength represents an elegant mechanism by which both “wired” axonal release of fast neurotransmitters and the more temporally imprecise “unwired” volume transfer of a neuromodulatory factor like OT could have prominent effects upon cognitive processes that require millisecond precision. It should be noted that this inhibition-dependent tightening of transmission fidelity may not be restricted to OT or CA1, as frequency-dependent depression of inhibitory transmission is a phenomenon also present in inhibitory interneurons in the dentate gyrus (Kraushaar and Jonas, 2000) and the neocortex (Hestrin and Galarreta, 1998). Moreover, while the correct functioning of this and similar processes may underpin OT’s physiological roles in social recognition, familial bonding, and the cognitive changes associated with the rearing of offspring, disruption of this mechanism may also be linked to this hormone’s role in the etiology of autism spectrum disorder (Guastella and Hickie, 2016), as this condition is additionally linked to aberrations in both FS interneuronal function (Gogolla et al., 2009) and transmission fidelity (Dinstein et al., 2012). In addition to information processing within the hippocampus, OT’s electrogenic effects may also play a role in the gating and filtering of information entry into this limbic structure. For example, the activation of OTRs in the dentate gyrus similarly depolarizes hilar interneurons (Harden and Frazier, 2016), neurons that exert

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inhibitory control over mossy cells, an enigmatic glutamatergic population that has emerging roles in anxiety and associative learning (Scharfman, 2016). While the underlying postsynaptic inward current bore many similarities to that described earlier, it remains, nevertheless, unidentified (Harden and Frazier, 2016). AVP has also been reported to have a permissive effect on learning and memory (Alescio-Lautier and Soumireu-Mourat, 1998), and V1A receptor activation has also been shown to excite hippocampal interneurons, though these responses are to be found in the CA1 stratum radiatum, with the resultant excitation being underpinned by a markedly different ion channeldependent mechanism. Here, AVP application resulted not in the activation of a mixed cationic current but rather the inhibition of a K2P-dependent, constitutively active, Kþ leak current. However, the ensuing enhancement of local inhibitory drive fails to inhibit CA1 pyramidal cells, with this effect being masked by a putative direct excitation of these neurons, though the mechanism remains unknown (Ramanathan et al., 2012). Accordingly, given its enhancement of inputindependent spiking or “noise,” it seems unlikely that this V1A-dependent mechanism mimics the effect of OTR activation upon transmission fidelity described earlier, and as such, its role in AVP-dependent learning remains to be fully explored, though this mechanism of postsynaptic excitation and simultaneous enhancement presynaptic inhibitory tone, a recurring theme with AVP as we shall see, has nevertheless been suggested to play a role in the fine tuning of CA1 neuronal output (Ramanathan et al., 2012). Despite this, alternative roles for AVP’s engagement of inhibitory interneurons in the hippocampus have also been proffered, including functioning as neuroprotective mechanism during the hypoxic conditions at the point of birth (Spoljaric et al., 2017). The lateral septum (LS), an inhibitory nucleus also heavily implicated in the role of AVP and OT in social recognition and aggression, receives its most prominent input from the hippocampus (Risold and Swanson, 1997), in addition to AVP-ergic and OT-ergic inputs from the amygdala and parvocellular PVH, respectively. Application of AVP to this structure recapitulates the effects seen in the hippocampus, directly exciting a proportion of LS neurons, while enhancing GABAergic tone in almost all (Allaman-Exertier et al., 2007). Furthermore, recent work has demonstrated that a hippocampal CA2 to LS projection selectively excites dorsal LS neurons that provide inhibitory input to ventral LS neurons. In turn, ventral LS neurons tonically inhibit the central aggression locus found within the VMH, thus forming a hippocampally driven, polysynaptic disinhibitory circuit that promotes social aggression (Leroy et al., 2018). Moreover, both the strength of excitatory

inputs to (Pagani et al., 2015) and excitatory outputs from CA2 pyramidal cells are enhanced by the activation of V1B receptors, which in turn triggers social aggression (Leroy et al., 2018). Given the near universal enhancement of inhibitory tone within the LS in response to bath application of AVP, it is tempting to speculate once again as to divergent roles in this mechanism for focal axonal release and volume transfer, with axonal release perhaps selectively enhancing mnemonic information from CA2, while volume transmission could more broadly impact the LS, though further work is required to identify the functional roles of the neurons postsynaptically excited by AVP in both these structures. Nevertheless, despite these gaps in our understanding, these data demonstrate a clear role for AVP in the septo-hippocampal processing of social memory and the gating of aggressive behavior. Hypothalamus (TRPC): The transductional modulation of ion channels by OT and AVP receptor activation has been reported in numerous hypothalamic nuclei, with perhaps the best characterized electrophysiological responses being found in the LHA and the TIDA system. When applied to oscillating TIDA neurons, OTdin a manner similar to that of PRLdinduces excitation via the OTR-dependent activation of a postsynaptic TRPClike mixed cationic current and a Ca2þ-dependent high-voltage component, which may involve the regulation of L-type and BK channels (Briffaud et al., 2015). This mechanism, in conjunction with PRL’s inhibition of OT-ergic neurons (Sirzen-Zelenskaya et al., 2011), represents an added layer of functionally coherent crosstalk in the hormonal regulation and temporal coordination of lactation and suckling. These now unambiguously integrated mechanisms could conceivably function to limit the metabolically demanding PRL-mediated production of milk to discrete packets of time orientated around the feeding, and if the parent is lucky, subsequent satiation of suckling offspring. However, given the apparent uncoupling of TIDA activity from DA release discussed before (Romano et al., 2013) and its potential replacement with a PRL-releasing factor, an additional scenario needs considering. It may be that this mechanism serves not to limit PRL release, and hence milk production, but rather to terminate milk ejection via PRL-mediated inhibition of neurohypophysial OT release. Under such circumstances, suckling-induced OT release coulddin tandem with initiating milk ejectiondenhance milk production via the stimulation of PRL release, which would after a vascular and transductional delay, subsequently terminate milk ejection via PRL feedback at OT-ergic MSNs. Such hormonal crosstalk could function to temporally distribute bouts of feeding while maintaining milk production capacity. Returning once again to the LHA, we shall now examine the effects of AVP and OT on the electrical

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excitability of wakefulness promoting Hcrt/Ox neurons and REM sleep promoting MCH neurons whichdlike both OT and AVPdregulate arousal and sleep/wake cycles, while also influencing energy balance and fluid intake. Both AVP and OT excite MCH neurons of the LHA, the optogenetic stimulation of which has been reported to enhance sleep (Konadhode et al., 2013). With MCH neurons expressing both OTR and V1A receptors, application of OT and AVP induced a postsynaptic inward current that was jointly mediated by the activation of a TRPC channel and the Naþ/Ca2þ exchanger, though it should be noted that the AVP depolarization was much greater, a potential consequence of its cumulatively enhanced agonist activity at both receptors (Yao et al., 2012). Both OT and AVP also excite neighboring Hcrt/ Ox neurons via the postsynaptic, V1A receptordependent activation of a TRPC-like mixed cationic conductance that was in part dependent upon Ca2þ release from intracellular stores (Tsunematsu et al., 2008). The excitatory action of AVP and OTdboth of which have anorexigenic effects (Sabatier et al., 2013; Pei et al., 2014)dupon two populations of neurons regarded as key orexigenic players in the central regulation of energy balance may at first seem paradoxical. However, as there is a major OT-ergic projection from PVH and SON to anorexigenic POMC neurons in the ARCdwhere OT application increases [Ca2þ]i in the majority of POMC neuronsdit may be that the resultant satiating effects predominate (Maejima et al., 2014). It therefore seems reasonable to suggest that the likely physiological role of these LHA effects may reside more with the impact these two neuronal populations have upon arousal, water homeostasis, and reproduction, though this interpretation too is not without complications. It becomes easier to envisage a role for AVP in LHA-mediated arousal when we consider recent work demonstrating rather than simply enhancing sleepda response at odds with the general effect of AVPd optogenetic stimulation of MCH neurons has been reported to have no effect on vigilance or the total sleep time, instead enhancing the proportion of sleep spent in the REM state (Tsunematsu et al., 2014), an effect mediated via inhibitory input to histaminergic neurons of the TMN (Jego et al., 2013). Thus for AVP, given the permissive effects of MCH on water ingestion and Hcrt/Ox on arousal and locomotion, the simultaneous activation of both groups of neurons has been proposed as a mechanism by which this hormone could drive water-seeking behavior during dehydration, a physiological response diminished in V1A receptor null mice (Tsunematsu et al., 2008; Yao et al., 2012). In contrast, while OT also has a role in water homeostasis, the activation of MCH neurons by this hormone has been proposeddas a consequence of MCH neuron’s inhibitory projection to preoptic/septal GnRH neurons (Wu et al., 2009)das a

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mechanism by which the fertility of nursing mothers is reduced, thus helping to maximize the successful rearing of offspring by coordinating the optimal timing of subsequent pregnancy (Yao et al., 2012). Oxytocin’s effects on Hcrt/Ox neurons are also difficult to interpret, but given the much smaller excitatory response of OT when compared to AVP and the extremely high dose required to achieve even this comparatively meagre effect, this process may well be state dependent. And indeed, administration of OT antagonists promotes arousal and wakefulness, while acute administration of OT delays sleep onset and reduces both REM and non-REM sleep. Thus, it may be under basal conditions that OT promotes sleep, while under circumstances of enhanced hormone elevationda situation where Hcrt/Ox neurons may well be recruiteddthis effect is reversed (Lancel et al., 2003). The spinal cord and brainstem (KATP, TRPV1, KV, TRPC, GIRK): Oxytocin and AVP have increasingly well-established roles in the electrogenic modulation of spinal cord circuits involved in nociceptive processing, motor output, and autonomic control. Nociceptive processing: Oxytocin and AVP, both of which have analgesic properties, exert numerous effects upon the membrane excitability of neurons in the DRG and the superficial and possibly deeper laminae of the dorsal horn of the spinal cord, important way points for the modulation and transmission of nociceptive information to the central nervous system. Primary sensory afferent neurons within the DRG possess both OTR and V1A receptors and also themselves represent prominent extrahypothalamic sources of AVP and OT, with the expression of the latter being powerfully upregulated during pregnancy (Dayanithi et al., 2018). Upon application of OT, nociceptive DRG neurons undergo a membrane potential hyperpolarization and a reduction in evoked AP amplitude and half-width. The underlying hyperpolarization was shown to be carried by KATP channels whose activation was the result of OTRdependent stimulation of nitric oxide synthase. While not explicitly demonstrated, the observed impact on DRG rheobase and AP waveform is most likely an additional consequence of enhanced KATP activation, further reducing the transmission capacity of these neurons (Gong et al., 2015). In addition to the activation of a Kþ current, it has also been reported that OT robustly inhibits heat- and capsaicin-activated TRPV1 currents and acid-sensing ion channels via the activation of a V1A receptor, the absence or blockade of which powerfully curtails both OT’s and AVP’s analgesic effects (Qiu et al., 2014a; Han et al., 2018). Moreover, in a fascinating development, OT has recently been shown to directly activate TRPV1 channels independent of receptor-mediated signal transduction. While the functioning of TRPV1 as an effective ionotropic OT receptor

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may appear at odds with this hormone’s antinociceptive properties, this direct gating of TRPV1 by OT was shown to result in rapid and prolonged channel desensitization, powerfully diminishing its capacity for subsequent activation by noxious stimuli (Nersesyan et al., 2017). Thus, given that TRPV1 enables the sensation of noxious heat, pH, and inflammation, and is in fact used as a molecular marker for nociceptive neuronal components, OT’s antinociceptive effects via this mechanism may well extend beyond the DRG. Within the superficial laminae of the dorsal hornd which receives dense OT-ergic innervation (Rousselot et al., 1990)dOT induces a complicated array of often divergent electrophysiological effects. These OTRdependent responses include inhibiting the capacity for repetitive discharge by simultaneously reducing both IA and IDR, an ostensibly excitatory response that results in inhibition by diminishing the pool of Naþ channels relieved of voltage-dependent block and available for subsequent AP initiation (Breton et al., 2009), and excitation by the closure of a Kþ current and/or the activation of TRPC-like mixed cationic current (Jiang et al., 2014). While the identity of the neurons affected remains ambiguous, the generation of an exact circuit model for OT’s and potentially AVP’s effects within this dorsal horn will prove difficult. Nevertheless, it has been suggested that OT-induced excitations recruit local glutamatergic neurons, which in turn engage GABAergic and glycinergic interneurons (which may themselves also be directly excited) to enhance local inhibitory tone. This, in conjunction with KV channeldependent suppression of excitability in putative projection neurons of lamina I, functions to curtail the transmission of nociceptive information across this structure and onward to supraspinal sites in the CNS (Breton et al., 2008). Furthermore, OT’s pain modulating effects appear not to be limited to the superficial laminae, as a recently identified population of OT-ergic parvocellular neurons in the PVH has been shown to project to both MSNs in the SON and to a population of neurokinin1 receptor-positive “wide dynamic range” (WDR) cells: pain processing projection neurons located in laminae IV and V. Optogenetic stimulation of this OTergic population simultaneously initiates the vascular release of OT from the SON and synaptic release of OT onto WDR neurons, where OTR activation induces a postsynaptic reduction in excitability thought to resemble the KV-dependent mechanism observed in the superficial lamina (Breton et al., 2009; Eliava et al., 2016). Thus, distinct projections from this small population of OT-ergic neurons are thought to engage a temporally segregated, two-tier analgesic response, composed of rapid release within the spinal cord and slow vascular release from SON. Collectively, this body of research demonstrates the ability of OT’s electrogenic effects to

diminish nociceptive signals as this information is trafficked to the CNS, highlighting a multicomponent process that can account for the analgesia associated with neurohypophysial hormones and the potential for reduced nociception during circumstances in which their release is elevated, e.g., childbirth. In addition to the inhibition of afferent signals traveling to the spinal cord, particularly those of a nociceptive nature, there is considerable evidence to demonstrate that neurohypophysial hormones exert the opposite effects on the autonomic and somatic efferent signals leaving this structuredwith the boosting of such signals likely mediated by their electrophysiological impact on motoneurons and preganglionic neurons in the brain stem and spinal corddboth of which receive prominent projections from parvocellular populations in the PVH. Autonomic control: In sympathetic preganglionic neurons (SPNs), located in the intermediolateral (IML) column of the thoracic spinal cord, AVP application universally results in excitation and the commencement of AP discharge. Electrophysiological analysis suggests the observed depolarization is the consequence of postsynaptic V1A receptor activation, resulting in a compound effect, being driven singularly, or in combination, by the suppression of a Ba2þ-sensitive Kþ conductance and the activation of a mixed cationic current, electrophysiological events most likely mediated by the modulation of a constitutively active GIRK channel and a TRP channel, respectively (Kolaj and Renaud, 1998). This excitatory mechanism is predicted to enhance sympathetic outflow and contribute to AVPinduced stimulation of heart rate and associated vascular and renal effects. Of note, OT has also been reported to excite SPNs via V1A receptors, and though this effect was robust, it was an order of magnitude less potent than the AVP responses described in the same study and so may be of less physiological relevance (Sermasi and Coote, 1994). In contrast, OT has been shown to excite brainstem vagal neurons, cells whose axons terminate in the parasympathetic ganglia of the thoracic and abdominal viscera. Application of OT to identified parasympathetic preganglionic neurons in the DMV induces an OTR-dependent membrane depolarization and commencement of AP discharge. The underlying ionic conductance is thought to be postsynaptic, Naþ dependent anddgiven the negative slope conductance of its I/V relationshipdmost likely carried by TRP channels (Dubois-Dauphin et al., 1992; Raggenbass and Dreifuss, 1992; Alberi et al., 1997). Moreover, in the NTSdwhich forms part of a reflex arc, relaying “organ-topic” sensory afferent information arriving via the solitary tract (ST) to appropriate parasympathetic preganglionic neurons in the DMV and elsewhere in the brainstemdOT-ergic fibers have been shown to make synaptic contact with

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neurons receiving cardiopulmonary inputs. And indeed, OT application excites a proportion of NTS neurons, both increasing postsynaptic excitability by the closure of a Kþ current and presynaptically enhancing glutamate release probability from the ST. Moreover, AVP opposes this potentiation of excitatory inputs in the NTS, both reducing glutamate release probability and promoting transmission failure along the ST (Bailey et al., 2006), while enhancing inhibitory transmission onto cardiac parasympathetic preganglionic neurons in the nucleus ambiguus (Wang et al., 2002). Thus, in a manner similar to that described for nociception earlier in the text, AVP and OT act at multiple points within the brain stem and spinal corddoften in tandemdto regulate both parasympathetic and sympathetic outflow to influence a multitude of physiological processes including cardiovascular and gastrointestinal function. Motor control: The receptors of both neurohypophysial hormones are to be found in somatic motoneurons of the brain stem, while V1A receptors are found in both interneurons and motoneurons of the ventral horn of the spinal cord, with the expression of this receptor being particularly high at birth and declining with age (Tribollet et al., 1991; Liu et al., 2003). In neonatal rats, AVP application to antidromically identified motoneurons in the ventral horn of the thoracolumbar spinal cord results in a slow-onset, long-lasting, membrane potential depolarization driven by the closure of a constitutively active Kþ conductance. This excitatory response was sufficient to elicit AP discharge and was the result of V1A receptor activation. Moreover, this postsynaptic effect was associated with the pronounced enhancement of presynaptic inputs, with increases in both excitatory glutamatergic and inhibitory GABAergic and glycinergic transmission. These premotor, network effects are most likely driven by the AVP-induced excitation of putative interneurons within the ventral horn, a proportion of which are excited by a similar mechanism to that of motoneurons, with the remaining neurons being depolarized via the activation of a TRPC-like mixed cationic current (Oz et al., 2001; Liu et al., 2003). It has been hypothesized that by increasing the membrane excitability, and thus responsiveness of spinal motoneurons to excitatory inputs, hypothalamic AVP may behave as a regulator of motor function and muscular contraction. However, given the excitatory nature of glycinergic and GABAergic inputs in very young animals and the developmental decline of V1A receptor expression in the ventral horn, the primary role of hypothalamic AVP in this structure may be more concerned with the early developmental tuning and maturation of neuronal circuits regulating motor control, rather than modulating their established function in the adult (Singer et al., 1998; Reymond-Marron et al., 2006). Interestingly, one population of motoneurons that fails to undergo

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developmental downregulation of V1A receptors are the sexually dimorphic pudendal motoneurons. Found in most mammals within a single neuronal group, Onuf’s nucleus, pudendal motoneurons straddle spinal segments L5 and L6 anddinnervating the striated muscles of the pelvis involved in penile erection and ejaculation in the male and the external sphincters of the urethra and anus in both males and femalesdhave critical roles in eliminative and reproductive functions (McKenna and Nadelhaft, 1986). Application of AVP to identified pudendal motoneurons results in membrane depolarization and AP discharge, which, like the excitatory responses of premotor interneurons in the more rostral lumbar segments described before, has been attributed to either the closure of a constitutively active Kþ conductance or the activation of a TRPC-like mixed cationic current (Ogier et al., 2006). Given the enduring presence of V1A receptors in these neurons, it therefore seems likely that AVP’s electrogenic effects may regulate the coordination and force of muscular contraction underpinning both reproductive and eliminative activity. Therefore, as a consequence of segregated receptor expression patterns and projection fields, it appears that the two neurohypophysial hormones have prominent, yet distinct, roles in the regulation of information flow across the spinal cord, with OT impeding the passage of sensory afferent information in the dorsal horn, while AVP enhances efferent motor output from the ventral horn. Despite this apparent separation of function, however, and the absence of OTRs in the ventral horn, a potential role for OT in the regulation of motor outputs may nevertheless exist, as application of OTR selective agonists can both excite lumbar motoneurons and promote and coordinate fictive locomotion in an isolated spinal cord preparation (Pearson et al., 2003; Dose et al., 2014). These OTR-dependent effects are presynaptic in origin and, being unlikely to originate from premotor interneurons within the ventral horn itself, are possibly derived from OTR-mediated excitation of interneurons in laminae I and II that collateralize to lumbar motoneurons. It has been suggested that this modulation of locomotor output by OXY and AVP serves an evolutionary conserved role in the generation of the coordinated motor behavior associated with reproduction, such as lordosis in the female rat (Wagenaar et al., 2010; Stoop, 2012). Additional motor outputs that exhibit developmental flux in V1A receptor expression can be found in both the facial motor nucleus (VII) and the hypoglossal nucleus (XII) (Tribollet et al., 1991; Liu et al., 2003). Motoneurons within these brainstem structures project to the musculature of the face and tongue and accordingly function to regulate the motor patterning of behaviors such as chewing, swallowing, breathing, and vocalization. Application of AVP to both facial and hypoglossal

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motoneurons in newly born rats results in pronounced depolarizations and commencement of AP discharge, with the underlying postsynaptic mixed cationic current, while not definitively identified, manifesting a number of properties associated with a TRPC-like conductance (Raggenbass et al., 1991; PalouzierPaulignan et al., 1994; Reymond-Marron et al., 2006). Unlike their counterparts in the facial motor nucleus, a subpopulation of hypoglossal motoneurons express functional OTRs, and application of selective OTR agonists also results in the postsynaptic activation of an inward current (Palouzier-Paulignan et al., 1994). In addition to postsynaptic effects, both OTR and V1A receptor activation in the hypoglossal nucleus results in the enhancement of glycinergic, GABAergic, and glutamatergic inputs to motoneurons (Wrobel et al., 2010). Once again, while these effects may have a role in coupling autonomic activity to motor output in the adult, given the developmental plasticity of V1A receptors in these neurons, the primary impact of the neurohypophysial hormones on hypoglossal and facial motor output may well be the activity-dependent maturation of developing motor circuits or the promotion of motor activity specifically associated with the perinatal period such as suckling. The preceding data clearly identifies neurohypophysial hormones as powerful regulators of neuronal ion channel function, who by initiating predominantly excitatory events are able to influence not only neuronal excitability but also network performance across a wide range of neuronal structures.

4. CONCLUDING REMARKS With the evolution of the patch clamp technique in the early 1980s (Hamill et al., 1981), there has been an explosion of research into the molecular correlates of membrane excitability and their rapid modulation by signaling factors, including hormones. This powerful biophysical approach and its myriad configurations were the products of decades of incremental experimental refinement (Verkhratsky et al., 2006), whichdculminating in the development of the gigaohm sealdfinally armed researchers with a toolset capable of producing the signal-to-noise ratios that are required to detect the vanishingly small currents passing through individual ion channels. This hurdling of previously intractable technical barriers, and the resultant highresolution recording of single-channel and whole-cell events (Sakmann and Neher, 1984), rapidly led to the electrophysiological characterization of the various members of the VSIC superfamily and, when these techniques have been applied to brain tissue, the ability to eavesdrop upon neuronal communication with

unprecedented clarity. The “signals intelligence” gathered with these techniques has not only afforded us an improved understanding of how these proteins dictate neuronal function and network processing but, when coupled with the parallel application of histochemical, pharmacological, and molecular approaches, has firmly established the rapid transductional regulation of neuronal ion channel function as a mainstay of hormonal signaling within the brain. And if we return to the conversational analogy proffered during the introduction, we can see from the examples given earlier that this rapid arm of hormone signaling has dramatic effects upon neuronal dialogue, and that these effects, being shaped by mode of transmission, transductional mechanism, and ultimately channel target, have powerful impact upon neuronal voice, influencing who speaks, what is said, and who is heard. Transmission mode: Hormones arrive at their neuronal receptors via two principal transmission modes, wired, or network intrinsic release, and unwired, or network extrinsic release, with the former’s spatiotemporal influence largely determined by connectomic constraints. When acting in an orthodox fashion, being delivered to the brain via the peripheral circulation, or from within the central nervous system via volume transfer, unwired hormonal signals are able to simultaneously activate multiple sites, anddlike a tide affecting all shipsdfloat or sink targets depending upon the electrogenic mechanisms engaged. This concurrent, spatially indiscrete signaling, akin to a shout heard by many, can be contrasted with the wired synaptic release of centrally synthesized hormones, which acting with greater spatiotemporal precision, more closely resembles a whisper, a sotto voce message heard by only a select, axonally targeted few. Despite obvious differences in their temporal and anatomical scope, these distinct transmission modes, and their impact on ion channel activity, do not necessarily subserve segregated functions, anddas demonstrated by the antinociceptive action of OT in the spinal cord detailed beforedmay in fact operate in concert to produce functionally coordinated physiological effects. The opposite scenario also arises, as the different delivery mechanisms are capable of inducing equally different, even opposing results, in the same system: one such example being the regulation of pituitary PRL release by the hypophysiotropic hormone TRH. Here the axonal release of TRH within the hypothalamus excites TIDA neurons and therefore inhibits PRL secretion, while vascular delivery of this hormone to the anterior pituitary exerts the adverse effect. Signal transduction: The preceding pages have detailed numerous examples of specific hormone receptors being able to recruit different ion channels in different cellular populations. However, as we have seen with insulin and leptin, the signal transduction

4. CONCLUDING REMARKS

mechanisms linking receptor activation to channel activity are also themselves highly variable and can even differ according to prevailing physiological state. Moreover, this signaling plasticity occurs not just in terms of strength, whereby electrogenic responses are diminished or absent, but also in terms of the ion channel to which receptor activation is coupled, with distinct or even opposing conductances recruited by the same receptor in the same neuronal population in a historyspecific fashion. Importantly, it remains to be seen the whether this form of state-dependent transductional plasticity is limited to a discrete number of factors, such as leptin and insulin, or is a feature common to the majority of hormones. Channel target: As a consequence of the sheer variety of neuronal ion channels hormones can engaged channels with varying charge carriers and reversal potentials, voltages of activation, gating kinetics, and topographical distributionsdmembers of this diverse family of signaling molecules can affect equally variegated changes in membrane excitability, signal processing, and transmission fidelity. In many of the ex vivo experiments described in this chapter the hormonal activation or inhibition of subthreshold currents was often able to initiate or extinguish AP discharge and presumed transmitter release. However, given the relatively high concentrations usually administered and the methods and duration of application employed, the image painted by these results may be one of extreme potential rather than an accurate portrait of the physiological situation. It may therefore be, that under physiological conditions, these tonic metabotropic effects, rather than representing noncontingent signals in and of themselves, more closely resemble neuromodulation as envisaged by Irving Kupfermann (Kupfermann, 1979), and enhance, or diminish, the likelihood of AP initiation in response to rapid ionotropic inputs mediated by fast neurotransmission. Such gain modulation, whereby the postsynaptic neuron is made deaf or more attentive to its presynaptic partners as membrane potential is moved away or toward threshold, is necessarily frame-shifted according to the transductional delay of hormone receptor-channel coupling. And while this signaling cunctation is unlikely to impact hormone signals delivered via unwired transmission, it may be of particular importance to those arriving via axonal release, where network intrinsic neuromodulatory effects are restricted in time to noncoincident or asynchronous events occurring subsequent to hormone exocytosis. Moreover, the onset delay and duration of what may well be a highly plastic postrelease window would presumably preclude, or at least dictate, the degree to which the electrogenic effects of hormones and cotransmitters simultaneously released from the same source may interact. Nevertheless, as revealed by the

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action of OT in the hippocampus, the comparatively slow tonic currents typically activated by hormone signaling are still capable of regulating network processes reliant upon phasic fast neurotransmission and demanding millisecond precision. In addition to the regulation of subthreshold conductancesdwhose activation increases or decreases the valence of presynaptic inputsdhormone signaling is also able to rapidly regulate the activity of peri- and supra-threshold conductances. While, as a consequence of their role in setting spike threshold, the hormonal regulation of these ion channels may also modulate input valence, their transductional manipulation is, perhaps most importantly, a key determinant of neuronal output. Accordingly, hormone-induced modulation of the channels responsible for the upshoot and subsequent repolarization of the AP dictate intrinsic excitability, impacting properties such as waveform, spike frequency adaptation, and interspike interval. This sculpting of AP shape not only determines the number of APs a stimulus may produce, but by regulating AP duration or Ca2þ channels directly, powerfully influences presynaptic Ca2þ influx and the likelihood of AP-induced transmitter release. What is more, in addition to resultant variation in inputeoutput relationships and transmission fidelity, these changes in discharge pattern and Ca2þ dynamics, supplemented by hormone-induced alterations in the biosynthesis of transmitters and neuromodulators, means hormone signaling can potentially regulate not only exocytotic patterning butdas a consequence of differential Ca2þ dependencedthe type of transmitter released, effectively changing not only how loudly a neuron speaks, but what it says and how it says it (Nusbaum et al., 2017). Hormones and neuromodulation: Collectively, these neuromodulatory effects are of vital importance to the computational range of neuronal circuits, enabling a dynamic richness that is reliant upon electrogenic, rather than time- and resource-consuming structural change. This rapid and efficient responsorial flexibility, which permits an anatomically constant connectome, irrespective of complexity, to implement context-specific processing rules, dramatically affects the outcome of the game of neuronal telephone we described in the introduction, elevating the clarity of certain conversations, while diminishing that of others according to prevailing circumstance. Such hormone-induced network alterations and the resultant defense of systemic balance can be considered a form of physiological gattopardismo, an adaptive process where “everything needs to change, so that everything can stay the same.” And indeed, it is these situation-appropriate reconfigurations of circuit output that underpin a great deal of the brain’s ability to drive homeostatic and allostatic mechanisms,

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the hormone-modulated processing dynamics that allow behaving animals to maintain functional integrity in the face of often dramatically changing internal and external challenges. Given the fundamental importance of these processes, considerable effort has been devoted to understanding their underlying neurophysiology: the what, where, when, and how of hormonal neuromodulation. In recent years the pursuit of this endeavor has been enhanced by a remarkable increase in the genetic, anatomical, and imaging tools at our disposal. And indeed, the application of this expanded toolset has been particularly successful in resolving the first three of these interrogatives, permitting not only the mapping of neuronal connectomes with singular clarity, but the probing of their functional and behavioral impact with an unprecedented degree of phenotypic and spatiotemporal precision. However, while the advent of these methodologies has undoubtedly ushered in a “gilded age” for circuit physiologists, we must nevertheless remember that the data generated by these exciting technologies still only represents a piece of the puzzle, and as important as an accurate circuit map and a measure of its network and behavioral outputs irrefutably are, if we are to have a complete understanding of hormone signaling within the brain, and fully capitalize upon the undoubted research and clinical utility such information represents, then at some stage, we must, in the words of Eve Marder, “look beyond the connectome” (Marder, 2012) and establish “how” these changes occur. And in terms of hormonal neuromodulation and its impact upon the complex conversations of neurons, for “how,” one must always look for the functional fingerprints of “nature’s most superb chemists” and identify the ion channels involved.

References Abbott, G.W., Sesti, F., Splawski, I., Buck, M.E., Lehmann, M.H., Timothy, K.W., Keating, M.T., Goldstein, S.A., 1999. MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia. Cell 97, 175e187. Available at: http://www.ncbi.nlm. nih.gov/pubmed/10219239. Abizaid, A., Liu, Z.-W., Andrews, Z.B., Shanabrough, M., Borok, E., Elsworth, J.D., Roth, R.H., Sleeman, M.W., Picciotto, M.R., Tscho¨p, M.H., Gao, X.-B., Horvath, T.L., 2006. Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite. J. Clin. Investig. 116, 3229e3239. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17060947. Adelman, J.P., Shen, K.Z., Kavanaugh, M.P., Warren, R.A., Wu, Y.N., Lagrutta, A., Bond, C.T., North, R.A., 1992. Calcium-activated potassium channels expressed from cloned complementary DNAs. Neuron 9, 209e216. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/1497890. Aime´, P., Duchamp-Viret, P., Chaput, M.A., Savigner, A., Mahfouz, M., Julliard, A.K., 2007. Fasting increases and satiation decreases olfactory detection for a neutral odor in rats. Behav. Brain Res. 179,

258e264. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 17367877. Aime´, P., Hegoburu, C., Jaillard, T., Degletagne, C., Garcia, S., Messaoudi, B., Thevenet, M., Lorsignol, A., Duchamp, C., Mouly, A.-M., Julliard, A.K., 2012. A physiological increase of insulin in the olfactory bulb decreases detection of a learned aversive odor and abolishes food odor-induced sniffing behavior in rats. PLoS One 7, e51227. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/23251461. Akemann, W., Kno¨pfel, T., 2006. Interaction of Kv3 potassium channels and resurgent sodium current influences the rate of spontaneous firing of Purkinje neurons. J. Neurosci. 26, 4602e4612. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16641240. Akopian, A.N., Sivilotti, L., Wood, J.N., 1996. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 379, 257e262. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/8538791. Akopian, A.N., Souslova, V., England, S., Okuse, K., Ogata, N., Ure, J., Smith, A., Kerr, B.J., McMahon, S.B., Boyce, S., Hill, R., Stanfa, L.C., Dickenson, A.H., Wood, J.N., 1999. The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat. Neurosci. 2, 541e548. Available at: http://www.ncbi.nlm. nih.gov/pubmed/10448219. Al Koborssy, D., Palouzier-Paulignan, B., Canova, V., Thevenet, M., Fadool, D.A., Julliard, A.K., 2019. Modulation of olfactory-driven behavior by metabolic signals: role of the piriform cortex. Brain Struct. Funct. 224 (1), 315e336. Available at: http://www.ncbi. nlm.nih.gov/pubmed/30317390. https://doi.org/10.1007/s00429018-1776-0. Epub 2018 Oct 13. PMID: 30317390. Al-Qassab, H., Smith, M.A., Irvine, E.E., Guillermet-Guibert, J., Claret, M., Choudhury, A.I., Selman, C., Piipari, K., Clements, M., Lingard, S., Chandarana, K., Bell, J.D., Barsh, G.S., Smith, A.J.H., Batterham, R.L., Ashford, M.L.J., Vanhaesebroeck, B., Withers, D.J., 2009. Dominant role of the p110b isoform of PI3K over p110a in energy homeostasis regulation by POMC and AgRP neurons. Cell Metabol. 10, 343e354. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/19883613. Alberi, S., Dreifuss, J.J., Raggenbass, M., 1997. The oxytocin-induced inward current in vagal neurons of the rat is mediated by G protein activation but not by an increase in the intracellular calcium concentration. Eur. J. Neurosci. 9, 2605e2612. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9517466. Alescio-Lautier, B., Soumireu-Mourat, B., 1998. Role of vasopressin in learning and memory in the hippocampus. Prog. Brain Res. 119, 501e521. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 10074809. Allaman-Exertier, G., Reymond-Marron, I., Tribollet, E., Raggenbass, M., 2007. Vasopressin modulates lateral septal network activity via two distinct electrophysiological mechanisms. Eur. J. Neurosci. 26, 2633e2642. Available at: http://doi.wiley.com/10.1111/j.1460-9568. 2007.05866.x. Amaral, M.D., Pozzo-Miller, L., 2007. TRPC3 channels are necessary for brain-derived neurotrophic factor to activate a nonselective cationic current and to induce dendritic spine formation. J. Neurosci. 27, 5179e5189. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 17494704. Anderson, G.M., Beijer, P., Bang, A.S., Fenwick, M.A., Bunn, S.J., Grattan, D.R., 2006. Suppression of prolactin-induced signal transducer and activator of transcription 5b signaling and induction of suppressors of cytokine signaling messenger ribonucleic acid in the hypothalamic arcuate nucleus of the rat during late pregnancy and lactation. Endocrinology 147, 4996e5005. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/16857756. Andrews, Z.B., Erion, D., Beiler, R., Liu, Z.-W., Abizaid, A., Zigman, J., Elsworth, J.D., Savitt, J.M., DiMarchi, R., Tschop, M., Roth, R.H., Gao, X.-B., Horvath, T.L., 2009. Ghrelin promotes and protects

REFERENCES

nigrostriatal dopamine function via a UCP2-dependent mitochondrial mechanism. J. Neurosci. 29, 14057e14065. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19906954. Angioni, L., Cocco, C., Ferri, G.-L., Argiolas, A., Melis, M.R., Sanna, F., 2016. Involvement of nigral oxytocin in locomotor activity: a behavioral, immunohistochemical and lesion study in male rats. Horm. Behav. 83, 23e38. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/27189764. Ashcroft, F.M., Rorsman, P., 1989. Electrophysiology of the pancreatic beta-cell. Prog. Biophys. Mol. Biol. 54, 87e143. Available at: http://www.ncbi.nlm.nih.gov/pubmed/2484976. Ashford, M.L., Boden, P.R., Treherne, J.M., 1990. Glucose-induced excitation of hypothalamic neurones is mediated by ATP-sensitive Kþ channels. Pflu¨gers Archiv. 415, 479e483. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/2315006. Atzori, M., Lau, D., Tansey, E.P., Chow, A., Ozaita, A., Rudy, B., McBain, C.J., 2000. H2 histamine receptor-phosphorylation of Kv3.2 modulates interneuron fast spiking. Nat. Neurosci. 3, 791e798. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10903572. Augustine, R.A., Ladyman, S.R., Bouwer, G.T., Alyousif, Y., Sapsford, T.J., Scott, V., Kokay, I.C., Grattan, D.R., Brown, C.H., 2017. Prolactin regulation of oxytocin neurone activity in pregnancy and lactation. J. Physiol. 595, 3591e3605. Available at: http://www. ncbi.nlm.nih.gov/pubmed/28211122. Bailey, T.W., Jin, Y.-H., Doyle, M.W., Smith, S.M., Andresen, M.C., 2006. Vasopressin inhibits glutamate release via two distinct modes in the brainstem. J. Neurosci. 26, 6131e6142. Available at: http://www. ncbi.nlm.nih.gov/pubmed/16763021. Bajic, D., Hoang, Q.V., Nakajima, S., Nakajima, Y., 2004. Dissociated histaminergic neuron cultures from the tuberomammillary nucleus of rats: culture methods and ghrelin effects. J. Neurosci. Methods 132, 177e184. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/14706715. Bakowska, J.C., Morrell, J.I., 1997. Atlas of the neurons that express mRNA for the long form of the prolactin receptor in the forebrain of the female rat. J. Comp. Neurol. 386, 161e177. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9295145. Bakowska, J.C., Morrell, J.I., 2003. The distribution of mRNA for the short form of the prolactin receptor in the forebrain of the female rat. Brain Res. Mol. Brain Res. 116, 50e58. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/12941460. Banks, W.A., Tscho¨p, M., Robinson, S.M., Heiman, M.L., 2002. Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J. Pharmacol. Exp. Ther. 302, 822e827. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/12130749. Bannister, R.A., Beam, K.G., 2013. CaV1.1: the atypical prototypical voltage-gated Ca2þ channel. Biochim. Biophys. Acta Biomembr. 1828, 1587e1597. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/22982493. Baquero, A.F., de Solis, A.J., Lindsley, S.R., Kirigiti, M.A., Smith, M.S., Cowley, M.A., Zeltser, L.M., Grove, K.L., 2014. Developmental switch of leptin signaling in arcuate nucleus neurons. J. Neurosci. 34, 9982e9994. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/25057200. Baver, S.B., Hope, K., Guyot, S., Bjorbaek, C., Kaczorowski, C., O’Connell, K.M.S., 2014. Leptin modulates the intrinsic excitability of AgRP/NPY neurons in the arcuate nucleus of the hypothalamus. J. Neurosci. 34, 5486e5496. Available at: http://www.ncbi.nlm.nih. gov/pubmed/24741039. Bayliss, D.A., Viana, F., Berger, A.J., 1992. Mechanisms underlying excitatory effects of thyrotropin-releasing hormone on rat hypoglossal motoneurons in vitro. J. Neurophysiol. 68, 1733e1745. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1479442. Beck, P., Urbano, F.J., Williams, D.K., Garcia-Rill, E., 2013. Effects of leptin on pedunculopontine nucleus (PPN) neurons. J. Neural Transm.

269

120, 1027e1038. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/23263542. Ben-Jonathan, N., Hnasko, R., 2001. Dopamine as a prolactin (PRL) inhibitor. Endocr. Rev. 22, 724e763. Available at: http://www. ncbi.nlm.nih.gov/pubmed/11739329. Berkefeld, H., Fakler, B., 2008. Repolarizing responses of BKCa-Cav complexes are distinctly shaped by their Cav subunits. J. Neurosci. 28, 8238e8245. Available at: http://www.ncbi.nlm. nih.gov/pubmed/18701686. Berkefeld, H., Sailer, C.A., Bildl, W., Rohde, V., Thumfart, J.-O., Eble, S., Klugbauer, N., Reisinger, E., Bischofberger, J., Oliver, D., Knaus, H.G., Schulte, U., Fakler, B., 2006. BKCa-Cav channel complexes mediate rapid and localized Ca2þ-activated Kþ signaling. Science 314, 615e620. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17068255. Biel, M., Wahl-Schott, C., Michalakis, S., Zong, X., 2009. Hyperpolarization-activated cation channels: from genes to function. Physiol. Rev. 89, 847e885. Available at: http://www. ncbi.nlm.nih.gov/pubmed/19584315. Birnbaumer, M., 2000. Vasopressin receptors. Trends Endocrinol. Metabol. 11, 406e410. Available at: http://www.ncbi.nlm.nih. gov/pubmed/11091117. Blake, C.B., Smith, B.N., 2012. Insulin reduces excitation in gastricrelated neurons of the dorsal motor nucleus of the vagus. Am. J. Physiol. Integr. Comp. Physiol. 303, R807eR814. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22914748. Bloodgood, B.L., Sabatini, B.L., 2007. Nonlinear regulation of unitary synaptic signals by CaV2.3 voltage-sensitive calcium channels located in dendritic spines. Neuron 53, 249e260. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17224406. Breton, J.-D., Veinante, P., Uhl-Bronner, S., Vergnano, A.M., FreundMercier, M.J., Schlichter, R., Poisbeau, P., 2008. Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition. Mol. Pain 4, 19. Available at: http://journals.sagepub. com/doi/10.1186/1744-8069-4-19. Breton, J.D., Poisbeau, P., Darbon, P., 2009. Antinociceptive action of oxytocin involves inhibition of potassium channel currents in lamina II neurons of the rat spinal cord. Mol. Pain 5, 63. Available at: http://journals.sagepub.com/doi/10.1186/1744-8069-5-63. Briffaud, V., Williams, P., Courty, J., Broberger, C., 2015. Excitation of tuberoinfundibular dopamine neurons by oxytocin: crosstalk in the control of lactation. J. Neurosci. 35, 4229e4237. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25762669. Broberger, C., McCormick, D.A., 2005. Excitatory effects of thyrotropinreleasing hormone in the thalamus. J. Neurosci. 25, 1664e1673. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15716402. Brown, H.F., Giles, W., Noble, S.J., 1977. Membrane currents underlying activity in frog sinus venosus. J. Physiol. 271, 783e816. Available at: http://www.ncbi.nlm.nih.gov/pubmed/303699. Brown, R.S.E., Kokay, I.C., Herbison, A.E., Grattan, D.R., 2010. Distribution of prolactin-responsive neurons in the mouse forebrain. J. Comp. Neurol. 518, 92e102. Available at: http://www.ncbi. nlm.nih.gov/pubmed/19882722. Bru¨ning, J.C., Gautam, D., Burks, D.J., Gillette, J., Schubert, M., Orban, P.C., Klein, R., Krone, W., Mu¨ller-Wieland, D., Kahn, C.R., 2000. Role of brain insulin receptor in control of body weight and reproduction. Science 289, 2122e2125. Available at: http://www. ncbi.nlm.nih.gov/pubmed/11000114. Buraei, Z., Yang, J., 2010. The ß subunit of voltage-gated Ca2þ channels. Physiol. Rev. 90, 1461e1506. Available at: http://www.physiology. org/doi/10.1152/physrev.00057.2009. Cabral, A., Valdivia, S., Fernandez, G., Reynaldo, M., Perello, M., 2014. Divergent neuronal circuitries underlying acute orexigenic effects of peripheral or central ghrelin: critical role of brain accessibility. J. Neuroendocrinol. 26, 542e554. Available at: http://www.ncbi. nlm.nih.gov/pubmed/24888783.

270

11. HORMONES AND THE REGULATION OF NEURONAL VOLTAGE-SENSING ION CHANNELS

Cantrell, A.R., Catterall, W.A., 2001. Neuromodulation of Naþ channels: an unexpected form of cellular plasticity. Nat. Rev. Neurosci. 2, 397e407. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/11389473. Catterall, W.A., 2011. Voltage-gated calcium channels. Cold Spring Harb. Prespect. Biol. 3 a003947ea003947 Available at: http:// www.ncbi.nlm.nih.gov/pubmed/21746798. Catterall, W.A., Goldin, A.L., Waxman, S.G., 2005. International union of pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacol. Rev. 57, 397e409. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 16382098. Catterall, W.A., Leal, K., Nanou, E., 2013. Calcium channels and shortterm synaptic plasticity. J. Biol. Chem. 288, 10742e10749. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23400776. Chen, C., Bharucha, V., Chen, Y., Westenbroek, R.E., Brown, A., Malhotra, J.D., Jones, D., Avery, C., Gillespie, P.J., KazenGillespie, K.A., Kazarinova-Noyes, K., Shrager, P., Saunders, T.L., Macdonald, R.L., Ransom, B.R., Scheuer, T., Catterall, W.A., Isom, L.L., 2002. Reduced sodium channel density, altered voltage dependence of inactivation, and increased susceptibility to seizures in mice lacking sodium channel beta 2-subunits. Proc. Natl. Acad. Sci. U.S.A. 99, 17072e17077. Available at: http://www.ncbi.nlm. nih.gov/pubmed/12481039. Chen, C., Westenbroek, R.E., Xu, X., Edwards, C.A., Sorenson, D.R., Chen, Y., McEwen, D.P., O’Malley, H.A., Bharucha, V., Meadows, L.S., Knudsen, G.A., Vilaythong, A., Noebels, J.L., Saunders, T.L., Scheuer, T., Shrager, P., Catterall, W.A., Isom, L.L., 2004. Mice lacking sodium channel 1 subunits display defects in neuronal excitability, sodium channel expression, and nodal architecture. J. Neurosci. 24, 4030e4042. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/15102918. Chen, X., Talley, E.M., Patel, N., Gomis, A., McIntire, W.E., Dong, B., Viana, F., Garrison, J.C., Bayliss, D.A., 2006. Inhibition of a background potassium channel by Gq protein -subunits. Proc. Natl. Acad. Sci. U.S.A. 103, 3422e3427. Available at: http://www.ncbi. nlm.nih.gov/pubmed/16492788. Chen, R.-S., Deng, T.-C., Garcia, T., Sellers, Z.M., Best, P.M., 2007. Calcium channel gamma subunits: a functionally diverse protein family. Cell Biochem. Biophys. 47, 178e186. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/17652770. Chen, S.-R., Chen, H., Zhou, J.-J., Pradhan, G., Sun, Y., Pan, H.-L., Li, D.-P., 2017. Ghrelin receptors mediate ghrelin-induced excitation of agouti-related protein/neuropeptide Y but not proopiomelanocortin neurons. J. Neurochem. 142, 512e520. Available at: http://www.ncbi.nlm.nih.gov/pubmed/28547758. Cheng, W., Sun, C., Zheng, J., 2010. Heteromerization of TRP channel subunits: extending functional diversity. Protein Cell 1, 802e810. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/21203922. Cheung, C.C., Clifton, D.K., Steiner, R.A., 1997. Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus. Endocrinology 138, 4489e4492. Available at: http://www.ncbi.nlm.nih. gov/pubmed/9322969. Chiu, S., Wise, P.M., 1994. Prolactin receptor mRNA localization in the hypothalamus by in situ hybridization. J. Neuroendocrinol. 6, 191e199. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 8049718. Chou, T.C., Scammell, T.E., Gooley, J.J., Gaus, S.E., Saper, C.B., Lu, J., 2003. Critical role of dorsomedial hypothalamic nucleus in a wide range of behavioral circadian rhythms. J. Neurosci. 23, 10691e10702. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/14627654. Ciofi, P., Crowley, W.R., Pillez, A., Schmued, L.L., Tramu, G., Mazzuca, M., 1993. Plasticity in expression of immunoreactivity for neuropeptide Y, enkephalins and neurotensin in the

hypothalamic tubero-infundibular dopaminergic system during lactation in mice. J. Neuroendocrinol. 5, 599e602. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8680430. Clapham, D.E., Runnels, L.W., Stru¨bing, C., 2001. The TRP ion channel family. Nat. Rev. Neurosci. 2, 387e396. Available at: http://www. ncbi.nlm.nih.gov/pubmed/11389472. Clement, J.P., Kunjilwar, K., Gonzalez, G., Schwanstecher, M., Panten, U., Aguilar-Bryan, L., Bryan, J., 1997. Association and stoichiometry of K(ATP) channel subunits. Neuron 18, 827e838. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9182806. Coetzee, W.A., Amarillo, Y., Chiu, J., Chow, A., Lau, D., McCormack, T., Moreno, H., Nadal, M.S., Ozaita, A., Pountney, D., Saganich, M., Vega-Saenz de Miera, E., Rudy, B., 1999. Molecular diversity of Kþ channels. Ann. N. Y. Acad. Sci. 868, 233e285. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10414301. Cone, J.J., Roitman, J.D., Roitman, M.F., 2015. Ghrelin regulates phasic dopamine and nucleus accumbens signaling evoked by foodpredictive stimuli. J. Neurochem. 133, 844e856. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25708523. Cowley, M.A., Smart, J.L., Rubinstein, M., Cerda´n, M.G., Diano, S., Horvath, T.L., Cone, R.D., Low, M.J., 2001. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411, 480e484. Available at: http://www.ncbi. nlm.nih.gov/pubmed/11373681. Cowley, M.A., et al., 2003. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 37, 649e661. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12597862. Cravo, R.M., Frazao, R., Perello, M., Osborne-Lawrence, S., Williams, K.W., Zigman, J.M., Vianna, C., Elias, C.F., 2013. Leptin signaling in Kiss1 neurons arises after pubertal development. PLoS One 8, e58698. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/23505551. Crill, W.E., 1996. Persistent sodium current in mammalian central neurons. Annu. Rev. Physiol. 58, 349e362. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/8815799. Crunelli, V., To´th, T.I., Cope, D.W., Blethyn, K., Hughes, S.W., 2005. The ‘window’ T-type calcium current in brain dynamics of different behavioural states. J. Physiol. 562, 121e129. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/15498803. Cueni, L., Canepari, M., Luja´n, R., Emmenegger, Y., Watanabe, M., Bond, C.T., Franken, P., Adelman, J.P., Lu¨thi, A., 2008. T-type Ca2þ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites. Nat. Neurosci. 11, 683e692. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18488023. Cui, J., Cox, D.H., Aldrich, R.W., 1997. Intrinsic voltage dependence and Ca2þ regulation of mslo large conductance Ca-activated Kþ channels. J. Gen. Physiol. 109, 647e673. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/9154910. Cui, R.J., Li, X., Appleyard, S.M., 2011. Ghrelin inhibits visceral afferent activation of catecholamine neurons in the solitary tract nucleus. J. Neurosci. 31, 3484e3492. Available at: http://www.ncbi.nlm. nih.gov/pubmed/21368060. Cvetkovic-Lopes, V., Eggermann, E., Uschakov, A., Grivel, J., Bayer, L., Jones, B.E., Serafin, M., Mu¨hlethaler, M., 2010. Rat hypocretin/ orexin neurons are maintained in a depolarized state by TRPC channels. PLoS One 5, e15673. Available at: http://www.ncbi. nlm.nih.gov/pubmed/21179559. Dayanithi, G., Forostyak, O., Forostyak, S., Kayano, T., Ueta, Y., Verkhratsky, A., 2018. Vasopressin and oxytocin in sensory neurones: expression, exocytotic release and regulation by lactation. Sci. Rep. 8, 13084. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/30166555. Decher, N., Bundis, F., Vajna, R., Steinmeyer, K., 2003. KCNE2 modulates current amplitudes and activation kinetics of HCN4: influence of KCNE family members on HCN4 currents. Pflu¨gers Arch e Eur J

REFERENCES

Physiol 446, 633e640. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/12856183. Dhar, M., Wayman, G.A., Zhu, M., Lambert, T.J., Davare, M.A., Appleyard, S.M., 2014. Leptin-induced spine formation requires TrpC channels and the CaM kinase cascade in the hippocampus. J. Neurosci. 34, 10022e10033. Available at: http://www.ncbi.nlm. nih.gov/pubmed/25057204. Dhillon, H., Zigman, J.M., Ye, C., Lee, C.E., McGovern, R.A., Tang, V., Kenny, C.D., Christiansen, L.M., White, R.D., Edelstein, E.A., Coppari, R., Balthasar, N., Cowley, M.A., Chua, S., Elmquist, J.K., Lowell, B.B., 2006. Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal bodyweight homeostasis. Neuron 49, 191e203. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/16423694. DiFrancesco, D., 1993. Pacemaker mechanisms in cardiac tissue. Annu. Rev. Physiol. 55, 455e472. Available at: http://www.ncbi.nlm.nih. gov/pubmed/7682045. Dinstein, I., Heeger, D.J., Lorenzi, L., Minshew, N.J., Malach, R., Behrmann, M., 2012. Unreliable evoked responses in autism. Neuron 75, 981e991. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/22998867. Diogenes, A., Patwardhan, A.M., Jeske, N.A., Ruparel, N.B., Goffin, V., Akopian, A.N., Hargreaves, K.M., 2006. Prolactin modulates TRPV1 in female rat trigeminal sensory neurons. J. Neurosci. 26, 8126e8136. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 16885226. Dolphin, A.C., 2013. The a2d subunits of voltage-gated calcium channels. Biochim. Biophys. Acta Biomembr. 1828, 1541e1549. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23196350. Dolphin, A.C., 2016. Voltage-gated calcium channels and their auxiliary subunits: physiology and pathophysiology and pharmacology. J. Physiol. 594, 5369e5390. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/27273705. Dolphin, A.C., Wyatt, C.N., Richards, J., Beattie, R.E., Craig, P., Lee, J.H., Cribbs, L.L., Volsen, S.G., Perez-Reyes, E., 1999. The effect of alpha2-delta and other accessory subunits on expression and properties of the calcium channel alpha1G. J. Physiol. 519 (Pt 1), 35e45. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 10432337. Donato, J., Silva, R.J., Sita, L.V., Lee, S., Lee, C., Lacchini, S., Bittencourt, J.C., Franci, C.R., Canteras, N.S., Elias, C.F., 2009. The ventral premammillary nucleus links fasting-induced changes in leptin levels and coordinated luteinizing hormone secretion. J. Neurosci. 29, 5240e5250. Available at: http://www.ncbi.nlm. nih.gov/pubmed/19386920. Dose, F., Zanon, P., Coslovich, T., Taccola, G., 2014. Nanomolar oxytocin synergizes with weak electrical afferent stimulation to activate the locomotor CPG of the rat spinal cord in vitro. PLoS One 9, e92967. Available at: https://dx.plos.org/10.1371/journal. pone.0092967. Dubois-Dauphin, M., Raggenbass, M., Widmer, H., Tribollet, E., Dreifuss, J.J., 1992. Morphological and electrophysiological evidence for postsynaptic localization of functional oxytocin receptors in the rat dorsal motor nucleus of the vagus nerve. Brain Res. 575, 124e131. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 1324083. Edwards, A., Abizaid, A., 2017. Clarifying the ghrelin system’s ability to regulate feeding behaviours despite enigmatic spatial separation of the GHSR and its endogenous ligand. Int. J. Mol. Sci. 18, 859. Available at: http://www.mdpi.com/1422-0067/18/4/859. Eliava, M., et al., 2016. A new population of parvocellular oxytocin neurons controlling magnocellular neuron activity and inflammatory pain processing. Neuron 89, 1291e1304. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/26948889. Elmquist, J.K., Bjørbaek, C., Ahima, R.S., Flier, J.S., Saper, C.B., 1998. Distributions of leptin receptor mRNA isoforms in the rat brain.

271

J. Comp. Neurol. 395, 535e547. Available at: http://www.ncbi. nlm.nih.gov/pubmed/9619505. Emerick, M.C., Stein, R., Kunze, R., McNulty, M.M., Regan, M.R., Hanck, D.A., Agnew, W.S., 2006. Profiling the array of Cav3.1 variants from the human T-type calcium channel gene CACNA1G: alternative structures, developmental expression, and biophysical variations. Protiens Struct. Funct. Bioinform 64, 320e342. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16671074. Espinosa, F., Torres-Vega, M.A., Marks, G.A., Joho, R.H., 2008. Ablation of Kv3.1 and Kv3.3 potassium channels disrupts thalamocortical oscillations in vitro and in vivo. J. Neurosci. 28, 5570e5581. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18495891. Faber, E.S.L., Sah, P., 2003. Calcium-activated potassium channels: multiple contributions to neuronal function. Neuroscience 9, 181e194. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15065814. Fadool, D.A., Tucker, K., Pedarzani, P., 2011. Mitral cells of the olfactory bulb perform metabolic sensing and are disrupted by obesity at the level of the Kv1.3 ion channel. PLoS One 6, e24921. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21966386. Freeman, M.E., Kanyicska, B., Lerant, A., Nagy, G., 2000. Prolactin: structure, function, and regulation of secretion. Physiol. Rev. 80, 1523e1631. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 11015620. Fry, M., Ferguson, A.V., 2009. Ghrelin modulates electrical activity of area postrema neurons. Am. J. Physiol. Integr. Comp. Physiol. 296, R485eR492. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/19118100. Gao, Y., Yao, T., Deng, Z., Sohn, J.-W., Sun, J., Huang, Y., Kong, X., Yu, K., Wang, R., Chen, H., Guo, H., Yan, J., Cunningham, K.A., Chang, Y., Liu, T., Williams, K.W., 2017. TrpC5 mediates acute leptin and serotonin effects via pomc neurons. Cell Rep. 18, 583e592. Available at: http://www.ncbi.nlm.nih.gov/pubmed/28099839. Gavello, D., Carbone, E., Carabelli, V., 2016. Leptin-mediated ion channel regulation: PI3K pathways, physiological role, and therapeutic potential. Channels 10, 282e296. Available at: http://www.ncbi. nlm.nih.gov/pubmed/27018500. Gibson, H.E., Edwards, J.G., Page, R.S., Van Hook, M.J., Kauer, J.A., 2008. TRPV1 channels mediate long-term depression at synapses on hippocampal interneurons. Neuron 57, 746e759. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18341994. Gilg, S., Lutz, T.A., 2006. The orexigenic effect of peripheral ghrelin differs between rats of different age and with different baseline food intake, and it may in part be mediated by the area postrema. Physiol. Behav. 87, 353e359. Available at: http://www.ncbi.nlm.nih. gov/pubmed/16356516. Gimpl, G., Fahrenholz, F., 2001. The oxytocin receptor system: structure, function, and regulation. Physiol. Rev. 81, 629e683. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11274341. Glowatzki, E., Fakler, G., Bra¨ndle, U., Rexhausen, U., Zenner, H. -p., Ruppersberg, J.P., Fakler, B., 1995. Subunit-dependent assembly of inward-rectifier Kþ channels. Proc. R. Soc. Lond. Ser. B Biol. Sci. 261, 251e261. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/7568278. Goforth, P.B., Leinninger, G.M., Patterson, C.M., Satin, L.S., Myers, M.G., 2014. Leptin acts via lateral hypothalamic area neurotensin neurons to inhibit orexin neurons by multiple GABAindependent mechanisms. J. Neurosci. 34, 11405e11415. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25143620. Gogolla, N., LeBlanc, J.J., Quast, K.B., Su¨dhof, T.C., Fagiolini, M., Hensch, T.K., 2009. Common circuit defect of excitatoryinhibitory balance in mouse models of autism. J. Neurodev. Disord. 1, 172e181. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 20664807. Goldin, A.L., Snutch, T., Lu¨bbert, H., Dowsett, A., Marshall, J., Auld, V., Downey, W., Fritz, L.C., Lester, H.A., Dunn, R., 1986. Messenger RNA coding for only the alpha subunit of the rat brain Na channel

272

11. HORMONES AND THE REGULATION OF NEURONAL VOLTAGE-SENSING ION CHANNELS

is sufficient for expression of functional channels in Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 83, 7503e7507. Available at: http://www.ncbi.nlm.nih.gov/pubmed/2429308. Goldman, D.E., 1943. Potential, impedance, and rectification in membranes. J. Gen. Physiol. 27, 37e60. Available at: http://www. ncbi.nlm.nih.gov/pubmed/19873371. Goldstein, S.A., Price, L.A., Rosenthal, D.N., Pausch, M.H., 1996. ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 93, 13256e13261. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8917578. Gong, L., Gao, F., Li, J., Li, J., Yu, X., Ma, X., Zheng, W., Cui, S., Liu, K., Zhang, M., Kunze, W., Liu, C.Y., 2015. Oxytocin-induced membrane hyperpolarization in pain-sensitive dorsal root ganglia neurons mediated by Ca2þ/nNOS/NO/KATP pathway. Neuroscience 289, 417e428. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 25617653. Gonon, F., 1997. Prolonged and extrasynaptic excitatory action of dopamine mediated by D1 receptors in the rat striatum in vivo. J. Neurosci. 17, 5972e5978. Available at: http://www.ncbi.nlm. nih.gov/pubmed/9221793. Gonza´lez, J.A., Horjales-Araujo, E., Fugger, L., Broberger, C., Burdakov, D., 2009. Stimulation of orexin/hypocretin neurones by thyrotropin-releasing hormone. J. Physiol. 587, 1179e1186. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi? artid¼2674990&tool¼pmcentrez&rendertype¼abstract. Grabauskas, G., Wu, X., Lu, Y., Heldsinger, A., Song, I., Zhou, S.-Y., Owyang, C., 2015. K ATP channels in the nodose ganglia mediate the orexigenic actions of ghrelin. J. Physiol. 593, 3973e3989. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26174421. Grattan, D.R., Kokay, I.C., 2008. Prolactin: a pleiotropic neuroendocrine hormone. J. Neuroendocrinol. 20, 752e763. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/18601698. Gravati, M., Busnelli, M., Bulgheroni, E., Reversi, A., Spaiardi, P., Parenti, M., Toselli, M., Chini, B., 2010. Dual modulation of inward rectifier potassium currents in olfactory neuronal cells by promiscuous G protein coupling of the oxytocin receptor. J. Neurochem. 114. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20557424. Greene, D.L., Hoshi, N., 2017. Modulation of Kv7 channels and excitability in the brain. Cell. Mol. Life Sci. 74, 495e508. Available at: http://www.ncbi.nlm.nih.gov/pubmed/27645822. Guastella, A.J., Hickie, I.B., 2016. Oxytocin treatment, circuitry, and autism: a critical review of the literature placing oxytocin into the autism context. Biol. Psychiatry 79, 234e242. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/26257243. Guatteo, E., Chung, K.K.H., Bowala, T.K., Bernardi, G., Mercuri, N.B., Lipski, J., 2005. Temperature sensitivity of dopaminergic neurons of the substantia nigra pars compacta: involvement of transient receptor potential channels. J. Neurophysiol. 94, 3069e3080. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16014800. Gulledge, A.T., Kampa, B.M., Stuart, G.J., 2005. Synaptic integration in dendritic trees. J. Neurobiol. 64, 75e90. Available at: http://www. ncbi.nlm.nih.gov/pubmed/15884003. Gutman, G.A., et al., 2003. International union of pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels. Pharmacol. Rev. 55, 583e586. Available at: http://www.ncbi.nlm. nih.gov/pubmed/14657415. Halliwell, J.V., Adams, P.R., 1982. Voltage-clamp analysis of muscarinic excitation in hippocampal neurons. Brain Res. 250, 71e92. Available at: http://www.ncbi.nlm.nih.gov/pubmed/6128061. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflu¨gers Archiv. 391, 85e100. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/6270629.

Han, R.T., Kim, H.-B., Kim, Y.-B., Choi, K., Park, G.Y., Lee, P.R., Lee, J., Kim, H young, Park, C.-K., Kang, Y., Oh, S.B., Na, H.S., 2018. Oxytocin produces thermal analgesia via vasopressin-1a receptor by modulating TRPV1 and potassium conductance in the dorsal root ganglion neurons. Korean J. Physiol. Pharmacol. 22, 173. Available at: http://www.ncbi.nlm.nih.gov/pubmed/29520170. Hanson, J.E., Smith, Y., Jaeger, D., 2004. Sodium channels and dendritic spike initiation at excitatory synapses in globus pallidus neurons. J. Neurosci. 24, 329e340. Available at: http://www.ncbi.nlm.nih. gov/pubmed/14724231. Hansson, C., Alvarez-Crespo, M., Taube, M., Skibicka, K.P., Schmidt, L., Karlsson-Lindahl, L., Egecioglu, E., Nissbrandt, H., Dickson, S.L., 2014. Influence of ghrelin on the central serotonergic signaling system in mice. Neuropharmacology 79, 498e505. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24373901. Hara, J., Gerashchenko, D., Wisor, J.P., Sakurai, T., Xie, X., Kilduff, T.S., 2009. Thyrotropin-releasing hormone increases behavioral arousal through modulation of hypocretin/orexin neurons. J. Neurosci. 29, 3705e3714. Available at: http://www.jneurosci.org/cgi/doi/ 10.1523/JNEUROSCI.0431-09.2009. Harden, S.W., Frazier, C.J., 2016. Oxytocin depolarizes fast-spiking hilar interneurons and induces GABA release onto mossy cells of the rat dentate gyrus. Hippocampus 26, 1124e1139. Available at: http://www.ncbi.nlm.nih.gov/pubmed/27068005. Hashiguchi, H., Sheng, Z., Routh, V., Gerzanich, V., Simard, J.M., Bryan, J., 2017. Direct versus indirect actions of ghrelin on hypothalamic NPY neurons. PLoS One 12, e0184261. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/28877214. Havrankova, J., Roth, J., Brownstein, M., 1978. Insulin receptors are widely distributed in the central nervous system of the rat. Nature 272, 827e829. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/205798. Hekerman, P., Zeidler, J., Bamberg-Lemper, S., Knobelspies, H., Lavens, D., Tavernier, J., Joost, H.-G., Becker, W., 2005. Pleiotropy of leptin receptor signalling is defined by distinct roles of the intracellular tyrosines. FEBS J. 272, 109e119. Available at: http://www. ncbi.nlm.nih.gov/pubmed/15634336. Heldsinger, A., Grabauskas, G., Song, I., Owyang, C., 2011. Synergistic interaction between leptin and cholecystokinin in the rat nodose ganglia is mediated by PI3K and STAT3 signaling pathways. J. Biol. Chem. 286, 11707e11715. Available at: http://www.ncbi. nlm.nih.gov/pubmed/21270124. Hell, J.W., Westenbroek, R.E., Warner, C., Ahlijanian, M.K., Prystay, W., Gilbert, M.M., Snutch, T.P., Catterall, W.A., 1993. Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits. J. Cell Biol. 123, 949e962. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 8227151. Herna´ndez-Ochoa, E.O., Schneider, M.F., 2018. Voltage sensing mechanism in skeletal muscle excitation-contraction coupling: coming of age or midlife crisis? Skelet. Muscle 8, 22. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/30025545. Hestrin, S., Galarreta, M., 1998. Frequency-dependent synaptic depression and the balance of excitation and inhibition in the neocortex. Nat. Neurosci. 1, 587e594. Available at: http://www.ncbi.nlm. nih.gov/pubmed/10196566. Hill, J.W., Williams, K.W., Ye, C., Luo, J., Balthasar, N., Coppari, R., Cowley, M.A., Cantley, L.C., Lowell, B.B., Elmquist, J.K., 2008. Acute effects of leptin require PI3K signaling in hypothalamic proopiomelanocortin neurons in mice. J. Clin. Investig. 118, 1796e1805. Available at: http://www.jci.org/articles/view/32964. Hindmarch, C.C.T., Ferguson, A.V., 2016. Physiological roles for the subfornical organ: a dynamic transcriptome shaped by autonomic state. J. Physiol. 594, 1581e1589. Available at: http://www.ncbi. nlm.nih.gov/pubmed/26227400.

REFERENCES

Hirschberg, B., Maylie, J., Adelman, J.P., Marrion, N,V., 1998. Gating of recombinant small-conductance Ca-activated Kþ channels by calcium. J Gen Physiol 111 (4), 565e581. Hiyama, T.Y., Watanabe, E., Ono, K., Inenaga, K., Tamkun, M.M., Yoshida, S., Noda, M., 2002. Nax channel involved in CNS sodium-level sensing. Nat. Neurosci. 5, 511e512. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11992118. Hodgkin, A.L., Huxley, A.F., 1946. Potassium leakage from an active nerve fibre. Nature 158, 376. Available at: http://www.ncbi.nlm. nih.gov/pubmed/21065150. Hodgkin, A.L., Huxley, A.F., 1952. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117, 500e544. Available at: http://www.ncbi. nlm.nih.gov/pubmed/12991237. Hodgkin, A.L., Katz, B., 1949. The effect of sodium ions on the electrical activity of giant axon of the squid. J. Physiol. 108, 37e77. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18128147. Hodgkin, A.L., Huxley, A.F., Katz, B., 1952. Measurement of currentvoltage relations in the membrane of the giant axon of Loligo. J. Physiol. 116, 424e448. Available at: http://www.ncbi.nlm.nih. gov/pubmed/14946712. Holst, B., Cygankiewicz, A., Jensen, T.H., Ankersen, M., Schwartz, T.W., 2003. High constitutive signaling of the ghrelin receptor d identification of a potent inverse agonist. Mol. Endocrinol. 17, 2201e2210. Available at: https://academic.oup.com/mend/ article-lookup/doi/10.1210/me.2003-0069. Hou, L., Zhou, X., Chen, Y., Qiu, D., Zhu, L., Wang, J., 2012. Thyrotropin-releasing hormone causes a tonic excitatory postsynaptic current and inhibits the phasic inspiratory inhibitory inputs in inspiratory-inhibited airway vagal preganglionic neurons. Neuroscience 202, 184e191. Available at: http://www.ncbi.nlm. nih.gov/pubmed/22198018. Huang, Y., He, Z., Gao, Y., Lieu, L., Yao, T., Sun, J., Liu, T., Javadi, C., Box, M., Afrin, S., Guo, H., Williams, K.W., 2018. Phosphoinositide 3-kinase is integral for the acute activity of leptin and insulin in male arcuate NPY/AgRP neurons. J. Endocr. Soc. 2, 518e532. Available at: http://www.ncbi.nlm.nih.gov/pubmed/29850651. Hull, J.M., Isom, L.L., 2018. Voltage-gated sodium channel b subunits: the power outside the pore in brain development and disease. Neuropharmacology 132, 43e57. Available at: http://www.ncbi. nlm.nih.gov/pubmed/28927993. Hung, L.W., Neuner, S., Polepalli, J.S., Beier, K.T., Wright, M., Walsh, J.J., Lewis, E.M., Luo, L., Deisseroth, K., Do¨len, G., Malenka, R.C., 2017. Gating of social reward by oxytocin in the ventral tegmental area. Science 357, 1406e1411. Available at: http://www.sciencemag.org/lookup/doi/10.1126/science. aan4994. Ilango, A., Kesner, A.J., Keller, K.L., Stuber, G.D., Bonci, A., Ikemoto, S., 2014. Similar roles of substantia nigra and ventral tegmental dopamine neurons in reward and aversion. J. Neurosci. 34, 817e822. Available at: http://www.jneurosci.org/lookup/doi/10.1523/ JNEUROSCI.1703-13.2014. Inanobe, A., Fujita, A., Ito, M., Tomoike, H., Inageda, K., Kurachi, Y., 2002. Inward rectifier K þ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses. Am. J. Physiol. Cell Physiol. 282, C1396eC1403. Available at: http://www.ncbi.nlm.nih. gov/pubmed/11997254. Ishibashi, H., Nakahata, Y., Eto, K., Nabekura, J., 2009. Excitation of locus coeruleus noradrenergic neurons by thyrotropin-releasing hormone. J. Physiol. 587, 5709e5722. Available at: http://www. ncbi.nlm.nih.gov/pubmed/19840999. Ishii, T.M., Silvia, C., Hirschberg, B., Bond, C.T., Adelman, J.P., Maylie, J., 1997. A human intermediate conductance calciumactivated potassium channel. Proc. Natl. Acad. Sci. U.S.A. 94, 11651e11656. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/9326665.

273

Isom, L.L., De Jongh, K.S., Catterall, W.A., 1994. Auxiliary subunits of voltage-gated ion channels. Neuron 12, 1183e1194. Available at: http://www.ncbi.nlm.nih.gov/pubmed/7516685. Jego, S., Glasgow, S.D., Herrera, C.G., Ekstrand, M., Reed, S.J., Boyce, R., Friedman, J., Burdakov, D., Adamantidis, A.R., 2013. Optogenetic identification of a rapid eye movement sleep modulatory circuit in the hypothalamus. Nat. Neurosci. 16, 1637e1643. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/24056699. Jensen, B.S., Strobaek, D., Christophersen, P., Jorgensen, T.D., Hansen, C., Silahtaroglu, A., Olesen, S.P., Ahring, P.K., 1998. Characterization of the cloned human intermediate-conductance Ca2þactivated Kþ channel. Am. J. Physiol. 275, C848eC856. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9730970. Jia, Y., Nguyen, T., Desai, M., Ross, M.G., 2008. Programmed alterations in hypothalamic neuronal orexigenic responses to ghrelin following gestational nutrient restriction. Reprod. Sci. 15, 702e709. Available at: http://journals.sagepub.com/doi/10.1177/1933719108316982. Jiang, C.-Y., Fujita, T., Kumamoto, E., 2014. Synaptic modulation and inward current produced by oxytocin in substantia gelatinosa neurons of adult rat spinal cord slices. J. Neurophysiol. 111, 991e1007. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24335211. Johnston, J., Forsythe, I.D., Kopp-Scheinpflug, C., 2010. Going native: voltage-gated potassium channels controlling neuronal excitability. J. Physiol. 588, 3187e3200. Available at: http://www. ncbi.nlm.nih.gov/pubmed/20519310. Kaczmarek, L.K., Zhang, Y., 2017. Kv3 channels: enablers of rapid firing, neurotransmitter release, and neuronal endurance. Physiol. Rev. 97, 1431e1468. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/28904001. Katz, B., 1949. Les constantes electriques de La membrane du muscle. Arch. Sci. Physiol. 3, 285e300. Kaupp, U.B., Seifert, R., 2001. Molecular diversity of pacemaker ion channels. Annu. Rev. Physiol. 63, 235e257. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/11181956. Kim, J., Nakajima, K., Oomura, Y., Wayner, M.J., Sasaki, K., 2009. Electrophysiological effects of ghrelin on pedunculopontine tegmental neurons in rats: an in vitro study. Peptides 30, 745e757. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19118591. Kimura, T., Makino, Y., Saji, F., Takemura, M., Inoue, T., Kikuchi, T., Kubota, Y., Azuma, C., Nobunaga, T., Tokugawa, Y., 1994. Molecular characterization of a cloned human oxytocin receptor. Eur. J. Endocrinol. 131, 385e390. Available at: http://www.ncbi.nlm.nih. gov/pubmed/7921228. King, B., Rizwan, A.P., Asmara, H., Heath, N.C., Engbers, J.D.T., Dykstra, S., Bartoletti, T.M., Hameed, S., Zamponi, G.W., Turner, R.W., 2015. IKCa channels are a critical determinant of the slow AHP in CA1 pyramidal neurons. Cell Rep. 11, 175e182. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25865881. Ko¨hler, M., Hirschberg, B., Bond, C.T., Kinzie, J.M., Marrion, N.V., Maylie, J., Adelman, J.P., 1996. Small-conductance, calciumactivated potassium channels from mammalian brain. Science 273, 1709e1714. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/8781233. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402, 656e660. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/10604470. Kokay, I.C., Bull, P.M., Davis, R.L., Ludwig, M., Grattan, D.R., 2006. Expression of the long form of the prolactin receptor in magnocellular oxytocin neurons is associated with specific prolactin regulation of oxytocin neurons. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R1216eR1225. Available at: http://www.physiology. org/doi/10.1152/ajpregu.00730.2005. Kolaj, M., Renaud, L.P., 1998. Vasopressin-induced currents in rat neonatal spinal lateral horn neurons are G-protein mediated and

274

11. HORMONES AND THE REGULATION OF NEURONAL VOLTAGE-SENSING ION CHANNELS

involve two conductances. J. Neurophysiol. 80, 1900e1910. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9772248. Kolaj, M., Zhang, L., Renaud, L.P., 2016. L-type calcium channels and MAP kinase contribute to thyrotropin-releasing hormone-induced depolarization in thalamic paraventricular nucleus neurons. Am. J. Physiol. Integr. Comp. Physiol. 310, R1120eR1127. Available at: http://www.ncbi.nlm.nih.gov/pubmed/27009047. Konadhode, R.R., Pelluru, D., Blanco-Centurion, C., Zayachkivsky, A., Liu, M., Uhde, T., Glen, W.B., van den Pol, A.N., Mulholland, P.J., Shiromani, P.J., 2013. Optogenetic stimulation of MCH neurons increases sleep. J. Neurosci. 33, 10257e10263. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/23785141. Ko¨nner, A.C., Janoschek, R., Plum, L., Jordan, S.D., Rother, E., Ma, X., Xu, C., Enriori, P., Hampel, B., Barsh, G.S., Kahn, C.R., Cowley, M.A., Ashcroft, F.M., Bru¨ning, J.C., 2007. Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production. Cell Metabol. 5, 438e449. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17550779. Koyama, S., Brodie, M.S., Appel, S.B., 2007. Ethanol inhibition of Mcurrent and ethanol-induced direct excitation of ventral tegmental area dopamine neurons. J. Neurophysiol. 97, 1977e1985. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16956995. Koyrakh, L., Luja´n, R., Colo´n, J., Karschin, C., Kurachi, Y., Karschin, A., Wickman, K., 2005. Molecular and cellular diversity of neuronal Gprotein-gated potassium channels. J. Neurosci. 25, 11468e11478. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16339040. Kraushaar, U., Jonas, P., 2000. Efficacy and stability of quantal GABA release at a hippocampal interneuron-principal neuron synapse. J. Neurosci. 20, 5594e5607. Available at: http://www.ncbi.nlm. nih.gov/pubmed/10908596. Kubo, Y., Baldwin, T.J., Nung Jan, Y., Jan, L.Y., 1993. Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362, 127e133. Available at: http://www.ncbi. nlm.nih.gov/pubmed/7680768. Kuczewski, N., Fourcaud-Trocme´, N., Savigner, A., Thevenet, M., Aime´, P., Garcia, S., Duchamp-Viret, P., Palouzier-Paulignan, B., 2014. Insulin modulates network activity in olfactory bulb slices: impact on odour processing. J. Physiol. 592, 2751e2769. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24710056. Kukkonen, J.P., 2011. A me´nage a` trois made in heaven: G-proteincoupled receptors, lipids and TRP channels. Cell Calcium 50, 9e26. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21616532. Kupfermann, I., 1979. Modulatory actions of neurotransmitters. Annu. Rev. Neurosci. 2, 447e465. Available at: http://www.ncbi.nlm.nih. gov/pubmed/44174. Lakhi, S., Snow, W., Fry, M., 2013. Insulin modulates the electrical activity of subfornical organ neurons. Neuroreport 24, 329e334. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23481267. Lancel, M., Kro¨mer, S., Neumann, I.D., 2003. Intracerebral oxytocin modulates sleep-wake behaviour in male rats. Regul. Pept. 114, 145e152. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12832103. Lau, D., Vega-Saenz de Miera, E.C., Contreras, D., Ozaita, A., Harvey, M., Chow, A., Noebels, J.L., Paylor, R., Morgan, J.I., Leonard, C.S., Rudy, B., 2000. Impaired fast-spiking, suppressed cortical inhibition, and increased susceptibility to seizures in mice lacking Kv3.2 Kþ channel proteins. J. Neurosci. 20, 9071e9085. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/11124984. Le Tissier, P.R., Hodson, D.J., Martin, A.O., Romano`, N., Mollard, P., 2015. Plasticity of the prolactin (PRL) axis: mechanisms underlying regulation of output in female mice. In: Advances in Experimental Medicine and Biology, pp. 139e162. Available at: http://www.ncbi. nlm.nih.gov/pubmed/25472537. Lee, C.-H., MacKinnon, R., 2017. Structures of the human HCN1 hyperpolarization-activated channel. Cell 168, 111e120 e11 Available at: http://www.ncbi.nlm.nih.gov/pubmed/28086084.

Lee, S.J., Verma, S., Simonds, S.E., Kirigiti, M.A., Kievit, P., Lindsley, S.R., Loche, A., Smith, M.S., Cowley, M.A., Grove, K.L., 2013. Leptin stimulates neuropeptide Y and cocaine amphetamine-regulated transcript coexpressing neuronal activity in the dorsomedial hypothalamus in diet-induced obese mice. J. Neurosci. 33, 15306e15317. Available at: http://www.jneurosci. org/cgi/doi/10.1523/JNEUROSCI.0837-13.2013. Lee, S., Lee, J., Kang, G.M., Kim, M.-S., 2018. Leptin directly regulate intrinsic neuronal excitability in hypothalamic POMC neurons but not in AgRP neurons in food restricted mice. Neurosci. Lett. 681, 105e109. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/29857041. Leinninger, G.M., Opland, D.M., Jo, Y.-H., Faouzi, M., Christensen, L., Cappellucci, L.A., Rhodes, C.J., Gnegy, M.E., Becker, J.B., Pothos, E.N., Seasholtz, A.F., Thompson, R.C., Myers, M.G., 2011. Leptin action via neurotensin neurons controls orexin, the mesolimbic dopamine system and energy balance. Cell Metabol. 14, 313e323. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21907138. Leng, G., 2018. The endocrinology of the brain. Endocr. Connect. 7, R275eR285. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/30352398. Leng, G., Ludwig, M., 2008. Neurotransmitters and peptides: whispered secrets and public announcements. J. Physiol. 586, 5625e5632. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18845614. Leroy, F., Park, J., Asok, A., Brann, D.H., Meira, T., Boyle, L.M., Buss, E.W., Kandel, E.R., Siegelbaum, S.A., 2018. A circuit from hippocampal CA2 to lateral septum disinhibits social aggression. Nature 564, 213e218. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/30518859. Li, Y., Jia, Y.-C., Cui, K., Li, N., Zheng, Z.-Y., Wang, Y., Yuan, X., 2005. Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor. Nature 434, 894e898. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15758952. Lin, S.-S., Tzeng, B.-H., Lee, K.-R., Smith, R.J.H., Campbell, K.P., Chen, C.-C., 2014. Cav3.2 T-type calcium channel is required for the NFAT-dependent Sox9 expression in tracheal cartilage. Proc. Natl. Acad. Sci. U.S.A. 111, E1990eE1998. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/24778262. Lipscombe, D., Andrade, A., Allen, S.E., 2013. Alternative splicing: functional diversity among voltage-gated calcium channels and behavioral consequences. Biochim. Biophys. Acta Biomembr. 1828, 1522e1529. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/23022282. Lipski, J., Park, T.I.H., Li, D., Lee, S.C.W., Trevarton, A.J., Chung, K.K.H., Freestone, P.S., Bai, J.-Z., 2006. Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices. Brain Res. 1077, 187e199. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16483552. Liu, X., Tribollet, E., Ogier, R., Barberis, C., Raggenbass, M., 2003. Presence of functional vasopressin receptors in spinal ventral horn neurons of young rats: a morphological and electrophysiological study. Eur. J. Neurosci. 17, 1833e1846. Available at: http://www.ncbi. nlm.nih.gov/pubmed/12752783. Lopatin, A.N., Makhina, E.N., Nichols, C.G., 1994. Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification. Nature 372, 366e369. Available at: http://www.ncbi. nlm.nih.gov/pubmed/7969496. Lo´pez Soto, E.J., Agosti, F., Cabral, A., Mustafa, E.R., Damonte, V.M., Gandini, M.A., Rodrı´guez, S., Castrogiovanni, D., Felix, R., Perello´, M., Raingo, J., 2015. Constitutive and ghrelin-dependent GHSR1a activation impairs CaV 2.1 and CaV 2.2 currents in hypothalamic neurons. J. Gen. Physiol. 146, 205e219. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26283199. Lu, Y., Dang, S., Wang, X., Zhang, J., Zhang, L., Su, Q., Zhang, H., Lin, T., Zhang, X., Zhang, Y., Sun, H., Zhu, Z., Li, H., 2018. NO involvement in the inhibition of ghrelin on voltage-dependent

REFERENCES

potassium currents in rat hippocampal cells. Brain Res. 1678, 40e46. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 28987626. Ludwig, M., Sabatier, N., Bull, P.M., Landgraf, R., Dayanithi, G., Leng, G., 2002. Intracellular calcium stores regulate activitydependent neuropeptide release from dendrites. Nature 418, 85e89. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 12097911. Ludwig, M., Apps, D., Menzies, J., Patel, J.C., Rice, M.E., 2016. Dendritic release of neurotransmitters. In: Comprehensive Physiology. John Wiley & Sons, Inc, Hoboken, NJ, USA, pp. 235e252. Available at: http://www.ncbi.nlm.nih.gov/pubmed/28135005. Luja´n, R., 2010. Organisation of potassium channels on the neuronal surface. J. Chem. Neuroanat. 40, 1e20. Available at: http://www. ncbi.nlm.nih.gov/pubmed/20338235. Lyons, D.J., Broberger, C., 2014. Tidal waves: network mechanisms in the neuroendocrine control of prolactin release. Front. Neuroendocrinol. 35, 420e438. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/24561279. Lyons, D.J., Horjales-Araujo, E., Broberger, C., 2010. Synchronized network oscillations in rat tuberoinfundibular dopamine neurons: switch to tonic discharge by thyrotropin-releasing hormone. Neuron 65, 217e229. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/20152128. Lyons, D.J., Hellysaz, A., Broberger, C., 2012. Prolactin regulates tuberoinfundibular dopamine neuron discharge pattern: novel feedback control mechanisms in the lactotrophic axis. J. Neurosci. 32, 8074e8083. Available at: http://www.jneurosci.org/cgi/doi/10. 1523/JNEUROSCI.0129-12.2012. Ma, F.Y., Anderson, G.M., Gunn, T.D., Goffin, V., Grattan, D.R., Bunn, S.J., 2005. Prolactin specifically activates signal transducer and activator of transcription 5b in neuroendocrine dopaminergic neurons. Endocrinology 146, 5112e5119. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/16123156. Maccaferri, G., Mangoni, M., Lazzari, A., DiFrancesco, D., 1993. Properties of the hyperpolarization-activated current in rat hippocampal CA1 pyramidal cells. J. Neurophysiol. 69, 2129e2136. Available at: http://www.physiology.org/doi/10.1152/jn.1993.69.6.2129. Macica, C.M., von Hehn, C.A.A., Wang, L.-Y., Ho, C.-S., Yokoyama, S., Joho, R.H., Kaczmarek, L.K., 2003. Modulation of the kv3.1b potassium channel isoform adjusts the fidelity of the firing pattern of auditory neurons. J. Neurosci. 23, 1133e1141. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12598601. MacKenzie, G., Franks, N.P., Brickley, S.G., 2015. Two-pore domain potassium channels enable action potential generation in the absence of voltage-gated potassium channels. Pflu¨gers Arch e Eur J Physiol 467, 989e999. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/25482670. Maejima, Y., Sakuma, K., Santoso, P., Gantulga, D., Katsurada, K., Ueta, Y., Hiraoka, Y., Nishimori, K., Tanaka, S., Shimomura, K., Yada, T., 2014. Oxytocinergic circuit from paraventricular and supraoptic nuclei to arcuate POMC neurons in hypothalamus. FEBS Lett. 588, 4404e4412. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/25448678. Magee, J.C., 1999. Dendritic Ih normalizes temporal summation in hippocampal CA1 neurons. Nat. Neurosci. 2, 508e514. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10448214. Maimaiti, S., Frazier, H.N., Anderson, K.L., Ghoweri, A.O., Brewer, L.D., Porter, N.M., Thibault, O., 2017. Novel calciumrelated targets of insulin in hippocampal neurons. Neuroscience 364, 130e142. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/28939258. Mancini, M., Soldovieri, M.V., Gessner, G., Wissuwa, B., Barrese, V., Boscia, F., Secondo, A., Miceli, F., Franco, C., Ambrosino, P., Canzoniero, L.M.T., Bauer, M., Hoshi, T., Heinemann, S.H., Taglialatela, M., 2014. Critical role of large-conductance calcium-

275

and voltage-activated potassium channels in leptin-induced neuroprotection of N-methyl-d-aspartate-exposed cortical neurons. Pharmacol. Res. 87, 80e86. Available at: http://www.ncbi.nlm. nih.gov/pubmed/24973659. Mangoni, M.E., Traboulsie, A., Leoni, A.-L., Couette, B., Marger, L., Le Quang, K., Kupfer, E., Cohen-Solal, A., Vilar, J., Shin, H.-S., Escande, D., Charpentier, F., Nargeot, J., Lory, P., 2006. Bradycardia and slowing of the atrioventricular conduction in mice lacking CaV3.1/alpha1G T-type calcium channels. Circ. Res. 98, 1422e1430. Available at: https://www.ahajournals.org/doi/10. 1161/01.RES.0000225862.14314.49. Manning, M., Misicka, A., Olma, A., Bankowski, K., Stoev, S., Chini, B., Durroux, T., Mouillac, B., Corbani, M., Guillon, G., 2012. Oxytocin and vasopressin agonists and antagonists as research tools and potential therapeutics. J. Neuroendocrinol. 24, 609e628. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22375852. Mantegazza, M., Catterall, W.A., 2012. Voltage-Gated Naþ Channels: Structure, Function, and Pathophysiology. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/22787615. Marder, E., 2012. Neuromodulation of neuronal circuits: back to the future. Neuron 76, 1e11. Available at: http://www.ncbi.nlm.nih. gov/pubmed/23040802. Martı´nez Damonte, V., Rodrı´guez, S.S., Raingo, J., 2018. Growth hormone secretagogue receptor constitutive activity impairs voltagegated calcium channel-dependent inhibitory neurotransmission in hippocampal neurons. J. Physiol. 596, 5415e5428. Available at: http://www.ncbi.nlm.nih.gov/pubmed/30199095. Marty, A., 1981. Ca-dependent K channels with large unitary conductance in chromaffin cell membranes. Nature 291, 497e500. Available at: http://www.ncbi.nlm.nih.gov/pubmed/6262657. McCormick, D a, Bal, T., 1997. Sleep and arousal: thalamocortical mechanisms. Annu. Rev. Neurosci. 20, 185e215. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9056712. McGregor, G., Harvey, J., 2018. Regulation of hippocampal synaptic function by the metabolic hormone, leptin: implications for health and neurodegenerative disease. Front. Cell. Neurosci. 12, 340. Available at: http://www.ncbi.nlm.nih.gov/pubmed/30386207. McKenna, K.E., Nadelhaft, I., 1986. The organization of the pudendal nerve in the male and female rat. J. Comp. Neurol. 248, 532e549. Available at: http://www.ncbi.nlm.nih.gov/pubmed/3722467. McKinley, M.J., McAllen, R.M., Davern, P., Giles, M.E., Penschow, J., Sunn, N., Uschakov, A., Oldfield, B.J., 2003. The sensory circumventricular organs of the mammalian brain. Adv. Anat. Embryol. Cell Biol. 172 (IIIeXII), 1e122 back cover Available at: http://www. ncbi.nlm.nih.gov/pubmed/12901335. McManus, O.B., Magleby, K.L., 1991. Accounting for the Ca(2þ)dependent kinetics of single large-conductance Ca(2þ)-activated Kþ channels in rat skeletal muscle. J. Physiol. 443, 739e777. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1822543. McNay, E.C., Recknagel, A.K., 2011. Brain insulin signaling: a key component of cognitive processes and a potential basis for cognitive impairment in type 2 diabetes. Neurobiol. Learn. Mem. 96, 432e442. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 21907815. Meera, P., Wallner, M., Jiang, Z., Toro, L., 1996. A calcium switch for the functional coupling between alpha (hslo) and beta subunits (KV,cabeta) of maxi K channels. FEBS Lett. 385, 127e128. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8641456. Mercer, J.G., Hoggard, N., Williams, L.M., Lawrence, C.B., Hannah, L.T., Trayhurn, P., 1996. Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett. 387, 113e116. Available at: http://www.ncbi.nlm.nih. gov/pubmed/8674530. Merchenthaler, I., 1993. Induction of enkephalin in tuberoinfundibular dopaminergic neurons during lactation. Endocrinology 133,

276

11. HORMONES AND THE REGULATION OF NEURONAL VOLTAGE-SENSING ION CHANNELS

2645e2651. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 7694844. Miller, C., 2000. Ion channels: doing hard chemistry with hard ions. Curr. Opin. Chem. Biol. 4, 148e151. Available at: http://www. ncbi.nlm.nih.gov/pubmed/10742182. Molineux, M.L., McRory, J.E., McKay, B.E., Hamid, J., Mehaffey, W.H., Rehak, R., Snutch, T.P., Zamponi, G.W., Turner, R.W., 2006. Specific T-type calcium channel isoforms are associated with distinct burst phenotypes in deep cerebellar nuclear neurons. Proc. Natl. Acad. Sci. U.S.A. 103, 5555e5560. Available at: http://www.ncbi.nlm. nih.gov/pubmed/16567615. Mrejeru, A., Wei, A., Ramirez, J.M., 2011. Calcium-activated nonselective cation currents are involved in generation of tonic and bursting activity in dopamine neurons of the substantia nigra pars compacta. J. Physiol. 589, 2497e2514. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/21486760. Mu¨hlethaler, M., Charpak, S., Dreifuss, J.J., 1984. Contrasting effects of neurohypophysial peptides on pyramidal and non-pyramidal neurones in the rat hippocampus. Brain Res. 308, 97e107. Available at: http://www.ncbi.nlm.nih.gov/pubmed/6478205. Munzberg, H., Jobst, E.E., Bates, S.H., Jones, J., Villanueva, E., Leshan, R., Bjornholm, M., Elmquist, J., Sleeman, M., Cowley, M.A., Myers, M.G., 2007. Appropriate inhibition of orexigenic hypothalamic arcuate nucleus neurons independently of leptin receptor/STAT3 signaling. J. Neurosci. 27, 69e74. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17202473. Muroya, S., Funahashi, H., Yamanaka, A., Kohno, D., Uramura, K., Nambu, T., Shibahara, M., Kuramochi, M., Takigawa, M., Yanagisawa, M., Sakurai, T., Shioda, S., Yada, T., 2004. Orexins (hypocretins) directly interact with neuropeptide Y, POMC and glucose-responsive neurons to regulate Ca2þ signaling in a reciprocal manner to leptin: orexigenic neuronal pathways in the mediobasal hypothalamus. Eur. J. Neurosci. 19, 1524e1534. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15066149. Myers, M.G., Mu¨nzberg, H., Leinninger, G.M., Leshan, R.L., 2009. The geometry of leptin action in the brain: more complicated than a simple ARC. Cell Metabol. 9, 117e123. Available at: http://www.ncbi. nlm.nih.gov/pubmed/19187770. Nanou, E., Catterall, W.A., 2018. Calcium channels, synaptic plasticity, and neuropsychiatric disease. Neuron 98, 466e481. Available at: http://www.ncbi.nlm.nih.gov/pubmed/29723500. Nernst, W., 1889. Die elektromotorische Wirksamkeit der Ionen. Z. Phys. Chem. 4, 129e181. Nersesyan, Y., Demirkhanyan, L., Cabezas-Bratesco, D., Oakes, V., Kusuda, R., Dawson, T., Sun, X., Cao, C., Cohen, A.M., Chelluboina, B., Veeravalli, K.K., Zimmermann, K., Domene, C., Brauchi, S., Zakharian, E., 2017. Oxytocin modulates nociception as an agonist of pain-sensing TRPV1. Cell Rep. 21, 1681e1691. Available at: https://linkinghub.elsevier.com/retrieve/pii/ S2211124717315115. Nichols, C.G., 2016. Adenosine triphosphate-sensitive potassium currents in heart disease and cardioprotection. Card. Electrophysiol. Clin. 8, 323e335. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/27261824. Nilius, B., Szallasi, A., 2014. Transient receptor potential channels as drug targets: from the science of basic research to the art of medicine. Pharmacol. Rev. 66, 676e814. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/24951385. Nishida, M., MacKinnon, R., 2002. Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 A resolution. Cell 111, 957e965. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/12507423. Nishida, M., Cadene, M., Chait, B.T., MacKinnon, R., 2007. Crystal structure of a Kir3.1-prokaryotic Kir channel chimera. EMBO J. 26, 4005e4015. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/17703190.

Nishino, S., Arrigoni, J., Shelton, J., Kanbayashi, T., Dement, W.C., Mignot, E., 1997. Effects of thyrotropin-releasing hormone and its analogs on daytime sleepiness and cataplexy in canine narcolepsy. J. Neurosci. 17, 6401e6408. Available at: http://www. ncbi.nlm.nih.gov/pubmed/9236248. Niswender, K.D., Morrison, C.D., Clegg, D.J., Olson, R., Baskin, D.G., Myers, M.G., Seeley, R.J., Schwartz, M.W., 2003. Insulin activation of phosphatidylinositol 3-kinase in the hypothalamic arcuate nucleus: a key mediator of insulin-induced anorexia. Diabetes 52, 227e231. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 12540590. Noda, M., Shimizu, S., Tanabe, T., Takai, T., Kayano, T., Ikeda, T., Takahashi, H., Nakayama, H., Kanaoka, Y., Minamino, N., 1984. Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312, 121e127. Available at: http://www.ncbi.nlm.nih.gov/pubmed/6209577. Nolan, M.F., Dudman, J.T., Dodson, P.D., Santoro, B., 2007. HCN1 channels control resting and active integrative properties of stellate cells from layer II of the entorhinal cortex. J Neurosci 27 (46), 12440e12451. Notomi, T., Shigemoto, R., 2004. Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain. J. Comp. Neurol. 471, 241e276. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 14991560. Nusbaum, M.P., Blitz, D.M., Marder, E., 2017. Functional consequences of neuropeptide and small-molecule co-transmission. Nat. Rev. Neurosci. 18, 389e403. Available at: http://www.ncbi.nlm.nih. gov/pubmed/28592905. Obici, S., Feng, Z., Karkanias, G., Baskin, D.G., Rossetti, L., 2002. Decreasing hypothalamic insulin receptors causes hyperphagia and insulin resistance in rats. Nat. Neurosci. 5, 566e572. Available at: http://www.nature.com/doifinder/10.1038/nn861. Ogaya, M., Kim, J., Sasaki, K., 2011. Ghrelin postsynaptically depolarizes dorsal raphe neurons in rats in vitro. Peptides 32, 1606e1616. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21763741. Ogier, R., Tribollet, E., Suarez, P., Raggenbass, M., 2006. Identified motoneurons involved in sexual and eliminative functions in the rat are powerfully excited by vasopressin and tachykinins. J. Neurosci. 26, 10717e10726. Available at: http://www.ncbi.nlm. nih.gov/pubmed/17050711. Ohta, H., Kato, Y., Matsushita, N., Shimatsu, A., Kabayama, Y., Imura, H., 1985. Central inhibitory action of TRH on prolactin secretion in the rat. Proc. Soc. Exp. Biol. Med. 179, 9e12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/3921974. Oshima, N., Onimaru, H., Matsubara, H., Uchida, T., Watanabe, A., Imakiire, T., Nishida, Y., Kumagai, H., 2017. Direct effects of glucose, insulin, GLP-1, and GIP on bulbospinal neurons in the rostral ventrolateral medulla in neonatal wistar rats. Neuroscience 344, 74e88. Available at: https://linkinghub.elsevier.com/ retrieve/pii/S0306452216307394. Osterstock, G., Escobar, P., Mitutsova, V., Gouty-Colomer, L.-A., Fontanaud, P., Molino, F., Fehrentz, J.-A., Carmignac, D., Martinez, J., Guerineau, N.C., Robinson, I.C.A.F., Mollard, P., Me´ry, P.-F., 2010. Ghrelin stimulation of growth hormonereleasing hormone neurons is direct in the arcuate nucleus. PLoS One 5, e9159. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/20161791. Owen, S.F., Tuncdemir, S.N., Bader, P.L., Tirko, N.N., Fishell, G., Tsien, R.W., 2013. Oxytocin enhances hippocampal spike transmission by modulating fast-spiking interneurons. Nature 500, 458e462. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23913275. Oz, M., Kolaj, M., Renaud, L.P., 2001. Electrophysiological evidence for vasopressin V(1) receptors on neonatal motoneurons, premotor and other ventral horn neurons. J. Neurophysiol. 86, 1202e1210. Available at: http://www.physiology.org/doi/10.1152/jn.2001.86.3. 1202.

REFERENCES

O’Malley, H.A., Isom, L.L., 2015. Sodium channel b subunits: emerging targets in channelopathies. Annu. Rev. Physiol. 77, 481e504. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25668026. O’Malley, D., Shanley, L.J., Harvey, J., 2003. Insulin inhibits rat hippocampal neurones via activation of ATP-sensitive Kþ and large conductance Ca2þ-activated Kþ channels. Neuropharmacology 44, 855e863. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 12726817. Pagani, J.H., Zhao, M., Cui, Z., Williams Avram, S.K., Caruana, D.A., Dudek, S.M., Young, W.S., 2015. Role of the vasopressin 1b receptor in rodent aggressive behavior and synaptic plasticity in hippocampal area CA2. Mol. Psychiatry 20, 490e499. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/24863146. Palouzier-Paulignan, B., Dubois-Dauphin, M., Tribollet, E., Dreifuss, J.J., Raggenbass, M., 1994. Action of vasopressin on hypoglossal motoneurones of the rat: presynaptic and postsynaptic effects. Brain Res. 650, 117e126. Available at: http://www.ncbi. nlm.nih.gov/pubmed/7953662. Pape, H.-C., McCormick, D.A., 1989. Noradrenaline and serotonin selectively modulate thalamic burst firing by enhancing a hyperpolarization-activated cation current. Nature 340, 715e718. Available at: http://www.ncbi.nlm.nih.gov/pubmed/2475782. Parmentier, R., Kolbaev, S., Klyuch, B.P., Vandael, D., Lin, J.-S., Selbach, O., Haas, H.L., Sergeeva, O.A., 2009. Excitation of histaminergic tuberomamillary neurons by thyrotropin-releasing hormone. J. Neurosci. 29, 4471e4483. Available at: http://www. ncbi.nlm.nih.gov/pubmed/19357273. Patil, M.J., Ruparel, S.B., Henry, M.A., Akopian, A.N., 2013. Prolactin regulates TRPV1, TRPA1, and TRPM8 in sensory neurons in a sex-dependent manner: contribution of prolactin receptor to inflammatory pain. Am. J. Physiol. Endocrinol. Metab. 305, E1154eE1164. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/24022869. Patil, M.J., Henry, M.A., Akopian, A.N., 2014. Prolactin receptor in regulation of neuronal excitability and channels. Channels 8, 193e202. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 24758841. Payandeh, J., Scheuer, T., Zheng, N., Catterall, W.A., 2011. The crystal structure of a voltage-gated sodium channel. Nature 475, 353e358. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 21743477. Pearson, S.A., Mouihate, A., Pittman, Q.J., Whelan, P.J., 2003. Peptidergic activation of locomotor pattern generators in the neonatal spinal cord. J. Neurosci. 23, 10154e10163. Available at: http://www.ncbi. nlm.nih.gov/pubmed/14602832. Pei, H., Sutton, A.K., Burnett, K.H., Fuller, P.M., Olson, D.P., 2014. AVP neurons in the paraventricular nucleus of the hypothalamus regulate feeding. Mol. Metab. 3, 209e215. Available at: http://www. ncbi.nlm.nih.gov/pubmed/24634830. Petersen, P.S., Woldbye, D.P.D., Madsen, A.N., Egerod, K.L., Jin, C., Lang, M., Rasmussen, M., Beck-Sickinger, A.G., Holst, B., 2009. In vivo characterization of high basal signaling from the ghrelin receptor. Endocrinology 150, 4920e4930. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/19819980. Phillips, M.S., Liu, Q., Hammond, H.A., Dugan, V., Hey, P.J., Caskey, C.T., Hess, J.F., 1996. Leptin receptor missense mutation in the fatty Zucker rat. Nat. Genet. 13, 18e19. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8673096. Pinto, S., Roseberry, A.G., Liu, H., Diano, S., Shanabrough, M., Cai, X., Friedman, J.M., Horvath, T.L., 2004. Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science 304, 110e115. Available at: http://www.sciencemag.org/cgi/doi/10.1126/science.1089459. Plum, L., et al., 2006. Enhanced PIP3 signaling in POMC neurons causes KATP channel activation and leads to diet-sensitive obesity. J. Clin. Investig. 116, 1886e1901. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/16794735.

277

Pocai, A., Lam, T.K.T., Gutierrez-Juarez, R., Obici, S., Schwartz, G.J., Bryan, J., Aguilar-Bryan, L., Rossetti, L., 2005. Hypothalamic KATP channels control hepatic glucose production. Nature 434, 1026e1031. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 15846348. Posner, B.I., van Houten, M., Patel, B., Walsh, R.J., 1983. Characterization of lactogen binding sites in choroid plexus. Exp. Brain Res. 49, 300e306. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 6832262. Pru¨ss, H., Derst, C., Lommel, R., Veh, R.W., 2005. Differential distribution of individual subunits of strongly inwardly rectifying potassium channels (Kir2 family) in rat brain. Mol. Brain Res. 139, 63e79. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 15936845. Qiu, J., Fang, Y., Ronnekleiv, O.K., Kelly, M.J., 2010. Leptin excites proopiomelanocortin neurons via activation of TRPC channels. J. Neurosci. 30, 1560e1565. Available at: http://www.ncbi.nlm. nih.gov/pubmed/20107083. Qiu, J., Fang, Y., Bosch, M.A., Rønnekleiv, O.K., Kelly, M.J., 2011. Guinea pig kisspeptin neurons are depolarized by leptin via activation of TRPC channels. Endocrinology 152, 1503e1514. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21285322. Qiu, F., Qiu, C.-Y., Cai, H., Liu, T.-T., Qu, Z.-W., Yang, Z., Li, J.-D., Zhou, Q.-Y., Hu, W.-P., 2014a. Oxytocin inhibits the activity of acid-sensing ion channels through the vasopressin, V 1A receptor in primary sensory neurons. Br. J. Pharmacol. 171, 3065e3076. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24641084. Qiu, J., Zhang, C., Borgquist, A., Nestor, C.C., Smith, A.W., Bosch, M.A., Ku, S., Wagner, E.J., Rønnekleiv, O.K., Kelly, M.J., 2014b. Insulin excites anorexigenic proopiomelanocortin neurons via activation of canonical transient receptor potential channels. Cell Metabol. 19, 682e693. Available at: http://www.ncbi.nlm. nih.gov/pubmed/24703699. Qiu, J., Wagner, E.J., Rønnekleiv, O.K., Kelly, M.J., 2018. Insulin and leptin excite anorexigenic pro-opiomelanocortin neurones via activation of TRPC5 channels. J. Neuroendocrinol. 30, e12501. Available at: http://www.ncbi.nlm.nih.gov/pubmed/28675783. Qu, J., Kryukova, Y., Potapova, I.A., Doronin, S.V., Larsen, M., Krishnamurthy, G., Cohen, I.S., Robinson, R.B., 2004. MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes. J. Biol. Chem. 279, 43497e43502. Available at: http://www.ncbi. nlm.nih.gov/pubmed/15292247. Raggenbass, M., 2008. Overview of cellular electrophysiological actions of vasopressin. Eur. J. Pharmacol. 583, 243e254. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18280467. Raggenbass, M., Dreifuss, J.J., 1992. Mechanism of action of oxytocin in rat vagal neurones: induction of a sustained sodium-dependent current. J. Physiol. 457, 131e142. Available at: http://www.ncbi. nlm.nih.gov/pubmed/1297830. Raggenbass, M., Goumaz, M., Sermasi, E., Tribollet, E., Dreifuss, J.J., 1991. Vasopressin generates a persistent voltage-dependent sodium current in a mammalian motoneuron. J. Neurosci. 11, 1609e1616. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1646297. Ramanathan, G., Cilz, N.I., Kurada, L., Hu, B., Wang, X., Lei, S., 2012. Vasopressin facilitates GABAergic transmission in rat hippocampus via activation of V1A receptors. Neuropharmacology 63, 1218e1226. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22884625. Reuter, H., 1983. Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 301, 569e574. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/6131381. Reymond-Marron, I., Tribollet, E., Raggenbass, M., 2006. The vasopressin-induced excitation of hypoglossal and facial motoneurons in young rats is mediated by V1a but not V1b receptors, and is independent of intracellular calcium signalling. Eur. J. Neurosci. 24, 1565e1574. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 17004920.

278

11. HORMONES AND THE REGULATION OF NEURONAL VOLTAGE-SENSING ION CHANNELS

Risold, P.Y., Swanson, L.W., 1997. Connections of the rat lateral septal complex. Brain Res. Brain Res. Rev. 24, 115e195. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9385454. Romano, N., Yip, S.H., Hodson, D.J., Guillou, A., Parnaudeau, S., Kirk, S., Tronche, F., Bonnefont, X., Le Tissier, P., Bunn, S.J., Grattan, D.R., Mollard, P., Martin, A.O., 2013. Plasticity of hypothalamic dopamine neurons during lactation results in dissociation of electrical activity and release. J. Neurosci. 33, 4424e4433. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23467359. Rousselot, P., Papadopoulos, G., Merighi, A., Poulain, D.A., Theodosis, D.T., 1990. Oxytocinergic innervation of the rat spinal cord. An electron microscopic study. Brain Res. 529, 178e184. Available at: http://www.ncbi.nlm.nih.gov/pubmed/2282492. Ruangkittisakul, A., Schwarzacher, S.W., Secchia, L., Poon, B.Y., Ma, Y., Funk, G.D., Ballanyi, K., 2006. High sensitivity to neuromodulatoractivated signaling pathways at physiological [Kþ] of confocally imaged respiratory center neurons in on-line-calibrated newborn rat brainstem slices. J. Neurosci. 26, 11870e11880. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17108160. Sabatier, N., Leng, G., Menzies, J., 2013. Oxytocin, feeding, and satiety. Front. Endocrinol. 4, 35. Available at: http://www.ncbi.nlm.nih. gov/pubmed/23518828. Sabatini, B.L., Regehr, W.G., 1997. Control of neurotransmitter release by presynaptic waveform at the granule cell to Purkinje cell synapse. J. Neurosci. 17, 3425e3435. Available at: http://www. ncbi.nlm.nih.gov/pubmed/9133368. Sakmann, B., Neher, E., 1984. Patch clamp techniques for studying ionic channels in excitable membranes. Annu. Rev. Physiol. 46, 455e472. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 6143532. Sausbier, U., Sausbier, M., Sailer, C.A., Arntz, C., Knaus, H.-G., Neuhuber, W., Ruth, P., 2006. Ca2þ-activated Kþ channels of the BK-type in the mouse brain. Histochem. Cell Biol. 125, 725e741. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16362320. Savigner, A., Duchamp-Viret, P., Grosmaitre, X., Chaput, M., Garcia, S., Ma, M., Palouzier-Paulignan, B., 2009. Modulation of spontaneous and odorant-evoked activity of rat olfactory sensory neurons by two anorectic peptides, insulin and leptin. J. Neurophysiol. 101, 2898e2906. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 19297511. Schaeffer, M., Langlet, F., Lafont, C., Molino, F., Hodson, D.J., Roux, T., Lamarque, L., Verdie´, P., Bourrier, E., Dehouck, B., Bane`res, J.-L., Martinez, J., Me´ry, P.-F., Marie, J., Trinquet, E., Fehrentz, J.-A., Pre´vot, V., Mollard, P., 2013. Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons. Proc. Natl. Acad. Sci. U.S.A. 110, 1512e1517. Available at: http://www.ncbi.nlm. nih.gov/pubmed/23297228. Scharfman, H.E., 2016. The enigmatic mossy cell of the dentate gyrus. Nat. Rev. Neurosci. 17, 562e575. Available at: http://www.ncbi. nlm.nih.gov/pubmed/27466143. Schewe, M., Nematian-Ardestani, E., Sun, H., Musinszki, M., Cordeiro, S., Bucci, G., de Groot, B.L., Tucker, S.J., Rapedius, M., Baukrowitz, T., 2016. A non-canonical voltage-sensing mechanism controls gating in K2P Kþ channels. Cell 164, 937e949. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26919430. Schreiber, M., Salkoff, L., 1997. A novel calcium-sensing domain in the BK channel. Biophys. J. 73, 1355e1363. Available at: http://www. ncbi.nlm.nih.gov/pubmed/9284303. Schreiber, M., Yuan, A., Salkoff, L., 1999. Transplantable sites confer calcium sensitivity to BK channels. Nat. Neurosci. 2, 416e421. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10321244. Sermasi, E., Coote, J.H., 1994. Oxytocin acts at V1 receptors to excite sympathetic preganglionic neurones in neonate rat spinal cord in vitro. Brain Res. 647, 323e332. Available at: http://www.ncbi. nlm.nih.gov/pubmed/7922507.

Shanley, L.J., Irving, A.J., Rae, M.G., Ashford, M.L.J., Harvey, J., 2002a. Leptin inhibits rat hippocampal neurons via activation of large conductance calcium-activated Kþ channels. Nat. Neurosci. 5, 299e300. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 11889470. Shanley, L.J., O’Malley, D., Irving, A.J., Ashford, M.L., Harvey, J., 2002b. Leptin inhibits epileptiform-like activity in rat hippocampal neurones via PI 3-kinase-driven activation of BK channels. J. Physiol. 545, 933e944. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 12482897. Sharif Naeini, R., Witty, M.-F., Se´gue´la, P., Bourque, C.W., 2006. An N-terminal variant of Trpv1 channel is required for osmosensory transduction. Nat. Neurosci. 9, 93e98. Available at: http://www. nature.com/articles/nn1614. Sharma, A.N., Elased, K.M., Garrett, T.L., Lucot, J.B., 2010. Neurobehavioral deficits in db/db diabetic mice. Physiol. Behav. 101, 381e388. Available at: http://linkinghub.elsevier.com/retrieve/pii/ S003193841000257X. Shen, W., Flajolet, M., Greengard, P., Surmeier, D.J., 2008. Dichotomous dopaminergic control of striatal synaptic plasticity. Science 321, 848e851. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 18687967. Shi, L., Bian, X., Qu, Z., Ma, Z., Zhou, Y., Wang, K., Jiang, H., Xie, J., 2013. Peptide hormone ghrelin enhances neuronal excitability by inhibition of Kv7/KCNQ channels. Nat. Commun. 4, 1435. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23385580. Shipston, M.J., 2001. Alternative splicing of potassium channels: a dynamic switch of cellular excitability. Trends Cell Biol. 11, 353e358. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 11514177. Shmukler, B.E., Bond, C.T., Wilhelm, S., Bruening-Wright, A., Maylie, J., Adelman, J.P., Alper, S.L., 2001. Structure and complex transcription pattern of the mouse SK1 K(Ca) channel gene, KCNN1. Biochem. Biophys. Acta 1518, 36e46. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11267657. Singer, J.H., Talley, E.M., Bayliss, D.A., Berger, A.J., 1998. Development of glycinergic synaptic transmission to rat brain stem motoneurons. J. Neurophysiol. 80, 2608e2620. Available at: http://www. physiology.org/doi/10.1152/jn.1998.80.5.2608. Sinnegger-Brauns, M.J., Huber, I.G., Koschak, A., Wild, C., Obermair, G.J., Einzinger, U., Hoda, J.-C., Sartori, S.B., Striessnig, J., 2009. Expression and 1,4-dihydropyridine-binding properties of brain L-type calcium channel isoforms. Mol. Pharmacol. 75, 407e414. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/19029287. Sirzen-Zelenskaya, A., Gonzalez-Iglesias, A.E., Boutet de Monvel, J., Bertram, R., Freeman, M.E., Gerber, U., Egli, M., 2011. Prolactin induces a hyperpolarising current in rat paraventricular oxytocinergic neurones. J. Neuroendocrinol. 23, 883e893. Available at: http://doi.wiley.com/10.1111/j.1365-2826.2011.02207.x. Sohn, J.-W., Oh, Y., Kim, K.W., Lee, S., Williams, K.W., Elmquist, J.K., 2016. Leptin and insulin engage specific PI3K subunits in hypothalamic SF1 neurons. Mol. Metab. 5, 669e679. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/27656404. Song, K., Wang, H., Kamm, G.B., Pohle, J., Reis, F. d. C., Heppenstall, P., Wende, H., Siemens, J., 2016a. The TRPM2 channel is a hypothalamic heat sensor that limits fever and can drive hypothermia. Science 353, 1393e1398. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/27562954. Song, Z., Borland, J.M., Larkin, T.E., O’Malley, M., Albers, H.E., 2016b. Activation of oxytocin receptors, but not arginine-vasopressin V1a receptors, in the ventral tegmental area of male Syrian hamsters is essential for the reward-like properties of social interactions. Psychoneuroendocrinology 74, 164e172. Available at: https:// linkinghub.elsevier.com/retrieve/pii/S0306453016306357.

REFERENCES

Spanswick, D., Smith, M.A., Groppi, V.E., Logan, S.D., Ashford, M.L.J., 1997. Leptin inhibits hypothalamic neurons by activation of ATPsensitive potassium channels. Nature 390, 521e525. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9394003. Spanswick, D., Smith, M.A., Mirshamsi, S., Routh, V.H., Ashford, M.L.J., 2000. Insulin activates ATP-sensitive Kþ channels in hypothalamic neurons of lean, but not obese rats. Nat. Neurosci. 3, 757e758. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 10903566. Spiegelman, B.M., Flier, J.S., 2001. Obesity and the regulation of energy balance. Cell 104, 531e543. Available at: http://www.ncbi.nlm.nih. gov/pubmed/11239410. Spoljaric, A., Seja, P., Spoljaric, I., Virtanen, M.A., Lindfors, J., Uvarov, P., Summanen, M., Crow, A.K., Hsueh, B., Puskarjov, M., Ruusuvuori, E., Voipio, J., Deisseroth, K., Kaila, K., 2017. Vasopressin excites interneurons to suppress hippocampal network activity across a broad span of brain maturity at birth. Proc. Natl. Acad. Sci. U.S.A. 114, E10819eE10828. Available at: http://www. ncbi.nlm.nih.gov/pubmed/29183979. Stanfield, P.R., Nakajima, S., Nakajima, Y., 2002. Constitutively active and G-protein coupled inward rectifier Kþ channels: Kir2.0 and Kir3.0. Rev. Physiol. Biochem. Pharmacol. 145, 47e179. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12224528. Steriade, M., Datta, S., Pare´, D., Oakson, G., Curro´ Dossi, R.C., 1990. Neuronal activities in brain-stem cholinergic nuclei related to tonic activation processes in thalamocortical systems. J. Neurosci. 10, 2541e2559. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 2388079. Stocker, M., Pedarzani, P., 2000. Differential distribution of three Ca2þactivated Kþ channel subunits, SK1, SK2, and SK3, in the adult rat central nervous system. Mol. Cell. Neurosci. 15, 476e493. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10833304. Stoop, R., 2012. Neuromodulation by oxytocin and vasopressin. Neuron 76, 142e159. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/23040812. Striessnig, J., Pinggera, A., Kaur, G., Bock, G., Tuluc, P., 2014. L-type Ca 2þ channels in heart and brain. Wiley Interdiscip. Rev. Membr. Transp. Signal. 3, 15e38. Available at: http://www.ncbi.nlm.nih. gov/pubmed/24683526. Stuart, G., Spruston, N., 1998. Determinants of voltage attenuation in neocortical pyramidal neuron dendrites. J. Neurosci. 18, 3501e3510. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 9570781. Suda, Y., Kuzumaki, N., Sone, T., Narita, M., Tanaka, K., Hamada, Y., Iwasawa, C., Shibasaki, M., Maekawa, A., Matsuo, M., Akamatsu, W., Hattori, N., Okano, H., Narita, M., 2018. Downregulation of ghrelin receptors on dopaminergic neurons in the substantia nigra contributes to Parkinson’s disease-like motor dysfunction. Mol. Brain 11, 6. Available at: http://www.ncbi.nlm. nih.gov/pubmed/29458391. Sun, Y., Lu, X., Gershengorn, M.C., 2003. Thyrotropin-releasing hormone receptors e similarities and differences. J. Mol. Endocrinol. 30, 87e97. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 12683933. Sun, Y., Shi, N., Li, H., Liu, K., Zhang, Y., Chen, W., Sun, X., 2014. Ghrelin suppresses Purkinje neuron P-type Ca2þ channels via growth hormone secretagogue type 1a receptor, the bg subunits of Go-protein, and protein kinase A pathway. Cell. Signal. 26, 2530e2538. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/25049077. Szentirmai, E´., 2012. Central but not systemic administration of ghrelin induces wakefulness in mice. PLoS One 7, e41172. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22815958. Tache´, Y., Adelson, D., Yang, H., 2014. TRH/TRH-R1 receptor signaling in the brain medulla as a pathway of vagally mediated gut responses during the cephalic phase. Curr. Pharm. Des. 20,

279

2725e2730. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 23886382. Taddese, A., Bean, B.P., 2002. Subthreshold sodium current from rapidly inactivating sodium channels drives spontaneous firing of tuberomammillary neurons. Neuron 33, 587e600. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11856532. Takahashi, M., Seagar, M.J., Jones, J.F., Reber, B.F., Catterall, W.A., 1987. Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle. Proc. Natl. Acad. Sci. U.S.A. 84, 5478e5482. Available at: http://www.ncbi.nlm.nih.gov/pubmed/2440051. Takakusaki, K., Saitoh, K., Harada, H., Okumura, T., Sakamoto, T., 2004. Evidence for a role of basal ganglia in the regulation of rapid eye movement sleep by electrical and chemical stimulation for the pedunculopontine tegmental nucleus and the substantia nigra pars reticulata in decerebrate cats. Neuroscience 124, 207e220. Available at: http://www.ncbi.nlm.nih.gov/pubmed/14960352. Takano, S., Kim, J., Ikari, Y., Ogaya, M., Nakajima, K., Oomura, Y., Wayner, M.J., Sasaki, K., 2009. Electrophysiological effects of ghrelin on laterodorsal tegmental neurons in rats: an in vitro study. Peptides 30, 1901e1908. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/19646496. Talley, E.M., Lei, Q., Sirois, J.E., Bayliss, D.A., 2000. TASK-1, a two-pore domain Kþ channel, is modulated by multiple neurotransmitters in motoneurons. Neuron 25, 399e410. Available at: http://www.ncbi. nlm.nih.gov/pubmed/10719894. Talley, E.M., Solorzano, G., Lei, Q., Kim, D., Bayliss, D.A., 2001. CNS distribution of members of the two-pore-domain (KCNK) potassium channel family. J. Neurosci. 21, 7491e7505. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11567039. Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., Numa, S., 1987. Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328, 313e318. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/3037387. Tang, Y., Chen, Z., Tao, H., Li, C., Zhang, X., Tang, A., Liu, Y., 2014. Oxytocin activation of neurons in ventral tegmental area and interfascicular nucleus of mouse midbrain. Neuropharmacology 77, 277e284. Available at: https://linkinghub.elsevier.com/retrieve/ pii/S0028390813004784. Tesauro, M., Schinzari, F., Caramanti, M., Lauro, R., Cardillo, C., 2010. Cardiovascular and metabolic effects of ghrelin. Curr. Diabetes Rev. 6, 228e235. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 20459393. Tomizawa, K., Iga, N., Lu, Y.-F., Moriwaki, A., Matsushita, M., Li, S.-T., Miyamoto, O., Itano, T., Matsui, H., 2003. Oxytocin improves longlasting spatial memory during motherhood through MAP kinase cascade. Nat. Neurosci. 6, 384e390. Available at: http://www. ncbi.nlm.nih.gov/pubmed/12598900. Tong, Q., Ye, C.-P., Jones, J.E., Elmquist, J.K., Lowell, B.B., 2008. Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance. Nat. Neurosci. 11, 998e1000. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid¼266 2585&tool¼pmcentrez&rendertype¼abstract. Townsend, J., Cave, B.J., Norman, M.R., Flynn, A., Uney, J.B., Tortonese, D.J., Wakerley, J.B., 2005. Effects of prolactin on hypothalamic supraoptic neurones: evidence for modulation of STAT5 expression and electrical activity. Neuroendocrinol. Lett. 26, 125e130. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 15855883. Travagli, R.A., Gillis, R.A., Vicini, S., 1992. Effects of thyrotropinreleasing hormone on neurons in rat dorsal motor nucleus of the vagus, in vitro. Am. J. Physiol. 263, G508eG517. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1329553. Tribollet, E., Goumaz, M., Raggenbass, M., Dubois-Dauphin, M., Dreifuss, J.J., 1991. Early appearance and transient expression of vasopressin receptors in the brain of rat fetus and infant. An

280

11. HORMONES AND THE REGULATION OF NEURONAL VOLTAGE-SENSING ION CHANNELS

autoradiographical and electrophysiological study. Brain Res. Dev. Brain Res. 58, 13e24. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/1826642. Tscho¨p, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908e913. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/11057670. Tsunematsu, T., Fu, L.-Y., Yamanaka, A., Ichiki, K., Tanoue, A., Sakurai, T., van den Pol, A.N., 2008. Vasopressin increases locomotion through a V1a receptor in orexin/hypocretin neurons: implications for water homeostasis. J. Neurosci. 28, 228e238. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18171940. Tsunematsu, T., Ueno, T., Tabuchi, S., Inutsuka, A., Tanaka, K.F., Hasuwa, H., Kilduff, T.S., Terao, A., Yamanaka, A., 2014. Optogenetic manipulation of activity and temporally controlled cellspecific ablation reveal a role for MCH neurons in sleep/wake regulation. J. Neurosci. 34, 6896e6909. Available at: http://www. ncbi.nlm.nih.gov/pubmed/24828644. Turner, R.W., Zamponi, G.W., 2014. T-type channels buddy up. Pflu¨gers Arch e Eur J Physiol 466, 661e675. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/24413887. Turner, R.W., Kruskic, M., Teves, M., Scheidl-Yee, T., Hameed, S., Zamponi, G.W., 2015. Neuronal expression of the intermediate conductance calcium-activated potassium channel KCa3.1 in the mammalian central nervous system. Pflu¨gers Arch e Eur J Physiol 467, 311e328. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/24797146. Ulrich, D., 2002. Dendritic resonance in rat neocortical pyramidal cells. J. Neurophysiol. 87, 2753e2759. Available at: http://www.ncbi. nlm.nih.gov/pubmed/12037177. Ulrich, D., Huguenard, J.R., 1997. GABAa-receptor-mediated rebound burst firing and burst shunting in thalamus. J. Neurophysiol. 78, 1748e1751. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 9310462. van den Pol, A.N., 2012. Neuropeptide transmission in brain circuits. Neuron 76, 98e115. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/23040809. van den Pol, A.N., Acuna-Goycolea, C., Clark, K.R., Ghosh, P.K., 2004. Physiological properties of hypothalamic MCH neurons identified with selective expression of reporter gene after recombinant virus infection. Neuron 42, 635e652. Available at: http://www.ncbi. nlm.nih.gov/pubmed/15157424. van den Top, M., Lee, K., Whyment, A.D., Blanks, A.M., Spanswick, D., 2004. Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus. Nat. Neurosci. 7, 493e494. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15097991. van den Top, M., Lyons, D.J., Lee, K., Coderre, E., Renaud, L.P., Spanswick, D., 2007. Pharmacological and molecular characterization of ATP-sensitive K(þ) conductances in CART and NPY/ AgRP expressing neurons of the hypothalamic arcuate nucleus. Neuroscience 144, 815e824. Available at: http://linkinghub. elsevier.com/retrieve/pii/S0306452206013091. Vennekens, R., Menigoz, A., Nilius, B., 2012. TRPs in the brain. In: Reviews of Physiology, Biochemistry and Pharmacology, vol. 163. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 27e64. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23184016. Verkhratsky, A., Krishtal, O.A., Petersen, O.H., 2006. From Galvani to patch clamp: the development of electrophysiology. Pflu¨gers Arch e Eur J Physiol 453, 233e247. Available at: http://www.ncbi.nlm. nih.gov/pubmed/17072639. Villanueva, E.C., Myers, M.G., 2008. Leptin receptor signaling and the regulation of mammalian physiology. Int. J. Obes. 32, S8eS12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19136996. Voets, T., Owsianik, G., Janssens, A., Talavera, K., Nilius, B., 2007. TRPM8 voltage sensor mutants reveal a mechanism for integrating thermal and chemical stimuli. Nat. Chem. Biol. 3, 174e182. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17293875.

Wagenaar, D.A., Hamilton, M.S., Huang, T., Kristan, W.B., French, K.A., 2010. A hormone-activated central pattern generator for courtship. Curr. Biol. 20, 487e495. Available at: https:// linkinghub.elsevier.com/retrieve/pii/S0960982210002113. Wainwright, A., Rutter, A.R., Seabrook, G.R., Reilly, K., Oliver, K.R., 2004. Discrete expression of TRPV2 within the hypothalamoneurohypophysial system: implications for regulatory activity within the hypothalamic-pituitary-adrenal axis. J. Comp. Neurol. 474, 24e42. Available at: http://doi.wiley.com/10.1002/cne.20100. Walsh, R.J., Slaby, F.J., Posner, B.I., 1987. A receptor-mediated mechanism for the transport of prolactin from blood to cerebrospinal fluid. Endocrinology 120, 1846e1850. Available at: https:// academic.oup.com/endo/article-lookup/doi/10.1210/endo-1205-1846. Wang, J., Irnaten, M., Venkatesan, P., Evans, C., Mendelowitz, D., 2002. Arginine vasopressin enhances GABAergic inhibition of cardiac parasympathetic neurons in the nucleus ambiguus. Neuroscience 111, 699e705. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/12031355. Wang, J.-H., Wang, F., Yang, M.-J., Yu, D.-F., Wu, W.-N., Liu, J., Ma, L.Q., Cai, F., Chen, J.-G., 2008. Leptin regulated calcium channels of neuropeptide Y and proopiomelanocortin neurons by activation of different signal pathways. Neuroscience 156, 89e98. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18588949. Wang, J., Ou, S.-W., Wang, Y.-J., 2017. Distribution and function of voltage-gated sodium channels in the nervous system. Channels 11, 534e554. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/28922053. Weiss, N., Zamponi, G.W., 2013. Control of low-threshold exocytosis by T-type calcium channels. Biochim. Biophys. Acta Biomembr. 1828, 1579e1586. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 22885170. Wheeler, D.B., Randall, A., Tsien, R.W., 1994. Roles of N-type and Qtype Ca2þ channels in supporting hippocampal synaptic transmission. Science 264, 107e111. Available at: http://www. ncbi.nlm.nih.gov/pubmed/7832825. Wheeler, D.G., Groth, R.D., Ma, H., Barrett, C.F., Owen, S.F., Safa, P., Tsien, R.W., 2012. CaV1 and CaV2 channels engage distinct modes of Ca2þ signaling to control CREB-dependent gene expression. Cell 149, 1112e1124. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/22632974. Wickman, K., Clapham, D.E., 1995. Ion channel regulation by G proteins. Physiol. Rev. 75, 865e885. Available at: http://www. ncbi.nlm.nih.gov/pubmed/7480165. Wicks, N.L., Wong, T., Sun, J., Madden, Z., Young, E.C., 2011. Cytoplasmic cAMP-sensing domain of hyperpolarization-activated cation (HCN) channels uses two structurally distinct mechanisms to regulate voltage gating. Proc. Natl. Acad. Sci. U.S.A. 108, 609e614. Available at: http://www.pnas.org/cgi/doi/10.1073/ pnas.1012750108. Willesen, M.G., Kristensen, P., Rømer, J., 1999. Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat. Neuroendocrinology 70, 306e316. Available at: https://www.karger.com/Article/FullText/54491. Williams, K.W., Smith, B.N., 2006. Rapid inhibition of neural excitability in the nucleus tractus solitarii by leptin: implications for ingestive behaviour. J. Physiol. 573, 395e412. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/16581866. Williams, S.R., Stuart, G.J., 2000. Site independence of EPSP time course is mediated by dendritic I h in neocortical pyramidal neurons. J. Neurophysiol. 83, 3177e3182. Available at: http://www.ncbi. nlm.nih.gov/pubmed/10805715. Williams, K.W., Zsombok, A., Smith, B.N., 2007. Rapid inhibition of neurons in the dorsal motor nucleus of the vagus by leptin. Endocrinology 148, 1868e1881. Available at: http://www.ncbi.nlm.nih. gov/pubmed/17194747.

REFERENCES

Williams, K.W., Margatho, L.O., Lee, C.E., Choi, M., Lee, S., Scott, M.M., Elias, C.F., Elmquist, J.K., 2010. Segregation of acute leptin and insulin effects in distinct populations of arcuate proopiomelanocortin neurons. J. Neurosci. 30, 2472e2479. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20164331. Williams, K.W., Scott, M.M., Elmquist, J.K., 2011a. Modulation of the central melanocortin system by leptin, insulin, and serotonin: Coordinated actions in a dispersed neuronal network. Eur. J. Pharmacol. 660, 2e12. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/21211525. Williams, K.W., Sohn, J.-W., Donato, J., Lee, C.E., Zhao, J.J., Elmquist, J.K., Elias, C.F., 2011b. The acute effects of leptin require PI3K signaling in the hypothalamic ventral premammillary nucleus. J. Neurosci. 31, 13147e13156. Available at: http://www. ncbi.nlm.nih.gov/pubmed/21917798. Wittekind, D.A., Kluge, M., 2015. Ghrelin in psychiatric disorders e a review. Psychoneuroendocrinology 52, 176e194. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25459900. Woo, S.K., Kwon, M.S., Ivanov, A., Gerzanich, V., Simard, J.M., 2013. The sulfonylurea receptor 1 (Sur1)-Transient receptor potential melastatin 4 (Trpm4) channel. J. Biol. Chem. 288, 3655e3667. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23255597. Wrobel, L.J., Reymond-Marron, I., Dupre´, A., Raggenbass, M., 2010. Oxytocin and vasopressin enhance synaptic transmission in the hypoglossal motor nucleus of young rats by acting on distinct receptor types. Neuroscience 165, 723e735. Available at: http://www.ncbi. nlm.nih.gov/pubmed/19896520. Wu, M., Dumalska, I., Morozova, E., van den Pol, A., Alreja, M., 2009. Melanin-concentrating hormone directly inhibits GnRH neurons and blocks kisspeptin activation, linking energy balance to reproduction. Proc. Natl. Acad. Sci. U.S.A. 106, 17217e17222. Available at: http://www.pnas.org/cgi/doi/10.1073/pnas.0908200106. Xia, X.M., Fakler, B., Rivard, A., Wayman, G., Johnson-Pais, T., Keen, J.E., Ishii, T., Hirschberg, B., Bond, C.T., Lutsenko, S., Maylie, J., Adelman, J.P., 1998. Mechanism of calcium gating in smallconductance calcium-activated potassium channels. Nature 395, 503e507. Available at: http://www.nature.com/articles/26758. Xia, X.-M., Zeng, X., Lingle, C.J., 2002. Multiple regulatory sites in large-conductance calcium-activated potassium channels. Nature 418, 880e884. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/12192411. Xiao, L., Priest, M.F., Nasenbeny, J., Lu, T., Kozorovitskiy, Y., 2017. Biased oxytocinergic modulation of midbrain dopamine systems. Neuron 95, 368e384 e5 Available at: http://www.ncbi.nlm.nih. gov/pubmed/28669546. Xu, A.W., Kaelin, C.B., Takeda, K., Akira, S., Schwartz, M.W., Barsh, G.S., 2005. PI3K integrates the action of insulin and leptin on hypothalamic neurons. J. Clin. Investig. 115, 951e958. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15761497. Xu, J., Bartolome, C.L., Low, C.S., Yi, X., Chien, C.-H., Wang, P., Kong, D., 2018. Genetic identification of leptin neural circuits in energy and glucose homeostases. Nature 556, 505e509. Available at: http://www.ncbi.nlm.nih.gov/pubmed/29670283. Yamanaka, A., Beuckmann, C.T., Willie, J.T., Hara, J., Tsujino, N., Mieda, M., Tominaga, M., Yagami, K ichi, Sugiyama, F., Goto, K., Yanagisawa, M., Sakurai, T., 2003. Hypothalamic orexin neurons regulate arousal according to energy balance in mice. Neuron 38, 701e713. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12797956. Yanagihara, K., Irisawa, H., 1980. Inward current activated during hyperpolarization in the rabbit sinoatrial node cell. Pflu¨gers Archiv. 385, 11e19. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 7191093. Yang, M.-J., Wang, F., Wang, J.-H., Wu, W.-N., Hu, Z.-L., Cheng, J., Yu, D.-F., Long, L.-H., Fu, H., Xie, N., Chen, J.-G., 2010. PI3K integrates the effects of insulin and leptin on large-conductance Ca2þ-activated Kþ channels in neuropeptide Y neurons of the hypothalamic arcuate nucleus. Am. J. Physiol. Endocrinol. Metab. 298.

281

E193-201 Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 19671839. Yao, Y., Fu, L.-Y., Zhang, X., van den Pol, A.N., 2012. Vasopressin and oxytocin excite MCH neurons, but not other lateral hypothalamic GABA neurons. Am. J. Physiol. Regul. Integr. Comp. Physiol. 302, R815eR824. Available at: http://www.physiology.org/doi/10. 1152/ajpregu.00452.2011. Yu, F.H., Catterall, W.A., 2004. The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis. Sci. Signal. 2004, re15. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/15467096. Yu, H., Wu, J., Potapova, I., Wymore, R.T., Holmes, B., Zuckerman, J., Pan, Z., Wang, H., Shi, W., Robinson, R.B., El-Maghrabi, M.R., Benjamin, W., Dixon, J., McKinnon, D., Cohen, I.S., Wymore, R., 2001. MinK-related peptide 1: a beta subunit for the HCN ion channel subunit family enhances expression and speeds activation. Circ. Res. 88, E84eE87. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/11420311. Yu, F.H., Yarov-Yarovoy, V., Gutman, G.A., Catterall, W.A., 2005. Overview of molecular relationships in the voltage-gated ion channel superfamily. Pharmacol. Rev. 57, 387e395. Available at: http:// pharmrev.aspetjournals.org/cgi/doi/10.1124/pr.57.4.13. Yu, X., Ye, Z., Houston, C.M., Zecharia, A.Y., Ma, Y., Zhang, Z., Uygun, D.S., Parker, S., Vyssotski, A.L., Yustos, R., Franks, N.P., Brickley, S.G., Wisden, W., 2015. Wakefulness is governed by GABA and histamine cotransmission. Neuron 87, 164e178. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26094607. Zamponi, G.W., 2003. Regulation of presynaptic calcium channels by synaptic proteins. J. Pharmacol. Sci. 92, 79e83. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12832834. Zaninetti, M., Raggenbass, M., 2000. Oxytocin receptor agonists enhance inhibitory synaptic transmission in the rat hippocampus by activating interneurons in stratum pyramidale. Eur. J. Neurosci. 12, 3975e3984. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/11069593. Zhang, X., van den Pol, A.N., 2012. Thyrotropin-releasing hormone (TRH) inhibits melanin-concentrating hormone neurons: implications for TRH-mediated anorexic and arousal actions. J. Neurosci. 32, 3032e3043. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/22378876. Zhang, X., van den Pol, A.N., 2015. Dopamine/tyrosine hydroxylase neurons of the hypothalamic arcuate nucleus release GABA, communicate with dopaminergic and other arcuate neurons, and respond to dynorphin, met-enkephalin, and oxytocin. J. Neurosci. 35, 14966e14982. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/26558770. Zhang, X., van den Pol, A.N., 2016. Hypothalamic arcuate nucleus tyrosine hydroxylase neurons play orexigenic role in energy homeostasis. Nat. Neurosci. 19, 1341e1347. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/27548245. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., Friedman, J.M., 1994. Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425e432. Available at: http://www.ncbi.nlm.nih.gov/pubmed/7984236. Zhang, L., Kolaj, M., Renaud, L.P., 2013. GIRK-like and TRPC-like conductances mediate thyrotropin-releasing hormone-induced increases in excitability in thalamic paraventricular nucleus neurons. Neuropharmacology 72, 106e115. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/23632082. Zhou, Y., Wang, X., Cao, T., Xu, J., Wang, D., Restrepo, D., Li, A., 2017. Insulin modulates neural activity of pyramidal neurons in the anterior piriform cortex. Front. Cell. Neurosci. 11, 378. Available at: http://www.ncbi.nlm.nih.gov/pubmed/29234275. Zigman, J.M., Jones, J.E., Lee, C.E., Saper, C.B., Elmquist, J.K., 2006. Expression of ghrelin receptor mRNA in the rat and the mouse brain. J. Comp. Neurol. 494, 528e548. Available at: http://www. ncbi.nlm.nih.gov/pubmed/16320257.

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12 Hormonal Regulation of Epithelial Sodium Channel (ENaC) and Other Nonneuronal Epithelial Ion Channels Gerald Litwack Formerly Institute for Regenerative Medicine, Texas A&M Health Science Center, Temple, TX, United States

1. THE EPITHELIUM ENaC is located in the membranes of epithelial cells. The epithelium consists of layers of cells that line the outer surfaces of organs and blood vessels as well as the inner surfaces of lumens in several internal organs (e.g., kidney tubule and intestinal tract). Epithelial cells function to protect the skin, filter and secrete substances from glandular cells or kidney cells, and line the digestive tract, the esophagus, alveolar sacs of the lungs, and the stomach. Substances entering or leaving the body pass one or more layers of epithelial cells, A model of the epithelial cell is shown in Fig. 12.1.

2. STRUCTURE OF ENAC CHANNEL The location and function of the ENaC channel is diagrammed in Fig. 12.2. In this model, sodium ion from the lumen (e.g., lumen of the GI tract or the kidney tubule) enters the apical side of the cell facing the lumen, crosses the cell to the basolateral side, and is exported from the cell, powered by a sodium/potassium ATPase (Derfoul et al., 1998; Kolla and Litwack, 2000), to the extracellular space and finally to the bloodstream. The epithelial cell is polarized with the apical side facing the lumen containing cilia, the absorptive surface, and at the other end, the basolateral side, via energy from Naþ/Kþ ATPase, exporting ions to the extracellular space for access to the bloodstream. In Fig. 12.3 is shown a predicted structure or oligomerized human ENaC (hENaC).

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00012-2

The subunits of ENaC thus form a perpendicular central channel through which sodium ions stream from the luminal (alveolar) side of the cell across the cell to the basolateral side, where the sodium ion is expelled through the energy released from the action of a sodium/potassium ATPase. In Fig. 12.4 is shown a schematic illustration of the transmembrane localization of an ENaC subunit. As shown in this figure for the a subunit, each subunit contains two transmembrane domains. Mature ENaC is a heterotrimer in which there are three nonidentical subunits, a, b, and g (1:1:1) with all three subunits present in the ENaC in cortical/medullary kidney, distal colon, and lung (Canessa et al., 1994). A fourth subunit, d, has been discovered that can replace the a-subunit in the trimeric ENaC, and this substitution creates different pharmacological and biophysical properties (Ji et al., 2012).

