The Pituitary (Pituitary (Melmed))

The Pituitary

Notice: The indications and dosages of all drugs in this book have been recommended in the medical literature and conform to the practices of the general community. The medications described do not necessarily have specific approval by the Food and Drug Administration for use in

the diseases and dosages for which they are recommended. The package insert for each drug should be consulted for use and dosage as approved by the FDA. Because standards for usage change, it is advisable to keep abreast of revised recommendations, particularly those concerning new drugs.

The Pituitary EDITED BY

SHLOMO MELMED, Senior Vice-President, Academic Affairs Cedars-Sinai Medical Center Professor and Director, Burns & Allen Research Institute UCLA School of Medicine Los Angeles, California

SECOND EDITION

Blackwell Science

MD

© 2002 by Blackwell Science, Inc. Editorial Offices: Commerce Place, 350 Main Street, Malden, Massachusetts 02148, USA Osney Mead, Oxford OX2 0EL, England 25 John Street, London WC1N 2BS, England 23 Ainslie Place, Edinburgh EH3 6AJ, Scotland 54 University Street, Carlton, Victoria 3053, Australia Other Editorial Offices: Blackwell Wissenschafts-Verlag GmbH, Kurfürstendamm 57, 10707 Berlin, Germany Blackwell Science KK, MG Kodenmacho Building, 7-10 Kodenmacho Nihombashi, Chuo-ku, Tokyo 104, Japan Iowa State University Press, A Blackwell Science Company, 2121 S. State Avenue, Ames, Iowa 50014-8300, USA

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Contents

Contributors vii Preface

Section 3. PITUITARY TUMORS

x

10.

Pituitary Surgery 405 Rudolf Fahlbusch, Michael Buchfelder and Panos Nomikos

11.

Acromegaly 419 Shlomo Melmed

12.

Prolactinoma 455 Mark E. Molitch

13.

Cushing’s Disease 496 Xavier Bertagna, Marie-Charles Raux-Demay, Brigitte Guilhaume, François Girard and Jean-Pierre Luton

14.

Thyrotropin-secreting Pituitary Tumors 561 Yona Greenman and Shlomo Melmed

15.

Gonadotroph Adenomas Peter J. Snyder

16.

Nonpituitary Tumors of the Sellar Region 592 Steffen Albrecht, Juan M. Bilbao and Kalman Kovacs

Section 1. HYPOTHALAMIC–PITUITARY FUNCTION 1.

2.

Functional Anatomy of the Hypothalamic Pituitary Axis 3 Sylvia L. Asa, Kalman Kovacs and Shlomo Melmed Adrenocorticotropin 45 Mark A. Herman and Joseph A. Majzoub

3.

Growth Hormone 79 Vivien S. Herman-Bonert and Shlomo Melmed

4.

Prolactin 119 Mark E. Molitch

5.

Thyroid-stimulating Hormone 172 Virginia D. Sarapura, Mary H. Samuels and E. Chester Ridgway

6.

Follicle-stimulating Hormone and Luteinizing Hormone 216 Shalender Bhasin, Charles E. Fisher and Ronald S. Swerdloff

7.

The Posterior Pituitary Daniel G. Bichet

279

Section 2. HYPOTHALAMIC–PITUITARY DYSFUNCTION 8.

The Hypothalamus 317 Glenn D. Braunstein

9.

Anterior Pituitary Failure Charles F. Abboud

349

575

Section 4. PITUITARY DISEASE IN SYSTEMIC DISORDERS 17.

Pituitary Function in Systemic Disorders 613 Harold E. Carlson

18.

The Pituitary Gland in Pregnancy and the Puerperium 628 Harold E. Carlson

19.

Drugs and Pituitary Function Harold E. Carlson

642 v

vi

Contents

Section 5.

DIAGNOSTIC PROCEDURES

20.

Pituitary Imaging 663 Barry D. Pressman

21.

Neuro-ophthalmologic Evaluation of Pituitary Disorders 687 Anthony C. Arnold

22. Index

Evaluation of Normal Pituitary Function 709 Gillian L. Booth, Afshan Zahedi and Shereen Ezzat 727

Contributors

Charles F. Abboud, MD David Eisenberg Professor of Medicine Consultant, Internal Medicine and Endocrinolgy Mayo Clinic, Mayo Medical School Rochester, MN Steffen Albrecht, MD FRCP(C) Department of Pathology Sir Mortimer B. Davis Jewish General Hospital Montreal, Canada Anthony C. Arnold, MD Associate Professor of Ophthalmology and Chief, Neuro-Ophthalmology Division Jules Stein Eye Institute, UCLA Department of Ophthalmology Los Angles, CA Sylvia L. Asa, MD, PhD Associate Professor of Pathology Mount Sinai Hospital University of Toronto Toronto, Canada Xavier Bertagna, MD Professor of Endocrinology Clinique des Maladies Endocriniennes et Metaboliques Hôpital Cochin Paris, France Shalender Bhasin, MD Chief, Division of Endocrinology, Metabolism, and Molecular Medicine Professor of Medicine Charles R. Drew University School of Medicine and Science Los Angeles, CA

Daniel G. Bichet, MD, FRCP(C) Service de néphrologie et Centre de recherche Hôpital du Sarcé-Coeur de Montréal Department of Medicine Université de Montréal Montréal, Quebec Canada Juan M. Bilbao, MD, FRCP(C) Neuropathologist, Department of Pathology Saint Michael’s Hospital University of Toronto Toronto, Canada Gillian L. Booth, MD Department of Medicine University of Toronto Mount Sinai Hospital Toronto, Canada Glenn D. Braunstein, MD Chairman, Department of Medicine Cedars-Sinai Medical Center Professor of Medicine UCLA School of Medicine Los Angles, CA Michael Buchfelder, MD Associate Professor Department of Neurosurgery University of Erlangen-Nürnberg Erlangen, Germany vii

viii

Contributors

Harold E. Carlson, MD Chief, Endocrinology Section Northport Veterans Affairs Hospital, Northport Professor of Medicine State University of New York at Stony Brook Stony Brook, NY

Kalman Kovacs, MD, PhD, DSc, FRCP(C), FCAP, FRC Path. Professor of Pathology Saint Michael’s Hospital University of Toronto Toronto, Canada

Shereen Ezzat, MD, FRCP(C), FACP Assistant Professor of Medicine University of Toronto Consultant Endocrinologist Wellesley Hospital Toronto, Canada

Jean-Pierre Luton, MD Professor of Endocrinology and Director Clinique des Maladies Endocriniennes et Metaboliques Hôpital Cochin Dean, Cochin Medical School University René Descartes Paris, France

Rudolf Fahlbusch, MD Professor of Neurosurgery Department of Neurosurgery University of Erlangen-Nürnberg Erlangen, Germany Charles Ellis Fisher, MD Division of Endocrinology and Metabolism King Drew Medical Center Los Angeles, CA François Girard, MD Professor of Physiology and Director Laboratoire d’Explorations Fonctionnelles Endocriniennes Hôpital Trousseau Paris, France Yona Greenman, MD Tel-Aviv Sourasky Medical Center Tel-Aviv, Israel Brigitte Guilhaume, MD Clinique des Maladies Endocriniennes et Metaboliques Hôpital Cochin Paris, France Mark A. Herman, MD Fellow in Medicine Department of Internal Medicine University of Texas Southwestern Medical Center Dallas, Texas Vivien S. Herman-Bonert, MD Division of Endocrinology Cedars-Sinai Medical Center Associate Professor of Medicine UCLA School of Medicine Los Angeles, CA

Joseph A. Majzoub, MD Chief, Division of Endocrinology Department of Pediatrics Children’s Hospital Professor of Pediatrics Harvard Medical School Boston, MA Shlomo Melmed, MD Senior Vice-President, Academic Affairs Director, Burns & Allen Research Institute Associate Dean, UCLA School of Medicine Cedars-Sinai Medical Center Los Angeles, CA Mark E. Molitch, MD Professor of Medicine Center for Endocrinology, Metabolism, and Molecular Medicine Northwestern University Medical School Chicago, IL Panos Nomikos, MD Neurosurgical Fellow Department of Neurosurgery University of Erlangen-Nürnberg Erlangen, Germany Barry D. Pressman, MD, FACR Chief, Section of Neuroradiology and Chairman, Department of Imaging Cedars-Sinai Medical Center Los Angles, CA Marie-Charles Raux-Demay, MD Laboratoire d’Explorations Fonctionnelles Endocriniennes Hôpital Trousseau Paris, France

Contributors

Chester E. Ridgway, MD Professor of Medicine and Head, Division of Endocrinology University of Colorado Health Sciences Center Denver, CO Mary H. Samuels, MD Assistant Professor of Medicine Division of Endocrinology Oregon Health Science University Portland, OR Virginia D. Sarapura, MD Assistant Professor of Medicine Department of Medicine, Division of Endocrinology University of Colorado Health Sciences Center Denver, CO

Peter J. Snyder, MD Professor of Medicine University of Pennsylvania School of Medicine Philadelphia, PA Ronald S. Swerdloff, MD Professor of Medicine and Chief, Division of Endocrinology Department of Medicine Harbor/UCLA Medical Center UCLA School of Medicine Torrance, CA Afshan Zahedi, MD Department of Medicine University of Toronto Mount Sinai Hospital Toronto, Canada

ix

Preface

The second edition of The Pituitary follows the successful initial 1995 debut of this comprehensive text devoted to understanding pathogenesis and treatment of pituitary disorders. The new edition is extensively revised to reflect the wealth of novel information derived from advances in molecular biology, biochemistry and therapeutics as they apply to the pituitary gland. Notably, a new chapter devoted to pituitary surgery has been added to complement a comprehensive overview of management options for patients harboring pituitary tumors. The wide spectrum of clinical disorders emanating from disordered function of the “master gland” are described in detail by experts in the field. Furthermore, fundamental mechanisms underlying disease pathogenesis are presented to provide the reader with an in-depth understanding of mechanisms underlying pituitary hormone secretion and action.

Thus the volume continues to reflect the cogent blend of basic science and clinical medicine which was the successful hallmark of the first edition. I am especially indebted to my erudite colleagues for their scholarly contributions and dedicated efforts in compiling this body of knowledge for physicians and scientists dedicated to caring for patients with pituitary disorders. Our desire is to provide medical students, clinical and basic endocrinology trainees, endocrinologists, internists pediatricians, gynecologists and neurosurgeons with a comprehensive single text devoted to the science and art of pituitary medicine. Shlomo Melmed, MD Los Angeles 2002

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Hypothalamic–Pituitary Function

C h a p t e r

1 Functional Anatomy of the Hypothalamic Pituitary Axis Sylvia L. Asa Kalman Kovacs Shlomo Melmed

INTRODUCTION

Historical Review Descartes was the first to recognize that the brain was an organ integrating the functions of the mind and body [1]. The association was reinforced by Zander [2] who noted the connection between the adrenals and the brain, referring to observations on the absence of the adrenal cortex in anencephaly as recorded by Morgagni in 1733, Soemmering in 1792, and Meckel in 1802. It was only in 1849 that direct evidence of a role for the hypothalamus in endocrinology was provided by Claude Bernard [3] when he demonstrated that injury to the floor of the fourth ventricle, the “piqûre diabetique,’ caused polyuria and glucosuria; subsequently, numerous studies in the late 19th century and early 20th century confirmed that the hypothalamic–posterior pituitary system was the site of production of a major osmoregulatory substance. Galen (129–201 ad) had described the pituitary as the site of drainage of phlegm from the brain to the nasopharynx. Soon after the description of acromegaly by Pierre Marie in 1886 [4], the association of acromegaly with pituitary tumor was noted by Minkowski in 1887 [5], and the recognition of endocrine functions of the pituitary followed rapidly thereafter, with major contributions by Cushing [6] and Simmonds [7]. The early part of the 20th century saw the identification, isolation and characterization of the hormones of the anterior pituitary. Their regulation by the hypothalamus was the subject of a landmark monograph by Harris in 1948 [8]. The concept that neurons could release secretory materials into the bloodstream, proposed by Du Bois Reymond

in 1877 [9] and Schiefferdecker in 1905 [10], was revolutionary. The anatomic basis of a hypothalamic–pituitary vascular connection was already known to exist in the form of a system of capillaries, which were identified joining the medial basal hypothalamus to the anterior pituitary by Lieutaud in 1742 [11] and von Luschka in 1860 [12] and given the name “portal plexus” by Popa and Fielding in 1930 [13]. These vessels were thought at first to carry blood from the pituitary upwards to the hypothalamus; however, subsequent studies confirmed that they provided the main blood supply to the adenohypophysis. The concept of neurosecretion was suggested in the 1940s by Scharrer and Scharrer [14] and Bargmann and Scharrer [15], who demonstrated that peptide synthesized by neurons in the supraoptic and paraventricular nuclei of the hypothalamus passed through nerve fibers, was stored in nerve endings in the posterior pituitary and subsequently was released into the general circulation; the analysis and synthesis of oxytocin and vasopressin in 1954 [16] led to a Nobel Prize for du Vigneaud. In the 1950s and 1960s, evidence for the presence of hypothalamic releasing and inhibiting factors accumulated [17]. The isolation and characterization of the first of these yielded shared Nobel prizes for Guillemin and Schally in 1977 and by the mid 1980s, the isolation and characterization of many had been achieved [18–26].

Methodology Progress in the understanding of hypothalamic and pituitary endocrinology has utilized numerous techniques which have allowed the identification of hormones, their isolation and characterization, their localization, and recognition of their cell of origin. At the beginning of the 20th century, Benda [27], Erdheim [28], Cushing [6], and others [29,30] utilized histologic staining methods for the investigation of the human pituitary. Morphologic studies of the hypothalamus were 3

4

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Hypothalamic–Pituitary Function

dependent on conventional neurohistologic methods to demonstrate neurosecretory material and the silver stains that allowed visualization of neuronal cell processes [31]. The tinctorial properties of cell cytoplasm using hematoxylin–eosin, hematoxylin–phloxine, or hematoxylin– phloxine–saffron methods allowed the distinction of three cell types in the adenohypophysis. Clinical observations, correlated with morphologic changes, led to the classification of acidophils as cells thought to produce growth hormone (GH), basophils as those believed to contain adrenocorticotropin (ACTH) and chromophobes as functionally inactive cells. In the 1930s, Rasmussen [32] quantitated the three cell types in relation to sex, age, and body weight and determined their distributions within the gland. In 1940, Romeis [33] used a modification of the trichrome technique to establish that more than three cell types were identifiable in the adenohypo-physis. Application of the periodic acid–Schiff (PAS) method distinguished the PASpositive corticotrophs, thyrotrophs and gonadotrophs; similar results were obtained with lead hema-toxylin. Various trichrome stains, the aldehyde fuchsin and aldehyde thionin techniques were used to separate basophils [34] and the addition of Herlant’s erythrosin [35] and Brookes’ carmoisine [36] were useful to distinguish lactotrophs from the orange G-positive somatotrophs. Physiologic and biochemical studies provided evidence that six different hormones are produced by the anterior pituitary; however, histologic techniques failed to correlate cell type with clinical hormone production or secretory function. The search for correlations between structure and function led to the application of novel techniques including immunocytology [37,38] and electron microscopy [39,40] to the study of pituitary morphology. Ultrastructural immunocytochemistry [41], greatly improved by the use of the immunogold technique [40,42], and the application of the reverse hemolytic plaque assay [43], tissue culture methods [44] and molecular probes [45], have made pituitary and hypothalamic morphology significant for assessing pituitary function and pathology. HYPOTHALAMUS AND POSTERIOR PITUITARY

Topographic Anatomy The hypothalamus is a phylogenetically primitive structure. Understanding of its role in endocrine regulation is largely derived from animal studies; early data regarding functional localization were based on selective stimulation and ablation experiments. However, there are considerable anatomic and physiologic species variations which make extrapolation to the human difficult. Only the advent of sophisticated neurophysiologic methods and the recent applications of immunocytochemistry, as well as experiments of nature in which careful analyses of the location of pathologic lesions are performed, have allowed structure–function correlations in the human hypothalamus.

The hypothalamus is a poorly defined anatomic region which constitutes less than 1% of brain volume and weighs approximately 5 g [46,47]. The superior border of this region is the hypothalamic sulcus which courses from the interventricular foramen to the cerebral aqueduct, demarcating the hypothalamus from the thalamus (Fig. 1.1). The anterior delineation is roughly defined as a line through the anterior commissure, lamina terminalis, and optic chiasm. Posteriorly, it is bordered on the superior aspect by the midbrain tegmentum and inferiorly by the mamillary bodies. The lateral borders are defined conventionally as the substantia innominata, the internal capsule, the subthalamic nucleus, and the cerebral peduncle. The inferior aspect of the hypothalamus, known as the tuber cinereum, contains the median eminence and gives rise to the infundibulum, the neural stalk and the posterior lobe of the pituitary.

Vascular Supply A significant number of afferent and efferent signals of the hypothalamus are not neural, but rather represent bloodborne information, such as temperature, osmotic pressure, hormones, and glucose levels, thus endorsing the importance of the vascular supply of this area. The blood supply of the hypothalamus is derived from small arterial branches of the circle of Willis and the superior hypophysial artery. It is divided into three parts: (i) an anterior group which arises from the internal carotid artery, the anterior cerebral artery, and the anterior portion of the posterior communicating artery; (ii) an intermediate group from the posterior communicating artery; and (iii) a posterior group from the posterior communicating artery, posterior cerebral, and basilar arteries. Individual hypothalamic nuclei receive blood from more than one small artery. The microvascular pattern of the hypothalamus resembles that of the remainder of the brain with the exception of the magnocellular nuclei, the supraoptic and paraventricular nuclei. These are some of the most richly vascularized structures in the brain, in which the neurons are intimately associated with capillaries. The capillary endothelium is fenestrated and the blood–brain barrier is absent in many areas of the hypothalamus, including the subfornical organ, the organum vasculosum of the lamina terminalis (OVLT), the median eminence, and the neurohypophysis. The arterial supply of the median eminence and posterior pituitary has been extensively studied [48–51] (Fig. 1.2). It is derived from two (or three, in some species) paired arteries that arise from the intracranial portions of the internal carotid arteries. The superior hypophysial arteries branch into a plexus of small arteries that surrounds the upper half of the stalk (the external plexus), giving rise to a mesh of capillaries, and the gomitoli (the internal plexus). The latter are unique vascular structures composed of a central muscular artery surrounded by a spiral of capillaries, the arteriole communicating with the capillaries by way of small

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

5

FIGURE 1.1. Schematic representation of the human hypothalamus and pituitary, the disposition of hypothalamic nuclei and principal fiber tracts. From Scheithauer [47]

orifices surrounded by muscular sphincters. The gomitoli, which measure 1–2 mm in length and 0.1 mm in width, are found in large numbers in the infundibulum and proximal hypophysial stalk. Flow through these complex structures continues on through the portal vessels to the adenohypophysial capillaries. Although their function is not certain, the unique structure of gomitoli suggests that they may affect the rate of blood flow to the anterior pituitary, thereby influencing the entry of hypothalamic regulatory hormones into the portal circulation. In some individuals, a pair of middle hypophysial arteries also contributes to the trabecular or loral arteries which descend from the superior hypophysial arteries along the external surface of the pituitary stalk. These then divide to contribute to the subcapsular artery and the artery of the fibrous core (which provide a minor contribution to the blood supply of the adenohypophysis) and return upwards along the pituitary stalk as the long stalk arteries. The inferior hypophysial

arteries enter the sella just beneath its diaphragm and supply the pituitary capsule, the neural lobe, and the lower pituitary stalk. They enter the intralobar groove where they divide into ascending and descending branches that join their opposite partners to form an arterial circle about the neural lobe. They also give off a branch to the lower pituitary stalk, the communicating artery, which anastomoses with the trabecular arteries. Notably, the capillaries of the neurohypophysis are fenestrated and lie outside the blood–brain barrier. The earliest studies of the hypophysial portal vasculature assumed that blood flowed from the pituitary upwards to the hypothalamus [13]. Subsequently, it was found that the predominant blood flow is from the hypothalamus to the adenohypophysis, carrying hypothalamic regulatory factors. Recent studies suggest that the direction of blood flow is variable and there may be a component of reverse flow within the neurohypophysis, providing a route by which

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FIGURE 1.2. Schematic representation of the blood supply of the hypothalamus and pituitary. From Scheithauer [47]

neuro- and adenohypophysial hormones gain access to the long portal system, the pars tuberalis of the adenohypophysis, the median eminence, and the cerebrospinal fluid of the third ventricle [50,51].

Nerve Supply A number of afferent neural pathways provide the hypothalamus with input from the forebrain, limbic system, visual cortex, thalamus, and brain stem [47,52]. The median forebrain bundle, a large unmyelinated structure lateral to the fornix, carries information to and from the brain stem and provides important input from the limbic system and olfactory forebrain structures. Olfactory connections are more extensively from the piriform cortex and

amygdaloid nuclear complex via the stria terminalis and ventral amygdalofugal pathway. The hippocampal–hypothalamic tract originates in the hippocampus, a limbic structure, and enters the anterolateral portion of the mamillary bodies. The amygdalo–hypothalamic tract provides entry of emotional data from the amygdaloid nucleus into the hypothalamus. The retino–hypothalamic tract originates in the ganglion cells of the retina and affects the pineal gland, playing a role in the regulation of circadian rhythms. In addition, a small number of cortico–hypothalamic fibers from the posterior orbital gyrus of the frontal lobe is a potential source of input from the neocortex into the hypothalamus.

Chapter 1

The hypothalamus sends efferent projections to the forebrain, brain stem, spinal cord and neurohypophysis. The median forebrain bundle carries fibers to the septal nuclei, integrating visceral and olfactory functions. The dorsal longitudinal fasciculus carries efferents to the midbrain tegmentum where it innervates visceral and sympathetic motor neurons. Fibers emanating from the mamillary body divide into the mamillo–thalamic tract, serving the anterior nucleus of the thalamus and memory function, and the mamillotegmental tract which projects to the midline tegmentum. Efferents from the ventral medial nucleus project to the amygdaloid nucleus via the stria terminalis. The hypothalamo–hypophysial tract consists primarily of nerve fibers from the supraoptic and paraventricular nuclei carrying vasopressin, oxytocin and their neurophysins to the posterior lobe of the pituitary, where the hormones are released into capillaries. The tubero–infundibular tract, originating from neurosecretory neurons which produce hypophysiotropic hormones, projects from several nuclei to the median eminence where the hormones are released into the hypophysial portal vascular system.

Nuclei and Nucleoinfundibular Pathways Within the hypothalamus, distinct clusters of neurons are called “nuclei.” The hypothalamic nuclei are generally divided into four anatomic regions, the preoptic, supraoptic and lateral, tuberal, and mamillary regions. In general, the nuclei are topographically discrete in many species and, although relatively well demarcated in the human fetus, they are poorly defined in the mature human hypothalamus [46]. In a few cases, the existence of some nuclei is inferred entirely from studies of experimental animals. A schematic illustration is provided in Fig. 1.1. Structure–function correlations are difficult because of cellular heterogeneity of many hypothalamic nuclei. Any given hypothalamic hormone is often produced in more than one nucleus and, in many cases, a single nucleus may express more than one hormone. The physiologic roles of many nuclei remain unknown. The most anterior nuclei are the paired medial and lateral nuclei that have been associated with autonomic function, particularly temperature control and olfaction. The suprachiasmatic nucleus is found in the preoptic area dorsal to the optic chiasm and anterior to the supraoptic nucleus. This sexually dimorphic cell group shows a striking decrease in volume and cell number with age [53]. It is located in an area that is essential for gonadotropin release and sexual behavior in lower animals and may play a role in maintaining circadian rhythms. It is thought that the nucleus also plays a role in the sexual differentiation of the brain which, in the absence of male gonadal hormones, remains female but, if exposed to these at a critical stage in development, becomes male [54]. It is also associated with fibers of the supraoptic commissure, and receives afferents from the retina and the lateral geniculate bodies.

Functional Anatomy of the Hypothalamic Pituitary Axis

7

The anterior hypothalamic nucleus is composed of small neurons which mediate parasympathetic effects; it has connections to the insular cortex, substantia innominata and thalamus. The lateral hypothalamic nuclei are poorly defined areas composed of larger neurons which receive fibers from and contribute efferents to the median forebrain bundle. The paraventricular nuclei lie adjacent to the third ventricle and ventromedial to the fornix. They are composed mainly of large “magnocellular” neurons along with the supraoptic nuclei and also contain a number of “parvicellular” neurons. Efferent fibers to the pituitary stalk terminate in the posterior lobe of the pituitary. These nuclei are a major site of oxytocin and vasopressin synthesis. In patients with traumatic or surgical stalk section and in those with long-standing hypopituitarism, there is a marked reduction in the number of magnocellular neurons and stalk nerve fibers [46]; the parvicellular component remains. The supraoptic nuclei are the other paired magnocellular nuclei of the hypothalamus; they have no significant parvicellular component. They overlie the anterior optic tract and posteriorly are divided by the optic tract into anterolateral and posteromedial components, united by a thin stream of cells. Patients with stalk section or long-standing hypopituitarism also have atrophy of these nuclei [55]. The dorsomedial and ventromedial nuclei are involved in autonomic function and emotional behavior. They are situated between the tuber cinereum and paraventricular nuclei. Stimulation of the dorsomedial and destruction of the ventromedial nuclei produces rage in experimental animals. The ventromedial nuclei are concerned with control of balance between hunger and satiety. Destruction of the ventromedial nucleus results in obesity [56,57]; conversely, destruction of the ventrolateral nucleus, known as the “feeding center,” is associated with anorexia and cachexia [56]. These nuclei show a large number of afferent connections from olfactory and retinal fibers, the reticular formation, and the nucleus of the solitary tract which receives input from the vagus. Afferents from the cortex enter by way of the thalamus. Ventral to the third ventricle and paraventricular nuclei lies the arcuate (infundibular) nucleus which is another important component of the hypophysiotropic region and plays a major role in the modulation of anterior pituitary function. The subventricular nucleus lies on the floor of the third ventricle posterior to the arcuate and anteromedial to the tuberal nuclei. This usually parvicellular nucleus shows marked hypertrophy of the neurons which become magnocellular in postmenopausal women [58,59], in young women suffering from postpartum hypopituitarism with complete gonadal atrophy [60], in hypogonadal men and women, in starvation [61], after hypophysectomy [62], and in late pregnancy. The neurons develop a distinctive nucleolar change considered a manifestation of feedback effect; a similar change is also observed in neurons of the arcuate

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Hypothalamic–Pituitary Function

nucleus [60]. In some of these conditions, the change is thought to reflect lack of estrogens [59,63,64]. The tuberal nuclei consist of irregularly grouped masses of large neurons inferior to the lateral nuclei that give rise to efferent fibers of the hypothalamus. After stalk section, a slight increase in coarsely granular basophilic cytoplasmic material may occur [46]. The posterior hypothalamic nucleus, situated between the third ventricle and the mamillo–thalamic tract, lies superior to the mamillary bodies. When stimulated, it produces sympathetic effects; it may play a role in temperature regulation and its large neurons may be the source of hypothalamic efferents that descend to the reticular formation of the brain stem. The paired mamillary nuclei and a variety of minor nuclei in the supramamillary area, including the nucleus intracalatus, form the most posterior portion of the hypothalamus. These nuclei integrate incoming information from the limbic system and the midbrain tegmentum and send out efferent fibers to the anterior nucleus of the thalamus and to the brain stem.

Embryology The forebrain of the human embryo can be identified at 4 weeks postconception; by 5 weeks it has divided into a central diencephalon and two lateral telencephalic structures. Three swellings appear in the diencephalon which represent the anlages of epithalamus, thalamus, and hypothalamus. The nuclei of the hypothalamus and the supraoptico-hypophysial tract are recognizable at 8 weeks of gestation [65]. The nerve cells originate from proliferating neuroblasts and form several closely associated nuclei in the hypothalamus. At 6 weeks, a thickening of neuroblasts projects caudally to become the median eminence and infundibulum and establishes direct contact with the upward invagination of the roof of the primitive oral cavity, Rathke’s pouch. By 3 months, the rudimentary neural lobe consists of a short wide sac with a thick wall and a lumen lined by columnar ependymal cells [66]. Proliferation of spongioblasts gives rise to the modified neuroglia of the neurohypophysis known as pituicytes while the ependymal cells become dispersed in the neuroglial substance. Downgrowth of unmyelinated axons of neurons of magnocellular nuclei of the hypothalamus reaches the median eminence by 3 months, passes through the stalk and terminates in the neural lobe by 6 months of gestation. Monoamine fluoresence can be detected at 10 weeks in hypothalamic nuclei and at 13 weeks in the median eminence [67]. The supraoptic nucleus matures earlier than the paraventricular nucleus; vasopressin is demonstrated earlier and in higher concentrations than oxytocin in the neural lobe [68]. Arginine vasopressin is identified by radioimmunoassay at 12 weeks of gestation [69]. Intracellular granules have been demonstrated in hypothalamic nuclei at 19 weeks and in the posterior lobe at 23 weeks of gestation. The

neurosecretory material is clearly visible in the supraoptic and paraventricular nuclei as well as the posterior lobe at 23–28 weeks of gestation [70–72]. Ultrastructurally, clusters of small clear vesicles and axon profiles have been identified in a fetus at 7.5 weeks of age. Granular vesicles become apparent in the axon profiles at 8.5 weeks of gestation. Fenestration of capillaries and expansion of perivascular spaces are noted with the appearance of intraaxonal vesicles [73]. Growth hormone-releasing hormone (GHRH) has been identified by immunohistochemistry in the fetal hypothalamus at 18 weeks of gestation [74]. Somatostatin (somatotropin release-inhibiting hormone; SRIH) is identified in fetal hypothalami in a large molecular form at 10 weeks of gestation and there is a gradual increase in both the number of somatostatin-containing neurons and the 14-amino acid form of this peptide with fetal age [75,76]. Dopamine is present in high concentrations in the fetal hypothalamus by 15 weeks of gestation [67]. Corticotropin-releasing hormone (CRH) has been detected in fetal median eminence at 16 weeks of gestation [77]. Thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH) are the first adenohypophysiotropic substances, being detectable in fetal hypothalami at 4.5 weeks and present in significant amounts by 10 weeks of gestation [78,79]. GnRH is localized by immunocytochemistry at 13 weeks and GnRH-containing nerve terminals are detected on portal vessels at 16 weeks [80]. Sexual dimorphism of GnRH concentration has been documented and correlates with sexual dimorphism of fetal gonadotropic hormones [81].

Functional Anatomy and Control of Anterior Pituitary Function Control of anterior pituitary hormone secretion is under a complex set of central and peripheral neural and chemical signals. The hypothalamus synthesizes and secretes unique releasing and inhibitory hormones that control the anterior pituitary hormones (Fig. 1.3). The polypeptide hormones are secreted into the hypothalamic pituitary portal vasculature, impinge upon specialized anterior pituitary cells and bind to specific surface receptors. The cell signal elicits regulation of pituitary trophic hormone gene transcription, translation, and/or secretion. The resultant secretion of anterior pituitary hormones into the systemic circulation depends upon a carefully controlled dynamic balance of hypothalamic hormone input, feedback regulation from peripheral target hormones, nonhormonal neurotransmitter agents, and paracrine or autocrine pituitary growth factors and peptides. These factors all contribute to the amount of each pituitary hormone secreted, as well as to their pattern of secretion. Complex feedback regulation loops interact to regulate the hypothalamic–pituitary axis. The target gland peripheral hormone usually exerts negative feedback regulation directly at the level of pituitary synthesis or secretion of the respective trophic hormone (Fig. 1.4). Pituitary

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

9

FIGURE 1.3. Amino acid sequences of human hypothalamic hormones. CRH, corticotropin-releasing hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; SRIH, somatostatin; TRH, thyrotropin-releasing hormone.

FIGURE 1.4. General control of anterior pituitary hormone release by central and hypothalamic inputs and feedback regulation by peripheral and trophic hormones. ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; DA, dopamine; FSH, folliclestimulating hormone; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; IGF-I, insulin-like growth factor-I; LH, luteinizing hormone; PRL, prolactin; SRIH, somatostatin; TRH, thyrotropin-releasing hormone; TSH, thyrotropin; VIP, vasoactive intestinal polypeptide.

trophic hormones may feed back in a very short loop to autoregulate their own secretion. They may also participate in short loop feedback regulation of the secretion of their respective hypothalamic releasing or inhibiting hormone. Finally, peripheral hormones may participate indirectly by feedback regulation of hypothalamic releasing and inhibiting hormones.

The central nervous system also influences the control of hypothalamic hormonal secretion by efferent signals emanating from exteroceptive organs including the ears, eyes, and skin, and from interoceptive senses of indices of metabolic and electrolyte homeostasis. The transduction of neuronal inputs to neurochemical signaling by the hypothalamus appears to occur in an oscil-

10

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latory fashion. The temporal nature of pituitary hormone secretion is a manifestation of this rhythmicity resulting in at least two patterns of circadian rhythm including sleeprelated hormone secretory cycles and 24-hour rhythm cycles entrained by a light–dark cycle. The peptidergic neurons of the hypothalamus are not classified into distinct cell types and there is no known correlation between cell structure and function. In fact, neurons of any anatomic area often contain more than one neuropeptide or candidate neurotransmitter. The functional anatomy of the hypothalamus is, therefore, best approached by discussion of each hormone or peptide. GHRH

The presence of the factor responsible for stimulating release of growth hormone (GH) was known for many years, but it was only in 1982 that GHRH was isolated and characterized [25,26]. It is now known to have two forms, a 40- and a 44-amino acid peptide; the active portion of the molecule is the 29-amino acid N-terminus. GHRH binds to the surface of the somatotrophs. The intracellular actions of GHRH are probably mediated by cyclic adenosine monophosphate (cAMP), resulting in stimulation of GH gene transcription, translation, and secretion. Somatostatin attenuates the secretion of GH by the somatotroph. GH stimulates predominantly the liver to produce insulin-like growth factor-I (IGF-I) which acts as a target hormone to induce tissue growth. GH itself also acts directly on the chondrocytes and possesses antiinsulin metabolic activity. Immunocytochemical studies have identified GHRH in neuronal cell bodies of the arcuate nucleus [82,83] (Fig. 1.5). Nerve fibers from this nucleus project mainly to the median eminence, are found in the external zone, and terminate on capillaries of the plexus of the hypothalamohypophysial portal system. In monkey hypothalamus, small groups of immunoreactive neurons are also found in ventral

portions of the ventromedial nucleus. In the rat, scattered cells are also seen in the lateral basal hypothalamus, the medial and lateral portion of the ventromedial nucleus, and the dorsal medial and paraventricular nuclei [84,85]. The primary anatomic localization of GHRH to the arcuate nucleus is in agreement with earlier physiologic studies which indicated that a region including the ventromedial and arcuate nuclei was facilitatory in GH regulation. Extrapituitary effects of GHRH have been reported in the rat. Administration of small doses of the peptide into the cerebral ventricles induces behavioral sedation as well as an increase in food intake [86]. No extra-hypothalamic sites of GHRH synthesis are known to exist in the human under normal circumstances, but endocrine tumors of lung, pancreas, gut, adrenal medulla, and C-cells of thyroid can give rise to GHRH excess and acromegaly [87] (see Chapter 11). Somatostatin (SRIH)

A hypothalamic factor with GH release-inhibiting activity was first described in 1968 [88]. In 1973, Brazeau and colleagues [23] isolated and characterized a 14-amino acid peptide which they called somatostatin, or somatotropin release-inhibiting hormone (SRIH). This peptide was also shown to inhibit thyrotropin (thyroid-stimulating hormone; TSH) secretion and to produce a variety of other effects not related to its action on the pituitary. The hypothalamic distribution of SRIH shows marked species variation [89–92]. In the human hypothalamus, SRIH-containing neurons are concentrated in the anterior portion of the arcuate nucleus [90]. No positivity has been found in the paraventricular nucleus, unlike the distribution observed in rodents where SRIH is found in the paraventricular nucleus in a narrow field extending 1–2 mm from the midline. Nerve fibers staining for SRIH are observed in the medial basal hypothalamus; nerve endings are concentrated in the external zone of the median eminence and the neurovascular zone of the pituitary stalk. Fibers containing SRIH are also localized in the ventromedial, arcuate, suprachiasmatic, and paraventricular nuclei. Somatostatincontaining neurons store the hormone in dense core cytoplasmic vesicles which measure 80–110 nm in diameter and are found in both the cell bodies and the nerve endings [92]. Somatostatin is also found in extrahypothalamic regions of the rat brain, including the preoptic region, central gray mesencephalon, amygdala, olfactory area, and spinal cord. In humans it is detectable in cortex, brain stem, pineal, retina, optic nerve, auditory nerve, and spinal cord. Somatostatin is also found in pancreatic islet cells, gut endocrine cells, thyroid C-cells, salivary gland, and placenta [93–95]. Prolactin-inhibiting Factor

FIGURE 1.5. Neurons containing immunoreactive growth hormone-releasing hormone are localized by immunocytochemistry in the arcuate nucleus. (Magnification ¥64)

Prolactin (PRL) secretion by the adenohypophysis is known to be under tonic inhibition by the hypothalamus [96]. The PRL-inhibiting activity of dopamine is well documented and, currently, it is widely accepted that dopaminergic

Chapter 1

control is the main PRL regulatory function of the hypothalamus. Dopamine is the only nonpeptidergic hypothalamic compound with a well-defined hypophysiotropic function. It exerts primarily inhibitory effects on the lactotroph and inhibits both PRL gene transcription and lactotroph mitotic activity. PRL acts to induce lactation in concert with other peripheral hormones. Although TRH also stimulates lactotroph function, its effect is probably not physiologic. Vasoactive intestinal peptide (VIP) has also been shown to stimulate the production of PRL although its physiologic role has not yet been clarified. Dopamine has been localized to the arcuate and periventricular nuclei of the mediobasal hypothalamus as well as being widespread throughout the brain [97,98]. The medium-sized dopaminergic neurons are scattered among other neurosecretory cells and are further subdivided into the tuberoinfundibular group, with terminals in the median eminence and pituitary stalk, and the tuberohypophysial group, with ends terminating in the neural and intermediate lobes of the pituitary. The dopamine projections to the intermediate and neural lobes of the pituitary originate primarily in the anterior and central portions of the arcuate nucleus, respectively. Dopamine-containing axon terminals are abundant in the zona externa of the median eminence and are characterized by dense core vesicles that measure 15–120 nm in diameter. They do not appear to form true synaptic connections. CRH

Despite being the first hypothalamic regulatory factor postulated, CRH was among the last identified. In 1981, a 41-amino acid peptide was extracted from the ovine hypothalamus, characterized and shown to stimulate specifically the release of ACTH and other pituitary peptides derived from proopiomelanocortin (POMC) [24]. CRH binds to the surface of the corticotroph and elicits transcription of the POMC gene, whose products include ACTH and bendorphin. ACTH stimulates the adrenal gland to synthesize corticosteroids which are critical for essential stress responses and cellular homeostasis. Antibodies against CRH have localized this peptide to neuronal cell bodies, mainly in the parvocellular portion of the paraventricular nucleus but also in a few magnocellular neurons [99–104]. In the rat hypothalamus, scattered cell bodies containing CRH are also detected in the lateral preoptic and hypothalamic areas and in some portions of the dorsal medial nucleus. CRH-immunoreactive fibers are observed in the area of the paraventricular nucleus, the neurovascular zone of the median eminence, the pituitary stalk, and the neurohypophysis. CRH is colocalized with arginine vasopressin in a subpopulation of magnocellular neurons of the paraventricular nucleus [103,104]; it is found together with enkephalin, peptide histidine isoleucine (PHI, a member of the secretin–glucagon family) and neurotensin in parvocellular neurons as well as in a discrete subset of oxytocinergic magnocelluar neurons [104]. Adrenalectomy

Functional Anatomy of the Hypothalamic Pituitary Axis

11

increases CRH immunoreactivity in parvocellular neurons of the paraventricular nucleus, consistent with established negative feedback effects of adrenal steroids on CRH production [104]. Vasopressin can also be found in the majority of CRH-containing parvocellular neurons after adrenalectomy [104]; it has been suggested that this plasticity allows for synergy of stimulation of ACTH secretion by the two peptides [104]. CRH is also widely distributed throughout the brain in the cerebral cortex, limbic system, brain stem, and spinal cord [98–101]. It is also found extracerebrally, particularly in placenta; some investigators have also localized CRH in endocrine cells of the pancreas, gut and lung; in liver, adrenal [105], and testicular Leydig cells [106]. TRH

The first adenohypophysiotropic hypothalamic hormone identified was the tripetide TRH [17,18]. At first demonstrated to release TSH, TRH was rapidly also shown to release PRL. TRH binds to specific receptors on the thyrotroph and stimulates the transcription and secretion of TSH. TSH stimulates the thyroid gland to secrete primarily thyroxine, and triiodothyronine. The major source of triiodothyronine, however, is hepatic deiodination of thyroxine. These hormones act to regulate thermogenesis and protein synthesis. Immunocytochemical studies have localized TRH in the rat brain, in neuronal cell bodies of the preoptic nucleus, the parvocellular portion of the paraventricular nucleus, the perifornical region, dorsomedial nucleus, and basolateral hypothalamus [107,108]. TRH-positive terminals that contain dense core vesicles are found in high concentrations in the median eminence as well as in several of the hypothalamic nuclei, which also contain TRH-positive neuronal cell bodies. The tripetide is widely distributed throughout the brain and spinal cord, suggesting that it may act as a neurotransmitter in other areas [107,108]. Several effects of TRH not mediated by the pituitary gland have been demonstrated, including hyperthermia, behavioral excitation and the ability to reverse or prevent anesthesia from barbiturates and other depressants [109]. TRH has been reported in fetal and neonatal pancreatic islet cells [110] and gut endocrine cells [111], where it is thought to inhibit gastric secretion, gastric motility, and exocrine pancreatic secretion; it has also been localized in several human tumors [112]. GnRH

The second hypothalamic adenohypophysiotropic peptide to be characterized was originally called luteinizing hormone (LH)-releasing hormone (LRH) but was subsequently shown to release both gonadotropins, follicle stimulating hormone (FSH), and LH, by the pituitary gland [20–22]. The gonads are thereby stimulated to produce sex steroids and secondary sex characteristics. GnRH is also thought to stimulate sexual activity in experimental animals of both

12

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Hypothalamic–Pituitary Function

sexes, an effect that appears to be independent of its hypophysiotropic action [113]. GnRH has been localized in the hypothalami of several species [113–115]. In primates, GnRH neurons are located in large areas extending caudally from the preoptic region to the premamillary nucleus [96]. The neurons are bipolar or multipolar cells with multiple dendrites. The highest concentration of these cells is found in the mediobasal hypothalamus (infundibular and premamillary nuclei) and in the preoptic area. These neurons give rise to an infundibular GnRH pathway ending on the capillaries of the pituitary portal plexus as well as to a preopticoterminal tract projecting to the capillaries of the OVLT. In rodents, the majority of neuronal cell bodies are located in the septal preoptic regions and the anterior hypothalamus, and are particularly in the dorsal and medial septal nuclei, the nucleus of the diagonal band of Broca and anterior hypothalamic nuclei, and the OVLT. These neurons are usually elongated with eccentric nuclei and are unipolar. They project to the external zone of the median eminence as well as to the OVLT. Immunoelectron microscopy has documented localization of GnRH in dense core vesicles which measure 75–95 nm in diameter and are found in cell bodies as well as nerve endings [115]. Extrahypothalamic localization of GnRH includes several areas of the limbic system (septum, hippocampus, and olfactory bulb), the breast, and placenta [116]. Vasopressin and Oxytocin

Vasopressin, oxytocin, and their binding proteins, neurophysins I and II, are detected by immunohistochemistry in the perikarya of magnocellular neurons in the supraoptic (Fig. 1.6) and paraventricular (Fig. 1.7) nuclei [117,118]. Fibers emanating from these neurons converge towards the median eminence, pass caudally through its internal zone and end in dilated nerve terminals of the pars nervosa of

the pituitary known as Herring bodies (Fig. 1.8). The nerve fibers, their axonal swellings and the exceedingly large and characteristic swellings which can exceed 20 mm in diameter contain secretory granules of variable electron density and electron-lucent vesicles (Fig. 1.9). The secretory granules originate in the perikarya of the neurons and mature, growing from 60 to 220 nm in diameter, as they travel to the nerve endings [119,120]. It has been clearly shown that vasopressin and oxytocin are produced by different magnocellular neurons in both nuclei. Immunostaining for vasopressin is markedly increased after adrenalectomy in the external zone but not the internal zone of the rat median eminence [121,122]. Vasopressin is colocalized in some neurons with CRH and is additive with that substance in the regulation of ACTH release. Interestingly, it is found in high concentrations in portal blood draining to the anterior pituitary, suggesting that it may play a physiologic role in ACTH release [104]. A subset of oxytocin-containing neurons also may contain CRH. Immunoreactive vasopressin has been demonstrated in various other areas of the central nervous system as well as testis, ovary, uterus, adrenal, and thymus [123]. VIP

This 28-amino acid peptide, first isolated from intestine, has been localized in mammalian brain [124]. Immunoreactive fibers are present in high concentrations in the cortex, hippocampus, dentate gyrus, amygdala, nucleus of the stria terminalis, suprachiasmatic nucleus, and periaqueductal gray matter. Fibers containing immunoreactive VIP and its related PHI are also detected in areas that do not contain cell bodies. In the hypothalamus, VIP has been localized in the ventromedial nucleus, where it may affect appetite [56], in the suprachiasmatic nucleus, where it is thought to play a role in circadian rhythmicity, and in parvocellular neurons of the paraventricular nucleus [125]. It is known to modulate secretion of GH, PRL, and LH at the level of their hypothalamic regulation [126–128] and also stimulates PRL release directly [126]; some investigators have suggested that VIP is the physiologic PRL-releasing factor. After adrenalectomy, the number and intensity of VIP-containing small neurons in the paraventricular nucleus increase, suggesting a possible role for this substance in PRL regulation during stress. Cholecystokinin (CCK)

FIGURE 1.6. Perikarya and axonal processes of magnocellular neurons in the supraoptic nucleus contain strong immunopositivity for vasopressin. (Magnification ¥64)

This hormone, which was first discovered in the gut, was detected in the brain by immunocytochemistry [129–131]. CCK-containing cell bodies are prominent in the cerebral cortex and are also widely distributed throughout the olfactory and limbic systems. In the hypothalamus, they are found in several nuclei, including the magnocellular paraventricular and supraoptic nuclei. The midbrain also contains a striking concentration of CCK-immunoreactive cell bodies in the ventral tegmentum and raphe. This peptide modulates dopaminergic, noradrenergic, and opioid systems. It also inhibits feeding, probably at the level

Chapter 1

Functional Anatomy of the Hypothalamic Pituitary Axis

13

FIGURE 1.7. Adjacent to the third ventricle (V), magnocellular neurons in the paraventricular nucleus demonstrate strong immunoreactivity for vasopressin. (Magnification (a) ¥26; (b) ¥64)

FIGURE 1.8. The posterior lobe of the pituitary contains nerve fibers of the hypothalamohypophysial tract with axonal swellings and globular Herring bodies that stain with aldehyde thionin (a) and contain intense immunopositivity for vasopressin (b). (Magnification (a) ¥102; (b) ¥64)

of the ventromedial hypothalamus, and alters the pain threshold [132].

release, and to enhance GH release in response to GHRH in humans and rats [141], suggesting that hypothalamic galanin may modulate secretion of those hormones.