2.1 Amiloride Inhibitor and ENaC Current ENaC is an amiloride-sensitive channel. Amiloride is a Kþ-sparing diuretic that binds to ENaC and makes physical contact with all three subunits at serine and glycine residues (aSer-583, bGly-525 and gGly-542), all of which are present at a homologous site within the three subunits of ENaC. The aSer-583 likely has a role in ion permeation through ENaC. Mutations of the b and g-glycines greatly weaken the amiloride block (Kashlan et al., 2005). Amiloride blocks ENaC with 50% inhibition at a concentration of 0.1 mM and inhibits the reabsorption of Naþ in the late distal

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FIGURE 12.1 Model of an epithelial cell showing types of junctions between cells. From a comprehensive approach to Life Science, Division of Advanced Education in Science, Komaba Organization for Educational Excellence, College of Arts and Sciences, The University of Tokyo, http://csls-text3.c.utokyo.ac.jp/active/11_06.html.

FIGURE 12.2 Schematic illustration of the location and function of ENaC in epithelia. Reproduced with permission from Hanugloku, I., Hanugloku, A., 2016. Epithelial sodium channel (ENaC) family: phylogeny, structure-function, tissue distribution, and associated inherited diseases. Gene 579, 95e132.

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FIGURE 12.3 Predicted structure of oligomerized hENaC. View of the ribbon structure of the predicted heteromeric hENaC perpendicular (A) and parallel (B; from the extracellular side) to the molecular threefold axis. Adapted alpha- (red), beta- (yellow), and gamma- (blue) hENaC model using the 2QTS structural coordinates for the A, B, and C subunits of the ASIC1 homotrimer. Predicted ENaC heterotrimer with filled surfaces perpendicular (C) and parallel (D) to the molecular threefold axis highlighting fenestrations between TM1 (pale yellow) and TM2 (pale red) of adjacent monomers, and the numerous cavities and crevices formed by the subunits. Subunit coloring is the same as in A and B. The enlarged area in D shows the opening at the top of the channel (pointed to by white arrow) to a tunnel that runs the length of the extracellular region of the ˚ wide. At its hENaC to the channel pore down the center of the threefold axis perpendicular to the membrane. At its opening, the tunnel is 14 A ˚ wide. Reproduced, with permission, from Fig. 3 of Stockand, J.D., Staruschenko, A., Porchynyuk, O., Booth, R.E., Silverthorn, D.U., narrowest, it is 4 A 2008. Insight toward epithelial Naþ channel mechanism revealed by the acid-sensing ion channel1 structure. IUMB Life 60, 620e628.

convoluted tubule of the kidney. In Fig. 12.5 is shown the chemical structure of amiloride, the normal currents produced in the channel, and the inhibition of the current by amiloride. When the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed on the same membrane with ENaC, CFTR inhibits the activity of ENaC. CFTR is activated by protein kinase A (PKA), and in the presence of activated CFTR and ENaC, there is an initial influx of Cl and an increase of luminal (apical)

intracellular Cl concentration. Activation CFTR Cl conductance leads to parallel inhibition of epithelial sodium channels (Kunzelmann et al., 2001).

2.2 Regulation of ENaC Mainly by Aldosterone and Other Hormones Proteins induced by the action of aldosterone generally are genomic and are mediated by the mineralocorticoid receptor (MR). Some actions of aldosterone occur

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FIGURE 12.4 Schematic illustration of the transmembrane localization of an ENaC subunit. The sequence shown is the human alpha-subunit. All homologous ENaC subunits have two transmembrane segments and 10 extracellular loops. The transmembrane segments for this figure were predicted by the Phobius program. The extracellular domain includes about 70% of the sequence of amino acids of an ENaC subunit. TM, transmembrane. Reproduced with permission from Fig. 3 of Hanugloku, I., Hanugloku, A., 2016. Epithelial sodium channel (ENaC) family: phylogeny, structure-function, tissue distribution, and associated inherited diseases. Gene 579, 95e132.

too quickly to be the result of genomic action and occur through nongenomic means. The genomic actions of aldosterone are mediated by the MR. The ligandbinding domain of the human MR is shown in Fig. 12.6. Aldosterone acts through the MR to generate genomic expression. In the kidney, the mRNA for the a-subunit is induced by the hormone. The b- and g-subunits are increased by the action of the hormone in the colon (Stokes and Sigmund, 1998). In addition, aldosterone increases the expression of other proteins, through indirect transcriptional pathways, that modulate ENaC. These proteins are glucocorticoid regulated kinase-1 (SGK1) (Shigaev et al., 2000), glucocorticoid-induced leucine zipper protein (GILZ) (Robert-Nicoud et al., 2001), and connector enhancer of kinase suppressor of Ras-3 (CNKSR3) (Ziera et al., 2009). Fig. 12.7 shows the action of aldosterone to express SGK1 leading to the phosphorylation of an ENaC subunit. This figure shows the genomic expression of SGK1 by aldosterone functioning through its activated receptor, MR. SGK1 upregulates the activity of ENaC through the phosphorylation (green dot) of ENaC to increase its activity, as described in the legend for Fig. 12.7 (Valinsky et al., 2019). The effect of

SGK1 in enhancing the activity of ENaC could be focused to the C-terminus of a-ENaC because a mutation of Ser621 of the C-terminus negated the SGK1 effect (Diakov and Korbmacher, 2004). Another kinase involved in the regulation of ENaC (and also of ROMK, discussed later) is WNK (with no lysine). These kinases lack the lysine residue essential for ATP binding. There are five WNK kinases: WNK1, WNK2, WNK3, WNK4, and kidney-specific WNK (KSWNK). WNKs interact with signaling cascades throughout the nephron and determine the activity of central Naþ/Kþ transporters. In the distal nephron, WNKs are involved in the regulation of the Naþ/Cl cotransporter and in the regulation of ENaC in the connecting tubule (CNT) and collecting duct (CD). In these locations, the phosphorylation of ENaC is upregulated by both WNK1 and KS-WNK1 and inhibited by WNK4 (Fig. 12.7). WNK1 acts indirectly on ENaC by its activation of SGK1 through the phosphatidylinositol 3-kinase (PI3K), wherein SGK1 reverses the WNK4 inhibition of ENaC (Fig. 12.7). Finally, both potassium loading and aldosterone increase the level of mRNAs for KS-WNK1 and WNK1 (Hoom et al., 2011).

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FIGURE 12.5 Structure of amiloride is shown at the top. The figure shows the current-voltage curves obtained using a cut-open oocyte technique. (A) current recordings obtained from a cut-open oocyte perfused with intracellular and extracellular solutions containing 50 mM Naþ during a series of 175 ms square voltage pulses ranging from 140 to þ60 mV. The exposed membrane (at the vegetal pole of the oocyte) had a diameter of -500 mm. (B) current recordings are as in A, obtained after application of 5 mM amiloride. (C) amiloride-sensitive currents (i.e., AeB). (D) Currentevoltage relationships for the whole membrane current (solid circles), residual current after application of amiloride (solid squares), and amiloride-sensitive current (Iamil, open circles). The currents were measured at 150 ms after the beginning of the voltage pulse. Vmax, membrane potential. Reproduced with permission from Fig. 3 of Abriel, H. and Horisberger, J.-D., 1999. Feedback inhibition of rat amiloride-sensitive epithelial sodium channels expressed in Xenopus laevis oocytes. J. Physiol. 516.1, 31e43.

In addition to SGK1, other proteins, expressed transcriptionally, have been shown to affect the expression of ENaC. These proteins form a multiprotein complex that includes SGK1. One of these components is the GILZ (D’Adamio et al., 1997), which also has been shown to be increased more potently by aldosterone than by glucocorticoid (Muller et al., 2003). GILZ is required for the formation of the multiprotein complex, and it stimulates the action of ENaC. Thus, a model (Soundararajan et al., 2012) can be presented to show that the formation of the complex results in the activation of the channel (Fig. 12.8). The inhibited form of ENaC is rendered suppressed by the attachment of Nedd4-2 and Raf-1. Aldosterone, when present, elevates the level of CNKSR3 (a scaffolding protein) expressed in

the kidney connecting tubule (CNT) and cortical collecting duct (CCD) (Ziera et al., 2009). Aldosterone also elevates the levels of GILZ and SGK1, directly or indirectly, so a new complex is formed in which the inhibition of ENaC function by Nedd4-2 and Raf-1 is abolished. The expression of all of these proteins that become elevated by the actions of aldosterone occurs through the genome. There is a nongenomic mechanism in which aldosterone increases the amount of phosphorylated protein kinase D1 (PKD1) (McEneaney et al., 2007), which was later shown to be a prerequisite for the intracellular trafficking of ENaC to membrane-bound vesicles (McEneaney et al., 2008). This activity was unaffected by inhibitors of transcription or of translation but was

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inhibited by the MR antagonist, spironolactone, indicating independence from the genomic pathway but dependence on the MR (or an MR-like protein inhibitable by spironolactone). Earlier data showed that measurements of the efflux of 22Naþ from rat tail arterial smooth muscle, apparently requiring the action of the basolateral Naþ/Kþ ATPase, was independent of transcription (Moura and Worcel, 1984). Aldosterone could increase the activity of the Naþ/Kþ ATPase pump within 15 min and was not inhibited by actinomycin D (Mihailidou et al., 1998). In other studies by Mihailidou, nongenomic actions of aldosterone involved the regulation of intracellular sodium homeostasis by protein kinase C (PKC) (Mihailidou et al., 2004). PKC differentially regulates ENaC subunit levels by decreasing the levels of b and g but not aENaC protein (Stockand et al., 2000). FIGURE 12.6 Crystal structure of the ligand-binding domain (LBD) of the mineralocorticoid receptor (MR) bound to aldosterone. The aldosterone model is shown as a stick model in the lower center. The overall structure of the LBD of the MR is similar to the other steroid receptors. Reproduced with permission from Fig. 3A of Bledsoe, R.K., Madauss, K.P., Holt, J.A. et al., 2005. A ligand-mediated hydrogen bond network required for the activation of the mineralocorticoid receptor. J. Biol. Chem. 280, 31283e31293.

2.3 Bradykinin Bradykinin (BK), a peptide hormone of the kallikreinkinin system, increases sodium excretion through its control of ENaC-mediated reabsorption in the distal nephron (Mamenko et al., 2015). BK consists of nine amino acids (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg9)

FIGURE 12.7 Interactions of aldosterone, SGK1, and ENaC. Aldosterone freely enters cells and forms a high affinity interaction with the cytosolic mineralocorticoid receptor (MR) (step 1). The aldosterone-MR complex translocates to the nucleus, binds to hormone response elements, and initiate the transcription of aldosterone-regulated genes, including SGK1 (2). Newly synthesized SGK1 mRNA is translated into protein, where it upregulates ENaC activity. Such pathways include reduced ENaC ubiquitination via bisphosphorylation of NEDD-4-2 (3), prevention of ENaC endocytosis by phosphorylation of WNK4 (4), recruitment of silent ENaC channels into active channels by direct ENaC phosphorylation (5), and inhibition of transcriptional suppressor complex Dot1a-AF9 via phosphorylation of AF9 (6). Green dot, phosphate aI., 2019, Large green dot, aldosterone. Reproduced with permission from Fig. 1 of Valinsky, W.C., Touyz, R.M., Shrier, A., 2019. Aldosterone and ion channels. Vitam. Horm. 109, 105e132.

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reduces Naþ reabsorption in the principal cells of the distal renal nephron, leading to excretion of Naþ, Kþ, and water and resulting in lowered blood pressure. This occurs through the acute inhibition of ENaC via stimulation of B2Rs and following the depletion of PIP2 but not increases in Ca2þ (Zaika et al., 2011).

2.4 Endothelin

FIGURE 12.8 Formation of an ENaC multiprotein complex (ERC) resulting in the activation of the epithelial sodium channel (ENaC). In the basal state, while ENaC is associated with Nedd4-2 and Raf-1, which inhibit the activity of the channel, its function is suppressed. Under the inducing action of aldosterone, a multiprotein complex is formed consisting of Nedd4-2 and Raf-1 (as before) plus SGK1, GILZ1, and CNK3 (connector enhancer of kinase suppressor of Ras3 (CNKSR3)), which is a scaffolding protein expressed I the kidney connecting tubule) (Ziera et al., 2009). SGK1, GILZ, and CNK3 appear to be induced by aldosterone directly or indirectly. The model is based on Soundararajan, R., Melters, D., Shih, I.C., Wang, J., Pearce, D., 2009. Epithelial sodium channel regulated by differential composition of a signaling complex. Proc. Natl. Acad. Sci. U. S. A. 106, 7804e7809 and Fig. 2 of Soundararajan, R., Pearce, D., Ziera, T., 2012. The role of ENaC-regulatory complex in aldosterone-mediated sodium transport. Mol. Cell. Endocrinol. 350, 242-247; with permission.

with a molecular weight of about 1060; it is encoded by the human gene KNG1. There are two separate receptors that recognize kinins at the plasma membrane classified as B1R and B2R (Blaukat, 2003; Hall, 1997). BK binds preferentially to B2R, whereas B1R recognizes carboxy terminally truncated peptides desArg9-BK and desArg10-kallidin (Blaukat, 2003). The signaling pathway for B2R includes the binding of BK to B2R (a Gq-coupled receptor); B2R activates phospholipase C (PLC), which hydrolyzes PIP2 (phosphatidylinositol 4,5-bisphospate) in the plasma membrane to the cytoplasmic products diacylglycerol (DAG) and IP3 (inositol trisphosphate), resulting in the release of microsomal Ca2þ via IP3 and the activation of protein kinase C (PKC). PKC activates MAPK, which enters the cell nucleus to activate transcription (Litwack, 2017d). BK

Another hormonal protein that acts on ENaC is endothelin (ET). There are three members of the endothelin family: ET-1, ET-2, and ET-3. ET-1 is secreted by renal cells, and it affects several targets in different parts of the nephron. ET-1, as with ET-2 and ET-3, has a 21 amino acid sequence; the sequence of ET-1 is Cys1-Ser-Cys3-SerSer-Leu-Met-Asp-Lys-Gly-Cys11-Val-Tyr-Phe-Cys15-HisLeu-Asp-Ile-Ile-Trp21. Cys1 and Cys 15 form a disulfide bond, as do Cys3 and Cys11. ET-1 appears to be an important inhibitor of ENaC in vivo (Sorokin and Staruschenko, 2015). The production of ET-1 from its preproendothelin precursor is controlled at the genomic level (Forschi et al., 2001). In native rat CD principal cells, ET-1, operating through its receptor (ETRB), decreases ENaC P0 (P0, open probability) by about threefold within 5 minutes, suggesting a nongenomic pathway. Subsequent intracellular pathways involved Src tyrosine kinase activity and MAPK1/2 signaling (Bugaj et al., 2008). Additionally, experiments in which endothelin receptors (ETRA, ETRB, and ETRA/ETRB) were knocked out in mice confirmed that ET-1 regulation of ENaC, mediated by ETB receptors, contributes to the antihypertensive and natriuretic effects of the local endothelial system in the kidney CDs (Bugaj et al., 2012). When PP2, the Src family kinase inhibitor, was used in cell culture experiments, ENaC was completely blocked. Also, the inhibition of MAPK abolished the ET-1 effects on ENaC, confirming the ET-1 signaling pathway for ENaC inhibition (Bugaj et al., 2008). SGK (serum and glucocorticoid regulated kinase, a serine-threonine kinase) and PKA have been shown to upregulate ENaC activity (Baines, 2013). Additional kinases that upregulate ENaC activity are CK2, GRK2, IKKb, and PKD1. PKC, ERK1/2, and AMPK are inhibitory kinases (Baines, 2013). Aldosterone-induced SGK1 appears to be a principal regulator of ENaC (Pao, 2012; Soundararajan et al., 2012).

3. RENAL OUTER MEDULLARY KD (ROMK OR KIR1) CHANNEL ROMK channel is a renal outer medullary channel that transports potassium out of cells and is inwardly rectifying ATP regulated. Human ROMK channel is

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The kinases, WNK1 and WNK4, both inhibit ROMK by interacting with the scaffolding protein intersection to increase clathrin-mediated endocytosis (Hoom et al., 2011). ROMK is a tetrameric channel, as shown in Fig. 12.11. Two nonadjacent subunits of the four subunits are illustrated in Fig. 12.12. The structures are based on the crystal structure of the bacterial form of ROMK, KirBac1.1. Here, the side view of the membrane-spanning region is shown. Two nonadjacent subunits are shown to illustrate the permeation path in the channel. The path is completely obstructed by the four L160 residues in each of the four subunits. The opening and closing of the gate possibly occur through a rotation of adjacent subunits or by movement through a hinge of the inner helices.

4. POTASSIUM CHANNELS ACTIVATED BY CALCIUM, BK CHANNEL (MAXI-K CHANNEL)

FIGURE 12.9 Location of ROMK along the nephron. Expression of major sodium and potassium transport proteins in the distal nephron, including WNK kinases and associated regulatory proteins. Reproduced with permission from Fig. 1 of Hoorn, E.J., Nelson, J.H., McCormick, J.A., Ellison, D.H., 2011. The WNK kinase network regulating sodium, potassium, and blood pressure. J. Am. Soc. Nephrol. 22, 605e614.

encoded by the KCNJ1 gene. Different transcripts arising by alternative splicing (Boim et al., 1995) are expressed as three ROMKs: ROMK 1, ROMK 2, and ROMK 3. These three channels are differentially expressed along the aldosterone-sensitive distal nephron, as shown in Fig. 12.9. The cortical connecting duct expresses ROMK 1 and 2; the connecting tubule expresses ROMK 2 and the distal convoluted tubule expresses ROMK 2 and 3 (Boim et al., 1995). Adrenalectomy downregulates cortical ROMK but upregulates the medullary ROMK. Potassium deficiency downregulates ROMK mRNA in the kidney cortex and medulla. Aldosterone stimulates the activity of cortical ROMK through its genomic action in inducing SGK1 (as indicated before) and the eventual phosphorylation of ROMK by SGK1. This is illustrated in Fig. 12.10.

BK channels are involved in a wide variety of physiologic functions, including its function in the nephron. Aldosterone upregulates the calcium-activated potassium channel in vascular epithelial cells (Zhao et al., 2012). In support of this conclusion, the blockade of MR inhibited expression of BK in the distal nephron (Wen et al., 2013). Additional studies confirm that aldosterone secretion is key to upregulation of BK (Cornelius et al., 2015). The relationships of calcium uptake in the principal cell of the CCD of the kidney to the activation of the BK channel are shown in Fig. 12.13 (Latorre et al., 2017). In the principal cell of the CCD, Ca2þ is taken up through the TRPV4 channel into the cytoplasm. The cytosolic Ca2þ activates the BK channel to stimulate the outward flow of Kþ. ROMK is also involved in the outward flow of Kþ from the cell. Aspects of the structure of the BK channel are illustrated in Fig. 12.14. The BK channel acts as a negative feedback system that limits increases in intracellular calcium to outward hyperpolarizing potassium currents, and therefore, in addition to actions in the kidney, regulates smooth muscle tone, release of neurotransmitters, and neuronal excitability (Feletou, 2009). 17b-Estradiol also has been shown to activate BK channels, and this activation depends on the presence of the b-1 subunit (Valverde et al., 1999; Dick and Sanders, 2001). In addition, there are molecules produced endogenously that increase the activity of the BK channel: arachidonic acid, metabolites of cytochrome P450, epoxygenase, and lipoxygenase (Bentzen et al., 2014).

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FIGURE 12.10 Interactions of aldosterone, SGK1, and ROMK. As described in Fig. 12.7, aldosterone binds to the MR and enhances SGK1 mRNA expression, resulting in SGK1 protein. The newly synthesized SGK1 phosphorylates POMK, resulting in increased NHERF2-dependent trafficking (1) as well as increased channel function (2). In addition, SGK1 decreases ROMK endocytosis via bis-phosphorylation of WNK4 (3). NHERF2, Naþ/Hþ exchange regulatory cofactor; WNK4 Ser-Thr protein kinase. Reproduced with permission from Fig. 2 of Valinsky, W.C., Touyz, R.M., Shrier, A., 2019. Aldosterone and ion channels. Vitam. Horm. 109, 105e132.

4.1 Magnesium Channels (TRPM6 and TRPM7) These channels are the significant members of a large family. They are transient receptor potential (TRP) channels having a tetrameric structure and are cationselective ion channels that respond to various forms of sensory input. The TRPM members (TRPM6 and TRPM7) have a self-assembling tetrameric C-terminal cytoplasmic coilecoil domain that underlies channel assembly and trafficking (Fujiwara and Minor, 2008). TRPM6 (transient receptor potential melastatin 6) is found mainly in the kidney, cecum, colon, and lung, indicating that these are the primary organs involved in Mg2þ reabsorption. TRPM6 contains a C-terminal a-kinase domain; this domain can bind RACK1 (receptor of activated protein C kinase1) to inhibit channel activity in a phosphorylation-dependent manner (Cao et al., 2008). TRPM6 is regulated by dietary Mg2þ and by 17b-estradiol (which represents the gatekeeper function of this epithelial Mg2þ channel). The regulation of bodily Mg2þ balance is served principally by the kidney where TRPM6 is the gatekeeper in transepithelial Mg2þ transport. As opposed to the limited organ locations of TRPM6, TRPM7 is involved in cellular Mg2þ homeostasis and is distributed in most cells. 17b-Estradiol regulates the levels of TRPM6 mRNA (but not TRPM7 mRNA) in the kidney. In ovariectomized rats, renal TRPM6 mRNA falls, and in these animals, administration of 17b-estradiol raises TRPM6

mRNA levels to normal (Groenestege et al., 2006; Wouter et al., 2006). A repressor of estradiol receptor activity (REA) is a protein that binds to the sixth, seventh and eighth b-sheets in the a-kinase domain of TRPM6 (but REA does not interact with TRPM7). Both REA and TRPM6 are coexpressed in the kidney Mg2þ transporting distal convoluted tubule (DCT) (the DCT is depicted in Fig. 12.9). Estradiol treatment dissociates the binding of REA to TRPM6 a-kinase, and 17b-estradiol also stimulates TRPM6-mediated current in HEK293 cells. Consequently, REA is a negative feedback inhibitor of TRPM6 in the regulation of active Mg2þ reabsorption (Gao et al., 2009). TRPM7, the homolog of TRPM6, is a 220 kDa ion channel (antiport) that transports monovalent cations out of cells and carries divalent cations into cells (Nadler et al., 2001). This channel has a selectivity in the following order: Zn2þ ¼ Ni2þ > Ba2þ > Co2þ > Mg2þ > Mn2þ > Sr2þ > Cd2þ > Ca2þ (Li et al., 2006). The structure of TRPM7 is shown in Fig. 12.15. In Fig. 12.16 is shown the TRPM family. TRPM6 has a 52% homology to TRPM7. TRPM7 is located primarily in intracellular vesicles rather than having its major site in the plasma membrane, although there exists a much smaller population of TRPM7 molecules in the plasma membrane (Valinsky et al., 2019). Its function, if any, in the vesicles, apart from storage, is not yet understood (Abiria et al., 2017). Considerable evidence suggests that TRPM7 is not relevant to the function of magnesium reabsorption

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FIGURE 12.11 Residues involved in the pH sensing by the tetrameric ROMK channel. (A) Cartoon model of ROMK showing the N-terminus, slide helix (SH), first transmembrane domain (M1), pore helix (PH), second transmembrane domain (M2), and the C-terminus. The RKR sites are shown for the three ROMK splice variants. The ROMK1 C-terminal R311 is shown together with the salt bridge partner E302. B-D0 N-terminal (B and C) and C-terminal (D) alignments are shown for members of the Kir inwardly rectifying potassium channel family. Important residues are indicated by residue numbers and arrows. The bacterial Kir ortholog, KirBac1.1 is also shown for comparison. Reproduced with permission from Fig. 8 of Leng, Q., MacGregor, G.G., Dong, K., Giebisch, G., Hebert, S.C., 2006. Subunit-subunit interactions are critical for proton sensitivity of ROMK: evidence in support of an intermolecular gating mechanism. Proc. Soc. Natl. Acad. Sci. U. S. A 103, 1982e1987. Copyright (2006) National Academy of Sciences, United States.

in the kidney (Valinsky et al., 2019). Aldosterone, however, increases the population of TRPM7 molecules in the plasma membrane and whole cell current in HEK293 cells overexpressing TRPM7. These effects require both the MR and an SGK1-dependent mechanism where SGK1 operates through the a-kinase domain (Valinsky et al., 2016). When phosphotransferase was inactive in a mutant (K1648R) cell system, the action of aldosterone in moving TRPM7 to the plasma membrane requiring the MR and SGK1 was abolished, again implying that SGK1 acts on the a-kinase domain (Valinsky et al., 2016). Aldosterone increased magnesium reactive oxygen species and proinflammatory mediator

expression; however, the proinflammatory mediator expression was observed only in HEK293 cells expressing a-kinase defective mutants, not in the wild-type TRPM7, implying that the a-kinase takes part in an antiinflammatory function (Yogi et al., 2013). In the case of TRPM6, it was observed in a mouse model of hypomagnesemia that aldosterone downregulates TRPM6 (Sontia et al., 2008; Yogi et al., 2013). This was also the case in the human where TRPM6 expression decreases in patients with hypomagnesemia (Jiang et al., 2014). Because hyperaldosteronism also causes hypomagnesemia, the expression of TRPM6 also may be affected in that condition (Valinsky et al., 2019).

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basolateral membrane, causing Mg2þ wasting (Muallem and Orson, 2007).

5. EPITHELIAL BASOLATERAL NAD/KD ATPASE PUMP

FIGURE 12.12 Homology model of ROMK based on the known crystal structure of KirBac1.1. Side view, showing two of four subunits. Shown is the permeation of Kþ ions (K). The putative pH gate consists of four Leu1 60 (Kir1.1b) residues, projecting into the permeation path at the cytoplasmic edge of the membrane, near the apical crossing of the inner transmembrane helices. Opening and closing of the gate could occur via rotation of adjacent subunits relative to each other or by a hinge movement of the inner helices. Reproduced with permission from Fig. 8 of Sackin, H., Narazashvili, M., Palmer, L.G., Krambis, M., Walters, D.C., 2005. Structural locus of the pH gate in the Kir1.1 inward rectifier channel. Biophys. J. 88, 2597e2606.

Xie et al. have shown that phosphatidylinositol bisphosphate (PIP2) is required for TRPM6 channel function. Thus, hydrolysis of PIP2 by phospholipase C (PLC)-coupled hormones is an important pathway for TRPM6 gating and magnesium homeostasis (Xie et al., 2011). Consequently, many peptide hormones, such as parathyroid hormone (PTH), calcitonin, glucagon, and vasopressin may be involved. Epidermal growth factor (EGF) has been shown to be a hormonal regulator of TRPM6 activity. Accordingly, mutations in EGF could explain familial hypomagnesemia. An inhibitor of EGF receptor has been shown to cause hypomagnesemia by inhibiting EGF signaling (Groenestege et al., 2007). Additionally, EGF stimulates magnesium reabsorption in the renal DCT by engaging its receptor on the basolateral side of DCT cells and activation of the TRPM6 magnesium channel in the apical membrane. A point mutation in pro-EGF retains EGF secretion to the apical membrane but not to the

The Naþ/Kþ ATPase pump of the basolateral plasma membrane generates electrochemical gradients for sodium and potassium, exchanging three Naþ ions for two Kþ ions (antiport) across the membrane during each cycle of the hydrolysis of ATP (Marth et al., 2007). This pump is the driving force for the secondary active transport of solutes, such as amino acids, sugars, and phosphate across the renal tubules (Bertorello and Katz, 1993). The Naþ/Kþ-ATPase contains motifs for phosphorylation by protein kinase A (PKA) and for protein kinase C (PKC). Generally, phosphorylation by PKA results in increased activity of the pump; however the phosphorylation by PKC may be inhibitory, although these effects are complex. For example, PTH activates PKC and reduces the activity of the Naþ/Kþ ATPase pump in the proximal convoluted tubule (PCT) (Satoh et al., 1993). The pump contains highly conserved phosphorylation motifs involving threonine: 369 DKTGTLT375 and 608MVTGD612 (Laursen et al., 2013). A number of hormones in various tissues regulate the Naþ/Kþ ATPase, and these hormones, operating through kinases, produce both short-term and long-term control of the enzyme. Long-term regulation is caused by thyroid hormone and aldosterone, while short-term regulation caused by catecholamines is mediated by phosphorylationedephosphorylation of the alpha-subunit. Alterations of the subcellular distribution of the pump can be caused, in some tissues, by the rapid action of insulin and aldosterone (Beguin et al., 1994). Humans produce endogenous cardiotonic steroids (hormones) similar to ouabain and digitalis, and these circulate in the bloodstream. These substances bind to the Naþ/Kþ ATPase, producing inhibition. Both the alpha-subunit and the beta-subunit of the ATPase are regulated at the genomic level by aldosterone and glucocorticoids operating through their respective receptors (MR and GR) (Derfoul et al., 1998; Kolla et al., 1999; Kolla and Litwack, 2000). Moreover, it appears that the N-terminal domain of the MR can modulate the inhibition of both the MR- and GR-mediated transactivation from the Naþ/Kþ ATPase beta1 target gene promoter (Derfoul et al., 2000). While the alpha-subunit of Naþ/ Kþ ATPase has all of the functional domains of the enzyme, the function of the beta-subunit is required so the alpha-subunit can reach the correct, stable conformation that is needed for the alpha-subunit to acquire its functional properties as well as its exit from the

The BK channel plays a key role in renal Kþ flux within the nephron. BK channels in the apical membrane of epithelial cells of the cortical collecting duct (CCD) are activated by tubule fluid flow-induces shear stress Ca2þ entry (possibly through TRPV4 channel) and cell swelling. In basal conditions, ROMK is thought to be primarily responsible for Kþ secretion demanded by higher flow high aldosterone, or others; it is proposed that both ROMK and BK play a role. The physiologic role of BK in the proximal cells (PC) or intercalated cells (IC) in the distal nephron is defined by the b-subunit coexpressed with the channel. Arrows illustrate places where higher Kþ flux occurs in the nephron. PCT, proximal convoluted tubule; I CT, initial collecting tubule; CCD, cortical collecting duct. Reproduced with permission from Fig. 3 of Latorre, R., Castillo, K., Carrasquel-Ursulaez, W., Sepulveda, R.V., Gonzalez-Nilo, F., Gonzalez, C., Alvarez, O., 2017. Molecular determinants of BK channel functional diversity and functioning. Physiol. Rev. 97, 38e87.

FIGURE 12.13

FIGURE 12.14 The modular nature of the BK channel. (A) Topography of the BK channel predited by hydrophobicity profiles. Alpha helices are represented as rectangles. So to S4 form the voltage sensor domain. The turret (T) is the loop joining S5 with the pore helix (P). SF is the selectivity filter. SF and S6 form the internal entryway. NH2 terminal is extracellular, and COOH terminal is cytosolic. Intracellular domain consists of a pair of RCK domains. (B) Full homotetrameric BK channel. The transmembrane region comprised the pore domain (yellow) and the voltage sensors (orange). The large cytosolic region contains the calcium sensors, which are composed of two regulators of potassium conductance domains, RCK1 and RCK2 (violet and green, respectively). (C) Structure (top view) of the transmembrane region. D. Structure (top view) of the calcium-bound gating ring with the RCK1 in violet and the RCK2 in green. E. A modular model for BK channel proposed that each domain corresponds to a specific structural domain and they interact with one another. Details of alignment, segment building, and rendering are given in the original paper. Reproduced with permission from Fig. 1 of Latorre, R., Castillo, K., Carrasquel-Ursulaez, W., Sepulveda, R.V., Gonzalez-Nilo, F., Gonzalez, C., Alvarez, O., 2017. Molecular determinants of BK channel functional diversity and functioning. Physiol. Rev. 97, 38e87.

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FIGURE 12.15 A cartoon model of TRPM7 showing the relationship between the antiparallel assembly domain, transmembrane domains, and dimeric kinase domains. The kinase domain dimer is shown linked to the C-terminal ends of antiparallel assembly domain strands. Reproduced with permission from Fig. 7A of Fujiwara and Minor (2008).

FIGURE 12.16 Cartoon of the TRPM family subunits. Conserved TRP domain (green box) and coiled-coil domain (red box) are indicated. Kinase domains of TRPM6 and TRPM7 and the TRPM2NUTd9 motif are shown as ovals. Reproduced with permission from Fig. 1A of Fujiwara and Minor (2009).

endoplasmic reticulum (Geering, 1991). The crystal structure of the Naþ/Kþ ATPase is available and locates the several sites for the binding of cardiotonic steroids and other molecules as well as depicting the sites for phosphorylation (Laursen et al., 2013). The crystal structure is shown in Fig. 12.17.

6. APICAL BRUSH BORDER RENAL PHOSPHATE CHANNELS (SLC34 TRANSPORTERS, NPT2 AND NPT3) The main proteins mediating apical phosphate uptake are NPT2A (encoded by SLC34A1 gene) and

NPT2C (SLC34A3) (Gattineni and Friedman, 2015). Expression of Npt2a (lower case indicates nonhuman form) and Npt2c in the kidney is limited to the proximal tubules. Npt2a is located in S1, S2, and S3 segments of the proximal nephron, but Npt2c is found only in S1 segments (Nowik et al., 2008; Picard et al., 2010). Npt2a mediates about 70% of apical inorganic phosphate (Pi) uptake. NPT2A is a 12 membraneespanning protein. It transports three sodium ions per molecule of HPO42. The remaining 30% of Na-dependent uptake following ablation of Npt2a is due to Npt2c, an electroneutral uptake moving 2Naþ ions for each molecule of Pi uptake. Both channels preferentially transport divalent HPO42. Mutations of NPT2C cause hereditary hypophosphatemic rickets with hypercalciuria. NPT2C is more important in phosphate economy in humans than in mice (the preceding discussion is from Gattinena and Friedman, 2015).

6.1 Parathyroid Hormone Npt2a contains many structural motifs for the binding of regulatory molecules (Hernando et al., 2005). Npt2a is phosphorylated constitutively in spite of its content of serines, threonines, and lysines (Jankowski et al., 2001). PTH leads to the dephosphorylation of Npt2a, but inhibition of phosphatase did not affect PTH downregulation of Npt2a, suggesting that its induction of Npt2a dephosphorylation does not play a role in the inhibition of Pi transport. Neither

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FIGURE 12.17 The high affinity Naþ/Kþ ATPase E2P-ouabain complex. The E2B-ouabain complex is depicted using the following color codes: red [nucleotide-binding (N) domain], blue [phosphorylation (P) domain], light yellow [actuator (A) domain], orange [aM1-2], teal [aM3-4], purple [aM5-6], brown [aM7-10], green [b-subunit], and deep pink [g-subunit]. The CTS (cardiosteroid binding site) binding site is marked CTS. Ouabain is represented by yellow sticks, Mgþþ and the three water molecules by light green and red spheres, respectively. The cholesterol-phospholipid site is marked CLR, with cholesterol and phosphatidylserine depicted in orange and black sticks, respectively. The carbohydrate moieties are depicted in light pink sticks, confirming the three glycosylation sites at Asn158, Asn193, and Asn265 at the b-subunit. Reproduced with permission from Fig. 1A of Laursen, M., Yatime, L., Nissen, P. and Fedosova, N.U., 2013. Crystal structure of the high affinity Naþ,Kþ-ATPase-ouabain complex with Mg2þ bound in the cation binding site. Proc. Natl. Acad. Sci. U. S. A. https://doi. org/10.1073/pnas.1222308110.

phosphorylation nor ubiquitination are important in the behavior or fate of the transporter (Gattinena and Friedman, 2015). PTH is a polypeptide consisting of 84 amino acids: N-Terminus-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-

His-Asn10-LeuGly-Lys-His-Leu-Asn-Ser-Met-GluArg 20 -Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp 30 Val-His-Asn-Phe-Val-Ala-Leu-Gly-Ala-Pro40-Leu-AlaPro-Arg-Asp-Ala-Gly-Ser-Gln-Arg50-Pro-Arg-Lys-LysGlu-Asp-Asn-Val-Leu-Val60-Glu-Ser-His-Glu-Lys-SerLeu-Gly-Glu-Ala70-Asp-Lys-Ala-Asp-Val-Asn-Val-LeuThr-Lys80-Ala-Lys-Ser-Gln-C-Terminus. PTH (3e34) lacks the cAMP-activating region but is able to internalize Npt2a, whereas PTH (1e34) can operate from both (apical and basolateral) epithelial membranes (Traebert et al., 2000). The luminal (apical) surface PTH inhibits Na-Pi cotransport primarily through a PKC-dependent pathway. However, basolateral activity of PTH proceeds through PKA primarily. PTH affects Pi transport at the apical membrane about 10 times more than its action at the basolateral membrane (Reshkin et al., 1990). PKC inhibitors block PTHdependent Pi transport, confirming the role of PKC at the apical side (Cunningham et al., 2009). In any case, there are a number of contradictions in the literature concerning which is the dominant protein kinase involved. It seems that both PKC and PKA pathways are involved to different degrees (Lederer et al., 1998). Aside from these protein kinases, PTH stimulation leads to the activation of extracellular signal-regulated kinases-1 and -2 (ERK1/2) (Cole, 1999; Lederer et al., 2000; Sneddon et al., 2000). Blockade of ERK1/2 downregulates PTH-inhibitable Npt2a endocytosis in mouse kidney and internalization induced by direct activation of PKA, PKC, or cGMP pathways (Bacic et al., 2003), indicating that ERK1/2 is downstream of these pathways and that all three pathways converge on ERK1/ 2 (Gattinena and Friedman, 2015). Accumulated evidence supports the conclusion that both PKA and PKC pathways are involved in the PTH-dependent downregulation of mRNA for Npt2a.

6.2 Fibroblast Growth Factor 23 There is a large family of fibroblast growth factors (FGFs). FGF15/19, FGF21, and FGF23 are considered to be hormones (Gattinena and Friedman, 2015). All FGFs have a heparin-binding sequence (with different affinities). The nonhormonal FGFs require heparin sulfate (HS) proteoglycans as cofactors to bind to FGF receptors (FGFRs) in a complex of FGF-FGFR-HS. FGF23 and FGF19 (hormones), on the other hand, have decreased affinity for HS so they can circulate in the bloodstream as hormones (Goetz et al., 2007; Beenken and Mohammadi, 2012). The FGF hormones form the hormone receptor complex weakly, if at all, when the coreceptor (HS) is absent. However, klotho (a novel b-glucuronidase, ceramidase, hormone (soluble form), scaffolding protein, antiaging protein (Litwack, 2016)) can act as a coreceptor (in place of HS) for FGF23 and

7. AQUAPORINS

FGF19 and facilitate binding of the hormonal FGFs to their receptors (Kurosu and Kuro, 2008; Kurosu et al., 2006; Haussler et al., 2016). The functioning of klotho as a coreceptor of FGF23 generates the FGFR-klotho complex that reduces the reabsorption of phosphorus and inhibits the synthesis of 1,25-dihydroxyvitaminD3 in renal tubules. This complex also suppresses PTH synthesis in the parathyroid glands. Ectodomain shedding of transmembrane klotho is mediated by ADAM10/17 secretase or by other secretases releasing soluble klotho into the extracellular space, accessing the circulation. Soluble klotho can bind to FGF23 in the bloodstream to form a mobile receptor complex capable of activating FGFR on cells where transmembrane klotho is absent or nearly so. Through its b-glucuronidase activity, soluble klotho can alter channel occupancy in the plasma membrane, so either internalization or retention results via binding to galactin-1. In addition to these effects, klotho has many cytoprotective effects (Tan et al., 2014). The many effects of klotho are illustrated in Fig. 12.18.

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The FGFR contains 228 amino acid residues and has a molecular weight of 22.5 kDa. It is a member of a subgroup of tyrosine kinase receptors. There are four such high-affinity receptors, two of which have alternatively spliced forms. These receptors form dimers and are phosphorylated constitutively in the absence of ligand and are active either as homodimers or heterodimers. The functional domains of the dimeric FGFR and the FGFR in complex with FGF23 and klotho are shown in Fig. 12.19. When FGF23 is secreted as a soluble protein, it acts as a powerful phosphaturic hormone causing kidney phosphate wasting by decreasing the renal brush border expression of Npt2a and Npt2c in the proximal tubules (Grattinena and Friedman, 2015). FGF23 also decreases the expression of 1a-hydroxylase in the kidney and increases the level of 24-hydroxylase (liver) resulting in lowered levels of 1,25-dihydroxyvitamin D3 in the bloodstream (Gattineni et al., 2009; Shimada et al., 2004). Other hormones, such as glucocorticoids, estradiol, and insulin-like growth factor-1 can play a role in the homeostasis of phosphate (Gattinena and Friedman, 2015).

FIGURE 12.18 Mechanisms of klotho action. FGF23-klotho signaling in the kidney. In the proximal renal tubules, blood-borne FGF23 binds to a receptor complex consisting of FGFRs and aklotho and activates a signaling cascade involving ERL1/2 and SGK1. SGK1, in turn, phosphorylates NHERF-1 (Naþ/Hþ exchanger regulatory factor), leading to internalization and degradation of NaPi-2a (sodium-phosphate cotransporter) in proximal tubule cell membrane that transports phosphate from the urine across the membrane to the bloodstream. FGF23 signaling also may involve Janus kinase-3 (JAK-3). PTH binds to the PTH receptor (PTHR), leading to activation of both kinases, PKA and PKC, and subsequent phosphorylation of NHERF-1. FGF23- and PTH-induced phosphorylation of NHERF-1 decreases the membrane abundance of NaPi-2a and leads to increased urinary phosphate excretion. The FGF23 signaling-induced mechanisms downstream of ERK1/2, which suppress the transcription of 1a-hydroxylase in proximal renal tubules, are unknown. In distal renal tubules, FGF23 circulating in blood binds to the FGFRklotho receptor complex and activates ERK1/2, SGK1, and the WNK1/4 complex. Activation of WNK signaling increases the luminal membrane abundance of glycosylated TRPV5 and of NCC (Naþ/Cl cotransporter), leading to increased distal tubular calcium and sodium reabsorption. Reproduced with permission from Fig. 1 of Erben, R.G., Andrukhova, O., 2017. FGF23-klotho signaling axis in the kidney. Bone 100, 62e68, https://doi.org/10. 1016/j.bone.2016.09.010.

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FIGURE 12.19 The FGF1-FGFR2-heparin complex. (A) View perpendicular to the approximate dyad of the complex. FGFR2 domains 2 (D2) and 3 (D3) are cyan and magenta, respectively, and FGF1 is green. The heparin molecule is in CPK representation. (B) View along the dyad. (Bottom) Overall topology of the FGF23-FGFR1cecto and a-klothoecto complex. Surface representation of the ternary complex structure. a-Klotho, KL1 (cyan), and KL2 (blue) domains are joined by a short proline-rich linker (not visible in this structure). FGF23 is in orange with its proteolytic cleavage motif in gray. RBA, receptor binding arm. FGFR1c is in green. (A) Reproduced with permission from Pellegrini, L., Burke, D.F., von Delft, F., Mulloy, B. and Blundell, T.L., “Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin.” Nature, 407: 1029e1034, 2000. (B) Reproduced, with permission, from part of Fig. 1 of Chen, G., Liu, Y., Goetz, R., Fu, L., Jayaraman, S., Hu, M.-C., Moe, O.W., Liang, G., Li, X., Mohammadi, M., 2018. aKlotho is a non-enzymatic molecular scaffold for FGF23 hormone signaling. Nature 553, 461e466.

7. AQUAPORINS Aquaporins (AQPs) are channels in the apical and basolateral plasma membranes of the distal kidney. They can be formed in the apical plasma membrane, following the action of vasopressin that activates PKA to phosphorylate aquaporin subunits in the cytoplasm. The phosphorylated subunits are inserted into the apical membrane, forming channels through which water is taken up, and the water molecules are transported to the basolateral side of the cell and moved to the extracellular space and finally to the bloodstream. There are 12 known aquaporins, of which four aquaporins are

located in the kidney: AQP1 is located in the kidney capillary endothelia; AQP2 is located in the intracellular and apical kidney CD cells; AQP3 is located in the kidney; and AQP11 is located in the proximal tubule. Some of these are located in other tissues, as well. AQP2 is the signal transporter of water in the kidney and will be emphasized. A simplified drawing of an AQP is shown in Fig. 12.20. The locations of AQP1, AQP2, AQP3, and AQP4 in the kidney are shown in Fig. 12.21. The rapid formation of AQP2 in the distal kidney is under the control of vasopressin (Arg-vasopressin, AVP) that is released from the posterior pituitary when water needs to be reabsorbed from the urine. Response of other AQPs, AQP3 and AQP4, which are continuously expressed in the basolateral membrane, do not respond to AVP acutely (Ikeda and Matsuzaki, 2015). Arginine vasopressin (AVP, or antidiuretic hormone, ADH) is a 9-amino acid peptide having the sequence: NH2-Cys1-Tyr-Phe-Gln-Asn5-Cys6-Pro-Arg-Gly9COOH. The two Cys residues (Cys1-Cys6) form a disulfide bond. AVP is secreted from the posterior pituitary in response to osmolality receptors in the hypothalamus. These receptors detect and increase in blood osmolality (increase of substances in blood, such as: Naþ, Kþ, Cl, urea, and glucose) or a decrease in blood volume. In addition to osmolality receptors in the hypothalamus, other organs sense increases in osmolality, such as the vascular organ of the lamina terminalis (located in the median part of the wall of the forebrain) and the macula densa region of the juxtaglomerular apparatus in the kidney. AVP is secreted together with another protein, neurophysin II, which protects the integrity of AVP until the complex dissociates in the blood before AVP alone binds to its receptor in the basolateral membrane of principal cells in the renal CD. There are three AVP receptor subtypes, V1a (vascular smooth muscle, cardiac myocytes, brain, testis, superior cervical ganglion, liver, platelets, and renal medulla), V1b (or V3, in anterior pituitary, pancreas, and adrenal medulla), and V2 (basolateral cells of principal cells of the renal CD and to a lesser extent in the thick ascending limb of the loop of Henle and the vascular endothelium) (Ikeda and Matsuzaki, 2015). AVP, dissociated from neurophysin II, binds to and activates its V2R receptor in the principal cell basolateral membrane of the renal CD. This leads to the conversion of ATP to cAMP, which then coverts PKA to its active form. PKA phosphorylates subunits of AQP2 and AQP4 and then forms the AQP2 channel in the apical membrane. Four subunits constitute the AQP channel. This facilitates the uptake of water molecules through AQP2 that cross the cell cytoplasm to the basolateral membrane. Water molecules then pass through

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FIGURE 12.20 Simplified drawing of an aquaporin channel. Water molecules enter the channel (top) and flow to the center of the channel with the oxygen (red) facing downward. After further descent, the orientation of the oxygens is inverted by the central positively charged gate that attracts the oxygen (O2 when ionized), so the oxygens of the water molecule are facing upward. Reproduced with permission from Fig. 9 of Litwack, G., 2017a. Human Biochemistry. Academic Press.

FIGURE 12.21 The locations of specific aquaporins (AQPs) in the kidney. Reproduced with permission from Fig. 13, page 47 of Human Biochemistry by G. Litwack (2017d).

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FIGURE 12.23 Overall structure of aquaporin2 (AQP2). (A) Overview of the AQP2 tetramer with half helices formed by loop B and E highlighted in yellow. (B) Overview of the AQP2 tetramer from the intracellular side. The color scheme for each of the protomers is A, purple; B, magenta; C, pink; D, light pink. Reproduced with permission from Fig. 1 of Frick, A., Eriksson, U.K., de Mattia, F., Oberg, F., Hedfalk, K., Neutze, R., de Grip, W.J., Deen, P.M.T., Tornroth-Horsefield, S., 2014. X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking. Proc. Natl. Acad. Sci. U. S. A. 111, 6305e6310.

FIGURE 12.22

Mechanisms of short- and long-term regulations of aquaporin 2 (AQP2). Arginine vasopressin (AVP) released from the posterior pituitary binds to the V2 receptor (V2R) on the basolateral membrane of the principal cells of the renal collecting duct. Activation of the receptor stimulates adenylate cyclase activity to produce cAMP, which activates PKA, which phosphorylates AQP2. AQP2 then accumulates on the apical membrane through vesicular trafficking from intracellular storage vesicles expressing AQp2. Water molecules flowing into collecting duct cells through AQP2 flow out to the interstitium via the basolateral water channels AQP3 and/or AQP4. PKS also phosphorylates CRE-binding protein (CREB). Phosphorylated CREB stimulates transcription of the AQP2 gene. AVP also regulates the abundance of renal AQP2 via excretion of exosomal AQP2. Responding to AVP, multivesicular bodies (MVBs) fuse with the plasma membrane, and intraluminal vesicles (exosomes) are released to the urinary space. Reproduced with permission from Fig. 4 of Ikeda, M., Matsuzaki, T., 2015. Regulation of aquaporins by vasopressin in the kidney. Vitam. Horm. 98, 307e337.

AQP3 and AQP4 channels residing in the basolateral membrane, and water molecules enter the bloodstream, expanding blood volume. This scenario is recapitulated in Fig. 12.22. In Fig. 12.23 is shown the X-ray crystal structure of human tetrameric AQP2.

Transport of Caþþ from the intestinal lumen into the enterocyte, across the cytoplasm through the basolateral membrane under conditions of low or high levels of Ca2þ in the diet. When the amount of Ca2þ in the diet is low, Ca2þ is transported into the intestinal cell through the TRPV6 apical channel, moving Ca2þ into the cytosol, where it is bound to the transport protein calbindin, whose synthesis results from the action of the vitamin D receptor (VDR) to produce calbindin mRNA. Calbindin-Ca2þ moves to the basolateral side of the cell where calcium is transported through the PMCA channel and released to the extracellular space and then to the bloodstream. When the diet contains high levels of calcium, VDR may not be required for the passive transport of Ca2þ. Reproduced with permission from Fig. 19.53 of Litwack, G., 2017b. Human Biochemistry. Academic Press, Figure 3.9, p. 45.

FIGURE 12.24

8. TRPV EPITHELIAL CALCIUM TRANSPORT CHANNELS (TRPV5 AND TRPV6)

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FIGURE 12.25 Above (A, B): Surface structure of the human TRPV6 at 3.6 A˚ cryo-electron microscope reconstruction with density shown at 0.035 threshold level (UCSF Chimera) representing hTRPV6 subunits colored green, cyan, pink, and yellow; lipid in purple and ions in red. Cartoons represent the structural changes associated with TRPV6 channel gating. Transition from the closed to open state, stabilized by the formation of salt bridges (dashed lines), leads to permeation of ions (green spheres) and is accompanied by a local a-to-p helical transition in the S6 that maintains the selectivity filter conformation, while the lower part of S6 bends by 11 and rotates by 100 . These movements result in a different set of residues (blue vs. pink symbols) lining the pore in the vicinity of the channel gate. Reproduced with permission from Fig. 1d and e from McGoldrick, L.L., Singh, A.K., Saotome, K., Yelshanskya, M.V., Twomey, E.C., Grassucci, R.A., Sobolevsky, A.I., 2018. Opening of the human epithelial calcium channel TRPV6. Nature 553, 2333e237. Reproduced with permission from Fig. 5 of McGoldrick, L.L., Singh, A.K., Saotome, K., Yelshanskya, M.V., Twomey, E.C., Grassucci, R.A., Sobolevsky, A.I., 2018. Opening of the human epithelial calcium channel TRPV6. Nature 553, 2333e237.

8. TRPV EPITHELIAL CALCIUM TRANSPORT CHANNELS (TRPV5 AND TRPV6) The regulation of Ca2þ homeostasis is essential for several physiologic functions. TRP proteins comprise a large family of polymodal nonselective cation channels. The TRP vanilloid (TRPV) subfamily consists of six homologous members with diverse functions. Vanilloid refers to the content of a vanillin group representing compounds like capsaicin. TRPV1 through TRPV4 are nonselective cation channels that are involved in nociception. TRPV5 and TPPV6 channels are involved in epithelial Ca2þ homeostasis. Fig. 12.24 shows the movement of Ca2þ from the intestinal lumen into the enterocyte. Ca2þ is transported into the intestinal cell through the apical TRPV6 channel, and the cytosolic

Ca2þ binds to the transport protein calbindin, whose synthesis is induced by the action of the vitamin D receptor (VDR). Calbindin-Ca2þ crosses the cytoplasm to the basolateral side of the cell where Ca2þ is extruded into the extracellular space through the PMCA1b channel and gains access to the circulation. Fig. 12.25 shows the X-ray crystal structure of TRPV6. The TRP structures contain repeats of ankyrin. The regulation of TRPV6 in the kidney is by dietary Mg2þ content and by 17b-estradiol. 1,25Dihydroxyvitamin D3 and PTH do not regulate the level of renal TRPV6 mRNA. 17b-Estradiol upregulates the level of renal TRPV6 mRNA, but the level of mRNA for TRPV7 is unaffected. In ovariectomized rats, the level of TRPV6 mRNA is greatly reduced, and after estradiol treatment, the mRNA level is normalized (Groenestege et al., 2006).

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FIGURE 12.26 Model of the CFTR channel. The CFTR consists of 1480 amino acid residues with a molecular weight of 170,000. It is a membrane-bound glycoprotein and a member of the ATP-binding cassette (ABC) superfamily. In addition to CFTR, this superfamily includes such clinically important proteins as P-glycoprotein, multidrug resistance associated protein, and the TAP transporters. CFTR contains six membrane-spanning regions, each of which is connected to an ATPbinding domain (nucleotide-binding domains NBD1 and NBD2). Between NBD1 and NBD2 is a regulatory R domain that is unique to CFTR among members of the ABC superfamily. The R domain contains protein kinase A (PKA) and protein kinase C (PKC) activities and is phosphorylated to activate the ion channel. There are several sites in the R domain for phosphorylation by cAMP-activated PKA or PKC. Reproduced with permission from Fig. 1 of Litwack, G., 2017c. Human Biochemistry. Academic Press, Figure 19.53, p. 633.

The main sites of Mg2þ reabsorption occur principally in the kidney and to a lesser extent in the lung, cecum, and colon. During dietary Mg2þ restriction, 17b-estradiol increased renal TRPV6 mRNA, whereas a Mg2þenriched diet stimulated TRPV6 mRNA in the colon, in support of the concept of a gatekeeper function of TRPV6 in transepithelial Mg2þ transport (Groenestege et al., 2006).

9. CHLORIDE TRANSPORT, CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CFTR is an ABC transporter-class (ATP-binding cassette family) ion channel protein that primarily conducts Cl ions across epithelial cell membranes. Mutation of the gene that encodes CFTR leads to cystic fibrosis. The epithelial surface hydration and regulation of luminal pH is maintained by CFTR chloride channel. This is especially important in the airways, the GI tract, and the sweat and salivary glands. Inactive CFTR causes impaired Cl transport with elevated Naþ absorption into airway epithelial cells. In the apical surface of duodenal villus epithelial cells, the insertion of the CFTR channel from

storage vesicles is stimulated by vasoactive intestinal peptide (VIP) (Ameen et al., 1999). CFTR is regulated by estrogen in the uterine epithelial cell in vitro and in vivo (Rochwerger and Buchwald, 1993; Rochwerger et al., 1994). Consistent with the cyclic profile of ovarian hormones, there is an estrous cycle-dependent expression of CFTR at proestrous in the vagina and cervix, and at estrus in the uterus. CTFR is downregulated by progesterone (Mularoni et al., 1995). Fig. 12.26 shows a model of the CFTR channel. The CFTR channel is activated by phosphorylation of the R subunit by PKA. Endogenous membrane-attached phosphatases can partly dephosphorylate the R domain, but as long as ATP is present the channel will remain open. Phosphorylation of the R domain by PKA is required for the gating action of the channel, and this phosphorylation increases channel activity at least 100fold. Both PKA and PKC are able to phosphorylate the R domain; however, the phosphorylation by PKC produces meager channel opening; the activity of PKC may be required for the subsequent channel activation by PKA (Gadsby et al., 2006). Besides its function as a Cl channel, CFTR is able to regulate other channels and transporters. It can mediate the cAMP regulation of the amiloridesensitive Naþ channels, outwardly rectifying Cl channels, Cl/HCO3 exchanger, and the ROMK Kþ channel either through direct or indirect interactions. There are some proteins that are able to couple CFTR to the cytoskeleton and may be involved in apical membrane localization; these proteins are PDZbinding domain proteins (PDZ motif, T/S-X-V/L), including the Naþ/Hþ exchanger regulatory factor and ezrin-binding protein 50 that bind to the Cterminus of CFTR (Akabas, 2000).

10. PENDRIN, APICAL IODIDE TRANSPORTING CHANNEL IN THYROID FOLLICULAR CELL (THYROCYTE) The thyrocyte transports iodide ion (I) from the bloodstream through the symporter, NIS (sodium/iodide symport), in its basolateral membrane along with 2Naþ. NIS function is powered, on the basolateral membrane, by Naþ/Kþ ATPase. The iodide ion crosses the cytoplasm of the thyrocyte to the apical membrane where it is transported into the follicular lumen by the pendrin channel. Thyroglobulin is also passively transported from the cytoplasm to the follicular lumen, where it serves as the precursor of the thyroid hormones, thyroxine and triiodothyronine. Intermediate steps involve thyroid peroxidase (TPO). Eventually the thyroid hormones efflux through the basolateral

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FIGURE 12.27 Main steps in thyroid hormone synthesis. At the basolateral membrane of thyroid follicular cells, which form the follicles, iodide is transported into thyrocytes by the NIS (Naþ/I symporter). NIS is dependent on the sodium gradient created by the Naþ/Kþ ATPase. At the apical membrane, iodide efflux is, in part, mediated by pendrin (PDS) channel. At the cell-colloid interface, iodide is oxidized by TPO (thyroid peroxidase) in the presence of H2O2. H2O2 is produced by the calcium- and reduced-nicotinamide adenine dinucleotide phosphate-dependent (NADPH) enzyme DUOX2 (dual oxidase 2). DUOX2 requires a specific maturation factor DUOXA2. Thyroglobulin (TG), which is secreted into the follicular lumen, serves as matrix for synthesis of T4 (thyroxine) and T3 (triiodothyronine). First, TPO catalyzes iodination of selected tyrosyl residues (organification), which results in the formation of MIT (monoiodotyrosine) and DIT (diiodotyrosine). Subsequently, two iodotyrosines are coupled to form either T4 or T3 in a reaction that also is catalyzed by TPO. Iodinated thyroglobulin is stored as colloid in the follicular lumen. Upon a demand for thyroid hormone secretion, substituted thyroglobulin is internalized into the follicular cell by pinocytes and digested in lysosomes, which generates T4 and T3 that are released into the bloodstream through unknown mechanisms. The unused MIT and DIT are retained in the cell and deiodinated by the iodotyrosine dehalogenase (DEHAL1). The released iodide is recycled for future thyroid hormone synthesis. Reproduced with permission from Fig. 1 of Bizhanova, A., Koop, P., 2009. The sodium-iodide symporter NIS and pendrin in iodide homeostasis of the thyroid. Endocrinology 150, 1084e1090.

membrane, perhaps by passive diffusion, although this function is not well understood. However, in certain cells, the multidrug transporter (MDR) has been shown to extrude triiodothyronine (Mitchell et al., 2005). The thyroid hormones circulate in the bloodstream and enter target cells where triiodothyronine (the active hormone) binds to the thyroid hormone receptor in the cell nucleus to activate gene expression. Thyroxine, whose blood concentration far exceeds that of triiodothyronine, can be converted intracellularly to

triiodothyronine by the action of cytoplasmic deiodinases (also, dehalogenases) in target cells. Fig. 12.27 outlines the movements and fates of iodide ion in the thyrocyte. In addition to the thyroid gland, pendrin also is found in the inner ear, mammary gland, kidney, and airways. Deficiency of pendrin action can lead to the Pendred syndrome, which is nonsyndromic deafness and thyroid goiter. In the mammary gland, pendrin accounts for the iodide content of milk. Prolactin (PRL) in

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FIGURE 12.28 Regulation of epithelial Naþ channel (ENaC) and pendrin by aldosterone in the collecting duct. In a state of aldosterone excess, aldosterone binds to the mineralocorticoid receptor (MR) and activates ENaC in principal cells, which promotes electrogenic Naþ reabsorption and lumen-negative potential in the collecting duct. This drives Kþ secretion through apical Kþ channel renal outer medullary Kþ channel (ROMK) in principal cells, resulting in the decrease in plasma Kþ levels. Hypokalemia causes MRs842p dephosphorylation in b-intercalated cells, allowing aldosterone binding to MR in these cells and increasing pendrin at the plasma membrane. Reproduced with permission from Fig. 6 of Xu, N., Hirohama, D., Ishizawa, K., Chang, W.X., Shimosawa, T., Fujita, T., Uchida, S., Shibata, S., 2017. Hypokalemia and pendrin induction by aldosterone. Hypertension 69, 855e862.

physiologic concentrations has been shown to increase the expression of the pendrin transporter in mammary tissues taken from 12- to 14-day pregnant mice. The PRL enhanced expression of pendrin in mammary tissues could be negated by the pendrin transport inhibitors furosemide, probenecid, and DIDS (4,40 -diisothiocyanatostilbene-2,2-cisulfonate). Thus, PRL stimulation of pendrin activity is an essential element in the PRL stimulation of iodide uptake into milk (Rillema and Hill, 2003). The pendrin transporter is comprised of 780 amino acid residues and has a molecular weight of 73 kDa.

together with hypokalemia can induce pendrin in the kidney. Also, decreased Kþ levels in the circulation cause the Naþ/Cl cotransporter (ENaC) levels to

10.1 Aldosterone The apical membrane of intercalated cells of the connecting tubule and CCD of the kidney cortex contain pendrin. These cells have high levels of pendrin mRNA. Although pendrin transports iodide ion in the thyrocyte apical membrane, in the kidney cortex, pendrin is a chloride ion-bicarbonate ion transporter (Cl/HCO3 transporter). Aldosterone

FIGURE 12.29

The crystal structure of the sodium/iodide symporter (NIS). Reproduced from Fig. 1b of Ravera, S., Reyna-Neyra, A., Ferrandino, G., Amzel, L.M., Carrasco, N., 2017. The sodium/iodide symporter (NIS): molecular physiology and preclinical and clinical applications. Annu. Rev. Physiol. 79, 261e289.

10. PENDRIN, APICAL IODIDE TRANSPORTING CHANNEL IN THYROID FOLLICULAR CELL (THYROCYTE)

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FIGURE 12.30 Crystal structure of the MDR P-glycoprotein. Upper figure shows the front view of the P-glycoprotein. Transmembrane (TM) domains 1e6 are shown. The N- and C-terminal halves of the molecule are colored yellow and blue, respectively. TM4-5 and TM10-11 (not shown) cross over to form intertwined interfaces that stabilize the inward-facing conformation. Horizontal bars represent the approximate positioning of the lipid bilayer. The N- and C- termini are labeled. The nucleotide-binding domains (NBDs) are labeled. The bottom figures show a model of substrate transport by P-glycoprotein (MDR). (A) Substrate (magenta) partitions into the bilayer from outside of the cell to the inner leaflet and enters the internal drug-binding pocket through an open portal. The residues in the drug-binding pocket (cyan spheres) interact with OZ59 compounds and verapamil in the inward-facing conformation. (B) ATP (yellow) binds to the NBDs, causing a large conformational change, presenting the substrates and drug-binding site(s) to enter the outer leaflet/extracellular space. In this model of P-glycoprotein, which is based on the outward-facing conformation of MsbA and Sav 1866 (two other compounds), exit of the substrate to the inner leaflet is sterically occluded, providing unidirectional transport to the outside. Reproduced with permission from Fig. 1 in part of Aller, S.G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., Harrell, P.M., Trinh, Y.T., Zhang, Q., Urbatsch, I.L., Chang, G., 2009. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 323, 1718e1722. (B) Reproduced with permission from Fig. 4 of Aller, S.G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., Harrell, P.M., Trinh, Y.T., Zhang, Q., Urbatsch, I.L., Chang, G., 2009. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 323, 1718e1722.

increase. Phosphorylation of the MR prevents the binding of its ligand, aldosterone. Hypokalemia prevents phosphorylation of MR in intercalated cells. This results in enhanced pendrin response to aldosterone and explains the coordinated action of aldosterone and hypokalemia in the regulation of pendrin. Furthermore, in pendrin knockout mice, the plasma

concentration of Kþ is lowered significantly after infusion of aldosterone, which demonstrates that pendrin alleviates hypokalemia when aldosterone is present in excess. Lowered circulatory Kþ levels enhance pendrin induction by aldosterone together with the Naþ/Cl cotransporter (ENaC). While this counteracts progression of hypokalemia, hypertension is present in

306

12. HORMONAL REGULATION OF EPITHELIAL SODIUM CHANNEL (ENAC) AND OTHER NONNEURONAL EPITHELIAL ION CHANNELS

primary aldosterone excess (Xu et al., 2017). These activities are illustrated in Fig. 12.28

11. SODIUM/IODIDE TRANSPORTER The sodium/iodide transporter (NIS) transports iodide ion (I) in the basolateral membrane of the thyrocyte, but NIS also mediates active iodide ion transport in salivary glands, gastric mucosa, and lactating mammary gland. Thyroid stimulating hormone (TSH) increases NIS mRNA in FRTL-5 thyrocyte cells at 3e6 h and reaches a maximum in 24 h where NIS has increased six times the basal level. Elevation of NIS concentration in these cells was completely blocked by inhibitors of transcription, confirming a time-course characteristic of genomic expression. Either this is a direct result of the action of TSH or it is the result of a TSH-regulated factor (Kogai et al., 1997). The crystal structure of NIS is shown in Fig. 12.29.

12. MDR, MULTIDRUG RESISTANCE CHANNEL, P-GLYCOPROTEIN MDR is an efflux pump that moves drugs and xenobiotics out of the cell, and its specificity is broad. In the mouse genome, there are three genes encoding MDR: mdr1, mdr2, and mdr3. In the human, there are two genes encoding MDR: MDR1 and MDR2. In the human, only MDR1 can confer multidrug resistance by transfection into otherwise drug-sensitive cells. In mouse hepatoma cells, dexamethasone elevated the mRNAs of mdr1 and mdr3, but there was no induction of mdr2 mRNA. In human hepatoma HepG2 cells, the mRNA for MDR1 was induced by treatment with dexamethasone. Dexamethasone was inactive in nonhepatoma HeLa cells (Zhao et al., 1993). Experiments in the human have been carried out where an antitumor drug is attached to an antibody specific for a unique cell surface antigen, coupled to a transfecting protein. The antitumor drug enters the tumor cell but is quickly eliminated from the cell by the efflux action of MDR1 abolishing any antitumor activity. In the uterine secretory epithelium, MDR mRNA and P-glycoprotein are induced at high levels by estrogen combined with progesterone, the major steroid hormones of pregnancy (Arceci et al., 1990). The crystal structure of the MDR P-glycoprotein is shown in Fig. 12.30. Although there are other epithelial ion channels, reports of their regulatory hormones and mechanisms appear to be at an early stage. For that reason, the discussion of their hormonal regulation has not been reviewed.

References Abiria, S.A., Krapivinsky, G., Sah, R., Santa-Cruz, A.G., Chaudhuri, D., Zhang, J., Clapham, D.E., 2017. TRPM7 senses oxidative stress to release Zn(2þ) from unique intracellular vesicles. Proc. Natl. Acad. Sci. U. S. A. 114, E6079eE6088. Abriel, H., Horisberger, J.-D., 1999. Feedback inhibition of rat amiloride-sensitive epithelial sodium channels expressed in Xenopus laevis oocytes. J. Physiol. 516 (1), 31e43. Akabas, M.H., 2000. Cystic fibrosis transmembrane conductance regulator. Structure and function of an epithelial chloride channel. J. Biol. Chem. 275, 3729e3732. Aller, S.G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., Harrell, P.M., Trinh, Y.T., Zhang, Q., Urbatsch, I.L., Chang, G., 2009. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 323, 1718e1722. Ameen, N.A., Martensson, B., Bourguinon, L., Marino, C., Isenberg, J., McLaughlin, G.E., 1999. CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo. J. Cell Sci. 112, 887e894. Arceci, R.J., Baas, F., Raponi, R., Horwitz, S.B., Housman, D., Croop, J.M., 1990. Multidrug resistance gene expression is controlled by steroid hormones in the secretory epithelium of the uterus. Mol. Reprod. Dev. 25, 101e109. Bacic, D., Schulz, N., Biber, J., Kaissling, B., Murer, H., Wagner, C.A., 2003. Involvement of the MAPK-kinase pathway in the PTHmediated regulation of the proximal tubule type IIa Naþ/Pi cotransporter in mouse kidney. Pflu¨gers Arch. 446, 52e60. Baines, D., 2013. Kinases as targets for ENaC regulation. Curr. Mol. Pharmacol. 6, 50e64. Beguin, P., Beggah, A.T., Chibalin, A.V., Burgener-Kairuz, P., Jaisser, F., Mathews, P.M., Rossier, B.C., Cotecchia, S., Geering, K., 1994. Phosphorylation of the Na,K ATPase alphasubunit by PKA and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation. J. Biol. Chem. 269, 24437e24445. Beenken, A., Mohammadi, M., 2012. The structural biology of the FGF19 subfamily. Adv. Exp. Med. Biol. 728, 1e24. Bertorello, A.M., Katz, A.I., 1993. Short-term regulation of renal Na-KATPase activity: physiological relevance and cellular mechanisms. Ren. Physiol. 265, F743eF755. Bentzen, B.H., Olesen, S.-P., Ronn, L.C.B., Grunnet, M., 2014. BK channel activators and their therapeutic perspectives. Front. Physiol. 5. https://doi.org/10.3389/fphys.2014.00389. Bizhanova, A., Koop, P., 2009. The sodium-iodide symporter NIS and pendrin in iodide homeostasis of the thyroid. Endocrinology 150, 1084e1090. Blaukat, A., 2003. Mechanisms of ENaC regulation and clinical implications. J. Am. Soc. Nephrol. 19, 1845e1854. Bledsoe, R.K., Madauss, K.P., Holt, J.A., et al., 2005. A ligandmediated hydrogen bond network required for the activation of the mineralocorticoid receptor. J. Biol. Chem. 280, 31283e31293. Boim, M.A., Ho, K., Shuck, M.E., Bienkowski, M.J., Block, J.H., Slightom, J.L., Hebert, S.C., 1995. ROMK inwardly rectifying ATPsensitive Kþ channel II. Cloning and distribution of alternative forms. Am. J. Physiol. 268 (6 Pt 2), F1132eF1140. Bugaj, V., Mironova, E., Kohan, D.E., Stockand, J.D., 2012. Collecting duct-specific endothelin B receptor knockout increases ENaC activity. Am. J. Physiol. Cell Physiol. 302, C188eC194. Bugaj, V., Pochynyuk, O., Mironova, E., Vandewalle, A., Medina, J.L., Stockand, J.D., 2008. Regulation of the epithelial Naþ channel by endothelin-1 in rat collecting duct. Am. J. Physiol. Renal. Physiol. 295, F1063eF1070. Canessa, C.M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J.D., Rossier, B.C., 1994. Amiloride-sensitive epithelial

REFERENCES

Naþ channel is made of three homologous subunits. Nature 367, 463e467. Cao, G., Thebault, S., van der Wijst, J., van der Kemp, A., Lasonder, E., Bindels, R.J., Hoenderop, J.G., 2008. RACK1 inhibits TRPM6 activity via phosphorylation of the fused alpha-kinase domain. Curr. Biol. 18, 168e176. Chen, G., Liu, Y., Goetz, R., Fu, L., Jayaraman, S., Hu, M.-C., Moe, O.W., Liang, G., Li, X., Mohammadi, M., 2018. aKlotho is a non-enzymatic molecular scaffold for FGF23 hormone signaling. Nature 553, 461e466. Cole, A., 1999. Parathyroid hormone activates mitogen-activated protein kinase in opossum kidney cells. Endocrinology 140, 5771e5779. Cornelius, R.J., Wen, D., Li, H., Yuan, Y., Wang-France, J., Warner, P.C., Sansom, S.C., 2015. Low Na, high K diet and the role of aldosterone in BK-mediated K excretion. PLoS One 10, e0115515. https:// doi.org/10.1371/journal.pone.0115515. Cunningham, R., Biswas, R., Brazie, M., Steplock, D., Shenolikar, S., Weinman, E.J., 2009. Signaling pathways utilized by PTH and dopamine to inhibit phosphate transport in mouse renal proximal tubule cells. Am. J. Physiol. Renal. Physiol. 296, F355eF361. D’Adamio, F., Zollo, O., Moraca, R., Ayoldi, E., Bruscoli, S., Barytoli, A., Riccardi, C., 1997. A new dexamethasone-induced gene of the leucine zipper family protects T lymphocytes from TCR/CD3activated cell death. Immunity 7, 803e812. Derfoul, A., Robertson, N.M., Hall, D.J., Litwack, G., 2000. The N-terminal domain of the mineralocorticoid receptor (MR) modulates inhibition of both MR and the glucocorticoid receptor mediated transactivation from the native Na/K ATPase beta 1 target gene promoter. Endocrine 13, 287e295. Derfoul, A., Robertson, N.M., Lingrel, J.B., Hall, D.J., Litwack, G., 1998. Regulation of the human Na/K-ATPase beta1 gene promoter by mineralocorticoid and glucocorticoid receptors. J. Biol. Chem. 273, 20702e20711. Diakov, A., Korbmacher, C., 2004. A novel pathway of epithelial sodium channel activation involves a serum- and glucocorticoidinducible kinase consensus motif in the C-terminus of the channel’s alpha-subunit. J. Biol. Chem. 270, 38134e38142. Dick, G.M., Sanders, K.M., 2001. (Xeno)estrogen sensitivity of smooth muscle BK channels conferred by the regulatory b1 subunit. J. Biol. Chem. 276, 44835e44840. Erben, R.G., Andrukhova, O., 2017. FGF23-klotho signaling axis in the kidney. Bone 100, 62e68. https://doi.org/10.1016/ j.bone.2016.09.010. Feletou, M., 2009. Calcium-activated potassium channels and endothelial dysfunction: therapeutic options? Br. J. Pharacol. 156, 545e562. Forschi, M., Sorokin, A., Pratt, P., McGinty, A., La Villa, G., Franchi, F., et al., 2001. PreproEndothelin-1 expression in human mesangial cells: evidence for a p38 mitogen-activated protein-kinase/protein kinases-C-dependent mechanism. J. Am. Soc. Nephrol. 12, 1137e1150. Frick, A., Eriksson, U.K., de Mattia, F., Oberg, F., Hedfalk, K., Neutze, R., de Grip, W.J., Deen, P.M.T., Tornroth-Horsefield, S., 2014. X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking. Proc. Natl. Acad. Sci. U. S. A. 111, 6305e6310. Fujiwara, Y., Minor Jr., D.L., 2008. X-ray crystal structure of a TRPM assembly domain reveals an anti-parallel four-stranded coiled-coil. J. Mol. Biol. 383, 854e870. Gadsby, D.C., Vergani, P., Csanady, L., 2006. The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 440, 477e483. Gattineni, J., Bates, C., Twombley, K., Dwarakanath, V., Robinson, M.L., Goetz, R., et al., 2009. FGF23 decreases renal Na-Pi-2a and Na-Pi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor. Am. J. Physiol. Renal. Physiol. 297, F282eF291. Gattineni, J., Friedman, P.A., 2015. Regulation of hormone-sensitive renal phosphate transport. Vitam. Horm. 98, 249e306.

307

Gao, G., van der Wijst, J., van der Kemp, A.-M., van Zeeland, F., Bindels, R.J., Hoenderop, J.G., 2009. Regulation of the epithelial Mg2þ channel TRPM6 by estrogen and the associated repressor protein of estrogen receptor activity (REA). J. Biol. Chem. 284, 14788e14795. Geering, K., 1991. The functional role of the b-subunit in the maturation and intracellular transport of Na-K-ATPase. FEBS Lett. 285, 189e193. Goetz, R., Bennken, A., Ibrahimi, O.A., Kalinina, J., Olsen, S.K., Eliseenkova, A.V., et al., 2007. Molecular insights into the klothodependent, endocrine mode of action of fibroblast growth factor 19 subfamily members. Mol. Cell Biol. 27, 3417e3428. Groenestege, W.M.T., Hoenderop, J.G., van den Heuvel, L., Knoers, N., Bindels, R.J., 2006. The epithelial magnesium channel transient receptor potential melastatin 6 is regulated by dietary magnesium content and estrogens. J. Am. Soc. Nephrol. 17, 1035e1043. Groenestege, W.M.T., Thebault, S., van der Wijst, J., van den Berg, D., Janssen, R., Tejpar, S., van den Heuvel, L.P., van Cutsem, E., Hoenderop, J.G., Knoers, N.V., Bindels, R.J., 2007. Impaired basolateral sorting of pro-EGF causes isolated recessive renal hypomagnesemia. J. Clin. Investig. 117, 2260e2267. Hall, J.M., 1997. Bradykinin receptors. Gen. Pharmacol. 28, 1e6. Hanugloku, I., Hanugloku, A., 2016. Epithelial sodium channel (ENaC) family: phylogeny, structure-function, tissue distribution, and associated inherited diseases. Gene 579, 95e132. Haussler, M.R., Whitfield, G.K., Haussler, C.A., Sabir, M.S., Khan, Z., Sandoval, R., Juruka, P.W., 2016. 1,25-Dihydroxyvitamin D and klotho: a tale of two renal hormones coming of age. Vitam. Horm. 100, 165e230. Hernando, N., Biber, J., Forster, I., Murer, H., 2005. Recent advances in renal phosphate transport. Ther. Apher Dial 9 (4), 323e327. Hoorn, E.J., Nelson, J.H., McCormick, J.A., Ellison, D.H., 2011. The WNK kinase network regulating sodium, potassium, and blood pressure. J. Am. Soc. Nephrol. 22, 605e614. Ikeda, M., Matsuzaki, T., 2015. Regulation of aquaporins by vasopressin in the kidney. Vitam. Horm. 98, 307e337. Jankowski, M., Hilfiker, H., Biber, J., Murer, H., 2001. The opossum kidney cell type IIa NaP(i) cotransporter is a phosphoprotein. Kidney Blood. Press. Res. 24, 1e4. Ji, H.L., Zhao, R.Z., Chen, Z.X., Shetty, S., Idell, S., Matalon, S., 2012. Delta-ENaC: a novel divergent amiloride-inhibitable sodium channel. Am. J. Physiol. Lung Cell Mol. Physiol. 303, L1013eL1026. Jiang, L., Chen, C., Yuan, T., Qin, Y., Hu, M., Li, X., Xing, X., Lee, X., Chen, L., 2014. Clinical severity of Gitelman syndrome determined by serum magnesium. Am. J. Nephrol. 39, 357e366. Kashlan, O.B., Sheng, S., Kleyman, T.R., 2005. On the interaction between amiloride and its putative a-subunit epithelial Naþ channel binding site. J. Biol. Chem. 280, 26206e26215. Kogai, T., Endo, T., Saito, T., Miyazaki, A., Kawaguchi, A., Onaga, T., 1997. Regulation by thyroid-stimulating hormone of sodium/iodide symporter gene expression and protein levels in FRTL-5 cells. Endocrinology 138, 2227e2232. Kolla, V., Litwack, G., 2000. Transcriptional regulation of the human Na/K ATPase via the human mineralocorticoid receptor. Mol. Cell. Biochem. 204, 35e40. Kolla, V., Robertson, N.M., Litwack, G., 1999. Identification of a mineralocorticoid/glucocorticoid response element in the human Na/K ATPase alpha1 gene promoter. Biochem. Biophys. Res. Commun. 266, 5e14. Kunzelmann, K., Schreiber, R., Boucherot, A., 2001. Mechanisms of the inhibition of epithelial Naþ channels by CFTR and purigenic stimulation. Kidney Int. 60, 455e461. Kurosu, H., Kuro-o, M., 2008. The klotho gene family and the endocrine fibroblast growth factors. Curr. Opin. Nephrol. Hypertens. 17, 72e78. Kurosu, H., Ogawa, Y., Miyoshi, M., Yamamoto, M., Nandi, A., Rosenblatt, K.P., et al., 2006. Regulation of fibroblast growth factor-23 signaling by klotho. J. Biol. Chem. 281, 6120e6123.

308

12. HORMONAL REGULATION OF EPITHELIAL SODIUM CHANNEL (ENAC) AND OTHER NONNEURONAL EPITHELIAL ION CHANNELS

Latorre, R., Castillo, K., Carrasquel-Ursulaez, W., Sepulveda, R.V., Gonzalez-Nilo, F., Gonzalez, C., Alvarez, O., 2017. Molecular determinants of BK channel functional diversity and functioning. Physiol. Rev. 97, 38e87. Laursen, M., Yatime, L., Nissen, P., Fedosova, N.U., 2013. Crystal structure of the high affinity Naþ,Kþ-ATPase-ouabain complex with Mg2þ bound in the cation binding site. Proc. Natl. Acad. Sci. U. S. A. https://doi.org/10.1073/pnas.1222308110. Lederer, E.D., Sohi, S.S., Mathiesen, J.M., Klein, J.B., 1998. Regulation of expression of type II sodium-phosphate cotransporters by protein kinases A and C. Am. J. Physiol. Renal. Physiol. 275, F270eF277. Lederer, E.D., Sohi, S.S., McLeish, 2000. Parathyroid hormone stimulates extracellular signal-regulated kinase (ERK) activity through two independent signal transduction pathways: role of ERK in sodium-phosphate transport. J. Am. Soc. Nephrol. 11, 222e231. Leng, Q., MacGregor, G.G., Dong, K., Giebisch, G., Hebert, S.C., 2006. Subunit-subunit interactions are critical for proton sensitivity of ROMK: evidence in support of an intermolecular gating mechanism. Proc. Natl. Acad. Sci. U. S. A 103, 1982e1987. Li, M., Jiang, J., Yue, L., 2006. Functional characterization of homo- and heteromeric channel kinases TRPM6 and TRPM7. J. Gen. Physiol. 127, 525e537. Litwack, G., 2017a. Human Biochemistry. Academic Press. Litwack, G., 2017b. Human Biochemistry. Figure 3.9. Academic Press, p. 45. Litwack, G., 2017c. Human Biochemistry. Figure 19.53. Academic Press, p. 633. Litwack, G., 2017c. Human Biochemistry. Figure 18.1. Academic Press, p. 554. Litwack, G., 2017d. Human Biochemistry, Figure 13. Academic Press, 47. Litwack, G. (Ed.), 2016. Klotho. Academic Press. Mamenko, M., Zaika, O., Boukelmoune, N., Madden, E., Pochynyuk, O., 2015. Control of ENaC-mediated sodium reabsorption in the distal nephron by bradykinin. Vitam. Horm. 98, 137e154. Marth, J.P., Pedersen, B.P., Toustrup-Jensen, M.S., Sorensen, T.L.-M., Petersen, J., Andersen, J.P., Vilsen, B., Nissen, P., 2007. Nature 450, 1043e1049. McEneaney, V., Harvey, B.J., Thomas, W., 2007. Aldosterone rapidly activates protein kinase D via a mineralocorticoid receptor/EGFR trans-activation pathway of the M1 kidney CCD cell line. J. Steroid Biochem. Mol. Biol. 107, 180e190. McEneaney, V., Harvey, B.J., Thomas, W., 2008. Aldosterone regulates rapid trafficking of epithelial sodium channel subunits in renal cortical collecting duct cells via protein kinase D activation. Mol. Endocrinol. 22, 881e892. McGoldrick, L.L., Singh, A.K., Saotome, K., Yelshanskya, M.V., Twomey, E.C., Grassucci, R.A., Sobolevsky, A.I., 2018. Opening of the human epithelial calcium channel TRPV6. Nature 553, 2333-237. Mihailidou, A.S., Buhagiar, K.A., Rasmussen, H.H., 1998. Naþ influx and Na(þ)-K(þ) pump activation during short-term exposure of cardiac myocytes to aldosterone. Am. J. Physiol. 274, C175eC181. Mihailidou, A.S., Mardini, M., Funder, J.W., 2004. Rapid, nongenomic effects of aldosterone in the heart mediated by epsilon protein kinase C. Endocrinology 145, 773e780. Mitchell, A.M., Tom, M., Mortimer, R.H., 2005. Thyroid hormone export from cells: contribution of P-glycoprotein. J. Endocrinol. 185, 93e98. Moura, A.M., Worcel, M., 1984. Direct action of aldosterone on transmembrane 22Na efflux from arterial smooth muscle. Rapid and delayed effects. Hypertension 6, 425e430.

Muallem, S., Orson, W.M., 2007. When EGF is offside, magnesium is wasted. J. Clin. Investig. 117, 2086e2089. Mularoni, A., Beck, L., Sadir, R., Adessi, G.L., Nicollier, M., 1995. Down-regulation by progesterone of cystic fibrosis transmembrane regulator expression in endometrial epithelial cells: a study by competitive RT-PCR. Biochem. Biophys. Res. Commun. 217, 1105e1111. Muller, O.G., Parnova, R.G., Centeno, G., Rossier, B.C., Firsov, D., Horisberger, J., 2003. Mineralocorticoid effects in the kidney: correlation between alpha-ENaC, GILZ, and SGK1 mRNA expression and urinary excretion of Naþ and Kþ. J. Am. Soc. Nephrol. 14, 1107e1115. Nadler, M.J., Hermosura, M.C., Inabe, K., Perraud, A.L., Zhu, Q., Stokes, A.J., Kurosaki, T., Kinet, J.P., Penner, R., Scharenberg, A.M., Fleig, A., 2001. Nature 411, 590e595. Nowik, M., Picard, N., Stange, G., Capuano, P., Tenenhouse, H.S., Biber, J., et al., 2008. Renal phosphaturia during metabolic acidosis revisited: molecular mechanisms for decreased renal phosphate reabsorption. Pflu¨gers Archiv 457, 539e549. Pao, A.C., 2012. SGK regulation of renal sodium transport. Curr. Opin. Nephrol. Hypertens. 21, 534e540. Picard, N., Capuano, P., Stange, G., Mihailova, M., Kaissling, B., Murer, H., Biber, J., Wagner, C.A., 2010. Acute parathyroid hormone differentially regulates renal brush border membrane phosphate cotransporters. Pflugers Arch 460, 677e687. Ravera, S., Reyna-Neyra, A., Ferrandino, G., Amzel, L.M., Carrasco, N., 2017. The sodium/iodide symporter (NIS): molecular physiology and preclinical and clinical applications. Annu. Rev. Physiol. 79, 261e289. Reshkin, S.J., Forgo, J., Murer, H., 1990. Functional asymmetry in phosphate transport and its regulation in opossum kidney cells: parathyroid hormone inhibition. Pflu¨gers Archiv: Eur. J. Physiol. 416, 624e631. Rillema, R., Hill, M.A., 2003. Prolactin regulation of the pendrin-iodide transporter in the mammary gland. Endocrinol. Metab. https:// doi.org/10.1152/ajpendo.00383. Robert-Nicoud, M., Flahaut, M., Elalouf, J.M., Nicod, M., Salinas, M., Bens, M., Firsov, D., 2001. Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin. Proc. Natl. Acad. Sci. U. S. A. 98 (5), 2712e2816. Rochwerger, L., Buchwald, M., 1993. Stimulation of cystic fibrosis transmembrane regulator expression by estrogen in vivo. Endocrinology 133, 921e930. Rochwerger, L., Dhos, S., Parker, L., Foskett, J.K., Buchwald, M., 1994. J. Cell Sci. 107, 2439e2448. Satoh, T., Cohen, H.T., Katz, A.I., 1993. Different mechanisms of renal Na-K-ATPase regulation by protein kinases in proximal and distal nephron. Ren. Physiol. 265, F399eF405. Sackin, H., Narazashvili, M., Palmer, L.G., Krambis, M., Walters, D.C., 2005. Structural locus of the pH gate in the Kir1.1 inward rectifier channel. Biophys. J. 88, 2597e2606. Shigaev, A., Asher, C., Latter, H., Garty, H., Reuveny, E., 2000. Regulation of sgk by aldosterone and its effects on the epithelial Na(þ) channel. Am. J. Physiol. Renal. Physiol. 278 (4), F613eF619. Shimada, T., Hasegawa, H., Yamazaki, Y., Hino, R., Takeuchi, Y., et al., 2004. FGF23 is a potent regulator of vitamin D metabolism and phosphate homeostasis”. J. Bone Miner. Res. 19, 429e435. Sneddon, W.B., Gesek, F.A., Friedman, P.A., 2000. Obligate MAP kinase activation in parathyroid hormone stimulation of calcium transport but not calcium signaling. Endocrinology 141, 4185e4193. Blockade of ERK1/2. Sontia, B., Montezano, A., Paravicini, T., Tabet, F., Touyz, R.M., 2008. Downregulation of renal TRPM7 and increased inflammation and fibrosis in aldosterone-infused mice: effects of magnesium. Hypertension 51, 915e921.

FURTHER READING

Sorokin, A., Staruschenko, A., 2015. Inhibition of ENaC by endothelin1. Vitam. Horm. 98, 155e187. Soundararajan, R., Melters, D., Shih, I.C., Wang, J., Pearce, D., 2009. Epithelial sodium channel regulated by differential composition of a signaling complex”. Proc. Natl. Acad. Sci. U. S. A. 106, 7804e7809. Soundararajan, R., Pearce, D., Ziera, T., 2012. The role of ENaCregulatory complex in aldosterone-mediated sodium transport. Mol. Cell. Endocrinol. 350, 242e247. Stockand, J.D., Bao, H.-F., Schenck, J., Malik, B., Middleton, P., Schlanger, L.E., Eaton, D.C., 2000. Differential effects of protein kinase C on the levels of epithelial Naþ channel subunit proteins. J. Biol. Chem. 275 (33), 2576. Stockand, J.D., Staruschenko, A., Porchynyuk, O., Booth, R.E., Silverthorn, D.U., 2008. Insight toward epithelial Naþ channel mechanism revealed by the acid-sensing ion channel1 structure. IUMB Life 60, 620e628. Stokes, J.B., Sigmund, R.D., 1998. Regulation of ENaC mRNA by dietary NaCl and steroids: organ, tissue and steroid heterogeneity. Am. J. Physiol. 274 (6Pt 1), C1699eC1707. Tan, S.-J., Smith, E.R., Hewitson, T.D., Holt, S.G., Toussaint, N.D., 2014. The importance of klotho in phosphate metabolism and kidney disease. Nephrology 19, 439e449. Traebert, M., Vokl, H., Biber, J., Murer, H., Kaissling, B., 2000. Luminal and contraluminal action of 1-34 and 3-34 PTH peptides on renal type IIa Na-Pi cotransporter. Am. J. Physiol. Renal. Physiol. 278, F148eF154. Valinsky, W.C., Jolly, A., Miquel, P., Touyz, R.M., Shrier, A., 2016. Aldosterone upregulates transient receptor potential melastatin 7 (TRPM7). J. Biol. Chem. https://doi.org/10.1074/jbc.M116.735175. Valinsky, W.C., Touyz, R.M., Shrier, A., 2019. Aldosterone and ion channels. Vitam. Horm. 109, 105e132. Valverde, M.A., Rojas, P., Amigo, J., Cosmelli, D., Orio, P., Bahamonde, M.L., Mann, G.E., Vergara, C., Latorre, R., 1999. Acute activation of maxi-k channels (hSlo) by estradiol binding to the b subunit. Science 285, 1929e1931. Wen, D., Cornelius, R.J., Yuan, Y., Sansom, S.C., 2013. Regulation of BKalpha expression in the distal nephron by aldosterone and urine pH. Am. J. Physiol. Renal. Physiol. 305, F463eF476. Wouter, M., Groenstege, T., Hoenderop, J.G., van den Heuvel, L., Knoers, N., Bindels, R.J., 2006. The epithelial Mg2þ channel

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transient receptor potential melastatin 6 is regulated by dietary Mg2þ content and estrogens. J. Am. Soc. Nephrol. 17, 1035e1043. Xie, J., Sun, B., Du, J., Yang, W., Chen, H.-C., Overton, J.D., Runnels, L.W., Yu, L., 2011. Phosphatidylinositol 4,5-bisphosphate (PIP2) controls magnesium gatekeeper TRPM6 activity. Nat-Sci. Rep article 146. Xu, N., Hirohama, D., Ishizawa, K., Chang, W.X., Shimosawa, T., Fujita, T., Uchida, S., Shibata, S., 2017. Hypokalemia and pendrin induction by aldosterone. Hypertension 69, 855e862. Yogi, A., Callera, G.E., O’Connor, S., Antunes, T.T., Valinsky, W., Miquel, P., Touyz, R.M., 2013. Aldosterone signaling through transient receptor potential melastatin 7 (TRPM7) and its alpha-kinase domain. Cell. Signal. 25 (11), 2163e2175. https://doi.org/S08986568(13)00191-5 [pii]. https://doi.org/10.1016/j.cellsig.2013.07.002. Zaika, O., Mamenko, M., O’Neil, R.G., Pochynyk, O., 2011. Bradykinin acutely inhibits activity of epithelial Naþ channel in mammalian aldosterone-sensitive distal nephron. Am. J. Physiol. Renal. Physiol. 300, F1105eF1115. Zhao, M., Celerier, I., Jeanny, J.C., Jonet, I., Savoldelli, M., BeharCohen, F., 2012. Mineralocorticoid receptor is involved in rat and human ocular chorioretinopathy. J. Clin. Investig. 122, 2672e2679. Zhao, J.Y., Ikeguchi, M., Eckersberg, T., Kuo, M.T., 1993. Modulation of multidrug resistance gene expression by dexamethasone in cultured hepatoma cells. Endocrinology 133, 521e528. Ziera, T., Irlbacher, H., Fromm, A., Latouche, C., Krug, S.M., Fromm, M., Borden, S.A., 2009. Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key role in the regulation of the epithelium sodium channel. FASEB J. 23 (11) https://doi.org/ 10.1096/fj.09-134759.

Further Reading CSLS (Editor), Introduction to Life Science. The University of Tokyo, 11.6 Cellular Adhesion and Tissue Architecture, csis-text3.c.utokyo.ac.jp. Saotome, K., Singh, A.K., Yelshanskaya, M.V., Sobolevsky, A., 2016. Crystal structure of the epithelial calcium channel TRPV6. Nature 534, 506e511.

C H A P T E R

13 Thymosins Haruka Kobayashi1,2, Yue Yu1,3, David E. Volk1 1

Institute of Molecular Medicine, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; 2Tokushima University Faculty of Medicine, Tokushima, Tokushima, Japan; 3Shanghai Jiao Tong University School of Medicine, Shanghai, China

1. STRUCTURES OF THYMOSIN PROTEINS The functional alpha and beta thymosins are very short proteins consisting of only 28 amino acids in the case of a-thymosin-1 (Ta1), also called thymalfasin, and around 43 amino acids for the b-thymosins (Hoch and Volk, 2016). The lack of structure in the thymosins can be attributed to their lack of aromatic amino acids and sulfur-containing amino acids, as well as their high content of acidic amino acids. Although their names suggest that they all are related, they are not.

1.1 Structures of Uncomplexed Thymosin Proteins 1.1.1 Prothymosin-a (ProTa), Parathymosin, and a-Thymosin-1 (Ta1) The active Ta1 peptide first discovered by Goldstein in calf thymus (Goldstein et al., 1977) is derived from a 110-amino acid precursor protein called prothymosin a (ProTa), which contains 54 acidic amino acids and no aromatic amino acids (Haritos et al., 1984; Goodall et al., 1986). Under physiologic pH and native conditions, ProTa and Ta1 have random coil structures (Watts et al., 1990; Cordero et al., 1992; Gast et al., 1995). However, secondary structure or structural collapse can be induced by low pH (Watts et al., 1990; Uversky et al., 1999), and helical content (up to 69% for ProTa) is enhanced in the presence of an organic solvent like 50% trifluoroethanol (Gast et al., 1995). Charge neutralization of ProTa by Zn2þ ions also induces helical structure, about 12%, in ProTa at 12.5 mM concentration, although many other metal ions do not have

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00013-4

this effect even at much higher concentrations (Uversky et al., 2000). Parathymosin (Zn2þ-binding protein, macromolecular translocation inhibitor II), a protein with high homology to and some identity with ProTa (Haritos et al., 1985), has a very different tissue distribution in the body (Clinton et al., 1989) compared to ProTa but behaves structurally very similar to ProTa. That is, it is natively unstructured but can bind with other proteins, such as the glucocorticoid receptor (Okamoto and Isohashi, 2000, 2005). The shorter Ta1 peptide behaves much like its larger precursor protein ProTa (Grottesi et al., 1998; ElizondaRiojas et al., 2011; Volk et al., 2012; Nepravishta et al., 2015; Hoch and Volk, 2016). Under native conditions Ta1 is a disordered peptide, but in the presence of trifluoroethanol (Grottesi et al. 1998; Elizondo-Riojas et al., 2011) or sodium dodecylsulfate (Nepravishta et al., 2015), two helices are formed (Fig. 13.1) ranging approximately from amino acids A3eE10 and from K14eE27. A more detailed description of these and other thymosin structures is available (Hoch and Volk, 2016). 1.1.2 Beta Thymosin Proteins 1.1.2.1 Thymosin Beta 4 The high charges and identity present in beta thymosins b4, b5, b9, b10, b11, b12, and b15 (see Section 2) leads them to exhibit similar structural behavior showing little to no structure under physiologic conditions (Zarbock et al., 1990; Czisch et al., 1993). However, structure can be induced by low temperatures or by interactions with other proteins. Thymosin b4 is a 43 amino acid peptide that is unstructured at ambient temperatures in aqueous buffers, but Czisch et al. (1993) found two helical areas, namely

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FIGURE 13.1 NMR model structure of thymosin a with the N-terminus at the left and the right terminus at the right. Atomic coordinates were obtained from the Protein Data Bank (PDB ID 2MNQ, Nepravishta et al., 2015).

residues 5 to 19 and 30 to 37, at low temperatures (> CNP; NPR2 ¼ CNP >> ANP > BNP; NPR3 ¼ ANP > CNP > BNP (Suga et al., 1992).

3. PHYSIOLOGIC FUNCTIONS OF NPS In physiologic conditions, cardiac NPs regulate cardiovascular homeostasis and metabolic activities (Schlueter et al., 2014). ANP is predominantly synthesized in atrial cardiomyocytes. BNP is synthesized in ventricular cardiomyocytes and remarkably upregulated in the patients with heart failure (Mukoyama et al., 1991). The synthesis of ANP and BNP is finely tuned in the heart to maintain the circulatory homeostasis. Therefore, plasma ANP and BNP reflect the entire circulatory systems, cardiac contractility, and size of the hearts. Heterozygous ANP-deficient (Nppaþ/) mice show salt-sensitive hypertension (John et al., 1995), while BNP-deficient (Nppb/) mice do not (Tamura et al., 2000). Nppb/ mice show cardiac fibrosis, suggesting BNP acts as a paracrine factor in physiologic conditions.

3.1 Secretion of ANP ANP is synthesized in atrial cardiomyocytes and subsequently stored in specific atrial granules. ANP secretion is stimulated by the wall stretch resulting from increased blood volume (Bruneau et al., 1997). The details of molecular mechanisms for how stretch stimulation exerts secretion of cardiac granules have not been fully clarified. Stretch-activated ANP secretion is partly mediated through pertussis toxin-sensitive Gi/o proteins (Bensimon et al., 2004). Stretch-sensitive ion channels might function as mechanosensors in cardiomyocytes and might contribute to ANP secretion (Han et al., 2008; Zhang et al., 2008). ANP secretion is also induced

by the activation of G protein-coupled receptor agonists, such as endothelin1 (ET1) and alpha adrenergic agents (Bruneau et al., 1996; Fukuda et al., 1988). Those agonist-stimulated ANP secretions are mediated through the activation of Gq proteins (Bensimon et al., 2004).

3.2 Clearance and Degradation of NPs NPs are removed from circulatory systems. NPR3 facilitates internalization of NPs followed by lysosomal degradation (Maack et al., 1987; Potter, 2011). NPs are also degraded by extracellular proteases, such as neprilysin, dipeptidyl peptidase-4, and insulin-degrading enzyme (Brandt et al., 2006; Kenny et al., 1993; Ralat et al., 2011). The half-life of ANP is about 2.5 min, while that of BNP is about 22.6 min (Holmes et al., 1993; Yandle et al., 1986). Lower affinity of BNP to NPR3 and neprilysin than ANP accounts for the longer plasma half-life of BNP than ANP (Potter, 2011).

3.3 NPs Inhibit Cardiac Hypertrophy ANP inhibits cardiac hypertrophy in autocrine/paracrine fashions. Treatment of NPR1 antagonist results in an increase in cell size of cultured neonatal rat cardiomyocytes (Horio et al., 2000). NPR1-deficient (Npr1/) mice show cardiac hypertrophy as well as systemic hypertension (Oliver et al., 1997). Moreover, cardiomyocyte-specific Npr1/ mice show cardiac hypertrophy without showing hypertension (Holtwick et al., 2003). These data suggest that NPs directly inhibit cardiac hypertrophy.

3.4 NPs Regulate Cardiomyocyte Proliferation NPs show an antiproliferative effect on cardiomyocytes in autocrine/paracrine fashion. ANP treatment

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suppresses angiotensin II-stimulated (ANGIIstimulated) proliferation of cultured fetal sheep cardiomyocytes through an NPR1/cGMP-dependent pathway (O’Tierney et al., 2010). On the other hand, there is a controversial report demonstrating that NPs promote cardiomyocyte proliferation through NPR3/Gi/o protein-mediated reduction of intracellular cAMP accumulation. In developing zebrafish, cardiomyocyte proliferation is increased by simultaneous knockdown of npr1 and npr2, whereas it is decreased by knockdown of npr3. In cultured neonatal rat cardiomyocytes, a low concentration (10 nM) of ANP triggers an NPR3dependent pathway to promote cardiomyocyte proliferation, while a high concentration (10 mM) of ANP induces NPR1- and NPR2-dependent pathways to inhibit cardiomyocyte proliferation (Becker et al., 2014).

3.5 NPs Inhibit Cardiac Fibrosis NPs regulate cardiac fibrosis in paracrine fashions. NPs inhibit the proliferation of cardiac fibroblasts and their collagen synthesis in a cGMP-dependent manner (Kapoun et al., 2004; Redondo et al., 1998). The endogenous ANP released from cultured cardiomyocytes inhibits collagen synthesis in cardiac fibroblasts (Maki et al., 2000). Consistently, both Npr1/ mice and Nppb/ mice exhibit more cardiac fibrosis than the control (Tamura et al., 2000; Oliver et al., 1997). Cardiac fibrosis is also regulated by the expression of matrix metalloproteinases (MMPs). The expression of MMP2 and MMP9 is increased in the Npr1/ mouse hearts. Cardiac fibrosis in Npr1/ mice is reduced by the treatment of an MMP inhibitor (Vellaichamy et al., 2005). These data suggest that ANP suppresses MMPmediated cardiac fibrosis.

3.6 NPs Decrease Blood Pressure NPs regulate blood pressure in endocrine fashions. NPs decrease intravascular volume and blood pressure by NPR1/cGMP-mediated natriuretic and vasodilator effects. In the kidney, NPs regulate electrolyte and fluid balance. NPs increase glomerular filtration rate by dilation of afferent arteriolar and constriction of efferent arteriolar (Marin-Grez et al., 1986). NPs also decrease sodium reabsorption in the collecting ducts through the inhibition of cGMP-bound ion channels, such as amiloride-sensitive Naþ channel and Naþ-Kþ-ATPase (Sonnenberg et al., 1986; Zeidel et al., 1988). In addition, NPs decrease intravascular volume by inhibiting the renin-angiotensin-aldosterone-system and by a direct effect on endothelial permeability (Atarashi et al., 1985; Burnett et al., 1984; Sabrane et al., 2005).

NPs reduce vascular tone through a relaxant effect on vascular smooth muscle cells (Holtwick et al., 2002). PKG activation leads to a decrease of intracellular Ca2þ concentrations through the regulation of Ca2þ channels at the sarcoplasmic reticulum and the plasma membrane (Potter et al., 2006). PKG also decreases calcium sensitivity of the contractile systems through the phosphorylation of myosin light chain phosphatase (Nakamura et al., 1999). Furthermore, NPs regulate blood pressure by reducing sympathetic tone and by suppressing the secretion of ET1, a potent vasoconstrictor (Clerico et al., 2011).

3.7 NPs Regulate Metabolism NPs activate lipolysis, thermogenesis, and muscular oxidative capacity in endocrine fashions. NPs directly act on adipose tissues, resulting in the inhibition of the proliferation of human primary adipocytes (Sarzani et al., 2008a). NPs induce the synthesis of free fatty acid (FFA) through promoting lipolysis in human adipose tissues. NP-induced lipolysis is mediated by NPR1/cGMP/PKG-dependent activation of hormonesensitive lipase (Sengenes et al., 2000, 2003). Indeed, ANP infusion increases plasma FFA and glycerol in young men. These increments are independent of the activation of the sympathetic nervous system (Birkenfeld et al., 2006). NP-induced lipolysis is limited to primates because other mammalian adipocytes predominantly express NPR3 (Sengenes et al., 2002). Therefore, NPR3-deficiency restores NP-induced lipolytic properties in mice (Bordicchia et al., 2012). Of note, NPs induce thermogenesis. White adipose tissue (WAT) is the main fat reservoir, while brown adipose tissue (BAT) is another fat reservoir. BAT can generate heat through mitochondria-uncoupled respiration. NPs induce a transition from WAT to BAT-like tissue. In both BAT and WAT, NPs increase the expression of thermogenic genes, such as peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1a) and uncoupling protein 1 (UCP1). The expression of those genes by NPs is mediated by an NPR1/ cGMP/PKG/p38 mitogen-activated protein kinase (p38 MAPK) and subsequent activating transcription factor 2-dependent (ATF2-dependent) pathway (Bordicchia et al., 2012). These data suggest that NPs might increase energy expenditure through the regulation of lipolysis and thermogenesis. The heart needs a huge energy supply to maintain continuous beating. The adult heart mainly uses either FFA or glucose as its energy source to generate ATP. Under normal conditions, most of the ATP is generated from mitochondrial oxidation of FFA (Lionetti et al., 2011). NPs increase FFA availability and mitochondrial biogenesis. These effects

4. PATHOLOGIC FUNCTIONS OF NPS

might contribute to efficient FFA oxidation in the heart (Sarzani et al., 2008b). NPs also control oxidative capacity of skeletal muscles. Mice overexpressing BNP or PKG in skeletal muscles show higher oxygen consumption, greater FFA oxidation, and higher expression of mitochondrial oxidative genes (Miyashita et al., 2009). Interestingly, exercise training upregulates NPR1 transcripts in human skeletal muscles (Engeli et al., 2012). Moreover, exercise training improves ANP-induced lipolysis in human adipose tissues (Moro et al., 2005, 2009). Therefore, NP signaling may also contribute to exercise traininginduced fat oxidation and lipolysis.

3.8 NPs Control Satiety NPs might regulate the gastrointestinal system through modulating satiety hormone levels. ANP/ NPR1 signaling stimulates somatostatin secretion from the rat gastric oxyntic mucosa (Gower et al., 2003). Somatostatin reduces plasma concentrations of ghrelin, a gut-derived hormone (Shimada et al., 2003). Ghrelin increases appetite and regulates energy balance (Kojima et al., 1999; Tschop et al., 2000). Considering these data, NPs might indirectly inhibit ghrelin secretion through somatostatin secretion. Indeed, intravenous BNP administration inhibits the fasting-induced increment of plasma ghrelin and decreases the subjective rating of hunger in healthy volunteers (Vila et al., 2012). These results support the existence of a mutual regulation mediated by peptide hormones between the heart (NPs) and gut (ghrelin).

4. PATHOLOGIC FUNCTIONS OF NPS 4.1 NP Secretion in Pathologic Conditions ANP secretion from cardiomyocytes is increased under pathologic conditions, such as hypertension, ventricular hypertrophy, heart failure, and myocardial infarction (MI) (Burnett et al., 1986; Clerico et al., 2011; Nagaya et al., 1998; Nishikimi et al., 1996). In these conditions, not only wall stretch but also hypoxia induces ANP secretion. Under hypoxic condition, hypoxia inducible factor 1 alpha promotes ANP transcription in neonatal rat cardiomyocytes (Chun et al., 2003). In addition, cardiomyocytes under hypoxic conditions develop intracellular acidosis. The intracellular acidosis-induced ion imbalance results in an increase of ANP secretion (Chen, 2005). ANP and BNP are co-stored in the same secretory granules in cardiomyocytes (Nakamura et al., 1991). They are co-released from cardiomyocytes of the hearts with congestive heart failure, suggesting ANP and BNP

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share common secretory mechanisms (Bialik et al., 2001). Plasma BNP reflects the severity of the pathologic conditions much more sensitively than plasma ANP (Mukoyama et al., 1990). BNP expression is selectively promoted by specific cytokines that are increased in pathologic conditions. Tumor necrosis factor alpha (TNFa) and interleukin 1 beta (IL1b) selectively promote BNP expression through a p38 MAPK-dependent pathway (Ma et al., 2004). In addition, BNP synthesis and secretion might be regulated by endoplasmic reticulum (ER) stress. ER stress under pathologic conditions promotes cardiomyokine secretion. ER stress promotes the expression of transcription factors, such as ATF4, ATF6, and X-box binding protein 1 (XBP1). These factors induce the expression of the genes involved in protein folding and secretion as well as the genes encoding cardiomyokines (Doroudgar and Glembotski, 2011; Belmont et al., 2008; Ron and Walter, 2007; Thuerauf et al., 2006). In failing human hearts, ER stress induces BNP expression through an XBP1-dependent pathway (Sawada et al., 2010).

4.2 NPs Suppress Inflammation NPs exhibit antiinflammatory actions in endocrine fashions. NPs inhibit the expression of proinflammatory enzymes. Activated macrophages produce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) to destroy microorganisms. An excess of NO production, however, can cause damage in neighboring tissues (Murray and Wynn, 2011). ANP inhibits lipopolysaccharide-induced (LPS-induced) expression of iNOS in macrophages (Kiemer and Vollmar, 1998). ANP also inhibits LPSinduced prostaglandin E2 release by the reduction of cyclooxygenase 2 expression in murine macrophage (Kiemer et al., 2002). NPs inhibit the expression of proinflammatory cytokines. Expression of proinflammatory cytokines is increased in the Npr1/ mouse hearts, while it is decreased in the NPR1 gene-duplicated mouse hearts (Vellaichamy et al., 2014). In addition, ANP inhibits the secretion of proinflammatory cytokines, chemokines, and adipokines from cultured human adipose tissues (Moro et al., 2007). Similarly, BNP has protective effects on acute lung, kidney, and intestinal tissue injury by downregulating the expression of proinflammatory cytokines, such as TNFa and IL6. These antiinflammatory effects of NPs are mediated through the suppression of nuclear factor kappa B (NFkB) expression and NFkB inhibitor (IkB) phosphorylation (Li et al., 2014; Song et al., 2013, 2015; Yang et al., 2014). Because the chronic low-grade inflammation state is a risk factor for cardiovascular and metabolic diseases (Moro et al., 2007), those antiinflammatory effects of NPs on circulatory system and metabolic organs seem

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to be beneficial to suppress the onset of diseases caused by chronic inflammation.

4.3 NPs Suppress Cardiovascular Diseases Cardiac NPs have antipathogenic actions against cardiovascular diseases. Cardiac NPs appear to play an antihypertensive action in an endocrine fashion. Intravenous infusion of ANP induces natriuretic and diuretic effects in spontaneous hypertensive rats compared with normotensive control rats (Pollock and Arendshorst, 1990). NPs also attenuate salt-sensitive hypertension. While Nppaþ/ mice exhibit normal blood pressures under a standard salt diet, they become hypertensive when fed a high-salt diet (John et al., 1995). Clinical studies demonstrate the relationship between NPs and hypertension. Human common variants at the NPPA-NPPB locus that increase circulating ANP and BNP concentrations are associated with lower blood pressure and reduced odds of hypertension (Newton-Cheh et al., 2009). Cardiac NPs inhibit cardiac remodeling, such as hypertrophy, fibrosis, and inflammation through autocrine/paracrine effects (Volpe, 2014). The NPPA gene promoter variant responsible for a significant decrease of plasma proANP was associated with left ventricular hypertrophy in patients with essential hypertension independently of blood pressure (Rubattu et al., 2006).

4.4 NPs Affect Metabolic Diseases Cardiac NPs regulate metabolic diseases in endocrine fashions. There are several clinical studies showing that plasma NP concentrations are associated with metabolic disorders, such as obesity, metabolic syndrome, and type 2 diabetes. Obese individuals have higher odds of low plasma BNP and low plasma ANP compared with lean individuals (Wang et al., 2004). Incidence of metabolic syndrome was associated with both lower and higher BNP concentrations (Musani et al., 2013). The risk for type 2 diabetes is inversely correlated with baseline quartiles of NPs (Lazo et al., 2013; Magnusson et al., 2012). Although mechanisms behind the association between the concentration of NPs and the risks of metabolic diseases are not completely understood, roles played by NPs in metabolic effect may explain this correlation. BNPoverexpressing mice fed with a high-fat diet are protected from obesity and insulin resistance (Miyashita et al., 2009). BNP treatment in type 2 diabetic mice result in an improvement of the metabolic profile, such as a reduction in fat content, increased insulin sensitivity, improved glucose tolerance, and lower blood glucose, despite increased food intake (Plante

et al., 2014). In obese and insulin-resistant subjects, NPR3 expression in the adipose tissue is markedly elevated. It might result in reduced ANP and BNP levels (Gruden et al., 2014). Therefore, NP-increasing therapy might be useful for the treatment of metabolic diseases.

4.5 NPs as Biomarkers NPs are well established markers of acute and chronic heart failure. NT-proBNP and BNP tests are recommended by guidelines for diagnosis, prognosis, and guided therapy of heart failure (Fu et al., 2018). Increased levels of BNP and NT-proBNP are also associated with cardioembolic stroke (Rost et al., 2012), acute MI (Morita et al., 1993), and atrial fibrillation (Schnabel et al., 2010). However, due to the heterogeneity of cardiovascular diseases and the diversity of BNP forms, such as proBNP, glycosylated BNP, and neprilysin degraded BNP, current immunoassays may not truly reflect cardiac function. BNP assays should be improved to enhance their diagnostic, therapeutic, and prognostic values (Fu et al., 2018).

4.6 NPs as Therapeutic Targets Recombinant ANP (carperitide) and recombinant BNP (nesiritide) are approved for treatment of the patients with acute decompensated congestive heart failure in Japan and in the United States, respectively. Although the recombinant NPs are currently considered to improve clinical symptoms and cardiac function, there have been no reports proving that they extend prognosis of the patients with heart failure (Kerkela et al., 2015). Novel NP-based therapeutic approaches have been developed. For example, a compounding drug of an angiotensin receptor inhibitor and a neprilysin inhibitor (LCZ696) is approved as a novel drug in heart failure. It increase NP concentrations by inhibiting NP degradation by neprilysin (Fu et al., 2018). LCZ696 lowers blood pressure greater than angiotensin receptor inhibitor alone. NPR3-blocking drug might also improve both metabolic and cardiovascular diseases (Sarzani et al., 2017). Characterization of the regulatory mechanism of NP processing may open the possibilities of novel therapeutic options. O-glycosylation of T71 residue in ProBNP attenuates its prospecting into active BNP (Tonne et al., 2011). In patients with heart failure, the processing of proBNP is altered (Huntley et al., 2015). Plasma active NP levels also depend on corin levels. A SNP in corin gene is correlated with hypertension (Dries et al., 2005). A proprotein convertase subtilisin/kexin type 6, a corin activator, may become a target to regulate NP levels (Fu et al., 2018).

5. OTHER CARDIOMYOKINES

5. OTHER CARDIOMYOKINES Recently, several cardiomyokines have been demonstrated to be secreted from cardiomyocytes. Among them, some exert endocrine activity. We summarize how cardiomyokines besides NPs work as heartderived hormones.

5.1 Follistatin-Like 1 Follistatin-like 1 (FSTL1) is also referred to as transforming growth factor beta-stimulated (TGFb-stimulated) clone-36 (TSC-36). FSTL1 is a secreted extracellular glycoprotein identified originally as a molecule induced by TGFb stimulation (Shibanuma et al., 1993). FSTL1 belongs to a follistatin family because it shares a domain structure that is called the FS domain (Tanaka et al., 2010). FSTL1 has low sequence homology compared to other follistatin family members. Therefore, FSTL1 has little or no functional overlap with other members of the follistatin family (Ouchi et al., 2010; Planavila et al., 2017). FSTL1 binds to Disco-interacting protein 2 homolog A and subsequently activates phosphoinositide 3 kinase (PI3K)/AKT serine/threonine kinase (AKT) signaling pathway (Ouchi et al., 2010). In neonatal rat cardiomyocytes, FSTL1 induces extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation and AMPactivated protein kinase (AMPK) activation (Ogura et al., 2012; Oshima et al., 2008). In addition, FSTL1 binds to TGFb superfamily proteins and antagonizes BMP signaling (Tanaka et al., 2010; Sylva et al., 2011). FSTL1 is secreted from cardiomyocytes via secretory granules (Doroudgar and Glembotski, 2011).The expression of Fstl1 is ubiquitous in early mouse embryos, whereas it becomes restricted in the mesenchymal tissue later during development (Adams et al., 2007). Although FSTL1 is expressed in epicardial cells of the normal adult mouse heart, its expression is observed in cardiomyocytes but not in epicardial cells after MI (Wei et al., 2015). FSTL1 protein in the heart and plasma is increased in mice with a pathologic condition, such as MI, transverse aortic constriction (TAC), and ischemia/reperfusion (I/R) injury models (Ogura et al., 2012; Shimano et al., 2011) In addition, FSTL1 expression is increased in patients with heart failure (El-Armouche et al., 2011; Oshima et al., 2008). FSTL1 prolongs cardiomyocyte survival and inhibits cardiac hypertrophy in autocrine/paracrine fashions. In neonatal rat cardiomyocytes, FSTL1 knockdown exacerbates hypoxia/reoxygenation-induced apoptosis through an AKT-dependent pathway (Oshima et al., 2008). Cardiac-specific FSTL1-deficient (Fstl1/) mice display exacerbation of cardiac hypertrophy following

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TAC. Cardiac FSTL1 inhibits TAC-induced cardiac hypertrophy through an AMPK-dependent mechanism (Shimano et al., 2011). On the other hand, epicardial cells, but not cardiomyocyte-derived FSTL1, improve cardiac functions after MI (Wei et al., 2015). Although the role of cardiomyocyte-derived FSTL1 remains to be resolved, these data indicate that FSTL1 is a cardioprotective cardiomyokine. FSTL1 also works as an antiinflammatory endocrine factor. In mice with kidney injury, plasma FSTL1 is increased. Cardiac-specific Fstl1/ mice show the exacerbation of renal injury after nephrectomy. Cardiomyocyte-derived FSTL1 exerts antiinflammatory effects in kidneys via an AMPK-dependent mechanism (Hayakawa et al., 2015). Thus, FSTL1 is considered to function as a mediator involved in interorgan communication between the heart and kidney.

5.2 Secreted Phospholipase A2 Secreted phospholipase A2 (sPLA2) is a class of enzyme that catalyze sn-2 ester of glycerophospholipids to release FFAs and lysophospholipids. PLA2 family consists of intracellular PLA2 and sPLA2 (Lambeau and Gelb, 2008). In mammals, there are 11 sPLA2s (groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA, and XIIB) (Murakami and Taketomi, 2015). sPLA2s can bind to cell membranes and provide arachidonic acid for eicosanoid generation. sPLA2s are stored in secretory granules. Group V sPLA2 (PLA2G5) is mainly expressed in the heart (Murakami et al., 2001). The release of sPLA2 from hearts is enhanced by proinflammatory C-C motif chemokine ligand 7 (CCL7). CCL7 binds to MMP2 and is inactivated by MMP2. In MMP2-deficient (Mmp2/) mice, the plasma sPLA2 presumably released from cardiomyocytes is elevated (Berry et al., 2015; Hernandez-Anzaldo et al., 2015). sPLA2s works as a proinflammatory autocrine/paracrine factor. The elevation of cardiomyocyte-derived sPLA2s in the Mmp2/ mice results in cardiac inflammation. In the Mmp2/ mouse hearts, proinflammatory cytokines are increased. The cytokine expression is downregulated by the knockdown of Pla2g5 gene and the treatment of a pan-sPLA2 inhibitor, respectively (Berry et al., 2015). Therefore, cardiomyocyte-derived sPLA2s is thought to induce cardiac inflammation. sPLA2s also induce inflammation and metabolic dysregulation in endocrine fashions. sPLA2s derived from the Mmp2/ mouse hearts induce hepatic inflammation and metabolic dysregulation. A pan-sPLA2 inhibitor normalizes the expression of lipid metabolic genes and proinflammatory cytokines in the liver of Mmp2/ mice (Hernandez-Anzaldo et al., 2015). Therefore, cardiac sPLA2s regulate liver functions in the Mmp2/ mice.

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5.3 Fibroblast Growth Factors Fibroblast growth factor (FGF) family consists of 22 FGFs, FGF1eFGF23 (FGF15 and FGF19 are orthologous peptides). FGFs can be classified as paracrine, endocrine, and intracrine FGFs (Itoh and Ohta, 2013). Seven major FGF receptors (FGFRs) are translated from four FGFR genes, FGFR1eFGFR4 (Zhang et al., 2006). Paracrine FGFs bind to FGFRs with heparin/heparin sulfate as a cofactor. Endocrine FGFs require either aKlotho or bKlotho as a cofactor to bind to FGFRs. Because of their low heparin-binding affinity, endocrine FGFs can target remote organs through the blood stream (Goetz et al., 2007). Intracrine FGFs are intracellular proteins that regulate voltage-gated sodium channels through intracrine fashions. FGFs mainly regulate development and systemic metabolism. Among FGFs, FGF2, FGF9, FGF10, FGF16, and FGF21 act on the heart in pathologic conditions as paracrine factors (Itoh et al., 2016). FGF21, an endocrine FGF, activates FGFR1c, FGFR2c, and FGFR3c with bKlotho (Itoh and Ohta, 2013). Cardiomyocytes express both FGFR1 and bKlotho (Itoh et al., 2016). FGF21 signaling activates ERK1/2, p38 MAPK, AMPK, and PI3K/AKT pathways in mouse hearts and in rat cardiomyocytes (Patel et al., 2014; Zhang et al., 2015). FGF21 is expressed in the liver and acts as a metabolic regulator. FGF21 increases glucose uptake, regulates lipid metabolism, and improves insulin sensitivity in the liver and adipose tissues (Planavila et al., 2017; Degirolamo et al., 2016). Because FGF21 is also produced in cardiomyocytes, it is regarded as a cardiomyokine (Planavila et al., 2017). Its expression is upregulated in H9C2 cardiomyotubes by ER stress and in mouse hearts with mitochondrial dysfunction, respectively (Brahma et al., 2014; Dogan et al., 2014). FGF21 mRNAs in mouse hearts are also upregulated after isoproterenol-induced cardiac hypertrophy, TAC, and MI (Planavila et al., 2013). FGF21 exerts a protective effect on cardiomyocytes in autocrine/paracrine fashions. Cardiomyocyte-derived FGF21 prevents cardiac hypertrophy through the inhibition of metabolic dysregulation and proinflammatory signaling in cardiomyocytes. FGF21 activates FFA oxidation through an ERK1/2-, cAMP-responsive element binding protein-, and PGC1a-dependent pathway. FGF21 suppresses proinflammatory gene expression through the inhibition of NFkB activity (Planavila et al., 2013). FGF21 protects the heart from oxidative stress through upregulating antioxidant factors, including UCP2, UCP3, and superoxide dismutase 2 (Planavila et al., 2015). Additionally, FGF21 inhibits diabetesinduced cardiac apoptosis in an ERK1/2-, p38 MAPK-, and AMPK-dependent manner (Zhang et al., 2015). FGF21 might control systemic metabolism in endocrine fashions. Mice with cardiac-specific FGF21

overexpression show increased plasma FGF21 and decreased body weight (Brahma et al., 2014). Moreover, mitochondrial dysfunction-induced FGF21 upregulation in the mouse heart seems to be responsible for systemic changes in metabolism (Dogan et al., 2014). These data suggest that cardiomyocyte-derived FGF21 has a potential to regulate systemic metabolism as an endocrine factor.

5.4 Others Osteocrin (OSTN) is originally identified in muscles and bones by signal sequence trap methods (Nishizawa et al., 2004; Thomas et al., 2003). OSTN is proposed to belong to the NP family because it has two NP-like motifs. Because NP-like motifs in OSTN lack disulfide cysteine bridges that are essential for the circle structure, OSTN binds with high affinity to NPR3, but not to NPR1 and NPR2 (Potter et al., 2006; Moffatt et al., 2007). Recently, we reported the possibility that cardiomyocyte-derived OSTN might regulate bone formation in zebrafish. OSTN is expressed in cardiomyocytes in zebrafish. OSTN-deficient fish showed bone shortening. The phenotype was rescued by the cardiacspecific overexpression of OSTN. Although it is unclear whether the amount of endogenous cardiomyocytederived OSTN is enough to regulate bone formation, these data suggest the cardiomyocyte-derived peptide has a potential to regulate bone growth at least in zebrafish (Chiba et al., 2017). Growth differentiation factor 15 (GDF15) is a member of TGFb superfamily and is identified as an NO-induced gene in cardiomyocytes (Kempf et al., 2006). GDF15 is expressed broadly in multiple tissues. The expression of GDF15 is induced in the human heart with ischemia or infarction (Kempf et al., 2006). Cardiac-specific GDF15 overexpression protects the heart from hypertrophy (Xu et al., 2006). GDF15 binds to its receptor GDNF family receptor a-like with coreceptor RET and subsequently activates PI3K/AKT, ERK1/2, and phospholipase C/protein kinase C pathways (Mullican and Rangwala, 2018). Estrogen-related receptor alpha- and gamma-deficient mice show plasma GDF15 upregulation, liver growth hormone inhibition, and body growth inhibition. These phenotypes are rescued by cardiacspecific GDF15 knockdown. GDF15 derived from cardiomyocytes acts on the liver to inhibit body growth (Wang et al., 2017). Myostatin (MSTN) is a member of TGFb superfamily. It classically act as an inhibitor of skeletal muscle growth (Planavila et al., 2017). MSTN promotes SMAD2/3dependent gene expression through activin receptor IIB/activin receptor-like kinase 4 or 5 complex (Sartori et al., 2009). MSTN is mainly expressed in skeletal

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muscles. Its expression is also induced in failing cardiomyocytes (George et al., 2010). Cardiac-specific MSTN deficiency rescues skeletal muscle wasting in heart failure. Cardiomyocyte-derived MSTN contributes to skeletal muscle atrophy in heart failure (Heineke et al., 2010).

6. CONCLUSIONS AND FUTURE DIRECTIONS The discovery of ANP established the fact that heart is a hormone-secreting organ. Since the discovery of ANP, a lot of proteins have been reported to be secreted from cardiomyocytes. Although most of such cardiomyokines act as autocrine or paracrine factors, several cardiomyokines target remote organs as heart hormones. ANP is probably the only heart hormone that acts on remote organs in physiologic conditions. ANP regulates not only cardiovascular homeostasis but also metabolic activity. In pathologic conditions, both ANP and BNP exert cardioprotective roles and metabolismimproving roles. Therefore, therapeutic agents increasing the plasma NPs are expected to improve both cardiovascular and metabolic diseases. BNP is a good biomarker and indicator in the management of cardiovascular or metabolic diseases, although the assay for BNP can be improved to precisely monitor active BNP in the patients with diseases. Cardiomyokines are synthesized and released in response to cardiac stress and inflammation. Cardiomyokines seem to become therapeutic targets to regulate systemic inflammatory and metabolism. Although there are many cardiomyokines that have potentials to regulate homeostasis, their endocrine functions are not fully demonstrated because of the lack of cardiac-specific knockout data. Further investigation will lead to a better understanding.

References Adams, D., Larman, B., Oxburgh, L., 2007. Developmental expression of mouse Follistatin-like 1 (Fstl1): dynamic regulation during organogenesis of the kidney and lung. Gene Expr. Patterns 7, 491e500. Anand-Srivastava, M.B., Sehl, P.D., Lowe, D.G., 1996. Cytoplasmic domain of natriuretic peptide receptor-C inhibits adenylyl cyclase. Involvement of a pertussis toxin-sensitive G protein. J. Biol. Chem. 271, 19324e19329. Atarashi, K., Mulrow, P.J., Franco-Saenz, R., 1985. Effect of atrial peptides on aldosterone production. J. Clin. Investig. 76, 1807e1811. Becker, J.R., Chatterjee, S., Robinson, T.Y., Bennett, J.S., Panakova, D., Galindo, C.L., Zhong, L., Shin, J.T., Coy, S.M., Kelly, A.E., Roden, D.M., Lim, C.C., MacRae, C.A., 2014. Differential activation of natriuretic peptide receptors modulates cardiomyocyte proliferation during development. Development 141, 335e345. Belmont, P.J., Tadimalla, A., Chen, W.J., Martindale, J.J., Thuerauf, D.J., Marcinko, M., Gude, N., Sussman, M.A., Glembotski, C.C., 2008.

335

Coordination of growth and endoplasmic reticulum stress signaling by regulator of calcineurin 1 (RCAN1), a novel ATF6inducible gene. J. Biol. Chem. 283, 14012e14021. Bensimon, M., Chang, A.I., de Bold, M.L., Ponce, A., Carreras, D., De Bold, A.J., 2004. Participation of G proteins in natriuretic peptide hormone secretion from heart atria. Endocrinology 145, 5313e5321. Berry, E., Hernandez-Anzaldo, S., Ghomashchi, F., Lehner, R., Murakami, M., Gelb, M.H., Kassiri, Z., Wang, X., FernandezPatron, C., 2015. Matrix metalloproteinase-2 negatively regulates cardiac secreted phospholipase A2 to modulate inflammation and fever. J. Am. Heart Assoc. 4. Bialik, G.M., Abassi, Z.A., Hammel, I., Winaver, J., Lewinson, D., 2001. Evaluation of atrial natriuretic peptide and brain natriuretic peptide in atrial granules of rats with experimental congestive heart failure. J. Histochem. Cytochem. 49, 1293e1300. Birkenfeld, A.L., Boschmann, M., Moro, C., Adams, F., Heusser, K., Tank, J., Diedrich, A., Schroeder, C., Franke, G., Berlan, M., Luft, F.C., Lafontan, M., Jordan, J., 2006. Beta-adrenergic and atrial natriuretic peptide interactions on human cardiovascular and metabolic regulation. J. Clin. Endocrinol. Metab. 91, 5069e5075. Bordicchia, M., Liu, D., Amri, E.Z., Ailhaud, G., Dessi-Fulgheri, P., Zhang, C., Takahashi, N., Sarzani, R., Collins, S., 2012. Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes. J. Clin. Investig. 122, 1022e1036. Brahma, M.K., Adam, R.C., Pollak, N.M., Jaeger, D., Zierler, K.A., Pocher, N., Schreiber, R., Romauch, M., Moustafa, T., Eder, S., Ruelicke, T., Preiss-Landl, K., Lass, A., Zechner, R., Haemmerle, G., 2014. Fibroblast growth factor 21 is induced upon cardiac stress and alters cardiac lipid homeostasis. J. Lipid Res. 55, 2229e2241. Brandt, I., Lambeir, A.M., Ketelslegers, J.M., Vanderheyden, M., Scharpe, S., De Meester, I., 2006. Dipeptidyl-peptidase IV converts intact B-type natriuretic peptide into its des-SerPro form. Clin. Chem. 52, 82e87. Bruneau, B.G., Piazza, L.A., de Bold, A.J., 1996. Alpha 1-adrenergic stimulation of isolated rat atria results in discoordinate increases in natriuretic peptide secretion and gene expression and enhances Egr-1 and c-Myc expression. Endocrinology 137, 137e143. Bruneau, B.G., Piazza, L.A., de Bold, A.J., 1997. BNP gene expression is specifically modulated by stretch and ET-1 in a new model of isolated rat atria. Am. J. Physiol. 273, H2678eH2686. Burnett Jr., J.C., Granger, J.P., Opgenorth, T.J., 1984. Effects of synthetic atrial natriuretic factor on renal function and renin release. Am. J. Physiol. 247, F863eF866. Burnett Jr., J.C., Kao, P.C., Hu, D.C., Heser, D.W., Heublein, D., Granger, J.P., Opgenorth, T.J., Reeder, G.S., 1986. Atrial natriuretic peptide elevation in congestive heart failure in the human. Science 231, 1145e1147. Chang, M.S., Lowe, D.G., Lewis, M., Hellmiss, R., Chen, E., Goeddel, D.V., 1989. Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases. Nature 341, 68e72. Chen, Y.F., 2005. Atrial natriuretic peptide in hypoxia. Peptides 26, 1068e1077. Chiba, A., Watanabe-Takano, H., Terai, K., Fukui, H., Miyazaki, T., Uemura, M., Hashimoto, H., Hibi, M., Fukuhara, S., Mochizuki, N., 2017. Osteocrin, a peptide secreted from the heart and other tissues, contributes to cranial osteogenesis and chondrogenesis in zebrafish. Development 144, 334e344. Chinkers, M., Garbers, D.L., Chang, M.S., Lowe, D.G., Chin, H.M., Goeddel, D.V., Schulz, S., 1989. A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature 338, 78e83. Chun, Y.S., Hyun, J.Y., Kwak, Y.G., Kim, I.S., Kim, C.H., Choi, E., Kim, M.S., Park, J.W., 2003. Hypoxic activation of the atrial natriuretic peptide gene promoter through direct and indirect actions of hypoxia-inducible factor-1. Biochem. J. 370, 149e157.

336

14. HEART HORMONES

Clerico, A., Giannoni, A., Vittorini, S., Passino, C., 2011. Thirty years of the heart as an endocrine organ: physiological role and clinical utility of cardiac natriuretic hormones. Am. J. Physiol. Heart Circ. Physiol. 301, H12eH20. de Bold, A.J., 1985. Atrial natriuretic factor: a hormone produced by the heart. Science 230, 767e770. de Bold, A.J., Flynn, T.G., 1983. Cardionatrin I e a novel heart peptide with potent diuretic and natriuretic properties. Life Sci. 33, 297e302. de Bold, A.J., Borenstein, H.B., Veress, A.T., Sonnenberg, H., 1981. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci. 28, 89e94. Degirolamo, C., Sabba, C., Moschetta, A., 2016. Therapeutic potential of the endocrine fibroblast growth factors FGF19, FGF21 and FGF23. Nat. Rev. Drug Discov. 15, 51e69. Dogan, S.A., Pujol, C., Maiti, P., Kukat, A., Wang, S., Hermans, S., Senft, K., Wibom, R., Rugarli, E.I., Trifunovic, A., 2014. Tissuespecific loss of DARS2 activates stress responses independently of respiratory chain deficiency in the heart. Cell Metabol. 19, 458e469. Doroudgar, S., Glembotski, C.C., 2011. The cardiokine story unfolds: ischemic stress-induced protein secretion in the heart. Trends Mol. Med. 17, 207e214. Dries, D.L., Victor, R.G., Rame, J.E., Cooper, R.S., Wu, X., Zhu, X., Leonard, D., Ho, S.I., Wu, Q., Post, W., Drazner, M.H., 2005. Corin gene minor allele defined by 2 missense mutations is common in blacks and associated with high blood pressure and hypertension. Circulation 112, 2403e2410. El-Armouche, A., Ouchi, N., Tanaka, K., Doros, G., Wittkopper, K., Schulze, T., Eschenhagen, T., Walsh, K., Sam, F., 2011. Follistatinlike 1 in chronic systolic heart failure: a marker of left ventricular remodeling. Cric. Heart Fail. 4, 621e627. Engeli, S., Birkenfeld, A.L., Badin, P.M., Bourlier, V., Louche, K., Viguerie, N., Thalamas, C., Montastier, E., Larrouy, D., Harant, I., de G, I., Lieske, S., Reinke, J., Beckmann, B., Langin, D., Jordan, J., Moro, C., 2012. Natriuretic peptides enhance the oxidative capacity of human skeletal muscle. J. Clin. Investig. 122, 4675e4679. Flynn, T.G., de Bold, M.L., de Bold, A.J., 1983. The amino acid sequence of an atrial peptide with potent diuretic and natriuretic properties. Biochem. Biophys. Res. Commun. 117, 859e865. Fu, S., Ping, P., Zhu, Q., Ye, P., Luo, L., 2018. Brain natriuretic peptide and its biochemical, analytical, and clinical issues in heart failure: a narrative review. Front. Physiol. 9, 692. Fukuda, Y., Hirata, Y., Yoshimi, H., Kojima, T., Kobayashi, Y., Yanagisawa, M., Masaki, T., 1988. Endothelin is a potent secretagogue for atrial natriuretic peptide in cultured rat atrial myocytes. Biochem. Biophys. Res. Commun. 155, 167e172. Fuller, F., Porter, J.G., Arfsten, A.E., Miller, J., Schilling, J.W., Scarborough, R.M., Lewicki, J.A., Schenk, D.B., 1988. Atrial natriuretic peptide clearance receptor. Complete sequence and functional expression of cDNA clones. J. Biol. Chem. 263, 9395e9401. George, I., Bish, L.T., Kamalakkannan, G., Petrilli, C.M., Oz, M.C., Naka, Y., Sweeney, H.L., Maybaum, S., 2010. Myostatin activation in patients with advanced heart failure and after mechanical unloading. Eur. J. Heart Fail. 12, 444e453. Glembotski, C.C., 2011. Functions for the cardiomyokine, MANF, in cardioprotection, hypertrophy and heart failure. J. Mol. Cell. Cardiol. 51, 512e517. Goetz, R., Beenken, A., Ibrahimi, O.A., Kalinina, J., Olsen, S.K., Eliseenkova, A.V., Xu, C., Neubert, T.A., Zhang, F., Linhardt, R.J., Yu, X., White, K.E., Inagaki, T., Kliewer, S.A., Yamamoto, M., Kurosu, H., Ogawa, Y., Kuro-o, M., Lanske, B., Razzaque, M.S., Mohammadi, M., 2007. Molecular insights into the klothodependent, endocrine mode of action of fibroblast growth factor 19 subfamily members. Mol. Cell Biol. 27, 3417e3428. Gower Jr., W.R., McCuen, R.W., Arimura, A., Coy, D.A., Dietz, J.R., Landon, C.S., Schubert, M.L., 2003. Reciprocal paracrine pathways

link atrial natriuretic peptide and somatostatin secretion in the antrum of the stomach. Regul. Pept. 110, 101e106. Gruden, G., Landi, A., Bruno, G., 2014. Natriuretic peptides, heart, and adipose tissue: new findings and future developments for diabetes research. Diabetes Care 37, 2899e2908. Han, J.H., Bai, G.Y., Park, J.H., Yuan, K., Park, W.H., Kim, S.Z., Kim, S.H., 2008. Regulation of stretch-activated ANP secretion by chloride channels. Peptides 29, 613e621. Hayakawa, S., Ohashi, K., Shibata, R., Kataoka, Y., Miyabe, M., Enomoto, T., Joki, Y., Shimizu, Y., Kambara, T., Uemura, Y., Yuasa, D., Ogawa, H., Matsuo, K., Hiramatsu-Ito, M., van den Hoff, M.J., Walsh, K., Murohara, T., Ouchi, N., 2015. Cardiac myocyte-derived follistatin-like 1 prevents renal injury in a subtotal nephrectomy model. J. Am. Soc. Nephrol. 26, 636e646. Heineke, J., Auger-Messier, M., Xu, J., Sargent, M., York, A., Welle, S., Molkentin, J.D., 2010. Genetic deletion of myostatin from the heart prevents skeletal muscle atrophy in heart failure. Circulation 121, 419e425. Hernandez-Anzaldo, S., Berry, E., Brglez, V., Leung, D., Yun, T.J., Lee, J.S., Filep, J.G., Kassiri, Z., Cheong, C., Lambeau, G., Lehner, R., Fernandez-Patron, C., 2015. Identification of a novel heart-liver axis: matrix metalloproteinase-2 negatively regulates cardiac secreted phospholipase A2 to modulate lipid metabolism and inflammation in the liver. J. Am. Heart Assoc. 4. Holmes, S.J., Espiner, E.A., Richards, A.M., Yandle, T.G., Frampton, C., 1993. Renal, endocrine, and hemodynamic effects of human brain natriuretic peptide in normal man. J. Clin. Endocrinol. Metab. 76, 91e96. Holtwick, R., Gotthardt, M., Skryabin, B., Steinmetz, M., Potthast, R., Zetsche, B., Hammer, R.E., Herz, J., Kuhn, M., 2002. Smooth muscle-selective deletion of guanylyl cyclase-A prevents the acute but not chronic effects of ANP on blood pressure. Proc. Natl. Acad. Sci. U. S. A. 99, 7142e7147. Holtwick, R., van, E.M., Skryabin, B.V., Baba, H.A., Bubikat, A., Begrow, F., Schneider, M.D., Garbers, D.L., Kuhn, M., 2003. Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A. J. Clin. Investig. 111, 1399e1407. Horio, T., Nishikimi, T., Yoshihara, F., Matsuo, H., Takishita, S., Kangawa, K., 2000. Inhibitory regulation of hypertrophy by endogenous atrial natriuretic peptide in cultured cardiac myocytes. Hypertension 35, 19e24. Huntley, B.K., Sandberg, S.M., Heublein, D.M., Sangaralingham, S.J., Burnett Jr., J.C., Ichiki, T., 2015. Pro-B-type natriuretic peptide-1108 processing and degradation in human heart failure. Cric. Heart Fail. 8, 89e97. Inoue, K., Naruse, K., Yamagami, S., Mitani, H., Suzuki, N., Takei, Y., 2003. Four functionally distinct C-type natriuretic peptides found in fish reveal evolutionary history of the natriuretic peptide system. Proc. Natl. Acad. Sci. U. S. A. 100, 10079e10084. Itoh, N., Ohta, H., 2013. Pathophysiological roles of FGF signaling in the heart. Front. Physiol. 4, 247. Itoh, N., Ohta, H., Nakayama, Y., Konishi, M., 2016. Roles of FGF signals in heart development, health, and disease. Front. Cell Dev. Biol. 4, 110. John, S.W., Krege, J.H., Oliver, P.M., Hagaman, J.R., Hodgin, J.B., Pang, S.C., Flynn, T.G., Smithies, O., 1995. Genetic decreases in atrial natriuretic peptide and salt-sensitive hypertension. Science 267, 679e681. Kangawa, K., Matsuo, H., 1984. Purification and complete amino acid sequence of alpha-human atrial natriuretic polypeptide (alphahANP). Biochem. Biophys. Res. Commun. 118, 131e139. Kapoun, A.M., Liang, F., O’Young, G., Damm, D.L., Quon, D., White, R.T., Munson, K., Lam, A., Schreiner, G.F., Protter, A.A., 2004. B-type natriuretic peptide exerts broad functional opposition to transforming growth factor-beta in primary human cardiac

REFERENCES

fibroblasts: fibrosis, myofibroblast conversion, proliferation, and inflammation. Circ. Res. 94, 453e461. Kawakoshi, A., Hyodo, S., Yasuda, A., Takei, Y., 2003. A single and novel natriuretic peptide is expressed in the heart and brain of the most primitive vertebrate, the hagfish (Eptatretus burgeri). J. Mol. Endocrinol. 31, 209e220. Kawakoshi, A., Hyodo, S., Nozaki, M., Takei, Y., 2006. Identification of a natriuretic peptide (NP) in cyclostomes (lamprey and hagfish): CNP-4 is the ancestral gene of the NP family. Gen. Comp. Endocrinol. 148, 41e47. Kempf, T., Eden, M., Strelau, J., Naguib, M., Willenbockel, C., Tongers, J., Heineke, J., Kotlarz, D., Xu, J., Molkentin, J.D., Niessen, H.W., Drexler, H., Wollert, K.C., 2006. The transforming growth factor-beta superfamily member growth-differentiation factor-15 protects the heart from ischemia/reperfusion injury. Circ. Res. 98, 351e360. Kennedy, B.P., Marsden, J.J., Flynn, T.G., de Bold, A.J., Davies, P.L., 1984. Isolation and nucleotide sequence of a cloned cardionatrin cDNA. Biochem. Biophys. Res. Commun. 122, 1076e1082. Kenny, A.J., Bourne, A., Ingram, J., 1993. Hydrolysis of human and pig brain natriuretic peptides, urodilatin, C-type natriuretic peptide and some C-receptor ligands by endopeptidase-24.11. Biochem. J. 291 (Pt 1), 83e88. Kerkela, R., Ulvila, J., Magga, J., 2015. Natriuretic peptides in the regulation of cardiovascular physiology and metabolic events. J. Am. Heart Assoc. 4, e002423. Kiemer, A.K., Vollmar, A.M., 1998. Autocrine regulation of inducible nitric-oxide synthase in macrophages by atrial natriuretic peptide. J. Biol. Chem. 273, 13444e13451. Kiemer, A.K., Lehner, M.D., Hartung, T., Vollmar, A.M., 2002. Inhibition of cyclooxygenase-2 by natriuretic peptides. Endocrinology 143, 846e852. Kishimoto, I., Tokudome, T., Nakao, K., Kangawa, K., 2011. Natriuretic peptide system: an overview of studies using genetically engineered animal models. FEBS J. 278, 1830e1841. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402, 656e660. Lambeau, G., Gelb, M.H., 2008. Biochemistry and physiology of mammalian secreted phospholipases A2. Annu. Rev. Biochem. 77, 495e520. Lazo, M., Young, J.H., Brancati, F.L., Coresh, J., Whelton, S., Ndumele, C.E., Hoogeveen, R., Ballantyne, C.M., Selvin, E., 2013. NH2-terminal pro-brain natriuretic peptide and risk of diabetes. Diabetes 62, 3189e3193. Li, N., Jin, H.X., Song, Z., Bai, C.Z., Cui, Y., Gao, Y., 2014. Protective effect of recombinant human brain natriuretic peptide on acute renal injury induced by endotoxin in canines. Cell Biochem. Biophys. 70, 1317e1324. Lionetti, V., Stanley, W.C., Recchia, F.A., 2011. Modulating fatty acid oxidation in heart failure. Cardiovasc. Res. 90, 202e209. Ma, K.K., Ogawa, T., de Bold, A.J., 2004. Selective upregulation of cardiac brain natriuretic peptide at the transcriptional and translational levels by pro-inflammatory cytokines and by conditioned medium derived from mixed lymphocyte reactions via p38 MAP kinase. J. Mol. Cell. Cardiol. 36, 505e513. Maack, T., Suzuki, M., Almeida, F.A., Nussenzveig, D., Scarborough, R.M., McEnroe, G.A., Lewicki, J.A., 1987. Physiological role of silent receptors of atrial natriuretic factor. Science 238, 675e678. Magnusson, M., Jujic, A., Hedblad, B., Engstrom, G., Persson, M., Struck, J., Morgenthaler, N.G., Nilsson, P., Newton-Cheh, C., Wang, T.J., Melander, O., 2012. Low plasma level of atrial natriuretic peptide predicts development of diabetes: the prospective Malmo Diet and Cancer study. J. Clin. Endocrinol. Metab. 97, 638e645.

337

Maki, T., Horio, T., Yoshihara, F., Suga, S., Takeo, S., Matsuo, H., Kangawa, K., 2000. Effect of neutral endopeptidase inhibitor on endogenous atrial natriuretic peptide as a paracrine factor in cultured cardiac fibroblasts. Br. J. Pharmacol. 131, 1204e1210. Marin-Grez, M., Fleming, J.T., Steinhausen, M., 1986. Atrial natriuretic peptide causes pre-glomerular vasodilatation and post-glomerular vasoconstriction in rat kidney. Nature 324, 473e476. Misono, K.S., Philo, J.S., Arakawa, T., Ogata, C.M., Qiu, Y., Ogawa, H., Young, H.S., 2011. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase. FEBS J. 278, 1818e1829. Miyashita, K., Itoh, H., Tsujimoto, H., Tamura, N., Fukunaga, Y., Sone, M., Yamahara, K., Taura, D., Inuzuka, M., Sonoyama, T., Nakao, K., 2009. Natriuretic peptides/cGMP/cGMP-dependent protein kinase cascades promote muscle mitochondrial biogenesis and prevent obesity. Diabetes 58, 2880e2892. Moffatt, P., Thomas, G., Sellin, K., Bessette, M.C., Lafreniere, F., Akhouayri, O., St-Arnaud, R., Lanctot, C., 2007. Osteocrin is a specific ligand of the natriuretic peptide clearance receptor that modulates bone growth. J. Biol. Chem. 282, 36454e36462. Morita, E., Yasue, H., Yoshimura, M., Ogawa, H., Jougasaki, M., Matsumura, T., Mukoyama, M., Nakao, K., 1993. Increased plasma levels of brain natriuretic peptide in patients with acute myocardial infarction. Circulation 88, 82e91. Moro, C., Pillard, F., De G, I., Harant, I., Riviere, D., Stich, V., Lafontan, M., Crampes, F., Berlan, M., 2005. Training enhances ANP lipid-mobilizing action in adipose tissue of overweight men. Med. Sci. Sports Exerc. 37, 1126e1132. Moro, C., Klimcakova, E., Lolmede, K., Berlan, M., Lafontan, M., Stich, V., Bouloumie, A., Galitzky, J., Arner, P., Langin, D., 2007. Atrial natriuretic peptide inhibits the production of adipokines and cytokines linked to inflammation and insulin resistance in human subcutaneous adipose tissue. Diabetologia 50, 1038e1047. Moro, C., Pasarica, M., Elkind-Hirsch, K., Redman, L.M., 2009. Aerobic exercise training improves atrial natriuretic peptide and catecholamine-mediated lipolysis in obese women with polycystic ovary syndrome. J. Clin. Endocrinol. Metab. 94, 2579e2586. Mukoyama, M., Nakao, K., Saito, Y., Ogawa, Y., Hosoda, K., Suga, S., Shirakami, G., Jougasaki, M., Imura, H., 1990. Increased human brain natriuretic peptide in congestive heart failure. N. Engl. J. Med. 323, 757e758. Mukoyama, M., Nakao, K., Hosoda, K., Suga, S., Saito, Y., Ogawa, Y., Shirakami, G., Jougasaki, M., Obata, K., Yasue, H., 1991. Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide. J. Clin. Investig. 87, 1402e1412. Mullican, S.E., Rangwala, S.M., 2018. Uniting GDF15 and GFRAL: therapeutic opportunities in obesity and beyond. Trends Endocrinol. Metabol. 29, 560e570. Murakami, M., Taketomi, Y., 2015. Secreted phospholipase A2 and mast cells. Allergol. Int. 64, 4e10. Murakami, M., Koduri, R.S., Enomoto, A., Shimbara, S., Seki, M., Yoshihara, K., Singer, A., Valentin, E., Ghomashchi, F., Lambeau, G., Gelb, M.H., Kudo, I., 2001. Distinct arachidonatereleasing functions of mammalian secreted phospholipase A2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms. J. Biol. Chem. 276, 10083e10096. Murray, P.J., Wynn, T.A., 2011. Protective and pathogenic functions of macrophage subsets. Nat. Rev. Immunol. 11, 723e737. Musani, S.K., Vasan, R.S., Bidulescu, A., Liu, J., Xanthakis, V., Sims, M., Gawalapu, R.K., Samdarshi, T.E., Steffes, M., Taylor, H.A., Fox, E.R., 2013. Aldosterone, C-reactive protein, and plasma B-type natriuretic peptide are associated with the development of metabolic syndrome and longitudinal changes in metabolic syndrome

338

14. HEART HORMONES

components: findings from the Jackson heart study. Diabetes Care 36, 3084e3092. Nagaya, N., Nishikimi, T., Goto, Y., Miyao, Y., Kobayashi, Y., Morii, I., Daikoku, S., Matsumoto, T., Miyazaki, S., Matsuoka, H., Takishita, S., Kangawa, K., Matsuo, H., Nonogi, H., 1998. Plasma brain natriuretic peptide is a biochemical marker for the prediction of progressive ventricular remodeling after acute myocardial infarction. Am. Heart J. 135, 21e28. Nakamura, S., Naruse, M., Naruse, K., Kawana, M., Nishikawa, T., Hosoda, S., Tanaka, I., Yoshimi, T., Yoshihara, I., Inagami, T., 1991. Atrial natriuretic peptide and brain natriuretic peptide coexist in the secretory granules of human cardiac myocytes. Am. J. Hypertens. 4, 909e912. Nakamura, M., Ichikawa, K., Ito, M., Yamamori, B., Okinaka, T., Isaka, N., Yoshida, Y., Fujita, S., Nakano, T., 1999. Effects of the phosphorylation of myosin phosphatase by cyclic GMPdependent protein kinase. Cell. Signal. 11, 671e676. Newton-Cheh, C., Larson, M.G., Vasan, R.S., Levy, D., Bloch, K.D., Surti, A., Guiducci, C., Kathiresan, S., Benjamin, E.J., Struck, J., Morgenthaler, N.G., Bergmann, A., Blankenberg, S., Kee, F., Nilsson, P., Yin, X., Peltonen, L., Vartiainen, E., Salomaa, V., Hirschhorn, J.N., Melander, O., Wang, T.J., 2009. Association of common variants in NPPA and NPPB with circulating natriuretic peptides and blood pressure. Nat. Genet. 41, 348e353. Nishikimi, T., Yoshihara, F., Morimoto, A., Ishikawa, K., Ishimitsu, T., Saito, Y., Kangawa, K., Matsuo, H., Omae, T., Matsuoka, H., 1996. Relationship between left ventricular geometry and natriuretic peptide levels in essential hypertension. Hypertension 28, 22e30. Nishizawa, H., Matsuda, M., Yamada, Y., Kawai, K., Suzuki, E., Makishima, M., Kitamura, T., Shimomura, I., 2004. Musclin, a novel skeletal muscle-derived secretory factor. J. Biol. Chem. 279, 19391e19395. O’Tierney, P.F., Chattergoon, N.N., Louey, S., Giraud, G.D., Thornburg, K.L., 2010. Atrial natriuretic peptide inhibits angiotensin II-stimulated proliferation in fetal cardiomyocytes. J. Physiol. 588, 2879e2889. Ogawa, T., de Bold, A.J., 2014. The heart as an endocrine organ. Endocr. Connect. 3, R31eR44. Ogura, Y., Ouchi, N., Ohashi, K., Shibata, R., Kataoka, Y., Kambara, T., Kito, T., Maruyama, S., Yuasa, D., Matsuo, K., Enomoto, T., Uemura, Y., Miyabe, M., Ishii, M., Yamamoto, T., Shimizu, Y., Walsh, K., Murohara, T., 2012. Therapeutic impact of follistatinlike 1 on myocardial ischemic injury in preclinical models. Circulation 126, 1728e1738. Oliver, P.M., Fox, J.E., Kim, R., Rockman, H.A., Kim, H.S., Reddick, R.L., Pandey, K.N., Milgram, S.L., Smithies, O., Maeda, N., 1997. Hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor A. Proc. Natl. Acad. Sci. U. S. A. 94, 14730e14735. Oshima, Y., Ouchi, N., Sato, K., Izumiya, Y., Pimentel, D.R., Walsh, K., 2008. Follistatin-like 1 is an Akt-regulated cardioprotective factor that is secreted by the heart. Circulation 117, 3099e3108. Ouchi, N., Asaumi, Y., Ohashi, K., Higuchi, A., Sono-Romanelli, S., Oshima, Y., Walsh, K., 2010. DIP2A functions as a FSTL1 receptor. J. Biol. Chem. 285, 7127e7134. Patel, V., Adya, R., Chen, J., Ramanjaneya, M., Bari, M.F., Bhudia, S.K., Hillhouse, E.W., Tan, B.K., Randeva, H.S., 2014. Novel insights into the cardio-protective effects of FGF21 in lean and obese rat hearts. PLoS One 9, e87102. Planavila, A., Redondo, I., Hondares, E., Vinciguerra, M., Munts, C., Iglesias, R., Gabrielli, L.A., Sitges, M., Giralt, M., van, B.M., Villarroya, F., 2013. Fibroblast growth factor 21 protects against cardiac hypertrophy in mice. Nat. Commun. 4, 2019.

Planavila, A., Redondo-Angulo, I., Ribas, F., Garrabou, G., Casademont, J., Giralt, M., Villarroya, F., 2015. Fibroblast growth factor 21 protects the heart from oxidative stress. Cardiovasc. Res. 106, 19e31. Planavila, A., Fernandez-Sola, J., Villarroya, F., 2017. Cardiokines as modulators of stress-induced cardiac disorders. Adv. Protein Chem. Struct. Biol. 108, 227e256. Plante, E., Menaouar, A., Danalache, B.A., Broderick, T.L., Jankowski, M., Gutkowska, J., 2014. Treatment with brain natriuretic peptide prevents the development of cardiac dysfunction in obese diabetic db/db mice. Diabetologia 57, 1257e1267. Pollock, D.M., Arendshorst, W.J., 1990. Exaggerated natriuretic response to atrial natriuretic factor in rats developing spontaneous hypertension. Hypertension 16, 72e79. Potter, L.R., 2011. Natriuretic peptide metabolism, clearance and degradation. FEBS J. 278, 1808e1817. Potter, L.R., Abbey-Hosch, S., Dickey, D.M., 2006. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr. Rev. 27, 47e72. Ralat, L.A., Guo, Q., Ren, M., Funke, T., Dickey, D.M., Potter, L.R., Tang, W.J., 2011. Insulin-degrading enzyme modulates the natriuretic peptide-mediated signaling response. J. Biol. Chem. 286, 4670e4679. Redondo, J., Bishop, J.E., Wilkins, M.R., 1998. Effect of atrial natriuretic peptide and cyclic GMP phosphodiesterase inhibition on collagen synthesis by adult cardiac fibroblasts. Br. J. Pharmacol. 124, 1455e1462. Rinne, A., Vuolteenaho, O., Jarvinen, M., Dorn, A., Arjamaa, O., 1986. Atrial natriuretic polypeptides in the specific atrial granules of the rat heart: immunohistochemical and immunoelectron microscopical localization and radioimmunological quantification. Acta Histochem. 80, 19e28. Ron, D., Walter, P., 2007. Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell Biol. 8, 519e529. Rost, N.S., Biffi, A., Cloonan, L., Chorba, J., Kelly, P., Greer, D., Ellinor, P., Furie, K.L., 2012. Brain natriuretic peptide predicts functional outcome in ischemic stroke. Stroke 43, 441e445. Rubattu, S., Bigatti, G., Evangelista, A., Lanzani, C., Stanzione, R., Zagato, L., Manunta, P., Marchitti, S., Venturelli, V., Bianchi, G., Volpe, M., Stella, P., 2006. Association of atrial natriuretic peptide and type a natriuretic peptide receptor gene polymorphisms with left ventricular mass in human essential hypertension. J. Am. Coll. Cardiol. 48, 499e505. Sabrane, K., Kruse, M.N., Fabritz, L., Zetsche, B., Mitko, D., Skryabin, B.V., Zwiener, M., Baba, H.A., Yanagisawa, M., Kuhn, M., 2005. Vascular endothelium is critically involved in the hypotensive and hypovolemic actions of atrial natriuretic peptide. J. Clin. Investig. 115, 1666e1674. Sartori, R., Milan, G., Patron, M., Mammucari, C., Blaauw, B., Abraham, R., Sandri, M., 2009. Smad2 and 3 transcription factors control muscle mass in adulthood. Am. J. Physiol. Cell Physiol. 296, C1248eC1257. Sarzani, R., Marcucci, P., Salvi, F., Bordicchia, M., Espinosa, E., Mucci, L., Lorenzetti, B., Minardi, D., Muzzonigro, G., DessiFulgheri, P., Rappelli, A., 2008. Angiotensin II stimulates and atrial natriuretic peptide inhibits human visceral adipocyte growth. Int. J. Obes. 32, 259e267. Sarzani, R., Salvi, F., Dessi-Fulgheri, P., Rappelli, A., 2008. Reninangiotensin system, natriuretic peptides, obesity, metabolic syndrome, and hypertension: an integrated view in humans. J. Hypertens. 26, 831e843. Sarzani, R., Spannella, F., Giulietti, F., Balietti, P., Cocci, G., Bordicchia, M., 2017. Cardiac natriuretic peptides, hypertension and cardiovascular risk. High Blood Press. Cardiovasc. Prev. 24, 115e126.

REFERENCES

Sawada, T., Minamino, T., Fu, H.Y., Asai, M., Okuda, K., Isomura, T., Yamazaki, S., Asano, Y., Okada, K., Tsukamoto, O., Sanada, S., Asanuma, H., Asakura, M., Takashima, S., Kitakaze, M., Komuro, I., 2010. X-box binding protein 1 regulates brain natriuretic peptide through a novel AP1/CRE-like element in cardiomyocytes. J. Mol. Cell. Cardiol. 48, 1280e1289. Schlueter, N., de, S.A., Willmes, D.M., Spranger, J., Jordan, J., Birkenfeld, A.L., 2014. Metabolic actions of natriuretic peptides and therapeutic potential in the metabolic syndrome. Pharmacol. Ther. 144, 12e27. Schnabel, R.B., Larson, M.G., Yamamoto, J.F., Sullivan, L.M., Pencina, M.J., Meigs, J.B., Tofler, G.H., Selhub, J., Jacques, P.F., Wolf, P.A., Magnani, J.W., Ellinor, P.T., Wang, T.J., Levy, D., Vasan, R.S., Benjamin, E.J., 2010. Relations of biomarkers of distinct pathophysiological pathways and atrial fibrillation incidence in the community. Circulation 121, 200e207. Schulz, S., Singh, S., Bellet, R.A., Singh, G., Tubb, D.J., Chin, H., Garbers, D.L., 1989. The primary structure of a plasma membrane guanylate cyclase demonstrates diversity within this new receptor family. Cell 58, 1155e1162. Semenov, A.G., Tamm, N.N., Seferian, K.R., Postnikov, A.B., Karpova, N.S., Serebryanaya, D.V., Koshkina, E.V., Krasnoselsky, M.I., Katrukha, A.G., 2010. Processing of pro-B-type natriuretic peptide: furin and corin as candidate convertases. Clin. Chem. 56, 1166e1176. Sengenes, C., Berlan, M., De G, I., Lafontan, M., Galitzky, J., 2000. Natriuretic peptides: a new lipolytic pathway in human adipocytes. FASEB J. 14, 1345e1351. Sengenes, C., Zakaroff-Girard, A., Moulin, A., Berlan, M., Bouloumie, A., Lafontan, M., Galitzky, J., 2002. Natriuretic peptide-dependent lipolysis in fat cells is a primate specificity. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283, R257eR265. Sengenes, C., Bouloumie, A., Hauner, H., Berlan, M., Busse, R., Lafontan, M., Galitzky, J., 2003. Involvement of a cGMPdependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes. J. Biol. Chem. 278, 48617e48626. Shibanuma, M., Mashimo, J., Mita, A., Kuroki, T., Nose, K., 1993. Cloning from a mouse osteoblastic cell line of a set of transforminggrowth-factor-beta 1-regulated genes, one of which seems to encode a follistatin-related polypeptide. Eur. J. Biochem. 217, 13e19. Shimada, M., Date, Y., Mondal, M.S., Toshinai, K., Shimbara, T., Fukunaga, K., Murakami, N., Miyazato, M., Kangawa, K., Yoshimatsu, H., Matsuo, H., Nakazato, M., 2003. Somatostatin suppresses ghrelin secretion from the rat stomach. Biochem. Biophys. Res. Commun. 302, 520e525. Shimano, M., Ouchi, N., Nakamura, K., van, W.B., Ohashi, K., Asaumi, Y., Higuchi, A., Pimentel, D.R., Sam, F., Murohara, T., van den Hoff, M.J., Walsh, K., 2011. Cardiac myocyte follistatinlike 1 functions to attenuate hypertrophy following pressure overload. Proc. Natl. Acad. Sci. U. S. A. 108, E899eE906. Shimano, M., Ouchi, N., Walsh, K., 2012. Cardiokines: recent progress in elucidating the cardiac secretome. Circulation 126, e327ee332. Silberbach, M., Roberts Jr., C.T., 2001. Natriuretic peptide signalling: molecular and cellular pathways to growth regulation. Cell. Signal. 13, 221e231. Song, Z., Cui, Y., Ding, M.Z., Jin, H.X., Gao, Y., 2013. Protective effects of recombinant human brain natriuretic peptide against LPSinduced acute lung injury in dogs. Int. Immunopharmacol. 17, 508e512. Song, Z., Zhao, X., Liu, M., Jin, H., Wang, L., Hou, M., Gao, Y., 2015. Recombinant human brain natriuretic peptide attenuates trauma-/ haemorrhagic shock-induced acute lung injury through inhibiting oxidative stress and the NF-kappaB-dependent inflammatory/ MMP-9 pathway. Int. J. Exp. Pathol. 96, 406e413.

339

Sonnenberg, H., Honrath, U., Chong, C.K., Wilson, D.R., 1986. Atrial natriuretic factor inhibits sodium transport in medullary collecting duct. Am. J. Physiol. 250, F963eF966. Sudoh, T., Kangawa, K., Minamino, N., Matsuo, H., 1988. A new natriuretic peptide in porcine brain. Nature 332, 78e81. Sudoh, T., Minamino, N., Kangawa, K., Matsuo, H., 1990. C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Biochem. Biophys. Res. Commun. 168, 863e870. Suga, S., Nakao, K., Hosoda, K., Mukoyama, M., Ogawa, Y., Shirakami, G., Arai, H., Saito, Y., Kambayashi, Y., Inouye, K., 1992. Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide. Endocrinology 130, 229e239. Sylva, M., Li, V.S., Buffing, A.A., van Es, J.H., van den Born, M., van d, V., Gunst, Q., Koolstra, J.H., Moorman, A.F., Clevers, H., van den Hoff, M.J., 2011. The BMP antagonist follistatin-like 1 is required for skeletal and lung organogenesis. PLoS One 6, e22616. Takei, Y., 2008. Exploring novel hormones essential for seawater adaptation in teleost fish. Gen. Comp. Endocrinol. 157, 3e13. Tamura, N., Ogawa, Y., Chusho, H., Nakamura, K., Nakao, K., Suda, M., Kasahara, M., Hashimoto, R., Katsuura, G., Mukoyama, M., Itoh, H., Saito, Y., Tanaka, I., Otani, H., Katsuki, M., 2000. Cardiac fibrosis in mice lacking brain natriuretic peptide. Proc. Natl. Acad. Sci. U. S. A. 97, 4239e4244. Tanaka, M., Murakami, K., Ozaki, S., Imura, Y., Tong, X.P., Watanabe, T., Sawaki, T., Kawanami, T., Kawabata, D., Fujii, T., Usui, T., Masaki, Y., Fukushima, T., Jin, Z.X., Umehara, H., Mimori, T., 2010. DIP2 disco-interacting protein 2 homolog A (Drosophila) is a candidate receptor for follistatin-related protein/follistatin-like 1danalysis of their binding with TGF-beta superfamily proteins. FEBS J. 277, 4278e4289. Thomas, G., Moffatt, P., Salois, P., Gaumond, M.H., Gingras, R., Godin, E., Miao, D., Goltzman, D., Lanctot, C., 2003. Osteocrin, a novel bone-specific secreted protein that modulates the osteoblast phenotype. J. Biol. Chem. 278, 50563e50571. Thuerauf, D.J., Marcinko, M., Gude, N., Rubio, M., Sussman, M.A., Glembotski, C.C., 2006. Activation of the unfolded protein response in infarcted mouse heart and hypoxic cultured cardiac myocytes. Circ. Res. 99, 275e282. Tonne, J.M., Campbell, J.M., Cataliotti, A., Ohmine, S., Thatava, T., Sakuma, T., Macheret, F., Huntley, B.K., Burnett Jr., J.C., Ikeda, Y., 2011. Secretion of glycosylated pro-B-type natriuretic peptide from normal cardiomyocytes. Clin. Chem. 57, 864e873. Tschop, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908e913. van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K.S., Yee, V.C., 2000. Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor. Nature 406, 101e104. Vellaichamy, E., Khurana, M.L., Fink, J., Pandey, K.N., 2005. Involvement of the NF-kappa B/matrix metalloproteinase pathway in cardiac fibrosis of mice lacking guanylyl cyclase/natriuretic peptide receptor A. J. Biol. Chem. 280, 19230e19242. Vellaichamy, E., Das, S., Subramanian, U., Maeda, N., Pandey, K.N., 2014. Genetically altered mutant mouse models of guanylyl cyclase/natriuretic peptide receptor-A exhibit the cardiac expression of proinflammatory mediators in a gene-dose-dependent manner. Endocrinology 155, 1045e1056. Vila, G., Grimm, G., Resl, M., Heinisch, B., Einwallner, E., Esterbauer, H., Dieplinger, B., Mueller, T., Luger, A., Clodi, M., 2012. B-type natriuretic peptide modulates ghrelin, hunger, and satiety in healthy men. Diabetes 61, 2592e2596. Volpe, M., 2014. Natriuretic peptides and cardio-renal disease. Int. J. Cardiol. 176, 630e639.

340

14. HEART HORMONES

Wang, T.J., Larson, M.G., Levy, D., Benjamin, E.J., Leip, E.P., Wilson, P.W., Vasan, R.S., 2004. Impact of obesity on plasma natriuretic peptide levels. Circulation 109, 594e600. Wang, T., Liu, J., McDonald, C., Lupino, K., Zhai, X., Wilkins, B.J., Hakonarson, H., Pei, L., 2017. GDF15 is a heart-derived hormone that regulates body growth. EMBO Mol. Med. 9, 1150e1164. Wei, K., Serpooshan, V., Hurtado, C., Diez-Cunado, M., Zhao, M., Maruyama, S., Zhu, W., Fajardo, G., Noseda, M., Nakamura, K., Tian, X., Liu, Q., Wang, A., Matsuura, Y., Bushway, P., Cai, W., Savchenko, A., Mahmoudi, M., Schneider, M.D., van den Hoff, M.J., Butte, M.J., Yang, P.C., Walsh, K., Zhou, B., Bernstein, D., Mercola, M., Ruiz-Lozano, P., 2015. Epicardial FSTL1 reconstitution regenerates the adult mammalian heart. Nature 525, 479e485. Xu, J., Kimball, T.R., Lorenz, J.N., Brown, D.A., Bauskin, A.R., Klevitsky, R., Hewett, T.E., Breit, S.N., Molkentin, J.D., 2006. GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation. Circ. Res. 98, 342e350. Yan, W., Wu, F., Morser, J., Wu, Q., 2000. Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptideconverting enzyme. Proc. Natl. Acad. Sci. U. S. A. 97, 8525e8529. Yandle, T.G., Richards, A.M., Nicholls, M.G., Cuneo, R., Espiner, E.A., Livesey, J.H., 1986. Metabolic clearance rate and plasma half life of

alpha-human atrial natriuretic peptide in man. Life Sci. 38, 1827e1833. Yang, H., Song, Z., Jin, H., Cui, Y., Hou, M., Gao, Y., 2014. Protective effect of rhBNP on intestinal injury in the canine models of sepsis. Int. Immunopharmacol. 19, 262e266. Zeidel, M.L., Kikeri, D., Silva, P., Burrowes, M., Brenner, B.M., 1988. Atrial natriuretic peptides inhibit conductive sodium uptake by rabbit inner medullary collecting duct cells. J. Clin. Investig. 82, 1067e1074. Zhang, X., Ibrahimi, O.A., Olsen, S.K., Umemori, H., Mohammadi, M., Ornitz, D.M., 2006. Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J. Biol. Chem. 281, 15694e15700. Zhang, Y.H., Youm, J.B., Earm, Y.E., 2008. Stretch-activated nonselective cation channel: a causal link between mechanical stretch and atrial natriuretic peptide secretion. Prog. Biophys. Mol. Biol. 98, 1e9. Zhang, C., Huang, Z., Gu, J., Yan, X., Lu, X., Zhou, S., Wang, S., Shao, M., Zhang, F., Cheng, P., Feng, W., Tan, Y., Li, X., 2015. Fibroblast growth factor 21 protects the heart from apoptosis in a diabetic mouse model via extracellular signal-regulated kinase 1/2-dependent signalling pathway. Diabetologia 58, 1937e1948.

C H A P T E R

15 Stomach Hormones Helge Waldum Faculty of Medicine, Norwegian University of Science and Technology and St. Olav’s University Hospital, Trondheim, Norway The concept of hormones, and thus endocrinology, started within the upper gastrointestinal tract by Bayliss and Starling showing that acidification of denervated duodenum/pancreas resulted in a profuse stimulation of the secretion of water and bicarbonate (Bayliss and Starling, 1902). They concluded that a chemical signal substance was released from the duodenal mucosa and carried with the blood to the pancreas, where it stimulated secretion. Accordingly, they named this signal substance secretin. A few years later, Edkins postulated that another similar signal substance, gastrin, was released from the antral mucosa and transported by the blood to the oxyntic mucosa, and there stimulated acid secretion (Edkins, 1906). Substances being released at one place and carried with the blood to another place/organ, where they influence the function, are generally called hormones. Both the secretin- and gastrin-producing cells occur in mucosae spread among exocrine cells. Insulin, on the other hand, is produced in b cells located among other endocrine cells found as the islands of Langerhans in the exocrine pancreas. Endocrine organs like the pituitary gland, the parathyroid glands, and the thyroid gland represent a further step in the separation of endocrine cells from other cell types. The endocrine cells are, together with neurons, central in the regulation of other cells and organs. There are many similarities between these regulatory cells, like secretory granules with connected proteins (granins) (Deftos, 1991) and synaptic vesicles with their specific proteins/peptides like synaptophysin (Wiedenman et al., 1988). In phenotype, there is a continuous spectrum from neurons with axons, through neuroendocrine (NE) cells with shorter elongations approaching their nearby target cells for delivery of their signal substances, to round endocrine cells releasing their hormones to the blood. Based upon all these similarities and the gradual changes in

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00015-8

phenotype, it was not surprising when Pearse proposed that all these cells were embryonically connected, claiming that the NE cells derived from the neural crest (Pearse and Polak, 1971). In the last decades, this theory has not been experimentally supported (Thompson et al., 1990a,b), but there are also recent data supporting the neural crest origin of NE cells (Thompson et al., 1990a,b). Some signal substance-producing cells have direct contact with the gastrointestinal lumen, and their function can accordingly be influenced directly (cells of the open type), whereas other cells do not reach the lumen (cells of the closed type) and can therefore be affected only by nerves, hormones, and paracrine substances. In this overview, I will cover the cells in the stomach, producing signal substances. However, only the real hormones, transported by the blood (gastrin and ghrelin), (Table 15.1) will be described in detail, in contrast to signal substances affecting function via the paracrine route.

1. GASTRIC CELLS PRODUCING SIGNAL SUBSTANCES First, the gastric mucosa differs according to localization: (1) the oxyntic mucosa producing acid, localized to the corpus and fundus; (2) the antral (pyloric) mucosa where gastrin, the main regulator of gastric acid secretion, is produced; and finally, (3) the cardiac mucosa that normally covers only a few millimeters (Fig. 15.1). However, it has been recognized that oxyntic glands may be found in the antral mucosa (Choi et al., 2014). Thus, the ECL cell is also naturally occurring in the antrum. Furthermore, there is a dispute whether cardiac mucosa is a separate entity or just a metaplastic mucosa (Choandrasoma et al., 2000). Anyhow, since there are no

341

Copyright © 2020 Elsevier Inc. All rights reserved.

342 TABLE 15.1

15. STOMACH HORMONES

Gastric Hormones. Producing Cell

Receptor, Cellular, Anatomic Localization

Structure

Localization

Gastrin

Peptide

Antral mucosa

G Cell

Gastrin/CCKB R localized on ECL cell and in the brain

Stimulation of ECL cell histamine release and proliferation; neurotransmitter

Regulation and stimulation of gastric acidity

The pathogenesis of peptic ulcer disease and gastric carcinogenesis

Ghrelin

Peptide

Oxyntic mucosa

A-like cell

GHS-R

Stimulation of GH release, activation of orexigenic neuronal pathways

Food intake Regulation of growth

Obesity A factor in emaciation after gastrectomy

FIGURE 15.1 Gastric lumen is covered by two (three) different mucosae.

signal-producing cells occurring solely in the cardiac mucosa, cardiac mucosa will not be further dealt with in this overview.

2. METHODS FOR IDENTIFYING CELLS PRODUCING SIGNAL SUBSTANCES In the later part of the 19th century and the first part of the 20th, cellular heterogeneity of the gastrointestinal mucosa was recognized. By light microscopy with or without specific staining reagents, it was realized that the gastrointestinal mucosa contains cells with different qualities compared to the dominating exocrine cells (Sundler and Ha˚kanson, 1991). Chrome salts accumulate in such cells, and they were therefore named enterochromaffin (EC) cells. The same cell type also has the ability to reduce Agþþ to Ag and to accumulate metallic silver, and Masson thus showed them to be argentaffine (Masson, 1914). Without specific staining, Feyrter recognized

Primary Function

Biologic Function

Hormone

Role in Disease

the endocrine cells to be paler than the others, and he accordingly called them clear cells (Feurter, 1938). Serotonin (5-hydroxytryptophane) was later recognized as the substance within the EC cells reducing silver and chrome (Sundler and Ha˚kanson, 1991). Based upon morphologic similarities to EC cells, but without argentaffinity, another type of endocrine cells was suspected to be present in the oxyntic mucosa. These cells could pick up metallic silver from a solution of silver nitrate containing a reducing agent. They were named argyrophilic, and Grimelius developed a method to detect such cells (Grimelius, 1968). Another silver method described by Sevier and Munger (1965) was shown to stain only EC cells, and this new type of endocrine cells, occurring principally only in the oxyntic mucosa, was subsequently called enterochromaffin-like (ECL) cells, and they were shown to contain histamine in the rat (Ha˚kanson and Owman, 1969). Subsequently, a histofluorescence method for the detection of monoamines in tissue was developed (Falck et al., 1962), and by a specific substance (o-phthalaldehyde (OPT) as vapor reagent), Ha˚kanson could show that rat ECL cells contained histamine (Ha˚kanson, 1970). Shortly afterward, immunocyto-histochemistry was established, and this improved specificity as well as sensitivity for the localization of molecules in tissue. Using the rate-limiting enzyme for histamine synthesis, histidine decarboxylase (HDC), as well as histamine as antigens for the production of antibodies, it became evident that the ECL cell was histamine producing not only in rodents, but also in other species, including man (Ha˚kanson et al., 1986b). Soon, it was established that there are many different types of endocrine cells in the gastrointestinal mucosa (Fig. 15.1), and the same cell may produce more than one signal substance (Sykaras et al., 2014). Besides the characterization of endocrine cells by their hormones, they are also classified ultrastructurally (Vasallo et al., 1969; Capella et al., 1991). By introducing immune-electron microscopy, the ultrastructural

4. THE EC CELL AND 5-HYDROXYTRYPATAMINE (5-HT) (SEROTONIN)

localization of the signal substances in the secretory granules may be detected (Ravazzola and Orci, 1980). The aforementioned methods have been used to classify the different endocrine cells in the gastrointestinal mucosa.

3. TYPES OF “ENDOCRINE” CELLS IN THE GASTRIC MUCOSA, THEIR LOCALIZATION AND DISTRIBUTION Although there is no sharp border between the oxyntic and antral mucosa, the distinction between these two types of mucosa is important (Fig. 15.1). In the antrum, the gastrin producing G cell is localized, often in close proximity to the somatostatin-producing D cell. Both these cells are of the open type (having contact with gastric lumen), so they may respond directly to the gastric luminal content. Moreover, the antral D cell sends axon-like projections toward the G cell, thus allowing directed release of somatostatin to the G cell (Larsson et al., 1979). In the oxyntic mucosa, the D cells are of the closed type, in contrast to the antral ones. The serotonin producing EC cell occurs as a cell of the open type in the antrum. In the oxyntic mucosa, they are of the closed type, and there is a marked species difference in EC cell density in the oxyntic mucosa, where they are virtually lacking in the rat, whereas they are quite common in man (Sundler and Ha˚kanson, 1991). This implies that the Sevier-Munger method, staining only EC and ECL cells, may be used as a rather specific method for ECL cells in the rat stomach. In contrast to the antrum, all the “endocrine” cells in the oxyntic mucosa are of the closed type. Among the “endocrine” cells in the oxyntic mucosa, the histamine-producing ECL cell TABLE 15.2

343

is the most prevalent in both rodents and man, although quantitatively much more prevalent in the rat (around 65% of all endocrine cells) compared to man (around 35%) (Sundler and Ha˚kanson, 1991). This difference in ECL cell density reflects the differences in 24-h gastrin levels between these two species, with much higher gastrin values in the rat, eating all the time and thus having food in the stomach continuously, compared to man, having a few meals a day. Animals eating only once a day have even lower ECL density than man, and interestingly, histamine is more potent in stimulating gastric acid secretion in those species with low density of ECL cells (Waldum et al., 1993). The second most prevalent “endocrine” cell in the oxyntic mucosa is the A-like cell, named after its ultrastructural similarities to glucagon producing a-cell in the pancreas. A-like cells produce the hormone ghrelin (Date et al., 2000), taking part in the regulation of food intake (Nakazato et al., 2001). The different cells in the gastric mucosa producing signal substances are summarized in Table 15.2.

4. THE EC CELL AND 5HYDROXYTRYPATAMINE (5-HT) (SEROTONIN) The EC cell occurs in both the oxyntic and the antral mucosa, being of the closed type in the oxyntic mucosa and of the open type in the antrum. In the oxyntic mucosa, there are large species differences in the prevalence of EC cells, accounting for about 25% of the NE cells in man and virtually not present in the rat oxyntic mucosa (Sundler and Ha˚kanson, 1991). Like many monoamines, 5-HT found in abundance in EC cells is a reducing agent,

Gastric Signal Substance Producing Cells.

Cell

Localization

Type

Signal Substance

Type of Delivery

Main Receptors

ECL cell

Oxyntic mucosa

Closed

Histamine

Paracrine

CCCKB R, PAC1 R, somatostatin2R

A-like cell

Mainly oxyntic

Closed

Ghrelin (obestatin)

Hormonal

GPR120

D cell

Oxyntic

Closed

Somatostatin

Paracrine

Neurotransmitter receptors (muscarinic-3 (M-3)) GLP-1, CGPR, somatostatin-2 R

D cell

Antral

Open

Somatostatin

Paracrine

Putative Hþ receptor (CaR), CCKB (gastrin) receptor

G cell

Antral

Open

Gastrin

Hormonal

GPR92, CaR, GRPR, somatostatin2R

EC cell

Oxyntic

Closed

Serotonin

Paracrine

Chemosensor

EC cell

Antral

Open

Serotonin

Paracrine

Chemosensor

344

15. STOMACH HORMONES

making such cells brownish when exposed to a silversalt solution or more yellow with a chrome-salt. These methods were the initial ones used to characterize NE cells before immunohistochemistry was established, using antibodies toward the signal substance (5-HT in the EC cell) or general markers like granins, particularly chromogranin A. Granins are found in secretory granules only, so granins have become useful general markers for the NE cells. The main signal substance of the EC cell, 5-HT, is released together with the granins stored in the same secretory granules. Granins probably play a role in binding the small molecular signal substances, thus reducing the osmolality in the granules. Neither granins themselves nor their split products seem to play any important biologic role extracellularly, although many effects have been described (D’amico et al., 2014). EC cells may show elongations, which seem to be a general trait of NE cells (Gustafsson et al., 2006). Based upon ultrastructural appearance of the secretory granules, a classification of NE cells was established. The secretory granules of the EC cells are pleomorphic and may be rod-like, possibly reflecting a biconcave shape (Solcia et al., 1975). Serotonin (5-HT), the most important signal substance produced in EC cells, is not a hormone since it is rapidly destructed in the blood, particularly in the liver. This is exemplified by the fact that EC cell carcinoids give rise to overproduction symptoms (diarrhea, flushing, and asthma), only after having metastasized to the liver. The number of EC cells in the stomach is only a small fraction of the total number of EC cells in the gastrointestinal tract. There may be differences between EC cells in different locations. Leptin may also be found in gastric EC cells (Le Beyec et al., 2014). Locally, however, 5-HT may affect the function of muscle and exocrine cells, as reflected by the symptoms seen in carcinoid syndrome. 5-HT from EC cells and enteric plexus reduces/stimulates gastric acid secretion in a modulatory way (Lai et al., 2009). The mechanism by which 5-HT influences acid secretion is, however, not clarified. Fibrosis of the heart valves is also a complication of EC cell carcinoid syndrome, and serotonin released from platelets could play a role in the pathogenesis of heart valve disease in general. However, there are inherent problems detecting serotonin in blood due to the escape of serotonin from platelets during the blood sampling (Zeinali et al., 2013). Recently, the EC cell was reported to serve as a chemosensor coupled to sensory nerves (Bellono et al., 2017). EC cells express voltage-gated channels, which upon activation, leads to serotonin release (Bellono et al., 2017). In the stomach, acidification has been reported to increase the serotonin level (Yu et al., 2001). We have previously shown that EC cell axons have a connection to neural cells resembling a synapsis (Gustafsson et al., 2006). The EC cell probably plays a role in mucosal

inflammation in general, and the blocking of peripheral serotonin synthesis by telotristat reduced gut inflammation in mice (Kim et al., 2015). Inflammation and serotonin release are important factors in nausea after cytotoxic treatment for cancer. Moreover, rotavirus may infect EC cells in the small intestine, resulting in increased release of serotonin (Bialowas et al., 2016). It is likely that at least EC cells in the antral mucosa, being of the open type, may similarly be affected by this virus. Only a few studies have addressed the regulation of serotonin release from gastric EC cells. In the isolated rat stomach, luminal acidification was reported to increase gastric serotonin release (Yu et al., 2001). Serotonin has been reported to inhibit both pentagastrin-stimulated (Bech and Andersen, 1985) and histamine-stimulated (Bech, 1986) acid secretion in the dog. Since there are many more EC cells in the gut than in the stomach, it may be presumed that gut EC cells are the most important ones in these respects. There are many different receptors on the EC cell that, however, will not be dealt with, since the EC cell is mainly a cell of the intestine.

5. THE D CELL AND SOMATOSTATIN The somatostatin-producing D cells (Fig. 15.2) occur in the mucosa throughout the gastrointestinal tract, as well as in the pancreas. The secretory granules of the D cells are round and homogenous. In contrast to other NE cells in the stomach, granins are not abundant in the secretory granules of the D cells. Only antibodies toward a few sequences of the granin molecules react with the secretory granules of the D cells (Norlen et al., 2001; Portela-Gomes and Stridsberg, 2002). However, in a recent comprehensive study, granins, including chromogranin A expression, were detected (Egerod et al., 2015). D cells were the first NE cells shown to have axon-like projections (Larsson et al., 1979), approaching neighboring cells of different types. The D cell shape thus reflects their local regulatory role, releasing somatostatin in this modified paracrine way. Somatostatin has probably no endocrine function in the normal situation. However, a syndrome due to a somatostatin-producing tumor, somatostatinoma, has been described, with diabetes, malabsorption, and cholelithiasis attributable to the endocrine effects of somatostatin (Larsson et al., 1977). Somatostatinomas are very rare and often connected to neurofibromatosis type 1 or multiple endocrine neoplasia (Garbrecht et al., 2008), as well as to mutations of hypoxia-inducible factors (Yang et al., 2013). Gastric somatostatinoma is an extremely rare tumor. Based upon the negative trophic effect of gastrin on oxyntic D cells (Chen et al., 1992), we examined tumors developing in the oxyntic mucosa of patients after antral resection, many decades earlier (Waldum et al., 1994). We could classify one of 20 tumors

6. THE G CELL AND ITS REGULATION

345

FIGURE 15.2 A pyloric gland showing the interaction between somatostatin from the D cell and gastrin release from the G cell. Both the D and G cell have luminal contact and respond to Hþ. The G cell also responds to luminal peptides and amino acids. In the figure, only receptors on the basolateral membrane are depicted.

as D cell derived in a Billroth II operated patient (Waldum et al., 1994). Otherwise, to my knowledge, there is only one other described gastric somatostatinoma (Prchayakui et al., 2013). Besides somatostatin, the D cell produces amylin (Beales and Calam, 2003) and PYY (Egerod et al., 2015). In the stomach, the D cells are of the open type in the antrum and of the closed type in the oxyntic mucosa. Somatostatin has a general inhibitory effect on exocrine and endocrine cells, affecting their functions by interacting with five different receptors (Moller et al., 2003). The elongations of the D cells allow release of somatostatin in proximity to the target cells, thus ensuring a high local concentration. In the antrum the D cells are in close connection with the gastrinproducing G cells, both cells being of the open type, thus able to respond to intraluminal changes (Fig. 15.2). The quantitative role of somatostatin versus a direct effect of intraluminal factors on the G cell in the regulation of gastrin release is still not clarified. However, studies on isolated innervated antral pouches showed that luminal administration of acid resulted in a concomitant fall in gastrin release and increase in somatostatin release, as well as a fall in gastrin release upon intraarterial infusion of somatostatin (Holst et al., 1992). The calcium-sensing receptor (CaR), which may be the intragastric Hþ receptor, is located on the D cell (Egerod et al., 2015). When located on the luminal part of the D cell membrane, it may regulate acid secretion by affecting gastrin release (Fig. 15.2). Moreover, the gastric D cell expresses

somatostatin receptors type 1 and 2, having a negative effect on release of somatostatin, thereby acting as a negative feedback loop (Egerod et al., 2015). A receptor for a signal substance, localized to the cell producing the same substance, will probably have a negative effect. The role of somatostatin in the regulation of the gastrin release will also be handled under the heading for the G cell. D cells in the oxyntic mucosa may influence acid secretion both by inhibiting ECL cell histamine release and by a direct effect on the acid-producing parietal cell (Sandvik and Waldum, 1988). Interestingly, baseline histamine release was unaffected by somatostatin. This is also supported by the lack of any effect on baseline acid secretion when knocking out the somatostatin-2 receptor (Piqueras and Martinez, 2004). The latter finding also indicates that somatostatin does not have any direct effect on the parietal cell in the basal state.

6. THE G CELL AND ITS REGULATION The G cell (Fig. 15.4) was identified as the gastrinproducing cell by immunofluorescence, using gastrin antibodies, in the late 1960s (McGuigin, 1968). The G cells are located in the antral glands, but the dept within the glands varies between species (Sundler and Ha˚kanson, 1991). In man, they occur mainly in the mid layer. The G cells have a flask shape, reaching the lumen

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FIGURE 15.3 The most important receptors on the ECL cell and the regulation of gastric acid secretion. There is no gastrin receptor on the parietal cell.

FIGURE 15.4 Gastrin stimulates the ECL cell to release histamine and to proliferate. Whether the general trophic effect of gastrin is mediated by direct stimulation of stem cells or via histamine or other signal substances from the ECL cell is not yet clear. Reproduced with permission from Waldum, H.L., Brenna, E., Sandvik, A.K., 1998c. Relationship of ECL cells and gastric neoplasia. Yale J. Biol. Med. 71, 325e335.

with microvilli on their luminal membrane. The secretory granules may be vesicular or compact. By immunogold-cytochemistry the secretory granules have been shown to contain gastrins, as well as granins. Inconsistently, and with species differences, the G cells have also been described to produce other peptide

hormones, like ACTH, neurotensin, and glycoprotein hormone alpha-subunit (Sundler and Ha˚kanson, 1991), but the physiologic role, if any, of the gastric production of these peptides is unknown. Such sporadic production of signal substances in NE cells makes it, on the other hand, easier to understand the occurrence of diversified symptoms of hormonal overproduction, which may occur in NE tumors. Gastrin release has been examined in vivo, as well as in isolated G cells, and in intact conscious rats by microdialysis (Ericsson et al., 2010). Proteins, peptides, and amino acids stimulate (Lichtenberger et al., 1982) and Hþ inhibits gastrin release (Walsh et al., 1975). The peptone receptor GPR92 is an actual candidate for the receptor by which peptides stimulate gastrin release (Rettenberger et al., 2015) and subsequently acid secretion. Moreover, an additional candidate is the amino acid receptor GPRC6A (Haid et al., 2011). On the other hand, the receptor sensing intraluminal acidity has been difficult to characterize. The calcium-sensing receptor (CaR) is a candidate for this function (Goo et al., 2010). CaR-null mice have low gastrin values, and neither did their gastrin release increase during neutralization of gastric content (Feng et al., 2010). Furthermore, human gastrinoma cells also express CaR (Itami et al., 2001). Concerning the role of the D cell versus the G cell itself, in the regulation of gastrin release (Fig. 15.2), it is interesting that basal gastrin and gastrin levels during neutralization of gastric content were not affected when knocking out the somatostatin-2 receptor

7. THE ECL CELL

(Piqueras and Martinez, 2004). The G cell and the antral D cell differ from each other in respect to intracellular signaling (Haid et al., 2012). These results suggest that there is no restraint on gastrin release by somatostatin in the baseline situation. The G cell probably has a secretin receptor as well, since secretin affects the concentration of gastrin in blood of healthy individuals (Brady et al., 1987), and a secretin receptor has been shown to be present in gastrinomas (Long et al., 2007). Initially, secretin was reported to reduce blood gastrin levels in subjects without gastrinoma, whereas it induced a marked increase in patients with gastrinoma (Isenberg et al., 1972). However, this so-called paradoxical effect by secretin in patients with gastrinoma may rather be classified as an exaggeration since secretin also increases gastrin levels in healthy individuals (Brady et al., 1987). It has to be recalled that gastric acid is the main stimulator of secretin release (Boden et al., 1974). In patients with gastrinoma, on the other hand, secretin stimulates gastrin release and acid secretion more markedly, possibly due to an increased G cell mass. Gastrin release is also stimulated by gastrin-releasing peptide (GRP) via the receptor GRPR localized on the G cell (Varnet et al., 1981). We found that stimulation of peroxisome proliferatoractivated receptors of type a (PPAR-a) induced hypergastrinemia in rats, an effect not reduced by concomitant administration of the long-acting somatostatin analog octreotide (Bakke et al., 2000). We detected PPAR-a in G cells in the antrum, which could indicate that a PPAR-a ligand could have a direct effect on antral G cells, explaining its hypergastrinemic effect (Martinsen et al., 2005).

7. THE ECL CELL Since Popielsky described that histamine-stimulated gastric acid secretion, there has been a continuous dispute about the physiologic role of histamine with regard to the regulation of acid secretion, and the possible interactions between gastrin and histamine. Komarov showed that the gastric secretagogue in the antral mucosa was not histamine (Komarov, 1938). Moreover, the traditional histamine blockers did not affect gastric acid secretion. Gregory and Tracy extracted and purified gastrin (Gregory and Tracy, 1961), which was shown to be a potent stimulator of gastric acid secretion (Maklouf et al., 1966). Shortly afterward, McGuigan, by the aid of radioimmunoassay, described that food intake resulted in gastrin increase in the blood (McGuigan and Trudeau, 1970). Thus, there seemed to be no role for histamine (Johnson, 1971). This changed, of course, when Black and coworkers described the presence of a histamine-2

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receptor and showed the first histamine-2 blocker to be a potent inhibitor of acid secretion (Black et al., 1971). Curiously, the mast cell, a cell without a fixed position in the tissue, was thought to be the cell producing the histamine taking part in the regulation of gastric acid secretion, until Ha˚kanson described that the ECL cell in rat oxyntic mucosa was histamine producing (Ha˚kanson and Owman, 1969). Even after this finding, the mast cell was thought to be the cell producing the histamine participating in the regulation of acid secretion in other species, until methods with improved sensitivity allowed the Ha˚kanson group to detect histamine in ECL cells from all species examined (Ha˚kanson et al., 1986b). The ECL cell is argyrophilic but lacks monoamines, so it cannot reduce silver (not argentaffin), but it is argyrophilic positive for both Grimelius and Sevier-Munger staining (Sevier and Munger, 1965). Immunocytochemistry using antibodies toward histamine, showed positivity for histamine in ECL cells in all species examined (Ha˚kanson et al., 1986b). Histamine is synthetized from histidine by the action of histidine decarboxylase (HDC), which is also a marker for the ECL cell. After synthesis of histamine in the cytoplasm, the vesiculo-membrane transporter type 2 (VMAT-2) pumps histamine into secretory vesicles (Dimaline and Struthers, 1996), where it is stored together with granins. VMAT-2 is a relatively specific marker for the ECL cell. The ECL cell is restricted to the oxyntic glands, and in mammals, they are mainly localized toward the lower part of the glands (Fig. 15.3). Rodents have a much higher density of ECL cells than other mammals, probably reflecting differences in eating habits. Thus, rodents tend to eat continuously so have food in the stomach all the time, and therefore have continuously high gastrin values, the most important trophic factor of the ECL cell. In species with a high ECL cell density, histamine tends to have low potency as gastric acid secretagogue (Waldum et al., 1993). The ECL cell has axon-like projections, often in close proximity to the parietal cell (Gustafsson et al., 2011). Ultrastructurally, the secretory granules of the ECL cell are quite typical, being vesicular with an eccentric core. Besides histamine, the ECL cell produces calbindin (Buffa et al., 1989) and the a subunit of chorionic gonadotropin (a-hCG) (Bordi et al., 1988). Whereas histamine is central in the stimulation of the parietal cell, the functions of calbindin and a-hCG are still obscure. The CCK B (gastrin) receptor is localized to the ECL cell, where it has a central role in the regulation of the gastric acid secretion (Blair et al., 1987; Bakke et al., 2001; Fig. 15.3). The CCK B receptor (CCKBR) was initially cloned from isolated oxyntic mucosal cells enriched to high purity in parietal cells (Kopin et al., 1992). This was taken as a proof that there is a CCKBR

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on the parietal cell, which had been disputed for decades. Shortly afterward the CCKBR was also cloned from a gastric neuroendocrine tumor (NET) originating from the ECL cell in Mastomys natalensis (Nakata et al., 1992). From a functional point of view, the effect of gastrin on acid secretion may be explained by its stimulatory effect on histamine release from the ECL cell (Waldum and Sandvik, 1993). In binding studies, applying a gastrin analog at a concentration in the upper physiologic range, in the isolated rat stomach, we found binding to the ECL cell but not to the parietal cell (Bakke et al., 2001). The binding affinity to a specific receptor should be independent of the cell type localization. This strongly indicates that there is no CCKB R on the parietal cell and that the CCKB R cloned from oxyntic mucosal cells, enriched in parietal cells (Kopin et al., 1992), nevertheless contained contaminating ECL cells. The CCKB R belongs to the seven-transmembrane type of receptors. The binding site of the CCKB R is the five C-terminal amino acid residues that gastrins/CCKs have in common. Accordingly, the CCKB R does not discriminate between gastrins and CCKs. However, it may be that the larger molecular forms have a slightly reduced affinity to the receptor, manifested by somewhat reduced potency (Sandvik and Waldum, 1991). The CCKs have a sulfate group in the C-terminal amino acid residue in position 7, and the CCKA R has a 500 times higher affinity for CCKs compared to gastrins, which by definition has no sulfatation in this position (Wank, 1988). After ligand binding to its receptor, the signal is transmitted to the interior of the cell by interaction with G proteins and further down by activation of the phospholipase C, diacylglycerol, Caþþ, protein kinase C chain (80). Gastrin antagonists bind to the CCKB R without inducing activating conformal changes of the receptor. The CCKB R is found in the central nervous system (CNS) and in the stomach localized to the ECL cell. There are reports that there is a CCKB R on the stem cells in oxyntic mucosa (Kazumori et al., 2001), mediating the general trophic effect of gastrin on this mucosa. However, the general trophic effect on oxyntic mucosa may also be an indirect one, mediated by signal substances released from the ECL cell upon stimulation. In this respect, histamine could be a candidate, but such an effect is not mediated by a histamine-2 receptor, since the histamine-2 agonist, impromidine, did not affect stem cell proliferation (Brenna et al., 1995). On the other hand, the REG I protein stimulating oxyntic stem cell proliferation (Fukui et al., 1998) is released from the ECL cell, and it is therefore an actual candidate (Sekikawa et al., 2005). There are also reports on the presence of the CCKB R in other organs in rodents, particularly the pancreas (Berna et al., 2010), but

also the kidneys (de Weerth et al., 1998). However, as there is a close correlation between the functional and trophic effects of gastrin on the ECL cell, such a connection probably also exists in other organs. Moreover, the lack of a positive trophic effect on these organs caused by long-term hypergastrinemia (Ha˚kanson et al., 1986a) does not support a role of gastrin in the carcinogenesis outside the stomach. The pituitary adenylate cyclase-activating peptide (PACAP) does not have any direct effect on the parietal function, but it is a potent stimulator of ECL cell histamine release (Fig. 15.3), and it has an additive effect on gastrin-stimulated histamine release (Sandvik et al., 2001). A PACAP antagonist partly inhibited acid secretion, showing that PACAP is involved in the vagal stimulation of acid secretion, mediated via the ECL cell (Sandvik et al., 2001). The ECL cell expresses the highaffinity PAC1 receptor, and in isolated ECL cells, PACAP stimulated both histamine release and proliferation (Oh et al., 2005). Similar to gastrin, PACAP therefore seems to have parallel functional and trophic effects on the ECL cell. Gastrin and PACAP were reported to stimulate histamine release from ECL cells by induction of intracellular increase in Caþþ, by an interaction between different Ca channels (Lindstro¨m et al., 2001). Unilateral vagotomy in the rat induced a marked reduction in ECL density in the vagotomized mucosa compared to the innervated side, showing that vagal activity has a trophic tone on the ECL cell (Ha˚kanson et al., 1984). In isolated rat ECL cells, PACAP was even found to stimulate proliferation to a higher degree than gastrin (La¨uffer et al., 1999). However, unilateral vagotomy did not prevent the development of ECL cell neoplasia in Mastomys (Wa¨ngberg et al., 1996). The role of the vagal nerves in human gastric tumorigenesis is accordingly not clear, but there are no indications that vagotomy predisposes to gastric cancer (Lundega˚rdh et al., 1994; Sagatun et al., 2014). Since there is no method for the quantification of long-term vagal activity, any direct gastric tumor-promoting effect of the vagal nerves has not been shown. Somatostatin interacts with five different receptors (SSTR1-5) coupled to G proteins. In the oxyntic mucosa, SSTR2 is expressed on both parietal and ECL cells (Allen et al., 2002). Somatostatin has an inhibitory effect on isolated parietal cells (Chew, 1983) as well as ECL cells (Prinz et al., 1994), thus inhibiting two steps of the secretion of gastric acid. Not only D cells but also neurons release somatostatin (Grider, 1989), and since somatostatin is not a circulating hormone, but a messenger released from axons and axon-like elongations on D cells, there may nevertheless be a differentiated response.

8. GASTRIN

8. GASTRIN 8.1 History Gastrin was the second hormone postulated to exist, and it was among the first purified, sequenced, and of which a biologic active ligand (pentagastrin) was synthetized (Gregory and Tracy, 1961). Moreover, McGuigan and Trudeau (1970) established a radioimmunoassay for the determination of gastrin some years after the original description of an insulin radioimmunoassay (Yalow and Berson, 1960). Food intake induced gastrin release (McGuigan and Trudeau, 1970), and pentagastrin was a potent stimulator of gastric acid secretion (Maklouf et al., 1966). Therefore, gastrin in many ways fulfilled the requirements for being the sole humoral stimulator of gastric acid secretion, so histamine was dismissed as a physiologic regulator of acid secretion (Johnson, 1971). However, in the 1960s, Kahlson showed that food intake and gastrin reduced oxyntic mucosal histamine concentration and concomitantly stimulated histamine synthesis (Kahlson et al., 1964). When Black and coworkers shortly afterward described the histamine-2 (H-2) receptor and the effect of H-2 blockers on baseline as well as pentagastrinstimulated gastric acid secretion (Black et al., 1971), it became evident that histamine was important in the regulation of gastric acid secretion. As pointed out earlier, the ECL cell was subsequently accepted as the target cell for gastrin, producing and releasing histamine. The main stimulus for gastrin release from G cells (Fig. 15.6) is intraluminal proteins/peptides/amino acids, whereas Hþ is a brake, inhibiting gastrin release at luminal pH below 3.0 (Walsh et al., 1975). The G cells are mainly localized to the antral mucosa, but they are also found in the duodenum. The receptors controlling gastrin release have been discussed in the G cell paragraph.

8.2 Structure The gastrin gene consists of three exons resulting, in humans, in a peptide of 101 amino acid residues, which, after splitting off the signal peptide from preprogastrin, gives progastrin, a peptide of 80 amino acid residues. This is posttranslationally processed by cleavage during passage through the Golgi apparatus. In the secretory vesicles, gastrins of different length are activated by C-terminal amidation into active compounds (Rehfeld and Johnsen, 1993). The determination of progastrin may have higher sensitivity than measuring amidated gastrin in the diagnosis of gastrinoma (Bardham, 1990), but any separate biologic role of nonamidated

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gastrin precursors has not been shown, although it was claimed some decades ago (Seva et al., 1994). Gastrin belongs to the CCK/gastrin family of peptides. All these peptides have the same carboxyamidated tetrapeptide sequence, Trp-Met-Asp-Phe-NH2, which is also the sequence binding to the CCK B (gastrin) receptor and eliciting the response. CCKs are in contrast to gastrins, sulfated in C-terminal amino acid residue 7, which is important for the binding to the CCK A receptor (Dufresne et al., 2006). Therefore, all CCKs have gastrin activity, whereas gastrin has only a faint CCK activity. These peptide hormones are synthetized in the endoplasmic reticulum, and after removal of the N-terminal signal sequence, the resulting progastrin is modified by phosphorylation and sulfatation during transport through the Golgi apparatus to the secretory vesicles, where they are shortened by proteolytic cleavage and amidated C terminally into biologically active hormones (Rehfeld and Johnsen, 1993). Gastrins are rapidly destroyed in the circulation by endopeptidases. In healthy, fasting individuals, gastrin in the blood is generally below 10 pmol/L, and food intake results in an increase, seldom exceeding three to four times the baseline value. To obtain biologically meaningful results in gastrin immunoassays, it is important to use an antibody directed toward the C-terminal active sequence of the gastrin molecule (Rehfeld et al., 1972; Rehfeld, 1981). Since not only gastrins, but also CCKs, have this sequence, an immunoassay with antibodies directed toward the active site will also include the CCKs. However, since CCKs have full gastrin activity, an immunoassay determining both these peptides is unproblematic and reflecting true biologic gastrin activity. Moreover, CCKs circulate in much lower concentrations than the gastrins, so the CCK contribution to the gastrin values determined by immunoassays will be low anyway. We have for nearly 40 years used antibodies (donated by Rehfeld) directed toward the active gastrin site, and we have experienced a stable and reliable gastrin immunoassay (Kleveland et al., 1985).

8.3 Physiology The cellular localization of the CCKB R in the oxyntic mucosa, and the interaction between the three major gastric acid secretagogues, and particularly regarding histamine and gastrin, was disputed for decades. In the 1970s, Grossman and Code, working in the same institution, had opposite views in this subject (Grossman and Konturek, 1974; Code, 1956). Studying acid secretion indirectly in isolated parietal cells, Berglindh et al. found no effect of gastrin when studying rabbit oxyntic glands, in contrast to the other two secretagogues (histamine and a cholinergic compound) (Berglindh et al.,

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1976), whereas Soll by the same method claimed a faint effect of gastrin on the aminopyrine uptake in canine parietal cells (Soll, 1980). However, the stimulation reported by Soll was inconstant and of doubtful statistical significance. Since dog parietal cells are particularly sensitive toward histamine in respect to stimulation of acid secretion, the effect Soll claimed may have been due to an effect of histamine released from ECL cells by gastrin. In the isolated rat stomach, we found that gastrin, in contrast to a cholinergic compound, did not augment maximal histamine-stimulated acid secretion (Kleveland et al., 1987), and when we infused a fluorescein-labeled gastrin analog at the concentration of 100 pmol/L, we found binding to the ECL cell but not to the parietal cell (Bakke et al., 2001). Similarly, Athmann and coworkers found that a histamine-2 blocker could prevent an increase in intracellular parietal cell Caþþ, evoked by gastrin up to the concentration of 1000 p/mol (Athmann et al., 2000). They reported, however, that gastrin at the concentration of 10,000 pmol/L induced an increase in Caþþ that could not be blocked by a histamine-2 blocker, and therefore speculated that there, nevertheless, could be a gastrin receptor on the parietal cell. The most likely explanation to the latter finding is that the effect of the nonphysiologic concentration was due to an interaction with another type of receptor. In the rat, we detected a stimulation of histamine release by gastrin at a concentration of 2 pmol/L (Sandvik and Waldum, 1990, 1991), reflecting the high affinity of the ligand to its receptor. CCKA R has been described in the oxyntic mucosa, mainly in the basal parts (Reubi et al., 1997). Gastrin has an affinity of 1:500 to the CCKA R compared to CCK, and thereby very high gastrin concentrations can possibly influence the function of cells with a CCKA R. Also, antral stem cells have been claimed to harbor the CCKB R, so gastrin may be a factor in antral carcinogenesis (Hayakawa et al., 2015). However, long-term hypergastrinemia in rats was not reported to induce any positive trophic effect in the antrum (Ha˚kanson et al., 1986a), and in patients with gastrinoma, there is a sharp border between the hypertrophic oxyntic mucosa and the apparently normal antral mucosa. Accordingly, the expression and function of the CCKB R in the antral mucosa needs further studies. In the CNS, both CCKA R and CCKB R are expressed. In peripheral tissue, it seems to be accepted that there is a CCKB R on the C cells in the thyroid, based upon the pentagastrin stimulation test of release of calcitonin (Cooper et al., 1971). CCKB R was also described in the normal thyroid gland and medullary thyroid carcinoma, both by mRNA expression and immunohistochemistry (Bla¨ker et al., 2002). It is, however, a paradox that endogenous hypergastrinemia does not lead to increased concentrations of calcitonin

(Brandsborg et al., 1980). Moreover, in the rat, cholecystokinins and not gastrin-17 release calcitonin from thyroid C cells (Persson et al., 1988). In the pentagastrin stimulation test, the concentration of pentagastrin will be so high that it probably would activate a CCKA R as well. The receptor mediating the calcitonin response of the C cells is accordingly not completely settled. Anyhow, gastrin is an important stimulator of the gastric acid secretion by releasing histamine from the ECL cell (Sandvik et al., 1987; Brenna and Waldum, 1991; Prinz et al., 1993; Prinz et al., 1994; Figs. 15.3 and 15.4). In the isolated rat stomach, we could show that the maximal gastrin-stimulated acid secretion was lower than the maximal histamine-stimulated acid secretion, and that gastrin in contrast to a cholinergic compound did not augment maximal histamine-stimulated acid secretion (Kleveland et al., 1987). Thus, the maximal gastric acid secretory capacity is not reached by gastrin stimulation alone (Qvigstad et al., 1999a; Waldum et al., 1998a). Chronic overstimulation with gastrin induces ECL cell hyperplasia and thus increased capacity to release histamine (Waldum et al., 1991a) and acid secretion. This is the main mechanism of the increase in gastric acid secretion due to hypergastrinemia, seen after a period with profound drug inhibition of gastric acid secretion (rebound acid hypersecretion) (Waldum et al., 1996) and in patients with gastrinoma (Ojeaburu et al., 2011).

8.4 Role in Disease The interaction between gastrin and Helicobacter pylori (Hp) is very complicated but also interesting. It was a major breakthrough when Marshall and Warren (Marshall and Warren, 1984) showed that Hp was the main cause of gastritis and peptic ulcer disease. Hypersecretion of gastric acid had for long been known to be associated with duodenal ulcer (Wormsley and Grossman, 1965), and it was soon described that Hp infection, localized to the antral mucosa, resulted in a slight hypergastrinemia, which got normalized after Hp eradication and concomitantly reduced gastric acid secretion (Levi et al., 1989; El Omar et al., 1993). Initially, it was proposed that the increase in blood gastrin was due to antral alkalization, caused by NH3 produced by the Hp urease. However, although the hypothesis was attractive, it was difficult to prove experimentally (El Nujumi et al., 1991). When going through all the available data, we could nevertheless conclude that local NH3 production in the neighborhood of the antral G cells was the most probable mechanism for the faint hypergastrinemia, which via an increased acid secretion predisposed to duodenal ulcer (Waldum et al., 2016). Blood gastrin in patients with duodenal ulcer secondary

8. GASTRIN

to antral Hp infection is generally within the normal range, but nevertheless inappropriately high in relation to gastric acidity (Smith et al., 1990). When Hp infection affects the oxyntic mucosa, the mucous and bicarbonate secretions from the surface cells, representing the defense mechanisms of the gastric mucosa, are reduced, thereby predisposing for gastric peptic ulcer, although gastric acid secretion may be normal or even reduced (Wormsley and Grossman, 1965). As Hp-induced inflammation in the oxyntic mucosa develops, also affecting the glands, oxyntic mucosal atrophy leads to reduced gastric acid secretion, reduced gastric acidity, and secondary hypergastrinemia. Before the description of Hp as the major cause of gastritis, it was known that gastric cancer occurred only in a stomach with gastritis (Morson, 1955). Therefore, soon after Marshall and Warren had incriminated Hp as the main cause of gastritis, the role of Hp in gastric carcinogenesis was recognized (Parsonnet et al., 1991). The exact mechanism by which Hp infection causes gastric cancer, has, however, not been found, in spite of 25 years of research. Interestingly, Hp infection protects against gastric cancer when the infection is confined to the antral mucosa, and since duodenal ulcer seems to reduce the risk of gastric cancer (Hansson et al., 1996), Hp infection only predisposes to gastric cancer after having provoked atrophy of the oxyntic mucosa (Walker et al., 1971; Tatsuta et al., 1993; Fossmark et al., 2015). Atrophic oxyntic gastritis reduces gastric acid secretion, which induces hypergastrinemia. Based upon our studies on the role of the ECL cell in the gastric carcinogenesis (Waldum et al., 1991a, 1998b,c; Qvigstad et al., 1999b), as well as work on the classification of gastric carcinomas (Qvigstad et al., 2000; Sørdal et al., 2013; Waldum and Sørdal, 2016), we postulated that gastrin is involved in Hpinduced gastric carcinogenesis (Waldum et al., 2015). The well-known risk of not only gastric cancer but also ECL cell carcinoids in patients with so-called autoimmune gastritis (Zamcheck et al., 1955; Kokkola et al., 1998) is also an argument for incriminating the atrophic gastritis and not specific Hp factors in Hp gastric carcinogenesis. Autoimmune gastritis affects only the oxyntic mucosa, and since gastric neoplasia occurring in such patients are found in this mucosa, and not in the cardiac and antral mucosae, the carcinogenic mechanism of oxyntic atrophy is probably not due to a secondary infectious agent. When reexamining gastric carcinomas from patients with anacidity due to autoimmune gastritis, we found that nearly all of them were of the diffuse type and expressed ECL cell markers, indicating that they had developed from the gastrin target cell (Qvigstad et al., 2002). Thus, gastrin could be the mediator of Hp-induced gastric cancer of the diffuse type (Waldum et al., 1991a, 1998a; Bakkelund et al., 2006) but also plays a role in the pathogenesis of the

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intestinal type, as some of them also express neuroendocrine markers. Even though gastric carcinomas of the intestinal type probably mainly originate from stem cells, gastrin could have a pathogenic role, either via a possible CCKB R on the stem cell (Kazumori et al., 2001) or via mediators released from the ECL cell, like the REG protein (Fukui et al., 1998). The final proof that hypergastrinemia alone is so central in human gastric carcinogenesis would be the development of carcinomas in patients with hypergastrinemia and normal or increased gastric acid secretion. To my knowledge, there is no such description. However, there is no report of any patient with gastrinoma living decades with hypergastrinemia, which is required to develop gastric carcinoma (Calvete et al., 2015). On the other hand, patients with gastrinoma, not only secondary to multiple endocrine syndrome type I (MEN I) but also those having a so-called spontaneous gastrinoma, develop gastric NETs (Cadiot et al., 1995), which may be regarded as a precursor to gastric carcinoma. During tumorigenesis/ carcinogenesis the differentiated cell of origin would be expected to gradually lose specific markers, as depicted in Fig. 15.5. Curiously, the role of gastrin in the pathogenesis of many abdominal malignancies not originating from the oxyntic mucosa, like antral carcinomas (Hayakawa et al., 2015), and particularly the colon carcinomas (Smith et al., 1989; Thorburn et al., 1998), has been extensively studied. In spite of great efforts, gastrin has not been firmly incriminated in the pathogenesis of tumors in any of these organs. This is in agreement with the rat study by the group of Ha˚kanson, not finding any trophic effect of long-term hypergastrinemia in any of these organs (Ha˚kanson et al., 1986a,b). Moreover, the same group did not detect gastrin receptor mRNA in the gut, including colon, or in the pancreas (Monstein et al., 1996). The idea behind, for instance, a role of gastrin in colonic neoplasia, was that the tumor cells by mutations of the gastrin receptor gene increased receptor activity, thereby promoting tumorigenesis (Willard et al., 2012). However, such a mechanism is not very probable since the presence of a gastrin receptor on colonic cells has not been established. It may therefore be concluded that gastrin probably has a central position in the carcinogenesis in the oxyntic mucosa, whereas a role in the development of neoplasia in other locations is very doubtful. In conclusion, gastrin has been extensively studied for the more than 100 years after it was postulated to exist. It has a central role in the regulation of gastric acidity, keeping the stomach content sufficiently acidic to kill swallowed microorganisms, which probably is the main biologic function of gastric juice. Acidic gastric juice plays an important role in the pathogenesis of peptic ulcer disease and reflux esophagitis. Intake of inhibitors of

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1976). The A-like cell was so named because of ultrastructural resemblance to the secretory granules in the pancreatic glucagon (A) cell. They were also called X cells because the appearance of their secretory granules is small, round, and compact, and at that time without known production of a signal substance. The A-like cells in most species do not produce glucagon and glucagonrelated peptides. However, in the dog and cat, glucagon production in A-like cells has been described (Solcia et al., 1975; Sundler et al., 1976). The A-like cells are mainly localized to the oxyntic mucosa, but A-like cells are also found in other locations of the gastrointestinal tract.

9.2 Structure

FIGURE 15.5 The changes of a differentiated cell during tumorigenesis/carcinogenesis, leading to loss of specific markers, making it increasingly difficult to recognize the cell of origin. Reproduced with permission from Waldum, H.L., Brenna, E., Sandvik, A.K., 1998c. Relationship of ECL cells and gastric neoplasia. Yale J. Biol. Med. 71, 325e335.

gastric acid secretion and inflammation of the oxyntic mucosa cause reduced gastric acid secretion and reduced gastric acidity leading to secondary hypergastrinemia, which in the long-term may lead to gastric neoplasia. Treatment with a gastrin antagonist may be a promising concept in gastric cancer.

9. A-LIKE CELLS AND GHRELIN/ OBESTATIN 9.1 History The A-like cells were recognized as an entity before their specific hormone production (Sundler et al.,

The signal substance of the A-like cell was described by Kojima et al. (1999). They showed that a new 28 amino acid residue peptide (ghrelin) (Fig. 15.6) purified from the oxyntic mucosa stimulated growth hormone (GH) release by interaction with the so-called GH secretagogue (GHS) receptor (GHS-R), which until then had been an orphan receptor. Ghrelin is modified by octanoylation, a modification not known for other peptides in mammals. There are species differences in the amino acid sequence, but, nevertheless, ghrelin has been well conserved during evolution. Particularly, the N-terminal sequence has been preserved across species, suggesting an important biologic role (Sato et al., 2012). Besides stimulating GH release, ghrelin also stimulates food intake and development of obesity (Tschop et al., 2000). Gastrectomy reduces circulating ghrelin to about one-third (Koizumi et al., 2014), in agreement with the high concentration of A-like cells particularly in the oxyntic mucosa, but it also shows that ghrelin is produced in other organs, since it is still present and even shows tendency to recover over time. Ghrelin is acylated posttranslationally and catalyzed by ghrelin-O-acyltransferase (GOAT), which is important for receptor binding and activation. Nonacylated ghrelin, des-acyl ghrelin, does not bind to GHS-R but, nevertheless stimulates food intake (Toshinai et al., 2006), which could indicate an effect mediated via another receptor. GHS-R was early identified as a seventransmembrane G proteinecoupled receptor (Howard et al., 1996). GHS-R has some similarities to the motilin receptor (Smith et al., 2001).

9.3 Physiology Ghrelin has also been described to increase gastric acid secretion, possibly via the CNS (Date et al., 2001). The stimulatory effect on gastric acid secretion has only been described for acylated ghrelin (Sakurada

10. NEURAL INFLUENCE ON GASTRIC SECRETORY AND SIGNAL-PRODUCING CELLS

et al., 2010). Ghrelin together with leptin and glucagonlike peptide 1, interacts with the GLP-1 receptor localized on vagal afferent neurons and thereby modulates feeding behavior (Ronveaux et al., 2015). Ghrelin concentrations are high during starvation, but low when there is sufficient access to nutrition, independent of whether feeding is oral or via the intravenous route (Qader et al., 2005), which also would be expected taking into consideration that the A-like cell is of the closed type. The exact receptor mediating this effect during feeding is not known, but the fatty acid receptor GPR120 is a candidate (Widmayer et al., 2017) as well as the lactate receptor GPR81 and the CaS R (Engelstoft et al., 2013; Fig. 15.7). Furthermore, lactate via the GPR81 receptor and somatostatin inhibit ghrelin release, whereas adrenalin, GIP, and secretin stimulate ghrelin release (Engelstoft et al., 2013). From a physiologic point of view, it therefore seems that inhibition by mealrelated substances might be most important in the regulation of ghrelin release.

9.4 Obestatin Interestingly, the ghrelin gene also codes for another secreted peptide, obestatin, with claimed biologic activity. This peptide was reported to counteract the effects of ghrelin. Thus, it inhibits food intake as well as motor contractions in the upper part of the small intestine and reduces body weight (Zhang et al., 2005; Ataka et al., 2008). However, nobody has identified an obestatin receptor mediating these effects. Two G proteine coupled receptors (GPR), GPR39 (Dong et al., 2009) and GLP-1R (Gargantini et al., 2016), have been suggested as obestatin receptors. GPR39 is an orphan receptor, while GLP-1R was known to be the receptor for glucagon-like peptide-1. However, others (Sato et al., 2012; Yamamoto et al., 2007) have not reproduced the claimed biologic effects of obestatin.

353

FIGURE 15.6 Ghrelin is a 28 amino acid residue peptide acylated with octanoic acid in position 3 by the action of ghrelin-O-acyltransferase (GOAT). Based upon Sato, T., Nakamura, Y., Shiimura, Y., Ohgusu, H., Kangawa, K., Kojima, M., 2012. Structure, regulation and function of ghrelin. J. Biochem. 151, 119e128.

10. NEURAL INFLUENCE ON GASTRIC SECRETORY AND SIGNAL-PRODUCING CELLS The importance of the CNS in the regulation of gastric acid secretion was demonstrated by Pawlow (1901). The stimulatory phases of gastric acid secretion are divided into two phases, the CNS and gastric ones, whereas the intestinal phase is mainly inhibitory. The quantitative relationship between the two stimulatory phases varies between species, but generally, in species eating and tolerating more contaminated food, the CNS phase will prevail, reflecting that the stomach is prepared for the food before starting to eat. Neurons regulate gastric acid secretion at different levels of the regulatory chain. The acetylcholine analog carbachol stimulates the parietal cell to produce acid (Berglindh et al., 1976) via the muscarinic-3 receptor (Kajimura et al., 1992). PACAP, on the other hand, stimulates histamine release from the ECL cell via the PACE1 receptor (La¨uffer et al.,

FIGURE 15.7 The most important receptors on the ghrelin-producing A-like cell. Based upon Engelstoft, M.S., Park, W.M., Sakata, J., Kristensen, L.V., Husted, A.S., Osborne-Lawrence, S., et al., 2013. Seven transmembrane G-protein-coupled receptor repertoire of gastric ghrelin cells. Mol. Metabol. 2, 376e392.

354

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1999), and like gastrin, PACAP has a positive trophic effect on the ECL cell (Oh et al., 2005). PACAP is released from vagal nerves and neural plexuses in the stomach wall. Likewise, GRP from nerves stimulates gastrin release from the G cell via a GRP receptor. The axon-like elongations that seem to be a common trait of neuroendocrine cells in general make it possible to approach and influence not only effector cells but also sensory neurons in a synaptic way. Thus serotonin from EC cells (Bellono et al., 2017) and ghrelin from A-like cells (Ronveaux et al., 2015) may affect the CNS function via afferent vagal fibers.

11. CONCLUSION The neuroendocrine cells function as relay cells, getting information from nerves and circulating hormones, and a proportion of them, being of the open type, have direct contact with the lumen of hollow organs. Thus, they get information from the whole body, as well as locally, which they integrate into a response transmitted either by delivering a signal substance to the blood (a hormone) affecting the function of an organ or cells far away, or via the paracrine route to neighbor cells, often via neuron-like projections. As a rule, the functional effect of a signal substance on a certain cell type also affects the proliferation of the same cell. Overstimulation of the breast by estrogen, and the prostate by androgens, predisposes to cancer development. Hitherto, peptide hormones have not in the same way been incriminated in carcinogenesis, but recent findings suggest that also these hormones may play an important role in the development of neoplasia by stimulation of cell proliferation. Furthermore, disturbances in concentrations of some of these signal substances most likely will lead to functional problems. Few, if any, hormones have been so carefully studied as gastrin, since modern physiology in many ways started with studies on the regulation of gastric acid secretion, and gastrin was the second hormone to be postulated to exist and the second hormone where a reliable method for the determination of its concentration in blood was established. Moreover, due to its role as an efficient disinfectant, by keeping the gastric juice sufficiently acidic, very few microorganisms, except Hp, can survive there, thus reducing confounding factors. Last, but not least, gastric juice can easily be collected both in animals and man. From the interaction between Hp and gastrin, the pathogenesis of peptic ulcer disease was understood, allowing the disease to be treated. The role of gastrin in gastric carcinogenesis will probably lead to progress in the treatment of gastric cancer, a very severe type of cancer.

References Allen, J.P., Canty, A.J., Schulz, S., Humphrey, P.P., Emson, P.C., Young, H.M., 2002. Identification of cells expressing somatostatin receptor 2 in the gastrointestinal tract of Sstr2 knockout/lacZ knockin mice. J. Comp. Neurol. 454, 329e340. Ataka, K., Inui, A., Asakawa, A., Kato, I., Fujimiya, M., 2008. Obestatin inhibits motor activity in the antrum and duodenum in the fed state of conscious rats. Am. J. Physiol. Gastrointest. Liver Physiol. 294, G1210eG1218. Athmann, C., Zeng, N., Scott, D.R., Sachs, G., 2000. Regulation of parietal cell calcium signaling in gastric glands. Am. J. Physiol. Gastrointest. Liver Physiol. 279, G1048eG1058. Bakke, I., Qvigstad, G., Sandvik, A.K., Waldum, H.L., 2001. The CCK-2 receptor is located on the ECL cell, but not on the parietal cell. Scand. J. Gastroenterol. 36, 1128e1133. Bakke, I., Sandvik, A.K., Waldum, H.L., 2000. Octreotide inhibits the enterochromaffin-like cell but not peroxisome proliferatorinduced hypergastrinemia. J. Mol. Endocrinol. 25, 109e119. Bakkelund, K., Fossmark, R., Nordrum, I., Waldum, H.L., 2006. Signet ring cells in gastric carcinomas are derived from neuroendocrine cells. J. Histochem. Cytochem. 54, 615e621. Bardram, L., 1990. Progastrin in serum from Zollinger-Ellison patients. Gastroenterology 98, 1420e1426. Beales, I.L., Calam, J., 2003. Regulation of amylin release from cultured rabbit gastric fundic mucosal cells. BMC Physiol. 3, 13. https:// doi.org/10.1186/1472-6793-3-13. PMID 14572315 PMCID: PMC269984. Bayliss, W.M., Starling, E.H., 1902. The mechanism of pancreatic secretion. J. Physiol. 28, 325e353. Bech, K., 1986. Exogenous serotonin and histamine-stimulated gastric acid and pepsin secretion in dogs. Scand. J. Gastroenterol. 21, 1205e1210. Bech, K., Andersen, D., 1985. Effect of serotonin on pentagastrinstimulated gastric acid secretion and gastric antral motility in dogs with gastric fistula. Scand. J. Gastroenterol. 20, 1115e1123. Bellono, N.W., Bayrer, J.R., Leitch, D.B., Castro, J., Zhang, C., ´ Donnell, T.A., et al., 2017. Enterochromaffin cells are gut chemoO sensors that couple to sensory neural pathways. Cell 170, 185e198. ¨ brink, K.J., 1976. Effects of secretaBerglindh, T., Helander, H.F., O gogues on oxygen consumption, aminopyrine accumulation and morphology in isolated gastric glands. Acta Physiol. Scand. 97, 401e414. Berna, M.J., Seiz, O., Nast, J.F., Benten, D., Bla¨ker, M., Koch, J., et al., 2010. CCK1 and CCK 2 receptors are expressed on pancreatic stellate cells and induce collagen production. J. Biol. Chem. 285, 38905e38914. Bialowas, S., Hagbom, M., Nordgren, J., Karlsson, T., Sharma, S., Magnusson, K.E., Svensson, L., 2016. Rotavirus and serotonin cross-talk in diarrhoea. PLoS One 11 (7), eO159660. https:// doi.org/10.1371/journal.pone.0159660. Black, J.W., Duncan, W.A., Durant, C.J., Ganellin, C.R., Parsons, E.M., 1971. Definition and antagonism of histamine H2- receptors. Nature 236, 385e390. Blair III, A.J., Feldman, M., Barnett, C., Walsh, J.H., Richardson, C.T., 1987. Detailed comparison of basal and food-stimulated gastric acid secretion rates and serum gastrin concentrations in duodenal ulcer patients and normal subjects. J. Clin. Investig. 79, 582e587. Blaker, M., dr Weerth, A., Tometten, M., Schulz, M., Ho¨ppner, W., Arlt, D., et al., 2002. Expression of the cholecystokinin2-receptor in normal human thyroid gland and medullary thyroid carcinoma. Eur. J. Endocrinol. 146, 89e96. Boden, G., Essa, N., Owen, O.E., Reichle, F.A., 1974. Effects of intraduodenal administration of HCl and glucose on circulating immunoreactive secretin and insulin concentrations. J. Clin. Investig. 53, 1185e1193.

REFERENCES

Bordi, C., Pilato, F.P., Bertele´, A., D’Adda, T., Missale, G., 1988. Expression of glycoprotein hormone alpha- subunit by endocrine cells of the oxyntic mucosa is associated with hypergastrinemia. Hum. Pathol. 19, 580e585. Brady III, C.E., Utts, S.J., Dev, J., 1987. Secretin provocation in normal and duodenal ulcer subjects. Is the gastrin rise in Zollinger-Ellison syndrome paradoxic or exaggeration? Dig. Dis. Sci. 32, 232e238. Brandsborg, M., Nielsen, H.E., Brandsborg, O., Olsen, K.J., Løvgreen, N.A., 1980. The role of serum gastrin in the secretion of calcitonin: studies in patients with pernicious anaemia and healthy subjects. Scand. J. Gastroenterol. 15, 23e28. Brenna, E., Waldum, H.L., 1991. Studies on isolated parietal and enterochromaffin-like cells from the rat. Scand. J. Gastroenterol. 26, 1295e1306. Brenna, E., Tielemans, Y., Kleveland, P.M., Sandvik, A.K., Willems, G., Waldum, H.L., 1995. Effect of the histamine-2 agonist impromidine on stem cell proliferation of the rat oxyntic mucosa. Scand. J. Gastroenterol. 30, 311e314. Buffa, R., Mare, P., Salvadore, M., Solcia, E., Furness, J.B., Lawson, D.E., 1989. Calbindin 28 kDa in endocrine cells of known or putative calcium-regulating function. Thyro-parathyroid C cells, gastric ECL cells, intestinal secretin and enteroglucagon cells, pancreatic glucagon, insulin and PP cells, adrenal medullary NA cells and some pituitary (TSH?) cells. Histochemistry 91, 107e113. Cadiot, G., Vissuzaine, C., Potet, F., Mignon, M., 1995. Fundic argyrophil carcinoid tumour in patient with sporadic-type ZollingerEllison syndrome. Dig. Dis. Sci. 40, 1275e1278. Calvete, O., Reyes, J., Zunı´ga, S., Paumard-Herna´ndez, B., Ferna´ndez, V., Bujanda, L., et al., 2015. Exome sequencing identifies ATP4A gene as responsible of an atypical familial type I gastric neuroendocrine tumour. Hum. Mol. Genet. 24, 2914e2922. Capella, C., Finzi, G., Cornaggia, M., Usellini, L., Luinetti, O., Buffa, R., Solcia, E., 1991. Ultrastructural typing of gastric endocrine cells. In: Ha˚kanson, R., Sundler, F. (Eds.), The Stomach as an Endocrine Organ. Elsevier, pp. 27e51. Chen, D., Uribe, A., Ha˚kanson, R., Sundler, F., 1992. Somatostatin cells in the oxyntic mucosa of hypo- or hypergastrinemic rats. Scand. J. Gastroenterol. 27, 479e482. Chew, C.S., 1983. Inhibitory action of somatostatin on isolated gastric glands and parietal cells. Am. J. Physiol. 245, G221eG229. Chandrasoma, P.T., Der, R., Ma, Y., Dalton, P., Taira, M., 2000. Histology of the gastroesophageal junction: an autopsy study. Am. J. Surg. Pathol. 24, 402e409. ´ Neal, R.O., Rich, A.E., Nam, K.T., Choi, E., Roland, J.T., Barlow, B.J., O et al., 2014. Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum. Gut 63, 1711e1720. Code, C.F., 1956. Histamine and gastric secretion. In: Wolstenholme, G.E.W., O’Conner, C.J. (Eds.), Ciba Foundation Symposium on Histamine. J. & A. Churchill, London, pp. 189e219. Cooper, C.W., Schwesinger, W.H., Mahgoub, A.M., Ontjes, D.A., 1971. Thyrocalcitonin: stimulation of secretion by pentagastrin. Science 172, 1238e1240. Da´mico, M.A., Ghinassi, B., Izzicupo, P., Manzoli, L., Di Baldassarre, A., 2014. Biological function and clinical relevance of chromogranin A and derived peptides. Endocr. Connect. 3, R45eR54. Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M.S., Suganuma, T., et al., 2000. Ghrelin, a novel GH-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141, 4255e4261. Date, Y., Nakazato, M., Murakami, N., Kojima, M., Kangawa, K., Matsukura, S., 2001. Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochem. Biophys. Res. Commun. 280, 904e907.

355

de Weerth, A., Jonas, L., Schade, R., Scho¨neberg, T., Wolf, G., Pace, A., et al., 1998. Gastrin/cholecystokinin type B receptors in the kidney: molecular, pharmacological, functional characterization, and localization. Eur. J. Clin. Investig. 28, 592e601. Deftos, L.J., 1991. Chromogranin A: its role in endocrine function and as an endocrine and neuroendocrine tumor marker. Endocr. Rev. 12, 181e187. Dimaline, R., Struthers, J., 1996. Expression and regulation of a vesicular monoamine transporter in rat stomach: a putative histamine transporter. J. Physiol. 490, 249e256. Dong, X.Y., He, J.M., Tang, S.Q., LI, H.Y., Jiang, Q.Y., Zou, X.T., 2009. Is GPR39 the natural receptor of obestatin? Peptides 30, 431e438. Dufresne, M., Seva, C., Fourmy, D., 2006. Cholecystokinin and gastrin receptors. Physiol. Rev. 86, 805e847. Edkins, J., 1906. The chemical mechanism of gastric secretion. J. Physiol. 34, 133e144. Egerod, K.L., Engelstoft, M.S., Lund, M.L., Grunddal, K.V., Zhao, M., Barir-Jensen, D., et al., 2015. Transcriptional and functional characterization of the G protein-coupled receptor repertoire of gastric somatostatin cells. Endocrinology 156, 3909e3923. el Nujumi, A.M., Dorrian, C.A., Chittajallu, R.S., Neithercut, W.D., McColl, K.E., 1991. Effect of inhibition of Helicobacter pylori urease activity by acetohydroxamic acid on serum gastrin in duodenal ulcer subjects. Gut 32, 866e870. el-Omar, E., Penman, I., Dorrian, C.A., Ardill, J.E., McColl, K.E., 1993. Eradicating Helicobacter pylori infection lowers gastrin and acid secretion by two thirds in patients with duodenal ulcer. Gut 34, 1060e1065. Engelstoft, M.S., Park, W.M., Sakata, J., Kristensen, L.V., Husted, A.S., Osborne-Lawrence, S., et al., 2013. Seven transmembrane Gprotein-coupled receptor repertoire of gastric ghrelin cells. Mol. Metabol. 2, 376e392. Ericsson, P., Ha˚kanson, R., Norle´n, P., 2010. Gastrin response to candidate messengers in intact conscious rats monitored by antrum microdialysis. Regul. Pept. 163, 24e30. ˚ ., Thieme, G., Torp, A., 1962. Fluorescence of catFalck, B., Hillarp, N.-A echolamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10, 348e354. Feng, J., Petersen, C.D., Coy, D.H., Jiang, J.K., Thomas, C.J., Pollak, M.R., Wank, S.A., 2010. Calcium-sensing receptor is a physiologic multimodal chemosensor regulating gastric G-cell growth and gastrin secretion. Proc. Natl. Acad. Sci. U.S.A. 107, 17791e17796. ¨ ber Diffuse Endocrine Epiheliale Organe. J.A. Barth Feurter, F., 1938. U Publisher, Leipzig. Fossmark, R., Sagatun, L., Nordrum, I.S., Sandvik, A.K., Waldum, H.L., 2015. Hypergastrinemia is associated with adenocarcinoma in the gastric corpus and shorter patient survival. APMIS 123, 509e514. Fukui, H., Kinoshita, Y., Maekawa, T., Okada, A., Waki, S., Hassan, S., et al., 1998. Regenerating gene protein may mediate gastric mucosal proliferation induced by hypergastrinemia in rats. Gastroenterology 115, 1483e1493. Garbrecht, N., Anlauf, M., Schmitt, A., Henopp, T., Sipos, B., Raffel, A., et al., 2008. Somatostatin-producing neuroendocrine tumors of the duodenum and pancreas: incidence, types, biological behavior, association with inherited syndromes, and functional activity. Endocr. Relat. Cancer 15, 229e241. Gargantini, E., Lazzari, L., Settanni, E., Taliano, M., Trovato, L., Gesmundo, I., et al., 2016. Obestatin promotes proliferation and survival of adult hippocampal progenitors and reduces amyloidb-induced toxicity. Mol. Cell. Endocrinol. 422, 18e30. Goo, T., Akiba, Y., Kaunitz, J.D., 2010. Mechanism of intragastric pH sensing. Curr. Gastroenterol. Rep. 12, 465e470. Gregory, R.A., Tracy, H.J., 1961. The preparation and properties of gastrin. J. Physiol. 156, 523e543.

356

15. STOMACH HORMONES

Grider, J.R., 1989. Somatostatin release from isolated ganglia of the myenteric plexus. Am. J. Physiol. 257, G313eG315. Grimelius, L., 1968. A silver nitrate stain for a-2 cells in the pancreatic islets. Acta Soc. Med. Ups. 73, 243e270. Grossman, M.I., Konturek, S.J., 1974. Inhibition of acid secretion in dog by metiamide, a histamine antagonist acting on H2 receptors. Gastroenterology 66, 517e521. Gustafsson, B.I., Bakke, I., Tømmera˚s, K., Waldum, H.L., 2006. A new method for visualization of gut mucosal cells, describing the enterochromaffin cell in the rat gastrointestinal tract. Scand. J. Gastroenterol. 41, 390e395. Gustafsson, B.I., Bakke, I., Hauso, Ø., Kidd, M., Modlin, I.M., Fossmark, R., Brenna, E., Waldum, H.L., 2011. Parietal cell activation by arborization of ECL cell cytoplasmic projections is likely the mechanism for histamine induced secretion of hydrochloric acid. Scand. J. Gastroenterol. 46, 531e537. Haid, D.C., Jordan-Biegger, C., Widmayer, P., Breer, H., 2012. Receptors responsive to protein breakdown products in g-cells and d-cells of mouse, swine and human. Front. Physiol. 3, 65. https://doi.org/ 10.3389/fphys.2012.00065. Haid, D., Widmayer, P., Breer, H., 2011. Nutrient sensing receptors in gastric endocrine cells. J. Mol. Histol. 42, 355e364. Ha˚kanson, R., 1970. New aspects of the formation and function of histamine, 5-hydroxytryptamine and dopamine in gastric mucosa. Acta Physiol. Scand. (Suppl. 340), 1e34. Ha˚kanson, R., Blom, H., Carlsson, E., Larsson, H., Ryberg, B., Sundler, F., 1986a. Hypergastrinaemia produces trophic effects in the stomach but not in pancreas and intestines. Regul. Pept. 13, 225e233. Ha˚kanson, R., Bo¨ttcher, G., Ekblad, E., Panula, P., Simonsson, M., Dohlsten, M., et al., 1986b. Histamine in endocrine cells in the stomach; a survey of several species using a panel of histamine antibodies. Histochemistry 86, 5e17. Ha˚kanson, R., Owman, C., 1969. Argyrophilic reaction of histaminecontaining epithelial cells in murine gastric mucosa. Experientia 25, 625e626. Ha˚kanson, R., Vallgen, S., Ekelund, M., Rehfeld, J.F., Sundler, F., 1984. The vagus exerts trophic control of the stomach in the rat. Gastroenterology 86, 28e32. Hansson, L.E., Nyren, O., Hsing, A.W., Bergstro¨m, R., Josefsson, S., Chow, W.H., et al., 1996. The risk of stomach cancer in patients with gastric or duodenal ulcer disease. N. Engl. J. Med. 335, 242e249. Hayakakawa, Y., Jin, G., Wang, H., Chen, X., Westphalen, C.B., Asafaha, S., et al., 2015. CCK2R identifies and regulates gastric antral stem cell states and carcinogenesis. Gut 64, 544e553. Holst, J.J., Orskov, C., Seier-Poulsen, S., 1992. Somatostatin is an essential paracrine link in acid inhibition of gastrin secretion. Digestion 51, 95e102. Howard, A.D., Feighner, S.D., Cully, D.F., Arena, J.P., Liberator, P.A., Rosenblum, C.I., et al., 1996. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273, 974e977. Isenberg, J.I., Walsh, J.H., Passaro Jr., E., Moore, E.W., Grossman, M.I., 1972. Unusual effect of secretin on serum gastrin, serum calcium and gastric acid secretion in a patient with suspected ZollingerEllison syndrome. Gastroenterology 62, 626e631. Itami, A., Kato, M., Komoto, I., Doi, R., Hosotani, R., Shimada, Y., Imamura, M., 2001. Human gastrinoma cells express calciumsensing receptor. Life Sci. 70, 119e129. Johnson, L.R., 1971. Control of gastric secretion: no room for histamine. Gastroenterology 61, 106e118. Kahlson, G., Rosengren, E., Svahn, D., Thunberg, R., 1964. Mobilization and formation of histamine in the gastric mucosa as related to acid secretion. J. Physiol. 174, 400e416. Kajimura, M., Reuben, M.A., Sachs, G., 1992. The muscarinic receptor gene expressed in rabbit parietal cells is the m3 subtype. Gastroenterology 103, 870e875.

Kazumori, H., Ishihara, S., Kawashima, K., Fukuda, R., Chiba, T., Kinoshita, Y., 2001. Analysis of gastrin receptor gene expression in proliferating cells in the neck zone of gastric fundic glands using laser capture microdissection. FEBS Lett. 489, 208e214. Kim, J.J., Wang, H., Terc, J.D., Zambrowicz, B., Yang, Q.M., Khan, W.I., 2015. Blocking peripheral serotonin synthesis by telotristat etiprate (LX1032/LX1606) reduces severity of both chemical-and infection induced intestinal inflammation. Am. J. Physiol. Gastrointest. Liver Physiol. 309, G455eG465. Kleveland, P.M., Haugen, S.E., Waldum, H.L., 1985. The preparation of bioactive 125I-Gastrin, using Iodo-gen as oxidizing agent, and the use of this tracer in receptor studies. Scand. J. Gastroenterol. 20, 569e576. Kleveland, P.M., Waldum, H.L., Larsson, H., 1987. Gastric acid secretion in the totally isolated, vascularly perfused rat stomach. A selective muscarinic-1 agent does whereas gastrin does not augment maximal histamine-stimulated acid secretion. Scand. J. Gastroenterol. 22, 705e713. Koizumi, M., Hosoya, Y., Dezaki, K., Yada, T., Hosoda, H., Kangawa, K., et al., 2014. Serum ghrelin levels partially recover with the recovery of appetite and food intake after total gastrectomy. Surg. Today 44, 2131e2137. Kojima, M., Hosoda, H., Dale, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth-hormone-releasing acylated peptide from the stomach. Nature 402, 656e660. Kokkola, A., Sjo¨blom, S.M., Haapiainen, R., Sipponen, P., Puolakkainen, P., Ja¨rvinen, H., 1998. The risk of gastric carcinoma and carcinoid tumours in patients with pernicious anaemia. A prospective follow-up study. Scand. J. Gastroenterol. 33, 88e92. Komarov, S.A., 1938. Gastrin. Soc. Exp. Biol. Med. 38, 514e516. Kopin, A.S., Lee, Y.M., McBride, E.W., Miller, L.J., Lu, M., Lin, H.Y., et al., 1992. Expression, cloning and characterization of the canine parietal cell gastrin receptor. Proc. Natl. Acad. Sci. U.S.A. 89, 3605e3609. Lai, Y.C., Ho, Y., Huang, K.H., Tsai, L.H., 2009. Effects of serotonin on acid secretion in isolated rat stomach: the role of 5-HT3 receptors. Chin. J. Physiol. 52 (5 Suppl. l), 395e405. Larsson, L.I., Goltermann, N., de Magistris, L., Rehfeld, J.F., Schwartz, T.W., 1979. Somatostatin cell processes as pathways for paracrine secretion. Science 205, 1393e1395. Larsson, L.I., Hirsch, M.A., Holst, J.J., Ingemansson, S., Ku¨hl, C., Jensen, S.L., et al., 1977. Pancreatic somatostatinoma. Clinical features and physiological implications. Lancet 1, 666e668. La¨uffer, J.M., Modlin, I.M., Hinoue, T., Kidd, M., Zhang, T., Schmid, S.W., Tang, L.H., 1999. Pituitary adenylate cyclaseactivating polypeptide modulates gastric enterochromaffin-like cell proliferation in rats. Gastroenterology 116, 623e635. Le Beyec, J., Pelletier, A.L., Arapis, K., Hourseau, M., Cluzeaud, F., Descatoire, V., et al., 2014. Overexpression of gastric leptin precedes adipocyte leptin during high-fat diet and is linked to 5HTcontaining enterochromaffin cells. Int. J. Obes. 38, 1357e1364. Levi, S., Beardshall, K., Haddad, G., Playford, R., Ghosh, P., Calam, J., 1989. Campylobacter pylori and duodenal ulcers: the gastrin link. Lancet 1, 1167e1168. Lichtenberger, L.M., Delansorne, R., Graziani, L.A., 1982. Importance of amino acid uptake and decarboxylation in gastrin release from isolated G cells. Nature 295, 698e700. Lindstro¨m, E., Eliassson, L., Bjo¨rkqvist, M., Ha˚kanson, R., 2001. Gastrin and neuropeptide PACAP evoke secretion from rat stomach histamine-containing (ECL) cells by stimulating influx of Ca2þ through different Ca2þ channels. J. Physiol. 535, 663e677. Long, S.H., Berna, M.J., Thill, M., Pace, A., Pradhan, T.K., Hoffman, K.M., et al., 2007. Secretin-receptor and secretinreceptor-variant expression in gastrinomas: correlation with clinical and tumoral features and secretin and calcium provocative test results. J. Clin. Endocrinol. Metab. 92, 4394e4402.

REFERENCES

Lundega˚rdh, G., Ekbom, A., McLaughlin, J.K., Nyre´n, O., 1994. Gastric cancer risk after vagotomy. Gut 35, 946e949. Makhlouf, G.M., McManus, J.P., Card, W.I., 1966. Action of the pentapeptide (ICI 50123) on gastric secretion in man. Gastroenterology 51, 455e465. Marshall, B.J., Warren, J.R., 1984. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1, 1311e1315. Martinsen, T.C., Bakke, I., Chen, D., Sandvik, A.K., Zahlsen, K., Aamo, T., Waldum, H.L., 2005. Ciprofibrate stimulates the gastrin-producing cell by acting luminally on antral PPAR-alpha. Am. J. Physiol. Gastrointest. Liver Physiol. 289, G1052eG1060. Masson, P., 1914. La glande endocrine de lintestine chez lhomme. C. R. Acad. Sci. 158, 52e61. McGuigan, J.E., Trudeau, W.L., 1970. Studies with antibodies to gastrin. Radioimmunoassay in human serum and physiological studies. Gastroenterology 58, 139e150. McGuigan, J.E., 1968. Gastric mucosal intracellular localization of gastrin by immunofluorescence. Gastroenterology 55, 315e327. Møller, L.N., Stidsen, C.E., Hartmann, B., Holst, J.J., 2003. Somatostatin receptors. Biochim. Biophys. Acta 1616, 1e84. Monstein, H.J., Nylander, A.G., Salehi, D., Chen, D., Lundquist, I., Ha˚kanson, R., 1996. Cholecystokinin-A and cholecystokinin-B/ gastrin receptor mRNA expression in the gastrointestinal tract and pancreas of the rat and man. A polymerase chain reaction study. Scand. J. Gastroenterol. 31, 383e390. Morson, B.C., 1955. Intestinal metaplasia of the gastric mucosa. Br. J. Canc. 9, 365e376. Nakata, H., Matsui, T., Ito, M., Taniguchi, T., Naribayashi, Y., Arima, N., et al., 1992. Cloning and characterization of gastrin receptor from ECL carcinoid of Mastomys natalensis. Biochem. Biophys. Res. Commun. 187, 1151e1157. Nakazato, M., Murakami, N., Date, Y., Kojima, M., Matsuo, H., Kangawa, K., Matsukura, S., 2001. A role for ghrelin in the central regulation of feeding. Nature 409, 194e198. Norle´n, P., Curry, W.J., Bjo¨rkqvist, M., Maule, A., Cunningham, R.T., Hogg, R.B., et al., 2001. Cell-specific processing of chromogranin A in endocrine cells of the rat stomach. J. Histochem. Cytochem. 49, 9e18. Oh, D.S., Lieu, S.N., Yamaguchi, D.J., Tachiki, K., Lambrecht, N., Ohning, G.V., et al., 2005. PACAP regulation of secretion and proliferation of pure populations of gastric ECL cells. J. Mol. Neurosci. 26, 85e97. Ojeaburu, J.V., Ito, T., Crafa, P., Bordi, C., Jensen, R.T., 2011. Mechanism of acid hypersecretion post curative gastrinoma resection. Dig. Dis. Sci. 56, 139e154. Parsonnet, J., Friedman, G.D., Vandersteen, D.P., Chang, Y., Vogelman, J.H., Orentreich, N., Sibley, R.K., 1991. Helicobacter pylori infection and the risk of gastric carcinoma. N. Engl. J. Med. 325, 1127e1131. Pawlow, J.-P., 1901. Le Travail Des Glandes Digestives, pp. 1e29. Pearse, A.G., Polak, J.M., 1971. Neural crest origin of the endocrine polypeptide (APUD) cells of the gastrointestinal tract and pancreas. Gut 12, 783e788. Persson, P., Grunditz, T., Axelson, J., Sundler, F., Ha˚kanson, R., 1988. Cholecystokinins but not gastrin-17 release calcitonin from thyroid C-cells in the rat. Regul. Pept. 21, 45e56. Piqueras, L., Martinez, V., 2004. Role of somatostatin receptors on gastric acid secretion in wild-type and somatostatin receptor 2 knockout mice. Naunyn-Schmiedeberg’s Arch. Pharmacol. 370, 510e520. Portela-Gomes, G.M., Stridsberg, M., 2002. Chromogranin A in the human gastrointestinal tract: an immunocytochemical study with region-specific antibodies. J. Histochem. Cytochem. 50, 1487e1492.

357

Prchayakui, V., Aswakul, P., Deesomsak, M., Pongpaibul, A., 2013. Gastric somatostatinoma: an extremely rare cause of upper gastrointestinal bleeding. Clin. Endosc. 46, 582e585. Prinz, C., Kajimura, M., Scott, D.R., Mercier, F., Helander, H.P., Sachs, G., 1993. Histamine secretion from rat enterochromaffinlike cells. Gastroenterology 105, 449e461. Prinz, C., Sachs, G., Walsh, J.H., Coy, D.H., Wu, S.V., 1994. The somatostatin receptor subtype on rat enterochromaffinlike cells. Gastroenterology 107, 1067e1074. Qader, S.S., Salehi, A., Ha˚kanson, R., Lundquist, I., Ekelund, M., 2005. Long-term infusion of nutrients (total parenteral nutrition) suppresses circulating ghrelin in food-deprived rats. Regul. Pept. 131, 82e88. Qvigstad, G., Bjørgaas, M., Eide, I., Sandvik, A.K., Waldum, H.L., 1999a. Vagal stimulation augments maximal (penta)gastrinstimulated acid secretion in humans. Acta Physiol. Scand. 165, 277e281. Qvigstad, G., Falkmer, S., Westre, B., Waldum, H.L., 1999b. Clinical and histopathological tumour progression in ECL cell carcinoids (“ECLomas”). APMIS 107, 1085e1092. Qvigstad, G., Qvigstad, T., Westre, B., Sandvik, A.K., Brenna, E., Waldum, H.L., 2002. Neuroendocrine differentiation in gastric carcinomas associated with severe hypergastrinemia and/or pernicious anemia. APMIS 110, 132e139. Qvigstad, G., Sandvik, A.K., Brenna, E., Aase, S., Waldum, H.L., 2000. Detection of chromogranin A in human gastric adenocarcinomas using a sensitive immunohistochemical technique. Histochem. J. 32, 551e556. Ravazzola, M., Orci, L., 1980. Glucagon and glicentin immunoreactivity are topologically segregated in the alpha-granules of human pancreatic A cell. Nature 284, 66e67. Rehfeld, J.F., Johnsen, A.H., 1993. Structure of the bioactive gastrins. In: Walsh, J.H. (Ed.), Gastrin. Raven Press, New York, pp. 1e14. Rettenberger, A.T., Schulze, W., Breer, H., Haid, D., 2015. Analysis of the protein related receptor GPR92 in G-cells. Front. Physiol. 6 https://doi.org/10.3389/fphys, 261:261. Rehfeld, J.F., 1981. A, unique high-titer antiserum to gastrin. Scand. J. Clin. Lab. Invest. 41, 723e727. Rehfeld, J.F., Stadil, F., Rubin, B., 1972. Production and evaluation of antibodies for the radioimmunoassay of gastrin. Scand. J. Clin. Lab. Invest. 30, 221e232. Reubi, J.C., Waser, B., La¨derach, U., Stettler, C., Friess, H., Halter, F., Schmassmann, A., 1997. Localization of cholecystokinin A and cholecystokinin B-gastrin receptors in the human stomach. Gastroenterology 112, 1197e1205. Ronveaux, C.C., Tome´, D., Raybould, H.E., 2015. Glucagon-like peptide 1 interacts with ghrelin and leptin to regulate glucose metabolism and food intake through vagal afferent neuron signaling. J. Nutr. 145, 672e680. Sagatun, L., Jianu, C.S., Fossmark, R., Ma˚rvik, R., Nordrum, I.S., Waldum, H.L., 2014. The gastric mucosa 25 years after proximal gastric vagotomy. Scand. J. Gastroenterol. 49, 1173e1180. Sakurada, T., Ro, S., Onouchi, T., Ohno, S., Aoyama, T., Chinen, K., et al., 2010. Comparison of the actions of acylated and desacylated ghrelin on acid secretion in the rat stomach. J. Gastroenterol. 45, 1111e1120. Sandvik, A.K., Cui, G., Bakke, I., Munkvold, B., Waldum, H.L., 2001. PACAP stimulates gastric acid secretion in the rat by inducing histamine release. Am. J. Physiol. Gastrointest. Liver Physiol. 281. G9997-G1003. Sandvik, A.K., Waldum, H.L., Kleveland, P.M., Schulze Søgnen, B., 1987. Gastrin produces an immediate and dose-dependent histamine release preceding acid secretion in the totally isolated, vascularly perfused rat stomach. Scand. J. Gastroenterol. 22, 803e808.

358

15. STOMACH HORMONES

Sandvik, A.K., Waldum, H.L., 1988. The effect of somatostatin on baseline and stimulated acid secretion and vascular histamine release from the totally isolated vascularly perfused rat stomach. Regul. Pept. 20, 233e239. Sandvik, A.K., Waldum, H.L., 1991. CCK-B (gastrin) receptor regulates gastric histamine release and acid secretion. Am. J. Physiol. 260, G925eG928. Sandvik, A.K., Waldum, H.L., 1990. Rat gastric histamine release: a sensitive gastrin bioassay. Life Sci. 46, 453e459. Sato, T., Nakamura, Y., Shiimura, Y., Ohgusu, H., Kangawa, K., Kojima, M., 2012. Structure, regulation and function of ghrelin. J. Biochem. 151, 119e128. Sekikawa, A., Fukui, H., Fujii, S., Takeda, J., Nanakin, A., Hisatsune, H., et al., 2005. REG I alpha protein may function as a trophic and/or anti-apoptotic factor in the development of gastric cancer. Gastroenterology 128, 642e653. Seva, C., Dickinson, C.J., Yamada, T., 1994. Growth-promoting effects of glycine-extended progastrin. Science 265, 410e412. Sevier, A.C., Munger, B.L., 1965. A silver method for paraffin sections of neural tissue. J. Neuropathol. Exp. Neurol. 24, 130e135. Smith, R.G., Leonard, R., Bailey, A.R., Palyha, O., Feighner, S., Tan, C., et al., 2001. Growth hormone secretagogue receptor family members and ligands. Endocrine 14, 9e14. Smith, J.T., Pounder, R.E., Nwokolo, C.U., Lanzon-Miller, S., Evans, D.G., Graham, D.Y., Evans Jr., D.J., 1990. Inappropriate hypergastrinemia in asymptomatic healthy subjects infected with Helicobacter pylori. Gut 31, 522e525. Smith, J.P., Wood, J.G., Solomon, T.E., 1989. Elevated gastrin levels in patients with colon cancer or adenomatous polyps. Dig. Dis. Sci. 34, 171e174. Solcia, E., Capella, C., Vassallo, G., Buffa, R., 1975. Endocrine cells of the gastric mucosa. Int. Rev. Cytol. 42, 223e286. Soll, A.H., 1980. Secretagogue stimulation of (14C)aminopyrine accumulation by isolated canine parietal cells. Am. J. Physiol. 238, G366eG375. Sykaras, A.G., Demenis, C., Cheng, L., Pisitkun, T., McLaughlin, J.T., Fenton, R.A., Smith, C.P., 2014. Duodenal CCK cells from male mice express multiple hormones including ghrelin. Endocrinology 155, 3339e3351. Sørdal, Ø., Qvigstad, G., Nordrum, I.S., Gustafsson, B., Waldum, H.L., 2013. In situ hybridization in human and rodent tissue by the use of a new and simplified method. Appl. Immunohistochem. Mol. Morphol. 21, 185e189. Sundler, F., Alumets, J., Holst, J., Larsson, L.-I., Ha˚kanson, R., 1976. Ultrastructural identification of glucagon-producing cells (A cells) in dog gastric mucosa. J. Cell Biol. 69, 455e464. Sundler, F., Ha˚kanson, R., 1991. Gastric endocrine cell typing in the light microscopic level. In: Ha˚kanson, R., Sundler, F. (Eds.), The Stomach as an Endocrine Organ. Elsevier, pp. 9e26. Tatsuta, M., Iishi, H., Nakaizumi, A., et al., 1993. Fundal atrophic gastritis as a risk factor for gastric cancer. Int. J. Cancer 53, 70e74. Thompson, E.M., Price, Y.E., Wright, N.A., 1990a. Kinetics of enteroendocrine cells with implications for their origin: a study of cholecystokinin and gastrin subpopulations combining tritiated thymidine labelling and immunocytochemistry in the mouse. Gut 31, 406e411. Thompson, M., Fleming, K.A., Evans, D.J., Fundele, R., Surani, M.A., Wright, N.A., 1990b. Gastric endocrine cells share a clonal origin with other gut cell lineages. Development 110, 477e481. Thorburn, C.M., Friedman, G.D., Dickinson, C.J., Vogelman, J.H., Orentreich, N., Parsonnet, J., 1998. Gastrin and colorectal cancer: a prospective study. Gastroenterology 115, 275e280. Toshinai, K., Yamaguchi, H., Sun, Y., Smith, R.G., Yamanaka, A., Sakurai, T., et al., 2006. Des-acyl ghrelin induces food intake by a mechanism independent of the growth hormone secretagogue receptor. Endocrinology 147, 2306e2314.

Tscho¨p, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908e913. Varner, A.A., Modlin, I.M., Walsh, J.H., 1981. High potency of bombesin for stimulation of human gastrin release and gastric acid secretion. Regul. Pept. 1, 289e296. Vassallo, G., Solcia, E., Capella, G., 1969. Light and electron microscopic identification of -several types of endocrine cells in the gastrointestinal tract of the cat. Z Zellforsch. 98, 333e356. Waldum, H.L., Aase, S., Kvetnoi, I., et al., 1998a. Neuroendocrine differentiation in human gastric carcinoma. Cancer 83, 435e444. Waldum, H.L., Arnestad, J.S., Brennna, E., Syversen, U., Sandvik, A.K., 1996. Marked increase in gastric acid secretory capacity after omeprazole treatment. Gut 39, 649e653. Waldum, H.L., Brenna, E., Sandvik, A.K., 1998b. Maximal gastric acid secretion in man: a concept that needs precision. Scand. J. Gastroenterol. 33, 1009e1015. Waldum, H.L., Brenna, E., Sandvik, A.K., 1998c. Relationship of ECL cells and gastric neoplasia. Yale J. Biol. Med. 71, 325e335. Waldum, H.L., Haugen, O., Brenna, E., 1994. Do neuroendocrine cells, particularly the D-cell, play a role in the development of gastric stump cancer? Cancer Detect. Prev. 18, 431e436. Waldum, H.L., Haugen, O.A., Isaksen, C., Mecsei, R., Sandvik, A.K., 1991a. Are diffuse gastric carcinomas neuroendocrine tumours? (ECL-omas). Eur. J. Gastroenterol. Hepatol. 3, 245e249. Waldum, H.L., Hauso, Ø., Sørdal, Ø.F., Fossmark, R., 2015. Gastrin may mediate the carcinogenic effect of Helicobacter pylori infection of the stomach. Dig. Dis. Sci. 60, 1522e1527. Waldum, H.L., Kleveland, P.M., Sørdal, Ø.F., 2016. Helicobacter pylori and gastric acid: an intimate and reciprocal relationship. Therap. Adv. Gastroenterol. 9, 836e844. Waldum, H.L., Lehy, T., Brenna, E., Sandvik, A.K., Petersen, H., Søgnen, B.S., Bonfils, S., Lewin, M.J.M.W., 1991b. Effect of the histamine-1 antagonist astemizole alone or combined with omeprazole on rat gastric mucosa. Scand. J. Gastroenterol. 26, 23e35. Waldum, H.L., Sandvik, A.K., Brenna, E., 1993. Gastrin: histaminereleasing activity. In: Walsh, J.H. (Ed.), Gastrin. Raven Press, New York, pp. 259e271. Waldum, H.L., Sandvik, A.K., 1993. Gastric functional and trophic receptors for CCK. In: Smith, T. (Ed.), Aspects of Synaptic Transmission. Taylor and Francis, London, pp. 157e172. Waldum, H.L., Sørdal, Ø.F., 2016. Classification of epithelial malignant tumors-the differentiation between adenocarcinomas and neuroendocrine carcinomas: why rely on nonspecific histochemistry and dismiss specific methods like immunohistochemistry and in situ hybridization? Appl. Immunohistochem. Mol. Morphol. 24, 309e312. Walker, I.R., Strickland, R.G., Ungar, B., Mackay, I.R., 1971. Simple atrophic gastritis and gastric carcinoma. Gut 12, 906e911. Walsh, J.H., Richardson, C.T., Fordtran, J.S., 1975. pH dependence of acid secretion and gastrin release in normal and ulcer subjects. J. Clin. Investig. 55, 462e468. Wa¨ngberg, B., Nilsson, O., Theodorsson, E., Modlin, I.M., Dahlstro¨m, A., Ahlman, H., 1996. The effect of vagotomy on enterochromaffin-like cells in Mastomys natalensis. J. Auton. Nerv. Syst. 59, 133e139. Wank, S.A., 1988. G protein-coupled receptors in gastrointestinal physiology. I. CCK receptors: an exemplary family. Am. J. Physiol. 274, G607eG613. Widmayer, P., Kusmakshi, S., Ha¨gele, F.A., Boehm, U., Breer, H., 2017. Expression of the fatty acid receptors GPR84 and GPR120 and cytodifferentiation of epithelial cells in gastric mucosa of mouse pups in the course of dietary transition. Front. Physiol. 8, 601. https:// doi.org/10.3389/fphys.2017.00601. Wiedenmann, B., Waldherr, R., Buhr, H., Hille, A., Rosa, P., Huttner, W.B., 1988. Identification of gastroenteropancreatic

REFERENCES

neuroendocrine cells in normal and neoplastic human tissue with antibodies against synaptophysin, chromogranin A, secretogranin I (chromogranin B) and secretogranin II. Gastroenterology 95, 1364e1374. Willard, M.D., Lajiness, M.E., Wulur, I.H., Feng, B., Swearingen, M.L., Uhlik, M.T., et al., 2012. Somatic mutations in CCK2R alter receptor activity that promote oncogenic phenotype. Mol. Canc. Res. 10, 739e749. Wormsley, K.G., Grossman, M.I., 1965. Maximal histalog test in control subjects and patients with peptic ulcer. Gut 6, 427e435. Yalow, R.S., Berson, S.A., 1960. Immunoassay of endogenous plasma insulin in man. J. Clin. Investig. 39, 1157e1175. Yamamoto, D., Ikeshita, N., Daito, R., Herningtyas, E.H., Toda, K., Takahashi, K., et al., 2007. Neither intravenous nor intracerebroventricular administration of obestatin affects the secretion of GH, PRL, TSH, and ACTH in rats. Regul. Pept. 138, 141e144. Yang, C., Sun, M.G., Matro, J., Huynh, T.T., Rahimpour, S., Prchal, J.T., et al., 2013. Novel HIF2A mutations disrupt oxygen sensing,

359

leading to polycythemia, paragangliomas, and somatostatinomas. Blood 121, 2563e2566. Yu, P.L., Fujimura, M., Hayashi, N., Nakamura, T., Fujimiya, M., 2001. Mechanisms in regulating the release of serotonin from the perfused rat stomach. Am. J. Physiol. Gastrointest. Liver Physiol. 280, 1099e1105. Zamcheck, N., Grable, E., Ley, A., Norman, L., 1955. Occurrence of gastric cancer among patients with pernicious anemia at the Boston city Hospital. N. Engl. J. Med. 252, 1103e1155. Zeinali, F., Fossmark, R., Hauso, Ø., Wiseth, R., Hjertner, Ø., Waldum, H.L., 2013. Serotonin in blood: assessment of its origin by concomitant determination of b-thromboglobulin (platelets) and chromogranin A (enterochromaffin cells). Scand. J. Clin. Lab. Invest. 73, 148e153. Zhang, J.V., Ren, P.G., Avisan-Kretchmer, O., Luo, C.W., Rauch, R., Klein, C., Hsueh, A.J., 2005. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 310, 996e999.

C H A P T E R

16 Intestinal Hormones Giulia Cantini1, Martina Trabucco1, Ilaria Dicembrini1,2, Edoardo Mannucci1,2, Michaela Luconi1,2 1

Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy; 2 Careggi University Hospital (AOUC), Florence, Italy

1. OVERVIEW OF THE INTESTINAL HORMONES Energy homeostasis is a finely regulated process, where food intake is the main source of energy to maintain the physiological functions of the organism and is counterbalanced by energy expenditure. When the balance between energy intake and energy expenditure fails, it triggers metabolic pathologies. Several genes have been identified to encode gut hormones involved in regulating metabolic physiology and appetite and intestinal nutrient absorption (Schwartz et al., 2000; Sahu, 2004). The endocrine activity of the intestine has been the object of intense studies for several decades, starting from the pioneering studies on secretin (Drucker et al., 2017). At the beginning of the 20th century, Bayliss and Starling by discovering this first gut hormone, secretin, in dog intestinal extract (Bayliss and Starling, 1902) founded not only gastrointestinal endocrinology but, more widely, endocrinology: in fact, in the Starling’s Croonian lecture in 1905 (Starling, 1905) the word hormone (from Greek hormoa) was coined. For many years the composition and the different functions of the factors present in the intestinal crude extract remained unclear, and only in the late 1990s the different hormones were identified. These hormones act through a strict crosstalk between intestine, where the majority of the nutrient absorption takes place, and other organs such as the brain, pancreas, liver, and heart to control food intake, intestinal absorption, and glucose homeostasis. The main gut hormones, their activities, and properties are summarized in Table 16.1. The gutebrain axis can be considered a complex neurohormonal communication network pivotal for the metabolic homeostasis. For the neural part, it

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00016-X

consists of the central nervous system (CNS), the local enteric nervous system, the autonomous nervous system, and its associated sympathetic and parasympathetic arms; whereas in the endocrine component, it comprehends the enteroendocrine cells dispersed in the stomach and intestine. In addition, the immunological system integrated in the mucosa and the microbiota populating the gut contribute to modulate the axis activity (Bliss and Whiteside, 2018). In the axis, sensory information, nutrients, and factors produced by microbiota are converted into neural, hormonal, and immunological signals, which are relayed back and forth from the CNS to the gut and vice versa (Mayer et al., 2015). Gastric emptying is prolonged by the vagal activation and by the release of gut hormones, such as cholecystokinin (CCK), peptide YY (PYY), and glucagon-like peptide 1 (GLP-1) through a negative feedback to the brain or their local action in reducing food intake within 6 minutes of feeding in fasting re-fed rats (Davis and Smith, 1990) (Fig. 16.1).

2. THE INTESTINAL ENTEROENDOCRINE CELLS AND THE ENTEROENDOCRINE SYSTEM Distribution of enteroendocrine cells (ECC) and their secretion in the human intestinal mucosa were elegantly described in pioneer immunocytochemical studies in the early 1980s (Sjo¨lund et al., 1983). Endocrine cells mainly concentrate in the small intestine, although the large intestine has also more recently been described to express some hormone-secreting cells (Engelstoft et al., 2013). Together with the gastric tract, the intestine can be considered the largest and phylogenetically oldest endocrine organ in the organism. Functional and

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FIGURE 16.1

Gutebrain axis. Peptide hormones released from gut (GLP-1, PYY, GIP, CCK) regulate food intake via the vagus nerve or by acting on the hypothalamus and brainstem. CCK, cholecystokinin; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagonlike peptide-1; PYY, peptide YY.

molecular characterization of the intestinal endocrine cells has remained elusive for a long time due to the difficulties in identifying these cells scattered in the intestinal epithelium, which is mainly formed by the overwhelming number of absorptive/secreting nonendocrine enterocytes. Initial immunohistochemical studies resulted in the classification of enteroendocrine cells on the basis of the release of a single peptide hormone associated with a single peptide hormone precursor from each type of cells (see Table 16.1). More recently, generation of transgenic reporter mice for several individual enteroendocrine cell types, based on transcriptional regulatory elements for the peptide hormone precursors associated with fluorescence sorting techniques followed by qPCR, gene chip, and RNA sequencing analyses, have shown that the dogma of the unicity of one cell type-one secretory peptide is no longer valid (Egerod et al., 2012; Engelstoft et al., 2013). A broad spectrum of intestinal peptide hormones can be coexpressed by the same cells producing different hormone precursors based on their lineage. Egerod et al. demonstrated the potential of coexpression of six gut

hormones, CCK, GLP-1, glucose insulinotropic peptide (GIP), PYY, neurotensin, and secretin, in intestinal cells belonging to the lineage nonexpressing somatostatin (which, conversely, identifies a second enteroendocrine subtype specification, Beucher et al., 2012). These related enteric hormones are all involved in metabolic regulation by inducing appetite suppression, gastric emptying, and insulin secretion stimulation associated to glucagon repression (Egerod et al., 2012). Even more interestingly, ablation studies in transgenic reporter mice have shown that the expression of intestinal hormones and their combination in each cell are controlled by a hierarchy of transcription factors with specific spatial and temporal expression patterns (Habib et al., 2012). These findings, together with the evidence of the short life span and the high renewal and differentiation rate of intestinal endocrine cells, result in the new concept that gene expression in the enteroendocrine cells is deeply regulated by cell localization in the intestinal tract. Moreover, lineage-related and spatially close enteroendocrine cells can switch their hormone secretion profile upon specific stimulation or following damage of this highly adaptive organ. Bariatric surgery in the rat model has also been demonstrated to induce such plasticity, resulting in GLP-1 secretion increase and secretory cell expansion after surgery (Mumphrey et al., 2013). These findings were confirmed in morbid obese patients after Roux-en-Y gastric bypass (RYGB), which induced an eightfold increase in GLP-1, GLP-2, and PYY (Jørgensen et al., 2013), suggesting that the rapid transit and absorption of nutrients in more distal small intestine segments as a consequence of the surgery stimulates plastic adaptation and remodeling of intestinal enteroendocrine cells and of their secretions (Holst, 2013). Interestingly, RYGB selectively increases the density of incretinproducing cell populations in the jejunum (Nerga˚rd et al., 2015), and this effect is maintained for long term, therefore opening up the possibility to obtain a stable stimulation of GLP-1 production to treat type 2 diabetes (T2D) and obesity. A switch in the production of glucagon-like peptides has been recently described in alpha cells in prediabetic NOD mice, where the prohormone convertase (PC) PC1/3 is atypically expressed instead of PC2, leading to endogenous production of GLP-1 rather than glucagon (Kilimnik et al., 2010). Endogenous pancreatic GLP-1 would act in a paracrine manner to enhance beta cells survival in response to the metabolic injury (Liu et al., 2011). The enteroendocrine cell appears as a flask-shaped polar-oriented structure with an apical sensory pole decorated with numerous microvilli extruding in the intestinal lumen, and a basal pole releasing hormones in the circulation (Fig. 16.2). This peculiar structure makes the endocrine cell a food and gastric-derived factors sensor, enabling a tuned modulation of the cell

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3. THE MAIN INTESTINAL HORMONES

TABLE 16.1

Main Intestinal Hormones.

Intestinal Hormone

Secretory Cell Type

Secretin

S

CCK

Main Intestinal Distribution of Secreting Cells

Action

Target Tissue

Duodenum and jejunum

Bicarbonate and water release, gastric acid secretion, gallbladder contraction, colon motility, pancreatic growth, insulin secretion

Pancreas, stomach

I

Duodenum and jejunum

Gallbladder contraction, inhibits stomach emptying, pancreatic enzyme secretion and food intake, stimulates pancreatic enzyme and HCO3 secretion

Gallbladder, pancreas, gastric smooth muscle

Neurotensin

N

Jejunum and ileum, colon

Gastric acid secretion, biliary secretion, intestinal mucosal growth, intestinal peristalsis, gastric emptying, fat absorption, smooth muscle contraction

Brain, stomach, smooth muscle cells

PYY

L

Jejunum and ileum

Nutrient uptake, intestinal motility, appetite regulation, insulin release, inhibits glucagon release, slows gastric emptying

Endocrine pancreas, brain stomach

GLP-1

L

Jejunum and ileum, colon

Nutrient uptake, intestinal motility, appetite regulation, insulin release, inhibits glucagon release, slows gastric emptying

Endocrine pancreas, brain, liver, adipose tissue, heart

GLP-2

L

Jejunum and ileum, colon

Intestine growth and homeostasis

Endocrine pancreas

GIP

K

Duodenum and jejunum

Insulin release, pancreatic beta cell proliferation, gastric acid secretion, LPL activity and fatty acid synthesis

Pancreatic beta cells, adipose tissue, stomach

The intestinal hormones, the secretory cell types, and their localization in the intestine, their main actions, and target organs are indicated.

hormone-release activity in response to the transit of the digestion products (Mace et al., 2015). Moreover, the intestine is densely innervated by fibers from vagal and splanchnic nerves, with a greater number of afferent fibers that contribute to the enteroendocrine cellregulated secretion. Vagal fibers extend into the lamina propria of the intestinal villi and at the basolateral endocrine cell membrane. They express receptors for the local intestinal hormones further contributing to the gute brain axis at a local level (Dockray, 2013). Finally, the local enteronervous system with neurons close to both enteroendocrine cells and afferent fibers also contribute to the transmission of local signaling of the axis (Fig. 16.1). G proteinecoupled receptors (GPCRs) are expressed on the majority of enteroendocrine cells and regulate EEC secretory output (Fig. 16.2). Physiologically, the products of digestion (glucose and fructose, amino acids and oligopeptides, and fatty acids), microbiota products and metabolites, triglycerides derivatives, inflammatory cytokines, systemic hormones, and neurotransmitters are detected by GPCRs expressed by EECs to regulate hormone secretion (Reimann et al., 2012). Among those GPCRs expressed in EECs, the majority belong to class A. The crystal structures of members of class A, B, and

C have recently been characterized together with the downstream signaling events (Mace et al., 2015). Mainly, activation of the cAMP/PKA system represents the second messenger pathway downstream of the classical GLP1-R, though cGMP pathways and other signalings have also been described to act in extrapancreatic tissues expressing GLP-1R (Cantini et al., 2016).

3. THE MAIN INTESTINAL HORMONES 3.1 Secretin Discovered in 1902 by Bayliss and Starling from the dog intestinal extract and later purified and sequenced from homogenates of porcine small intestinal mucosa (Mutt et al., 1970), secretin is a 27-amino-acid hormone highly conserved and able to stimulate pancreatic exocrine activity. Together with GLPs, glucagon, pituitary adenylate cyclaseeactivating peptide (PACAP), vasoactive intestinal peptide (VIP), growth hormonee releasing hormone (GHRH), and secretin belong to a large superfamily of related peptides (secretin-like peptides) that control metabolic homeostasis (Vaudry et al., 2009). Secretin-producing cells are mainly localized in

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FIGURE 16.2 GLP-1 pleiotropic actions on peripheral tissues. GLP-1 is secreted by intestinal endocrine L cells a few minutes after a meal. It exerts effects on endocrine pancreas, stomach, brain, heart, liver, muscle, and adipose tissue.

the upper small intestine, in the duodenum, and jejunum Table 16.1 (Polak et al., 1971). Moreover, gastric cells (Chey and Chang, 2003) and developing pancreas and gonads (Sherwood et al., 2000) are positive for secretin and can secrete it as a neuropeptide in the brain (Wang et al., 2018), heart, lung, and kidney (Sherwood et al., 2000). The intestinal secretion of secretin is stimulated upon gastric acid delivery into the duodenal lumen, biliary acids, and products of fat and protein digestion. Secretin induces the secretion of gastric pepsin and inhibits gastric acid secretion and gut motility (Sherwood et al., 2000). In addition, it induces a peripheral stimulation of water and bicarbonate secretion at the pancreatic level, and an inhibition of gastric emptying and acid secretion. Moreover, some central effects on fatty acid metabolism and glucose homeostasis control have also been described in the brain. In fact, secretin and its receptors have been found across hypothalamus, cerebellum, and the limbic system, where secretin contributes to water and food homeostasis, spatial memory, and motor learning (Wang et al., 2018). A specific receptor has been cloned and characterized for humans (Jiang and Ulrich, 1995; Patel et al., 1995).

3.2 Cholecystokinin In 1928, two scientists demonstrated that infusion of lipids into the small intestine released a substance able to induce gallbladder contraction. The factor was named “cholecystokinin” due to its capacity to promote the gallbladder motility (Ivy and Oldberg, 1928). Only later in 1971, CCK was purified from porcine small intestine extracts and identified as a peptide of about 33 amino acids. CCK is synthesized as a large precursor molecule of 95 amino acids and after posttranslational modification is cleaved to produce a number of biologically active forms of CCK (CCK-5, CCK-8, CCK-22, CCK-33, CCK-39, CCK-58, Polak et al., 1975; Buffa et al., 1976). CCK-8 is the most abundant form of CCK in the human brain, while CCK-58, CCK-33, and CCK-22 are found in the human intestine and circulation (Little et al., 2005). CCK is secreted by the enteroendocrine cells (I-cells) placed in the mucosa of duodenum, jejunum, and proximal ileum and by specific neurons located in the myenteric plexus and brain (Little et al., 2005). In the CNS, CCK is mainly produced by the dopamine-containing neurons in the mesencephalon (Ho¨kfelt et al., 1980). CCK plays a number of physiological effects, such as stimulation of gastric acid and pancreatic secretion, as

3. THE MAIN INTESTINAL HORMONES

well as an increase in gallbladder and gastrointestinal motility (Little et al., 2005). Moreover, it plays a key role through the gutebrain axis in controlling the food intake (Fig. 16.1). In fact, it induces a slowing of gastric emptying, modulating the glucose utilization and reducing the energy intake. CCK is the initial gut hormone to be involved in appetite control, and it is released in response to the assumption of a meal enriched in fat and protein from EECs within the duodenum and jejunum (Bliss and Whiteside, 2018). Its concentration increases within 15 min after meal and, due to its short half-life (few minutes), CCK acts within a limited period of time through CCK-1/2 receptors, expressed in the gastrointestinal tract and in the brain (CNS, vagal nerve, and hypothalamus, Bliss and Whiteside, 2018).

3.3 Neurotensin Neurotensin (NT) is a 13 amino acid peptide (pGlue LeueTyreGlueAsneLyseProeArgeArgeProeTyre IleeLeueOH) originally isolated from acid-acetone extracts of bovine hypothalami (Carraway and Leeman, 1973, 1975). Subsequently, using radioimmunoassay, NT production was determined in extracts of tissues from rats. The majority (85%) of the tridecapeptide was found in the intestine, and only a small percentage (10%) was located in the brain. More specifically, the highest concentration of NT was detected in the jejuno-ileal section, whereas lower concentrations were also present in the esophagus, stomach, duodenum, and large intestine (Carraway and Leeman, 1976). Like all neuropeptides, NT is synthesized as part of a larger precursor, proneurotensin, which also contains neuromedin N (NN), a six amino acid neurotensin-like peptide (Vincent et al., 1999). The precursor protein is cleaved by PCs to produce one of two peptides that could differently be liberated in a number of tissues and physiological conditions (Kislauskis et al., 1988; Schroeder and Leinninger, 2018). NT is contained in secretory granules located in the basal portion of the small intestinal enteroendocrine cells named N-cells. These granules represent the storage sites for the tridecapeptide that is released consistently with an exocytotic process in response to luminal stimuli (Polak et al., 1977; Zhao and Pothaulakis, 2006). Plasma levels of NT increase several minutes after meals, and fat intake is the most potent stimulus for its release, primarily from the distal small intestine. It fulfills many central and peripheral functions through its interaction with three known specific receptors (NTSRs). Two of them belong to the family of GPCRs, whereas the third one is a 100-kDa protein with a single transmembrane domain (Vincent et al., 1999). NT has been shown to regulate several gastrointestinal functions, including inhibition of small bowel and

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gastric motility. Additional physiological effects include stimulation of the pancreatic and biliary secretion, colonic motility, small and large intestine, pancreas, and stomach growth. In addition, NT can affect the postprandial uptake and distribution of fat in several tissues (Pothoulakis et al., 1998; Zhao and Pothoulakis, 2006).

3.4 Peptide YY3L36 PYY is a small peptide of 36 amino acids secreted by intestinal L cells of the gastrointestinal tract postprandial (Batterham et al., 2002, 2006). It belongs to the "PP fold" family of peptides, which includes pancreatic polypeptide (PP) and neuropeptide Y (NPY) (Troke et al., 2014). The active endocrine form PYY3 36 results from the cleavage of the tyrosine-proline residue NH2-terminal of the secreted form, PYY1e36, by the enzyme dipeptidyl-peptidase 4 (DPP-4) (Steinert et al., 2017). Endogenous concentrations of PYY start to rise within 15 min of a meal in response to intestinal nutrient sensing (Oesch et al., 2006), and it is released proportionally to calories ingested (Gibbons et al., 2013). Unlike GLP-1 (cosecreted from endocrine L cells), its half-life is rather long, and its circulating levels remain elevated for several hours postprandial (Batterham et al., 2003a). PYY binds to Y2 receptors, members of the NPY receptor family, which are found throughout both the peripheral and central nervous system, with a greater concentration in the arcuate nucleus of the hypothalamus (ARC) (Troke et al., 2014). Therefore, PYY plays a key role in the regulation of appetite, reducing food intake mainly by peripheral binding to its Y2 receptors in the vagal afferent fibers to the nucleus tractus solitarius (Na¨slund and Hellstro¨m, 2007). Additionally, it elicits direct activation of the proopiomelanocortin (POMC) and inhibition of the orexigenic NPY neurons within the melanocortin system in the CNS (Bauer et al., 2016). Moreover, it induces a reduction in gastric emptying and a delay in intestinal transit. PYY infusion has also been shown to reduce the levels of ghrelin, which is an orexigenic hormone (Batterham et al., 2003b).