Galanin

Galanin is a 29-amino acid peptide that was originally isolated from intestine [133]. Immunoreactive galanin has been demonstrated in the central nervous system [134]; in hypothalamus, it colocalizes with dopamine and g-aminobutyric acid (GABA) [135]. It is also present in peripheral neural elements of the respiratory tract, gastrointestinal tract, pituitary, pancreas, urogentital tract, and adrenal medulla of humans and several mammalian species [136–139]. Galaninpositive neurons have been observed in several hypothalamic tumors [139]. Galanin is thought to act as a neurotransmitter although its functional roles have not been fully clarified. It has been shown to inhibit dopamine [140], thereby stimulating PRL

Gastrin

Gastrin is a gut peptide which is known to be produced in stomach and in numerous endocrine tumors of gut and pancreas. Specific antibodies have also localized gastrin in cerebral cortex, hypothalamus, and neurohypophysis [142,143]. Gastrin is known to modulate GH secretion at the hypothalamic level, either by reducing somatostatin release or by enhancing GHRH secretion [144]. Glucagon

Glucagon is expressed in the pancreatic islets and intestine, and has also been reported in other tissues, including thymus, thyroid, and adrenal medulla. A growing body of

14

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Hypothalamic–Pituitary Function

levels of NPY immunoreactivity are found in hypophysial portal blood. This peptide stimulates appetite when injected into the hypothalamus and appears to modulate the secretion of both LH and ACTH [150]. NPY-containing fibers also are known to contact TRH-synthesizing perikarya and neuronal processes in the paraventricular nucleus where it may mediate the neuroendocrine regulation of TRH [151]. Neurotensin

This 13-amino acid gut–brain peptide is found in the highest concentration in the ileum and hypothalamus [152]. In the rat, it is localized in the medial preoptic region, infundibular nucleus, paraventricular nucleus, and lateral hypothalamus with fibers most dense in the paraventricular and periventricular regions and in the median eminence. It has been shown to colocalize in neurons with catecholamines, possibly dopamine, in the periventricular and arcuate regions, and with CRH in the paraventricular nucleus. Some investigators have shown that it alters PRL release and blunts the response of that hormone to TRH [153]; it may also play a role in the regulation of GH and LH. Substance P

FIGURE 1.9. By electron microscopy, the normal human posterior lobe of pituitary contains numerous axonal processes filled with neurosecretory material (arrows). A large, dilated nerve terminal is the ultrastructural equivalent of the Herring body. (Magnification ¥7960)

evidence suggests that the brain is a site of glucagon biosynthesis. Neurons staining for glucagon and glucagon-like peptides have been detected in different regions of the brain including retina, hypothalamus, and medulla oblongata [145,146] and glucagon gene expression has been demonstrated in the brain stem and hypothalamus [147]. Intracerebral injections of glucagon produce dosedependent hyperglycemia, suggesting that this substance plays a role in the regulation of glucose metabolism centrally as well as peripherally [148]. Neuropeptide Y (NPY)

This 36-amino acid peptide is found by immunocytochemistry in a widely distributed pattern throughout the brain [149,150]. NPY-containing neurons are particularly numerous in the arcuate nucleus of the hypothalamus, striatum, hippocampus, and cortex. Nerve fibers are abundant in the paraventricular and suprachiasmastic nuclei and fibers are found in the internal zone of the median eminence. High

Substance P has been found in neurons in various regions of the brain. There are multiple substance P-containing afferents to the hypothalamus from the amygdala via the stria terminalis. Hypothalamic neurons containing this peptide are abundant in the arcuate, dorsomedial, ventromedial, and premamillary nuclei [152,154]. It is colocalized with vasopressin, is probably involved in pain perception within the limbic system, and may be secreted into the hypophysial portal vasculature where it is thought to affect blood flow. Substance P has been shown to alter GH regulation in vivo, but this is postulated to act via regulation of somatostatin neurons. It has variable effects on release of PRL, TSH, and LH. Bombesin/Gastrin-releasing Peptide (GRP)/Neuromedin

Bombesin was originally isolated from the skin of amphibians and stimulated great interest when it was shown to have potent biologic activity in mammals. Mammalian counterparts were soon discovered and given the names gastrin-releasing peptide and neuronedin. Bombesin-like immunoreactivity was found in neurons of the external zone of the median eminence near small blood vessels and colocalized with CRH [155]. Pharmacologic studies showed that bombesin can alter the release of adenohypophysial hormones including GH, PRL, and LH. The discovery of immunoreactivity for homologous peptides in hypothalamus suggests a possible role for these substances in the regulation of pituitary function. Calcitonin Gene-related Peptide (CGRP)

This 37-amino acid peptide is derived from the calcitonin gene by alternative messenger RNA (mRNA) processing.

Chapter 1

It is detected primarily in nervous tissue; CGRPimmunoreactive cell bodies are found in the supraoptic, paraventricular, and infundibular nuclei of the human hypothalamus [156]. CGRP inhibits GH release both in vitro and in vivo. It may also affect PRL release [157]. POMC-derived Peptides b-Endorphin

and other POMC-derived peptides are widespread in the central nervous system, their highest concentration being in the arcuate nucleus of the hypothalamus [158]. Nerve fibers containing b-endorphin and melanocyte-stimulating hormone-a (MSH-a) extend throughout many regions and innervate the median eminence, amygdala, preoptic area, ventromedial nucleus, and capillaries of the median eminence and portal system. There is another distinct and complex network of fibers containing the opiate methionine enkephalin (met-enkephalin). In addition to the multiple effects of these peptides on behavior, pain perception, immunomodulation, and many other functions too numerous to review here, they are also thought to modulate pituitary function [159], by inhibiting ACTH and LH release and stimulating PRL secretion. The effect on PRL is likely both direct and by reducing dopaminergic inhibition; the inhibition of LH appears to be only indirect via GnRH inhibition. The pituitary actions of endogenous opioids are complex, since they are themselves regulated by gonadal steroids and opioids may play a role in the negative-feedback effects of gonadal steroids on gonadotropins. Opioids also suppress the release of vasopressin and oxytocin, directly and possibly via noradrenergic regulation [160]. Cytokines

The close link between the neuroendocrine and immune systems has become widely recognized. Hormones are known to modulate the reactivity of immunocompetent cells and cytokines secreted by members of the immune system simulate secretion of hormones, most notably in the hypothalamus–pituitary–adrenal axis [161]. Interleukins are a family of peptides that mediate the immune response. Interleukin-1 (IL-1) is a monokine produced by macrophages in response to antigenic challenge. IL-1 stimulates ACTH and suppresses GH, LH and TSH secretion [162]. IL-1b immunoreactivity has been reported in neuronal fibers of the human brain, including the subfornical organ, stria terminalis, ventromedial nucleus of the hypothalamus, the posterior hypothalamus, and around the vessels of the median eminence. It is found in areas containing CRH-immunoreactive neurons and is known to increase CPH-mediated ACTH but is not found in the same synaptic vesicles as CRH. In fact, it is not released by depolarizing stimuli and it is not clear that IL-1b is even produced by neurons [161]. It also stimulates somatostatin biosynthesis in cultures of fetal rat brain [163], providing a

15

Functional Anatomy of the Hypothalamic Pituitary Axis

likely explanation for the in vivo reduction of GH and TSH secretion. Other interleukins are not known to be hypothalamic peptides but affect pituitary function. Interleukin-6 (IL-6) stimulates release of GH, PRL, and LH; tumor necrosis factor-a (TNF-a) and interferon-g also affect release of PRL and LH [164–166]. This information has significant neuroendocrinologic implications in the study of reproduction, autoimmunity, and other diseases. Growth Factors

A number of peptide growth factors, classified into several major families, are known to modulate cell proliferation. [167] Some are also known to affect hormone production (Table 1.1) and some are, in turn, modulated by hormones. Only a few have been identified in the hypothalamus and are thought to play a physiologic role in pituitary regulation; these include fibroblast growth factor (FGF) [168] and transforming growth factor-a (TGF-a) [169]. The former increases release of PRL and reduces basal GH; it also modifies the response of pituitary hormone release to TRH [170]. The latter has been localized by immunocytochemistry mainly in magnocellular neurons of the paraventricular and supraoptic nuclei of the hypothalamus. It is structurally homologous to epidermal growth factor (EGF) and may mediate some EGF effects on adenohypophysial production of GH, PRL, and TSH as well as cell proliferation [171].

Classification of Hypothalamic and Neurohypophysial Pathology Numerous pathologic processes involve the hypothalamus and neurohypophysis [46,47], giving rise to various symptoms, signs, and clinical syndromes. Among these, endocrine changes may be conspicuous. The local symptoms, such as visual defects, nerve palsy, and headache, may be secondary

Table 1.1. factors

Hormone regulation by pituitary-derived growth

Peptide

GH

PRL

EGF Activin FGF IGF-I IGF-II Insulin Endothelin Interleukin-1

Ø Ø

≠

Ø

FSH/LH

ACTH ≠

≠ ≠

Ø Ø Ø

TSH

≠

≠ ≠

≠ ≠

≠

GH, growth hormone; PRL, prolactin; TSH, thyroid-stimulating hormone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; ACTH, adrenocorticotropic hormone; EGF, epidermal growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor.

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to increased intracranial pressure or may be due to compression, invasion, injury, or destruction of tissue components. Involvement of certain parts of the hypothalamus can give rise to nonendocrine pathology, including abnormalities of appetite and temperature regulation. The endocrine sequela of hypothalamic disorders can be classified into two categories: (i) those giving rise to hypothalamic hormone deficiency; and (ii) those giving rise to hypothalamic hormone excess. The major pathologic processes causing these disorders are reviewed briefly and their features are fully discussed in ensuing chapters. Hypothalamic Hormone Deficiency

Insufficient secretion of hypothalamic adenohypophysiotropic hormones may be caused by destructive lesions of the hypothalamus. Lesions such as granulomas, meningitis or other inflammatory processes, pituitary adenomas, craniopharyngiomas, lymphomas, histiocytosis-X, metastatic carcinomas, craniocerebral trauma or hypoxic tissue injury may impair the synthesis of numerous hormones, including vasopressin, oxytocin, and the various adenohypophysiotropic hormones. These lesions give rise to true hypothalamic hypopituitarism. Similarly, lesions affecting the pituitary stalk, such as surgical or traumatic sectioning of the hypophysial stalk or its interruption by various pathologic processes, block the transport of the hypothalamic hypophysiotropic hormones from their sites of production in the hypothalamus to the anterior pituitary. It is obvious that any disconnection of the direct link between the hypothalamus and pituitary results in various degrees of hypopituitarism. Hypocortisolism, hypogonadism, and/or hypothyroidism have been demonstrated conclusively in patients with destructive diseases of the hypothalamus. Deficiency of GHRH usually results in growth retardation or in dwarfism [172]. Destructive lesions may manifest as growth retardation of hypothalamic origin in which patients respond to administration of GHRH. Idiopathic GH deficiency may be associated with other pituitary insufficiency and hypothalamic hormone abnormalities. Of particular interest is an autosomal recessive form of hypothalamic dwarfism due to failure to produce GHRH [173,174]; this disorder represents approximately 10% of cases of inherited growth retardation. The limited morphologic studies of patients with GHRH deficiency have documented that pituitary somatotrophs have normal morphology and GH content and administration of GHRH elicits a normal GH response [175]. Destructive lesions of the hypothalamus may impair the synthesis and/or release of dopamine and therefore may give rise to hyperprolactinemia [96]. Elevated blood PRL levels are also found in patients with disruption of, or damage to, the pituitary stalk. As a result of decreased transport of dopamine, lactotrophs are relieved from their tonic hypothalamic inhibition, leading to hyperprolactinemia. Hypothalamic hypothyroidism is usually not an isolated deficiency; it is most often associated with multiple hypo-

thalamic hormone deficiencies caused by organic hypothalamic pathology. Hypothalamic hypogonadism may be attributed to lack of GnRH or to nonpulsatile release of that hormone [176]. It may be an isolated defect of GnRH neurons [177]; in patients with hemochromatosis it is attributable to a combination of iron-induced damage to GnRH neurons and pituitary abnormalities [178]. Kallmann’s syndrome is an uncommon variant of hypothalamic hypogonadism which occurs in both men and women. The hypogonadism is associated with anosmia [179]. These deficits are due to defective development of the GnRH-secreting part of the hypothalamus associated with hypoplasia or aplasia of the olfactory area of the brain that can be documented by magnetic resonance imaging (MRI) [180]. Kallman’s syndrome is now known to be attributed to deletion or mutations of the KAL gene, located at Xp22.3; this gene encodes a 680 amino acid protein, anosmin, that has homology to other molecules implicated in neural development [181]. The pathology of the hypothalamus in this disorder has been thoroughly described in a histologic study in which abnormalities were found in various hypothalamic nuclei [182]; the lateral tuberal nuclei were underdeveloped and the subventricular nucleus exhibited neuronal hypertrophy. The number of pituitary gonadotrophs was markedly decreased as documented by immunohistochemistry and electron microscopy [182]. Detailed pathologic examination of the hypothalamus is yet to be reported in most patients with no major organic hypothalamic disease and apparently idiopathic deficiency of hypothalamic adenohypophysiotropic hormones. Diabetes insipidus is an uncommon but distinct clinical syndrome characterized by polyuria and polydipsia and results from vasopressin deficiency [46,47]. The extent of disease varies with the location of tissue destruction. Because vasopressin is transported through the pituitary stalk to the posterior lobe where it is stored in nerve endings, destruction of the posterior pituitary may interfere with storage and release of the hormone, resulting in its deficiency. This situation is usually transient and selective lesions of the posterior lobe usually cause only mild and temporary polyuria and polydipsia. Destruction high in the infundibulum results in permanent diabetes insipidus due to retrograde axonal degeneration and atrophy of neurons in the supraoptic and paraventricular nuclei [183]. Destruction of the supraoptic and/or paraventricular nuclei gives rise to permanent disease; approximately 90% destruction is required to cause significant and permanent disease [184], explaining why section low in the stalk, which creates atrophy of a minority of neuronal axons, causes only a transient disorder. Rare forms of hereditary idiopathic diabetes insipidus are usually unassociated with gross destruction of the hypothalamus [185]; detailed histologic studies reveal severe atrophy of neurons in the supraoptic and paraventricular nuclei in most patients but the etiology of these disorders remains unknown. In a few instances, neurons of the

Chapter 1

two magnocellular nuclei have been microscopically normal but devoid of vasopressin [186]. Some studies have suggested that this disorder may in some cases represent autoimmune destruction of vasopressin-producing neurons [187]. Oxytoxin deficiency, resulting from a decrease in the production of oxytocin, causes no major clinical abnormalities. Hypothalamic Hormone Excess

Hypersecretion of hypothalamic hormones, although rare, can result in endocrine symptomatology. Certain endocrine diseases characterized by hyperfunction of the anterior pituitary may be due primarily to adenohypophysiotropic hormone excess as a result of excessive secretion of hypothalamic releasing hormones. As yet, there is no evidence that hypersecretion of hypothalamic inhibiting hormones causes pituitary hypofunction. Acromegaly, a syndrome resulting from sustained GH hypersecretion, is almost always the result of a pituitary adenoma. Only one case of proven ectopic GH production has been reported [188]. More numerous are cases of production of GHRH by tumors that result in somatotroph hyperplasia or adenoma in the pituitary and produce clinical features of GH excess [87]. Extrahypothalamic GHRH production has been documented in endocrine tumors of lung, pancreas, thymus, medullary thyroid carcinoma, and pheochromocytoma. Primary tumors of the hypothalamus, called variously hypothalamic neuronal hamartomas, hypothalamic gangliocytomas and adenohypophysial neuronal choristomas, are lesions consisting of neurons with hypothalamic differentiation. Some of these tumors have been shown to contain GHRH and have been associated with pituitary somatotroph adenoma and acromegaly [189]. Several have been shown to be plurihormonal, containing GnRH, glucagon, gastrin, and somatostatin. Gigantism with pituitary GH excess has been described occasionally in the McCune–Albright syndrome [190]; this disorder is characterized by polyostotic fibrous dysplasia, pigmentary abnormalities and a variety of endocrine abnormalities, most commonly precocious puberty in female children. The pituitary may show mammosomatotroph hyperplasia that is attributed to activation of the signal transduction pathway generating cyclic AMP in these cells. These various manifestations are now known to be due to activating mutations of the Gs alpha gene (gsp mutations) [191,192]. Mammosomatotroph hyperplasia has also been reported as a cause of gigantism in the absence of McCune–Albright syndrome [193]. No proof of GHRH excess, either of hypothalamic or ectopic origin, has been offered in these patients; however, the pathogenetic role of GHRH in mammosomatotroph proliferation has been proven in mice transgenic for GHRH who have mammosomatotroph hyperplasia resembling that seen in these human disorders [194]. Cerebral gigantism (Sotos’ syndrome) has also been postulated to be a primary hypothalamic endocrinopathy. However, in patients with this

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disorder, hormone levels are normal and a morphologic study of the hypothalamus and pituitary showed no specific abnormality [47]. Cushing’s disease is known to be associated with pituitary corticotroph hyperplasia in some cases [195]; it has been suggested that the corticotroph hyperplasia may be secondary to excess production of CRH [196]. Ectopic production of CRH has been shown with increasing frequency and can cause Cushing’s disease; it must be considered in the differential diagnosis of that disorder [197,198]. In some cases, CRH has been elaborated in association with ACTH, suggesting autocrine regulation of some cases of ectopic ACTH syndrome. Rarely, a hypothalamic CRH-producing neuronal tumor has been reported in association with pituitary corticotroph hyperplasia or adenoma and Cushing’s disease [196,199–201]. Precocious puberty is an uncommon clinical syndrome which is in most instances idiopathic or attributable to androgen- or estrogen-producing neoplasms. Idiopathic precocious puberty is assumed to be hypothalamic in origin despite the absence of any anatomic lesion; less than 10% of cases are due to demonstrable hypothalamic pathology. Electroencephalographic abnormalities and behavioral disturbances have been observed in otherwise neurologically normal patients with this disorder [202]. Some patients respond to therapy directed against hypothalamic stimulation, but the disease may also be GnRH independent [203]. Structural lesions in and around the hypothalamus associated with precocious puberty fall into two groups. Some lesions actively participate in the production of precocious puberty by elaborating GnRH; these consist primarily of hypothalamic neuronal hamartomas which have been shown to contain GnRH in some cases [204,205]. The second group consists of hormonally inactive lesions which nonspecifically stimulate or inhibit hypothalamic centers involved in the regulation of sexual maturation. These are primarily lesions in the region of the posterior hypothalamus and include tumors of the pineal and hypothalamus, astrocytic gliomas, infections, hydrocephalus, head trauma, arachnoid cysts, and, occasionally, craniopharyngiomas. Precocious puberty is a prominent feature of the McCune–Albright syndrome [200]; in this disease, there is no demonstrable hypothalamic lesion and the patients may develop normal sexual function in adulthood. The hypothalamus has been implicated as the cause of some cases of hyperthyroidism but this has not been proven [206]. Vasopressin excess may cause water intoxication. The syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) may be due to idiopathic hypothalamic vasopressin excess or may be associated with various disease, including malignant tumors, most often bronchogenic carcinoma, and tuberculosis [207]. In the latter instances, this syndrome may be due to secretion of vasopressin or a similar peptide by tumoral or diseased pulmonary tissue [208,209]. In patients in whom no underlying cause is found, the

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idiopathic syndrome is likely due to posterior pituitary hyperfunction but no morphologic characteristics of this disorder have been described. The pathologic process in the brain may be in a number of areas; inhibitory osmoreceptors are found diffusely in the brain, as far away as the brain stem, and lesions can reduce the tonic inhibition of vasopressin, thereby increasing its release. Evidence of this widespread control is found in the increase in vasopressin release associated with congestive heart failure, cirrhosis, renal disease, brain trauma, positive pressure ventilation, and neurosurgical manipulation of the pituitary and hypothalamus. Age Related Changes in the Hypothalamus

Age related changes in the hypothalamus and neurohypophysis are few. The most striking is a morphologic curiosity, the accumulation of corpora amylacea, most often seen in the region of the supraoptic nuclei. The magnocellular nuclei are remarkably unaffected by age; immunohistochemistry shows no significant change in staining of vasopressin and oxytocin in older subjects [210]. The volume of the mamillary bodies is reduced but cell numbers appear normal [211]. Lipofuscin accumulation is commonly seen in the aging brain but does not appear to affect hypothalamic neuronal function. The suprachiasmatic nucleus decreases in volume and cell number with age [53]. The subventricular nucleus, in contrast, undergoes marked hypertrophy of its usually parvicellular neurons in postmenopausal women, as well as in starvation, posthypophysectomy, late pregnancy, and in patients suffering from prolonged postpartum hypopituitarism [58–64]. ADENOHYPOPHYSIS

Anatomy The adenohypophysis is composed of three parts, the pars distalis, the pars intermedia, and the pars tuberalis (Fig.

(a)

1.10). The pars distalis constitutes the largest portion of the gland; it is also known as the anterior lobe or the pars glandularis. The pars intermedia is rudimentary in the human pituitary; it is the vestigial posterior limb of Rathke’s pouch and is found in its underdeveloped form behind the medial cleft of the pituitary. It is also known as the intermediate lobe. The pars tuberalis is an upward extension of a few layers of adenohypophysial cells surrounding the external aspect of the lower hypophysial stalk; it is also known as the pars infundibularis. The adult human pituitary is bean shaped, bilaterally symmetrical, and measures approximately 13 mm transversely, 9 mm anteroposteriorly, and 6 mm vertically. It weighs approximately 0.6 g. The anterior lobe comprises about 80% of the pituitary. The pituitary of women is somewhat larger than that of men; moreover, the pituitary is heavier in multiparous than in nulliparous women. The increased size is attributable to the marked hyperplasia of the pituitary during pregnancy and lactation, which may increase the weight to 1 g or more; postlactational involution occurs but the gland does not return to its prepregnancy size. A slight to moderate weight reduction is seen with advancing age. The hypophysis is found within the sella turcica where it is lined by dura mater, a dense layer of connective tissue. The uppermost portion of the adenohypophysis is covered by the diaphragma sellae, a reflection of the dura that constitutes the roof of the sella turcica. The sellar diaphragm has a small central opening for the hypophysial stalk and protects the pituitary from the pressure of cerebrospinal fluid (CSF). Defective development or absence of this structure may lead to the so-called empty sella syndrome in which the pituitary is compressed by CSF pressure. The increased pressure results in enlargement of the sella turcica and flattening of the pituitary, which may be compressed to a thin layer of tissue at the bottom of the sella turcica. This lesion is usually unassociated with functional

(b)

FIGURE 1.10. (a) Sagittal section of an adult human pituitary obtained at autopsy illustrates the adenohypophysis (A) and neurohypophysis (N) attached to the pituitary stalk. (b) Horizontal cross-section of an adult human autopsy pituitary allows accurate identification of the neural lobe (N) and the anterior lobe (A) with subtle distinction between the two lateral lobes and the median wedge. The cystic remnants of Rathke’s cleft that represent the pars intermedia in humans are indistinct (arrow head).

Chapter 1

hypophysial abnormalities [206] but is important in differential diagnosis. Other anatomic variations in the shape of the hypophysis, the size and width of the sellar diaphragm, and relation to pituitary structures have been reported [207]. Minor changes have no endocrine significance.

Vascular Supply The blood circulation of the human hypophysis has major implications for the regulation of adenohypophysial hormone secretion [48–51]. The gland receives blood through the hypophysial portal circulation which carries the hypothalamic stimulatory and inhibitory hormones from the infundibulum to adenohypophysial cells. As described above (see Fig. 1.2), the branches of the superior hypophysial arteries penetrate the stalk, form a superficial network of vessels (external plexus) and give rise to a mesh of capillaries and gomitoli (internal plexus). The latter presumably regulate blood flow, thereby affecting transport of hypothalamic hormones to the adenohypophysis. The long portal vessels that arise from the infundibular plexuses and the short portal vessels that originate in the lower pituitary stalk and posterior lobe enter the pars distalis carrying adenohypophysiotropic hormones; the former carry 70–90% of the blood flow while only 10–30% originates in the short portal vessels. The trabecular or loral arteries transport blood to the adenohypophysis via the neural lobe, but, in addition, some arterial blood is directed to the adenohypophysis via two branches of the inferior hypophysial artery: (i) the capsular artery, which serves the connective tissue of the pituitary capsule and penetrates to the superficial cell rows of the adenohypophysis; and (ii) the artery of the fibrous core. In some individuals, the middle hypophysial artery may vascularize the adenohypophysis directly [212]. The volume of veins leading away from the adenohypophysis is considerably smaller than that of portal vessels entering the gland. The meager venous connections drain adenohypophysial and neurohypophysial blood to the cavernous sinus. It has been shown that the short portal vessels also serve as efferent channels, giving rise to reversal of blood flow in the neurohypophysial vascular bed. This implies that secretory products of the adenohypophysis may also enter the neurohypophysis and, by reverse flow to the median eminence, play a role in the regulation of hypothalamic factors via blood flow [50,51]. Pituitary capillaries are lined by fenestrated endothelium with a thin subendothelial space. Hormones released by adenohypophysial cells must therefore pass through the basement membrane of their cell of origin, capillary basement membrane, subendothelial space, and the endothelial cell layer to reach the bloodstream.

Nerve Supply The adenohypophysis has no direct nerve supply, apart from small sympathetic nerve fibers which are associated with and

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presumably innervate capillaries [195]. In some species, the intermediate lobe has rich innervation [213]. Thus, although neural connections may affect blood flow to the adenohypophysis, it is unlikely that they are involved in the regulation of adenohypophysial hormone secretion, which is predominantly neurohumoral.

Embryology All three portions of the adenohypophysis are assumed to derive from Rathke’s pouch, an endodermal pouch of the primitive oral cavity. At the third week of gestation, endoderm from the roof of the stomodeum thickens and begins to invaginate; by 5 weeks, Rathke’s pouch is a long tube with a narrow lumen and a thick wall composed of stratified cuboidal epithelium. By 6 weeks, the connection with the oropharynx is totally obliterated and Rathke’s pouch establishes direct contact with the downward extension of the hypothalamus that gives rise to the infundibulum. The two tissues are enclosed by the cartilage anlage of the sphenoid bone, separating them from the stomodeum; the sella turcica is formed by 7 weeks [66]. It was suggested that Rathke’s pouch arises from the ventral neural ridge in the pharyngeal region, thus sharing with the hypothalamus and posterior pituitary a common neuroectodermal origin [214–217]. Some features of adenohypophysial cells suggest that they are members of the APUD (amine precursor uptake and decarboxylation) cell system. The use of avian allografts, biologic markers, and serial sections of early chick embryos has provided indirect evidence for this theory; however, further proof is required for its validation. As the cells of Rathke’s pouch proliferate, the anterior portion forms the pars distalis and pars tuberalis whereas the posterior wall lies in direct contact with the posterior lobe anlage and becomes the pars intermedia [218,219]. The growth of the anterior limb extends laterally and follows a triradiate pattern; the lateral borders become the lateral wings of the adult gland and the midline portion becomes the anteromedial “mucoid wedge.” By midgestation, the medial cleft becomes a residual lumen and growth of the pars nervosa reverses the convexity of the posterior wall of the cleft to a concave structure. The border between Rathke’s pouch and the pars nervosa becomes indistinct; it consists of remnants of the obliterating lumen, and a few cystic cavities lined by cuboidal or columnar epithelium. This represents the rudimentary pars intermedia of the human hypophysis. The pituitary grows rapidly in early fetal life: the mean weight at 10–14 weeks of gestation is 3 mg, at 25–29 weeks 50 mg, and at term approximately 100 mg [218]. The pituitary portal vascular system begins to form before 7 weeks of gestation, and by 12 weeks the anterior pituitary and median eminence are well vascularized. Portal vessels are recognized at 11.5–14 weeks, are well developed by 15–16 weeks, and are fully established by 18–20 weeks [71,72].

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The first cell type to develop in the human fetal pituitary is the corticotroph [219–221]; at 6 weeks of gestation, cells with ultrastructural features of differentiating corticotrophs are found, and by 7 weeks, ACTH immunoreactivity is detectable. At 8 weeks of gestation, somatotrophs are identified by ultrastructural criteria and contain intense GH immunoreactivity. a-Subunit of the glycoprotein hormones is found in cells which have features of the glycoprotein hormone cell line; differentiated thyrotrophs and gonadotrophs are found subsequently at 12 weeks when b-subunits of those hormones are immunolocalized. Gonadotrophs show sexual dimorphism in the fetus; the differences are most marked from 15–25 weeks when this cell type is more numerous in female glands. Throughout gestation, LH-containing cells predominate in male glands, unlike the more equal distribution of LH- and FSHcontaining cells in female tissues [220]. Lactotrophs are the last cells to differentiate in the human fetal pituitary. PRL is found only at 12 weeks of gestation and is localized in mammosomatotrophs, bihormonal cells which appear to be the sole source of PRL until 24 weeks of gestation [221]. Differentiated lactotrophs are found after that time and undergo a striking hyperplasia in the late third trimester, analogous to that seen in late gestation and lactation in the pituitary of the mother. Novel transcription factors that play a role in anterior primordial development are being identified at a rapid pace. Many of these are implicated in early pituitary organogenesis, including the bicoid-related pituitary homeobox factor Ptx1 [222], Pituitary homeobox factor 2 (Ptx2), structurally related to Ptx1 [223], two members of the Lhx gene family, a group of LIM homeobox genes, Lhx3 and Lhx4 [224] and P-LIM, another LIM homeobox protein transcription factor that is selectively expressed in the pituitary with highest levels at the early stages of Rathke’s pouch development [225]. Another early marker of pituitary differentiation is the Rathke’s pouch homeobox (Rpx) protein which is identified in the pituitary primordium prior to the onset of known pituitary hormone production [226]. The Prophet of Pit-1 (PROP-1) is a paired-like homeodomain protein that is expressed early in pituitary development. It induces Pit-1 expression and plays a role in downregulation of Rpx [227,228]. Inactivating mutations of PROP-1 have been identified as the cause of Pit-1 deficiency in Ames dwarf mice [227] and in humans with combined pituitary hormone deficiency [229,230]. Id, a member of the helix-loop-helix (HLH) family of transcription factors is also found early in development and in some pituitary tumor cell lines but is decreased or absent in differentiated cells [231]. Its role in pituitary cytodifferentiation remains unclear. The molecular factors that determine hormone production have now been identified as transcription factors that target specific hormone genes. These factors have clarified three main pathways of cell differentiation [232,233]. ACTHproducing corticotrophs are determined by a novel T box

factor in cooperation with Ptx1 and corticotropin upstream transcription-binding element (CUTE) proteins including neuroD1/beta2 [234,235]. Bihormonal gonadotrophs require expression of steroidogenic factor (SF)-1 [236,237]. Pit-1 [238] directs differentiation of a complex family of cells that can mature into somatotrophs, mammosomatotrophs, lactotrophs or thyrotrophs [239–241] with the additional expression of estrogen receptor (ER) a [242], which enhances PRL secretion, or thyrotroph embryonic factor (TEF) which stimulates TSH-beta production [243]. The recognition of these molecular determinants of adenohypophysial cytodifferentiation has clarified the patterns of plurihormonality which have been recognized in pituitary adenomas and provide a framework for classification of these tumors. In rodents and humans, differentiation and/or maintenance of somatotroph, lactotroph and thyrotroph phenotypes are dependent on expression of a functional pit-1 gene; mutations in the pit-1 gene result in hypopituitarism [244–247] and hypoplasia of somatotrophs, lactotrophs and thyrotrophs [244]. An interesting observation is that Pit-1 mRNA and protein are highly expressed during human pituitary development at 17–19 weeks, when GH levels are extremely high, and near term when there is proliferation of lactotrophs [248]. These data suggest that Pit-1 plays an important role not only in the differentiation process, but also in the regulation of hormonal activity and possibly also of cell proliferation. Gonadotroph differentiation likewise requires SF-1; mice with disruption of SF-1 fail to develop pituitary gonadotrophs [236] as well as a ventromedial nucleus of the hypothalamus and steroidogenic glands [249].

Functional Anatomy In contrast with the hypothalamus, the cell types of the adenohypophysis are highly characterized with respect to structure and function. It is notable that with the exception of ACTH and a-subunit of the glycoprotein hormones, ectopic production of pituitary hormones is rare. Somatotrophs

Approximately 50% of the cells of the adenohypophysis produce GH. Most of these are located in the lateral wings of the anterior lobe; scattered somatotrophs are found in the median wedge [195]. These cells can usually be identified by light microscopy as medium-sized acidophilic cells that stain with eosin, phloxin, and orange G. They are spherical or oval in shape, with central, spherical nuclei. Immunohistochemistry reveals intense positivity for GH distributed throughout the cytoplasm [38] and for Pit-1 in the nucleus [221,250]. Occasionally, smaller cells contain GH positivity in a globular structure which represents the Golgi complex; these may be sparsely granulated, actively secreting cells. In situ hybridization localizes GH mRNA to both densely granulated acidophils and to occasional chromophobes (Fig. 1.11). By electron microscopy, somatotrophs are spher-

Chapter 1

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FIGURE 1.11. In situ hybridization localizes growth hormone messenger RNA in the normal pituitary in large cells with acidophilic cytoplasm and in occasional chromophobes. (Magnification ¥102)

ical or oval cells with centrally located spherical nuclei and cytoplasm of relatively low electron density [40]. The prominence of rough endoplasmic reticulum and Golgi regions varies with the secretory activity of the cell; active cells generally have well-developed, lamellar rough endoplasmic reticulum, and a large Golgi complex, whereas less active ones tend to have less conspicuous synthetic organelles. In the majority of somatotrophs, the cytoplasm is occupied by spherical, evenly electron-dense secretory granules which store hormone (Fig. 1.12). The limiting membrane of the granules is tightly opposed. The secretory granules range widely in size from 150 to 800 nm in diameter with an average of 350–500 nm. Secretory granules may be found accumulated at the cell periphery but granule extrusions are not detected. Ultrastructural immunocytology confirms the presence of GH in secretory granules and within the Golgi region of actively secreting cells. A subset of these cells has been recognized to contain a-subunit of glycoprotein hormones [40]. The incidence, distribution, morphology, and hormone content of somatotrophs are remarkably constant in the human pituitary. They do not appear to be affected by age, sex, various disease states, or drug therapies. Even in glands containing GH-producing adenomas the nontumorous somatotrophs show no evidence of suppression and cannot be distinguished from those in normal glands [40]. An exception to this morphologic uniformity is the case of long-standing hypothyroidism in which some degranulation of somatotrophs may occur; this change is much less marked than the almost complete degranulation of somatotrophs due to hypothyroidism which has been documented in rodents [250]. In pituitary adenomas, somatotrophs display a striking dimorphism [195]. Despite the morphologic differences between densely granulated and sparsely granulated soma-

FIGURE 1.12. A nontumorous somatotroph contains short profiles of rough endoplasmic reticulum (arrow head), a moderately developed Golgi complex (G) and numerous spherical, evenly electron-dense secretory granules with tightly fitted limiting membranes. (Magnification ¥5500)

totroph adenomas, both types have similar clinical features with elevated blood GH levels and acromegaly. Although the GH concentration in the tumor is proportional to the degree of granularity, neither of these correlate with blood GH levels. The morphine variant does, however, predict response to octreotide therapy [251]. Densely granulated somatotroph adenomas are composed of intensely acidophilic cells which contain strong immunoreactivity for GH throughout their cytoplasm. The tumor cells resemble resting nontumorous somatotrophs, with welldeveloped rough endoplasmic reticulum, prominent Golgi complexes and numerous spherical, evenly electron-dense secretory granules which measure 300–600 nm in diameter. These tumors may contain immunoreactivity for prolactin and/or a-subunit of glycoprotein hormones. The presence of these substances and GH in the same secretory granules has been documented by ultrastructural immunocytology using the double immunogold technique [252]. The use of keratin immunocytochemistry distinguishes these cells from

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FIGURE 1.13. A sparsely granulated adenomatous somatotroph harbors a characteristic juxtanuclear fibrous body composed of intermediate filaments that trap smooth endoplasmic reticulum, Golgi saccules (arrowheads), centrioles (arrows) and pleomorphic forming secretory granules. (Magnification ¥11390)

other somatotroph adenomas. Densely granulated cells have perinuclear low molecular weight cytokeratin filaments. Sparsely granulated somatotroph adenomas are composed predominantly of chromophobic or only slightly acidophilic cells which may show considerable nuclear pleomorphism. The nuclei are generally concave and binucleate; multinucleate cells are easily found. By light microscopy, spherical light areas can be seen adjacent to the nuclei; these correspond to the fibrous bodies seen by electron microscopy and are known to contain immunoreactive keratin [254]. The application of cytokeratin staining allows recognition of fibrous bodies that characterize this tumor [253]. GH positivity is found generally in the juxtanuclear Golgi region, while secretory granules, which are few, can be seen usually only with oil immersion. By electron microscopy, this tumor has a highly characteristic ultrastructure which differs significantly from that of nontumorous somatotrophs (Fig. 1.13). The tumor cells are irregularly shaped with numerous indentations and have lobulated pleomorphic and eccentrically located nuclei. The globular cytoplasmic

fibrous body, found in up to 95% of cells in sparsely granulated somatotroph adenomas, consists of intermediate-sized filaments that accumulate in the Golgi region, close to the concave side of an indented eccentric nucleus. The nearly perfectly globular structure traps tubular smooth endoplasmic reticulum, Golgi saccules, centrioles, mitochondria, lysosomes, and secretory granules. The secretory granules in the cells are scattered throughout the cytoplasm and are smaller than in densely granulated cells, measuring 100 – 250 nm in diameter. No exocytoses are seen. GHRH has been shown to stimulate proliferation of GH-containing cells in vitro during long-term incubations [255]. The effects of chronic exposure to GHRH in humans can be deduced from the study of pituitaries of patients with tumors producing GHRH [40,87]. These patients have marked somatotroph hyperplasia; the somatotrophs are generally large with very well-developed rough endoplasmic reticulum profiles and large Golgi complexes. The hyperplastic cells have numerous large electron-dense secretory granules characteristic of nontumorous cells; sparsely granulated cells with fibrous bodies are generally not seen. Although short-term exposure to GHRH is known to deplete the GH pool [256] and to convert densely granulated cells to more sparsely granulated forms [257], cells of densely granulated somatotroph adenomas chronically exposed to GHRH in vitro show an increase in the cytoplasmic volume densities of rough endoplasmic reticulum and Golgi complexes and a decrease in cytoplasmic volume density of secretory granules. However, they do not assume the characteristics of sparsely granulated cells and do not contain fibrous bodies [258]. Sparsely granulated adenomas undergo similar alterations during long-term exposure to GHRH in vitro and retain their characteristic fibrous bodies [258]. Although GHRH stimulates somatotroph proliferation in patients with GHRH-producing tumors, the hyperplastic adenohypophysial cell population in mice transgenic for this hormone is dominated by mammosomatotrophs as well as somatotrophs [194]. The presence of mammosomatotrophs may be attributed to exposure to GHRH excess in these animals during fetal life, at which time mammosomatotrophs are numerous, in contrast to the usual onset of tumoral GHRH excess in adulthood. Further studies are required to clarify the complex regulation of somatotroph and mammosomatotroph differentiation and proliferation by GHRH. The morphologic effects of somatostatin exposure on nontumorous somatotrophs has not been documented. The use of somatostatin analogs, such as SMS 201–995 (Sandostatin or Octreotide) in the therapy of acromegaly has allowed morphologic study of the effects of that substance on somatotroph adenoma cells. Both in vivo [259–261] and in vitro [262], somatostatin and its long acting analogs induce inconsistent morphologic alterations. In some tumors, there is no change, while in others, cell size is reportedly reduced. In some cases, there is an increase in the cytoplasmic volume density of lysosomes unassociated with significant alteration