3.5 Incretins For several years, it has been postulated that the gut can secrete factors able to stimulate the pancreatic release of substances that reduce blood glucose levels and urine sugars in diabetic patients (Baggio and Drucker, 2007; Bayliss and Starling, 1902; Moore, 1906). These glucose-lowering intestinal factors were purified from gut extracts and named incretins (La Barre, 1932). Incretin hormones include those intestinal peptides

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(GIP, GLP-1) that stimulate insulin secretion in response to nutrient intake, being responsible for the so-called incretin effect observed as the two- to threefold increase in insulin secretory response to oral compared to intravenous glucose administration. As a consequence, the increased suppression of glucagon secretion from pancreatic alpha cells can be included in the concept of incretin effects (Nauck and Meier, 2018). Therefore, these two hormones and the strategies for their stimulation have become pivotal in the treatment of T2D and potentially in other metabolic disorders such as obesity (see later). A widening of the concept of incretin hormones to all the intestinal secretions stimulated by food intake has recently been proposed, not limiting incretin hormones to GIP and GLP-1, on the basis of multiple interactions and secretion between gut hormones in a cooperative manner that can ultimately affect pancreatic insulin secretion (Rehfeld, 2018). 3.5.1 Glucose-Dependent Insulinotropic Peptide The first incretin to be identified was the glucosedependent insulinotropic peptide (GIP, isolated from porcine intestinal extracts by John Brown in the 1970s, Brown and Bryburgh, 1971), with a weak inhibitory effect on gastric acid secretion but a strong insulinotropic action. GIP is a 42-amino acid polypeptide hormone synthesized in the K cells of the small intestine and primarily secreted in response to glucose or fat intake, with the activity of potentiating the glucose-stimulated insulin

secretion (Meier and Nauck, 2004), Fig. 16.4A. Despite its potent insulinotropic action, inactivation of this incretin from gut extracts did not remove the effect, providing evidence for the existence of additional peptides with incretin-like activity (Ebert et al., 1983). 3.5.2 Glucagon-Like Peptide 1 Ten years later, a second incretin hormone, GLP-1, was identified following the cloning and characterization of the proglucagon gene (Bell et al., 1983). This gene encodes a large prohormone precursor able to generate by tissue-specific posttranslational cleavage of all glucagon-like peptides, including GLP-1, GLP-2, glucagon, and oxyntomodulin (Fig. 16.3) and some fragments with unclear roles, such as glicentin, glicentin-related polypeptide (GRPP), and major glucagon fragment (MPGF) (Sandoval and D’Alessio, 2015). Production of the different peptides from the unique precursor (Fig. 16.3) depends on the differential tissue expression of the PC. PC1/3 is mainly expressed in CNS and L intestinal cells, thus GLP-1, GLP-2, and oxyntomodulin are the main products in these tissues (Mojsov et al., 1986; Larsen et al., 1997; Tucker et al., 1996; Vrang and Larsen, 2010; Vrang et al., 2007). Conversely, high levels of PC2 in a-cells elicit a prevailing expression of glucagon in the pancreas (Holst et al., 1994). A low amount of PC2 has been also found in neural cells; moreover, a-cells express PC1/3, although at low levels,

FIGURE 16.3 Preproglucagon processing. Schematic cartoon representing the processing of preproglucagon from gene expression to the proglucagon-derived proteins. EECs, enteroendocrine cells; PC, prohormone convertase.

3. THE MAIN INTESTINAL HORMONES

FIGURE 16.4

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GLP-1 analog structures. (A) General structure and functional residues of GLP-1 and aminoacidic sequence of GLP-1 related peptides. Aminoacidic residues involved in DPP-4 cleavage are in indicated in red/black, conserved residues in deep blue, while specific different amino acids are colored in light blue. (B) GLP-1RA aminoacidic sequences.

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suggesting that GLP-1 is secreted not only in the brain and gut but can also be produced from pancreatic islets (Larsen et al., 1997). In the intestine, GLP-1 is synthesized as an inactive 37 amino acid product of proglucagon cleavage GLP1(1e37), and then further processed to generate a family of GLP-1 endogenous active forms. Plasma levels of GLP-1 are reduced in the fasted state (range 5e10 pmol/L), while increasing rapidly after eating (15e50 pmol/L). The circulating concentrations of the hormone diminish rapidly not only for the renal clearance but also because of the enzymatic action of DPP-4 (Orskov et al., 1993). In fact, both forms of GLP1 contain an alanine residue at position 2, which is the cleavage site of the DPP-4, which rapidly degrades both GLP-1s to a truncated form, GLP-1(9-36) amide and GLP-1 (9-37), respectively, within 1e3 min from their release from L cells (Deacon et al., 1995; Kieffer et al., 1995), Fig. 16.4A. The truncated forms are unable to bind the GLP-1R and to elicit the glucose metabolism regulation in the pancreas. Although an alternative receptor for GLP-1(9e36) amide has not yet been identified, evidence in rodent models supports a role for this truncated peptide in protection against postmyocardial infarction (Ban et al., 2010; Robinson et al., 2016). Direct effects of this peptide have also been described in human adipose precursor cells resulting in remodeling of adipose differentiation and restraining of precursor proliferation (Cantini et al., 2017). Pancreatic and extrapancreatic pleiotropic activities of GLP-1 depend on tissue distribution of its classical and alternative receptor/mechanism(s). In the pancreas, it acts on alpha cells to suppress glucagon secretion, on gamma cells to stimulate somatostatin secretion, and in particular on beta cells, where together with the other incretin hormone GIP, GLP-1 stimulates insulin biosynthesis and secretion. A trophic effect on beta cells has also been described, as GLP-1 induces cell proliferation while inhibiting apoptosis and endoplasmic reticulum (ER) stress. Among the cardiovascular effects of GLP-1, a rapid increase in glucose and decrease in fatty acid utilization is accompanied by amelioration of cardiac function, vasoprotection, and decreased inflammation. Notably, classical GLP-1 receptors are present only in atrial and not in ventricular cardiomyocytes (Kim et al., 2013), suggesting the existence of alternative receptors (Cantini et al., 2016) (Fig. 16.2). GLP-1Rs are expressed in several areas of human and mouse brain, including the arcuate and paraventricular nuclei of the hypothalamus involved in the regulation of appetite and satiety. GLP-1 and GLP-1RAs can rapidly cross the bloodebrain barrier after peripheral

administration and are also locally produced by the nucleus of the solitary tract in rats and primates (Vrang et al., 2007). In addition to inhibition of food intake, GLP-1 action in the brain also includes a trophic effect, neuroprotection, and decrease in addictive behavior. However, due to rapid cleavage of endogenous GLP-1 by DPP-4, it is unclear how much of the peripherally secreted hormone is bioavailable for GLP-1Rs in the CNS. A direct effect of native GLP-1, its synthetic analog liraglutide and its truncated form on adipose remodeling in an in vitro human adipose stem cell model (Cantini et al., 2015, 2017) has also been described, which can contribute to the weight loss effect of GLP-1 in addition to the central mechanism previously described (Sisley et al., 2014). To go deeply inside the pleiotropic pancreatic and extrapancreatic activity of GLP1, see Tuduri et al. (2016) and Cantini et al. (2016), respectively.

3.6 Glucagon-Like Peptide 2 Glucagon-like peptide-2 (GLP-2) is a 33-amino-acid derived from specific posttranslational cleavage of proglucagon peptide and secreted from enteroendocrine L cells (Figs. 16.3 and 16.4A). GLP-2 plasma levels are low in the fasting period and rise rapidly after food ingestion and in response to intestinal damage. GLP-2 production and activity seem selectively restricted to the intestine (Drucker and Yusta, 2014), where GLP-2 has been described to increase mesenteric blood flow and stimulate proabsorptive pathways enhancing nutrient absorption, delaying gastric transit. Moreover, it exerts a proproliferative action and cytoprotection in the small bowel, also ameliorating gut barrier functions (Drucker and Yusta, 2014). GLP-2 receptors also enhance gut barrier function and induce proliferative and cytoprotective pathways in the small bowel. The actions of GLP-2 are mediated by a single GPCR (GLP-2R) of 553 amino acids encoded in humans by Glp2r gene in 17p13.3 locus, and mainly expressed within the gastrointestinal tract. This receptor belongs to the class B GPCR superfamily that signal through adenylate cyclase (Mayo et al., 2003). Extraintestinal effects of GLP-2 have also been described on the pancreas and bone with different results observed across species. In humans, exogenous GLP-2 rapidly increases plasma glucagon levels with no change in glycemia in healthy subjects (Meier et al., 2006), this stimulatory effect being preserved in type 1 diabetes (Christensen et al., 2010) but reduced in T2D (Lund et al., 2011). Bone reabsorption is reduced in response to GLP-2 injection, in agreement with the physiological increase observed during fasting and decrease

4. THE PARADIGM OF COEVOLUTION OF GLUCAGON-LIKE RECEPTORS AND THEIR LIGANDS

following food intake; also, increased spinal bone mineral density and intestinal calcium absorption is stimulated after prolonged administration of GLP-2 (Haderslev et al., 2002). The effects of exogenous administration of GLP-2 on the brain and brain indirect activity on food intake and gastric emptying still remain to be elucidated. Due to its intestinal trophic effects and stimulation of nutrient absorption in the intestine, GLP-2 may represent a therapeutic agent in different diseases such as short bowel syndrome, Crohn disease, cancer, and intestinal injury. The therapeutic actions of GLP-2 and teduglutide, a degradation-resistant human GLP-2 analog, are under intensive study in preclinical experiments as well as in human subjects (Jeppesen et al., 2012; Buchman et al., 2010).

3.7 Oxyntomodulin Like GLP-1, oxyntomodulin, a 37-amino-acidic peptide, is one of the several products of proglucagon peptide after a posttranslational processing (Baggio et al., 2004; Habib et al., 2012) (Figs. 16.3 and 16.4A). It is secreted from L cells in response to nutrient ingestion, and it is released into the circulation, concurrently with GLP1 and PYY, reaching the circulating peak concentration within 30 min postprandial, before being rapidly degraded by DPP-4 (Bliss and Whiteside, 2018). Firstly characterized as an inhibitor of gastric acid secretion like GLP-1, oxyntomodulin also reduces food intake (Cohen et al., 2003) and decreases gastric acid secretion in the gut and CNS, whereas in the pancreas, it improves glucose metabolism with an incretin action by binding to glucagon receptor (Maida et al., 2008). Oxyntomodulin binds to the GLP-1 receptor, and although the affinity of oxyntomodulin for the GLP-1 receptor is much lower than that of GLP-1, oxyntomodulin and GLP-1 share a similar efficacy in inhibiting food intake. However, a specific oxyntomodulin receptor has been described (Murphy et al., 2006). Both central and peripheral administration of oxyntomodulin induces repletion and weight loss in rats (Dakin et al., 2001, 2004; Baggio et al., 2004). Subsequent studies reported that intravenous infusion of oxyntomodulin to supraphysiological levels reduces food intake and increases energy expenditure in humans, leading to weight loss in human subjects (Cohen et al., 2003; Wynne et al., 2006). Preliminary data suggest that oxyntomodulin may prove useful as an obesity drug (Cohen et al., 2003). Moreover, it has been reported that oxyntomodulin could be one of the key drivers of the metabolic benefits of bariatric surgery, since its secretion in response to oral glucose is increased after RYGB (Tan and Bloom, 2013; Laferre`re et al., 2010).

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4. THE PARADIGM OF COEVOLUTION OF GLUCAGON-LIKE RECEPTORS AND THEIR LIGANDS The similarity and the presence of extended conserved regions among the numerous members of the glucagon-like receptor family belonging to the GPCR class B1, and among their ligands, suggest that the ligands and their receptors are likely to have emerged each by common ancestor genes with sequential phenomena of duplication and differentiation in paralogue structures coevolving together, the ligands and their receptors (Irwin, 2005; Hwang et al., 2014). For evolution of the wide glucagon-like receptor family, two rounds (2R) of all genome duplication have been proposed to have occurred early in the history of vertebrate evolution, associated with local gene duplications in the chromosome, both phenomena accounting for the expansion and diversification of the family members starting from a single common gene (Irwin, 2005; Hwang et al., 2014). These amplification phenomena have then been followed by specific sequence changes and gene loss that coevolved with ligand diversification, resulting in the variety of receptors and functions present inside and between species (Fig. 16.5): glucagon receptor (GCGR), glucagon-like peptide 1 receptor (GLP-1R), glucagon-like peptide 2 receptor (GLP2R), glucose-dependent insulinotropic peptide receptor (GIP-R), and glucagon-related peptide receptor (GCRPR) genes. The reconstruction of the phylogenetic tree of the GCGR subfamily (Fig. 16.5) indicates that the first branch to separate during evolution was that of GLP2R from one side and the other one comprising GLP-1R, GIP-R, GCGR, and GCRPR from the other side. In humans, GCRPR was likely lost, but was maintained and diversified in other species, while GLP-1R subsequently evolved separately from GCGR and GIP-R, which finally also diverged. This means that phylogenetically, GIP-R and GCGR are more strictly related than GLP-1R, and GLP2R was the first one to diverge during the family evolution. Phylogenetic and genome synteny analyses showed that GLP2R, GLP-1R, and an ancestor for GCGR/GIP-R/GCRPR arose by a local duplication in the chromosome before 2R, while GCGR, GIP-R, and GCRPR were the result of a genome 2R. Notably, this duplication scheme supporting the receptor evolution is also mirrored by what is supposed to have occurred for the corresponding ligand genes. In fact, GCG-, GLP1-, and GLP2-coding exons emerged as duplication occurred inside the same ancestor GCG gene, as suggested by the fact that in most vertebrate, GCG, GLP1, and GLP2 are encoded as consecutive exons on a proglucagon gene (Fig. 16.3). GCG, GIP, and GCRP then arose through a

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FIGURE 16.5 Phylogenetic tree of GCG-like receptor family genes. Human (hu), mouse (mo), chicken (ch), anole lizard (an), Xenopus (xe), zebrafish (zf), medaka (md), fugu (fu), stickleback (sb), tetraodon (to), and lamprey (lam). Bootstrap figures indicate 100 replicates. Different colors are associated with the different receptor genes: glucagon-related peptide receptor (GCRPR), insulinotropic peptide receptor (GIP-R), glucagon receptor (GCGR), glucagon-like peptide 1 receptor (GLP-1R), glucagon-like peptide 2 receptor (GLP2R) genes.

2R duplication mechanism similar to what occurred for the receptors (Irwin and Prentice, 2011; Hwang et al., 2014). Then, the occurrence of specific amino acid sequence changes in both receptor and ligand genes drove the selectivity for ligandereceptor interactions. Recent studies have demonstrated that interactions between residues evolved from ortholog-specific peptides, and receptors enhanced recognition of a peptide for the cognate receptor, while maintaining discrimination by other receptors (Moon et al., 2012a). Mammalian genomes have six genes encoding glucagon-like sequences (Sherwood et al., 2000). Moreover, some of these genes that encode the proglucagon sequence are organized in exons for different glucagon-like peptides (GLP-1, GLP-2, oxyntomodulin, glucagon, see Fig. 16.3), thus enhancing the number of glucagon-like peptides that can be generated.

In addition to the proglucagon genes, in fact, other members of this family are genes, each of which generates secretin, GIP, and GHRH. Other different genes encoding PACAP and VIP and secretin can be together considered to belong to the secretin-like superfamily. Finally, two additional long distance-related members of the glucagon-like gene family are only found in some vertebrate genomes. In particular, the lizard Gila monster has two genes selectively expressed in the salivary glands and encoding the two hormones exendin 4 (Fig. 16.4A) and helodermin (Pohl and Wank, 1998; Irwin, 2012) with about 50% homology to GLP-1 and VIP, respectively. For its ability to bind the GLP-1R and mimic the incretin effect of GLP-1, exendin 4 captured the attention of the diabetologists, becoming the basic molecule for the development of antidiabetic drugs, such as exenatide, where a glycine substitution at the 2 position makes exendin 4 resistant to the enzymatic action of DPP-4, thus extending its half-life in vivo. To further introduce differences among species in this complex receptoreligand system, some receptors or some ligands generated by the duplication plus divergence mechanism have been lost during evolution in difference species, and their cognate ligand effects are mediated by other maintained members of the family. For instance, in zebrafish, GLP-1R was lost, and GLP-1 binds glucagon receptor sharing its gluconeogenic activity, whereas humans do not possess the GCRPR receptor separated from the GIP-R/GCGR branch (Fig. 16.5). This promiscuity in the binding and effects between members may suggest that also in humans, depending on tissue specificity of receptor expression, ligand activity could be mediated by receptors different from the cognate or by heterodimerization of different members of the family, thus activating signaling different from the canonic ones observed in other tissues (Cantini et al., 2016). By comparing the mechanisms of ligand production in EECs across the evolution, it appears that mammalians produce different ligands from a single proglucagon mRNA by a posttranslational modification on the prohormone protein exerted by different PCs specifically expressed in the secreting cell. Therefore, nonmammalians produce different peptides by tissuespecific splicing of proglucagon mRNA transcripts coded by duplicate genes (Fig. 16.6, Irwin, 2005). Interestingly, teleost fishes have an intermediate step between posttranslational (mammalians) or posttranscriptional (nonmammalians) splicing of the protein or of the mRNA, so they generate two different mRNAs that are translated in two different forms of proglucagon protein, each redundant for glucagon and GLP-1. Consequently, these proglucagon precursors are finally differentially spliced in the pancreas to generate glucagon and GLP-1, and in the intestine where

5. THE GLUCAGON-LIKE PEPTIDE-1 RECEPTOR

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FIGURE 16.6 Differences between mammalian and nonmammalian (teleost fish) proglucagon peptide synthesis regulation. (A) Mammalian proglucagon genes are composed of six exons that are transcribed into a single messenger RNA (mRNA) and translated into a single protein in all tissues where the gene is expressed (pancreatic islets, intestinal L cells, and selected neurons). The proglucagon precursor protein is processed by proteolytic cleavage in a tissue-specific manner to generate mainly glucagon in the islets, while glucagon-like peptides 1 and 2 (GLP1 and GLP-2) are the main products in the intestine and neurons. Glucagon acts to regulate blood sugar levels. GLP-1 is an incretin hormone that increases insulin secretion in the presence of glucose. GLP-2 is an intestinal growth factor. (B) Teleost fishes have two or more proglucagon genes composed of four or six exons. The longer six-exon proglucagon gene is alternatively spliced in a tissue-specific manner to generate two mRNA transcripts, the longer form generated in the intestine and the shorter transcript in the pancreas. The shorter four-exon proglucagon gene generates a transcript similar to the shorter alternatively spliced six-exon gene transcript. The two mRNA transcripts encode different proglucagon precursors, with only the longer intestinal transcript encoding GLP-2. Tissue-specific proteolytic processing liberates glucagon and GLP-1 in the pancreas, while GLP-1 and probably GLP-2 are generated in the intestine. Glu, glucagon gene. From Irwin, D.M., July 1, 2005. Evolution of hormone function: proglucagon-derived peptides and their receptors. Bioscience 55 (7), 583e591 with permission.

they produce GLP-1 and GLP-2 (Fig. 16.6, Irwin, 2005). Some vertebrate species such as fishes have two proglucagon genes, due to genome duplication, that exhibit differing coding potentials for the glucagon-like peptides. On the other hand, chickens possess a single proglucagon gene that expresses multiple mRNA transcripts with different coding potentials (Richards and McMurtry, 2008, 2009). The significance of acquisition of different mechanisms of control of glucagon-related peptides during evolution is still unclear, but it may be hypothesized that the posttranslational control present in

mammalians may enable a rapid switch in the peptide production in the secretory cell.

5. THE GLUCAGON-LIKE PEPTIDE-1 RECEPTOR The receptor for GLP-1 (GLP-1R), identified for the first time through radiolabeling experiments in rat and human islets (Donnelly, 2012), was cloned from cDNA pancreatic libraries derived from rat in the 1992 (Thorens, 1992), and the following year in humans

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(Dillon et al., 1993; Thorens et al., 1993). After cloning, GLP-1R has been identified as a member of the GPCR superfamily, named "Class B" (or "secretin receptorlike") (Hwang et al., 2013). These GPCRs include other seven-transmembrane domain proteins like secretin, parathyroid hormone, and calcitonin receptors, as well as all the glucagon-related peptide receptors (GCG receptors) (Fig. 16.7) (Segre and Goldring, 1993). GLP-1R and its related paralogous receptors have a high degree of identity since they derive from duplication and divergence of a common ancestor receptor (see earlier). All ligands of this receptor family, on their side, have conserved regions and share typical three-dimensional structures to facilitate the receptor binding (Sherwood et al., 2000). These receptors have a distinctive extracellular N-terminal domain (NTD) of about 100e150 residues, which is joined together to the integral membrane core domain of GPCRs. GLP-1R is widely expressed in the human and mouse pancreas, lung, brain, stomach, heart, and kidney, while its expression in tissues involved in glucose metabolism, such as the liver, skeletal muscle, and fat, is still debated (Dunphy et al., 1998; de Graaf et al., 2016). The first pioneer research study elucidating the molecular structure of the integral membrane domains of GLP-1R was conducted at the end of the 1990s (Frimurer

and Bywater, 1999). Human GLP-1R is a 463-residue glycoprotein containing an N-terminal extracellular domain (ECD), characterized by an a-helix followed by four b-strand motifs, and the C-terminal transmembrane domain organized in seven-transmembrane helices (TM1-TM7), being connected by three intra (ICL1-ICL3) and extracellular loops (ECL1-ECL3) (Runge et al., 2008; Donnelly, 2012, Fig. 16.7). The presence of a short leader sequence (23 amino acids long) in the NTD results to be essential for the correct processing and trafficking of the receptor. In fact, cleavage of this signal peptide or single amino acidic mutations in the leader sequence could compromise the correct assembly of the receptor at the plasma membrane by trapping it in the reticulum (Huang et al., 2010; Thompson and Kanamarlapudi, 2014). In addition, the Nterminus contains some amino acidic residues (in particular, six cysteine residues) essential for the ligand binding, together with others in the extracellular loop between TM1 and TM3 (Runge et al., 2008). In particular, the amino acidic residues involved in ligand-receptor binding have been identified by demonstrating the spatial approximations between the 24 and 25 C-terminal residues of GLP-1 and the Glu133 and Glu125 residues within the N-terminus of GLP-1R (Chen et al., 2009). While the N-terminus of the receptor

FIGURE 16.7 GLP-1R structure. A snake plot of human GLP-1R amino acidic sequence. ECLs, extracellular loops; ICLs, intracellular loops. Modified from http://www.gpcrdb.org/protein/glp1r_human/.

6. THE GLUCAGON-LIKE PEPTIDE-1 RECEPTOR LIGANDS

is essential for the ligand binding, conversely, distinct domains within the third intracellular loop in the C-terminal position play a critical role in the selective docking of specific G proteins (Thompson and Kanamarlapudi, 2015). GLP-1R mediates the incretin action of GLP-1 through the intestinal-pancreatic connection. In fact, stimulation of the receptor triggers insulin secretion from b-cells, in addition to all the others known effects described before. Like other GPCRs, GLP-1R pleiotropically binds and signals through G proteinedependent and eindependent mechanisms. Most of the studies focused on GLP-1R signaling were performed on b-cells, where it mediates increased insulin secretion, storage, and synthesis, as well as increased b-cell mass. The glucose-dependent insulin secretion via GLP-1R signaling involves the activation of Gas protein, upregulation of cAMP levels, and subsequent activation of protein kinase A (PKA) and exchange protein activated by cAMP (Epac) (Coopman et al., 2010; de Graaf et al., 2016; Tudurı´ et al., 2016). The increased insulin storage in the b-cells occurs in both cAMP/PKA-dependent and -independent manners (Baggio and Drucker, 2007). The prosurvival action of GLP-1 in the b pancreatic cells involves the phosphatidyloinositol-3- kinase (PI3K) signaling pathway and downstream activates Akt/protein kinase B (PKB) or p38/ERK mitogenactivated protein kinase (MAPK) (Bastien-Dionne et al., 2011; de Graaf et al., 2016; Tudurı´ et al., 2016).

6. THE GLUCAGON-LIKE PEPTIDE-1 RECEPTOR LIGANDS Glucagon-related peptides share a common 30e40 amino acidic structure, demonstrated by nuclear magnetic resonance and crystallography (Underwood et al., 2010). The N-terminal contains six/seven amino acidic residues forming a random coil structure with more than 70% amino acid sequence identity, while the following alpha-helical structure has a moderate sequence similarity, accounting for the specificity of ligand-receptor recognition (Thornton and Gorenstein, 1994; Neidigh et al., 2001; Alan˜a et al., 2007; Parthier et al., 2007; Underwood et al., 2010). Some residues with different lengths follow in the C-terminus region, forming a helix N-capping motif not necessary for the receptor recognition and activation (Hinke et al., 2003; Moon et al., 2010), but essential to stabilize the helical structure (Neumann et al., 2008). While the N-terminus and the first half of the central alpha-helical structures are responsible for interaction with the receptor core domain, the second half of the alpha-helical region binds directly to the ECD of the receptor (Parthier et al., 2007; Underwood et al., 2010; Moon et al., 2012a), Fig. 16.4A.

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Despite the high level of conservation among members of the glucagon-like peptide family, selectivity of the interaction with the cognate receptor supporting the specific physiological functions of each peptide receptor pair (Moon et al., 2012b; Park et al., 2013) is determined by differences in specific amino acid residues. His-1, Gly-4, Phe-6, Thr-7, and Asp-9 are pivotal for maintaining the peptide secondary structure necessary for interaction with the receptor core (Runge et al., 2003; Adelhorst et al., 1994; Gallwitz et al., 1994). His-1 and Thr/Ser-7 are conserved in all GLP-1 peptides (GLP-1, GLP-2, GCG, and GCRP) but GIP, which conversely displays a Tyr-1 and Ile-7 substitution, suggesting that these two residues are likely important for the selective interaction with GLP-1R and discriminate GIP-R. By introducing His-1 and Thr-7 into GIP, the resulting chimeric peptide binds and activates GLP-1R with a relatively high affinity and potency (Moon et al., 2010, 2012a). At 17-20 positions, GLP-1-related peptides exhibited marked variations in amino acid sequence, suggesting that the selectivity of recognition of ECD in the cognate receptor is achieved through interactions of these ortholog-specific residues of the alpha-helix (Parthier et al., 2007; Underwood et al., 2010). GLP-1 family is cleaved and inactivated by activity of ubiquitously expressed DPP-4 enzymes that recognize Pro-2 or Ala-2. Generation of DPP-4-resistant GLP-1modified molecules with increased half-life has been pivotal for the treatment of T2D (see Fig. 16.4B). It has been extensively reviewed that GPCRs could exist as homo- or heterodimers at the cell surface (Bulenger et al., 2005; Gurevich and Gurevich, 2008; Devi, 2001). Due to the relatively high level of sequence identity between GLP-1R and GIP-R, their similar expression profiles, and physiological functions, it has been suggested that GLP-1R might form a functional complex with the GIP-R (Whitaker et al., 2012). An alternative nonclassical GLP-1R has been suggested to mediate the nonpancreatic effects described in the heart (Ban et al., 2008) and adipose tissue (Cantini et al., 2016) for the truncated GLP-1(9-37) form, where the receptor knockout only partially eliminates the effects of GLP-1 (Ussher et al., 2014). Alternatively, these effects in the absence of the classical GLP-1R might be mediated by related GCGRs (Cantini et al., 2016). However, the specific actions of both homo- and heterodimers compared to the single GLP-1R molecules are far from been elucidated. Distribution of GLP-1R and related receptors are important for activities of these ligands other than in the pancreas. For extrapancreatic effects of GLP1, see Cantini et al. (2016). mRNA for classical GLP-1R was identified in the lung, pancreatic islets, stomach, kidney, in the

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hypothalamus and heart, but not in the adipose tissue, liver, and skeletal muscle. Furthermore, GLP-1R forms expressed in the kidney and heart have been suggested to be structural variants of the pancreatic classical receptor. Within the pancreas, GLP-1R is predominantly localized in the b and, to a lesser extent, in a and d cells. In addition, the receptor is not present in the ventricular myocardium, while it is expressed at the atrial level and sinoatrial myocytes (Kim et al., 2013). Expression of classical GLP-1R in the adipose tissue is still controversial, despite the evidence of GLP-1 effects (Cantini et al., 2015, 2016, 2017). Indeed, some of GLP-1 effects in this tissue may be mediated by alternative receptors in the family, including the glucagon receptor, whose activation mediates the lipolytic effects of glucagon (Bertin et al., 2001).

7. INTESTINAL HORMONES IN THE PATHOGENESIS OF T2D GLP-1. Some studies reported a reduction of circulating levels of GLP-1 in patients with T2D (Mannucci et al., 2000; Vilsbøll et al., 2001), although other investigators obtained contrasting results (Nauck et al., 1993). An impairment of the GLP-1 axis could contribute to the reduction of first-phase postprandial insulin secretion that is typical of T2D, leading to hyperglycemia after meals. Studies in first-degree relatives of patients with T2D were inconclusive (Nauck et al., 2004). Differences in results can be, at least partly, determined by the heterogeneity of experimental methods or by diversities in case mix. It is possible that some minor alterations of GLP-1 secretion occur early in the natural history of T2D, contributing to its pathogenesis. However, available data also suggests that a decline of circulating levels of active GLP-1 could be the consequence of chronic hyperglycemia, establishing a vicious cycle; in fact, high glucose levels induce hyperexpression and hyperactivity of DPP-4, which inactivates GLP-1, both in vitro and in vivo (Pala et al., 2003; Mannucci et al., 2005). An impairment of the GLP-1 axis in diabetes could also be due to reduced GLP-1 sensitivity. In fact, in patients with established diabetes the insulin secretory response to GLP-1 is reduced (Nauck et al., 1993); this could be the consequence of the reduction of b-cell functional mass, rather than a specific defect of GLP-1 signal transmission. The known polymorphism of GLP-1 receptor gene does not seem to be associated with the risk of diabetes (Koole et al., 2013), nor with response to GLP-1 receptor agonists in diabetic patients (Lin et al., 2015). GIP. Although GIP plays a crucial role in the regulation of insulin and glucagon secretion in the postprandial phase, few studies have explored its possible involvement in the pathogenesis of T2D. Early reports

showed a reduction of active GIP levels in patients with T2D, when compared to healthy controls (Nauck et al., 1993). The GIP response has been found to be significantly increased following a mixed meal but not after glucose ingestion, whereas GLP-1 concentrations were unaffected by meal composition, suggesting that the increased GIP response to nutrients other than glucose may predispose to glucose intolerance (Vollmer et al., 2008). CCK. CCK has been reported to stimulate b-cell neogenesis and inhibit b-cell apoptosis in animal models. Therefore, it could have a theoretical role in the pathogenesis of T2D; however, no studies have confirmed any association between CCK secretion and T2D in humans.

8. INTESTINAL HORMONES IN THE TREATMENT OF T2D GLP-1. GLP-1 as such cannot be used for treating diabetes in humans because of its unfavorable kinetics. Several long-acting GLP-1 receptor agonists, which can be administered twice daily, once daily, or once weekly, have been developed and are routinely used in the treatment of T2D. Available molecules for clinical use include exendin-4-like molecules, such as exenatide and lixisenatide, and human GLP-1-based analogs, such as liraglutide, dulaglutide, albiglutide, and semaglutide (Fig. 16.4B). Exenatide is administered twice daily or once weekly, depending on the pharmaceutical formulation, whereas lixisenatide and liraglutide are used once daily, and dulaglutide, albiglutide, and semaglutide once weekly. Shorter acting agents (i.e., exenatide b.i.d. and lixisenatide) have a greater efficacy on postprandial glucose, but they are less effective than longer-acting agents on fasting glucose and glycated hemoglobin (HbA1c). Long-acting GLP-1 receptor agonists are more effective on hyperglycemia than any other therapeutic agent described so far, without inducing hypoglycemia, unless associated with insulin and/or sulfonylureas (Mannucci and Dicembrini, 2012). In addition, they induce a remarkable weight loss (see later), and they reduce cardiovascular morbidity and/or mortality (Marso et al, 2016a, 2016b; Holman et al., 2017). Current formulations of GLP-1 receptor agonists are administered as subcutaneous injections, but oral preparations are under development (Davies et al., 2017). An alternative to GLP-1 receptor agonists is represented by DPP-4 inhibitors, which inhibit the inactivation of both GLP-1 and GIP, together with other hormones with a similar cleavage structure. These small molecules increase circulating concentrations of active GLP-1 by three to four times, while circulating levels of injectable GLP-1 receptor agonists can exceed 10 times

11. CONCLUSIONS AND FUTURE DIRECTIONS

those of endogenous GLP-1 (DeFronzo et al., 2008). DPP4 inhibitors, which are administered orally, provide a moderate reduction of hyperglycemia, particularly in the postprandial phase, with a very good tolerability profile, and without increasing cardiovascular risk (Scirica et al., 2014; Zannad et al., 2015; Bethel et al., 2015). GIP. Although GIP contributes, at least as much as GLP-1, to the incretin effect under physiological conditions, in T2D, these two incretins seem to have different insulinotropic effects: while pharmacological levels of GLP-1 promote a relevant glucose-dependent insulin secretion, the increase of GIP levels has a much smaller insulinotropic effect (Nauck et al., 1993). On the other hand, GIP seems to be more effective than GLP-1 in inhibiting glucagon secretion (Christensen, 2016). Some dual GLP-1/GIP receptor agonists, which combine the effects of GLP-1 on insulin secretion and those of GIP on glucagon secretion, are currently under development for the treatment of T2D (Frias et al., 2017; Sa´nchez-Garrido et al., 2017; Skow et al., 2016; Mu¨ller et al., 2018).

9. INTESTINAL HORMONES IN THE PATHOGENESIS OF OBESITY GLP-1. Recent research indicates that the incretin effect is also impaired in obesity (Knop et al., 2012). Together with a glucose-dependent insulin stimulation and glucagon suppression, GLP-1 also exerts a plethora of extraglycemic effects (see Cantini et al., 2016). In the gastrointestinal tract, GLP-1 decelerates gut motility and gastric emptying and thereby delays nutrient absorption by the gastrointestinal tract. GLP-1 has a key role also in satiation signaling, reducing appetite, and food intake, through both central and peripheral activation of GLP-1 receptors. Animal studies showed that GLP-1 is used as a neurotransmitter by a population of neurons in the nucleus of the solitary tract that receives signals from the vagal nerve and project their terminations to the arcuate and paraventricular nuclei of the hypothalamus; this pathway transmits signals from the gastrointestinal tract to centers regulating food intake. In addition, GLP-1 receptors in the hypothalamus are accessible to circulating (peripheral) GLP-1 (Iepsen et al., 2014); therefore, the hormonal gut signals can directly regulate food intake. GIP, CCK, and PYY. Other intestinal peptides such as GIP, CCK, and PYY contribute to the inhibitory effect of GLP-1 on gastric emptying and to the so-called “ileal brake,” which represents the feedback inhibitory control of gastrointestinal transit by intestinal hormones, elicited by the presence of nutrients in the gut (Iepsen et al., 2015). Evidence suggesting a link between GIP and obesity comes from genetic studies in humans:

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polymorphisms in adjacent regions of the GIP receptor gene may contribute to genetic predisposition to obesity (Vogel et al., 2009). PYY also has a potent inhibitory effect on food intake, likely acting via Y2 receptors in the arcuate nucleus of hypothalamus, which seems to play a major role in the regulation of appetite (Troke et al., 2014), Fig. 16.1.

10. INTESTINAL HORMONES IN THE TREATMENT OF OBESITY GLP-1. Significant weight loss has been reported with GLP-1 receptor agonists (GLP-1RAs) in both people with T2D and obese people without diabetes. In 2015, the European Medicines Agency approved the GLP1RA liraglutide for chronic weight management in addition to a reduced calorie diet and physical activity. The drug is approved for the use in adults with a BMI of 30 kg/m2 or greater (obesity) or 27 kg/m2 or greater (overweight) in the presence of at least one weightrelated comorbidity such as hypertension, T2D, dyslipidemia, or obstructive sleep apnea. The dose is 3.0 mg daily in contrast to the 1.8 mg daily for T2D management. The SCALE trial, performed on 864 T2D and obese patients, showed a 6% weight loss after 56 weeks of treatment with liraglutide 3 mg daily (Davies et al., 2015). In obese prediabetic subjects, glucose tolerance was normalized in the majority after 2 years of treatment, suggesting that GLP-1RA therapy may prevent T2D in high-risk population (Astrup et al., 2012). Other GLP-1RAs, such as semaglutide, are currently under development for the treatment of obesity (Blundell et al., 2017), Fig. 16.4B. GIP. The association between elevated GIP levels and the development of obesity and insulin resistance has prompted interest in GIP antagonism; to date, only data coming from animal models seems to confirm potentially benefits of GIP antagonism in obesityrelated diseases (Paschetta et al., 2011).

11. CONCLUSIONS AND FUTURE DIRECTIONS The high level of redundancy and variety of ligands and cognate receptors belonging to same families, as well as the high conservation of this system in the evolution, confirm the pivotal role exerted in the metabolic control by the intestinal hormones, in particular by glucagon-like peptides. Moreover, the recently demonstrated extrapancreatic pleiotropic activity exerted by GLP-1 on several organs makes GLP-1 and its synthetic analogs extremely appealing for developing novel and

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more physiologic therapies for metabolic and cardiovascular pathologies. The impairment of the incretin system, which seems to play a major role in the pathogenesis of both obesity and T2D, underlines the potential of incretin-based therapies in the prevention and treatment of these diseases. However, despite already promising effects with GLP-1 receptor agonists, better results seem to be obtained by combining the effects of more than one intestinal hormone. Interestingly, pharmacology of T2D and obesity has recently moved toward the synthesis of dual/triple agonists of receptors for intestinal ligands (Sa´nchez-Garrido et al., 2017; Skow et al., 2016; Mu¨ller et al., 2018), which show a potentiated effect due to the presence of more than one effective ligand. Moreover, some of these molecules take advantage to be selectively driven to the cellular/tissue targets due to the selectivity of the interaction between one of the ligand molecules and its tissue-specific receptor.

References Adelhorst, K., Hedegaard, B.B., Knudsen, L.B., Kirk, O., March 4, 1994. Structure-activity studies of glucagon-like peptide-1. J. Biol. Chem. 269 (9), 6275e6278. Alan˜a, I., Malthouse, J.P., O’Harte, F.P., Hewage, C.M., July 1, 2007. The bioactive conformation of glucose-dependent insulinotropic polypeptide by NMR and CD spectroscopy. Proteins 68 (1), 92e99. Astrup, A., Carraro, R., Finer, N., Harper, A., Kunesova, M., Lean, M.E., Niskanen, L., Rasmussen, M.F., Rissanen, A., Ro¨ssner, S., Savolainen, M.J., Van Gaal, L., NN8022-1807 Investigators, June 2012. Safety, tolerability and sustained weight loss over 2 years with the once-daily human GLP-1 analog, liraglutide. Int. J. Obes. 36 (6), 843e854. Baggio, L.L., Drucker, D.J., May 2007. Biology of incretins: GLP-1 and GIP. Gastroenterology 132 (6), 2131e2157. Baggio, L.L., Huang, Q., Brown, T.J., Drucker, D.J., August 2004. Oxyntomodulin and glucagon-like peptide-1 differentially regulate murine food intake and energy expenditure. Gastroenterology 127 (2), 546e558. Ban, K., Kim, K.H., Cho, C.K., Sauve´, M., Diamandis, E.P., Backx, P.H., Drucker, D.J., Husain, M., April 2010. Glucagon-like peptide (GLP)1(9-36)amide-mediated cytoprotection is blocked by exendin(9-39) yet does not require the known GLP-1 receptor. Endocrinology 151 (4), 1520e1531. Ban, K., Noyan-Ashraf, M.H., Hoefer, J., Bolz, S.S., Drucker, D.J., Husain, M., May 6, 2008. Cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are mediated through both glucagon-like peptide 1 receptor-dependent and eindependent pathways. Circulation 117 (18), 2340e2350. Bastien-Dionne, P.O., Valenti, L., Kon, N., Gu, W., Buteau, J., December 2011. Glucagon-like peptide 1 inhibits the sirtuin deacetylase SirT1 to stimulate pancreatic b-cell mass expansion. Diabetes 60 (12), 3217e3222. Batterham, R.L., Cohen, M.A., Ellis, S.M., Le Roux, C.W., Withers, D.J., Frost, G.S., Ghatei, M.A., Bloom, S.R., September 4, 2003a. Inhibition of food intake in obese subjects by peptide YY3-36. N. Engl. J. Med. 349 (10), 941e948. Batterham, R.L., Cowley, M.A., Small, C.J., Herzog, H., Cohen, M.A., Dakin, C.L., Wren, A.M., Brynes, A.E., Low, M.J., Ghatei, M.A.,

Cone, R.D., Bloom, S.R., August 8, 2002. Gut hormone PYY(3-36) physiologically inhibits food intake. Nature 418 (6898), 650e654. Batterham, R.L., Heffron, H., Kapoor, S., Chivers, J.E., Chandarana, K., Herzog, H., Le Roux, C.W., Thomas, E.L., Bell, J.D., Withers, D.J., September 2006. Critical role for peptide YY in protein-mediated satiation and body-weight regulation. Cell Metabol. 4 (3), 223e233. Batterham, R.L., Le Roux, C.W., Cohen, M.A., Park, A.J., Ellis, S.M., Patterson, M., Frost, G.S., Ghatei, M.A., Bloom, S.R., August 2003b. Pancreatic polypeptide reduces appetite and food intake in humans. J. Clin. Endocrinol. Metab. 88 (8), 3989e3992. Bauer, P.V., Hamr, S.C., Duca, F.A., February 2016. Regulation of energy balance by a gut-brain axis and involvement of the gut microbiota. Cell. Mol. Life Sci. 73 (4), 737e755. Bayliss, W.M., Starling, E.H., September 12, 1902. The mechanism of pancreatic secretion. J. Physiol. 28 (5), 325e353. Bell, G.I., Sanchez-Pescador, R., Laybourn, P.J., Najarian, R.C., July 28e August 3, 1983. Exon duplication and divergence in the human preproglucagon gene. Nature 304 (5924), 368e371. Bertin, E., Arner, P., Bolinder, J., Hagstro¨m-Toft, E., March 2001. Action of glucagon and glucagon-like peptide-1-(7-36) amide on lipolysis in human subcutaneous adipose tissue and skeletal muscle in vivo. J. Clin. Endocrinol. Metab. 86 (3), 1229e1234. Bethel, M.A., Green, J.B., Milton, J., Tajar, A., Engel, S.S., Califf, R.M., Holman, R.R., TECOS Executive Committee, April 2015. Regional, age and sex differences in baseline characteristics of patients enrolled in the trial evaluating cardiovascular outcomes with sitagliptin (TECOS). Diabetes Obes. Metab. 17 (4), 395e402. Beucher, A., Gjernes, E., Collin, C., Courtney, M., Meunier, A., Collombat, P., Gradwohl, G., 2012. The homeodomain-containing transcription factors Arx and Pax4 control enteroendocrine subtype specification in mice. PLoS One 7 (5), e36449. Bliss, E.S., Whiteside, E., July 12, 2018. The gut-brain Axis, the human gut microbiota and their integration in the development of obesity. Front. Physiol. 9, 900. Blundell, J., Finlayson, G., Axelsen, M., Flint, A., Gibbons, C., Kvist, T., Hjerpsted, J.B., September 2017. Effects of once-weekly semaglutide on appetite, energy intake, control of eating, food preference and body weight in subjects with obesity. Diabetes Obes. Metab. 19 (9), 1242e1251. Brown, J.C., Dryburgh, J.R., August 1971. A gastric inhibitory polypeptide. II. The complete amino acid sequence. Can. J. Biochem. 49 (8), 867e872. Buchman, A.L., Katz, S., Fang, J.C., Bernstein, C.N., Abou-Assi, S.G., Teduglutide Study Group, June 2010. Teduglutide, a novel mucosally active analog of glucagon-like peptide-2 (GLP-2) for the treatment of moderate to severe Crohn’s disease. Inflamm. Bowel Dis. 16 (6), 962e973. Buffa, R., Solcia, E., Go, V.L., April 1976. Immunohistochemical identification of the cholecystokinin cell in the intestinal mucosa. Gastroenterology 70 (4), 528e532. Bulenger, S., Marullo, S., Bouvier, M., March 2005. Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation. Trends Pharmacol. Sci. 26 (3), 131e137. Cantini, G., Di Franco, A., Mannucci, E., Luconi, M., January 5, 2017. Is cleaved glucagon-like peptide 1 really inactive? Effects of GLP-1(936) on human adipose stem cells. Mol. Cell. Endocrinol. 439, 10e15. Cantini, G., Di Franco, A., Samavat, J., Forti, G., Mannucci, E., Luconi, M., February 15, 2015. Effect of liraglutide on proliferation and differentiation of human adipose stem cells. Mol. Cell. Endocrinol. 402, 43e50. Cantini, G., Mannucci, E., Luconi, M., June 2016. Perspectives in GLP-1 research: new targets, new receptors. Trends Endocrinol. Metabol. 27 (6), 427e438. Carraway, R., Leeman, S.E., November 25, 1976. Characterization of radioimmunoassayable neurotensin in the rat. Its differential

REFERENCES

distribution in the central nervous system, small intestine, and stomach. J. Biol. Chem. 251 (22), 7045e7052. Carraway, R., Leeman, S.E., March 10, 1975. The amino acid sequence of a hypothalamic peptide, neurotensin. J. Biol. Chem. 250 (5), 1907e1911. Carraway, R., Leeman, S.E., October 10, 1973. The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J. Biol. Chem. 248 (19), 6854e6861. Chen, Q., Pinon, D.I., Miller, L.J., Dong, M., 2009. Molecular basis of glucagon-like peptide 1 docking to its intact receptor studied with carboxyl-terminal photolabile probes. J. Biol. Chem. 284 (49), 34135e34144. Chey, W.Y., Chang, T.M., 2003. Secretin, 100 years later. J. Gastroenterol. 38 (11), 1025e1035. Christensen, M., Knop, F.K., Vilsbøll, T., Aaboe, K., Holst, J.J., Madsbad, S., Krarup, T., August 9, 2010. Glucagon-like peptide-2, but not glucose-dependent insulinotropic polypeptide, stimulates glucagon release in patients with type 1 diabetes. Regul. Pept. 163 (1e3), 96e101. Christensen, M.B., April 2016. Glucose-dependent insulinotropic polypeptide: effects on insulin and glucagon secretion in humans. Dan. Med. J. 63 (4). Cohen, M.A., Ellis, S.M., Le Roux, C.W., Batterham, R.L., Park, A., Patterson, M., Frost, G.S., Ghatei, M.A., Bloom, S.R., October 2003. Oxyntomodulin suppresses appetite and reduces food intake in humans. J. Clin. Endocrinol. Metab. 88 (10), 4696e4701. Coopman, K., Huang, Y., Johnston, N., Bradley, S.J., Wilkinson, G.F., Willars, G.B., September 1, 2010. Comparative effects of the endogenous agonist glucagon-like peptide-1 (GLP-1)-(7-36) amide and the small-molecule ago-allosteric agent “compound 2” at the GLP-1 receptor. J. Pharmacol. Exp. Ther. 334 (3), 795e808. Dakin, C.L., Gunn, I., Small, C.J., Edwards, C.M., Hay, D.L., Smith, D.M., Ghatei, M.A., Bloom, S.R., October 2001. Oxyntomodulin inhibits food intake in the rat. Endocrinology 142 (10), 4244e4250. Dakin, C.L., Small, C.J., Batterham, R.L., Neary, N.M., Cohen, M.A., Patterson, M., Ghatei, M.A., Bloom, S.R., June 2004. Peripheral oxyntomodulin reduces food intake and body weight gain in rats. Endocrinology 145 (6), 2687e2695. Davies, M., Pieber, T.R., Hartoft-Nielsen, M.L., Hansen, O.K.H., Jabbour, S., Rosenstock, J., October 17, 2017. Effect of oral semaglutide compared with placebo and subcutaneous semaglutide on glycemic control in patients with type 2 diabetes: a randomized clinical trial. J. Am. Med. Assoc. 318 (15), 1460e1470. Davies, M.J., Bergenstal, R., Bode, B., Kushner, R.F., Lewin, A., Skjøth, T.V., Andreasen, A.H., Jensen, C.B., DeFronzo, R.A., NN8022-1922 Study Group, August 18, 2015. Efficacy of liraglutide for weight loss among patients with type 2 diabetes: the SCALE diabetes randomized clinical trial. J. Am. Med. Assoc. 314 (7), 687e699. Davis, J.D., Smith, G.P., December 1990. Learning to sham feed: behavioral adjustments to loss of physiological postingestional stimuli. Am. J. Physiol. 259 (6 Pt 2), R1228eR1235. Deacon, C.F., Johnsen, A.H., Holst, J.J., March 1995. Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo. J. Clin. Endocrinol. Metab. 80 (3), 952e957. DeFronzo, R.A., Okerson, T., Viswanathan, P., Guan, X., Holcombe, J.H., MacConell, L., October 2008. Effects of exenatide versus sitagliptin on postprandial glucose, insulin and glucagon secretion, gastric emptying, and caloric intake: a randomized, cross-over study. Curr. Med. Res. Opin. 24 (10), 2943e2952. Devi, L.A., October 2001. Heterodimerization of G-protein-coupled receptors: pharmacology, signaling and trafficking. Trends Pharmacol. Sci. 22 (10), 532e537.

377

Dillon, J.S., Tanizawa, Y., Wheeler, M.B., Leng, X.H., Ligon, B.B., Rabin, D.U., Yoo-Warren, H., Permutt, M.A., Boyd 3rd, A.E., October 1993. Cloning and functional expression of the human glucagon-like peptide-1 (GLP-1) receptor. Endocrinology 133 (4), 1907e1910. Dockray, G.J., December 2013. Enteroendocrine cell signalling via the vagus nerve. Curr. Opin. Pharmacol. 13 (6), 954e958. Donnelly, D., May 2012. The structure and function of the glucagonlike peptide-1 receptor and its ligands. Br. J. Pharmacol. 166 (1), 27e41. Drucker, D.J., Habener, J.F., Holst, J.J., December 1, 2017. Discovery, characterization, and clinical development of the glucagon-like peptides. J. Clin. Investig. 127 (12), 4217e4227. Drucker, D.J., Yusta, B., 2014. Physiology and pharmacology of the enteroendocrine hormone glucagon-like peptide-2. Annu. Rev. Physiol. 76, 561e583. Dunphy, J.L., Taylor, R.G., Fuller, P.J., June 25, 1998. Tissue distribution of rat glucagon receptor and GLP-1 receptor gene expression. Mol. Cell. Endocrinol. 141 (1e2), 179e186. Ebert, R., Unger, H., Creutzfeldt, W., June 1983. Preservation of incretin activity after removal of gastric inhibitory polypeptide (GIP) from rat gut extracts by immunoadsorption. Diabetologia 24 (6), 449e454. Egerod, K.L., Engelstoft, M.S., Grunddal, K.V., Nøhr, M.K., Secher, A., Sakata, I., Pedersen, J., Windeløv, J.A., Fu¨chtbauer, E.M., Olsen, J., Sundler, F., Christensen, J.P., Wierup, N., Olsen, J.V., Holst, J.J., Zigman, J.M., Poulsen, S.S., Schwartz, T.W., December 2012. A major lineage of enteroendocrine cells coexpress CCK, secretin, GIP, GLP-1, PYY, and neurotensin but not somatostatin. Endocrinology 153 (12), 5782e5795. Engelstoft, M.S., Egerod, K.L., Lund, M.L., Schwartz, T.W., December 2013. Enteroendocrine cell types revisited. Curr. Opin. Pharmacol. 13 (6), 912e921. Frias, J.P., Bastyr 3rd, E.J., Vignati, L., Tscho¨p, M.H., Schmitt, C., Owen, K., Christensen, R.H., DiMarchi, R.D., August 1, 2017. The sustained effects of a dual GIP/GLP-1 receptor agonist, NNC0090-2746, in patients with type 2 diabetes. Cell Metabol. 26 (2), 343e352.e2. Frimurer, T.M., Bywater, R.P., June 1, 1999. Structure of the integral membrane domain of the GLP1 receptor. Proteins 35 (4), 375e386. Gallwitz, B., Witt, M., Paetzold, G., Morys-Wortmann, C., Zimmermann, B., Eckart, K., Fo¨lsch, U.R., Schmidt, W.E., November 1, 1994. Structure/activity characterization of glucagon-like peptide-1. Eur. J. Biochem. 225 (3), 1151e1156. Gibbons, C., Caudwell, P., Finlayson, G., Webb, D.L., Hellstro¨m, P.M., Na¨slund, E., Blundell, J.E., May 2013. Comparison of postprandial profiles of ghrelin, active GLP-1, and total PYY to meals varying in fat and carbohydrate and their association with hunger and the phases of satiety. J. Clin. Endocrinol. Metab. 98 (5), E847eE855. Graaf, C., Donnelly, D., Wootten, D., Lau, J., Sexton, P.M., Miller, L.J., Ahn, J.M., Liao, J., Fletcher, M.M., Yang, D., Brown, A.J., Zhou, C., Deng, J., Wang, M.W., October 2016. Glucagon-like peptide-1 and its class B G protein-coupled receptors: a long march to therapeutic successes. Pharmacol. Rev. 68 (4), 954e1013. Gurevich, V.V., Gurevich, E.V., May 2008. How and why do GPCRs dimerize? Trends Pharmacol. Sci. 29 (5), 234e240. Habib, A.M., Richards, P., Cairns, L.S., Rogers, G.J., Bannon, C.A., Parker, H.E., Morley, T.C., Yeo, G.S., Reimann, F., Gribble, F.M., July 2012. Overlap of endocrine hormone expression in the mouse intestine revealed by transcriptional profiling and flow cytometry. Endocrinology 153 (7), 3054e3065. Haderslev, K.V., Jeppesen, P.B., Hartmann, B., Thulesen, J., Sorensen, H.A., Graff, J., Hansen, B.S., Tofteng, F., Poulsen, S.S., Madsen, J.L., Holst, J.J., Staun, M., Mortensen, P.B., April 2002. Short-term administration of glucagon-like peptide-2. Effects on

378

16. INTESTINAL HORMONES

bone mineral density and markers of bone turnover in short-bowel patients with no colon. Scand. J. Gastroenterol. 37 (4), 392e398. Hinke, S.A., Gelling, R., Manhart, S., Lynn, F., Pederson, R.A., Ku¨hnWache, K., Rosche, F., Demuth, H.U., Coy, D., McIntosh, C.H., March 2003. Structure-activity relationships of glucose-dependent insulinotropic polypeptide (GIP). Biol. Chem. 384 (3), 403e407. Ho¨kfelt, T., Rehfeld, J.F., Skirboll, L., Ivemark, B., Goldstein, M., Markey, K., June 12, 1980. Evidence for coexistence of dopamine and CCK in meso-limbic neurones. Nature 285 (5765), 476e478. Holman, R.R., Bethel, M.A., Mentz, R.J., Thompson, V.P., Lokhnygina, Y., Buse, J.B., Chan, J.C., Choi, J., Gustavson, S.M., ¨ hman, P., Pagidipati, N.J., Iqbal, N., Maggioni, A.P., Marso, S.P., O Poulter, N., Ramachandran, A., Zinman, B., Hernandez, A.F., EXSCEL Study Group, September 28, 2017. Effects of once-weekly exenatide on cardiovascular outcomes in type 2 diabetes. N. Engl. J. Med. 377 (13), 1228e1239. Holst, J.J., Bersani, M., Johnsen, A.H., Kofod, H., Hartmann, B., Orskov, C., July 22, 1994. Proglucagon processing in porcine and human pancreas. J. Biol. Chem. 269 (29), 18827e18833. Holst, J.J., December 2013. Enteroendocrine secretion of gut hormones in diabetes, obesity and after bariatric surgery. Curr. Opin. Pharmacol. 13 (6), 983e988. Huang, Y., Wilkinson, G.F., Willars, G.B., January 2010. Role of the signal peptide in the synthesis and processing of the glucagonlike peptide-1 receptor. Br. J. Pharmacol. 159 (1), 237e251. Hwang, J.I., Moon, M.J., Park, S., Kim, D.K., Cho, E.B., Ha, N., Son, G.H., Kim, K., Vaudry, H., Seong, J.Y., May 2013. Expansion of secretin-like G protein-coupled receptors and their peptide ligands via local duplications before and after two rounds of whole-genome duplication. Mol. Biol. Evol. 30 (5), 1119e1130. Hwang, J.I., Yun, S., Moon, M.J., Park, C.R., Seong, J.Y., June 2014. Molecular evolution of GPCRs: GLP1/GLP1 receptors. J. Mol. Endocrinol. 52 (3), T15eT27. Iepsen, E.W., Torekov, S.S., Holst, J.J., 2015. Liraglutide for type 2 diabetes and obesity: a 2015 update. Expert Rev. Cardiovasc Ther. 13 (7), 753e767. Iepsen, E.W., Torekov, S.S., Holst, J.J., December 2014. Therapies for inter-relating diabetes and obesityeGLP-1 and obesity. Expert Opin. Pharmacother. 15 (17), 2487e2500. Irwin, D.M., Prentice, K.J., October 2011. Incretin hormones and the expanding families of glucagon-like sequences and their receptors. Diabetes Obes. Metab. 13 (Suppl. 1), 69e81. Irwin, D.M., July 1, 2005. Evolution of hormone function: proglucagonderived peptides and their receptors. Bioscience 55 (7), 583e591. Irwin, D.M., 2012. Origin and convergent evolution of exendin genes. Gen. Comp. Endocrinol. 175 (1), 27e33. Ivy, A.C., Oldberg, E.A., 1928. A hormone mechanism for gallbladder contraction and evacuation. Am. J. Physiol. 86, 599e613. Jeppesen, P.B., Pertkiewicz, M., Messing, B., Iyer, K., Seidner, D.L., O’keefe, S.J., Forbes, A., Heinze, H., Joelsson, B., December 2012. Teduglutide reduces need for parenteral support among patients with short bowel syndrome with intestinal failure. Gastroenterology 143 (6), 1473e1481 e3. Jiang, S., Ulrich, C., February 27, 1995. Molecular cloning and functional expression of a human pancreatic secretin receptor. Biochem. Biophys. Res. Commun. 207 (3), 883e890. Jørgensen, N.B., Dirksen, C., Bojsen-Møller, K.N., Jacobsen, S.H., Worm, D., Hansen, D.L., Kristiansen, V.B., Naver, L., Madsbad, S., Holst, J.J., September 2013. Exaggerated glucagon-like peptide 1 response is important for improved b-cell function and glucose tolerance after Roux-en-Y gastric bypass in patients with type 2 diabetes. Diabetes 62 (9), 3044e3052. Kieffer, T.J., McIntosh, C.H., Pederson, R.A., August 1995. Degradation of glucose-dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology 136 (8), 3585e3596.

Kilimnik, G., Kim, A., Steiner, D.F., Friedman, T.C., Hara, M., Maye June 2010. Intraislet production of GLP-1 by activation of prohormone convertase 1/3 in pancreatic a-cells in mouse models of ßcell regeneration. Islets 2 (3), 149e155. Kim, M., Platt, M.J., Shibasaki, T., Quaggin, S.E., Backx, P.H., Seino, S., Simpson, J.A., Drucker, D.J., May 2013. GLP-1 receptor activation and Epac2 link atrial natriuretic peptide secretion to control of blood pressure. Nat. Med. 19 (5), 567e575. Kislauskis, E., Bullock, B., McNeil, S., Dobner, P.R., April 5, 1988. The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences. J. Biol. Chem. 263 (10), 4963e4968. Knop, F.K., Aaboe, K., Vilsbøll, T., Vølund, A., Holst, J.J., Krarup, T., Madsbad, S., June 2012. Impaired incretin effect and fasting hyperglucagonaemia characterizing type 2 diabetic subjects are early signs of dysmetabolism in obesity. Diabetes Obes. Metab. 14 (6), 500e510. Koole, C., Savage, E.E., Christopoulos, A., Miller, L.J., Sexton, P.M., Wootten, D., August 2013. Minireview: signal bias, allosterism, and polymorphic variation at the GLP-1R: implications for drug discovery. Mol. Endocrinol. 27 (8), 1234e1244. La Barre, J., 1932. Sur les possibilite´s d’un traitement du diabe`te par l’incre´tine. Bull. Acad. R. Med. Belg. 12, 620e634. Laferre`re, B., Swerdlow, N., Bawa, B., Arias, S., Bose, M., Oliva´n, B., Teixeira, J., McGinty, J., Rother, K.I., August 2010. Rise of oxyntomodulin in response to oral glucose after gastric bypass surgery in patients with type 2 diabetes. J. Clin. Endocrinol. Metab. 95 (8), 4072e4076. Larsen, P.J., Tang-Christensen, M., Holst, J.J., Orskov, C., March 1997. Distribution of glucagon-like peptide-1 and other preproglucagon-derived peptides in the rat hypothalamus and brainstem. Neuroscience 77 (1), 257e270. Lin, C.H., Lee, Y.S., Huang, Y.Y., Hsieh, S.H., Chen, Z.S., Tsai, C.N., 2015. Polymorphisms of GLP-1 receptor gene and response to GLP-1 analogue in patients with poorly controlled type 2 diabetes. J. Diabetes Res. 2015, 176949. Little, T.J., Horowitz, M., Feinle-Bisset, C., November 2005. Role of cholecystokinin in appetite control and body weight regulation. Obes. Rev. 6 (4), 297e306. Liu, Z., Stanojevic, V., Avadhani, S., Yano, T., Habener, J.F., August 2011. Stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis activation induces intra-islet glucagon-like peptide-1 (GLP-1) production and enhances beta cell survival. Diabetologia 54 (8), 2067e2076. Lund, A., Vilsbøll, T., Bagger, J.I., Holst, J.J., Knop, F.K., June 2011. The separate and combined impact of the intestinal hormones, GIP, GLP-1, and GLP-2, on glucagon secretion in type 2 diabetes. Am. J. Physiol. Endocrinol. Metab. 300 (6), E1038eE1046. Mace, O.J., Tehan, B., Marshall, F., August 2015. Pharmacology and physiology of gastrointestinal enteroendocrine cells. Pharmacol. Res. Perspect. 3 (4), e00155. Maida, A., Lovshin, J.A., Baggio, L.L., Drucker, D.J., November 2008. The glucagon-like peptide-1 receptor agonist oxyntomodulin enhances beta-cell function but does not inhibit gastric emptying in mice. Endocrinology 149 (11), 5670e5678. Mannucci, E., Dicembrini, I., May 2012. Incretin-based therapies and cardiovascular risk. Curr. Med. Res. Opin. 28 (5), 715e721. Mannucci, E., Ognibene, A., Cremasco, F., Bardini, G., Mencucci, A., Pierazzuoli, E., Ciani, S., Fanelli, A., Messeri, G., Rotella, C.M., October 2000. Glucagon-like peptide (GLP)-1 and leptin concentrations in obese patients with type 2 diabetes mellitus. Diabet. Med. 17 (10), 713e719. Mannucci, E., Pala, L., Ciani, S., Bardini, G., Pezzatini, A., Sposato, I., Cremasco, F., Ognibene, A., Rotella, C.M., June 2005. Hyperglycaemia increases dipeptidyl peptidase IV activity in diabetes mellitus. Diabetologia 48 (6), 1168e1172.

REFERENCES

Marso, S.P., Bain, S.C., Consoli, A., Eliaschewitz, F.G., Jo´dar, E., Leiter, L.A., Lingvay, I., Rosenstock, J., Seufert, J., Warren, M.L., Woo, V., Hansen, O., Holst, A.G., Pettersson, J., Vilsbøll, T., SUSTAIN-6 Investigators, November 10, 2016b. Semaglutide and cardiovascular outcomes in patients with type 2 diabetes. N. Engl. J. Med. 375 (19), 1834e1844. Marso, S.P., Daniels, G.H., Brown-Frandsen, K., Kristensen, P., Mann, J.F., Nauck, M.A., Nissen, S.E., Pocock, S., Poulter, N.R., Ravn, L.S., Steinberg, W.M., Stockner, M., Zinman, B., Bergenstal, R.M., Buse, J.B., LEADER Steering Committee, LEADER Trial Investigators, July 28, 2016a. Liraglutide and cardiovascular outcomes in type 2 diabetes. N. Engl. J. Med. 375 (4), 311e322. Mayer, E.A., Tillisch, K., Gupta, A., March 2, 2015. Gut/brain axis and the microbiota. J. Clin. Investig. 125 (3), 926e938. Mayo, K.E., Miller, L.J., Bataille, D., Dalle, S., Go¨ke, B., Thorens, B., Drucker, D.J., March 2003. International Union of Pharmacology. XXXV. The glucagon receptor family. Pharmacol. Rev. 55 (1), 167e194. Meier, J.J., Nauck, M.A., Pott, A., Heinze, K., Goetze, O., Bulut, K., Schmidt, W.E., Gallwitz, B., Holst, J.J., January 2006. Glucagonlike peptide 2 stimulates glucagon secretion, enhances lipid absorption, and inhibits gastric acid secretion in humans. Gastroenterology 130 (1), 44e54. Meier, J.J., Nauck, M.A., NovembereDecember 2004. GIP as a potential therapeutic agent? Horm. Metab. Res. 36 (11e12), 859e866. Mojsov, S., Heinrich, G., Wilson, I.B., Ravazzola, M., Orci, L., Habener, J.F., September 5, 1986. Preproglucagon gene expression in pancreas and intestine diversifies at the level of posttranslational processing. J. Biol. Chem. 261 (25), 11880e11889. Moon, M.J., Kim, H.Y., Kim, S.G., Park, J., Choi, D.S., Hwang, J.I., Seong, J.Y., August 2010. Tyr1 and Ile7 of glucose-dependent insulinotropic polypeptide (GIP) confer differential ligand selectivity toward GIP and glucagon-like peptide-1 receptors. Mol. Cell. 30 (2), 149e154. Moon, M.J., Kim, H.Y., Park, S., Kim, D.K., Cho, E.B., Park, C.R., You, D.J., Hwang, J.I., Kim, K., Choe, H., Seong, J.Y., 2012a. Evolutionarily conserved residues at glucagon-like peptide-1 (GLP-1) receptor core confer ligand-induced receptor activation. J. Biol. Chem. 287 (6), 3873e3884. Moon, M.J., Park, S., Kim, D.K., Cho, E.B., Hwang, J.I., Vaudry, H., Seong, J.Y., November 19, 2012b. Structural and molecular conservation of glucagon-like peptide-1 and its receptor confers selective ligand-receptor interaction. Front. Endocrinol. 3, 141. Moore, B., 1906. On the treatment of diabetus mellitus by acid extract of duodenal mucous membrane. Biochem. J. 1 (1), 28e38. Mu¨ller, T.D., Clemmensen, C., Finan, B., DiMarchi, R.D., Tscho¨p, M.H., October 2018. Anti-obesity therapy: from rainbow pills to polyagonists. Pharmacol. Rev. 70 (4), 712e746. Mumphrey, M.B., Patterson, L.M., Zheng, H., Berthoud, H.R., January 2013. Roux-en-Y gastric bypass surgery increases number but not density of CCK-, GLP-1-, 5-HT-, and neurotensin-expressing enteroendocrine cells in rats. Neurogastroenterol. Motil. 25 (1), e70ee79. Murphy, K.G., Dhillo, W.S., Bloom, S.R., December 2006. Gut peptides in the regulation of food intake and energy homeostasis. Endocr. Rev. 27 (7), 719e727. Mutt, V., Jorpes, J.E., Magnusson, S., September 1970. Structure of porcine secretin. The amino acid sequence. Eur. J. Biochem. 15 (3), 513e519. Na¨slund, E., Hellstro¨m, P.M., September 10, 2007. Appetite signaling: from gut peptides and enteric nerves to brain. Physiol. Behav. 92 (1e2), 256e262. Nauck, M.A., El-Ouaghlidi, A., Gabrys, B., Hu¨cking, K., Holst, J.J., Deacon, C.F., Gallwitz, B., Schmidt, W.E., Meier, J.J., November 15, 2004. Secretion of incretin hormones (GIP and GLP-1) and

379

incretin effect after oral glucose in first-degree relatives of patients with type 2 diabetes. Regul. Pept. 122 (3), 209e217. Nauck, M.A., Heimesaat, M.M., Orskov, C., Holst, J.J., Ebert, R., Creutzfeldt, W., January 1993. Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus. J. Clin. Investig. 91 (1), 301e307. Nauck, M.A., Meier, J.J., February 2018. Incretin hormones: their role in health and disease. Diabetes Obes. Metab. 20 (Suppl. 1), 5e21. Neidigh, J.W., Fesinmeyer, R.M., Prickett, K.S., Andersen, N.H., November 6, 2001. Exendin-4 and glucagon-like-peptide-1: NMR structural comparisons in the solution and micelle-associated states. Biochemistry 40 (44), 13188e13200. Nerga˚rd, B.J., Lindqvist, A., Gislason, H.G., Groop, L., Ekelund, M., Wierup, N., Hedenbro, J.L., NovembereDecember 2015. Mucosal glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide cell numbers in the super-obese human foregut after gastric bypass. Surg. Obes. Relat. Dis. 11 (6), 1237e1246. Neumann, J.M., Couvineau, A., Murail, S., Lacape`re, J.J., Jamin, N., Laburthe, M., July 2008. Class-B GPCR activation: is ligand helixcapping the key? Trends Biochem. Sci. 33 (7), 314e319. Oesch, S., Ru¨egg, C., Fischer, B., Degen, L., Beglinger, C., May 30, 2006. Effect of gastric distension prior to eating on food intake and feelings of satiety in humans. Physiol. Behav. 87 (5), 903e910. Orskov, C., Wettergren, A., Holst, J.J., May 1993. Biological effects and metabolic rates of glucagonlike peptide-1 7-36 amide and glucagonlike peptide-1 7-37 in healthy subjects are indistinguishable. Diabetes 42 (5), 658e661. Pala, L., Mannucci, E., Pezzatini, A., Ciani, S., Sardi, J., Raimondi, L., Ognibene, A., Cappadona, A., Vannelli, B.G., Rotella, C.M., October 10, 2003. Dipeptidyl peptidase-IV expression and activity in human glomerular endothelial cells. Biochem. Biophys. Res. Commun. 310 (1), 28e31. Park, C.R., Moon, M.J., Park, S., Kim, D.K., Cho, E.B., Millar, R.P., Hwang, J.I., Seong, J.Y., June 11, 2013. A novel glucagon-related peptide (GCRP) and its receptor GCRPR account for coevolution of their family members in vertebrates. PLoS One 8 (6), e65420. Parthier, C., Kleinschmidt, M., Neumann, P., Rudolph, R., Manhart, S., Schlenzig, D., Fangha¨nel, J., Rahfeld, J.U., Demuth, H.U., Stubbs, M.T., August 28, 2007. Crystal structure of the incretinbound extracellular domain of a G protein-coupled receptor. Proc. Natl. Acad. Sci. U.S.A. 104 (35), 13942e13947. Paschetta, E., Hvalryg, M., Musso, G., October 2011. Glucosedependent insulinotropic polypeptide: from pathophysiology to therapeutic opportunities in obesity-associated disorders. Obes. Rev. 12 (10), 813e828. Patel, D.R., Kong, Y., Sreedharan, S.P., March 1995. Molecular cloning and expression of a human secretin receptor. Mol. Pharmacol. 47 (3), 467e473. Pohl, M., Wank, S.A., April 17, 1998. Molecular cloning of the helodermin and exendin-4 cDNAs in the lizard. Relationship to vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide and glucagon-like peptide 1 and evidence against the existence of mammalian homologues. J. Biol. Chem. 273 (16), 9778e9784. Polak, J.M., Bloom, S., Coulling, I., Pearse, A.G., August 1971. Immunofluorescent localization of secretin in the canine duodenum. Gut 12 (8), 605e610. Polak, J.M., Bloom, S.R., Rayford, P.L., Pearse, A.G., Buchan, A.M., Thompson, J.C., November 22, 1975. Identification of cholecystokinin-secreting cells. Lancet 2 (7943), 1016e1018. Polak, J.M., Sullivan, S.N., Bloom, S.R., Buchan, A.M., Facer, P., Brown, M.R., Pearse, A.G., November 10, 1977. Specific localisation of neurotensin to the N cell in human intestine by radioimmunoassay and immunocytochemistry. Nature 270 (5633), 183e184.

380

16. INTESTINAL HORMONES

Pothoulakis, C., Castagliuolo, I., Leeman, S.E., May 1, 1998. Neuroimmune mechanisms of intestinal responses to stress. Role of corticotropin-releasing factor and neurotensin. Ann. N.Y. Acad. Sci. 840, 635e648. Rehfeld, J.F., July 16, 2018. The origin and understanding of the incretin concept. Front. Endocrinol. 9, 387. Reimann, F., Tolhurst, G., Gribble, F.M., April 4, 2012. G-proteincoupled receptors in intestinal chemosensation. Cell Metabol. 15 (4), 421e431. Richards, M.P., McMurtry, J.P., April 1, 2008. Expression of proglucagon and proglucagon-derived peptide hormone receptor genes in the chicken. Gen. Comp. Endocrinol. 156 (2), 323e338. Richards, M.P., McMurtry, J.P., September 1, 2009. The avian proglucagon system. Gen. Comp. Endocrinol. 163 (1e2), 39e46. Robinson, E., Tate, M., Lockhart, S., McPeake, C., O’Neill, K.M., Edgar, K.S., Calderwood, D., Green, B.D., McDermott, B.J., Grieve, D.J., April 14, 2016. Metabolically-inactive glucagon-like peptide-1(9-36)amide confers selective protective actions against post-myocardial infarction remodelling. Cardiovasc. Diabetol. 15, 65. Runge, S., Gram, C., Brauner-Osborne, H., Madsen, K., Knudsen, L.B., Wulff, B.S., July 25, 2003. Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus. J. Biol. Chem. 278 (30), 28005e28010. Runge, S., Thøgersen, H., Madsen, K., Lau, J., Rudolph, R., April 25, 2008. Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain. J. Biol. Chem. 283 (17), 11340e11347. Sahu, A., June 2004. Minireview: a hypothalamic role in energy balance with special emphasis on leptin. Endocrinology 145 (6), 2613e2620. Sa´nchez-Garrido, M.A., Brandt, S.J., Clemmensen, C., Mu¨ller, T.D., DiMarchi, R.D., Tscho¨p, M.H., October 2017. GLP-1/glucagon receptor co-agonism for treatment of obesity. Diabetologia 60 (10), 1851e1861. Sandoval, D.A., D’Alessio, D.A., April 2015. Physiology of proglucagon peptides: role of glucagon and GLP-1 in health and disease. Physiol. Rev. 95 (2), 513e548. Schroeder, L.E., Leinninger, G.M., March 2018. Role of central neurotensin in regulating feeding: implications for the development and treatment of body weight disorders. Biochim. Biophys. Acta 1864 (3), 900e916. Schwartz, M.W., Woods, S.C., Porte Jr., D., Seeley, R.J., Baskin, D.G., April 6, 2000. Central nervous system control of food intake. Nature 404 (6778), 661e671. Scirica, B.M., Braunwald, E., Raz, I., Cavender, M.A., Morrow, D.A., Jarolim, P., Udell, J.A., Mosenzon, O., Im, K., UmezEronini, A.A., Pollack, P.S., Hirshberg, B., Frederich, R., Lewis, B.S., McGuire, D.K., Davidson, J., Steg, P.G., Bhatt, D.L., SAVOR-TIMI 53 Steering Committee and Investigators, October 28, 2014. Heart failure, saxagliptin, and diabetes mellitus: observations from the SAVOR-TIMI 53 randomized trial. Circulation 130 (18), 1579e1588. Segre, G.V., Goldring, S.R., December 1993. Receptors for secretin, calcitonin, parathyroid hormone (PTH)/PTH-related peptide, vasoactive intestinal peptide, glucagonlike peptide 1, growth hormone-releasing hormone, and glucagon belong to a newly discovered G-protein-linked receptor family. Trends Endocrinol. Metabol. 4 (10), 309e314. Sherwood, N.M., Krueckl, S.L., McRory, J.E., December 2000. The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocr. Rev. 21 (6), 619e670. Sisley, S., Gutierrez-Aguilar, R., Scott, M., D’Alessio, D.A., Sandoval, D.A., Seeley, R.J., June 2014. Neuronal GLP1R mediates liraglutide’s anorectic but not glucose-lowering effect. J. Clin. Investig. 124 (6), 2456e2463.

Sjo¨lund, K., Sande´n, G., Ha˚kanson, R., Sundler, F., November 1983. Endocrine cells in human intestine: an immunocytochemical study. Gastroenterology 85 (5), 1120e1130. Skow, M.A., Bergmann, N.C., Knop, F.K., September 2016. Diabetes and obesity treatment based on dual incretin receptor activation: ‘twincretins’. Diabetes Obes. Metab. 18 (9), 847e854. Starling, E.H., 1905. The croonian lectures on the chemical correlation of the function of the body. Lecture 1. Lancet 2, 339e341. Steinert, R.E., Feinle-Bisset, C., Asarian, L., Horowitz, M., Beglinger, C., Geary, N., January 2017. Ghrelin, CCK, GLP-1, and PYY(3-36): secretory controls and physiological roles in eating and glycemia in health, obesity, and after RYGB. Physiol. Rev. 97 (1), 411e463. Tan, T., Bloom, S., December 2013. Gut hormones as therapeutic agents in treatment of diabetes and obesity. Curr. Opin. Pharmacol. 13 (6), 996e1001. Thompson, A., Kanamarlapudi, V., September 15, 2015. Distinct regions in the C-Terminus required for GLP-1R cell surface expression, activity and internalisation. Mol. Cell. Endocrinol. 413, 66e77. Thompson, A., Kanamarlapudi, V., December 15, 2014. The regions within the N-terminus critical for human glucagon like peptide-1 receptor (hGLP-1R) cell surface expression. Sci. Rep. 4, 7410. Thorens, B., September 15, 1992. Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc. Natl. Acad. Sci. U.S.A. 89 (18), 8641e8645. Thorens, B., Porret, A., Bu¨hler, L., Deng, S.P., Morel, P., Widmann, C., 1993. Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor. Diabetes 42 (11), 1678e1682. Thornton, K., Gorenstein, D.G., March 29, 1994. Structure of glucagonlike peptide (7-36) amide in a dodecylphosphocholine micelle as determined by 2D NMR. Biochemistry 33 (12), 3532e3539. Troke, R.C., Tan, T.M., Bloom, S.R., January 2014. The future role of gut hormones in the treatment of obesity. Ther. Adv. Chronic. Dis 5 (1), 4e14. Tucker, J.D., Dhanvantari, S., Brubaker, P.L., April 9, 1996. Proglucagon processing in islet and intestinal cell lines. Regul. Pept. 62 (1), 29e35. Tudurı´, E., Lo´pez, M., Die´guez, C., Nadal, A., Nogueiras, R., May 2016. Glucagon-like peptide 1 analogs and their effects on pancreatic islets. Trends Endocrinol. Metabol. 27 (5), 304e318. Underwood, C.R., Garibay, P., Knudsen, L.B., Hastrup, S., Peters, G.H., Rudolph, R., Reedtz-Runge, S., January 1, 2010. Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor. J. Biol. Chem. 285 (1), 723e730. Ussher, J.R., Baggio, L.L., Campbell, J.E., Mulvihill, E.E., Kim, M., Kabir, M.G., Cao, X., Baranek, B.M., Stoffers, D.A., Seeley, R.J., Drucker, D.J., May 9, 2014. Inactivation of the cardiomyocyte glucagon-like peptide-1 receptor (GLP-1R) unmasks cardiomyocyte-independent GLP-1R-mediated cardioprotection. Mol. Metab. 3 (5), 507e517. Vaudry, D., Falluel-Morel, A., Bourgault, S., Basille, M., Burel, D., Wurtz, O., Fournier, A., Chow, B.K., Hashimoto, H., Galas, L., Vaudry, H., September 2009. Pituitary adenylate cyclaseactivating polypeptide and its receptors: 20 years after the discovery. Pharmacol. Rev. 61 (3), 283e357. Vilsbøll, T., Krarup, T., Deacon, C.F., Madsbad, S., Holst, J.J., March 2001. Reduced postprandial concentrations of intact biologically active glucagon-like peptide 1 in type 2 diabetic patients. Diabetes 50 (3), 609e613. Vincent, J.P., Mazella, J., Kitabgi, P., July 1999. Neurotensin and neurotensin receptors. Trends Pharmacol. Sci. 20 (7), 302e309. Vogel, C.I., Scherag, A., Bro¨nner, G., Nguyen, T.T., Wang, H.J., Grallert, H., Bornhorst, A., Rosskopf, D., Vo¨lzke, H., Reinehr, T., Rief, W., Illig, T., Wichmann, H.E., Scha¨fer, H., Hebebrand, J.,

FURTHER READING

Hinney, A., March 2, 2009. Gastric inhibitory polypeptide receptor: association analyses for obesity of several polymorphisms in large study groups. BMC Med. Genet. 10, 19. Vollmer, K., Holst, J.J., Baller, B., Ellrichmann, M., Nauck, M.A., Schmidt, W.E., Meier, J.J., March 2008. Predictors of incretin concentrations in subjects with normal, impaired, and diabetic glucose tolerance. Diabetes 57 (3), 678e687. Vrang, N., Hansen, M., Larsen, P.J., Tang-Christensen, M., May 29, 2007. Characterization of brainstem preproglucagon projections to the paraventricular and dorsomedial hypothalamic nuclei. Brain Res. 1149, 118e126. Vrang, N., Larsen, P.J., November 2010. Preproglucagon derived peptides GLP-1, GLP-2 and oxyntomodulin in the CNS: role of peripherally secreted and centrally produced peptides. Prog. Neurobiol. 92 (3), 442e462. Wang, R., Chow, B.K.C., Zhang, L., June 7, 2018. Distribution and functional implication of secretin in multiple brain regions. J. Mol. Neurosci. Whitaker, G.M., Lynn, F.C., McIntosh, C.H., Accili, E.A., 2012. Regulation of GIP and GLP1 receptor cell surface expression by Nglycosylation and receptor heteromerization. PLoS One 7 (3), e32675. Wynne, K., Park, A.J., Small, C.J., Meeran, K., Ghatei, M.A., Frost, G.S., Bloom, S.R., December 2006. Oxyntomodulin increases energy

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expenditure in addition to decreasing energy intake in overweight and obese humans: a randomised controlled trial. Int. J. Obes. 30 (12), 1729e1736. Zannad, F., Cannon, C.P., Cushman, W.C., Bakris, G.L., Menon, V., Perez, A.T., Fleck, P.R., Mehta, C.R., Kupfer, S., Wilson, C., Lam, H., White, W.B., EXAMINE Investigators, May 23, 2015. Heart failure and mortality outcomes in patients with type 2 diabetes taking alogliptin versus placebo in EXAMINE: a multicentre, randomised, double-blind trial. Lancet 385 (9982), 2067e2076. Zhao, D., Pothoulakis, C., October 2006. Effects of NT on gastrointestinal motility and secretion, and role in intestinal inflammation. Peptides 27 (10), 2434e2444.

Further Reading Baggio, L.L., Drucker, D.J., October 2014. Glucagon-like peptide-1 receptors in the brain: controlling food intake and body weight. J. Clin. Investig. 124 (10), 4223e4226. Chey, W.Y., Chang, T.M., March 2014. Secretin: historical perspective and current status. Pancreas 43 (2), 162e182. Drucker, D.J., March 2016. Evolving concepts and translational relevance of enteroendocrine cell biology. J. Clin. Endocrinol. Metab. 101 (3), 778e786.

C H A P T E R

17 Pancreatic Hormones Pierre De Meyts1,2, Pierre J. Lefe`bvre3 1

Department of Cell Signalling, de Duve Institute, Brussels, Belgium; 2Department of Stem Cell Research, Novo Nordisk A/S, Ma˚løv, Denmark; 3Division of Diabetes, Nutrition and Metabolic Diseases, Department of Medicine, CHU, Lie`ge, Belgium

1. THE ENDOCRINE PANCREAS 1.1 History The history of medical discoveries regarding the pancreas from antiquity to today has been extensively covered in the masterly treatise of Howard and Hess (2002). The exocrine function of the pancreas was elucidated long before its endocrine function. The exocrine pancreatic duct was discovered in 1642 by Johann Georg Wirsu¨ng, and the digestive function of the organ well established by Claude Bernard. The islands of endocrine cells (small, irregular polygonal structures) were discovered by Paul Langerhans, a student of Rudolph Virchow, and described in his thesis (Langerhans, 1869), which provided the first careful description of the histology of the pancreas (see the splendid biography by Bjo¨rn M. Hausen, 1988). Langerhans did not know the role of those “Ha¨utchen” (little heaps), although Virchow had stated in 1854 that he believed that “the pancreas not only has a secretion outwards but also inwards into the blood.” Edouard Laguesse renamed in 1893 these spots as “islets of Langerhans,” showed that they contained granules, and speculated that they were the site of an internal secretion (Laguesse, 1893). Lane in 1907 was the first to distinguish a and b cells (Lane, 1907). In 1909, the Belgian physiologist Jean De Meyer postulated that the islets were the origin of the internal secretion and proposed to name this internal secretion “insuline” (De Meyer, 1909). John J.R. McLeod in Toronto established in 1922 that the islets were the source of insulin by studying teleost fishes where the islets are separate from the pancreatic acini (McLeod, 1922). A connection between diabetes mellitus and the pancreas was first suggested by Etienne Lancereaux, based on histologic changes associated with the presence of glycosuria (Lancereaux, 1880). It was definitely

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00017-1

established in 1889 when Oskar Minkowski and Joseph von Mering at the Hoppe-Seyler Institute of Pharmacology in Strasbourg showed that total pancreatectomy caused polyuria in a dog and that the urine contained glucose (von Mering and Minkowski, 1890).

1.2 Evolution of the Endocrine Pancreas Multiple insulin-like peptides (ILPs, numbering from 1 to 40) exist in invertebrates such as insects and the worm Caenorhabditis elegans (C. elegans) (for review, see Na¨ssel et al., 2015; Gro¨nke et al., 2010, and references therein). Remarkably, the first identified ILPs of insects are produced by median neurosecretory cells of the brain, making these ILPs neurohormones, although some are also produced in other tissues such as the midgut. Likewise the 40 C. elegans ILPs are primarily produced in neurons including sensory neurons. For more detailed review of the evolution of the insulinlike peptide family, see Chan and Steiner (2000), Conlon (2001), and De Meyts (2004). The phylogeny and ontogeny of the pancreas in vertebrates has been reviewed previously (Falkmer, 1995; Madsen, 2007). In protochordates like amphioxus, insulin is produced by scattered cells of the intestinal tissue. In the most primitive vertebrates (hagfish and lampreys), the first sign of a new organ is found as collections of endocrine cells around the area of the bile duct connection with the duodenum (99% insulinproducing beta cells and 1% somatostatin-producing delta cells). This migration forms, thus, a new organ involved in sensing blood glucose rather than gut glucose. Later, in fish evolution, the beta cells are joined by exocrine tissue and glucagon-producing alpha cells. From sharks and on, the islet PP cells join the group. The beta cell is thus the phylogenetic founder of the pancreas (Madsen, 2007).

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1.3 Developmental Aspects The pancreas is of endodermal origin and forms from the embryonic foregut. Pancreatic development begins with the formation of a ventral and a dorsal pancreatic bud, each communicating with the foregut through a duct. The dorsal pancreatic bud forms the head, neck, body, and tail, whereas the ventral pancreatic bud forms the uncinate process. Differential rotation and fusion of the ventral and dorsal pancreatic buds results in the formation of the definitive pancreas. All the cell lineages of the pancreas (acinar, ductal, and endocrine) derive from multipotent progenitor cells (for reviews, see Madsen, 2007; Gittes, 2009, and references therein). The multipotent pancreatic progenitor cells have the capacity to differentiate into any of the pancreatic cells: acinar cells, endocrine cells, and ductal cells. These progenitor cells are characterized by the coexpression of the homeodomain transcription factors Pdx1 and Nkx61. Under the influence of transcription factors neurogenin-3 (Ngn3) and Isl1, but in the absence of Notch signaling, these cells differentiate to form two lines of committed endocrine precursor cells. The first line, under the direction of Pax6, forms a and PP cells, which secrete glucagon and pancreatic polypeptide, respectively. The second line, influenced by Pax4, produces b cells and d cells, which secrete insulin and somatostatin, respectively. Other transcription factors such as Aristaless related homeobox (Arx) and forkhead box A2 (Foxa2) are implicated in the initial or terminal differentiation of a cells (Gosmain et al., 2011). Interestingly, further studies have shown that “endocrine cell reprogramming” is possible with conversion of pancreatic a cells into cells displaying a b-cell phenotype, thus potentially offering a totally new avenue for the treatment (or cure) of diabetes (Thorel et al., 2010; Courtney et al., 2011). This will be discussed further in the final Section 5.

1.4 Cellular Architecture of the Islets of Langerhans The islets are richly vascularized: they occupy 1%e2% of the pancreas volume but receive 10%e15% of its blood flow. The architecture and composition of the islets in multiple species has been thoroughly reviewed by Steiner et al. (2010), demonstrating a great degree of plasticity. The prototypical view of islet organization and cellular composition derives mostly from studies of rodent (especially murine) islets. The insulin- and amylin-producing b cells (60%e80% of total cells) (Fig. 17.1) are grouped in the core region. All other cell types are disposed around this core in the peripheral mantle: glucagon-producing a cells (15%e20%),

FIGURE 17.1 3-D reconstruction of whole cell electron tomography of the beta cell. 3-D models revealing key compartments involved in insulin production and release by beta cells following serial section electron tomographic reconstruction. In the picture on the left, the mature insulin granules shown in dark blue on the right picture are omitted to visualize better the other compartments. Plasma membrane: purple; nucleus: yellow; main Golgi ribbon: gray; transmost Golgi cisterna: red; penultimate trans-Golgi cisterna: gold; mitochondria: green; multivesicular bodies: orange; immature granules: light blue. This cell contains 3370 mature insulin granules and w700 immature granules. For details on methodology, see Noske et al. (2008). From Noske, A.B., Costin, A.J., Morgan, G.P., Marsh, B., 2008. Expedited approaches to whole cell electron tomography and organelle mark-up in situ in highpressure frozen pancreatic islets. J. Struct. Biol. 161, 298e313, used with permission under Creative Commons Attribution 4.0 International License (CC-BY).

somatostatin-producing d cells (fewer than 10%), pancreatic polypeptide PP cells (fewer than 1%), and more recently discovered ghrelin-producing ε cells (Andralojc et al., 2009; Wierup et al., 2013) (fewer than 1%). However, studies in other species have questioned this pattern as being the dominant one. For recent reviews focused on human islets (the description of which has been quite controversial), see Bosco et al. (2010) and Da Silva Xavier (2018). Human islets show a more scattered distribution with a, b, and d cells being randomly distributed, with a higher proportion of a cells. According to Bosco et al. (2010), human islets have a unique architecture allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between b and a cells, while permitting homologous contacts between b cells. In contrast, Bonner-Weir et al. (2015) found that human and rodent islets are quite similar. Marsupial islets have more a cells than b cells, bird islets more d cells, and some reptiles have no distinct islets. Islet composition is also very dependent on physiologic conditions (Steiner et al., 2010). Islet cell innervation is also an important aspect of its organization (as already described by Langerhans) with both extrinsic adrenergic, cholinergic, and peptidergic innervation, and intrinsic cholinergic and peptidergic innervation.

2. INSULIN

2. INSULIN Insulin is an anabolic peptide hormone secreted by the b cells of the pancreas acting through a receptor located in the membrane of target cells, the major one being liver (where it promotes glucose storage into glycogen and decreases glucose output), as well as skeletal muscle and fat (where it stimulates glucose transport through translocation of the GLUT4 transporter), but also b cells, brain cells, and in fact most cells, where it has pleiotropic effects (De Meyts, 2016).

2.1 History In the nearly 100 years since the first successful isolation and purification in Toronto by Frederic G. Banting, Charles H. Best, John J.R. McLeod, and James B. Collip of an active pancreatic extract suitable for administration to diabetic patients in 1921e22 (Banting and Best, 1922), much has been written about historical aspects of the discovery of insulin and subsequent milestones in establishing its structure, biosynthesis, and mechanism of action, and we will not elaborate much more here. The definitive account of the discovery itself and its surrounding controversies is the riveting book of Michael Bliss (25th anniversary edition, 2007). For review of subsequent research milestones, see De Meyts (2004), Ward and Lawrence (2011), and Weiss et al. (2014). These research milestones have resulted so far in three Nobel prizes: to Banting and McLeod in 1923 for the discovery of insulin, to Fred Sanger for insulin’s sequence in 1955 (Fig. 17.2, reviewed in Sanger, 1988), and to Rosalyn Yalow in 1977 for insulin’s radioimmunoassay (reviewed in Yalow, 1992). For recent detailed reviews on the structure, biosynthesis, and processing, secretion, and mechanisms of action of insulin, see Weiss et al. (2014), De Meyts (2016), Haeusler et al. (2018), Rorsman and Ashcroft (2018), and Petersen and Shulman (2018).

2.2 Structure of Insulin The circulating (and biologically active) form of insulin is a monomer (Fig. 17.3 left) consisting of two chains, an A chain of 21 amino acids and a B chain of 30 amino acids (in man), linked by two disulfide bridges, A7eB7 and A20eB19. Its MW is ca. 6000 Da. The A chain contains an intrachain disulfide bridge between A6 and

FIGURE 17.2 Amino acid sequence of human insulin.

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A11. At micromolar concentrations, insulin dimerizes and, in the presence of zinc ions, further associates into hexamers (for a discussion of insulin’s selfassembly, see multiple chapters in Federwisch et al., 2002). In the 2-Zn hexamer solved by Dorothy Hodgkin and her colleagues (Adams et al., 1969; Blundell et al., 1971, 1972; Baker et al., 1988), the A chain has an N-terminal helix, A1eA8, linked to an antiparallel C-terminal helix, A12eA20. The B chain has a central helix B8eB19, extended by N- and C-terminal strands. This crystallographic conformation is referred to as the T conformation (Fig. 17.3 left). In the 2-Zn crystal form, all six monomers are in the T conformation (T6). An alternative monomer conformation exists in which the B chain helix extends all the way to the N-terminal (B1eB19, Fig. 17.3 right), referred to as the R conformation. In the 4-Zn hexamer generated by high chloride concentrations, three of the monomers are in the R form and three in the T form (R3T3) (Bentley et al., 1976). In phenol-containing crystals, all six monomers are in the R form (R6) (Derewenda et al., 1989). This structure has also been solved in solution by NMR (Chang et al., 1997). The T-R transition, a true allosteric equilibrium, has been extensively studied in solution (Wollmer, 2002; Bloom et al., 1997). It plays an important role in the pharmaceutical formulations of insulin where phenol is used as an antimicrobial agent and chloride as an isotonic agent. Around its hydrophobic core, the insulin monomer has two extensive nonpolar surfaces (Fig. 17.4) (Blundell et al., 1972; Baker et al., 1988). One is flat and mainly aromatic and buries upon dimer formation in an antiparallel beta sheet structure. The other is more extensive and is buried when the dimers assemble to form hexamers. As discussed subsequently, not surprisingly for a small globular protein, insulin uses the same surfaces for binding to its receptor.

2.3 Biosynthesis, Processing, and Secretion A milestone in understanding the biosynthesis, processing and secretion of insulin was the discovery by Don Steiner in the late 1960s of a precursor protein, proinsulin (MW ca. 9000 Da) (Steiner and Oyer, 1967; Steiner et al., 1967, 1969). The single chain proinsulin (Fig. 17.5) contains the B chain, a connecting domain (C-domain) of 31 residues in man, followed by the A chain. The C-domain is flanked at each end by dibasic

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FIGURE 17.3 3D structure of insulin monomers: T form (left); R form (right). A chain is red; B chain is yellow. See text for explanation. PDB accession numbers: 9INS, 1ZNJ. Graphics program: DSViewerPro from Accelrys.

FIGURE 17.4 Dimer- and hexamer-forming surfaces of the insulin monomer. The dimer-forming surface is shown in yellow. It is made of amino acids B8 Gly, B9 Ser, B12 Val, B13 Glu, B16 Tyr, B23 Gly, B24 Phe, B25 Phe, B26 Tyr, and B27 Thr. The hexamer-forming surface is shown in red. It is made of amino acids B1 Phe, B2 Val, B4 Gln, B13 Glu, B14 Ala, B17 Leu, B18 Val, B19 Cys, B20 Gly, A13 Leu, A14 Tyr, and A17 Glu. Backbone is shown in blue. PDB accession number: 9INS. Graphics program: DSViewerPro from Accelrys.

residues that are cleaved by two trypsin-like and carboxypeptidase B-like enzymes (the prohormone convertases PC2 and PC1/3), then by carboxypeptidase E, to release the mature insulin and a free C-peptide cosecreted with insulin. For details of this process, see Hutton (1994) and Weiss et al. (2014). The human insulin gene, located on chromosome 11 (11p15.5), was cloned by Graeme Bell and colleagues in 1980 (Bell et al., 1980). Its three exons encode a larger precursor than that described before, preproinsulin

(Fig. 17.5), where proinsulin’s B domain is preceded by a 24-residue signal peptide of hydrophobic residues characteristic of proteins that enter the secretory pathway and are cleaved during passage through the endoplasmic reticulum by a signal peptidase (Weiss et al., 2014). There proinsulin undergoes rapid folding and disulfide bond formation to generate the native tertiary structure. Newly synthesized insulin is transported as 2-Zn insulin crystals into the secretory granules by a zinc transporter (ZnT8) (Lemaire et al., 2009). High ambient glucose concentration in the islets promotes insulin biosynthesis and is the primary regulator of secretion (for review, see Weiss et al., 2014, Rorsman and Braun, 2013, and the encyclopedic review by Rorsman and Ashcroft, 2018). Calcium-dependent exocytosis of secretory granules is the main mechanism of secretion in both glucose-stimulated and basal states (Tanese et al., 1970; Wollheim and Sharp, 1981; Rhodes and Halban, 1987). In brief, elevation of glucose concentration to greater than 8e10 mM results in depolarization of the b cell. Glucose is taken up into the b cell by the GLUT2 transporter (in rodents, GLUT1 in humans) and metabolized via glucokinase, glycolysis, and generates ATP in mitochondria. This alters the ATP/ADP ratio, which closes KATP channels and depolarizes the b cell via decreased Kþ permeability. These potassium channels (Kir 1.6) belong to the inward rectifier family (Kir) and are associated with the sulfonylurea receptor SUR1 that is required for their function (AguilarBryan and Bryan, 1999). ATP inhibits KATP channels, while ADP opens them. Membrane depolarization opens voltage-dependent Ca2þ channels; the resulting Ca2þ influx leads to the exocytosis of the glucoseregulated secretory granules and insulin secretion. Insulin secretion is also regulated by other hormones: stimulated by glucagon, glucagon-like peptide 1 (GLP-1), gastric inhibitory polypeptide, now called glucose-dependent insulinotropic peptide (GIP), and cholecystokinin, and inhibited by catecholamines and somatostatin (Weiss et al., 2014).

2.4 Insulin Clearance and Inactivation The stages of the journey of insulin in the body from its biosynthesis and secretion to its degradation in the kidney have been recently described in great detail in the excellent review of Tokarz et al. (2018). Insulin is undetectable in the circulation 30 min after its release from the pancreas, and its half-life in the circulation is w6 min. The liver is the first organ that insulin encounters after pulsatile secretion from the pancreas into the portal vein. In humans, 50%e80% of insulin reaching the liver is degraded during first-pass hepatic clearance (Meier

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PC1/3 + carboxypeptidase E

Signal peptidase Signal peptide 24 aa

PC2

B-chain

C-peptide

A-chain

30 aa

31 aa

21 aa

Dibasic residues Arg31 Arg32

Dibasic residues Lys64 Arg 65

FIGURE 17.5 Structure of human preproinsulin. The sites of cleavage by the various processing enzymes are indicated. PC, prohormone convertase.

et al., 2005). After binding to insulin receptors in hepatocytes, receptor-bound insulin is degraded by extracellular insulin-degrading enzyme (IDE) (Terris et al., 1979; Duckworth, 1988; Duckworth et al., 1998). After internalization by ligand-mediated endocytosis (Carpentier, 1994), both receptor-bound and dissociated free insulin are degraded in the endosomes by intracellular IDE; any remaining intact insulin or fragments progress to the lysosomes for final proteolytic degradation (Tokarz et al., 2018). A similar inactivation process occurs in peripheral target cells (muscle, adipose tissue, second pass hepatic clearance) after insulin passes from the liver into the general circulation and interstitium (Terris et al., 1979; Sonne, 1988). The brunt of the degradation of circulating insulin remaining after second pass through the liver occurs in the kidney (Tokarz et al., 2018), through a double internalization mechanism involving renal epithelial cells (through low-affinity binding sites on megalin and cubilin, proteins that recover a number of proteins by endocytosis) as well as renal tubular cells through insulin receptors.

2.5 The Insulin Receptor and Its Apo Structure The concept that insulin acts by promoting glucose transport across the membrane of target cells (rather than acting directly on enzymes of intermediary metabolism of glucose) was established in 1949 by the iconic experiment of Rachmiel Levine et al. (1949), who showed that insulin markedly increased the volume of distribution of nonmetabolizable galactose in eviscerated nephrectomized dogs from 45% of body weight to 75%, a percentage close to that of total body water. From this finding they proposed the following working hypothesis: “Insulin acts upon the cell membrane of certain tissues (skeletal muscle, etc.) in such a manner that the transfer of hexoses (and perhaps other substances) from the extracellular fluid into the cell is facilitated. The intracellular fate of the hexoses depends upon the availability of metabolic systems for their

transformation. In the case of glucose, dissimilation, glycogen storage, and transformation to fat are secondarily stimulated by the rapidity of its entry into the cell.” This major conceptual advance paved the way to the notion that insulin acts on a specific cell membrane receptor. The receptor was first characterized by radioligand binding studies in the early 1970s (House and Weidemann, 1970; Freychet et al., 1971; Cuatrecasas et al., 1971), and by detailed biochemical studies in the early 1980s that established the glycoprotein nature and (ab)2 subunit structure of the receptor. The early steps in insulin receptor research have been reviewed elsewhere (De Meyts, 2004). Following the demonstration in 1982 by Ora Rosen’s group that a tyrosine kinase was closely associated with the insulin receptor (Petruzelli et al., 1982), several groups showed that the insulin receptor itself is a tyrosine kinase, an enzyme that catalyzes the transfer of the g phosphate of ATP to tyrosine residues on protein substrates, the first being the receptor itself (Kasuga et al., 1982a, 1982b, 1983; Avruch et al., 1982; Stadtmauer and Rosen, 1983). The cloning of the insulin receptor cDNA in 1985 by the groups of Axel Ullrich and Bill Rutter (Ullrich et al., 1985; Ebina et al., 1985) established that the insulin receptor indeed belongs to the superfamily of receptor tyrosine kinases (RTKs) (Hubbard and Miller, 2007; Lemmon and Schlessinger, 2010; De Meyts, 2015a). The insulin receptor is encoded by a gene (located on chromosome 19) with 22 exons and 21 introns (Seino et al., 1989, Fig. 17.6), suggesting that the receptor has a modular structure. The short exon 11 that encodes a 12-amino acid sequence is alternatively spliced, resulting in two receptor isoforms (A and B) that differ slightly in affinity for insulin (Seino and Bell, 1989; Mosthaf et al., 1990; Knudsen et al., 2011). The B isoform binds the IGFs with at least 100 times lower affinity than insulin, while the A isoform has significantly higher affinity than the B isoform for IGF-I and especially for IGF-II (Belfiore et al., 2009) and may play a role in tumorigenesis. The receptor is synthesized as single chain preproreceptor that is processed by a furin-like proteolytic enzyme, glycosylated, folded, and dimerized to yield the mature (ab)2 receptor. In cells expressing both

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FIGURE 17.6 Modular structure of the insulin receptor. Domain layout of the (ab)2 linear structure of the insulin receptor, drawn to scale. On the left half of the receptor, spans of the 22 exon-encoded sequences. On the right half, spans of predicted protein modules. Module boundaries mostly correspond to exon boundaries. Module 1 on the left corresponds to the signal peptide. C, C-terminal tail; CR, Cys-rich domain; FnIII-1, FnIII-2, FnIII-3, fibronectin III domains; ID, insert domain in FnIII-2; JM, juxtamembrane domain; L1 and L2, large domains 1 and 2 (leucine-rich repeats); TM, transmembrane domain; TK, tyrosine kinase domain. Orange dots: N-glycosylation sites. Black dots: ligand binding “hotspots” identified by single amino acid site-directed mutagenesis. The two a-subunits are linked by a disulfide bond between the two Cys 524 in the first FnIII domain. One to three of the triplet Cys at 682, 683, and 685 in the insert within the second FnIII domain are also involved in aea disulfide bridges. There is a single disulfide bridge between a and b subunits between Cys 647 in the second FnIII domain and Cys 872 (nomenclature of the B isoform). The alternatively spliced exon 11 is highlighted. aCT: the C-terminal peptide (16 aa) of the a-subunit, in the insert domain, is a critical tandem binding element of binding site 1 (see text for explanations). Adapted from De Meyts, P., Whittaker, J., 2002. Structural biology of insulin and IGF1 receptors: implications for drug design. Nat. Rev. Drug Disc. 1, 769e783.

insulin and IGF-I receptors (which have a similar structure), hybrid receptors are formed stochastically, consisting of halves of each receptor (Soos et al., 1993). It took 35 years after the first demonstrations of the existence of an insulin receptor to obtain three-

dimensional structural data for the extracellular domain (including the ligand binding sites) of this complicated multiglycosylated membrane protein. Meanwhile, extensive biochemical studies (reviewed in De Meyts and Whittaker, 2002) had established a model of the modular structure of the receptor (see legend of Fig. 17.6 for a detailed description). The structure of the insulin receptor tyrosine kinase domain was determined in 1994 (Hubbard et al., 1994; Hubbard, 1997); it will be described in the next section on the mechanism of receptor activation. The stepwise progress over the past 12 years in determining the structure of the human insulin receptor ectodomain (Lou et al., 2006; McKern et al., 2006; Smith et al., 2010; Whittaker et al., 2012; Croll et al., 2016) has been recently reviewed (Ward et al., 2013; De Meyts, 2015b, 2016), and I will focus here on the most salient features, ˚ ) strucbased on the most recent higher-resolution (3.3 A ture of the hormone-free ectodomain determined in complex with four monoclonal antibody Fab fragments (Soos et al., 1986) and with its refinement aided by a novel interactive molecular dynamics strategy (Croll et al., 2016, Fig. 17.7). The ectodomain homodimer has a twofold symmetric inverted “V” conformation, with the FnIII-1-2-3 modules protruding in a linear fashion from the cell membrane and the upstream L1-CR-L2 module folding over downwards. L1 and L2 are leucine-rich repeat domains. The L1-CR-L2 module of one receptor monomer packs against the FnIII-1,-2,-3 modules of the alternate receptor monomer. Of utmost importance for insulin binding is a w15-amino-acid helical segment (aCT) lying at the C-terminus of the a chain component of the insert domain of one monomer that packs against the central b-sheet of the L1 domain of the alternate receptor monomer in the hormone-free structure. This tandem L1/aCT element (Smith et al., 2010) forms the major insulin binding site (site 1, see later). See also legend of Fig. 17.7 for further description.

2.6 Mechanism of Insulin Receptor Binding The mechanism and kinetics of insulin binding to its receptor have been extensively studied for over 45 years using radioligands (De Meyts, 2004). Insulin binding is complex and displays negative cooperativity, as shown by curvilinear Scatchard plots and acceleration of the dissociation of a prebound insulin tracer in an “infinite” dilution in the presence of cold insulin (De Meyts et al., 1973). Only one insulin molecule binds to the receptor dimer with high affinity; additional insulin binding is of lower affinity, presumably due to ligand-induced asymmetry. It was proposed in 1994 that high-affinity insulin receptor binding results from insulin having two binding sites 1 and 2 that crosslink two binding sites (1

2. INSULIN

389

FIGURE 17.7 Architectural assembly of the unbound insulin receptor ectodomain (apoIRDb). This figure is based on the latest higher ˚ resolution (Croll et al., 2016). (A) The a subunit. Domains are labeled as in Fig. 17.6. FnIII-2a is the resolution structure of the apo receptor at 3.3-A a subunit component of the FnIII-2 domain. (B) The b subunit. Domains are labeled as in Fig. 17.6. FnIII-2b is the b subunit component of the FnIII2 domain. (C) The ab monomer. The monomer shows an inverted V-shaped structure. The arrow denotes the site of proteolytic cleavage of the proreceptor. This structure is the A isoform of the insulin receptor. Twelve amino acids at the end of the insert domain of the b subunit are missing from the structure. The B isoform would have 12 more amino acids encoded by exon 11 at the end of the aCT domain of the a subunit. (D) The (ab) 2 dimer. The arrow indicates the location of the triplet of disulfide bonds between Cys 682, 683, and 685 of the two a subunits. (E) The tandem insulin binding site 1 made of the aCT helical segment of one a subunit binding in trans to the beta sheet surface of the L1 domain of the second a subunit. The rest of the a subunit insert (ID), which is not part of the binding site, is also shown for orientation. (F) Idem, rotated 90 . Drawn using DSViewerPro from PDB file Model-S1 2, kindly provided by Mike Lawrence, based on PDB file 4ZXB complemented using IMDFF (Croll et al., 2016). From De Meyts, P., 2016. The insulin receptor and its signal transduction network. In: De Groot, L.J., et al. (Eds.), Endotext [Internet]. MDText.com, Inc., South Darthmouth, MA, 2000. © MDText.com, Inc., available under Creative Commons License (CC-BY-NC-ND).

and 20 , or 10 and 2) located on the two separate receptor a subunits (Scha¨ffer, 1994; De Meyts, 1994). To explain the negative cooperativity, De Meyts postulated that the two a subunits of the receptor should have an antiparallel arrangement in order for sites 1 and 20 (or 10 and 2) to be apposed (De Meyts, 1994), so that alternative crosslinking at each subsite pair may occur upon ligand binding, followed by ligand-induced asymmetry. The antiparallel symmetry of the receptor monomers was

subsequently validated in the apo receptor crystal structure (McKern et al., 2006). Site 1 and site 2 on the insulin molecule have been mapped over the last 4 decades by a variety of biochemical approaches, including alanine-scanning mutagenesis (Fig. 17.8, see legend for details). As mentioned earlier, these sites overlap respectively the dimer- and hexamer-forming surfaces of the insulin molecule.

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Putative identification of sites 1 and 2 on the insulin receptor a-subunits has been achieved by a variety of biochemical approaches including chimeric receptors, site-directed mutagenesis, and photoaffinity crosslinking (for review, see De Meyts and Whittaker, 2002; De Meyts, 2004). Residues involved in site 1 binding affinity were mapped by alanine scanning to the L1 domain and to the distal aCT segment. These locations were supported by photoaffinity crosslinking experiments. The most remarkable result of such experiments was the finding that contiguous residues Phe B24 and Phe B25 of insulin crosslinked to very distant epitopes of the receptor a subunit, B24 to the N-terminal L1 domain, and B25 to the C-terminal aCT segment, suggesting that these two domains somehow come close together in the folded receptor structure (Kurose et al., 1994; Xu et al., 2004). Complementation experiments showed that the interaction between L1 and aCT occurs in trans between the two a subunits rather than in cis within the same subunit (Chan et al., 2007), constituting the tandem binding element ultimately resolved by X-ray crystallography (Smith et al., 2010). Finally, the critical insulin A3 residue was shown also to crosslink to aCT (Huang et al., 2004). Receptor site 2 was putatively mapped by alanine scanning mutagenesis to the loop regions near the junction of the FnIII1-FnIII2 regions of the a subunit (Whittaker et al., 2008).

FIGURE 17.8 Receptor binding sites 1 and 2 on the insulin molecule. The residues in site 1 are mapped in yellow, site two in red, backbone in blue. Spheres and tube shown at 0.7 van der Waals radius. Site 1: Gly A1, Ile A2, Val A3, Glu A4, Tyr A19, Asn 21, Gly B8, Ser B9, Leu B11, Val B12, Tyr B16, Phe B24, Phe B25, Tyr B26. Site 2: Thr A8, Ile A10, Ser A12, Leu A13, Glu A17, His B10, Glu B13, Leu B17. PDB file 9INS. Modeled using DSViewerPro from Accelrys. From De Meyts, P., 2016. The insulin receptor and its signal transduction network. In: De Groot, L.J., et al. (Eds.), Endotext [Internet]. MDText.com, Inc., South Darthmouth, MA, 2000. © MDText.com, Inc., available under Creative Commons License (CC-BY-NC-ND).

A quantitative mathematical model of the alternative two-site crosslinking model, based on the concept of the receptor as a harmonic oscillator, was shown to describe accurately all the thermodynamic and kinetic properties of the insulin (and IGF-I) receptor interaction (Kiselyov et al., 2009).

2.7 Structure of the Site 1 Insulin Receptor Complex Four crystal structures of insulin bound to truncated ˚ resolution, in insulin receptor constructs at 3.9e4.4 A the presence of exogenous aCT peptide and monoclonal antibody Fab fragments, were determined by Menting et al., in 2013. The major surprise of the four new structures was that, contrary to predictions, insulin barely interacts with L1. Most of the residues from the L1 b sheet surface that had been mapped by alanine scanning in fact engage aCT, not insulin (Fig. 17.9), with most of the site 1 residues of insulin being in intimate contact with aCT. The exceptions are Val B12 and Tyr B16; these latter contacts are, however, essential for high affinity. See Table 1 in De Meyts (2015b) for a detailed description of insulin residues contacts with the receptor residues, as well as the impact of alanine scanning of those residues on binding affinity. Overwhelming evidence supports a critical role of some of the C-terminal residues (B24eB26) of insulin’s B chain in binding affinity (Weiss and Lawrence, 2018), as well as the occurrence of negative cooperativity (De Meyts et al., 1978). However, the second surprise was that the B22 to B30 segment appeared disordered in the 2013 structures. It was clear, however, that if it kept the same conformation as in native insulin, there would be a steric clash with aCT, supporting the earlier concept that the B chain C-terminal must “detach” to uncover A2 and A3 (Hua et al., 1991; Ludvigsen et al., 1998). The last missing piece of the site 1 complex puzzle fell into place in a more recent publication (Menting et al., 2014, reviewed in De Meyts, 2015b), reporting a refinement in one of the 2013 structures that finally revealed the conformation of insulin B chain’s C-terminal B20eB27 (residues B28eB30 being unimportant for binding). As expected, residues B24eB26 had to rotate away by 50 from the core of the molecule to make room for the aCT segment and inserting between the first strand of the central L1 b sheet and aCT residues 715e718, supporting the detachment model.

2.8 Structure of the Insulin Receptor Kinase and Mechanism of Activation The crystal structure of the insulin receptor tyrosine kinase domain was determined by Stevan Hubbard and colleagues, both in the inactive state (Hubbard

2. INSULIN

FIGURE 17.9

Structure of the site 1 insulin receptor complex. Detailed view of insulin’s site 1 and site 2 residues in the ternary complex between insulin, L1, and aCT that includes insulin’s B chain C-terminus. The figure shows the structural relationship of insulin’s site 1 (in magenta) and site 2 (in red) residues with the receptor’s aCT and L1 surfaces. See Table 1 in De Meyts (2015b) for more detailed description of the contacts. Drawn by Marek Brzozowski using the CCP4MG program. PDB file: 4OGA. Modified from De Meyts P., 2015b. Insulin/receptor binding: the last piece of the puzzle? Bioessays 37, 389e397.

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et al., 1994) and in the activated state (in the presence of a peptide substrate, a stable ATP analog and Mg2þ) (Hubbard, 1997) (Fig. 17.10). Like in all kinases, the architecture features two structurally distinct lobes, the N-terminal lobe and the C-terminal lobe (Fig. 17.10), which form the catalytic site of the kinase where ATP, bivalent cations, and the substrate tyrosine residue get together. The two lobes are linked by a linker region that forms a hinge that enables the relative motion of the two lobes. The structures revealed a novel autoinhibition mechanism (described in Fig. 17.10) whereby an “activation loop” behaves as a pseudosubstrate that blocks the active site in the basal state (closed configuration) and is stabilized in the open position after transphosphorylation of three tyrosines. More recent data (Cabail et al., 2015) have shown that the activated insulin and IGF-I receptor kinases are functional dimers, and that, in addition to the activation loop phosphorylation, an allosteric stabilization takes place involving an exchange of the juxtamembrane regions proximal to the kinase domain. For review of the structural aspects of the insulin receptor and other kinases, see Hubbard and Miller (2007), Hubbard (2013), and Su¨veges and Jura (2015). The mechanism whereby the tyrosine kinase is activated is thus by dimerization followed by transphosphorylation of the activation loop freeing the active site to phosphorylate tyrosine residues on the receptor, creating binding sites for signaling proteins containing SH2 or PTB domains (see next section).

FIGURE 17.10 Structure of the inactive and activated insulin receptor tyrosine kinase (with bound ATP analog AMP-PNP, peptide substrate, and Mg2D). This figure illustrates the autoinhibition mechanism whereby Tyr 1162, one of the three tyrosines that are autophosphorylated in the activation loop (shown in white) in response to insulin (1158, 1162, 1163) is bound in the active site, hydrogen bonded to a conserved Asp 1132 in the catalytic loop (left). Tyr 1162 in effect competes with protein substrates before autophosphorylation. In the activated state (right), the activation loop is tris-phosphorylated and moves out of the active site. Tyr 1163 becomes hydrogen bonded to a conserved Arg 1155 in the beginning of the activation loop, which stabilizes the repositioned loop. Also shown is the peptide substrate with the WMXM motif. Originally published in De Meyts, P., Whittaker, J., 2002. Structural biology of insulin and IGF1 receptors: implications for drug design. Nat. Rev. Drug Disc. 1, 769e783.

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Despite the considerable recent progress in the structures of the extracellular and kinase domains of the insulin and IGF-I receptors, no high-resolution structure of the full-length unliganded and liganded receptors is available, and therefore we lack the details of the precise mechanism whereby extracellular ligand binding results in apposition and activation of the kinase domains. The distance between the membrane insertions of the stems of the ˚ extracellular domain of the insulin apo receptor is 117 A (Croll et al., 2016). This is too far apart for the kinase domains to be apposed if they remain close to the transmembrane segments, if that distance is maintained in the activated receptor. Ward et al. speculated (Ward et al., 2013) that a ligand-induced conformational change causes the descent of the kinase domains (like a yo-yo) from a constrained position where they are partially wrapped up by the juxtamembrane region and lie near the membrane. This release allows the kinases to appose and transphosphorylate. In contrast with this model, Lee et al. (2014) suggested that insulin binding prises apart the transmembrane domains in a receptor where they are held close together in the inactive receptor. This is based on the finding that a 24-residue peptide having the IR transmembrane domain sequence (but not the IGF-IR sequence) activated the IR tyrosine kinase. There were no structural studies to substantiate either of these claims. A mechanism quite different from the aforementioneddbacked up by convincing structural and biochemical datadwas proposed by Kavran et al. (2014). They concluded that the molecular mechanism for IGF-1R activation (and hence likely for IR as well) involves relaxation of the separation of the transmembrane domains (otherwise enforced in the unliganded ectodomain and which maintains the receptors in an inhibited state), i.e., the opposite of the mechanism proposed in Lee et al. (2014). Ligand binding relieves the inhibition by disrupting the L1-FnIII20 -30 interaction that stabilizes the transmembrane domain separation, freeing the transmembrane domains to associate, and allowing autophosphorylation of the kinase domains. In their view, ligand binding does not stimulate the kinase activity of the phosphorylated receptor; rather, the role of the IR/IGF1R extracellular domain is to inhibit activity in the absence of ligand rather than promote activity in the presence of ligand. Maruyama (2015) has proposed a common mechanism for all dimeric transmembrane receptors whereby ligand binding to the extracellular domain of receptor dimers induces a rotation of transmembrane domains, followed by rearrangement and/or activation of intracellular domains (the “rotation model”). Ye et al. (2017) have proposed in an insightful review a “bow and arrow” model to explain the role of aCT in the insulin-induced conformational change. Recently, Scapin et al. (2018) have proposed a model of IR activation based on single-particle cryo-electron microscopy analysis of

the soluble ectodomain that postulates that the aCT segment is located away from L1 in the absence of insulin and recruited to it upon insulin binding, a displacement ˚ . In their structure, the extracellular domain of 55e70 A binds two insulin molecules to the respective site 1 site 2 pairs, in contrast to what has been observed in in vitro binding studies of the soluble ectodomain that suggests two low-affinity site 1-bound insulins, while a single insulin molecule binds to high-affinity constructs (De Meyts and Whittaker, 2002). Also, their mapping of site 2 does not correspond to the mapping by alanine scanning (Whittaker et al., 2008). This model should thus perhaps be taken with a grain of salt. A recent study (Gutmann et al., 2018) of the structure of an insulinbound glycosylated full-length human IR, reconstituted into lipid nanodiscs and obtained by single-particle negative-stain electron microscopy, showed that a single insulin molecule binding to the dimeric receptor converts its ectodomain from an inverted V-shaped conformation to a T-shaped conformation (Fig. 17.11). This structural rearrangement of the ectodomain propagates to the transmembrane domains by apposing them, facilitating transphosphorylation of the cytoplasmic kinase domains. Finally, Weis et al. (2018) recently reported a cryoelectron microscopy structure of a physiologically relevant, high-affinity version of the insulin-bound receptor ectodomain, the latter generated through attachment of a C-terminal leucine zipper (Hoyne et al., 2000) to overcome the conformational flexibility associated with receptor ectodomain truncation. It shows a single insulin molecule bound. This higher resolution structure reveals how the membrane proximal domains of the receptor come together to effect signaling and how insulin’s negative cooperativity of binding arises through a ligand-induced asymmetry between the filled and empty insulin binding sites (Fig. 17.12). Interestingly, a recent crystal structure of the related IGF-I receptor (Xu et al., 2018) both in unliganded and liganded form showed that while the overall structure was very similar to that of the insulin apo receptor ectodomain, the stems of the reverse V-shaped structure ˚ ). Strikingly, were much closer than in the IR (67 vs. 117 A IGF-I was able to make its way inside the closed receptor binding domain by soaking the crystal, suggesting an induced fit component in the ligand binding process. The harmonic oscillator model (Kiselyov et al., 2009) was adapted to account for this new mechanistic insight.

2.9 Insulin Intracellular Signaling Pathways Following the activation of the insulin receptor tyrosine kinase by triphosphorylation of its activation loop, the kinase phosphorylates tyrosine residues outside the kinase domain of the receptor, thus creating binding

2. INSULIN

FIGURE 17.11 Mechanism of insulin receptor activation as suggested by single-particle negative-stain electron microscopy of the insulin holoreceptor embedded in lipid nanodiscs (Gutmann et al., 2018). ECD, extracellular domain; TMD, transmembrane domain; TKD, tyrosine kinase domain. © 2018. Gutmann et al., used with permission. Originally published in J. Cell Biol. http://doi.org/10.1083/jcb.201711047.

sites for signaling protein partners containing SH2 (src homology 2) domains (Pawson, 2004) or PTB (phosphotyrosine-binding) domains. Unlike other RTKs, the insulin (and IGF-I) receptors do not bind signaling proteins directly, but instead bind to the phosphorylated juxtamembrane domain residue Tyr 960 (IRA numbering) a family of large docking proteins called IRS (insulin receptor substrate) 1e6, the first of which was cloned in 1991 (Sun et al., 1991; White, 1998, 2003),

FIGURE 17.12 Mechanism of insulin receptor activation as suggested by cryo-electron microscopy of a high-affinity insulinbound receptor ectodomain zipped by a leucine zipper (IRDb-zipInsFv, on the right) (Weis et al., 2018), compared to the unbound unzipped construct apoIRDb (on the left). The complex with only one insulin bound shows a striking rearrangement with the C-termini of ˚ to being apposed to the a-subunits getting from a distance of 117 A ˚ , which should appose the tyrosine kinase domains for trans15 A phosphorylation. The single bound insulin molecule induces binding sites asymmetry that explains the negative cooperativity in binding. See Weis et al. (2018) for more detailed explanation. Used with permission. Originally published in Nat. Commun. https://doi.org/10.1038/S41467-01806826-6 under Creative Common Attribution 4.0 international license.

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as well as the adapter Shc (Src homology 2 domain containing) (Ravishandran, 2001). These form the nucleus for the assembly of a signal transduction particle that is the starting hub of the various intracellular signaling cascades. A detailed inventory of all the signaling proteins involved in insulin signal transduction pathways (Fig. 17.13) is beyond the scope of this review. For more detailed reviews, see White (2003, 2012), Avruch (1998), Taniguchi et al. (2006), Boucher et al. (2014), White and Copps (2016), Haeusler et al. (2018), and Petersen and Shulman (2018). Most insulin effects appear to be mediated through the interaction of IRS-1 and -2, and Shc with the insulin receptor (White, 1998, 2003; Taniguchi et al., 2006). IRS proteins contain an N-terminal pleckstrin homology (PH) domain (which attaches to the plasma membrane phospholipids) adjacent to a PTB domain that binds to a phosphorylated NPXY (Y 960) motif in the receptor’s juxtamembrane domain. The central and C-terminal parts of the IRS proteins contain up to 20 potential phosphorylation sites that when phosphorylated by the insulin receptor bind to signaling proteins that contain SH2 domains. The two main pathways of insulin signaling emanating from the insulin receptor-IRS complex are the phosphatidylinositol 3-kinase (PI3K, a lipid kinase)/AKT (also known as PKB or protein kinase B) pathway (Sheperd et al., 1998; Cantley, 2002) and the Raf/Ras/MEK/ MAPK (mitogen activated protein kinase, also known as ERK or extracellular signal regulated kinase) pathway (Avruch, 2007). The PI3K pathway is responsible for most metabolic effects of insulin, and it is connected exclusively through IRS, while the MAPK pathway emanates from both IRS and Shc and is involved in the regulation of gene expression and, in cooperation with the PI3K pathway, in the control of cell growth (“mitogenesis”) and differentiation (Taniguchi et al., 2006). Four of the critical downstream substrates of AKT/ PKB are mTOR, mammalian target of rapamycin, involved in the regulation of protein synthesis (Harris and Lawrence, 2003); GSK3 (glycogen synthase kinase 3), involved in the regulation of glycogen synthesis (Cohen, 2001); FoxO (forkhead box-containing protein, O subfamily) transcription factors, especially FoxO1, involved in the regulation of gluconeogenic and adipogenic genes (Accili and Arden, 2004), and AS160 (AKT substrate of 160 kDa), involved in glucose transport through GLUT4 translocation (Sano et al., 2003) (Fig. 17.13). mTOR is a serine/threonine kinase that acts as a nutrient sensor; it is the catalytic subunit of two structurally distinct complexes, mTORC1 and mTORC2 (Harris and Lawrence, 2003). It stimulates protein synthesis by phosphorylating eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and p70 ribosomal protein S6 kinase (p70S6K).

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Insulin

GLUT4 translocation GLUT4

PIP3

IR PIP2 SHC IRS

PDK

PI3K

Shc

Grb2

SOS

RAF PTP 1B

PTEN AKT

RAS

MEK

GSK3

Glycogen synthesis GLUT4

ERK Cell growth and differentiation

FoxO mTOR Metabolism Cell cycle progression FIGURE 17.13

Protein synthesis

p90RSK

Canonical insulin receptor signaling pathways. See text for detailed explanations.

GSK3 is a serine/threonine protein kinase that inhibits glycogen synthase (but is also involved in other cellular processes); it is inhibited when phosphorylated by AKT/PKB (Cohen, 2001). FoxO1 is a transcription factor that translocates to the nucleus in the absence of insulin signal and stimulates the expression of genes such as PEPCK (phosphoenolpyruvate carboxykinase), the key enzyme in gluconeogenesis (Accili and Arden, 2004), as well as cyclin G2, an atypical cyclin that blocks the cell cycle and is inhibited by insulin (Svendsen et al., 2014), and it appears to play a key role in insulin (and IGF-I)-induced mitogenesis. FoxO1 is sequestered in the cytoplasm when phosphorylated by AKT. FoxO1 is highly conserved in evolution and, under the name daf16, plays a major role in metabolism and longevity in C. elegans (Accili and Arden, 2004). The prototypical metabolic effect of insulin is the stimulation of glucose transport in adipose tissue and skeletal and cardiac muscle (White, 2012). Glucose disposal into muscle is the major component of insulin action that prevents postprandial hyperglycemia. This is accomplished through the translocation by exocytosis of the insulin-sensitive glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (Fig. 17.13), by a mechanism that is still far from completely understood (Sano et al., 2003; for review, see Khan and Pessin, 2002; Huang and Czech, 2007). For a detailed review of the insulin signaling pathways in muscle, liver, and fat and their potential role in insulin resistance, see the magistral review of Petersen and Shulman (2018).

A multitude of mechanisms (for review, see De Meyts, 2016) are in place to attenuate or terminate the signal induced by insulin, both at the receptor and postreceptor level (Taniguchi et al., 2006). The insulin receptor and IRS proteins are negatively regulated by ligand-induced downregulation due to clathrindependent endocytosis (Gavin et al., 1974; Carpentier, 1994; Bergeron et al., 2016) by tyrosine protein phosphatases (Enuka et al., 2015) and by serine phosphorylation of IRS proteins (Taniguchi et al., 2006; Zick, 2005). Insulin receptor signaling is also attenuated by several proteins that bind to the insulin receptor: suppressor of cytokine signaling (SOCS) proteins 1, 3, 6, and 7 (Emmanuelli et al., 2000; Howard and Flier, 2006) and the adaptors Grb10 and Grb 14 that bind to the kinase activation loop (Desbuquois et al., 2013). Finally, insulin receptor signaling is inhibited by PC-1, also known as the enzyme ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1), and this was shown to be dependent on its enzymatic activity (Chin et al., 2009).

2.10 Pathophysiologic and Clinical Implications of Insulin Signaling We will not attempt here to provide a general discussion of the complex pathophysiology of the various forms of human diabetes mellitus but will instead focus on the topic of this book, which is the role of hormonal signaling. With the considerable progress in unraveling the insulin receptor signaling network in recent years,

2. INSULIN

numerous studies have addressed the possible dysregulation of this network in causing insulin resistance (see the extensive recent review by Petersen and Shulman, 2018 with close to 1000 references). In particular, gene invalidation and transgenic approaches in mice have been used extensively to explore the role of individual signaling molecules in the maintenance of normal insulin sensitivity, as well as, for some, in normal beta cell function (Lamothe et al., 1998; White and Copps, 2016). The importance of the insulin receptor in metabolic homeostasis was clearly demonstrated by the finding of various compound heterozygous insulin receptor mutations (about 100 so far) in human syndromes associated with extreme insulin resistance, so-called receptoropathies: type A insulin resistance with acanthosis nigricans, leprechaunism (Donohue syndrome), Rabson Mendenhall syndrome (Taylor, 1992; Semple et al., 2016; Brierly et al., 2018), and congenital fibertype disproportion myopathy (Vorwerk et al., 1999). Depending on the location of the mutation in the receptor structure, the functional consequences have been ranked in five categories (Taylor, 1992): (1) impaired synthesis, (2) impaired transport to the plasma membrane, (3) impaired insulin binding, (4) impaired transmembrane signaling, and (5) impaired endocytosis, recycling, and degradation. The complete knockout of the two alleles of the insulin receptor in mice led to major metabolic alterations soon after suckling. They were born with moderate growth impairment (5%). They developed a marked postnatal growth retardation and skeletal muscle hypotrophy and died within a week after birth of acute diabetic ketoacidosis (Accili et al., 1996; Joshi et al., 1996). Reported cases of human patients with homozygous insulin receptor deletion are exceptional, likely due to embryonic lethality. Interestingly though, the four reported human cases (Wertheimer et al., 1993; Krook et al., 1993; Psiachou et al., 1993; Jospe et al., 1996) showed a milder phenotype (leprechaunism) than the mice double knockouts and lived several months to a year after birth like most leprechauns, showing that in humans, life is compatible with lack of insulin receptors. Following the demonstration in cell culture of insulin receptor downregulation in the presence of increased insulin concentrations in the culture medium in vitro in 1974 (Gavin et al., 1974), studies showed that the insulin receptor was also downregulated in vivo in hyperinsulinemic resistant states in animal models (Soll et al., 1975a, 1975b; Friedman et al., 1997) as well as in circulating monocytes (Bar et al., 1976) or adipocytes (Olefsky, 1976) of obese humans, although some human studies dissented (Misbin et al., 1979; Amatruda et al., 1975). This led to the notion that receptor downregulation in vivo was a major determinant of insulin resistance. However, it was later shown that heterozygous null

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mutants (IRþ/) for the insulin receptor that had only half the normal complement of receptors did not present with any major metabolic abnormalities and had normal glucose tolerance following an intraperitoneal glucose tolerance test, and had normal insulin levels (Accili et al., 1996), suggesting that downregulation of the insulin receptor alone is not sufficient to cause insulin resistance, and that postreceptor defects may be required, as had been previously postulated (Kolterman et al., 1981). Obvious candidates for a postreceptor defect in insulin resistance are the IRS proteins, considering the critical role of this node in both insulin and IGF-I signal transduction (Taniguchi et al., 2006). Defects in muscle IRS1 expression and function have been reported in insulin-resistant states such as obesity and type 2 diabetes (reviewed in Sesti et al., 2001). Surprisingly, the homozygous deletion of IRS1 in mice by two different groups (Tamimoto et al., 1994; Araki et al., 1994) induced a rather moderate metabolic phenotype with no type 2 diabetes, normal fasting glycemia, and normal glucose tolerance in one case (Tamimoto et al., 1994) but significant hyperglycemia after intraperitoneal glucose load in the other (Araki et al., 1994). A mild degree of insulin (and IGF-I) resistance was found in both studies. Both showed marked intrauterine and postnatal growth retardation. In contrast, mice with homologous deletion of IRS2 (Withers et al., 1998) were 10% smaller and mildly insulin resistant at birth, but developed a marked glucose intolerance and had full-blown diabetes at 10 weeks with both marked insulin resistance and beta cell deficiency (more than twofold decrease in beta cell mass as early as 4 weeks) with decreased insulin secretion in response to glucose. These and further data demonstrated that IRS2 plays a critical integrative role in pancreatic beta cell plasticity and function (for review, see White, 2012; White and Copps, 2016) and support the concept that the beta cell is not only the source of insulin, but an important target of its actions (Leibiger et al., 2008; Oakie and Wang, 2018). Tissue-specific knockout of the insulin receptor gene in the b cell caused a defect in their early secretory response similar to that in type 2 diabetes (Kulkarni et al., 1999). Among the numerous total or tissue-specific knockouts of insulin pathway components, we will just emphasize a few of the unexpected results that challenged our current wisdom regarding diabetes pathophysiology. The targeted disruption of the p85 regulatory subunit of PI3K paradoxically caused hypoglycemia due to increased sensitivity and increased basal and insulinstimulated glucose disposal in peripheral tissues with a preferential localization of GLUT4 in the plasma membrane (Tarauchi et al., 1998; Mauvais-Jarvis et al., 2002; see Taniguchi et al., 2006 for review).

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Surprisingly, homozygous deletion of GLUT4 did not produce a diabetic phenotype (Katz et al., 1995). Females had normal glucose levels in fasted and fed state, while males had a 34% lower fasting glucose and 20% increase in fed glycemia. Both sexes were insulin resistant and hyperinsulinemic, showed growth retardation, and had disturbed lipid metabolism. They had a shorter life span linked to significant cardiac hypertrophy. Seventy percent of insulin-induced glucose disposal goes to skeletal muscle. Tissue-specific knockout of the insulin receptor in skeletal muscle (MIRKO, Bru¨ning et al., 1998) resulted as expected in a marked reduction of insulin-stimulated glucose transport in skeletal muscle. Surprisingly, the animals showed no hyperglycemia, hyperinsulinemia, or evidence for any impairment of glucose homeostasis. They had disturbed lipid metabolism. Also somewhat counterintuitively, mice with tissuespecific insulin receptor knockout in adipose tissue (FIRKO, Blu¨her et al., 2002) showed a markedly reduced fat mass and whole-body triglyceride stores and were protected from gold thioglucose-induced and agerelated obesity as well as from the obesity-associated glucose intolerance. Finally, the third major target tissue for insulin, the liver, was also targeted by tissue-specific knockout of the insulin receptor (LIRKO, Michael et al., 2000). The liver accounts for 30% of the disposal of an oral glucose load through glucose uptake (insulin independent) and storage into glycogen, and it accounts for the major part of insulin clearance through receptor-mediated endocytosis. The knockout mice showed severe primary insulin resistance and a defect in insulin clearance. Thus, isolated liver insulin resistance is sufficient to cause severe defects in glucose homeostasis. The mice also developed alterations in liver function and cellular morphology. We will not review here the extensive work done in knocking out the insulin receptor in nonclassical target tissues (the beta cell has been discussed previously), except to mention the critical finding based on neuronspecific knockout mice (NIRKO) that the insulin receptor in the brain (the presence of which was reported years earlier (Posner et al., 1974; Havrankova et al., 1978)) plays an important role in the control of body weight and reproduction (Bru¨ning et al., 2000). Thus from all these studies, the hepatocyte and the b cell have emerged as the major insulin-responsive cells where loss of insulin signaling induces the major phenotypic alterations in glucose homeostasis found in type 2 diabetes. Major efforts have been devoted in recent years by large consortia to pick potential susceptibility gene loci in human type 2 diabetes by genome-wide association scans (GWAS). So far, 243 established susceptibility loci have been defined, explaining 18% of T2D risk,

most of which pointing to b cell dysfunction rather than insulin resistance (Billings and Flores, 2010; Scott et al., 2017; Zhao et al., 2017; Mahajan et al., 2018a, 2018b). No smoking gun has been detected in insulin signaling with the exception of Grb14 discussed before as a negative regulator of insulin signaling (Morris et al., 2012).

3. GLUCAGON 3.1 History Discovered in 1923 as a contaminant of early insulin preparations causing transient hyperglycemia (Murlin et al., 1923; Kimball and Murlin, 1923; Sutherland and de Duve, 1948; de Duve and Vuylsteke, 1953), glucagon (whose name is a contraction of “glucose agonist”) has long been a neglected hormone. As previously reviewed (Lefe`bvre, 2011), it was among the very first polypeptide hormones to be isolated, purified, sequenced, and synthesized (de Duve, 1953; Staub et al., 1955; Bromer et al., 1957; Wu¨nsch and Wendlbeger, 1968; Shanghai Protein Synthesis Group, 1975; Merrifield and Mosjov, 1983). Thanks to the pioneering work of Roger Unger, it was the first polypeptide hormone to become measurable by radioimmunoassay, almost 1 year before insulin (Unger et al., 1959). It has served two Nobel Prize winners as a unique tool, which permitted Earl Sutherland and his associates to discover the second messenger cyclic AMP (cAMP) (Robison et al., 1968) (Nobel Prize 1971), and Martin Rodbell, Al Gilman, and their coworkers to discover the role of G proteins in the activation of cell membrane receptors (Rodbell, 1980; Gilman, 1984) (Nobel Prize 1994). Subsequently, the nucleotide sequence of the glucagon gene has been determined (Heinrich et al., 1984; White and Saunders, 1986; for review, see Philippe, 1991), and the structure of the human glucagon precursor (preproglucagon) has been deduced. This discovery has been fundamental for clarifying the relationships of glucagon itself with various other peptides derived from the same common precursor and originating from both the pancreas and the gut. The physiology and pharmacology of glucagon in health and disease has been covered in recent reviews (Sandoval and d’Alessio, 2015; Campbell and Drucker, 2015; Muller et al., 2017). Unger and Orci (2010) suggested that diabetes should be seen as a paracrinopathy of the islets of Langerhans in which intra-islet insulin deficiency would induce an excessive glucagon release from the neighboring a cells, and the resulting hyperglucagonemia would be a critical factor in the pathophysiology of diabetes. This proposal elicited an intense debate that

3. GLUCAGON

concluded that indeed insulin and glucagon should be considered “partners for life” both in physiology and pathophysiology (Holst et al., 2017). Inhibiting glucagon secretion or antagonizing the effects of glucagon are currently seen as innovative approaches in the treatment of diabetes (Lefe`bvre et al., 2015; Scheen et al., 2017).

3.2 Amino Acid Sequence, Evolution, and Structure The amino acid sequence of glucagon (Bromer et al., 1957) isolated from the pancreas of pigs, cattle, and humans is identical (Fig. 17.14). Guinea pig glucagon is different from all other glucagons that have been isolated, while avian glucagons differ from the predominant mammalian glucagon only by a few conservative replacements. Glucagon belongs to the pituitary adenylate cyclase activating polypeptide (PACAP)/glucagon superfamily that includes nine hormones that are related by structure, distribution (especially the brain and the gut), function (often by activation of cAMP), and receptors (a subset of seven transmembrane (7TM) or G proteinecoupled receptors (GPCRs)) (Sherwood et al., 2000). The nine hormones include glucagon, GLP-1 (glucagon-like peptide 1), GLP-2 (glucagon-like peptide 2), GIP, GRF (growth hormoneereleasing factor), PHM (peptide histidine methionine), PACAP, secretin, and VIP (vasoactive intestinal peptide). Secretin was the first factor identified as a hormone (Bayliss and Starling, 1902a). The origin of the ancestral superfamily members is at least as old as the invertebrates. The most ancient and tightly conserved members are PACAP and glucagon. Evidence to date suggests that the superfamily began with a gene or exon duplication and then continued to diverge with some gene duplications in vertebrates (Sherwood et al., 2000). In dilute aqueous solutions, glucagon has little defined secondary structure and certainly exists as a population of conformers in equilibrium. Circular dichroism and fluorescence are consistent with 10%e15% of a helix. Proton NMR studies are consistent with a largely unstructured and flexible chain (for review, see Blundell, 1983). In concentrated solutions, there is an equilibrium between a largely unstructured polypeptide and a partly helical trimer. On increasing concentrations, these trimers become involved in further intermolecular

interactions, leading to an increased a helical content and crystallization (Blundell, 1983). In contrast, on standing in acid solutions, glucagon forms fibrils of b-pleated sheet conformers (Blundell, 1983). The ability of glucagon to form both a helices and b-pleated sheets had been predicted by Chou and Fasman (1975). Glucagon forms rhombic dodecahedral crystals containing 12 molecules packed with cubic symmetry, in which a helical rods are packed as two different forms of trimers (Fig. 17.15), as shown by X-ray crystallog˚ resolution (Sasaki et al., 1975; Blundell, raphy at 3-A 1983). The helical conformers feature two different hydrophobic patches, one N-terminal (Phe 6, Tyr 10, Tyr 13, and Leu 14), and one C-terminal (Ala 19, Phe 22, Val 23, Trp 25, Leu 26, and Met 27), which are responsible for the molecular interactions that stabilize the trimeric crystal form (Blundell, 1983), as well as providing two binding sites for the glucagon receptor where glucagon adopts a similar a helix rod conformation (Zhang et al., 2018) (see later).

3.3 Biosynthesis and Processing The glucagon gene consists of six exons and five introns spanning 10 kb; the human glucagon gene is located on chromosome 2 (Heinrich et al., 1984; White and Saunders, 1986, for review, see Philippe, 1991). It encodes for a preprohormone of 180 amino acids that contains not only glucagon but several other peptides including two glucagon-like peptides (Irwin, 2001) whose amino acid structures are distinct from, but closely resemble, that of glucagon and other members of the glucagon superfamily of peptides. The glucagon gene is expressed in the a cells of the islets of Langerhans, the intestinal L cells, and in some parts of the brain. While the functional relevance of glucagon gene expression in the brain is still poorly understood, a clear image has emerged regarding the processing of proglucagon into several bioactive peptides in the pancreas and in the gut (Fig. 17.16) (Drucker, 2002). In the pancreas, glucagon is the predominant peptide produced, together with glucagon-related polypeptide (GRPP), while the glucagon-like peptides remain in an incompletely processed prohormone fragment (Patzelt and Schiltz, 1984). In the gut, two glucagon-like peptides (GLP-1 and GLP-2) are produced, while glucagon remains, in part, as a prohormone fragment, glicentin.

1 His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-ArgAla-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr 29

FIGURE 17.14 Amino acid sequence of human glucagon.

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However, glicentin can be further processed to oxyntomodulin and GRPP (Mosjov et al., 1986). Oxyntomodulin (Scholdager et al., 1988) and glicentin (Kirkegaard et al., 1982) are implicated in the physiologic negative control of gastric acid secretion. GLP-1 is a so-called incretin. The incretin effect refers to the fact that oral glucose administration promotes a much greater degree of insulin secretion compared to a parenteral isoglycemic glucose infusion, postulating the existence of gut-derived factors that enhance glucose-stimulated insulin secretion from the islet b-cell. This was first proposed in the early 1900s (Bayliss and Starling, 1902b; Moore et al., 1906), and the term incretin was coined by the Belgian pharmacologist Jean La Barre (1932). The first identified incretin hormone was GIP (gastric inhibitory polypeptide, now called glucose-dependent insulinotropic peptide) (Dupre et al., 1973). For the history of further developments, see the website maintained by Daniel J. Drucker, www.glucagon.com. For a review of the therapeutic potential of gut hormones, see Tan and Bloom (2013). Glucagon-like peptide GLP-1-(7e37), a peptide of 31 amino acids, or the equally potent iso-peptide GLP-1(7e36), an amide of 30 amino acids, has a major insulinotropic action on pancreatic b cells (Drucker, 2002; Phillips and Prins, 2011). This peptide binds to specific receptors on islet b cells, stimulating cAMP formation, insulin release, proinsulin gene transcription, and proinsulin biosynthesis, all in a glucose-dependent manner. GLP-1 is also a strong inhibitor of glucagon secretion. GLP-1 also exerts numerous extrapancreatic actions, including inhibition of food intake, promotion of satiety, cardioprotection, vasodilation, and possibly beneficial effects on endothelial function and inflammation (Herzlinger and Horton, 2013; Drucker et al., 2017). GLP-1 is rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-

4) (Deacon et al., 1995). GLP-1 analogs, such as exenatide and liraglutide, and DPP-4 inhibitors, such as vildagliptin, sitagliptin, saxagliptin, linagliptin, and alogliptin are now largely used in the treatment of type 2 diabetes (Drucker et al., 2017; Scheen, 2018). GLP-2 is recognized as a major regulator of intestinal growth and function. Its secretion from the intestinal L cells is essentially stimulated by nutrient intake. It has specific trophic effects on the gut that appear to be mediated by stimulation of mucosal cell proliferation and inhibition of apoptosis and proteolysis (Drucker, 2001). Additional effects of GLP-2 on the digestive tract include stimulation of enterocyte glucose transport and GLUT-2 expression, increased nutrient absorption, reduction of intestinal permeability, stimulation of intestinal blood flow, relaxation of intestinal smooth muscle, and inhibition of gastric emptying and gastric acid secretion (Dude and Brubaker, 2007; Rowland and Brubaker, 2011). Further effects of GLP-2 include stimulation of glucagon release from the islet a cells and modulation of islet adaptation to metabolic stress. GLP-2 has been reported to reduce bone resorption and to significantly increase bone mineral density in postmenopausal women. Numerous studies indicate a great therapeutic potential of GLP-2 in total parenteral nutrition, short bowel syndrome following major intestinal resection, nonsteroidal drugeinduced enteritis, inflammatory bowel disease, and ischemic bowel. Teduglutide, a degradationresistant GLP-2 analog, has been approved for the treatment of short bowel syndrome (Jeppesen, 2012). Both glucagon and oxyntomodulin are further processed into N-terminal and C-terminal fragments by cleavage at a dibasic site (Arg17-Arg18) (Bataille et al., 1988; Blache et al., 1990). The C-terminal fragments are of particular interest: glucagon-(19e29) modulates the plasma membrane calcium flow, whereas

FIGURE 17.15 Crystal structure of human glucagon. The structure shows an a helical monomer engaged in two kinds of trimers. From Sasaki, K., Dockerill, S., Adamiak, D.A., Tickle, I.J., Blundell, T.L., 1975. X-ray analysis of glucagon and its relationship to receptor binding. Nature 257, 751e757 and Blundell, T.L., 1983. The conformation of glucagon. In: Glucagon, I., Lefe`bvre, P.J., (Eds.), Handbook of Experimental Pharmacology, 66/1. SpringerVerlag, Berlin Heidelberg; New York; Tokyo, pp. 37e56, used with permission from Springer Nature.

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3. GLUCAGON

Glucagon

GRPP 1

30 33

Major ProglucagonFragment

IP1

61 64 69 72

Pancreas α cells 158

PC2 processing Proglucagon 31 32

1

62 63 70 71 77

109 110

159 160

124 125

PC1/3 processing Glicentin 69

1

GRPP 1

IP2

GLP-1 78

111 107

123 126

Gut L cells

GLP-2 158

Oxyntomodulin 30 33

FIGURE 17.16

69

Preproglucagon processing. See text for detailed explanations.

oxyntomodulin-(19e37) inhibits gastric acid secretion, as does oxyntomodulin itself. Glucagon processing to glucagon-(19e29) (mini-glucagon) is probably essential for the positive inotropic effect of glucagon on heart contraction. Thus, the concept has emerged that glucagon and oxyntomodulin are first released into the blood and then processed at the level of their respective targets into the corresponding biologically active C-terminal fragments. Interestingly, mini-glucagon inhibits insulin secretion at picomolar concentrations. It has been proposed that mini-glucagon acts as a local inhibitory regulator of insulin release by turning off the main external calcium source for islet b cells via a specific receptor linked to ion channels that control cell polarity (Dalle et al., 1999, 2002). As illustrated by Fig. 17.16, the tissue-specific liberation of proglucagon is controlled by cell-specific expression of enzymes known as prohormone convertases (PC). A major defect in the processing of proglucagon to mature pancreatic glucagon is seen in the PC-2 knockout mouse. These animals exhibit mild hypoglycemia and hyperplasia of the a cells in the islets of Langerhans (Furuta et al., 1997, 2001). These abnormalities are corrected by glucagon replacement via a micro-osmotic pump. The transcription of proglucagon, and therefore the maintenance of a-cell function, is regulated by several factors, including forkhead box A1 (Foxa1), also known as hepatocyte nuclear factor a (HNF-3a), paired box 6 (Pax6), brain 4 (Brn4), and islet-1 (isl-1) (reviewed in Gosmain et al., 2011).

3.4 The Glucagon Receptor The glucagon receptor is expressed in the liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart, pancreatic b cells, and placenta (Charron and Vuguin, 2015). There have been major recent advances in the structural biology of the glucagon receptor as well as receptors for other secretin peptide hormones (Hollenstein et al., 2014; Parthier et al., 2009; Mann et al., 2008; Liang et al., 2017; Zhang et al., 2017b; Jazayeri et al., 2017; Zhang et al., 2017, 2018; Yang et al., 2015; Siu et al., 2013). The glucagon receptor belongs to the class B G proteinecoupled receptors (GPCRs), also called seven transmembrane (7TM) receptors. The class B is also referred to as secretin class of receptors. Besides glucagon, this class comprises among other ligands (15 in all) GLP-1, GIP, secretin, calcitonin, parathyroid hormone, CRH, growth hormonee releasing hormone (GHRH), VIP, and PACAP. These receptors feature an extracellular domain (ECD) and a transmembrane domain of seven helical structures (TMD). The ligands adopt an N- to C-terminal a-helical rod structure when bound to the receptor (Parthier et al., 2009), similar to that shown for glucagon in the crystal structure (Sasaki et al., 1975; Blundell, 1983). Structural work started a decade ago (Mann et al., 2008; Parthier et al., 2009; Hollenstein et al., 2014) had suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal

region of the peptide binds to the TMD binding pocket. Further work at rather low resolution supported this concept (Liang et al., 2017; Zhang et al., 2017a; Jazayeri et al., 2017). Two recent crystal structures of the full-length ˚ resolution, one in an inacglucagon receptor at 3.0-A tive state in complex with a negative allosteric modulator (Zhang et al., 2017a) and one with a glucagon low-potency partial agonist analog (Zhang et al., 2018) (Fig. 17.17), have unequivocally defined the mechanism of ligand binding and receptor activation. The glucagon a-helical rod takes place on the receptor like a sausage in a hot dog. This causes a major structural change compared to the inactive receptor conformation (Zhang et al., 2018): notably the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with two sites on the peptide. The glucagon receptor as well as the GLP-1 receptor exhibit negative cooperativity in ligand binding, as shown by an acceleration in the dissociation rate of a bound tracer of radiolabeled ligand in an “infinite dilution” in the presence of unlabeled ligand (Roed et al., 2012), as first demonstrated for the insulin receptor, a receptor tyrosine kinase (see the insulin section) (De Meyts et al., 1973). This property was subsequently shown to be shared by many GPCRs (Limbird et al., 1975; De Meyts, 1976; Gao et al., 2009; Urizar et al., 2005; Svendsen, 2008a, 2008b; Springael et al., 2006; for review, see Roed et al., 2012). This property appears to correlate with the ability of GPCRs including the glucagon receptor to homo- and heterodimerize or oligomerize (Milligan, 2001) as shown among others by bioluminescence resonance energy transfer; for review, see Roed et al. (2012).

3.5 Glucagon Receptor Inactivation and Mutations Elimination of signaling through the glucagon receptor has been investigated in depth in mice (Parker et al., 2002; Charron and Vuguin, 2015). Glucagon receptor knockout (Gcgr/) mice are viable but exhibit a number of striking phenotypes including an expected mild hyperglycemia and improved glucose tolerance. Other consequences include increase in total pancreatic weight, marked a-cell hyperplasia, and extreme elevations of circulating plasma levels of glucagon and GLP1. These mice also exhibit multiple defects in islet cell phenotypes, implying a complex, and still poorly understood, role of glucagon in islet development. Another striking observation is an increased susceptibility of

Extracellular domain

17. PANCREATIC HORMONES

Extracellular loop 1

Stalk

Glucagon analogue

Transmembrane domain

400

Coupling to G protein Helix VIII

β subunit

FIGURE 17.17 Crystal structure of the glucagon receptor in complex with a glucagon analog and low-potency partial agonist, NNC1702. The extracellular domain (residues Q27eD124), stalk (residues G125eK136), transmembrane domain (residues M137eY202 and V221eE246) and extracellular loop 1 (residues S203eA220) of the receptor, and the peptide ligand NNC1702, are colored orange, green, blue, magenta, and red, respectively. Glycan modifications in the extracellular domain and disulfide bonds are displayed as orange and yellow sticks, respectively. Adapted from Zhang, H., Qiao, A., Yang, L., et al., 2018. Structure of the glucagon receptor in complex with a glucagon analogue. Nature 553, 106e110.

Gcgr/ hepatocytes to apoptotic injury, suggesting that glucagon signaling is essential for cell survival. Furthermore, Gcgr/ hepatocytes exhibit profound defects in lipid oxidation and accumulate excessive lipid during fasting. Last, Gcgr/ mice exhibit reduced fetal weight, increased fetal demise at the end of gestation, and excessive abnormalities in the placenta, suggesting that glucagon signaling is essential in fetal and placental development. The most striking effect of elimination of glucagon signaling is the lack of diabetes after alloxan or streptozotocin destruction of islet b cells in Gcgr/ mice (Lee et al., 2011), as will be discussed subsequently. Rare cases of mutation of the human glucagon receptor have been reported (Zhou et al., 2009; Yu et al., 2012; Ro et al., 2013; Larger et al., 2016; Li et al., 2018). This syndrome is sometimes referred to as Mahvash disease (Yu, 2018). Among other abnormalities, these patients exhibit marked a-cell hyperplasia, extreme hyperglucagonemia, and significant hyperaminoacidemia, but no disruption of glucose homeostasis. Interestingly, due to

401

3. GLUCAGON

(Rodbell, 1980) made of three subunits, a, b, and g (Clapham and Neer, 1997; Hurowitz et al., 2000; Neves et al., 2002; Svoboda´ et al., 2004). The G protein also changes conformation, resulting in the replacement of GDP bound to the Ga subunit by GTP, the receptor thus playing the role of a guanine nucleotide exchange factor. The GTP-bound Ga subunit then dissociates from the bg pair and the receptor and goes on to activate the enzyme adenylyl cyclase (also known as adenylate cyclase and adenyl cyclase), an integral membrane enzyme with 12 transmembrane domains (Hanoune and Defer, 2001). The Ga subunit, which is a GTPase, then inactivates itself by hydrolyzing GTP into GDP and rebinds to a Gbg dimer, restarting the cycle (Svoboda´ et al., 2004). This last process is facilitated by RGS (regulators of G protein signaling) proteins (Neitzel and Hepler, 2006). Adenylyl cyclase cyclizes ATP into the “second messenger” 30 ,50 cyclic AMP (cAMP) with release of pyrophosphate. cAMP activates protein kinase A (PKA, cAMP-dependent protein kinase). PKA in turn activates phosphorylase kinase, which then phosphorylates glycogen phosphorylase b and converts it into the active phosphorylase a, the enzyme that releases glucose-1phosphate from glycogen polymers (Stalmans, 1983). PKA also phosphorylates the bifunctional enzyme fructose 2,6-bisphosphatase/phosphofructokinase-2, activating the former and inhibiting the latter, resulting

inactive receptors, the marked hyperglucagonemia does not cause symptoms associated with glucagonomas (necrolytic migratory erythema, diarrhea, stomatitis, usually mild diabetes mellitus, anemia, weight loss, venous thrombosis, and frequently, neuropsychiatric symptoms, reviewed in Wermers et al., 1996). Pancreatic neuroendocrine tumors (Ro et al., 2013) are usually but not always present. The data suggest that amino acids may provide the feedback link between the liver and the pancreatic a cells (Larger et al., 2016). These observations cast some doubt about the feasibility of using glucagon receptor blockers in the treatment of diabetes (see later).

3.6 Glucagon Receptor Signaling Pathways and Endpoint Metabolic Effects The signal transduction pathways of glucagon signaling (Fig. 17.18) are probably among the best established signaling networks for any hormone. The hepatocyte is a major target cell of glucagon. For reviews on glucagon actions on hepatic metabolism, see Stalmans (1983), Habegger et al. (2010), Ramnanan et al. (2011), and Miller and Birnbaum (2016). Upon glucagon binding, the receptor undergoes a conformational change that allows it to couple to a heterotrimeric G protein (guanine nucleotide binding protein)

Glucose

Glucagon

Adenylyl cyclase 2

GCGR

α

β

GTP

Catalytic domains

γ

GDP cAMP

ATP

PPK

PPK ATP ADP

PYG b

Pi

Glucokinase

β

γ Gs

GTP exchange cAMP-independent actions

P

Glycogen P GYS a

P PYG a

GYS b

ATP ADP UDP-glucose

Uridyltransferase Glucose-1-phosphate Glucose-6-Pase Phosphoglucomutase

ATP ADP

FIGURE 17.18

GDP

PKA

PKA

Glucose

PLC PDEs Ion channels

α

Glucose-6-phosphate

Glucagon signaling pathways to glycogenolysis in the liver. GCGR, glucagon receptor; GYS, glycogen synthase; PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; PLC, phospholipase C; PPK, phosphorylase kinase; PYG, glycogen phosphorylase. See text for details.

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in slowing down the formation of fructose-2,6bisphosphate (a potent activator of glycolysis, Hers, 1984; Hue and Rider, 1987) and inhibition of glycolysis, with resulting predominance of gluconeogenesis. Glucagon stimulation of PKA also inactivates the glycolytic enzyme pyruvate kinase in hepatocytes (Feliu et al., 1976). The main effect of glucagon on the liver is to increase glucose output, due to inhibition of glycogen synthesis and stimulation of liver glycogenolysis and gluconeogenesis. Cherrington and his colleagues in Nashville (Hendrick et al., 1992; Ramnanan et al., 2011) have established the importance of basal glucagon in maintaining hepatic glucose production during a prolonged fast and have shown that gluconeogenesis and glycogenolysis are equally sensitive to stimulation by glucagon in vivo. However, Chibber et al. (2000) using mass isotopomers suggested that low doses of glucagon stimulate only nongluconeogenic glucose release, while higher doses stimulate both the gluconeogenic and nongluconeogenic pathways. Interestingly, hyperglucagonemia stimulated endogenous glucose production during fructose infusion, but this effect was not secondary to stimulation of gluconeogenesis but rather to channeling of glucose-6-phosphate toward systemic release (Paquot et al., 1996). Most of these effects are probably mediated by cAMP, but part of the glycogenolytic effect of glucagon may occur by a cAMP-independent mechanism. In vitro studies have shown that pulsatile delivery of glucagon is more efficient than continuous exposure to stimulate hepatic glucose production. Similarly, pulsatile delivery of glucagon in humans has greater effects in stimulating endogenous glucose production than continuous infusion (Paolisso et al., 1989, 1990). Furthermore, when both insulin and glucagon are delivered intermittently and out of phase, the effect of glucagon in stimulating glucose production prevails over the effect of insulin in inhibiting this parameter (Paolisso et al., 1989). Another major effect of glucagon on the liver is to stimulate ketogenesis. McGarry and Foster (1983) have convincingly shown that liver ketogenesis depends upon both the flux of free fatty acids (FFA) into the liver and the pathway status of this organ, which is influenced in a crucial manner by the glucagon/insulin ratio in the blood perfusing the liver. These authors have shown that a high glucagon/insulin ratio increases the intracellular level of cAMP, reduces glycolysis and acetyl-CoA carboxylase activity, and reduces the intracellular concentration of malonyl-CoA. This fall in malonyl-CoA brings fatty acid synthesis to a halt and causes derepression of the enzyme carnitine acyltransferase, so incoming fatty acids (made abundant through stimulation of lipolysis) are efficiently converted into the ketone bodies acetoacetate and 3-hydroxybutyrate.

Surprisingly, recent evidence suggests that glucagon inhibits hepatic production through effects on the brain (mediobasal hypothalamus) in rodents, thereby limiting the direct stimulatory effect of this hormone on the liver (Mighiu et al., 2013, commented by Edgerton and Cherrington, 2013). The effects of glucagon on the adipocyte markedly depend upon the species considered (for review, see Lefe`bvre, 1983 and references therein). Although glucagon is a potent lipolytic hormone in birds and in rodents, its effects on the human adipose cell have long been disputed. Glucagon has recently been shown to be strongly lipolytic in the human adipocyte in vitro, but this effect is difficult to demonstrate using incubation of adipocytes or adipose tissue pieces because glucagon is rapidly destroyed by a proteolytic activity associated with those cells. When perfusion techniques are used, the lipolytic effect of glucagon on human adipocytes can easily be demonstrated. Paolisso et al. (1990) have shown in humans that in the presence of somatostatin-induced insulin deficiency, pulsatile glucagon exerts greater effects than its continuous delivery not only on blood glucose (see earlier) but also on plasma FFA, glycerol, and 3-hydroxybutyrate levels. Interestingly, in the older population the lipolytic and ketogenic, but not the hyperglycemic, responses to pulsatile glucagon are significantly reduced. Observations of Carlson et al. (1993) in healthy volunteers have shown that moderate hyperglucagonemia undoubtedly stimulates the rate of appearance in the plasma of both glycerol and FFA, while microdialysis studies performed by others failed to demonstrate a lipolytic effect of glucagon (Gravholt et al., 2001; Bertin et al., 2001). Glucagon regulates amino acid metabolism by increasing ureagenesis. Interestingly, it has been recently shown that, in turn, the plasma level of amino acids regulates the secretion of glucagon (a liver-a-cell axis). In case of hepatic insulin resistance, reflecting hepatic steatosis, the feedback cycle is disrupted, leading to hyperaminoacidemia and hyperglucagonemia (Wewer Albrechtsen et al., 2018; Knop, 2018). The glucagonalanine index has thus been suggested as a marker for hepatic glucagon signaling. The glucagon receptor superfamily peptides have also actions on blood vessels, including control of blood flow, blood pressure, angiogenesis, atherosclerosis, and vascular inflammation (for review, see Pujadas and Drucker, 2016).

3.7 Control of Secretion The control of glucagon secretion is multifactorial and involves direct effects of nutrients on the a-cell stimulussecretion coupling as well as paracrine regulation by

3. GLUCAGON

insulin, somatostatin, and possibly, other mediators such as zinc, g-amino-butyric acid (GABA), or glutamate (Gromada et al., 2007, 2018; Walker et al., 2011). Glucagon secretion is also regulated by circulating hormones and the autonomic nervous system (reviews in Thorens, 2011; Holst et al., 2011). The main physiologic or pathophysiologic stimulators of glucagon release are hypoglycemia (insulininduced, associated with starvation or intense muscular exercise), hyperaminoacidemia (the rise in plasma glucagon levels after a balanced meal is probably due mainly to amino acideinduced glucagon release), stimulation of the adrenergic system (stress, exercise, and possibly hypoglycemia), and stimulation of the vagal system (which together with hormones like GIP and CCK-PZ probably participate in the mixed mealinduced glucagon rise). The main physiologic inhibitors of glucagon release are probably hyperglycemia and hyperinsulinemia (in a glucose-rich or carbohydrate-rich meal) and high circulating levels of FFA. It has been suggested that glucokinase may serve as a metabolic glucose sensor in pancreatic a cells, and hence constitute a mechanism for direct regulation of glucagon release by extracellular glucose. In their pioneer work, Samols et al. (1983) have emphasized the delicate mechanisms by which intraislet insulin, glucagon, and somatostatin release may be interrelated. In such paracrine mechanisms, further data have suggested that the oscillatory pattern of islet hormone release may be particularly important (Lefe`bvre et al., 1996; Tian et al., 2011). Using isolated perifused human islets, Hellman et al. (2009) have shown that glucose generates coincident insulin and somatostatin pulses and clear antisynchronous glucagon pulses. The periodicity of these pulses is 7 to 8 min. The fact that these pulses occur in isolated islets demonstrates that their origin is the islets themselves and independent of external metabolic, hormonal, or neuronal signals. The nature of the intra-islet signal(s) coordinating the secretion of the various endocrine cells of the islets of Langerhans is still the subject of intense investigation (Brereton et al., 2015; Hutchens and Piston, 2015).

3.8 Physiologic Functions The liver is the main site at which moment-to-moment control of glucose homeostasis takes place, and in normal humans, glucagon is the major glucose counterregulatory hormone. By antagonizing the suppressive effects of insulin on glucose production and by stimulating glucose production when appropriate, glucagon not only defends the organism against hypoglycemia, but it also restores normoglycemia if hypoglycemia occurs. Perturbation of

403

the mechanisms controlling hypoglycemia-induced glucagon release in some diabetic patients markedly increases the risk of severe hypoglycemia in these subjects. Other hormones, such as epinephrine (acutely) and growth hormone and cortisol (more slowly), also participate in the counterregulation of the effects of insulin, but glucagon constitutes the first line of defense against hypoglycemia (Gerich, 1979). Glucagon levels increase progressively during prolonged exercise (Luyckx et al., 1978), during which blood glucose remains relatively constant thanks to a fine balance between muscle glucose uptake and liver glucose production. Hyperglucagonemia and hyperglycemia are classic features of stress. They occur mainly as a result of the b-adrenergic stimulation associated with stress. Glucagon has a crucial role in neonatal glucose homeostasis (Hume et al., 2005). Furthermore, glucagon has an important role in thermogenic regulation (Kinoshita et al., 2014). Stimulation of the glucagon receptor in the hypothalamus may suppress appetite, while peripheral administration of glucagon and GLP-1 increases energy expenditure in humans (Tan et al., 2013). These observations suggest that signaling through the glucagon receptor might be useful for controlling body weight (Campbell and Drucker, 2015).

3.9 Pathophysiologic and Therapeutic Implications for Diabetes Mellitus and Obesity Glucagon is an effective means of correcting insulininduced hypoglycemia in diabetic patients. Plasma levels of glucagon are increased in all experimental and clinical forms of diabetes mellitus. This disturbance undoubtedly contributes to the hyperglycemia of type 1 and type 2 diabetes and to the excessive ketogenesis of diabetic coma (reviewed by Lefe`bvre et al., 2015). The main abnormality in the islet cell population of diabetics is a decrease in the b cells with an expansion of the a-cell mass (Henquin and Rahier, 2011). Unger and Orci (2010) proposed that diabetes is a “paracrinopathy” of the islets of Langerhans, based on the concept that the very high concentrations of insulin inside the stimulated islets exert, directly or by proxy through somatostatin-producing cells, a major inhibitory effect on glucagon secretion from the neighboring a cells. Conversely, a reduction in intra-islet insulin concentrations permits glucagon release from the a cells. Disruption of this mechanism was proposed as a key factor in the pathophysiology of diabetes (Unger and Cherrington, 2012; Holst et al., 2017). In type 1 diabetes, a cells lack the constant action of high insulin levels from juxtaposed b cells. Replacement with exogenous insulin injected subcutaneously does

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not approach the paracrine levels of insulin, except with high doses that “overinsulinize” the peripheral insulin targets, thereby promoting glycemic volatility (Unger and Orci, 2010). In type 2 diabetes, the a-cell dysfunction may result from the failure of the juxtaposed b cells to secrete the first phase of insulin or from the loss of the intra-islet pulsatile secretion of insulin. Observations made in experimental diabetes in minipigs (Meier et al., 2006) and confirmed in human type 2 diabetes (Menge et al., 2011) are in support of the second mechanism. To determine unambiguously if suppression of glucagon action eliminates manifestations of diabetes, Lee et al. (2012) expressed glucagon receptors in livers of glucagon receptor-null (Gcgr/) mice before and after b-cell destruction by high doses of streptozotocin. Wildtype mice developed fatal diabetic ketoacidosis after streptozotocin, whereas Gcgr/ mice remained clinically normal without hyperglycemia, impaired glucose tolerance, or hepatic glycogen depletion. Restoration of receptor expression using adenovirus containing the Gcgr cDNA restored hepatic Gcgr. Phospho-cAMP response element binding protein (P-CREB) and phosphoenolpyruvate carboxykinase, markers of glucagon action, rose dramatically and severe hyperglycemia appeared. When Gcgr mRNA spontaneously disappeared 7 days later, P-CREB declined and hyperglycemia disappeared. In this experimental setting, the metabolic manifestations of diabetes cannot occur without glucagon action and, once present, disappear promptly when glucagon action is abolished. However, Steenberg et al. (2016) reached opposite conclusions when disrupting glucagon secretion or action in a mouse model of severe diabetes by (1) a-cell elimination, (2) glucagon immunoneutralization, and (3) glucagon receptor antagonism. They concluded that insulin lack is the major factor underlying hyperglycemia in b-cell-deficient diabetes. In view of such conflicting results, the European Association for the Study of Diabetes convened in 2016 a consensus panel of leaders in glucagon biology to evaluate the potential therapeutic benefit of glucagon blockade in diabetes. The consensus was that antagonizing glucagon may be of great benefit for the treatment of diabetes; however, sufficient levels of basal insulin are required for their therapeutic efficacy (Holst et al., 2017). Lefe`bvre and Luyckx, in a 1979 review reappraising the then controversial role of glucagon in diabetes, had already concluded that a search for selective glucagon inhibitors represents an attractive new way in diabetes management, with the caveat that the role of glucagon in countering insulin-induced hypoglycemia must be preserved. At the time of writing this chapter, there are still no selective glucagon inhibitors used in the treatment of

diabetes. Numerous peptide and nonpeptide glucagon receptor antagonists have been developed and have been shown to reduce blood glucose in animal models. The benefits and limitations of reducing glucagon action to treat type 2 diabetes have been reviewed by Ali and Drucker (2009). Nine different glucagon receptor antagonists (peptides, small molecules, GCGR antisense, and a monoclonal IgG4 antibody) have been in human phase I and phase II clinical trials (for reviews, see Scheen et al., 2017; Nunez and D’Alessio, 2017); five have been discontinued due to adverse effects (disturbed lipid profile, ALT/AST elevation, increased blood pressure, increased body weight, hepatic steatosis, increased circulating gluconeogenic amino acids). It is known that glucagon induces hypolipidemic effects in multiple species and that Gcgr/ mice exhibit significant defects in lipid synthesis, secretion, and oxidation (Longuet et al., 2008). Consequently, marked attenuation of glucagon signaling potentially increases the risk of hepatosteatosis and hepatocellular injury. Furthermore, glucagon receptor signaling is an important regulator of hepatocyte survival (Sinclair et al., 2008). Last, marked inhibition of glucagon signaling in rodents results in islet hyperplasia, increased endocrine cell proliferation, and significant increases in pancreatic weight (Drucker, 2013). As mentioned before, similar findings have been reported in humans with mutations of the glucagon receptor (Zhou et al., 2009; Yu et al., 2012; Larger et al., 2016). Rather than blocking glucagon action, reducing excessive glucagon secretion or glucagon production may be a more realistic approach in the treatment of diabetes (Young, 2005; Lefe`bvre et al., 2015; Evans et al., 2015). Interestingly, besides insulin, several drugs already used today in the management of diabetes appear to exert their effect in part by inhibiting glucagon secretion (GLP1 receptor agonists, dipeptidyl peptidase-4 inhibitors, a-glucosidase inhibitors, and possibly, sulfonylureas) (Lefe`bvre et al., 2015). As discussed by Campbell and Drucker (2015), by increasing energy expenditure and reducing food intake, glucagon has some potential in the management of obesity. In fact, a new paradigm arose with the development of a GLP-1/glucagon receptor coagonist (Day et al., 2009) and even a GLP-1/GIP (glucose-dependent insulinotropic polypeptide)/glucagon receptor tri-agonist (Finan et al., 2015); see Muller et al. (2017) for review. These newly designed peptides showed remarkable effects in rodent models of diabetes and obesity by improving glucose metabolism and inducing weight loss. In this approach, activation of the glucagon receptor drastically contrasts with previous attempts to inhibit glucagon secretion or action, as described here. The theory is that glucagon could have some beneficial

4. OTHER PANCREATIC HORMONES

activities by increasing energy expenditure and promoting weight loss, whereas its diabetogenic effects might be counteracted by coactivation of the GLP-1 and GIP receptors (Scheen and Paquot, 2015). These promising results in rodents have been partly confirmed in humans. Indeed, Tan et al. (2013) reported that coadministration of GLP-1 during glucagon infusion results in increased energy expenditure and amelioration of hyperglycemia, while Cegla et al. (2014) reported that coadministration of glucagon and GLP-1, at doses that are individually subanorectic, significantly reduces food intake.

4. OTHER PANCREATIC HORMONES 4.1 Somatostatin 4.1.1 History Somatostatin is a cyclic tetradecapeptide produced in the hypothalamus and the d cells of the pancreas and gastrointestinal tract that inhibits the secretion of a multitude of hormones. The d cells were discovered in 1931 (Bloom, 1931). The existence of a growth hormone release inhibitor from the hypothalamus was first postulated in 1968 by Krulich and colleagues from Southwestern Medical School in Dallas (Krulich et al., 1968). The actual discovery of the peptide was achieved in 1972 by Roger Guillemin and colleagues at the Salk Institute, in the process of characterizing pituitary hormoneereleasing hormones from sheep hypothalamus (reviewed in Guillemin, 2008). They found that minute doses of hypothalamic extracts inhibited the secretion of immunoreactive growth hormone by dispersed rat pituitary cells in monolayer cultures. The compound, initially called SRIF (somatotropin release inhibitory factor), was purified, sequenced, and synthesized and renamed somatostatin. The synthetic peptide was shown to also inhibit growth hormone secretion by dispersed cells from the pituitary gland of an acromegalic patient (Brazeau et al., 1973). Roger Guillemin shared the 1977 Nobel Prize in Medicine with Andrew Schally, for their work on the peptide hormone production of the brain, and Rosalyn Yalow (for insulin radioimmunoassay). The 1973 Science paper reporting the discovery (Brazeau et al., 1973) concluded: “Should SRIF be active in humans, its possible clinical significance, particularly in the treatment of acromegaly and in the management of juvenile diabetes, has not escaped our attention.” That same year, George Alberti, Hans Ørskov, and colleagues in Aarhus showed that somatostatin inhibited insulin secretion in normal humans as well as perfused canine pancreas, suggesting a direct effect on the beta cell (Alberti et al., 1973). Subsequently, it was found that Guillemin’s peptide also decreased insulin and glucagon secretion in baboons, and independently Lelio Orci at

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University of Geneva with Maurice Dubois and colleagues at INRA in France (Orci et al., 1975) and Rolf Luft and Thomas Ho¨kfelt and colleagues at Karolinska in Stockholm (Luft et al., 1974; Elde et al., 1978) showed the presence of immunoreactive somatostatin in the d cells (then of unknown function) of the endocrine pancreas (reviewed in Guillemin, 2008). Today a number of stable analogs like octreotide, lanreotide, and pasireotide have clinical uses in treatment of acromegaly, gastroenteropancreatic tumors, and neuroendocrine tumors, as well as in diagnostic procedures (Sun and Coy, 2016; Ando, 2016). For more details on somatostatin actions, see the special issue on somatostatin in Mol Cell Endocrinol (Castan˜o et al., 2008) and the reviews by Morisset (2015) and Rorsman and Huising (2018) focused on the endocrine pancreas. 4.1.2 Amino Acid Sequence, Evolution, and Structure (Ando, 2016) The 14 amino acid sequence of the mature vertebrate somatostatin (SS1 or SS-14) form is shown in Fig. 17.19. The peptide is cyclized by a disulphide bond between Cys 3 and Cys 14. This sequence is fully conserved in all vertebrates. An N-terminally extended form of 28 amino acid residues (SS-28) is produced in the gastrointestinal system and shares 40%e60% identity with its counterpart in fish (Pradayrol et al., 1980). Molecular cloning has revealed six paralog genes in vertebrates (SS1-6). Zebrafish has all six. These six genes arose in vertebrate evolution from an ancestor gene through three whole-genome duplications along with local duplications in teleost fish, during which some genes were lost. Humans have only SS1 (SST) and SS2/CST encoding an SS-related cyclic peptide, 14e17 amino acids-long cortistatin (de Lecea et al., 1996). In addition, in humans, another SS-related peptide is encoded by the somatostatin gene, 13 amino acids-long neuronostatin (Samson et al., 2008), which is not cyclic. 4.1.3 Biosynthesis and Processing (Ando, 2016) The human preprosomatostatin gene SS1 (SST) located on chromosome 3 consists of two exons, and encodes a 116 amino acid precursor (Fig. 17.20) that is converted to SS-14 and SS-28 by specific proprotein convertases (Andrews and Dixon, 1986; Camier et al., 1986). A variety of other peptides are also generated (Benoit et al., 1990). SS-14 is the predominant form in the brain, both in the hypothalamus (ventromedial nucleus) and in extrahypothalamic regions. SS-28 is mainly produced by D cells at several locations in the digestive system, namely the pyloric antrum and the duodenum, while the d cells of the pancreatic islets of Langerhans secrete SS-14 (Rorsman and Huising, 2018), where it has also paracrine and autocrine effects. d cells have

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SS-14: Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys

SS-28: Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-(SS-14)

FIGURE 17.19

Amino acid sequence of human somatostatins 14 and 28.

long, neurite-like processes that make close contacts with a cells, b cells, and other d cells, thereby enabling an extensive paracrine network (Rorsman and Huising, 2018). The SST2/CST gene is expressed in the cortex and hippocampus. Neuronostatin is localized in the hypothalamus (Samson et al., 2008), the pancreatic d cells (Elrick et al., 2016), as well as parietal cells of the gastric mucosa. Our understanding of the cellular control of somatostatin secretion remains fragmentary (Rorsman and Huising, 2018). The synthesis and release of hypothalamic somatostatin are regulated by growth hormone, GHRH, and glucose. Somatostatin secretion by the D cells of the gastrointestinal tract (responsible for 65% of total body somatostatin) is regulated by the autonomous nervous system (inhibited by the vagus nerve; Holst et al., 1992) and by various gut regulatory peptides such as gastrin, cholecystokinin (CCK), and substance P. For review of enteroendocrine cells, see Gribble and Reimann (2016) and Mace et al. (2015). The cell physiology of somatostatin secretion by the pancreatic d cells has been recently reviewed in detail by Rorsman and Huising (2018). Stimulants include glucose, leucine, arginine, urocortin III (see later), ghrelin, GLP-1, glucagon, and acetylcholine. Inhibitors include nonesterified FFA, adrenaline, acetylcholine, and somatostatin (for the list of receptors involved, see Rorsman and Huising, 2018). Insulin has been reported as both a stimulant and an inhibitor. Among pharmacologic agents, sulfonylureas are stimulants and diazoxide, Ca2þ channel blockers, and SERCA inhibitors are inhibitors. Pancreatic somatostatin is primarily a paracrine regulator in the islets by activation of somatostatin receptors (SSTRs) in other islet cells 1

25 31 SIGNAL PEPTIDE

43 N

NEURONOSTATIN-13

and not likely to have a major systemic effect, SS-14 being only 5%e10% of plasma somatostatin (Rorsman and Huising, 2018). Recent data suggest that activation of the beta cells under conditions of high glucose (using an optogenetic method) propagates to the d cells via gap junctions, and the resulting stimulation of somatostatin secretion inhibits a-cell electrical activity by a paracrine mechanism (Briant et al., 2018; see their Fig. 9). 4.1.4 Somatostatin Receptors and Signaling Pathways Somatostatin receptors belong to the class A, rhodopsin-like G proteinecoupled receptors (GPCRs) and show high sequence identity with urotensin II receptors (Tostivint et al., 2014). Urotensin II is a somatostatin-like peptide that is a potent vasoconstrictor (Pearson et al., 1980; Ross et al., 2010). There are five subtypes of somatostatin receptors (SSTR1eSSTR5), all of which bind to somatostatin and cortistatin with high affinity (Tostivint et al., 2014). Their genes are located on different chromosomes: 14 (SSTR1), 17 (SSTR2), 22 (SSTR3), 20 (SSTR4), 16 (SSTR5). They are all widely distributed, with all five expressed in the brain, pituitary, and islets (Ando, 2016). Understanding the molecular basis of action of multiple natural and synthetic agonists through this family of receptors, which are able to homo- and heterodimerize, is quite challenging, and the literature is mildly confusing (Strowski and Blake, 2008). As far as the endocrine pancreas is concerned, in vivo and in vitro studies in rodents including knockout models suggest that SSTR1 and SSTR5 appear to be involved in inhibiting insulin secretion and SSTR2 in inhibiting glucagon secretion. In humans, SSTR2 seems to be the main inhibitor of both insulin and glucagon 89

103

116

SS SS-28 SS-14

FIGURE 17.20

Preprosomatostatin processing.

4. OTHER PANCREATIC HORMONES

secretion (Strowski and Blake, 2008), but the picture is far from crystal clear. For Rorsman and Huising (2018), transcriptomic analyses support that SSTR2 and SSTR3 are the predominant receptor subtypes in mouse and human a cells and SSTR3 in b cells. SSTR2 knockout mice showed a markedly reduced binding of radiolabeled SS-14 to almost all brain structures, indicating that SST2R accounts for most of somatostatin binding in mouse brain (Viollet et al., 2000). The five somatostatin GPCRs couple to the Gi/Go class of trimeric G proteins, which inhibits adenylyl cyclase and cyclic AMP-dependent protein kinase (Catalan et al., 1979, 1983), opens Kþ channels via b/g subunits, and closes Ca2þ channels. Consequently, these receptors antagonize the actions of multiple cAMPstimulating hormones. 4.1.5 Physiologic Actions (Huang, 1997; Ando, 2016) Somatostatin has been described as an “universal off switch” because it inhibits most of the organ and cellular functions with which it has been associated (Morisset, 2015). In the anterior pituitary gland, hypothalamic somatostatin inhibits the release of growth hormone (GH), thyroid-stimulating hormone, corticotropin, and prolactin. Somatostatin-deficient mice show increased plasma GH levels without significant increase in body size. However, the sexual dimorphism in GH pulsatile secretion and GH-regulated hepatic gene expression is abolished (Low et al., 2001). In the pancreas, somatostatin inhibits insulin, glucagon, and PP release. Somatostatin also blocks the exocrine secretory action of the pancreas through both direct and indirect effects (Heintges et al., 1994). In the gastrointestinal system, somatostatin inhibits the release of gastrointestinal hormones: gastrin, CCK, secretin, motilin, VIP, GIP, and glucagon-like peptide 1 (GLP-1). In the latter case, it is clearly demonstrated that this is a paracrine effect of SS-28 through SSTR5 (Chisholm and Greenberg, 2002). In addition, somatostatin inhibits various gastrointestinal functions, such as gastric acid secretion (an indirect effect of inhibiting gastrin, secretin, and histamine secretion), gastric emptying, intestinal motility, and blood flow within the intestine. Somatostatin also has antiproliferative actions inducing cell-cycle arrest and apoptosis (CuevasRamos and Fleseriu, 2014). Cortistatin depresses neuronal activity and enhances slow-wave sleep, while somatostatin increases REM sleep (de Lecea et al., 1996). Neuronostatin affects neuronal function, cardiac function, blood pressure, food intake, and drinking behavior (Samson et al., 2008; Vainio et al., 2012). It also stimulates glucagon

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secretion and inhibits insulin secretion (Salvatori et al., 2014). It acts through the orphan G proteine coupled receptor GPR107 (Yosten et al., 2012; Elrick et al., 2016). A more detailed discussion of these latter two peptides is beyond the scope of this review. 4.1.6 Pathophysiologic and Therapeutic Implications (Ando, 2016) Somatostatinoma is a rare malignant tumor arising from transformed d cells in the pancreatic islets or duodenum, and it is associated with malabsorption, diabetes mellitus, steatorrhea, and cholelithiasis. A variety of long-acting somatostatin analogs as well as nonpeptidergic SSTR ligands are available for treatment of acromegaly and pituitary adenomas (Cuevas-Ramos and Fleseriu, 2014), gastroenteropancreatic tumors, neuroendocrine tumors, and other gastrointestinal disorders such as secretory diarrhea and gastrointestinal bleeding. Radioactively labeled somatostatin analogs are used in imaging and radiotherapy. Somatostatin is widely used in experimental metabolic studies to control hormone actions, such as in the euglycemic clamp (Møller et al., 1995). The d cells emerging as master regulators within the islets suggests that they may represent an interesting and novel pharmaceutical target through which dysregulated insulin and glucagon secretion in diabetes mellitus may be corrected, e.g., by combining SSTR2 antagonists and insulin therapy (Rorsman and Huising, 2018).

4.2 Pancreatic Polypeptide (Williams, 2014) 4.2.1 History Pancreatic polypeptide (PP) is a 36-amino acid linear peptide produced and secreted by PP cells (originally called F cells) in the periphery of the islets of Langerhans. It is part of the neuropeptide Y family (Takei, 2016), which consists of neuropeptide Y (NPY), peptide YY (PYY), and PP. PP was the first member discovered in 1968 by Joe Kimmel and colleagues at University of Kansas as a contaminant (named APP) of insulin isolation from the chicken pancreas (Kimmel et al., 1968), and its sequence was determined by the same group in 1975 (Kimmel et al., 1975). Kimmel and colleagues localized APP to a specific pancreatic islet cell type in 1974 (Larsson et al., 1974), soon confirmed in humans (Larsson et al., 1975). A high-resolution crystal structure of APP was published in 1983 by Tom Blundell’s group (Glover et al., 1983). NPY, produced in the CNS, stimulates appetite and reduces energy expenditure, while PYY and PP, produced in peripheral endocrine cells in response to food intake, act in an opposite way as satiety factors (Yulyaningsih et al.,

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2014). For reviews, see Lonovics et al. (1981), Williams, (2014), Takei (2016), Katsuura and Inui (2016), and Vinik et al. (2017). 4.2.2 Amino Acid Sequence, Evolution, and Structure PP has been isolated and purified from multiple vertebrate species. All PPs possess 36 amino acids with a C-terminal amide (Fig. 17.21), which are highly conserved in mammals but much less so in other vertebrates (Williams, 2014). It has about 50% homology to NPY and PYY. From the N-terminus, the sequence includes a polyproline helix, a b-turn, and an a-helix followed by the C-terminal hexapeptide (Williams, 2014). Its biologic activity resides in the C-terminal hexapeptide and requires the C-terminal amide. NPY and PYY exist in ancestral vertebrates, but PP has been found only in tetrapods (Takei, 2016). The crystal structure (Fig. 17.22) shows a compact globular structure with a hydrophobic core, from which the C-terminal hexapeptide extends out from the globular portion (Glover et al., 1983). It has a characteristic U-shaped PP-fold consisting of an extended polyproline helix and an a-helix connected by a b-turn. The molecule forms symmetrical dimers linked through zinc atoms. The molecule exhibits considerable flexibility. 4.2.3 Biosynthesis, Processing, and Secretion The human PP gene is localized with the NPY gene on chromosome 17 and consists of four exons (Katsuura and Inui, 2016). It is processed from a larger prepro-PP of 95 amino acids in humans, comprising a 29-amino acid signal peptide, a 36-amino acid mature PP, and a 30-amino acid C-terminal peptide that gives rise to a conserved icosapeptide of unknown function (Williams, 2014). PP in plasma exhibits a rapid increase after a protein meal ingestion. The vagal nerve is a major stimulator, also mediating PP secretion stimulation by insulininduced hypoglycemia (Williams, 2014). CCK and gastrin are also good stimulants, partially through the vagus (Williams, 2014). Secretion is inhibited by somatostatin, ghrelin, and obestatin.

4.2.4 PP Receptors and Signaling Pathways Receptors for the NPY family comprise five receptors (Y receptors Y1, Y2, Y4, Y5, and Y6) belonging to the subfamily nine of class A GPCRs that couple to pertussis toxinesensitive GI/O protein (Michel et al., 1998). They inhibit cAMP formation and cAMP-dependent kinase (Takei, 2016). PP has high affinity for Y4, expressed in brain loci involved in the regulation of appetite, circadian rhythm, and anxiety: brainstem, hypothalamus, and amygdala (Michel et al., 1998; Takei, 2016; Katsuura and Inui, 2016). Recent work suggests that PP is also the primary ligand for Y6 in the suprachiasmatic nucleus involved in energy homeostasis in mice (see later) (Williams, 2014; Yulyaningsih et al., 2014). 4.2.5 PP Receptors Inactivation Deficiency of the Y4 receptor in mice results in decreased food intake and body weight, as well as anxiolytic behavior, consistent with the anxiogenic effect of overexpression of the PP gene (Katsuura and Inui, 2016). The data suggest that Y4 receptors and PP regulate food intake via hypothalamic orexin and brain-derived neurotropic factor-dependent pathways (Sainsbury et al., 2010). Recent work from the group of Herbert Herzog at the Garvan Institute in Sydney has shed light on the role of the Y6 receptor in mice on energy homeostasis (Yulyaningsih et al., 2014). The Y6 receptor, encoded by the Npy6r gene, is less conserved than the other Y receptors. It is truncated in primates including humans due to a single-base deletion resulting in a frameshift mutation and an early stop codon. Npy6r is totally absent from the rat genome. The phenotype of the Npy6r/ mice demonstrates that Npy6r is a critical regulator of energy homeostasis and body composition, with reduced body weight and lean body mass and an age-dependent increased adiposity that is exacerbated by high-fat feeding, likely due to lack of Y6 signaling in the suprachiasmatic nucleus to influence the VIP-GH/IGF-1 axis (Yulyaningsih et al., 2014). It is still unclear whether the truncated human Y6 receptor is functional at all since it has not been successfully expressed (Michel et al., 1998).

1 Ala-Pro-Leu-Glu-Pro-Val-Tyr-Pro-Gly-Asp-Asn-Ala-Thr-Pro-Glu-Gln-Met-AlaGln-Tyr-Ala-Ala-Asp-Leu-Arg-Arg-Tyr-Ile-Asn-Met-Leu-Thr-Arg-Pro-Arg-Tyr-NH2 36

FIGURE 17.21 Amino acid sequence of human pancreatic polypeptide.

4. OTHER PANCREATIC HORMONES

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clinically as therapeutic agents (Katsuura and Inui, 2016).

4.3 Amylin 4.3.1 History Amylin (also called islet amyloid polypeptide, IAPP) belongs to the calcitonin (CT)/calcitonin gene-related peptide family that also includes calcitonin, calcitonin gene-related peptide, and adrenomedullin 1, 2, and 5 (Suzuki, 2016). It is responsible for the deposition of amyloid in the islets of Langerhans in type 2 diabetes (Westermark, 2011). It was isolated in 1987 by two different groups from an insulinoma and islets of diabetic cats (Westermark et al., 1987) and from amyloidrich pancreases from type 2 diabetic patients (Cooper et al., 1987). It is produced in the b cells, stored in the granules and released together with insulin. It contributes to lowering blood glucose levels, and one of its analogs, under the name of pramlintide, is used in the treatment of type 1 and type 2 diabetes (Ryan et al., 2005). FIGURE 17.22

Crystal structure of avian pancreatic polypeptide. From Glover, I., Haneef, I., Pitts, J., Wood, S., Moss, D., Tickle, I., Blundell, T., 1983. Conformational flexibility in a small globular hormone: X-ray ˚ resolution. Biopolymers analysis of avian pancreatic polypeptide at 0.98-A 22, 293e304, used with permission from Wiley.

4.2.6 Physiologic Actions While the preceding data suggest a role for PP in energy homeostasis in rodents, a clearcut biologic role for PP in humans has not been established, with the exception of inhibition of gallbladder contraction and exocrine pancreatic enzyme secretion (Vinik et al., 2017; Williams, 2014). These effects do not seem to be direct but mediated through PP receptors in the CNS resulting in inhibition of vagal excitatory output of the pancreas. A potential role as a satiety factor (Vinik et al., 2017) has been suggested by the observation that PP secretion is almost abolished in obese children with Prater-Willi syndrome and that food intake was reduced by bovine PP infusion both in PradereWilli syndrome (Berntson et al., 1993) and in normal humans (Batterham et al., 2003). PP levels are increased in anorexia nervosa (Batterham et al., 2003). 4.2.7 Pathophysiologic and Therapeutic Implications The variable symptomatology associated with PPomas and tumors of the pancreas with increased PP levels has been reviewed by Vinik et al. (2017). No PP-related peptides nor compounds have been used

4.3.2 Amino Acid Sequence and Structure Mature amylin is a 37-amino acid peptide (Fig. 17.23). There is a disulphide bridge between cysteine 2 and cysteine 7, and the C-terminus is amidated. The sequence 20e29 is considered responsible for forming fibrils in type 2 diabetes and is conserved in cats but not rodents (who do not have amyloid). The rest of amylin’s sequence is highly conserved in vertebrates (Ogoshi, 2016). The amyloid monomer is unstructured but forms amyloid fibrils that are proposed to be a double hairpin with three b-strands (Ogoshi, 2016). A crystal structure of amylin bound to the IDE, a Zn2þ metalloproteinase involved in the clearance of insulin and amyloid beta, shows the formation of b strands between amylin and the enzyme (Shen et al., 2006). 4.3.3 Biosynthesis, Processing, and Secretion (Ogoshi, 2016) Human amylin is derived from a 67-amino acid proamylin by the prohormone convertases PC2 and PC1/3 to generate the 37-amino acid sequence of mature amylin. The human amylin gene (IAPP) is located on chromosome 12 and is made of three exons. It is regulated by transcription factor PDX-1. Mammalian IAPP is expressed in b cells, in d cells, in the gastrointestinal tract, and in sensory neurons. Proamylin is processed in the Golgi complex, and amylin is stored in the insulin secretory granules of pancreatic b cells. It is released together with insulin in response to elevated glucose in a 20:1 M ratio of insulin

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Lys - Cys - Asn - Thr - Ala - Thr - Cys - Ala - Thr - Gln - Arg - Leu - Ala – Asn – Phe - Leu - Val - His - Ser - Ser - Asn - Asn - Phe - Gly - Ala - Ile - Leu - Ser – Ser - Thr - Asn - Val - Gly - Ser - Asn - Thr - Tyr

FIGURE 17.23

Amino acid sequence of human amylin.

to amylin. Like insulin’s, amylin secretion is defective in type 1 diabetes. 4.3.4 The Amylin Receptor and Signaling Pathways (Ogoshi, 2016) Amylin binds to the calcitonin receptor (CTR), a member of the class B of G proteinecoupled (seven transmembrane domains) receptors like the glucagon receptor (see before). It binds to the CTR with low affinity, but the affinity is markedly enhanced by complexing with receptor activity-modifying (single transmembrane) proteins (RAMP)-1, -2, or -3, with RAMP-3 generating the highest affinity. The crystal structure of amylin bound to the CTR and RAMP has not been determined. The CTR is coupled to Gs, and amylin stimulates cAMP production in target cells. CTR also activates phospholipase C (PLC) (Suzuki, 2016). 4.3.5 Physiologic Functions Amylin lowers blood glucose by slowing gastric emptying, inhibiting glucagon secretion, increasing first-phase insulin release after a meal, and it promotes satiety via hypothalamic receptors (Ryan et al., 2005).

4.4 Ghrelin Ghrelin is a growth hormone-releasing and orexigenic acylated peptide originally isolated from rat stomach in 1999 by Masayasu Kojima and colleagues from Kuruma University, as the endogenous ligand of the GH secretagogue receptor (Kojima et al., 1999). The original Nature paper has been cited over 8700 times. It was also isolated as a mouse mRNA by Tomasetto et al. (2000) under the name motilin-related peptide (MTLRP). It turned out to be a highly multifunctional hormone, as described in a recent magistral review cosigned by 57 leading endocrinologists (Mu¨ller et al., 2015). Ghrelin is primarily a stomach hormone and as such is reviewed in the chapter on stomach hormones in this book. We will focus here on the presence and role of ghrelin cells as an insulinostatic hormone in the islets of Langerhans

of the pancreas, discovered 16 years ago (Wierup et al., 2002; for review see Wierup et al., 2013). The source of pancreatic ghrelin was subsequently identified as previously ultrastructurally characterized A-like cells in rats and P/D1-cells in humans not known to express a hormone (see Wierup et al., 2013 for review), now named ε cells or “ghrelin cells.” They are as a rule located at the peripheral rim of islets, and sometimes in fetal and neonatal islets form a continuous cell layer embracing the other islet cells (Wierup et al., 2013). These cells are highly developmentally regulated. In humans the cells are most abundant in fetal and neonatal islets (10% of all islet cells) but in adults still present (1%). In fact, in humans, 35%e45% of circulating ghrelin remains after total gastrectomy. The pancreas appears to be the main source of circulating ghrelin during human fetal development. In contrast with humans, ghrelin is undetectable in the pancreas in mice and rats after 2 weeks of age and in adults, and it appears to be exclusively a fetal pancreatic hormone, when the gastric ghrelin cell density is still low. The exact lineage of these cells is still an unresolved puzzle (Wierup et al., 2013). There is lot of evidence that ghrelin inhibits insulin secretion (Dezaki et al., 2008). Oral administration of a ghrelin antagonist improved glucose homeostasis in rodents (Esler et al., 2007). Considering also the orexigenic effects of ghrelin, such antagonists may have potential to treat type 2 diabetes and obesity (Wierup et al., 2013).

4.5 Urocortin III Urocortin III (Ucn III, also called stresscopin), related to CRF (corticotropin-releasing factor), was identified in 2001 by the group of Willie Vale at the Salk Institute as a 38-amino acid neuropeptide expressed in the hypothalamus, amygdala, and brainstem (Lewis et al., 2001). Subsequently Vale’s group showed that Ucn III is expressed in pancreatic b cells and in the mouse b-cell line MIN6. Ucn III stimulates insulin and glucagon secretion in rats and from isolated rat islets and raises blood glucose in rats (Li et al., 2003). This effect is mediated through the class B GPCR CRF2 receptor that activates adenylyl

5. CONCLUSIONS AND PERSPECTIVES

cyclase. Ucn III also stimulates somatostatin secretion by the d cells (Rorsman and Huising, 2018). Secretion of Ucn III from b cells is stimulated by high potassium, forskolin, or high glucose. Interestingly Ucn III and insulin are located in separate areas of the beta cell, suggesting they are not cosecreted. Unlike in rodents, in primates Ucn III appears to be also expressed in a cells (Van der Meulen et al., 2012).

4.6 Stanniocalcin 2 Stanniocalcin (STC) is a secreted disulphide-bonded dimeric glycoprotein that plays a major role in the calcium and phosphate homeostasis of fish (for review, see Moore et al., 1999). It is produced in the corpuscles of Stannius, endocrine organs that are associated with fish kidneys. Two human homologs, STC1 and STC2, were identified about 20 years ago. STC1 is widely expressed and has been suggested to play a role in calcium and phosphate homeostasis (Moore et al., 1999). STC2 showed 30%e36% homology to both the fish STC and mammalian STC1. It was initially found to be most highly expressed in the a cells of the pancreas and postulated to play a role in glucose homeostasis (Moore et al., 1999). However, to this day the precise physiologic role and biochemical actions of mammalian STCs are far from clear. Interestingly, mice overexpressing STC2 are reduced 45% in size, whereas STC2 knockout mice are 15% larger than wild-type littermates (reviewed in Jepsen et al., 2015). Jepsen at al. showed that, in fact, STC2 interferes with the insulin-like growth factor axis by inhibiting the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) that cleaves IGF-binding proteins (IGFBPs) 2, 4, and 5 and thus releases bioactive IGFs from the inactive complex with IGFBPs.

4.7 Serotonin-Producing Enterochromaffin Cells and Gastrin-Producing G Cells Besides the cell types described previously, less frequently occurring, sometimes transiently present during embryonic development and species-specific islet cell types have been described (reviewed in Wierup et al., 2013), such as serotonin-producing enterochromaffin (EC) cells and gastrin-producing G cells. Little is known about their functional significance, and we will not elaborate here.

4.8 Gamma-Aminobutyric Acid (GABA) Production by b cells (Braun et al., 2010) g-aminobutyric acid (GABA), best known as an inhibitory neurotransmitter, is present in human pancreatic

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islets at concentrations similar to those found in the brain (Braun et al., 2010). Human b cells contain high concentrations of GABA. GABA released from b cells inhibits glucagon secretion in rodent islets by activating GABAA receptors in a cells but appears to play a more diverse role in human islets. Braun et al. (2010) have demonstrated the existence of an autocrine excitatory feedback loop by GABA on GABAA receptors in b cells stimulating insulin secretion. It also induces somatostatin secretion by a paracrine effect; human d cells express high levels of GABAA receptors. Most importantly, the group of Patrick Collombat in Nice has recently shown that long-term administration of GABA induces b-cell neogenesis from a cells, which could represent an unprecedented hope toward improved therapies for diabetes (Ben-Othman et al., 2017). This is further discussed in the next section of this chapter.

5. CONCLUSIONS AND PERSPECTIVES It is clear through the discoveries of the past few decades that the prevalent concept that held up through the 1960s of the islets of Langerhans of the endocrine pancreas being an isolated endocrine system regulating glucose homeostasis through the antagonistic balance between insulin from the b cells and glucagon form the a cells under the control of glucose and nutrient levels needed a major overhaul. It is clear now that the islets of Langerhans contain at least five cell types and secrete at least eight peptide hormones (some shared with other endocrine sources) plus some neurotransmitters like serotonin and GABA. Complex interactions exist between the various islet cell subtypes, the hypothalamus, the autonomous nervous system, and the gastrointestinal system. It is not always easy to decipher the role of direct versus indirect effects of hormones, nor distant endocrine versus local paracrine or autocrine effects. Adding to the complexity is the existence of multiple ligands cross-reacting with multiple receptor subtypes, opening therapeutic windows for multiagonist ligands. Despite considerable progress in unraveling the complex mechanisms of insulin signal transduction and those of insulin secretion, the classical dichotomy between insulin resistance and b cell dysregulation as being the major cause of the metabolic alterations of type 2 diabetes remains largely unresolved. As mentioned before, both massive insulin resistance and glucose intolerance are associated with loss of insulin signaling in the liver, and an insulin secretion defect results from loss of insulin signaling in the b cell. New concepts are emerging that both may result from common mechanisms, the loss of powerful signal integrators that are IRS2 (White, 2012) and FOXO1 in both the beta cell

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and the liver (Pajvani and Accili, 2015). In their aptly called “new biology of diabetes,” Accili and colleagues have recently proposed a plausible liver- and b cellcentric hypothesis of diabetes pathophysiology linked to progressive loss of FOXO function (Pajvani and Accili, 2015). FOXO integrates a surprisingly diverse array of biologic functions in both the liver and the b cell. In the liver, FOXO prevents excessive glucose production and increased lipid synthesis and secretion. In the b cell, FOXO is required to maintain b cell differentiation. Diabetes progression involves a gradual loss of FOXO function that results in b cell dedifferentiation (Talchai et al., 2012; Cinti et al., 2016), which is proposed to be the principal cause of b cell failure and conversion to non-b endocrine cells. Finally, an important emerging concept is that islet cell subtypes may enjoy considerably more plasticity than previously estimated. Besides the concept of b-cell dedifferentiation just discussed, it appears that a cells may be able to transform relatively easily into b cells (Gromada et al., 2018), since they have remarkably similar patterns of gene expression. Emerging data from Patrick Collombat and colleagues in Nice (Ben Othman et al., 2017) suggest that long-term administration of GABA (now known to be a natural product of the islets) may facilitate this interconversion. Nearly a century after the discovery of insulin and glucagon, the islet of Langerhans is far from having revealed all its secrets.

Acknowledgments Mike Lawrence is acknowledged for many enlightening scientific discussions and for critical comments on the insulin receptor sections.

References Accili, D., Arden, K.C., 2004. FoxOs at the crossroads of cellular metabolism, differentiation and transformation. Cell 117, 421e426. Accili, D., Drago, J., Lee, E.J., Johnson, M.D., Cool, M.H., Salvatore, P., Asico, L.D., Jose´, P.A., Taylor, S.I., Westphal, A., 1996. Early neonatal death in mice homozygous for a null allele of the insulin receptor gene. Nat. Genet. 12, 106e109. Adams, M.J., Blundell, T.L., Dodson, E.J., Dodson, G.G., Vijayan, M., Baker, E.N., Hardine, M.M., Hodgkin, D.C., Rimer, B., Sheet, S., 1969. Structure of rhombohedral 2 zinc insulin crystals. Nature 224, 491e495. Aguilar-Bryan, L., Bryan, J., 1999. Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr. Rev. 20, 101e135. Alberti, K.G., Christensen, N.J., Christensen, S.E., Prange Hansen, A.A., Iversen, J., Lundbæk, K., Seyer-Hansen, K., Ørsov, H., 1973. Inhibition of insulin secretion by somatostatin. Lancet 302, 1299e1301. Ali, S., Drucker, D.J., 2009. Benefits and limitations of reducing glucagon action for the treatment of type 2 diabetes. Am. J. Physiol. Endocrinol. Metab. 296, E415eE421.

Amatruda, J.M., Livingston, J.N., Lockwood, D.H., 1975. Insulin receptor role in the resistance of human obesity to insulin. Science 188, 264e266. Ando, H., 2016. Somatostatin. In: Takei, Y., Ando, H., Tsutsui, K. (Eds.), Handbook of Hormones. Comparative Endocrinology for Basic and Clinical Research. Academic Press, pp. 36e38. Andralojc, K.M., Mercalli, A., Nowak, K.W., Albarello, L., Calcagno, R., Luzi, L., Bonifacio, E., Doglioni, C., Remonti, L., 2009. Ghrelinproducing epsilon cells in the developing and adult human pancreas. Diabetologia 52, 486e493. Andrews, P.C., Dixon, J.E., 1986. Biosynthesis and processing of the somatostatin family of peptide hormones. Scand. J. Gastroenterol. 21 (Suppl. 119), 22e28. Araki, E., Lipes, M.A., Patti, M.-E., Bru¨ning, J.C., Haag III, B., Johnson, R.S., Kahn, C.R., 1994. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372, 186e190. Avruch, J., 1998. Insulin signal transduction through protein kinase cascades. Mol. Cell. Biochem. 182, 3e11. Avruch, J., 2007. MAP kinase pathways: the first twenty years. Biochim. Biophys. Acta 1773, 1150e1160. Avruch, J., Nemenoff, A., Blackshear, P.J., Pierce, M.W., Osathanondh, R., 1982. Insulin-stimulated tyrosine phosphorylation of the insulin receptor in detergent extracts of human placental membranes. Comparison to epidermal growth factor-stimulated phosphorylation. J. Biol. Chem. 257, 15162e15166. Baker, E.N., Blundell, T.L., Cutfield, J.F., Cutfield, S.M., Dodson, E.J., Dodson, G.G., Hodgkin, D.M., Hubbard, R.E., Isaacs, N.W., ˚ Reynolds, C.D., 1988. The structure of 2Zn pig insulin at 1.5A resolution. Phil. Trans. R. Soc. Lond. B 19, 369e456. Banting, F.G., Best, C.H., 1922. The internal secretion of the pancreas. J. Lab. Clin. Med. VII, 5, 256e271. Bar, R.S., Gorden, P., Roth, J., Kahn, C.R., De Meyts, P., 1976. Fluctuation in the affinity and concentration of insulin receptors on circulating monocytes of obese subjects. J. Clin. Invest. 58, 1123e1135. Bataille, D., Jarrousse, C., Blache, P., et al., 1988. Oxyntomodulin and glucagon: are the whole molecules and their C-terminal fragments different biological entities? Biomed. Res. 9 (Suppl. 3), 169e179. Batterham, R.L., Le Roux, C.W., Cohen, M.A., Park, A.J., Ellis, S.M., Patterson, M., Frost, G.S., Ghatei, M.A., Bloom, S.R., 2003. Pancreatic polypeptide reduces appetite and food intake in humans. J. Clin. Endocrinol. Metab. 88, 3989e3992. Bayliss, W.M., Starling, E.H., 1902a. The mechanism of pancreatic secretion. J. Physiol. 28, 325e353. Bayliss, W.M., Starling, E.H., 1902b. On the causation of the so-called “peripheral reflex secretion” of the pancreas. Proc. R. Soc. Lond. (Biol.) 69, 352e353. Belfiore, A., Frasca, F., Pandini, G., Sciacca, L., Vigneri, R., 2009. Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease. Endocr. Rev. 30, 586e623. Bell, G.I., Pictet, R.L., Rutter, W.J., Cordell, B., Tischer, E., Goodman, H.M., 1980. Sequence of the human insulin gene. Nature 284, 26e32. Ben-Othman, N., Vieira, A., Courtney, M., Record, F., Gjernes, E., Avolio, F., Hadzic, B., Druelle, N., Napolitano, T., NavarroSanz, S., Silvano, S., Al-Hasani, K., Pfeifer, A., Lacas-Gervais, S., Leuckx, G., Marroqui, L., The´venet, J., Madsen, O.D., Eizirik, D.L., Heimberg, H., Kerr-Comte, J., Pattou, F., Mansouri, A., Collombat, P., 2017. Long-term GAGA administration induces alpha-cell-mediated beta-like cell neogenesis. Cell 168, 73e85. Benoit, R., Esch, F., Bennett, H.P.J., Ling, N., Ravazzola, M., Orci, L., 1990. Processing of prosomatostatin. Metabolism 39, 22e25.

REFERENCES

Bentley, G., Dodson, E.J., Dodson, G.G., Hodgkin, D.C., Mercola, D.A., 1976. Structure of insulin in 4-Zn insulin. Nature 261, 166e168. Bergeron, J.J., Di Guglielmo, G.M., Dahan, S., Dominguez, M., Posner, B.I., 2016. Spatial and temporal regulation of receptor tyrosine kinase activation and intracellular signal transduction. Annu. Rev. Biochem. 85, 573e597. Berntson, G., Zipf, W.B., O’Dorisio, T.M., Hoffman, J., Chance, R., 1993. Pancreatic polypeptide infusions reduce food intake in Prader-Willi Syndrome. Peptides 14, 497e503. Bertin, E., Arner, P., Bolinder, J., Hagstrøm-Toft, E., 2001. Action of glucagon and glucagon-like peptide -1-(7-36) amide on lipolysis in human subcutaneous tissue and skeletal muscle in vivo. J. Clin. Endocrinol. Metab. 86, 1229e1234. Billings, L.K., Flores, J.C., 2010. The genetics of type 2 diabetes: What have we learned from GWAS? Ann. N.Y. Acad. Sci. 1212, 59e72. Blache, P., Kervran, A., Dufour, M., Martinez, J., Le-Nguyen, D., Lotersztajn, S., Pecker, F., Bataille, D., 1990. Glucagon-(19-29), a Ca2þ pump inhibitory peptide, is processed from glucagon in the rat liver plasma membrane by a thiol endopeptidase. J. Biol. Chem. 265, 21514e21519. Bliss, M., 2007. The Discovery of Insulin, 25th anniversary ed. University of Toronto Press, Toronto. Bloom, W., 1931. A new type of granular cells in the islets of Langerhans of man. Anatom. Record 49, 363e371. Bloom, C.R., Kaarsholm, N.C., Ha, J., Dunn, M.F., 1997. Half-site reactivity, negative cooperativity, and positive cooperativity: quantitative considerations of a plausible model. Biochemistry 36, 12759e12765. Blu¨her, M., Michael, M.D., Peroni, O.D., Ueki, K., Carter, N., Kahn, B.B., Kahn, C.R., 2002. Adipose-tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance. Dev. Cell 3, 25e38. Blundell, T.L., 1983. The conformation of glucagon. In: Glucagon, I., Lefe`bvre, P.J. (Eds.), Handbook of Experimental Pharmacology, vol. 66/1. Springer-Verlag, Berlin Heidelberg New York Tokyo, pp. 37e56. Blundell, T.L., Cutfield, J.F., Cutfield, S.M., Dodson, E.J., Dodson, G.G., Hodgkin, D.C., Mercola, D.A., Vijayan, M., 1971. Atomic positions in rhombohedral 2-zinc insulin crystals. Nature 231, 506e511. Blundell, T.L., Dodson, G.G., Hodgkin, D.C., Mercola, D.A., 1972. Insulin: the structure in the crystal and its reflection in chemistry and biology. Adv. Prot. Chem. 26, 279e402. Bonner-Weir, S., Sullivan, B.A., Weir, G.C., 2015. Human islet cell morphology revisited: human and rodent islets are not so different after all. J. Histochem. Cytochem. 63, 604e612. Bosco, D., Armanet, M., Morel, P., Niclauss, N., Sgroi, A., Muller, Y.D., Giovannoni, L., Parnaud, G., Berney, T., 2010. Unique arrangement of a- and b-cells in human islets of Langerhans. Diabetes 59, 1202e1210. Boucher, J., Kleinridders, A., Kahn, C.R., 2014. Insulin receptor signaling in normal and insulin-resistant states. Cold Spring Harb. Perspect. Biol. 6, a009191. Braun, M., Ramracheya, R., Bengtsson, M., Clark, A., Walker, J.N., Johnson, P.R., Rorsman, P., 2010. Gamma-aminobutyric acid (GABA) is an autocrine excitatory transmitter in human pancreatic beta-cells. Diabetes 59, 1694e1701. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., Guillemin, R., 1973. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179, 77e79. Brereton, M.F., Vergari, E., Zhang, Q., Clark, A., 2015. Alpha, delta and PP-cells: are they the architectural cornerstone of islet structure and coordination? J. Histochem. Cytochem. 63, 575e591. Briant, L.B.J., Reinbothe, T.M., Spiliotis, I., Miranda, C., Rodriguez, B., Rorsman, P., 2018. d-cells and b-cells are electrically coupled

413

and regulate a-cell activity via somatostatin. J. Physiol. 596, 197e215. Brierly, G.V., Siddle, K., Semple, R.K., 2018. Evaluation of anti-insulin receptor antibodies as potential novel therapies for human human insulin receptoropathy using cell culture models. Diabetologia 61, 1662e1675. Bromer, W.W., Sinn, L.G., Staub, A., Behrens, O.K., 1957. The amino acid sequence of glucagon. Diabetes 6, 234e238. Bru¨ning, J.C., Michael, M.D., Winnay, J.N., Hayashi, T., Horsch, D., Accili, D., Goodyear, L.J., Kahn, C.R., 1998. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol. Cell 2, 559e569. Bru¨ning, J.C., Gautam, D., Burks, D.J., Gillette, J., Schubert, M., Orban, P.C., Klein, R., Krone, W., Mu¨ller-Wieland, D., Kahn, C.R., 2000. Role of brain insulin receptor in control of body weight and reproduction. Science 289, 2122e2125. Cabail, M.Z., Li, S., Lemmon, E., Bowen, M.E., Hubbard, S.R., Miller, W.T., 2015. The tyrosine kinase domain of the insulin and IGF-I receptors are functional dimers in the activated state. Nat. Commun. 6, 6406. Camier, M., Barre, N., Morel, A., Cohen, P., 1986. In vivo synthesis and processing of rat hypothalamic prosomatostatin. FEBS Lett. 196, 14e18. Campbell, J.E., Drucker, D.J., 2015. Islet a cells and glucagon e critical regulators of energy homeostasis. Nat. Rev. Endocrinol. 11, 329e338. Cantley, L.C., 2002. The phosphoinositide 3-kinase pathway. Science 296, 1655e1657. Carlson, M.G., Snead, W.L., Campbell, P.J., 1993. Regulation of free fatty acid metabolism by glucagon. J. Clin. Endocrinol. Metab. 77, 11e15. Carpentier, J.L., 1994. Insulin receptor internalization: molecular mechanisms and pathophysiological implications. Diabetologia 37 (Suppl. 2), S117eS124. Special issue: somatostatin, corticostatin and their receptors in health and disease. In: Castan˜o, J.P., Ghizo, E., Kineman, R.D., de Lecea, L., Malago´n, M.M., Vaudry, H. (Eds.), Mol. Cell. Endocrinol. 286 (1 and 2). Catalan, R.E., Aragones, M.D., Martinez, A.M., 1979. Somatostatin effect on cyclic AMP and cyclic GMP levels in rat brain. Biochem. Biophys. Acta 586, 213e216. Catalan, R.E., Martinez, A.M., Aragones, M.D., 1983. Inhibition of cyclic AMP dependent protein kinase by somatostatin in slices of mouse brain: dependence on extracellular calcium. Neuropharmacology 22, 641e645. Cegla, J., Troke, R.C., Jones, B., Tharakan, G., Kenkre, J., McCullough, K.A., Lim, C.T., Parvizi, N., Hussein, M., Chambers, E.S., Minnion, J., Cuenco, J., Ghatei, M.A., Meeran, K., Tan, T.M., Bloom, S.R., 2014. Coinfusion of GLP-1 and glucagon in man results in a reduction of food intake. Diabetes 63, 3711e3720. Chan, S.J., Steiner, D.F., 2000. Insulin through the ages: phylogeny of a growth-promoting and metabolic regulatory hormone. Am. Zool. 40, 213e222. Chan, S.J., Nakagawa, S., Steiner, D.F., 2007. Complementation analysis demonstrates that insulin crosslinks both a subunits in a truncated receptor dimer. J. Biol. Chem. 282, 13754e13758. Chang, X., Jorgensen, A.M., Bardrum, P., Led, J.J., 1997. Solution structures of the R6 human insulin hexamer. Biochemistry 36, 9409e9422. Charron, M.J., Vuguin, P.M., 2015. Lack of glucagon receptor signaling and its implications beyond glucose homeostasis. J. Endocrinol. 224, R123eR130. Chibber, V.L., Soriano, C., Tayek, J.A., 2000. Effects of low-dose and high-dose glucagon on endogenous glucose production and gluconeogenesis in humans. Metabolism 49, 39e46.

414

17. PANCREATIC HORMONES

Chin, C.N., Dallas-Yang, Q., Liu, F., Ho, T., Ellsworth, K., Fischer, P., Natasha, T., Ireland, C., Lu, P., Li, C., Wang, I.M., Strohl, W., Berger, J.P., An, Z., Zhong, B.B., Jiang, G., 2009. Evidence that inhibition of insulin receptor signalling activity by PC-1/ENPP1 is dependent on its enzymatic activity. Eur. J. Pharmacol. 606, 17e24. Chisholm, C., Greenberg, G.R., 2002. Somatostatin-28 regulates GLP-1 secretion via somatostatin receptor subtype 5 in rat intestinal cultures. Am. J. Physiol. Endocrinol. Metab. 283, E311eE317. Chou, P.Y., Fasman, G.D., 1975. The conformation of glucagon: predictions and consequences. Biochemistry 14, 2536e2541. Cinti, F., Bouchl, R., Kim-Muller, J.Y., Ohmura, Y., Sandoval, P.R., Masini, M., Ratner, L.E., Marchetti, P., Accili, D., 2016. Evidence of beta cell dedifferentiation in human type 2 diabetes. J. Clin. Endocrinol. Metab. 101, 1044e1054. Clapham, D.E., Neer, E.J., 1997. G protein beta gamma subunits. Ann. Rev. Pharmacol. Toxicol. 37, 167e203. Cohen, P., 2001. The renaissance of GSK3. Nat. Rev. Mol. Cell Biol. 2, 767e776. Conlon, J.M., 2001. Evolution of the insulin molecule: insights into structure-activity and phylogenetic relationships. Peptides 22, 1183e1193. Cooper, G.J., Willis, A.C., Clark, A., Turner, R.C., Sim, R.B., Reid, K.B., 1987. Purification and characterization of a peptide from amyloidrich pancreases of type 2 diabetic patients. Proc. Natl. Acad. Sci. U.S.A. 84, 8628e8632. Courtney, M., Pfeifer, A., Al-Hasany, K., 2011. In vivo conversion of adult a-cells into b-like cells: a new research avenue in the context of type 1 diabetes. Diab Obes Metab 13 (Suppl. 1), 47e52. Croll, T.I., Smith, B.J., Margetts, M.B., Whittaker, J., Weiss, M.A., Ward, C.W., Lawrence, M.C., 2016. Higher-resolution structure of the human insulin receptor ectodomain: muælti-modal inclusion of the insert domain. Structure 24, 469e476. Cuatrecasas, P., Desbuquois, B., Krug, F., 1971. Insulin receptor interactions in liver cell membranes. Biochem. Biophys. Res. Comm. 44, 333e339. Cuevas-Ramos, D., Fleseriu, M., 2014. Somatostatin receptor ligands and resistance to treatment in pituitary adenomas. J. Mol. Endocrinol. 52, R223eR240. Da Silva Xavier, G., 2018. The cells of the islets of Langerhans. J. Clin. Med. 7, 54. https://doi.org/10.3390/jcm7030054. Dalle, S., Smith, P., Blache, P., Le Nguyen, D., LeBrigand, L., Bergeron, F., Ashcroft, F.M., Bataille, D., 1999. Mini-glucagon (glucagon 19e29), a potent and efficient inhibitor of secretagogueinduced insulin release through a Ca2þ pathway. J. Biol. Chem. 274, 10869e10876. Dalle, S., Fonte´s, G., Lajoix, A.D., LeBrigand, L., Gross, R., Ribes, G., Dufour, M., Barry, L., Le Nguyen, D., Bataille, D., 2002. Miniglucagon (glucagon 19e29). A novel regulator of the pancreatic islet physiology. Diabetes 51, 406e412. Day, J.W., Ottaway, N., Patterson, J.T., Gelfanov, V., Smiley, D., Gidda, J., Findeisen, H., Bruemmer, D., Drucker, D.J., Chaudhary, N., Holland, J., Hembree, J., Abplanalp, W., Grant, E., Ruehl, J., Wilson, H., Kirchner, H., Lockie, S.H., Hofmann, S., Woods, S.C., Nogueiras, R., Pfluger, P.T., Perez-Tilve, D., DiMarchi, R., Tscho¨p, M.H., 2009. A new glucagon and GLP-1 coagonist eliminates obesity in rodents. Nat. Chem. Biol. 5, 749e757. de Duve, C., 1953. Glucagon, the hyperglycemic glycogenolytic factor of the pancreas. Lancet 265, 99e104. de Duve, C., Vuylsteke, C.A., 1953. Purification of glucagon (hyperglycemic-glycogenolytic factor of the pancreas). Arch. Int. Physiol. 61, 107e108. De Lecea, L., Criado, J.R., Prospero-Garcia, O., Gautvik, K.M., Schweitzer, P., Danielson, P.E., Dunlop, C.L., Siggins, G.R., Henrikson, S.J., Sutcliffe, J.G., 1996. A cortical neuropeptide with neuronal depressant and sleep-modulating properties. Nature 381, 242e245.

De Meyer, J., 1909. Action de la se´cre´tion interne du pancreas sur diffe´rents organes et en particulier sur la se´cre´tion re´nale. Arch. di Fysiol. 7, 96. De Meyts, P., 1976. Cooperative properties of hormone receptors in cell membranes. J. Supramol. Struct. 4, 241e258. De Meyts, P., 1994. The structural basis of insulin and insulin-like growth factor-I (IGF-I) receptor binding and negative cooperativity, and its relevance to mitogenic versus metabolic signalling. Diabetologia 37, S135eS148. De Meyts, P., 2004. Insulin and its receptor: structure, function and evolution. Bioessays 26, 1351e1362. De Meyts, P., 2015a. Receptor tyrosine kinase signal transduction and the molecular basis of signalling specificity. In: Wheeler, D.L., Yarden, Y. (Eds.), Receptor Tyrosine Kinases: Structure, Functions and Role in Human Disease. Humana Press Springer, New York; Heidelberg; Dodrecht; London, pp. 51e76. De Meyts, P., 2015b. Insulin/receptor binding: the last piece of the puzzle? Bioessays 37, 389e397. De Meyts, P., 2016. The insulin receptor and its signal transduction network. In: De Groot, L.J., et al. (Eds.), Endotext [Internet]. MDText.com, Inc., South Darthmouth, MA, 2000. De Meyts, P., Whittaker, J., 2002. Structural biology of insulin and IGF1 receptors: implications for drug design. Nat. Rev. Drug Disc. 1, 769e783. De Meyts, P., Roth, J., Neville Jr., D.M., Gavin III, J.R., Lesniak, M.A., 1973. Insulin interactions with its receptors. Experimental evidence for negative cooperativity. Biochem. Biophys. Res. Commun. 55, 154e161. De Meyts, P., Van Obberghen, E., Roth, J., Wollmer, A., Brandenburg, D., 1978. Mapping of the residues responsible for the negative cooperativity of the receptor-binding region of insulin. Nature 273, 504e509. Deacon, C.F., Nauck, M.A., Toft-Nielsen, N.I., Pridal, L., Willms, B., Holst, J.J., 1995. Both subcutaneously and intravenously administered glucagon-like peptide 1 are rapidly degraded from the NH2 terminus in type 2 diabetic subjects and in healthy subjects. Diabetes 44, 1126e1131. Derewenda, U., Derewenda, Z., Dodson, E.J., Dodson, G.G., Reynolds, C.D., Smith, G.D., Sparks, C., Swenson, D., 1989. Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer. Nature 338, 594e596. Desbuquois, B., Carre´, N., Burnol, A.F., 2013. Regulation of insulin and type 1 insulin-like growth factor signalling and action by the Grb10/14 and SH2B1/B2 adaptor proteins. FEBS J. 280, 794e816. Dezaki, K., Sone, H., Yada, T., 2008. Ghrelin is a physiological regulator of insulin release in pancreatic islets and glucose homeostasis. Pharmacol. Therapeut. 118, 239e249. Drucker, D.J., 2001. Glucagon-like peptide 2. J. Clin. Endocr. Metab. 86, 1759e1764. Drucker, D.J., 2002. Biological actions and therapeutic potential of the glucagon-like peptides. Gastroenterology 122, 252e271. Drucker, D.J., 2013. Incretin action in the pancreas: potential promise, possible perils, and pathological pitfalls. Diabetes 62, 3316e3323. Drucker, D.J., Habener, J.F., Holst, J.J., 2017. Discovery, characterization and clinical development of the glucagon-like peptides. J. Clin. Invest. 127, 4217e4227. Duckworth, W.C., 1988. Insulin degradation: mechanisms, products, and significance. Endocr. Rev. 9, 319e345. Duckworth, W.C., Bennett, R.G., Hamel, F.G., 1998. Insulin degradation: progress and potential. Endocr. Rev. 19, 608e624. Dude, P.E., Brubaker, P.L., 2007. Frontiers in glucagon-like peptide 2: multiple actions, multiple mediators. Am. J. Physiol. Endocrinol. Metab. 293, E460eE465.

REFERENCES

Dupre, J., Ross, S.A., Watson, D., Brown, J.C., 1973. Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J. Clin. Endocrinol. Metab. 37, 826e828. Ebina, Y., Ellis, L., Jamagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y.W., Goldfine, I.D., Roth, R.A., Rutter, W.J., 1985. The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling. Cell 40, 747e758. Edgerton, D.S., Cherrington, A.D., 2013. Glucagon yin and yang effects on hepatic glucose production. Nat. Med. 19, 674e675. Elde, R., Ho¨kfelt, T., Johansson, O., Schultzberg, M., Efendic, S., Luft, R., 1978. Cellular localization of somatostatin. Metabolism 27 (Suppl. 1), 1151e1159. Elrick, M.M., Samson, W.K., Corbett, J.A., Salvatori, A.S., Stein, L.M., Kolar, G.R., Naatz, A., Yosten, G.L.C., 2016. Neuronostatin acts via GPR107 to increase cAMP-independent PKA phosphorylation and proglucagon mRNA accumulation in pancreatic a-cells. Am. J. Physiol. Regul. Integr. Comp. Physiol. 310, R143eR155. Emmanuelli, B., Peraldi, P., Filloux, C., Sawka-Verhelle, D., Hilton, D., Van Obberghen, E., 2000. SOCS3 is an insulininduced negative regulator of insulin signalling. J. Biol. Chem. 275, 15985e15991. Enuka, Y., Feldman, M.E., Yarden, Y., 2015. Computational and modeling aspects of RTK networks. In: Wheeler, D.L., Yarden, Y. (Eds.), Receptor Tyrosine Kinases: Structure, Function and Role in Human Disease. Humana Press Springer, New York; Heidelberg; Dordrecht; London, pp. 111e132. Esler, W.P., Rudolph, J., Claus, T.H., Tang, W., Barucci, N., Brown, S.E., Bullock, W., Daly, M., Decarr, L., Li, Y., Milardo, L., Molstad, D., Zhu, J., Gardell, S.J., Livingston, J.N., Sweet, L.J., 2007. Smallmolecule ghrelin receptor antagonists improve glucose tolerance, suppress appetite, and promote weight loss. Endocrinology 148, 5175e5185. Evans, M.R., Wei, S., Posner, B.A., Unger, R.H., Roth, M.G., 2015. J. Biomol. Screen 21, 325e332. Falkmer, S., 1995. Origin of the parenchymal cells of the endocrine pancreas: some phylogenetic and ontogenetic aspects. In: Mignon, M., Jensen, R.T. (Eds.), Endocrine Tumors of the Pancreas: Frontiers in Gastrointestinal Research. Karger, Basel, pp. 2e29. Federwisch, M., Dieken, M.L., De Meyts, P., 2002. Insulin and Related Proteins e Structure to Function and Pharmacology. Kluwer Academic Publishers, Dordrecht/Boston/London. Feliu, J.E., Hue, L., Hers, H.G., 1976. Hormonal control of pyruvate kinase activity and of gluconeogenesis in isolated hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 73, 2762e2766. Finan, B., Yang, B., Ottaway, N., Smiley, D.L., Ma, T., Clemmensen, C., Chabenne, J., Zhang, L., Habegger, K.M., Fischer, K., Campbell, J.E., Sandoval, D., Seeley, R.J., Bleicher, K., Uhles, S., Riboulet, W., Funk, J., Hertel, C., Belli, S., Sebokova, E., Conde-Knape, K., Konkar, A., Drucker, D.J., Gelfanov, V., Pfluger, P.T., Mu¨ller, T.D., Perez-Tilve, D., DiMarchi, R.D., Tscho¨p, M.H., 2015. A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents. Nat. Med. 21, 27e36. Freychet, P., Roth, J., Neville Jr., D.M., 1971. Insulin receptors in the liver: specific binding of 125I-insulin to the plasma membrane and its relation to insulin bioactivity. Proc. Natl. Acad. Sci. U.S.A. 68, 1833e1837. Friedman, J.E., Ishizuka, T., Liu, S., Farrell, C.T., Bedol, D., Koletsky, R.J., Kung, H.L., Einsberger, P., 1997. Reduced insulin receptor signaling in the obese spontaneously hypertensive Koletsky rat. Am. J. Physiol. 273, E1014eE1023. Furuta, M., Yano, H., Zhou, A., Rouille´, Y., Holst, J.J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H., Steiner, D.F., 1997. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc. Natl. Acad. Sci. U.S.A. 94, 6646e6651.

415

Furuta, M., Zhou, A., Webb, G., Carroll, R., Ravazzola, M., Orci, L., Steiner, D.F., 2001. Severe defect in proglucagon processing in islet A-cells of prohormone convertase 2 null mice. J. Biol. Chem. 276, 27197e27202. Gao, F., Harikumar, K.G., Dong, M., Lam, P.C.H., Sexton, P.M., Christopoulos, A., Bordner, A., Abagyan, R., Miller, L.J., 2009. Functional importance of a structurally distinct homodimeric complex of the family B G protein-coupled secretin receptor. Mol. Pharmacol. 76, 264e274. Gavin III, J.R., Roth, J., Neville Jr., D.M., De Meyts, P., Buell, D.N., 1974. Insulin-dependent regulation of insulin receptor concentration: a direct demonstration in cell culture. Proc. Natl. Acad. Sci. U.S.A. 71, 84e88. Gerich, J.E., 1979. In: Proc. 2nd Internat. Symposium on Hypoglycemia, Rome, January 1979. Academic Press London, New York. Gilman, A.G., 1984. G proteins and dual control of adenylate cyclase. Cell 36, 577e579. Gittes, G.K., 2009. Developmental biology of the pancreas. A comprehensive review. Dev. Biol. 326, 4e35. Glover, I., Haneef, I., Pitts, J., Wood, S., Moss, D., Tickle, I., Blundell, T., 1983. Conformational flexibility in a small globular hormone: X-ray ˚ resolution. Bioanalysis of avian pancreatic polypeptide at 0.98-A polymers 22, 293e304. Gosmain, Y., Cheyssac, C., Heddad Masson, M., Dibner, C., Philippe, J., 2011. Glucagon gene expression in the endocrine pancreas: the role of the transcription factor Pax6 in a-cell differenciation, glucagon biosynthesis and secretion. Diab. Obes. Metab. 13 (Suppl. 1), 31e38. Gravholt, C.H., Møller, N., Jensen, M.D., Christiansen, J.S., Schmitz, O., 2001. Physiological levels of glucagon do not influence lipolysis in abdominal adipose tissue as assessed by microdialysis. J. Clin. Endocrinol. Metab. 86, 2085e2089. Gribble, F.M., Reimann, F., 2016. Enteroendocrine cells: chemosensors in the intestinal epithelium. Annu. Rev. Physiol. 78, 277e299. Gromada, J., Franklin, I., Wollheim, C.B., 2007. A-cells of the endocrine pancreas: 35 years of research but the enigma remains. Endocr. Rev. 28, 84e116. Gromada, J., Chabosseau, P., Rutter, G.A., 2018. The a-cell in diabetes mellitus. Nat. Rev. Endocrinol. 14, 694e704. Gro¨nke, S., Clarke, D.-F., Broughton, S., Andrews, T.D., Partridge, L., 2010. Molecular evolution and functional characterisation of Drosophila insulin-like peptides. PLoS Genet. 6, e1000857. Guillemin, R., 2008. Somatostatin: the beginnings, 1972. In: Castan˜o, J.P., Ghizo, E., Kineman, R.D., de Lecea, L., Malago´n, M.M., Vaudry, H. (Eds.), Special issue: somatostatin, cortistatin and their receptors in health and disease. Mol. Cell. Endocrinol., 286, pp. 3e4. ¨ ., 2018. Gutmann, T., Kim, K.H., Grzybek, M., Walz, T., Coskun, U Visualization of ligand-induced transmembrane signalling in the full-length human insulin receptor. J. Cell Biol. 217, 1643e1649. Habegger, K.M., Heppner, K.M., Geary, N., Bartness, T.J., DiMarchi, R., Tscho¨p, M.H., 2010. The metabolic actions of glucagon revisited. Nat. Rev. Endocrinol. 6, 689e697. Haeusler, R.A., McGraw, T.E., Accili, D., 2018. Biochemical and cellular properties of insulin receptor signalling. Nat. Rev. Mol. Cell Biol. 19, 31e44. Hanoune, J., Defer, N., 2001. Regulation and role of adenylyl cyclase isoforms. Ann. Rev. Pharmacol. Toxicol. 41, 145e174. Harris, T.E., Lawrence Jr., J.C., 2003. TOR signalling. Sci. STKE 2003, RE 15. Hausen, B.M., 1988. Die Inseln des Paul Langerhans. In: Eine biographie in Bildern und Dokumenten. Ueberreuter Wissenschaft, Wien e Berlin.

416

17. PANCREATIC HORMONES

Havrankova, J., Roth, J., Brownstein, M., 1978. Insulin receptors are widely distributed in the central nervous system of the rat. Nature 272, 827e8829. Heinrich, G., Gros, P., Habener, J.F., 1984. Glucagon gene sequence. Four of the six exons encode separate functional domains of rat pre-proglucagon. J. Biol. Chem. 259, 14082e14087. Heintges, T., Lu¨then, R., Niederau, C., 1994. Inhibition of exocrine pancreatic secretion by somatostatin and its analogues. Digestion 55 (Suppl. 1), 1e9. Hellman, B., Salehi, A., Gylfe, E., Dansk, H., Grapengiesser, E., 2009. Glucose generates coincident insulin and somatostatin pulses and anti-synchronous pulses from human pancreatic islets. Endocrinology 150, 5334e5340. Hendrick, G.K., Wasserman, D.H., Frizzel, R.T., Williams, D.E., Lacy, D.B., Jaspan, J.B., Cherrington, A.D., 1992. Importance of basal glucagon in maintaining hepatic glucose production during a prolonged fast in conscious dogs. Am. J. Physiol. Endocrinol. Metab. 263, E541eE549. Henquin, J.C., Rahier, J., 2011. Pancreatic alpha cell mass in European subjects with type 2 diabetes. Diabetologia 54, 1720e1725. Hers, H.G., 1984. The discovery and the biological role of fructose 2,6bisphosphate. Biochem. Soc. Trans. 12, 729e735. Herzlinger, S., Horton, E.S., 2013. Extraglycemic effects of GLP-1-based therapeutics: addressing metabolic and cardiovascular risks associated with type 2 diabetes. Diabetes Res. Clin. Practice 100, 1e10. Hollenstein, K., de Graaf, C., Bortolato, A., Wang, M.W., Marshall, F.H., Stevens, R.C., 2014. Insights into the structure of class B GPCRs. Trends Pharmacol. Sci. 35, 12e22. Holst, J.J., Skak-Nielsen, T., Orskov, C., Seier-Poulsen, S., 1992. Vagal control of the release of somatostatin, vasoactive intestinal polypetide, gastrin-releasing peptide, and HCl from porcine non-antral stomach. Scand. J. Gastroenterol. 27, 677e685. Holst, J.J., Christensen, M., Lund, A., 2011. Regulation of glucagon secretion by incretins. Diabetes Obes. Metab. 13 (Suppl. 1), 89e94. Holst, J.J., Holland, W., Gromada, J., Lee, Y., Unger, R.H., Yan, H., Sloop, K.W., Kieffer, T.J., Diamond, N., Herrera, P.L., 2017. Insulin and glucagon: partners for life. Endocrinology 158, 696e701. House, P.D., Weidemann, M.J., 1970. Characterisation of a 125I-insulin binding plasma membrane fraction form rat liver. Biochem. Biophys. Res. Commun. 41, 541e548. Howard, J.K., Flier, J.S., 2006. Attenuation of leptin and insulin signalling by SOCS proteins. Trends Endocrinol. Metab. 17, 365e371. Howard, J.M., Hess, W., 2002. History of the Pancreas. Mysteries of a Hidden Organ. Kluwer Academic/Plenum Publishers, New York. Hoyne, P.A., Cosgrove, L.J., McKern, N.M., Bentley, J.D., Ivancic, N., Elleman, T.C., Ward, C.W., 2000. High affinity binding by soluble insulin receptor extracellular domain fused to a leucine zipper. FEBS Lett. 479, 15e18. Hua, Q.X., Shoelson, S.E., Kochoyan, M., Weiss, M.A., 1991. Receptor binding redefined by a structural switch in a mutant human insulin. Nature 354, 238e241. Huang, X.-Q., 1997. Somatostatin: likely the most widely effective gastrointestinal hormone in the human body. World J. Gastroenterol. 3, 201e204. Huang, S., Czech, M.P., 2007. The GLUT4 glucose transporter. Cell Metab. 5, 237e252. Huang, K., Chan, S.J., Hua, Q.X., Chu, Y.C., Wang, R.Y., Klaproth, B., Jia, W., Whittaker, J., De Meyts, P., Nakagawa, S.H., Steiner, D.F., Katsoyannis, P.G., Weiss, M.A., 2004. The A-chain of insulin contacts the insert domain of the insulin receptor: photocrosslinking and mutagenesis of a diabetes-related crevice. J. Biol. Chem. 282, 5337e5349. Hubbard, S.R., 1997. Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 16, 5572e5581.

Hubbard, S.R., 2013. The insulin receptor: both a prototypical and atypical receptor tyrosine kinase. Cold Spring Harbor Perspect. Biol. 5, a008946. Hubbard, S.R., Miller, W.T., 2007. Receptor tyrosine kinases: mechanisms of activation and signalling. Curr. Opin. Cell Biol. 19, 117e123. Hubbard, S.R., Wei, L., Ellis, L., Hendrickson, W.A., 1994. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 372, 746e754. Hue, L., Rider, M.H., 1987. Role of fructose 2,6-bisphosphate in the control of glycolysis in mammalian tissues. Biochem. J. 245, 313e324. Hume, R., Burchell, A., Williams, F.L., Koh, D.K., 2005. Glucose homeostasis in the newborn. Early Hum. Dev. 81, 95e101. Hurowitz, E.H., Melnyk, J.M., Chen, Y.J., Kouros-Mehr, H., Simon, M.I., Shizuya, H., 2000. Genomic characterization of the human heterotrimeric G protein alpha, beta and gamma subunit genes. DNA Res. 7, 111e120. Hutchens, T., Piston, D.W., 2015. Eph4 receptor forward signaling inhibits glucagon secretion from a-cells. Diabetes 64, 3839e3851. Hutton, J.C., 1994. Insulin secretory granule biogenesis and the proinsulin-processing endopeptidases. Diabetologia 37 (Suppl. 2), S48eS56. Irwin, D.M., 2001. Molecular evolution of proglucagon (2001). Regul. Pept. 98, 1e12. Jazayeri, A., Rappas, M., Brown, A.J.H., Kean, J., Errey, J.C., Robertson, N.J., Fiez-Vandal, C., Andrews, S.P., Congreve, M., Bortolato, A., Mason, J.S., Baig, A.H., Teobald, I., Dore´, A.S., Weir, M., Cooke, R.M., Marshall, F.H., 2017. Cryo-EM structure of the activated GLP-1 receptor bound to a peptide agonist. Nature 546, 254e258. Jeppesen, P.B., 2012. Teduglutide, a novel glucagon-like peptide 2 analog, in the treatment of patients with short bowel syndrome. Therap. Adv. Gastroenterol. 5, 159e171. Jepsen, M.R., Kløverpris, S., Mikkelsen, J.H., Pedersen, J.H., Fu¨chtbauer, E.-M., Laursen, L.S., Oxvig, C., 2015. Stanniocalcin-2 inhibits mammalian growth by proteolytic inhibition of the insulinlike growth factor axis. J. Biol. Chem. 290, 3430e3439. Joshi, R.L., Lamothe, B., Cordonnier, N., Mesbah, K., Monthioux, E., Jami, J., Bucchini, D., 1996. Targeted disruption of the insulin receptor gene results in neonatal lethality. EMBO J. 15, 1542e1547. Jospe, N., Kaplowitz, P.B., Furlanetto, R.W., 1996. Homozygous nonsense mutation in the insulin receptor gene in a patient with severe congenital insulin resistance, leprechaunism and the role of the insulin-like growth factor receptor. Clin. Endocrinol. 45, 229e255. Kasuga, M., Karlsson, F.A., Kahn, C.R., 1982a. Insulin stimulates the phosphorylation of the 95.000 dalton subunit of its own receptor. Science 215, 185e187. Kasuga, M., Zick, Y., Karlsson, F.A., Ha¨ring, H.U., Kahn, C.R., 1982b. Insulin stimulation of phosphorylation of the beta subunit of the insulin receptor. Formation of both phosphoserine and phosphotyrosine. J. Biol. Chem. 257, 9891e9894. Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D.L., Kahn, C.R., 1983. Tyrosine-speciific protein kinase activity is associated with the purified insulin receptor. Proc. Natl. Acad. Sci. U.S.A. 80, 2137e2141. Katsuura, G., Inui, A., 2016. Pancreatic polypeptide. In: Takei, Y., Ando, H., Tsutsui, K. (Eds.), Handbook of Hormones. Comparative Endocrinology for Basic and Clinical Research. Academic Press, pp. 213e214. Katz, E.B., Stenbit, A.E., Hatton, K., DePinho, R., Charron, M.J., 1995. Cardiac and adipose tissue abnormalities but not diabetes in mice deficient in GLUT4. Nature 377, 151e155.

REFERENCES

Kavran, J.M., McCabe, J.M., Byrne, P.O., Connache, M.K., Wang, Z., Ramek, A., Sarabipour, S., Shan, Y., Shaw, D.E., Hristova, K., Cole, P.A., Leahy, D., 2014. How IGF-I activates its receptor. eLife. https://doi.org/10.7554/eLife 03772. Khan, A.H., Pessin, J.E., 2002. Insulin regulation of glucose uptake: a complex interplay of intracellular signalling pathways. Diabetologia 45, 1475e1483. Kimball, C., Murlin, G., 1923. Aqueous extracts of pancreas III. Some precipitation reactions of insulin. J. Biol. Chem. 58, 337e348. Kimmel, J.R., Pollock, H.G., Hazelwood, R.L., 1968. Isolation and characterization of chicken insulin. Endocrinology 83, 1323e1330. Kimmel, J.R., Hayden, L.J., Pollock, H.G., 1975. Isolation and characterization of a new pancreatic polypeptide hormone. J. Biol. Chem. 250, 9369e9376. Kinoshita, K., Ozaki, N., Tagaki, Y., Murata, Y., Oshida, Y., Hayashi, Y., 2014. Glucagon is essential for adaptative thermogenesis in brown adipose tissue. Endocrinology 155, 3484e3492. Kirkegaard, P., Moody, A.J., Holst, J.J., Loud, F.B., Skov Olsen, P., Christiansen, J., 1982. Glicentin inhibits gastric acid secretion in the rat. Nature 297, 156e157. Kiselyov, V.V., Versteyhe, S., Gauguin, L., De Meyts, P., 2009. Harmonic oscillator model of the insulin and IGF1 receptor’s allosteric binding and activation. Mol. Sys. Biol. 5, 243. Knop, F.P., 2018. A gut feeling about glucagon. Eur. J. Endocrinol. 178, R267eR280. Knudsen, L., De Meyts, P., Kiselyov, V.V., 2011. Insight into the molecular basis for the kinetic differences between the two insulin receptor isoforms. Biochem. J. 440, 297e403. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kanagawa, K., 1999. Ghrelin is a growth hormone-releasing acylated peptide from stomach. Nature 402, 656e660. Kolterman, O.G., Gray, R.S., Griffin, J., 1981. Receptor and postreceptor defects contribute to the insulin resistance in non insulin dependent diabetes mellitus. J. Clin. Invest. 68, 957e969. Krook, A., Brueton, L., O’Rahilly, S., 1993. Homozygous nonsense mutation in the insulin receptor gene in infant with leprechaunism. Lancet 342, 277e278. Krulich, I., Dhariwal, A.P.S., McCann, S.M., 1968. Stimulatory and inhibitory effects of purified hypothalamic extracts on growth hormone release from rat pituitary in vitro. Endocrinology 83, 783e790. Kulkarni, R.N., Bru¨ning, J.C., Winnay, J.N., Postic, C., Magnuson, M.A., Kahn, C.R., 1999. Tissue-speciific knock-out of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell 96, 329e339. Kurose, T., Pashmforoush, M., Yoshimasa, Y., Carroll, R., Schwartz, G.P., Burke, G.T., Katsoyannis, P.G., Steiner, D.F., 1994. Crosslinking of a B25 azidophenylalanine insulin derivative to the carboxy-terminal region of the a-subunit of the insulin receptor. Biochemistry 43, 8356e8372. La Barre, J., 1932. Sur les possibilite´s d’un traitement du diabe`te par l’incre´tine. Bull. Acad. R. Med. Belg. 12, 620e634. Laguesse, E., 1893. Sur la formation des ˆılots de Langerhans dans le pancre´as. C.R. Soc. Biol. Paris 30, 819e820. Lamothe, B., Baudry, A., Desbois, P., Lamotte, L., Bucchini, D., De Meyts, P., Joshi, R.L., 1998. Genetic engineering in mice: impact on insulin signaling and action. Biochem. J. 335, 193e204. Lancereaux, E., 1880. Le diabe`te maigre, ses symptoˆmes, son e´volution, son prognostic et son traitement. Union Me´d. 29, 161. Lane, M.A., 1907. The cytological characters of the areas of Langerhans. Am. J. Anat. 7, 409e422. Langerhans, P., 1869. Beitra¨ge zur mikroskopischen Anatomie der Bauchspeicheldru¨se (Inaugural dissertation). G. Lange, Berlin. Larger, E., Wewer Albrechtsen, N.J., Hansen, L.H., Gelling, R.W., Capeau, J., Deacon, C.F., Madsen, O.D., Yakushiji, F., De Meyts, P., Holst, J.J., Nishimura, E., 2016. Pancreatic a-cell hyperplasia and

417

hyperglucagonemia due to a receptor splice mutation. Endocrinol Diabetes Metab Case Report 2016 pii:16-0081. Larsson, L.I., Sundler, F., Hakanson, R., Pollock, H.G., Kimmel, R., 1974. Localization of APP, a postulated new hormone, to a pancreatic endocrine cell type. Histochemistry 42, 377e382. Larsson, L.I., Sundler, F., Hakanson, R., 1975. Immunohistochemical localization of human pancreatic polypeptide (HPP) to a population of islet cells. Cell Tissue Res. 156, 167e171. Lee, Y., Wang, M.-Y., Du, X.Q., Charron, M., Unger, R.H., 2011. Glucagon receptor knock out prevents insulin-deficient type 1 diabetes in mice. Diabetes 60, 391e397. Lee, Y., Berglund, E.D., Wang, M.Y., Fu, X., Yu, X., Charron, M.J., Burgess, S.C., Unger, R.H., 2012. Metabolic manifestations of insulin deficiency do not occur without glucagon action. Proc. Natl. Acad. Sci. U.S.A. 109, 14972e14976. Lee, J., Miyazaki, M., Romeo, G.R., Shoelson, S.E., 2014. Insulin activation with transmembrane ligands. J. Biol. Chem. 289, 19769e19777. Lefe`bvre, P.J., 1983. Glucagon and adipose tissue lipolysis. In: Glucagon, I., Lefe`bvre, P.J. (Eds.), Handbook of Experimental Pharmacology, vol. 66/1. Springer-Verlag, Berlin; Heidelberg; New York; Tokyo, pp. 419e440. Lefe`bvre, P.J., 2011. Early milestones in glucagon research. Diabetes Obes. Metab. 13 (Suppl. 1), 1e4. Lefe`bvre, P.J., Luyckx, A.S., 1979. Glucagon and diabetes: a reappraisal. Diabetologia 16, 347e354. Lefe`bvre, P.J., Paolisso, G., Scheen, A.J., 1996. Pulsatility of glucagon. In: Glucagon III, , Lefe`bvre, P.J. (Eds.), Handbook of Experimental Pharmacology. Springer-Verlag, Berlin; Heidelberg; New York; Tokyo, pp. 105e113. Lefe`bvre, P.J., Paquot, N., Scheen, A.J., 2015. Inhibiting or antagonizing glucagon: making progress in diabetes care. Diabetes Obes. Metab. 17, 720e725. Leibiger, I.B., Leibiger, B., Berggren, P.O., 2008. Insulin signaling in the pancreatic beta cell. Annu. Rev. Nutr. 28, 233e251. Lemaire, K., Ravier, M.A., Schraenen, A., Creemers, J.W., Van de Plas, R., Granvic, M., Van Lommel, L., Waelkens, E., Chimienty, F., Rutter, G.A., Gilon, P., Int’Veld, P.A., Schuit, F.C., 2009. Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice. Proc. Natl. Acad. Sci. U.S.A. 106, 14872e14877. Lemmon, M.A., Schlessinger, J., 2010. Cell signalling by receptor tyrosine kinases. Cell 141, 117e1134. Levine, R., Goldstein, M., Klein, S., Huddelston, B., 1949. The action of insulin on the distribution of galactose in eviscerated and nephrectomised dogs. J. Biol. Chem. 179, 985e986. Lewis, K., Li, C., Perrin, M.H., Blount, A., Kunitake, K., Donaldson, C., Vaughan, J., Reyes, T.M., Gulyas, J., Fischer, W., Bilezikjian, L., Rivier, J., Sawchenko, P.E., Vale, W.W., 2001. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc. Natl. Acad. Sci. U.S.A. 98, 7570e7575. Li, C., Chen, P., Blount, A., Chen, A., Jamison, P.M., Rivier, J., Smith, M.S., Vale, W., 2003. Urocortin III is expressed in pancreatic beta cells and stimulates insulin and glucagon secretion. Endocrinology 144, 3216e3224. Li, H., Zhao, L., Singh, R., Ham, J.N., Fadoyu, D.O., Bean, L.J.H., Zhang, Y., Xu, Y., Xu, H.E., Gambello, M.J., 2018. The first pediatric case of glucagon receptor defect due to biallelic mutations in GCRG is identified by newborn screening of elevated arginine. Mol. Genet. Metab. Rep. 17, 46e52. Liang, Y.L., Khoshouei, M., Radjainia, M., Zhang, Y., Glukhova, A., Tarrasch, J., Thal, D.M., Furness, S.G.B., Christopoulos, G., Coudrat, T., Danev, R., Baumeister, W., Miller, L.J., Christopoulos, A., Kobilka, B.K., Wooten, D., Skiniotis, G.,

418

17. PANCREATIC HORMONES

Sexton, P.M., 2017. Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 546, 248e253. Limbird, L.E., De Meyts, P., Lefkowitz, R.J., 1975. Beta-adrenergic receptors: evidence for negative cooperativity. Biochem. Biophys. Res. Commun. 64, 1160e1168. Longuet, C., Sinclair, E.M., Maida, A., Baggio, L.L., Maziarz, M., Charron, M.J., Drucker, D.J., 2008. The glucagon receptor is required for the adaptative metabolic response to fasting. Cell Metab. 8, 359e371. Lonovics, J., Devitt, P., Watson, L.C., Rayford, P.L., Thompson, J.C., 1981. Pancreatic polypeptide. A review. Arch. Surg. 116, 1256e1264. Lou, M., Garrett, T.P., McKern, N.M., Hoyne, P.A., Epa, V.C., Bentley, J.D., Lovrecz, G.O., Cosgrove, J.L., frenkel, M.J., Ward, C.W., 2006. Crystal structure of the first three domains of the human insulin receptor reveals major differences from the IGF-1 receptor in the regions governing ligand specificity. Proc. Natl. Acad. Sci. U.S.A. 103, 12429e12434. Low, M.J., Otero-Corchon, V., Parlow, A.F., Ramirez, J.L., Kumar, U., Patel, Y.C., Rubinstein, M., 2001. Somatostatin is required for masculinization of growth hormone-regulated hepatic gene expression but not of somatic growth. J. Clin. Invest. 107, 1571e1580. Ludvigsen, S., Olsen, H.B., Kaarsholm, N.C., 1998. A structural switch in a mutant insulin exposes key residues for receptor binding. J. Mol. Biol. 279, 1e7. Luft, R., Efendic, S., Ho¨kfelt, T., Johansson, O., Arimura, A., 1974. Immunohistochemical evidence for the localization of somatostatin-like immunoreactivity in a cell population of the pancreatic islets. Med. Biol. 52, 428e430. Luyckx, A.S., Pirnay, F., Lefe`bvre, P.J., 1978. Effect of glucose on plasma glucagon and free fatty acids during prolonged exercise. Eur. J. Appl. Physiol. 39, 53e61. Mace, O.J., Tehan, B., Marshall, F., 2015. Pharmacology and physiology of gastrointestinal enteroendocrine cells. Pharmacol. Res. Persp. 3, e00155. Madsen, O.D., 2007. Pancreas phylogeny and ontogeny in relation to a “pancreatic stem cell”. C. R. Biol. 330, 534e537. Mahajan, A., et al., 2018a. Fine-mapping type 2 diabetes loci to singlevariant resolution using high-density imputation and islet-specific epigenome maps. Nat. Genet. 50, 1505e1513. Mahajan, A., et al., 2018b. Refining the accuracy of validated target identification through coding variant fine-mapping in type 2 diabetes. Nat. Genet. 50, 559e571. Mann, R., Wigglesworth, M.J., Donnelly, D., 2008. Ligand-receptor interactions at the parathyroid hormone receptors. Subtype binding selectivity is mediated via an interaction between residue 23 on the ligand and residue 41 on the receptor. Mol. Pharmacol. 74, 605e613. Maruyama, I.N., 2015. Activation of transmembrane cell-surface receptors via a common mechanism? The rotation model. Bioessays 37, 959e967. Mauvais-Jarvis, F., Fruman, D.A., Hirshman, M.F., Sakamoto, K., Goodyear, L.J., Iannacone, M., Accili, D., Cantley, L.C., Kahn, C.R., 2002. Reduced expression of the murine p85a subunit of phosphoinositide-3 kinase improves insulin signaling and ameliorates diabetes. J. Clin. Invest. 109, 141e149. McGarry, J.D., Foster, D.W., 1983. Glucagon and ketogenesis. In: Glucagon, I., Lefe`bvre, P.J. (Eds.), Handbook of Experimental Pharmacology, vol. 66/1. Springer-Verlag, Berlin; Heidelberg; New York; Tokyo, pp. 383e398. McKern, N.M., Lawrence, M.C., Streltsov, V.A., Lou, M.-Z., Adams, T.E., Lovrecz, G.O., Elieman, T.C., Richards, K.M., Bentley, J.D., Piling, P.A., Hoyne, P.A., Cartledge, K.A., Pham, T.M., Lewis, J.L., Sankovich, S.E., Stoichevska, V., Da Silva, E., Robinson, C.P., Frenkel, M.J., Sparrow, L.G., Fernley, A.T., Epa, V.C., Ward, C.W., 2006. Structure of the insulin

receptor ectodomain reveals a folded-over conformation. Nature 443, 218e221. McLeod, J.J.R., 1922. Source of insulin: study of effect produced on blood sugar by extracts of pancreas on principle islets of fishes. J. Metab. Res. 2, 149e172. Meier, J.J., Veldhuis, J.D., Butler, P.C., 2005. Pulsatile insulin secretion dictates systemic insulin deliveryby regulating hepatic insulin extraction in humans. Diabetes 54, 1649e1656. Meier, J.J., Kjems, L.L., Veldhuis, J.D., Lefe`bvre, P., Butler, P.C., 2006. Postprandial suppression of glucagon secretion depends on intact pulsatile insulin secretion: further evidence for the intraislet hypothesis. Diabetes 55, 1051e1056. Menge, B.A., Gru¨ber, L., Jørgensen, S.M., Deacon, C.F., Schmidt, W.E., Veldhuis, J.D., Holst, J.J., Meier, J.J., 2011. Loss of inverse relationship between pulsatile insulin and glucagon secretion in patients with type 2 diabetes. Diabetes 60, 2160e2168. Menting, J.G., Whittaker, J., Margetts, M.B., Whittaker, L., Kong, G.K., Smith, B.J., Watson, C.J., Zakova, L., Kletvikova, E., Jiracek, J., Chan, S.J., Steiner, D.F., Dodson, G.G., Brzozowski, A.M., Weiss, M.A., Ward, C.W., Lawrence, M.C., 2013. How insulin engages its primary binding site on the insulin receptor. Nature 493, 241e245. Menting, J.G., Yang, Y., Chan, S.J., Philips, N.B., Smith, B.J., Whittaker, J., Wickramasinghe, N.P., Whittaker, L.J., Pandyarajan, V., Wan, Z.I., Yadav, S.P., Carroll, J.M., Strokes, N., Roberts Jr., C.T., Ismail-Belgi, F., Milewski, W., Steiner, D.F., Chauhan, V.S., Ward, C.W., Weiss, M.A., Lawrence, M.C., 2014. Protective hinge in insulin opens to enable its receptor engagement. Proc. Natl. Acad. Sci. U.S.A. 111, E3395eE3404. Merrifield, R.B., Mosjov, S., 1983. The chemical synthesis of glucagon. In: Glucagon, I., Lefe`bvre, P.J. (Eds.), Handbook of Experimental Pharmacology, vol. 66/1. Springer Verlag, Berlin; Heidelberg; New York; Tokyo, pp. 23e35. Michael, M.D., Kulkarni, R.N., Postic, C., Previs, S.F., Shulman, G.I., Magnuson, M.A., Kahn, C.R., 2000. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol. Cell 6, 87e97. Michel, M.C., Beck-Sickinger, A., Cox, H., Doods, H.N., Herzog, H., Larhammer, D., Quirion, R., Schwartz, T., Westfall, T., 1998. XVI international union of pharmacology e Recommendations for the nomenclature of neuropeptide Y, peptide YY and pancreatic polypeptide receptors. Pharmacol. Rev. 50, 143e150. Mighiu, P.I., Yue, J.T., Filippi, B.M., Abraham, M.A., Chari, M., Lam, C.K., Yang, C.S., Christian, N.R., Charron, M., Lam, T.K., 2013. Hypothalamic glucagon signaling inhibits hepatic glucose production. Nat. Med. 19, 766e772. Miller, R.A., Birnbaum, M.J., 2016. Glucagon: acute actions on hepatic metabolism. Diabetologia 39, 1376e1381. Milligan, G., 2001. Oligomerization of G-protein-coupled receptors. J. Cell Sci. 14, 1265e1271. Misbin, R.I., O’Leary, D., Palkinnen, A., 1979. Insulin receptor binding in obesity: a reassessment. Science 205, 1003e1004. Møller, N., Bagger, J.P., Schmitz, O., Jørgensen, J.O., Ovesen, P., Møller, J., Alberti, K.G., Ørskov, H., 1995. Somatostatin enhances insulin-stimulated glusose uptake in the perfused human forearm. J. Clin. Endocrinol. 80, 1789e1793. Moore, B., Edies, E.S., Abram, J.H., 1906. On the treatment of diabetes mellitus by acid extract of duodenal mucous membranes. Biochem. J. 1, 28e38. Moore, E.E., Kuestner, R.E., Conklin, D.C., Whitmore, T.E., Downey, W., Buddle, M.M., Adams, R.L., Bell, L.A., Thompson, D.L., Wolf, A., Chen, L., Stamm, M.R., Grant, F.J., Lok, S., Ren, H., De Jongh, K.S., 1999. Stanniocalcin 2: characterization of the protein and its localization to human pancreatic alpha cells. Horm. Metab. Res. 31, 406e414.

REFERENCES

Morisset, J., 2015. Somatostatin. The Pancreapedia. Exocrine Pancreas Knowledge Base. https://doi.org/10.3998/panc.2015.43. www. pancreapedia.org. Morris, A.P., et al., 2012. Large scale association analysis provides insight into the genetic architecture and pathophysiology of type 2 diabetes. Nat. Genet. 44, 981e990. Mosjov, S., Heinrich, G., Wilson, I.B., Ravazzola, M., Orci, L., Habener, J., 1986. Preproglucagon gene expression in pancreas and intestine diversifies at the level of posttranslational processing. J. Biol. Chem. 261, 11880e11889. Mosthaf, L., Giako, K., Dull, T.J., Coussens, L., Ullrich, A., McClain, D.A., 1990. Functionally distinct insulin receptors generated by tissue specific alternative splicing. EMBO J. 9, 2409e2413. Mu¨ller, T.D., , et al.(57 authors), 2015. Ghrelin. Mol Metab 21, 437e460. Muller, T.D., Finan, B., Clemmensen, C., DiMarchi, R.D., Tscho¨p, M.H., 2017. The new biology and pharmacology of glucagon. Physiol. Rev. 97, 721e766. Murlin, J.R., Clough, H.D., Gibbs, C.B.F., Stokes, A.M., 1923. Aqueous extracts of the pancreas. I. Influence on the carbohydrate metabolism of depancreatized animals. J. Biol. Chem. 56, 253e296. Na¨ssel, D.R., Liu, Y., Luo, J., 2015. Insulin/IGF signalling and its regulation in Drosophila. Gen. Compar. Endocrinol. 221, 255e266. Neitzel, K.L., Hepler, K., 2006. Cellular mechanisms that determine selective RGS protein regulation of G protein-coupled signaling. Semin. Cell Dev. Biol. 17, 383e389. Neves, S.R., Ram, P.T., Iyengar, R., 2002. G protein pathways. Science 296, 1636e1639. Noske, A.B., Costin, A.J., Morgan, G.P., Marsh, B., 2008. Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets. J. Struct. Biol. 161, 298e313. Nunez, D.J., D’Alessio, D., 2017. Glucagon receptor as a drug target: a witche’s brew of eye of newt (peptides) and toe of frog (receptors). Diabetes Obes. Metab. 2017, 1e5. Oakie, A., Wang, R., 2018. b-cell receptor tyrosine kinases in controlling insulin receptor secretion and exocytic machinery: c-Kit and insulin receptor. Endocrinology 59, 3813e3821. Ogoshi, M., 2016. Amylin. In: Takei, Y., Ando, H., Tsutsui, K. (Eds.), Handbook of Hormones. Comparative Endocrinology for Basic and Clinical Research. Academic Press, pp. 245e246. Olefsky, J.M., 1976. Decreased insulin binding to adipocytes and circulating monocytes from obese subjects. J. Clin. Invest. 57, 1165e1172. Orci, L., Baetens, D., Dubois, M.P., Rufener, C., 1975. Evidence for the D-cell of the pancreas secreting somatostatin. Horm. Metab. Res. 7, 400e402. Pajvani, U.B., Accili, D., 2015. The new biology of diabetes. Diabetologia 58, 2459e2468. Paolisso, G., Scheen, A.J., Albert, A., Lefe`bvre, P.J., 1989. Effects of pulsatile delivery of insulin and glucagon in humans. Am. J. Physiol. Endocrinol. Metab. 257, E686eE696. Paolisso, G., Buonocore, S., Gentile, S., Sgambado, S., Varrichio, M., Scheen, A., D’Onofrio, F., Lefe`bvre, P.J., 1990. Pulsatile glucagon has greater hyperglycemic, lipolytic and ketogenic effects than continuous hormone delivery in man: effect of age. Diabetologia 33, 272e277. Paquot, N., Schneiter, P., Je´quier, E., Gaillard, R., Lefe`bvre, P.J., Scheen, A., Tappy, L., 1996. Effects of ingested fructose and infused glucagon on endogenous glucose production in obese NIDDM patients, obese non-diabetic subjects, and healthy subjects. Diabetologia 39, 580e586. Parker, J.C., Andrews, K.M., Allen, M.R., Stock, J.L., McNeish, J.D., 2002. Glycemic control in mice with targeted disruption of the glucagon receptor gene. Biochem. Biophys. Res. Commun. 290, 839e843.

419

Parthier, C., Reedtz-Runge, S., Rudolph, R., Stubbs, M.T., 2009. Passing the baton in class B GPCRs: peptide hormone activation via helix induction? Trends Biochem. Sci. 34, 303e310. Patzelt, C., Schiltz, E., 1984. Conversion of proglucagon in pancreatic alpha cells: the major endproducts are glucagon and a single peptide, the major proglucagon fragment, that contains two glucagonlike sequences. Proc. Natl. Acad. Sci. U.S.A. 81, 5007e5011. Pawson, T., 2004. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116, 191e203. Pearson, D., Shively, J.E., Clark, B.R., Geschwind II, Barkley, M., Nishioka, R.S., Bern, H.A., 1980. Urotensin II: a somatostatin-like peptide in the caudal neurosecretory system of fishes. Proc. Natl. Acad. Sci. U.S.A. 77, 5021e5024. Petersen, M.C., Shulman, G.I., 2018. Mechanisms of insulin action and insulin resistance. Physiol. Rev. 98, 2133e2223. Petruzelli, L.M., Ganguly, S., Smith, C.J., Cobb, M.H., Rubin, C.S., Rosen, O.M., 1982. Insulin activates a tyrosine-specific protein kinase in extracts of 3T3-L1 adipocytes and human placenta. Proc. Natl. Acad. Sci. U.S.A. 79, 6792e6796. Philippe, J., 1991. Structure and pancreatic expression of the insulin and glucagon genes. Endocr. Rev. 12, 252e271. Phillips, L.K., Prins, J.B., 2011. Update on incretin hormones. Ann. N.Y. Acad. Sci. 1243, E55eE74. Posner, B.I., Kelly, P.A., Shiu, R.P.C., Friesen, H.G., 1974. Studies of insulin, growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinology 95, 321e531. Pradayrol, L., Jornvall, H., Mutt, V., Ribet, A., 1980. N-terminally extended somatostatin: the primary structure of somatostatin-28. FEBS Lett. 109, 55e58. Psiachou, H., Mitton, S., Alaghband-Zadeh, J., Hone, J., Taylor, S.I., Sinclair, L., 1993. Leprechaunism and homozygous nonsense mutation in the insulin receptor gene. Lancet 342, 924. Pujadas, G., Drucker, D.J., 2016. Vascular biology of glucagon receptor superfamily peptides: mechanistic and clinical relevance. Endocrine Rev. 37, 554e583. Ramnanan, C.J., Edgerton, D.S., Kraft, G., Cherrington, A.D., 2011. Physiologic action of glucagon on liver glucose metabolism. Diabetes Obes. Metab. 13 (Suppl. 1), 118e125. Ravishandran, K.S., 2001. Signaling via Shc family adapter proteins. Oncogene 20, 6322e6330. Rhodes, C.J., Halban, P.A., 1987. Newly synthesised proinsulin/insulin and stored insulin are released from pancreatic b cells predominantly via a regulated, rather than a constitutive, pathway. J. Cell Biol. 105, 145e153. Ro, C., Chai, W., Yu, V.E., Yu, R., 2013. Pancreatic neuroendocrine tumors: biology, diagnosis and treatment. Chinese J. Cancer 32, 312e324. Robison, G.A., Butcher, R.W., Sutherland, E.W., 1968. Cyclic AMP. Annu. Rev. Biochem. 37, 149e174. Rodbell, M., 1980. The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 284, 17e22. Roed, S.N., Orgaard, A., Jorgensen, R., De Meyts, P., 2012. Receptor oligomerization in family B1 of G-protein-coupled receptors: focus on BRET investigations and the link between GPCR oligomerization and binding cooperativity. Front. Endocrinol. 3, 62. https:// doi.org/10.3389/fendo.2012.00062. Rorsman, P., Ashcroft, F.M., 2018. Pancreatic b cell electrical activity and insulin secretion: of mice and men. Physiol. Rev. 98, 117e214. Rorsman, P., Braun, M., 2013. Regulation of insulin secretion in human pancreatic islets. Annu. Rev. Physiol. 75, 155e179. Rorsman, P., Huising, M.O., 2018. The somatostatin-secreting pancreatic d-cell in health and disease. Nat. Rev. Endocrinol. 14, 404e414. Ross, B., McKendy, K., Glaid, A., 2010. Role of urotensin II in health and disease. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298, R1156eR1172.

420

17. PANCREATIC HORMONES

Rowland, K.J., Brubaker, P.E., 2011. The “cryptic” mechanism of action of glucagon-like peptide 2. Am. J. Physiol. Gastrointest. Liver Physiol. 301, G1eG8. Ryan, G.J., Jobe, L.J., Martin, R., 2005. Pramlintide in the treatment of diabetes mellitus. BioDrugs 22, 375e386. Sainsbury, A., Shi, Y.C., Zhang, L., Aljanova, A., Lin, Z., Nguyen, A.D., Herzog, H., Lin, S., 2010. Y4 receptors and pancreatic polypeptide regulate food intake via hypothalamic orexin and brain-derived neurotropic factor dependent pathways. Neuropeptides 44, 261e268. Salvatori, A.S., Elrick, M.M., Samson, W.K., Corbett, J.A., Yosten, G.L., 2014. Neuronostatin inhibits glucose-stimulated insulin secretion via direct action on the pancreatic a cell. Am. J. Physiol. Endocrinol. Metab. 306, E1257eE1263. Samols, E., Weir, G.C., Bonner-Weir, S., 1983. Intra-islet insulinglucagon somatostatin relationships. In: Glucagon, I.I., Lefe`bvre, P.J. (Eds.), Handbook of Experimental Pharmacology, vol. 66/1. Springer-Verlag, Berlin; Heidelberg; New York; Tokyo, pp. 133e173. Samson, W.K., Zhang, J.V., Avsian-Kretchmer, O., Cui, K., Yosten, G.L., Klein, C., Lyn, R.M., Wang, Y.X., Chen, X.Q., Yang, J., Price, C.J., Hoyda, T.D., Ferguson, A.V., Yuan, X.B., Chang, J.K., Hsueh, A.J., 2008. Neuronostatin encoded by the somatostatin gene regulates neuronal, cardiovascular, and metabolic functions. J. Biol. Chem. 283, 31949e31959. Sandoval, D.A., D’Alessio, D.A., 2015. Physiology of proglucagon peptides: role of glucagon and GLP-1 in health and disease. Physiol. Rev. 95, 513e548. Sanger, F., 1988. Sequences, sequences, and sequences. Ann. Rev. Biochem. 57, 1e28. Sano, H., Kane, S., Sano, E., Miner, C.P., Asara, J.M., Lane, W.S., Garner, C.W., Lienhard, G.E., 2003. Insulin-stimulated phosphorylation of Rab GTPase-activating protein regulates GLUT4 translocation. J. Biol. Chem. 278, 14599e14602. Sasaki, K., Dockerill, S., Adamiak, D.A., Tickle, I.J., Blundell, T.L., 1975. X-ray analysis of glucagon and its relationship to receptor binding. Nature 257, 751e757. Scapin, G., Dandey, V.P., zhang, Z., Prosise, W., Hruza, A., Kelly, T., Mayhood, T., Strickland, C., Potter, C.S., Carragher, B., 2018. Structure of the insulin receptor complex by single-particle cryo-EM analysis. Nature 556, 122e125. Scha¨ffer, L., 1994. A model for insulin binding to the insulin receptor. Eur. J. Biochem. 221, 1127e1132. Scheen, A.J., 2018. Cardiovascular safety of DPP-4 inhibitors compared with sulphonylureas: results of randomized control trials and observational studies. Diabetes Metab. 44, 386e392. Scheen, A.J., Paquot, N., 2015. Obesity. A new paradigm for treating obesity and diabetes mellitus. Nat. Rev. Endocrinol. 11, 196e198. Scheen, A.J., Paquot, N., Lefe`bvre, P.J., 2017. Investigational glucagon receptor antagonists in phase I and II clinical trials for diabetes. Expert Opin. Invest. Drugs 26, 1373e1389. Scholdager, B.T.G., Baldissera, F.G.A., Mortensen, P.E., Holst, J.J., Christiansen, J., 1988. Oxyntomodulin: a potential hormone from the distal gut. Pharmacokinetics and effects on gastric acid and insulin secretion in man. Eur. J. Clin. Invest. 18, 499e503. Scott, R.A., et al., 2017. An expanded genome-wide association study of type 2 diabetes in Europeans. Diabetes 66, 2888e2902. Seino, S., Bell, G.I., 1989. Alternative splicing of human insulin receptor messenger RNA. Biochem. Biophys. Res. Comm. 159, 312e316. Seino, S., Seino, M., Nishi, S., Bell, G.I., 1989. Structure of the human insulin receptor gene and characterisation of its promoter. Proc. Natl. Acad. Sci. U.S.A. 86, 114e118. Semple, R.K., Savage, D.B., Brierly, G.V., O’Rahilly, S., 2016. Syndromes of severe insulin resistance and/or lipodystrophy. In: Weiss, R.E.,

Refetoff, S. (Eds.), Genetic Diagnosis of Endocrine Disorders, second ed. Acedemic Press, Cambridge, MA, pp. 307e324. Sesti, G., Federici, M., Hribal, M.L., Lauro, D., Sbraccia, P., Lauro, R., 2001. Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. FASEB J. 15, 2099e2111. Shanghai protein synthesis group, 1975. Total synthesis of crystalline glucagon by the method of solid-phase condensation of fragments. Sci Sinica 18, 745e768. Shen, Y., Jiachimiak, A., Rosner, M.R., Tang, W.J., 2006. Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature 443, 870e874. Sheperd, P., Withers, D.J., Siddle, K., 1998. Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. Biochem. J. 333, 471e490. Sherwood, N.M., Krueckl, S.L., McRory, J.E., 2000. The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocr. Rev. 21, 619e670. Sinclair, E.M., Yusta, B., Streutker, C., Baggio, L.L., Kohler, J., Charron, M., Drucker, D.J., 2008. Glucagon receptor signaling bis essential for control of murine hepatocyte survival. Gastroenterology 135, 2096e2106. Siu, F.Y., He, M., de Graaf, C., Han, G.W., Yang, D., Zhang, Z., Zhon, C., Xu, Q., Wacker, D., Joseph, J.S., Liu, W., Lau, J., Cherezov, V., Katritch, V., Wang, M.W., Stevens, R.C., 2013. Structure of the human glucagon class B G-protein-coupled receptor. Nature 499, 444e449. Smith, B.J., Huang, K., Kong, G., Chan, S.J., Nakagawa, S., Menting, J.G., Hu, S.-Q., Whittaker, J., Steiner, D.F., Kaatsoyannis, P.G., Ward, C.W., Weiss, M.A., Lawrence, M.C., 2010. Structural resolution of a tandem hormone-binding element in the insulin receptor and its implication for the design of peptide agonists. Proc. Natl. Acad. Sci. U.S.A. 107, 6771e6776. Soll, A.H., Kahn, C.R., Neville Jr., D.M., 1975a. Insulin binding to liver plasma membranes in the obese hyperglycemic (ob/ob) mouse. Demonstration of a decreased number of functionally normal receptors. J. Biol. Chem. 250, 4702e4707. Soll, A.H., Kahn, C.R., Neville Jr., D.M., Roth, J., 1975b. Insulin receptor deficiency in genetic and acquired obesity. J. Clin. Invest. 56, 769e780. Sonne, O., 1988. Receptor-mediated endocytosis and degradation of insulin. Physiol. Rev. 68, 1129e1196. Soos, M.A., Siddle, K., Baron, M.D., Heward, J.M., Luzio, P., Bellatin, J., Lennox, E.S., 1986. Monoclonal antibodies reacting with multiple epitopes of the insulin receptor. Biochem. J. 235, 199e208. Soos, M., Field, C.E., Siddle, K., 1993. Purified hybrid insulin/insulinlike growth factor-I receptors bind insulin-like growth factor-I, but not insulin, with high affinity. Biochem. J. 290, 419e426. Springael, J.Y., Le Minh, P.N., Urizar, E., Costagliola, S., Vassart, G., Parmentier, M., 2006. Allosteric modulation of binding properties between units of chemokine receptors homo- and heterooligomers. Mol. Pharmacol. 69, 1652e1661. Stadtmauer, L.A., Rosen, O.M., 1983. Phosphorylation of exogenous substrates by the insulin receptor-asociated protein kinase. J. Biol. Chem. 258, 6682e6685. Stalmans, W., 1983. Glucagon and liver glycogen metabolism. In: Glucagon, I., Lefe`bvre, P.J. (Eds.), Handbook of Experimental Pharmacology, vol. 66/1. Springer-Verlag, Berlin; Heidelberg; New York; Tokyo, pp. 291e314. Staub, A., Sinn, L., Behrens, O.K., 1955. Purification and crystallization of glucagon. J. Biol. Chem. 214, 619e632. Steenberg, V.R., Jensen, S.M., Pedersen, J., Madsen, A.N., Windeløv, J.A., Holst, B., Quistorff, B., Poulsen, S.S., Holst, J.J., 2016. Acute disruption of glucagon secretion or action does not improve glucose tolerance in an insulin-deficient mouse model of diabetes. Diabetologia 59, 363e370.

REFERENCES

Steiner, D.F., Oyer, P.E., 1967. The biosynthesis of insulin and a probable precursor of insulin by a human islet cell adenoma. Proc. Natl. Acad. Sci. U.S.A. 57, 473e480. Steiner, D.F., Cunningham, D., Spigelman, L., Aten, B., 1967. Insulin biosynthesis: evidence for a precursor. Science 157, 697e700. Steiner, D.F., Clark, J.L., Nolan, C., Rubenstein, A.H., Margoliash, E., Aten, B., Oyer, P.E., 1969. Proinsulin and the biosynthesis of insulin. Recent Prog. Horm. Res. 25, 207e282. Steiner, D.F., Kim, A., Miller, K., Hara, M., 2010. Pancreatic islets plasticity: interspecies comparison of islet architecture and composition. Islets 2, 135e145. Strowski, M.Z., Blake, A.D., 2008. Function and expression of somatostatin receptors of the endocrine pancreas. Special issue: somatostatin, corticostatin and their receptors in health and disease. In: Castan˜o, J.P., Ghizo, E., Kineman, R.D., de Lecea, L., Malago´n, M.M., Vaudry, H. (Eds.), Special issue: somatostatin, cortistatin and their receptors in health and disease. Mol. Cell. Endocrinol., 286, pp. 169e179. Sun, L., Coy, D.H., 2016. Somatostatin and its analogs. Curr. Drug Targets 17, 529e537. Sun, X.J., Rothenberg, P., Kahn, C.R., Backer, J.M., Araki, E., Wilden, P.A., Cahill, D.A., Goldstein, B.J., White, M.F., 1991. Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature 352, 73e77. Sutherland, E.W., de Duve, C., 1948. Origin and distribution of the hyperglycemic-glucogenolytic factor of the pancreas. J. Biol. Chem. 175, 663e674. Su¨veges, D., Jura, N., 2015. Structural features of the kinase domain. In: Wheeler, D.L., Yarden, Y. (Eds.), Receptor Tyrosine Kinases: Structure, Functions and Role in Human Disease. Humana Press Springer, New York; Heidelberg; Dordrecht; London, pp. 195e223. Suzuki, N., 2016. Calcitonin/calcitonin gene related peptide family. In: Takei, Y., Ando, H., Tsutsui, K. (Eds.), Handbook of Hormones. Comparative Endocrinology for Basic and Clinical Research. Academic Press, pp. 230e231. Svendsen, A.M., Vrecl, M., Ellis, T.M., Heding, A., Kristensen, J.B., Wade, J.D., Bathgate, R.A.D., De Meyts, P., Nohr, J., 2008a. Cooperative binding of insulin-like peptide 3 to a dimeric relaxin family peptide receptor 2. Endocrinology 149, 1113e1120. Svendsen, A.M., Zalesko, A., Konig, J., Vrecl, M., Heding, A., Kristensen, J.B., Wade, J.D., Bathgate, R.A.D., De Meyts, P., Nohr, J., 2008b. Negative cooperativity in H2 relaxin binding to a dimeric relaxin family peptide receptor 1. Mol. Cell. Endocrinol. 296, 10e17. Svendsen, A.M., Winge, S.B., Zimmermann, M., Lindvig, A.B., Warzecha, C.B., Sajid, W., Horne, M.C., De Meyts, P., 2014. Downregulation of cyclin G2 by insulin, IGF-I (insulin-like growth factor 1) and X10 (Asp B10 insulin): role in mitogenesis. Biochem. J. 457, 69e77. Svoboda´, P., Teisinger, J., Novotny´, J., Bourova´, L., Drmota, T., Hejnova´, L., Moravcova´, Z., Lisy´, V., Rudajev, V., Sto¨hr, J., Vokurkova´, A., Svandova´, I., Durcha´nkova´, D., 2004. Biochemistry of transmembrane signaling mediated by trimeric G proteins. Physiol. Rev. 53 (Suppl. 1), S141eS152. Takei, Y., 2016. Neuropeptide Y family. In: Takei, Y., Ando, H., Tsutsui, K. (Eds.), Handbook of Hormones. Comparative Endocrinology for Basic and Clinical Research. Academic Press, pp. 211e212. Talchai, C., Xuan, S., Lin, H.V., Sussel, L., Accili, D., 2012. Pancreatic beta cell dedifferentiation as a mechanism of diabetic beta cell failure. Cell 150, 1223e1234. Tamimoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakava, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekiraha, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yasaki, Y., Aizawa, S., 1994. Insulin resistance and

421

growth retardation in mice lacking insulin receptor substrate 1. Nature 372, 182e186. Tan, T., Bloom, S.R., 2013. Gut hormones as therapeutic agents in treatment of diabetes and obesity. Curr. Opin. Pharmacol. 13, 996e1001. Tan, T.M., Field, B.C., McCullough, K.A., Troke, R.C., Chambers, E.S., Salem, V., Gonzales Maaffe, J., Baynes, K.C., De Silva, A., Viardot, A., Alsafi, A., Frost, G.S., Ghatei, M., Bloom, S.R., 2013. Coadministration of glucagon-like peptide-1 during glucagon infusion in man results in increased energy expenditure and amelioration of hyperglycemia. Diabetes 62, 1131e1138. Tanese, T., Lazarus, N.R., Devrim, S., Recant, L., 1970. Synthesis and release of proinsulin and insulin by isolated rat islets of Langerhans. J. Clin. Invest. 49, 1394e1404. Taniguchi, C.M., Emmanuelli, B., Kahn, C.R., 2006. Critical nodes in signaling pathways: insights into insulin action. Nat. Rev. Mol. Cell Biol. 7, 85e96. Tarauchi, Y., Satoh, S., Minowa, H., Murakami, K., Okuno, A., Inukai, K., Asano, T., Kaburagi, Y., Ueki, K., Nakajima, H., Hanafusa, T., Matsurawa, Y., Sekiraha, H., Yin, Y., Barrett, J.C., Oda, H., Ishikawa, T., Akahuma, Y., Kimuro, I., Suzuki, M., Yamamura, K., Kodama, T., Suzuki, H., Koyasu, S., Aizawa, S., Tobe, K., Fukui, Y., Yasaki, Y., Kadowaki, T., 1998. Increased insulin sensitivity and hypoglycemia in mice lacking the p85a subunit of phosphoinosiitide-3 kinase. Nature Genet. 21, 230e235. Taylor, S.I., 1992. Lilly lecture: molecular mechanisms of insulin resistance: lessons from patienets with mutations in the insulin receptor gene. Diabetes 41, 1473e1490. Terris, S., Hofmann, C., Steiner, D.F., 1979. Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells. Can. J. Biochem. 57, 459e468. Thorel, F., Ne´pote, V., Avril, I., 2010. Conversion of adult pancreatic a-cells to b-cells after extreme b-cell loss. Nature 464, 1149e1154. Thorens, B., 2011. Brain glucose sensing and neural regulation of insulin and glucagon secretion. Diabetes Obes. Metab. 13 (Suppl. 1), 89e94. Tian, G., Sandler, G., Gylfe, E., Tengholm, A., 2011. Glucose and hormone-induced cAMP oscillations in a- and b-cells within pancreatic islets. Diabetes 60, 1535e1543. Tokarz, V.L., MacDonald, P.E., Klip, A., 2018. The cell biology of systemic insulin function. J. Cell Biol. 217, 2273e2289. Tomasetto, C., Karam, S.M., Ribieras, S., Masson, R., Lefe`bvre, O., Staub, A., Alexander, G., Chenard, M.P., Rio, M.C., 2000. Identification and characterization of a novel gastric peptide hormone: the motilin-related peptide. Gastroenterology 119, 395e405. Tostivint, H., Ocampo Daza, D., Bergqvist, C.A., Quan, F.B., Bougerol, M., Lihrmann, I., Larhammar, D., 2014. Molecular evolution of GPCRs: somatostatin/urotensin II receptors. J. Mol. Endocrinol. 52, T61eT86. Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzelli, L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O.M., Ramachandran, J., 1985. Human insulin receptor and its relationship to the tyrosine family of oncogenes. Nature 313, 756e761. Unger, R.H., Cherrington, A.D., 2012. Glucagonocentric restructuring of diabetes: a pathophysiologic and therapeutic makeover. J. Clin. Invest. 122, 4e12. Unger, R.H., Orci, L., 2010. Paracrinology of islets and the paracrinopathy of diabetes. Proc. Natl. Acad. Sci. U.S.A. 107, 16009e16012. Unger, R.H., Eisentraut, A.M., McCall, M.S., Keller, S., Lanz, H.C., Madison, L.L., 1959. Glucagon antibodies and their use for immunoassay for glucagon. Proc. Soc. Exp. Biol. Med. 102, 621e623. Urizar, E., Montanelli, L., Loy, T., Bonomi, M., Swillens, S., Gales, C., Bouvier, M., Smits, G., Vassart, G., Costagliola, S., 2005. Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity. EMBO J. 24, 1954e1964.