Chapter 1

of other subcellular organelles. A few studies have indicated that these substances may induce fibrosis; others have found vascular changes which may cause cell necrosis and tumor shrinkage but direct cytotoxic effects have not been documented in vitro. No inhibition of hormone synthesis has been identified, as indicated by reduction in the cytoplasmic volume density of rough endoplasmic reticulum or Golgi complexes; this suggestion confirms molecular studies which have shown no suppression of mRNA in somatostatinexposed nontumorous adenohypophysial cells [263] or tumors [264]. The morphologic studies suggest that somatostatin suppresses hormone release and may stimulate lysosomal degradation of stored hormones. There is, however, differential response to octreotide in vivo by the two somatotroph adenoma types; densely granulated tumors exhibit greater reduction of growth hormone secretion than do sparsely granulated tumors [251]. Lactotrophs

Lactotrophs, or PRL cells, represent approximately 15% of the cells of the adenohypophysis. However, the number of these cells shows wide variation, related to age, sex, and parity in women. In adult men and nulliparous women they constitute approximately 9% of adenohypophysial cells, whereas in multiparous women they represent up to 31% of the cell population [266]. PRL cells are randomly distributed throughout the anterior lobe but are most numerous in the posteromedial portions, with large numbers extending to the posterolateral aspect [195]. Using conventional stains, PRL cells are acidophils that are indistinguishable from somatotrophs; however, Herlant’s erythrosin and Brookes’ carmoisine allow selective visualization of densely granulated lactotrophs, although these techniques are inconsistent and are not

FIGURE 1.14. Immunocytochemistry identifies prolactin diffusely in the cytoplasm of polyhedral and elongated densely granulated cells (arrow), and in the juxtanuclear globular Golgi region of sparsely granulated cells (arrow head). (Magnification ¥102)

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sufficiently sensitive to detect sparsely granulated forms. Immunohistochemistry reveals larger numbers of PRLcontaining cells among which two populations can be distinguished [38] (Fig. 1.14). Densely granulated, polyhedral, or elongated cells are frequently found close to capillaries randomly distributed throughout the anterior lobe; they have abundant cytoplasm almost completely filled with dense granular positivity corresponding to secretory granules. Sparsely granulated cells, in contrast, are more numerous and are found predominantly in clusters at the posterolateral portion of the gland. They are elongated or angular cells with long cytoplasmic processes and strong immunoreactivity for PRL in a juxtanuclear globular Golgi complex. It has been postulated that the densely granulated cells store PRL, whereas the sparsely granulated cells are actively secreting forms. In addition to nuclear Pit-1 reactivity these cells may also exhibit staining for estrogen receptor a [265a,265b]. By electron microscopy, the two cell types are readily distinguishable [40] (Fig. 1.15). Densely granulated cells are

FIGURE 1.15. A nontumorous lactotroph has abundant cytoplasm with highly developed rough endoplasmic reticulum in parallel arrays (*) and few, small secretory granules. (Magnification ¥6080)

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rare in the adult pituitary but are more common in childhood and adolescence. They have ovoid or elongated cell bodies, well-developed rough endoplasmic reticulum at the cell periphery and a spherical or oval Golgi complex containing a few forming granules. The cell cytoplasm is almost completely filled with spherical, oval, or irregularly shaped large granules that have evenly electron-dense cores and measure up to 650 nm in diameter. Granule extrusions are seen occasionally, usually on the cell surfaces overlying basement membrane. The majority of lactotrophs in the adult gland are sparsely granulated, elongated or polygonal cells which may have multiple cell processes extending from the center of acini to the basement membrane and intimately surrounding gonadotrophs. They contain richly developed rough endoplasmic reticulum found in parallel arrays and occasionally forming concentric structures known as “Nebenkern” formations. The Golgi apparatus is prominent and contains pleomorphic immature secretory granules. Scattered secretory granules are fewer and smaller than in storing cells; the diameters range from 150 to 250 nm. Granule extrusions are common and are found not only at the basal cell surface but also on the lateral cell borders, distant from capillaries and basement membranes; the term “misplaced exocytosis” is used to designate this form of granule extrusion. Ultrastructural immunocytology localizes PRL in secretory granules of both cell types; cloudy positivity can also be found in the Golgi region of sparsely granulated forms. In the fetus [220] and newborn as well as during pregnancy and lactation [266,267] there is marked hyperplasia of PRL cells which has been attributed to stimulation by estrogens. During pregnancy and lactation the weight of the pituitary gland may increase to more than 1 g and almost 50% of the total pituitary cell population is composed of PRLcontaining cells. Exogenous estrogens are known to induce only mild hyperplasia of lactotrophs in the human pituitary; the increased cell population is composed predominantly of the small sparsely granulated cell type [268a]. With sustained stimulation of PRL synthesis due to decreased dopamine inhibition or increased estrogen levels, there is a progressive increase in cytoplasmic volume and accumulation of highly organized rough endoplasmic reticulum, enlarged Golgi, numerous forming granules, and few small stored granules [40]. In animals lacking the dopamine D2 receptor, there is extensive lactotroph hyperplasia that progresses to adenoma [268b]. Suppressed lactotrophs are found in some pituitary glands containing PRL-producing adenoma or in patients treated with dopamine agonists. These inactive cells have a reduced nuclear volume, irregular indented nucleus with coarse clumped heterochromatin, small cytoplasm with poorly developed organelles, and secretory granules which measure 50–300 nm; the granules have an increased relative cytoplasmic volume due to the markedly reduced total cytoplasmic volume. Occasional exocytoses may be found at the basal cell surface. These suppressed lactotrophs are identified by immunocytology at the electron microscopic level; they

cannot be characterized on the basis of their ultrastructural features alone, which are not unlike those of null cells. PRL cell adenomas also occur in densely and, more commonly, in sparsely granulated varieties [195]. There is no correlation between morphology and blood PRL levels, age, or sex of the patient. The cells resemble the densely and sparsely granulated cells of the nontumorous pituitary. Effects of dopamine agonists on the cells of lactotroph adenomas include significant reduction of cytoplasmic, nuclear, and nucleolar areas [269]. There is also a marked reduction in the cytoplasmic volume densities of rough endoplasmic reticulum and Golgi complexes, whereas secretory granules increase in size and cytoplasmic volume density. The size reduction of bromocriptine-treated prolactinomas is therefore attributable to reduction of individual cell size rather than to a decrease in cell number. Protracted therapy may result in fibrosis which can adversely affect surgical outcome [270]. Mammosomatotrophs

The existence of bihormonal cells containing both GH and PRL in the nontumorous human pituitary has been recognized only in the last few years [40]. This cell type was initially identified in pituitary tumors [195]; it was subsequently found to be the cell of origin of PRL in the fetal pituitary during the first half of gestation [221]. Mammosomatotrophs are thought to be randomly distributed throughout the anterior lobe. They cannot be recognized using conventional histologic techniques; they are acidophils and are indistinguishable from somatotrophs. Immunohistochemistry reveals intense GH content within these densely granulated, polyhedral cells. PRL content is also identified but staining is usually less intense. Nuclear Pit-1 and ER staining is usually strong [239,240,265a,265b] and these cells often contain a-subunits [253]. By electron microscopy these cells resemble densely granulated somatotrophs (Fig. 1.16a). They are fairly large, polyhedral cells with ovoid or slightly irregular nuclei. They have abundant electron-lucent cytoplasm which contains well-developed rough endoplasmic reticulum and a prominent Golgi apparatus which often harbors numerous immature secretory granules. The mitochondria vary from ovoid to rod-shaped; they have a light matrix and lamellar cristae. The distinctive feature of these cells is their unique population of secretory granules. Some are small, spherical, or slightly ovoid, electron-dense granules which measure 150–400 nm; they have tightly fitting limiting membranes. The larger granules are irregular, often extremely elongated structures which can measure from 350 to 2000 nm; they contain secretory material of variable electron density and are bound by a loosely fitting membrane. These secretory granules frequently show misplaced exocytosis with fusion of the limiting membrane to the cell membrane. Unlike the granule extrusions of lactotrophs, the contents that are emptied into the intercellular space persist and retain electron density. Ultrastructural

Chapter 1

(a)

Functional Anatomy of the Hypothalamic Pituitary Axis

25

(b)

FIGURE 1.16. (a) By electron microscopy, mammosomatotrophs resemble densely granulated somatotrophs but have numerous conspicuous large secretory granules of irregular shape and variable electron density, as well as the hallmark of prolactin secretion, the misplaced exocytosis (arrow). (Magnification ¥7300) (b) For the definitive evidence of bihormonal differentiation of mammosomatotrophs, ultrastructural immunocytology localizes both growth hormone (15 nm gold particles) and prolactin (40 nm gold particles) to the same cell, often within the same secretory granule. (Magnification ¥20160) (b) From Felix et al. [271]

immunocytology using the double immunogold technique documents the presence of both GH and PRL in a single cell, frequently within the same secretory granule (Fig. 1.16b). Hyperplasia of mammosomatotroph cells has been identified as a cause of gigantism of early onset [193] and has been associated with acromegaly and hyperprolactinemia in the McCune–Albright syndrome [190]. Although the pathogenesis of this lesion in human patients remains unclear, an animal model for this disorder is provided by giant mice transgenic for GHRH in whom chronic exposure to excess GHRH causes mammosomatotroph hyperplasia [194]; continued long-term GHRH stimulation leads to the development of mammosomatotroph adenoma in old animals [272]. In human patients, tumors composed of mammosomatotrophs are more common than assumed in the past; they are slowly growing adenomas most often found in young patients with gigantism and mild hyperprolactinemia [195,252]. Corticotrophs

A single cell type in the human pituitary is responsible for the production of the POMC molecule and its various derivatives, including ACTH, MSH, lipotropic hormone (LPH), and endorphins [195]. Corticotrophs comprise approximately 15–20% of the adenohypophysial cell popu-

lation. The vast majority of these cells are found in clusters in the central mucoid wedge of the adenohypophysis; occasional scattered cells are also found in the lateral wings of the anterior lobe. Corticotrophs are also the predominant cell type in the poorly developed intermediate lobe of the human pituitary where they are found scattered in follicular structures. By light microscopy these medium-sized cells have varying degrees of cytoplasmic basophilia and stain strongly with PAS; the affinity is attributed to the carbohydrate moiety present in ACTH precursors. They also stain with lead hematoxylin. The most reliable method of identifying corticotrophs is the immunoperoxidase technique which reveals strong granular cytoplasmic positivity for ACTH. The presence of a large unstained perinuclear lysosomal vacuole known as the “enigmatic body” may be helpful in identifying these cells. Corticotrophs can also be identified by in situ hybridization which localizes the POMC mRNA [273]. Immunocytologic studies have indicated that almost all corticotrophs contain immunoreactive MSH, LPH, endorphins and other fragments of the POMC molecule which are derived by differing posttranslational processing [158]. By electron microscopy (Fig. 1.17), corticotroph cells are oval or slightly angular medium-sized cells with spherical or oval eccentric nuclei and a spherical nucleolus which is

26

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Hypothalamic–Pituitary Function

FIGURE 1.18. In the pituitary of a patient treated with high doses of glucocorticoids, corticotrophs with Crooke’s hyaline change have glassy cytoplasm and their periodic acid–Schiff (PAS) positive secretory material is pushed to the rim of the cell; the large juxtanuclear globule (arrow head) is the enigmatic body. (PAS stain; magnification ¥256)

FIGURE 1.17. The nontumorous corticotroph has widely dispersed short rough endoplasmic reticulum membranes, a prominent Golgi complex (G) and numerous secretory granules of variable size, shape and electron density. The two highly characteristic features of this cell type are the perinuclear and cytoplasmic bundles of intermediate filaments (arrows) and the large lysosomal enigmatic body (EB). (Magnification ¥5150)

usually attached to the nuclear membrane [40]. The cytoplasm has varying electron density and is frequently moderately electron-opaque. The rough endoplasmic reticulum is moderately developed and takes the form of widely dispersed membranes. Numerous free ribosomes can be found. The Golgi apparatus is spherical or flattened and is often displaced by the “enigmatic body.” This large structure is membrane-bound and has an electron-dense periphery that exhibits acid phosphatase activity, confirming its lysosomal nature [274]. Mitochondria are spherical or oviod, with lamellar or tubular cristae and a moderately electron-dense matrix. Variable numbers of intermediate filaments of the cytokeratin type [275], previously described as type I microfilaments, are found in small bundles usually adjacent to the nucleus; they measure about 7 nm in width and show no periodicity. They vary considerably in amount and are not numerous under physiologic conditions. The secretory granules are usually numerous and extremely variable in size,

shape, and electron density. They may be spherical, flattened, dented, heart shaped, or tear-drop shaped. They vary in electron density and may be 150–700 nm in diameter, most measuring 150–400 nm. While secretory granules may be found lined up along the cell membrane, exocytosis is not described in this cell type. Immunoelectron microscopy identifies the various POMC-derived peptides in the secretory granules of corticotrophs; however, there is no evidence that the morphologic differences of granule populations reflect their content [38]. Corticotrophs are the first cell type to differentiate in the fetal pituitary [220,221]; however, they are found in significantly decreased numbers in anencephaly [276]; it has been suggested that after autonomous differentiation, they are dependent on hypothalamic factors for normal growth and development. Their numbers do not vary with age or changes in the hormonal environment; however, they do develop specific morphologic features that reflect changes in endocrine homeostasis. In the human pituitary exposed to glucocorticoid excess due to administration of exogenous corticosteroids or any cause of endogenous glucocorticoid hypersecretion (including ectopic secretion of ACTH), corticotrophs undergo a distinctive morphologic alteration known as Crooke’s hyaline change [195]. By light microscopy, the cells accumulate a glassy, homogeneous, slightly acidophilic substance in the cytoplasm; the PAS positivity and ACTH immunoreactivity, corresponding to secretory granules, are displaced to the perinuclear rim and the periphery of the cell [38] (Fig. 1.18). The hyaline material does not contain immunoreactive POMC derivatives. This material has been shown to be composed of keratin filaments and stains with several antibodies directed against low-molecular-weight keratin proteins [275]. By electron microscopy, Crooke’s hyaline

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27

FIGURE 1.20. Corticotrophs invade the neural lobe of the pituitary (N) in a normal age-related phenomenon that is unassociated with endocrinopathy. This case, the same as that illustrated in Fig. 1.18, shows that the population of corticotrophs participating in “basophil invasion,” like those lining the pars intermedia cysts (arrow head), do not undergo Crooke’s hyaline change. (Periodic acid–Schiff stain; magnification ¥102)

FIGURE 1.19. Corticotroph with Crooke’s change in the pituitary of a patient with cortisol excess has a ring of intermediate filaments filling the cytoplasm, surrounding the nucleus and trapping numerous secretory granules in a juxtanuclear location. (Magnification ¥5090)

material is composed of an accumulation of intermediate filaments, resembling the smaller bundles seen in nontumorous corticotrophs (Fig. 1.19). The accumulation of microfilaments can be so extensive as to occupy almost the entire cytoplasm, leaving only a small juxtanuclear Golgi region and a thin rim of secretory granules adjacent to the cell membrane. Crooke’s hyaline change is reversible [195]. In the pituitaries of patients with inadequately treated adrenal insufficiency, the corticotrophs become hypertrophied, with large nuclei, prominent nucleoli, and a poorly granulated cytoplasm. In cases of long-standing Addison’s disease, focal nodular hyperplasia of corticotrophs and corticotroph adenomas may be present [277]. The ultrastructural appearance of these “adrenalectomy” cells in the human pituitary has not been well documented. However, in adrenalectomized rats, the markedly enlarged cells contain abundant rough endoplasmic reticulum and Golgi membranes and an increased number of secretory granules [278]. Chronic administration of CRH to rats increases the number of ACTH-containing cells, but no significant ultra-

structural changes have been documented [279]. Morphometric analysis indicated that there was no change in cell area; the nuclear area increased slightly and the secretory granule diameter was greater in corticotrophs of CRHexposed animals. These data suggest that the proliferation of corticotrophs in Addison’s disease may be mediated by CRH, but the morphologic characteristics of adrenalectomy cells cannot be attributed solely to CRH excess. The pars intermedia corticotrophs also are strongly (PASpositive and exhibit intense immunostaining for ACTH and other POMC derivatives [195]. The border between the pars intermedia and the pars distalis is often indistinct; occasionally a thin layer of connective tissue is noticeable between these two portions of the adenohypophysis. On the posterior aspect of the pars intermedia, basophil cells are frequently identified, often in clusters within the neuropil of the pars nervosa. These clusters vary in number and in some cases they may be found spreading deeply into the neural lobe (Fig. 1.20). This process, known as “basophil invasion,” is found in older individuals; it is more frequent and pronounced with advancing age and is said to be more prominent in men than in women. Its functional significance is not known but it is not thought to be associated with endocrine abnormalities. Some studies have suggested that the presence of a-MSH immunoreactivity is indicative of intermediate lobe differentiation [280,281]. By electron microscopy, corticotrophs of the pars intermedia are smaller, more dense, and contain fewer intermediate filaments [192]. They are also known to be less sensitive to the feedback effect of glucocorticoids, in most cases showing absent or only mild filament accumulation in patients with glucocorticoid excess and lacking the Crooke’s hyaline change that is found in pars distalis corticotrophs of those patients [195].

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The corticotrophs of the pars intermedia and those which form the basophil invasion of the pars nervosa may give rise to a specific group of corticotroph adenomas. These tumors may represent one form of the silent corticotroph adenomas which are unassociated with signs or symptoms of ACTH excess [252]; they are probably capable of secreting biologically and immunologically active substances that remain uncharacterized. As is the case with pars intermedia corticotrophs, these tumors do not show striking accumulations of intermediate filaments and, in some cases, may lack filaments totally. The derivation of corticotroph cell adenomas from this subpopulation of corticotrophs is difficult to prove. The morphology of corticotrophs in most adenomas associated with ACTH excess resembles that of nontumorous corticotrophs [195,252]. Occasional tumors are composed of cells with Crooke’s hyaline change [252], suggesting that the adenomatous cells are sensitive to feedback inhibition by glucocorticoids. Morphologic studies of functioning corticotroph adenomas in vitro [282] confirm the response of most such tumors to CRH stimulation associated with slight ultrastructural changes indicative of increased hormone synthesis. Incubation of tumor cells with glucocorticoids inhibits ACTH release and causes accumulation of intermediate filaments; however, it remains to be seen if the threshold for suppression is altered in tumor cells.

idase technique reveals granular positivity for TSH; this stain reveals the characteristic angular shape of these cells and their long cytoplasmic processes that establish contact with the basement membrane (Fig. 1.21). Ultrastructurally, these cells are characterized by their marked angularity and well-developed cytoplasmic processes [40] (Fig. 1.22). The nucleus is spherical and often eccentric while the cytoplasm contains numerous short, slightly dilated rough endoplasmic reticulum profiles, a globoid Golgi complex and small, spherical secretory granules that measure 100–200 nm in diameter. When scanty, the secretory granules characteristically are aligned under the plasmalemma and may contain rod-shaped or irregularly electron-dense contents; more densely granulated cells have granules scattered throughout the cytoplasm. Although the numbers of thyrotrophs do not appear to vary with age, these cells undergo morphologic changes in association with altered hormonal status. In patients with primary hyperthyroidism, thyrotrophs are few and small;

Thyrotrophs

The least common cell type in the adenohypophysis is the thyrotroph. These comprise approximately 5% of the total adenohypophysial cell population and are found primarily singly or in small clusters in the anteromedial portion of the gland. These medium-sized cells are basophilic when stained with conventional dyes, contain PAS positivity, stain with aldehyde fuchsin and aldehyde thionin. The immunoperox-

FIGURE 1.21. Immunohistochemical localization of thyrotropin identifies scattered angular thyrotrophs in the nontumorous human autopsy pituitary. (Magnification ¥102)

FIGURE 1.22. Nontumorous thyrotrophs have welldeveloped cytoplasm with short, slightly dilated profiles of rough endoplasmic reticulum (arrow heads), a large Golgi region (G), small secretory granules lined up along the plasma membrane and small numerous secondary lysosomes (L). (Magnification ¥7440)

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29

subset of altered somatotrophs which are bihormonal [283]; the morphologic changes are reversible in the experimental animal. Gonadotrophs

FIGURE 1.23. In a patient with long-standing hypothyroidism, the cytoplasm of these “thyroidectomy cells” is dominated by the abundant widely dilated rough endoplasmic reticulum with flocculent contents; they also contain expanded Golgi complexes (G), few small secretory granules and prominent lysosomes (L). (Magnification ¥4380)

their ultrastructural features are not well documented. In cases of untreated primary hypothyroidism, thyrotrophs are released from the negative feedback effects of thyroid hormones and TSH secretion is increased. The number and size of thyrotrophs are increased; the enlarged cytoplasm is less strongly positive using the PAS, aldehyde fuchsin and aldehyde thionin stains, and TSH immunoreactivity is faint but diffuse. These cells exhibit strong nuclear positivity for Pit-1 [195,239]. By electron microscopy, these stimulated thyrotrophs, known as “thyroidectomy cells” or “thyroid deficiency cells,” have abundant dilated rough endoplasmic reticulum cisternae and an expanded Golgi complex, whereas the number of secretory granules is reduced [40] (Fig. 1.23). Large cytoplasmic spherical, PAS-positive lysosomal globules are prominent. With prolonged stimulation, nodular hyperplasia and thyrotroph adenomas may occur [195]; TRH may be implicated in the pathogenesis of this cell proliferation [252]. In the rat, there is evidence that at least some of the thyroidectomy cells may derive from a

The cells which produce the gonadotropins, FSH and LH are the gonadotrophs; these probably represent up to 10% of the human adenohypophysial cell population [195]. No reliable cell count is, however, available. In rats, the number of gonadotrophs containing each hormone varies with age, sex, and hormonal status [40]. Gonadotrophs are scattered throughout the pars distalis and comprise the major constituent of the pars tuberalis [284]. They stain with basic dyes, the PAS technique, aldehyde thionin, and aldehyde fuchsin. The immunoperoxidase technique reveals cytoplasmic positivity for FSH and LH, often both present in the same cell, indicating that one cell type is capable of producing both gonadotropins. There are, however, variations in the number of cells containing FSH and LH, indicating that some gonadotrophs contain only one of these two hormones and there are likely fluctuations with functional demand. Nuclear reactivity for SF-1 defines the gonadotroph population [237]. The ultrastructural features of gonadotrophs reveal a single cell population which is responsible for both FSH and LH production (Fig. 1.24). The large oval or elongated cells have spherical, eccentric nuclei which are oriented with distinct polarity at the base of the cell [40,252]. Their abundant, slightly dilated rough endoplasmic reticulum profiles contain a flocculent electron-lucent substance. The Golgi is prominent and globoid, composed of sacculi and vesicles associated with immature secretory granules. Mature secretory granules are unevenly distributed in the cytoplasm and fall into two groups which vary in size and density. The distribution of these two granule populations may show sexual dimorphism. In men, smaller secretory granules are usually numerous; they have an average diameter of 250 nm. In women, larger secretory granules predominate and measure 300–600 nm in diameter. Ultrastructural immunocytology localizes FSH and LH within the same cells and, in some cases, within the same secretory granule. Gonadotrophs are found in close proximity to the basement membrane and exhibit an intriguing intimate contact with lactotrophs. The latter frequently extend cell processes around gonadotrophs and there are intercellular junctions between the two cell types [285], suggesting paracrine interactions which are not well understood. Gonadotrophs are found in the pituitaries of fetuses and show sex-related dimorphism during gestation [220]. Between 15 and 25 weeks of gestation, pituitaries of female fetuses contain more numerous gonadotrophs than pituitaries of male fetuses. Throughout gestation, LH-containing cells predominate in male pituitaries, whereas the number of LHand FSH-containing cells are almost equal in females. This dimorphism correlates with differences in the levels of hypothalamic GnRH at the same stages of gestation. In the adult

30

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Hypothalamic–Pituitary Function

FIGURE 1.24. A nontumorous gonadotroph is large and oval with short profiles of dilated rough endoplasmic reticulum containing electron-lucent contents (*), ring-like Golgi complex (G) and large, dense but irregular secretory granules. (Magnification ¥8170)

pituitary, no such dimorphism of gonadotroph numbers has been identified; however, the ultrastructural differences in males and females have been described above. Gonadotroph adenomas of the adult pituitary show an even more striking fine-structural sexual dimorphism [195,286]. Gonadotroph adenomas in women are usually well-differentiated tumors consisting of polyhedral cells with long cytoplasmic processes, dilated profiles of well-developed endoplasmic reticulum, and the characteristic “honeycomb” Golgi apparatus, a large saccular structure associated with developing secretory granules. The secretory granules in this tumor type are spherical and membrane-bound with varying electron density; they measure about 400–450 nm in diameter. The tumors in adult men are less differentiated, with poorly to moderately developed rough endoplasmic reticulum profiles, a moderately developed Golgi complex and fewer, generally smaller secretory granules. Strong nuclear SF-1 positivity is a reliable marker [237]. Gonadotrophs of the pars distalis show morphologic changes which reflect their hormonal environment; in con-

trast, the gonadotrophs of the pars tuberalis show signs of functional inactivity in most circumstances. In patients treated with pharmacologic doses of estrogen, the gonadotroph cells are small and dense [195]. During pregnancy, the number of cells immunoreactive for FSH and/or LH is also significantly reduced [264]. Gonadotrophs are morphologically abnormal in Kallmann’s syndrome, an uncommon variant of hypothalamic hypogonadism due to GnRH deficiency and associated with anosmia [182]; there is a marked decrease in the number of these cells and the remaining small cells are only weakly positive for FSH and LH. Castration leads to characteristic morphology in gonadotrophs [40,195,287]. Prolonged lack of the negative feedback effect of gonadal steroids results in stimulation of gonadotrophs to secrete FSH and LH in higher quantities. These stimulated cells are enlarged and are found in increased numbers. They have vacuolated cytoplasm which may take the form of several small vacuoles or one large vacuole; the latter displaces the nucleus to the cell periphery, giving the cell a “signet ring” appearance. By electron microscopy, these so-called “gonadectomy cells,” “gonadal deficiency cells,” or “castration cells” (Fig. 1.25) have markedly dilated rough endoplasmic reticulum, which is responsible for the cytoplasmic vacuoles seen by light microscopy. The Golgi complex is enlarged and hormone storage is decreased but not lost, despite the marked increase in secretory activity, since secretory granules are present in reduced numbers. It has been suggested that secretory granule formation is bypassed and that discharge of hormone takes place in an unconventional way not discernible by electron microscopy. An unusual prominence of large active gonadotrophs has been noted in the nontumorous adenohypophysis of some women with PRL-producing adenomas [40]. The factors underlying this change are not clear, but it has been suggested as the explanation for the LH hyperresponse to GnRH which has been found in these patients. Follicular Cells

Follicular cells are found throughout the adenohypophysis surrounding follicles, lumina lined mainly by agranular or poorly granulated cells that are joined at their apex by junctional complexes [195,288]. By electron microscopy, it was shown that granulated adenohypophysial cells can form follicles around damaged cells which have disrupted cell membranes. The surrounding cells form specialized intercellular attachments and the participating cells undergo degranulation and dedifferentiation. These follicular cells may derive from somatotrophs, lactotrophs, or corticotrophs. The follicular cells form specialized junctions, macula adhaerens, between each other and with adjacent granulated adenohypophysial cells; in contrast, granulated adenohypophysial cells form only the less prominent zonulae adhaerentes. Follicles are found in areas with increased cell destruction, particu-

Chapter 1

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31

FIGURE 1.26. Immunolocalization of S-100 protein identifies a pituitary stellate cell with long branched cytoplasmic processes surrounding granulated adenohypophysial cells. (Magnification ¥256)

FIGURE 1.25. After castration, “gonadectomy cells” have markedly dilated rough endoplasmic reticulum and large globoid Golgi regions (G) which harbor forming secretory granules. (Magnification ¥4620)

been implicated as the source of FGF and vascular endothelial growth factor [293], but are not found in the pituitaries of anencephalics [294]. They are numerous in the compressed adenohypophysis at the periphery of adenomas. Some investigators have found no S-100-reactive sustentacular cells within pituitary adenomas [290]; in contrast, others have identified S-100-containing cells within tumors of several types [295]. They are also found in large numbers at the periphery of other pituitary lesions, such as abscesses, amyloid deposits and in the residual hypophysis after surgery, but not adjacent to metastatic tumor deposits, infarcts, or Rathke’s cleft cysts [296]. Null Cells

larly surrounding tumors [40]. They are assumed to play a role in isolating and processing cell debris. Some pituitary adenomas contain follicles. Folliculostellate Cells

Immunocytochemical studies have localized S-100 protein to a specific subtype of cells in the normal human pituitary gland [289,290]. These cells are agranular and do not contain immunoreactive hormones. Some also contain immunoreactivity for glial fibrillary acidic protein (GFAP). They have a characteristic morphology, with long, branched cytoplasmic processes embracing granulated adenohypophysial cells (Fig. 1.26). Because of confusion with the follicular cells described above, some authors have suggested that these cells be called “stellate cells” [289]. They are believed to have a supportive role similar to that played by the S-100-positive sustentacular cells of the adrenal medulla and carotid body. In addition, they are thought to play a role in paracrine regulation [291] and have been shown to produce IL-6, a cytokine which may participate in local regulation of hormone secretion [292]. These cells have also

The term null cell was used to identify cells that show evidence of adenohypophysial differentiation, possessing organelles required for hormone synthesis and storage, but whose differentiation is incomplete and does not resemble any of the known adenohypophysial cell types [297]. The term was applied initially to a group of tumors which possessed no histologic, immunocytochemical, or ultrastructural markers revealing their cellular differentiation and which were unassociated with clinical evidence of hormone excess. Application of increasingly sensitive techniques of cell identification have shown that most tumors with morphologic features of null cells are in fact gonadotroph adenomas [298,237]; however, only a small subpopulation of tumor cells may be hormonally active at any one time, as indicated by the reverse hemolytic plaque assay [299]. Immunohistochemical analyses [221] and studies of hormone mRNA [300] have shown that the majority of these tumors contain cells capable of producing glycoprotein hormone subunits. Focal ultrastructural differentiation along several lines has also been documented [301]. It has been suggested that these tumors may originate from uncommitted or

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committed stem cells or from dedifferentiated adenohypophysial cells. Null cells may also represent unstimulated or resting cells that are not actively engaged in hormone production and cannot be recognized with currently available investigative techniques [302]. The nontumorous pituitary contains cells that cannot be identified conclusively by ultrastructural criteria. These may represent resting cells, uncommitted or committed stem cells. In the fetal pituitary, a population of cells with features of the glycoprotein hormone cell line can be identified prior to the recognition of differentiated thyrotrophs or gonadotrophs [221]; a-subunit immunoreactivity is present in fetal glands at the same stage of gestation and b-subunits are not yet detected [220]. It has been suggested that these primitive cells which resemble null cells may represent the source of a-subunit and may be precursors of the glycoprotein hormone cell line; they may be the cell of origin of some null cell adenomas. The null cells are chromophobic and by immunocytochemistry, usually contain no hormones [195,252]. A small number of these cells may contain various pituitary hormones, most commonly a-subunit of the glycoprotein hormones followed by b-subunits of FSH and/or LH. They usually exhibit SF-1 nuclear staining, indicating gonadotrophin differentiation [237]. By electron microscopy, the cells are polyhedral with irregular nuclei. The poorly developed cytoplasm contains short, scattered rough endoplasmic reticulum profiles, an inconspicuous or only moderately developed Golgi apparatus and small, dense, rodshaped mitochondria. Secretory granules are few and small, measuring less than 250 nm in diameter, frequently with central electron-dense cores, peripheral electron-lucent halos, and a prominent limiting membrane (Fig. 1.27). Oncocytes

Oncocytes are large cells which have abundant acidophilic granular cytoplasm due to the numerous mitochondria that take up acid dyes [195,252]. By electron microscopy, they are characterized by a striking increase in the number and size of mitochondria. A varying number of oncocytic cells may be found in the normal human hypophysis [303]. The number of these cells appears to increase with advancing age, a phenomenon also found in other organs including thyroid, parathyroid, and salivary glands. Cells in hormonally active adenomas only rarely show oncocytic transformation; the tumors usually retain their fine structural and immunocytochemical markers and can be recognized as showing various degrees of oncocytic change. In contrast, null cells, which have no other identifying markers, more frequently undergo oncocytic change. Gonadotroph adenomas regularly contain a varying number of cells with oncocytic change, and the number of oncocytes in any given tumor often increases with tumor recurrence [195]. Oncocytomas are found more commonly in elderly patients. In vitro studies using tissue culture techniques [298] and the reverse hemolytic plaque

FIGURE 1.27. A null cell in the nontumorous human pituitary has poorly developed organelles in a scant cytoplasm. However, there are short profiles of rough endoplasmic reticulum (arrow heads) and a few small secretory granules, indicating the potential for hormone synthesis. This cell has no features which allow recognition of its differentiation. (Magnification ¥5950)

assay [299] have indicated that there is no functional difference between null cell adenomas and oncocytomas of the human pituitary. The factors underlying oncocytic change are unknown, but they may lie in abnormalities of the mitochondrial DNA which is independent of the cellular DNA [304]. Oncocytes are large polyhedral cells with abundant granular cytoplasm that varies from strongly acidophilic to chromophobic. These cells may show ill-defined purple coloration with trichrome stains using aniline blue; this may lead to misinterpretation as basophilia. The phosphotungstic acid–hematoxylin method is helpful to demonstrate mitochrondrial abundance. Oncocytes showing ultrastructural features of differentiated adenohypophysial cells may contain the appropriate immunoreactive hormone. The majority of oncocytes, which are thought to be derived from null cells of gonadotrophs contain no immunoreactive hormones or may contain a-subunit, FSH LH. They often have nuclear SF-1 reactivity [237].

Chapter 1

FIGURE 1.28. In the nontumorous human pituitary, an isolated oncocyte has abundant cytoplasm which is almost totally occupied by numerous spherulated mitochondria. Rough endoplasmic reticulum (arrow heads), a small Golgi complex (G) and small secretory granules suggest that the cell is capable of hormone synthesis and storage. (Magnification ¥5590)

By electron microscopy, oncocytes are readily identified as large, polyhedral cells with irregular nuclei and a cytoplasm almost totally filled with numerous mitochondria. Histologically some oncocytes are dark or acidophilic; these cells contain tightly packed oblong mitochondria with granular matrix and numerous transverse cristae. Other oncocytes may be histologically light, i.e., only slightly acidophilic or chromophobic; their crowded mitochondria appear swollen and rarefied, with loss of cristae. Oncocytes contain small, short profiles of rough endoplasmic reticulum, inconspicuous Golgi regions and few scattered secretory granules (Fig. 1.28). Others

Acidophil Stem Cells are recognized primarily in pituitary adenomas producing both GH and PRL [195,305]. They are rare and are not known to occur in the nontumorous pituitary. The cell type is assumed to derive from a common precursor of somatotrophs and lactotrophs; while

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mammosomatotrophs have been recognized as the source of GH and PRL early in gestation, no cells resembling tumorous acidophil stem cells have yet been identified in the human fetal pituitary [221]. Immunohistochemistry reveals the presence of PRL and GH in the cells. The positivity for PRL is usually stronger and patients with tumors composed of these cells have hyperprolactinemia; GH production may be reflected in the phenomenon known as “fugitive acromegaly.” By electron microscopy, these cells resemble sparsely granulated lactotrophs and have some features of somatotrophs. They are irregular in shape and have large nuclei containing fine chromatin and inconspicuous nucleoli. The cytoplasm contains scattered rough endoplasmic reticulum profiles and relatively small Golgi regions. The Golgi sacculi are flat and may contain a few forming granules. The mitochondria may be abundant, indicating oncocytic transformation, and often show focal cavitation as well as a unique mitochondrial gigantism not identified in other pituitary cell types. Giant mitochondria may reach the size of a nucleus and be visible by light microscopy as cytoplasmic vacuoles. The mitochondria retain their double limiting membranes but lose their cristae and are filled with electron-lucent granular matrix. Occasionally, they contain electron-dense tubular structures resembling tubules of centrioles and cilia. The secretory granules in this cell type are sparse and small, ranging from 50 to 300 nm. Characteristically, granule extrusions or “misplaced exocytoses” are identified, similar to those of sparsely granulated lactotrophs. In addition, these cells have a structural marker of sparsely granulated somatotrophs, the fibrous body, a juxtanuclear aggregate of smooth endoplasmic reticulum membranes, and keratin immunoreactive intermediate filaments. These features of lactotrophs and somatotrophs are frequently found in the same cell; ultrastructural immunocytochemistry confirms the localization of GH and prolactin in the same cell. A tumor type identified as silent subtype III adenoma is composed of chromophobic or slightly acidophilic cells which contain immunocytochemical evidence of multiple hormones. Staining for ACTH is generally weak, bendorphin immunoreactivity may be strong and the tumor cells may contain GH, PRL, and/or a-subunit immunoreactivity. In some cases, the tumors contain no recognizable hormones. By electron microscopy, large, elongated cells have an eccentric nucleus, a prominent nucleolus which may harbor spheridia, and a highly differentiated cytoplasm with abundant rough and smooth endoplasmic reticulum profiles and a well-developed Golgi apparatus. The secretory granules are small, measuring less than 200 nm in diameter, and have variable electron density; they are mainly spherical but occasionally irregular or dropshaped granules are found. These cells bear no similarity to any of the known nontumorous adenohypophysial cells and their cytodifferentiation is unknown. Other unclassified plurihormonal cells have been identified in the nontumorous pituitary and adenomas [40,195]. The

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most common types produce GH, a-subunit, TSH, and/or PRL in any combination. Some of these cells have ultrastructural features of somatotrophs or mammosomatotrophs, while others resemble thyrotrophs [40]. Studies of the rat pituitary have confirmed the existence of these cells and indicate a role for them in some pathologic states; for example, some somatotrophs transform into a population of thyroidectomy cells in hypothyroidism [283]. This common pattern of plurihormonality is easily explained by the expression of Pit-1, the transcription factor implicated in activation of the genes encoding GH, PRL and bTSH. However, occasionally other patterns of plurihormonality have been reported. Very rarely, cells producing POMC derivatives with ultrastructural features of corticotrophs may produce gonadotropins or other hormones both in human tumors [195,306] and in the rat nontumorous pituitary [307]. Conversely, cells with gonadotroph morphology may contain ACTH as well as gonadotropins [308]; it is intriguing, however, that ACTH, which is so commonly expressed in extrapituitary tumors, is rarely found in plurihormonal cells of the adenohypophysis.

Localization of Other Peptides in the Adenohypophysis A number of substances not initially thought to be of pituitary origin are detectable in adenohypophysial cells. Hypothalamic Adenohypophysiotropic Hormones

GHRH, somatostatin, TRH, and GnRH have been identified in pituitary adenohypophysial cells [308–311]. Large amounts of GHRH and somatostatin were measured in culture media of perifused normal pituitaries and GHsecreting adenomas and their release was stimulated by other peptides [309]. Preprosomatostatin mRNA has been detected in nontumorous adenohypophysis as well as several types of pituitary adenomas [310]. It is known that GnRH binds to gonadotrophs and becomes internalized [311]; however, it has also been shown that TRH and GnRH are released by pituitary cells maintained in culture for a prolonged period [308]. GnRH is colocalized with b-LH and some gonadotrophs also contain TRH. These data suggest production of hypothalamic peptides by de novo synthesis in a multipotential pituitary cell. Angiotensin II

Angiotensin II, as well as renin and angiotensin converting enzyme, have been localized in pituitary gonadotrophs [312]. These substances colocalize with b-LH. Angiotensin II has been shown to stimulate PRL release in vitro; this is a dosedependent phenomenon. It has also been found to stimulate ACTH and b-endorphin release. Lactotrophs and corticotrophs have high-affinity binding sites for angiotensin II. Bombesin/GRP

Bombesin-like immunoreactivity has been found in the pituitary gland [313]; however, cell localization has not been

demonstrated. Bombesin-related peptides stimulate pituitary hormone release, mediated by the hypothalamus. CGRP

CGRP is found in the pituitary in small amounts and the concentration is known to increase with age. It is unclear if this substance is present in adenohypophysial cells or in nerve terminals of the posterior lobe [314], since CGRP is found in the hypothalamus (see above). CCK

CCK is found in the adenohypophysis but in a form which differs from that present in other CCK-synthesizing tissues [315]. The pituitary is devoid of CCK-8 but contains substantial amounts of three large nonamidated pro-CCK fragments and small amounts of a-amidated CCK (CCK-58, CCK-33, component I). No immunocytochemical localization has been reported but CCK has been found in corticotroph adenomas in large amounts and in some tumors associated with acromegaly which also contained ACTH. It therefore seems that pituitary CCK derives from corticotrophs [316]. The form found in tumors is smaller and less sulfated than in the normal pituitary; tumors contain CCK-8-like forms similar to those found in brain and gut [315]. Galanin

In humans, galanin has been localized in corticotrophs of the nontumorous pituitary, including Crooke’s cells and cells of basophil invasion in the posterior lobe. Galanin is also present in some corticotroph adenomas [139,317]. In contrast, in rats, galanin mRNA is found in estrogen-induced pituitary tumors [318] and galanin has been localized in PRL- and GH-producing cells as well as some TSHcontaining cells [319]. The reason for this species-specific regulation is unknown. In the rat, galanin expression shows sexual dimorphism [319], is inducible by estrogen [318–320] and is altered by changes in adrenal, thyroid, or gonadal status [319]. It appears to be involved in the modulation of pituitary GH and PRL secretion, probably mediated by alterations in their hypothalamic regulation [140,141]. Gastrin

The neurohypophysis is known to contain gastrin [142], but there appears to be species-specific variation in the adenohypophysis. The human adenohypophysis contains only traces of this substance which is localized to corticotrophs [321] and corticotroph adenomas [322]. Analogy has been drawn to cosynthesis with POMC in other tissues; the significance of this colocalization is unknown and gastrin is not thought to act directly on the pituitary but rather in the hypothalamus. NPY

NPY immunoreactivity and mRNA are found in the pituitary and can be localized by immunocytochemistry

Chapter 1

in adenohypophysial cells consistent with a subset of thyrotrophs [323]. These cells increase in size and NPYimmunoreactivity after thyroidectomy. NPY may have autocrine or paracrine activity in the modulation of release of GH and gonadotropins. Neurotensin

Neurotensin has been localized to the anterior pituitary and is thought to be present in gonadotrophs. It is also known to be affected by thyroid status, being found in reduced amounts in both hypothyroidism and hyperthyroidism [151,324]. The role of neurotensin in the pituitary is not known. Substance P

Immunoreactive substance P has been found in the rodent anterior pituitary. In rats it is localized in gonadotrophs and lactotrophs [325], but in the guinea pig it is found in thyrotrophs [326]. Thyroidectomy increases and thyroid hormone treatment decreases the amount of substance P in the pituitary [152]; the amount also varies with levels of gonadal steriods. It is not clear if thyroid hormone and corticosteroids act directly to influence pituitary content. Substance P may play a local or paracrine role in the adenohypophysis. VIP

There is evidence that VIP is synthesized in the pituitary [327] in lactotrophs [127,328]; it is also inducible by estrogen and may be altered by other changes in the hormonal environment. Recently, it was found to be present in increased amounts in hypothyroidism [329] and VIP production by adenohypophysial cells in vitro was found to be stimulated by TRH as well as GHRH, but not by CRH or GnRH. Tissue culture studies have shown that VIP regulates tonic PRL secretion but does not affect TRH- or GHRHinduced PRL release [329]. Cytokines

Cytokines are known to modulate pituitary function and some have been shown to be produced in the hypothalamus (see above and references 161–166). IL-6 has been demonstrated in stellate cells within the pituitary [292] and is known to modulate GH and PRL responses to appropriate stimuli [164]. Growth Factors

Growth factors regulate cell replication and functional differentiation by directly altering the expression of specific genes. Several polypeptide growth factors regulate anterior pituitary hormone secretion. Growth factor action in the pituitary, however, must be understood in the context that the pituitary itself is a site of both synthesis and action of growth factors [330,331]. Several pituitary-derived growth factors are also regulated within the pituitary by peripheral hormones including triiodothyronine and hydrocortisone.

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Pituitary-derived growth factors include EGF, FGF, TGF-a and TGF-b, IGF, endothelin, and IL-1 [331]. Several partially characterized growth factors have also been described, including thyroid hormone-inducible growth factor, vascular endothelial growth factor mammary cell growth factor, chondrocyte growth factor, mammary cell growth factor, chondrocyte growth factor, and adipocyte growth factor [331]. EGF, a potent stimulator of PRL and ACTH secretion in vitro, also attenuates GH secretion [332–334]. EGF may also play a role in the regulation of LH secretion [335]. EGF has been localized in the pituitary within cells that produce glycoprotein hormones. EGF-receptor is expressed by normal human anterior pituitary and is upregulated in some adenomas, usually recurrent somatotroph tumors [336]. It has been speculated that EGF may mediate proliferation of thyrotrophs in hypothyroidism and of lactotrophs in the lactotroph hyperplasia of pregnancy [337]. TGF-a is expressed by pituitary cells where it has a membrane-anchored localization [338,339]; by immunohistochemistry it colocalizes with PRL and possibly with GH in the bovine pituitary [340]. TGF-a interacts with the EGF receptor and inhibits replication of rat pituitary tumor cells [341]. The regulation of lactotroph growth and differentiation by this substance suggests a potential role in promotion or facilitation of pituitary tumorigenesis [339] and in transgenic mice, targeting of TGF-a overexpression to lactotrophs results in lactotroph adenoma formation [343]. Activin, a member of the TGF-b gene family, suppresses GH secretion and somatotroph replication, and stimulates FSH secretion [343–346]. The inhibin subunits which comprise activin and inhibin and their mRNAs have been localized in pituitary gonadotrophs [309]. Activin-binding protein, which shows homology to follistatin, has been isolated in the rat pituitary and appears to block the action of activin [347]. Follistatin is expressed by normal gonadotrophs and is reduced or absent in gonadotroph adenomas, suggesting a role for this growth factor in the tumorigenic process [348]. The pituitary contains high concentrations of basic FGF [349]. There are several indications that the FGF family plays an important role in the regulation of pituitary function. It may be an important paracrine regulator of PRL secretion [170]; nonmitogenic concentrations of FGF stimulate TRHinduced PRL and TSH secretion in pituitary cells. Pituitaryderived FGF stimulates the replication of human pituitary cells in vitro [350]. FGF has also been implicated in the new arteriolar vessel formation reported to accompany prolactinomas [351]. FGF expression is upregulated in aggressive pituitary adenomas and this growth factor has been shown to be released by the adenoma cells [352], indicating its potential role in the control of adenohypophysial cell proliferation. Moreover, altered FGF receptors have been implicated in the dysregulated growth of pituitary tumors [353].

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Endothelin, a powerful vasoconstrictor, is expressed in the pituitary and stimulates FSH and LH secretion [354]. Pituitary-derived endothelin-3 is stimulated by IGF-I [355]. IGF-I mediates most of the peripheral growth promoting actions of GH [356]. GH-dependent IGF-I is secreted predominantly by the liver, and IGF-I participates in feedback regulation of GH gene transcription and secretion [357]. Regulation of pituitary IGF-I receptors also determines the action of the growth factor on somatotroph function [356]. IGF-I also stimulates the in vitro secretion of hypothalamic SRIH, further delineating its role in feedback regulation of GH secretion [358]. Pituitary expression of GH, IGF-I and IGF-I receptors have been documented [359,360] and may therefore mutually interact by a paracrine or autocrine feedback mechanism [361]. Regulation of endogenous or circulating pituitary growth factors and their respective receptors may be important paracrine or autocrine determinants of pituitary cell function and trophic hormone secretion. The role of these growth factors in the development of pituitary tumors is being intensively studied [232].

Classification of Adenohypophysial Pathology Many diseases can affect tissues in and adjacent to the sella turcica, giving rise to a wide spectrum of clinical pathology. The clinical problems may be manifest as pituitary hormone deficiency or pituitary hormone excess. In addition, associated problems arise from compression, invasion, injury, or destruction of other tissue components; symptoms such as visual field defects, nerve palsies and headaches are characteristic of mass effects on the hypothalamus and neurohypophysis. Adenohypophysial Hormone Deficiency

The clinical problem of pituitary hormone deficiency may be due to decreased secretion or to increased degradation of hormone. Characteristic adenohypophysial changes are often associated with abnormalities of hormone secretion. Hypopituitarism involving one, two, or more adenohypophysial hormones is usually attributed to either a decreased number of hormone-secreting cells or to reduced endocrine activity. Organic lesions such as massive necrosis, inflammatory, or neoplastic processes can replace large portions of the pituitary and account for reduction of hormone-secreting parenchyma [195]. Reduced endocrine activity can result from target gland hormone excess or administration of exogenous hormones feedback negatively to suppress hypophysial function. Alternatively, it may be due to decreased hypothalamic stimulation or interference with receptors which govern secretion of hormones. Various developmental abnormalities can reduce the amount of adenohypophysial parenchyma. Some of these have been reviewed above (see embryology). It is now recognized that mutations of PROP-1 or Pit-1 result in various patterns of pituitary hormone deficiency and hypoplasia

[229,230,233,245,247]. In pituitary aplasia, there is total absence of the adenohypophysis due to a defect in the formation of Rathke’s pouch; pituitary hypoplasia is a milder variant of this disorder. In anencephaly, the brain, including the hypophysiotropic hypothalamus, is missing; due to lack of neurohumoral control, the adenohypophysis is smaller than normal and there is marked reduction in the number of corticotrophs associated with a less striking reduction of other adenohypophysial cell types [276a]. Cornelia de Lange syndrome, a rare congenital cyst of Rathke’s pouch, may compress the adenohypophysis and lead to various degrees of hypopituitarism. Pituitary dystopia or ectopia rarely affects function. Tissue destruction leads to a decrease or absence of pituitary hormone-producing parenchyma. This may be caused by tumors, inflammatory or infiltrative processes, or vascular lesions. Symptoms of hypopituitarism develop only with reduction of more than 60% of anterior lobe tissue and is clinically significant when there is more than a 90% reduction of hormone-producing cells. Hormonally inactive tumors of the adenohypophysis represent approximately 25% of surgically removed pituitary adenomas [195]. The null cell adenomas and oncocytomas fall into this category; these cell types have been discussed above. Craniopharyngiomas are less common than pituitary adenomas [195]. They represent 3–5% of intracranial neoplasms and occur usually in children or young adults. They are more frequently suprasellar than intrasellar and can cause varying degrees of hypopituitarism due to compression or destruction of the adenohypophysis, pituitary stalk, or hypophysiotropic hypothalamus. Other primary neoplasms in and around the sella turcica include fibromas, angiomas, gliomas, meningiomas, granular cell tumors, paragangliomas, chordomas, and many others (see Chapter 15) [195]. These tumors produce no hormones. The majority are benign and may be discovered incidentally at postmortem examination. Occasionally they cause hypopituitarism and local symptoms. Primary sarcomas have been reported in patients who have previously received irradiation for the treatment of pituitary adenomas [363]. Metastatic malignancies are found in patients with widely disseminated neoplasms; reported incidences vary from 1 to 14% [47,362]; however, the true incidence of adenohypophysial involvement is not known, since small deposits are easily overlooked. Pituitary involvement is rarely manifest before patients are overwhelmed by the malignancy. Inflammatory conditions in the sella turcica may occur as acute processes, with purulent hypophysitis and abscess formation, due to severe septicemia or direct extension from adjacent tissues. Granulomatous inflammation may be seen in tuberculosis, syphilis, sarcoidosis, and giant-cell granuloma. The latter is an uncommon disorder of unknown etiology which may involve the pituitary gland selectively; it may represent localized sarcoidosis [364]. Lymphocytic hypophysitis is an autoimmune disorder which may cause

Chapter 1

extensive destruction of the adenohypophysis and subsequent hypopituitarism [365,366]. Vascular lesions such as hemorrhages may occur as complications of traumatic head injuries or in tumors. Infarction is seen focally as an incidental autopsy finding but significant lesions may be associated with diabetes mellitus, generalized carcinomas, cranio-cerebral trauma, increased intracranial pressure, infection, and in patients maintained on mechanical ventilators. Postpartum pituitary necrosis, known as Sheehan’s syndrome [367], occurs in pregnancy, during which time the pituitary is predisposed to vasospasm and infarction. When damage is significant, hypopituitarism is proportional to the degree of tissue destruction. Infiltrative disorders such as amyloidosis, hemochromatosis, and mucopolysaccharidosis, may impair adenohypophysial cell function. Hemochromatosis can give rise to selective hypogonadism due to preferential deposition of iron in pituitary gonadotrophs [368]. Feedback inhibition may cause cytologic alterations in the adenohypophysis. These changes have been described above. Selective deficiencies of adenohypophysial hormones may result from inherited genetic abnormalities or selective damage to one cell type. Isolated GH deficiencies are a genetically heterogeneous group of disorders that cause dwarfism. In most cases, there is a normal pituitary with normal numbers of granulated somatotrophs; these are attributed to GHRH deficiency or abnormality [172–175]. Selective deficiency of a specific pituitary transcription factor, Pit-1, causes selective congenital GH and PRL deficiency associated with TSH deficiency [369–371]; several animal models of pituitary dwarfism, the Snell, Jackson and Ames dwarf mice, have also been shown to have specific abnormalities of the Pit-1 gene [372]. Congenital isolated ACTH deficiency has been attributed to T pit mutations. Lymphocytic hypophysitis has been reported to cause selective destruction of corticotrophs; antibodies to other pituitary cell types are also detectable in patients with endocrine autoimmune diseases and may give rise to isolated pituitary hormone deficiency [365,366]. Short stature with features of isolated GH deficiency but elevated GH blood levels was found to be due to a deficiency of GH-binding protein, the extracellular domain of the GH receptor [373,374]. Adenohypophysial Hormone Excess

Excess hormone production by the pituitary is usually due to a pituitary tumor [195]. The most common lesion is adenoma; carcinoma occurs exceedingly rarely and may be associated with hormone excess. Hyperplasia and hypertrophy of hormone-secreting cells may result from increased stimulation by hypothalamic releasing hormones, or from loss of inhibition by hypothalamic-inhibiting hormones, or excess target organ hormones. The cytologic features of each adenoma type have been described above. The features of hyperplastic adenohypophysial cells have also been provided. Tumor-like lesions may also be associated with hypersecretion of adenohypophysial hormones. Lymphocytic

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hypophysitis may give rise to a mass lesion of the sella turcica associated with hyperprolactinemia [365,366]. The elevated PRL levels may be due to inflammation, injury of the stalk or hypothalamus or compression of those structures by the enlarged, inflamed gland. The disorder is frequently associated with pregnancy and it has been suggested that antibodies directed against hyperplastic lactotrophs may stimulate PRL release, interfere with dopamine receptors, or damage PRL cells causing release of stored PRL. Rarely, pituitary hormone excess has been associated with normal pituitary morphology [195]. The hyperfunction may be caused by intrinsic abnormalities of the cells which produce subtle changes that may not be detectable by conventional morphologic techniques.

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Functional Anatomy of the Hypothalamic Pituitary Axis

41

213 Cox BM, Baizman ER, Su T-P et al. Further studies on the nature and function of pituitary endorphins. Adv Biochem Psychopharmacol 1978;18: 183–189. 214 Ferrand R, Hraoui S. Origine exlusivement ectodermique de l’adénohypophyse chez la caille et le poulet; demonstration par la méthode des associations tissulaires interspécifiques. C R Soc Biol 1973;167:740–743. 215 Ferrand R, Pearse AGE, Polak JM, Le Douarin NM. Immunohistochemical studies on the development of avian embryo pituitary corticotrophs under normal and experimental conditions. Histochemistry 1974;38:133–141. 216 LeDouarin N. Particularités du noyau interphasique chez la caille japonaise (Coturnix coturnix japonica). Utilisation de ces particularités comme “marque biologique” dans les recherches sur les interactions tissulaires et les migrations cellulaires au cours de l’ontogénèse. Bull Biol Fr Belg 1969;103: 435–452. 217 Takor Takor T, Pearse AGE. Neuroectodermal origin of avian hypothalamohypophyseal complex: the role of the ventral neural ridge. J Embryol Exp Morphol 1975;34:311–325. 218 Daikoku S. Studies on the human foetal pituitary. 1. Quantitative observations. Tokushima J Exp Med 1958;5:200–213. 219 Baker BL, Jaffe RB. The genesis of cell types in the adenohypophysis of the human fetus as observed by immunocytochemistry. Am J Anat 1975;143: 137–162. 220 Asa SL, Kovacs K, Laszlo FA et al. Human fetal adenohypophysis. Histologic and immunocytochemical analysis. Neuroendocrinology 1986;43:308–316. 221 Asa SL, Kovacs K, Horvath E et al. Human fetal adenohypophysis. Electron microscopic and ultrastructural immunocytochemical analysis. Neuroendocrinology 1988;48:423–431. 222 Tremblay JJ, Lanctot C, Drouin J. The pan-pituitary activator of transcription, Ptxl (pituitary homeobox 1) acts in synergy with SF-1 and Pit-1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol 1998;12:428–441. 223 Gage PJ, Camper SA. Pituitary Homeobox 2, a novel member of the bicoidrelated family of homeobox genes, is a potential regulator of anterior structure formation. Hum Mol Genet 1997;6:457–464. 224 Sheng HZ, Moriyama K, Yamashita T et al. Multistep control of pituitary organogenesis. Science 1997;278:1809–1812. 225 Bach I, Rhodes SJ, Pearse RV et al. P-Lim, a LIM homeodomain factor, is expressed during pituitary organ and cell commitment and synergizes with Pit-1. Proc Natl Acad Sci USA 1995;92:2720–2724. 226 Hermesz E, Machem S, Mahon KA. Rpx: a novel anterior-restricted homeobox gene progressively activated in the prechordal plate, anterior neural plate and Rathke’s pouch of the mouse embryo. Develop 1996;122:41–52. 227 Sornson MW, Wu W, Dasen JS et al. Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 1996;384:327–333. 228 Gage PJ, Brinkmeier MLSLM, Knapp LT. The Ames dwarf gene, df, is required early in pituitary ontogeny for the extinction of Rpx transcription and initiation of lineage-specific cell proliferation. Mol Endocrinol 1996;10: 1570–1581. 229 Wu W, Cogan JD, Pfäffle RW et al. Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nature Genet 1998;18:147–149. 230 Fofanova O, Takmura N, Kinoshita E. Compound heterozygous deletion of the prop-1 gene in children with combined pituitary hormone deficiency. J Clin Endocrinol Metab 1998;83:2601–2604. 231 Jackson SM, Barnhart KM, Mellon P et al. Helix-loop proteins are present and differentially expressed in different cell lines from the anterior pituitary. Mol Cell Endocrinol 1993;96:167–176. 232 Asa SL, Ezzat S. The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev 1998;19:798–827. 233 Asa SL, Ezzat S. Molecular determinants of pituitary cytodifferentiation. Pituitary 1999;1:159–168. 234 Lamolet B, Pulichino AM, Lamonerie T et al. A pituitary cell-restricted T box factor, T pit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 2001;104:849–859. 235 Poulin G, Turgeon B, Drouin J. NeuroD1/beta2 contributes to cell-specific transcription of the proopiomelanocortin gene. Mol Cell Biol 1997;17: 6673–6682. 236 Ingraham HA, Lala DS, Ikeda Y et al. The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 1994;8:2302–2312. 237 Asa SL, Bamberger A-M, Cao B et al. The transcription activator steroidogenic factor-1 is preferentially expressed in the human pituitary gonadotroph. J Clin Endocrinol Metab 1996;81:2165–2170.

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SECTION 1

Hypothalamic–Pituitary Function

238 Rosenfeld MG. POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev 1991;5:897–907. 239 Asa SL, Puy LA, Lew AM et al. Cell type-specific expression of the pituitary transcription activator Pit-1 in the human pituitary and pituitary adenomas. J Clin Endocrinol Metab 1993;77:1275–1280. 240 Friend KE, Chiou Y-K, Laws ER Jr et al. Pit-1 messenger ribonucleic acid is differentially expressed in human pituitary adenomas. J Clin Endocrinol Metab 1993;77:1281–1286. 241 Pellegrini I, Barlier A, Gunz G et al. Pit-1 gene expression in the human pituitary and pituitary adenomas. J Clin Endocrinol Metab 1994;79:189–196. 242 Day RN, Koike S, Sakai M et al. Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. Mol Endocrinol 1990;4:1964–1971. 243 Drolet DW, Scully KM, Simmons DM et al. TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper proteins. Genes Dev 1991;5:1739–1753. 244 Li S, Crenshaw EB III, Rawson EJ et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 1990;347:528–533. 245 Tatsumi K, Miyai K, Notomi T et al. Cretinism with combined hormone deficiency caused by a mutation in the Pit-1 gene. Nature Genet 1992;1:56–58. 246 Pfäffle RW, DiMattia GE, Parks JS et al. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118–1121. 247 Radovick S, Nations M, Du Y et al. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257:1115–1118. 248 Puy LA, Asa SL. The ontogeny of pit-1 expression in the human fetal pituitary gland. Neuroendocrinology 1996;63:349–355. 249 Ikeda Y, Luo X, Abbud R et al. The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 1995;9:478–486. 250 Yang H-J, Ozawa H, Kurosumi K. Ultrastructural changes in growth hormone cells in the rat anterior pituitary after thyroidectomy as studied by immunoelectron microscopy and enzyme nistochemistry. J Clin Electr Microsc 1989;22:269–283. 251 Ezzat S, Kontogeorgos G, Redelmeier DA et al. In vitro responsiveness of morphological variants of growth hormone-producing pituitary adenomas to octreotide. Eur J Endocrinol 1995;133:686–690. 252 Horvath E, Kovacs K. The adenohypophysis. In: Kovacs K, Asa SL, eds. Functional Endocrine Pathology 2nd ed. Boston: Blackwell Science, 1998; 247–281. 253 Sano T, Ohshima T, Yanad S. Expression of glycoprotein hormones and intracytoplasmic distribution of cytokeratin on growth hormone-producing pituitary adenomas. Pathol Res Pract 1991;187:530–533. 254 Neumann PE, Goldman JE, Horoupian DS, Hess MA. Fibrous bodies in growth hormone-secreting adenomas contain cytokeratin filaments. Arch Pathol Lab Med 1985;109:505–508. 255 Billestrup N, Swanson L, Vale W. Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro. Proc Natl Acad Sci USA 1986;83: 6854–6857. 256 Richardson SB, Twente S. Evidence that diminished pituitary responsitivity to GHRF is secondary to intracellular GH pool depletion. Am J Physiol 1988;254:E358–364. 257 Loras B, Yi JY, Durand A et al. GRF et adénomes somatotropes humains. Correlations in vivo et in vitro entre la libération de GH et les aspects morphologiques et immunocytochimiques. Ann Endocrinol 1985;46: 373–382. 258 Kawakita S, Asa SL, Kovacs K. Effects of growth hormone-releasing hormone (GHRH) on densely granulated somatotroph adenomas and sparsely granulated somatotroph adenomas in vitro: a morphological and functional investigation. J Endocrinol Invest 1989;12:443–448. 259 Beckers A, Stevenaert A, Kovacs K et al. The treatment of acromegaly with SMS 201–995. Advanc Biosci 1988;69:227–228. 260 Barkan AL, Lloyd RV, Chandler WF et al. Preoperative treatment of acromegaly with long-acting somatostatin analog SMS 201–995: shrinkage of invasive pituitary macroadenomas and improved surgical remission rate. J Clin Endocrinol Metab 1988;67:1040–1048. 261 Ezzat S, Snyder PJ, Young WF et al. Octreotide treatment of acromegaly. A randomized, multicenter study. Ann Intern Med 1992;117:711–718. 262 Asa SL, Felix I, Kovacs K, Ramyar L. Effects of somatostatin on somatotroph adenomas of the human pituitary: an in vitro functional and morphological study. Endocr Pathol 1990;1:228–235.

263 Baringa M, Bilezikjian LM, Vale WW, Rosenfeld MG, Evans RM. Independent effects of growth hormone releasing factor on growth hormone release and gene transcription. Nature 1985;314:279–281. 264 Herman V, Weiss M, Becker D, Melmed S. Hypothalamic hormonal regulation of human growth hormone gene expression in somatotroph adenoma cell cultures. Endocr Pathol 1990;1:236–244. 265a Zafar M, Ezzat S, Ramyar L et al. Cell specific expression of estrogen receptor in the human pituitary and its adenomas. J Clin Endocrinol Metab 1995;80:3621–3627. 265b Friend KE, Chiou TK, Lopes MBS et al. Estrogen receptor expression in human pituitary. J Clin Endocrinol Metab 1994;78:1497–1504. 266 Asa SL, Penz G, Kovacs K, Ezrin C. Prolactin cells in the human pituitary: a quantitative immunocytochemical analysis. Arch Pathol Lab Med 1982;106: 360–363. 267 Scheithauer BW, Sano T, Kovacs K et al. The pituitary gland in pregnancy: a clinicopathologic and immunohistochemical study of 69 cases. Mayo Clin Proc 1990;65:461–474. 268a Scheithauer BW, Kovacs K, Randall RV, Ryan N. Effects of estrogen on the human pituitary: a clinicopathologic study. Mayo Clin Proc 1989;64: 1077–1084. 268b Asa SL, Kelly MA, Grandy DK, Low MJ. Pituitary lactotroph adenomas develop after prolonged lactotroph hyperplasia in dopamine D2 receptordeficient mice. Endocrinology 1999;140:5348–5355. 269 Tindall GT, Kovacs K, Horvath E, Thorner MO. Human prolactin-producing adenomas and bromocriptine: a histological, immunocytochemical, ultrastructural, and morphometric study. J Clin Endocrinol Metab 1982;55: 1178–1183. 270 Esiri MM, Bevan JS, Burke CW, Adams CBI. Effect of bromocriptine treatment on the fibrous tissue component of prolactin-secreting and nonfunctioning macroadenomas of the pituitary gland. J Clin Endocrinol Metab 1986;63:383–388. 271 Felix IA, Horvath E, Kovacs K et al. Mammosomatotroph adenoma of the pituitary associated with gigantism and hyperprolactinemia. Acta Neuropathol 1986;71:76–82. 272 Asa SL, Kovacs K, Stefaneanu L et al. Pituitary adenomas in mice transgenic for growth hormone-releasing hormone. Endocrinology 1992;131:2083–2089. 273 Lewis ME, Sherman TG, Burke S et al. Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe. Proc Natl Acad Sci USA 1986;83:5419–5423. 274 Horvath E, Ilse G, Kovacs K. Enigmatic bodies in human corticotroph cells. Acta Anat 1977;98:427–433. 275 Neumann PE, Horoupian DS, Goldman JE, Hess MA. Cytoplasmic filaments of Crooke’s hyaline change belong to the cytokeratin class. An immunocytochemical and ultrastructural study. Am J Pathol 1984;116:214–222. 276a Asa SL, Kovacs K. Functional morphology of the human fetal pituitary. Pathol Annu 1984;19(1):275–315. 276b Pilardzic D, Kovacs K, Asa SL. Pituitary morphology in human anencephalic fetuses. Neuroendocrinology 1997;65:164–172. 277 Scheithauer BW, Kovacs K, Randall RV. The pituitary gland in untreated Addison’s disease. A histologic and immunocytologic study of 18 adenohypophyses. Arch Pathol Lab Med 1983;107:484–487. 278 Siperstein ER, Allison VF. Fine structures of the cells responsible for secretion of adrenocorticotropin in the adrenalectomized rat. Endocrinology 1965;76: 70–79. 279 Asa SL, Kovacs K, Hammer GD et al. Pituitary corticotroph hyperplasia in rats implanted with a medullary thyroid carcinoma cell line transfected with a corticotropin-releasing hormone complementary deoxyribonucleic acid expression vector. Endocrinology 1992;131:715–720. 280 Coates PJ, Doniach I, Hale AC, Rees LH. The distribution of immunoreactive a-melanocyte-stimulating hormone cells in the adult human pituitary gland. J Endocrinol 1986;111:335–342. 281 McNicol AM. A study of intermediate lobe differentiation in the human pituitary gland. J Pathol 1986;150:169–173. 282 Horvath SE, Asa SL, Kovacs K et al. Human pituitary corticotroph adenomas in vitro: morphologic and functional reponses to corticotropin-releasing hormone and cortisol. Neuroendocrinology 1990;51:241–248. 283 Horvath E, Lloyd RV, Kovacs K. Propylthiouracyl-induced hypothyroidism results in reversible transdifferentiation of somatotrophs into thyroidectomy cells: a morphologic study of the rat pituitary including immunoelectron microscopy. Lab Invest 1990;63:511–520. 284 Asa SL, Kovacs K, Bilbao JM. The pars tuberalis of the human pituitary: a histologic immunohistochemical, ultrastructural and immunoelectron microscopic analysis. Virchows Archiv (Pathol Anat) 1983;399:49–59.

Chapter 1 285 Horvath E, Kovacs K. Gonadotroph adenomas of the human pituitary: sexrelated fine structural dichotomy. A histologic, immunocytochemical and electron microscopic study of 30 tumors. Am J Pathol 1984;117:429–440. 286 Denef C. Paracrine interactions in the anterior pituitary. Clin Endocrinol Metab 1986;15:1–32. 287 Kovacs K, Horvath E. Gonadotrophs following removal of the ovaries: a fine structural study of human pituitary glands. Endokrinologie 1975;66:1–8. 288 Horvath E, Kovacs K, Penz G, Ezrin C. Origin, possible function and fate of “follicular cells” in the anterior lobe of the human pituitary. Am J Pathol 1974;77:199–212. 289 Girod C, Trouillas J, Dubois MP. Immunocytochemical localization of S-100 protein in stellate cells (folliculo-stellate cells) of the anterior lobe of the normal human pituitary. Cell Tissue Res 1985;241:505–511. 290 Höfler H, Walter GF, Denk H. Immunohistochemistry of folliculo-stellate cells in normal human adenohypophyses and in pituitary adenomas. Acta Neuropathol 1984;65:35–40. 291 Baes M, Allaerts W, Denef C. Evidence for functional communication between folliculo-stellate cells and hormone-secreting cells in perifused anterior pituitary aggregates. Endocrinology 1987;120:685–691. 292 Vankelecom A, Carmeliet P, Van Damme J et al. Production of interleukin-6 by folliculo-stellate cells of the anterior pituitary gland in a histiotypic cell aggregate culture system. Neuroendocrinology 1989;49:102–106. 293 Ferrara N, Schweigerer L, Neufeld G et al. Pituitary follicular cells produce basic fibroblast growth factor. Proc Natl Acad Sci USA 1987;84: 5773–5777. 294 Coates PJ, Doniach I. Development of folliculo-stellate cells in the human pituitary. Acta Endocrinol 1988;119:16–20. 295 Lauriola L, Cocchia D, Sentinelli S et al. Immunohistochemical detection of folliculo-stellate cells in human pituitary adenomas. Virchows Archiv (Cell Pathol) 1984;47:189–197. 296 Nishioka H, Llena JF, Hirano A. Immunohistochemical study of folliculostellate cells in pituitary lesions. Endocr Pathol 1991;2:155–160. 297 Kovacs K, Horvath E, Ryan N, Ezrin C. Null cell adenomas of the human pituitary. Virchows Archiv (Pathol Anat) 1980;387:165–174. 298 Asa SL, Cheng Z, Ramyar L et al. Human pituitary null cell adenomas and oncocytomas in vitro: effects of adenohypophysiotropic hormones and gonadal steroids on hormone secretion and tumor cell morphology. J Clin Endocrinol Metab 1992;74:1128–1134. 299 Yamada S, Asa SL, Kovacs K et al. Analysis of hormone secretion by clinically nonfunctioning human pituitary adenomas using the reverse hemolytic plaque assay. J Clin Endocrinol Metab 1989;68:73–80. 300 Jameson JL, Klibanski A, Black PMcL et al. Glycoprotein hormone genes are expressed in clinically nonfunctioning pituitary adenomas. J Clin Invest 1987;80:1472–1478. 301 Kontogeorgos G, Horvath E, Kovacs K et al. Null cell adenoma of the pituitary with features of plurihormonality and plurimorphous differentiation. Arch Pathol Lab Med 1991;115:61–64. 302 Kovacs K, Asa SL, Horvath E et al. Null cell adenomas of the pituitary: attempts to resolve their cytogenesis. In: Lechago J, Kameya T, eds. Endocrine Pathology Update. Philadelphia: Field and Wood, 1990;17–31. 303 Kovacs K, Horvath E, Bilbao JM. Oncocytes in the anterior lobe of the human pituitary gland. A light and electron microscopic study. Acta Neuropathol 1974;27:43–54. 304 Carafoli E. Mitochondrial pathology: an overview. Ann NY Acad Sci 1986; 488:1–18. 305 Horvath E, Kovacs K, Singer W et al. Acidophil stem cell adenoma of the human pituitary: clinicopathological analysis of 15 cases. Cancer 1981;47: 761–771. 306 Sano T, Kovacs K, Asa SL, Smyth HS. Immunoreactive luteinizing hormone in functioning corticotroph adenomas of the pituitary. Immunohistochemical and tissue culture studies of two cases. Virchows Archiv (Pathol Anat) 1990;471: 361–367. 307 Moriarty GC, Garner LL. Immunocytochemical studies of cells in the rat adenohypophysis containing both ACTH and FSH. Nature 1977;265:356–358. 308 May V, Wilber JF, U’Prichard DC, Childs GV. Persistence of immunoreactive TRH and GnRH in long-term primary anterior pituitary cultures. Peptides 1987;8:543–558. 309 Joubert (Bression) D, Benlot C, Lagoguey A et al. Normal and growth hormone (GH)-secreting adenomatous pituitaries release somatostatin and GH-releasing hormone. J Clin Endocrinol Metab 1989;68:572–577. 310 Pagesy P, Li JY, Rentier-Delrue F et al. Evidence of pre-prosomatostatin mRNA in human normal and tumoral anterior pituitary gland. Mol Endocrinol 1989;3:1289–1294.

Functional Anatomy of the Hypothalamic Pituitary Axis

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311 Duello TM, Nett TM, Farquhar MG. Fate of a gonadotropin releasing hormone agonist internalized by rat pituitary gonadotrophs. Endocrinology 1983;112:1–10. 312 Deschepper CF, Crumrine DA, Ganong WF. Evidence that the gonadotrophs are the likely site of production of angiotensin II in the anterior pituitary of the rat. Endocrinology 1986;119:36–43. 313 Major J, Ghatei MA, Bloom SR. Bombesin-like immunoreactivity in the pituitary gland. Experientia 1983;39:1158–1159. 314 Zaidi M, Abeyasekera G, MacIntyre I. Calcitonin gene-related peptide: endocrine distribution and characterization of circulating forms. J Endocrinol Invest 1989;12:699–704. 315 Rehfeld JF. Preprocholecystokinin processing in the normal human anterior pituitary. Proc Natl Acad Sci USA 1987;84:3019–3023. 316 Rehfeld JF, Lindholm J, Andersen BN et al. Pituitary tumors containing cholecystokinin. N Engl J Med 1987;316:1244–1247. 317 Vrontakis ME, Sano T, Kovacs K, Friesen HG. Presence of galanin-like immunoreactivity in nontumorous corticotrophs and corticotroph adenomas of the human pituitary. J Clin Endocrinol Metab 1990;70:747–751. 318 Vrontakis ME, Peden LM, Duckorth MC, Friesen HG. Isolation and characterization of a complementary DNA (galanin) clone from estrogeninduced pituitary tumor messenger RNA. J Biol Chem 1987;262: 16755–16758. 319 O’Halloran DJ, Jones PM, Steel JH et al. Effect of endocrine manipulation on anterior pituitary galanin in the rat. Endocrinology 1990;127: 467–475. 320 Kaplan LM. Gabriel SM, Koenig JI et al. Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 1988;85:7408–7412. 321 Larson LI, Rehfeld JF. Pituitary gastrins occur in corticotrophs and melanotrophs. Science 1981;213:768–770. 322 Bardram L, Lindholm J, Rehfeld JF. Gastrin in pituitary tumors. Acta Endocrinol 1987;115:419–422. 323 Jones PM, Ghatei MA, Steel J et al. Evidence for neuropeptide-Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin. Endocrinology 1989;125:334–341. 324 Goedert M, Lightman SL, Nagy IT et al. Neurotensin in the rat anterior pituitary gland. Nature 1982;298:163–165. 325 Morel G, Chayvialle JA, Kerdelhue B, Dubois PM. Ultrastructural evidence for endogenous substance P-like immunoreactivity in the rat pituitary gland. Neuroendocrinology 1982;35:86–92. 326 de Palatis LR, Fiorindo RP, Ho RH. Substance P immunoreactivity in the anterior pituitary gland of the guinea pig. Endocrinology 1982;110: 282–284. 327 Arnaout MA, Garthwaite TL, Martinson DR, Hagen TC. Vasoactive intestinal polypeptide is synthesized in anterior pituitary tissue. Endocrinology 1986;119: 2052–2057. 328 Steel JH, Gon G, O’Halloran DJ et al. Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression. Histochemistry 1989;93:183–189. 329 Lam SK, Reichlin S. Pituitary vasoactive intestinal peptide regulates prolactin secretion in the hypothyroid rat. Neuroendocrinology 1989;50: 524–528. 330 Webster J, Ham J, Bevan JS, Scanlon MF. Growth factors and pituitary tumors. Trends Endocrinol Metab 1989;1:95–98. 331 Ezzat S, Melmed S. The role of growth factors in the pituitary. J Endocrinol Invest 1990;13:691–698. 332 White BA, Bancroft FC. Epidermal growth factor and thyrotropin-releasing hormone interact synergistically with calcium to regulate prolactin mRNA levels. J Biol Chem 1983;258:4618–4622. 333 Murdoch GH, Potter E, Nicolaisen AK et al. Epidermal growth factor rapidly stimulates prolactin gene transcription. Nature 1982;300:192–194. 334 Polk DH, Ervin MG, Padbury JF et al. Epidermal growth factor acts as a corticotropin-releasing factor in chronically catheterized fetal lambs. J Clin Invest 1987;79:984–988. 335 Miyake A, Tasaka K, Otsuka S et al. Epidermal growth factor stimulates secretion of rat pituitary luteinizing hormone in vitro. Acta Endocrinol 1985;108:175–178. 336 Leriche VK, Asa SL, Ezzat S. Epidermal growth factor and its receptor (EGFR) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness. J Clin Endocrinol Metab 1996;81:656–662. 337 Kasselberg AG, Orth DN, Gray ME, Stahlman MT. Immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues. J Histochem Cytochem 1985;33:315–322.

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338 Mueller SG, Kobrin MS, Paterson AJ, Kudlow JE. Transforming growth factor-a expression in the anterior pituitary gland: regulation by epidermal growth factor and phorbol ester in dispersed cells. Mol Endocrinol 1989;3:976–983. 339 Ezzat S, Walpola IA, Ramyar L et al. Membrane-anchored expression of transforming growth factor-≤ in human pituitary adenoma cells. J Clin Endocrinol Metab 1995;80:534–539. 340 Kobrin MS, Asa SL, Samoondar J, Kudlow JE. a-Transforming growth factor in the bovine anterior pituitary gland: secretion by dispersed cells and immunohistochemical localization. Endocrinology 1987;121:1412–1416. 341 Ramsdell JS. Transforming growth factor-a and -b are potent and effective inhibitors of GH4 pituitary tumor cell proliferation. Endocrinology 1991;128: 1981–1990. 342 McAndrew J, Paterson AJ, Asa SL et al. Targeting of transforming growth factor-a expression to pituitary lactotrophs in transgenic mice results in selective lactotroph proliferation and adenomas. Endocrinology 1995;136: 4479–4488. 343 Ying S-Y. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 1988;9:267–293. 344 Roberts V, Meunier H, Vaughan J et al. Production and regulation of inhibin subunits in pituitary gonadotropes. Endocrinology 1989;124:552–554. 345 Billestrup N, González-Manchón C, Potter E, Vale W. Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro. Mol Endocrinol 1990;4:356–362. 346 Corrigan AZ, Bilezikjian LM, Carroll RS et al. Evidence for an autocrine role of activin B within rat anterior pituitary cultures. Endocrinology 1991;128:1682–1684. 347 Kogawa K, Nakamura T, Sugino K et al. Activin-binding protein is present in pituitary. Endocrinology 1991;128:1434–1440. 348 Penabad JL, Bashey HM, Asa SL et al. Decreased follistatin gene expression in gonadotroph adenomas. J Clin Endocrinol Metab 1996;81:3397–3403. 349 Gospodarowicz D, Ferrara N, Schweigener L, Neufeld G. Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 1987;8:95–114. 350 Prysor-Jones RA, Silverlight JJ, Jenkins JS. Oestradiol, vasoactive intestinal peptide and fibroblast growth factor in the growth of human pituitary tumour cells in vitro. J Endocrinol 1989;120:171–177. 351 Elias KA, Weiner RI. Direct arterial vascularization of estrogen-induced prolactin-secreting anterior pituitary tumors. Proc Natl Acad Sci USA 1984;81:4549–4553. 352 Ezzat S, Smyth HS, Ramyar L, Asa SL. Heterogeneous in vivo and in vitro expression of basic fibroblast growth factor by human pituitary adenomas. J Clin Endocrinol Metab 1995;80:878–884. 353 Ezzat S, Zheng L, Zhu XF, Wu GE, Asa SL. Targeted expression of a human pituitary tumor-derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis. J Clin Invest 2002;109:69–78. 354 Stojlkovic SS, Merelli F, Iida T et al. Endothelin stimulation of cystolic calcium and gonadotropin secretion in anterior pituitary cells. Science 1990;248:1663–1666. 355 Matsumoto H, Suzuki N, Shiota K et al. Insulin-like growth factor-I stimulates endothelin-3 secretion from rat anterior pituitary cells in primary culture. Biochem Biophys Res Commun 1990;172:661–668.

356 Prager D, Melmed S, Fagin J. Feedback regulation of growth hormone gene expression by insulin-like growth factor I. In: LeRoith D, Raizada MK, eds. Molecular and Cellular Biology of Insulin-like Growth Factors and Their Receptors. New York: Plenum Press, 1989:57–71. 357 Yamashita S, Melmed S. Insulin-like growth factor I regulation of growth hormone gene transcription in primary rat pituitary cells. J Clin Invest 1987;79:449–452. 358 Berelowitz M, Szabo M, Frohman LA et al. Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science 1982;212:1279. 359 Han VKM, Lund PK, Lee DC, D’Ercole AJ. Expression of somatomedin/ insulin-like growth factor messenger ribonucleic acids in the human fetus: identification, characterization, and tissue distribution. J Clin Endocrinol Metab 1998;66:422–429. 360 Yamashita S, Weiss M, Melmed S. Insulin-like growth factor I regulates GH secretion and mRNA levels in human pituitary tumor cells. J Clin Endocrinol Metab 1986;63:730–735. 361 Asa SL, Coschigano KT, Bellush L et al. Evidence for growth hormone (GH) autoregulation in pituitary somatotrophs in GH antagonist-transgenic and GH receptor-deficient mice. Am J Pathol 2000;156:1009–1015. 362 Asa SL, Kovacs K. Histological classification of pituitary disease. J Clin Endocrinol Metab 1983;12:567–596. 363 Shi T, Farrel MA, Kaufmann JCL. Fibrosarcoma Complicating irradiated pituitary adenoma. Surg Neurol 1984;22:277–283. 364 Del Pozo JM, Roda JE, Montoya JG et al. Intrasellar granuloma. Case Report. J Neurosurg 1980;53:717–719. 365 Asa SL, Bilbao JM, Kovacs K et al. Lymphocytic hypophysitis of pregnancy resulting in hypopituitarism. A distinct clinicopathologic entity. Ann Intern Med 1981;95:166–171. 366 Thodou E, Asa SL, Kontogeorgos G et al. Lymphocytic hypophysitis: Clinicopathologic findings. J Clin Endocrinol Metab 1995;80:2302–2311. 367 Sheehan HL, Stanfield JR. The pathogenesis of post-partum necrosis of the anterior lobe of the pituitary gland. Acta Endocrinol 1961;37:479–510. 368 Bergeron C, Kovacs K. Pituitary siderosis. A histologic and immunocytologic study. Arch Intern Med 1979;139:248-249. 369 Pfäffle RW, DiMattia GE, Parks JS et al. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118–1121. 370 Radovick S, Nations M, Du Y et al. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257:1115–1118. 371 Tatsumi K, Miyai K, Notomi T et al. Cretinism with a combined hormone deficiency caused by a mutation in the Pit-1 gene. Nature Gen 1992;1:56–58. 372 Li S, Crenshaw EB III, Rawson EJ et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 1990;347:528–533. 373 Daughaday WH, Trivedi B. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism). Proc Natl Acad Sci USA 1987;84:4636–4640. 374 Baumann G, Shaw MA, Winter RJ. Absence of the plasma growth hormonebinding protein in Laron-type dwarfism. J Clin Endocrinol Metab 1987;65: 814–816.

C h a p t e r

2 Adrenocorticotropin Mark A. Herman Joseph A. Majzoub

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

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

Adult Anatomy

Molecular Embryology

Corticotrophs were initially identified by their basophilic staining. This has been subsequently found to be due to the presence of complex sugars in corticotrophs, principally

Tremendous progress has been made in recent years concerning the molecular mechanisms controlling pituitary organogenesis and pituitary cell-lineage specification in

CELLS OF ORIGIN

Fetal Anatomy

45

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animal models. A complex network of transcriptional events mediated by extrinsic and intrinsic signals has been implicated in the determination and a stereotypical spatiotemporal differentiation of the five trophic cell types of the mature anterior pituitary gland. Intrinsic Signals

RPX (for Rathke’s pouch homeobox), also known as HESX1 (for homeobox gene expression in embryonic stem cells) is the earliest known marker for the pituitary primordium [6]. HESX1/RPX is a member of the paired-like class of homeobox genes. Early HESX1/RPX expression occurs in the anterior midline endoderm and prechordal plate precursor, followed by expression in the cephalic neural plate [6]. Expression becomes progressively restricted anteriorly in the cephalic neural plate in a distribution consistent with tissue known by fate mapping studies to form the primordium of the anterior pituitary [7–10]. HESX1/RPX expression ultimately becomes restricted to Rathke’s pouch, and down regulation of HESX1/RPX in the pouch coincides with the differentiation of pituitary-specific cell types [6]. Embryonic mice lacking Rpx demonstrate variable abnormalities of hypothalamic and pituitary morphogenesis, reduced prosencephalon, anophthalmia or microophthalmia, and defective olfactory development. Neonatal mice have abnormalities in the corpus callosum, the anterior and hippocampal commisures, and the septum pellucidum [11]. These abnormalities are reminiscent of defects seen in a heterogeneous group of human disorders known as septooptic dysplasia (SOD). Deficits in SOD include optic nerve hypoplasia, abnormalities of the midline brain structures, and abnormalities of the hypothalamic-pituitary axis. Patients commonly present with endocrinopathies including hypoglycemia and adrenal crisis, which may signal growth hormone, ACTH, or thryoid-stimulating hormone deficiency [12]. Dattani and colleagues have identified two siblings with SOD who are homozygous for missense mutations within the HESX1 homeodomain which prevents it from binding target DNA [11]. The siblings identified with HESX1 mutations presented with hypoglycemia hours after birth and demonstrated hypopituitarism, substantiating a role for HESX1 in human pituitary development [11,13]. The corticotroph lineage appears to diverge relatively early from the other cell types of the anterior pituitary. LHX3, a LIM-type homeodomain protein, is essential for growth of Rathke’s pouch and determination of pituitary cell lineages [14]. In Lhx3-deficient mice, proopiomelanocortin (Pomc) was detected in a small cohort of cells at the ventral base of the pouch remnant which roughly corresponds to the position of the first presumptive corticotroph cells to differentiate in the wild-type pouch [14]. Although some pouch cells were able to differentiate and express Pomc, these cells failed to proliferate. An insufficient or non-functional corticotroph cell mass may have caused the hypoplastic adrenal cortices noted in these animals [14].