422

17. PANCREATIC HORMONES

Vainio, L., Perjes, A., Ryti, N., Magga, A., Alakoski, T., Serpi, R., Kaikkonen, L., Piuhola, J., Szokodi, L., Ruskoaho, H., Kerkela, R., 2012. Neuronostatin, a novel peptide encoded by the somatostatin gene, regulates cardiac contractile fonction and cardiomyocyte survival. J. Biol. Chem. 287, 4572e4580. Van der Meulen, T., Xie, R., Kelly, O.G., Vale, W.W., Sander, M., Huising, M.O., 2012. Urocortin 3 marks mature human primary and embryonic stem cell-derived pancreatic alpha and beta cells. PLoS One 7 (12), e52181. Vinik, A., Feliberti, E., Perry, R.R., 2017. Pancreatic polypeptide (PPoma). In: De Groot, L.J., et al. (Eds.), Endotext [Internet]. MDText.com, Inc, South Darthmouth, MA, 2000. Viollet, C., Vaillend, C., Videau, C., Bluet-Pajot, M.T., Ungerer, A., L’He´ritier, A., Kopp, C., Potier, B., Billard, J., Schaeffer, J., Smith, R.G., Rohrer, S.P., Wilkinson, H., Zheng, H., Epelbaum, J., 2000. Involvement of sst2 somatostatin receptor in locomotor, exploratory activity and emotional reactivity in mice. Eur. J. Neurosci. 12, 3761e3770. Von Mering, J., Minkowski, O., 1890. Diabetes mellitus nach Pankreasexstirpation. Arch. Exp. Pathol. Pharm. 26, 371e387. Vorwerk, P., Christoffersen, C.T., Mu¨ller, J., Vestergaard, H., Pedersen, O., De Meyts, P., 1999. Alternative splicing of exon 17 and a missense mutation in exon 20 of the insulin receptor gene in two siblings with a novel syndrome of insulin resistance (congenital fiber-type disproportion myopathy). Horm. Res. 52, 211e220. Walker, J.N., Ramracheva, R., Zhang, Q., Johnson, P.R., Braun, M., Rorsman, P., 2011. Regulation of glucagon secretion by glucose: paracrine, intrinsic or both? Diabetes Obes. Metab. 13 (Suppl. 1), 95e105. Ward, C.W., Lawrence, M.C., 2011. Landmarks in insulin research. Front. Endocrinol. https://doi.org/10.3389/fendo.2011.00076. Ward, C.W., Menting, J.G., Lawrence, M.C., 2013. The insulin receptor changes conformation in unforeseen ways on ligand binding: sharpening the picture of insulin receptor activation. Bioessays 35, 945e954. Weis, F., Menting, J.G., Margetts, M.B., Chan, S.J., Xu, Y., Tennagels, N., Wohlfart, P., Langer, T., Mu¨ller, C.W., Dreyer, M.K., Lawrence, M.C., 2018. The signaling conformation of the insulin receptor ectodomain. Nat. Commun. 9, 4420. Weiss, M.A., Lawrence, M.C., 2018. A thing of beauty: structure and function of insulin’s “aromatic triplet”. Diabetes Obes. Metab. 20 (Suppl. 2), 51e63. Weiss, M., Steiner, D.F., Philipson, L.H., 2014. Insulin biosynthesis, secretion, structure and structure-activity relationships. In: De Groot, L.J., et al. (Eds.), Endotext [Internet]. MDText.com, Inc, South Darthmouth (MA), 2000. Wermers, R.A., Fatoucheri, V., Wynne, A.G., Kvols, L.K., Lloyd, R.V., 1996. The glucagonoma syndrome. Clinical and pathologic features in 21 patients. Medicine (Baltim.) 75, 53e63. Wertheimer, E., Lu, S.P., Backeljauw, P.F., Davenport, M.L., Taylor, S.I., 1993. Homozygous deletion of the human insulin receptor results in leprechaunism. Lancet 342, 277e278. Westermark, P., 2011. Amyloid in the islets of Langerhans: thoughts and some historical aspects. Ups. J. Med. Sci. 116, 81e89. Westermark, P., Wernstedt, C., Wilander, E., Haayden, D.W., O’Brien, T.D., Johnson, K.K., 1987. Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells. Proc. Natl. Acad. Sci. U.S.A. 84, 3881e3885. Wewer Albrechtsen, N.J., Faerch, K., Jensen, T.M., Witte, D.R., Pedersen, J., Mahendran, Y., Jonsson, A.E., Galsgaard, K.D., Winther-Sørensen, M., Torekov, S.S., Lauritzen, T., Pedersen, O., Knop, F.K., Hansen, T., Jørgensen, M.E., Vistisen, D., Holst, J.J., 2018. Evidence of a liver-alpha cell axis in humans: hepatic insulin

resistance attenuates relationships between fasting plasma glucagon and glucagonotropic amino acids. Diabetologia 61, 671e680. White, M.F., 1998. The IRS signalling system: a network of docking proteins that mediate insulin action. Mol. Cell. Biochem. 182, 3e11. White, M.F., 2003. Insulin signaling in health and disease. Science 302, 1710e1711. White, M.F., 2012. Mechanisms of insulin action. In: Skyler, J.S. (Ed.), Atlas of Diabetes, fourth ed. Springer ScienceþBusiness Media LLC. https://doi.org/10.1007/978-1-4614-1028-7_2. White, M.F., Copps, K.D., 2016. The mechanisms of insulin action. In: Jameson, J.L., De Groot, L., de Kretser, D.M., Giudice, L.C., Grossman, A.B., Melmed, S., Potts Jr., J.T., Weir, G.C. (Eds.), Endocrinology, Adult and Pediatric, seventh ed., vol. 1. Elsevier Saunders, pp. 556e585. White, J.W., Saunders, G.F., 1986. Structure of the human glucagon gene. Nucl. Acid Res. 14, 4719e4730. Whittaker, L., Hao, C., Fu, W., Whittaker, J., 2008. High affinity binding: insulin interacts with two receptor ligand binding sites. Biochemistry 47, 12900e12909. Whittaker, J., Whittaker, L.J., Roberts Jr., C.T., Phillips, N.B., IsmailBeigi, F., Lawrence, M.C., Weiss, M.A., 2012. Proc. Natl. Acad. Sci. U.S.A. 109, 11166e11171. Wierup, N., Svensson, H., Mulder, H., Sundler, F., 2002. The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas. Regul. Peptides 107, 63e69. Wierup, N., Sundler, F., Heller, R.S., 2013. The islet ghrelin cells. J. Mol. Endocrinol. 52, R35eR49. Williams, J.A., 2014. Pancreatic polypeptide. Pancreapedia. Exocrine Pancreas Knowledge Base. https://doi.org/10.3998/panc.2014.4. www.pancreapedia.org. Withers, D., Guttierez, J.S., Towery, H., Burks, D.J., Ren, J.M., Pravis, S., Zhang, Y., Bernal, D., Pons, S., Shulman, G.I., Bonner-Weir, S., White, M.F., 1998. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391, 900e904. Wollheim, C.B., Sharp, G.W., 1981. Regulation of insulin release by calcium. Physiol. Rev. 61, 914e973. Wollmer, A., 2002. T-R transition. In: Federwisch, M., Dieken, M.L., De Meyts, P. (Eds.), Insulin and Related Proteins e Structure to Function and Pharmacology. Kluwer Academic Publishers, Dordrecht/Boston/London, pp. 77e89. Wu¨nsch, E., Wendlberger, G., 1968. Zur Synthese des Glucagons XVIII. Darstellung der Gesamtesequenz. Chem. Ber. 101, 3659e3663. Xu, B., Hu, S.Q., Huang, K., Natagawa, S.H., Whittaker, J., Katsoyannis, P.G., Weiss, M.A., 2004. Diabetes associated mutations in insulin: consecutive residues in the B chain contact distinct domains of the insulin receptor. Biochemistry 43, 8356e8372. Xu, Y., Kong, G.K.-W., Menting, J.G., Margetts, M.B., Delaine, C.A., Jenkin, L.M., Kiselyov, V.V., De Meyts, P., Forbes, B.E., Lawrence, M.C., 2018. How ligand binds to the type 1 insulin-like growth factor receptor. Nat. Commun. 9, 82. Yalow, R.S., 1992. The Nobel lectures in immunology. The Nobel prize for physiology or medicine 1977 awarded to Rosalyn S. Yalow. Scand. J. Immunol. 35, 1e23. Yang, L., Yang, D., de Graaf, C., Moeller, A., West, G.M., Dharmarajan, V., Wang, C., Siu, F.Y., Song, G., Reedtz-Runge, S., Pascal, B.D., Wu, B., Potter, C.S., Zhon, H., Griffin, P.R., Carragher, B., Yang, H., Wang, M.W., Stevens, R.C., Jiang, H., 2015. Conformational states of the full-length glucagon receptor. Nat. Commun. 6, 7859. Ye, L., Maji, S., Sanghera, N., Gopalasingam, P., Gorbunov, E., Tarasov, S., Epstein, O., Klein-Seetharaman, J., 2017. Structure and dynamics of the insulin receptor: implications for receptor activation and drug discovery. Drug Disc. Today 22, 1092e1102. Yosten, G.L., Redlinger, L.J., Samson, W.K., 2012. Evidence for an interaction of neuronostatin with the orphan G protein-coupled receptor GPR107. Am. J. Physiol. 303, R941eR949.

REFERENCES

Young, A., 2005. Inhibition of glucagon secretion. Adv. Pharmacol. 52, 151e171. Yu, R., 2018. Mahvash disease, 10 years after discovery. Pancreas 47, 511e515. Yu, R., Wawrowsky, K., Zhou, C., 2012. A natural inactivating mutant of human glucagon receptor exhibits multiple abnormalities in processing and signaling. Endocrinol. Nutr. 58, 258e266. Yulaningsih, E., Loh, K., Lin, S., Lau, J., Zhang, L., Shi, Y., Berning, B., Enriquez, R., Driessler, F., Macia, L., Khor, E., Qi, J., Baldock, P., Sainsbury, A., Herzog, H., 2014. Pancreatic polypeptide controls energy homeostasis via Npy6r signaling in the suprachiasmatic nucleus in mice. Cell Metab. 19, 58e72. Zhang, H., Qiao, A., Yang, D., Yang, L., Dai, A., de Graaf, C., ReedtzRunge, S., Dharmarajan, V., Zhang, H., Han, G.W., Grant, T.D., Sierra, R.G., Weierstall, U., Nelson, G., Liu, W., Wu, Y., Ma, L., Cai, X., Lin, G., Wu, X., Geng, Z., Dong, Y., Song, G., Griffin, P.R., Lau, J., Cherezov, V., Yang, H., Hanson, M.A., Stevens, R.C., Zhao, Q., Jiang, H., Wang, M.W., Wu, B., 2017a. Structure of the

423

full length glucagon class B G-protein-coupled receptor. Nature 546, 259e264. Zhang, Y., Sun, B., Feng, D., Hu, H., Chu, M., Qu, Q., Tarrasch, J.T., Li, S., Sun Kobilka, T., Kobilka, B.K., Skiniotis, G., 2017b. CryoEM structure of the activated GLP-1 receptor in complex with a G-protein. Nature 546, 248e253. Zhang, H., Qiao, A., Yang, L., et al., 2018. Structure of the glucagon receptor in complex with a glucagon analogue. Nature 553, 106e110. Zhao, W., et al., 2017. Identification of new susceptibility loci for type 2 diabetes and shared etiological pathways with coronary heart disease. Nat. Genet. 49, 1450e1457. Zhou, C., Dhall, D., Nissen, N.N., Chen, C.R., Yu, R., 2009. Homozygous P86S mutation of the human glucagon receptor is associated with hyperglucagonemia, alpha cell hyperplasia, and islet cell tumor. Pancreas 38, 941e946. Zick, Y., 2005. Ser/Thr phosphorylation of IRS proteins: a molecular basis for insulin resistance (2005). Sci. STKE 2005, PE4.

C H A P T E R

18 Liver Hormones Sila Cocciolillo1, Giada Sebastiani1, Mark Blostein2, Kostas Pantopoulos2 1

Royal Victoria Hospital, McGill University Health Center, and Department of Medicine, McGill University, Montreal, QC, Canada; 2Lady Davis Institute for Medical Research, Jewish General Hospital and Department of Medicine, McGill University, Montreal, QC, Canada

1. ANGIOTENSINOGEN Angiotensinogen (AGT) is a prohormone and the unique substrate of the renin-angiotensin system (RAS), an enzyme-linked hormonal cascade that regulates blood pressure and fluid homeostasis (Sparks et al., 2014). Through sequential cleavages by classic or alternative pathways, AGT gives rise to a spectrum of angiotensin peptides, with angiotensin II (Ang II) being the major vasoconstrictive hormone (Fig. 18.1). Circulating AGT is mainly produced in the liver by hepatocytes. AGT expression in other tissues, and particularly the kidney, does not appear to have systemic effects (Matsusaka et al., 2012). A detailed historical account on the discovery of the RAS can be found elsewhere (Basso and Terragno, 2001). The hepatic origin of the renin plasma substrate was established in the early 1940s (Page et al., 1941; Leloir et al., 1942). The protein was originally termed “hypertensinogen” and was later renamed to AGT (Braun-Menendez and Page, 1958). The AGT cDNA was first cloned in 1983 from rat (Ohkubo et al., 1983). The human AGT gene is localized on chromosome 1q42.2 and contains five exons. The murine Agt gene is localized on chromosome 8 (72.81 cM) and contains, likewise, five exons.

1.1 Structure AGT is synthesized as a preprotein that matures to AGT upon removal of an N-terminal signal peptide. In humans, pre-AGT consists of 485 amino acids and the signal peptide of 33 amino acids. Plasma AGT exhibits heterogeneity due to variable glycosylation at four asparagine residues (N4, N137, N217, and N295). Kidney-derived renin cleaves AGT between a leucine

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00018-3

and a valine residue close to its N-terminus, liberating angiotensin I (AngI) from the remaining protein, which is known as des(angI)AGT. AngI is a decapeptide [Ang(1e10)] with the sequence DRVYIHPFHL. It undergoes further processing by the angiotensin converting enzyme (ACE) on the surface of the lung and kidney epithelial cells. ACE removes the C-terminal histidine and leucine residues of AngI to generate AngII, the biologically active hormone. Thus, the sequence of the octapeptide AngII [or Ang(1e8)] is DRVYIHPF. AngI and AngII are sources of additional angiotensin derivatives (Fig. 18.2). These are generated by pathways involving angiotensin converting enzyme 2 (ACE2) or other proteases, such as aminopeptidase A (APA) or aminopeptidse N (APN), and include the peptides AngIII [or Ang(1-7) with the sequence DRVYIHP], Ang(1e9) (DRVYIHPFH), but also Ang(2e8) (RVYIHPF) and AngIV [or Ang(3-8) with the sequence VYIHPF]. Another angiotensin peptide is AngA (ARVYIHPF), which differs from AngII by having an alanine in the first amino acid position instead of aspartic acid (Jankowski et al., 2007). The rate-limiting step for generation of AngII and the relatively minor angiotensin derivative peptides is the initial renin-mediated cleavage of AGT, yielding AngI. Crystallographic studies revealed that the cleavage site of AGT is buried within its N-terminal domain and is therefore inaccessible to renin (Zhou et al., 2010) (Fig. 18.3). Exposure of the cleavage site to renin requires a structural rearrangement of AGT following oxidation of two conserved cysteine residues (at positions 18 and 138 in human AGT), which form a disulfide bridge. It has been proposed that the renin-mediated generation of AngI involves a redox switch in C18eC138 of AGT (Zhou et al., 2010). Nevertheless, in vivo studies with mouse models were not

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FIGURE 18.2

AGT is the precursor of Ang II and other angio-

tensive peptides.

FIGURE 18.1 Schematic diagram of the renin-angiotensin system (RAS). The major vasoconstrictive hormone angiotensin II (Ang II) is generated by sequential cleavages of angiotensinogen (AGT).

supportive (Wu et al., 2015), and further work is needed to clarify this issue. Other proteolytic enzymes such as cathepsin D, cathepsin G, kallikrein, pepsin, tissue-plasminogen activator, tonin, or trypsin can also cleave AGT to form angiotensin peptides; however, the physiologic relevance of these in vitro findings is not known.

1.2 Evolution AGT belongs to the serpin superfamily of proteins, which mainly consists of serine protease inhibitors, such as antithrombin or a1-antitrypsin. Serpins interact with and inhibit proteases via a reactive center loop

FIGURE 18.3 Crystal structure of AGT (PDB ID: 2WXW). The sequence corresponding to Ang I is highlighted in yellow. The arrow indicates the renin cleavage site.

(RCL). AGT is member of the noninhibitory branch of the serpin superfamily that includes proteins lacking an RCL, such as ovalbumin. As component of the RAS, AGT is conserved in all vertebrates from teleosts to humans and even has homologues in some invertebrates (Fournier et al., 2012). The critical cysteines at positions 18 and 138 that are thought to regulate cleavage of AGT by renin are conserved in all vertebrates. The cleavage of

1. ANGIOTENSINOGEN

mature AGT by renin exhibits remarkable species specificity. Interestingly, recombinant AGT from the lamprey Lampetra fluviatilis was shown to possess antithrombin activity (Wang and Ragg, 2011), indicating preservation of an active serpin RCL in the AGT molecule of this ancient jawless fish. Phylogenetic studies suggest that the RCL of AGT gradually evolved from inhibitory in lamprey to noninhibitory in humans over a period of 500 million years (Kumar et al., 2014). These findings also highlight a common evolutionary origin in serpinmediated regulation of blood coagulation and blood pressure (Wang et al., 2014).

427

Ang(1e7) antagonizes AngII-mediated effects and operates by binding to the G proteinecoupled receptor Mas (McKinney et al., 2014) (Fig. 18.5). This activates the phosphoinositol-3-kinase/Akt pathway and leads to phosphorylation and induction of vasodilatory endothelial NO synthase. The Ang(1e7)/Mas interaction also stimulates phospholipase A2 to release arachidonic acid, which increases levels of cAMP via prostacyclin and prostaglandin E2. In addition, it inhibits MAP kinase signaling. These responses promote vasodilation and reduce proliferation and migration of vascular smooth muscle cells. Ang(1e7)/Mas signaling also inhibits pathways promoting inflammation and fibrosis.

1.3 Biochemical Reactions AngII binds to AngII type 1 receptors (AT1R) and to AngII type 2 receptors (AT2R). These are homologous G proteinecoupled receptors with seventransmembrane domains. AT1R accounts for most of the known actions of AngII, while AT2R appears to mitigate AT1R-mediated responses (Nguyen Dinh Cat et al., 2013). The binding of AngII to AT1R in vascular smooth muscle cells activates phospholipase C (PLC) via the coupling of heterotrimeric Gq proteins (Fig. 18.4). Subsequently, PLC catalyzes the generation of inositol 1,4,5trisphosphate and diacylglycerol, promoting elevation in intracellular calcium levels. This triggers phosphorylation of myosin light chain 20, leading to contraction. In addition, AngII/AT1R signaling stimulates contraction via the RhoA/Rho kinase pathway that increases calcium sensitivity by inhibiting myosin light chain phosphatase. Moreover, AngII/AT1R signaling induces formation of reactive oxygen species via NADPH oxidases, which activate MAPK pathways, but also further stimulate AT1R. These responses trigger vasoconstriction and vascular remodeling.

FIGURE 18.4

1.4 Physiological Functions The physiologic functions of AGT are tightly linked to AngII, the principal AGT derivative, via the RAS. AngII acts on various target tissues, and its activities lead to increased blood pressure (Fig. 18.1). Thus, AngII directly promotes vasoconstriction in venous and arterial smooth muscle cells. On the adrenal gland cortex, AngII triggers secretion of aldosterone, a steroid hormone that controls blood pressure by regulating sodium homeostasis in the kidney, salivary glands, sweat glands, and the colon. AngII also acts on the hypothalamus to stimulate production of the antidiuretic hormone vasopressin, which constricts arterioles and increases the amount of solute-free water reabsorbed from the kidney. Other aspects of AGT physiology have received less attention due to the traditional view of this molecule as a passive substrate of renin. Nevertheless, experiments with mouse models offered new insights and suggested that AGT also possesses AngII-independent functions.

Ang II signaling via the AT1R in vascular smooth muscle cells.

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18. LIVER HORMONES

validated that AGT operates as a component of the RAS, but also raised the possibility that increased circulating AGT concentrations may contribute to the pathogenesis of hypertension.

1.6 AGT in Human Disease Association of single nucleotide polymorphisms (SNPs) of the AGT gene with human disease has been reported. Thus, the M235T polymorphism, linked to approximately 15% higher plasma AGT levels, was proposed to be causally related to essential hypertension (Jeunemaitre et al., 1992). However, in subsequent studies the M235T as well as other AGT polymorphisms were not consistently associated with hypertension or other clinical conditions (Lu et al., 2016). Computational analysis of 1092 human genomes from 14 different populations revealed a total of 690 genetic variants in AGT, including 613 SNPs, 36 somatic single nucleotide variants, 29 deletions, and 19 insertions (Kumar et al., 2014). Possible association of at least some of these variants with hypertension or other pathologies awaits further investigation. FIGURE 18.5 Ang(1e7) signaling via the Mas receptor in vascular smooth muscle cells.

1.5 Mouse Models with Altered AGT Expression Agt/ mice exhibit global AGT deficiency, which leads to low neonatal survival and hypotension, as well as impaired growth and renal development (Tanimoto et al., 1994; Smithies and Kim, 1994). Mice with hepatocyte-specific disruption of AGT recapitulate the low blood pressure phenotype (Matsusaka et al., 2012; Wu et al., 2015). In low-density lipoprotein receptoredeficient background, these animals also develop reduced obesity, atherosclerosis, and liver steatosis in response to high-fat diet (Wu et al., 2015). Interestingly, weight gain and steatosis were increased following adenoviral administration of des(angI)AGT (Lu et al., 2016). This finding suggests that des(angI) AGT, the core serpin domain of AGT, is not a mere byproduct of the enzymatic cleavage of AGT by renin, but it exhibits metabolic functions in regulating dietinduced obesity and liver steatosis. The underlying mechanisms are not well understood. Transgenic overexpression of human AGT in mice had no functional implications, in spite of a 150-fold increase in circulating AGT levels. However, infusion of human renin to the animals promoted a rapid elevation in blood pressure (Yang et al., 1994). These data

1.7 Conclusions and Clinical Applications AGT is a liver-derived prohormone and essential component of the RAS. It provides the substrate of renin and is the unique precursor of AngII and other angiotensin peptides that control blood pressure and cardiac contractility, as well as renal sodium and water absorption. While dysregulation of the RAS leads to hypertension, there is little evidence for pathogenicity of AGT dysregulation or genetic inactivation. Nevertheless, the direct pharmacological targeting of AGT expression may offer an alternative tool for the control of blood pressure, at least complementing standard downstream approaches.

2. HEPCIDIN Hepcidin is a liver peptide hormone that controls systemic iron homeostasis. It was first isolated in 2000 from human blood ultrafiltrates as a liver-derived cysteinerich peptide with antimicrobial activity (Krause et al., 2000); hence, it was named LEAP-1 (liver-expressed antimicrobial peptide 1). Shortly thereafter, the same antimicrobial peptide was isolated from human urine (Park et al., 2001), and the cDNA of its murine homologue was cloned by a suppressive subtractive hybridization screen to identify iron-inducible genes in the mouse liver (Pigeon et al., 2001). Since then the peptide has been known as hepcidin, a name that reflects its

2. HEPCIDIN

hepatic origin and antimicrobial properties. The human hepcidin gene (HAMP) is located on chromosome 19q13.12 and contains three exons. Murine Hamp has a similar genomic organization but is located on chromosome 7 (19.27 cM). Circulating hepcidin is almost exclusively produced by hepatocytes. Extrahepatic hepcidin is miniscule and appears to lack any systemic iron regulatory function (Zumerle et al., 2014).

2.1 Structure Hepcidin is expressed as prepro-peptide of 84 amino acids. Prepro-hepcidin is processed to prohepcidin upon removal of its 24 amino acid long N-terminal endoplasmic reticulum targeting sequence. Finally, prohepcidin is cleaved by the prohormone convertase furin at the C-terminus to yield mature hepcidin. The bioactive hormone is cysteine-rich (8 C residues) and consists of 25 amino acids. Cleavage products of 22 or 20 amino acids can be detected in urine. The sequence of human hepcidin is DTHFPICIFCCGCCHRSKCGMCCKT. Structural studies with two-dimensional 1H NMR spectroscopy suggested that hepcidin folds to a distorted b-sheet with an uncommon disulfide bond between adjacent C13eC14 at the turn of a hairpin loop; the structure is stabilized by another three disulfide bonds between C7eC23, C10eC22, and C11eC19 (Hunter et al., 2002) (Fig. 18.6A). An alternative structure with different disulfide bond connectivity (C7eC23, C10eC13, C11eC19, and C14eC22) was proposed based on biochemical studies, NMR spectroscopy, and X-ray crystallography (Jordan et al., 2009) (Fig. 18.6B). The N-terminal fragment of hepcidin DTHFPICIF retains substantial hormonal activity (Preza et al., 2011), indicating that the structural organization supported by

FIGURE18.6

429

disulfide bonds is redundant for hormonal iron regulation. Nevertheless, it is likely important for antimicrobial activity.

2.2 Evolution Hepcidin is related to families of cysteine-rich cationic antimicrobial peptides, such as a- and b-defensins, protegrins, and others, which mediate innate immune responses to infection in plants, insects, fish, amphibians, and mammals. Insects produce antimicrobial peptides in the fat body, the equivalent of the vertebrate liver. Hepcidin homologues have been found in mammals and teleosts, as well as in some reptiles and amphibians (Hilton and Lambert, 2008). With the exception of mice, mammals have a single copy of the hepcidin gene. Mice harbor a second hepcidin gene (Hamp2) on chromosome 7 as a result of gene duplication, which yields a peptide devoid of iron regulatory activity (Lou et al., 2004). Clusters of two or more hepcidin genes are present in the genome of some fish, but it appears that only one of them operates as hormonal iron regulator.

2.3 Biochemical Reactions Hepcidin operates by binding to the ferrous iron (Fe2þ) exporter ferroportin in tissue macrophages, duodenal enterocytes, and other target cells (Fig. 18.7). The binding of hepcidin promotes ubiquitination, internalization, and degradation of ferroportin in lysosomes (Nemeth et al., 2004). The interaction involves the N-terminal portion of hepcidin DTHFPICIF and a hepcidin-binding site in ferroportin that is predicted to be located within a central cavity. A model based on crystallographic data with a bacterial ferroportin

Crystal structures of hepcidin. (A) Structure with disulfide bonds between C7eC23, C10eC22, C13eC14, and C11eC19 (PDB ID: 1M4F). (B) Alternative structure with disulfide bonds between C7eC23, C10eC13, C11eC19, and C14eC22 (PDB ID: 2KEF). The N-terminal aminoacids that are essential for binding to ferroportin are highlighted in yellow.

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18. LIVER HORMONES

FIGURE 18.7 Hepcidin targets ferroportin in intestinal enterocytes and tissue macrophages. Hepcidin-mediated degradation of ferroportin inhibits iron efflux from these cells into plasma.

homologue suggests that the binding of hepcidin arrests the conformational transition of ferroportin from an outward-facing to an inward-facing state (Taniguchi et al., 2015). This presumably prevents opening of the molecular gate that allows iron efflux; moreover, it exposes the ubiquitination domain of ferroportin to ubiquitin ligases.

2.4 Physiological Functions Hepcidin negatively regulates iron efflux from cells to the bloodstream (Fig. 18.7), which is critical for body iron homeostasis. This needs to be tightly controlled

because iron is an essential nutrient but also a potential biohazard. The adult human body contains approximately 3e5 g of iron, with the largest fraction (>70%) utilized for heme biosynthesis during erythropoiesis (Papanikolaou and Pantopoulos, 2017). Senescent red blood cells are cleared by tissue macrophages, which degrade heme and export iron to the bloodstream via ferroportin for reutilization (Fig. 18.8). Plasma iron is captured by transferrin and delivered to the bone marrow and other tissues. Transferrin contains a very small (w0.1%) but highly dynamic fraction of body iron that turns over >10 times/day to satisfy the iron need for erythropoiesis (20e30 mg/day). The transferrin

FIGURE 18.8 Dynamics of systemic iron balance in humans. Iron efflux to the bloodstream is negatively regulated by hepcidin.

2. HEPCIDIN

iron pool is primarily replenished with iron recycled during erythrophagocytosis, and to a small extent with iron absorbed from the diet or, in case of iron deficiency, mobilized from body iron stores. In adults, dietary iron absorption (typically 1e2 mg/day) compensates for nonspecific iron losses via bleeding or cell desquamation. In iron-deficient states, high metabolic needs for iron can be covered by increased dietary iron absorption in the duodenum and, if necessary, by mobilization of liver iron stores. Similar to macrophages, enterocytes and hepatocytes release iron to the bloodstream via ferroportin in a process that is tightly regulated by hepcidin. Hepcidin-mediated internalization and degradation of ferroportin promotes hypoferremia and iron retention within ferroportin-expressing cells. Hepcidin expression is regulated by various stimuli; the major positive regulators are iron and inflammatory signals (Sangkhae and Nemeth, 2017). When systemic iron levels increase, hepcidin is induced to prevent iron overload and inhibit dietary iron absorption (Ganz, 2013). This has practical implications for oral iron supplementation therapy. Thus, intake of an iron pill induces hepcidin, which in turn inhibits further iron absorption for up to 24 h (Moretti et al., 2015). These findings suggested that administration of iron supplements on alternate days and in single doses is more efficacious than daily or twice-daily, a conclusion further validated in two randomized controlled trials (Stoffel et al., 2017). Iron intake triggers hepcidin induction following secretion of bone morphogenetic protein 6 (BMP6) from liver sinusoidal endothelial cells. These cells also

431

secrete BMP2, which appears to control basal hepcidin expression. The binding of BMP6 or BMP2 to cell surface BMP receptors promotes phosphorylation of regulatory SMADs, recruitment of SMAD4, and translocation of the complex to the nucleus for transcriptional activation of the hepcidin promoter (Fig. 18.9). The BMP/SMAD signaling pathway requires upstream auxiliary factors: (A) hemojuvelin (HJV), a BMP coreceptor; (B) the hemochromatosis protein HFE, an atypical major histocompatibility complex class 1 type molecule; and (C) transferrin receptor 2 (TfR2), a sensor of iron-loaded plasma transferrin. The cascade is negatively regulated by the serine protease matriptase-2 (TMPRSS6). Hepcidin is also induced during infection; the ensuing hypoferremia is considered an innate immune response to deprive invading bacteria from iron (Ganz and Nemeth, 2015). Inflammatory induction of hepcidin involves the IL-6/STAT3 pathway (Fig. 18.9). Binding of the inflammatory cytokine IL-6 to the receptor on hepatocytes triggers STAT3 phosphorylation by JAK1/2 kinases, and translocation of phospho-STAT3 to the nucleus for transcriptional activation of hepcidin. Experimental evidence suggests that this pathway requires active SMAD signaling. Under conditions of iron deficiency or hypoxemia, hepcidin levels drop to facilitate iron mobilization for erythropoiesis (Papanikolaou and Pantopoulos, 2017). A key suppressor of hepcidin during stress erythropoiesis is erythroferrone (ERFE), a hormone produced by erythroblasts in response to erythropoietin (Kautz et al., 2014). Erythroferrone inhibits hepcidin expression by

FIGURE 18.9 Signaling pathways leading to transcriptional regulation of hepcidin.

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18. LIVER HORMONES

interacting with BMP6, thereby inhibiting its signaling activity (Arezes et al., 2018) (Fig. 18.9).

2.5 Hormone Inactivation 2.5.1 Hereditary Hemochromatosis Genetic inactivation of iron signaling to hepcidin causes hereditary hemochromatosis, an endocrine disorder of systemic iron overload (Brissot et al., 2018). Hepcidin deficiency promotes increased dietary iron absorption (up to 8e10 mg/day) and uncontrolled release of iron to the bloodstream due to overexpression of ferroportin in duodenal enterocytes and tissue macrophages. This results in gradual saturation of transferrin and formation of unshielded nontransferrin-bound iron, which accumulates in tissue parenchymal cells. Clinical complications of hemochromatosis include liver cirrhosis, hepatocellular carcinoma, cardiomyopathy, diabetes, endocrinopathy, arthritis, and osteoporosis. Hereditary hemochromatosis is genetically heterogenous, and its severity correlates with the degree of hepcidin suppression relative to body iron stores. Four distinct types of the disease have been described (Table 18.1): (1) Type I hemochromatosis is linked to mutations in HFE (especially C282Y) and constitutes the most frequent genetic disorder in Caucasians. Nevertheless, the clinical penetrance of the predominant C282Y mutation is variable and depends on further genetic and environmental factors. HFErelated hemochromatosis is relatively mild, and symptoms typically develop after the fourth decade of life. (2) Type II or juvenile hemochromatosis is very rare and linked to inactivation of either HJV or hepcidin. This causes early onset iron overload in the late teens or early twenties. (3) Type III hemochromatosis is caused by inactivation of TfR2, which yields an TABLE 18.1

intermediate clinical phenotype compared to the other forms of the disease. (4) Type IV hemochromatosis or ferroportin disease is a distinct clinical entity linked to mutations in ferroportin. It is characterized by autosomal dominant pattern of inheritance, contrary to all other forms of hemochromatosis, which are autosomal recessive. There are two subtypes of ferroportin disease, caused by either loss or gain of ferroportin function. Loss-of-function mutations inhibit intracellular trafficking of ferroportin and result in macrophage iron loading. Conversely, gain-of-function mutations inhibit the binding of hepcidin to ferroportin and cause hepcidin resistance, which is associated with parenchymal iron overload. 2.5.2 Iron-Loading Anemias Sustained suppression of hepcidin by erythroferrone and other erythroid regulators is a pathogenic factor in iron-loading anemias. These are hereditary anemias associated with bone marrow hyperplasia and ineffective erythropoiesis, such as thalassemias, congenital dyserythropoietic anemias, or refractory anemia with ring sideroblasts (Papanikolaou and Pantopoulos, 2017). In transfusion-dependent forms of the diseases, secondary iron overload due to repeated blood transfusions is further aggravated by increased iron absorption due to hepcidin suppression. 2.5.3 Chronic Liver Diseases Hepcidin deficiency is also often observed in chronic liver diseases due to alcohol abuse or infection with hepatitis C virus. It is mostly linked to oxidative stress and promotes excessive hepatocellular iron deposition, which aggravates the underlying liver disease (Pietrangelo, 2016).

Genetic and Clinical Features of Hereditary Hemochromatosis Variants.

Disease Name

Type

Gene

Locus

Transmission

Liver Pathology

Laboratory Features

Clinical Expression

Hereditary hemochromatosis

1

HFE

6p21.3

Recessive

Hepatocellular iron loading

[ serum ferritin and transferrin saturation

Hepatic

Juvenile hemochromatosis

2A

HJV

1q21

Recessive

Hepatocellular iron loading

[ serum ferritin and transferrin saturation

Cardiac and endocrine

Juvenile hemochromatosis

2B

HAMP

19q13

Recessive

Hepatocellular iron loading

[ serum ferritin and transferrin saturation

Cardiac and endocrine

Hereditary hemochromatosis

3

TFR2

7q22

Recessive

Hepatocellular iron loading

[ serum ferritin and transferrin saturation

Hepatic

Ferroportin disease

4A

SLC40A1 2q32

Dominant

Mainly Kupffer cell iron loading

[[ serum ferritin, normal transferrin saturation

Articular and hepatic

Ferroportin disease

4B

SLC40A1 2q32

Dominant

Hepatocellular iron loading

[ serum ferritin and transferrin saturation

Hepatic

3. INSULIN-LIKE GROWTH FACTORS

2.6 Hormone Hyperactivation 2.6.1 Anemias with Iron-Restricted Erythropoiesis Sustained hyperhepcidinemia sequesters iron in macrophages, which reduces its availability to erythroid progenitor cells and causes anemia. Genetic hyperactivation of hepcidin is the hallmark of iron-refractory iron deficiency anemia (IRIDA), a disease caused by mutations in TMPRSS6, a suppressor of signaling to hepcidin (De Falco et al., 2013). Importantly, iron-restricted erythropoiesis is a common side effect of chronic inflammation. When inflammatory upregulation of hepcidin remains unresolved, hyperhepcidinemia contributes to anemia of inflammation, also known as anemia of chronic disease (Weiss, 2015). This is a frequent complication in patients with inflammatory bowel disease, rheumatic or chronic kidney disease, malignancies, or in frail, elderly persons. In fact, it constitutes the most prevalent anemia among hospitalized patients, and the second most prevalent anemia worldwide. 2.6.2 Chronic Liver Diseases Aberrant hepcidin activation has been observed in some patients with nonalcoholic steatohepatitis (NASH), a disease characterized by metabolic defects, inflammation, and endoplasmic reticulum stress. This can lead to iron deposition in Kupffer cells, which exacerbates liver pathology (Pietrangelo, 2016).

2.7 Conclusions and Clinical Applications Hepcidin is the master hormonal regulator of systemic iron homeostasis. It inhibits iron efflux to plasma by binding to and inactivating ferroportin, the sole

FIGURE 18.10

433

iron exporter. Hepcidin was discovered in the early 2000s, and its characterization revolutionized our understanding on iron metabolism in health and disease. Maintenance of hepcidin concentrations within a physiologic window is imperative for health. Dysregulation of hepcidin is associated with a wide spectrum of hepcidinopathies ranging from iron overload to anemias with iron-restricted erythropoiesis (summarized in Fig. 18.10). Pharmacological targeting of hepcidin pathways can provide etiologic cure to hereditary hemochromatosis or IRIDA and offer therapeutic benefits to ironloading anemias or the anemia of inflammation. Several drugs have been developed and validated in animal models, and some of them are further evaluated in clinical trials. It is likely that hepcidin agonists and antagonists will reach the clinic in the near future. A major challenge for the use of hepcidin therapeutics is to maintain physiologic concentrations of circulating hepcidin, and thereby avoid conditions leading to hepcidinopathies.

3. INSULIN-LIKE GROWTH FACTORS Insulin-like growth factors 1 and 2 (IGF-1 and IGF2, respectively) are homologous protein hormones that control growth, anabolic activities, metabolism, and aging. They are produced in many tissues for paracrine/autocrine functions, but the majority of circulating hormones are secreted from the liver and have endocrine functions. The expression of IGF-1 peaks during the puberty growth spurt and declines with age; its levels also depend on nutrition and various

Pathologies associated with hepcidin deficiency or excess.

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18. LIVER HORMONES

pathophysiological stimuli. IGF-2 primarily operates during gestation to stimulate fetal growth. Nevertheless, IGF-2 is approximately four times more abundant than IGF-1 throughout the life-span in humans, but not in rodents (Holly and Perks, 2012). The levels of IGF-2 are stable and show little variation, if any, in any conditions. IGF-1 and IGF-2 were first identified in 1957 as a “sulphation factor” stimulating 35S incorporation into rat cartilage (Salmon and Daughaday, 1957). The “sulphation factor” was later purified and shown to be induced by growth hormone (GH); therefore, it was renamed “somatomedin” (Daughaday et al., 1972). Another group independently reported a nonsuppressible insulin-like activity (NSILA) of two soluble serum components (NSILA I and II) (Froesch et al., 1963). Following their isolation and characterization, these proteins were shown to be identical to “somatomedin” and to share w50% homology with proinsulin (Rinderknecht and Humbel, 1976, 1978). Thus, they were renamed IGF-1 and IGF-2. In humans, the IGF-1 gene (IGF1) is located on chromosome 12q23.2 and contains seven exons, while the IGF-2 gene (IGF2) is located on chromosome 11p15.5 and contains nine exons. In mice, Igf1 and Igf2 are located on chromosomes 10 (43.70 cM) and 7 (87.99 cM), and contain 12 and eight exons, respectively. IGF1 can be alternatively spliced into several transcripts, encoding hepatocyte-derived circulating IGF-1 and tissue-specific isoforms (IGF-1Ea-c). The IGF-1Eb isoform, known as mechano-growth factor, is strongly upregulated in the skeletal muscle following mechanical loading (Matheny et al., 2010).

FIGURE 18.11

3.1 Structure IGF-1 is synthesized as a preproprotein of 158 amino acids with an N-terminal signal peptide of 21 amino acids that is cleaved to form pro-IGF-1. Mature IGF-1 contains 70 amino acids and is formed upon cleavage of a C-terminal peptide from pro-IGF-1. Likewise, prepro-IGF-2 of 180 amino acids undergoes N- and C-terminal cleavage to form mature IGF-2, which contains 66 amino acids. IGF-1 and IGF-2 share approximately 67% sequence identity and have similar ternary structures with three helical segments connected by a 12-residue linker (Fig. 18.11A). The linker (C-region) extends away from the core of the polypeptide and contains residues involved in receptor binding (Vajdos et al., 2001).

3.2 Evolution The insulin/IGF signaling pathway is highly conserved across the evolutionary scale. Thus, it operates in metazoans, while precursors of the pathway have been described in yeast (Barbieri et al., 2003). IGF-1 is well conserved among vertebrates from teleosts, reptiles, and birds to mammals (Rotwein, 2018). This applies particularly to the structural organization of the IGF1 gene and to exons encoding the mature IGF-1 peptide. By contrast, some regulatory elements found in the mammalian IGF1 promoter, such as Stat5b-binding enhancers, are absent in nonmammalian vertebrates and exhibit variation among mammalian species (Rotwein, 2017). IGF-2 is likewise conserved in vertebrates from

Crystal structure of IGF-1. (A) IGF-1 alone (PDB ID: 1GZZ). The 12-residue linker is highlighted in yellow. (B) IGF-1 (arrows) in complex with the ectodomain of the IGF-1R dimer (PDB ID: 5U8Q).

3. INSULIN-LIKE GROWTH FACTORS

teleosts to mammals (O’Neill et al., 2007). Interestingly, the IGF2 gene is paternally imprinted in humans and mice, and the imprinting mechanism is conserved among placental and marsupial mammals (Smits et al., 2008).

435

protein, which is thought to act as a clearance receptor of IGF-2 via lysosomes, rather than a traditional ligandactivated signaling receptor. Thus, IGF-2 signaling mainly involves the IGF-1R.

3.4 Physiological Functions

3.3 Biochemical Reactions The binding of IGF-1 or IGF-2 to the a chains of the IGF-1 receptor (IGF-1R) leads to a conformational change in the b subunit, which induces tyrosine kinase activity. Crystallographic studies with the ectodomain of the IGF-1R in the apo-form and in complex with IGF-1 (Fig. 18.11B) suggested an induced fit mechanism (Xu et al., 2018). The activated receptor kinase phosphorylates several cytoplasmic substrates, such as insulin receptor substrates (IRSs) and Src-homology collagen (Shc) (Fig. 18.12). These are subsequently recognized by adaptor proteins containing SH2 domains, such as Grb2, SHP2/Syp, or p85, the regulatory subunit of phosphatidylinositol 3-kinase. Binding of the adaptor proteins activates the phosphoinositol-3-kinase/Akt and MAP kinase signaling cascades (Hakuno and Takahashi, 2018). The IGF-1R forms homodimers or heterodimers with the insulin receptor. Both homo- and heterodimeric forms of the IGF-1R can also bind insulin, albeit with 50- to 100-fold lower affinity compared to IGF-1 (IC50: 0.2e0.8 nM) and IGF-2 (IC50: 0.5e4.4 nM). The IGF-2 receptor (IGF-2R) is a distinct monomeric transmembrane

FIGURE18.12

IGF-1 and IGF-2 play a critical role in regulating somatic growth according to nutritional availability. Thus, they stimulate growth, development, and maturation to reproductive state when food supply is high. This includes placental and fetal growth, neuronal, cardiovascular, and renal development, hematopoiesis, muscle and bone growth, and development of the reproductive system (Fig. 18.13), often at the expense of reduced lifespan. Conversely, the IGF signaling pathway is suppressed when nutrients are scarce, which elicits opposite biologic responses. The association of IGF signaling and longevity has been demonstrated in flies, worms, and mice and can be attributed to effects on genetic stability, telomere shortening, stress resistance, and metabolic control. However, experimental data with these model organisms cannot be extrapolated to primates (Sell, 2015), where maintenance of an older, postreproductive generation can have evolutionary advantages by passing on experience and wisdom to the young. Nevertheless, the age-dependent lowering of IGF-1 levels may be associated with some aging hallmarks, such as gradual reduction in muscle mass and bone mineral density, and cognitive decline. Moreover, low IGF-1 levels in

IGF-1/IGF-1R signaling pathways.

436

18. LIVER HORMONES

FIGURE 18.13

Physiologic functions of IGF-1.

the elderly may be associated with cardiovascular, metabolic, or neurodegenerative disorders. IGF-1 expression is stimulated by the pituitary GH. Some nutrients directly affect IGF-1 expression in hepatocytes, while others indirectly modulate it by altering levels of insulin, GH or growth hormone receptor (GH-R). Elevated IGF-1 levels inhibit GH secretion in pituitary gland, in a feedback control mechanism. Circulating IGF-1 and IGF-2 bind to IGF-binding proteins (IGFBPs). These interactions generally protect the hormones from proteolysis but inhibit hormonal function because IGF-1 and IGF-2 bind with higher affinities to IGFBPs compared to IGF-1R (Allard and Duan, 2018). Approximately 5% of serum IGF-1 is free and has a short half-life of 1e2 min (Fig. 18.14). Another small fraction (20%) is bound in binary complexes to IGFBPs, which extend its half-life to 20e30 min. Most of circulating IGF-1 (75%) forms ternary complexes with IGFBP-3 and the 88 kDa protein acid labile subunit (ALS), which further prolong its half-life up to 16e24 h. Hence, the IGFBPs maintain a large depot of circulating IGF-1 and IGF-2 that reach up to 1000 higher concentrations than those of insulin. Nevertheless, most of

circulating IGF-1 and IGF-2 are at a dormant state, and therefore their biologic activities are uncoupled from their secretion rates and steady-state concentrations. Activation of IGF-1 or IGF-2 occurs in proximity to IGF-1R and involves degradation of IGFBPs by proteases present in the circulation or in extravascular fluids. These include kallikreins, cathepsins, matrix metalloproteinases, the pregnancy-associated plasma protein-A, or the prostate-specific antigen. The IGFBPdegrading proteases are suppressed by specific inhibitors, and their activation requires elimination of the inhibitors. IGFBPs are critical regulators of IGF-1 and IGF-2 because they control their bioavailability, distribution and interaction with IGF-1R. They comprise an evolutionary conserved family of proteins with 200e300 amino acids. There are six well-characterized types (IGFBP1e6), which consist of conserved cysteine-rich N- and C-terminal domains, and a linker with low sequence conservation. Additional IGFBP-related proteins exhibit structural and functional similarities to IGFBPs; however, their affinities to IGF-1 and IGF-2 are lower, and their physiologic roles are not well

3. INSULIN-LIKE GROWTH FACTORS

437

FIGURE 18.14 Stabilization of circulating IGF-1 and IGF-2 by IGFBPs. Based on Baxter, R.C., 2014. Nat. Rev. Cancer 14, 329e341.

understood. It appears that the diversity of IGFBPs serves for temporospatial fine-tuning of IGF-1 and IGF-2 signaling activities.

3.5 Hormone Inactivation 3.5.1 Growth Disorders Genetic inactivation of IGF-1 or IGF-2 leads to intrauterine and postnatal growth restriction (Backeljauw and Chernausek, 2012). Severe IGF-2 deficiency is also associated to the Silver-Russell syndrome, a type of dwarfism (Begemann et al., 2015). The Laron syndrome, another type of dwarfism, is caused by GH insensitivity due to inactivating mutations in the GH-R, which in turn results in severe IGF-1 deficiency. GH insensitivity additionally develops in response to STAT5b deficiency, since STAT5b is a downstream component of the GH-R signaling pathway. Patients with STAT5b deficiency also exhibit severe immune defects due to STAT5b involvement in cytokine signaling pathways. Other forms of growth disorders are linked to IGF resistance due to inactivating mutations in IGF-1R, or deficiency in the IGF-binding protein ALS. 3.5.2 Metabolic Disorders Overfeeding and sedentary lifestyle can downregulate or inhibit IGF-1 expression and lead to metabolic dysfunction. This is reflected in reduced peripheral glucose and lipid uptake, insulin resistance, increased liver glucose production, increased stored triglyceride hydrolysis, and elevated circulating glucose and free fatty acid levels. These responses are tightly linked to development of the metabolic syndrome. Low serum levels of IGF-1 have been associated with the severity of inflammation and fibrosis in NASH and to cardiovascular disease.

3.6 Hormone Hyperactivation 3.6.1 Acromegaly Excessive IGF-1 expression is observed in acromegaly, a disease caused by hyperproduction of GH in the pituitary gland. In most cases, this is triggered by a pituitary adenoma. Acromegaly is characterized by abnormal enlargement in bones of the hands, feet and head. 3.6.2 Cancer The IGF signaling pathway plays a key role in tumorigenesis, as well as in survival and growth of cancer cells (Weroha and Haluska, 2012). In line with this notion, patients with acromegaly exhibit a twofold increased risk for gastrointestinal cancers, while Laron syndrome patients appear to be protected against cancer development (Laron et al., 2017). Nevertheless, the association of increased circulating IGF-1 or IGF-2 levels with cancer risk is controversial, and conflicting results have been reported. IGFBPs have also been linked to cancer pathways, but the underlying mechanisms are not well understood and may also involve IGF-independent functions.

3.7 Conclusions and Clinical Applications IGF-1 and IGF-2 are ancient master regulators of growth and development. They have critical physiologic functions in virtually all organs of the body, and their functional concentrations are tightly controlled by IGFBPs. Recent data suggested that circulating IGF-1 levels can also be affected by the gut microbiota (Yan and Charles, 2018), but more work is required to elucidate underlying mechanisms and to appreciate pathophysiological implications. Genetic defects leading to IGF deficiency or resistance are causatively linked to growth disorders.

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IGF-1 replacement therapy has been approved by health authorities for the treatment of pediatric patients with severe primary IGF-1 deficiency (Backeljauw and Chernausek, 2012). This involves the use of recombinant human IGF-1 (rhIGF-1 or mecasermin) alone or complexed with recombinant human IGFBP-3 (rhIGFBP-3 or rinfabate). Even though rhIGF-1 may promote hypoglycemia due to its insulin-like activity, as well as other side effects, its overall safety profile is acceptable. Nevertheless, long-term studies are needed to assess potential cancer-related risks. Further work is also required for optimization of rhIGF-1 administration and sustained expression. To date, rhIGF-1 is exclusively used (and offers the only option) for the treatment of severe primary IGF-1 deficiency. It is also being evaluated for the treatment of anorexia or severe insulin resistance. The pharmacological potential of rhIGF-1 in the context of other clinical settings awaits future investigation. It should be noted that due to its anabolic properties, rhIGF-1 is one of the most abused doping agents (Guha et al., 2009), and it is included in the list of banned drugs by the World Anti-Doping Agency.

4. THROMBOPOIETIN Thrombopoietin (TPO) is a liver protein hormone that controls megakaryocyte differentiation and platelet production. As far back as 1954, a physiologic regulator of platelet production was proposed in an analogous manner as erythropoietin, a known regulator of red cell mass. This accounts for its nomenclature (Hitchcock and Kaushansky, 2014). Nevertheless, TPO was only discovered in 1994 by five independent research groups using different approaches (Bartley et al., 1994; de Sauvage et al., 1994; Kaushansky et al., 1994; Kuter et al., 1994; Lok et al., 1994). The human TPO gene (THPO) is located on chromosome 3q27.1 and contains seven exons. Murine Thpo is located on chromosome 16 (12.51 cM) and contains nine exons. Circulating TPO is predominantly produced by hepatocytes, and at lower levels by renal proximal tubule epithelial cells and bone marrow stromal cells.

4.1 Structure TPO is synthesized as a precursor with an N-terminal leader sequence of 21 amino acids that is cleaved to form a secreted mature glycosylated protein of 332 amino acids. The N-terminal residues 1e153 of TPO are critical for receptor binding and share some homology with those of erythropoietin; nevertheless, each cytokine can only bind to its respective receptor. The crystal structure

FIGURE 18.15 Crystal structure of the receptor-binding domain of TPO (arrow), complexed to neutralizing antibody Tn1 Fab (PDB ID: 1V7N).

of the N-terminus has an antiparallel four-helix bundle fold (Fig. 18.15) and exhibits 1:2 ligand-receptor stoichiometry with high (109 M) and low (106 M) binding affinities (Feese et al., 2004). The C-terminal domain comprising residues 154e332 is carbohydrate-rich and responsible for maintaining a sufficient circulatory half-life; in addition, it facilitates proper folding of the TPO polypeptide.

4.2 Evolution TPO belongs to the four-helix-bundle cytokine superfamily. It is evolutionary conserved among vertebrate organisms. TPO homologues have been found in mammals, teleosts, amphibians, and reptiles (Svoboda et al., 2014).

4.3 Biochemical Reactions TPO binds to the c-Mpl receptor expressed on platelets and megakaryocytes, as well as on hematopoietic precursors (Fig. 18.16). The c-Mpl receptor was actually discovered as a homologue of the murine myeloproliferative leukemia virus protein v-Mpl. It is a member of the hematopoietic receptor superfamily that includes receptors for erythropoietin, interleukin 3 and granulocyte colony stimulating factor (Cosman, 1993). The binding of TPO promotes dimerization of c-Mpl and subsequent activation of JAK2-STAT3/5, phosphoinositol-3-kinase/ Akt, MAP kinase, and other signaling pathways.

4. THROMBOPOIETIN

439

FIGURE 18.16 TPO/c-MPL signaling pathways.

4.4 Physiological Functions TPO signaling induces cell survival and proliferation, as well as cell differentiation, particularly in platelets and megakaryocytes (Hitchcock and Kaushansky, 2014) (Fig. 18.17). TPO is the only hormone that increases platelet counts from basal levels as TPO (or c-Mpl) knock-out mice have platelets, but at a reduced number (Solar et al., 1998). TPO does not activate platelets directly but reduces the threshold of activation by other agonists by approximately 50%. TPO also increases the ploidy, number, and survival of megakaryocytes (Harker et al., 1996; Kaushansky et al., 1994). Administration of TPO to nonhuman primates increased megakaryocyte ploidy and number for approximately 4 days, as platelet counts began to rise after 5 days. They remained elevated throughout the treatment but returned back to baseline after withdrawal of TPO (Harker et al., 1996). There is no particular defined regulation of TPO levels (Hitchcock and Kaushansky, 2014). TPO is produced constitutively and degraded upon binding to its receptor. Hence, platelet mass, which reflects abundance of the c-Mpl receptor, determines TPO levels

(Fig. 18.18). Regulated TPO expression has been described in reactive/inflammatory thrombocythemia, where IL-6 induces hepatic TPO mRNA (Kaser et al., 2001). Interestingly, the TPO mRNA harbors an unusual 50 untranslated region with seven upstream AUG codons, which indicates poor and inefficient translation.

4.5 Hormone Inactivation 4.5.1 Congenital Amegakaryocytic Thrombocytopenia Congenital amegakaryocytic thrombocytopenia (CAMT) is a severe genetically heterogenous pediatric disease that is primarily caused by inactivating mutations in the c-Mpl receptor. Other, rare forms of this disease are causatively linked to loss-of-function TPO mutations. For instance, the R17C and R119C TPO mutations impair the interaction of TPO with the c-Mpl receptor and attenuate downstream signaling (Dasouki et al., 2013; Pecci et al., 2018). The R99W and R157X TPO mutations are likewise associated with CAMT (Seo et al., 2017).

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FIGURE 18.17

FIGURE 18.18

Regulation of megakaryocytopoiesis by TPO.

Expression of TPO in the liver and circulating TPO levels are determined by platelet counts. Based on Kuter, D.J., 1996. Oncologist 1, 98e106.

4. THROMBOPOIETIN

4.5.2 Thrombocytopenia in Liver Disease

441

Thrombocytopenia is a common hematological abnormality in chronic liver disease. While historically it was attributed to hypersplenism, it appears that low TPO levels due to compromised liver function contribute to this condition (Afdhal et al., 2008).

more common and sporadic myeloproliferative neoplasms (Varghese et al., 2017). Some of them are promoted by mutated variants of calreticulin, which binds to c-Mpl via its extracellular N-glycosylated domain and promotes its aberrant and persistent activation.

4.5.3 Exogenous TPO-Induced Immune Thrombocytopenia

4.7 Conclusions and Clinical Applications

There have been reports of thrombocytopenia due to TPO antibodies that developed during treatment with pegylated recombinant TPO (Basser et al., 2002; Li et al., 2001), which led to the discontinuation of its use. The appearance of such TPO antibodies was only transient and the affected patients ultimately recovered with normal platelet counts.

4.6 Hormone Hyperactivation 4.6.1 Congenital Thrombocythemia Several mutations in the 50 untranslated region of TPO mRNA causing more efficient translation are associated with congenital thrombocythemia (Hitchcock and Kaushansky, 2014). A distinct variant of congenital thrombocythemia was linked to increased TPO levels in response to a heterozygous loss-of-function R102P mutation in c-Mpl (Bellanne-Chantelot et al., 2017). 4.6.2 Myeloproliferative Neoplasms Other c-Mpl, but not TPO, mutations causing pathologic c-Mpl activation by noncanonical ligands underlie

The discovery of TPO as a liver hormone that controls platelet homeostasis had a major impact on understanding pathophysiology of diseases characterized by thrombocytopenia or thrombocythemia. Moreover, it has led to the development of therapeutics targeting the TPO/c-Mpl axis (Hitchcock and Kaushansky, 2014). Exogenous stimulation of c-Mpl has been used to treat immune thrombocytopenia, a disorder of premature immunologic destruction of platelets and megakaryocytes. Attempts at ameliorating chemotherapy had mixed success. Moderate improvement in chemotherapy-induced thrombocytopenia has been demonstrated for certain solid tumors, whereas no benefit for TPO was demonstrated in thrombocytopenia induced by autologous stem cell transplantation. Given the severe thrombocytopenia caused by antibodies against exogenous TPO, further development of TPO as a platelet stimulating agent was abandoned. Rather, alternative strategies involving TPO mimetics have been employed. First, a 14 amino acid peptide called romiplostim (Fig. 18.19A) was discovered from a peptide library screen and produced as a dimer to

FIGURE 18.19 The TPO mimetic drugs romiplostim (A) and eltrombopag (B).

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mimic the dimerization properties upon binding to the c-Mpl receptor. Further modifications include incorporation of the IgG heavy chain moiety to increase halflife. A second strategy employing high-throughput screening of small molecule libraries led to the discovery of eltrombopag (Fig. 18.19B). Romiplostim and eltrombopag have been approved by health authorities for the management of immune thrombocytopenia. Interestingly, romiplostim has also been successfully used for the treatment of patients with CAMT due to TPO loss-of-function mutations (Pecci et al., 2018; Seo et al., 2017). It should be noted that romiplostim and eltrombopag activate c-Mpl through different mechanisms, which implies that they can potentially be used synergistically. Moreover, nonresponse to one drug does not preclude response to the other. Further drug optimization or development of novel therapeutics targeting the TPO/c-Mpl axis remain future challenges.

References Afdhal, N., Mchutchison, J., Brown, R., Jacobson, I., Manns, M., Poordad, F., Weksler, B., Esteban, R., 2008. Thrombocytopenia associated with chronic liver disease. J. Hepatol. 48, 1000e1007. Allard, J.B., Duan, C., 2018. IGF-binding proteins: why do they exist and why are there so many? Front. Endocrinol. 9, 117. Arezes, J., Foy, N., Mchugh, K., Sawant, A., Quinkert, D., Terraube, V., Brinth, A., Tam, M., Lavallie, E., Taylor, S., Armitage, A.E., Pasricha, S.R., Cunningham, O., Lambert, M., Draper, S.J., Jasuja, R., Drakesmith, H., 2018. Erythroferrone inhibits the induction of hepcidin by BMP6. Blood 132, 1473e1477. Backeljauw, P.F., Chernausek, S.D., 2012. The insulin-like growth factors and growth disorders of childhood. Endocrinol Metab. Clin. N. Am. 41, 265e282 v. Barbieri, M., Bonafe, M., Franceschi, C., Paolisso, G., 2003. Insulin/IGFI-signaling pathway: an evolutionarily conserved mechanism of longevity from yeast to humans. Am. J. Physiol. Endocrinol. Metab. 285, E1064eE1071. Bartley, T.D., Bogenberger, J., Hunt, P., Li, Y.S., Lu, H.S., Martin, F., Chang, M.S., Samal, B., Nichol, J.L., Swift, S., et al., 1994. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 77, 1117e1124. Basser, R.L., O’flaherty, E., Green, M., Edmonds, M., Nichol, J., Menchaca, D.M., Cohen, B., Begley, C.G., 2002. Development of pancytopenia with neutralizing antibodies to thrombopoietin after multicycle chemotherapy supported by megakaryocyte growth and development factor. Blood 99, 2599e2602. Basso, N., Terragno, N.A., 2001. History about the discovery of the renin-angiotensin system. Hypertension 38, 1246e1249. Begemann, M., Zirn, B., Santen, G., Wirthgen, E., Soellner, L., Buttel, H.M., Schweizer, R., van Workum, W., Binder, G., Eggermann, T., 2015. Paternally inherited IGF2 mutation and growth restriction. N. Engl. J. Med. 373, 349e356. Bellanne-Chantelot, C., Mosca, M., Marty, C., Favier, R., Vainchenker, W., Plo, I., 2017. Identification of MPL R102P mutation in hereditary thrombocytosis. Front. Endocrinol. 8, 235. Braun-Menendez, E., Page, I.H., 1958. Suggested revision of nomenclature–angiotensin. Science 127, 242. Brissot, P., Pietrangelo, A., Adams, P.C., de Graaff, B., Mclaren, C.E., Loreal, O., 2018. Haemochromatosis. Nat. Rev. Dis. Primers 4, 18016.

Cosman, D., 1993. The hematopoietin receptor superfamily. Cytokine 5, 95e106. Dasouki, M.J., Rafi, S.K., Olm-Shipman, A.J., Wilson, N.R., Abhyankar, S., Ganter, B., Furness, L.M., Fang, J., Calado, R.T., Saadi, I., 2013. Exome sequencing reveals a thrombopoietin ligand mutation in a Micronesian family with autosomal recessive aplastic anemia. Blood 122, 3440e3449. Daughaday, W.H., Hall, K., Raben, M.S., Salmon Jr., W.D., van den Brande, J.L., Van Wyk, J.J., 1972. Somatomedin: proposed designation for sulphation factor. Nature 235, 107. de Falco, L., Sanchez, M., Silvestri, L., Kannengiesser, C., Muckenthaler, M.U., Iolascon, A., Gouya, L., Camaschella, C., Beaumont, C., 2013. Iron refractory iron deficiency anemia. Haematologica 98, 845e853. de Sauvage, F.J., Hass, P.E., Spencer, S.D., Malloy, B.E., Gurney, A.L., Spencer, S.A., Darbonne, W.C., Henzel, W.J., Wong, S.C., Kuang, W.J., et al., 1994. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 369, 533e538. Feese, M.D., Tamada, T., Kato, Y., Maeda, Y., Hirose, M., Matsukura, Y., Shigematsu, H., Muto, T., Matsumoto, A., Watarai, H., Ogami, K., Tahara, T., Kato, T., Miyazaki, H., Kuroki, R., 2004. Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment. Proc. Natl. Acad. Sci. U. S. A. 101, 1816e1821. Fournier, D., Luft, F.C., Bader, M., Ganten, D., Andrade-Navarro, M.A., 2012. Emergence and evolution of the renin-angiotensin-aldosterone system. J. Mol. Med. (Berl.) 90, 495e508. Froesch, E.R., Buergi, H., Ramseier, E.B., Bally, P., Labhart, A., 1963. Antibody-suppressible and nonsuppressible insulin-like activities in human serum and their physiologic significance. An insulin assay with adipose tissue of increased precision and specificity. J. Clin. Investig. 42, 1816e1834. Ganz, T., 2013. Systemic iron homeostasis. Physiol. Rev. 93, 1721e1741. Ganz, T., Nemeth, E., 2015. Iron homeostasis in host defence and inflammation. Nat. Rev. Immunol. 15, 500e510. Guha, N., Dashwood, A., Thomas, N.J., Skingle, A.J., Sonksen, P.H., Holt, R.I., 2009. IGF-I abuse in sport. Curr. Drug Abuse Rev. 2, 263e272. Hakuno, F., Takahashi, S.I., 2018. IGF1 receptor signaling pathways. J. Mol. Endocrinol. 61, T69eT86. Harker, L.A., Marzec, U.M., Hunt, P., Kelly, A.B., Tomer, A., Cheung, E., Hanson, S.R., Stead, R.B., 1996. Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates. Blood 88, 511e521. Hilton, K.B., Lambert, L.A., 2008. Molecular evolution and characterization of hepcidin gene products in vertebrates. Gene 415, 40e48. Hitchcock, I.S., Kaushansky, K., 2014. Thrombopoietin from beginning to end. Br. J. Haematol. 165, 259e268. Holly, J.M., Perks, C.M., 2012. Insulin-like growth factor physiology: what we have learned from human studies. Endocrinol Metab. Clin. N. Am. 41, 249e263 v. Hunter, H.N., Fulton, D.B., Ganz, T., Vogel, H.J., 2002. The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis. J. Biol. Chem. 277, 37597e37603. Jankowski, V., Vanholder, R., van der Giet, M., Tolle, M., Karadogan, S., Gobom, J., Furkert, J., Oksche, A., Krause, E., Tran, T.N., Tepel, M., Schuchardt, M., Schluter, H., Wiedon, A., Beyermann, M., Bader, M., Todiras, M., Zidek, W., Jankowski, J., 2007. Massspectrometric identification of a novel angiotensin peptide in human plasma. Arterioscler. Thromb. Vasc. Biol. 27, 297e302. Jeunemaitre, X., Soubrier, F., Kotelevtsev, Y.V., Lifton, R.P., Williams, C.S., Charru, A., Hunt, S.C., Hopkins, P.N., Williams, R.R., Lalouel, J.M., et al., 1992. Molecular basis of human hypertension: role of angiotensinogen. Cell 71, 169e180.

REFERENCES

Jordan, J.B., Poppe, L., Haniu, M., Arvedson, T., Syed, R., Li, V., Kohno, H., Kim, H., Schnier, P.D., Harvey, T.S., Miranda, L.P., Cheetham, J., Sasu, B.J., 2009. Hepcidin revisited, disulfide connectivity, dynamics, and structure. J. Biol. Chem. 284, 24155e24167. Kaser, A., Brandacher, G., Steurer, W., Kaser, S., Offner, F.A., Zoller, H., Theurl, I., Widder, W., Molnar, C., Ludwiczek, O., Atkins, M.B., Mier, J.W., Tilg, H., 2001. Interleukin-6 stimulates thrombopoiesis through thrombopoietin: role in inflammatory thrombocytosis. Blood 98, 2720e2725. Kaushansky, K., Lok, S., Holly, R.D., Broudy, V.C., Lin, N., Bailey, M.C., Forstrom, J.W., Buddle, M.M., Oort, P.J., Hagen, F.S., et al., 1994. Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 369, 568e571. Kautz, L., Jung, G., Valore, E.V., Rivella, S., Nemeth, E., Ganz, T., 2014. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat. Genet. 46, 678e684. Krause, A., Neitz, S., Magert, H.J., Schulz, A., Forssmann, W.G., schulzKnappe, P., Adermann, K., 2000. LEAP-1, a novel highly disulfidebonded human peptide, exhibits antimicrobial activity. FEBS Lett. 480, 147e150. Kumar, A., Sarde, S.J., Bhandari, A., 2014. Revising angiotensinogen from phylogenetic and genetic variants perspectives. Biochem. Biophys. Res. Commun. 446, 504e518. Kuter, D.J., Beeler, D.L., Rosenberg, R.D., 1994. The purification of megapoietin: a physiological regulator of megakaryocyte growth and platelet production. Proc. Natl. Acad. Sci. U. S. A. 91, 11104e11108. Laron, Z., Kauli, R., Lapkina, L., Werner, H., 2017. IGF-I deficiency, longevity and cancer protection of patients with Laron syndrome. Mutat. Res. Rev. Mutat. Res. 772, 123e133. Leloir, L.F., Mun˜oz, J.M., Taquini, A.C., Braun-Mene´ndez, E., Fasciolo, J.C., 1942. La formacio´n del angiotensino´geno. Rev. Argent. Cardiol. 9. Li, J., Yang, C., Xia, Y., Bertino, A., Glaspy, J., Roberts, M., Kuter, D.J., 2001. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood 98, 3241e3248. Lok, S., Kaushansky, K., Holly, R.D., Kuijper, J.L., Lofton-Day, C.E., Oort, P.J., Grant, F.J., Heipel, M.D., Burkhead, S.K., Kramer, J.M., et al., 1994. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 369, 565e568. Lou, D.Q., Nicolas, G., Lesbordes, J.C., Viatte, L., Grimber, G., Szajnert, M.F., Kahn, A., Vaulont, S., 2004. Functional differences between hepcidin 1 and 2 in transgenic mice. Blood 103, 2816e2821. Lu, H., Cassis, L.A., Kooi, C.W., Daugherty, A., 2016. Structure and functions of angiotensinogen. Hypertens. Res. 39, 492e500. Matheny Jr., R.W., Nindl, B.C., Adamo, M.L., 2010. Minireview: mechano-growth factor: a putative product of IGF-I gene expression involved in tissue repair and regeneration. Endocrinology 151, 865e875. Matsusaka, T., Niimura, F., Shimizu, A., Pastan, I., Saito, A., Kobori, H., Nishiyama, A., Ichikawa, I., 2012. Liver angiotensinogen is the primary source of renal angiotensin II. J. Am. Soc. Nephrol. 23, 1181e1189. McKinney, C.A., Fattah, C., Loughrey, C.M., Milligan, G., Nicklin, S.A., 2014. Angiotensin-(1-7) and angiotensin-(1-9): function in cardiac and vascular remodelling. Clin. Sci. (Lond.) 126, 815e827. Moretti, D., Goede, J.S., Zeder, C., Jiskra, M., Chatzinakou, V., Tjalsma, H., Melse-Boonstra, A., Brittenham, G., Swinkels, D.W., Zimmermann, M.B., 2015. Oral iron supplements increase hepcidin and decrease iron absorption from daily or twice-daily doses in iron-depleted young women. Blood 126, 1981e1989. Nemeth, E., Tuttle, M.S., Powelson, J., Vaughn, M.B., Donovan, A., Ward, D.M., Ganz, T., Kaplan, J., 2004. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 306, 2090e2093.

443

Nguyen Dinh Cat, A., Montezano, A.C., Burger, D., Touyz, R.M., 2013. Angiotensin Ii, NADPH oxidase, and redox signaling in the vasculature. Antioxid. Redox Signal. 19, 1110e1120. O’Neill, M.J., Lawton, B.R., Mateos, M., Carone, D.M., Ferreri, G.C., Hrbek, T., Meredith, R.W., Reznick, D.N., O’Neill, R.J., 2007. Ancient and continuing Darwinian selection on insulin-like growth factor II in placental fishes. Proc. Natl. Acad. Sci. U. S. A. 104, 12404e12409. Ohkubo, H., Kageyama, R., Ujihara, M., Hirose, T., Inayama, S., Nakanishi, S., 1983. Cloning and sequence analysis of cDNA for rat angiotensinogen. Proc. Natl. Acad. Sci. U. S. A. 80, 2196e2200. Page, I.H., Mcswain, B., Knapp, G.M., Andrus, W.D., 1941. The origin of renin activator. Am. J. Physiol. 135, 214e222. Papanikolaou, G., Pantopoulos, K., 2017. Systemic iron homeostasis and erythropoiesis. IUBMB Life 69, 399e413. Park, C.H., Valore, E.V., Waring, A.J., Ganz, T., 2001. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J. Biol. Chem. 276, 7806e7810. Pecci, A., Ragab, I., Bozzi, V., DE Rocco, D., Barozzi, S., Giangregorio, T., Ali, H., Melazzini, F., Sallam, M., Alfano, C., Pastore, A., Balduini, C.L., Savoia, A., 2018. Thrombopoietin mutation in congenital amegakaryocytic thrombocytopenia treatable with romiplostim. EMBO Mol. Med. 10, 63e75. Pietrangelo, A., 2016. Iron and the liver. Liver Int. 36 (Suppl. 1), 116e123. Pigeon, C., Ilyin, G., Courselaud, B., Leroyer, P., Turlin, B., Brissot, P., Loreal, O., 2001. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J. Biol. Chem. 276, 7811e7819. Preza, G.C., Ruchala, P., Pinon, R., Ramos, E., Qiao, B., Peralta, M.A., Sharma, S., Waring, A., Ganz, T., Nemeth, E., 2011. Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload. J. Clin. Investig. 121, 4880e4888. Rinderknecht, E., Humbel, R.E., 1976. Polypeptides with nonsuppressible insulin-like and cell-growth promoting activities in human serum: isolation, chemical characterization, and some biological properties of forms I and II. Proc. Natl. Acad. Sci. U. S. A. 73, 2365e2369. Rinderknecht, E., Humbel, R.E., 1978. The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J. Biol. Chem. 253, 2769e2776. Rotwein, P., 2017. Diversification of the insulin-like growth factor 1 gene in mammals. PLoS One 12, e0189642. Rotwein, P., 2018. Insulinlike growth factor 1 gene variation in vertebrates. Endocrinology 159, 2288e2305. Salmon Jr., W.D., Daughaday, W.H., 1957. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J. Lab. Clin. Med. 49, 825e836. Sangkhae, V., Nemeth, E., 2017. Regulation of the iron homeostatic hormone hepcidin. Adv. Nutr. 8, 126e136. Sell, C., 2015. Minireview: the complexities of IGF/insulin signaling in aging: why flies and worms are not humans. Mol. Endocrinol. 29, 1107e1113. Seo, A., Ben-Harosh, M., Sirin, M., Stein, J., Dgany, O., Kaplelushnik, J., Hoenig, M., Pannicke, U., Lorenz, M., Schwarz, K., Stockklausner, C., Walsh, T., Gulsuner, S., Lee, M.K., Sendamarai, A., Sanchez-Bonilla, M., King, M.C., Cario, H., Kulozik, A.E., Debatin, K.M., Schulz, A., Tamary, H., Shimamura, A., 2017. Bone marrow failure unresponsive to bone marrow transplant is caused by mutations in thrombopoietin. Blood 130, 875e880. Smithies, O., Kim, H.S., 1994. Targeted gene duplication and disruption for analyzing quantitative genetic traits in mice. Proc. Natl. Acad. Sci. U. S. A. 91, 3612e3615.

444

18. LIVER HORMONES

Smits, G., Mungall, A.J., Griffiths-Jones, S., Smith, P., Beury, D., Matthews, L., Rogers, J., Pask, A.J., Shaw, G., Vandeberg, J.L., Mccarrey, J.R., Consortium, S., Renfree, M.B., Reik, W., Dunham, I., 2008. Conservation of the H19 noncoding RNA and H19-IGF2 imprinting mechanism in therians. Nat. Genet. 40, 971e976. Solar, G.P., Kerr, W.G., Zeigler, F.C., Hess, D., Donahue, C., DE Sauvage, F.J., Eaton, D.L., 1998. Role of c-mpl in early hematopoiesis. Blood 92, 4e10. Sparks, M.A., Crowley, S.D., Gurley, S.B., Mirotsou, M., Coffman, T.M., 2014. Classical Renin-Angiotensin system in kidney physiology. Comp. Physiol. 4, 1201e1228. Stoffel, N.U., Cercamondi, C.I., Brittenham, G., Zeder, C., GeurtsMoespot, A.J., Swinkels, D.W., Moretti, D., Zimmermann, M.B., 2017. Iron absorption from oral iron supplements given on consecutive versus alternate days and as single morning doses versus twice-daily split dosing in iron-depleted women: two openlabel, randomised controlled trials. Lancet Haematol. 4, e524ee533. Svoboda, O., Stachura, D.L., Machonova, O., Pajer, P., Brynda, J., Zon, L.I., Traver, D., Bartunek, P., 2014. Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin. Blood 124, 220e228. Taniguchi, R., Kato, H.E., Font, J., Deshpande, C.N., Wada, M., Ito, K., Ishitani, R., Jormakka, M., Nureki, O., 2015. Outward- and inwardfacing structures of a putative bacterial transition-metal transporter with homology to ferroportin. Nat. Commun. 6, 8545. Tanimoto, K., Sugiyama, F., Goto, Y., Ishida, J., Takimoto, E., Yagami, K., Fukamizu, A., Murakami, K., 1994. Angiotensinogen-deficient mice with hypotension. J. Biol. Chem. 269, 31334e31337. Vajdos, F.F., Ultsch, M., Schaffer, M.L., Deshayes, K.D., Liu, J., Skelton, N.J., DE Vos, A.M., 2001. Crystal structure of human insulin-like growth factor-1: detergent binding inhibits binding protein interactions. Biochemistry 40, 11022e11029.

Varghese, L.N., Defour, J.P., Pecquet, C., Constantinescu, S.N., 2017. The thrombopoietin receptor: structural basis of traffic and activation by ligand, mutations, agonists, and mutated calreticulin. Front. Endocrinol. 8, 59. Wang, Y., Koster, K., Lummer, M., Ragg, H., 2014. Origin of serpinmediated regulation of coagulation and blood pressure. PLoS One 9, e97879. Wang, Y., Ragg, H., 2011. An unexpected link between angiotensinogen and thrombin. FEBS Lett. 585, 2395e2399. Weiss, G., 2015. Anemia of chronic disorders: new diagnostic tools and new treatment strategies. Semin. Hematol. 52, 313e320. Weroha, S.J., Haluska, P., 2012. The insulin-like growth factor system in cancer. Endocrinol Metab. Clin. N. Am. 41, 335e350 vi. Wu, C., Xu, Y., Lu, H., Howatt, D.A., Balakrishnan, A., Moorleghen, J.J., Vander Kooi, C.W., Cassis, L.A., Wang, J.A., Daugherty, A., 2015. Cys18-Cys137 disulfide bond in mouse angiotensinogen does not affect AngII-dependent functions in vivo. Hypertension 65, 800e805. Xu, Y., Kong, G.K., Menting, J.G., Margetts, M.B., Delaine, C.A., Jenkin, L.M., Kiselyov, V.V., DE Meyts, P., Forbes, B.E., Lawrence, M.C., 2018. How ligand binds to the type 1 insulin-like growth factor receptor. Nat. Commun. 9, 821. Yan, J., Charles, J.F., 2018. Gut microbiota and IGF-1. Calcif. Tissue Int. 102, 406e414. Yang, G., Merrill, D.C., Thompson, M.W., Robillard, J.E., Sigmund, C.D., 1994. Functional expression of the human angiotensinogen gene in transgenic mice. J. Biol. Chem. 269, 32497e32502. Zhou, A., Carrell, R.W., Murphy, M.P., Wei, Z., Yan, Y., Stanley, P.L., Stein, P.E., Broughton Pipkin, F., Read, R.J., 2010. A redox switch in angiotensinogen modulates angiotensin release. Nature 468, 108e111. Zumerle, S., Mathieu, J.R., Delga, S., Heinis, M., Viatte, L., Vaulont, S., Peyssonnaux, C., 2014. Targeted disruption of hepcidin in the liver recapitulates the hemochromatotic phenotype. Blood 123, 3646e3650.

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19 The Endocrine Kidney: Local and Systemic Actions of Renal Hormones Robert T. Mallet, Rong Ma Department of Physiology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX, United States Abbreviations ACE AM ANP, BNP, CNP AngII cAMP, cGMP ATR [Ca2D]i CGRP cMGP CRF CRLR DBP EMT ER ET-1 ETAR, ETBR GFR HIF HO-1 IMD IP3 NOS NPR PKA, PKC PTH RAMP ROS UII UTR VDR

angiotensin converting enzyme; adrenomedullin atrial, brain, C-type natriuretic peptides angiotensin II cyclic AMP, GMP angiotensin receptor intracellular Ca2þ concentration calcitonin gene-related peptide g-carboxyglutamic acid protein corticotropin-releasing factor calcitonin receptor-like receptor vitamin D binding protein epithelial-mesenchymal transition endoplasmic reticulum endothelin-1 endothelin receptor type A, B glomerular filtration rate hypoxia-inducible factor heme oxygenase-1 intermedin inositol 1,4,5-trisphosphate nitric oxide synthase natriuretic peptide receptor protein kinase A, C parathyroid hormone receptor activity modifying protein reactive oxygen species urotensin II urotensin II receptor vitamin D receptor

1. INTRODUCTION Bones can break, muscles can atrophy, glands can loaf, even the brain can go to sleep, without immediately endangering our survival, but when the kidneys fail to manufacture the proper kind of blood neither bone, muscle, gland nor brain can carry on. Homer W. Smith: William Harvey Welch Lecture, 1943

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00019-5

The mammalian kidney maintains the volume and composition of the body fluids, thereby preserving the milieu inte´rieur essential for physiologic function. This complex and indispensable task is accomplished by two distinct processes: bulk ultrafiltration of essentially protein-free plasma from the glomerular capillaries into Bowman’s space, and transformation of this filtrate into urine by selective tubular reabsorption and secretion of electrolytes, organic compounds, Hþ, and water. Glomerular filtration rate (GFR) and tubular transport are exquisitely regulated by a battery of hormones. Although some of these hormones are products of the classical endocrine system, an expanding number are generated within the kidneys. This chapter presents these renal hormones, the factors governing their production and activity, their receptors and signaling mechanisms, their physiologic functions within and beyond the kidneys, and their favorable or adverse actions in the settings of renal and systemic diseases. The body’s Naþ content determines extracellular fluid volume and, thus, arterial pressure, and an array of neurohumoral factors regulate glomerular filtration and tubular reabsorption of Naþ. In the healthy kidney, this remarkable system adjusts urinary Naþ excretion to stabilize blood pressure against broad variations in dietary Naþ intake. Although the renin-angiotensin-aldosterone system is the prototypical regulator of renal Naþ balance, in recent decades, several more hormones have been found to modulate renal Naþ handling, including atrial (ANP) and brain (BNP) natriuretic peptides and their intrarenal counterpart urodilatin, the powerful vasoconstrictors endothelin-1 (ET-1) and urotensin II (UII), and the vasodilators adrenomedullin (AM) and intermedin (IMD). The kidneys also produce calcitriol, essential for Ca2þ and phosphate (PO4)

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balance, and erythropoietin, activator of erythrocyte production to maintain O2 delivery for aerobic metabolism. These diverse hormones enable the kidneys to sustain homeostasis, yet their dysfunction elicits renal and systemic disease.

2009), as well as AngII’s proinflammatory, profibrotic, and proliferative actions in diseased kidney (Simo˜es e Silva et al., 2013).

2. RENIN-ANGIOTENSIN SYSTEM

G proteinecoupled type 1 AngII receptors (AT1R) mediate AngII-induced vasoconstriction and Naþ-retention. Via AT1R, AngII activates phospholipase Cg1 to hydrolyze phosphatidylinositol 4,5-bis phosphate, yielding inositol 1,4,5-tris phosphate (IP3) and diacylglycerol (Schieffer et al., 1996). IP3 mobilizes intracellular Ca2þ to contract vascular smooth muscle and mesangial cells, and diacylglycerol activates protein kinase C (PKC) and its complex signaling cascades (Berry et al., 2001). PKCinitiated signaling causes mesangial cells to proliferate and deposit extracellular matrix (Sorokin and Kohan, 2003). PKC also mediates AngII activation of proximal tubular Na/H exchanger (Li and Zhuo, 2011) and Na,K ATPase (Gomes et al., 2005), as well as collecting duct Naþ channels (Sun et al., 2012). Via a second ATR subtype, AT2R, AngII triggers formation of vasodilators NO, bradykinin, and prostaglandin F2a, dampening AT1R-mediated vasoconstriction (Berry et al., 2001).

Angiotensin II (AngII) is the best-characterized regulator of renal Naþ balance. Upon its secretion by the juxtaglomerular cells of the afferent arterioles, the proteolytic enzyme renin cleaves circulating angiotensinogen, liberating the decapeptide angiotensin I. Further cleavage of this inactive prohormone by angiotensin converting enzyme 1 (ACE1) yields the octapeptide AngII. This powerful hormone exerts several diverse effects that collectively raise arterial pressure: powerful constriction of systemic resistance vessels, including the renal afferent and efferent arterioles; sympathetic activation within the brain (Leenen, 2014); induction of vasopressin and aldosterone secretion from the pituitary and adrenal cortex, respectively; increased renal tubular Naþ reabsorption; and glomerular mesangial cell contraction. Although crucial to stabilize blood pressure during hypovolemia, AngII also is pivotal to the maladaptive cycle of circulatory congestion, inflammation, and fibrosis in heart failure (Sayer and Bhat, 2014). Three mechanisms provoke renin secretion by the juxtaglomerular cells: (1) direct stimulation by sympathetic neurons; (2) functioning as “intrarenal baroreceptors” the juxtaglomerular cells secrete renin in response to decreased hydrostatic pressure in the adjacent afferent arterioles, e.g., during constriction of the upstream vascular smooth muscle; (3) in the tubuloglomerular feedback mechanism, decreased Naþ delivery to the macula densa at the distal end of Henle’s loop initiates an electrochemical signal that suppresses Ca2þ entry into the juxtaglomerular cells, permitting renin secretion. Although AngII production in the pulmonary vasculature is well recognized, ACE1, angiotensinogen, and renin are abundant in the kidneys, comprising a fully functional, intrarenal AngII-generating system (Yang and Xu, 2017). A second enzyme, ACE2 cleaves AngII’s C-terminal phenylalanine, yielding Ang 1e7 (Dilauro and Burns, 2009). ACE2 is abundant in the tubular epithelium and also present in podocytes and the renal microcirculation (Soler et al., 2013). Ang 1e7 generally opposes AngII’s actions: it dampens AngII-induced Naþ reabsorption in the proximal tubule and loop of Henle and water reabsorption in the collecting duct (Dilauro and Burns,

2.1 Angiotensin II-Activated Cellular Signaling

2.2 Adverse Effects of AngII in Chronic Kidney Disease Although AngII’s responses to hypovolemia are crucial to stabilize effective circulating volume and blood pressure, chronically elevated AngII, e.g., in heart failure, diabetes mellitus, and cardiorenal syndrome, is harmful. Systemic hypertension raises hydrostatic pressure in the glomerular capillaries, which over time damages the capillary endothelium, and increased glomerular filtration raises ATP requirements for tubular reabsorption of Naþ and other solutes. Elevated AngII provokes mesangial cell proliferation and deposition of extracellular matrix, culminating in glomerular fibrosis (Komers and Plotkin, 2016). In tubular epithelium, AngII activates NADPH and xanthine oxidases to overproduce reactive oxygen species (ROS) that damage cellular components (Sung et al., 2013), deplete cytoprotective NO (Mende, 2010), and provoke epithelial-mesenchymal transition (EMT) and tubulointerstitial fibrosis (Prunotto et al., 2012). AngII also mobilizes the transcription factor NFkB to drive tubular epithelial expression of proinflammatory cytokines (Simo˜es e Silva et al., 2013). Accordingly, ACE1 inhibitors and AT1R antagonists, mainstays of heart failure treatment, also are efficacious for management of chronic kidney disease (Kolesnyk et al., 2010),

3. VASOCONSTRICTORS: ENDOTHELIN-1 AND UROTENSIN II

and AngII antagonist Ang 1e7 is another promising intervention (Simo˜es e Silva et al., 2013).

3. VASOCONSTRICTORS: ENDOTHELIN-1 AND UROTENSIN II 3.1 Endothelin-1 Endothelins are powerful vasoconstrictors produced by vascular endothelium. The predominant endothelin in kidney, ET-1, is generated from a precursor, big ET-1, either directly by endothelin converting enzyme-1 (ECE-1) or via chymase-catalyzed formation of an intermediary, ET1-31, which then is cleaved by neutral endopeptidase yielding ET-1 (Kuruppu et al., 2013). These pathways intersect in the tubular epithelium, where ET1-31 mobilizes intracellular Ca2þ to promote ECE-1 synthesis (Huang et al., 2003). Immunohistochemistry of human kidney revealed ECE-1 in endothelium of arcuate and interlobular arteries and vasa recta, and in the tubular epithelium of the thin limbs of Henle’s loop and the medullary collecting duct (Pupilli et al., 1997). 3.1.1 Endothelin Receptors and Signaling Mechanisms Many renal cells produce ET-1 and have G proteine coupled ET receptors (ETR), including microvascular endothelium, mesangial cells, podocytes, and epithelial cells of several tubular segments (Kohan, 1997; Wesson et al., 1998; Larivie`re and Lebel, 2003). ETAR is the main subtype in vascular smooth muscle and mesangial cells, while ETBR predominates in renal tubules (Larivie`re and Lebel, 2003). Vascular endothelium also harbors ETBR, which mediates formation of vasodilator eicosanoids and NO (Campese et al., 2006). ETAR and ETBR bind ET-1 with Kd w100 p.m. (Sorokin and Kohan, 2003). Circulating ET-1 concentrations are normally only 1e2 pM, so achieving higher concentrations in microenvironments (e.g., within renal corpuscles or tubules) may be crucial for ET-1 signaling. Endothelin-1 activates both rapid and sustained renal vasoconstriction (Fellner and Arendshorst, 2007). In the rapid mechanism, ET-1, acting on ETAR and ETBR in vascular smooth muscle, generates IP3, which triggers endoplasmic reticular Ca2þ release and contraction. In the sustained mechanism, ETAR and ETBR activation opens sarcolemmal Ca2þ channels, causing protracted increases in intracellular Ca2þ concentration ([Ca2þ]i). In mesangial cells, ETAR activation, in the same fashion as AT1R, elicits diacylglycerol and IP3 formation, culminating in contraction, proliferation, matrix accumulation, and fibrosis (Sorokin and Kohan, 2003).

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3.1.2 Modulators of ET-1 Myriad factors activate renal ET-1 production, including AngII, vasopressin, epinephrine, ROS, hyperglycemia, inflammatory cytokines, and shear stress (Kohan, 1997; Campese et al., 2006). These factors trigger protein kinase signaling converging on the ET-1 gene promoter. The vasodilators ANP, BNP, NO, prostaglandins E2 and I2, and bradykinin inhibit ET-1 synthesis via guanylyl cyclase, cyclic GMP (cGMP), and protein kinase G. Medullary interstitial osmolality modulates tubular ET-1 secretion. High osmolality activates ET-1 production and binding to ETBR in the thick ascending limb, thereby activating nitric oxide synthase (NOS) (Herrera and Garvin, 2005). NOS generates NO that dampens Naþ-reabsorbing Na,K,2Cl cotransport and Na/H exchange (Garvin et al., 2011), and triggers cGMP formation (Kohan, 1997) to suppress protein kinase A (PKA)dependent water retention (Fig. 19.1A). Mechanosensitive Ca2þ channels at the base of the principal cell’s primary cilium transduce collecting duct flow to a [Ca2þ]i signal (Weinbaum et al., 2010), which activates PKC, Ca2þ-dependent calmodulin kinase, calcineurin, and finally, ET-1 expression (Kohan et al., 2011). Hypoxia also augments renal ET-1. Hypoxia induced ET-1 in rat inner medullary collecting duct (Miller and Kohan, 1998), and in dogs, increased urinary ET-1 and Naþ excretion (Nir et al., 1994). Hypoxiainducible factor-1 (HIF-1) mediates hypoxia induction of ET-1. Suppression of HIF-1’s a subunit disrupted pressure natriuresis and increased salt sensitivity of rats (Li et al., 2008). Angiotensin II dampens medullary ET-1 activity. AngII blunted ETAR and ETBR expression in medullary collecting duct (Wong and Tsui, 2001) and natriuretic response to medullary ET-1 in rats (Kittikulsuth et al., 2012). ACE inhibitors increased medullary ET-1 and ETR expression in hypertensive rats (Hale et al., 2011) and cardiomyopathic hamsters (Wong et al., 1998). Thus, increased medullary AngII in hypertensive rats (Kujal et al., 2010) and humans (Kobori et al., 2010) may worsen hypertension by impeding ET-1dependent natriuresis and diuresis. 3.1.3 Actions of ET-1 ET-1 exerts important effects in mesangial cells (Kohan, 1997), cortical and medullary blood vessels (Neuhofer and Pittrow, 2006), and renal tubules (Fig. 19.1). ET-1 is a powerful vasoconstrictor in kidneys, where resistance vessels are tenfold more ET-1-sensitive than any other vascular bed (Madeddu et al., 1989). Abundant in renal vascular smooth muscle, ETARs respond to ET-1 from the adjacent endothelium. ET-1 constricts arcuate and interlobular arteries, afferent

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FIGURE 19.1 Physiologic (panel A) and pathologic (panel B) signaling mechanisms of endothelin-1 (ET-1) in kidney. Panel A: ET-1, acting via ETB receptors (ETBRs) activates mechanisms (blue) that suppress vasopressin-induced (green) water reabsorption and aldosterone-induced (purple) Naþ reabsorption in the distal nephron, culminating in natriuresis and diuresis that limit ET-1 increases in arterial pressure. Red lines and borders indicate processes inhibited by these mechanisms. Panel B: Acting via ETA receptors, ET-1 triggers signaling cascades that provoke mesangial cell contraction and proliferation and synthesis of extracellular matrix, culminating in maladaptive remodeling of the renal corpuscles in chronic kidney disease. See text for details and definitions of abbreviations.

and efferent arterioles, and vasa recta (Kohan, 1997). ETAR activation increased [Ca2þ]i in arteriolar smooth muscle (Fellner and Arendshorst, 2007). The superoxide scavenger tempol attenuated the [Ca2þ]i increase by 60%, implicating ROS in the [Ca2þ]i increase. Nicotinamide and 8-Br-cyclic ADP-ribose also blunted ET-1increased [Ca2þ]i, implying ET-1 activated formation of cyclic ADP-ribose, a mobilizer of sarcoplasmic reticular Ca2þ (Fellner and Arendshorst, 2007). ETAR-mediated vasoconstriction and mesangial cell contraction lower GFR, while ETBR-dependent production of vasodilators NO, AM, and prostaglandin I2 increases GFR. Several factors limit ET-1-induced vasoconstriction (Kohan, 1997). In vascular smooth muscle, the ET-1induced increase in [Ca2þ]i provokes sarcolemmal depolarization, which activates Ca2þ-dependent Kþ channels, repolarizing the sarcolemma and dampening contraction. Activation of vascular endothelial ETBRs elicits formation and release of NO to exert its familiar vasodilatory effect. ET-1 also activates synthesis of eicosanoids that relax vascular smooth muscle.

Endothelin-1 promotes mesangial cell contraction, proliferation, and hypertrophy (Fig. 19.1B), stimulates their production of proinflammatory cytokines and extracellular matrix, and potentiates AngII formation. Although ET-1 dampens juxtaglomerular renin release, it constricts afferent arterioles and lowers GFR, initiating tubuloglomerular feedback to release renin (Kohan, 1997). Like AngII, ET-1 generates IP3, which raises [Ca2þ]I, causing mesangial contraction that lowers GFR. ET-1-induced formation of eicosanoids via phospholipase A2 (Fig. 19.1A) and cGMP via NO-induced guanylyl cyclase (Kohan, 1997) moderates the contraction. Endothelin’s effects in the proximal tubule are complex. Low ET-1 concentrations selectively activate PKC, which increases Na-PO4 and NaeHCOe 3 cotransport and Na/H exchange to effect Naþ and HCOe 3 reabsorption. Higher ET-1 activates cyclooxygenase and lipoxygenase to produce Na, K ATPase inhibitors, and more intense PKC activity also inhibits Na, K ATPase. These actions promote natriuresis and diuresis, limiting

3. VASOCONSTRICTORS: ENDOTHELIN-1 AND UROTENSIN II

the increase in blood pressure produced by elevated circulating ET-1 (Kohan, 1997). In the collecting ducts, ET-1 suppresses Naþ and H2O reabsorption via phospholipase C and Ca2þ signaling, respectively (Fig. 19.1A). 3.1.4 Endothelin-Enhanced Hþ excretion ET-1 activates Hþ excretion to compensate for metabolic acidosis. Consumption of a protein-rich, Hþyielding diet (Khanna et al., 2004) increases tubular and microvascular ET-1 production (Wesson et al., 1998). Acting on ETBR, ET-1 activates proximal tubular Na/H exchange, promoting secretion and excretion of excess Hþ (Laghmani et al., 2001). By suppressing Na,K,2Cl cotransport in the thick ascending limb (Garvin et al., 2011), ET-1 augments Naþ delivery to the collecting duct, activating Hþ secretion by Na/H exchange. ET-1 also inhibits HCOe 3 secretion by collecting duct b-intercalated cells (Tsuruoka et al., 2006). 3.1.5 Endothelin-I in Chronic Kidney Diseases ET-1 is implicated in mesangial and tubulointerstitial fibrosis and proliferation (Fig. 19.1B), important contributors to chronic kidney disease. ET-1 and other profibrotic factors including AngII and TGF-b activate mesangial cells and peritubular fibroblasts to produce extracellular matrix. Guanine nucleotide exchange factor bPix and adaptor protein p66shc mediate ET-1 inactivation of transcription factor FOXO3a and cell cycle inhibitor p27kip1, enabling cell proliferation (Sorokin, 2011). Acting via ETAR, ET-1 provokes interstitial cell proliferation and matrix deposition in rat renal medulla (Zhuo et al., 1998). In animal models of chronic kidney disease, ACE inhibitors and AT1R blockers suppressed ET-1 formation, vasoconstriction, mesangial proliferation, and extracellular matrix buildup (Larivie`re and Lebel, 2003). ET-1 induction in mesangial cells contrasted to AngII’s suppression of ET-1 in collecting duct. ETAR blockade attenuated renal failure, hypertension, and cardiac hypertrophy in a rat partial nephrectomy model (Larivie`re and Lebel, 2003). In diabetic nephropathy patients, ET-generating chymase was elevated several-fold in vascular smooth muscle, mesangial cells, and peritubular interstitium (Huang et al., 2003). Excess ET-1 may exacerbate renal injury by constricting the microcirculation, thus making the tissue ischemic, and by inducing extracellular matrix overproduction. Conversely, tubulointerstitial diseases selectively impair tubular ET-1 production, natriuresis, and diuresis, causing hypervolemia and hypertension (Kohan, 1997).

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3.2 Urotensin II The potent vasoconstrictor UII and its G proteine coupled urotensin receptors (UTRs) are expressed in the urinary, cardiovascular, pulmonary, and central nervous systems (Chen et al., 2009; Ross et al., 2010). UTRs are abundant in inner medullary collecting ducts (Song et al., 2006; Disa et al., 2006). Renal arterioles and thin limbs of Henle’s loop also harbor UTRs (Song et al., 2006). UII’s myriad actions include vasoconstriction, proliferation of vascular smooth muscle, fibroblasts and cancer cells, induction of proatherosclerotic foam cells, inflammatory cell chemotaxis, cardiac hypertrophy, insulin resistance, disruption of normal sleep cycle and food intake, and in the kidneys, tubular Naþ retention and decreased GFR (Ross et al., 2010). The kidneys are major producers of UII, which exerts autocrine/paracrine effects on tubular transport and renal hemodynamics. Renal UII originates mainly in proximal tubules and medullary collecting ducts (Song et al., 2006). In rats, UII is 1650-fold more concentrated in urine than plasma, suggesting renal UII remains in the kidneys (Song et al., 2006). In rats, the UTR antagonist urantide increased GFR and Naþ and water excretion (Song et al., 2006), suggesting that endogenous UII promotes Naþ and water retention. UII infusion lowered renal Naþ clearance without altering fractional excretion of filtered Naþ, implying decreased GFR was the key factor lowering Naþ clearance (Song et al., 2006). 3.2.1 Ca2þ-Mediated UII Signaling Adebiyi (2014) examined UII-induced contraction in a mouse mesangial cell line. UII increased sarcoplasmic reticular Ca2þ release, store-operated Ca2þ entry, and contraction, in a manner blunted by UTR and IP3 receptor antagonists and inhibitors of sarcoplasmic reticular Ca2þ ATPase and store-operated Ca2þ channels. Regulator of G protein signaling (RGS2) modulates UTR activity and UII-activated mesangial cell contraction (Adebiyi, 2014). UII doubled RGS2 membrane content and increased the RGS2$UTR interaction, while siRNA knockdown of RGS2 increased UII-induced contraction and [Ca2þ]. Thus, the UTR: RGS2 interaction moderates UII-induced mesangial cell contraction. Ca2þ signaling also may mediate UII induction of mesangial cell proliferation and extracellular matrix deposition in response to hyperglycemia (Soni and Adebiyi, 2017). Exposure of cultured mouse glomerular mesangial cells to UII and high glucose activated storeoperated Ca2þ entry via transient receptor potential cation 4 channels, followed by sequential activation of

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Ca2þ-calmodulin dependent protein kinase and cAMP response element binding protein, culminating in proliferation and synthesis of extracellular matrix proteins type IV collagen and fibronectin. 3.2.2 Urotensin II in Chronic Diseases Serum UII is elevated in heart failure, systemic and pulmonary hypertension, atherosclerosis, hepatic cirrhosis, diabetes, and chronic kidney disease (Russell, 2004; Ong et al., 2005; Ross et al., 2010). UII contributes to disease progression by provoking cardiomyocyte hypertrophy, extracellular matrix deposition, vasoconstriction, vascular smooth muscle hyperplasia, and endothelial hyperpermeability (Russell, 2004). Increased vascular expression of UII and UTR have been identified in animal models of myocardial infarct and heart failure, and in patients with hypertension, atherosclerosis, and diabetic cardiomyopathy (Zhu et al., 2006). Its association with hypercholesterolemia and hyperglycemia implicates UII in the development of insulin resistance in metabolic syndrome (Thanassoulis et al., 2004). Urotensin II is implicated in diabetic nephropathy (Wassef et al., 2004). UII and UTR mRNA increased 45and c.2000-fold, respectively, in renal biopsies from diabetic patients with proteinuria and decreased GFR vs. control subjects, and the tubular epithelium had intense UII immunoreactivity and UTR binding activity (Langham et al., 2004). UII provoked ER stress and tubular EMT in diabetic mice, and it activated synthesis of extracellular matrix proteins fibronectin and fibroblast-specific protein 1 (Pang et al., 2016). As noted before, UII-induced [Ca2þ]i may mediate hyperglycemia-induced mesangial proliferation and extracellular matrix accumulation. Renovascular inflammatory diseases, e.g., atherosclerosis, hypertension, and glomerulonephritis, may modulate UII formation. In the inflammatory glomerular diseases membranoproliferative glomerulonephritis and focal segmental glomerulosclerosis, UII accumulated in glomerular basement membrane, mesangium, and tubular epithelium (Disa et al., 2006). Interestingly, UII may exert cardioprotective effects in the settings of coronary artery disease and chronic renal failure (Zoccali and Mallamaci, 2008). UII preserved cardiac contractility in chronically volume-overloaded rats, and UII pretreatment minimized ischemia-reperfusion injury in isolated rat hearts (Prosser et al., 2008). Low serum UII is associated with cardiomyopathy, cardiovascular complications, and increased mortality in patients with chronic kidney disease (Zoccali and Mallamaci, 2008); conversely, end-stage renal disease patients with higher UII had increased left ventricular systolic function and decreased left atrial volume (Zoccali et al., 2008). UII’s myriad effects in chronic diseases

underscore the urgent need for further research of the actions and mechanisms of this pleiotropic peptide.

4. THE NATRIURETIC PEPTIDES: ANP, BNP, AND URODILATIN Natriuretic peptides lower arterial blood pressure by dilating resistance vessels throughout the systemic circulation and by increasing urinary Naþ and water excretion. Natriuresis and diuresis are effected by increasing GFR while suppressing Naþ and water reabsorption (Zeidel, 1993; Endlich and Steinhausen, 1997). Circulating ANP and BNP increase GFR by dilating arcuate and interlobular arteries and afferent arterioles, while constricting efferent arterioles (Endlich et al., 1995). Urodilatin, produced in the renal tubules and a powerful inhibitor of Naþ and water reabsorption, is effectively absent from the systemic circulation (Drummer et al., 1993). All three natriuretic peptides ANP, BNP, and urodilatin contain a 17-amino acid ring (Endlich and Steinhausen, 1997). An N-terminal extension makes urodilatin highly resistant to proteolysis (Meyer et al., 1998). ANP and BNP are stored in myoendocrine cells in the cardiac atria and ventricles, respectively (Forssmann et al., 1998). Plasma volume expansion stretches the atria, triggering ANP secretion (Forssmann et al., 1998). In a similar fashion, ventricular distention provokes BNP secretion, making this peptide a heart failure biomarker (Oremus et al., 2014). The renal tubules are the exclusive source of urodilatin and also produce ANP. Urodilatin is secreted by the distal convoluted tubule and carried to the inner medullary collecting duct, where it binds apical membrane receptors to inhibit Naþ and water reabsorption (Forssmann et al., 2001). Circulating ANP and BNP are small peptides that undergo glomerular filtration and are then degraded at the proximal tubular brush border by a metalloproteinase, neprilysin. Resistant to proteolysis, urodilatin is cleared by urinary excretion (Forssmann et al., 1998).

4.1 Natriuretic Peptide Signaling Natriuretic peptides activate guanylyl cyclasee coupled natriuretic peptide receptors (NPR)(Endlich and Steinhausen, 1997). ANP, BNP, and urodilatin bind NPR-A with similar affinities greatly exceeding that of C-type natriuretic peptide (CNP). Conversely, CNP has far greater affinity for NPR-B than do the other ligands. NPR-C binds all four peptides with similar affinity (Endlich and Steinhausen, 1997). NPR-C, which lacks guanylyl cyclase, may mediate proximal tubular endocytosis of ANP and BNP to initiate their degradation.

5. CALCITONIN GENE-RELATED PEPTIDES: ADRENOMEDULLIN AND INTERMEDIN

4.2 Renal Actions of Natriuretic Peptides ANP dilates afferent and constricts efferent arterioles (Endlich and Steinhausen, 1997), producing robust increases in glomerular filtration of Naþ and water. ANP, BNP, and urodilatin suppress Naþ reabsorption in the inner medullary collecting duct by inhibiting apical Naþ channels and basolateral Na,K ATPase (Bełtowski and Wo´jcicka, 2002). NPR activation also increases [Ca2þ]i in the tubular epithelium (Meyer et al., 1998); Ca2þ activates NOS, generating NO that inactivates the Naþ channels (Garvin et al., 2011). The natriuretic peptides also dampen Naþ reabsorption in the thick ascending limb by generating cGMP, which suppresses cAMP-activation of Na,K,2Cl cotransport (Bailly, 1998). The peptides suppress vasopressin-induced water reabsorption in the collecting duct by inhibiting the V2 receptor signaling cascade at a point downstream of cAMP formation (Inoue et al., 2001). A mediator of the diuretic effect is cGMP (Meyer et al., 1998; Drummer, 2001), which inhibits vasopressin’s effector, PKA, by competing with cAMP for PKA’s activation domain.

4.3 Natriuretic Peptides: Potential Interventions for Heart Failure Management None of the natriuretic peptides were effective natriuretic or diuretic agents in heart failure patients (Vesely, 2007), in all likelihood due to the massive increases in circulating Naþ- and water-retaining hormones in heart failure. Another factor is the decreased GFR, providing less Naþ and water for excretion. Conversely, it has been argued (Gassanov et al., 2012; Namdari et al., 2016) that despite the decreased GFR, the natriuretic peptides might be effective for managing advanced heart failure by virtue of their combination of diuresis, vasodilation, and suppression of renin-AngII. In 1995, human recombinant ANP was approved for acute heart failure in Japan, and in 2001, human recombinant BNP was approved by the U.S. Food and Drug Administration for management of acute heart failure. Recombinant human urodilatin is undergoing a phase 3 clinical trial for the same indication (Torres-Couchoud and Chen, 2016).

5. CALCITONIN GENE-RELATED PEPTIDES: ADRENOMEDULLIN AND INTERMEDIN 5.1 Adrenomedullin First identified in human pheochromocytoma (Kitamura et al., 1993), adrenomedullin (AM), a member

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of the calcitonin gene-related peptide (CGRP) family, is antiproliferative, vasorelaxant, and natriuretic. AM is abundantly expressed in kidney (Takahasi et al., 2009) as well as heart, vasculature, lung, endocrine organs, and central nervous system (Eto et al., 1999; Taylor and Samson, 2002). AM circulates as inactive AMglycine and active AM-mature (Jougasaki et al., 1995). Chronic kidney disease increases serum AM-glycine and AM-mature, but it lowers urinary excretion of both, indicating serum and urinary AM have different origins (Bunton et al., 2004). Adrenomedullin is produced in many renal cell types (Takahashi et al., 2009). Glomerular capillary endothelium, mesangial cells, podocytes, distal convoluted tubule, and collecting duct secrete AM (Lai et al., 1998). Human kidney contains strong AM immunoreactivity (Eto et al., 2003), especially in distal nephron segments (Asada et al., 1999). PKC and cAMP activate the AM propeptide gene. The presence of an HIF-1 consensus site in the promoter suggests that hypoxia may activate AM expression (Cormier-Regard et al., 1998). Exercise, sympathetic activity, ANP, and AngII also may induce AM (Eto et al., 2003). AM production (Fig. 19.2A) is activated by proinflammatory cytokines (Sugo et al., 1995; Isumi et al., 1998), glucocorticoids (Imai et al., 1995), and ROS (Ando et al., 1998; Chun et al., 2000). 5.1.1 Adrenomedullin Signaling Mechanisms AM activates calcitonin receptor-like receptors (CRLR) (Mukoyama et al., 2001). Receptor activity modifying proteins (RAMPs) combine with CRLR; the specific RAMP determines CRLR’s ligand binding affinity and character. RAMP1 imparts CGRP selectivity; RAMPs 2 and 3, both expressed in kidney, make CRLR more AM-responsive. Vasopressin activates RAMP3 expression, which, by augmenting AM-induced natriuresis, may limit expansion of extracellular fluid volume (Mukoyama et al., 2001). In rat aortic smooth muscle cells (Liu et al., 2007), AM attenuated AngII-activated ROS formation by NADPH oxidase. The cAMP analog dibutyryl cAMP also blocked ROS production, while PKA inhibitors abrogated AM suppression of AngII-induced ROS. The CRLR antagonist CGRP8-37 also blunted AM’s antioxidant effect, as did expression of constitutively active Src. COOH-terminal Src kinase, i.e., Csk, phosphorylates and inactivates Src at Tyr527, while AngII phosphor activates Src at Tyr416 (Liu et al., 2007). AM suppresses ROS via the cAMP-PKA pathway in rat mesangial cells (Chini et al., 1997). Collectively, these findings support a mechanism whereby AM binding to CRLR$RAMP sequentially activates cAMP formation, PKA, and Csk, which, by inactivating Src, suppresses ROS formation by NADPH oxidase (Fig. 19.2B).

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FIGURE 19.2 Renoprotective mechanisms of adrenomedullin and intermedin. Panel A: Adrenomedullin (AM) induction by oxidative stress and fluid shear stress activates nitric oxide synthase (NOS) and NO production, which dilates the renal microcirculation, increasing renal perfusion. Panel B: Adrenomedullin and intermedin activate intracellular signaling cascades (blue) that suppress reactive oxygen species (ROS) formation and tubular epithelial apoptosis induced by unilateral ureteral obstruction (UUO; green), a rat model of chronic renal injury. Red lines and borders indicate processes inhibited by these mechanisms. See text for details.

5.1.2 Physiologic Actions of Adrenomedullin AM lowers blood pressure and provokes natriuresis and diuresis (Edwards et al., 1997). Renal arterial AM infusion in rats and dogs increased renal blood flow and GFR, dampened mesangial cell ROS formation and proliferation, and lowered serum vasopressin, ET1, and aldosterone (Jougasaki et al., 1995; Mukoyama et al., 2001; Fujisawa et al., 2004). Collectively, these actions elicited copious natriuresis and diuresis. Extrarenal effects of AM include vasodilation, cardiac inotropy, and aldosterone suppression (Eto et al., 1999; Nagaya et al., 2000). In human subjects, AM infusion increased resting heart rate and cardiac output, the latter by 70% (Lainchbury et al., 2000), and augmented serum norepinephrine and renin without increasing aldosterone (Nicholls et al., 2001). In IgA nephropathy patients, AM infusion increased serum AngII and norepinephrine, lowered systemic arterial pressure, and increased heart rate, diuresis, and natriuresis (McGregor et al., 2001). 5.1.3 Renoprotective Mechanisms of Adrenomedullin AM dampens renal AngII signaling (Takahashi et al., 2009), ROS formation (Chini et al., 1997), and mesangial proliferation (Parameswaran et al., 2001). Puromycin aminonucleoside generated ROS that induced apoptosis of murine podocytes (Oba et al., 2008); AM attenuated the ROS and apoptosis by activating PKA (Fig. 19.2B) in a manner abrogated by a CRLR antagonist. In AM-

deficient mice, AngII infusion and high-Naþ diet increased urinary oxidative stress markers and provoked more intense perivascular fibrosis and intimal hyperplasia than in wild-type mice (Shimosawa et al., 2002). Tunicamycin, an ER stress activator, caused tubular cell death prevented by AM in wild-type but not RAMP2þ/ mice (Uetake et al., 2014), showing CRLR$RAMP2 to mediate AM cytoprotection. AM also suppressed mesangial proliferation induced by ET-1 or increased glomerular capillary transmural pressure (Chini et al., 1997). By suppressing mesangial cell proliferation, a hallmark of glomerular disease, AM may ameliorate glomerulonephritis (Parameswaran et al., 2001). 5.1.4 Antihypertensive Actions of Adrenomedullin AM is elevated in acute and chronic cardiorenal diseases including chronic renal disease, myocardial infarction, hypertension, congestive heart failure, diabetes, and sepsis (Edwards et al., 1997; Eto et al., 2003; Bunton et al., 2004). Chronic AM administration improved renal function and decreased glomerular injury in Dahl saltsensitive rats fed a high-salt diet (Nishikimi et al., 2002). In Wistar rats, a high-salt diet increased serum AM 2.5-fold, and induced adrenal and renal AM, CRLR, and RAMP expression (Cao et al., 2003), likely to compensate for the high salt intake. In hypertensive humans, plasma AM activity paralleled the increased arterial pressure, renal injury, and

5. CALCITONIN GENE-RELATED PEPTIDES: ADRENOMEDULLIN AND INTERMEDIN

cardiac and arterial wall hypertrophy (Sogbe-Dı´az and Dı´az-Lo´pez, 2016). AM infusion increased cardiac index, urine volume, and Naþ excretion, and lowered pulmonary arterial and capillary wedge pressures and plasma aldosterone activity in heart failure patients (Nagaya et al., 2000). In heart failure, increased AM content in myocardium, kidney, and plasma (Nicholls et al., 2003) may blunt vasoconstriction by elevated catecholamines, ET-1, and AngII.

5.2 Intermedin Intermedin (IMD), i.e., adrenomedullin-2, is a ligand for all three CRLR$RAMP combinations (Takahashi et al., 2009). Brain, pituitary, heart, and renal tubular epithelium express IMD mRNA (Takahashi et al., 2006). In humans, IMD is most abundant in the neurohypophysis and renal cortex (Morimoto et al., 2007). IMD also is present in natriuretic peptide-secreting cardiomyocytes and colocalizes with vasopressin in the hypothalamus (Takahashi et al., 2006). IMD’s locations bespeak its role in both central and peripheral regulation of circulating volume. 5.2.1 Physiologic Effects of Intermedin IMD increased phosphorylation of ERK, Akt, and eNOS, NO formation, and synthesis of proangiogenic vascular endothelial growth factor and its receptor. In rats, renal arterial IMD infusion increased renal blood flow, urine volume, and Naþ excretion, without altering arterial pressure or GFR (Fujisawa et al., 2004), while NOS inhibition blunted IMD’s vasodilatory effects (Jolly et al., 2009). In conscious sheep, intravenous IMD increased heart rate and cardiac output, yet the vasodilation lowered arterial pressure (Charles et al., 2006). IMD increased circulating natriuretic peptides, plasma renin, and AngII. 5.2.2 Intermedin’s Actions Against Renal and Cardiovascular Disease Kidney-directed delivery of the human IMD gene suppressed inflammation and fibrosis in rats with IgA nephropathy (Wang et al., 2016). In salt-sensitive, hypertensive rats, IMD delivery increased GFR, urine volume, and tubular NO formation, lowered proinflammatory cytokines, adhesion molecules, and inflammatory cell infiltration, and mitigated fibrosis and damage to renal corpuscles and tubules (Hagiwara et al., 2008). Conversely, persons with a homozygous deletion near IMD’s N-terminus had hypertension, elevated serum creatinine, and heightened risk of chronic kidney disease, lacunar brain infarcts, and cerebral small vessel disease (Hirose et al., 2011).

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Chronic kidney disease is associated with Ca2þ deposition in atherosclerotic plaques, i.e., vascular calcification. This pathology was modeled in rats by administering calcitriol and nicotine, which decreased aortic IMD content and increased Ca2þ deposition while lowering smooth muscle lineage markers in the vascular wall (Cai et al., 2010). IMD administration attenuated Ca2þ deposition and preserved smooth muscle lineage markers and the Ca2þ-sequestering protein, g-carboxyglutamic acid protein (cMGP). Pretreatment with anti-cMGP small interfering RNA abrogated IMD suppression of vascular calcification. Thus, IMD ameliorates vascular calcification by inducing cMGP expression in vascular smooth muscle. Qiao et al. (2013) examined renoprotective mechanisms of kidney-specific IMD overexpression in rats subjected to renal artery occlusion-reperfusion after unilateral nephrectomy. The ischemic insult increased serum creatinine, and activities of ROS-generating myeloperoxidase and proapoptotic caspase 3, and lowered activity of the antioxidant enzyme, superoxide dismutase. Tubular expression of ET-1 and the inflammatory mediators ICAM-1 and P-selectin increased markedly after ischemia. Renal ischemia also induced ER stress. Adenoviral delivery of the IMD gene preserved superoxide dismutase and attenuated ROS, inflammation, ET-1 content, apoptosis, and ER stress (Qiao et al., 2013). These investigators also examined IMD’s renoprotective mechanisms (Fig. 19.2B) in a rat unilateral ureteral obstruction model of renal fibrosis (Qiao et al., 2017). In these rats, adenoviral-delivered IMD decreased tubular injury, interstitial collagen and fibronectin accumulation, proinflammatory cytokine production, and macrophage infiltration and attenuated ROS and EMT. IMD also augmented renal activity of an antioxidant enzyme, heme oxygenase-1 (HO-1). The HO-1 inhibitor zinc protoporphyrin abrogated all of IMD’s renoprotective actions (Qiao et al., 2017), indicating HO-1 mediated IMD suppression of EMT and fibrosis (Fig. 19.2B). Like AM, IMD exerts several potentially beneficial cardiovascular effects, including systemic and pulmonary vasodilation, increased cardiac contractility, suppression of cardiomyocyte hypertrophy, and cardioprotection against ROS (Bell and McDermott, 2008). IMD vasodilates by acting on the same receptors as AM (Jolly et al., 2009). In a rat model of hindlimb ischemia, delivery of the human IMD gene to the ischemic muscle caused progressive IMD accumulation, increased capillary and arteriole densities and enhanced perfusion (Smith et al., 2009).

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6. CALCITRIOL AND CA2D METABOLISM Although the steroid hormone calcitriol is involved in insulin secretion, blood pressure regulation, skeletal myocyte differentiation, and immune responses (Jones, 2007), its primary mission is maintenance of Ca2þ balance. To accomplish this task, calcitriol activates absorption of dietary Ca in the duodenum and upper jejunum (Tudpor et al., 2008), bone resorption to release Ca2þ into the circulation, and Ca2þ reabsorption in the distal convoluted tubule, where calcitriol activates expression of apical membrane Ca2þ channels (Hoenderop et al., 2001). Active calcitriol, 1,25(OH)2D3, is produced by 25hydroxylation of vitamin D in the liver and then 1a-hydroxylation in the renal tubules. Vitamin D binding protein (DBP) carries 25(OH)D3 in plasma. After glomerular filtration, the 25(OH)D3$DBP complex binds megalin and cubulin on the proximal tubular apical membrane and undergoes endocytosis. In the inner mitochondrial membrane, 25(OH)D3 is hydroxylated by 1a- or 24-hydroxylase, yielding active calcitriol or inactive calcitroic acid, 24,25(OH)2D3, respectively (Chesney, 2016).

6.1 Regulation of Calcitriol Activity Although widely distributed in the body, 1a-hydroxylase is most abundant in proximal tubule (Murayama et al., 1999). Calcitriol binds cytosolic vitamin D receptor (VDR), forming a complex that suppresses 1a-hydroxylase mRNA expression (Fig. 19.3) (Takeyama et al., 1997;

Turunen et al., 2007) and evokes expression of the competing 24-hydroxylase to dampen calcitriol production (Wang et al., 2015). Calcitriol$VDR also activates expression of Ca2þ-sensitive receptors, which suppress tubular Ca2þ reabsorption during hypercalcemia (Bai and Favus, 2006). In a similar fashion, calcitriol modulates 1a-hydroxylase, 24-hydroxylase, and Ca2þ-sensitive receptors in intestine, bone, and parathyroid cells. Concordant with its task of raising serum [Ca2þ], parathyroid hormone (PTH) activates 1a-hydroxylase expression (Fig. 19.3), increasing calcitriol production capacity in the proximal tubule (Armbrecht et al., 2003). PTH-activated PKA activates the transcription factor CREB, which promotes 1a-hydroxylase expression (Armbrecht et al., 2003). Genetic deletion of VDR intensified 1a-hydroxylase induction by PTH and its Ca2þ-regulating partner, calcitonin (Murayama et al., 1999). Calcitonin induces 1a-hydroxylase (Shinki et al., 1999) by mobilizing another transcription factor, C/EBPb (Zhong et al., 2009).

6.2 FGF23-Klotho Calcitriol and PO4 activate FGF23 secretion by skeletal osteoblasts and osteocytes. FGF23 inhibits proximal tubular 1a-hydroxylase, thereby decreasing calcitriol synthesis (Torres et al., 2009). In distal tubule, FGF23 increases membrane-associated and soluble content of a cell surface protein, klotho (Takenaka et al., 2016). Metalloproteinases release klotho’s large extracellular domain into the interstitium from where it is delivered to the systemic circulation. Soluble klotho cleaves the FGF

FIGURE 19.3 Regulation of renal tubular calcitriol synthesis and its renal and systemic actions. Parathyroid hormone (PTH) induces

1a-hydroxylase, the final step in calcitriol activation. Calcitriol$vitamin D receptor (VDR) complexes inhibit 1a-hydroxylase and activate Ca2þsensitive receptors (CaSR) to dampen bone resorption, intestinal Ca2þ absorption, and renal Ca2þ reabsorption. Calcitriol and phosphate stimulate FGF23 secretion from bone, initiating FGF23/klotho suppression of calcitriol to prevent hypercalcemia and hyperphosphatemia. Mechanisms opposing increases in serum Ca2þ and PO4 are shown in red.

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receptor, increasing its FGF23 affinity and augmenting FGF23-induced PO4 and Ca2þ excretion. Klotho also complexes with FGF23 in the proximal tubular epithelium, where the FGF23$klotho complex decreases calcitriol by blocking PTH induction of 1a-hydroxylase and by activating 24a-hydroxylase (Fig. 19.3). Calcitriol induces klotho and FGF23 (Umbach et al., 2015) to limit further calcitriol formation.

6.3 Calcitriol in Renal Disease Calcitriol may be renoprotective in the setting of kidney disease (Tan et al., 2007). The hormone exerts several renoprotective effects: it suppresses inflammatory cell infiltration, renin expression, and extracellular matrix production by mesangial cells and fibroblasts; it prevents effacement of the podocyte epithelium, and it activates expression of the tight junction protein Ecadherin to prevent EMT (Tan et al., 2007). Calcitriol decreased fibrotic lesions in mice with unilateral ureteral obstruction (Tan et al., 2006), and it lowered profibrotic expression of types I and III collagen, fibronectin, and TGF-b1 in the obstructed kidney. Patients with renal disease often have low serum calcitriol (Jones, 2007), and an inverse relationship between serum calcitriol and renal inflammation has been reported (Zehnder et al., 2008). Inflammatory damage to the glomerular membrane in chronic kidney disease permits massive amounts of protein to enter the nephrons, saturating endocytosis at the expense of 25(OH)D3$DBP and calcitriol$DBP uptake (Fukagawa et al., 2012). Diabetic (Ogasawara et al., 2012) and IgA nephropathy (Seki et al., 2014) in humans are associated with shedding and urinary excretion of megalin and its fragments; the loss of these DBP receptors disables proximal tubular uptake of calcitriol$DBP. The resultant calcitriol deficiency in renal failure is associated with bone mineral loss, i.e., renal osteodystrophy (Mazzaferro et al., 2010).

6.4 Estrogen Replacement and Calcitriol In postmenopausal women, estrogen replacement therapy increased serum PTH by 38% and calcitriol by 24%, lowered urinary Ca2þ excretion by 33%, and decreased deoxypyridinoline, a serum marker of bone resorption, by 20% (McKane et al., 1995). Estrogen replacement also lowered fractional excretion of filtered Ca2þ from 2.4% to 1.3%. By suppressing bone resorption, estrogen lowers serum Ca2þ, provoking PTH secretion that activates tubular Ca2þ reabsorption and calcitriol production. Thus, estrogen replacement minimizes osteodystrophy in postmenopausal women by increasing renal retention of Ca2þ.

7. ERYTHROPOIETIN The ATP demands of the brain, heart, and other internal organs can only be met by oxidative phosphorylation, which consumes vast amounts of O2. Tissues have a limited O2 storage capacity, so O2 must be delivered to respiring cells via the circulation. Adequate O2 delivery requires hemoglobin, packaged in erythrocytes. Circulating erythrocytes survive for 70e140 days (Mock et al., 2011), and they must be continually replenished by hematopoiesis. The glycoprotein erythropoietin inhibits apoptosis of erythroid progenitors in bone marrow, permitting their maturation to erythrocytes (Malik et al., 2013) to maintain the blood’s O2-carrying capacity.

7.1 O2 regulation of Renal Erythropoietin Production The kidneys generate 90% of circulating erythropoietin in adults. Specialized peritubular fibroblasts at the corticomedullary border secrete erythropoietin (Wenger and Hoogewijs, 2010), which circulates to the bone marrow. HIF-1-driven transcription of the erythropoietin gene regulates erythropoietin production. O2 exerts robust control over HIF-1 by regulating cellular content of its a subunit (Myllyharju, 2013). In the presence of O2, Fe2þ, and a-ketoglutarate, prolyl hydroxylase catalyzes hydroxylation of two prolyl residues, targeting HIF-1a for proteasomal degradation and suppressing erythropoietin expression. This mechanism is exquisitely O2sensitive: even modest declines in interstitial O2, e.g., at altitude, stabilize HIF-1a to produce erythropoietin. Renal disease disrupts this elegant mechanism (Wenger and Hoogewijs, 2010). As GFR falls and less Naþ enters the nephrons, proximal tubular reabsorption of Naþ declines, which lowers the epithelium’s ATP consumption, decreasing aerobic metabolism and O2 extraction from the peritubular capillaries. Interstitial O2 concentration increases, causing HIF-1a degradation, decreased HIF-1-driven gene expression, and impaired erythropoietin production, culminating in anemia. Tubulointerstitial diseases damaging the proximal tubules also lower O2 extraction, with similar outcomes. Thus, decreased proximal tubular O2 demand, not loss of erythropoietin-secreting fibroblasts, impairs erythropoietin production in renal failure. Angiotensin II activates erythropoiesis in response to hypovolemia or hemorrhage (Jelkmann, 2011). AngII infusions increased circulating erythropoietin in moderately exsanguinated male subjects, in a manner blunted by AT1R antagonists (Freudenthaler et al., 1999). AngII-induced ROS (Chen et al., 2005) may activate erythropoietin expression by depleting Fe2þ, the

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electron donor for prolyl hydroxylase, thereby stabilizing HIF-1a (Li et al., 2014). Erythropoietin production is not limited to the kidneys. Erythropoietin formation in brain (Noguchi et al., 2007; Ryou et al., 2012) and heart (Ryou et al., 2009) may serve to protect these organs from ischemic injury (Noguchi et al., 2007; Nguyen et al., 2014; Mallet and Ryou, 2017). However, this extrarenal erythropoietin production is but a fraction of the output from healthy kidneys, and it cannot replace the lost production in renal failure.

7.2 Erythropoietin: Renoprotective Hormone? Preclinical studies have demonstrated renoprotective actions of erythropoietin. Exogenous erythropoietin decreased tubular and microvascular damage in murine models of adriamycin nephrotoxicity (Nakazawa et al., 2013) and lipopolysaccharide-induced acute kidney injury (Stoyanoff et al., 2014). Pretreatment with human erythropoietin protected glomerular capillaries and podocytes in a rat cardiopulmonary bypass model (Liu et al., 2013). Also in rats, erythropoietin administration 2 d before the nephrotoxic anticancer agent cisplatin minimized increases in serum creatinine and renal cellular apoptosis and protein markers of ER stress (Kong et al., 2013). These renoprotective effects were independent of hematocrit. However, clinical trials of erythropoietin for anemia, renal transplantation, and renoprotection during surgery have not demonstrated consistent improvement in renal function, graft survival, or renal injury (Kim et al., 2016; Elliott et al., 2017). The alternative strategy of augmenting renal erythropoietin production for renoprotection has not been tested and merits investigation.

8. SUMMARY In addition to the externally generated hormones impacting renal function, including AngII from the pulmonary circulation, aldosterone from the adrenal cortex, vasopressin from the hypothalamus, and natriuretic peptides from the heart, an expanding roster of hormones produced by the kidneys has been identified. These renal hormones contribute extensively to the physiologic functions of the kidneys and, in many cases, other organs, and have been implicated in mechanisms of renal disease and/or renoprotection. The endocrine kidney offers exciting opportunities for development of novel interventions for renal and systemic diseases.

Acknowledgments While completing this work Dr. Ma was supported by grants 1-R01DK-115424 from the U.S. National Institute of Diabetes and Digestive and Kidney Diseases and 16GRNT27780043 from American Heart Association Southwest Affiliate, and Dr. Mallet by grants from the Institute for Cardiovascular and Metabolic Diseases and the Office of Research and Innovation, University of North Texas Health Science Center.

References Adebiyi, A., 2014. RGS2 regulates urotensin II-induced intracellular Ca2þ elevation and contraction in glomerular mesangial cells. J. Cell. Physiol. 229, 502e511. Ando, K., Ito, Y., Kumada, M., Fujita, T., 1998. Oxidative stress increases adrenomedullin mRNA levels in cultured rat vascular smooth muscle cells. Hypertens. Res. 21, 187e191. Armbrecht, H.J., Hodam, T.L., Boltz, M.A., 2003. Hormonal regulation of 25-hydroxyvitamin D3-1a-hydroxylase and 24-hydroxylase gene transcription in opossum kidney cells. Arch. Biochem. Biophys. 409, 298e304. Asada, Y., Hara, S., Marutsuka, K., Kitamura, K., Tsuji, T., Sakata, J., Sato, Y., Kisanuki, A., Eto, T., Sumiyoshi, A., 1999. Novel distribution of adrenomedullin-immunoreactive cells in human tissues. Histochem. Cell Biol. 112, 185e191. Bai, S., Favus, M.J., 2006. Vitamin D and calcium receptors: links to hypercalciuria. Curr. Opin. Nephrol. Hypertens. 15, 381e385. Bailly, C., 1998. Transducing pathways involved in the control of NaCl reabsorption in the thick ascending limb of Henle’s loop. Kidney Int. Suppl. 65, S29eS35. Bell, D., McDermott, B.J., 2008. Intermedin (adrenomedullin-2): a novel counter-regulatory peptide in the cardiovascular and renal systems. Br. J. Pharmacol. 153 (Suppl. 1), S247eS262. Berry, C., Touyz, R., Dominiczak, A.F., Webb, R.C., Johns, D.G., 2001. Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide. Am. J. Physiol. Heart Circ. Physiol. 281, H2337eH2365. Bełtowski, J., Wo´jcicka, G., 2002. Regulation of renal tubular sodium transport by cardiac natriuretic peptides: two decades of research. Med. Sci. Monit. 8, RA39e52. Bunton, D.C., Petrie, M.C., Hillier, C., Johnston, F., McMurray, J.J., 2004. The clinical relevance of adrenomedullin: a promising profile? Pharmacol. Ther. 103, 179e201. Cai, Y., Xu, M.J., Teng, X., Zhou, Y.B., Chen, L., Zhu, Y., Wang, X., Tang, C.S., Qi, Y.F., 2010. Intermedin inhibits vascular calcification by increasing the level of matrix g-carboxyglutamic acid protein. Cardiovasc. Res. 85, 864e873. Campese, V.M., Mitra, N., Sandee, D., 2006. Hypertension in renal parenchymal disease: why is it so resistant to treatment? Kidney Int. 69, 967e973. Cao, Y.N., Kitamura, K., Kato, J., Kuwasako, K., Ito, K., Onitsuka, H., Nagoshi, Y., Uemura, T., Kita, T., Eto, T., 2003. Chronic salt loading upregulates expression of adrenomedullin and its receptors in adrenal glands and kidneys of the rat. Hypertension 42, 369e372. Charles, C.J., Rademaker, M.T., Richards, A.M., 2006. Hemodynamic, hormonal, and renal actions of adrenomedullin-2 in normal conscious sheep. Endocrinology 147, 1871e1877. Chen, T.H., Wang, J.F., Chan, P., Lee, H.M., 2005. Angiotensin II stimulates hypoxia-inducible factor 1a accumulation in glomerular mesangial cells. Ann. N. Y. Acad. Sci. 1042, 286e293.

REFERENCES

Chen, Y.H., Yandle, T.G., Richards, A.M., Palmer, S.C., 2009. Urotensin II immunoreactivity in the human circulation: evidence for widespread tissue release. Clin. Chem. 55, 2040e2048. Chesney, R.W., 2016. Interactions of vitamin D and the proximal tubule. Pediatr. Nephrol. 31, 7e14. Chini, E.N., Chini, C.C., Bolliger, C., Jougasaki, M., Grande, J.P., Burnett Jr., J.C., Dousa, T.P., 1997. Cytoprotective effects of adrenomedullin in glomerular cell injury: central role of cAMP signaling pathway. Kidney Int. 52, 917e925. Chun, T.H., Itoh, H., Saito, T., Yamahara, K., Doi, K., Mori, Y., Ogawa, Y., Yamashita, J., Tanaka, T., Inoue, M., Masatsugu, K., Sawada, N., Fukunaga, Y., Nakao, K., 2000. Oxidative stress augments secretion of endothelium-derived relaxing peptides, C-type natriuretic peptide and adrenomedullin. J. Hypertens. 18, 575e580. Cormier-Regard, S., Nguyen, S.V., Claycomb, W.C., 1998. Adrenomedullin gene expression is developmentally regulated and induced by hypoxia in rat ventricular cardiac myocytes. J. Biol. Chem. 273, 17787e17792. Dilauro, M., Burns, K.D., 2009. Angiotensin-(1-7) and its effects in the kidney. ScientificWorldJournal 9, 522e535. Disa, J., Floyd, L.E., Edwards, R.M., Douglas, S.A., Aiyar, N.V., 2006. Identification and characterization of binding sites for human urotensin-II in Sprague-Dawley rat renal medulla using quantitative receptor autoradiography. Peptides 27, 1532e1537. Drummer, C., 2001. Involvement of the renal natriuretic peptide urodilatin in body fluid regulation. Semin. Nephrol. 21, 239e243. Drummer, C., Fiedler, F., Bub, A., Kleefeld, D., Dimitriades, E., Gerzer, R., Forssmann, W.G., 1993. Development and application of a urodilating (CDD/ANP-95-126)-specific radioimmunoassay. Pflu¨gers Archiv 423, 372e377. Edwards, R.M., Trizna, W., Aiyar, N., 1997. Adrenomedullin: a new peptide involved in cardiorenal homeostasis? Exp. Nephrol. 5, 18e22. Elliott, S., Tomita, D., Endre, Z., 2017. Erythropoiesis stimulating agents and reno-protection: a meta-analysis. BMC Nephrol. 18, 14. Endlich, K., Steinhausen, M., 1997. Urodilatin and the control of renal microcirculation. Blood Purif. 15, 323e333. Endlich, K., Forssmann, W.G., Steinhausen, M., 1995. Effects of urodilatin in the rat kidney: comparison with ANF and interaction with vasoactive substances. Kidney Int. 47, 1558e1568. Eto, T., Kitamura, K., Kato, J., 1999. Biological and clinical roles of adrenomedullin in circulation control and cardiovascular diseases. Clin. Exp. Pharmacol. Physiol. 26, 371e380. Eto, T., Kato, J., Kitamura, K., 2003. Regulation of production and secretion of adrenomedullin in the cardiovascular system. Regul. Pept. 112, 61e69. Fellner, S.K., Arendshorst, W., 2007. Endothelin-A and -B receptors, superoxide, and Ca2þ signaling in afferent arterioles. Am. J. Physiol. Renal. Physiol. 292, F175eF184. Forssmann, W.G., Richter, R., Meyer, M., 1998. The endocrine heart and natriuretic peptides: histochemistry, cell biology, and functional aspects of the renal urodilatin system. Histochem. Cell Biol. 110, 335e357. Forssmann, W., Meyer, M., Forssmann, K., 2001. The renal urodilatin system: clinical implications. Cardiovasc. Res. 51, 450e462. Freudenthaler, S.M., Schreeb, K., Korner, T., Gleiter, C.H., 1999. Angiotensin II increases erythropoietin production in healthy human volunteers. Eur. J. Clin. Investig. 29, 816e823. Fujisawa, Y., Nagai, Y., Miyatake, A., Takei, Y., Miura, K., Shoukouji, T., Nishiyama, A., Kimura, S., Abe, Y., 2004. Renal effects of a new member of adrenomedullin family, adrenomedullin2, in rats. Eur. J. Pharmacol. 497, 75e80. Fukagawa, M., Komaba, H., Hamano, T., 2012. Vitamin D supplementation in renal disease: is calcitriol all that is needed? Scand. J. Clin. Lab. Invest. Suppl. 243, 120e123.