Ptx1 is a bicoid-related homeobox transcription factor identified based upon its ability to activate transcription of Pomc in the corticotroph derived AtT-20 cell line [15] and its ability to interact with the transactivation domain of Pit-1 [16]. It is expressed in the primordial Rathke’s pouch, oral epithelium, first branchial arch, the duodenum, and hindlimb [16]. Ptx1 is expressed in all mature pituitary cells, but is differentially expressed during pituitary development in different cell types [17]. In the mature pituitary, the highest levels of expression are found within corticotrophs [15]. In addition to activating transcription of Pomc, Ptx1 is required for sustained expression of Lhx3 [18], directly activates transcription of the common a-glycoprotein subunit (a-GSU) [16], synergizes with Pit-1 on the growth hormone and prolactin promoter [18], and synergizes with SF-1 on the promoter of the lutenizing hormone b (LH-b) gene [18]. Ptx2, also known as RIEG, is an additional bicoid-related homeobox gene 97% identical to Ptx1 [19]. Ptx2 is also differentially expressed in the pituitary and is excluded from corticotrophs [20], suggesting that some of the functions ascribed to Ptx1 may be performed by Ptx2. Expression of Pit-1, a POU domain transcription factor, is restricted to the anterior pituitary and required for the development of thyrotophs, lactotrophs, and somatotrophs [21]. Consistent with this, patients with mutations in PIT-1 have normal hypothalamic-pituitary-advanced (HPA) axis function [22]. Prophet of PIT-1 (PROP-1) is required for PIT-1 expression, and mutation of its gene is also associated with deficiencies in the development of thyrotrophs, lactotrophs, and somatotrophs in the Ames dwarf mouse and in humans. In a small number of patients with mutations in PROP1, modest impairment of ACTH secretion has been reported [23]. Developmentally essential, cell line-restricted factors have been identified for a number of different pituitary cell types. SF-1, an orphan nuclear receptor, is necessary for the development of gonadotrophs [24]. However, no essential corticotroph specific factor has yet been clearly identified. NeuroD, also known as Beta2, is a cell-restricted basic helixloop-helix (bHLH) transcription factor implicated in late neuronal differentiation [25] which has also been isolated as a cell-specific transcription factor of the insulin gene [26]. NeuroD is expressed in a number of tissues including pancreatic endocrine cells, the intestine, and brain including the mouse corticotroph AtT-20 cell line, but not the rat somatotroph pituitary cell line (GH3) [26]. Drouin and colleagues have recently demonstrated that NeuroD expression does appear to be restricted to corticotrophs in the mouse pituitary, and that NeuroD, in association with ubiquitous bHLH dimerization partners, specifically recognizes and activates transcription from the POMC promoter E box that confers transcriptional specificity of POMC to corticotrophs [27,28]. However, the necessity of this transcription factor for corticotroph specification or differentiation is still undetermined. Recently, a corticotroph-restricted transcription factor. TPIT, has been identified, which interacts with PTX1 and is required for POMC transcription, as discussed subse-

Chapter 2

quently. However, its role in corticotroph development is not known, and awaits further study. Because no instrinsic factor has yet been identified that is absolutely required for the development of corticotrophs in situ, it is possible that they arise elsewhere during fetal life and migrate into the anterior pituitary. One possible source could be neural crest, as this is the origin of POMC-producing melanocytes which migrate to the skin [29]. Extrinsic Signals

The oral ectoderm, from which Rathke’s pouch forms, and the neural ectoderm of the ventral diencephalon, from which the hypothalamus arises, are in direct contact at the time of the formation of Rathke’s pouch [30]. Classical explant experiments have indicated that inductive signals arising from mesenchyme and neural ectoderm adjacent to Rathke’s pouch are required for pituitary organogenesis and cell line specification [31]. The necessity for extrinsic signals for development of corticotrophs is supported by the demonstration that the expression of POMC in ectoderm explants requires coculture with mesoderm [32]. Direct evidence confirming the necessity of the ventral diencephalon for proper pituitary organogenesis has recently been obtained from examination of mice bearing null mutations in the Nkx2.1 homeobox gene, also known as T/EBP or TTF1 [33]. This gene is not expressed in the oral ectoderm or in the developing pituitary at any time during embryogenesis [34], but Nkx2.1-null mice demonstrate severe defects in the development of the diencephalon and also fail to develop a pituitary gland [33]. The nature of the extrinsic signals that promote pituitary organogenesis and cause cell-line specification is an area of active investigation. A number of secreted factors have been implicated in pituitary development and cell-line specification including bone morphogenic proteins (BMP4, BMP2), fibroblast growth factor 8 (FGF8), Sonic hedgehog (Shh) and Wnt5a [35–37]. A model of coordinate control of anterior pituitary progenitor cell identity, proliferation and differentiation imposed by FGF8 secreted from the dorsally located infundibulum and BMP2 from ventrally located mesenchyme has been proposed [37]. It has been suggested that corticotroph progenitors progress to a definitive corticotroph state only after escaping both FGF8 and BMP2 signaling [37]. While the precise signals and interactions required for pituitary organogenesis and corticotroph specification are incompletely understood, it is clear that signals from the mature hypothalamus like corticotropin releasing hormone (CRH) and vasopressin (AVP) are not required for corticotroph specification. The anterior pituitary appears to develop normally in a Crh-null mouse [38]. Furthermore, deletion of the class III POU transcription Brn-2 results in failed maturation of Avp-, Crh-, and oxytocin producing neurons of the hypothalamus and failed maturation of the posterior pituitary with no apparent defect in the maturation of any anterior pituitary cell type [39].

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Localization of Non-ACTH Peptides with Corticotrophs Many other neuropeptides have been found to be colocalized with ACTH within corticotrophs, although in most cases it is not clear whether this is due to synthesis or binding of the peptide within the cell. Neurophysin (but not vasopressin) colocalizes with ACTH in both normal and adenomatous corticotrophs [40]. Neurophysin immunocytochemical staining is especially prominent in corticotrophs in the zona intermedia which appear to project into the posterior pituitary. Chromogranin A has been described in the majority of corticotroph adenomas, although only a fraction of these patients have elevations of circulating plasma chromogranin A levels [41]. Galanin is present in all normal corticotrophs as well as in the majority of corticotroph adenomas which have been examined [42]. Galanin has also been found in those corticotrophs in the zona intermedia which appear to be migrating into the posterior pituitary. It has also been described in corticotrophs which have undergone Crooke’s hyalinization. Calpastatin, an inhibitor of the calcium-dependent cysteine proteases, calpain I, and calpain II, has been found in all ACTH-containing cells of the anterior pituitary, including those in the median wedge as well as those extending into the posterior pituitary [43]. Vasoactive intestinal peptide has been found in corticotroph adenomas, but only rarely in normal corticotrophs [44]. Normal corticotrophs contain small amounts of cytokeratin, whereas corticotrophs which have undergone Crooke’s hyalinization are markedly positive for this protein [45]. Corticotrophs appear to have few structural characteristics associated with neuronal cell types, for they are negative for neurofilament, vimentin, glial fibrillary acidic protein and desmon [45,46]. Likewise, corticotrophs in the zona intermedia, unlike those in the pars intermedia of rodents, appear not to be innervated by neurons, since they do not stain with any of these neuron-specific markers [47]. Similarly, synaptophysin, a 38 kD integral membrane glycoprotein found in presynaptic vesicles of neurons stains only weakly in corticotrophs [48]. These findings, together with a lack in humans of an anatomically discrete intermediate lobe with large numbers of a-MSH-containing cells, has led most investigators to conclude that there is no functional counterpart of the rodent pituitary intermediate lobe in humans.

Extra-pituitary Localization of ACTH and Related Peptides Although the vast majority of ACTH is synthesized in anterior pituitary corticotrophs, it is also expressed in several nonpituitary human tissues, both within and outside of the central nervous system. Within the central nervous system, ACTH and its related peptides are expressed to the greatest degree in cell bodies of the infundibular nucleus of the basal hypothalamus (analogous to the rodent arcuate nucleus) [49–51]. (See POMC Biosynthesis and Processing,

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pp. 52–53.) The cell bodies of these hypothalamic ACTH neurons are located at the base of the third ventricle, adjacent to the median eminence and pituitary stalk. These neurons project to limbic, diencephalic, mesencephalic, and amygdaloid sites [52]. Lesser amounts of ACTH and related peptides are found in substantia nigra, periventricular grey matter, and hippocampus [53]. Interestingly, POMC expression in the hypothalamus occurs in areas also known to express the orexigenic neuropeptides NPY and AGRP and project to many of the same hypothalamic targets [54,55]. The rodent arcuate nucleus prominently expresses the leptin receptor, implicating Pomc products in the regulation of appetite and energy homeostasis (see Melanocortin Receptor 4, p. 60) [56,57]. The brain areas containing ACTH also coincide with areas mediating stimulation analgesia, suggesting that expression of ACTH, or another product of the POMC gene (such as b-endorphin) in these sites may regulate pain perception [52]. Outside of the central nervous system, ACTH and other POMC gene products, including a-MSH and b-endorphin, are synthesized in a large number of human tissues, including in descending order of abundance, adrenal, testis, peripheral mononuclear cells, spleen, kidney, ovary, lung, thyroid, liver, colon, and duodenum [58,59]. In most of these tissues, POMC peptides are translated from truncated messenger RNAs lacking signal peptide sequences, suggesting that they cannot be secreted extracellularly [60,61], raising the question of their functional significance. However, adrenal and testis in addition express full length POMC mRNA, suggesting that these tissues may also secrete POMC-related peptides [58]. Recently, several additional cell types have been shown to produce POMC peptides including monocytes [62], astrocytes [63], gastrin-producing cells of the gastrointestinal tract [64], keratinocytes [65], skin melanocytes [66], and atrial myocytes [67]. GENE STRUCTURE ACTH is derived from a 266 amino acid precursor, proopiomelanocortin (POMC), so named because it encodes opioid, melanotropic, and corticotropic activities [68]. The human POMC gene is a single copy gene located on chromosome 2p23 [69]. It and the highly homologous opioid peptides, preproenkephalin A and preproenkephalin B (dynorphin), are all located on different chromosomes [70]. The human POMC gene is 8 kilobases (kb) long (Fig. 2.1). It consists of a promoter of at least 400 basepairs (bp) at the 5¢ end of the gene, followed by three exons, 86 (exon 1), 152 (exon 2), and 833 (exon 3) bp long, and two introns, 3708 (intron 1) and 2886 (intron 2) bp in length [69]. Exon 1 is not translated. Perhaps because of this, exon 1 of the human and other mammalian POMC genes are less than 50% identical. The initiator methionine is located 20 bp into exon 2, and is followed by a 26 amino acid hydrophobic signal peptide. Except for the signal peptide and 18 amino acids of the amino-terminal glycopeptide, the majority of

the POMC precursor is encoded by exon 3 [71]. Exon 2 is close to 90% identical between the POMC genes of humans and other mammals. Within exon 3 of POMC are located all known peptide products of the gene, including N-terminal glycopeptide, gamma-melanotropin (gammaMSH), joining peptide, a-MSH, ACTH, corticotropin-like intermediate lobe peptide (CLIP), b-lipotropin (b-LPH), b-MSH, and b-endorphin. Within exon 3, the regions encoding the N-terminal glycopeptide, a-MSH, ACTH, and b-endorphin, are greater than 95% identical between the human and other mammals [71]. In contrast, joining peptide, the region between the N-terminal glycopeptide and ACTH, is very poorly conserved among mammals [72], which has suggested to some workers that it does not encode a biologically important function [73].

POMC Gene Promoter Structure The promoter of the human POMC gene contains typical TATA and CAAT boxes 28 and 62 bp, respectively, upstream from the transcription start site. Using in vitro transcription [74] and transfection of the human POMC gene into heterologous cells [75], the POMC gene promoter has been shown to contain DNA elements which mediate increased transcription by cyclic AMP and decreased transcription by glucocorticoids. Although these DNA elements have not been precisely localized in the human POMC gene promoter, they are present with in the 700 bp 5¢ to the transcription start site [75]. The rat Pomc gene promoter has been studied more extensively. Drouin and coworkers, using DNA-mediated gene transfer into transgenic mice and tissue culture cells, found that the DNA sequences needed for corticotroph-specific expression and negative transcriptional regulation by glucocorticoid are contained within 543 bp of the transcription start site. These workers have reported the presence of a DNA sequence which mediates negative regulation by glucocorticoids (nGRE), located in the region of the CAAT box, which also binds nuclear proteins of the chicken ovalbumin upstream promoter (COUP) family. Using DNAase footprint and gel retardation analysis of the rat Pomc gene promoter, multiple synergistic DNA elements have been reported to be necessary for correct pituitaryspecific expression of the gene [76]. Recently, these workers have identified NeuroD, Ptx1, and Tpit as transcription factors which cooperate to cause corticotroph-specific expression of POMC [77].

POMC mRNA Transcription, Splicing, and Polyadenylation In addition to the major transcriptional initiation site present in corticotrophs, at least six other start sites have been found in several non-pituitary tissues (Fig. 2.1), including adrenal, thymus, and testes of the human [60] and rat [78]. These sites are all between 41 and 162 nucleotides downstream from the 5¢ and of exon 3. The mRNAs transcribed from

Chapter 2

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FIGURE 2.1. Structure of pro-opiomelanocortin (POMC) gene, messenger RNA (mRNA), and peptide products. (a) schematic diagram of gene (top), mRNA (middle) and POMC peptide (bottom). The gene consists of the promoter, 3 exons (thick bar, 1–3) separated by two exons (thin broken lines). Translated regions are stippled. The mRNA consists of 3 exons and the polyadenylate (AAAA) tail. The POMC precursor can be divided into 3 domains: I (N-terminal glycopeptide, joining peptide), II (adrenocorticotropic hormone; ACTH); and III (b-lipotropin; b-LPH). (b) Structure of individual products of POMC. Individual peptides are enclosed in different boxes depending on proteolytic cleavage specificities: = similar processing in pituitary and brain; = pituitary pattern of processing; = brain pattern of processing. = dibasic amino acids at proteolytic cleave sites. = glycosylation sites. = N-terminal acetylation sites. CLIP, corticotropin-like intermediate lobe peptide; a-MSH, a-melanocyte stimulating hormone.

these sites thus would be intronless, and the only truncated molecules that might be translated would be devoid of a signal peptide, and therefore could not be secreted [78]. Tissues containing these shorter forms of POMC mRNA, including adrenal, testis, spleen, kidney, ovary, lung, thyroid, and gastrointestinal tract, express ACTH, N-terminal glycopeptide1–61, and b-endorphin [58]. These truncated mRNAs are capable of being translated both in cell-free translation systems and in heterologous cells transfected with the appropriate fragment of the human POMC gene, although the peptide products are not secreted [61]. The significance of the expression of these truncated forms of POMC mRNA and their translated peptide products in human nonpituitary tissue is not clear.

A canonical polyadenylation signal is present in human POMC mRNA 23 bases upstream from the poly (A) addition site [79]. The length of the polyadenylate tail, attached to the 3¢ end of POMC mRNA and believed to play a role in translational efficiency of mRNA stability, is much longer in hypothalamic compared with pituitary cells in the rat [78], although this has not been studied in the human. GENE REGULATION In human anterior pituitary corticotrophs, POMC mRNA levels are increased by CRH and inhibited by glucocorticoids [80]. Similar results are seen in normal and adenoma-

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tous corticotrophs, although the glucocorticoid inhibition of POMC mRNA expression is less in the latter cells [80]. The same results are seen in rat anterior pituitary corticotrophs, where the opposing effects of CRH and glucocorticoids are due to their opposite influence on the transcription rate of the POMC gene [81]. Of interest, glucocorticoids have no effect on the POMC mRNA content of rat intermediate pituitary lobe corticotrophs [81]. CRH, acting via the type 1 CRH receptor [82] increases cAMP content in anterior pituitary corticotrophs [83]. CRH, vasopressin, and glucocotricoids all inhibit expression of CRH receptor 1 mRNA [84], which may limit the effect of these agents during the stress response, as discussed subsequently. The CRH-induced rise in cAMP is responsible for both the increase in POMC transcription and peptide synthesis as well as for the rise in intracellular calcium which results in ACTH secretion [85]. CRH mediates its stimulation of POMC transcription via the POMC CRH responsive element (PCRH-RE), which binds PCRH-RE binding protein [86]. The negative effect of glucocorticoids upon POMC gene transcription is thought to be mediated by a glucocorticoid–glucocorticoid receptor complex binding to cis-acting DNA sequences within the POMC promoter, although definitive evidence for this is lacking. The possibility exists that the glucocorticoid receptor complex does not bind directly to the POMC gene, but instead to another protein such as a positive transcription factor, and in this way mediates its negative effect on POMC gene expression [87]. Glucocorticoid stimulates, rather than inhibits, POMC gene expression [88] in the arcuate nucleus of the hypothalamus, the site of alphaMSH production. The significance of this is not clear, but could be involved in the inhibition of appetite by glucocorticoids in the postabsorptive state, when glucocorticoid levels are high. Recently, new insights into the development and regulation of the HPA axis have come from the discovery that leukemia-inhibitory factor (LIF) has an important role in these events [89]. LIF is a cytokine expressed in corticotrophs and folliculostellate cells beginning as early as 14 weeks gestation [90]. LIF stimulates transcription of POMC and expression of ACTH [90]. In many tissues, including pituitary, LIF expression is unregulated by inflammatory stimuli. The LIF receptor is a member of the class I cytokine receptor superfamily which heterodimerizes with gp130. In common with other family members, the LIF receptor signals through the Jak-STAT pathway, particularly utilizing Jak1 and STAT-3 [91]. As with other receptors coupled via these mediators, SOCS-3 inhibits POMC expression following its simulation by LIF [92]. Mice which overexpress a LIF transgene develop Cushing’s syndrome [93]. Their pituitary glands have corticotroph hyperplasia and multiple Rathke-like cysts lined by ciliated cells. Mice with targeted deletion of LIF have secondary adrenal insufficiency [94]. All of these data point towards LIF playing an important role in the regulation of ACTH secretion, perhaps most

importantly by immune and inflammatory stimuli [95]. In addition, the data suggest that LIF might be involved in the pathogenesis of Rathke-cleft tumors. Vasopressin potentiates the action of CRH on ACTH secretion, both in vitro [96] and in vivo [97]. Vasopressin’s effect is mediated by the vasopressin V1b (or V3) receptor and protein kinase C [98]. Despite this positive effect on ACTH secretion, vasopressin has been reported to decrease both basal and CRH-stimulated POMC mRNA levels in anterior pituitary cells [99]. b-adrenergic catecholamines, like CRH, also increase POMC mRNA levels in corticotrophs via a cAMP mechanism [100]. Insulin-induced hypoglycemia also causes an increase in POMC mRNA content in rat anterior pituitary corticotrophs [101,102], but whether this is secondary to an increase in hypothalamic CRH [101], vasopressin [103], or catecholamines [104] is not known. The inhibitory neurotransmitter GABA causes a decrease in POMC mRNA levels in intermediate, but not anterior pituitary corticotrophs [105]. POMC BIOSYNTHESIS AND PROCESSING The human POMC precursor has the potential to encode several overlapping peptides of biological importance (Fig. 2.1). Within the precursor, these peptides are separated from one another by two or more basic amino acids which serve as recognition sites for prohormone cleavage enzymes. In addition, POMC-derived peptides contain potential signals for amidation, glycosylation, acetylation and phosphorylation. Because the nomenclature of the various proteolytic products of POMC has been derived from both peptidemapping studies as well as molecular biological studies in which putative peptides had been predicted from inspection of nucleotide sequences [72], the terminology can be confusing. To avoid confusion, amino acid (aa) positions in this chapter are numbered as superscripts with reference to the 240 aa-long human POMC precursor, formed after removal of the 26 aa-long signal peptide. The 240 aa POMC precursor can be considered to be composed of three domains (Fig. 2.1). Domain I (aa 1–111), the N-terminal domain, encodes the 76 aa long Nterminal glycopeptide1–76 within its first 78 aa, and the 30 aa long C-terminal joining, or hinge, peptide ( JP79–108) within its last 33 aa. The middle Domain II (aa 112–152), 41 aa long, encodes the 39 aa ACTH112–150 peptide, which may be further processed to a-melanocyte stimulating hormone (MSH)112–124. The C-terminal Domain III, termed b-LPH153–240, is 88 aa long. It contains within it the 31 aa long b-endorphin210–240. Besides these peptides, several other products have been identified, although evidence for their existence and/or biological importance in man is not clear [106]. These include gamma1-MSH51–61, gamma2-MSH51–62, and gamma3-MSH51–76 in Domain I, corticotropin-like intermediate lobe peptide (CLIP)130–150 in Domain II, and gamma-LPH153–206 and b-MSH191–206 in Domain III (Fig. 2.1).

Chapter 2

Glycosylation of POMC Within the endoplasmic reticulum (ER), POMC undergoes initial glycosylation. Human POMC is glycosylated solely at two sites in the N-terminal glycopeptide1–76 of Domain I. Carbohydrate is added via an O-linked glycosylation to Thr45 and via an N-linked glycosylation to Asn65.

C-terminal Amidation of POMC Three human POMC products undergo C-terminal amidation [107]. These include N-terminal glycopeptide1–61, JP79–108, and -MSH112–124. In addition, these three products are also present in their Gly-extended forms, which may be incompletely-processed intermediates. C-terminal amidation is common among neuropeptides, and is usually essential for bioactivity [108]. This reaction is mediated by a bifunctional enzyme consisting of peptidylglycinea-amidating monooxygenese (PAM) and peptidly-ahydroxyglycine a-amidating lyase (PAL) activities, which transfer the amino group of a C-terminal Gly to the carboxyl group of the adjacent amino acid. Human PAM/ PAL exists in both a membrane-bound and free cytoplasmic form [109].

N-terminal Acetylation of POMC Two human POMC products undergo N-terminal acetylation. These are (as described below) a-MSH and bendorphin. In humans a-MSH exists predominantly in the nonacetylated form [110]. N-terminal acetylation of ACTH1–13-amide to form a-MSH results in increased melanotrophic activity [111] and decreased corticotrophic activity.

Proteolytic Processing of POMC POMC gives rise to several smaller, biologically active peptide products. These are generated by posttranslational cleavage of POMC by trypsin-like prohormone convertase endopeptidase enzymes which cleave the precursor on the C-terminal side of regions of two or more basic amino acid residues [112]. These basic amino acids are subsequently removed by a carboxypeptidase activity. Some POMC proteolytic products subsequently undergo amidation at their C-terminus or acetylation at their N-terminus, as described above. The posttranslational processing of POMC exhibits a remarkable degree of tissue-specificity, which recently has been postulated to be due to the differential distribution of processing enzymes in the various tissues which synthesize POMC (see below). Proteolytic Processing Enzymes

All proteolytic processing of human POMC occurs at either lys–arg or arg–arg residues (Fig. 2.1). Every lys–arg and arg– arg site within the human precursor is capable of being cleaved in vivo [106], whereas in the mouse and rat, additional arg–lys and lys–lys sequences at the N-termini of

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gamma1-MSH and b-MSH, respectively, appear to be utilized [106]. It is likely that the proteolytic digestion of human POMC at all sites is mediated by either of two structurally related endopeptidases, prohormone convertase 1 (PC1) [113] or prohormone convertase 2 (PC2) [113]. These enzymes, best studied in rodents thus far, are part of a seven member family of subtilisin/kexin-like mammalian proteinases [114], and are distributed specifically within endocrine cells and neurons. Both enzymes are capable of cleaving neuropeptide precursors, including POMC, proinsulin, and proglucagon, at dibasic sites, and each appears to manifest distinct preferences for different sites within the same precursor prohormone, with PC2 able to cleave at a wider selection of available dibasic sites than PC1 [115]. The tissue distributions of PC1 and PC2 mRNAs are distinctly different. PC1 is abundant in approximately 20% of anterior pituitary cells (presumably including corticotrophs), in all intermediate lobe pituitary cells (of the rodent), and in the supraoptic nucleus of the hypothalamus [116]. In contrast, PC2 is absent from the anterior pituitary, but is highly expressed in rodent pituitary intermediate lobe, multiple sites within the central nervous system, including cerebral cortex, hippocampus, and thalamus, and in pancreatic islet cells [116]. The differential tissue-specific distribution of these enzymes matches nicely with the known tissue-specific differences in POMC proteolytic processing (see below), suggesting that PC1 is responsible for the POMC cleavage products found in anterior pituitary corticotrophs, whereas PC2 cleaves POMC in pituitary intermediate lobe (of lower mammals) and in the brain. This suggestion is supported by studies of the differential processing of POMC by PC1 and PC2 [115,116]. Recently, a patient with severe childhood obesity and hyperproinsulinemia with postprandial hypoglycemia has been identified with compound heterozygous mutations in the PC1 gene [117]. The patient presented with multiple endocrine abnormalities including impaired glucose tolerance and postprandial hypoglycemia which has been attributed to the secretion of large amounts of proinsulin given its partial insulin-like action and long biological half-life. She also suffered from hypogonadotropic hypogonadism with primary amenorrhea, but otherwise normal development of secondary sexual characteristics. Ovulation was induced with exogenous gonadotropins, and the patient delivered healthy quadruplets. The pregnancy was complicated by gestational diabetes requiring insulin treatment. She also suffered from mild adrenal cortical insufficiency with complaints of fatigue and excessive daytime somnolence reversed by glucocorticoid administration. The adrenal cortical insufficiency was attributed to defective POMC processing with elevated levels of serum ACTH precursors confirming a role for PC1 in human POMC processing [117]. Following the generation of peptide products by prohormone convertase enzymes, the C-terminal basic amino acids are removed by carboxypeptidase activities. Carboxypeptidase E (Cpe) is required for the excision of paired

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dibasic residues of various peptide prohormone intermediates, including those derived from proinsulin and POMC [118]. The mutation in a strain of hyperproinsulinemic, late-onset obese mouse, the fat/fat mouse, has recently been mapped to the carboxypeptidase E gene [118]. A missense mutation has been identified in the Cpefat allele and these mice demonstrated a 20-fold decrease in Cpe enzymatic activity in pituitaries and isolated islets. The sorting of POMC into secretory granules is probably mediated by a signal “patch” [119], located within the tertiary structure of the molecule, which directs it to the granules. The presence of all POMC processed products in equimolar amounts within secretory granules suggests that this sorting precedes proteolytic cleavage of POMC. This suggestion is supported by data which demonstrates that initiation of proteolytic processing of POMC begins in the trans-Golgi system and continues in secretory vesicles, and is also consistent with data demonstrating localization of PC1 and PC2 within the TGN and/or dense core secretory granules [120]. Since POMC as well as PC1 and PC2 all contain signal peptides and are presumably colocalized throughout the endoplasmic reticulum, prohormone convertase activity must be inhibited in the endoplasmic reticulum and cis-Golgi regions. Proconvertase activity may be regulated by the local intracellular environment of these compartments and/or by control of catalytic activation of prohormone convertase. 7B2 is a small, hydrophobic acidic protein originally isolated from porcine and human anterior pituitary glands [121] that is widely distributed in neuroendocrine tissues [122] and found to associate specifically with PC2 [123]. The 27-kDa 7B2 precursor protein is cleaved to a 21-kDa protein and a small carboxy-terminal peptide (CT peptide) [124]. Interaction of 7B2, particularly the 21-kDa fragment, with proPC2 appears necessary for the generation of mature and active PC2 in the trans-Golgi region [125]. The 7B2 CT peptide is a nanomolar inhibitor of PC2 in vitro, but its role in vivo has not been defined. The role of 7B2 in activating proPC2 has been confirmed in vivo in 7B2-null mice [126]. 7B2-null mice are devoid of PC2 activity and deficiently process islet hormones and Pomc. The mice are hypoglycemic and demonstrate hyperproinsulinemia and hypoglucagonemia. These mice also demonstrate profound intermediate lobe ACTH hypersecretion with minimal conversion of this peptide to a-MSH, resulting in a severe Cushing’s syndrome that causes death by 9 weeks of age [126]. Curiously, PC2 null mice demonstrate similar islet cell dysfunction resulting in hypoglycemia, but do not abnormally produce a Cushingnoid syndrome [127]. This discrepancy suggests additional functional roles for 7B2, which are further suggested by the localization of 7B2 in regions of the brain lacking PC2 [128]. Recently, the suggestion that 7B2 may represent one member of a family of related convertase inhibitor proteins has been proposed with the identification of the protein proSAAS [129]. ProSAAS is a 26 kDa granin-like neuroen-

docrine peptide precursor isolated from rodents and humans with structural similarity to 7B2, including a proline-rich sequence in the first half of the molecule and a C-terminal peptide (SAAS CT peptide) following a dibasic cleavage sequence [129]. Overexpression of proSAAS in AtT-20 cells reduces the rate of Pomc processing and the SAAS CT peptide is a nanomolar competitive inhibitor of PC1, but not PC2 [129]. Tissue Specificity of POMC Processing

In human corticotrophs, POMC is processed predominantly into N-terminal glycopeptide1–76, joining peptide ( JP)79–108, ACTH112–150, and b-LPH153–240 (Fig. 2.1) [130]. Much smaller amounts of a-MSH112–124, CLIP130–150, bendorphin210–240, and a truncated form of N-terminal glycopeptide1–61, also known as “big” gamma-MSH, are also present [107]. There is no evidence for cleavage after arg50, and therefore no evidence for the presence of gamma1MSH51–61 in the human pituitary. JP79–108 exists as both a monomer and homodimer, most likely joined via disulfide bonding between the single Cys87 of two molecules [73]. Although JP79–108 has been postulated to stimulate adrenal androgen steroidogenesis [131], some studies do not support this hypothesis [73]. A function for N-terminal glycopeptide has also not been assigned, although one intriguing study reported that it is capable of stimulating aldosterone release from adrenal cells [130]. Whereas the production of distinct POMC peptide derivatives is clearly segregated between the anterior and intermediate lobes of the rodent, in the human, small, acetylated POMC peptide derivatives colocalize with larger desacetylated POMC peptides in corticotrophs of the anterior pituitary, suggesting that the strict dichotomy between corticotrope and melanotrope POMC processing observed in rodents and other species does not extend to human pituitaries [132]. Levels of desacetyl-a-MSH are elevated in pituitary corticotrophs [133] and plasma [134] of patients with Addison’s disease, Cushing’s disease, and Nelson’s syndrome. Desacetyla-MSH has approximately 75% of the melanotrophic activity as does a-MSH [111], whereas ACTH is only 5% as potent as a-MSH in this regard [111,135]. Because the serum levels of ACTH are 50- to 100-fold higher than levels of desacetyl-a-MSH in patients with Cushing’s disease, Addison’s disease, and Nelson’s syndrome, it is likely that the hyperpigmentation associated with these disorders is largely due to the melanotrophic effect of ACTH, and not MSH. Thus, in human anterior pituitary corticotrophs, the POMC precursor is predominantly cleaved at limited lys–arg sites into two peptides in Domain I (N-terminal glycopeptide1–76 and JP79–108), one peptide in Domain II (ACTH112–150) and one peptide in Domain III (b-LPH153–240) (see Fig. 2.1). As discussed above, human POMC is also expressed in several brain sites outside of the anterior pituitary, predominantly in the arcuate nucleus of the anterior hypothalamus. In these extra-pituitary locations, POMC is

Chapter 2

processed to a greater extent than in anterior pituitary. In brain, ACTH112–150 is cleaved to a-MSH112–124 and CLIP130–150 such that the amount of a-MSH relative to ACTH is 300-fold higher in hypothalamus, telencephalon, and mesencephalon than it is in anterior pituitary [136]. As in anterior pituitary, a-MSH is almost exclusively present in the desacetyl form [50]. Adding additional levels of complexity to the issue of tissue specificity of POMC processing are results from a dopamine D2 receptor (D2R) deficient mouse. Similar to dopaminergic tonic inhibitory control of prolactin expression in lactotrophs, POMC expression in the rodent intermediate lobe is under inhibitory dopaminergic control mediated via D2 receptors [137]. D2R-deficient mice demonstrate mild intermediate lobe hyperplasia accompanied by upregulation of both PC1 and PC2 [138]. These mice present with unexpectedly high levels of ACTH with corresponding adrenal hypertrophy and increases in corticosteroid secretion [138]. The altered prohormone convertase levels in these mice suggest the possibility of dynamic regulation of prohormone processing within specific tissues. HORMONE MEASUREMENT ACTH was one of the first substances to be measured by radioimmunoassay (RIA) [139]. In pioneering work using polyclonal antisera, Berson and Yalow described the measurement of ACTH and related peptides in normal persons and those with ectopic ACTH production by lung cancer [140]. These studies provided among the first data that ACTH was synthesized from larger precursors, which were termed “big” and “big–big” ACTH. The ACTH RIA can be performed on unextracted plasma, but suffers from a limit of sensitivity of approximately 25 pg/ml, and is therefore often unable to detect levels of plasma ACTH in the normal basal range. Radioimmunoassay remained the standard method for the measurement of plasma ACTH until the development of the immunoradiometric assay (IRMA). ACTH IRMAs employ two antibodies, one or both monoclonal, against ACTH. The solution-phase antibody is radiolabeled, and the solid-phase antibody is linked to a bead or other solid support. In general, the ACTH IRMA on unextracted plasma compares very favorably with RIA [141], being more sensitive, more reproducible, and more rapid [142]. Most IRMAs have lower limits of detection of 3–5 pg/ml and coefficients of variation of less than 10% up to 5000 pg/ml [141,142]. Depending on the antigenic specificity of the chosen antibodies, the ACTH IRMA may detect only intact ACTH [141], both ACTH and POMC precursor peptides [143,144], or only POMC precursors [145]. It is essential to know the sequence specificity of any IRMA in clinical use, for some, unlike most polyclonal RIAs, are incapable of detecting ACTH precursors which may be secreted by lung carcinomas [143]. Despite the wide availability of the ACTH IRMA, results from different laboratories are diffi-

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cult to compare because of lack of agreement on a suitable ACTH reference standard. An immunofluorometric assay (IFMA) for ACTH has also been developed [146]. Its sensitivity and accuracy are similar to those of the IRMA. Its principal advantage is its speed and the use of a nonradioactive label which is stable for over 1 year. Plasma assay of POMC products other than ACTH, including b-endorphin [147] and N-terminal glycopeptide, has been suggested as an adjunct in the evaluation of the hypothalamic-pituitary-adrenal (HPA) axis. In general, the levels of these hormones parallel that of ACTH in various HPA axis abnormalities [148]. However, except for use as a screening test for lung carcinoma associated with preferential secretion of N-terminal glycopeptide1–61 or proACTH1–150 [148], such tests are much less helpful than the ACTH IRMA because of the low concentration of other POMC peptide fragments compared with ACTH in most physiologic and pathologic settings. SECRETION OF ACTH: BIOCHEMISTRY AND PHARMACOLOGY Secretion of ACTH from the corticotrophs of the anterior pituitary is mediated by several factors (Fig. 2.2). CRH and vasopressin are the primary secretagogues for ACTH, although a number of other agents may also affect its release, and glucocorticoids are the major negative regulators of ACTH secretion. Once a ligand has bound to its receptor, release of ACTH from the corticortroph is mediated by second messengers through one of four signal transduction pathways, involving either protein kinase A, protein kinase C, glucocorticoids, or the Janus kinase/STAT system. (This

–

CRH

AVP

Catecholamines IL1β IL-6 TNFα

+

–

Cortisol

ACTH

Adrenal

+

+

LIF IL-6 IL1β TNFα

CRH IL1β IL-6 TNFα

FIGURE 2.2. Control of adrenocorticotropic hormone synthesis and secretion.

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last pathway, regulated by LIF, was described in a previous section, p. 50.) These pathways result in changes in the phosphorylation pattern of specific cellular proteins, and/ or in intracellular calcium levels, impacting on ACTH synthesis and release. Circulating ACTH then binds to receptors, primarily in the adrenal gland, leading to steroid biosynthesis.

Second Messenger Regulation of ACTH Secretion Protein Kinase A Pathway

CRH stimulates ACTH release via the cAMP-protein kinase A pathway. CRH stimulates adenylate cyclase activity, which then increases cAMP [83]. Forskolin, a direct stimulator of adenylate cyclase activity, and 8-bromo-cAMP, a cAMP analog, both markedly stimulate ACTH release, and increase CRH-stimulated ACTH release, but do not potentiate ACTH release from cells maximally stimulated by CRH [149]. The CRH-stimulated increase in cAMP activates cAMP-dependent protein kinase A [83]. Protein Kinase C Pathway

AVP V1b and V2 receptors are coupled to different second messenger systems. V1 receptors in the pituitary mobilize calcium through activation of phospholipase C, whereas V2 receptors activate adenylate cyclase, increasing cAMP [150]. AVP-stimulated ACTH release is mediated via the protein kinase C pathway [151]. Evidence that AVP acts through the protein kinase C pathway in the anterior pituitary includes the finding that AVP stimulates accumulation of inositol phosphates in rat anterior pituitary cells [152]. Phorbol esters, which activate protein kinase C directly by substituting for diacylglycerol and binding to protein kinase, induce ACTH release in rat pituitary cell cultures. Activation of protein kinase C appears more important in the sustained phase of AVP-stimulated ACTH release, not in the initial phase [153].

CRH CRH Stimulation of ACTH Secretion

Perfused human fetal pituitaries and cultured human fetal pituitary cells in culture secrete ACTH in response to CRH [2]. Rats have been used extensively in the study of ACTH secretion, as it is difficult to culture human corticotroph cells in vitro. In dispersed rat anterior pituitary cells, CRH stimulates a ninefold increase in ACTH release that is sustained for as long as the cells are exposed to CRH [83]. Even with maximal CRH stimulation, only 6% of cultured pituitary cells release ACTH [154]. CRH stimulates ACTH synthesis as well as release. In humans, the biphasic response may reflect secretion of a ready pool of ACTH, followed by later release of newly synthesized protein [155]. Exposing cells to CRH for a long period of time results in an increase in ACTH in the cell and in the medium. Thus, the sustained phase of ACTH

secretion may represent later release of newly synthesized ACTH peptide. Most of the CRH released into the hypophyseal blood is derived from the PVN [156]. Concentrations of oCRH that are similar to the concentrations of CRH found in rat portal plasma have been shown to increase secretion of ACTH in human fetal hemipituitaries in vitro [157]. The CRH content in the hypophyseal portal blood of anesthetized male rats is about 100 pM [158], which exceeds the in vitro threshold of 10–20 pM CRH to stimulate ACTH release [159]. In addition to stimulating ACTH expression and release, CRH can also directly stimulate secretion of glucocorticoids from the adrenal gland [160]. CRH Receptors

In human pituitaries, CRH has been shown to bind to sites in the anterior lobe [161]. The distribution of binding sites in human pituitary correlates with the distribution of corticotrophs [161]. CRH receptors in the anterior pituitary gland are low capacity, high affinity receptors, with a Kd for CRH binding of about 1 nM [162]. To date, two CRH receptor genes have been identified in humans and other mammals [163–165], with a third additional one being described in the catfish [166]. The type 1 receptor [163,165] is expressed predominantly in anterior pituitary corticotroph cells, whereas the type 2 receptor [164] is more widely distributed in the brain and periphery, particularly in cardiovascular tissue. The type 1 receptor binds and is activated by both CRH as well as the CRH-like peptide, urocortin [167]. This receptor mediates the actions of CRH at the corticoctroph. In addition, the type 1 receptor mediates fear and anxiety behaviors following stressors, even in CRHdeficient mice [168]. Mice with deletion of the CRH type 1 receptor gene show reduced fear and anxiety [169]. These data suggest that a CRH-related peptide, possibly urocortin or another unknown member of the CRH family, mediates fear responses via the CRH type 1 receptor. The CRH type 2 receptor binds urocortin with over 20-fold higher affinity compared to CRH. Its distribution, along with the hypotensive cardiovascular response to infused urocortin which is abolished in CRH type 2 receptor-deficient mice [170], suggests that the receptor may be involved in blood pressure control. This may underlie the hypotension observed during the CRH stimulation test. Recently, a nonpeptide, type 1-specific CRH antagonist has been developed which is orally active [171,172]. It should help to elucidate the role of CRH in ACTH regulation and other pathways.

AVP AVP Stimulation of ACTH Secretion

In rat anterior pituitary cells, AVP causes a twofold increase in ACTH release [173]. AVP elicits an initial rapid release of ACTH, observable within 5 seconds, reaching a maximum in less than 1 to 2 minutes, and lasting less than 3 to 6 minutes. This is followed by a second phase lasting

Chapter 2

for several hours. In humans, AVP infusion by itself has only a small effect on ACTH release. AVP is synthesized in the same parvocellular hypothalamic paraventricular nuclear neurons which express CRH, and appears to be coreleased with CRH at the median eminence into the portal hypophyseal system [174]. However, a substantial amount of AVP in the portal blood is released from projections from the supraoptic nuclei in the median eminence [156]. AVP from the posterior pituitary may also reach the anterior pituitary through portal vessels that connect the two [175]. This raises the possibility that increased vasopressin secretion from the posterior pituitary, in response to hyperosmolality, may also stimulate ACTH secretion. AVP Receptors

A single population of specific AVP receptors have been identified in rat anterior pituitaries, which are distinct from CRH receptors [176]. Most corticotrophs have AVP receptors, since 80% of the ACTH-secreting cells in the pituitary bind AVP [176]. The anterior pituitary AVP receptors are distinct from the V2 renal receptors and the V1 hepatic/pressor receptors [150]. This has led to the classification of hepatic/pressor receptors as V1a receptors and anterior pituitary receptors as V1b [150], or V3, receptors. V1a, V1b, and V2 receptors can be distinguished by their patterns of recognition of AVP analogues [177]. V1a binding sites in the rat anterior pituitary have a Kd of about 1 nM, and the minimal effective dose of AVP is 0.1 nM [173]. dDAVP (desmopressin), an AVP analog with V2 receptor affinity, has an insignificant effect on plasma ACTH levels, though it does increase, but is not additive to, CRHstimulated ACTH release [178]. During the past 6 years, the genes for all three vasopressin receptors (V1a, V2, and V1b) have been identified [179]. They are highly related members of the 7 transmembrane, G protein coupled, receptor family. As anticipated, V1b receptor mRNA is highly expressed in anterior pituitary corticotrophs, and is coupled to stimulation of POMC gene expression and ACTH secretion via the protein kinase C pathway [180,181].

Modulators of CRH and AVP Release CRH is the most important physiologic ACTH secretagogue [182]. Stressors, endogenous circadiam rhythms, and glucocorticoids influence CRH release [183]. In the rat, afferent inputs to the PVN may mediate the action of stressors by controlling the release of CRH [183]. Sources of neuronal afferents to the hypothalamus include the amygdala and hippocampus of the limbic system, and brainstem regions involved in autonomic functions [184]. In rats, acetylcholine, norepinephrine, angiotensin II, and possibly CRH itself, increase CRH concentrations in the hypophyseal portal plasma. On the other hand, AVP, b-endorphin, and GABA inhibit the ACTH response to stress [182,185–187].

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In rodents, central catecholamines stimulate ACTH release, and the effects of catecholamines on ACTH secretion appear to be mediated via secretion of CRH, and possibly AVP, into the hypophyseal portal circulation [188]. However, in humans, catecholamines have little direct effect on ACTH secretion from the pituitary. Peripheral catecholamines, increased by a variety of stresses, do not cross the blood–brain barrier to reach the hypothalamus, but do reach the pituitary, yet do not increase basal or CRHstimulated plasma ACTH levels. This suggests that the increased peripheral levels of epinephrine and norepinephrine generated during stress are probably not responsible for the increase in ACTH, and that catecholamines do not act directly on the pituitary to stimulate ACTH release [189]. In humans and other intact mammals, the impact of angiotensin II on basal or CRH-stimulated ACTH release is unclear. In humans, infusion of angiotensin II alone does not increase ACTH release [178]. However, in dispersed rat anterior pituitary cells, angiotensin II does stimulate release of ACTH [190]. Angiotensin II has a synergistic effect with CRH in stimulating ACTH release in humans in vivo [178]. Angiotensin II potentiates CRH-stimulate ACTH release from cultured anterior pituitary cells [191], although it is less effective than AVP, and potentiates the CRH-stimulated increase in cAMP. Angiotensin II and AVP do not potentiate each other’s effect on ACTH release, suggesting that they act via the same mechanism. Interleukin-1 beta stimulates ACTH release in conscious rats by acting on the hypothalamus [192] to induce secretion of CRH [193,194]. Interleukin-1 does not cross the blood–brain barrier [195], but activates noradrenergic neurons in the brainstem and hypothalamus, which may stimulate CRH secretion, especially in the median eminence. Prostaglandins may be involved in the response to interleukin-1, since ibuprofen, which blocks the formation of prostaglandins, blocks endotoxin-induced ACTH release in humans [196]. Tumor necrosis factor is a potent secretagogue for ACTH, and when administered to human subjects, leads to an increase in ACTH, cortisol, and AVP [197], but inhibits CRH-, AVP-, and angiotensin II-stimulated ACTH secretion [198]. Tumor necrosis factor may stimulate ACTH secretion by stimulating CRH release from the hypothalamus [199]. However, there is evidence that the site of action of tumor necrosis factor is peripheral to the pituitary and hypothalamus [199]. Bacterial endotoxin, when administered to human subjects, increases ACTH, cortisol, and AVP release [196]. Tumor necrosis factor may mediate the hormonal responses to endotoxin, since tumor necrosis factor levels increase after endotoxin administration [200]. Endotoxin does not increase ACTH release from cultured rat adenohypophyseal cells [201]. In rats and humans, interleukin-6 leads to ACTH secretion via CRH-dependent [202] and CRH-independent pathways, most likely via a prostaglandin-dependent pathway [203]. GABA inhibits the secretion ACTH by inhibiting

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the release of CRH and possibly AVP [184]. GABAnergic inputs into the PVN of the hypothalamus have been characterized in animals, including inputs from the hippocampus [184]. Opiates and opiate agonists decrease ACTH secretion, and may tonically inhibit ACTH secretion [204–211], although some studies demonstrate no effect of naloxone, an opiate antagonist, on the basal levels of cortisol [212,213]. Opiates inhibit CRH- and AVP-stimulated ACTH release, and different opiate agonists differentially effect CRH- versus AVP-stimulated release [214]. In humans, morphine blunts CRH-stimulated ACTH release without decreasing AVP or catecholamine levels [214]. Modulation of CRH-stimulated ACTH secretion by opiates most likely occurs at a level above the corticotroph [214]. Met-enkephalin analogs inhibit ACTH secretion, and controversy exists as to whether inhibition occurs at the hypothalamus or pituitary. In humans CRH-induced ACTH release is almost completely abolished with pretreatment with a met-enkephalin analog [215].

Synergism Between CRH and AVP Alone, AVP is a less important physiologic ACTH secretagogue than CRH [182]. AVP potentiates CRH-stimulated release of ACTH from cultivated rat anterior pituitary cells by twofold. When cells are first exposed to CRH, reaching the sustained phase of CRH-stimulated ACTH release, and AVP is then added, the usual initial spike of AVP-stimulated ACTH secretion is superimposed on top of the CRHinduced plateau of secretion. Despite continued exposure to AVP, ACTH secretion decreases down to the level of the plateau phase of CRH-stimulated secretion [216]. In cells exposed to AVP before CRH, CRH does not potentiate AVP-stimulated ACTH secretion [216]. This, together with the presence of AVP and CRH in the same parvocellular neurons of the PVN, suggests that the two neuropeptides cooperate to regulate ACTH release. However, this remains to be proven in humans, as there are no examples of persons with defects in AVP synthesis or release who have impaired ACTH secretion.

Oxytocin In humans, low-dose oxytocin perfusion decreases plasma ACTH and cortisol levels [217], and infusion of oxytocin completely inhibits CRH-stimulated ACTH release [218]. Oxytocin acts via a similar mechanism as AVP. Oxytocin binds competitively to AVP receptors in the anterior pituitary [219], but is much weaker than AVP at stimulating ACTH release [220]. Like AVP, oxytocin stimulates ACTH secretion through the protein kinase C pathway.

Glucocorticoids Glucocorticoids are the primary negative regulators of ACTH secretion. Glucocorticoids act on corticotrophs to

inhibit the secretion of ACTH induced by AVP and CRH, the synergism between CRH and AVP, and substances that provoke production of inositol phosphates and cAMP. Glucocorticoids’ negative impact on ACTH regulation is also due to their inhibition of the principal stimulators of ACTH, CRH and AVP. In the rat, glucocorticoid receptors are widely distributed in the brain, including the PVN. The PVN is a site for glucocorticoid negative feedback, since dexamethasone decreases the amount of basal CRH in the hypothalami explant and in the PVN, and the CRH response to secretagogues like serotonin and to stress. Glucocorticoids inhibit CRH release and decrease intracellular CRH in the rat PVN [182], and dexamethasone has a local effect on the hypothalamus, decreasing CRH mRNA expression [221], and preventing the rise in intracellular of CRH and AVP usually seen after adrenalectomy [183]. Glucocorticoids increase, and adrenalectomy decreases, the amount of GABA in the hypothalamus and the hippocampus [222]. Glucocorticoids appear to feedback to increase the GABA activity of the hippocampus and hypothalamus, and thus inhibiting CRH release [222]. In the anterior pituitary, glucocorticoid inhibition of ACTH secretion in vitro is mediated via glucocorticoid receptors, and lack of glucocorticoid effect in the intermediate lobe of the pituitary is most likely because functional receptors are not present in these cells. The negative effect of glucocorticoids on CRH-, AVP-, angiotensin II-, and norepinephrinestimulated ACTH release [223] is biphasic, which may reflect an initial inhibition of ACTH release, followed by an inhibition of POMC biosynthesis. Glucocorticoids inhibit POMC secretion, gene transcription and mRNA levels in the anterior pituitary. EFFECTS OF POMC-DERIVED PEPTIDES IN SKIN AND ADRENAL The melanocortin receptors are a family of seven transmembrane-spanning, G-protein coupled receptors that are activated by melanocortin derivatives of POMC including a-MSH and ACTH. Activation of all five receptors results in adenylate cyclase activity and cAMP production. Cloning of the melanocyte MSH receptor (melanocortin 1 receptor–MC1R) [224,225] and the adrenal ACTH receptor (melanocortin 2 receptor–MC2R) [225] were quickly followed by the cloning of three additional family members. The five known melanocortin receptors show distinct tissue distributions throughout the nervous system and periphery and distinct selectivities for the various melanocortin peptides. Prior to the cloning of this receptor family, the actions of melanocortins were primarily known through the effects of MSH on pigmentation and the effects of ACTH on glucocorticoid secretion from the adrenals. However, many additional roles including cognitive and behavioral effects, effects on the immune system, and effects on the cardiovascular system have also been attributed to the melanocortins.

Chapter 2

With the cloning of this family of receptors, the physiologic roles of ACTH, MSH and other melanocortin derivatives are beginning to be elucidated.

Ligand Specificity The pharmacology of melanocortin receptor activation with a large number of natural and synthetic melanocortin peptides is the subject of extensive investigation. All five melanocortin receptors are activated by ACTH. However, MC2R binds only ACTH and is not activated by other melanocortin peptides [226]. The synthetic agonist 4norleucine, 7-D-phenylalanine-a-MSH (NDP-MSH) is the most potent agonist of MC1R, MC3R, MC4R, and MC5R [227,228]. The endogenous non-ACTH melanocortin peptides generally bind the melanocortin receptors with an order of potency MC1R > MC3R > MC4R > MC5R when expressed in COS cells and measurements are obtained in competition with NDP-MSH [228–232]. g-MSH is relatively selective for MC3R over MC4R and MC5R [228,233]. Whether the differences in melanocortin receptor specificity for different melanocortin ligands are physiologically relevant are unknown. Interestingly, Org 2766 and BIM 22015, two ACTH4-10 analogs, have no activity at any of the cloned MCRs, but have potent effects on central and peripheral nervous systems suggesting the possibility of undiscovered melanocortin receptors [234,235].

Melanocortin 1 Receptor The MC1R was initially cloned from primary melanoma tumors [224,236]. MC1R gene expression has also been confirmed in primary human melanocytes by northern blotting [236]. Other cutaneous cells including keratinocytes [237] and dermal fibroblasts [238] have also been reported to express MC1R, although the presence of MC1R mRNA [239] and MC1R protein [240] in keratinocytes could not be confirmed in other studies. Recent evidence demonstrates that the MC1R is expressed in keratinocytes and that its expression is induced by calcium and UV light treatments [241]. Corresponding to its expression in the skin, its function is perhaps most fully understood with respect to its role in cutaneous pigmentation (for review see [242]). Activation of melanocyte MC1R, via activation of adenylate cyclase, stimulates tyrosinase activity, the rate-limiting enzyme in melanogenesis [239]. The activation of tyrosinase results in an increased proportion of eumelanin (brown–black pigment) formation over pheomelanin (red–yellow pigment) formation resulting in increased pigmentation [243]. Mutations and variant alleles in the MC1R gene have been linked to variation in mammalian pigmentation. The extension locus has long been known to regulate pigmentation in mammalian species. The extension locus has been shown to encode the mouse MC1R [244]. The recessive yellow allele (e) at this locus results from a frameshift that produces a prematurely terminated, nonfunctioning recep-

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tor [244]. The sombre (Eso and Eso–3j) alleles and tobacco darkening (Etob) alleles, which have dominant melanizing effects, result from point mutations that produce constitutively active or hyperactive receptors [244]. The human MC1R is highly polymorphic, and mutant alleles have been associated with fair skin and blond or red hair [245–247]. Analysis of five naturally occurring common variants of MC1R associated with fair skin and red hair have revealed decreased stimulation of cAMP synthesis with no changes in or only slightly reduced aMSH binding [248]. In addition, MC1R variants have recently been shown to determine sun-sensitivity in humans with dark hair [249]. Melanocytes, which express POMC and several of its processed peptides [250,251], are derived from the neural crest [29]. It is likely that a-MSH produced via POMC expression in melanocytes is responsible for the pigmenting effects of ultraviolet light, and together with variations in MC1R discussed above, for the different degrees of skin pigmentation observed among ethnic groups. MC1R mRNA [252] and protein [253] have been detected in Leydig cells of the testis, luteal cells of the corpus luteum, and in the placenta. In the central nervous system, detection of MC1R mRNA and protein has been confined to a few scattered neurons of the periaqueductal grey in rat and human brains [254]. With the cloning of the melanocortin receptors, the role of melanocortins as anti-inflammatory agents has gained renewed interest [255–257]. Systemic administration of a-MSH has been shown to be beneficial in animal models of arthritis [258], adult respiratory distress syndrome (ARDS) [259], and septic shock [260]. Consistent with this role, MC1R expression has been documented on macrophages and monocytes [261,262]. Treatment of activated macrophages with a-MSH resulted in decreased nitric oxide production by inhibiting the induction of nitric oxide synthase II [262]. Lipopolysaccharides (LPS) and interferon treatments of neutrophils induced increases in neutrophil MC1R mRNA and treatment with a-MSH inhibited neutrophil chemotaxis by a cAMP dependent process [263]. Constitutive expression of MC1R has also been found on dermal microvascular endothelial cells where its expression can be increased by stimulation with IL-1b or a-MSH itself [264]. A potential role for melanocortins to regulate local inflammation in the brain has been proposed based on the evidence that tumor necrosis factor (TNF)-alpha secretion by an anaplastic astrocytoma cell line (A-172) is decreased by aMSH and that these cells express MC1R [63].

Melanocortin 2 Receptor—the ACTH Receptor Eary in the study of receptor biology, Haynes demonstrated the action of ACTH in generating cyclic adenosine monophosphate (cAMP) in adrenal cells [265]. The ACTH receptor was the first receptor that was shown to bind its ligand with high affinity and specificity [266]. In human adrenal glands, the ACTH receptor has a Kd of 1.6 nmol/l

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and about 3500 sites/cell [267]. The ED50 of ACTH for cAMP production is 0.11 nmol/l, 20-fold less than the Kd for binding [267]. The ED50 of ACTH for cortisol production is 2 pmol/l, 720-fold less than the Kd for ACTH binding, and 35-fold less than the concentration of ACTH needed to obtain a half-maximal increase in cAMP production [267]. Only a small percentage of ACTH receptors need to be occupied to achieve a maximal effect on steroidogenesis, which occurs at an ACTH concentration of 0.01 nmol/l [268,269]. Because the adrenal gland may express more than one of the five melanocortin receptors (see below) it is not possible to assign the biochemical characterizations to a specific melanocortin receptor. The human ACTH receptor (MC2R) was cloned based on its homology to MC1R [225]. In situ hybridization with the adrenal gland of the rhesus monkey demonstrated the presence of messenger RNA in the zona glomerulosa and fasciculata cells, and a weaker signal in the zona reticularis [225]. Initial attempts at characterizing the ligand specificity and cAMP signal generation in response to ACTH and other melanocortins was confounded by either poor levels of expression or the presence of endogenous melanocortin receptors in transfected cells [225,270,271]. Recently, the human MC2R has been stably transfected into the Y6 cell line, a mutant derived from the mouse Y1 adrenocortical cell line, that fails to express any endogenous MC2R [272]. Y6 cells alone demonstrated no cAMP response to micromolar ACTH, and MC2R transfected cells displayed an EC50 of 6.8 nmol/l [272].

Adrenal Actions of Acth The critical role of ACTH in regulating the synthesis and secretion of steroids from the adrenal gland has long been recognized. In adrenocortical cells, ACTH regulates lipoprotein uptake by receptor-mediated endocytosis from the plasma to lipid droplets by stimulation of lipoprotein receptors [273]. Within the lipid droplets, ACTH regulates hydrolysis of cholesterol esters by activation of cholesterol esterases or suppression of cholesterol acyltransferase, through cAMP-dependent protein kinase [274]. ACTH stimulates the transport of cholesterol to the mitochondria [275–277], principally via stimulation of steroidogenic acute regulatory protein (StAR) [278]. The rate-limiting step in steroidogenesis is the side-chain cleavage of cholesterol to pregnenolone, and is catalyzed by cytochrome P450-side chain cleavage enzyme [279] in the inner membrane of mitochondria of the adrenal [280], probably on the matrix side [281]. ACTH stimulation results in long-term and short-term effects on steroid hormone biosynthesis in the mitochondria [282]. The long-term effect of ACTH leads to an increase in the amounts of steroid hormone enzymes by increasing transcription of these genes [282]. StAR is the key protein which regulates cholesterol transport into the mitochondion [278]. Mutations in this protein result in defects in adrenal and gonadal steroidogenesis, which had been previously attributed to defects in side chain cleavage

enzyme activity [278]. Beginning several hours after ACTH administration, ACTH increases the levels of steroidogenic enzyme mRNAs in primary cultures of human adrenals by several-fold, including cholesterol side chain cleavage enzyme, 17-a-hydroxylase/17,20-lyase, 11-b-hydroxylase/ 18-hydroxylase/18-methyl-oxidase, and 21-hydroxylase cytochrome P-450 enzyme [283]. ACTH has a positive regulatory effect on its own receptors, and on the cAMP response to binding of ACTH to the receptor [284]. With sustained stimulation, down-regulation does occur, but physiologically this effect is minor since ACTH causes proliferation as well as steroid secretion. In addition to its prominent role in regulating adrenal steroidogenesis, ACTH exerts profound trophic effects upon the adrenal. Hypophysectomy results in adrenal atrophy and ACTH replacement restores adrenal gland weight in a dosedependent manner [285–288]. While the role of ACTH in adrenal hypertrophy is well established, its role in adrenocortical mitogenesis and hyperplasia is incompletely understood. The absence of ACTH induces apoptotic cell death in the adrenal cortex [289]. Prolonged ACTH administration not only blocks apoptosis, but also increases the adrenal DNA content in the rat [290], ACTH increases mRNA levels for c-fos and b-actin, proteins involved in cellular proliferation [291]. However, ACTH paradoxically inhibits mitosis of adrenocortical cells in culture [292–294]. ACTH antiserum administered to intact rats caused a highly significant decrease in corticosterone levels, but had no effect on adrenal weight [295]. Furthermore, ACTH inhibits the rapid compensatory proliferation of the remaining adrenal that normally occurs after unilateral adrenalectomy [296]. Another anterior pituitary-derived candidate for the stimulation of adrenal proliferation is the 28 amino acid Nterminal pro-opiomelanocortin peptide (N-POMC). This peptide is mitogenic in vitro and in vivo for the adrenal cortex, and N-POMC antiserum significantly diminishes adrenal mitotic activity after enucleation [297,298]. The compensatory adrenal growth that occurs after unilateral adrenalectomy may be mediated by a neural reflex that includes afferent neurons originating from the disrupted adrenal gland, the ventromedial nuclei of the hypothalamus, and efferent neurons innervating the remaining gland [299,300]. In 1947, Albright coined the term adrenarche to denote the developmental increase in adrenal androgens that occurs several years before the onset of gonadal maturation [301]. Adrenal androgen secretion may be sufficient for the development of some secondary sexual changes, including the development of pubic and axillary hair [302] and the maturation of sebaceous glands [303]. A condition in which axillary and pubic hair develop prematurely as a result of early adrenal androgen secretion has been termed premature adrenarche [304–309]. The mechanisms controlling adrenarche have remained obscure. Many hypotheses have been advanced, and many factors including ACTH, estrogens, prolactin, gonadotropins, growth hormone, glucocorticoids, androgens, and other POMC-derived products have been

Chapter 2

suggested as modulators of adrenal androgen secretion (for review see [310]). ACTH is widely accepted as a modulator of adrenal androgen secretion although, after administration of corticotropin, increases in dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) tend to be small [311–313]. Furthermore, the increase in adrenal androgens that occurs in adrenarche is not accompanied by an increase in serum cortisol levels, leading to the suggestion that factors other than ACTH are responsible for adrenarche [307,309,314,315]. However, cortisol production rates do increase at the same time as does the increase in adrenal androgen secretion, suggesting a possible common link that could be due to ACTH stimulation of both steroid pathways [316]. Dissection of the physiology of adrenarche has proven difficult as the only animal showing an adrenarche similar to that of humans is the chimpanzee [317]. Recent data from patients with genetic defects in MC2R provide definitive evidence of the participation of this receptor in the process of adrenarche, as discussed subsequently. Familial Glucocorticoid Deficiency Syndrome

The familial glucocorticoid deficiency (FGD) syndrome is a rare autosomal recessive syndrome originally described by Shepard et al. in 1959 [318]. Patients typically present with symptoms resulting from their glucocorticoid insufficiency including hyperpigmentation, hypoglycemia, lethargy, and weakness [318–321]. The clinical course may be complicated by frequent infections [318,319]. Patients with FGD do not present with symptoms related to salt wasting, dehydration, or electrolyte disturbances, as the renin-aldosterone axis is preserved. The preservation of the renin-aldosterone axis clearly distinguishes this syndrome from childhood Addison’s disease. An unexplained feature of the syndrome is that many of the patients are reported to be unusually tall [318,319,321–325]. Patients with FGD typically have low or undetectable cortisol concentrations, but occasionally low, normal cortisol values, which respond subnormally to exogenous corticotropin [318,319,321–325]. Plasma ACTH levels are markedly elevated, often greater than 1000 pg/ml. The adrenal glands are atrophic, and only occasional cortical cells remained in the zona glomerulosa with no remnants of the zona fasciculata or reticularis, but the adrenal medulla appears normal [318,319,323]. This adrenal atrophy substantiates the physiologic relevance of ACTH’s trophic action on the adrenal gland. Recently, an examination of 11 patients with FGD revealed a discrepancy between partial glucocorticoid deficiency and significantly diminished DHEAS secretion confirming a significant contribution for ACTH (or at least for MC2R) in the onset of adrenarche [326]. A number of different homozygous or compound heterozygous missense and nonsense mutations in MC2R have been reported in patients with FGD, and in all cases, these mutations cosegregate with disease in the affected families [322,327–331]. Transient expression of the S741 mutant MC2R (ser74 Æ ile) in COS-7 cells resulted in a EC50 for cAMP production of 67 nmol/l, approximately 12-fold

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higher than the EC50 of the normal MC2R of 5.5 nmol/l. Expression of a Asp107 Æ Asn mutation in Cloudman S91 melanoma cells resulted in a six- to ninefold increase in the EC50 for cAMP generation. These results were confounded by endogenous melanocortin receptors making it difficult to determine whether the mutant receptor demonstrated defective ligand binding or defective coupling to adenylate cyclase [328]. Not all cases of the FGD syndrome can be accounted for by mutations in MC2R. Several families exhibiting the classic FGD signs and symptoms have no mutations in the coding regions of the MC2R gene [322,328,332,333]. These patients are clinically indistinguishable from patients with MC2R mutations except that they do not demonstrate increased height [334]. Recently, linkage of this form of FGD to a 12-cM region surrounding MC2R has been excluded in six families [332]. FGD, with ACTH resistance, also occurs in the Allgrove, also known as triple A (adrenal insufficiency, achalasia, and alacrima) syndrome [335]. Mutations in MC2R do not cause the resistance to ACTH. Recently, a novel gene, AAAS, has been identified on chromosome 12q13 in which several homozygous or compound heterozygous loss-of-function mutations are associated with this syndrome, in nine unrelated families [336,337]. This protein must in some way interfere with the expression or function of MC2R. ACTH-independent Activation of ACTH Receptor Pathways

Several rare causes of ACTH-independent Cushing’s syndrome are due to ACTH-independent pathological activation of ACTH receptor pathways. In the McCune-Albright syndrome, a mutation in the GTPase region of the stimulatory alpha subunit G protein, Gsa, can result in constitutive activation of protein kinase A in the adrenal cortex, leading to hypersecretion of cortisol and adrenal adenoma formation [338,339]. Ectopic expression of several other transmembrane G protein-coupled receptors, including those which bind gastric inhibitory polypeptide, luteinizing hormone, vasopressin, and catecholamines, in the adrenal cortex, may occur [340]. In this situation, Cushing’s syndrome results from the cognate hormone binding to the ectopically expressed receptor, leading to stimulation of adenylate cylase, activation of cyclic AMP, and cortisol hypersecretion [340]. Finally, patients with mutations in the regulatory subunit of protein kinase A, R1a, may develop Cushing’s syndrome secondary to micronodular adrenocortical hyperplasia [341,342]. This disorder, termed Carney complex, may be associated with myxomas of the cardiac atria and other tissues, and freckling of the skin [343]. EFFECTS OF POMC-DERIVED PEPTIDES OUTSIDE OF ADRENAL ACTH binds with high affinity to rat adipocytes and has potent lipolytic effects [344–347]. High levels of MC2R mRNA expression have been demonstrated in all murine

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adipose tissues examined, but MC5R mRNA expression was also found in a subset of these tissues [348]. Both MC2R and MC5R mRNA were identified in the 3T3-L1 cell line after these cells were induced to differentiate into adipocytes. The physiologic importance of the actions of melanocortins on adipose tissue is unclear. Primate adipose tissues have been reported to be insensitive to the lipolytic actions of ACTH [349]. MC2R mRNA expression was not detected in human adipose tissue [252]. In order to examine whether the MC2R might play roles in the hypothalamic–pituitary–adrenal axis in sites other than the adrenal gland, MC2R expression has been examined in the hypothalamus and the pituitary. MC2R mRNA could not be detected in the hypothalamus or the pituitary [350]. The expression of an ACTH receptor on human mononuclear leukocytes was suggested after a finding that high-affinity ACTH binding did not occur in a patient with FGD [351]. However, this finding is puzzling in retrospect as MC2R mRNA expression has not been demonstrated in leukocytes. Human skin cells express MC2R along with mRNA for three obligatory enzymes of steroid synthesis, the cytochromes P450scc (CYP11A1), P450c17 (CYP17), and P450c21 (CYP21A2) [352]. Slominski et al. have hypothesized that an equivalent of the HPA axis composed of locally produced CRH, CRH-receptor, POMC, and cortisol operate in mammalian skin as a local response to stress [353]. Recently, it was demonstrated that ACTH can induce DNA synthesis and cell proliferation in an oral keratinocyte cell line [354].

Melanocortin 3 Receptor Expression of the third cloned melanocortin receptor, MC3R, was originally identified in the brain, placenta, and gut [355]. Subsequently, MC3R expression was also documented in the heart [252]. Detailed mapping of the MC3R in the central nervous system by in situ hybridization localized the highest densities of MC3R mRNA to the hypothalamus and limbic system [356]. MC3R mRNA was found in the arcuate nucleus [356], the site of POMC expression within the hypothalamus. An autoradiographic approach using the non-selective [125I]NDP-MSH in competition with the relatively MC3R selective g-MSH or an MC4R selective synthetic agent HS014 has been used to visualize the MC3R and MC4R distributions within the central nervous system [357]. In the nucleus accumbens shell, the medial preoptic area, and the ventromedial nucleus of the hypothalamus, the MC3R dominates. In the lateral septum and the olfactory tubercle, both MC3R and MC4R seem to be present. The lack of overlap between the autoradiographic data and the data concerning MC3R mRNA expression may indicate the presence of MC3R on nerve terminals projecting from the arcuate nucleus. A physiologic role for MC3R has not yet been identified.

Recently, in a murine model of experimental gout, systemic treatment of mice with ACTH4–10 inhibited neutrophil accumulation [358]. This effect was blocked by he melanocortin receptor type 3/4 antagonist SHU9119. MC3R, but not MC4R, mRNA was detected in murine macrophages suggesting that MC3R may play a role in modulating inflammation. Recently, a mouse with targeted deletion of the MC3R gene has been created [359]. Although this mouse has a normal body weight, it has an increased fat to lean weight ratio, which seems to be due to an increased efficiency of converting ingested food into stored fuel. Thus, a function of melanocortins acting via MC3R may be to promote the conversion of energy in food into either lean body mass or forms of energy other than fat. This raises the possibility that in humans, mutations in MC3R may contribute to the “thrifty” genotype, as is found in Pima Indians [360].

Melanocortin 4 Receptor The MC4R is localized to the brain [361] and, in contrast to the MC3R, its expression has been documented throughout the central nervous system including the cortex, thalamus, hypothalamus, brainstem, and spinal cord [362]. In humans, the MC4R has not been detected in any peripheral tissue [252], although this distribution does not hold true for all animals as the MC4R is expressed in many peripheral tissues of the chicken [363]. The MC4R has garnered considerable attention as this melanocortin receptor subtype appears to play a central role in weight regulation. Mice homozygous for an Mc4r-null allele demonstrate autosomal dominant, maturity-onset obesity, hyperphagia, hyperinsulinemia, and hyperglycemia [364]. Heterozygotes have an intermediate phenotype. The Mc4r-null mouse also demonstrates increased linear growth [364], a feature unique to agouti yellow mice (discussed subsequently) and MC4Rnull mice among rodent obesity models. The MC4R has been implicated in leptin signaling as administration of the melanocortin receptor antagonist SHU9119 can block the reduction of food intake induced by central administration of leptin [365]. Leptin receptors are found on POMC expressing neurons in the arcuate nucleus and administration of exogenous leptin does not reverse obesity in Mc4rnull mice [366]. The role of MC4R in weight regulation in humans has been confirmed with the identification of individuals with dominantly inherited obesity segregating with mutations in the MC4R gene that result in frameshift errors [367–369]. Recent studies suggest that haploinsufficiency of MC4R may be a frequent, but incompletely penetrant, cause of human obesity [370,371]. The incomplete penetrance is highlighted by the absence of obesity in individuals with large deletions of chromosome 18q, a region that spans the MC4R gene [372]. A dominant-negative effect of mutant MC4R has been proposed, but cotransfection studies of mutant and wild-type MC4R in vitro showed that mutants

Chapter 2

affected neither signaling nor cell surface expression of wildtype MC4R [373].

Melanocortin 5 Receptor Expression of the fifth melanocortin receptor was originally recognized in the brain [374]. Subsequently, MC5R expression was documented widely at low levels in tissues including the adrenal glands, skin, adipocytes, skeletal muscle, kidneys, lung, stomach, liver, spleen, thymus, lymph nodes, mammary glands, ovary, pituitary, testis, and uterus [64,374–377]. In situ hybridization studies showed that within the adrenal, MC5R is predominantly expressed in the aldosterone-producing zona glomerulosa cells [376]. High levels of MC5R mRNA expression have been documented in the secretory epithelia of a number of exocrine and endocrine glands including Harderian, preputial, lacrimal, sebaceous, and prostate glands and pancreas [378,379]. Melanocortins have been reported to affect a number of exocrine glands. Removal of the neurointermediate lobe of the pituitary reduces sebaceous lipid production, and this reduction is restored by administration of a-MSH [380–382]. Exogenous ACTH and MSH increase secretion from the lacrimal gland [383,384]. Deletion of the murine Mc5r resulted in the loss of detectable binding of [125I]NDP-MSH to Harderian, lacrimal, and preputial glands, and skeletal muscle indicating that MC5R is the predominant melanocortin receptor in these tissues [378]. Development of the Mc5r-null mouse confirmed a physiologic role for the melanocortins in regulating exocrine gland function; the mice demonstrate severe deficits in water repulsion and thermoregulation as a result of decreased sebaceous lipid production [378]. MC5R also appears to be essential for hormonally regulated release of porphyrins from the Harderian gland [378]. The preputial gland is a specialized sebaceous gland implicated in pheromone production [385]. Exogenous a-MSH stimulates the release of a preputial odorant into the urine of male mice which stimulates aggressive attacks [386]. Chen and colleagues have hypothesized the existence of a hypothalamic– pituitary–exocrine axis which might provide a mechanism by which stress could alter behavior via the regulation of olfactory cues [378].

Diseases Caused by Melanocortin Receptor Ligands POMC Deficiency

Two individuals have been identified with genetic defects in the POMC gene [387] (Fig. 2.3). The first patient was a compound heterozygote for two mutations in exon 3 which interfere with appropriate synthesis of ACTH and a-MSH. The second patient was homozygous for a mutation in exon 2 which abolishes POMC mRNA translation. Both patients presented with early-onset obesity, adrenal insufficiency, and red hair pigmentation. The brother of patient one had died at the age of 7 months of hepatic failure following severe

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cholestasis and was found to have bilateral adrenal hypoplasia in the postmortem examination. All other anterior pituitary-derived hormones were normal. No symptoms related to b-endorphin deficiency have been noted. The heterozygous parents were normal in both families. Isolated ACTH deficiency of the pituitary due to other causes is also rare. It most commonly appears to be acquired in later life, and is more common in men [388–391]. Most cases have been reported in Japan, and are often associated with other autoimmune endocrinopathies, including Hashimoto’s thyroiditis and type 1 diabetes mellitus. It is likely that only pituitary ACTH, and not hypothalamic aMSH, is affected in these patients, as they are not obese and do not have red hair, unlike patients with POMC gene mutations [387]. Recently, the gene Tpit has been identified as a transcription factor specific to pituitary corticotrophs and melanotrophs, and absent in hypothalamic neurons which also express POMC [77]. It interacts with the POMC-specific transcription factor, Ptx1, to regulated POMC transcription. Two families with recessive mutations in TPIT have been described [77]. Consistent with its sites of expression, these patients have congenital, secondary adrenal insufficiency, but not obesity or red hair. Recently, a POMC-deficient mouse has been produced whose phenotype is similar to the human POMC-deficient syndrome and confirms the known functions of melanocortins [392]. The phenotype includes obesity, increased body length, yellow pigmentation, deficits in sebaceous gland function and thermoregulation, and adrenal hypoplasia and glucocorticoid deficiency. Adrenal glands could not be identified. In addition to undetectable corticosterone levels, aldosterone levels were also undetectable. The mutant mice lost 40% of their excess weight after 2 weeks of treatment with a stable a-MSH agonist. Although some of this weight loss was clearly attributable to decreased food intake, these same authors have clearly shown that a-MSH administration has additional lipolytic effects and results in increased energy expenditure [393]. Agouti and Agouti-related Protein

The agouti gene locus was identified 45 years ago as a genetic locus that controls the amount and distribution of eumelanin (brown–black) and pheomelanin (yellow–red) pigmentation in the mammalian coat [394]. However, analysis of mutations at the agouti gene locus have occupied investigators for nearly a century. The lethal yellow mutation at the agouti locus was the first murine embryonic lethal mutation and the first murine obesity syndrome to be characterized [395,396]. Agouti is a small 131 amino acid protein that is secreted by dermal papillae cells and acts to block melanocortin action on follicular melanocytes at Mc1r in the mouse [397,398]. The recombinant murine agouti protein is a potent nanomolar competitive antagonist for melanocortin receptors at Mc1r and Mc4r, relatively weaker at Mc3r, and only a micromolar inhibitor of Mc5r [397,399,400] and

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SECTION 1

Girl's weight

Boy's weight

35

–P97 82

Patient

–P90

30

72

25

–P75 –P50 –P25 –P10 –P3

62

20

–P97 –P90 –P75 –P50 –P25 –P10 –P3

15

52

Patient

42 32

10 22 5

0

12

0

3 6 9 12 18 24 30 36 42 48 Age (months)

2

0

2

4

6

8 10 12 14 16 18

Age (years)

represents the first known endogenous antagonist for a Gprotein coupled-receptor. In wild-type rodents, agouti expression is restricted to the hair follicle [398]. The human agouti gene which is 85% identical to the mouse gene is expressed much more widely including in adipose tissue, testis, ovary, and heart, and at lower levels in liver, kidney, and foreskin [401,402]. The human agouti protein, the agouti-signaling protein (ASIP), displays a similar pharmacologic profile for antagonism of melanocortin receptors as compared to the murine agouti protein except that ASIP may display both competitive and noncompetitive antagonism at MC4R and also appears to act as a noncompetitive antagonist of MC2R [403,404]. The heterozygous lethal yellow agouti mouse is characterized by a yellow coat-color, late onset obesity associated

FIGURE 2.3. Patients and weight curves of two unrelated patients with homozygous mutations in POMC. Note the obesity coupled with fair skin and red hair, as well as early onset obesity. From reference [387] with permission.

with hyperphagia, hyperinsulinemia and hyperglycemia, increased linear growth, and susceptibility to a wide variety of epithelial and mesenchymal tumors (reviewed in [405]). This phenotype is the result of a 170 kb deletion which removes all but the promoter and noncoding first exon of the Raly gene 5¢ to the exons of the agouti gene [406] and results in ubiquitous overexpression of agouti in all tissues examined to date [398,407]. The lethal yellow phenotype is presumably the result of agouti antagonism of the widely distributed melanocortin receptors and is consistent with the known actions of melanocortin receptors described above. An agouti-related protein (AGRP) gene was recently identified based on its homology to agouti and is expressed in the hypothalamus, adrenal medulla, and at low levels

Chapter 2

in the testis, lung, and kidney [408,409]. In contrast to the divergent expression patterns of murine and human agouti, the expression pattern of murine and human AGRP appears identical [408]. Within the hypothalamus, AGRP expression is confined to the arcuate nucleus and AGRP-immunoreactive terminals paralleled POMCimmunoreactive terminals projecting from the arcuate nucleus [55]. AGRP is a selective, nanomolar competitiveantagonist of MC3R and MC4R, clearly implicating it as the endogenous melanocortin antagonist involved in energy homeostasis [410–412]. Corticostatins are a family of related low molecular weight members of the defensin family of peptides that are competitive inhibitors of ACTH-induced steroidogenesis in the adrenal cortex, which act by blocking the ACTH receptor [413]. In rats, a dose of 8 mg/kg body weight blocks the corticosterone response to stress [414]. Corticostatins and the agouti peptides are the two known endogenous competitive inhibitors of melanocortin receptors, but do not appear to be structurally related [415].

Additional Potential Melanocortin Actions Additional physiologic roles for melanocortins have been proposed that do not clearly correlate with the known functions of specific melanocortin receptors as surmised from human or mouse mutants. A multitude of behavioral and psychological effects have been attributed to melanocortins (for review see [416]). Intracerebroventricular administration of ACTH or a-MSH elicits excessive grooming behavior [417], yawning, stretching, and penile erection [418]. The grooming, stretching, and yawning behavior, but not erectile function, may be mediated by MC4R as deduced through the use of MC4R selective antagonists [233,419,420]. Recently, in a double-blind, placebo controlled crossover study, a cyclic a-MSH analogue initiated erections in men with psychogenic erectile dysfunction [421]. Roles for the actions of melanocortins in neuromuscular development, promotion of the regeneration of crushed nerves, and CNS protection from injury have also been postulated [235,422–425]. A potential role for melanocortins in fetal growth and brain development has been demonstrated [426,427]. Data indicating a role for melanocortins as modulators of inflammation have been discussed previously. Centrally administered MSH is a potent antipyretic agent and, in an endogenous pyrogen-induced fever model in rabbits, is approximately 25,000-fold more potent than acetaminophen [428]. Physiologic roles for melanocortins in maintenance of cardiovascular homeostasis have also been proposed. Peripheral or central administration of g-MSH causes tachycardia and pressor effects [429,430]. Central administration of a-MSH results in bradycardia and depressor effects [429,430]. Melanocortins may further influence cardiovascular homeostasis through their effects on electrolyte regulation. a-MSH and g-MSH are potent natriuretics [431–434].

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SECRETION OF ACTH: PHYSIOLOGY Physiologically a number of factors interact to determine the final pattern of ACTH release, including circadian rhythms, stress, and negative feedback by glucocorticoids. These factors impact on each other in an integrated fashion to control ACTH release. The stage of development, from fetal life through puberty, and pregnancy and parturition, also impact on ACTH secretion. Finally, the immune system interacts with the HPA axis, adding another facet to the complexity of ACTH release.

Secretion Dynamics of ACTH in vivo ACTH secretion is characterized by pulsatile release of ACTH from the corticotroph in a burst-like pattern, with no interpulse secretion [435]. Fifteen-minute sampling reveals approximately 12 ACTH and cortisol pulses over a 24-hour period [436], whereas more frequent 10-minute sampling reveals 40 ACTH pulses in 24 hours [435]. Blood ACTH rises by an average of 24 pg/ml per pulse [435]. bendorphin secretion parallels the pulsatile release of ACTH [437]. Spontaneous ACTH and cortisol pulses correlate highly. There is a strong relationship between the magnitude of concomitant ACTH and cortisol pulses, particularly if a 15 minute phase delay in cortisol secretion is allowed for [435,438–440]. Not all spontaneous ACTH and cortisol pulses are concomitant: approximately 50% to 75% of spontaneous ACTH pulses are followed by a cortisol pulse, whereas approximately 60% to 90% of spontaneous cortisol pulses are preceded by an ACTH pulse [436,440]. The 24-hour pattern of ACTH pulses, but not the cortisol pattern, differs between males and females. Males have more pulses, greater mean peak ACTH amplitude, greater 24-hour ACTH secretion, and higher mean ACTH levels [438]. The sensitivity of the adrenal cortex, or the availability of ACTH to the adrenal cortex, may be greater in females. Alternatively, males and females may have different set points for cortisol feedback [438]. CRH may also be secreted in a pulsatile fashion, accounting for pulsatile ACTH secretion. When exogenous CRH is given continuously, there is a progressive desensitization of the ACTH response to CRH [441]. However, when CRH is given in a prolonged pulsatile manner, it does not desensitize the corticotroph CRH receptor, and the releasable ACTH pool is not depleted [436].

Negative Regulation of ACTH Secretion Negative feedback can be defined as long, short, or ultrashort depending on the location and nature of the hormone mediating the feedback. Long Feedback by Glucocorticoids

Long feedback refers to the effects of adrenal glucocorticoids on ACTH secretion at the pituitary and in the hypo-

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thalamus. Glucocorticoid-mediated negative feedback can be subdivided into fast and delayed, which can be further subdivided into intermediate and slow feedback. In animals, the initial fast negative effect of glucocorticoids occurs within seconds to minutes [183], involves inhibition of stimulated ACTH and CRH release, not synthesis, and occurs during the period when plasma glucocorticoid levels are increasing. Cortisol given to patients at the start of surgery attenuates the surgery-induced ACTH rise [211], and may be an example of fast feedback. Delayed feedback has two components, intermediate and slow [442]. Intermediate feedback is the component of delayed feedback that is due to inhibition of ACTH release, but not synthesis, and may be important after short durations of glucocorticoid exposure, or after noncontinuous, repeated exposures [442]. Intermediate delayed feedback develops after 45 to 120 minutes, and maximal inhibition occurs 2 to 4 hours after administration of one dose of glucocorticoids. Unlike ACTH, CRH synthesis as well as release may be affected by intermediate feedback [442]. The slow component of delayed feedback is most important after long exposures to a moderately high dose of glucocorticoid, and is a function of the total dose of glucocorticoids, the glucocorticoid level achieved, and the amount of time since the steroid was given [442]. Slow feedback occurs after more than 24 hours of exposure to glucocorticoids and can persist for days. POMC biosynthesis is inhibited, leading to inhibition of basal and stimulated ACTH secretion [442], and intracellular ACTH decreases implying decreased synthesis. In humans, negative feedback by glucocorticoids takes 30 to 60 minutes to manifest. When endogenous cortisol is suppressed with metyrapone, the effects of exogenous glucocorticoids on the morning ACTH rise, or on oCRHstimulated ACTH release, are not seen initially but appears 30 minutes after glucocorticoid administration [443]. In addition, glucocorticoids have little effect on the initial CRH-induced increase in ACTH release, but decrease CRH-induced ACTH release after 60 minutes. Cortisol given to patients at the start of surgery attenuates the surgery-induced ACTH rise [211]. Cortisol modulates the responsiveness of the pituitary. The corticotroph is dependent on CRH stimulation to maintain ACTH secretion, and glucocorticoids suppress CRH-induced ACTH secretion in vivo and in vitro. On the other hand, when endogenous cortisol levels are suppressed by metyrapone, basal ACTH and CRH-induced ACTH release are increased [444]. Glucocorticoid inhibition of ACTH secretion from the corticotroph may recover more quickly than CRH secretion from the hypothalamus [445]. Secondary adrenal insufficiency due to long-term glucocorticoid therapy may in part be due to continued suppression of hypothalamic CRH secretion 184. Adrenalectomized patients on exogenous glucocorticoid therapy have a blunted ACTH response to CRH that normalizes after several CRH boluses, suggesting that lack of stimula-

tion of the corticotroph by CRH suppresses the ACTH response [446]. On the other hand, corticotrophs of patients recovering from transsphenoidal surgery for Cushing’s disease are profoundly unresponsive to CRH, which can not be attributed solely to deficient CRH priming [447]. Glucocorticoids inhibit AVP secretion [448]. In most studies, patients with hypopituitarism or primary adrenal insufficiency are unable to maximally dilute their urine in response to a water load, and this is corrected by glucocorticoid administration [449]. However, it is unclear if the elevated AVP levels, or the lack glucocorticoids, is responsible for the inability to maximally dilute the urine in the hypocortisolemic state [448]. Glucocorticoids inhibit nitric oxide synthase, and nitric oxide is capable of stimulating the insertion of the water channel, aquaporin 2, in the luminal membrane of the renal collecting cell [450]. This may provide an explanation for why glucocorticoid deficiency is associated with decreased free water clearance. Short Feedback

Short feedback refers to the effect of pituitary ACTH to inhibit CRH release. In normal subjects, the administration of ACTH does not affect CRH levels, most likely because of the negative effects of cortisol present prior to ACTH administration [451]. However, in patients with elevated CRH levels due to Addison’s disease or hypopituitarism, ACTH decreases CRH and b-endorphin levels, suggesting that ACTH inhibits CRH secretion [451]. ACTH may act in the median eminence or in the hypothalamus to inhibit CRH release [451].