457

Garvin, J.L., Herrera, M., Ortiz, P.A., 2011. Regulation of renal NaCl transport by nitric oxide, endothelin, and ATP: clinical implications. Annu. Rev. Physiol. 73, 359e376. Gassanov, N., Biesenbach, E., Caglayan, E., Nia, A., Fuhr, U., Er, F., 2012. Natriuretic peptides in therapy for decompensated heart failure. Eur. J. Clin. Pharmacol. 68, 223e230. Gomes, C.P., Lea˜o-Ferreira, L.R., Caruso-Neves, C., Lopes, A.G., 2005. Adenosine reverses the stimulatory effect of angiotensin II on the renal Naþ-ATPase activity through the A2 receptor. Regul. Pept. 129, 9e15. Hagiwara, M., Bledsoe, G., Yang, Z.R., Smith Jr., R.S., Chao, L., Chao, J., 2008. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress. Am. J. Physiol. Renal. Physiol. 295, F1735eF1743. Hale, T.M., Bushfield, T.L., Woolard, J., Pang, J.J., Rees-Milton, K.J., Adams, M.A., 2011. Changes critical to persistent lowering of arterial pressure in spontaneously hypertensive rat occur early in antihypertensive treatment. J. Hypertens. 29, 113e122. Herrera, M., Garvin, J.L., 2005. A high-salt diet stimulates thick ascending limb eNOS expression by raising medullary osmolality and increasing release of endothelin-1. Am. J. Physiol. Renal. Physiol. 288, F58eF64. Hirose, T., Totsune, K., Nakashige, Y., Metoki, H., Kikuya, M., Ohkubo, T., Hara, A., Satoh, M., Inoue, R., Asayama, K., Kondo, T., Kamide, K., Katsuya, T., Ogihara, T., Izumi, S., Rakugi, H., Takahashi, K., Imai, Y., 2011. Influence of adrenomedullin 2/intermedin gene polymorphism on blood pressure, renal function and silent cerebrovascular lesions in Japanese: the Ohasama study. Hypertens. Res. 34, 1327e1332. Hoenderop, J.G., Mu¨ller, D., Van Der Kemp, A.W., Hartog, A., Suzuki, M., Ishibashi, K., Imai, M., Sweep, F., Willems, P.H., Van Os, C.H., Bindels, R.J., 2001. Calcitriol controls the epithelial calcium channel in kidney. J. Am. Soc. Nephrol. 12, 1342e1349. Huang, X.R., Chen, W.Y., Truong, L.D., Lan, H.Y., 2003. Chymase is upregulated in diabetic nephropathy: implications for an alternative pathway of angiotensin II-mediated diabetic renal and vascular disease. J. Am. Soc. Nephrol. 14, 1738e1747. Imai, T., Hirata, Y., Iwashina, M., Marumo, F., 1995. Hormonal regulation of rat adrenomedullin gene in vasculature. Endocrinology 136, 1544e1548. Inoue, T., Nonoguchi, H., Tomita, K., 2001. Physiological effects of vasopressin and atrial natriuretic peptide in the collecting duct. Cardiovasc. Res. 51, 470e480. Isumi, Y., Shoji, H., Sugo, S., Tochimoto, T., Yoshioka, M., Kangawa, K., Matsuo, H., Minamino, N., 1998. Regulation of adrenomedullin production in rat endothelial cells. Endocrinology 139, 838e846. Jelkmann, W., 2011. Regulation of erythropoietin production. J. Physiol. 589 (6), 1251e1258. Jolly, L., March, J.E., Kemp, P.A., Bennett, T., Gardiner, S.M., 2009. Mechanisms involved in the regional haemodynamic effects of intermedin (adrenomedullin 2) compared with adrenomedullin in conscious rats. Br. J. Pharmacol. 157, 1502e1513. Jones, G., 2007. Expanding role for vitamin D in chronic kidney disease: importance of blood 25-OH-D levels and extra-renal 1a-hydroxylase in the classical and nonclassical actions of 1a,25dihydroxyvitamin D3. Semin. Dial. 20, 316e324. Jougasaki, M., Wei, C.M., Aarhus, L.L., Heublein, D.M., Sandberg, S.M., Burnett Jr., J.C., 1995. Renal localization and actions of adrenomedullin: a natriuretic peptide. Am. J. Physiol. Renal. Physiol. 268, F657eF663. Khanna, A., Simoni, J., Hacker, C., Duran, M.J., Wesson, D.E., 2004. Increased endothelin activity mediates augmented distal nephron acidification induced by dietary protein. J. Am. Soc. Nephrol. 15, 2266e2275. Kim, J.E., Song, S.W., Kim, J.Y., Lee, H.J., Chung, K.H., Shim, Y.H., 2016. Effect of a single bolus of erythropoietin on renoprotection in

458

19. KIDNEY HORMONES

patients undergoing thoracic aortic surgery with moderate hypothermic circulatory arrest. Ann. Thorac. Surg. 101, 690e696. Kitamura, K., Kangawa, K., Kawamoto, M., Ichiki, Y., Nakamura, S., Matsuo, H., Eto, T., 1993. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 192, 553e560. Kittikulsuth, W., Pollock, J.S., Pollock, D.M., 2012. Loss of renal medullary endothelin B receptor function during salt deprivation is regulated by angiotensin II. Am. J. Physiol. Renal. Physiol. 303, F659eF666. Kobori, H., Urushihara, M., Xu, J.H., Berenson, G.S., Navar, L.G., 2010. Urinary angiotensinogen is correlated with blood pressure in men (Bogalusa Heart Study). J. Hypertens. 28, 1422e1428. Kohan, D.E., 1997. Endothelins in the normal and diseased kidney. Am. J. Kidney Dis. 29, 2e26. Kohan, D.E., Rossi, N.F., Inscho, E.W., Pollock, D.M., 2011. Regulation of blood pressure and salt homeostasis by endothelin. Physiol. Rev. 91, 1e77. Kolesnyk, I., Struijk, D.G., Dekker, F.W., Krediet, R.T., 2010. Effects of angiotensin II-converting enzyme inhibitors and angiotensin II receptor blockers in patients with chronic kidney disease. Neth. J. Med. 68, 15e23. Komers, R., Plotkin, H., 2016. Dual inhibition of renin-angiotensinaldosterone system and endothelin-1 in treatment of chronic kidney disease. Am. J. Physiol. Regul. Integr. Comp. Physiol. 310, R877eR884. Kong, D., Zhuo, L., Gao, C., Shi, S., Wang, N., Huang, Z., Li, W., Hao, L., 2013. Erythropoietin protects against cisplatin-induced nephrotoxicity by attenuating endoplasmic reticulum stress-induced apoptosis. J. Nephrol. 26, 219e227.  Vernerova´, Z., Walkowska, A., KompanowskaKujal, P., Cha´bova´, V.C., Jezierska, E., Sadowski, J., Va nourkova´, Z., Huskova´, Z., Opocensky´, M., Skaroupkova´, P., Schejbalova´, S., Kramer, H.J., Rakusan, D., Maly´, J., Netuka, I., Vaneckova´, I., Kopkan, L., Cervenka, L., 2010. Similar renoprotection after renin-angiotensindependent and -independent antihypertensive therapy in 5/6nephrectomized Ren-2 transgenic rats: are there blood pressure-independent effects? Clin. Exp. Pharmacol. Physiol. 37, 1159e1169. Kuruppu, S., Rajapakse, N.W., Smith, A.I., 2013. Endothelin-converting enzyme-1 inhibition and renoprotection in end-stage renal disease. Pflu¨gers Archiv 465, 929e934. Laghmani, K., Preisig, P.A., Moe, O.W., Yanagisawa, M., Alpern, R.J., 2001. Endothelin-1/endothelin-B receptor-mediated increases in NHE3 activity in chronic metabolic acidosis. J. Clin. Investig. 107, 1563e1569. Lai, K.N., Leung, J.C., Yeung, V.T., Lewis, L.K., Nicholls, M.G., 1998. Gene transcription and synthesis of adrenomedullin by cultured human renal cells. Biochem. Biophys. Res. Commun. 244, 567e572. Lainchbury, J.G., Troughton, R.W., Lewis, L.K., Yandle, T.G., Richards, A.M., Nicholls, M.G., 2000. Hemodynamic, hormonal, and renal effects of short-term adrenomedullin infusion in healthy volunteers. J. Clin. Endocrinol. Metab. 85, 1016e1020. Langham, R.G., Kelly, D.J., Gow, R.M., Zhang, Y., Dowling, J.K., Thomson, N.M., Gilbert, R.E., 2004. Increased expression of urotensin II and urotensin II receptor in human diabetic nephropathy. Am. J. Kidney Dis. 44, 826e831. Larivie`re, R., Lebel, M., 2003. Endothelin-1 in chronic renal failure and hypertension. Can. J. Physiol. Pharmacol. 81, 607e621. Leenen, F.H., 2014. Actions of circulating angiotensin II and aldosterone in the brain contributing to hypertension. Am. J. Hypertens. 27, 1024e1032. Li, X.C., Zhuo, J.L., 2011. Phosphoproteomic analysis of AT1 receptormediated signaling responses in proximal tubules of angiotensin IIinduced hypertensive rats. Kidney Int. 80, 620e632.

Li, N., Chen, L., Yi, F., Xia, M., Li, P.L., 2008. Salt-sensitive hypertension induced by decoy of transcription factor hypoxia-inducible factor1a in the renal medulla. Circ. Res. 102, 1101e1108. Li, Y.N., Xi, M.M., Guo, Y., Hai, C.X., Yang, W.L., Qin, X.J., 2014. NADPH oxidase-mitochondria axis-derived ROS mediate arsenite-induced HIF-1a stabilization by inhibiting prolyl hydroxylase activity. Toxicol. Lett. 224, 165e174. Liu, J., Shimosawa, T., Matsui, H., Meng, F., Supowit, S.C., DiPette, D.J., Ando, K., Fujita, T., 2007. Adrenomedullin inhibits angiotensin IIinduced oxidative stress via Csk-mediated inhibition of Src activity. Am. J. Physiol. Heart Circ. Physiol. 292, H1714eH1721. Liu, X., Zhang, T., Xia, W., Wang, Y., Ma, K., 2013. Recombinant human erythropoietin pretreatment alleviates renal glomerular injury induced by cardiopulmonary bypass by reducing transient receptor potential channel 6-nuclear factor of activated T-cells pathway activation. J. Thorac. Cardiovasc. Surg. 146, 681e687. Madeddu, P., Troffa, C., Glorioso, N., Pazzola, A., Soro, A., Manunta, P., Tonolo, G., Demontis, M.P., Varoni, M.V., Anania, V., 1989. Effect of endothelin on regional hemodynamics and renal function in awake normotensive rats. J. Cardiovasc. Pharmacol. 14, 818e825. Malik, J., Kim, A.R., Tyre, K.A., Cherukuri, A.R., Palis, J., 2013. Erythropoietin critically regulates the terminal maturation of murine and human primitive erythroblasts. Haematologica 98, 1778e1787. Mallet, R.T., Ryou, M.G., 2017. Erythropoietin: endogenous protection of ischemic brain. Vitam. Horm. 105, 197e232. Mazzaferro, S., Pasquali, M., Pirro`, G., Rotondi, S., Tartaglione, L., 2010. The bone and the kidney. Arch. Biochem. Biophys. 503, 95e102. McGregor, D.O., Troughton, R.W., Frampton, C., Lynn, K.L., Yandle, T., Richards, A.M., Nicholls, M.G., 2001. Hypotensive and natriuretic actions of adrenomedullin in subjects with chronic renal impairment. Hypertension 37, 1279e1284. McKane, W.R., Khosla, S., Burritt, M.F., Kao, P.C., Wilson, D.M., Ory, S.J., Riggs, B.L., 1995. Mechanism of renal calcium conservation with estrogen replacement therapy in women in early postmenopause e a clinical research center study. J. Clin. Endocrinol. Metab. 80, 3458e3464. Mende, C.W., 2010. Application of direct renin inhibition to chronic kidney disease. Cardiovasc. Drugs Ther. 24, 139e149. Meyer, M., Richter, R., Forssmann, W.G., 1998. Urodilatin, a natriuretic peptide with clinical implications. Eur. J. Med. Res. 3, 103e110. Miller, R.L., Kohan, D.E., 1998. Hypoxia regulates endothelin-1 production by the inner medullary collecting duct. J. Lab. Clin. Med. 131, 45e48. Mock, D.M., Matthews, N.I., Zhu, S., Strauss, R.G., Schmidt, R.L., Nalbant, D., Cress, G.A., Widness, J.A., 2011. Red blood cell (RBC) survival determined in humans using RBCs labeled at multiple biotin densities. Transfusion 51, 1047e1057. Morimoto, R., Satoh, F., Murakami, O., Totsune, K., Suzuki, T., Sasano, H., Ito, S., Takahashi, K., 2007. Expression of adrenomedullin2/intermedin in human brain, heart, and kidney. Peptides 28, 1095e1103. Mukoyama, M., Sugawara, A., Nagae, T., Mori, K., Murabe, H., Itoh, H., Tanaka, I., Nakao, K., 2001. Role of adrenomedullin and its receptor system in renal pathophysiology. Peptides 22, 1925e1931. Murayama, A., Takeyama, K., Kitanaka, S., Kodera, Y., Kawaguchi, Y., Hosoya, T., Kato, S., 1999. Positive and negative regulations of the renal 25-hydroxyvitamin D3 1a-hydroxylase gene by parathyroid hormone, calcitonin, and 1a,25(OH)2D3 in intact animals. Endocrinology 140, 2224e2231. Myllyharju, J., 2013. Prolyl 4-hydroxylases, master regulators of the hypoxia response. Acta Physiol. 208, 148e165. Nagaya, N., Satoh, T., Nishikimi, T., Uematsu, M., Furuichi, S., Sakamaki, F., Oya, H., Kyotani, S., Nakanishi, N., Goto, Y., Masuda, Y., Miyatake, K., Kangawa, K., 2000. Hemodynamic, renal,

REFERENCES

and hormonal effects of adrenomedullin infusion in patients with congestive heart failure. Circulation 101, 498e503. Nakazawa, Y., Noshino, T., Obata, Y., Nakazawa, M., Furusu, A., Abe, K., Miyazaki, M., Koji, T., Kohno, S., 2013. Recombinant human erythropoietin attenuates renal tubulointerstitial injury in murine adriamycin-induced nephropathy. J. Nephrol. 26, 527e533. Namdari, M., Eatemadi, A., Negahdari, B., 2016. Natriuretic peptides and their therapeutic potential in heart failure treatment: an updated review. Cell. Mol. Biol. (Noisy-Le-Grand) 62, 1e7. Neuhofer, W., Pittrow, D., 2006. Role of endothelin and endothelin receptor antagonists in renal disease. Eur. J. Clin. Investig. 36 (Suppl. 3), 78e88. Nguyen, A.Q., Cherry, B.H., Scott, G.F., Ryou, M.G., Mallet, R.T., 2014. Erythropoietin: powerful protection of ischemic and post-ischemic brain. Exp. Biol. Med. 239, 1461e1475. Nicholls, M.G., Lainchbury, J.G., Lewis, L.K., McGregor, D.O., Richards, A.M., Troughton, R.W., Yandle, T.G., 2001. Bioactivity of adrenomedullin and proadrenomedullin N-terminal 20 peptide in man. Peptides 22, 1745e1752. Nicholls, M.G., Charles, C.J., Lainchbury, J.G., Lewis, L.K., Rademaker, M.T., Richards, A.M., Yandle, T.G., 2003. Adrenomedullin in heart failure. Hypertens. Res. 26 (Suppl. l), S135eS140. Nir, A., Clavell, A.L., Heublein, D., Aarhus, L.L., Burnett Jr., J.C., 1994. Acute hypoxia and endogenous renal endothelin. J. Am. Soc. Nephrol. 4, 1920e1924. Nishikimi, T., Mori, Y., Kobayashi, N., Tadokoro, K., Wang, X., Akimoto, K., Yoshihara, F., Kangawa, K., Matsuoka, H., 2002. Renoprotective effect of chronic adrenomedullin infusion in Dahl saltsensitive rats. Hypertension 39, 1077e1082. Noguchi, C.T., Asavaritikrai, P., Teng, R., Jia, Y., 2007. Role of erythropoietin in the brain. Crit. Rev. Oncol. Hematol. 64, 159e171. Oba, S., Hino, M., Fujita, T., 2008. Adrenomedullin protects against oxidative stress-induced podocyte injury as an endogenous antioxidant. Nephrol. Dial. Transplant. 23, 510e517. Ogasawara, S., Hosojima, M., Kaseda, R., Kabasawa, H., YamamotoKabasawa, K., Kurosawa, H., Sato, H., Iino, N., Takeda, T., Suzuki, Y., Narita, I., Yamagata, K., Tomino, Y., Gejyo, F., Hirayama, Y., Sekine, S., Saito, A., 2012. Significance of urinary full-length and ectodomain forms of megalin in patients with type 2 diabetes. Diabetes Care 35, 1112e1118. Ong, K.L., Lam, K.S., Cheung, B.M., 2005. Urotensin II: its function in health and its role in disease. Cardiovasc. Drugs Ther. 19, 65e75. Oremus, M., McKelvie, R., Don-Wauchope, A., Santaguida, P.L., Ali, U., Balion, C., Hill, S., Booth, R., Brown, J.A., Bustaman, A., Sohel, N., Raina, P., 2014. BNP and NT-proBNP as prognostic markers in persons with chronic stable heart failure. Heart Fail. Rev. 19, 413e419. Pang, X.X., Bai, Q., Wu, F., Chen, G.J., Zhang, A.H., Tang, C.S., 2016. Urotensin II induces ER stress and EMT and increases extracellular matrix production in renal tubular epithelial cells in early diabetic mice. Kidney Blood Press. Res. 41, 434e449. Parameswaran, N., Nowak, W., Hall, C.S., Sparks, H.V., Spielman, W.S., 2001. Cellular and molecular actions of adrenomedullin in glomerular mesangial cells. Peptides 22, 1919e1924. Prosser, H.C., Forster, M.E., Richards, A.M., Pemberton, C.J., 2008. Urotensin II and urotensin II-related peptide (URP) in cardiac ischemiareperfusion injury. Peptides 29, 770e777. Prunotto, M., Budd, D.C., Gabbiani, G., Meier, M., Formentini, I., Hartmann, G., Pomposiello, S., Moll, S., 2012. Epithelialmesenchymal crosstalk alteration in kidney fibrosis. J. Pathol. 228, 131e147. Pupilli, C., Romagnani, P., Lasagni, L., Bellini, F., Misciglia, N., Emoto, N., Yanagisawa, M., Rizzo, M., Mannelli, M., Serio, M., 1997. Localization of endothelin-converting enzyme-1 in human kidney. Am. J. Physiol. Renal. Physiol. 273, F749eF756.

459

Qiao, X., Li, R.S., Li, H., Zhu, G.Z., Huang, X.G., Shao, S., Bai, B., 2013. Intermedin protects against renal ischemia-reperfusion injury by inhibition of oxidative stress. Am. J. Physiol. Renal. Physiol. 304, F112eF119. Qiao, X., Wang, L., Wang, Y., Su, X., Qiao, Y., Fan, Y., Peng, Z., 2017. Intermedin attenuates renal fibrosis by induction of heme oxygenase-1 in rats with unilateral ureteral obstruction. BMC Nephrol. 18, 232. Ross, B., McKendy, K., Giaid, A., 2010. Role of urotensin II in health and disease. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298, R1156eR1172. Russell, F.D., 2004. Emerging roles of urotensin-II in cardiovascular disease. Pharmacol. Ther. 103, 223e243. Ryou, M.G., Flaherty, D.C., Hoxha, B., Sun, J., Rodriguez, S., Bell, G., Olivencia-Yurvati, A.H., Mallet, R.T., 2009. Pyruvate-fortified cardioplegia evokes myocardial erythropoietin signaling in swine undergoing cardiopulmonary bypass. Am. J. Physiol. Heart Circ. Physiol. 297, H1914eH1922. Ryou, M.G., Liu, R., Ren, M., Sun, J., Mallet, R.T., Yang, S.H., 2012. Pyruvate protects brain against ischemia-reperfusion injury by activating erythropoietin signaling. Stroke 43, 1101e1107. Sayer, G., Bhat, G., 2014. The renin-angiotensin-aldosterone system and heart failure. Cardiol. Clin. 32, 21e32. Schieffer, B., Paxton, W.G., Marrero, M.B., Bernstein, K.E., 1996. Importance of tyrosine phosphorylation in angiotensin II type 1 receptor signaling. Hypertension 27, 476e480. Seki, T., Asanuma, K., Asao, R., Nonaka, K., Sasaki, Y., Oliva Trejo, J.A., Kurosawa, H., Hirayama, Y., Horikoshi, S., Tomino, Y., Saito, A., 2014. Significance of urinary full-length megalin in patients with IgA nephropathy. PLoS One 9, e114400. Shimosawa, T., Shibagaki, Y., Ishibashi, K., Kitamura, K., Kangawa, K., Kato, S., Ando, K., Fujita, T., 2002. Adrenomedullin, an endogenous peptide, counteracts cardiovascular damage. Circulation 105, 106e111. Shinki, T., Ueno, Y., DeLuca, H.F., Suda, T., 1999. Calcitonin is a major regulator for the expression of renal 25-hydroxyvitamin D3-1a-hydroxylase gene in normocalcemic rats. Proc. Natl. Acad. Sci. U. S. A. 96, 8253e8258. Simo˜es e Silva, A.C., Silveira, K.D., Ferreira, A.J., Teixeira, M.M., 2013. ACE2, angiotensin-(1-7) and Mas receptor axis in inflammation and fibrosis. Br. J. Pharmacol. 169, 477e492. Smith Jr., R.S., Gao, L., Bledsoe, G., Chao, L., Chao, J., 2009. Intermedin is a new angiogenic growth factor. Am. J. Physiol. Heart Circ. Physiol. 297, H1040eH1047. Sogbe-Dı´az, M.E., Dı´az-Lo´pez, E.E., 2016. Adrenomedullin in the kidney: physiology and pathophysiology. Investig. Clin. 57, 66e76 (in Spanish). Soler, M.J., Wysocki, J., Batlle, D., 2013. ACE2 alterations in kidney disease. Nephrol. Dial. Transplant. 28, 2687e2697. Song, W., Abdel-Razik, A.E., Lu, W., Ao, Z., Johns, D.G., Douglas, S.A., Balment, R.J., Ashton, N., 2006. Urotensin II and renal function in the rat. Kidney Int. 69, 1360e1368. Soni, H., Adebiyi, A., 2017. Urotensin II-induced store-operated Ca2þ entry contributes to glomerular mesangial cell proliferation and extracellular matrix protein production under high glucose conditions. Sci. Rep. 7, 18049. Sorokin, A., 2011. Endothelin signaling and actions in the renal mesangium. Contrib. Nephrol. 172, 50e62. Sorokin, A., Kohan, D.E., 2003. Physiology and pathology of endothelin-1 in renal mesangium. Am. J. Physiol. Renal. Physiol. 285, F579eF589. Stoyanoff, T.R., Todaro, J.S., Aguirre, M.V., Zimmermann, M.C., Brandan, N.C., 2014. Amelioration of lipopolysaccharide-induced acute kidney injury by erythropoietin: involvement of mitochondria-regulated apoptosis. Toxicology 318, 13e21.

460

19. KIDNEY HORMONES

Sugo, S., Minamino, N., Shoji, H., Kangawa, K., Kitamura, K., Eto, T., Matsuo, H., 1995. Interleukin-1, tumor necrosis factor and lipopolysaccharide additively stimulate production of adrenomedullin in vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 207, 25e32. Sun, P., Yue, P., Wang, W.H., 2012. Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney. Am. J. Physiol. Renal. Physiol. 302, F679eF687. Sung, C.C., Hsu, Y.C., Chen, C.C., Lin, Y.F., Wu, C.C., 2013. Oxidative stress and nucleic acid oxidation in patients with chronic kidney disease. Oxid Med Cell Longev 2013, 301982. Takahashi, K., Kikuchi, K., Maruyama, Y., Urabe, T., Nakajima, K., Sasano, H., Imai, Y., Murakami, O., Totsune, K., 2006. Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney. Peptides 27, 1383e1389. Takahashi, K., Hirose, T., Mori, N., Morimoto, R., Kohzuki, M., Imai, Y., Totsune, K., 2009. The renin-angiotensin system, adrenomedullins and urotensin II in the kidney: possible renoprotection via the kidney peptide systems. Peptides 30, 1575e1585. Takenaka, T., Inoue, T., Miyazaki, T., Hayashi, M., Suzuki, H., 2016. Xeno-klotho inhibits parathyroid hormone signaling. J. Bone Miner. Res. 31, 455e462. Takeyama, K., Kitanaka, S., Sato, T., Kobori, M., Yanagisawa, J., Kato, S., 1997. 25-Hydroxyvitamin D3 1a-hydroxylase and vitamin D synthesis. Science 277, 1827e1830. Tan, X., Li, Y., Liu, Y., 2006. Paricalcitol attenuates renal interstitial fibrosis in obstructive nephropathy. J. Am. Soc. Nephrol. 17, 3382e3393. Tan, X., Li, Y., Liu, Y., 2007. Therapeutic role and potential mechanisms of active Vitamin D in renal interstitial fibrosis. J. Steroid Biochem. Mol. Biol. 103, 491e496. Taylor, M.M., Samson, W.K., 2002. Adrenomedullin and the integrative physiology of fluid and electrolyte balance. Microsc. Res. Tech. 57, 105e109. Thanassoulis, G., Huyhn, T., Giaid, A., 2004. Urotensin II and cardiovascular diseases. Peptides 25, 1789e1794. Torres, P.U., Prie´, D., Beck, L., De Brauwere, D., Leroy, C., Friedlander, G., 2009. Klotho gene, phosphocalcic metabolism, and survival in dialysis. J. Ren. Nutr. 19, 50e56. Torres-Courchoud, I., Chen, H.H., 2016. B-type natriuretic peptide and acute heart failure: fluid homeostasis, biomarker and therapeutics. Rev. Clin. Esp. 216, 393e398. Tsuruoka, S., Watanabe, S., Purkerson, J.M., Fujimura, A., Schwartz, G.J., 2006. Endothelin and nitric oxide mediate adaptation of the cortical collecting duct to metabolic acidosis. Am. J. Physiol. Renal. Physiol. 291, F866eF873. Turunen, M.M., Dunlop, T.W., Carlberg, C., Va¨isa¨nen, S., 2007. Selective use of multiple vitamin D response elements underlies the 1 a,25dihydroxyvitamin D3-mediated negative regulation of the human CYP27B1 gene. Nucleic Acids Res 35, 2734e2747. Tudpor, K., Teerapornpuntakit, J., Jantarajit, W., Krishnamra, N., Charoenphandhu, N., 2008. 1,25-dihydroxyvitamin D3 rapidly stimulates the solvent drag-induced paracellular calcium transport in the duodenum of female rats. J. Physiol. Sci. 58, 297e307. Uetake, R., Sakurai, T., Kamiyoshi, A., Ichikawa-Shindo, Y., Kawate, H., Iesato, Y., Yoshizawa, T., Koyama, T., Yang, L., Toriyama, Y., Yamauchi, A., Igarashi, K., Tanaka, M., Kuwabara, T., Mori, K., Yanagita, M., Mukoyama, M., Shindo, T., 2014. Adrenomedullin-RAMP2 system suppresses ER stress-

induced tubule cell death and is involved in kidney protection. PLoS One 9, e87667. Umbach, A.T., Zhang, B., Daniel, C., Fajol, A., Velic, A., Hosseinzadeh, Z., Bhavsar, S.K., Bock, C.T., Kandolf, R., Pichler, B.J., Amann, K.U., Fo¨ller, M., Lang, F., 2015. Janus kinase 3 regulates renal 25-hydroxyvitamin D 1a-hydroxylase expression, calcitriol formation, and phosphate metabolism. Kidney Int. 87, 728e737. Vesely, D.L., 2007. Urodilatin: a better natriuretic peptide? Curr. Heart Fail. Rep. 4, 147e152. Wong, W.H., Wong, B.P., Wong, E.F., Hunag, M.H., Wong, N.L., 1998. Downregulation of endothelin B receptors in cardiomyopathic hamsters. Cardiology 89, 195e201. Wang, Y., Zhu, J., DeLuca, H.F., 2015. The vitamin D receptor in the proximal renal tubule is a key regulator of serum 1a,25dihydroxyvitamin D₃. Am. J. Physiol. Endocrinol. Metab. 308, E201eE205. Wang, Y., Tian, J., Guo, H., Mi, Y., Zhang, R., Li, R., 2016. Intermedin ameliorates IgA nephropathy by inhibition of oxidative stress and inflammation. Clin. Exp. Med. 16, 183e192. Wassef, L., Langham, R.G., Kelly, D.J., 2004. Vasoactive renal factors and the progression of diabetic nephropathy. Curr. Pharmaceut. Des. 10, 3373e3384. Weinbaum, S., Duan, Y., Satlin, L.M., Wang, T., Weinstein, A.M., 2010. Mechanotransduction in the renal tubule. Am. J. Physiol. Renal. Physiol. 299, F1220eF1236. Wenger, R.H., Hoogewijs, D., 2010. Regulated oxygen sensing by protein hydroxylation in renal erythropoietin-secreting cells. Am. J. Physiol. Renal. Physiol. 298, F1287eF1296. Wesson, D.E., Simoni, J., Green, D.F., 1998. Reduced extracellular pH increases endothelin-1 secretion by human renal vascular endothelial cells. J. Clin. Investig. 101, 578e583. Wong, N.L., Tsui, J.K., 2001. Angiotensin regulates endothelin-B receptor in rat inner medullary collecting duct. Metabolism 50, 661e666. Yang, T., Xu, C., 2017. Physiology and pathophysiology of the intrarenal renin-angiotensin system: an update. J. Am. Soc. Nephrol. 28, 1040e1049. Zehnder, D., Quinkler, M., Eardley, K.S., Bland, R., Lepenies, J., Hughes, S.V., Raymond, N.T., Howie, A.J., Cockwell, P., Stewart, P.M., Hewison, M., 2008. Reduction of the vitamin D hormonal system in kidney disease is associated with increased renal inflammation. Kidney Int. 74, 1343e1353. Zeidel, M.L., 1993. Hormonal regulation of inner medullary collecting duct sodium transport. Am. J. Physiol. Renal. Physiol. 265, F159eF173. Zhong, Y., Armbrecht, H.J., Christakos, S., 2009. Calcitonin, a regulator of the 25-hydroxyvitamin D3 1a-hydroxylase gene. J. Biol. Chem. 284, 11059e11069. Zhu, Y.C., Zhu, Y.Z., Moore, P.K., 2006. The role of urotensin II in cardiovascular and renal physiology and diseases. Br. J. Pharmacol. 148, 884e901. Zhuo, J., Dean, R., Maric, C., Aldred, P.G., Harris, P., Alcorn, D., Mendelsohn, F.A., 1998. Localization and interactions of vasoactive peptide receptors in renomedullary interstitial cells of the kidney. Kidney Int. Suppl. 67, S22eS28. Zoccali, C., Mallamaci, F., 2008. Urotensin II: a cardiovascular and renal update. Curr. Opin. Nephrol. Hypertens. 17, 199e204. Zoccali, C., Mallamaci, F., Benedetto, F.A., Tripepi, G., Pizzini, P., Cutrupi, S., Malatino, L., 2008. Urotensin II and cardiomyopathy in end-stage renal disease. Hypertension 51, 326e333.

C H A P T E R

20 Adipocyte-Derived Hormones Allison J. Richard1, Jacqueline M. Stephens1,2 1

Pennington Biomedical Research Center, Baton Rouge, LA, United States; 2Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, United States

1. INTRODUCTION Our understanding of adipocyte biology has changed substantially in the last 30 years. Adipose tissue was typically considered an inert tissue whose primary function was energy storage in the form of triacylglycerides. In the mid-1980s, a serine protease named adipsin was shown to be secreted from cultured adipocytes and reduced in mouse models of obesity (Cook et al., 1987). Additional research revealed that adipsin, a member of the alternative complement family, was not similarly regulated in humans. Acylation stimulating protein (ASP) is another member of the alternative complement family produced in adipose tissue (Cianflone et al., 1999) and implicated to have a role in lipid storage (Saleh et al., 2011). Although the endocrine role of these first identified adipose secretory products is still not well established, their discovery shed new light on adipocytes and adipose tissue, which are now recognized as bona fide sources of endocrine hormones. Adipose tissue is comprised of a variety of cell types, including endothelial cells, blood cells, fibroblasts, pericytes, preadipocytes, macrophages, and several types of immune cells (Saetang and Sangkhathat, 2018). Since adipose tissue is comprised of so many different cell types, it is important to consider the cellular source of adipose tissue hormones. During the isolation of adipose tissue cells, the adipocytes are typically separated from all other cell types, and this portion of nonadipocyte cells is commonly referred to as the stromal vascular fraction (SVF) of the adipose tissue. There are many hormones and cytokines that are produced from cells that comprise the SVF in adipose tissue. The ability of the proinflammatory cytokine tumor necrosis factor alpha (TNFa) to induce insulin resistance in adipocytes and inhibit the adipogenesis of preadipocytes has been known for several decades. In the early 1990s, it was

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shown that TNFa production was increased in adipose tissue during metabolic disease states, in particular in type 2 diabetes (Hotamisligil et al., 1993). But, it was not until 2003 that adipose tissue macrophages were identified as the primary cellular source of adipose tissue TNFa (Weisberg et al., 2003). An important feature of adipose tissue is that it has lot of cell types that make a variety of different bioactives ranging from microRNAs (miRNAs) to lipids to cytokines and hormones. Moreover, adipocytes, themselves, also make a variety of secreted products including miRNAs, lipids, proteins, and exosomes reviewed in Fasshauer and Blu¨her (2015) (refer to Fig. 20.1). Recent studies suggest that many circulating miRNAs are derived from adipocyte exosomes (Thomou et al., 2017). Moreover, adipocyte exosomes have been linked to lipid metabolism and obesity-related insulin resistance reviewed in Zhang et al. (2016). Although it is well accepted that adipocytes secrete a large variety of bioactive molecules that have a multitude of systemic effects and contribute to numerous physiologic and pathologic processes, the autocrine and paracrine actions of these molecules is complex, and our understanding of these processes is likely rudimentary. However, substantial progress has been made studying three endocrine hormones that are almost exclusively produced in adipocytes and have functions to regulate food intake, reproductive axis, insulin sensitivity, and immune responses. The primary source of circulating leptin, adiponectin, and resistin in mice comes from adipocytes present in adipose tissue. In humans, resistin is not produced in adipocytes, but there is substantial evidence that this hormone has similar functions in mouse and man. The discovery and study of these hormones over the past 25 years has shifted the knowledge base regarding the functions and capabilities of adipocytes. Dysregulation

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FIGURE 20.1 The adipocyte secretome. Adipocytes secrete numerous protein, lipid, and nucleic acid factors that can act on other nearby or distant tissues within the body in an endocrine manner. The “big three”dleptin, adiponectin, and resistindwhich are covered in this chapter, are exclusively secreted from mouse adipocytes, while the other factors can also be secreted from other cell types. The arrowheaded line to resistin is dashed because in humans, macrophages, not adipocytes, primarily produce resistin. BMPs, bone morphogenetic proteins; FFA, free fatty acid; FGF21, fibroblast growth factor 21; miRNA, microRNA; PAI-1, plasminogen activator inhibitor 1; RBP4, retinol binding protein 4.

of any one of these hormones can contribute to systemic metabolic dysfunction, as well as to the pathogenesis of other diseases, including cancer. This chapter will focus on the structure, function, and signaling of adipocytederived adiponectin, leptin, and resistin, and their important endocrine functions. The role of these hormones in disease states will also be considered.

2. LEPTIN Leptin comes from the Greek word leptos. Leptos means thin. Now, 25 years after the discovery of leptin, this definition can be considered ironic as the amount of circulating leptin positively correlates with the amount of fat mass. Leptin was the first bona fide endocrine hormone shown to be produced from adipocytes (Zhang et al., 1994). Although the pituitary and pancreas are well-recognized as endocrine tissues, the endocrine roles of adipocytes are still underappreciated after 2 decades of innovative research in adipose tissue biology. The discovery of leptin was arduous as it involved the positional cloning of the obese (ob) gene. Positional cloning is a laboratory method used to locate the position of a disease-associated gene within a chromosome and is an approach that can be used when little or no information is available about the biochemical basis of the disease. The first ob/ob mouse arose by chance at the Jackson Laboratories in 1949 (Ingalls et al., 1950). The mutation in the ob gene is recessive and mice with

mutations in both copies of the ob genes appear phenotypically similar at birth, but throughout their life, they gain excess fat mass that is associated with hyperglycemia and hyperinsulinemia (Lindstro¨m, 2007). The positional cloning of the obese gene revealed that leptin is predominately produced in adipocytes and revealed that the ob/ob mice do not produce leptin (Zhang et al., 1994). The first function attributed to leptin was the ability of this hormone to affect food intake (Friedman and Halaas, 1998). However, an endocrine role of the ob gene was known before its identification by positional cloning because of a parabiosis experiment. Parabiosis is the surgical joining of two organisms that allows exchange of whole blood between two animals. The use of parabiosis dates back to 1864 and has been used to test for involvement of circulating factors in feedback regulation of physiologic systems (Harris, 2013). The ob/ob mice were the first genetically obese mice used in a parabiosis experiment (Hausberger, 1958). In this study, ob/ob mice were parabiosed to nonobese littermates, and the weight gain of ob/ob partner was inhibited. This study suggested that a circulating factor could reduce weight gain in ob/ob mice. Over a decade later, another laboratory parabiosed ob/ob mice with wildtype, lean mice and showed that not only was weight gain reduced in the ob/ob mice, but there was a notable improvement in hyperglycemia, hyperinsulinemia, and insulin sensitivity (Chlouverakis, 1972). It was well over 30 years between the parabiosis experiment that lead to the discovery that a circulating factor was responsible for the phenotype of ob/ob mice and the positional cloning of leptin gene in 1994. As indicated later, parabiosis experiments were also performed with mice containing a mutation in the leptin receptor. Notably, the use of parabiosis had a large impact on increasing our understanding of leptin, even before this hormone was identified.

2.1 Leptin Structure The structures of leptin and its receptor have been extensively studied using a variety of techniques including X-ray crystallography, small-angle X-ray scattering, electron microscopy, homology modeling, and mutagenesis reviewed in Peelman et al. (2014). The leptin propolypeptide has 167 amino acids. The N-terminal 21 residues act as a signal sequence directing secretion of mature leptin, which has 146 amino acids and a molecular weight of 16 kDa. Human and mouse leptin are highly homologous, as they share approximately 85% identity and 95% similarity in their primary sequence. Since human leptin has a tendency to aggregate due to two solvent-exposed tryptophans, researchers created a W100E mutant protein with increased solubility and full biologic activity to solve

2. LEPTIN

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FIGURE 20.2 Structure of leptin. (A) Alignment of the primary amino acid sequences of human (H) and mouse (M) leptin is shown. Human and mouse leptin are the same length and share 85% sequence identity; residue differences are colored orange. Leptin folds into a structure mainly composed of a-helices and loops (shown as cylinders and lines above the sequence alignment). (B) A cartoon representation of the human leptin crystal structure [Protein Data Bank (PDB ID: 1ax8)] shows its folding into a 4-helix bundle. The numbers 1 to 4 on the helices correspond to the numbers in A, and the arrows demonstrate the antiparallel down-down-up-up orientation of the helices. Two highly conserved cysteines, marked pink or cyan in A and B, form a disulfide bond in the tertiary structure that is necessary for structural stability and biologic activity.

the first leptin crystal structure reported in 1997 (Zhang et al., 1997). Leptin folds into a tertiary structure similar to that of members of the long-chain cytokine family (such as interleukin 6, granulocyte colony stimulating factor, leukemia inhibitory factor, and ciliary neurotrophic factor) with an antiparallel down-down-up-up arrangement of a-helices comprising a four-helix bundle (Fig. 20.2). Two conserved cysteine residues in the mature polypeptide form a solvent-exposed intrachain disulfide bond that is crucial for biologic activity by forming a bridge and stabilizing the tertiary fold (Rock et al., 1996; Haglund et al., 2012) (Fig. 20.2). Leptin shares structural homology with the longchain cytokines, and by analogy the structure of its specific receptor is classified as a class I cytokine receptor. The leptin receptor (LR) exhibits many hallmark features of this receptor family including (1) an extracellular ectodomain comprised of multiple fibronectin type III (FNIII) domains, an immunoglobulin-like (IGD) domain, and one or more cytokine receptor homology (CRH) domains containing a characteristic WSXWS sequence; (2) a single membrane-spanning domain; (3) an intracellular cytoplasmic tail domain; and (4) a lack of intrinsic kinase activity. Since the LR is devoid of intrinsic kinase activity like other family members, it uses Janus kinases (JAKs) that interact with the cytoplasmic domain to transmit the extracellular hormone binding signal. Six LR isoforms generated by alternative splicing or ectodomain shedding have

been described, and they are designated LRaeLRf, with LRb being the main and longest isoform of LR that is capable of full signaling via the JAK/STAT (signal transducer and activator of transcription) pathway reviewed in Peelman et al. (2014). Leptin binding to a soluble recombinant extracellular domain has been investigated using a variety of techniques including isothermal titration calorimetry and surface plasmon resonance, and these studies indicate a KD of 0.2e15 nM for this interaction reviewed in Peelman et al. (2014). Domain mapping studies demonstrate that leptin binding to the CRH2 domain of the LR has a KD comparable to binding the full ectodomain and suggest that only the CRH2 domain is necessary for the association of leptin with its receptor (Fong et al., 1998; Sandowski et al., 2002). A substantial amount of structural modeling has been conducted to predict the site of interaction and specific contacts within both leptin and its receptor reviewed in Peelman et al. (2014), Zabeau et al. (2015). While these studies are useful and can aid in the design of potential small molecule or peptide antagonists of the LR for the treatment of obesityrelated diseases, higher resolution structures of the LR are needed, but are hindered by its complexity.

2.2 Leptin Signaling and Metabolic Function The obese gene is now referred to as the leptin gene, but there are other mutant genes associated with obesity

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and diabetes. Mice lacking both functional copies of the diabetes (db) gene due to a spontaneous single autosomal recessive gene mutation have early onset type 2 diabetes (Hummel et al., 1966). It is now established that the diabetes gene codes for the LR. Although parabiosis of db/db mice to wild-type mice did not improve diabetes and indicated that a defect in the hypothalamus of the diabetic mouse caused the normal parabiont mouse to starve to death, a full understanding of these observations would not come for at least 30 years. Because of previous studies with parabiosis of ob/ob mice and since the ob/ob mice and db/db mice each had single gene mutations that were present on different chromosomes, perhaps the most well-known parabiosis experiment was performed using the ob/ob and db/db mice to determine if these similar phenotypes could be distinguished with this surgical approach. The experiment demonstrated that the ob/ob mice had normal satiety centers that were likely responding to a humoral factor produced by db/db mice (Coleman, 1978). It was even speculated that the ob/ob mice were unable to produce the satiety factor, whereas db/db mice appeared to produce the endocrine mediator, but did not respond to it. Parabiosis of these two genetic models explained why mutations of two apparently unrelated genes produced identical phenotypes of overeating, obesity, and diabetes (Coleman, 1978). Importantly, another laboratory replicated these experiments (Harris, 1997, 1999). Despite intriguing results of parabiosis experiments, the identification of the diabetes gene did not occur until 1990 (Bahary et al., 1990). However, it was not classified as the LR until almost 5 years later, following the identification of the leptin hormone (Tartaglia et al., 1995). The LR is classified as a class 1 cytokine receptor and has substantial homology to glycoprotein 130 (gp130), a plasma membrane receptor that mediates the biologic actions of many cytokines. The receptor lacks any intrinsic enzymatic activity and like gp130 cytokines primarily uses the JAK-STAT signaling pathway for intracellular signaling reviewed in Peelman et al. (2014). One aspect of the complexity of leptin signaling is the existence of six LR isoforms. As indicated earlier, these receptors are designated by letters, LRaeLRf, and most of them have identical extracellular and transmembrane domains but differ in their intracellular portions. The longest form of the leptin receptor, LRb, is the best studied and is highly expressed in the arcuate nucleus of the hypothalamus where it functions to affect regulation of body weight. Leptin’s association with its receptor causes dimerization of the receptor and activation of the cytosolic tyrosine kinase, JAK2. JAK2 phosphorylates the LRb at multiple tyrosine residues that are associated with recruitment of several signaling molecules, including STAT3 and STAT5 reviewed in Peelman et al. (2014). These two STATs can be phosphorylated

by JAK2 and have distinct effects on gene expression (refer to Fig. 20.3). Elegant studies, using LR knockout mice with knock in of the LR that has a defective STAT3 binding site, have demonstrated that STAT3 plays a pivotal role in the ability of the leptin to control energy balance (Bates et al., 2003). Genetically modified mice that have a neuron-specific disruption of LR display obesity (Cohen et al., 2001), and neuronspecific restoration of LR reverses the obese phenotype of LR-null mice (de Luca et al., 2005; Morton et al., 2003), clearly demonstrating that the primary regulatory effect of leptin on body weight is controlled in the brain. In addition to JAK2 and STAT proteins, several other signaling molecules play a role in leptin signaling. SH2containing protein tyrosine phosphatase 2 (SHP2) has been implicated in the ability of JAK2 to enhance MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase) activation (Carpenter et al., 1998; Li and Friedman, 1999). Notably, a catalytically inactive SHP2 mutant can block leptin-induced ERK activation by the LR (Bjørbaek et al., 2001). The relevance of this pathway has been demonstrated in mice with a neuron-specific deletion of SHP2 that display early onset obesity and leptin resistance, and the pharmacological inhibition of ERK1/2 in the hypothalamus can reverse the anorectic and weight-reducing effects of leptin (Rahmouni et al., 2009; Zhang et al., 2004). Mice with a POMC (proopiomelanocortin) neuronspecific deletion of the SHP2 gene have increased adiposity, a loss of leptin sensitivity, and reduced energy expenditure (Banno et al., 2010). In the hypothalamus, leptin-mediated induction of mammalian target of rapamycin (mTOR) activity and subsequent ribosomal protein S6 phosphorylation have also been to be shown to be required for the anorectic effect of leptin (Cota et al., 2006; Villanueva et al., 2009). Studies have shown that reduced mTOR signaling in the hypothalamus can contribute to hyperphagia, weight gain, and leptin resistance (Cota et al., 2008). The insulin receptor substrate (IRS)/phosphoinositide 3-kinase (PI3K) pathway can also be involved in leptin signaling (Zu et al., 2008; Krajewska et al., 2008) (Fig. 20.3). Both IRS1 and PI3K have been shown to be recruited to LR via SHP2 (Wauman and Tavernier, 2011). In the hypothalamus, leptininduced activation of hypothalamic PI3K is impaired in diet-induced obesity (Metlakunta et al., 2008). In addition, pharmacological inhibition of PI3K activity substantially decreased the anorectic effect of leptin (Niswender et al., 2001; Zhao et al., 2002). A variety of mouse models have been used to understand the crosstalk between leptin and insulin signaling on PI3K activation in the brain (Wauman and Tavernier, 2011), and although the relative contributions of insulin and leptin in signaling in the brain are difficult to assess, the importance of the PI3K pathway in leptin action is apparent.

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FIGURE 20.3 Leptin signaling in the arcuate nucleus of the hypothalamus. Leptin receptors are found on the plasma membrane of the arcuate nucleus within the hypothalamus of the brain. Adipocyte-derived leptin primarily signals through JAK2/STAT3 to modulate systemic energy balance. Leptin action is also mediated via STAT5 to impact reproductive functions, as well as other signaling proteins, such as insulin receptor substrate (IRS) and phosphoinositide 3-kinase (PI3K). Leptin-induced activation of STAT proteins results in increased nuclear localization and transcription of signaling molecules, including POMC and SOCS3. Increased amounts of SOCS3 protein result in feedback inhibition of leptin signaling at the level of the receptor.

AMPK (50 -AMP-activated protein kinase) is a cytosolic serine threonine kinase that is known to be activated by elevated AMP/ATP ratios and acts as an energy sensor in a variety of cells types (Kahn et al., 2005). In some cells, leptin can activate AMPK activity, but leptin inhibits AMPK activity in several hypothalamic regions to reduce food intake (Andersson et al., 2004; Minokoshi et al., 2004; Gao et al., 2007). The specific mechanism of AMPK activation by leptin is unclear, but other signaling molecules, including CAMKK2 (Ca2þ/calmodulin (CaM)-dependent protein kinase kinase 2) and CRTC1 (CREB-regulated transcription coactivator 1) have been implicated in the actions of leptin-mediated AMPK activity in the hypothalamus (Wauman and Tavernier, 2011). Both SOCS3 (suppressor of cytokine signaling 3) and PTP1B (protein tyrosine phosphatase 1B) are signaling proteins that have been implicated to have a role in the negative feedback mechanisms the regulate leptin signaling. Typically, in most cells, the expression of SOCS family genes is acutely induced by the JAKSTAT pathway, and the newly transcribed and translated SOCS proteins negatively regulate receptor activity (refer to Fig. 20.3). This receptor/JAK2/STAT3/SOCS negative feedback pathway is likely to be of particular importance for LR signaling because of the welldescribed pervasiveness leptin resistance.

Leptin resistance is simply defined as the inability to respond to leptin despite sufficient or excess levels of circulating leptin. Leptin resistance is one reason why leptin is not a viable antiobesity therapeutic. It is well known that the amount of leptin in mice and man strongly correlates with the amount of fat mass and that a primary function of leptin is to inhibit food intake. However, obesity and elevated leptin levels are often associated with excess food intake, a condition that is indicative of leptin resistance. Leptin resistance during obesity is typically not a global resistance; instead, characteristically, only one arm of leptin action, its ability to inhibit food intake, is disrupted, leaving the other functions of leptin intact. The molecular mechanisms responsible for this “selective” leptin resistance are not well understand and are the focus of active investigation (Balland and Cowley, 2015). There is ample evidence in mice and humans that leptin resistance is highly related to food intake and is a strong contributor to metabolic dysfunction. SOCS3 has been proposed to be one of the primary mechanisms involved in the hypothalamic leptin resistance. Increased leptin induces SOCS3 expression that inhibits further leptin signaling (Bjørbæk et al., 1998, 1999). Elevated SOCS3 expression is significantly increased in the hypothalamus of several leptin-resistant animal

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models (Bjørbæk et al., 1998; Enriori et al., 2007). Also, the specific loss of SOCS3 in POMC neurons increases leptin sensitivity and results in improved glucose homeostasis (Kievit et al., 2006). Other SOCS family members, including CIS (cytokine-inducible SH2-containing protein) and SOCS2 have been shown to interact with the LR, but a physiologic role for these proteins in leptin signaling has not been established. PTP1B is a tyrosine phosphatase that negatively regulates the activity of several hormone and cytokine receptors. PTP1B has been shown to be expressed in leptin-responsive neurons of the hypothalamus (Zabolotny et al., 2002) and has been shown to inhibit leptin signaling by binding and dephosphorylating JAK2 (Zabolotny et al., 2002; Kaszubska et al., 2002). Evidence of an inhibitory role of PTP1B comes from studies that show either systemic or POMC neuronspecific deletion of PTP1B can improve leptin sensitivity and protect against diet-induced obesity (Cheng et al., 2002; Bence et al., 2006; Picardi et al., 2008). PTP1B has been implicated as driver of leptin resistance because PTP1B levels are elevated in hypothalamus of leptin-resistant animals (Morrison et al., 2007; Qatanani et al., 2009). Overexpression of SH2B1, an endogenous enhancer of leptin sensitivity, can counteract PTP1Bmediated inhibition of leptin signaling in cultured cells (Ren et al., 2005). PTP1B expression is increased by high-fat feeding and inflammation (Zabolotny et al., 2008), but how PTP1B expression and activity is regulated after leptin stimulation or in cases of dietinduced obesity is still unclear. In summary, there are several signaling pathways (see Fig. 20.3) that are involved in leptin-mediated responses. Although leptin responses that involve JAK2 and STAT3 to induce POMC via the long form of the LR are fairly well understood in some regions of the brain, the actions of the leptin in peripheral tissues by other forms of the LR are still largely unknown. In addition to modulation of food intake, leptin can contribute to other physiologic processes including reproduction, angiogenesis, bone homeostasis, wound healing, and regulation of immune responses (Wauman and Tavernier, 2011). Moreover, leptin can modulate glucose homeostasis and lipid metabolism independent of its central regulation of food intake by having direct effects on hepatocytes and pancreatic b-cells. The multiple functions of this adipocyte-derived hormone, as well as the six forms of the receptor and their differential tissue expression, coupled with a large array of signaling proteins, and the possibility of “selective” leptin resistance are factors that substantially complicate both experimental design to study the effects of leptin and our understanding of LR signaling.

2.3 Other Functions of Leptin In the brain, leptin acts on a diverse set of central circuits to regulate distinct aspects of energy homeostasis reviewed in Mu¨nzberg and Morrison (2015). Different brain regions have been mapped to reveal functions of leptin in anorectic circuits, glucose homeostasis, and hedonic feeding. Evidence that deletion of LRb (the long form of the receptor) in peripheral tissues does not affect energy homeostasis strongly suggest that the primary action of this hormone is centrally mediated (Guo et al., 2007). In addition to obesity and energy balance, leptin also has influential effects on a variety of neural circuits including those that modulate hormones controlling reproduction, reproductive behaviors, antidepressive behaviors, activity, thermoregulation, and stress (Friedman, 2014). The role of leptin in anxiety and depression is an expanding area of investigation. In addition to these numerous central functions, leptin also plays a role in innate and adaptive immunity (La Cava and Matarese, 2004) as well as bone formation (Ducy et al., 2000) and bone metabolism (Dalamaga et al., 2013; Chen and Yang, 2015). Moreover, there is evidence that leptin may also act on adipose tissue (Harris, 2014). Effects of leptin on skeletal muscle, liver, and intestinal function have likewise been described (Sa´inz et al., 2015). However, studies on leptin action in the periphery have not employed the state-of-the-art tools that have been used in the brain, and the expression and signaling of the other forms of the LR are not well characterized. Overall, the actions of leptin outside the brain are still not well understood, and the prominent effects of leptin on energy homeostasis are independent of the long form of the LR in peripheral tissues (Guo et al., 2007).

2.4 Leptin and Cancer Obesity, or excess lipid accumulation, is a risk factor for a variety of types of cancers. Since, leptin levels correlate with fat mass, it is not surprising that leptin has been implicated in variety of cancers. Dysregulation of leptin or LR signaling has been described in a variety of types of malignant cancers including breast, thyroid, endometrial, and gastrointestinal (Ghosh et al., 2012). JAK/STAT signaling proteins as well as PI3K and ERKs have been implicated to play a role in these cancers. In papillary thyroid cancer, increased circulating levels of leptin occur independent of body mass index and are typically accompanied by increased expression of the LR (Akinci et al., 2009; Uddin et al., 2010). Leptin is also overexpressed in colorectal cancers (Paik et al., 2009; Kim et al., 2012). Several

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studies have demonstrated that leptin levels correlate with the presence of endometrial cancer, but these correlations disappear when adjusted for body mass index, indicating that leptin is not likely a causative factor (Petridou et al., 2002; Yuan et al., 2004; Cymbaluk et al., 2008). Both leptin and its receptor are highly expressed in breast cancer, particularly in high-grade tumors. In addition, levels of leptin and its receptor are associated with enhanced progression and poor survival in breast cancer. Leptin regulates endothelial cell proliferation and promotes angiogenesis, but there are several other factors such as insulin and HER2 (human epidermal growth factor receptor 2) that are also likely involved in the relationship between leptin and breast cancer reviewed in Artac and Altundag (2012). LR antagonists are also used as therapeutic treatments for breast cancer (Garcı´a-Robles et al., 2013). To date in 2018, according to the entire PubMed collection, there have been nearly 600 papers published on leptin and breast cancer. These observations are consistent with studies in obese, postmenopausal women who have a 20%e40% increased risk of developing breast cancer compared to normal weight women (Munsell et al., 2014). Based on these observations and the rapidly expanding literature on leptin and cancer, it is likely that our understanding of the roles of leptin in cancer is in its infancy.

2.5 Future of Leptin The discovery of leptin has greatly enhanced our understanding of the regulation of energy homeostasis in mice and man by leading to the identification of central pathways that regulate food intake. Although not addressed in this chapter, studies in patients with mutations that altered leptin action (Farooqi and O’Rahilly, 2014) have revealed the importance of rodent studies in understanding the complex biology of body weight regulation in humans and clinically significant disorders like obesity. Future studies in humans and mice will be required to unveil the detailed neuronal networks that mediate the multiple effects of leptin on physiology and behavior. There is ample evidence that targeting leptin action could potentially have broad therapeutic benefits for patients with a variety of disease states ranging to metabolic dysfunction to several types of cancers.

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this hormone is likely still in its infancy. Adiponectin, also known as acrp30, GBP-28, ApM1, and Adipoq, is a 30 kDa peptide hormone that was discovered in 1995 by subtractive hybridization experiments where the goal was to identify genes regulated during adipocyte differentiation (Scherer et al., 1995). Notably, this hormone was independently identified by three other research groups that employed different approaches (Nakano et al., 1996; Maeda et al., 1996; Hu et al., 1996). One highly unique feature of adiponectin is that is circulates at levels more than a 1000-fold higher than other hormones (refer to Fig. 20.4). The circulating plasma concentrations of adiponectin in humans range from 2 to 20 mg/mL (Turer and Scherer, 2012). Another complexity is that there are various circulating forms of adiponectin with a range of biologic targets and functions (Ruan and Dong, 2016). As detailed later, adiponectin signaling is also complex and not completely understood. Unlike leptin levels that increase with fat mass and are elevated in obesity, circulating adiponectin levels were first observed to be decreased with obesity (Hu et al., 1996). In mice and humans, adiponectin levels are typically decreased with increased fat mass. A large body of experimental evidence indicates that adiponectin has antidiabetic, antiatherogenic, and antiinflammatory capabilities. The numerous complexities of this hormone and its signaling proteins have caused road blocks in developing therapeutic treatments that target adiponectin and its receptors.

3.1 Adiponectin Structure Adiponectin has structural similarity with other proteins including complement factor C1q, tumor necrosis factor-a, and collagens VIII and X. Human adiponectin consists of 244 amino acids that generate a collagenous

3. ADIPONECTIN Although leptin may be the best understood adipocyte-specific secretory factor, studies on adiponectin are also highly comprehensive. Besides being an adipocyte-derived hormone with a complex biology, adiponectin is very distinct, and our understanding of

FIGURE 20.4 Relative circulating blood concentrations of adipokines and insulin for normal weight, healthy humans.

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FIGURE 20.5 Structure of adiponectin. (A) Schematic of the adiponectin domain architecture. (B and C) Cartoon representations prepared from the crystal structure of the globular domain of human adiponectin (PDB ID: 4dou). The collagen-type domain and variable region are difficult to crystalize, so the only high-resolution structures of adiponectin are of the globular domain. A single polypeptide chain is presented in (B) and shows the 10-stranded b-sheet sandwich structure of the globular domain. In (C), the quaternary structure of the globular trimer head is shown. Mouse adiponectin adopts a nearly identical structure (not shown; PDB ID: 1c28). Adiponectin trimers further oligomerize in multiples of three to form hexamers and other higher molecular weight species. The collagen-type domain, which is predicted to form a helical-like stalk, is not shown in (B) or (C).

N-terminal domain and a globular C-terminal domain (Berg et al., 2002) (Fig. 20.5). In reducing and denaturing conditions, adiponectin migrates as a w30-kDa protein during gel electrophoresis (Scherer et al., 1995). Although the nomenclature across publications is diverse, studies consistently find that there are several secreted forms of adiponectin: low-molecular weight (LMW) trimers (the most basic form), medium-molecular weight (MMW) hexamers, and high-molecular weight oligomers (HMW; 12e18 monomeric units) (Wang and Scherer, 2016). These oligomers have been detected experimentally using a variety of techniques such as native gel electrophoresis (Mashalidis et al., 2013; Hada et al., 2007), velocity sedimentation centrifugation, and size exclusion chromatography (Waki et al., 2005; Schraw et al., 2008). There is also a cleaved form referred to as globular adiponectin (Waki et al., 2005; Fruebis et al., 2001). The majority of circulating adiponectin in humans is the HMW and MMW, and less than 30% of the serum adiponectin in humans is present as a 90-kDa LMW trimer. The HMW oligomer is 360e540 kDa and consists of four to six trimers of adiponectin molecules, while the MMW form is 180 kDa and comprised of six adiponectin molecules (a

trimer-dimer) (Hada et al., 2007; Waki et al., 2003; Combs et al., 2004). Hydroxylation followed by glycosylation of specific lysine residues (Wang et al., 2002, 2006) and cysteine disulfide bonds (Waki et al., 2003; Pajvani et al., 2003) have both been shown to be necessary for posttranslational assembly of the HMW oligomer and adiponectin biologic function in ex vivo and in vivo studies. HMW adiponectin is more associated with insulin sensitivity and adiponectin’s ability to lower glucose levels (Pajvani et al., 2004). Studies of adiponectin folding, oligomerization, and secretion via the endoplasmic reticulum (ER) and Golgi have revealed a complex set of regulatory steps and identified some of the critical players including the ER-resident chaperone, ERp44 (Wang et al., 2007a,b), and oxidoreductases, Ero1 (Wang et al., 2007a,b; Qiang et al., 2007) and DsbA-L (Liu et al., 2008, 2015). Each of these proteins has been shown to interact with adiponectin and assist with its multimerization and/or secretion. Notably, the levels of these ER proteins positively correlate with changes in the amount of secreted HMW adiponectin; levels decrease in obese conditions and are increased

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by treatment with antidiabetic thiazolidinediones (Wang et al., 2007a,b; Liu et al., 2008). Crystal structures of both mouse (Shapiro and Scherer, 1998) and human adiponectin (Min et al., 2012) have been reported, but each one only contains the globular portion because of difficulties in crystalizing the collagenous and hypervariable regions. Globular mouse adiponectin crystalized as an asymmetrical trimer comprised of b-sandwich protomers, each of which folds into a jelly-roll topology made up of 10 bestrands (Fig. 20.5). The tertiary and quaternary structures of the adiponectin protomers and trimer are remarkably similar to TNF family members, indicating an evolutionary link between the C1q and TNF families even in the absence of primary sequence homology (Wang and Scherer, 2016; Shapiro and Scherer, 1998). Human and mouse adiponectin share 91% amino acid identity, and as expected, their tertiary folds and trimeric structures are nearly identical, as demonstrated by their high-resolution crystal structures (Ding et al., 2012). Multiple adiponectin receptors have been identified and include T-cadherin and adiponectin receptor-1 and -2, referred to AdipoR1 and AdipoR2. The discovery and structures of AdipoR1 and R2 are discussed in the next section. Several crystal structures of human AdipoR1 and R2 have been reported (Tanabe et al., 2015; Vasiliauskaite´-Brooks et al., 2017). AdipoR1 and R2 share 66.7% amino acid sequence identity and exhibit similar tertiary structures. Although the receptors have seven-transmembrane domains (7TMD), their structures are distinct from 7TMD-containing G proteinecoupled receptors and novel compared to other proteins in the Protein Data Bank (PDB). Another unique feature of these receptors is that the N-terminal domain is cytosolic, while the C-terminal domain is extracellular; this architecture is opposite that of typical 7TMDcontaining proteins. The structures further suggest that the globular domain of adiponectin interacts with the C-terminal extracellular surface of the receptors (Wang and Scherer, 2016; Tanabe et al., 2015). The high-resolution crystal structures of adiponectin and its receptors as well as in vitro and ex vivo studies examining the posttranslational processing and multimerization have provided valuable insights into our understanding of adiponectin’s biologic function and signaling. Additional three-dimensional modeling will likely provide further information regarding adiponectin’s interactions with its receptor and aid in the development of potential therapeutic agonists.

3.2 Adiponectin Signaling In 2003 and 2004, three cell surface receptors for adiponectin were identified. The use of HMW adiponectin

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ligands led to the identification of T-cadherin as an adiponectin receptor, and studies using the globular and trimeric forms of adiponectin led to the identification of AdipoR1 and AdipoR2. Adiponectin binding to Tcadherin was shown using a tagged HMW adiponectin and a variety of in vitro methods (Hug et al., 2004). Tcadherin, which does not appear to interact with adiponectin trimers, belongs to a family of cell surface proteins involved in Ca2þ-mediated cellecell interactions (Vestal and Ranscht, 1992), and it is expressed in a variety of tissues including the liver (Chan et al., 2008). The globular domain of adiponectin has a Ca2þ binding site that plays a role in binding to T-cadherin (Schraw et al., 2008). Ca2þ chelators can inhibit the binding of T-cadherin, indicating a potential role of Ca2þ in hormoneereceptor interactions (Hug et al., 2004). Tcadherin knockout mice lack tissue-associated adiponectin, have an accumulation of adiponectin in circulation, and have a similar cardiovascular phenotype to the adiponectin knockout mice (Matsuda et al., 2015). The majority of studies indicate the primary role of the association between adiponectin and T-cadherin is protection against cardiovascular pathologies (Fujishima et al., 2017; Denzel et al., 2010; Parker-Duffen et al., 2013). AdipoR1 and R2 are two related adiponectin receptors that were identified from a human skeletal muscle expression library by their binding to globular adiponectin (Yamauchi et al., 2003). The receptors have high conservation of the seven membrane-spanning domains from yeast to mammals. AdipoR1 is expressed ubiquitously, but it has the highest expression in skeletal muscle, whereas AdipoR2 is expressed most abundantly in liver (Yamauchi et al., 2003). AdipoR1 appears to primarily bind the globular form of adiponectin, in contrast to AdipoR2 that primarily binds the trimeric form (Yamauchi et al., 2003). A variety of polymorphisms have been found in the Adipo R1 and R2 genes that are associated with insulin resistance and type 2 diabetes, but many of these observations have not been widely replicated across populations (Crimmins and Martin, 2007). AdipoR1 and AdipoR2 belong to the family of progesterone/adiponectin/adipoQ receptors (PAQR) that can stimulate intracellular ceramidase activity (Villa et al., 2009). A recent crystal structure study of human AdipoR1 and AdipoR2 has provided some mechanistic insights into their functions (Tanabe et al., 2015). Notably, their structures contain a large cavity enclosed by the seven-transmembrane helices that has three conserved histidine residues coordinated to a zinc ion (Tanabe et al., 2015). The zinc-binding motif has been implicated in adiponectin-stimulated activation of AMPK and PPARa (Tanabe et al., 2015). Both AdipoR1 and AdipoR2 can regulate metabolic gene expression and insulin sensitivity in insulin target

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tissues, and both receptors appear to be important in the pathophysiology of insulin resistance and type 2 diabetes (Yamauchi et al., 2007, 2014). APPLs (adaptor proteins containing a pleckstrin homology domain, a phosphotyrosine binding domain, and a leucine zipper motif) are multifunctional adaptor proteins that bind to various membrane receptors and other signaling proteins to regulate a large variety of biologic activities (Liu et al., 2017). APPL1, an isoform of APPL, was identified to interact with both AdipoR1 and AdipoR2 in mammalian cells, and the interaction is stimulated by adiponectin (Mao et al., 2006). Overexpression of APPL1 increases adiponectin signaling, while loss of APPL1 reduces its signaling and adiponectin-mediated effects, such as fat oxidation and glucose uptake (Mao et al., 2006). The adiponectininduced interaction between APPL1 and the small GTPase Rab5 can lead to increased glucose transporter 4 (GLUT4) translocation to the plasma membrane to facilitate insulin-stimulated glucose uptake (Mao et al., 2006). APPLs also act on insulin signaling pathways and are important mediators of insulin sensitization. APPL1 plays a role in the crosstalk between adiponectin signaling and insulin signaling pathways (Liu et al., 2017). These results demonstrate a key function for APPL1 in adiponectin signaling and provide a molecular mechanism for the insulin-sensitizing functions of adiponectin. The APPL2 isoform shares 54% amino acid homology with APPL1 (Miaczynska et al., 2004; Wang et al., 2009). APPL2 negatively modulates adiponectin signaling in skeletal muscle cells (Wang et al., 2009) and can bind both AdipoR1 and AdipoR2 to prevent the interaction of APPL1 with these adiponectin receptors. Hence, APPL2 blocks adiponectin signaling through AdipoR1 and AdipoR2 by competitive inhibition of APPL1 (Wang et al., 2009). Acute treatment with adiponectin activates AMPactivated kinase (AMPK) in a variety of cell types, particularly the liver (Brooks et al., 2007; Combs and Marliss, 2014). AMPK is a serine/threonine kinase that is the cellular energy sensor known to inhibit acetyl CoA carboxylase (ACC), a rate limiting enzyme in de novo lipogenesis (Towler and Hardie, 2007). AMPK-mediated inhibition of ACC lowers malonyl-CoA production and increases fatty acid oxidation. Adiponectin has been reported to activate AMPK through two independent pathways involving liver kinase B1 (LKB1) and CaMKK. Both purified HMW adiponectin and recombinant globular adiponectin trimers can activate CaMKK (Hattori et al., 2008; Zhou et al., 2009; Iwabu et al., 2010). Adiponectin stimulation of AdipoR1 mediates Ca2þ release in C2C12 myotubes that activates CaMKK (Zhou et al., 2009; Iwabu et al., 2010). Adiponectin can also increase mitochondrial density, mitochondrial DNA content, and peroxisome proliferator-activated receptor acoactivator 1a (PGC1-a)

expression (Pirvulescu et al., 2014; Kadowaki et al., 2011). The ability of adiponectin to increase fatty acid oxidation is mediated by both AMPK (Yamauchi et al., 2002) and peroxisome proliferator-activated receptor a (PPARa) (Yamauchi et al., 2003). An overview of adiponectin signaling is shown in Fig. 20.6.

3.3 Adiponectin Functions A large number of target tissues and functions have been attributed to adiponectin in the past 20 years. There is evidence of adiponectin effects on reproduction (Dos Santos et al., 2012) and preimplantation embryo devel s, 2012). In addition, functions of adipoopment (Ciko nectin have been described in bone and cartilage (Ruscica et al., 2012). There are also several studies that implicate potential autocrine function of adiponectin (Dadson et al., 2011). However, the primary effects of adiponectin appear to be mediated by its actions in the heart, liver, and skeletal muscle. Adiponectin is widely recognized as an insulin sensitizer, and studies in liver and muscle have revealed some of the underlying mechanisms involved in the ability of adiponectin to enhance insulin action (Ruan and Dong, 2016) (refer to Fig. 20.7). The following section will focus on adiponectin’s action in insulin-sensitive tissues and the cardiovascular system. The liver plays a critical role in the homeostasis of blood glucose concentrations. The release of glucose from the liver is important for providing a circulating fuel that can be used by other tissues. Both endocrine and neural mechanisms stimulate the liver during fasting, exercise, and pregnancy to meet the increased demand for glucose. However, the liver also plays a major role in preventing hyperglycemia. There are many metabolic pathways that contribute to hepatic glucose production, and it is known that an inability to shut down hepatic glucose output can lead to hyperglycemia. One of the prominent effects of adiponectin is to suppress hepatic glucose output. In cultured liver cells that release glucose into the media, adiponectin decreased glucose production by 20%e40% (Wang et al., 2002; Berg et al., 2001). Intraperitoneal injection of adiponectin lowers plasma glucose in healthy mice as well as in mice with either Type 1 or type 2 diabetes (Berg et al., 2001). Although adiponectin acts to lower blood glucose levels, there is no evidence that high doses of adiponectin cause hypoglycemia. Insulin lowers plasma glucose by both lowering glucose production and raising glucose disposal. The lack of hypoglycemia in adiponectininjected mice indicates that the glucose-lowering effect of adiponectin is primarily mediated through the inhibition of glucose production. Euglycemic clamp studies

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FIGURE 20.6 Intracellular adiponectin signaling. Adiponectin signaling is complex and an area of active investigation. Although many proteins have been reported to play a role, a few of the better understood key players are shown. Adiponectin, in its multimeric or globular form signals through three known plasma membrane receptors, T-cadherin, AdipoR1, and AdipoR2. Each of these receptors has distinct specificity for globular or multimeric adiponectin. Ca2þ signaling likely plays a role in adiponectin signaling via T-cadherin and AdiopR1. APPL proteins interact with both AdipoR1 and AdipoR2 to mediate intracellular signaling events. Other kinases, such as CaMKK and AMPK, and transcription factors, including PPARa, also mediate the intracellular signaling of adiponectin, which partially culminates in the expression of mitochondrial biogenesis genes.

FIGURE 20.7 Actions of adiponectin on insulin-sensitive tissues. Adiponectin exerts many of its physiologic actions on liver and skeletal muscle, which are both insulin-responsive tissues. The ability of adiponectin to modulate glucose and lipid metabolism and energy expenditure within these tissues protects against diabetes and other metabolic diseases associated with insulin resistance.

using isotopic tracers were conducted to determine whether the reduction of plasma glucose by adiponectin was mediated by a decrease in glucose production, an increase in glucose disposal, or if both of these pathways were important. Intravenous infusion of adiponectin in mice resulted in a substantial elevation in circulating adiponectin that lowered glucose production by 65% without having any notable effects on glucose disposal or glycolysis (Combs et al., 2001). Moreover, transgenic mice with increased adiponectin have improved insulin

sensitivity (Combs et al., 2004), and mice lacking adiponectin have decreased hepatic insulin sensitivity (Nawrocki et al., 2006). There is also evidence that adiponectin expression is critical for the full glucoselowering effects evoked by antidiabetic thiazolidinediones (Nawrocki et al., 2006; Kubota et al., 2006) or FGF21 (Holland et al., 2013; Lin et al., 2013). In addition to its potent insulin-sensitizing ability to decrease hepatic glucose production, adiponectin has other effects in the liver including the ability to increase fatty acid oxidation, decrease inflammation, promote cell survival, and reduce fibrosis (Combs and Marliss, 2014; Yan et al., 2013). In the liver, adiponectin upregulates several PPARa target genes including CD36 (cluster of differentiation 36), which modulates hepatic fatty acid uptake and metabolism and genes associated with fatty acid oxidation (Yamauchi et al., 2001). Adiponectin has been shown to offset a number of processes involved in the progression of nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). Of note, NAFLD patients have significantly lower plasma adiponectin levels and exhibit insulin resistance (Pagano et al., 2005; Buechler et al., 2011). Overall, adiponectin promotes numerous beneficial effects in the liver and protects against diabetes and liver diseases (refer to Fig. 20.7).

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The insulin-sensitizing effects of adiponectin in liver are well established, but muscle metabolic function is also central to maintaining insulin sensitivity (Pendergrass et al., 2007) and is responsible for up to 80% of insulin-mediated glucose uptake in healthy individuals (Thiebaud et al., 1982). Adiponectin promotes glucose update in both C2C12 and L6 cultured myotubes (Yamauchi et al., 2002; Ceddia et al., 2005). In C2C12 myocytes, adiponectin increases fatty acid oxidation by the sequential activation of AMPK, p38 MAPK, and PPARa (Yamauchi et al., 2002; Yoon et al., 2006). Treatment of L6 myotubes with adiponectin induces GLUT4 translocation and glucose uptake (Ceddia et al., 2005). In skeletal muscle, adiponectin can promote insulinstimulated tyrosine phosphorylation of IRS1 and AKT serine phosphorylation by inhibiting p70 S6Kmediated serine phosphorylation of IRS1, thereby increasing insulin sensitivity (Wang et al., 2007a,b). Evidence suggests that the ability of adiponectin to activate the LKB1/AMPK/TSC1/2 pathway is responsible for its inhibitory actions on the mTOR/p70 S6K pathway, which results in improved insulin signaling in cultured myotubes (Wang et al., 2007a,b). Adiponectin knockout mice are obese and insulinresistant, but exposure to adiponectin via several methods results in improved skeletal muscle insulin sensitivity (Yamauchi et al., 2001; Yano et al., 2008; Kandasamy et al., 2012). Adiponectin promotes fat oxidation via AMPK activation in human myotubes, and this pathway is impaired in myotubes from obese T2D patients (Chen et al., 2005). These studies indicate that adiponectin also has important effects in skeletal muscle in humans and indicates that impairment of adiponectin function in skeletal muscle may contribute in the development of insulin resistance. In summary, skeletal muscle is another important target tissue for adiponectin action, where it regulates glucose and fatty acid metabolism directly and by means of its insulin-sensitizing effects (refer to Fig. 20.7). In addition to the antidiabetic actions of adiponectin, this hormone is also known to be cardioprotective. Low levels of serum adiponectin are now appreciated as a risk factor for a variety of different cardiovascular diseases (CVD) including coronary artery disease, restenosis, and hypertension (Ding et al., 2012). Low adiponectin levels correlate significantly and independently with CVD (Kumada et al., 2003). In one longterm study, male subjects with adiponectin levels in the highest quintile had decreased risk of myocardial infarction compared to those within the lowest quintile (Pischon et al., 2004). Males with hypoadiponectinemia have a twofold increase in CVD prevalence, independent of well-known risk factors (Kumada et al., 2003; Ouchi et al., 1999). Adiponectin is lower in patients with CVD even in age-, gender-, and BMI-matched

controls (Ouchi et al., 1999). Circulating adiponectin levels in patients with acute coronary syndrome are significantly lower than normal control patients (Nakamura et al., 2006). Progression of coronary artery calcification correlates independently with low adiponectin levels (Maahs et al., 2005). Components of the adiponectin signaling pathway have been studied to elucidate the molecular mechanisms involved in the cardioprotective effects of adiponectin. Because of its role in sensing cellular energy levels and modulating metabolic responses, AMPK has been implicated as a key molecule in cardiac responses to both overload and ischemia (Kim et al., 2009). AMPK is a primary signaling protein that mediates many responses to adiponectin. There is evidence that adiponectin contributes to regulation of cardiomyocyte hypertrophy (Ding et al., 2007). Moreover, low adiponectin levels are linked to vascular endothelial dysfunction (Shimabukuro et al., 2003), and numerous studies indicate that adiponectin is beneficial to the healthy functioning of the endothelium (Ohashi et al., 2011). The AMPKeeNOS (endothelial nitric oxide synthase) eNO (nitric oxide) signaling pathway has been implicated in the beneficial effects of adiponectin on the vascular endothelium. NO is a key regulator of endothelial actions and vessel relaxation, and its inhibition can lead to hypertension. Adiponectin-mediated AMPK phosphorylation of eNOS promotes its activity. Mice lacking adiponectin have lower eNOS expression in their aortas and decreased plasma NO metabolites compared to control mice fed a high-salt diet. Exogenous adiponectin administration rescues this phenotype and restores the level of eNOS (Ohashi et al., 2006). The therapeutic potential of adiponectin is relevant to providing treatments for diabetes, cardiovascular disease, and NAFLD. Future studies to understand the actions of adiponectin will be critical for the identification of new drug targets that could mimic the protective effects of adiponectin. Several years ago, the first small molecule AdiopR agonist was identified by screening a compound library (Okada-Iwabu et al., 2013). This agonist, termed “AdipoRon,” activates both AMPK and PGC1a to improve lipid and glucose metabolism in vitro and improves metabolic parameters and prolongs life span in db/db mice, a genetic mouse model for diabetes (Okada-Iwabu et al., 2013). More recent studies have shown that AdipoRon can decrease ceramides and lipotoxicity, and ameliorate diabetic nephropathy (Choi et al., 2018). These studies and others indicate that small molecules capable of enhancing adiponectin signaling might serve as potentially viable options for the treatment of obesity-linked metabolic diseases including type 2 diabetes. However, the current challenge is that the binding affinity for small molecules such as AdipoRon to the AdipoR1 or

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AdipoR2 is much higher than that of the full-length or globular domain of adiponectin. To date, high-affinity small-molecule AdipoR activators have not been identified. Other challenges include the high endogenous level of circulating adiponectin as well as the multiple receptors that can be engaged in response to the presence of this hormone. In summary, future studies will be needed to reveal more information about adiponectin signaling and secretion, which will provide new strategies to target adiponectin as a therapeutic for a variety of diseases.

4. RESISTIN 4.1 Background on Resistin/FIZZ3/ADSF Resistin could be considered “the last of the big three” when it comes to hormones that are exclusively produced from adipocytes. Leptin and adiponectin were discovered many years before resistin, and unlike these two other adipocyte hormones, the expression of resistin differs in mouse and man. The adipocyte production and secretion of resistin, first published in 2001, was independently discovered by two laboratories that have made substantial contributions to our understanding of adipocyte biology. Researchers from the Lazar laboratory at University of Pennsylvania identified a novel protein that they named resistin for its ability to confer “resistance to insulin.” In their studies, the resistin gene was originally identified in a screen of genes whose expression was repressed by one of the antidiabetic thiazolidinedione drugs (Steppan et al., 2001a). Resistin, and other tissue-specific signaling molecules in this family, were referred to as RELMs for resistin-like molecules (Steppan et al., 2001b). Notably, the prototypical member of the family was originally identified a year earlier from bronchoalveolar lavage fluid of inflamed lungs and designated FIZZ1 (“found in inflammatory zone”). Also, the RELM/FIZZ family is not well conserved in evolution (Yang et al., 2003). The lack of a common origin of this gene family has been a substantial factor that has greatly complicated the study and interpretation of resistin studies in humans. Another well-known adipocyte biology laboratory at UC Berkeley, the Sul Lab, also independently discovered resistin. They named the secretory factor, ADSF, for adipose tissue-specific secretory factor, and showed that it could inhibit adipocyte differentiation in vitro (Kim et al., 2001).

4.2 Resistin Structure The structure of resistin is rather unique, and homologous only to other members of the RELM family of secreted mammalian proteins. The primary sequence

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of RELMs can be broken down into three domains: (1) an N-terminal export signal sequence, (2) a variable central region, and (3) a C-terminal consensus sequence that is highly conserved and characterized by a distinctive spacing of 10 cysteines (C-X11-C-X8-C-X-C-X3-C-X10-CX-C-X-C-X9-CC-X3-6-END) (Steppan et al., 2001b). In humans, resistin is synthesized as a propolypeptide that is 108 amino acids in length and has a predicted molecular weight of 11.4 kDa. While the crystal structure of human resistin has not yet been solved, two structures of mouse resistin have been reported PDB codes: 1rfx and ˚ 1rgx and have respective resolutions of 2.0 and 1.79 A (Patel et al., 2004). Mouse resistin is six aa longer than its human homolog and has a predicted molecular weight of 12.5 kD (Ghosh et al., 2003). It crystallized as a trimer-dimer hexamer, which corresponded to the size of serum circulating species as determined by size exclusion chromatography and nonreducing Western Blotting (Patel et al., 2004). As shown in Fig. 20.8, the hexamer is made up of two tail-to-tail trimers, and each trimer assembles from three parallel, coiled-coil individual chains. Each individual chain folds into two domains, an a-helical N-terminal tail domain and a globular, C-terminal head domain that is comprised of six antiparallel b-strands adopting a “jelly-roll” topology, which is held together by five intramolecular disulfide bonds. The crystal structure revealed several interesting structural features that to date have unknown importance or function in contributing to resistin’s structural integrity and/or molecular action: (1) a flexible neck region that allows movement of the head relative to the tail domain by w20 degrees; (2) electrostatic polarity in that the head domain has positive electrostatic surfaces, while the tail has negative electrostatic potential; and (3) highly solvent-exposed disulfide bonds (w60% exposed compared to less than 20% for all other proteins in the PDB) (Patel et al., 2004). Mouse and human resistin circulate mainly as hexamers and a smaller fraction of trimers that form as a result of interchain disulfides as well as ionic and hydrophobic interactions. However, it is unclear whether the mouse and human resistin have significant structural homology on the tertiary level since they share only w60% sequence identity at both the mRNA and amino acid levels, which is less homology than most endocrine proteins conserved across species (Ghosh et al., 2003; Schwartz and Lazar, 2011). Although they have divergent promoter regions (Yang et al., 2003; Ghosh et al., 2003) and mouse resistin is secreted from adipocytes, while human resistin mainly comes from macrophages (Patel et al., 2003), human resistin has been shown to generate similar inflammatory and metabolic responses in mice (Qatanani et al., 2009). Secondary structure predictions indicate that human resistin has more a-helices and fewer b-strands than its

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FIGURE 20.8 Structure of resistin. (A) Primary sequence and secondary structural features of human (h) and mouse (m) resistin are shown. a-Helices are shown as cylinders and b-strands as arrows. Conserved cysteine residues are shown in red and as vertical lines in the secondary structure schematic. (B) Cartoon representation of a single polypeptide chain from the mouse resistin crystal structure (PDB ID: 1rgx) is shown. Each polypeptide chain of resistin folds into a structure composed of an N-terminal a-helical tail and a C-terminal globular head domain that is made up of six antiparallel b-strands. (C) The tail-to-tail dimerization of two resistin trimers (dark blue or light blue) is shown in cartoon representation (prepared from PDB ID: 1rgx and 1rfx).

mouse homolog (Al Hannan and Culligan, 2015) (refer to Fig. 20.8). In addition to the 10 conserved cysteines, human and mouse resistin have in common one more cysteine (Cys22, human, and Cys26, mouse) (Fig. 20.8) within the N-terminal domain of the processed, secreted protein that is not shared by all RELM family members. The additional N-terminal cysteine in both human and mouse resistin is important in the ability of the hormone to form oligomers by participating in interchain disulfide bonds (Patel et al., 2004; Banerjee and Lazar, 2001; Raghu et al., 2004). In mice, when this cysteine is mutated, the proteins cannot oligomerize and have increased bioactivity in their ability to inhibit hepatic insulin sensitivity (Patel et al., 2004). However, other studies with human and/or mouse resistin have demonstrated that formation of higher order oligomers (likely hexamers) is necessary for the protein to stimulate proinflammatory cytokines (Aruna et al., 2008) and inhibit glucose uptake in mouse cardiomyocytes. Thus, understanding resistin biology is complicated by the ability of resistin to form trimers and other oligomers both with itself and other RELM family members as well as differences in bioactivity due to the specific oligomerization state, human or mouse species, and target cell type.

4.3 Complexities of Resistin Expression Leptin and adiponectin are both expressed in white and brown fat. For a period of time, resistin expression was largely thought to be white fat specific. However, numerous studies have shown that it is also expressed

in brown fat in mice. In addition, different white adipose tissue depots (epididymal, inguinal, mesenteric, and retroperitoneal) and brown adipose tissue (BAT) have differential ontogenic patterns of resistin mRNA expression (Oliver et al., 2003). One hurdle to studying resistin expression is clear documentation from several laboratories that resistin mRNA levels can be inversely related to resistin protein levels (Rajala et al., 2004). However, in some conditions, including during fasting and ER stress, there is similar inhibition of both mRNA and circulating hormone levels (Rajala et al., 2004; Lefterova et al., 2009). To further complicate the understanding of resistin, the primary cellular source for resistin expression differs in mouse and man. The expression of resistin observed during adipocyte development in mouse adipocytes seems largely driven by the proadipogenic transcription factor C/EBPa and a small and proximal 264-base pair region of the resistin promoter that has been shown to be sufficient to drive its expression in adipocytes in vitro (Hartman et al., 2002). In addition to the C/EBPa binding site in the proximal resistin promoter, studies to investigate the adipocyte-restricted expression of murine resistin identified an enhancer 8.8 kb upstream of the transcription factor start site that contains several C/EBPa binding sites as well as a binding site for PPARg (Tomaru et al., 2009). It is well known that PPARg is the most important transcription factor that controls adipocyte development (de Sa´ et al., 2017). Although PPARg ligands repress resistin transcription,

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activation of PPARg increases enhancer activity within the resistin promoter, indicating that this region is not directly involved in the negative regulation (Tomaru et al., 2009) but is important for overall resistin expression. Importantly, the region homologous to the mouse resistin enhancer in the human gene contains all three C/EBP elements, but it is not conserved for the sequence bound by PPARg. Furthermore, the human resistin enhancer displays little or no binding by PPARg in vitro. Unlike the expression of resistin in murine adipocytes, human resistin is highly expressed in peripheral blood mononuclear cells (Savage et al., 2001) and has enriched expression human macrophages (Patel et al., 2003). The lack of PPARg binding to the human resistin enhancer likely explains, at least in part, the absence of resistin expression in human adipocytes (Tomaru et al., 2009). Despite substantial progress on the study of resistin expression, the incongruent regulation of resistin mRNA and protein coupled with the different cellular source of the hormone in mouse and man have limited the interpretation of many resistin studies and hampered the progress on the biologic actions and pathologic roles of this hormone.

4.4 Resistin Signaling The search for the resistin receptor remained elusive for well over a decade past the initial discoveries and characterization of resistin. Several signaling molecules induced by resistin in adipocytes were identified prior to the identification of the plasma membrane mediators of resistin action. A schematic of the primary players involved in resistin signaling is shown in Fig. 20.9. To date, two different receptors have been shown to play a role in the physiologic effects of resistin. CAP1, adenylyl cyclase-associated protein 1, has been shown to be a functional receptor for human resistin that induces intracellular signaling pathways, which modulate its proinflammatory effects (Lee et al., 2014). Human resistin can directly bind to CAP1 and upregulate cyclic AMP (cAMP) concentrations, protein kinase A (PKA) activity, and NF-kB-related transcription of inflammatory cytokines in monocytes (Lee et al., 2014). Both knockout and overexpression studies of CAP1 have revealed that this receptor can mediate the proinflammatory effects of resistin (Lee et al., 2014). In addition to binding CAP1, resistin has been to shown to bind the toll-like receptor 4 (TLR4) and compete with its primary ligand, lipopolysaccharide (LPS), for receptor binding (Tarkowski et al., 2010). Resistin-activated TLR4 modulates chemokine production and monocyte recruitment, an event that is thought to play a role in macrophage infiltration in the atherosclerotic arterial wall (Pirvulescu et al., 2014). In addition, resistin

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enhances TLR4-induced inflammatory responses in acute lung injury (Jiang et al., 2014). Members of the MAPK family mediate the actions of dozens of hormones and cytokines. The ability of resistin to modulate chemokine production has been shown to involve both p38 and JNK (c-Jun N-terminal kinase) MAPKs as well as the proinflammatory transcription factor NF-kB (nuclear factor kappa-light-chainenhancer of activated B cells) (Manduteanu et al., 2009). In addition to NF-kB, resistin can induce the transcriptional activity of the AP-1 (activator protein 1) family of transcription factors in human endothelial cells (Manduteanu et al., 2010). The first signaling molecule shown to be induced by resistin was SOCS3, suppressor of cytokine signaling 3. SOCS3 can inhibit the signaling of a wide variety of cytokines and hormones, including insulin signaling. In vitro experiments demonstrated that the levels of resistin present in circulation (20 ng/mL) can induce both SOCS3 protein levels and the association of SOCS3 with the insulin receptor in mouse adipocytes (Steppan et al., 2005). Notably, the inhibition of SOCS3 function prevented resistin from antagonizing insulin action in adipocytes (Steppan et al., 2005). Resistin also activates SOCS3 and the upstream signaling molecule STAT3 in human endothelial cells (Pirvulescu et al., 2012). These studies showed that inhibition of SOCS3 prevented resistin-induced expression of cell adhesion molecules and activation of endothelial cells. These data also uncovered a new resistin-mediated mechanism in human endothelial cells and designated SOCS3 as a novel therapeutic target to modulate resistin-dependent inflammation in vessel wall diseases. AMPK plays an important role in cellular energy homeostasis. In relation to cancer, studies have shown that AMPK activation inhibits resistin’s ability to stimulate hepatocellular carcinoma cell adhesion (Yang et al., 2014). Resistin-induced inhibition of AMPK was previously implicated in its ability to regulate hepatic glucose production (Banerjee et al., 2004). Furthermore, “hyperresistinemia” is associated with both systemic insulin resistance and decreased levels of phospho-activated AMPK in liver, as well as in skeletal muscle and adipose tissue (Satoh et al., 2004). In summary, resistin acts through the CAP1 or TLR4 receptors and utilizes a variety of kinases (p38 and JNK MAPKs and PKA), transcription factors (NFk-B, AP1, and STAT3), and signaling molecules (SOCS3 and cAMP) to execute its physiologic and pathologic functions. Interestingly, inhibition of AMPK activity also appears to play an important role in mediating resistin’s action. As indicated subsequently and shown in Fig. 20.10, resistin has several target tissues. However, the primary

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FIGURE 20.9

Intracellular resistin signaling. Resistin acts through TLR4 (toll-like receptor 4) or CAP1 (adenylyl cyclase-associated protein 1) receptor and utilizes a variety of kinases (p38 and JNK MAPKs and PKA), transcription factors (NFk-B, AP-1, and STAT3), and signaling molecules (SOCS3 and cAMP) to execute its physiologic and pathologic functions. When resistin signals through CAP1, adenylyl cyclase is activated and converts ATP to cAMP, which then activates protein kinase A (PKA). Resistin signaling via either TLR4 or CAP1 can promote NFkB nuclear translocation and transcriptional activity by causing dissociation and degradation of the IkB inhibitory factor. Although the upstream mechanism is not known (indicated by the dashed line/arrow), resistin can also increase SOCS3 protein levels via STAT3-mediated transcriptional regulation. SOCS3 can then inhibit insulin receptor signaling.

FIGURE 20.10

Pathophysiological actions of resistin. Inflammation and disease states, such as obesity/type 2 diabetes (T2D) and heart disease, promote resistin expression and pathophysiological action in multiple cell types, including adipocytes, hepatocytes, endothelial cells, and cardiomyocytes. The action of resistin in these cells can lead to harmful biologic consequences.

cell types that have been studied thus far in terms of resistin signaling include adipocytes and endothelial cells where resistin induces inflammation-related signaling.

However, it should be noted, that under some conditions there is evidence that resistin is also capable of inhibiting the inflammatory role of LPS, and this effect could be

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relevant in some pathologic conditions, including parasitic infections and sepsis (Jang et al., 2017).

4.5 Physiological and Pathologic Roles of Resistin It is difficult to consider the physiologic actions of resistin without considering the pathology associated with this hormone. As indicated before, resistin was originally discovered as a secreted factor that was suppressed by an insulin-sensitizing drug (Steppan et al., 2001a) and as a hormone that likely acted in autocrine manner to suppress adipocyte development (Kim et al., 2001). The ability of resistin to inhibit adipogenesis has also been confirmed by other researchers (Blagoev et al., 2002). There is also evidence that one of the physiologic functions for resistin is the maintenance of blood glucose during fasting (Banerjee et al., 2004). Overall, the past 15 years of resistin research indicate that this hormone plays a role in obesity, insulin resistance, inflammation, and cardiovascular disease; reviewed in Schwartz and Lazar (2011). Many of the initial studies of resistin were performed in mice and were related to obesity, insulin resistance, and type 2 diabetes. Serum resistin levels are increased in several genetic forms of obesity and in diet-induced obesity in mice (Steppan et al., 2001a), and they are also increased in human obesity (Silha and Murphy, 2004; Degawa-Yamauchi et al., 2003). These studies are not simply correlative as immunoneutralization of circulating resistin improves glucose tolerance and insulin action in diet-induced obesity (Steppan et al., 2001b). Similar results were observed with in vivo expression of a dominant-negative form of resistin that could also improve insulin sensitivity (Kim et al., 2004). The role of resistin has also been examined in ob/ob mice. In this rodent model, a loss of resistin improved insulin sensitivity and glucose tolerance by increasing insulinregulated glucose disposal into adipose tissue and skeletal muscle (Qi et al., 2006). In the current gold-standard model of diet-induced obesity in C57BL/6J mice, the loss of resistin is accompanied by reduced hepatic glucose production as well as increased peripheral glucose uptake (Qi et al., 2006). There is also evidence for central actions of resistin on metabolic function. Normal mice with resistin delivered to the lateral cerebral ventricle exhibit increased endogenous glucose production in the context of a hyperinsulinemic-euglycemic clamp, strongly suggesting that resistin action in the brain could induce hepatic insulin resistance (Singhal et al., 2007). Resistin also induces neuropeptide Y (NPY) expression in the hypothalamus, and NPY neurons appear to be important in the ability of resistin to dampen hepatic insulin sensitivity

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(Singhal et al., 2007). These mechanistic studies are also accompanied by several epidemiological studies that suggest a role of human resistin in insulin resistance and diabetes (Schwartz and Lazar, 2011). It has been known for over a decade that human resistin is produced mainly by macrophages rather than adipocytes. This differential expression in mouse and man might suggest distinct functions in the different species. To address this issue, a humanized resistin mouse was generated by knocking out the endogenous resistin gene in mice and knocking in macrophage-specific expression of the human resistin gene (Qatanani et al., 2009). Use of this innovative mouse model revealed that humanized resistin mice fed a high-fat diet rapidly developed inflammation in white adipose tissue (WAT) that was accompanied by increased lipolysis and elevated serum levels of free fatty acids. These mice had other features of insulin resistance as well. Overall, these elegant studies revealed that although the cellular origin of resistin differs between species, that both mouse and human resistin promote WAT inflammation and enhance systemic insulin resistance (Qatanani et al., 2009). In addition to being a marker for obesity and type 2 diabetes, resistin has been shown to be an inflammatory-associated marker of atherosclerosis in humans (Reilly et al., 2005). The heart is a resistin target tissue, and in cardiomyocytes, both mouse and human resistin can directly impair glucose transport (Graveleau et al., 2005). Unlike actions on the liver, these effects of resistin require its oligomerization. Increased resistin levels have been associated with coronary calcification as well as systemic lupus erythematosus (Baker et al., 2011). Elevated resistin has also been documented in patients with other inflammatory diseases, including inflammatory bowel disease and rheumatoid arthritis (Filkova´ et al., 2009). Many studies have implicated a role for resistin in human atherosclerosis, coronary heart disease, and congestive heart failure (Ding et al., 2011; Lee and Kim, 2012). Collectively, studies in rodent models support a pathogenic role for resistin in insulin resistance and abnormal glucose metabolism. By nature, the human studies are not mechanistic and fail to reveal whether resistin is more than a biomarker for insulin resistance, diabetes, and cardiovascular disease.

4.6 Conclusions and Future Directions of Resistin Research The study of resistin was hampered for years by the lack of an identified receptor. Although both TLR4 and CAP1 have been implicated as resistin receptors, there are still many remaining questions regarding resistin signaling. For example, CAP1 lacks a transmembrane

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domain, so it is not completely clear how resistin increases its localization at the plasma membrane. Although the generation and study of a humanized resistin mouse revealed that human resistin has comparable ability to murine resistin to promote insulin resistance in mice, it is still unclear whether human resistin can confer these prominent metabolic effects in humans. There are several pathologic conditions ranging from cancer to type 2 diabetes that have implicated a role of resistin. The most consistent observations indicate increased levels of resistin in a variety of inflammatory conditions and diseases. Future studies involving the inhibition of resistin action in humans by targeting the hormone itself or one of its receptors could potentially have a large impact on our understanding of resistin biology. Tissue-specific knockouts of the reported resistin receptors may also provide some insight, but the interpretation of these studies will be hampered by the lack of specificity to resistin as these receptors are likely to be modulated by other endocrine mediators. Although all of the adipocyte-produced hormones are complicated, the expression and modulation of resistin seems particularly challenging to study.

2017). Both brown and white fat also produce and secrete miRNAs that can be free in circulation or contained within exosomes. These miRNAs have the ability to be taken up by other tissues where they can modulate gene expression. As indicated in Fig. 20.1, adipocytes make a large number of endocrine mediators in addition to leptin, resistin, and adiponectin. Obesity, or excess adipose tissue, is a global pandemic that has become a major health concern in developed and developing countries due to its comorbidities like type 2 diabetes, cardiovascular disease, and some cancers. The discovery of adipose tissue’s role as an endocrine gland able to secrete a variety of substances that can affect whole-body energy homeostasis has provided a pivotal breakthrough toward a better understanding of obesity and metabolic diseases. There is little doubt that the discovery and characterization of new adipocytederived factors is still in progress. Hopefully, the information in this review of three adipocyte polypeptide hormones has underscored the need for future research on the adipose tissue secretome.

Acknowledgments 5. FUTURE OUTLOOK FOR ADIPOKINES AND ADIPOSE TISSUE SECRETED FACTORS There is no question that our knowledge of adipocyte biology has changed substantially in the last 30 years. Adipose tissue is no longer viewed as inert tissue associated with energy storage, but as a bona fide endocrine organ that makes many hormones, which have systemic effects on a variety of physiologic processes. Adipose tissue is comprised of a variety of cell types, including endothelial cells, blood cells, fibroblasts, pericytes, preadipocytes, macrophages, and several types of immune cells. Since adipose tissue is comprised of so many different cell types, it is important to consider the cellular source of adipose tissue hormones. Although this chapter has focused on three peptide hormones that are largely produced in adipocytes, the endocrine function of adipocytes and nonadipocyte cells that comprise adipose tissue is immense and continually expanding. For example, immune cells in adipose tissue produce both pro- and antiinflammatory cytokines that act on adjacent adipocytes in a paracrine manner. Although most adipose tissue is WAT, many recent studies have explored new roles for brown fat in effecting metabolic health in humans. There is increasing evidence that brown fat has a secretory role that contributes to the systemic consequences of BAT activity. Several BAT-derived molecules, referred to as BATokines, can act in an endocrine manner (Villarroya et al.,

We would like to thank Hardy Hang for assisting with the figure preparation and his attention to detail. We also extend our gratitude to Christina Zunica for helping with the references.

References Akinci, M., Kosova, F., Cetin, B., Aslan, S., Ari, Z., Cetin, A., 2009. Leptin levels in thyroid cancer. Asian J. Surg. 32, 216e223. https:// doi.org/10.1016/S1015-9584(09)60397-3. Al Hannan, F., Culligan, K.G., 2015. Human resistin and the RELM of inflammation in diabesity. Diabetol. Metab. Syndr. 7, 54. https:// doi.org/10.1186/s13098-015-0050-3. Andersson, U., Filipsson, K., Abbott, C.R., Woods, A., Smith, K., Bloom, S.R., Carling, D., Small, C.J., 2004. AMP-activated protein kinase plays a role in the control of food intake. J. Biol. Chem. 279, 12005e12008. https://doi.org/10.1074/jbc.C300557200. Artac, M., Altundag, K., 2012. Leptin and breast cancer: an overview. Med. Oncol. 29, 1510e1514. https://doi.org/10.1007/s12032-0110056-0. Aruna, B., Islam, A., Ghosh, S., Singh, A.K., Vijayalakshmi, M., Ahmad, F., Ehtesham, N.Z., 2008. Biophysical analyses of human resistin: oligomer formation suggests novel biological function. Biochemistry 47, 12457e12466. https://doi.org/10.1021/bi801266k. Bahary, N., Leibel, R.L., Joseph, L., Friedman, J.M., 1990. Molecular mapping of the mouse db mutation. Proc. Natl. Acad. Sci. U.S.A. 87, 8642e8646. https://doi.org/10.1073/PNAS.87.21.8642. Baker, J.F., Morales, M., Qatanani, M., Cucchiara, A., Nackos, E., Lazar, M.A., Teff, K., Von feldt, J.M., 2011. Resistin levels in lupus and associations with disease-specific measures, insulin resistance, and coronary calcification. J. Rheumatol. 38, 2369e2375. https:// doi.org/10.3899/jrheum.110237. Balland, E., Cowley, M.A., 2015. New insights in leptin resistance mechanisms in mice. Front. Neuroendocrinol. 39, 59e65. https:// doi.org/10.1016/j.yfrne.2015.09.004.

REFERENCES

Banerjee, R.R., Lazar, M.A., 2001. Dimerization of resistin and resistinlike molecules is determined by a single cysteine. J. Biol. Chem. 276, 25970e25973. https://doi.org/10.1074/jbc.M103109200. Banerjee, R.R., Rangwala, S.M., Shapiro, J.S., Rich, A.S., Rhoades, B., Qi, Y., Wang, J., Rajala, M.W., Pocai, A., Scherer, P.E., Steppan, C.M., Ahima, R.S., Obici, S., Rossetti, L., Lazar, M.A., 2004. Regulation of fasted blood glucose by resistin. Science 303, 1195e1198. https://doi.org/10.1126/science.1092341. Banno, R., Zimmer, D., De Jonghe, B.C., Atienza, M., Rak, K., Yang, W., Bence, K.K., 2010. PTP1B and SHP2 in POMC neurons reciprocally regulate energy balance in mice. J. Clin. Investig. 120, 720e734. https://doi.org/10.1172/JCI39620. Bates, S.H., Stearns, W.H., Dundon, T.A., Schubert, M., Tso, A.W.K., Wang, Y., Banks, A.S., Lavery, H.J., Haq, A.K., Maratos-Flier, E., Neel, B.G., Schwartz, M.W., Myers, M.G., 2003. STAT3 signalling is required for leptin regulation of energy balance but not reproduction. Nature 421, 856e859. https://doi.org/10.1038/ nature01388. Bence, K.K., Delibegovic, M., Xue, B., Gorgun, C.Z., Hotamisligil, G.S., Neel, B.G., Kahn, B.B., 2006. Neuronal PTP1B regulates body weight, adiposity and leptin action. Nat. Med. 12, 917e924. https://doi.org/10.1038/nm1435. Berg, A.H., Combs, T.P., Du, X., Brownlee, M., Scherer, P.E., 2001. The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat. Med. 7, 947e953. https://doi.org/10.1038/90992. Berg, A.H., Combs, T.P., Scherer, P.E., 2002. ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism. Trends Endocrinol. Metab. 13, 84e89. http://www.ncbi.nlm.nih.gov/ pubmed/11854024. Bjørbaek, C., El-Haschimi, K., Frantz, J.D., Flier, J.S., 1999. The role of SOCS-3 in leptin signaling and leptin resistance. J. Biol. Chem. 274, 30059e30065. https://doi.org/10.1074/JBC.274.42.30059. Bjørbaek, C., Buchholz, R.M., Davis, S.M., Bates, S.H., Pierroz, D.D., Gu, H., Neel, B.G., Myers, M.G., Flier, J.S., 2001. Divergent roles of SHP-2 in ERK activation by leptin receptors. J. Biol. Chem. 276, 4747e4755. https://doi.org/10.1074/jbc.M007439200. Bjørbæk, C., Elmquist, J.K., Frantz, J.D., Shoelson, S.E., Flier, J.S., 1998. Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol. Cell 1, 619e625. https://doi.org/10.1016/S10972765(00)80062-3. Blagoev, B., Kratchmarova, I., Nielsen, M.M., Fernandez, M.M., Voldby, J., Andersen, J.S., Kristiansen, K., Pandey, A., Mann, M., 2002. Inhibition of adipocyte differentiation by resistin-like molecule a. J. Biol. Chem. 277, 42011e42016. https://doi.org/10.1074/ jbc.M206975200. Brooks, N.L., Trent, C.M., Raetzsch, C.F., Flurkey, K., Boysen, G., Perfetti, M.T., Jeong, Y.-C., Klebanov, S., Patel, K.B., Khodush, V.R., Kupper, L.L., Carling, D., Swenberg, J.A., Harrison, D.E., Combs, T.P., 2007. Low utilization of circulating glucose after food withdrawal in Snell dwarf mice. J. Biol. Chem. 282, 35069e35077. https://doi.org/10.1074/ jbc.M700484200. Buechler, C., Wanninger, J., Neumeier, M., 2011. Adiponectin, a key adipokine in obesity related liver diseases. World J. Gastroenterol. 17, 2801e2811. https://doi.org/10.3748/wjg.v17.i23.2801. Carpenter, L.R., Farruggella, T.J., Symes, A., Karow, M.L., Yancopoulos, G.D., Stahl, N., 1998. Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor. Proc. Natl. Acad. Sci. U.S.A. 95, 6061e6066. https:// doi.org/10.1073/PNAS.95.11.6061. Ceddia, R.B., Somwar, R., Maida, A., Fang, X., Bikopoulos, G., Sweeney, G., 2005. Globular adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen synthesis in rat skeletal muscle cells. Diabetologia 48, 132e139. https://doi.org/ 10.1007/s00125-004-1609-y.

479

Chan, D.W., Lee, J.M.F., Chan, P.C.Y., Ng, I.O.L., 2008. Genetic and epigenetic inactivation of T-cadherin in human hepatocellular carcinoma cells. Int. J. Cancer 123, 1043e1052. https://doi.org/ 10.1002/ijc.23634. Chen, X.X., Yang, T., 2015. Roles of leptin in bone metabolism and bone diseases. J. Bone Miner. Metab. 33, 474e485. https://doi.org/ 10.1007/s00774-014-0569-7. Chen, M.B., McAinch, A.J., Macaulay, S.L., Castelli, L.A., O’Brien, P.E., Dixon, J.B., Cameron-Smith, D., Kemp, B.E., Steinberg, G.R., 2005. Impaired activation of AMP-kinase and fatty acid oxidation by globular adiponectin in cultured human skeletal muscle of obese type 2 diabetics. J. Clin. Endocrinol. Metab. 90, 3665e3672. https://doi.org/10.1210/jc.2004-1980. Cheng, A., Uetani, N., Simoncic, P.D., Chaubey, V.P., Lee-Loy, A., McGlade, C.J., Kennedy, B.P., Tremblay, M.L., 2002. Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B. Dev. Cell 2, 497e503. https://doi.org/10.1016/S15345807(02)00149-1. Chlouverakis, C., 1972. Insulin resistance of parabiotic obesehyperglycemic mice (obob). Horm. Metab. Res. 4, 143e148. https://doi.org/10.1055/s-0028-1094088. Choi, S.R., Lim, J.H., Kim, M.Y., Kim, E.N., Kim, Y., Choi, B.S., Kim, Y.S., Kim, H.W., Lim, K.-M., Kim, M.J., Park, C.W., 2018. Adiponectin receptor agonist AdipoRon decreased ceramide, and lipotoxicity, and ameliorated diabetic nephropathy. Metabolism. https:// doi.org/10.1016/j.metabol.2018.02.004. Cianflone, K., Maslowska, M., Sniderman, A.D., 1999. Acylation stimulating protein (ASP), an adipocyte autocrine: new directions. Semin. Cell Dev. Biol. 10, 31e41. https://doi.org/10.1006/ SCDB.1998.0272.  s, S.,  2012. Adiponectin and its receptors in preimplantation emCiko bryo development. Vitam. Horm. 211e238. https://doi.org/ 10.1016/B978-0-12-398313-8.00009-9. Cohen, P., Zhao, C., Cai, X., Montez, J.M., Rohani, S.C., Feinstein, P., Mombaerts, P., Friedman, J.M., 2001. Selective deletion of leptin receptor in neurons leads to obesity. J. Clin. Investig. 108, 1113e1121. https://doi.org/10.1172/JCI13914. Coleman, D.L., 1978. Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 14, 141e148. http://www.ncbi.nlm.nih.gov/pubmed/350680. Combs, T.P., Marliss, E.B., 2014. Adiponectin signaling in the liver. Rev. Endocr. Metab. Disord. 15, 137e147. https://doi.org/10.1007/ s11154-013-9280-6. Combs, T.P., Berg, A.H., Obici, S., Scherer, P.E., Rossetti, L., 2001. Endogenous glucose production is inhibited by the adiposederived protein Acrp30. J. Clin. Investig. 108, 1875e1881. https:// doi.org/10.1172/JCI14120. Combs, T.P., Pajvani, U.B., Berg, A.H., Lin, Y., Jelicks, L.A., Laplante, M., Nawrocki, A.R., Rajala, M.W., Parlow, A.F., Cheeseboro, L., Ding, Y.-Y., Russell, R.G., Lindemann, D., Hartley, A., Baker, G.R.C., Obici, S., Deshaies, Y., Ludgate, M., Rossetti, L., Scherer, P.E., 2004. A transgenic mouse with a deletion in the collagenous domain of adiponectin displays elevated circulating adiponectin and improved insulin sensitivity. Endocrinology 145, 367e383. https://doi.org/10.1210/en.2003-1068. Cook, K.S., Min, H.Y., Johnson, D., Chaplinsky, R.J., Flier, J.S., Hunt, C.R., Spiegelman, B.M., 1987. Adipsin: a circulating serine protease homolog secreted by adipose tissue and sciatic nerve. Science 237, 402e405. http://www.ncbi.nlm.nih.gov/pubmed/ 3299705. Cota, D., Proulx, K., Smith, K.A.B., Kozma, S.C., Thomas, G., Woods, S.C., Seeley, R.J., 2006. Hypothalamic mTOR signaling regulates food intake. Science 312, 927e930. https://doi.org/10.1126/ science.1124147. Cota, D., Matter, E.K., Woods, S.C., Seeley, R.J., 2008. The role of hypothalamic mammalian target of rapamycin complex 1 signaling in

480

20. ADIPOCYTE HORMONES

diet-induced obesity. J. Neurosci. 28, 7202e7208. https://doi.org/ 10.1523/JNEUROSCI.1389-08.2008. Crimmins, N.A., Martin, L.J., 2007. Polymorphisms in adiponectin receptor genes ADIPOR1 and ADIPOR2 and insulin resistance. Obes. Rev. 8, 419e423. https://doi.org/10.1111/j.1467-789X.2007.00348.x. Cymbaluk, A., Chudecka-Głaz, A., Rzepka-Go´rska, I., 2008. Leptin levels in serum depending on body mass index in patients with endometrial hyperplasia and cancer. Eur. J. Obstet. Gynecol. Reprod. Biol. 136, 74e77. https://doi.org/10.1016/ J.EJOGRB.2006.08.012. Dadson, K., Liu, Y., Sweeney, G., 2011. Adiponectin action: a combination of endocrine and autocrine/paracrine effects. Front. Endocrinol. 2 https://doi.org/10.3389/fendo.2011.00062. Dalamaga, M., Chou, S.H., Shields, K., Papageorgiou, P., Polyzos, S.A., Mantzoros, C.S., 2013. Leptin at the intersection of neuroendocrinology and metabolism: current evidence and therapeutic perspectives. Cell Metabol. 18, 29e42. https://doi.org/10.1016/ J.CMET.2013.05.010. de Luca, C., Kowalski, T.J., Zhang, Y., Elmquist, J.K., Lee, C., Kilimann, M.W., Ludwig, T., Liu, S.-M., Chua Jr., S.C., 2005. Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes. J. Clin. Investig. 115, 3484e3493. https://doi.org/10.1172/JCI24059. de Sa´, P.M., Richard, A.J., Hang, H., Stephens, J.M., 2017. Transcriptional regulation of adipogenesis. In: Compr. Physiol. John Wiley & Sons, Inc., Hoboken, NJ, USA, pp. 635e674. https://doi.org/ 10.1002/cphy.c160022. Degawa-Yamauchi, M., Bovenkerk, J.E., Juliar, B.E., Watson, W., Kerr, K., Jones, R., Zhu, Q., Considine, R.V., 2003. Serum resistin (FIZZ3) protein is increased in obese humans. J. Clin. Endocrinol. Metab. 88, 5452e5455. https://doi.org/10.1210/jc.2002021808. Denzel, M.S., Scimia, M.-C., Zumstein, P.M., Walsh, K., RuizLozano, P., Ranscht, B., 2010. T-cadherin is critical for adiponectin-mediated cardioprotection in mice. J. Clin. Investig. 120, 4342e4352. https://doi.org/10.1172/JCI43464. Ding, G., Qin, Q., He, N., Francis-David, S.C., Hou, J., Liu, J., Ricks, E., Yang, Q., 2007. Adiponectin and its receptors are expressed in adult ventricular cardiomyocytes and upregulated by activation of peroxisome proliferator-activated receptor gamma. J. Mol. Cell. Cardiol. 43, 73e84. https://doi.org/10.1016/ j.yjmcc.2007.04.014. Ding, Q., White, S.P., Ling, C., Zhou, W., 2011. Resistin and cardiovascular disease. Trends Cardiovasc. Med. 21, 20e27. https://doi.org/ 10.1016/j.tcm.2012.01.004. Ding, M., Rzucidlo, E.M., Davey, J.C., Xie, Y., Liu, R., Jin, Y., Stavola, L., Martin, K.A., 2012. Adiponectin in the heart and vascular system. Vitam. Horm. 289e319. https://doi.org/10.1016/B978-0-12398313-8.00011-7. Dos Santos, E., Pecquery, R., de Mazancourt, P., Dieudonne´, M.-N., 2012. Adiponectin and reproduction. Vitam. Horm. 187e209. https://doi.org/10.1016/B978-0-12-398313-8.00008-7. Ducy, P., Amling, M., Takeda, S., Priemel, M., Schilling, A.F., Beil, F.T., Shen, J., Vinson, C., Rueger, J.M., Karsenty, G., 2000. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell 100, 197e207. https://doi.org/10.1016/S00928674(00)81558-5. Enriori, P.J., Evans, A.E., Sinnayah, P., Jobst, E.E., Tonelli-Lemos, L., Billes, S.K., Glavas, M.M., Grayson, B.E., Perello, M., Nillni, E.A., Grove, K.L., Cowley, M.A., 2007. Diet-induced obesity causes severe but reversible leptin resistance in arcuate melanocortin neurons. Cell Metabol. 5, 181e194. https://doi.org/10.1016/ J.CMET.2007.02.004. Farooqi, I.S., O’Rahilly, S., 2014. 20 years of leptin: human disorders of leptin action. J. Endocrinol. 223, T63eT70. https://doi.org/ 10.1530/JOE-14-0480.

Fasshauer, M., Blu¨her, M., 2015. Adipokines in health and disease. Trends Pharmacol. Sci. 36, 461e470. https://doi.org/10.1016/ j.tips.2015.04.014.  Filkova´, M., Haluzı´k, M., Gay, S., Senolt, L., 2009. The role of resistin as a regulator of inflammation: implications for various human pathologies. Clin. Immunol. 133, 157e170. https://doi.org/ 10.1016/J.CLIM.2009.07.013. Fong, T.M., Huang, R.R., Tota, M.R., Mao, C., Smith, T., Varnerin, J., Karpitskiy, V.V., Krause, J.E., Van der Ploeg, L.H., 1998. Localization of leptin binding domain in the leptin receptor. Mol. Pharmacol. 53, 234e240. http://www.ncbi.nlm.nih.gov/pubmed/9463481. Friedman, J., 2014. 20 years of leptin: leptin at 20: an overview. J. Endocrinol. 223, T1eT8. https://doi.org/10.1530/JOE-14-0405. Friedman, J.M., Halaas, J.L., 1998. Leptin and the regulation of body weight in mammals. Nature 395, 763e770. https://doi.org/ 10.1038/27376. Fruebis, J., Tsao, T.S., Javorschi, S., Ebbets-Reed, D., Erickson, M.R., Yen, F.T., Bihain, B.E., Lodish, H.F., 2001. Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc. Natl. Acad. Sci. U.S.A. 98, 2005e2010. https://doi.org/10.1073/ pnas.041591798. Fujishima, Y., Maeda, N., Matsuda, K., Masuda, S., Mori, T., Fukuda, S., Sekimoto, R., Yamaoka, M., Obata, Y., Kita, S., Nishizawa, H., Funahashi, T., Ranscht, B., Shimomura, I., 2017. Adiponectin association with T-cadherin protects against neointima proliferation and atherosclerosis. FASEB J. 31, 1571e1583. https://doi.org/ 10.1096/fj.201601064R. Gao, S., Kinzig, K.P., Aja, S., Scott, K.A., Keung, W., Kelly, S., Strynadka, K., Chohnan, S., Smith, W.W., Tamashiro, K.L.K., Ladenheim, E.E., Ronnett, G.V., Tu, Y., Birnbaum, M.J., Lopaschuk, G.D., Moran, T.H., 2007. Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake. Proc. Natl. Acad. Sci. U.S.A. 104, 17358e17363. https://doi.org/10.1073/pnas.0708385104. Garcı´a-Robles, M.J., Segura-Ortega, J.E., Fafutis-Morris, M., 2013. The biology of leptin and its implications in breast cancer: a general view. J. Interferon Cytokine Res. 33, 717e727. https://doi.org/ 10.1089/jir.2012.0168. Ghosh, S., Singh, A.K., Aruna, B., Mukhopadhyay, S., Ehtesham, N.Z., 2003. The genomic organization of mouse resistin reveals major differences from the human resistin: functional implications. Gene 305, 27e34. https://doi.org/10.1016/S0378-1119(02)01213-1. Ghosh, S., Mukhopadhyay, P., Pandit, K., Chowdhury, S., Dutta, D., 2012. Leptin and cancer: pathogenesis and modulation. Indian J. Endocrinol. Metab. 16, 596. https://doi.org/10.4103/22308210.105577. Graveleau, C., Zaha, V.G., Mohajer, A., Banerjee, R.R., DudleyRucker, N., Steppan, C.M., Rajala, M.W., Scherer, P.E., Ahima, R.S., Lazar, M.A., Abel, E.D., 2005. Mouse and human resistins impair glucose transport in primary mouse cardiomyocytes, and oligomerization is required for this biological action. J. Biol. Chem. 280, 31679e31685. https://doi.org/10.1074/ jbc.M504008200. Guo, K., McMinn, J.E., Ludwig, T., Yu, Y.-H., Yang, G., Chen, L., Loh, D., Li, C., Chua, S., Zhang, Y., 2007. Disruption of peripheral leptin signaling in mice results in hyperleptinemia without associated metabolic abnormalities. Endocrinology 148, 3987e3997. https://doi.org/10.1210/en.2007-0261. Hada, Y., Yamauchi, T., Waki, H., Tsuchida, A., Hara, K., Yago, H., Miyazaki, O., Ebinuma, H., Kadowaki, T., 2007. Selective purification and characterization of adiponectin multimer species from human plasma. Biochem. Biophys. Res. Commun. 356, 487e493. https://doi.org/10.1016/J.BBRC.2007.03.004. Haglund, E., Sułkowska, J.I., He, Z., Feng, G.-S., Jennings, P.A., Onuchic, J.N., 2012. The unique cysteine knot regulates the

REFERENCES

pleotropic hormone leptin. PLoS One 7, e45654. https://doi.org/ 10.1371/journal.pone.0045654. Harris, R.B., 1997. Loss of body fat in lean parabiotic partners of ob/ob mice. Am. J. Physiol. 272, R1809eR1815. https://doi.org/10.1152/ ajpregu.1997.272.6.R1809. Harris, R.B.S., 1999. Parabiosis between db/db and ob/ob or db/þ mice. Endocrinology 140, 138e145. https://doi.org/10.1210/ endo.140.1.6449. Harris, R.B.S., 2013. Contribution made by parabiosis to the understanding of energy balance regulation. Biochim. Biophys. Acta 1832, 1449e1455. https://doi.org/10.1016/J.BBADIS.2013.02.021. Harris, R.B.S., 2014. Direct and indirect effects of leptin on adipocyte metabolism. Biochim. Biophys. Acta 1842, 414e423. https:// doi.org/10.1016/j.bbadis.2013.05.009. Hartman, H.B., Hu, X., Tyler, K.X., Dalal, C.K., Lazar, M.A., 2002. Mechanisms regulating adipocyte expression of resistin. J. Biol. Chem. 277, 19754e19761. https://doi.org/10.1074/ jbc.M201451200. Hattori, Y., Nakano, Y., Hattori, S., Tomizawa, A., Inukai, K., Kasai, K., 2008. High molecular weight adiponectin activates AMPK and suppresses cytokine-induced NF-kB activation in vascular endothelial cells. FEBS Lett. 582, 1719e1724. https://doi.org/10.1016/ j.febslet.2008.04.037. Hausberger, F.X., 1958. Parabiosis and transplantation experiments in hereditary obese mice. Anat. Rec. 130, 313. Holland, W.L., Adams, A.C., Brozinick, J.T., Bui, H.H., Miyauchi, Y., Kusminski, C.M., Bauer, S.M., Wade, M., Singhal, E., Cheng, C.C., Volk, K., Kuo, M.-S., Gordillo, R., Kharitonenkov, A., Scherer, P.E., 2013. An FGF21-adiponectin-ceramide axis controls energy expenditure and insulin action in mice. Cell Metabol. 17, 790e797. https://doi.org/10.1016/j.cmet.2013.03.019. Hotamisligil, G.S., Shargill, N.S., Spiegelman, B.M., 1993. Adipose expression of tumor necrosis factor-alpha: direct role in obesitylinked insulin resistance. Science 259, 87e91. http://www.ncbi. nlm.nih.gov/pubmed/7678183. Hu, E., Liang, P., Spiegelman, B.M., 1996. AdipoQ is a novel adiposespecific gene dysregulated in obesity. J. Biol. Chem. 271, 10697e10703. http://www.ncbi.nlm.nih.gov/pubmed/8631877. Hug, C., Wang, J., Ahmad, N.S., Bogan, J.S., Tsao, T.-S., Lodish, H.F., 2004. T-cadherin is a receptor for hexameric and high-molecularweight forms of Acrp30/adiponectin. Proc. Natl. Acad. Sci. U.S.A. 101, 10308e10313. https://doi.org/10.1073/ pnas.0403382101. Hummel, K.P., Dickie, M.M., Coleman, D.L., 1966. Diabetes, a new mutation in the mouse. Science 153, 1127e1128. https://doi.org/ 10.1126/SCIENCE.153.3740.1127. Ingalls, A.M., Dickie, M.M., Snell, G.D., 1950. Obese, a new mutation in the house mouse. J. Hered. 41, 317e318. http://www.ncbi.nlm.nih. gov/pubmed/14824537. Iwabu, M., Yamauchi, T., Okada-Iwabu, M., Sato, K., Nakagawa, T., Funata, M., Yamaguchi, M., Namiki, S., Nakayama, R., Tabata, M., Ogata, H., Kubota, N., Takamoto, I., Hayashi, Y.K., Yamauchi, N., Waki, H., Fukayama, M., Nishino, I., Tokuyama, K., Ueki, K., Oike, Y., Ishii, S., Hirose, K., Shimizu, T., Touhara, K., Kadowaki, T., 2010. Adiponectin and AdipoR1 regulate PGC-1a and mitochondria by Ca2þ and AMPK/SIRT1. Nature 464, 1313e1319. https://doi.org/10.1038/nature08991. Jang, J.C., Li, J., Gambini, L., Batugedara, H.M., Sati, S., Lazar, M.A., Fan, L., Pellecchia, M., Nair, M.G., 2017. Human resistin protects against endotoxic shock by blocking LPSeTLR4 interaction. Proc. Natl. Acad. Sci. U.S.A. 114, E10399eE10408. https://doi.org/ 10.1073/pnas.1716015114. Jiang, S., Park, D.W., Tadie, J.-M., Gregoire, M., Deshane, J., Pittet, J.F., Abraham, E., Zmijewski, J.W., 2014. Human resistin promotes neutrophil proinflammatory activation and neutrophil extracellular

481

trap formation and increases severity of acute lung injury. J. Immunol. 192, 4795e4803. https://doi.org/10.4049/ jimmunol.1302764. Kadowaki, T., Yamauchi, T., Waki, H., Iwabu, M., Okada-Iwabu, M., Nakamura, M., 2011. Adiponectin, adiponectin receptors, and epigenetic regulation of adipogenesis. Cold Spring Harbor Symp. Quant. Biol. 76, 257e265. https://doi.org/10.1101/ sqb.2012.76.010587. Kahn, B.B., Alquier, T., Carling, D., Hardie, D.G., 2005. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metabol. 1, 15e25. https:// doi.org/10.1016/J.CMET.2004.12.003. Kandasamy, A.D., Sung, M.M., Boisvenue, J.J., Barr, A.J., Dyck, J.R.B., 2012. Adiponectin gene therapy ameliorates high-fat, highsucrose diet-induced metabolic perturbations in mice. Nutr. Diabetes 2, e45. https://doi.org/10.1038/nutd.2012.18. Kaszubska, W., Falls, H.D., Schaefer, V.G., Haasch, D., Frost, L., Hessler, P., Kroeger, P.E., White, D.W., Jirousek, M.R., Trevillyan, J.M., 2002. Protein tyrosine phosphatase 1B negatively regulates leptin signaling in a hypothalamic cell line. Mol. Cell. Endocrinol. 195, 109e118. https://doi.org/10.1016/S03037207(02)00178-8. Kievit, P., Howard, J.K., Badman, M.K., Balthasar, N., Coppari, R., Mori, H., Lee, C.E., Elmquist, J.K., Yoshimura, A., Flier, J.S., 2006. Enhanced leptin sensitivity and improved glucose homeostasis in mice lacking suppressor of cytokine signaling-3 in POMCexpressing cells. Cell Metabol. 4, 123e132. https://doi.org/ 10.1016/J.CMET.2006.06.010. Kim, K.H., Lee, K., Moon, Y.S., Sul, H.S., 2001. A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation. J. Biol. Chem. 276, 11252e11256. https://doi.org/10.1074/ jbc.C100028200. Kim, K.-H., Zhao, L., Moon, Y., Kang, C., Sul, H.S., 2004. Dominant inhibitory adipocyte-specific secretory factor (ADSF)/resistin enhances adipogenesis and improves insulin sensitivity. Proc. Natl. Acad. Sci. U.S.A. 101, 6780e6785. https://doi.org/10.1073/ pnas.0305905101. Kim, A.S., Miller, E.J., Young, L.H., 2009. AMP-activated protein kinase: a core signalling pathway in the heart. Acta Physiol. 196, 37e53. https://doi.org/10.1111/j.1748-1716.2009.01978.x. Kim, H.H., Kim, Y.S., Kang, Y.K., Moon, J.S., 2012. Leptin and peroxisome proliferator-activated receptor g expression in colorectal adenoma. World J. Gastroenterol. 18, 557e562. https://doi.org/ 10.3748/wjg.v18.i6.557. Krajewska, M., Banares, S., Zhang, E.E., Huang, X., Scadeng, M., Jhala, U.S., Feng, G.-S., Krajewski, S., 2008. Development of diabesity in mice with neuronal deletion of Shp2 tyrosine phosphatase. Am. J. Pathol. 172, 1312e1324. https://doi.org/10.2353/ AJPATH.2008.070594. Kubota, N., Terauchi, Y., Kubota, T., Kumagai, H., Itoh, S., Satoh, H., Yano, W., Ogata, H., Tokuyama, K., Takamoto, I., Mineyama, T., Ishikawa, M., Moroi, M., Sugi, K., Yamauchi, T., Ueki, K., Tobe, K., Noda, T., Nagai, R., Kadowaki, T., 2006. Pioglitazone ameliorates insulin resistance and diabetes by both adiponectin-dependent and independent pathways. J. Biol. Chem. 281, 8748e8755. https://doi.org/10.1074/jbc.M505649200. Kumada, M., Kihara, S., Sumitsuji, S., Kawamoto, T., Matsumoto, S., Ouchi, N., Arita, Y., Okamoto, Y., Shimomura, I., Hiraoka, H., Nakamura, T., Funahashi, T., Matsuzawa, Y., Osaka CAD Study Group, 2003. Coronary artery disease, association of hypoadiponectinemia with coronary artery disease in men. Arterioscler. Thromb. Vasc. Biol. 23, 85e89. http://www.ncbi. nlm.nih.gov/pubmed/12524229. La Cava, A., Matarese, G., 2004. The weight of leptin in immunity. Nat. Rev. Immunol. 4, 371e379. https://doi.org/10.1038/nri1350.

482

20. ADIPOCYTE HORMONES

Lee, S.E., Kim, H.-S., 2012. Human resistin in cardiovascular disease. J. Smooth Muscle Res. 48, 27e35. http://www.ncbi.nlm.nih.gov/ pubmed/22504487. Lee, S., Lee, H.C., Kwon, Y.W., Lee, S.E., Cho, Y., Kim, J., Lee, S., Kim, J.Y., Lee, J., Yang, H.M., Mook-Jung, I., Nam, K.Y., Chung, J., Lazar, M.A., Kim, H.S., 2014. Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes. Cell Metabol. 19, 484e497. https:// doi.org/10.1016/j.cmet.2014.01.013. Lefterova, M.I., Mullican, S.E., Tomaru, T., Qatanani, M., Schupp, M., Lazar, M.A., 2009. Endoplasmic reticulum stress regulates adipocyte resistin expression. Diabetes 58, 1879e1886. https://doi.org/ 10.2337/db08-1706. Li, C., Friedman, J.M., 1999. Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction. Proc. Natl. Acad. Sci. U.S.A. 96, 9677e9682. https://doi.org/10.1073/PNAS.96.17.9677. Lin, Z., Tian, H., Lam, K.S.L., Lin, S., Hoo, R.C.L., Konishi, M., Itoh, N., Wang, Y., Bornstein, S.R., Xu, A., Li, X., 2013. Adiponectin mediates the metabolic effects of FGF21 on glucose homeostasis and insulin sensitivity in mice. Cell Metabol. 17, 779e789. https://doi.org/ 10.1016/j.cmet.2013.04.005. Lindstro¨m, P., 2007. The physiology of obese-hyperglycemic mice [ob/ ob mice]. Sci. World J. 7, 666e685. https://doi.org/10.1100/ tsw.2007.117. Liu, M., Zhou, L., Xu, A., Lam, K.S.L., Wetzel, M.D., Xiang, R., Zhang, J., Xin, X., Dong, L.Q., Liu, F., 2008. A disulfide-bond A oxidoreductase-like protein (DsbA-L) regulates adiponectin multimerization. Proc. Natl. Acad. Sci. U.S.A. 105, 18302e18307. https://doi.org/10.1073/pnas.0806341105. Liu, M., Chen, H., Wei, L., Hu, D., Dong, K., Jia, W., Dong, L.Q., Liu, F., 2015. Endoplasmic reticulum (ER) localization is critical for DsbA-L protein to suppress ER stress and adiponectin down-regulation in adipocytes. J. Biol. Chem. 290, 10143e10148. https://doi.org/ 10.1074/jbc.M115.645416. Liu, Z., Xiao, T., Peng, X., Li, G., Hu, F., 2017. APPLs: more than just adiponectin receptor binding proteins. Cell. Signal. 32, 76e84. https:// doi.org/10.1016/j.cellsig.2017.01.018. Maahs, D.M., Ogden, L.G., Kinney, G.L., Wadwa, P., SnellBergeon, J.K., Dabelea, D., Hokanson, J.E., Ehrlich, J., Eckel, R.H., Rewers, M., 2005. Low plasma adiponectin levels predict progression of coronary artery calcification. Circulation 111, 747e753. https://doi.org/10.1161/01.CIR.0000155251.03724.A5. Maeda, K., Okubo, K., Shimomura, I., Funahashi, T., Matsuzawa, Y., Matsubara, K., 1996. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (adipose most abundant gene transcript 1). Biochem. Biophys. Res. Commun. 221, 286e289. https://doi.org/10.1006/bbrc.1996.0587. Manduteanu, I., Dragomir, E., Calin, M., Pirvulescu, M., Gan, A.M., Stan, D., Simionescu, M., 2009. Resistin up-regulates fractalkine expression in human endothelial cells: lack of additive effect with TNF-a. Biochem. Biophys. Res. Commun. 381, 96e101. https:// doi.org/10.1016/j.bbrc.2009.02.015. Manduteanu, I., Pirvulescu, M., Gan, A.M., Stan, D., Simion, V., Dragomir, E., Calin, M., Manea, A., Simionescu, M., 2010. Similar effects of resistin and high glucose on P-selectin and fractalkine expression and monocyte adhesion in human endothelial cells. Biochem. Biophys. Res. Commun. 391, 1443e1448. https://doi.org/ 10.1016/J.BBRC.2009.12.089. Mao, X., Kikani, C.K., Riojas, R.A., Langlais, P., Wang, L., Ramos, F.J., Fang, Q., Christ-Roberts, C.Y., Hong, J.Y., Kim, R.-Y., Liu, F., Dong, L.Q., 2006. APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function. Nat. Cell Biol. 8, 516e523. https://doi.org/10.1038/ncb1404. Mashalidis, E.H., Briggs, D.B., Zhou, M., Vergara, A.M., Chhun, J.J., Ellsworth, R.K., Giron, R.M., Rood, J., Bray, G.A., Smith, S.R.,

Wysocki, V.H., Tsao, T.-S., 2013. High-resolution identification of human adiponectin oligomers and regulation by pioglitazone in type 2 diabetic patients. Anal. Biochem. 437, 150e160. https:// doi.org/10.1016/J.AB.2013.02.008. Matsuda, K., Fujishima, Y., Maeda, N., Mori, T., Hirata, A., Sekimoto, R., Tsushima, Y., Masuda, S., Yamaoka, M., Inoue, K., Nishizawa, H., Kita, S., Ranscht, B., Funahashi, T., Shimomura, I., 2015. Positive feedback regulation between adiponectin and Tcadherin impacts adiponectin levels in tissue and plasma of male mice. Endocrinology 156, 934e946. https://doi.org/10.1210/ en.2014-1618. Metlakunta, A.S., Sahu, M., Sahu, A., 2008. Hypothalamic phosphatidylinositol 3-kinase pathway of leptin signaling is impaired during the development of diet-induced obesity in FVB/N mice. Endocrinology 149, 1121e1128. https://doi.org/ 10.1210/en.2007-1307. Miaczynska, M., Christoforidis, S., Giner, A., Shevchenko, A., Uttenweiler-Joseph, S., Habermann, B., Wilm, M., Parton, R.G., Zerial, M., 2004. APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment. Cell 116, 445e456. https://doi.org/10.1016/S0092-8674(04)00117-5. Min, X., Lemon, B., Tang, J., Liu, Q., Zhang, R., Walker, N., Li, Y., Wang, Z., 2012. Crystal structure of a single-chain trimer of human adiponectin globular domain. FEBS Lett. 586, 912e917. https:// doi.org/10.1016/j.febslet.2012.02.024. Minokoshi, Y., Alquier, T., Furukawa, N., Kim, Y.-B., Lee, A., Xue, B., Mu, J., Foufelle, F., Ferre´, P., Birnbaum, M.J., Stuck, B.J., Kahn, B.B., 2004. AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428, 569e574. https://doi.org/10.1038/nature02440. Morrison, C.D., White, C.L., Wang, Z., Lee, S.-Y., Lawrence, D.S., Cefalu, W.T., Zhang, Z.-Y., Gettys, T.W., 2007. Increased hypothalamic protein tyrosine phosphatase 1B contributes to leptin resistance with age. Endocrinology 148, 433e440. https://doi.org/ 10.1210/en.2006-0672. Morton, G.J., Niswender, K.D., Rhodes, C.J., Myers, M.G., Blevins, J.E., Baskin, D.G., Schwartz, M.W., 2003. Arcuate nucleus-specific leptin receptor gene therapy attenuates the obesity phenotype of Koletsky (fa(k)/fa(k)) rats. Endocrinology 144, 2016e2024. https://doi.org/ 10.1210/en.2002-0115. Munsell, M.F., Sprague, B.L., Berry, D.A., Chisholm, G., TrenthamDietz, A., 2014. Body mass index and breast cancer risk according to postmenopausal estrogen-progestin use and hormone receptor status. Epidemiol. Rev. 36, 114e136. https://doi.org/10.1093/epirev/mxt010. Mu¨nzberg, H., Morrison, C.D., 2015. Structure, production and signaling of leptin. Metabolism 64, 13e23. https://doi.org/ 10.1016/j.metabol.2014.09.010. Nakamura, T., Funayama, H., Kubo, N., Yasu, T., Kawakami, M., Saito, M., Momomura, S., Ishikawa, S.-E., 2006. Association of hyperadiponectinemia with severity of ventricular dysfunction in congestive heart failure. Circ. J. 70, 1557e1562. http://www.ncbi. nlm.nih.gov/pubmed/17127799. Nakano, Y., Tobe, T., Choi-Miura, N.-H., Mazda, T., Tomita, M., 1996. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. 120, 803e812. https://doi.org/10.1093/oxfordjournals.jbchem.a021483. Nawrocki, A.R., Rajala, M.W., Tomas, E., Pajvani, U.B., Saha, A.K., Trumbauer, M.E., Pang, Z., Chen, A.S., Ruderman, N.B., Chen, H., Rossetti, L., Scherer, P.E., 2006. Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to proliferator-activated receptor g agonists. J. Biol. Chem. 281, 2654e2660. https://doi.org/10.1074/jbc.M505311200. Niswender, K.D., Morton, G.J., Stearns, W.H., Rhodes, C.J., Myers, M.G., Schwartz, M.W., 2001. Intracellular signalling: key

REFERENCES

enzyme in leptin-induced anorexia. Nature 413, 794e795. https:// doi.org/10.1038/35101657. Ohashi, K., Kihara, S., Ouchi, N., Kumada, M., Fujita, K., Hiuge, A., Hibuse, T., Ryo, M., Nishizawa, H., Maeda, N., Maeda, K., Shibata, R., Walsh, K., Funahashi, T., Shimomura, I., 2006. Adiponectin replenishment ameliorates obesity-related hypertension. Hypertension 47, 1108e1116. https://doi.org/10.1161/ 01.HYP.0000222368.43759.a1. Ohashi, K., Ouchi, N., Matsuzawa, Y., 2011. Adiponectin and hypertension. Am. J. Hypertens. 24, 263e269. https://doi.org/ 10.1038/ajh.2010.216. Okada-Iwabu, M., Yamauchi, T., Iwabu, M., Honma, T., Hamagami, K., Matsuda, K., Yamaguchi, M., Tanabe, H., Kimura-Someya, T., Shirouzu, M., Ogata, H., Tokuyama, K., Ueki, K., Nagano, T., Tanaka, A., Yokoyama, S., Kadowaki, T., 2013. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity. Nature 503, 493e499. https://doi.org/10.1038/nature12656. Oliver, P., Pico´, C., Serra, F., Palou, A., 2003. Resistin expression in different adipose tissue depots during rat development. Mol. Cell. Biochem. 252, 397e400. https://doi.org/10.1023/A: 1025500605884. Ouchi, N., Kihara, S., Arita, Y., Maeda, K., Kuriyama, H., Okamoto, Y., Hotta, K., Nishida, M., Takahashi, M., Nakamura, T., Yamashita, S., Funahashi, T., Matsuzawa, Y., 1999. Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin. Circulation 100, 2473e2476. http://www.ncbi.nlm. nih.gov/pubmed/10604883. Pagano, C., Soardo, G., Esposito, W., Fallo, F., Basan, L., Donnini, D., Federspil, G., Sechi, L.A., Vettor, R., 2005. Plasma adiponectin is decreased in nonalcoholic fatty liver disease. Eur. J. Endocrinol. 152, 113e118. http://www.ncbi.nlm.nih.gov/pubmed/15762194. Paik, S.S., Jang, S.-M., Jang, K.-S., Lee, K.H., Choi, D., Jang, S.J., 2009. Leptin expression correlates with favorable clinicopathologic phenotype and better prognosis in colorectal adenocarcinoma. Ann. Surg. Oncol. 16, 297e303. https://doi.org/10.1245/s10434-008-0221-7. Pajvani, U.B., Du, X., Combs, T.P., Berg, A.H., Rajala, M.W., Schulthess, T., Engel, J., Brownlee, M., Scherer, P.E., 2003. Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin: implications for metabolic regulation and bioactivity. J. Biol. Chem. 278, 9073e9085. https://doi.org/ 10.1074/jbc.M207198200. Pajvani, U.B., Hawkins, M., Combs, T.P., Rajala, M.W., Doebber, T., Berger, J.P., Wagner, J.A., Wu, M., Knopps, A., Xiang, A.H., Utzschneider, K.M., Kahn, S.E., Olefsky, J.M., Buchanan, T.A., Scherer, P.E., 2004. Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity. J. Biol. Chem. 279, 12152e12162. https://doi.org/10.1074/jbc.M311113200. Parker-Duffen, J.L., Nakamura, K., Silver, M., Kikuchi, R., Tigges, U., Yoshida, S., Denzel, M.S., Ranscht, B., Walsh, K., 2013. T-cadherin is essential for adiponectin-mediated revascularization. J. Biol. Chem. 288, 24886e24897. https://doi.org/10.1074/ jbc.M113.454835. Patel, L., Buckels, A.C., Kinghorn, I.J., Murdock, P.R., Holbrook, J.D., Plumpton, C., Macphee, C.H., Smith, S.A., 2003. Resistin is expressed in human macrophages and directly regulated by PPARg activators. Biochem. Biophys. Res. Commun. 300, 472e476. https://doi.org/10.1016/S0006-291X(02)02841-3. Patel, S.D., Rajala, M.W., Rossetti, L., Scherer, P.E., Shapiro, L., 2004. Disulfide-dependent multimeric assembly of resistin family hormones. Science 304, 1154e1158. https://doi.org/10.1126/ science.1093466. Peelman, F., Zabeau, L., Moharana, K., Savvides, S.N., Tavernier, J., 2014. 20 years of leptin: insights into signaling assemblies of the leptin receptor. J. Endocrinol. 223, T9eT23. https://doi.org/10.1530/ JOE-14-0264.

483

Pendergrass, M., Bertoldo, A., Bonadonna, R., Nucci, G., Mandarino, L., Cobelli, C., DeFronzo, R.A., 2007. Muscle glucose transport and phosphorylation in type 2 diabetic, obese nondiabetic, and genetically predisposed individuals. Am. J. Physiol. Metab. 292, E92eE100. https://doi.org/10.1152/ajpendo.00617.2005. Petridou, E., Belechri, M., Dessypris, N., Koukoulomatis, P., Diakomanolis, E., Spanos, E., Trichopoulos, D., 2002. Leptin and body mass index in relation to endometrial cancer risk. Ann. Nutr. Metab. 46, 147e151. https://doi.org/10.1159/000063081. Picardi, P.K., Calegari, V.C., de Oliveira Prada, P., Contin Moraes, J., Arau´jo, E., Gomes Marcondes, M.C.C., Ueno, M., Carvalheira, J.B.C., Velloso, L.A., Abdalla Saad, M.J., 2008. Reduction of hypothalamic protein tyrosine phosphatase improves insulin and leptin resistance in diet-induced obese rats. Endocrinology 149, 3870e3880. https://doi.org/10.1210/en.2007-1506. Pirvulescu, M., Manduteanu, I., Gan, A.M., Stan, D., Simion, V., Butoi, E., Calin, M., Simionescu, M., 2012. A novel proinflammatory mechanism of action of resistin in human endothelial cells: up-regulation of SOCS3 expression through STAT3 activation. Biochem. Biophys. Res. Commun. 422, 321e326. https://doi.org/ 10.1016/J.BBRC.2012.04.159. Pirvulescu, M.M., Gan, A.M., Stan, D., Simion, V., Calin, M., Butoi, E., Manduteanu, I., 2014. Subendothelial resistin enhances monocyte transmigration in a co-culture of human endothelial and smooth muscle cells by mechanisms involving fractalkine, MCP-1 and activation of TLR4 and Gi/o proteins signaling. Int. J. Biochem. Cell Biol. 50, 29e37. https://doi.org/10.1016/j.biocel.2014.01.022. Pischon, T., Girman, C.J., Hotamisligil, G.S., Rifai, N., Hu, F.B., Rimm, E.B., 2004. Plasma adiponectin levels and risk of myocardial infarction in men. J. Am. Med. Assoc. 291, 1730e1737. https:// doi.org/10.1001/jama.291.14.1730. Qatanani, M., Szwergold, N.R., Greaves, D.R., Ahima, R.S., Lazar, M.A., 2009. Macrophage-derived human resistin exacerbates adipose tissue inflammation and insulin resistance in mice. J. Clin. Investig. 119, 531e539. https://doi.org/10.1172/JCI37273. Qi, Y., Nie, Z., Lee, Y.-S., Singhal, N.S., Scherer, P.E., Lazar, M.A., Ahima, R.S., 2006. Loss of resistin improves glucose homeostasis in leptin deficiency. Diabetes 55, 3083e3090. https://doi.org/ 10.2337/db05-0615. Qiang, L., Wang, H., Farmer, S.R., 2007. Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase Ero1-L. Mol. Cell. Biol. 27, 4698e4707. https://doi.org/10.1128/ MCB.02279-06. Raghu, P., Ghosh, S., Soundarya, K., Haseeb, A., Aruna, B., Ehtesham, N.Z., 2004. Dimerization of human recombinant resistin involves covalent and noncovalent interactions. Biochem. Biophys. Res. Commun. 313, 642e646. https://doi.org/10.1016/ J.BBRC.2003.11.156. Rahmouni, K., Sigmund, C.D., Haynes, W.G., Mark, A.L., 2009. Hypothalamic ERK mediates the anorectic and thermogenic sympathetic effects of leptin. Diabetes 58, 536e542. https://doi.org/10.2337/ db08-0822. Rajala, M.W., Qi, Y., Patel, H.R., Takahashi, N., Banerjee, R., Pajvani, U.B., Sinha, M.K., Gingerich, R.L., Scherer, P.E., Ahima, R.S., 2004. Regulation of resistin expression and circulating levels in obesity, diabetes, and fasting. Diabetes 53, 1671e1679. https://doi.org/10.2337/DIABETES.53.7.1671. Reilly, M.P., Lehrke, M., Wolfe, M.L., Rohatgi, A., Lazar, M.A., Rader, D.J., 2005. Resistin is an inflammatory marker of atherosclerosis in humans. Circulation 111, 932e939. https://doi.org/ 10.1161/01.CIR.0000155620.10387.43. Ren, D., Li, M., Duan, C., Rui, L., 2005. Identification of SH2-B as a key regulator of leptin sensitivity, energy balance, and body weight in mice. Cell Metabol. 2, 95e104. https://doi.org/10.1016/ J.CMET.2005.07.004.