Circadian Regulation of ACTH Secretion There is an endogenous circadian rhythm to the pulsatile pattern of ACTH secretion which leads to a circadian rhythm of glucocorticoid release. The function of this circadian rhythm in cortisol secretion is not known, although one hypothesis suggests that the early morning rise in cortisol causes a delayed-phase rise in insulin resistance, which may play a role in altered glucose metabolism [452]. For instance, since the brain does not require insulin for glucose uptake, peripheral insulin resistance might cause a rise in glucose levels, leading to greater uptake in the brain. The circadian rhythm is generated in the suprachiasmatic nucleus (SCN), and the signals travel via efferent inputs to the PVN to modulate CRH release [183,453]. This circadian rhythm is due to variation in ACTH pulse amplitude, not frequency [435]. The amount of ACTH secreted per pulse varies by 3.8-fold over a 24-hour period [435]. Basal ACTH and cortisol levels parallel each other and are the highest upon awakening in the morning between 0600 hours and 0900 hours, decline through the day to intermediate levels at 1600 hours, and are lowest between 2300 hours and 0300 hours [435]. From 2300 hours to 0200 hours, there is a quiescent period of minimal secretory activity, corresponding to the nadir of ACTH and cortisol levels [454]. Secretion of

Chapter 2

ACTH and cortisol abruptly increases in the early morning [454]. The diurnal secretory pattern is similar for free and total cortisol, although the relative increase in free cortisol is about 1.5 times greater than the relative increase in total cortisol [454]. Alterations in feeding and sleep impact on cortisol secretion [455]. Cortisol briefly increases postprandially, especially after the midday meal [454]. Exercise or administration of ACTH at 1000 hours leads to a rise in cortisol and blunts the midday cortisol surge, and at 1400 hours leads to a rise in cortisol [454]. Overall, the major features of the diurnal cortisol pattern persist under conditions of complete fasting, continuous feeding, or total sleep deprivation [456]. However, the circadian rhythm of cortisol secretion fully adapts to permanent changes in environmental time and the sleep–wake pattern. This adaptation takes about 3 weeks, the limiting factor being the time it takes for the quiescent period of secretion to fully adapt [456]. The acrophase adapts much more quickly and is partially synchronized after one day and totally synchronized after 10 days [456]. There is circadian regulation of the sensitivity of the response of the adrenal cortex to ACTH [457,458]. Injection of ACTH at 0700 hours, just at the time of the endogenous cortisol peak, causes a significant increase in cortisol. The absolute increment in cortisol secretion in response to an ACTH stimulation test is greater when the test is performed at circadian nadir compared to the peak, although in one study this was true in males only [459]. The incremental increase in cortisol secretion in response to a CRH stimulation test is also greatest at night when basal levels are lowest, although the total amount of cortisol released is greatest in the morning when the basal cortisol levels are highest [460]. The basal cortisol level, not the time of day, seems to be the important factor determining the cortisol response to oCRH, and the higher the basal cortisol level, the lower the peak cortisol response to oCRH [436]. On the other hand, the maximum ACTH blood level in response to oCRH occurs at 0700 hours, the time of the minimum cortisol increment to oCRH [436]. The factors governing the circadian rhythm in ACTH release in humans are not clear. On the one hand, it may be regulated by a diurnal rhythm in CRH secretion [461]. The highest CRH levels occur at 0600 hours (7.0 pg/ml) and the lowest at 1800 hours and 2200 hours (about 1.8 pg/ml), which parallels the pattern of ACTH and cortisol secretion [462]. Serotoninergic and cholinergic pathways may play a role in the CRH circadian rhythm, and display circadian periodicity in their hypothalamic concentrations [463]. On the other hand, when the pituitary is exposed to constant levels of CRH 30-fold higher than those found in portal hypophyseal blood, such as in pregnancy [464] and during CRH infusion [441], the circadian rhythms in ACTH and cortisol persist. CRH clearly plays a role in the ACTH rhythm, as CRH-deficient mice have an absent or markedly attenuated diurnal rise in ACTH [465]. However, in these mice, constant infusion of CRH restores the ACTH

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rhythm. This indicates that changes in CRH amplitude are not necessary to drive the ACTH rhythm, but that a tonic level of CRH is required to maintain ACTH responsiveness [466] to circadian cues.

Physical Stress Regulation of ACTH Secretion Glucocorticoid release mediated by ACTH, plays a major role in the response to stress. An interaction exists between stress-mediated ACTH release, which leads to glucocorticoid secretion, and glucocorticoid-mediated negative feedback, which inhibits further ACTH and glucocorticoid release. A number of stressful stimuli lead to ACTH secretion. Most physical stressors activate the HPA axis. The magnitude of the rise in ACTH and cortisol is dependent upon many factors, including the nature of the stress, its magnitude including the rapidity of its appearance, and the time of day it is experienced. In general, stressors have a larger impact on ACTH release when they develop rapidly, are of high magnitude, and occur during the circadian nadir in ACTH release. Hypoglycemia

In humans, insulin induced hypoglycemia is associated with a increase in plasma ACTH levels [467], from a basal level of about 40 pg/ml to a peak of 250 pg/ml at 45 minutes [467]. Cortisol levels increase over twofold, from a basal level of about 11 ug/dl to a peak of about 25 ug/dl at 60 to 90 minutes [467]. Insulin-induced hypoglycemia causes a four- to fivefold greater increase in ACTH secretion than oCRH alone, and a 1.3-fold greater increase than AVP plus CRH [97,468]. CRH may play a permissive rather than a dynamic role in the ACTH response to hypoglycemia [182], whereas AVP may play a more direct role [467]. AVP levels have been shown to increase 2.5- to sevenfold at 30 to 45 minutes after the insulin. When AVP levels are raised endogenously by saline infusion or lowered to undetectable levels by waterloading, the hypoglycemia-induced AVP increase is greater after saline, even though saline blunts the hypoglycemic response to insulin [469]. Catecholamines increase in response to hypoglycemia, and may act at the hypothalamus to mediate ACTH release. Epinephrine appears to play more of a role than norepinephrine, increasing at least 13fold at 30 minutes after insulin, whereas norepinephrine increases 2.4-fold at 60 minutes. Exercise

Exercise increases ACTH and b-endorphin levels, and the response is dependent on the intensity of exercise and the level of training [470]. Exercising to exhaustion, or exercise of short duration and high intensity, increases ACTH, bendorphin, and cortisol levels. Hypercortisolism is seen in highly trained athletes, who need a higher level of oxygen consumption to stimulate ACTH release, and have elevated basal ACTH levels [471]. Physical exercise and stress both

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lead to analgesia in man [472]. Naloxone reverses exerciseinduced analgesia from certain types of pain, suggesting a role for endorphins. Dexamethasone reverses exerciseinduced analgesia from other types of pain, like dental pain, that are not reversed by naloxone, suggesting a role for ACTH, although dexamethasone also suppresses bendorphin release. Lower Body Negative Pressure and Acute Hemorrhage

Lower body negative pressure in humans simulates acute hemorrhage. Lower body negative pressure increases ACTH secretion to peak values of 60–250 pg/ml at 2 to 10 minutes after cessation of the stimulus, and the increase in ACTH is reversed by dexamethasone [473]. Hypovolemia may be the physiologically important stress. In animals, hemorrhage stimulates ACTH secretion primarily mediated by CRH and, to some degree, by AVP [182]. Hypovolemia increases portal blood CRH, AVP, epinephrine, and oxytocin, whereas hypotension, also a component of hemorrhage, induced by nitroprusside, increases portal blood CRH only. Surgical Stress

Surgery induces a large increase in plasma ACTH levels [284]. Patients undergoing surgery may have increased sensitivity of the adrenal cortex to ACTH [474,475]. Fentanyl, an opiate agonist, attenuates the ACTH response to surgery [211]. Evidence in adrenalectomized primates suggests that supraphysiological doses of glucocorticoids are not necessary for the animal to withstand surgical stress, but that a minimal level is necessary [474,476].

Psychological and Emotional Stress Psychological and emotional stress play a role in the hormonal stress response. ACTH levels are high in patients awaiting an insulin-tolerance test. During physical exercise, psychological and physical stress may act synergistically to increase b-endorphin and ACTH levels [477]. With increasing experience in performing a set level of exhaustive physical exercise, plasma ACTH and cortisol levels declined [478].

Fetal and Postnatal Regulation of ACTH Secretion In utero, maternal cortisol influences alterations of fetal heart rate, movement, and behavioral states [479]. By 35 weeks of gestation, there is a circadian rhythm in fetal behavioral states that is altered if the maternal diurnal variation in ACTH and cortisol secretion are abolished by maternal administration of triamicinolone [479]. Neonates, even when born prematurely, have an endogenous cortisol rhythm. Minimally and severely stressed neonates in the neonatal intensive care unit, born between 23 and 38 weeks of gestation, have a significant diurnal rhythm in cortisol and endorphin secretion, although ACTH levels do not vary sig-

nificantly [463]. Others have suggested that diurnal rhythmicity is a function of maturation, and is not present before 6 months of age [463,480,481].

Regulation of ACTH Secretion During Puberty Normal children between the ages of 1 year and 16 years do not differ from adults in ACTH, b-endorphin, and cortisol responses to CRH, and the responses of boys do not differ from girls [482]. Some of the other adrenal steroids and adrenal androgens demonstrate basal and stimulated variation with age. The CRH-stimulated androstenedi one to 17-hydroxyprogesterone ratio increases with sexual maturation, suggesting that the 17,20-desmolase activity increases with puberty [483]. The dehydroepiandrosterone response to CRH increases as children progress from stage 1 to stage 5 of puberty, and by stage 5 of puberty, dehydroepiandrosterone levels do not differ from adults [482].

HPA-Axis-Immune Interactions The immune system and the neuroendocrine system communicate with each other, and share a common set of structurally identical hormones and receptors [484–488]. Cells of the immune system synthesize biologically active neuroendocrine peptide hormones, immune cells contain receptors for neuroendocrine hormones, neuroendocrine hormones modulate immune function, and lymphokines modulate neuroendorine function. The neuroendocrine system and the immune system work together in the regulation of both the stress and the immune response, and the components include lymphoid cells, cholinergic and adrenergic neurons, cytokines and lymphokines, hormones and neuropeptides released by the endocrine glands and the CNS, receptors, and higher CNS activity which modulates these responses [489,490]. Effect of Immune System on HPA Axis

Immune cells, particularly monocytes, macrophages, and lymphocytes, produce cytokines involved in the immune response, and these cytokines activate the HPA axis. Bacterial-derived endotoxin and lipopolysacchride stimulate the release of interleukin-1, interleukin-6, and TNF-a, which are regulated by glucocorticoid feedback. The effects of cytokines on the HPA axis at the level of the brain, pituitary, and adrenal gland, are the best examples of immune modulation of a neuroendocrine system. Interleukin-1 is produced by stimulated macrophages and monocytes, and stimulates CRH release. Interleukin-1 also directly stimulates the adrenal cortex. Tumor necrosis factor is produced primarily by activated monocytes [491], and has many of the same biological activities and responds to many of the same immune challenges as interleukin-1. Tumor necrosis factor stimulates ACTH release, most likely at an extra-pituitary site. Interleukin-6 has actions similar to interleukin-1 and

Chapter 2

TNF, and stimulates the HPA axis [202,491]. Interleukin-2 is synthesized by T cells after an antigenic challenge, and increases ACTH and cortisol levels in humans, although this may be indirect and due to the stress response generated by fever and chills. Gamma-interferon causes an increase in steroid production by adrenal cells. As discussed above, leukemia-inhibitory factor (LIF) may be a major activator of POMC gene transcription and ACTH release following immune or inflammatory stimulation. Effect of HPA Axis on Immune System

Stress suppresses immune function. Following an infection or most immunization procedures, and the presence of bacterial endotoxin, a stress-like response of the pituitary occurs leading to the release of ACTH and cortisol which tends to suppress the immune system. However, in humans, there is no clear evidence that the rise in cortisol following an immune or inflammatory stimulus plays a significant role in modulating the subsequent immune response. In fact, prior to the treatment of patients with primary adrenal insufficiency, there were no substantive reports that these patients suffered from overactivity of their immune systems. Receptors for CRH, ACTH, and glucocorticoids most likely mediate the effect of these hormones on the immune system. ACTH receptors on human peripheral monocytes have been characterized, and glucocorticoid receptors are present on human lymphocytes. Glucocorticoids inhibit many aspects of immune function, establishing a negative feedback loop between the immune and neuroendocrine systems. Glucocortiocoids block lymphocyte activation, block the production and action of interleukin-2, interleukin-1, gamma-interferon, TNF, and prostaglandins, and interfer with the interaction of certain effector molecules with target cells. HPA Axis Within Immune System

Cells of the immune system produce CRH, ACTH and endorphins. Human peripheral mononuclear leukocytes synthesize three molecular forms of immunoreactiveACTH, and lymphocytes produce b-endorphins. Mouse spleen macrophages and virally infected mouse splenocytes contain b-endorphin and POMC mRNA [492–494]. CRH mRNA and peptide are found in monocytes in acute inflammatory reactions [495] and in T cell lymphocytes [496]. Releasing hormones and cytokines interact to stimulate ACTH production from immune cells. CRH and AVP stimulate a dramatic increase in biologically active ACTH and b-endorphin from human peripheral leukocytes in some studies. Unstimulated leukocytes produce little ACTH. In humans, only B lymphocytes secrete b-endorphin in response to CRH and AVP, and monocyte-secreted interleukin-1 mediates the effect. A feedback loop exists between the immune and neuroendocrine systems [490]. Cytokines released by immune cells stimulate secretion of ACTH and glucocorticoids which are active in the fight

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against infection. Subsequently, these hormones suppress the further synthesis of cytokines. SECRETION OF ACTH: PATHOPHYSIOLOGY Abnormal secretion of ACTH occurs in ACTH-secreting pituitary adenomas responsible for Cushing’s disease or Nelson’s syndrome, and in Cushing’s syndrome due to an ectopic pituitary adenoma or an ectopic ACTH-secreting tumor. Decreased or absent ACTH secretion due to genetic or autoimmune causes has been discussed in the above section on melanocortin actions.

Pituitary Adenomas ACTH immunoreactivity in an abnormal pituitary is usually due to a single functional adenoma, but can be associated with nodular hyperplasia or a silent cortiocotropic adenoma. Diffuse or nodular corticotroph hyperplasia, which could result from an ectopic or hypothalmic CRH-producing tumor, or Addison’s disease, is a rare cause of a pituitary adenoma or ACTH hyperfunction. Pituitary adenomas constitute about 15% of intracranial tumors [3]. Approximately 56% of pituitary adenomas are active, and about one third of active pituitary adenomas produce ACTH. Ten to 15% of pituitary adenomas are pleurihormonal, some of which secrete ACTH. When discovered, ACTH-producing adenomas are often functional, small, highly vascular, and prone to hemorrhage [497]. Corticotroph adenomas tend to be located in the central portion of the adenohypophysis, in the “mucoid wedge,” and form micronodular aggregates [497]. Invasive adenomas are more frequently found among undifferentiated, extermely laterally localized, or large adenomas, and recur more frequently than non-invasive tumors. There is no correlation between the size of an adenoma and the cortisol level or rate of recurrence [498]. The majority of adenomas responsible for Cushing’s disease or Nelson’s syndrome are microadenomas. ACTH-producing adenomas are usually monoclonal, but may be polyclonal [499]. Pleurihormonal adenomas are usually polyclonal [499]. The biochemical structure and ultrastructure of ACTHsecreting pituitary adenoma cells differ from nonadenomatous pituitary cells [500]. The cells may look normal, but be increased in number [497]. Cells are oval to polygonal in shape, with eccentric spherical nuclei and well-developed rough endoplasmic reticulim. Crooke’s hyaline is characteristic of ACTH-producing tumors [501], is associated with either endogenous or exogenous hypercortisolism, and is due to massive accumulation of intermediate cytoplasmic filaments that are normally present in small numbers [3]. Clinically, an increased number of Crooke’s cells is correlated with a longer postoperative replacement dose of cortisol requirement [498]. Pituitary adenomas may produce products in addition to ACTH. Some ACTH-producing corticotroph adenomas contain a form of gastrin that is smaller than gastrin found

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in the normal adenophypophysis. 7B2 is a secretory granule-associated protein, which may be involved in proconvertase activtion [502], which is sometimes secreted by ACTH-producing tumors, it is secreted in the highest levels from nonfunctional pituitary tumor. A variant of Cushing’s disease is due to ACTHproducing pituitary adenomas occurring ectopically [5], and have been described to arise in the mucosa of the sphenoid sinus [5,503], within a benign cystic ovarian teratoma [504], and intrahemispherically in a 6-year-old boy [505]. Rarely, both Cushing’s disease and Nelson’s syndrome have been preceded by generalized glucocorticoid resistance due to a mutation in the glucocorticoid receptor [506,507]. In these cases, the high rate of ACTH secretion, stimulated by the generalized glucocorticoid resistance, in some way led to adenoma formation, perhaps following a second oncogenic transformation. Pituitary carcinomas are malignant pituitary tumors associated with extracranial metastases, including liver and lung [508]. Pituitary carcinomas cannot be histologically differentiated from adenomas [3]. A large number of pituitary carcinomas produce Cushing’s disease [3]. In patients with Cushing’s disease due to a pituitary carcinoma, the primary tumor and metastases stain immunochemically for ACTH, b-LPH, b-endorphin, and a-MSH [508], and production of both CRH and ACTH from a pituitary carcinoma has been described [509].

Most nonpituitary POMC-secreting tumors do not produce CRH [515]. However, some patients with ectopic ACTH syndrome due to lung cancer have tumors that produce ACTH and CRH, and secrete ACTH in response to CRH [516]. Bronchial carcinoid tumors may contain CRH and ACTH, and be associated with high plasma ACTH and CRH levels and Cushing’s syndrome, or with normal plasma CRH levels. Cushing’s syndrome has been associated with ectopic ACTH secretion from a unilateral adrenal pheochromocytoma [517], and from bilateral pheochromocytomas in a case of multiple endocrine neoplasia type 2A [518]. A number of other ectopic tumors have been described that secrete ACTH, including an adenoid cystic carcinoma of the lung [519], a renal cell carcinoma [520], a neuroendocrine tumor of the nasal roof [521], and an ACTHproducing tumor metastatic to the liver [522]. In general, POMC mRNA from nonpituitary tumors responsible for the ectopic ACTH syndrome is identical to normal and to POMC mRNA from pituitary tumors [523]. However, some tumors contain a larger POMC mRNA species that is increased in amount from 0.3% of the overall POMC mRNA in normal pituitaries, to up to 35 to 40% in the tumor, and is transcribed from an alternative upstream promoter [523]. Pancreatic islet cell tumors responsible for Cushing’s syndrome have been demonstrated to contain ACTH and b-endorphin [524], and to express POMC mRNA [524].

Ectopic ACTH-secreting Tumors Several neuroendocrine neoplasms occur in the bronchopulmonary tract, including small-cell neuroendocrine carcinomas, carcinoids, well-differentiated neuroendocrine carcinomas, and intermediate-cell neuroendocrine carcinomas [510]. These neoplasms express neuroendocrine markers including chromogranins and synaptophysin [510]. Thirtyfour percent of small-cell carcinomas of the lung show immunoreactivity to one or more peptide hormones [511], and patients with peptide-positive small-cell lung carcinomas have a shorter mean survival than in patients with nonreactive tumors. ACTH is by far the most common hormone present, and is seen in 24% of small-cell carcinomas [511]. However, 56% of small-cell lung cancer cell lines secrete significant concentrations of ACTH precursors, with little, if any, processing to ACTH. Oat-cell lung carcinomas may also produce ACTH, leading to Cushing’s syndrome [512]. Carcinoid tumors often produce more than one hormone, and can be responsible for the ectopic ACTH syndrome [513]. Bronchial carcinoids occasionally contain ACTH and related opioid peptides, which does not alter the overall favorable prognosis of these tumors. Recently, Cushing’s syndrome caused by ACTH secretion by pulmonary tumorlets has been described [514]. Upon radiologic imaging, such tumorlets, which may be over 100 in number, present a very unusual appearance.

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187 Jones MT, Hillhouse EW, Burden J. Effect on various putative neurotransmitters on the secretion of corticotrophin-releasing hormone from the rat hypothalamus in vitro – a model of the neurotransmitters involved. J Endocrinol 1976;69:1–10. 188 Rivier C, Vale W. Effects of corticotropin-releasing factor, neurohypophyseal peptides, and catecholamines on pituitary function. Fed Proc 1985;44: 189–195. 189 al-Damluji S. Adrenergic mechanisms in the control of corticotrophin secretion. J Endocrinol 1988;119:5–14. 190 Watanabe T, Oki Y, Orth DN. Kinetic actions and interactions of arginine vasopressin, angiotensin-II, and oxytocin on adrenocorticotropin secretion by rat anterior pituitary cells in the microperifusion system. Endocrinology 1989; 125:1921–1931. 191 Schoenenberg P, Gaillard RC, Kehrer P et al. cAMP-dependent ACTH secretagogues facilitate corticotropin releasing activity of angiotensin II on rat anterior pituitary cells in vitro. Acta Endocrinol (Copenh) 1987;114:118–123. 192 McCann SM, Rettori V, Milenkovic L et al. Role of monokines in control of anterior pituitary hormone release. Adv Exp Med Biol 1990;274:315–329. 193 Berkenbosch F, van Oers J, del Rey A et al. Corticotropin-releasing factorproducing neurons in the rat activated by interleukin-1. Science 1987;238: 524–526. 194 Sapolsky R, Rivier C, Yamamoto G et al. Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 1987;238:522–524. 195 Matta S, Singh J, Newton R et al. The adrenocorticotropin response to interleukin-1 beta instilled into the rat median eminence depends on the local release of catecholamines. Endocrinology 1990;127:2175–2182. 196 Michie HR, Majzoub JA, ODwyer ST et al. Both cyclooxygenase-dependent and cyclooxygenase-independent pathways mediate the neuroendocrine response in humans. Surgery 1990;108:254–259. 197 Michie HR, Spriggs DR, Manogue KR et al. Tumor necrosis factor and endotoxin induce similar metabolic responses in human beings. Surgery 1988;104:280–286. 198 Gaillard RC, Turnill D, Sappino P et al. Tumor necrosis factor alpha inhibits the hormonal response of the pituitary gland to hypothalamic releasing factors. Endocrinology 1990;127:101–106. 199 Sharp BM, Matta SG, Peterson PK et al. Tumor necrosis factor-alpha is a potent ACTH secretagogue: comparison to interleukin-1 beta. Endocrinology 1989;124:3131–3133. 200 Michie HR, Manogue KR, Spriggs DR et al. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 1988;318: 1481–1486. 201 Yasuda N, Greer MA. Evidence that the hypothalamus mediates endotoxin stimulation of adrenocorticotropic hormone secretion. Endocrinology 1978; 102:947–953. 202 Naitoh Y, Fukata J, Tominaga T et al. Interleukin-6 stimulates the secretion of adrenocorticotropic hormone in conscious, freely-moving rats. Biochem Biophys Res Commun 1988;155:1459–1463. 203 Navarra P, Tsagarakis S, Faria MS et al. Interleukins-1 and -6 stimulate the release of corticotropin-releasing hormone-41 from rat hypothalamus in vitro via the eicosanoid cyclooxygenase pathway. Endocrinology 1991;128:37–44. 204 Bochicchio D, Ambrosi B, Faglia G. Loperamide, an opiate analog, differently modifies the adrenocorticotropin responses to corticotropin-releasing hormone and lysine vasopressin in patients with Addison’s disease. Neuroendocrinology 1988;48:611–614. 205 Atkinson RL. Endocrine and metabolic effects of opiate antagonists. J Clin Psychiatry 1984;45:20–24. 206 Gaillard RC, Grossman A, Smith R et al. The effects of a met-enkephalin analogue on ACTH, beta-LPH, beta-endorphin and MET-enkephalin in patients with adrenocortical disease. Clin Endocrinol (Oxf ) 1981;14:471–478. 207 Blankstein J, Reyes FI, Winter JS et al. Effects of naloxone upon prolactin and costisol in normal women. Proc Soc Exp Biol Med 1980;164:363–365. 208 Grossman A, Gaillard RC, McCartney P et al. Opiate modulation of the pituitary–adrenal axis: effects of stress and circadian rhythm. Clin Endocrinol (Oxf) 1982;17:279–286. 209 Morley JE, Baranetsky NG, Wingert TD et al. Endocrine effects of naloxoneinduced opiate receptor blockade. J Clin Endocrinol Metab 1980;50:251–257. 210 Volavka J, Cho D, Mallya A et al. Naloxone increases ACTH and cortisol levels in man [letter]. N Engl J Med 1979;300:1056–1057. 211 Raff H, Flemma RJ, Findling JW. Fast cortisol-induced inhibition of the adrenocorticotropin response to surgery in humans. J Clin Endocrinol Metab 1988;67:1146–1148. 212 Spiler IJ, Molitch ME. Lack of modulation of pituitary hormone stress response by neural pathways involving opiate receptors. J Clin Endocrinol Metab 1980;50:516–520.

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264 Hartmeyer M, Scholzen T, Becher E et al. Human dermal microvascular endothelial cells express the melanocortin receptor type 1 and produce increased levels of IL-8 upon stimulation with alpha-melanocyte-stimulating hormone. J Immunol 1997;159:1930–1937. 265 Haynes RC. The activation of adrenal phosphorylase by the adrenocorticotropic hormone. J Biol Chem 1958;233:1220–1222. 266 Lefkowitz RJ, Roth J, Pricer W et al. ACTH receptors in the adrenal: specific binding of ACTH-125I and its relation to adenyl cyclase. Proc Natl Acad Sci USA 1970;65:745–752. 267 Catalano RD, Stuve L, Ramachandran J. Characterization of corticotropin receptors in human adrenocortical cells. J Clin Endocrinol Metab 1986;62: 300–304. 268 Buckley DI, Ramachandran J. Characterization of corticotropin receptors on adrenocortical cells. Proc Natl Acad Sci USA 1981;78:7431–7435. 269 Dallman MF, Akana SF, Jacobson L et al. Characterization of corticosterone feedback regulation of ACTH secretion. Ann NY Acad Sci 1987;512:402–14. 270 Weber A, Kapas S, Hinson J et al. Functional characterization of the cloned human ACTH receptor: impaired responsiveness of a mutant receptor in familial glucocorticoid deficiency. Biochem Biophys Res Commun 1993;197: 172–178. 271 Naville D, Penhoat A, Barjhoux L et al. Characterization of the human ACTH receptor gene and in vitro expression. Endocr Res 1996;22:337–348. 272 Elias LL, Huebner A, Pullinger GD et al. Functional characterization of naturally occurring mutations of the human adrenocorticotropin receptor: poor correlation of phenotype and genotype. J Clin Endocrinol Metab 1999;84: 2766–2770. 273 Brown MS, Kovanen PT, Goldstein JL. Receptor-mediated uptake of lipoprotein-cholesterol and its utilization for steroid synthesis in the adrenal cortex. Recent Prog Horm Res 1979;35:215–257. 274 Pedersen RC, Brownie AC. Adrenocortical response to corticotropin is potentiated by part of the amino-terminal region of procorticotropin/endorphin. Proc Natl Acad Sci USA 1980;77:2239–2243. 275 Jefcoate CR, Simpson ER, Boyd GS. Spectral properties of rat adrenalmitochondrial cytochrome P-450. Eur J Biochem 1974;42:539–551. 276 Privalle CT, Crivello JF, Jefcoate CR. Regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P-450 in rat adrenal gland. Proc Natl Acad Sci USA 1983;80:702–706. 277 Papadopoulos V, Brown AS, Hall PF. Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells. Mol Cell Endocrinol 1990;74:109–123. 278 Miller WL, Strauss JF. Molecular pathology and mechanism of action of the steroidogenic acute regulatory protein, StAR. J Steroid Biochem Mol Biol 2001; 69:131–141. 279 Simpson ER. Cholesterol side-chain cleavage, cytochrone P450, and the control of steroidogenesis. Mol Cell Endocrinol 1979;13:213–227. 280 Yago N, Ichii S. Submitochondrial distribution of components of the steroid 11 beta-hydroxylase and cholesterol sidechain-cleaving enzyme systems in hog adrenal cortex. J Biochem (Tokyo) 1969;65:215–224. 281 Churchill PF, Kimura T. Topological studies of cytochromes P-450scc and P-45011 beta in bovine adrenocortical inner mitochondrial membranes. Effects of controlled tryptic digestion. J Biol Chem 1979;254:10443–10448. 282 Orme-Johnson NR. Distinctive properties of adrenal cortex mitochondria. Biochim Biophys Acta 1990;1020:213–231. 283 Miller WL. Molecular biology of steroid hormone synthesis. [Review]. Endocrine Reviews 1988;9:295–318. 284 Saez JM, Begeot M, Durand P. [ACTH receptors]. Ann Endocrinol (Paris) 1989; 50:409–417. 285 Tepperman J, Engel FL, Long CNH. A review of adrenal cortical hypertrophy. Endocrinol 1943;32:373–402. 286 Smith PE. Hypophysectomy and a replacement therapy in the rat. Amer J Anta 1930;45:205–273. 287 Ney RL, Dexter RN, Davis WW et al. A study of mechanisms by which adrenocorticotropic hormone maintains adrenal steroidogenic responsiveness. J Clin Invest 1967;46:1916–1924. 288 Ingle DJ. The functional interrelationship of anterior pituitary and the adrenal cortex. Ann Intern Med 1951;35:652–672. 289 Wyllie AH, Kerr JF, Macaskill IA et al. Adrenocortical cell deletion: the role of ACTH. J Pathol 1973;111:85–94. 290 Farese RV, Reddy WJ. Observations on the interrelations between adrenal protein, RNA and DNA during prolonged ACTH administration. Biocem Biophys Acta 1963;76:145–148. 291 Imai T, Seo H, Murata Y et al. Adrenocorticotropin increases expression of c-fos and beta-actin genes in the rat adrenals. Endocrinology 1990;127: 1742–1747.

292 Gospodarowicz D, Ill CR, Hornsby PJ et al. Control of bovine adrenal cortical cell proliferation by fibroblast growth factor. Lack of effect of epidermal growth factor. Endocrinology 1977;100:1080–1089. 293 Masui H, Garren LD. Inhibition of replication in functional mouse adrenal tumor cells by adrenocorticotropic hormone mediated by adenosine 3¢:5¢cyclic monophosphate. Proc Natl Acad Sci USA 1971;68:3206–3210. 294 Ramachandran J, Suyama AT. Inhibition of replication of normal adrenocortical cells in culture by adrenocorticotropin. Proc Natl Acad Sci USA 1975;72:113–117. 295 Rao AJ, Long JA, Ramachandran J. Effects of antiserum to adrenocorticotropin on adrenal growth and function. Endocrinology 1978;102:371–378. 296 Dallman MF, Engeland WC, Holzwarth MA et al. Adrenocorticotropin inhibits compensatory adrenal growth after unilateral adrenalectomy. Endocrinology 1980;107:1397–1404. 297 Estivariz FE, Iturriza F, McLean C et al. Stimulation of adrenal mitogenesis by N-terminal proopiocortin peptides. Nature 1982;297:419–422. 298 Estivariz FE, Morano MI, Carino M et al. Adrenal regeneration in the rat is mediated by mitogenic N-terminal pro-opiomelanocortin peptides generated by changes in precursor processing in the anterior pituitary. J Endocrinol 1988;116:207–216. 299 Engeland WC, Shinsako J, Dallman MF. Corticosteroids and ACTH are not required for compensatory adrenal growth. Am J Physiol 1975;229: 1461–1464. 300 Dallman MF, Engelmand WC, Shinsako J. Compensatory adrenal growth: a neurally mediated reflex. Am J Physiol 1976;231:408–414. 301 Albright F. Osteoporosis. Annals of Internal Medicine 1947;27:861–882. 302 Ishihara F, Komatsu M, Yamada T et al. Role of dehydroepiandrosterone and dehydroepiandrosterone sulfate for the maintenance of axillary hair in women. Horm Metab Res 1993;25:34–36. 303 Stewart ME, Downing DT, Cook JS et al. Sebaceous gland activity and serum dehydroepiandrosterone sulfate levels in boys and girls. Archives of Dermatology 1992;128:1345–1348. 304 Rosenfield RL, Grossman BJ, Ozoa N. Plasma 17-ketosteroids and testosterone in prepubertal children before and after ACTH administration. J Clin Endocrinol Metab 1971;33:249–253. 305 Rosenfield RL. Plasma 17-ketosteroids and 17-beta hydroxysteroids in girls with premature development of sexual hair. J Pediatr 1971;79: 260–266. 306 August GP, Hung W, Mayes DM. Plasma androgens in premature pubarche: value of 17 alpha-hydroxyprogesterone in differentiation from congenital adrenal hyperplasia. J Pediatr 1975;87:246–249. 307 Korth-Schutz S, Levine LS, New MI. Serum androgens in normal prepubertal and pubertal children and in children with precocious adrenarche. J Clin Endocrinol Metab 1976;42:117–124. 308 Warne GL, Carter JN, Faiman C et al. Hormonal changes in girls with precocious adrenarche: a possible role for estradiol or prolactin. J Pediatr 1978;92:743–747. 309 Sizonenko PC, Paunier L. Hormonal changes in puberty III: Correlation of plasma dehydroepiandrosterone, testosterone, FSH, and LH with stages of puberty and bone age in normal boys and girls and in patients with Addison’s disease or hypogonadism or with premature or late adrenarche. J Clin Endocrinol Metab 1975;41:894–904. 310 Parker LN, Odell WD. Control of adrenal androgen secretion. Endocr Rev 1980;1:392–410. 311 Vaitukaitis JL, Dale SL, Melby JC. Role of ACTH in the secretion of free dehydroepiandrosterone and its sulfate ester in man. J Clin Endocrinol Metab 1969;29:1443–1447. 312 Rosenfeld RS, Hellman L, Roffwarg H et al. Dehydroisoandrosterone is secreted episodically and synchronously with cortisol by normal man. J Clin Endocrinol Metab 1971;33:87–92. 313 Irvine WJ, Toft AD, Wilson KS et al. The effect of synthetic corticotropin analogues on adrenocortical, anterior pituitary and testicular function. J Clin Endocrinol Metab 1974;39:522–529. 314 Migeon C, Keller A, Lawrence B et al. DHA and androsterone levels in human plasma. J Clin Endocrinol Metab 1957;17:1051–1062. 315 Parker LN, Sack J, Fisher DA et al. The adrenarche: prolactin, gonadotropins, adrenal androgens, and cortisol. J Clin Endocrinol Metab 1978;46:396–401. 316 Linder BL, Esteban NV, Yergey AL et al. Cortisol production rate in childhood and adolescence [see comments]. J Pediatr 1990;117:892–896. 317 Cutler Jr GB, Glenn M, Bush M et al. Adrenarche: a survey of rodents, domestic animals, and primates. Endocrinology 1978;103:2112–2118. 318 Shepard TH, Landing BH, Mason DG. Familial Addison’s disease. Case reports of two sisters with corticoid deficiency unassociated with hypoaldosteronism. Am J Dis Child 1959;97:154–162.

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398 Bultman SJ, Michaud EJ, Woychik RP. Molecular characterization of the mouse agouti locus. Cell 1992;71:1195–1204. 399 Kiefer LL, Veal JM, Mountjoy KG et al. Melanocortin receptor binding determinants in the agouti protein. Biochemistry 1998;37:991–997. 400 Willard DH, Bodnar W, Harris C et al. Agouti structure and function: characterization of a potent alpha-melanocyte stimulating hormone receptor antagonist. Biochemistry 1995;34:12341–12346. 401 Kwon HY, Bultman SJ, Loffler C et al. Molecular structure and chromosomal mapping of the human homolog of the agouti gene. Proc Natl Acad Sci USA 1994;91:9760–9764. 402 Wilson BD, Ollmann MM, Kang L et al. Structure and function of ASP, the human homolog of the mouse agouti gene. Hum Mol Genet 1995;4:223–230. 403 Yang YK, Ollmann MM, Wilson BD et al. Effects of recombinant agoutisignaling protein on melanocortin action. Mol Endocrinol 1997;11:274–280. 404 Kiefer LL, Ittoop OR, Bunce K et al. Mutations in the carboxyl terminus of the agouti protein decrease agouti inhibition of ligand binding to the melanocortin receptors. Biochemistry 1997;36:2084–2090. 405 Yen TT, Gill AM, Frigeri LG et al. Obesity, diabetes, and neoplasia in yellow A(vy)/-mice: ectopic expression of the agouti gene. Faseb J 1994;8:479–488. 406 Michaud EJ, Bultman SJ, Klebig ML et al. A molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow (Ay) mutation. Proc Natl Acad Sci USA 1994;91:2562–2566. 407 Miller MW, Duhl DM, Vrieling H et al. Cloning of the mouse agouti gene predicts a secreted protein ubiquitously expressed in mice carrying the lethal yellow mutation. Genes Dev 1993;7:454–467. 408 Shutter JR, Graham M, Kinsey AC et al. Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated in obese and diabetic mutant mice. Genes Dev 1997;11:593–602. 409 Ollmann MM, Wilson BD, Yang YK et al. Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein [published erratum appears in Science 1998 Sep 11;281(5383):1615]. Science 1997;278:135–138. 410 Rosenfeld RD, Zeni L, Welcher AA et al. Biochemical, biophysical, and pharmacological characterization of bacterially expressed human agouti-related protein. Biochemistry 1998;37:16041–16052. 411 Yang YK, Thompson DA, Dickinson CJ et al. Characterization of Agoutirelated protein binding to melanocortin receptors. Mol Endocrinol 1999; 13:148–155. 412 Fong TM, Mao C, MacNeil T et al. ART (protein product of agouti-related transcript) as an antagonist of MC-3 and MC-4 receptors. Biochem Biophys Res Commun 1997;237:629–631. 413 Zhu Q, Bateman A, Singh A et al. Isolation and biological activity of corticostatic peptides (anti-ACTH). Endocr Res 1989;15:129–149. 414 Cervini LA, Gray WR, Kaiser R et al. Rat corticostatin R4: synthesis, disulfide bridge assignment, and in vivo activity. Peptides 1995;16:837–842. 415 Tatro JB. Receptor biology of the melanocortins, a family of neuroimmunomodulatory peptides. [Review] [224 refs]. Neuroimmunomodulation 1996;3:259–284. 416 de Wied D, Jolles J. Neuropeptides derived from pro-opiocortin: behavioral, physiological, and neurochemical effects. Physiol Rev 1982;62:976–1059. 417 Dunn AJ. Studies on the neurochemical mechanisms and significance of ACTH-induced grooming. Ann NY Acad Sci 1988;525:150–168. 418 Bertolini A, Gessa GA, Ferrari W. Penile erection and ejaculation: a central effect of ACTH-like peptides in mammals. In: Sandler M, Gessa GA. eds. Sexual Behavior, Pharmacology and Biochemistry. NY: Raven Press, 1975: 247–257. 419 Vergoni AV, Bertolini A, Mutulis F et al. Differential influence of a selective melanocortin MC4 receptor antagonist (HS014) on melanocortin-induced behavioral effects in rats. Eur J Pharmacol 1998;362:95–101. 420 Argiolas A, Melis MR, Murgia S et al. ACTH- and alpha-MSH-induced grooming, stretching, yawning and penile erection in male rats: site of action in the brain and role of melanocortin receptors. Brain Res Bull 2000;51: 425–431. 421 Wessells H, Fuciarelli K, Hansen J et al. Synthetic melanotropic peptide initiates erections in men with psychogenic erectile dysfunction: double-blind, placebo controlled crossover study. J Urol 1998;160:389–393. 422 Strand FL, Stoboy H, Cayer A. A possible direct action of ACTH on nerve and muscle. Neuroendocrinology 1973;13:1–20. 423 Strand FL, Kung TT. ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides 1980;1:135–138. 424 Strand FL, Rose KJ, King JA et al. ACTH modulation of nerve development and regeneration. Prog Neurobiol 1989;33:45–85. 425 Gispen WH, Verhaagen J, Bar D. ACTH/MSH-derived peptides and peripheral nerve plasticity: neuropathies, neuroprotection and repair. Prog Brain Res 1994;100:223–229.