484

20. ADIPOCYTE HORMONES

Rock, F., Altmann, S., van Heek, M., Kastelein, R., Bazan, J., 1996. The leptin haemopoietic Cytokine fold is stabilized by an intrachain disulfide bond. Horm. Metab. Res. 28, 649e652. https://doi.org/ 10.1055/s-2007-979871. Ruan, H., Dong, L.Q., 2016. Adiponectin signaling and function in insulin target tissues. J. Mol. Cell Biol. 8, 101e109. https://doi.org/ 10.1093/jmcb/mjw014. Ruscica, M., Steffani, L., Magni, P., 2012. Adiponectin interactions in bone and cartilage biology and disease. Vitam. Horm. 321e339. https://doi.org/10.1016/B978-0-12-398313-8.00012-9. Saetang, J., Sangkhathat, S., 2018. Role of innate lymphoid cells in obesity and metabolic disease (review). Mol. Med. Rep. 17, 1403e1412. Sa´inz, N., Barrenetxe, J., Moreno-Aliaga, M.J., Martı´nez, J.A., 2015. Leptin resistance and diet-induced obesity: central and peripheral actions of leptin. Metabolism 64, 35e46. https://doi.org/10.1016/ J.METABOL.2014.10.015. Saleh, J., Al-Wardy, N., Farhan, H., Al-Khanbashi, M., Cianflone, K., 2011. Acylation stimulating protein: a female lipogenic factor? Obes. Rev. 12, 440e448. https://doi.org/10.1111/j.1467789X.2010.00832.x. Sandowski, Y., Raver, N., Gussakovsky, E.E., Shochat, S., Dym, O., Livnah, O., Rubinstein, M., Krishna, R., Gertler, A., 2002. Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain. J. Biol. Chem. 277, 46304e46309. https://doi.org/10.1074/jbc.M207556200. Satoh, H., Nguyen, M.T.A., Miles, P.D.G., Imamura, T., Usui, I., Olefsky, J.M., 2004. Adenovirus-mediated chronic “hyperresistinemia” leads to in vivo insulin resistance in normal rats. J. Clin. Investig. 114, 224e231. https://doi.org/10.1172/JCI20785. Savage, D.B., Sewter, C.P., Klenk, E.S., Segal, D.G., Vidal-Puig, A., V Considine, R., O’Rahilly, S., 2001. Resistin/Fizz3 expression in relation to obesity and peroxisome proliferator-activated receptorgamma action in humans. Diabetes 50, 2199e2202. https:// doi.org/10.2337/DIABETES.50.10.2199. Scherer, P.E., Williams, S., Fogliano, M., Baldini, G., Lodish, H.F., 1995. A novel serum protein similar to C1q, produced exclusively in adipocytes. J. Biol. Chem. 270, 26746e26749. https://doi.org/ 10.1074/JBC.270.45.26746. Schraw, T., Wang, Z.V., Halberg, N., Hawkins, M., Scherer, P.E., 2008. Plasma adiponectin complexes have distinct biochemical characteristics. Endocrinology 149, 2270e2282. https://doi.org/ 10.1210/en.2007-1561. Schwartz, D.R., Lazar, M.A., 2011. Human resistin: found in translation from mouse to man. Trends Endocrinol. Metab. 22, 259e265. https://doi.org/10.1016/j.tem.2011.03.005. Shapiro, L., Scherer, P.E., 1998. The crystal structure of a complement1q family protein suggests an evolutionary link to tumor necrosis factor. Curr. Biol. 8, 335e338. https://doi.org/10.1016/S09609822(98)70133-2. Shimabukuro, M., Higa, N., Asahi, T., Oshiro, Y., Takasu, N., Tagawa, T., Ueda, S., Shimomura, I., Funahashi, T., Matsuzawa, Y., 2003. Hypoadiponectinemia is closely linked to endothelial dysfunction in man. J. Clin. Endocrinol. Metab. 88, 3236e3240. https://doi.org/10.1210/jc.2002-021883. Silha, J.V., Murphy, L.J., 2004. Serum resistin (FIZZ3) protein is increased in obese humans. J. Clin. Endocrinol. Metab. 89, 1977. https://doi.org/10.1210/jc.2003-031988. Singhal, N.S., Lazar, M.A., Ahima, R.S., 2007. Central resistin induces hepatic insulin resistance via neuropeptide Y. J. Neurosci. 27, 12924e12932. https://doi.org/10.1523/JNEUROSCI.2443-07.2007. Steppan, C.M., Bailey, S.T., Bhat, S., Brown, E.J., Banerjee, R.R., Wright, C.M., Patel, H.R., Ahima, R.S., Lazar, M.A., 2001. The hormone resistin links obesity to diabetes. Nature 409, 307e312. https://doi.org/10.1038/35053000. Steppan, C.M., Brown, E.J., Wright, C.M., Bhat, S., Banerjee, R.R., Dai, C.Y., Enders, G.H., Silberg, D.G., Wen, X., Wu, G.D.,

Lazar, M.A., 2001. A family of tissue-specific resistin-like molecules. Proc. Natl. Acad. Sci. U.S.A. 98, 502e506. https:// doi.org/10.1073/pnas.98.2.502. Steppan, C.M., Wang, J., Whiteman, E.L., Birnbaum, M.J., Lazar, M.A., 2005. Activation of SOCS-3 by resistin. Mol. Cell. Biol. 25, 1569e1575. https://doi.org/10.1128/MCB.25.4.1569-1575.2005. Tanabe, H., Fujii, Y., Okada-Iwabu, M., Iwabu, M., Nakamura, Y., Hosaka, T., Motoyama, K., Ikeda, M., Wakiyama, M., Terada, T., Ohsawa, N., Hato, M., Ogasawara, S., Hino, T., Murata, T., Iwata, S., Hirata, K., Kawano, Y., Yamamoto, M., KimuraSomeya, T., Shirouzu, M., Yamauchi, T., Kadowaki, T., Yokoyama, S., 2015. Crystal structures of the human adiponectin receptors. Nature 520, 312e316. https://doi.org/10.1038/ nature14301. Tarkowski, A., Bjersing, J., Shestakov, A., Bokarewa, M.I., 2010. Resistin competes with lipopolysaccharide for binding to toll-like receptor 4. J. Cell Mol. Med. 14, 1419e1431. https://doi.org/10.1111/ j.1582-4934.2009.00899.x. Tartaglia, L.A., Dembski, M., Weng, X., Deng, N., Culpepper, J., Devos, R., Richards, G.J., Campfield, L.A., Clark, F.T., Deeds, J., Muir, C., Sanker, S., Moriarty, A., Moore, K.J., Smutko, J.S., Mays, G.G., Wool, E.A., Monroe, C.A., Tepper, R.I., 1995. Identification and expression cloning of a leptin receptor, OB-R. Cell 83, 1263e1271. https://doi.org/10.1016/0092-8674(95)90151-5. Thiebaud, D., Jacot, E., DeFronzo, R.A., Maeder, E., Jequier, E., Felber, J.P., 1982. The effect of graded doses of insulin on total glucose uptake, glucose oxidation, and glucose storage in man. Diabetes 31, 957e963. https://doi.org/10.2337/DIACARE.31.11.957. Thomou, T., Mori, M.A., Dreyfuss, J.M., Konishi, M., Sakaguchi, M., Wolfrum, C., Rao, T.N., Winnay, J.N., Garcia-Martin, R., Grinspoon, S.K., Gorden, P., Kahn, C.R., 2017. Adipose-derived circulating miRNAs regulate gene expression in other tissues. Nature 542, 450e455. https://doi.org/10.1038/nature21365. Tomaru, T., Steger, D.J., Lefterova, M.I., Schupp, M., Lazar, M.A., 2009. Adipocyte-specific expression of murine resistin is mediated by synergism between peroxisome proliferator-activated receptor g and CCAAT/enhancer-binding proteins. J. Biol. Chem. 284, 6116e6125. https://doi.org/10.1074/jbc.M808407200. Towler, M.C., Hardie, D.G., 2007. AMP-activated protein kinase in metabolic control and insulin signaling. Circ. Res. 100, 328e341. https://doi.org/10.1161/01.RES.0000256090.42690.05. Turer, A.T., Scherer, P.E., 2012. Adiponectin: mechanistic insights and clinical implications. Diabetologia 55, 2319e2326. https:// doi.org/10.1007/s00125-012-2598-x. Uddin, S., Bu, R., Ahmed, M., Hussain, A.R., Ajarim, D., AL-Dayel, F., Bavi, P., Al-kuraya, K.S., 2010. Leptin receptor expression and its association with PI3K/AKT signaling pathway in diffuse large B-cell lymphoma. Leuk. Lymphoma 51, 1305e1314. https://doi.org/ 10.3109/10428191003802365. Vasiliauskaite´-Brooks, I., Sounier, R., Rochaix, P., Bellot, G., Fortier, M., Hoh, F., De Colibus, L., Bechara, C., Saied, E.M., Arenz, C., Leyrat, C., Granier, S., 2017. Structural insights into adiponectin receptors suggest ceramidase activity. Nature 544, 120e123. https:// doi.org/10.1038/nature21714. Vestal, D.J., Ranscht, B., 1992. Glycosyl phosphatidylinositoleanchored T-cadherin mediates calcium-dependent, homophilic cell adhesion. J. Cell Biol. 119, 451e461. http://www.ncbi.nlm.nih.gov/pubmed/ 1400585. Villa, N.Y., Kupchak, B.R., Garitaonandia, I., Smith, J.L., Alonso, E., Alford, C., Cowart, L.A., Hannun, Y.A., Lyons, T.J., 2009. Sphingolipids function as downstream effectors of a fungal PAQR. Mol. Pharmacol. 75, 866e875. https://doi.org/10.1124/mol.108.049809. Villanueva, E.C., Mu¨nzberg, H., Cota, D., Leshan, R.L., Kopp, K., Ishida-Takahashi, R., Jones, J.C., Fingar, D.C., Seeley, R.J., Myers, M.G., 2009. Complex regulation of mammalian target of rapamycin complex 1 in the basomedial hypothalamus by leptin

REFERENCES

and nutritional status. Endocrinology 150, 4541e4551. https:// doi.org/10.1210/en.2009-0642. Villarroya, F., Cereijo, R., Villarroya, J., Giralt, M., 2017. Brown adipose tissue as a secretory organ. Nat. Rev. Endocrinol. 13, 26e35. https://doi.org/10.1038/nrendo.2016.136. Waki, H., Yamauchi, T., Kamon, J., Ito, Y., Uchida, S., Kita, S., Hara, K., Hada, Y., Vasseur, F., Froguel, P., Kimura, S., Nagai, R., Kadowaki, T., 2003. Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin. J. Biol. Chem. 278, 40352e40363. https://doi.org/10.1074/jbc.M300365200. Waki, H., Yamauchi, T., Kamon, J., Kita, S., Ito, Y., Hada, Y., Uchida, S., Tsuchida, A., Takekawa, S., Kadowaki, T., 2005. Generation of globular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1. Endocrinology 146, 790e796. https:// doi.org/10.1210/en.2004-1096. Wang, Z.V., Scherer, P.E., 2016. Adiponectin, the past two decades. J. Mol. Cell Biol. 8, 93e100. https://doi.org/10.1093/jmcb/mjw011. Wang, Y., Xu, A., Knight, C., Xu, L.Y., Cooper, G.J.S., 2002. Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin. Potential role in the modulation of its insulin-sensitizing activity. J. Biol. Chem. 277, 19521e19529. https://doi.org/10.1074/jbc.M200601200. Wang, Y., Lam, K.S.L., Chan, L., Chan, K.W., Lam, J.B.B., Lam, M.C., Hoo, R.C.L., Mak, W.W.N., Cooper, G.J.S., Xu, A., 2006. Posttranslational modifications of the four conserved lysine residues within the collagenous domain of adiponectin are required for the formation of its high molecular weight oligomeric complex. J. Biol. Chem. 281, 16391e16400. https://doi.org/10.1074/ jbc.M513907200. Wang, C., Mao, X., Wang, L., Liu, M., Wetzel, M.D., Guan, K.-L., Dong, L.Q., Liu, F., 2007a. Adiponectin sensitizes insulin signaling by reducing p70 S6 kinase-mediated serine phosphorylation of IRS1. J. Biol. Chem. 282, 7991e7996. https://doi.org/10.1074/ jbc.M700098200. Wang, Z.V., Schraw, T.D., Kim, J.-Y., Khan, T., Rajala, M.W., Follenzi, A., Scherer, P.E., 2007b. Secretion of the adipocyte-specific secretory protein adiponectin critically depends on thiol-mediated protein retention. Mol. Cell. Biol. 27, 3716e3731. https://doi.org/ 10.1128/MCB.00931-06. Wang, C., Xin, X., Xiang, R., Ramos, F.J., Liu, M., Lee, H.J., Chen, H., Mao, X., Kikani, C.K., Liu, F., Dong, L.Q., 2009. Yin-Yang regulation of adiponectin signaling by APPL isoforms in muscle cells. J. Biol. Chem. 284, 31608e31615. https://doi.org/10.1074/jbc.M109.010355. Wauman, J., Tavernier, J., 2011. Leptin receptor signaling: pathways to leptin resistance. Front. Biosci 16, 2771e2793. http://www.ncbi. nlm.nih.gov/pubmed/21622208. Weisberg, S.P., McCann, D., Desai, M., Rosenbaum, M., Leibel, R.L., Ferrante, A.W., 2003. Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Investig. 112, 1796e1808. https://doi.org/10.1172/JCI200319246. Yamauchi, T., Kamon, J., Waki, H., Terauchi, Y., Kubota, N., Hara, K., Mori, Y., Ide, T., Murakami, K., Tsuboyama-Kasaoka, N., Ezaki, O., Akanuma, Y., Gavrilova, O., Vinson, C., Reitman, M.L., Kagechika, H., Shudo, K., Yoda, M., Nakano, Y., Tobe, K., Nagai, R., Kimura, S., Tomita, M., Froguel, P., Kadowaki, T., 2001. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat. Med. 7, 941e946. https://doi.org/10.1038/90984. Yamauchi, T., Kamon, J., Minokoshi, Y., Ito, Y., Waki, H., Uchida, S., Yamashita, S., Noda, M., Kita, S., Ueki, K., Eto, K., Akanuma, Y., Froguel, P., Foufelle, F., Ferre, P., Carling, D., Kimura, S., Nagai, R., Kahn, B.B., Kadowaki, T., 2002. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMPactivated protein kinase. Nat. Med. 8, 1288e1295. https:// doi.org/10.1038/nm788.

485

Yamauchi, T., Kamon, J., Ito, Y., Tsuchida, A., Yokomizo, T., Kita, S., Sugiyama, T., Miyagishi, M., Hara, K., Tsunoda, M., Murakami, K., Ohteki, T., Uchida, S., Takekawa, S., Waki, H., Tsuno, N.H., Shibata, Y., Terauchi, Y., Froguel, P., Tobe, K., Koyasu, S., Taira, K., Kitamura, T., Shimizu, T., Nagai, R., Kadowaki, T., 2003. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423, 762e769. https:// doi.org/10.1038/nature01705. Yamauchi, T., Nio, Y., Maki, T., Kobayashi, M., Takazawa, T., Iwabu, M., Okada-Iwabu, M., Kawamoto, S., Kubota, N., Kubota, T., Ito, Y., Kamon, J., Tsuchida, A., Kumagai, K., Kozono, H., Hada, Y., Ogata, H., Tokuyama, K., Tsunoda, M., Ide, T., Murakami, K., Awazawa, M., Takamoto, I., Froguel, P., Hara, K., Tobe, K., Nagai, R., Ueki, K., Kadowaki, T., 2007. Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions. Nat. Med. 13, 332e339. https://doi.org/ 10.1038/nm1557. Yamauchi, T., Iwabu, M., Okada-Iwabu, M., Kadowaki, T., 2014. Adiponectin receptors: a review of their structure, function and how they work. Best Pract. Res. Clin. Endocrinol. Metabol. 28, 15e23. https://doi.org/10.1016/j.beem.2013.09.003. Yan, W., Zhang, H., Liu, P., Wang, H., Liu, J., Gao, C., Liu, Y., Lian, K., Yang, L., Sun, L., Guo, Y., Zhang, L., Dong, L., Lau, W.B., Gao, E., Gao, F., Xiong, L., Wang, H., Qu, Y., Tao, L., 2013. Impaired mitochondrial biogenesis due to dysfunctional adiponectinAMPK-PGC-1a signaling contributing to increased vulnerability in diabetic heart. Basic Res. Cardiol. 108, 329. https://doi.org/ 10.1007/s00395-013-0329-1. Yang, R.-Z., Huang, Q., Xu, A., McLenithan, J.C., Eisen, J.A., Shuldiner, A.R., Alkan, S., Gong, D.-W., Eison, J.A., 2003. Comparative studies of resistin expression and phylogenomics in human and mouse. Biochem. Biophys. Res. Commun. 310, 927e935. http://www.ncbi.nlm.nih.gov/pubmed/14550293. Yang, C.-C., Chang, S.-F., Chao, J.-K., Lai, Y.-L., Chang, W.-E., Hsu, W.H., Kuo, W.-H., 2014. Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin. BMC Cancer 14, 112. https://doi.org/10.1186/ 1471-2407-14-112. Yano, W., Kubota, N., Itoh, S., Kubota, T., Awazawa, M., Moroi, M., Sugi, K., Takamoto, I., Ogata, H., Tokuyama, K., Noda, T., Terauchi, Y., Ueki, K., Kadowaki, T., 2008. Molecular mechanism of moderate insulin resistance in adiponectin-knockout mice. Endocr. J. 55, 515e522. http://www.ncbi.nlm.nih.gov/pubmed/ 18446001. Yoon, M.J., Lee, G.Y., Chung, J.-J., Ahn, Y.H., Hong, S.H., Kim, J.B., 2006. Adiponectin increases fatty acid oxidation in skeletal muscle cells by sequential activation of AMP-activated protein kinase, p38 mitogen-activated protein kinase, and peroxisome proliferatoractivated receptor alpha. Diabetes 55, 2562e2570. https:// doi.org/10.2337/db05-1322. Yuan, S.-S.F., Tsai, K.-B., Chung, Y.-F., Chan, T.-F., Yeh, Y.-T., Tsai, L.-Y., Su, J.-H., 2004. Aberrant expression and possible involvement of the leptin receptor in endometrial cancer. Gynecol. Oncol. 92, 769e775. https://doi.org/10.1016/J.YGYNO.2003.11.043. Zabeau, L., Peelman, F., Tavernier, J., 2015. Leptin: from structural insights to the design of antagonists. Life Sci. 140, 49e56. https:// doi.org/10.1016/J.LFS.2015.04.015. Zabolotny, J.M., Bence-Hanulec, K.K., Stricker-Krongrad, A., Haj, F., Wang, Y., Minokoshi, Y., Kim, Y.-B., Elmquist, J.K., Tartaglia, L.A., Kahn, B.B., Neel, B.G., 2002. PTP1B regulates leptin signal transduction in vivo. Dev. Cell 2, 489e495. https://doi.org/10.1016/S15345807(02)00148-X. Zabolotny, J.M., Kim, Y.-B., Welsh, L.A., Kershaw, E.E., Neel, B.G., Kahn, B.B., 2008. Protein-tyrosine phosphatase 1B expression is induced by inflammation in vivo. J. Biol. Chem. 283, 14230e14241. https://doi.org/10.1074/jbc.M800061200.

486

20. ADIPOCYTE HORMONES

Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., Friedman, J.M., 1994. Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425e432. https:// doi.org/10.1038/372425a0. Zhang, F., Basinski, M.B., Beals, J.M., Briggs, S.L., Churgay, L.M., Clawson, D.K., DiMarchi, R.D., Furman, T.C., Hale, J.E., Hsiung, H.M., Schoner, B.E., Smith, D.P., Zhang, X.Y., Wery, J.-P., Schevitz, R.W., 1997. Crystal structure of the obese protein leptinE100. Nature 387, 206e209. https://doi.org/10.1038/387206a0. Zhang, E.E., Chapeau, E., Hagihara, K., Feng, G.-S., 2004. Neuronal Shp2 tyrosine phosphatase controls energy balance and metabolism. Proc. Natl. Acad. Sci. U.S.A. 101, 16064e16069. https://doi.org/10.1073/pnas.0405041101. Zhang, Y., Yu, M., Tian, W., 2016. Physiological and pathological impact of exosomes of adipose tissue. Cell Prolif. 49, 3e13. https:// doi.org/10.1111/cpr.12233.

Zhao, A.Z., Huan, J.-N., Gupta, S., Pal, R., Sahu, A., 2002. A phosphatidylinositol 3-kinaseephosphodiesterase 3Becyclic AMP pathway in hypothalamic action of leptin on feeding. Nat. Neurosci. 5, 727e728. https://doi.org/10.1038/nn885. Zhou, L., Deepa, S.S., Etzler, J.C., Ryu, J., Mao, X., Fang, Q., Liu, D.D., Torres, J.M., Jia, W., Lechleiter, J.D., Liu, F., Dong, L.Q., 2009. Adiponectin activates AMP-activated protein kinase in muscle cells via APPL1/LKB1-dependent and phospholipase C/Ca2þ/Ca2þ/ Calmodulin-dependent protein kinase kinase-dependent pathways. J. Biol. Chem. 284, 22426e22435. https://doi.org/ 10.1074/jbc.M109.028357. Zu, L., Jiang, H., He, J., Xu, C., Pu, S., Liu, M., Xu, G., 2008. Salicylate blocks lipolytic actions of tumor necrosis factor-alpha in primary rat adipocytes. Mol. Pharmacol. 73, 215e223. https://doi.org/ 10.1124/mol.107.039479.

C H A P T E R

21 Thyroid Hormones Yan-Yun Liu, Anna Milanesi, Gregory A. Brent Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Physiology David Geffen School of Medicine at UCLA Department of Medicine, VA Greater Los Angeles Healthcare System, Los Angeles, California, US

1. INTRODUCTION Thyroid hormone (TH) is essential for normal brain development and for metabolic regulation in the adult (Brent, 2012). Phylogenetically, TH appears early, as a regulator of metabolism as well as being required for amphibian metamorphosis (Seebacher, 2018). TH is derived from the amino acid tyrosine, but acts predominantly through a nuclear receptor, in a fashion similar to steroid-derived hormones. The regulation of TH action occurs at multiple levels, including the nuclear receptor isoform, tissue uptake and activation of the prohormone, thyroxine (T4), to the active hormone triiodothyronine (T3), in responsive tissues. Clinical manifestations in conditions of TH excess and deficiency, as well as genetic defects of TH signaling pathways, have provided important information on specific elements of the TH response.

1.1 Structure and Synthesis of Thyroid Hormone T4 was first isolated by E.C. Kendall from porcine thyroids and was thought to be the only hormone produced by the thyroid gland (Kendall, 1915). Thyroid gland extracts, however, were noted to have greater thermogenic potency than accounted for by T4 content alone, and the much more physiologically active T3 was later identified (Gross and Pitt-Rivers, 1952).The thyroid gland produces two major forms of TH, T4 and T3 (Fig. 21.1) (Gereben et al., 2008). TH is derived from the amino acid tyrosine. The structure consists of a phenyl ring, connected by an ether linkage to a tyrosine. Iodine is added to positions on the phenyl ring, three iodines for T3 and four iodines for T4 (Gereben et al., 2008). The two phenyl rings are at an angle of around 120 degrees at the ether oxygen and

Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00021-3

have been shown to rotate. The addition of iodine atoms at the three and five position limits movement and results in the two rings taking a position perpendicular to each other (Jorgensen, 1964). The position and number of iodides is important in the interaction of TH with its nuclear receptor (THR), and the loss of the outer ring iodine makes T3 have a much higher affinity for THR than T4 (Cheng et al., 2010). T4 and T3 are hydrophobic and circulate bound to proteins. The serum proteins that bind TH, in order of the total fraction of TH bound, are thyroxine binding globulin, transthyretin, and albumin (Bartalena and Robbins, 1992). The vast majority of T4 and T3 in humans, greater than 99%, circulates bound to serum proteins, and only the free fraction is active. Thyroid uptake into some tissues requires specific TH membrane transporters (Bernal et al., 2015). TH synthesis requires iodine, and the only source is dietary. Dietary sources vary by the iodine content of food ingested and can be limited, especially in mountainous areas and areas of glacial activity. There is a highly specialized system to concentrate iodine in the thyroid, gut, and lactating breast through the sodium iodide symporter (NIS), a sodium-dependent transporter. The thyroid gland and lactating breast concentrate iodine 30e35-fold above the circulating levels. The iodine is then organified, incorporated into protein by the thyroid peroxidase enzyme (TPO). Thyroglobulin is contained within the thyroid follicle and is the substrate for TH synthesis. The human thyroid gland secretes an average of 100 mg T4 daily (Gereben et al., 2008). Daily T3 production is 30 mg, of which 20% is secreted by the thyroid gland and 80% is converted from T4 by deiodinases in peripheral tissues. There are two enzymes that convert T4 to the active form, T3, 50 deiodinase type 1 (Dio1) and 50 -deiodinase type 2 (Dio2). 5-deiodinase type 3 (Dio3) converts T4 to the

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21. THYROID HORMONES

FIGURE 21.1 Thyroid hormones and deiodinase. Pathways of thyroid metabolism shown with pathways for activation, 50 -deiodinase type 1 (Dio 1) and 50 -deiodinase type 2 (Dio 2), which converts thyroxine (T4) to triiodothyronine (T3) by removal of an outer ring iodine. The inactivation is by the 5-deiodinase type 3 (Dio 3) that inactivates T3 to reverse T3 (rT3) by removal of an inner ring iodine.

inactive 3,30 ,50 -L- triiodothyronine (reverse T3 or rT3) and converts T3 to 3,30 -T2 (Mondal et al., 2016). All three deiodinases (Dio1, 2, and 3) catalyze deiodination to produce less active TH metabolites (reverse T3 and T2) and regulate the availability of T4 and T3 (Fig. 21.1). The physiological role of the deiodinases is discussed in the Section 1.7.

1.2 Evolution of Thyroid Hormone TH production and signaling pathways were first found in vertebrates (Taylor and Heyland, 2017). Thyroid hormones, however, were found much earlier, in invertebrates and even some prokaryotes. The synthesis of TH from thyroglobulin (storage precursor found in thyroid follicles) is only seen in vertebrates; however, TH synthesis and nuclear TR are found in invertebrates. Some postulate that invertebrates contain iodinated tyrosine compounds that they receive from food sources they consume, such as sea plants. NIS also concentrates iodine in the gut and in fish and other vertebrates that make TH but do not have a thyroid gland. In these animals, the gut is the primary mechanism to retain dietary iodine. The signaling pathways that are activated by TH compounds in invertebrates are not well worked out, but they may involve both genomic and nongenomic actions. Some of the mediators of nongenomic action, such as specific integrin membrane receptors, are found in invertebrates (Taylor and Heyland, 2017). TH action, mediated by nuclear receptors, first plays a major role in energy metabolism in vertebrates (Seebacher, 2018). TH is required for amphibian metamorphosis, which has been a useful model of TH action in development. The metamorphosis process in Xenopus laevis, both tail resorption and subsequent limb development, is controlled by TH, acting through THR/TH-

mediated gene expression pathways (Fu et al., 2018). Xenopus expresses THRA and THRB, which are highly homologous to human THRA and THRB. Zebrafish is another biologic system in which TH and thyroid hormone receptor (zTHR) controls organ development and function, including development of the hypothalamic-pituitary-thyroid (HPT) axis. Dominant negative forms of zTHR influence tissue patterning, CNS development, and pituitary function (Marelli et al., 2016). In terrestrial animals, TH is essential for normal development, growth, and metabolism.

1.3 Thyroid Hormone Transport The TH transporters are part of the solute transport family, which are found across all eukaryotes (Bernal et al., 2015; Vancamp and Darras, 2018). Multiple organic anion transporting polypeptides (OATPs) can function as TH transporters (Table 21.1). The TH transporters, monocarboxylate transporter 8 (MCT8) and OATP1C (also known as OATP14), are widely expressed. MCT8 and OATP1C1 transport TH across the bloodebrain barrier to reach the brain. In the human brain, MCT8 is the primary T3 transporter, and OATP1C1 is expressed at a low level (Fu et al., 2013). Mutation of the MCT8 gene in humans is associated with profound developmental neurologic deficit, spasticity, and hypermetabolism, referred to as the AllanHerndon-Dudley syndrome (Groeneweg et al., 2017). MCT8 is required for secretion of T4 out of the thyroid gland. When the Mct8 gene is mutated, T4 is not transported out of the gland, the serum T4 is low, and there is a compensatory increase in T3 production from T4, leading to hypermetabolism. OATP1C1 is highly expressed in rodent brain, primarily localized at the basal-lateral surface of the bloodebrain barrier maintaining normal levels of T4, even with mutation of the

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TABLE 21.1

Thyroid Hormone (TH) Transporters.

TH Transporter in Tissues

Transporter Gene Symbol

Transporter Protein Symbol

Liver

Slc10a1

NTCP

Slco1a2

OATP1A2

Slco1b1

OATP1B1

Slco1b3

OATP1B3

Slc16a2

MCT8

Slco1b2

OATP1B2

Slc7A5

LAT1

Slc7A8

LAT2

Slco1a2

OATP1A2

Slco4c1

OATP4C1

Slc16a2

MCT8

Slc10a10

MCT10

Slc7A5

LAT1

Slc7A8

LAT2

Slco1a2

OATP1A2

Slco1c1

OATP1C1

Slco3a1

OATP3A1

Slco2b1

OATP2B1

Slc16a2

MCT8

Slc10a10

MCT10

Slc7A5

LAT1

Slc7A8

LAT2

Slco1c1

OATP1C1

Slc7A5

LAT1

Heart

Slc16a2

MCT8

Friesema et al. (2003)

Bone

Slc10a10

MCT10

Williams (2013)

Skeletal muscle

Slc16a2

MCT8

Slco1c1

OATP1C1

Slc10a10

MCT10

Mayerl et al. (2018), Mebis et al. (2009), and Di Cosmo et al. (2013)

Slc7A5

LAT1

Slc7A8

LAT2

Kidney

Brain

Cochlea

Mct8 gene (Mayerl et al., 2012). T4 crosses the bloode brain barrier and is taken up by astrocytes and tanocytes, where T4 is converted to T3 by Dio2 (Bernal et al., 2015). The locally converted T3 is then transported into neurons by MCT8. OATP1B1 and OATP1B3 are liver-specific transporters mediating TH cellular entry in human and rodents. Deletion of these transporters downregulates

References van der Deure et al. (2010)

Friesema et al. (2003) and Trajkovic-Arsic et al. (2010)

Bernal et al. (2015)

Sharlin et al. (2011)

expression of T3 target genes in liver (Visser, 2000; van der Deure et al., 2010). MCT10 functions as the major TH transporter in bone (Williams, 2013). L-type amino acid transporter LAT1 transports T4 and rT3 10-fold greater than transport of T3 (Krause and Hinz, 2017; Zevenbergen et al., 2015). Many transporters have been found capable of TH transport, although their functional role is not established (Table 21.1).

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1.4 Thyroid Hormone Receptors Thyroid hormone receptor (THR): There are two types of THR, alpha and beta (THRA and THRB), encoded by separate genes, and the proteins are highly conserved in vertebrates (Brent, 2012; Cheng et al., 2010). The human THRA gene has alternative splice products to yield four transcription variants (variant 1, 2, 3, and 4), coding for three proteins. THRA1 is coded by variant1, THRA2 by variant 2 and 4, and THRA3 by variant 3. THRB gene produces three transcripts using alternative transcription starting site, THRB 1, THRB 2, and THRB 3. THRs are composed of several domains with specific functions (Fig. 21.2). The A/B domains contain an autonomous activation function 1 (AF1) region, which has hormone-independent transactivation activity. Although the biologic function is not established, it is thought to maintain basal transcription expression. The hinge region contains a nuclear localization signal (NLS). THRB has one NLS, and THRA contains two NLSs, one in the A/B domain and another in the hinge domain (Mavinakere et al., 2012). The DNA binding domain (DBD) consisting of two zinc fingers with 96% homology of amino acid residues among ligand binding receptor isoforms (Fig. 21.3A). THR DBD recognizes a DNA motif in the regulatory region of genes, with specific sequence and configuration, referred to as a TH response element (TRE). Several consensus TREs are

identified with different configurations, the most well characterized being a direct repeat of the AGGTCA consensus hexamer with a four base pair gap (DR4TRE), a palindromic arrangement AGGTCA-TGACCT, or an inverted palindrome with a six base pair gap IP6 (Table 21.2). The crystal structure of THRB DBD predicts that THR prefers binding to DR4TRE as a THR retinoid X receptor (RXR) heterodimerizer, rather than a homodimer or monomer (Fig. 21.3B). THR homodimers bind efficiently to an inverted palindrome (IP6) configuration (Chen and Young, 2010). The ligand binding domains (LBDs) of THRA and THRB have up to 80% amino acid sequence homology. LBD, in addition to the ligand-binding function, includes an interface for RXR heterodimerization and coactivator 2 (NCoA2) interaction, which are essential for hormone-dependent transactivation. THRA and THRB have an identical interface for NCOA2, involving nine residues from three helixes (H3: TVK, H4: CIL, H11: PLE) (Fig. 21.4A an B). The heterodimer interface involves 10 residues in TRHB and TRHA1 (H7: V, H9: E, and H10: MTDMACAS) (Kojetin et al., 2015) (Fig. 21.4C). THRA1 and all three THRB isoforms possess the biologic function of ligand-induced transactivation. THRA 2 and 3 do not bind ligand and lack the regions to interface with NCOA2 or RXR, as well as having a truncated LBD. THRA2 and THRA3 may function as endogenous inhibitors of T3 action.

FIGURE 21.2 Thyroid hormone receptors domains. Domains (A/B domain, DBD, hinge, LBD) of thyroid hormone receptors are shown. See text for details. Functional domains drawn based on Homo sapiens THRA and THRB proteins. GeneBank IDs: THRB1 NP_000452.2, THRb2* NP_033406.1, THRB3 NP_001341644.1, THRA3 NP_001177847.1, THRA2 NP_001177848.1, and THRA1 NP_955366.1. DBD, DNA binding domain; LBD, ligand binding domain; NLS, nuclear localization signal. *, Human THRB2 record is removed from Genebank because of standard genome annotation processing. The figure shows mouse THRB2. Human THRb2 has 100% homology to mouse from DBD to AF2 domains (residues from 105 to 475) based on previous human THRb2 record (accession #:XP_005265477).

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1. INTRODUCTION

FIGURE 21.3 THR DBD structure and DNA binding. (A) Schematic drawing of THRB DBD structure. The residue numbering is shown as THR DBD from 106 to 192. (B) Crystal structure of THR-RXR DBD homodimer binding on DR4TRE (CAGGTCAtttcAGGTCAG). TABLE 21.2

Consensus Thyroid Hormone Response Elements (TREs).

Consensus TRE

DNA Sequence and Configuration

DR4 (direct repeat with four nucleotide spacing)

AGGTCA nnnn AGGTCA

IP6 (inverted palindrome with six nucleotide spacing)

TGACCT nnnnnn AGGTCA

Palindrome (with no spacing)

AGGTCATGACCT

THR isoform tissue-specific distribution: THRA and THRB have distinct spatial and temporal expression in development, as well as tissue-specific expression in the adult (Brent, 2012). THRA mRNA represents up to 80% of total THR mRNA in the developing rodent brain (Wallis et al., 2010). In the adult animal, THRA remains the predominant THR isoform, but some regions express THRB. In the cerebellum, THRA is highly expressed in the granular layer, and THRB is primarily in the Purkinje cell layer (Wallis et al., 2010). THRB1 is expressed in liver and kidney, THRB2 in the hypothalamus, pituitary, retina, and cochlea, and THRB3 in the kidney, liver, and lung (Zhang et al., 2018). The TR isoforms tissue-

specific actions have been explained by a different tissue-specific expression of TRs and/or by difference in TR isoforms specificity for TRE. The THR isoform specificity tissue distribution is observed in humans as well. THRA expression is the predominant isoform in most of tissues, but THRB predominates in liver (Fig. 21.5). Genome-wide association study, using neural cells, found that a significant number of T3 target genes display a preference for either THRA or THRB, after T3 treatment (Chatonnet et al., 2013). Additionally, the chromatin occupancy for receptors varies by THR isoform.

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21. THYROID HORMONES

FIGURE 21.4 3D structure of thyroid hormone receptor (THR) ligand binding domain (LBD). (A) THRB and RXR heterodimer. (B) A closer view shows the interaction between nuclear receptor coactivator (NCOA 2, also called TIF 2) and THRB. THR interaction with NCOA2 involves nine residues (281T, 284V, 288K, 298C, 302I, 305L, 450P, 454L, 457E). (C) THReRXR interaction and residues of THR involved in the interaction. The structure source NCBI 4Zo1 (Kojetin et al., 2015).

THR isoforms selective agonists: TH agonists have been designed to selectively stimulate a THR isoform, especially THRB, to enhance desired cardiac and metabolic effects and minimize negative actions (Scanlan, 2008). Reduced activation of THRA minimizes side effects, especially in the heart and bone. Sobetirome (also known as GC-1) is a T3 analog with a high affinity for THRB (Kd ¼ 76  4 p.m.) and reduced affinity for THRA (Kd ¼ 440  120 p.m.) (Scanlan, 2010). The high selectivity for THRB is based on interaction with a single residue in the ligand binding cavity. The crystal structure of the THRB LBD identifies 20 amino acid residues surrounding the ligand binding cavity. In the ligand binding pocket, the residue serine (Ser) at 277 in THRA is equivalent to asparagine (Asn) at 331 in THRB (Fig. 21.6). Asn is more hydrophilic than Ser with a hydropathy index of 3.4 Asn compared to 0.8 for Ser. Asn provides hydrogen bonding and gives the flexibility of the polar region to interact with the carboxyl tail of sobetirome. The design principles of THRB-selective thyromimetics are based on the structure of ligand binding pocket and the TR isoform tissue distribution. Selective activation of THRB has also been associated with lipid lowering, improved cardiac function, and “browning” of white fat (Lin et al., 2015; Scanlan, 2010).

1.5 TH/THR Signaling Pathways and Mechanisms THR/TH-mediated signaling pathways: TH signaling is primarily by genomic action through nuclear THRmediated genes expression (Fig. 21.7), referred to as type 1 TH action (Flamant et al., 2017). TH production is regulated by the HPT axis. T4 provides negative feedback to the hypothalamus and pituitary. Local Dio2 converts T4 to T3, which then downregulates thyroid releasing hormone (TRH) in the hypothalamus and thyroid stimulating hormone (TSH) in the pituitary (Fig. 21.7). When T4 levels are low and feedback is reduced, TRH is increased in the hypothalamus and stimulates the pituitary to secrete TSH; TSH then stimulates the thyroid gland to concentrate iodide and synthesize TH. THR/T3 action (type 1 TH action): TH exerts its primary biologic and physiological functions through binding to its receptors. Crystal structures show that both T4 and T3 bind to THR (Fig. 21.6); however, T4 has 30-fold lower affinity for THR than T3. Additionally, the T4THR complex is less stable and dissociates quickly, in about 9 min, compared to T3, which does in 8.4 h; Similarly, T4-THRB complex dissociates in 3.6 min compared

1. INTRODUCTION

493

FIGURE 21.5 THRA1 and THRB1 mRNA expression level in human tissues by RNA-seq. The data was taken from GTEx portal (https://www. gtexportal.org) and reconstructed for 29 specific human tissues.

to T3, which is in 6.2 h (Sandler et al., 2004). T3-THR interactions correlate most strongly physiologically with TH signaling and function. T3/THR signaling via gene regulation: THR heterodimerizes with RXR on a TRE and recruits coactivators (e.g., SRC1, MED1, HAC, and CPB/P300) to remodel the chromatin (Brent, 2012; Cheng et al., 2010). Chromatin remodeling creates a platform for recruiting transcription machinery, including TATA-binding protein and its associated factors and RNA polymerase II to initiate transcription. In recent years, THR-mediated transcription and DNA binding have been studied using high-throughput sequencing. Genome-wide studies, using chromatin immunoprecipitation combined with next-generation

sequencing technology (ChIP-seq), provide a topography of THR DNA binding properties. TREs were additionally identified in the 50 -upstream enhancer region, and TR binding sites are scattered in nonpromoter regions such as 50 -URL, intron, and the downstream region of the gene (Ramadoss et al., 2014; Ayers et al., 2014; Chatonnet et al., 2013). It is not unusual to identify several THR binding sites (peaks of binding) on a single gene. THR binding is highly enriched in the promoter region (Ayers et al., 2014); however, THR binding to a TRE is not sufficient for transcription, which requires recruitment of coactivators and Pol II. ChIP-seq analysis of the THRB cistrome from the liver of hypothyroid and hyperthyroid mice show that THR binding sites are diverse, including binding to sites

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FIGURE 21.6 Crystal structure of THRA ligand binding domain (LBD) complexes. (A) The overall structure of THRA with two T3 molecules bound. The T3 bound to the second binding site is located in the upper region. (B) The overall structure of THRA with T3 and T4 molecules bound. The T4 bound to the second binding site is located between helix H9, H10, and H11. H12 is shown in blue in both structures. From: Souza, P.C., et al., 2014. Identification of a new hormone-binding site on the surface of thyroid hormone receptor. Mol. Endocrinol. 28 (4), 534e545. https://doi.org/10.1210/me. 2013-1359. Copyright © 2014 by The Endocrine Society.

beyond the consensus TREs (Ramadoss et al., 2014). The THR can bind to direct repeat hexamers with spacing from 0 to 10 bases. T3-mediated induction has a strong associated with THRB enriched on DR4-TRE. Whereas, T3-mediated repression is associated with THRB enrichment on DR0- and DR3-TRE.

THR/T3-mediated repression is complex, and the mechanism of genes negatively regulated by TH/THR has been difficult to establish (Brent, 2012; Cheng et al., 2010). In the absence of TH, THR stimulates gene transcription; in the presence of TH, THR changes conformation

FIGURE 21.7 Hypothalamic-pituitary-thyroid axis and thyroid hormone action. Thyroid releasing hormone (TRH) is secreted in the hypothalamus and stimulates thyroid stimulating hormone (TSH) secretion from the pituitary gland. TSH acts on thyroid follicular cells to stimulate thyroid hormone secretion. T4 and T3 subsequently inhibit secretion of TRH and TSH. Thyroid hormones enter target cells via specific membrane transporters, such as Mct9. The intracellular concentration of T3 is determined by the relative activities of the deiodinases, Dio 1 and Dio 2. T3 enters the nucleus and binds to nuclear thyroid hormone receptors (THRs) to regulate expression of T3 target genes. T3 and T4 have also a nongenomic action through TRs and other membrane receptors. CoA, coactivator; RXR, retinoic X receptor; TRE, thyroid response element; avb3, plasma membrane integrin receptor.

1. INTRODUCTION

and recruits corepressors (NCoRs) and histone deacetylase (HDAC), resulting in condensing chromatin and reducing transcription. However, there is evidence that both the NCoR interface and the coactivator interface (AF-2 domain) is required for repression by THRB. A mutation in THRB (E457A) at the AF-2 domain not only abolishes THRB interaction with SRC-1 but also significantly reduces T3-mediated repression of TSHbeta gene transcription (Ortiga-Carvalho et al., 2005). Mice with a double disruption (E457A  SRC1/) have further loss of THRB/T3-mediated repressive activity (Alonso et al., 2009). In some models of TH-mediated repression, direct DNA binding of THR is not required and indirect binding occurs, through other tethering factors, referred to as type 2 TH action. Genome-wide footprinting, using DNase-seq analysis of transcription factor recruitment to DNA and chromatin assembly, provides new insight into negative regulation by TH (Grontved et al., 2015). After TH stimulation, DNase I hypersensitive sites (DHS) are largely changed, indicating improved chromatin accessibility. De novo DHS after TH stimulation are mostly enriched in DR4 motifs, in agreement with ChIP-seq analysis that DR4-TRE is preferred THR binding sites. Although in vitro studies indicated the THR binds to DNA in the absence of T3, DNase-seq showed that THR is recruited to DNA after T3-induced chromatin remodeling (Grontved et al., 2015). Posttranslational modifications of THR: Posttranslational modification of THR plays an important role in TH/THR-mediated gene regulation. Phosphorylation of THRs by protein kinase A (PKA) inhibits THR monomer, but enhances THR/RXR heterodimer, DNA binding, and enhances TH/THR-mediated transcription (Liu and Brent, 2018). THRA acetylation at lysine (K) 128, 132, and 134 of the DBD increases receptor DNA binding. Mutation of these sites inactivate transcription (Sanchez-Pacheco et al., 2009). Acetylation of THR is associated with recruiting coactivator SRC-1 (Lin et al., 2005). THR have several sumoylation sites: THRA sites are at K283 and K389 and THRB at K50, K146, and K443. The sumoylation of THRB is ligand-dependent and that of THRB is ligandindependent (Liu et al., 2012) (Liu and Brent, 2018). Sumoylation of THR influences interactions with coactivator and corepressor and influences THR/TH- mediated transcription and repression (Liu et al., 2012). In human primary preadipocytes, mutation of sumoylation motifs of THRA and THRB impairs adipocyte proliferation and differentiation (Liu et al., 2015). Desumoylation of THR impairs THR interaction with transcription coregulators. TH/THR modulates transcription without direct DNA binding (type 2 TH action): THR can modulate gene expression without directly binding to DNA, referred

495

to as type 2 TH action. Tethered THR can either antagonize or facilitate the transcription factoremediated pathways. Examples are that THR interacts with AP-1, resulting in either positive or negative regulation, depending on the AP-1 site configuration (Lopez et al., 1993). Liganded THR tethers to CREB and inhibits CREB phosphorylation, which reduces CREB-mediated c-AMP production and its downstream signaling pathways (Mendez-Pertuz et al., 2003). THR can act indirectly to regulate cell proliferation and differentiation (Pascual and Aranda, 2013). TH inhibits TGF-beta signaling by reducing phosphorylation of SMADs and reduces fibrosis in the liver in an animal model (Alonso-Merino et al., 2016). Nongenomic action of TH mediated by THR (type 3 TH Action): Truncated and inactive forms of THRA, such as p30 THRA, have been identified in the cytoplasm and plasma membrane and regulate nongenomic effect of T3 in bone cells (Kalyanaraman et al., 2014). More recently, it has been demonstrated that a small subpopulation of THRB in the cytosol can interact with the p85 subunit of PI3K and activate the PI3K-Akt signaling (Cao et al., 2005). Nongenomic actions of TH without THR (type 4 TH action): A membrane-bound receptor that mediates nongenomic effects of TH, referred to as type 4 TH (Flamant et al., 2017), has been identified. TH binds to a plasma membrane receptor, integrin avb3, an essential regulator of cellecell and celleextracellular matrix (ECM) protein interactions. TH induces phosphorylation of specific proteins (PKC, MAPKs), resulting in activation of signal transduction (Davis et al., 2016). TH nongenomic action has been linked to important TH-regulated processes, including cellular respiration in mitochondria, angiogenesis, and cell proliferation (Davis et al., 2016). This plasma membrane receptor does not have structural similarity with the nuclear TRs, binds both T4 and T3, and leads to activation of phosphatidylinositol 3-OH kinase (PI3K) and ERK1/2 signaling pathways and mediates actions in tissues like heart, blood vessels, and brain (Bergh et al., 2005). Proliferation of human bone cells in vitro can be stimulated by T3 and T4 though the integrin aVb3 receptor (Scarlett et al., 2008). The integrin avb3-mediated effects have been also linked to hepatic fibrosis via nongenomic mechanism (Zvibel et al., 2010). This receptor can also regulate the intracellular trafficking of THRB, THRA, and MAPK. For this reason, integrin avb3 might be involved in the movement of TR isoforms to the various compartments within cells (Cao et al., 2009). THR/TH mediate gene transcription via crosstalk with nuclear receptors: Nuclear receptors, especially those that heterodimerize with RXR, are similar in structure (Liu and Brent, 2010). Genome-wide identification of nuclear receptor binding sites indicates similar consensus motifs for THR and liver X receptor (LXR), which binds to a

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DR4- LXRE (Boergesen et al., 2012), peroxisome proliferator-activated receptor (PPAR) binds to DR1PPRE (Nielsen et al., 2008), and farnesoid X receptor (FXR), a bile-acid activated transcription factor binds to DR1 motif (Thomas et al., 2010). The reported crosstalk is consistent with the role of these receptors in nutrient regulation. THRA (PV/þ) mutant competes with PPARg for binding to PPRE and inhibits PPARg-regulated gene expression in adipose tissue, resulting in impaired adipogenesis (Ying et al., 2007). Similarly, THRA P398H mutant interferes with PPARa-regulated genes controlling fatty acid oxidation, resulting in reduced fatty acid oxidation and increased fatty accumulation in mutant mice (Liu et al., 2007).

1.6 Physiological Actions Development and Tissue-Specific Expression of THR TH plays a key role in the regulation of development, growth, and metabolism. The action of TH in a given tissue depends on the intracellular availability of the active form of TH, T3, the activity of TH transporters in the cell membrane, the expression and distribution of the thyroid TR isoforms, activity of corepressors and coactivators, and the location the TR DNA sequence that binds TR, thyroid hormone response element (TRE) (Fig. 21.1) (Brent, 2012; Little, 2016). The HPT axis regulates TH production though a feedback loop (Mendoza and Hollenberg, 2017). TRH is produced by the hypothalamus and stimulates the anterior pituitary to secrete TSH, which stimulates the thyroid gland to synthesize and release TH. The human fetal thyroid gland develops and begins to produce TH around 12e14 weeks gestation. Prior to development of the thyroid gland, the developing fetus relies entirely on the maternal T4 supply that is transported across the placenta down the gradient from high on the maternal side to very low on the fetal side. Human and animal models of TR isoform specificity: Animal models and patients with THRA and THRB mutation-inducing TH resistance have demonstrated the different role of TR isoforms (Brent, 2012; Refetoff et al., 2014). Resistance to thyroid hormone (RTH) due to a mutation of the THRB gene is characterized by goiter, growth abnormalities, and elevated serum levels of T3 and T4 with inappropriately high level of TSH secondary to defective pituitary-hypothalamic feedback. These patients are euthyroid in organs with prevalent THRB expression, such as the liver, since the TH levels are elevated, and hyperthyroid in TRa-expressing tissue, such as heart, bone, and gastrointestinal tract. In contrast, patients with RTH due to mutation of THRA, RTH-alpha, have short stature, mild intellectual

disability, bradycardia, and constipation due to the prevalence of THRA expression in the bone, heart, gastrointestinal tract, and brain (Schoenmakers et al., 2013). These patients have mild changes in circulating thyroid hormones, consistent with the fact that TRb mediates the TH feedback at the HPT axis. Genome-wide studies on cell lines stably expressing THRA or THRB have shown TR isoform-specific signaling on some target genes and differential binding of the individual receptors (Lin et al., 2013; Chatonnet et al., 2013). THRB isoforms are crucial for the development of the retina photoreceptors and the inner ear (Ng et al., 2009), while THRA is important for oligodendrocyte differentiation in the cerebellum (Picou et al., 2012). Another site of specific action of TR isoforms during development is the bone. T3 acts though TRa in chondrocytes and osteoblasts to regulate ossification and control the rate of linear growth and bone maturation and mineralization (Duncan Bassett and Williams, 2018). Hypothyroidism in children leads to delayed skeletal development and growth and impairs bone mineralization, with skeletal dysplasia in severe cases (Huffmeier et al., 2007). Children with RTH-THRA have a similar bone phenotype, with variable features of skeletal dysplasia, from delayed bone age, delayed closure of the fontanelles, macrocephaly, flattened nasal bridge, and disproportionate short stature mainly affecting the lower limbs (Bochukova et al., 2012; Demir et al., 2016). Functional role of TR nuclear cofactors: T3 genomic action is also regulated by the influence of nuclear corepressors, NCoR and SMRT, and coactivators (Mendoza and Hollenberg, 2017). The importance of these factors has been demonstrated by several in vivo models. Mice with loss of both THRA and THRB have a milder hypothyroid phenotype, suggesting that the repressive action of the unliganded receptor is more significant than reduced ligand in the presence of receptor (Gothe et al., 1999). In the absence of ligand, TR interacts with corepressors and reduces the expression of positively regulated genes. Addition of ligand disrupts TR binding with NCoR and promotes binding of the coactivator. After introducing an NCoR with a mutation of the TR interacting factor in a mouse model of RTH secondary to THRB, the resistance phenotype significantly improves (Astapova et al., 2011), suggesting the essential function of the corepressor.

1.7 Regulation of Thyroid Hormone Metabolism TH metabolism involves processes for excretion and salvage of iodine by conjugation, as well as deiodination pathways that contribute to disposal, but also those

1. INTRODUCTION

involved as regulators that activate or inactivate TH. TH activation and inactivation by the deiodinase enzymes plays a major role in TH action. Thyroid Hormone disposal: TH is disposed by conjugation, which involves sulfation or glucuronidation of the phenolic hydroxyl group, increasing the water solubility and therefore facilitating urinary and biliary clearance (Docter et al., 1997). Iodothyronine glucuronides are excreted in the bile, and iodothyronine sulfates are further degraded by deiodination in the liver. Sulfation leads to the irreversible degradation of T4 and T3 by Dio1. After hydrolysis of the glucuronides in the intestines, part of the liberated iodothyronines are reabsorbed, resulting in an enterohepatic cycle of iodothyronines (Wu et al., 2005). Dio1 plays a scavenger role for iodine, deiodinating T4 and T3 in the bile and the urine (Schneider et al., 2006). Dio1 also plays an important role in the recovery of iodide from inactive compounds for the resynthesis of thyroidal hormone in the thyroid gland (Schneider et al., 2006). Deiodinase enzymes: Iodothyronine deiodinase enzymes are homodimeric thioredoxin foldecontaining selenoproteins (Gereben et al., 2008). The subcellular localization of deiodinases is a key factor in the regulation of TH signaling. Dio2 is anchored to the endoplasmic reticulum membrane, and the proximity to the nucleus influences nuclear concentration of T3. It exhibits low Km and Vmax value with a preference for T4 over rT3. In the other hand, Dio1 is located in the plasma membrane and has high Km and Vmax value and low affinity for T4, so Dio1-generated T3 rapidly diffuses and reaches the circulation. Dio3 is anchored to the plasma membrane, where it is internalized as part of early endosomes and can be recycled back to the plasma membrane. D3 has an intermediate Km and Vmax value, and the subcellular localization of D3 can change in response to hypoxia. The primary circulating T3 in the rodent is produced by Dio1 converting T4 in liver and kidney (Larsen and Zavacki, 2012). Dio2 is responsible for converting T4 to T3 in most of metabolic active tissues, such as skeleton muscle, heart, adipose tissues, and brain, and it produces the majority of T3 in humans. Developmental and tissue distribution of deiodinase enzymes: Deiodinase tissue-specific activity and distribution is important during development and postnatal life. Dio1 is primarily expressed in liver, kidney proximal tubular cells, and thyroid follicular cells, and it contributes to the circulating T3. Dio3 activity is high in the placenta as well as in the brain, gastrointestinal tract, skin, and liver, especially during embryonic development (Gereben et al., 2008). Dio2 and Dio3 activity is present in human fetal cerebral cortex, increasing from 11 to 25 weeks of gestation. Dio2 is important in the development of the inner ear, since Dio2KO mice are deaf. Dio3 is also expressed in the cochlea during development

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before Dio2 and Dio3KO mice develop auditory defects as well. However, Dio3KO mice have an accelerated cochlear differentiation because of the premature stimulation, while Dio2KO mice display a delay in cochlear development (Ng et al., 2009). Dio3 also has a crucial role in cerebellar development, and Dio3KO mice display abnormally accelerated cerebellar differentiation and locomotor behavioral defects, due to abnormal exposure to T3 during development (Peeters et al., 2013). Similarly, Dio3 protects cones to T3 exposure in the immature mouse retina, since D3KO mice have a loss of about 80% of cones (Ng et al., 2010). Dio3 plays also a critical role in testis development and protects pancreatic beta-cells from early T3 exposure during development (Medina et al., 2011). In the adult, Dio2 plays a key role in TH signaling involving energy homoeostasis and the function of CNS (Bianco and Kim, 2006). About 70% of the plasma T3 in humans is generated intracellularly by Dio2. Dio2 has a half-life of about 45 min. This short half-life is important to modulate and minimize the intracellular changes of T3 despite changes in systemic T4 and is due to a ubiquitination and deubiquitination process (Gereben et al., 2008). Dio2 activity is inversely related to serum T4 levels, so low serum T4 results in an increase in Dio2 activity. DIO2 ubiquitination is tissue specific. In the brain, DIO2 activity is sensitive to T4 in the hippocampus and cortex, but not in the hypothalamus (Werneck de Castro et al., 2015). Regulation of Dio2 expression is time- and tissuedependent and involves transcriptional, posttranscription, and posttranslational mechanisms. DIO2 is mostly active in pituitary, brain, brown adipose tissue (BAT), skeletal muscle, and human thyroid. Dio2 mRNA is also expressed in the human heart. Dio2 plays a crucial role in regulating the HPT, by transducing the changes of plasma T4 in intrapituitary T3 level appropriate to modulate TSH expression (Larsen, 1982). This role of DIO2 is supported by a mouse model with DIO2 knockdown (DIO2KO mouse) showing a central resistance for T4 (Schneider et al., 2001), and in humans by the observation of disruption of TSH mechanism in patients with a mutation of a protein important for the selenoprotein synthesis (SECIS-BP2). DIO2KO mice have increased TSH and T4 levels in agreement with the HPT feedback but normal T3 levels, suggesting not a significant role of Dio2 in maintaining serum T3. Dio2 is important for adaptive thermogenesis in response to cold exposure, inducing the production of local T3 in BAT. Dio2KO mice developed hypothermia after cold exposure due to impaired BAT thermogenesis. Dio2 expression in skeletal muscle has been shown to play a critical role in muscle phenotype and homeostasis (Dentice et al., 2013). Dio2 KO mice had impaired differentiation of muscle-derived stem cells to myotubes

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in vitro and defective in vivo muscle regeneration after injury (Dentice et al., 2010). Dio2 mRNA is expressed at a high level in the human heart, but not in the rodent heart (Dentice et al., 2003). Mice overexpressing Dio2 in the heart showed improved myocardial glucose metabolism and cardiac function after doxorubicin-induced cardiac damage (Hong et al., 2013). Dio2 KO mice also have decreased bone formation and increased mineralization because of reduced local T3 in osteoblasts (Ng et al., 2004). Several studies have shown reactivation of Dio3 expression in the early phase of tissue regeneration, indicating a possible action in adult stem cell differentiation. Deletion of Dio3 in muscle satellite cells leads to failure of skeletal muscle regeneration after injury (Dentice et al., 2014). D3 activities are also increased in a mouse model of myocardial infarction (Janssen et al., 2013). Moreover, several studies have indicated increased expression of D3 in disease, such as chronic inflammation, cancer, and heart failure. The overexpression of Dio3 in juvenile and adult hepatic hemangiomas leads to excessive degradation of circulating TH and systemic hypothyroidism, so-called “consumptive hypothyroidism” (Huang et al., 2000). Thyroid hormone status and thyroid hormone metabolism: Deiodinase activity is highly regulated by thyroid status through pre- and posttranslational mechanisms to maintain the local T3 content as normal as possible. When serum T4 is low, such as with iodine deficiency or primary hypothyroidism, Dio2 activity is upregulated, leading to an increase in T3 in Dio2-expressing tissues. These are the tissues most important for T3 action, such as brain, skeletal muscle, heart, and BAT. TSH is increased in primary hypothyroidism due to reduced T4 feedback and stimulates and increases in Dio1 activity in the thyroid gland, leading to increase T3 production and secretion. Interestingly, in Graves disease, Dio2 activity can increase by thyroid stimulating Ig, and this can contribute to increased T3 production and secretion (Toyoda et al., 1990). Nonthyroidal Illness: Severe illness and fasting are characterized by a reduction in plasma T3 level, and with more severe illness, a reduction in T4 level, as well as an increase in rT3 without a compensatory rise of TSH (Lee and Farwell, 2016). There is evidence of reduced hepatic Dio1 activity and induction of Dio3 activity in several tissues, especially liver and skeletal muscle, with increased conversion of T4 to rT3. In addition, fasting and illness in rodents have shown increased Dio2 activity in the hypothalamus with increased local T3 production leading to possible feedback suppression on TRH and TSH (Coppola et al., 2005). However, the role of the deiodinases in critical illness is still controversial, and some studies suggest an enhanced TH degradation more than decreased T4 conversion as the main role

in nonthyroidal illness (Debaveye et al., 2008). Moreover, D3KO mice, after induction of systemic illness, develop the same reduction in T4 and T3 level as the wild-type animal, calling into question the real role of the deiodinases (Boelen et al., 2009). Thyroid metabolism and drugs: A variety of drugs can also interfere with TH metabolism. Radiographic agents such as iopanoic acid and ipodate are competitive inhibitors of D1 and Dio2. High-dose beta blockers can reduce Dio1 activity. Propylthiouracil is an uncompetitive inhibitor of D1 and amiodarone inhibits Dio2 activity as well as tissue thyroid transport.

1.8 Functional Thyroid Diseases, Clinical Manifestations, and Pathophysiology Hypothyroidism: Hypothyroidism, reduced TH production, is the most common thyroid disorder. Hypothyroidism in most patients is the result of chronic autoimmune destruction, referred to as Hashimoto disease (Cooper and Biondi, 2012). The thyroid test pattern shows a reduced serum T4 concentration and elevated TSH, due to reduced feedback inhibition. Rarely, hypothyroidism is the result of reduced TSH production due to pituitary or hypothalamic disease, referred to as central hypothyroidism. In this condition, serum T4 is reduced, but serum TSH is not elevated. Hyperthyroidism: Hyperthyroidism, excess TH production, is most commonly due to autoantibodies that stimulate the TSH receptor, Graves disease, but can also be caused by autonomously functioning thyroid tissue (Brent, 2008). The pattern of thyroid studies is an elevated serum T4 and T3 and a suppressed serum TSH level. Rarely, hyperthyroidism is due to excess TSH secretion from a thyrotroph pituitary tumor, which produces a thyroid test pattern of elevated serum T4 and T3 but a nonsuppressed serum TSH (Cooper and Biondi, 2012). Overview: TH has actions on all tissues in the body (Table 21.3). The wide range of clinical manifestations with TH excess and deficiency provides the spectrum of TH action. Cardiovascular system: T3 has positive inotropic and chronotropic effects on the heart (Danzi and Klein, 2012). THRA is the major THR in the atria, and both THRA and THRB are expressed in the ventricle. Excess TH increases resting heart rate, blood volume, stroke volume, myocardial contractility, and ejection fraction. Heart failure may occur secondary to severe and chronic hyperthyroidism because of a decrease of myocardial contractile reserve secondary to tachycardia/arrhythmia and direct myocardial damage (Klein and Danzi, 2016). TH deficiency results in reduced heart rate and weakening of myocardial contraction and relaxation, with prolonged systolic and early diastolic intervals

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1. INTRODUCTION

TABLE 21.3

Thyroid Hormone Targets in Development and Regulatory Pathways.

Pathway

Action

References

CNS development and ffunction

• • • •

Bernal (2007), Ng et al. (2010, 2013), and Mariotti and Beck-Peccoz (2000)

Cardiovascular ffunction

• Cardiac chronotropy, atrial arrythmias with TH excess • Cardiac inotropy, strength of contraction, heart failure with TH deficiency

Biondi (2012b)

Metabolism

• Cholesterol: cholesterol synthesis and LDL clearance in liver • Lipid: fatty acid synthesis, fat mobilization and catecholamine-stimulated fatty acid metabolism in adipose tissues • Carbohydrate: reduced TH associated with reduced insulin secretion; increased gluconeogenesis and glycogenolysis; TH increases muscle glucose transport and reduces glycogen synthesis in the liver

Mullur et al. (2014), Lopez et al. (2013), and Sinha et al. (2018)

Growth

• Skeletal growth and maturation • Muscle contractile function • Muscle regeneration

Bassett and Williams (2016), Kim and Mohan (2013), Salvatore et al. (2014), and Leal et al. (2015)

Thermogenesis

• Adaptive: T3 regulates UCP- 1 and catecholamine sensitivity in brown adipose tissue • White adipose tissue browning • White adipose tissue lipolysis

Brent (2012) and Lopez et al. (2013)

Neural cell proliferation, differentiation, and migration Photoreceptor differentiation Cochlear nerve function Regulation of hormonal gene expression (TRH, TSH beta, GH)

(Biondi, 2012a). Overt hypothyroidism is associated with accelerated atherosclerosis and coronary artery disease, possibly secondary to the higher incidence of hypercholesterolemia and hypertension (Asvold et al., 2012). The beneficial therapeutic use of beta blockers in hyperthyroidism suggests a crosstalk of TH with adrenergic signaling in the cardiovascular system (Brent, 2012; Vargas- Uricoechea et al., 2014). At the molecular level, T3 upregulates expression of the sarcoplasmic reticulum calcium-activated ATPase (SERCA2) (Dillmann, 2010) and negatively regulates expression of phospholamban. This results in myocardial relaxation and increases the expression of the rapid contractile alpha myosin heavy chain protein (Dillmann, 2010) and contributes to increased myocardial contractility. Hyperthyroidism increases the expression of angiotensin II receptors in the myocardium. Several in vivo and in vitro studies suggest that thyroid hormones might modulate the renin-angiotensin system (RAS). Alterations of the RAS system as reflected in plasma and tissue levels may play an important role in TH-induced cardiac hypertrophy (Kumar et al., 2008). Metabolism: TH regulates metabolism in the adult through actions on the hypothalamus, skeletal muscle, fat, and pancreas. TH modulates body weight and energy expenditure (Fox et al., 2008; Iwen et al., 2013; Mullur et al., 2014). TH excess increases resting energy

expenditure, lipolysis, gluconeogenesis, and promotes weight loss (Brent, 2008). TH deficiency reduces resting energy expenditure, lipolysis, and gluconeogenesis, and it results in modest weight gain (Mullur et al., 2014). Hyperthyroidism enhances central sympathetic outflow and directly stimulates BAT, leading to increase thermogenesis and weight loss. T3 also induces white adipose tissue lipolysis though enhanced sensitivity to sympathetic nervous system signaling and local increase of norepinephrine. In skeletal muscle, T3 promotes a shift from slow type I fiber to faster type II fibers, by increasing myosin and SERCA expression, which leads to greater energy turnover and heat generation during physical activity (Simonides and van Hardeveld, 2008). In the liver, T3 plays an important role in lipid homeostasis. T3 promotes both lipolysis and lipogenesis, fatty acid b-oxidation, LDL receptor expression, cholesterol synthesis, and the reverse cholesterol transport pathway (Sinha et al., 2018). TR is also associated with crosstalk signaling with nutrient-activated nuclear receptors, such as LXR, PPAR alpha, and PPAR gamma (Mullur et al., 2014). Nervous system: Thyroid dysfunction is associated with a wide spectrum of neurological signs and symptoms. Both hyper- and hypothyroidism can lead to psychiatric symptoms including depression, anxiety, memory deficits, executive function deficits, and even psychosis. Common features of hypothyroidism are

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slowed mentation, poor concentration, decreased shortterm memory, and depression (Osterweil et al., 1992). Altered mental status and coma are features of myxedema coma, which is a rare and life-threatening manifestation of profound hypothyroidism (Beynon et al., 2008). Hyperthyroidism is associated with nervousness, irritability, depression, lack of concentration, and a decline in executive function (Yuan et al., 2014; Vogels et al., 2007). Most of these manifestations are reversed within several months of normalizing TH levels, but improvement in some of these manifestations can lag significantly from the time that circulating TH levels are normalized. T3 stimulates genes important for circadian rhythm, as well as a candidate gene in bipolar disorders (Dbp, D-site binding protein) (Niculescu et al., 2000). T3 controls the expression of genes with roles in terminal cell differentiation, regulating neural cell shape, and glia morphology and functions such as migration via complex mechanisms (Lima et al., 1998; Manzano et al., 2007). Therefore, TH dysfunction may impair glial as well as neuronal function, leading to neurocognitive deficits. Gastrointestinal system: TH excess increases gastrointestinal motility and leads to malabsorption, hyperdefecation, and rarely, steatorrhea (Ebert, 2010). In contrast, hypothyroidism reduces gut mobility, resulting in delayed gastric emptying (Gunsar et al., 2003), slowtransit constipation with colonic dilatation, and even ileus (Ramadoss et al., 2014). THRA is the primary TR receptor in the gastrointestinal tract, and patients with RTH due to a defect in the THRA gene have constipation typical of hypothyroidism (Moran and Chatterjee, 2015). THRA has been shown to directly control intestinal epithelial cell proliferation (Plateroti et al., 2006), and histological findings in hypothyroid patients show a decrease in colonic crypts, indicating mucosal atrophy. The liver is influenced by functional thyroid disease. Transaminases, alanine, and aspartate aminotransferases are elevated in up to 30% of hyperthyroid patients (Lin et al., 2017). Intrahepatic cholestasis has also been described (Kyriacou et al., 2015). The mechanism of liver injury is not well understood, but it is explained, in part, by hepatocyte apoptosis mediated by T3 excess through activation of the mitochondrial-dependent pathway (Upadhyay et al., 2004). Hypothyroidism causes mild alterations of liver function tests (Daher et al., 2009), reduced gallbladder motility, and bilirubin excretion, resulting in an increased risk of gallstones (Mansourian, 2013). T3 is important in several key steps in cholesterol and bile acids synthesis mostly thought THRB action, and hypothyroidism leads to hypercholesterolemia (Mullur et al., 2014). Skeletal muscle: Severe skeletal muscle damage, leading to rhabdomyolysis, has been described in both

hypo- and hyperthyroidism (Altay et al., 2005; Salehi et al., 2017; Lichtstein and Arteaga, 2006). The hypothyroid myopathy presents with myalgia, muscular pseudohypertrophy (myoedema), and exercise intolerance and weakness of proximal upper and/or lower extremities (Sindoni et al., 2016), and more that 50% of the hypothyroid patients had increased levels of creatine kinases. Hyperthyroidism is associated with proximal limb muscle weakness that improves after treatment and increased Ca2þ recycling (Brennan et al., 2006; Riis et al., 2005). THRA and THRB are both present in skeletal muscle with predominant expression of THRA, and intracellular T3 content is controlled by the expression of Dio2 (Dentice et al., 2010). T3 stimulates myogenin and MyoD, crucial for skeletal muscle differentiation (Muscat et al., 1994; Downes et al., 1993). A mouse model of RTH secondary to a THRA mutation showed impaired skeletal muscle regeneration after injury and sarcopenia with aging (Milanesi et al., 2016, 2017). T3 also controls the expression of myosin heavy chain (MHC) genes, the major components of the skeletal muscle myofibrils. Animal and human studies have shown that hypothyroidism is associated with reduction of fast twitch muscle fiber (type 2 MHC) and hypertrophy of the slow twitching fiber (type 1 MHC) with hypothyroidism (Norris and Panner, 1966; Sindoni et al., 2016), inducing a switching to a “slow” muscle phenotype. Bone: TH plays a crucial role in bone development and maintenance of adult bone. Both hypothyroidism and hyperthyroidism have been associated with an increased risk of fracture in large population studies (Vestergaard and Mosekilde, 2002; Vestergaard et al., 2005; Bauer et al., 2001). In hypothyroidism, there is an increased remodeling cycle, resulting in low bone turnover and gain in bone mass and mineralization. By contrast, bone resorption and formation are both accelerated in hyperthyroidism, leading to high bone turnover and osteoporosis (Eriksen et al., 1986). Even with a normal bone mineral density, large population studies have shown that hypothyroid individuals have a twoto threefold increased risk of fracture, 10 years after diagnosis (Vestergaard and Mosekilde, 2002; Vestergaard et al., 2005). T3 regulates osteoblast and osteoclast activities via complex pathways involving growth factors and cytokines. THRA expression is at least 10-fold greater than THRB in the bone (Bookout et al., 2006). Adult mice with THRA knockdown are euthyroid, but they have osteosclerosis and increased cortical and trabecular bone. In contrast, mice with THRB knockdown have elevated TH levels and are osteoporotic, indicating that THRA mediates the major action of T3 in adult bone (Bassett and Williams, 2009).

1. INTRODUCTION

Hemopoietic system: TH is important for human erythropoiesis. Hypothyroidism is associated with a normocytic anemia (Horton et al., 1976), and erythrocytosis is seen in hyperthyroid patients. A slightly reduced total leukocyte count, neutropenia, and thrombocytopenia have been reported in hypothyroidism (Lima et al., 2006). In hyperthyroidism, leukocytes count has been shown to be variable (from elevated to normal or slightly depressed) with only a relative decrease in the number of neutrophils and a relative increase in the number of eosinophils and mononuclear cells (Kawa et al., 2010). Mice lacking THRA show compromised fetal and adult erythropoiesis and defective spleen erythropoiesis (Kendrick et al., 2008). In patients with resistance to TH due to a THRA mutation, mild normochromic and normocytic anemia is uniformly found (van Gucht et al., 2017). Skin: TH regulates epidermal and dermal homeostasis. In hypothyroidism, the skin is coarse and dry due to eccrine gland secretion and tends to be pale because of increased dermal mucopolysaccharides and water content, especially in myxedema (Heymann, 1992). In addition, increased dermal carotene can give a prominent yellow color to palms, soles, and nasolabial folds. The hair is usually dry, brittle, and slow growing. Alopecia, partial or generalized, can be present and the loss of the lateral third of the eyebrow (madarosis) can be seen (Safer, 2012). In hyperthyroidism, the skin is warm because of increased blood flow, thin but not atrophic. The hair is often thin and soft, and nail changes can occur, including onycholysis (Plummer sign). Some of the dermal changes in hyperthyroid patients derive from autoimmunity rather than direct TH action (Safer, 2012). Dermatopathy in Graves disease has a complex pathogenesis that involves cellular, immunologic, molecular, and environmental factors. There is fibroblast proliferation and GAG production that result in expansion of the connective tissue and fluid retention (Bull et al., 1993). It is usually localized in the lower extremity, in the lateral aspect of pretibial area (Fatourechi, 2012), and it is mostly triggered by mechanical factors, such as position, slower lymphatic drainage, and possibly trauma.

1.9 Changes in the Thyroid and Thyroid Function with Age Aging is associated with changes in both thyroid structure and function (Faggiano et al., 2004). Autopsy studies of individuals over 50 years old demonstrate progressive thyroid atrophy and fibrosis, an increase in adipose tissue deposition, and an overall reduction in thyroid gland volume (Andersen-Ranberg et al., 1999; Gonczi et al., 1994). In addition to the aging-related physiological and morphologic changes of the thyroid

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gland, the prevalence of primary thyroid diseases increases, making it difficult to distinguish between physiological changes related to normal aging from those due to disease. Thyroid disorders, especially subclinical thyroid dysfunctions, are common in the elderly, and the prevalence of antithyroperoxidase and antithyroglobulin antibody positivity is increased with age, especially among women (Hollowell et al., 2002). Distinct from the increased incidence of disease in the elderly, there is evidence that during normal healthy aging, there is an increase in TSH levels. This has been observed in cross-sectional and longitudinal studies, even in the absence of thyroid disease or thyroid autoantibodies. With increasing age in the US population, the prevalence of serum TSH greater than 4.5 mIU/L, the upper limit of the population reference range, increases, and a serum TSH greater than 4.5 mIU/L is seen in 6% of the 70e79 year old group and 10% of the 80 year older group (Hollowell et al., 2002). This age shift in serum TSH has been reported in other US subpopulations of Caucasian, African-American, Hispanic-American, and Ashkenazi Jews (Surks and Boucai, 2010; Atzmon et al., 2009). Cross-sectional studies in Australia and Scotland have reported a similar age-related increase in serum TSH, without a significant decrease in serum T4 (Kahapola-Arachchige et al., 2012; Vadiveloo et al., 2013). Underlying factors that may contribute to the age-related increase in TSH include reduced TSH biologic activity with age due to altered TSH glycosylation or an age-related decrease in thyroid gland sensitivity to TSH (Vadiveloo et al., 2011, 2013).

1.10 Conclusions and Future Directions Acquired thyroid diseases and genetic disorders of TH uptake and signaling have contributed to understanding the various components involved in TH signaling. The relative importance, however, of the THR isoform, THR posttranslational modifications, ligand transport, and activation/deactivation by deiodinase enzymes, as well as THR cofactors, remains to be established. Negative regulation of genes by TH is incompletely characterized, but it is clearly distinct from the mechanisms of positive regulation. The broad availability of whole genome analysis as a clinical diagnostic tool in patients is certain to identify additional TH signaling defects that will provide further insights into the mechanisms of TH action. TH modulates the action of pathways important in lipid regulation and energy balance, including the adrenergic system, leptinmelanocortin, LXR, PPARa, and PPARg. Selective actions of TH in neural myelination, metabolic regulation, and cardiac function are important areas of development for new therapeutic targets.

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Acknowledgments This work was supported by grants from National Institutes of Health RO1 DK98576 (GB) and by Merit Reviews Award Numbers BX-001966 (GAB) and BX-003665 (AM) from the United States Department of Veterans Affairs Biomedical Laboratory R&D (BLRD) Service.

References Alonso, M., Goodwin, C., Liao, X., Ortiga-Carvalho, T., Machado, D.S., Wondisford, F.E., Refetoff, S., Weiss, R.E., 2009. In vivo interaction of steroid receptor coactivator (SRC)-1 and the activation function-2 domain of the thyroid hormone receptor (TR) beta in TRbeta E457A knock-in and SRC-1 knockout mice. Endocrinology 150, 3927e3934. Alonso-Merino, E., Martin Orozco, R., Ruiz-Llorente, L., MartinezIglesias, O.A., Velasco-Martin, J.P., Montero-Pedrazuela, A., FanjulRodriguez, L., Contreras-Jurado, C., Regadera, J., Aranda, A., 2016. Thyroid hormones inhibit TGF-beta signaling and attenuate fibrotic responses. Proc. Natl. Acad. Sci. U.S.A. 113, E3451eE3460. Altay, M., Duranay, M., Ceri, M., 2005. Rhabdomyolysis due to hypothyroidism. Nephrol. Dial. Transplant. 20, 847e848. Andersen-Ranberg, K., Jeune, B., Hoier-Madsen, M., Hegedus, L., 1999. Thyroid function, morphology and prevalence of thyroid disease in a population-based study of Danish centenarians. J. Am. Geriatr. Soc. 47, 1238e1243. Astapova, I., Vella, K.R., Ramadoss, P., Holtz, K.A., Rodwin, B.A., Liao, X.H., Weiss, R.E., Rosenberg, M.A., Rosenzweig, A., Hollenberg, A.N., 2011. The nuclear receptor corepressor (NCoR) controls thyroid hormone sensitivity and the set point of the hypothalamic-pituitary-thyroid axis. Mol. Endocrinol. 25, 212e224. Asvold, B.O., Bjoro, T., Platou, C., Vatten, L.J., 2012. Thyroid function and the risk of coronary heart disease: 12-year follow-up of the HUNT study in Norway. Clin. Endocrinol. 77, 911e917. Atzmon, G., Barzilai, N., Hollowell, J.G., Surks, M.I., Gabriely, I., 2009. Extreme longevity is associated with increased serum thyrotropin. J. Clin. Endocrinol. Metab. 94, 1251e1254. Ayers, S., Switnicki, M.P., Angajala, A., Lammel, J., Arumanayagam, A.S., Webb, P., 2014. Genome-wide binding patterns of thyroid hormone receptor beta. PLoS One 9, e81186. Bartalena, L., Robbins, J., 1992. Variations in thyroid hormone transport proteins and their clinical implications. Thyroid 2, 237e245. Bassett, J.H., Williams, G.R., 2009. The skeletal phenotypes of TRalpha and TRbeta mutant mice. J. Mol. Endocrinol. 42, 269e282. Bassett, J.H., Williams, G.R., 2016. Role of thyroid hormones in skeletal development and bone maintenance. Endocr. Rev. 37, 135e187. Bauer, D.C., Ettinger, B., Nevitt, M.C., Stone, K.L., Study of Osteoporotic Fractures Research Group, 2001. Risk for fracture in women with low serum levels of thyroid-stimulating hormone. Ann. Intern. Med. 134, 561e568. Bergh, J.J., Lin, H.Y., Lansing, L., Mohamed, S.N., Davis, F.B., Mousa, S., Davis, P.J., 2005. Integrin alphaVbeta3 contains a cell surface receptor site for thyroid hormone that is linked to activation of mitogen-activated protein kinase and induction of angiogenesis. Endocrinology 146, 2864e2871. Bernal, J., 2007. Thyroid hormone receptors in brain development and function. Nat. Clin. Pract. Endocrinol. Metabol. 3, 249e259. Bernal, J., Guadano-Ferraz, A., Morte, B., 2015. Thyroid hormone transportersefunctions and clinical implications. Nat. Rev. Endocrinol. 11, 406e417. Beynon, J., Akhtar, S., Kearney, T., 2008. Predictors of outcome in myxoedema coma. Crit. Care 12, 111. Bianco, A.C., Kim, B.W., 2006. Deiodinases: implications of the local control of thyroid hormone action. J. Clin. Investig. 116, 2571e2579.

Biondi, B., 2012a. Mechanisms in endocrinology: heart failure and thyroid dysfunction. Eur. J. Endocrinol. 167, 609e618. Biondi, B., 2012b. Natural history, diagnosis and management of subclinical thyroid dysfunction. Best Pract. Res. Clin. Endocrinol. Metabol. 26, 431e446. Bochukova, E., Schoenmakers, N., Agostini, M., Schoenmakers, E., Rajanayagam, O., Keogh, J.M., Henning, E., Reinemund, J., Gevers, E., Sarri, M., Downes, K., Offiah, A., Albanese, A., Halsall, D., Schwabe, J.W., Bain, M., Lindley, K., Muntoni, F., Vargha-Khadem, F., Dattani, M., Farooqi, I.S., Gurnell, M., Chatterjee, K., 2012. A mutation in the thyroid hormone receptor alpha gene. N. Engl. J. Med. 366, 243e249. Boelen, A., Kwakkel, J., Wieland, C.W., ST Germain, D.L., Fliers, E., Hernandez, A., 2009. Impaired bacterial clearance in type 3 deiodinase-deficient mice infected with Streptococcus pneumoniae. Endocrinology 150, 1984e1990. Boergesen, M., Pedersen, T.A., Gross, B., Van Heeringen, S.J., Hagenbeek, D., Bindesboll, C., Caron, S., Lalloyer, F., Steffensen, K.R., Nebb, H.I., Gustafsson, J.A., Stunnenberg, H.G., Staels, B., Mandrup, S., 2012. Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor alpha in mouse liver reveals extensive sharing of binding sites. Mol. Cell. Biol. 32, 852e867. Bookout, A.L., Jeong, Y., Downes, M., Yu, R.T., Evans, R.M., Mangelsdorf, D.J., 2006. Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network. Cell 126, 789e799. Brennan, M.D., Powell, C., Kaufman, K.R., Sun, P.C., Bahn, R.S., Nair, K.S., 2006. The impact of overt and subclinical hyperthyroidism on skeletal muscle. Thyroid 16, 375e380. Brent, G.A., 2008. Clinical practice. Graves’ disease. N. Engl. J. Med. 358, 2594e2605. Brent, G.A., 2012. Mechanisms of thyroid hormone action. J. Clin. Investig. 122, 3035e3043. Bull, R.H., Coburn, P.R., Mortimer, P.S., 1993. Pretibial myxoedema: a manifestation of lymphoedema? Lancet 341, 403e404. Cao, H.J., Lin, H.Y., Luidens, M.K., Davis, F.B., Davis, P.J., 2009. Cytoplasm-to-nucleus shuttling of thyroid hormone receptorbeta1 (Trbeta1) is directed from a plasma membrane integrin receptor by thyroid hormone. Endocr. Res. 34, 31e42. Cao, X., Kambe, F., Moeller, L.C., Refetoff, S., Seo, H., 2005. Thyroid hormone induces rapid activation of Akt/protein kinase Bmammalian target of rapamycin-p70S6K cascade through phosphatidylinositol 3-kinase in human fibroblasts. Mol. Endocrinol. 19, 102e112. Chatonnet, F., Guyot, R., Benoit, G., Flamant, F., 2013. Genome-wide analysis of thyroid hormone, receptors shared and specific functions in neural cells. Proc. Natl. Acad. Sci. U.S.A. 110, E766eE775. Chen, Y., Young, M.A., 2010. Structure of a thyroid hormone receptor DNA-binding domain homodimer bound to an inverted palindrome DNA response element. Mol. Endocrinol. 24, 1650e1664. Cheng, S.Y., Leonard, J.L., Davis, P.J., 2010. Molecular aspects of thyroid hormone actions. Endocr. Rev. 31, 139e170. Cooper, D.S., Biondi, B., 2012. Subclinical thyroid disease. Lancet 379, 1142e1154. Coppola, A., Hughes, J., Esposito, E., Schiavo, L., Meli, R., Diano, S., 2005. Suppression of hypothalamic deiodinase type II activity blunts TRH mRNA decline during fasting. FEBS Lett. 579, 4654e4658. Daher, R., Yazbeck, T., Jaoude, J.B., Abboud, B., 2009. Consequences of dysthyroidism on the digestive tract and viscera. World J. Gastroenterol. 15, 2834e2838. Danzi, S., Klein, I., 2012. Thyroid hormone and the cardiovascular system. Med. Clin. N. Am. 96, 257e268.

REFERENCES

Davis, P.J., Goglia, F., Leonard, J.L., 2016. Nongenomic actions of thyroid hormone. Nat. Rev. Endocrinol. 12, 111e121. Debaveye, Y., Ellger, B., Mebis, L., Visser, T.J., Darras, V.M., van den Berghe, G., 2008. Effects of substitution and high-dose thyroid hormone therapy on deiodination, sulfoconjugation, and tissue thyroid hormone levels in prolonged critically ill rabbits. Endocrinology 149, 4218e4228. Demir, K., van Gucht, A.L., Buyukinan, M., Catli, G., Ayhan, Y., Bas, V.N., Dundar, B., Ozkan, B., Meima, M.E., Visser, W.E., Peeters, R.P., Visser, T.J., 2016. Diverse genotypes and phenotypes of three novel thyroid hormone receptor-alpha mutations. J. Clin. Endocrinol. Metab. 101, 2945e2954. Dentice, M., Ambrosio, R., Damiano, V., Sibilio, A., Luongo, C., Guardiola, O., Yennek, S., Zordan, P., Minchiotti, G., Colao, A., Marsili, A., Brunelli, S., Del Vecchio, L., Larsen, P.R., Tajbakhsh, S., Salvatore, D., 2014. Intracellular inactivation of thyroid hormone is a survival mechanism for muscle stem cell proliferation and lineage progression. Cell Metabol. 20, 1038e1048. Dentice, M., Marsili, A., Ambrosio, R., Guardiola, O., Sibilio, A., Paik, J.H., Minchiotti, G., Depinho, R.A., Fenzi, G., Larsen, P.R., Salvatore, D., 2010. The FoxO3/type 2 deiodinase pathway is required for normal mouse myogenesis and muscle regeneration. J. Clin. Investig. 120, 4021e4030. Dentice, M., Marsili, A., Zavacki, A., Larsen, P.R., Salvatore, D., 2013. The deiodinases and the control of intracellular thyroid hormone signaling during cellular differentiation. Biochim. Biophys. Acta 1830, 3937e3945. Dentice, M., Morisco, C., Vitale, M., Rossi, G., Fenzi, G., Salvatore, D., 2003. The different cardiac expression of the type 2 iodothyronine deiodinase gene between human and rat is related to the differential response of the Dio2 genes to Nkx-2.5 and GATA-4 transcription factors. Mol. Endocrinol. 17, 1508e1521. di Cosmo, C., Liao, X.H., Ye, H., Ferrara, A.M., Weiss, R.E., Refetoff, S., Dumitrescu, A.M., 2013. Mct8-deficient mice have increased energy expenditure and reduced fat mass that is abrogated by normalization of serum T3 levels. Endocrinology 154, 4885e4895. Dillmann, W., 2010. Cardiac hypertrophy and thyroid hormone signaling. Heart Fail. Rev. 15, 125e132. Docter, R., Friesema, E.C., van Stralen, P.G., Krenning, E.P., Everts, M.E., Visser, T.J., Hennemann, G., 1997. Expression of rat liver cell membrane transporters for thyroid hormone in Xenopus laevis oocytes. Endocrinology 138, 1841e1846. Downes, M., Griggs, R., Atkins, A., Olson, E.N., Muscat, G.E., 1993. Identification of a thyroid hormone response element in the mouse myogenin gene: characterization of the thyroid hormone and retinoid X receptor heterodimeric binding site. Cell Growth Differ. 4, 901e909. Duncan Bassett, J.H., Williams, G.R., 2018. Analysis of physiological responses to thyroid hormones and their receptors in bone. Methods Mol. Biol. 1801, 123e154. Ebert, E.C., 2010. The thyroid and the gut. J. Clin. Gastroenterol. 44, 402e406. Eriksen, E.F., Mosekilde, L., Melsen, F., 1986. Kinetics of trabecular bone resorption and formation in hypothyroidism: evidence for a positive balance per remodeling cycle. Bone 7, 101e108. Faggiano, A., Coulot, J., Bellon, N., Talbot, M., Caillou, B., Ricard, M., Bidart, J.M., Schlumberger, M., 2004. Age-dependent variation of follicular size and expression of iodine transporters in human thyroid tissue. J. Nucl. Med. 45, 232e237. Fatourechi, V., 2012. Thyroid dermopathy and acropachy. Best Pract. Res. Clin. Endocrinol. Metabol. 26, 553e565. Flamant, F., Cheng, S.Y., Hollenberg, A.N., Moeller, L.C., Samarut, J., Wondisford, F.E., Yen, P.M., Refetoff, S., 2017. Thyroid hormone signaling pathways: time for a more precise nomenclature. Endocrinology 158, 2052e2057.

503

Fox, C.S., Pencina, M.J., D’Agostino, R.B., Murabito, J.M., Seely, E.W., Pearce, E.N., Vasan, R.S., 2008. Relations of thyroid function to body weight: cross-sectional and longitudinal observations in a community-based sample. Arch. Intern. Med. 168, 587e592. Friesema, E.C., Ganguly, S., Abdalla, A., manning fox, J.E., Halestrap, A.P., Visser, T.J., 2003. Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. J. Biol. Chem. 278, 40128e40135. Fu, J., Refetoff, S., Dumitrescu, A.M., 2013. Inherited defects of thyroid hormone-cell-membrane transport: review of recent findings. Curr. Opin. Endocrinol. Diabetes Obes. 20, 434e440. Fu, L., Wen, L., Shi, Y.B., 2018. Role of thyroid hormone receptor in Amphibian development. Methods Mol. Biol. 1801, 247e263. Gereben, B., Zavacki, A.M., Ribich, S., Kim, B.W., Huang, S.A., Simonides, W.S., Zeold, A., Bianco, A.C., 2008. Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling. Endocr. Rev. 29, 898e938. Gonczi, J., Szabolcs, I., Kovacs, Z., Kakosy, T., Goth, M., Szilagyi, G., 1994. Ultrasonography of the thyroid gland in hospitalized, chronically ill geriatric patients: thyroid volume, its relationship to age and disease, and the prevalence of diffuse and nodular goiter. J. Clin. Ultrasound 22, 257e261. Gothe, S., Wang, Z., Ng, L., Kindblom, J.M., Barros, A.C., Ohlsson, C., Vennstrom, B., Forrest, D., 1999. Mice devoid of all known thyroid hormone receptors are viable but exhibit disorders of the pituitarythyroid axis, growth, and bone maturation. Genes Dev. 13, 1329e1341. Groeneweg, S., Visser, W.E., Visser, T.J., 2017. Disorder of thyroid hormone transport into the tissues. Best Pract. Res. Clin. Endocrinol. Metabol. 31, 241e253. Grontved, L., Waterfall, J.J., Kim, D.W., Baek, S., Sung, M.H., Zhao, L., Park, J.W., Nielsen, R., Walker, R.L., Zhu, Y.J., Meltzer, P.S., Hager, G.L., Cheng, S.Y., 2015. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling. Nat. Commun. 6, 7048. Gross, J., Pitt-Rivers, R., 1952. The identification of 3:5:30 -Ltriiodothyronine in human plasma. Lancet 1, 439e441. Gunsar, F., Yilmaz, S., Bor, S., Kumanlioglu, K., Cetinkalp, S., Kabalak, T., Ozutemiz, O.A., 2003. Effect of hypo- and hyperthyroidism on gastric myoelectrical activity. Dig. Dis. Sci. 48, 706e712. Heymann, W.R., 1992. Cutaneous manifestations of thyroid disease. J. Am. Acad. Dermatol. 26, 885e902. Hollowell, J.G., Staehling, N.W., Flanders, W.D., Hannon, W.H., Gunter, E.W., Spencer, C.A., Braverman, L.E., 2002. Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): national health and nutrition examination survey (NHANES III). J. Clin. Endocrinol. Metab. 87, 489e499. Hong, E.G., Kim, B.W., Jung, D.Y., Kim, J.H., Yu, T., Seixas da Silva, W., Friedline, R.H., Bianco, S.D., Seslar, S.P., Wakimoto, H., Berul, C.I., Russell, K.S., Lee, K.W., Larsen, P.R., Bianco, A.C., Kim, J.K., 2013. Cardiac expression of human type 2 iodothyronine deiodinase increases glucose metabolism and protects against doxorubicininduced cardiac dysfunction in male mice. Endocrinology 154, 3937e3946. Horton, L., Coburn, R.J., England, J.M., Himsworth, R.L., 1976. The haematology of hypothyroidism. Q. J. Med. 45, 101e123. Huang, S.A., Tu, H.M., Harney, J.W., Venihaki, M., Butte, A.J., Kozakewich, H.P., Fishman, S.J., Larsen, P.R., 2000. Severe hypothyroidism caused by type 3 iodothyronine deiodinase in infantile hemangiomas. N. Engl. J. Med. 343, 185e189. Huffmeier, U., Tietze, H.U., Rauch, A., 2007. Severe skeletal dysplasia caused by undiagnosed hypothyroidism. Eur. J. Med. Genet. 50, 209e215. Iwen, K.A., Schroder, E., Brabant, G., 2013. Thyroid hormones and the metabolic syndrome. Eur. Thyroid J. 2, 83e92.

504

21. THYROID HORMONES

Janssen, R., Zuidwijk, M., Muller, A., Mulders, J., Oudejans, C.B., Simonides, W.S., 2013. Cardiac expression of deiodinase type 3 (Dio3) following myocardial infarction is associated with the induction of a pluripotency microRNA signature from the Dlk1-Dio3 genomic region. Endocrinology 154, 1973e1978. Jorgensen, E.C., 1964. Stereochemistry of thyroxine and analogues. Mayo Clin. Proc. 39, 560e568. Kahapola-Arachchige, K.M., Hadlow, N., Wardrop, R., Lim, E.M., Walsh, J.P., 2012. Age- specific TSH reference ranges have minimal impact on the diagnosis of thyroid dysfunction. Clin. Endocrinol. 77, 773e779. Kalyanaraman, H., Schwappacher, R., Joshua, J., Zhuang, S., Scott, B.T., Klos, M., Casteel, D.E., Frangos, J.A., Dillmann, W., Boss, G.R., Pilz, R.B., 2014. Nongenomic thyroid hormone signaling occurs through a plasma membrane-localized receptor. Sci. Signal. 7 ra48. Kawa, M.P., Grymula, K., Paczkowska, E., Baskiewicz-Masiuk, M., Dabkowska, E., Koziolek, M., Tarnowski, M., Klos, P., Dziedziejko, V., Kucia, M., Syrenicz, A., Machalinski, B., 2010. Clinical relevance of thyroid dysfunction in human haematopoiesis: biochemical and molecular studies. Eur. J. Endocrinol. 162, 295e305. Kendall, E.C., 1915. The isolation in crystalline form of the compound containing iodine, which occurs in the thyroid. J. Am. Med. Assoc. 64, 2042e2043. Kendrick, T.S., Payne, C.J., Epis, M.R., Schneider, J.R., Leedman, P.J., Klinken, S.P., Ingley, E., 2008. Erythroid defects in TRalpha/ mice. Blood 111, 3245e3248. Kim, H.Y., Mohan, S., 2013. Role and mechanisms of actions of thyroid hormone on the skeletal development. Bone Res. 1, 146e161. Klein, I., Danzi, S., 2016. Thyroid disease and the heart. Curr. Probl. Cardiol. 41, 65e92. Kojetin, D.J., Matta-Camacho, E., Hughes, T.S., Srinivasan, S., Nwachukwu, J.C., Cavett, V., Nowak, J., Chalmers, M.J., Marciano, D.P., Kamenecka, T.M., Shulman, A.I., Rance, M., Griffin, P.R., Bruning, J.B., Nettles, K.W., 2015. Structural mechanism for signal transduction in RXR nuclear receptor heterodimers. Nat. Commun. 6, 8013. Krause, G., Hinz, K.M., 2017. Thyroid hormone transport across L-type amino acid transporters: what can molecular modelling tell us? Mol. Cell. Endocrinol. 458, 68e75. Kumar, R., Singh, V.P., Baker, K.M., 2008. The intracellular reninangiotensin system: implications in cardiovascular remodeling. Curr. Opin. Nephrol. Hypertens. 17, 168e173. Kyriacou, A., Mclaughlin, J., Syed, A.A., 2015. Thyroid disorders and gastrointestinal and liver dysfunction: a state of the art review. Eur. J. Intern. Med. 26, 563e571. Larsen, P.R., 1982. Thyroid-pituitary interaction: feedback regulation of thyrotropin secretion by thyroid hormones. N. Engl. J. Med. 306, 23e32. Larsen, P.R., Zavacki, A.M., 2012. The role of the iodothyronine deiodinases in the physiology and pathophysiology of thyroid hormone action. Eur. Thyroid J. 1, 232e242. Leal, A.L., Albuquerque, J.P., Matos, M.S., Fortunato, R.S., Carvalho, D.P., Rosenthal, D., da Costa, V.M., 2015. Thyroid hormones regulate skeletal muscle regeneration after acute injury. Endocrine 48, 233e240. Lee, S., Farwell, A.P., 2016. Euthyroid sick syndrome. Comp. Physiol. 6, 1071e1080. Lichtstein, D.M., Arteaga, R.B., 2006. Rhabdomyolysis associated with hyperthyroidism. Am. J. Med. Sci. 332, 103e105. Lima, C.S., Zantut Wittmann, D.E., Castro, V., Tambascia, M.A., Lorand-Metze, I., Saad, S.T., Costa, F.F., 2006. Pancytopenia in untreated patients with Graves’ disease. Thyroid 16, 403e409. Lima, F.R., Goncalves, N., Gomes, F.C., de Freitas, M.S., Moura Neto, V., 1998. Thyroid hormone action on astroglial cells from distinct brain regions during development. Int. J. Dev. Neurosci. 16, 19e27.

Lin, H.Y., Hopkins, R., Cao, H.J., Tang, H.Y., Alexander, C., Davis, F.B., Davis, P.J., 2005. Acetylation of nuclear hormone receptor superfamily members: thyroid hormone causes acetylation of its own receptor by a mitogen-activated protein kinase-dependent mechanism. Steroids 70, 444e449. Lin, J.Z., Martagon, A.J., Cimini, S.L., Gonzalez, D.D., Tinkey, D.W., Biter, A., Baxter, J.D., Webb, P., Gustafsson, J.A., Hartig, S.M., Phillips, K.J., 2015. Pharmacological activation of thyroid hormone receptors elicits a functional conversion of white to brown fat. Cell Rep. 13, 1528e1537. Lin, J.Z., Sieglaff, D.H., Yuan, C., Su, J., Arumanayagam, A.S., Firouzbakht, S., Cantu Pompa, J.J., Reynolds, F.D., Zhou, X., Cvoro, A., Webb, P., 2013. Gene specific actions of thyroid hormone receptor subtypes. PLoS One 8, e52407. Lin, T.Y., Shekar, A.O., Li, N., Yeh, M.W., Saab, S., Wilson, M., Leung, A.M., 2017. Incidence of abnormal liver biochemical tests in hyperthyroidism. Clin. Endocrinol. 86, 755e759. Little, A.G., 2016. A review of the peripheral levels of regulation by thyroid hormone. J. Comp. Physiol. B 186, 677e688. Liu, Y.Y., Ayers, S., Milanesi, A., Teng, X., Rabi, S., Akiba, Y., Brent, G.A., 2015. Thyroid hormone receptor sumoylation is required for preadipocyte differentiation and proliferation. J. Biol. Chem. 290, 7402e7415. Liu, Y.Y., Brent, G.A., 2010. Thyroid hormone crosstalk with nuclear receptor signaling in metabolic regulation. Trends Endocrinol. Metabol. 21, 166e173. Liu, Y.-Y., Brent, G.A., 2018. Posttranslational modification of thyroid hormone nuclear receptor by sumoylation. Methods Mol. Bio. 1801, 47e59. Liu, Y.Y., Heymann, R.S., Moatamed, F., Schultz, J.J., Sobel, D., Brent, G.A., 2007. A mutant thyroid hormone receptor alpha antagonizes peroxisome proliferator-activated receptor alpha signaling in vivo and impairs fatty acid oxidation. Endocrinology 148, 1206e1217. Liu, Y.Y., Kogai, T., Schultz, J.J., Mody, K., Brent, G.A., 2012. Thyroid hormone receptor isoform-specific modification by small ubiquitin-like modifier (SUMO) modulates thyroid hormonedependent gene regulation. J. Biol. Chem. 287, 36499e36508. Lopez, G., Schaufele, F., Webb, P., Holloway, J.M., Baxter, J.D., Kushner, P.J., 1993. Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site. Mol. Cell. Biol. 13, 3042e3049. Lopez, M., Alvarez, C.V., Nogueiras, R., Dieguez, C., 2013. Energy balance regulation by thyroid hormones at central level. Trends Mol. Med. 19, 418e427. Mansourian, A.R., 2013. A review of literature on the adverse effects of thyroid abnormalities and liver disorders: an overview on liver dysfunction and hypothyroidism. Pak. J. Biol. Sci. 16, 1641e1652. Manzano, J., Bernal, J., Morte, B., 2007. Influence of thyroid hormones on maturation of rat cerebellar astrocytes. Int. J. Dev. Neurosci. 25, 171e179. Marelli, F., Carra, S., Agostini, M., Cotelli, F., Peeters, R., Chatterjee, K., Persani, L., 2016. Patterns of thyroid hormone receptor expression in zebrafish and generation of a novel model of resistance to thyroid hormone action. Mol. Cell. Endocrinol. 424, 102e117. Mariotti, S., Beck-Peccoz, P., 2000. Physiology of the hypothalamicpituitary-thyroid axis. In: de Groot, L.J., Chrousos, G., Dungan, K., Feingold, K.R., Grossman, A., Hershman, J.M., Koch, C., Korbonits, M., Mclachlan, R., New, M., Purnell, J., Rebar, R., Singer, F., Vinik, A. (Eds.), Endotext. South Dartmouth (MA). Mavinakere, M.S., Powers, J.M., Subramanian, K.S., Roggero, V.R., Allison, L.A., 2012. Multiple novel signals mediate thyroid hormone receptor nuclear import and export. J. Biol. Chem. 287, 31280e31297. Mayerl, S., Schmidt, M., Doycheva, D., Darras, V.M., Huttner, S.S., Boelen, A., Visser, T.J., Kaether, C., Heuer, H., von Maltzahn, J.,

REFERENCES

2018. Thyroid hormone transporters MCT8 and OATP1C1 control skeletal muscle regeneration. Stem Cell Rep. 10 (6), 1959e1974. Mayerl, S., Visser, T.J., Darras, V.M., Horn, S., Heuer, H., 2012. Impact of Oatp1c1 deficiency on thyroid hormone metabolism and action in the mouse brain. Endocrinology 153, 1528e1537. Mebis, L., Paletta, D., Debaveye, Y., Ellger, B., Langouche, L., D’hoore, A., Darras, V.M., Visser, T.J., van den Berghe, G., 2009. Expression of thyroid hormone transporters during critical illness. Eur. J. Endocrinol. 161, 243e250. Medina, M.C., Molina, J., Gadea, Y., Fachado, A., Murillo, M., Simovic, G., Pileggi, A., Hernandez, A., Edlund, H., Bianco, A.C., 2011. The thyroid hormone-inactivating type III deiodinase is expressed in mouse and human beta-cells and its targeted inactivation impairs insulin secretion. Endocrinology 152, 3717e3727. Mendez-Pertuz, M., Sanchez-Pacheco, A., Aranda, A., 2003. The thyroid hormone receptor antagonizes CREB-mediated transcription. EMBO J. 22, 3102e3112. Mendoza, A., Hollenberg, A.N., 2017. New insights into thyroid hormone action. Pharmacol. Ther. 173, 135e145. Milanesi, A., Lee, J.W., Kim, N.H., Liu, Y.Y., Yang, A., Sedrakyan, S., Kahng, A., Cervantes, V., Tripuraneni, N., Cheng, S.Y., Perin, L., Brent, G.A., 2016. Thyroid hormone receptor alpha plays an essential role in male skeletal muscle myoblast proliferation, differentiation, and response to injury. Endocrinology 157, 4e15. Milanesi, A., Lee, J.W., Yang, A., Liu, Y.Y., Sedrakyan, S., Cheng, S.Y., Perin, L., Brent, G.A., 2017. Thyroid hormone receptor alpha is essential to maintain the satellite cell niche during skeletal muscle injury and sarcopenia of aging. Thyroid 27, 1316e1322. Mondal, S., Raja, K., Schweizer, U., Mugesh, G., 2016. Chemistry and biology in the biosynthesis and action of thyroid hormones. Angew Chem. Int. Ed. Engl. 55, 7606e7630. Moran, C., Chatterjee, K., 2015. Resistance to thyroid hormone due to defective thyroid receptor alpha. Best Pract. Res. Clin. Endocrinol. Metabol. 29, 647e657. Mullur, R., Liu, Y.Y., Brent, G.A., 2014. Thyroid hormone regulation of metabolism. Physiol. Rev. 94, 355e382. Muscat, G.E., Mynett-Johnson, L., Dowhan, D., Downes, M., Griggs, R., 1994. Activation of myoD gene transcription by 3,5,30 -triiodo-Lthyronine: a direct role for the thyroid hormone and retinoid X receptors. Nucleic Acids Res. 22, 583e591. Ng, L., Goodyear, R.J., Woods, C.A., Schneider, M.J., Diamond, E., Richardson, G.P., Kelley, M.W., Germain, D.L., Galton, V.A., Forrest, D., 2004. Hearing loss and retarded cochlear development in mice lacking type 2 iodothyronine deiodinase. Proc. Natl. Acad. Sci. U.S.A. 101, 3474e3479. Ng, L., Hernandez, A., He, W., Ren, T., Srinivas, M., Ma, M., Galton, V.A., St Germain, D.L., Forrest, D., 2009. A protective role for type 3 deiodinase, a thyroid hormone-inactivating enzyme, in cochlear development and auditory function. Endocrinology 150, 1952e1960. Ng, L., Kelley, M.W., Forrest, D., 2013. Making sense with thyroid hormone e the role of T(3) in auditory development. Nat. Rev. Endocrinol. 9, 296e307. Ng, L., Lyubarsky, A., Nikonov, S.S., Ma, M., Srinivas, M., Kefas, B., St Germain, D.L., Hernandez, A., Pugh Jr., E.N.,, Forrest, D., 2010. Type 3 deiodinase, a thyroid-hormone-inactivating enzyme, controls survival and maturation of cone photoreceptors. J. Neurosci. 30, 3347e3357. Niculescu 3rd, A.B., Segal, D.S., Kuczenski, R., Barrett, T., Hauger, R.L., Kelsoe, J.R., 2000. Identifying a series of candidate genes for mania and psychosis: a convergent functional genomics approach. Physiol. Genom. 4, 83e91. Nielsen, R., Pedersen, T.A., Hagenbeek, D., Moulos, P., Siersbaek, R., Megens, E., Denissov, S., Borgesen, M., Francoijs, K.J., Mandrup, S., Stunnenberg, H.G., 2008. Genome-wide profiling of PPARgamma:RXR and RNA polymerase II occupancy reveals

505

temporal activation of distinct metabolic pathways and changes in RXR dimer composition during adipogenesis. Genes Dev. 22, 2953e2967. Norris Jr., F.H.,, Panner, B.J., 1966. Hypothyroid myopathy. Clinical, electromyographical, and ultrastructural observations. Arch. Neurol. 14, 574e589. Ortiga-Carvalho, T.M., Shibusawa, N., Nikrodhanond, A., Oliveira, K.J., Machado, D.S., Liao, X.H., Cohen, R.N., Refetoff, S., Wondisford, F.E., 2005. Negative regulation by thyroid hormone receptor requires an intact coactivator-binding surface. J. Clin. Investig. 115, 2517e2523. Osterweil, D., Syndulko, K., Cohen, S.N., Pettler-Jennings, P.D., Hershman, J.M., Cummings, J.L., Tourtellotte, W.W., Solomon, D.H., 1992. Cognitive function in non-demented older adults with hypothyroidism. J. Am. Geriatr. Soc. 40, 325e335. Pascual, A., Aranda, A., 2013. Thyroid hormone receptors, cell growth and differentiation. Biochim. Biophys. Acta 1830, 3908e3916. Peeters, R.P., Hernandez, A., Ng, L., Ma, M., Sharlin, D.S., Pandey, M., Simonds, W.F., St Germain, D.L., Forrest, D., 2013. Cerebellar abnormalities in mice lacking type 3 deiodinase and partial reversal of phenotype by deletion of thyroid hormone receptor alpha1. Endocrinology 154, 550e561. Picou, F., Fauquier, T., Chatonnet, F., Flamant, F., 2012. A bimodal influence of thyroid hormone on cerebellum oligodendrocyte differentiation. Mol. Endocrinol. 26, 608e618. Plateroti, M., Kress, E., Mori, J.I., Samarut, J., 2006. Thyroid hormone receptor alpha1 directly controls transcription of the beta-catenin gene in intestinal epithelial cells. Mol. Cell. Biol. 26, 3204e3214. Ramadoss, P., Abraham, B.J., Tsai, L., Zhou, Y., Costa-E-Sousa, R.H., Ye, F., Bilban, M., Zhao, K., Hollenberg, A.N., 2014. Novel mechanism of positive versus negative regulation by thyroid hormone receptor beta1 (TRbeta1) identified by genome-wide profiling of binding sites in mouse liver. J. Biol. Chem. 289, 1313e1328. Refetoff, S., Bassett, J.H., Beck-Peccoz, P., Bernal, J., Brent, G., Chatterjee, K., de Groot, L.J., Dumitrescu, A.M., Jameson, J.L., Kopp, P.A., Murata, Y., Persani, L., Samarut, J., Weiss, R.E., Williams, G.R., Yen, P.M., 2014. Classification and proposed nomenclature for inherited defects of thyroid hormone action, cell transport, and metabolism. J. Clin. Endocrinol. Metab. 99, 768e770. Riis, A.L., Jorgensen, J.O., Moller, N., Weeke, J., Clausen, T., 2005. Hyperthyroidism and cation pumps in human skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 288, E1265eE1269. Safer, J.D., 2012. Thyroid hormone action on skin. Curr. Opin. Endocrinol. Diabetes Obes. 19, 388e393. Salehi, N., Agoston, E., Munir, I., Thompson, G.J., 2017. Rhabdomyolysis in a patient with severe hypothyroidism. Am. J. Case Rep. 18, 912e918. Salvatore, D., Simonides, W.S., Dentice, M., Zavacki, A.M., Larsen, P.R., 2014. Thyroid hormones and skeletal muscleenew insights and potential implications. Nat. Rev. Endocrinol. 10, 206e214. Sanchez-Pacheco, A., Martinez-Iglesias, O., Mendez-Pertuz, M., Aranda, A., 2009. Residues K128, 132, and 134 in the thyroid hormone receptor-alpha are essential for receptor acetylation and activity. Endocrinology 150, 5143e5152. Sandler, B., Webb, P., Apriletti, J.W., Huber, B.R., Togashi, M., Cunha Lima, S.T., Juric, S., Nilsson, S., Wagner, R., Fletterick, R.J., Baxter, J.D., 2004. Thyroxine-thyroid hormone receptor interactions. J. Biol. Chem. 279, 55801e55808. Scanlan, T.S., 2008. Thyroid hormone analogues: useful biological probes and potential therapeutic agents. Ann. Endocrinol. 69, 157e159. Scanlan, T.S., 2010. Sobetirome: a case history of bench-to-clinic drug discovery and development. Heart Fail. Rev. 15, 177e182. Scarlett, A., Parsons, M.P., Hanson, P.L., Sidhu, K.K., Milligan, T.P., Burrin, J.M., 2008. Thyroid hormone stimulation of extracellular

506

21. THYROID HORMONES

signal-regulated kinase and cell proliferation in human osteoblastlike cells is initiated at integrin alphaVbeta3. J. Endocrinol. 196, 509e517. Schneider, M.J., Fiering, S.N., Pallud, S.E., Parlow, A.F., St Germain, D.L., Galton, V.A., 2001. Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol. Endocrinol. 15, 2137e2148. Schneider, M.J., Fiering, S.N., Thai, B., Wu, S.Y., ST Germain, E., Parlow, A.F., ST Germain, D.L., Galton, V.A., 2006. Targeted disruption of the type 1 selenodeiodinase gene (Dio1) results in marked changes in thyroid hormone economy in mice. Endocrinology 147, 580e589. Schoenmakers, N., Moran, C., Peeters, R.P., Visser, T., Gurnell, M., Chatterjee, K., 2013. Resistance to thyroid hormone mediated by defective thyroid hormone receptor alpha. Biochim. Biophys. Acta 1830, 4004e4008. Seebacher, F., 2018. The evolution of metabolic regulation in animals. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 224, 195e203. Sharlin, D.S., Visser, T.J., Forrest, D., 2011. Developmental and cellspecific expression of thyroid hormone transporters in the mouse cochlea. Endocrinology 152, 5053e5064. Simonides, W.S., van Hardeveld, C., 2008. Thyroid hormone as a determinant of metabolic and contractile phenotype of skeletal muscle. Thyroid 18, 205e216. Sindoni, A., Rodolico, C., Pappalardo, M.A., Portaro, S., Benvenga, S., 2016. Hypothyroid myopathy: a peculiar clinical presentation of thyroid failure. Review of the literature. Rev. Endocr. Metab. Disord. 17, 499e519. Sinha, R.A., Singh, B.K., Yen, P.M., 2018. Direct effects of thyroid hormones on hepatic lipid metabolism. Nat. Rev. Endocrinol. 14, 259e269. Surks, M.I., Boucai, L., 2010. Age- and race-based serum thyrotropin reference limits. J. Clin. Endocrinol. Metab. 95, 496e502. Taylor, E., Heyland, A., 2017. Evolution of thyroid hormone signaling in animals: non-genomic and genomic modes of action. Mol. Cell. Endocrinol. 459, 14e20. Thomas, A.M., Hart, S.N., Kong, B., Fang, J., Zhong, X.B., Guo, G.L., 2010. Genome-wide tissue-specific farnesoid X receptor binding in mouse liver and intestine. Hepatology 51, 1410e1419. Toyoda, N., Nishikawa, M., Horimoto, M., Yoshikawa, N., Mori, Y., Yoshimura, M., Masaki, H., Tanaka, K., Inada, M., 1990. Graves’ immunoglobulin G stimulates iodothyronine 50 -deiodinating activity in FRTL-5 rat thyroid cells. J. Clin. Endocrinol. Metab. 70, 1506e1511. Trajkovic-Arsic, M., Visser, T.J., Darras, V.M., Friesema, E.C., Schlott, B., Mittag, J., Bauer, K., Heuer, H., 2010. Consequences of monocarboxylate transporter 8 deficiency for renal transport and metabolism of thyroid hormones in mice. Endocrinology 151, 802e809. Upadhyay, G., Singh, R., Kumar, A., Kumar, S., Kapoor, A., Godbole, M.M., 2004. Severe hyperthyroidism induces mitochondria-mediated apoptosis in rat liver. Hepatology 39, 1120e1130. Vadiveloo, T., Donnan, P.T., Cochrane, L., Leese, G.P., 2011. The thyroid epidemiology, audit, and research study (TEARS): morbidity in patients with endogenous subclinical hyperthyroidism. J. Clin. Endocrinol. Metab. 96, 1344e1351. Vadiveloo, T., Donnan, P.T., Murphy, M.J., Leese, G.P., 2013. Age- and gender-specific TSH reference intervals in people with no obvious thyroid disease in Tayside, Scotland: the thyroid epidemiology, audit, and research study (TEARS). J. Clin. Endocrinol. Metab. 98, 1147e1153. van der Deure, W.M., Peeters, R.P., Visser, T.J., 2010. Molecular aspects of thyroid hormone transporters, including MCT8, MCT10, and

OATPs, and the effects of genetic variation in these transporters. J. Mol. Endocrinol. 44, 1e11. van Gucht, A.L.M., Meima, M.E., Moran, C., Agostini, M., TykiSzymanska, A., Krajewska, M.W., Chrzanowska, K., Efthymiadou, A., Chrysis, D., Demir, K., Visser, W.E., Visser, T.J., Chatterjee, K., van Dijk, T.B., Peeters, R.P., 2017. Anemia in patients with resistance to thyroid hormone a: A role for thyroid hormone receptor a in human erythrocytosis. J. Clin. Endocrinol. Metab 102, 3517e3525. Vancamp, P., Darras, V.M., 2018. From zebrafish to human: a comparative approach to elucidate the role of the thyroid hormone transporter MCT8 during brain development. Gen. Comp. Endocrinol. 265, 219e229. Vargas-Uricoechea, H., Bonelo-Perdomo, A., Sierra-Torres, C.H., 2014. Effects of thyroid hormones on the heart. Clin. Invest. Arterioscler. 26, 296e309. Vestergaard, P., Mosekilde, L., 2002. Fractures in patients with hyperthyroidism and hypothyroidism: a nationwide follow-up study in 16,249 patients. Thyroid 12, 411e419. Vestergaard, P., Rejnmark, L., Mosekilde, L., 2005. Influence of hyperand hypothyroidism, and the effects of treatment with antithyroid drugs and levothyroxine on fracture risk. Calcif. Tissue Int. 77, 139e144. Visser, T.J., 2000. Cellular uptake of thyroid hormones. In: De Groot, L.J., Chrousos, G., Dungan, K., Feingold, K.R., Grossman, A., Hershman, J.M., Koch, C., Korbonits, M., Mclachlan, R., New, M., Purnell, J., Rebar, R., Singer, F., Vinik, A. (Eds.), Endotext. South Dartmouth (MA). Vogels, R.L., Oosterman, J.M., Van Harten, B., Gouw, A.A., SchroederTanka, J.M., Scheltens, P., van der Flier, W.M., Weinstein, H.C., 2007. Neuroimaging and correlates of cognitive function among patients with heart failure. Dement. Geriatr. Cognit. Disord. 24, 418e423. Wallis, K., Dudazy, S., Van Hogerlinden, M., Nordstrom, K., Mittag, J., Vennstrom, B., 2010. The thyroid hormone receptor alpha1 protein is expressed in embryonic postmitotic neurons and persists in most adult neurons. Mol. Endocrinol. 24, 1904e1916. Werneck de Castro, J.P., Fonseca, T.L., Ueta, C.B., Mcaninch, E.A., Abdalla, S., Wittmann, G., Lechan, R.M., Gereben, B., Bianco, A.C., 2015. Differences in hypothalamic type 2 deiodinase ubiquitination explain localized sensitivity to thyroxine. J. Clin. Investig. 125, 769e781. Williams, G.R., 2013. Thyroid hormone actions in cartilage and bone. Eur. Thyroid J. 2, 3e13. Wu, S.Y., Green, W.L., Huang, W.S., Hays, M.T., Chopra, I.J., 2005. Alternate pathways of thyroid hormone metabolism. Thyroid 15, 943e958. Ying, H., Araki, O., Furuya, F., Kato, Y., Cheng, S.Y., 2007. Impaired adipogenesis caused by a mutated thyroid hormone alpha1 receptor. Mol. Cell. Biol. 27, 2359e2371. Yuan, L., Tian, Y., Zhang, F., Dai, F., Luo, L., Fan, J., Wang, K., 2014. Impairment of attention networks in patients with untreated hyperthyroidism. Neurosci. Lett. 574, 26e30. Zevenbergen, C., Meima, M.E., Lima De Souza, E.C., Peeters, R.P., Kinne, A., Krause, G., Visser, W.E., Visser, T.J., 2015. Transport of iodothyronines by human L-type amino acid transporters. Endocrinology 156, 4345e4355. Zhang, J., Roggero, V.R., Allison, L.A., 2018. Nuclear import and export of the thyroid hormone receptor. Vitam. Horm. 106, 45e66. Zvibel, I., Atias, D., Phillips, A., Halpern, Z., Oren, R., 2010. Thyroid hormones induce activation of rat hepatic stellate cells through increased expression of p75 neurotrophin receptor and direct activation of Rho. Lab. Investig. 90, 674e684.

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22 Parathyroid Hormones Carole Le Henaff, Nicola C. Partridge Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, NY, United States

1. INTRODUCTION This chapter focuses on the hormone produced by the parathyroid gland, parathyroid hormone (PTH), its structure, regulation, action through its known receptors, physiologic functions, and diseases. Because of space constraints, we have not dealt with its homolog, parathyroid hormone-related protein (PTHrP), which is synthesized not by a gland but by many tissues, especially in the developing, pregnant, and lactating animal and which has major roles in endochondral bone formation, where it is produced by articular chondrocytes and also osteoblasts. The latter function is described by Kovacs in the chapter on Bone Hormones.

2. STRUCTURES 2.1 Parathyroid Hormone In the 1970s, for the first time, two independent groups determined the structure of bovine, porcine, and human PTH by protein sequence analysis after accumulation of sufficient animal material and from surgically removed human tumors (Brewer et al., 1972; Brewer and Ronan, 1970; Keutmann et al., 1978; Niall et al., 1970, 1974; Sauer et al., 1974). Subsequently, bovine PTH (1e34), the first 34 amino-terminal amino acids of bovine full-length PTH that contains its biologic activity, was synthesized (Potts, 2005; Potts et al., 1971a, 1971b; Tregear et al., 1973). The human PTH gene is localized on the short arm of chromosome 11 at 11p15 (Antonarakis et al., 1983; Zabel et al., 1985). In mammals, PTH is encoded by a singlecopy gene that consists of three exons and two introns (Kronenberg et al., 1986). The 5 kb of DNA upstream of the start site of the human PTH gene is very finely Hormonal Signaling in Biology and Medicine https://doi.org/10.1016/B978-0-12-813814-4.00022-5

regulated. Unlike rat, the human and bovine genes have two functional TATA transcription start sites, which induce the synthesis of two human PTH gene transcripts (Igarashi et al., 1986). There is considerable homology among mammalian PTH genes, for example, 85% identity between human and bovine proteins and 75% identity between human and rat proteins (NavehMany, 2010; Naveh-Many and Nechama, 2007). Strikingly, even though fish do not have parathyroid glands, they synthesize two forms of PTH via two different genes. This was found in pufferfish, zebrafish, and elephant shark (Danks et al., 2003; Gensure et al., 2004; Guerreiro et al., 2007; Liu et al., 2010). 2.1.1 PTH Synthesis and Secretion from Parathyroid Glands The parathyroid glands were discovered in the late 19th century, and interest in their function increased during the first 25 years of the 20th century. The removal of all of the parathyroid glands in cats, dogs, and rodents was associated with death from tetany. The function of the parathyroid glands was discovered as distinct and separate from the thyroid. The relationship between these two kinds of glands is anatomic and not functional. Patients undergoing extensive thyroid surgery occasionally suffered from tetany followed by death, due to the loss of the parathyroid glands (Potts, 2005). Later, they found that tetany was induced by severe hypocalcemia induced by a lack of blood Ca2þ control by the parathyroid glands and could be prevented by Ca2þ infusion (Potts, 2005; MacCallum and Voegtlin, 1909; MacCallum and Vogel, 1913). But the administration of extracts of the parathyroid glands failed to reverse the tetany. The principal physiologic role of the parathyroid glands in Ca2þ regulation was established in 1925 by Collip, who attempted to extract PTH from these glands to prevent tetany. Only years later, Aurbach

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purified and characterized the active principle of these glands (Potts, 2005; Aurbach, 1959a, 1959b). PTH is secreted by the chief cells (also called parathyroid principal cells or simply parathyroid cells) of the parathyroid glands. These glands are part of the endocrine system. Most people have four pea-sized parathyroid glands embedded at the back of the thyroid gland in the neck. When the Ca2þ level in the blood is high, calcitonin from the thyroid glands is secreted, bone resorption is inhibited, and the Ca2þ level is lowered by depositing Ca2þ in bones. Inversely, when the level of Ca2þ in the blood is too low, PTH is released, and the level of Ca2þ is raised by its release from bone. Parathyroid cells have multiple methods of adapting to increased needs for PTH production: a secretion of prohormone within minutes and then, within hours, new PTH expression (Potts et al., 1997). PTH is synthesized as a prepropeptide with a 25amino acid presequence that forms a hydrophobic domain and facilitates its passage through the membrane of the endoplasmic reticulum (Habener et al., 1976). Then the prosequence composed of 6 amino acids is cleaved before the hormone is routed to secretory vesicles (Kemper et al., 1975). The 84-amino acid mature peptide is stored in secretory vesicles (Habener et al., 1984). A final cleavage occurs when PTH is released into the circulation: the N-terminal PTH or PTH (1e34), which contains its biologically active functions, and a biologically inactive C-terminal fragment. Interestingly, PTH (1e34) shows high homology throughout all species. The hormone is metabolized to aminoterminal and carboxyl-terminal fragments primarily in liver, also in the kidney and perhaps in the parathyroid glands and blood. The carboxyl-terminal fragments are cleared by glomerular filtration (Shaker et al., 2000). Full-length PTH has a circulating half-life of less than 5 min (Brown and MacLeod, 2001). PTH is secreted in three distinct ways: tonic secretion, in a bimodal diurnal rhythm (with a primary peak in the early morning, and a secondary peak in the late afternoon and nadirs in the morning and evening) (el-Hajj Fuleihan et al., 1997), and a pulsatility that appears to be stochastic (occurring unpredictably, 10 or more times a day). But acute hypocalcemia elicits an immediate pulsatile and a delayed tonic secretion of PTH. Acute hypercalcemia suppresses both the pulsatile and the tonic components of PTH secretion (Schmitt et al., 1996). Most PTH is secreted continuously (Bilezikian et al., 2017). 2.1.2 PTH Regulation by Serum Calcium and Phosphate Homeostasis A long-term replenishment of PTH stores is dependent on new PTH synthesis. This is controlled by the availability of PTH mRNA for ribosomal translation

into prepro-PTH and its posttranscriptional regulation. Conversely, rapid PTH release from secretory granules in hypocalcemic states is modulated by the binding of Ca2þ to the calcium-sensing receptor (CaSR) on chief cells (Kumar and Thompson, 2011). Under normal conditions, a small increase in circulating Ca2þ will instantly suppress PTH secretion (Bilezikian et al., 2018a). On the other hand, an imperceptible reduction in serum Ca2þ will immediately stimulate PTH secretion. This inverse sigmoidal association between PTH and serum Ca2þ is regulated by the CaSR (Bilezikian et al., 2018a). In humans, the CaSR is located on chromosome 3 and was first cloned from human kidney (Aida et al., 1995). Although the CaSR is found in a variety of tissues, the highest expression of the receptor is found in the parathyroid glands and kidneys (Goltzman and Hendy, 2015). It is a class C GPCR receptor. These receptors are typified by large extracellular domains of around 450e600 amino acids, a seven-pass transmembrane domain, and an intracellular carboxyl-terminal domain that has several phosphorylation sites (Bai et al., 1998). The N-terminal extracellular domain, typically, has a large bilobed, nutrient-binding Venus flytrap (VFT) domain composed of around 450e550 residues (Bai et al., 1999; Conigrave and Ward, 2013; Fan et al., 1998; Ward et al., 1998). The CaSR VFT domain forms homodimers or heterodimers with other class C GPCRs by noncovalent interactions. The receptor subunits form dimers upon insertion into the endoplasmic reticulum membrane prior to their transfer to the Golgi and plasma membrane (Conigrave and Ward, 2013). When Ca2þ binds to the CaSR, conformational changes in the extracellular domain of the receptor are transmitted through the seven-pass transmembrane domains and allow interactions between the intracellular domains of CaSR with heterotrimeric G protein subunits, Gqa/11a. These activate phospholipase Cb (PLCb) and result in hydrolysis of phosphatidylinositol-4,5,-bisphosphate and the formation of inositol 1,4,5-trisphosphate and diacylglycerol. 1,4,5-trisphosphate mobilizes intracellular Ca2þ. The receptor also activates MAPKs. Activation of the CaSR suppresses PTH secretion and parathyroid cell proliferation and stimulates PTH (1e84) degradation (Hofer and Brown, 2003). The CaSR also interacts with Gia to inhibit adenylate cyclase activity and reduce intracellular cAMP, which also inhibits PTH secretion (Kumar and Thompson, 2011; Hofer and Brown, 2003; Conigrave, 2016). The CaSR may also interact with GSa, and it is speculated that this occurs under conditions of low Ca2þ and high phosphate (Conigrave, 2016). The CaSR monitors serum Ca2þ and maintains it in the range 1.1e1.3 mM and regulates the secretion of PTH. The CaSR is involved and required for PTH secretion, PTH gene expression, and cellular proliferation in the parathyroid glands.

2. STRUCTURES

In addition to Ca2þ, the CaSR binds several metals, amino acids, antibiotics, and organic compounds that modulate its activity (Kumar and Thompson, 2011; Brown et al., 1991a, 1991b, 1999; Nemeth, 2002, 2004a, 2004b; Nemeth and Bennett, 1998). Calcimimetics bind the transmembrane CaSR and alter the conformation of the CaSR. This results in decreased PTH secretion, PTH mRNA levels, and parathyroid cell proliferation (Nemeth and Bennett, 1998; Colloton et al., 2005; Hu and Spiegel, 2003; Levi et al., 2006). Therefore, the CaSR and its signal transduction are central to parathyroid physiology and the maintenance of a normal serum PTH and intact Ca2þ homeostasis (Galitzer et al., 2009). Posttranscriptional effects of Ca2þ and phosphate on the PTH gene are induced by changes in protein/RNA interactions at the PTH mRNA 30 -UTR (30 untranslated region). Thus, hypocalcemia leads to increased binding of parathyroid cytosolic proteins to the PTH mRNA 30 UTR, whereas hypophosphatemia induces decreased binding (Silver et al., 1999). This was shown also in experimental kidney failure (Levi et al., 2006; Galitzer et al., 2009; Kilav et al., 2001; Moallem et al., 1998). This 26-nucleotide sequence, within the PTH mRNA ARE (adenine- and uridine-rich element), is conserved among species. This sequence is necessary and sufficient for protein binding and the regulation of PTH mRNA stability by dietary Ca2þ or phosphorus depletion (Galitzer et al., 2009; Kilav et al., 2001; Bell et al., 2005). In experimental animal models, a phosphate diet induced an increase in parathyroid gland function (Slatopolsky et al., 2005). Conversely, dietary phosphate restriction following phosphate overload in rats also led to an immediate decrease in PTH secretion. The half-life of PTH mRNA is balanced between a stabilizing complex AUF1/UNR (AU-rich elemente binding protein 1/upstream of N-ras) and a destabilizing protein KSRP (K-homology splicing regulatory protein) binding to the ARE in the 30 -UTR. Activation (i.e., increased Ca2þ) of the CaSR decreases PTH gene expression through the PTH mRNA 30 -UTR and the balanced interactions between KSRP and AUF1 in the parathyroid (Galitzer et al., 2009). Similarly, KSRPPTH mRNA interaction is increased in parathyroid glands from hypophosphatemic rats inducing an unstable PTH mRNA. Conversely, these interactions are decreased in parathyroids from hypocalcemic rats and with experimental renal failure, and the PTH mRNA is more stable. So the balanced interaction of PTH mRNA with AUF1 and KSRP determines PTH mRNA half-life and levels (Galitzer et al., 2009; Nechama et al., 2008). KSRP phosphorylation can be altered by a protein, peptidyl-prolyl isomerase, NIMA-interacting-1 (Pin1). This protein specifically binds serine/threoninee

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protein motifs. It binds to KSRP and prevents the phosphorylation of KSRP at serine residue 181. By this action, Pin1 enhances the binding of KSRP to the AREs in PTH mRNA (Kumar and Thompson, 2011). In vitro, inhibition or knockdown of Pin1 increases PTH mRNA by inhibiting its degradation, whereas overexpression of Pin1 reduces PTH mRNA by accelerating its decay (Kumar and Thompson, 2011; Nechama et al., 2009). 2.1.3 PTH Regulation by Magnesium Homeostasis After Ca2þ, the physiologically most important divalent cation activator of the CaSR is magnesium (Mg2þ), whose plasma concentration lies in the range 0.8e1.3 mM (Conigrave and Ward, 2013). Mg2þ is an essential divalent cation that has many roles in the body, particularly intracellularly, where it participates in energy metabolism and intracellular signaling through its involvement in multiple phosphorylation reactions (Quinn et al., 2013). Some 60%e70% of total body Mg is bound in the skeleton. In the presence of physiologic Ca2þ levels, Mg2þ positively modulates CaSR function. Genetic evidence has suggested that the CaSR may also serve not only as a Ca2þ sensor but also as a Mg2þ sensor in vivo (Quinn et al., 2013). Indeed, hypermagnesemia suppresses PTH secretion and can induce hypoparathyroidism. Patients with moderate to severe hypomagnesemia can also show impaired PTH secretion, and they can develop a secondary hypocalcemia (Chase and Slatopolsky, 1974). This appears to arise from an inhibitory effect of intracellular Mg2þ on G protein a-subunits (Conigrave and Ward, 2013). Strangely, both PTH secretion and sensitivity of bone to PTH are decreased with hypomagnesemia or alkalosis (Kopic and Geibel, 2013). 2.1.4 PTH Regulation by Fluoride Fluoride is an element that plays an important role in both bone formation and homeostasis of bone mineral metabolism (Mousny et al., 2006). In one mouse study, fluoride exposure resulted in early reductions in PTH levels and later increases. Fluoride seems to interact with the parathyroid glands directly through increased PTH gene expression and modulation of rapid PTH release from vesicles in the chief cells (Puranik et al., 2015). It has also been reported for years that fluoride induces a modulation of PTH secretion mediated by changes in Ca2þ levels. Decreased serum Ca2þ levels were observed after acute hydrofluoric acid inhalation in humans or injection in rats (Imanishi et al., 2009; Zierold and Chauviere, 2012; Santoyo-Sanchez et al., 2013). This decrease is hypothesized to be caused by the formation of CaF2 complexes. This results in a net reduction in Ca2þ levels, leading to a hypocalcemic response (Puranik et al., 2015).

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2.1.5 PTH Regulation by Vitamin D The other principal regulator of PTH secretion is 1,25-dihydroxyvitamin D3, a vitamin D metabolite. It is inversely associated with serum PTH concentrations. Vitamin D is derived from endogenous production in the skin or absorbed from the gut. It is transformed into its active form by two successive steps: hydroxylation in the liver to 25-hydroxyvitamin D3 followed by 1a-hydroxylation in the renal proximal tubule to 1,25-dihydroxyvitamin D3, also known as calcitriol (Liberman, 2000). In the parathyroid glands, 1,25-dihydroxyvitamin D3 feeds back to inhibit PTH transcription, secretion, and cell proliferation (Liberman, 2000; Cantley et al., 1985; Silver et al., 1985). Indeed, the promoter of the PTH gene has a vitamin D response element (VDRE) that binds the vitamin D receptor or VDR (Christakos et al., 1981; Demay et al., 1992; Hawa et al., 1996; Nishishita et al., 1998; Russell et al., 1999; Koszewski et al., 1999). VDR is expressed in parathyroid glands at the same concentration as the intestine but also in bone and kidney (Holick, 2004; Naveh-Many et al., 1990). 1,25dihydroxyvitamin D3 decreases PTH transcription by acting on the 50 -PTH gene region. Indeed, 1,25dihydroxyvitamin D3 bound to the VDR heterodimerizes with the retinoic acid receptor (RXR) to bind the VDRE of the PTH gene (Kumar and Thompson, 2011; Demay et al., 1992; MacDonald et al., 1994; Okazaki et al., 1988; Naveh-Many and Silver, 2017). In rats, 1,25-dihydroxyvitamin D3 injection induces decreased PTH mRNA levels without serum Ca2þ modifications (Silver et al., 1999; Naveh-Many et al., 1989). A combination of retinoic acid (ligand of RXR) and 1,25dihydroxyvitamin D3 induces a greater decrease in PTH secretion and PTH mRNA levels compared with each component alone (MacDonald et al., 1994). 1,25-Dihydroxyvitamin D3 can also amplify its effects on parathyroid cells by increasing the number of CaSR. In fact, human CaSR has VDREs in its promoter (Canaff and Hendy, 2002). Thus 1,25-dihydroxyvitamin D3 induces CaSR expression in the parathyroid gland, making it more sensitive to calcium. Then, Ca2þ alters the ability of 1,25-dihydroxyvitamin D3 to regulate PTH gene expression. For instance, animals placed on a low-calcium diet have an increase in PTH and 1,25dihydroxyvitamin D3 levels, indicating that the low calcium overrides the inhibition by 1,25-dihydroxyvitamin D3 on PTH secretion (Brown and Hebert, 1996; Mendoza et al., 2009). But this diet also induces an increase in calreticulin levels, in parathyroid cells. Calreticulin is a calcium-binding protein that is present in the endoplasmic reticulum and also may have a nuclear function. This protein regulates gene transcription via its ability to bind a protein motif in the DNA-binding domain of

nuclear sterol hormone receptors. As a result, calreticulin can bind and inhibit the VDR and thus prevents the feedback of 1,25-dihydroxyvitamin D3 (Naveh-Many et al., 1990; Dedhar et al., 1994; Russell et al., 1993; Wheeler et al., 1995). The parathyroid glands also express CYP27B1 (25hydroxyvitamin D3 1-a-hydroxylase), so they can produce their own 1,25-dihydroxyvitamin D3, which can act in an autocrine or paracrine fashion to regulate PTH production (Bikle, 2017; Ritter et al., 2012). 2.1.6 PTH Regulation by FGF23 The relationship between PTH and FGF23 (fibroblast growth factor 23) is complex, and gaps in our understanding still remain. PTH was considered the primary regulator of renal phosphate reabsorption until the discovery of FGF23. This is a bone-derived hormone that regulates phosphate homeostasis and vitamin D metabolism. This protein is synthesized as a fulllength, biologically active 32 kDa glycoprotein (Shimada et al., 2001; White et al., 2001; Liu et al., 2003; Fukumoto, 2005). It is expressed by many cell types but at the highest levels by osteocytes (Liu et al., 2003; Onal et al., 2018). In parathyroid cells, FGF23 directly inhibits PTH synthesis and secretion (Ben-Dov et al., 2007; Krajisnik et al., 2007). However, in renal failure, patients show a secondary hyperparathyroidism and elevated serum FGF23 levels. Therefore, clinical evidence is lacking to support an inhibitory effect of FGF23 on PTH secretion (Blau and Collins, 2015). By causing phosphorus excretion, FGF23 induces indirectly a reduction of PTH release, in addition to the possible direct inhibitory action of FGF23 on parathyroid secretory activity (Drueke, 2000). 2.1.7 Other PTH Fragments PTH (1e84) and PTH (1e34) are not the only circulating forms in the body. Various PTH fragments can be found. The most interesting is the large NH2-terminal truncated non-PTH (1e84) fragments with PTH (7e84) being the major member. They represent 20% of the circulating PTH and 50% in kidney disease (Brossard et al., 1993, 1996; Lepage et al., 1998; Herberth et al., 2006). In general, these fragments antagonize the effect of PTH: inhibition of PTH release, inhibition of 1,25dihydroxyvitamin D3 production, decrease in bone calcium release by blocking PTH action or osteoclast formation. These fragments act as competitive antagonists and are unable to activate cAMP/PKA signaling. But they may exert other effects on PTHR1 such as through its downregulation or its internalization. These fragments are secreted in response to hypercalcemia and act as a feedback mechanism (Kopic and Geibel, 2013; Cheloha et al., 2015).

2. STRUCTURES

2.1.8 PTH Mutations 2.1.8.1 Hyperparathyroidism Historically, Albright, in 1934 was the first to describe the hypercalcemic state caused by primary hyperparathyroidism as a disease of bone and kidney (Albright, 1948). After all these years, the central target organs for potential complications of this disorder continue to be the skeleton and kidneys (Bilezikian et al., 2018b). Hyperparathyroidism is an excess of PTH in the bloodstream due to overactivity of one or more of the four parathyroid glands. The incidence of this disease is estimated to be 3/1000 in the general population, and most cases are sporadic (Adami et al., 2002). It predominantly affects women, with a ratio of three to four women to one man (Bilezikian et al., 2018b). Two types of hyperparathyroidism exist. Almost 90% of the patients are found to have sporadic, nonfamilial, and nonsyndromic disease. Sporadic primary hyperparathyroidism is usually induced by a single gland adenoma (85% of patients), but it may also be caused by hyperplasia of all four glands (10%), double adenomas (2%e5%), or rarely, parathyroid carcinomas (