Chapter 2 426 Swaab DF, Visser M, Tilders FJ. Stimulation of intra-uterine growth in rat by alpha-melanocyte-stimulating hormone. J Endocrinol 1976;70:445–455. 427 Swaab DF, Martin JT. Functions of alpha-melanotropin and other opiomelanocortin peptides in labour, intrauterine growth and brain development. Ciba Found Symp 1981;81:196–217. 428 Murphy MT, Richards DB, Lipton JM. Antipyretic potency of centrally administered alpha-melanocyte stimulating hormone. Science 1983;221: 192–193. 429 Klein MC, Hutchins PM, Lymangrover JR et al. Pressor and cardioaccelerator effects of gamma MSH and related peptides. Life Sci 1985;36:769–775. 430 Li SJ, Varga K, Archer P et al. Melanocortin antagonists define two distinct pathways of cardiovascular control by alpha- and gamma-melanocytestimulating hormones. J Neurosci 1996;16:5182–5188. 431 Lymangrover JR, Buckalew VM, Harris J et al. Gamma-2MSH is natriuretic in the rat. Endocrinology 1985;116:1227–1229. 432 Orias R, McCann SM. Natriuretic effect of alpha melanocyte stimulating hormone (-MSH) in hypophysectomized or adrenalectomized rats. Proc Soc Exp Biol Med 1972;139:872–876. 433 Orias R, McCann SM. Natriuresis induced by alpha and beta melanocyte stimulating hormone (MSH) in rats. Endocrinology 1972;90:700–706. 434 Chen XW, Ying WZ, Valentin JP et al. Mechanism of the natriuretic action of gamma-melanocyte-stimulating hormone. Am J Physiol 1997;272: R1946–R1953. 435 Veldhuis JD, Iranmanesh A, Johnson ML et al. Amplitude, but not frequency, modulation of adrenocorticotropin secretory bursts gives rise to the nyctohemeral rhythm of the corticotropic axis in man. J Clin Endocrinol Metab 1990;71:452–463. 436 Desir D, Van Cauter E, Beyloos M et al. Prolonged pulsatile administration of ovine corticotropin-releasing hormone in normal man. J Clin Endocrinol Metab 1986;63:1292–1299. 437 Veldhuis JD, Iranmanesh A, Johnson ML et al. Twenty-four-hour rhythms in plasma concentrations of adenohypophyseal hormones are generated by distinct amplitude and/or frequency modulation of underlying pituitary secretory bursts. J Clin Endocrinol Metab 1990;71:1616–1623. 438 Horrocks PM, Jones AF, Ratcliffe WA et al. Patterns of ACTH and cortisol pulsatility over twenty-four hours in normal males and females. Clin Endocrinol (Oxf ) 1990;32:127–134. 439 Iranmanesh A, Lizarralde G, Johnson ML et al. Circadian, ultradian, and episodic release of beta-endorphin in men, and its temporal coupling with cortisol. J Clin Endocrinol Metab 1989;68:1019–1026. 440 Krishnan KR, Ritchie JC, Saunders W et al. Nocturnal and early morning secretion of ACTH and cortisol in humans. Biol Psychiatry 1990;28: 47–57. 441 Schulte HM, Chrousos GP, Gold PW et al. Continuous administration of synthetic ovine corticotropin-releasing factor in man. Physiological and pathophysiological implications. J Clin Invest 1985;75:1781–1785. 442 Keller Wood ME, Dallman MF. Corticosteroid inhibition of ACTH secretion. Endocr Rev 1984;5:1–24. 443 DeBold CR, Jackson RV, Kamilaris TC et al. Effects of ovine corticotropinreleasing hormone on adrenocorticotropin secretion in the absence of glucocorticoid feedback inhibition in man. J Clin Endocrinol Metab 1989;68:431–437. 444 Totani Y, Niinomi M, Takatsuki K et al. Effect of metyrapone pretreatment on adrenocorticotropin secretion induced by corticotropin-releasing hormone in normal subjects and patients with Cushing’s disease. J Clin Endocrinol Metab 1990;70:798–803. 445 Watson AC, Rosenfield RL, Fang VS. Recovery from glucocorticoid inhibition of the responses to corticotrophin-releasing hormone. Clin Endocrinol (Oxf ) 1988;28:471–477. 446 Schopohl J, Hauer A, Kaliebe T et al. Repetitive and continuous administration of human corticotropin releasing factor to human subjects. Acta Endocrinol (Copenh) 1986;112:157–165. 447 Avgerinos PC, Nieman LK, Oldfield EH et al. The effect of pulsatile human corticotropin-releasing hormone administration on the adrenal insufficiency that follows cure of Cushing’s disease. J Clin Endocrinol Metab 1989;68: 912–916. 448 Raff H. Glucocorticoid inhibition of neurohypophysial vasopressin secretion. Am J Physiol 1987;252:R635–R644. 449 Oelkers W. Hyponatremia and inappropriate secretion of vasopressin (antidiuretic hormone) in patients with hypopituitarism [see comments]. N Engl J Med 1989;321:492–496. 450 Bouley R, Breton S, Sun T et al. Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane insertion of aquaporin 2 in renal epithelial cells. J Clin Invest 2000;106:115–126.

Adrenocorticotropin

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451 Suda T, Tomori N, Yajima F et al. A short negative feedback mechanism regulating corticotropin-releasing hormone release. J Clin Endocrinol Metab 1987;64:909–913. 452 Van Cauter E, Polonsky K, Scheen AJ. Role of circadian rhythm and sleep in human glucose regulation. Endocr Rev 1997;18:716–738. 453 Moore RY, Eichler VB. Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic lesions in the rat. Brain Res 1972;42:201–206. 454 Follenius M, Brandenberger G. Plasma free cortisol during secretory episodes. J Clin Endocrinol Metab 1986;62:609–612. 455 Dallman MF, Strack AM, Akana SF et al. Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. [Review] [26 refs.] Front Neuroendocrinol 1993;14:303–347. 456 Van Cauter E, Refetoff S. Multifactorial control of the 24-hour secretory profiles of pituitary hormones. J Endocrinol Invest 1985;8:381–391. 457 Dallman MF, Engeland WC, Rose JC et al. Nycthemeral rhythm in adrenal responsiveness to ACTH. Am J Physiol 1978;235:R210–R218. 458 Dijkstra I, Binnekade R, Tilders FJ. Diurnal variation in resting levels of corticosterone is not mediated by variation in adrenal responsiveness to adrenocorticotropin but involves splanchnic nerve integrity. Endocrinology 1996;137:540–547. 459 Iannotta F, Magnoli L, Visconti G et al. Differences in cortisol, aldosterone and testosterone responses to ACTH 1–17 administered at two different times of day. Chronobiologia 1987;14:38–46. 460 Sensi S, Capani F, De Remigis P et al. Circadian time structure of pituitary and adrenal response to CRF, TRH and LHRH. Prog Clin Biol Res 1990;341:535–542. 461 Suda T, Tomori N, Yajima F et al. Immunoreactive corticotropin-releasing factor in human plasma. J Clin Invest 1985;76:2026–2029. 462 Watabe T, Tanaka K, Kumagae M et al. Diurnal rhythm of plasma immunoreactive corticotropin-releasing factor in normal subjects. Life Sci 1987;40:1651–1655. 463 Hindmarsh KW, Tan L, Sankaran K et al. Diurnal rhythms of cortisol, ACTH, and beta-endorphin levels in neonates and adults. West J Med 1989;151: 153–156. 464 Sasaki A, Shinkawa O, Yoshinaga K. Placental corticotrophin-releasing hormone may be a stimulator of maternal pituitary adrenocorticotropic hormone secretion in humans. J Clin Invest 1989;84:1997–2001. 465 Muglia LJ, Jacobson L, Weninger SC et al. Impaired diurnal adrenal rhythmicity restored by constant infusion of corticotropin-releasing hormone, in corticotropin-releasing hormone-deficient mice. Journal of Clinical Investigation 1997;99:2923–2929. 466 Muglia LJ, Jacobson L, Luedke C et al. Corticotropin-releasing hormone links pituitary adrenocorticotropin gene expression and release during adrenal insufficiency [see comments]. J Clin Invest 2000 May 2000;105:1269–1277. 467 Ellis MJ, Schmidli RS, Donald RA et al. Plasma corticotrophin-releasing factor and vasopressin responses to hypoglycaemia in normal man. Clin Endocrinol (Oxf ) 1990;32:93–100. 468 Decherney GS, DeBold CR, Jackson RV et al. Effect of ovine corticotropinreleasing hormone administered during insulin-induced hypoglycemia on plasma adrenocorticotropin and cortisol. J Clin Endocrinol Metab 1987;64: 1211–1218. 469 Adler GK, Majzoub JA. Influence of infused hypertonic saline on the response to insulin-induced hypoglycemia in man. J Clin Endocrinol Metab 1987;65: 116–121. 470 Luger A, Deuster PA, Gold PW et al. Hormonal responses to the stress of exercise. Adv Exp Med Biol 1988;245:273–280. 471 Deuster PA, Chrousos GP, Luger A et al. Hormonal and metabolic responses of untrained, moderately trained, and highly trained men to three exercise intensities. Metabolism 1989;38:141–148. 472 Janal MN, Colt EW, Clark WC et al. Pain sensitivity, mood and plasma endocrine levels in man following long-distance running: effects of naloxone. Pain 1984;19:13–25. 473 Pitts AF, Preston MA, II, Jaeckle RS et al. Simulated acute hemorrhage through lower body negative pressure as an activator of the hypothalamic–pituitary–adrenal axis. Horm Metab Res 1990;22:436–443. 474 Udelsman R, Chrousos GP. Hormonal responses to surgical stress. Adv Exp Med Biol 1988;245:265–272. 475 Udelsman R, Norton JA, Jelenich SE et al. Responses of the hypothalamic– pituitary–adrenal and renin-angiotensin axes and the sympathetic system during controlled surgical and anesthetic stress. J Clin Endocrinol Metab 1987; 64:986–994. 476 Udelsman R, Ramp J, Gallucci WT et al. Adaptation during surgical stress. A reevaluation of the role of glucocorticoids. J Clin Invest 1986;77: 1377–1381.

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Hypothalamic–Pituitary Function

477 Oltras CM, Mora F, Vives F. Beta-endorphin and ACTH in plasma: effects of physical and psychological stress. Life Sci 1987;40:1683–1686. 478 Voigt K, Ziegler M, Grunert Fuchs M et al. Hormonal responses to exhausting physical exercise: the role of predictability and controllability of the situation. Psychoneuroendocrinology 1990;15:173–184. 479 Arduini D, Rizzo G, Parlati E et al. Modifications of ultradian and circadian rhythms of fetal heart rate after fetal-maternal adrenal gland suppression: a double blind study. Prenat Diagn 1986;6:409–417. 480 Price DA, Close GC, Fielding BA. Age of appearance of circadian rhythm in salivary cortisol values in infancy. Arch Dis Child 1983;58:454–456. 481 Onishi S, Miyazawa G, Nishimura Y et al. Postnatal development of circadian rhythm in serum cortisol levels in children. Pediatrics 1983;72:399–404. 482 Attanasio A, Rosskamp R, Bernasconi S et al. Plasma adrenocorticotropin, cortisol, and dehydroepiandrosterone response to corticotropin-releasing factor in normal children during pubertal development. Pediatr Res 1987;22:41–44. 483 Ghizzoni L, Virdis R, Ziveri M et al. Adrenal steroid, cortisol, adrenocorticotropin, and beta-endorphin responses to human corticotropinreleasing hormone stimulation test in normal children and children with premature pubarche. J Clin Endocrinol Metab 1989;69:875–880. 484 McEwen BS. The neurobiology of stress: from serendipity to clinical relevance. [Review] [157 refs]. Brain Res 2000 Dec 15 2001;886:172–189. 485 Elenkov IJ, Wilder RL, Chrousos GP et al. The sympathetic nerve—an integrative interface between two supersystems: the brain and the immune system. [Review] [445 refs]. Pharmacol Rev 2000 Dec 2001;52:595–638. 486 Blalock JE. Proopiomelanocortin and the immune-neuroendocrine connection. [Review] [68 refs]. Ann NY Acad Sci 1999;885:161–172. 487 Ottaviani E, Franchini A, Genedani S. ACTH and its role in immuneneuroendocrine functions. A comparative study. Curr Pharm Des 1999;5: 673–681. 488 Elenkov IJ, Webster EL, Torpy DJ et al. Stress, corticotropin-releasing hormone, glucocorticoids, and the immune/inflammatory response: acute and chronic effects. [Review] [59 refs]. Ann NY Acad Sci 1999;876:1–11. 489 Cavagnaro J. Molecular basis for the bidirectional modulation of the neuroendocrine and the immune systems. Year Immunol 1986;2:303–322. 490 Reichlin S. Neuroendocrine-immune interactions. N Engl J Med 1993;329: 1246–1253. 491 Eskay RL, Grino M, Chen HT. Interleukins, signal transduction, and the immune system-mediated stress response. Adv Exp Med Biol 1990;274: 331–343. 492 Kavelaars A, Ballieux RE, Heijnen CJ. The role of IL-1 in the corticotropinreleasing factor and arginine- vasopressin-induced secretion of immunoreactive beta-endorphin by human peripheral blood mononuclear cells. J Immunol 1989;142:2338–2342. 493 Westly HJ, Kleiss AJ, Kelley KW et al. Newcastle disease virus-infected splenocytes express the proopiomelanocortin gene. J Exp Med 1986;163: 1589–1594. 494 Lolait SJ, Clements JA, Markwick AJ et al. Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of beta endorphin in spleen macrophages. J Clin Invest 1986;77:1776–1779. 495 Karalis K, Sano H, Redwine J et al. Autocrine or paracrine inflammatory actions of corticotropin-releasing hormone in vivo. Science 1991;254:421–423. 496 Muglia LJ, Jenkins NA, Gilbert DJ et al. Expression of the mouse corticotropin-releasing hormone gene in vivo and targeted inactivation in embryonic stem cells. Journal of Clinical Investigation 1994;93:2066–2072. 497 Scheithauer BW. Surgical pathology of the pituitary: the adenomas. Part II. Pathol Annu 1984;19:269–329. 498 Saeger W, Geisler F, Ludecke DK. Pituitary pathology in Cushing’s disease. Pathol Res Pract 1988;183:592–595. 499 Herman V, Fagin J, Gonsky R et al. Clonal origin of pituitary adenomas. J Clin Endocrinol Metab 1990;71:1427–1433. 500 Hori T, Nishiyama F, Anno Y et al. Differences in glycoconjugates of adrenocorticotropic hormone-secretory granules between nonadenomatous pituitary cells and adenoma cells as detected by double labeling. Neurosurgery 1988;23:52–57. 501 Challa VR, Marshall RB, Hopkins MB et al. Pathobiologic study of pituitary tumors: report of 62 cases with a review of the recent literature. Hum Pathol 1985;16:873–884.

502 Apletalina V, Muller L, Lindberg I. Mutations in the catalytic domain of prohormone convertase 2 result in decreased binding to 7B2 and loss of inhibition with 7B2 C-terminal peptide. J Biol Chem 2000;275:14667–14677. 503 Schteingart DE, Chandler WF, Lloyd RV et al. Cushing’s syndrome caused by an ectopic pituitary adenoma. Neurosurgery 1987;21:223–227. 504 Axiotis CA, Lippes HA, Merino MJ et al. Corticotroph cell pituitary adenoma within an ovarian teratoma: A new cause of Cushing’s syndrome. Am J Surg Pathol 1987;11:218–224. 505 Neilson K, de Chadarevian JP. Ectopic anterior pituitary corticotropic tumour in a six-year-old boy. Histological, ultrastructural and immunocytochemical study. Virchows Arch [A] 1987;411:267–273. 506 Karl M, Lamberts SW, Koper JW et al. Cushing’s disease preceded by generalized glucocorticoid resistance: clinical consequences of a novel, dominant-negative glucocorticoid receptor mutation. Proc Assoc Am Physicians 1996;108:296–307. 507 Karl M, Von Wichert G, Kempter E et al. Nelson’s syndrome associated with a somatic frame shift mutation in the glucocorticoid receptor gene. J Clin Endocrinol Metab 1996;81:124–129. 508 Gabrilove JL, Anderson PJ, Halmi NS. Pituitary pro-opiomelanocortin-cell carcinoma occurring in conjunction with a glioblastoma in a patient with Cushing’s disease and subsequent Nelson’s syndrome. Clin Endocrinol (Oxf ) 1986;25:117–126. 509 Nawata H, Higuchi K, Ikuyama S et al. Corticotropin-releasing hormone- and adrenocorticotropin-producing pituitary carcinoma with metastases to the liver and lung in a patient with Cushing’s disease. J Clin Endocrinol Metab 1990;71:1068–1073. 510 Gould VE, Lee I, Warren WH. Immunohistochemical evaluation of neuroendocrine cells and neoplasms of the lung. Pathol Res Pract 1988;183:200–213. 511 Kasurinen J, Syrjanen KJ. Peptide hormone immunoreactivity and prognosis in small-cell carcinoma of the lung. Respiration 1986;49:61–67. 512 Sieber SC, Gelfman NA, Dandurand R et al. Ectopic ACTH and adrenal myelolipoma. Conn Med 1989;53:7–10. 513 Herbst WM, Kummer W, Hofmann W et al. Carcinoid tumors of the thymus. An immunohistochemical study. Cancer 1987;60:2465–2470. 514 Arioglu E, Doppman J, Gomes M et al. Cushing’s syndrome caused by corticotropin secretion by pulmonary tumorlets. N Engl J Med 1998;339:883–886. 515 Raux Demay MC, Proeschel MF, de Keyzer Y et al. Characterization of human corticotrophin-releasing hormone and pro-opiomelanocortin-related peptides in a thymic carcinoid tumour responsible for Cushing’s syndrome. Clin Endocrinol (Oxf ) 1988;29:649–657. 516 Suda T, Kondo M, Totani R et al. Ectopic adrenocorticotropin syndrome caused by lung cancer that responded to corticotropin-releasing hormone. J Clin Endocrinol Metab 1986;63:1047–1051. 517 Sakurai H, Yoshiike Y, Isahaya S et al. A case of ACTH-producing pheochromocytoma. Am J Med Sci 1987;294:258–261. 518 Mendonca BB, Arnhold IJ, Nicolau W et al. Cushing’s syndrome due to ectopic ACTH secretion by bilateral pheochromocytomas in multiple endocrine neoplasia type 2A [letter]. N Engl J Med 1988;319:1610–1611. 519 Southgate HJ, Archbold GP, el Sayed ME et al. Ectopic release of GHRH and ACTH from an adenoid cystic carcinoma resulting in acromegaly and complicated by pituitary infarction. Postgrad Med J 1988;64: 145–148. 520 Watanobe H, Yoshioka M, Takebe K. Ectopic ACTH syndrome due to Grawitz tumor. Horm Metab Res 1988;20:453–456. 521 Werner S, Jacobsson B, Bostrom L et al. Cushing’s syndrome due to an ACTH-producing neuroendocrine tumour in the nasal roof. Acta Med Scand 1985;217:235–240. 522 Long SI. ACTH-producing apudoma metastatic to the liver. J Natl Med Assoc 1987;79:122–123. 523 de Keyzer Y, Bertagna X, Luton JP et al. Variable modes of proopiomelanocortin gene transcription in human tumors. Mol Endocrinol 1989;3: 215–223. 524 Melmed S, Yamashita S, Kovacs K et al. Cushing’s syndrome due to ectopic proopiomelanocortin gene expression by islet cell carcinoma of the pancreas. Cancer 1987;59:772–778.

C h a p t e r

3 Growth Hormone Vivien S. Herman-Bonert Shlomo Melmed

GROWTH HORMONE (GH) GENE STRUCTURE

Somatotroph Development and Differentiation

The human GH genomic locus spans approximately 66 kb and contains a cluster of five highly conserved genes [1] located on the long arm of human chromosome 17 at bands q22–24 [2]. The 5¢ to 3¢ arrangement of these genes is hGHN, hCS-L, hCS-A, hGH-V, and hCS-B [1], all of which have the same basic structure consisting of five exons separated by four introns [1]. The hGH-N gene is transcribed only in somatotrophs of the anterior pituitary while the hCS-A and hCS-B genes are expressed in placental trophoblasts [3]. hGH-N codes for a 22-kDa protein consisting of 191 amino acids. Approximately 10% of pituitary GH is presented as a 20-kDa variant lacking amino acid residues 32–46 [4,5], and probably arising as a result of an alternate splicing mechanism [6]. hGH-V is expressed by the syncytiotrophoblast of the placenta during the second and third trimesters of gestation [7,8], hGH-V messenger RNA (mRNA) encodes 22kDa protein secreted form which can be detected in the maternal circulation from midpregnancy [9] and a minor form hGH-V2 which is predicted to be a 26-kDa protein product [10]. The role of hGH-V is unknown, however, the rise in hGH-V concentrations in maternal serum correlates with a fall in hGH-N concentrations, suggesting the possibility of a feedback loop on the maternal hypothalamic pituitary axis [9]. Postpartum, GH-V levels drop rapidly and are undetectable in the circulation after one hour [9]. hGH-V has a greater binding affinity than hGH-N for somatogen vs lactogen receptors [11]. hGH-V and hGH-N also differ in their ratio of somatogen to lactogen bioactivities, with hGH-V having the greater ratio [12]. In addition, hGH-V has been shown to influence carbohydrate and fat metabolism in rat adipose tissues in a manner similar to that of hGH-N [13]. The hCS-L gene is not known to yield a product [14].

GH is specifically expressed in the somatotroph cell of the anterior pituitary which develops in a time-and-space dependent manner. Expression of the a-subunit transcript in the hypophyseal placode within ectoderm of the pharynx prior to the formation of Rathke’s pouch defines the onset of pituitary organogenesis [15]. The mammalian anterior pituitary develops from Rathke’s pouch during the early stages of embryonic development [16]. Its ventral epithelium serves as the anterior pituitary anlagen while the dorsal epithelium generates the intermediate lobe of the pituitary. A process of cytodifferentiation gives rise to the different hormone-producing cells [17]. Acidophils are the progenitors for both GH-producing somatotrophs and prolactin producing lactotrophs (PRL), while basophils give rise to cells which secrete adrenocorticotrophic hormone (ACTH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) [17]. Cells of the intermediate lobe produce pro-opiomelanocortin (POMC) gene-related peptides [17]. Experimental evidence obtained from transgenic mice studies suggests that most PRL-expressing cells arose from GH-producing cells [18]. Ablation of somatotrophs by expression of GH-diphtheria toxin and GH-thymidine kinase fusion genes inserted into the germ line of transgenic mice results in the elimination of the majority of lactotrophs; however a small percentage of lactotrophs escape destruction [18,19]. This suggests that the majority of PRLproducing cells arose from postmitotic somatotrophs. The Pit-I gene transcript and POUIFI protein are found in somatotrophs, lactotrophs and thyrotrophs [15]. This suggests an mRNA-specific translational control as part of the developmental process [15]. The actions of Pit-1 protein are complemented by other factors required to achieve the 79

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physiologic patterns of cell-specific gene activation [15]. The estrogen receptor, which is activated subsequent to the appearance of Pit-1, appears to mediate in part a progressive increase in PRL gene expression characteristic of mature lactotrophs. Inherited isolated GH deficiency and short stature may be attributed to mutations of transcription factors, the GH-1 gene, the GHRH receptor, the GH receptor or rarely IGF-related molecules.

Promoter Structure The hGH promoter region contains cis-elements which mediate both pituitary-specific and hormone-specific signaling [20,21]. DNase footprinting reveals several proteinbinding sites within 300 bp of the 5¢ flanking DNA. One one of these proteins is unique to pituitary cells, suggesting that it plays a major role in tissues-specific expression of the GH gene [20,22]. This factor, alternately named Pit-1 or GHF-1 [23,24], binds to two sites centered around sequences -80 and -120. Pit-1 is a 31- or 33-kDa protein important for GH promoter activity both in vivo and in vitro [23–25]. When transferred into nonexpressing cells, Pit-1 is capable of stimulating GH-directed gene expression in nonpituitary cells [20]. In addition, Pit-1 binds to the PRL promoter [26]. Pit-1 is a member of a family of transcription factors sharing two regions of homology [27]; a highly conserved POU-specific (POUs) domain and a more divergent homeodomain (POUHD). Initial recognition of this family resulted from the cloning of complementary DNAs (cDNAs) encoding several transactivators Pit-1 [23,24], Oct1 [28,29], Oct-2 [30,31], and unc-86 [32]. The POUs domain is unique to this family of proteins but the POUHD, which is divergent, shares sequence homology and the predicted a-helical structures with classic homeodomain-containing proteins [33]. Data from mutant Pit-1 proteins suggest that while POUHD is required and sufficient for low-affinity DNA binding, the POUs domain is necessary for highaffinity binding and accurate recognition of natural Pit-1 response elements. In addition, POU domain proteins, in contrast to homeodomain proteins, require that both the POUs and POUHD contact the DNA [27]. Transcriptional activity of Pit-1 is conferred by a serine- and threoninerich N-terminal region which is different to the transcriptional contacts of other types of activation domains [27]. A variant isoform of Pit-1, Pit-1B [34], contains a 26-amino acid insertion in the transactivation domain and, like the previously described Pit-I protein, is capable of inducing both GH and PRL promoters. PROP-1, a paired homeobox protein, is required for initial commitment of Pit-1 cell lineages [35]. PROP-1 represses Rpx expression, and missense and spliced mutations of PROP-1 leading to loss of DNA-binding or transactivation leads to pituitary failure with short stature and varying degrees of thyroid failure, hypogonadism, and ACTH deficiency [36].

POUIFI [37] mutations may also lead to pituitary failure. Patients with combined pituitary hormone deficiency have predominantly GH and PRL deficiency, with variable degrees of hypothyroidism [37]. Footprinting analysis reveals the presence of a second, ubiquitous factor binding to the distal Pit-1 site [38,39]. The protected template contains a consensus sequence for the binding of Sp1, a transcription factor that recognizes the glycine-cysteine box in several viral and eukaryotic gene promoters [40–42]. Pit-1 binds to its distal site with a lower affinity [23,24], and, in addition, Pit-1 and Sp1 bind in a mutually exclusive manner to the GH promoter [38,39]. In vivo, both binding sites contribute to rat GH promoter activation [39]. Mutation of the Sp1 binding site results in a 50% reduction of promoter activity, suggesting that activation of GH by Pit-1 and Sp1 may occur through a multistage mechanism. Sequences -266/-252 of the hGH promoter contain an 8 bp recognition site for the adenoviral major late transcription factor (MLTF), also termed upstream stimulating factor (USF) [43]. A USF-like protein also binds to the upstream hGH promoter [38,43,44]. This factor contributes to one of the protected sites in the third footprint of the hGH gene promoter. USF is a basal transcription factor initially identified in Hela cell extracts [45,46]. Known targets of USF include adenovirus-2 [45,46], fibrinogen-g and the metallothionein-1 genes [47,48]. The distal part of this protected region of the hGH promoter, -290/-272, binds an insulin-inducible nuclear factor [44] that may represent an NF1 family protein [49]. A locus control region (LCR) of the hGH gene located 5¢ to the transcription start site determines somatotroph and latotroph GH expression. This genetic control also involves appropriate regulation of a chromatin domain in these pituitary cell types [50].

Hormonal Regulation of the hGH Gene Synthesis and release of GH is under control of a variety of hormonal agents, including insulin growth factor-I (IGF-I), GH-releasing hormone (GHRH), Ghrelin, somatostatin, thyroid hormone, glucocorticoids, and insulin. IGF-I inhibits GH mRNA expression as well as GH secretion in dispersed human adenoma cell cultures [51]. This effect is blocked by the monoclonal IGF-I receptor antibody a-IR3 [51]. IGF-I also attenuates basal and stimulated GH gene expression when 500 bp of the hGH gene promoter was transfected into human choriocarcinoma cells ( JEG3) or HeLa cells [52]. GHRH stimulates the release of hGH, and cyclic adenosine monophosphate (cAMP) is thought to be the second messenger for this effect. Analysis of the hGH promoter for a cAMP-responsive element (CRE) fails to reveal a consensus sequence. Two groups have reported the presence of novel CREs within -83 and -150 bp of the 5¢ flanking DNA, respectively [21,53]. CREB binding protein (CBP) is also a co-factor for Pit-1 dependent human GH

Chapter 3

activation. CBP is phosphorylateral by protein kinase A, independently of CREB [54]. The role of thyroid hormone in hGH synthesis remains unclear. Release of hGH in response to provocative stimuli is usually reduced in hypothyroid patients; however, basal GH levels and responses may be normal [55,56]. In numerous hGH gene transfer studies, absence of a T3 response has been documented [57,58]. Glucocorticoid responsiveness of the hGH gene has been demonstrated with the strongest receptor binding site being located within the first intron of the hGH gene; however, a 5¢ GRE is also present and capable of weak induction [59–61]. Insulin suppresses hGH secretion by acromegaly tumor cells in culture [62], and also attenuates basal and stimulated transient expression of a transfected hGH gene [63]. Somatostatin is synthesized in the medial preoptic area of the hypothalamus and inhibits secretion of GH from human adenoma cell cultures [64]. Thus, numerous ubiquitous factors bind to the GH promoter. In the absence of Pit-1 the promoter region is inactive, and binding of Pit-1 to the transcriptional machinery facilitates the interaction between this factor and other ubiquitous activators already bound to the promoter or subsequently activated to enhance GH transcription. This model

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81

of cooperative interaction provides an explanation for tissuespecific expression of the hGH gene by a single cell typespecific activator. CONTROL OF GH SECRETION The GH molecule, a single-chain polypeptide hormone consisting of 191 amino acids (Fig. 3.1), is synthesized, stored, and secreted by somatotroph cells. The crystal structure of human GH and GH-binding protein reveals four alpha helixes, and two identical GHBP molecules occurring within a single GH molecule [65]. Somatotrophs are located predominantly in the lateral wings of the anterior pituitary gland. Somatotrophs comprise 35% to 45% of pituitary cells and the gland contains a total of 5–15 mg of GH [66]. Circulating GH molecules are a heterogenous group of peptides [67] which comprises at least three monomeric forms and several oligomers. The monomeric moieties include a 22- and 20-kDa form, acetylated 22K, and two desamido GH’s. The 22-kDa peptide is the major physiologic GH component. The 20-kDa GH has a slower metabolic clearance than the 22-kDa form [68], which accounts for the plasma 20 : 22 ratio being higher than in the pituitary gland.

FIGURE 3.1. Amino acid structure of human growth hormone (GH). GH is a 191amino acid single-chain 21.5 kDa polypeptide with two intramolecular disulfide bonds. Fifteen percent of GH is deleted from amino acid (32–46) and is secreted as a 20 kDa protein.

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The 22-kDa peptide retains growth-promoting activity, but lacks diabetogenic effects, which are more pronounced with the 20-kDa form. The relative proportions of circulating GH molecular forms in children of various ages are quantitatively and qualitatively identical in adults. Monomeric GH forms found in the plasma of acromegalic patients are also qualitatively similar to those found in normal plasma [69].

Ontogeny of GH Levels Circulating GH is first detectable in fetal serum at the end of the first trimester, peaks at a concentration of 100 to 150 ng/ml at 20 weeks of gestation, and subsequently falls to 30 ng/ml at birth. GH levels continue to fall during infancy. During childhood, levels are similar to those in adulthood, until puberty, when circulating levels are elevated. GH levels decline after adolescent growth and remain stable until mid-adulthood, when they decline progressively through old age [70]. Decreased sensitivity of the pituitary to GHRH action with aging may contribute to these GH fluctuations with advancing age [71].

Physiologic Factors Involved in GH Secretion GH secretion is pulsatile, the anterior pituitary gland secreting bursts of GH, with almost undetectable basal levels occurring between these peaks [72]. The number of GH pulses detected depends on the frequency of blood sampling. When sampling frequency is reduced to three or fewer samples per hour, less than 0.4 GH pulses/hr are detected, and this rate increases as sampling is intensified [72]. Integrated GH levels are higher in women than in men [73], and are also enhanced in postmenopausal women following estrogen replacement [74]. In children and young adults, maximum GH secretion occurs within an hour of the onset of deep sleep (stage III or IV), with subsequent smaller peaks appearing during later sleep [75]. Effects of Sleep

A major GH secretory pulse occurs shortly after the onset of sleep, associated with the first episode of slow wave sleep [76]. Sleep stimulates GH secretion and 60% to 70% of daily GH secretion occurs during early sleep, in association with slow wave sleep [77] Rapid eye movement (REM) sleep is reduced by approximately 50% after age 50 years with significant sleep fragmentation [78]. The decline in slow wave sleep from early adulthood to midlife is paralleled by a major decline in GH secretion. This suggests that the age-related alterations in the somatotropic axis may partially reflect decreased sleep quality. Although increased PRL and ACTH concentrations occur later during sleep, their secretion is not as tightly linked to sleep patterns as is GH secretion. “Jet lag” transiently increases the height of GH peaks during the day and night, resulting in a transient increase of 24-hour

GH secretion. Jet lag also shifts the major GH secretory spike from early to late sleep [79]. Exercise

Exercise increases GH secretion probably mediated by a cholinergic mechanism [80]. Stress

GH release is stimulated by physical stress, including trauma with hypovolemic shock [81] and sepsis [82]. However, chronic debilitating diseases, including cancer, are not associated with increased GH levels [83]. Increased GHRH release, mediated by adrenergic pathways, is thought to mediate stress-induced GH secretion [84]. Emotional deprivation is associated with suppressed GH secretion [85], and subnormal GH responses to provocative stimuli have been described in endogenous depression [86]. Nutritional and Metabolic Effects

Nutritional and metabolic factors profoundly influence GH secretion. Chronic malnutrition [87] and voluntary 5-day fasting [88] are associated with elevated GH levels. Both pulse frequency and amplitude of GH secretory peaks increase with fasting [88] (Table 3.1). Obesity decreases basal and stimulated GH secretion. Insulin-induced hypoglycemia stimulates GH release 30 to 45 minutes after the glucose trough, whereas acute hyperglycemia inhibits GH secretion [89]. Diabetic patients with chronic hyperglycemia, however, do not have suppressed GH levels and in fact many poorly controlled diabetic patients have increased basal [90] and exercise-induced GH levels [91]. Central nervous system glucoreceptors appear to sense fluctuations, rather than absolute glucose levels. However, glucose homeostasis is not the major determinant of GH secretion, this being overridden by effects of sleep, exercise, stress, and by random GH bursts. A high-protein meal, and single amino acids (including arginine and leucine) administered intravenously stimulate GH secretion. Arginine may suppress endogenous somatostatin secretion and thereby stimulate GH secretion [92]. Decreased serum free fatty acid (FFA) levels cause acute GH release [93] and increased serum FFA blunt the effects of various stimuli, including arginine infusion, [94] sleep, Ldopa, and exercise [95–98] on GHRH-stimulated GH release [98].

Table 3.1. Deconvolution analysis of growth hormone (GH) secretion in adult males. From Thorner et al. [88a]

24 hr secretion Secretory bursts GH/burst

Normal

Fasting

Obesity

Middle-age

540 ± 44 12 ± 1 45 ± 4

2171 ± 333 32 ± 2 64 ± 9

77 ± 20 3 ± 0.5 24 ± 5

196 ± 65 10 ± 1 20 ± 6

Chapter 3

Growth Hormone

83

FIGURE 3.2. Schematic representation of the actions of leptin. Leptin acts either directly or by activating specific centers in the central nervous system to decrease food intake, increase energy expenditure, influence glucose and fat metabolism, and alter neuroendocrine function. From Mantzoros [100a].

FIGURE 3.3. Schematic representation of feedback loops involving leptin. Leptin, an adipocyte-derived hormone, circulates in the serum either in free form or bound to leptinbinding proteins, activates specific receptors in the hypothalamus, and alters expression of several neuropeptides; these in turn decrease appetite, increase energy expenditure by altering sympathetic and parasympathetic tone, and alter neuroendocrine function. Increasing leptin levels activate the thyroid, growth hormone, and gonadal axes and suppress the pituitary–adrenal axis. Leptin, acting directly or indirectly (by altering the levels of other hormones and neuropeptides), also influences hemopoiesis and immune function and improves glucose and fat metabolism. From Mantzoros [100a].

Leptin

Leptin, a 167 amino acid cytokine, is the product of the ob gene, expressed in white adipose tissue, stomach, placenta and possible mammary gland [99]. Leptin is thought to play a key role in the regulation of body fat mass [100]. Regulating food intake and energy expenditure (Fig. 3.2). GH has several metabolic effects, including lipolytic and anabolic effects. In turn, alterations in nutritional status, such as obesity and food deprivation, influence GH secretion. In obese subjects, GH secretion is markedly impaired. The mechanism by which metabolic status regulates GH secretion is poorly understood. As leptin receptors are present on

the hypothalamus, and leptin plays a role in hypothalamic–pituitary function in fasted animals [101], leptin may act as a metabolic signal to regulate GH secretion (Fig. 3.3). Leptin antiserum administered to normal fed rats, decreased plasma GH levels [102], implying that physiologic leptin levels may facilitate normal spontaneous GH secretion. Furthermore leptin administered to fasted rats reversed the inhibitory effect of food deprivation on in vivo GH secretion [102]. Increased SRIF release may be a factor in the suppressed GH secretion observed in fasted rats [103], possibly mediated by neuropeptide Y, as hypothalamic neuropeptide Y

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release is increased in fasted rats [104]. Leptin receptors are present in neuropeptide Y producing hypothalamic neurons [105], which synapse with somatostatin neurones [104]. Furthermore both antisera to neuropeptide Y and somatostatin reverse starvation-induced GH release [103,104]. Further studies are needed to elucidate the effects of leptin on neuropeptide Y, and somatostatin and its role as a metabolic signal in the regulation of GH secretion. In GH deficient hypopituitary adults, leptin concentrations are more elevated than would be expected from the degree of obesity [106].

Cholinergic and Serotonergic Neurotransmitters

Cholinergic and serotoninergic neurons have been implicated in the etiology of sleep-induced GH secretion [119]. Opiods

Endorphins and enkephalins stimulate GH secretion in animals [120], probably via interaction with other neurotransmitters in both the pituitary and hypothalamus. Opioids increase rat GHRH concentrations [121], and endogenous opiates probably increase GH release during severe physical stress and extreme exercise [122]. Gastrointestinal Neuropeptides

Pharmacologic Control of GH Secretion The central neurogenic control of GH is complex. Neuropeptides, neurotransmitters and opiates impinge on the hypothalamus and modulate GHRH and somatostatin (SRIF) release. The net effect of these complex influences determines the final secretory pattern of GH.

Several gastrointestinal neuropeptides stimulate GH secretion in animal models, including substance P, neurotensin, vasoactive intestinal polypeptide, peptide histidine isoleucine amide (PHI), motilin, galanin, cholecystokinin, and glucagon [123]. HYPOTHALAMIC HORMONES

Dopaminergic Neurotransmitters

Dopamine (DA) is a precursor of epinephrine and norepinephrine. Apomorphine, a central dopamine receptor agonist, stimulates GH secretion [107]. GH-deficient children have been shown to increase their growth velocity after 6 months of levodopa treatment [108]. Sixty to 90 minutes after oral l-dopa administration, adults increase their serum GH levels from 0 to 5–20 ng/ml [109]. Phentolamine, an a-adrenergic blocking agent, inhibits l-dopa-induced GH release, indicating that l-dopa may, in addition, require conversion to epinephrine or norepinephrine to exert its effect on GH secretion [110]. Catecholaminergic Neurotransmitters

Norepinephrine increases GH secretion via a-adrenergic pathways and inhibits GH release via b-adrenergic pathways [111]. Insulin-induced hypoglycemia increases GH secretion via an a2-adrenergic pathway [112], whereas clonidine acts on a1-adrenergic receptors to increase GH secretion [113]. Arginine administration, exercise, l-dopa, and antidiuretic hormone (ADH) facilitate GH secretion by a-adrenergic effects [114]. Animal studies using pharmacologic agents and passive immunization show that a-adrenergic blockade simultaneously inhibits GHRH release while stimulating SRIF release, resulting in suppressed GH secretion [115]. b2-adrenergic receptor stimulation decreases basal GH levels [116]. b-adrenergic blockade increases GHRH-induced GH release, possibly due to a b-adrenergic effect at the pituitary level or via decreased hypothalamic somatostatin release [117]. b-blockade also enhances GH release elicited by insulin-induced hypoglycemia, ADH, glucagon, and L-dopa [114]. Epinephrine may regulate GH release by decreasing somatostatin release [118], and metabolites of g-aminobutyric acid (GABA) may alter GH secretion by enhancing catecholaminergic release [110].

Thyrotropin-releasing hormone (TRH) does not stimulate GH secretion in normal subjects [124]; it does, however, induce GH secretion in about 70% of patients with acromegaly [125]. TRH also stimulates in vitro GH secretion in cultured pituitary tumor cells [126]. The discordant GH response to TRH has also been demonstrated in patients with liver disease [127], renal disease, ectopic GHRH-releasing carcinoid tumors [128], anorexia nervosa [129], and depression [130]. Administration of intravenous corticotropin-releasing hormone (CRH) increases GH secretion in a small percentage of patients with acromegaly [131] and in patients with chronic depression [132]. Gonadotropin-releasing hormone (GnRH) stimulates GH secretion in one-third of patients with acromegaly, but the mechanism is unclear [133].

Hormones Facilitating GH Secretion Physiologic glucocorticoid concentrations increase GH secretion in human pituitary cultures [134]. In acromegalic patients, dexamethasone suppresses GH secretion in vivo [135]. Supraphysiologic serum glucocorticoid concentrations retard growth in humans [136]. Cushing’s disease is also associated with growth retardation, decreased serum GH [137], and decreased pituitary GH content in the tissue surrounding the adenoma [138]. A single dose of dexamethasone administered to normal subjects suppresses GHRH-induced GH release within 12 hours. Glucocorticoids administered to normal subjects produce a dose dependent inhibition of GHRH-stimulated GH secretion, identical to that seen in Cushing’s syndrome [139,140]. Glucocorticoids have a greater inhibitory action on GHRHinduced GH secretion than clonidine or arginine [139]. In chronic hypocortisolism, GHRH-induced GH secretion is reduced [141]. To elucidate the mechanism of the blocking

Chapter 3

action of glucocorticoids on GH release, increased somatostatin secretion seems unlikely, as pyridostigmine (which blocks hypothalamic somatostatin secretion) does not increase suppressed GH levels in Cushing’s syndrome [142]; neither can IGF-I be incriminated, as IGF-I levels are normal in Cushing’s syndrome [143]. The above data exclude decreased endogenous GHRH release as the mechanism. When the glucocorticoid effect is measured acutely, i.e., at 3 hours, stimulated GH levels are measured, which remain elevated for 2 hours [144]. Acute glucocorticoidinduced GH secretion occurs with several different corticosteroid preparations including hydrocortisone, deflazacort, and dexamethasone. The short-term stimulatory effect of glucocorticoids on GH secretion is one of the most potent and also the most delayed stimulus to GH secretion, as it takes 3 hours to occur [144]. Thus glucocorticoids have short-term stimulatory effects on GH secretion and delayed inhibitory effects. Specific glucocorticoid responsive elements have been identified on the GH gene. Currently, the mechanism of action of the dual stimulation and inhibition of glucocorticoid on GH secretion has not been elucidated. Thyroid hormone increases basal and stimulated rat GH synthesis, acting directly at the level of gene expression [145]. This effect appears to be synergistic with glucocorticoids. T3 binds to nuclear receptors in rat pituitary tumor cells [146] and stimulates GH gene transcription [147]. In vitro data confirm that the hGH promoter is not thyroid hormone-responsive, while T3 strongly induces the rat GH promoter [21,148]. In hyperthyroid patients, GH secretion rate is decreased, but normalizes when patients are rendered euthyroid [149], suggesting that thyroid hormone suppresses GH secretion from the normal human somatotroph in vivo. GHRH

Hypothalamic GHRH was characterized initially by two different groups from ectopic pancreatic GHRH-secreting tumors causing acromegaly [128,150]. Analysis of one tumor revealed a 44-amino acid GHRH residue [151]; the other contained 37-, 40-, and 44-amino acid forms [152]. GHRH (1–40) and GHRH (1–44) are both found in extracts derived from the human hypothalamus. GHRH is secreted

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85

from neurons in the hypothalamic arcuate nucleus and premammillary area, with axons that project to the median eminence [153]. The hGHRH gene encodes a 108-amino acid prepro-hormone for GHRH-44 [154,155], which has a free amino terminal and amidated carboxy terminal residue. The amino terminal tyrosine appears to bestow biologic activity on the GHRH molecule (Fig. 3.4). There is considerable structural homology between GHRH and several gut peptides. The highest is between GHRH and PHI, which have 12 amino acids in common in equivalent positions [156]. Varying degrees of homology exist between GHRH and VIP, glucagon, secretin, and GIP [157]. All of these peptides stimulate GH secretion in various physiologic systems, but with lower potency than GHRH. GHRH binds to specific sites on the somatotroph membrane, resulting in increased intracellular 3¢, 5¢cAMP [158]. The GHRH receptor gene has recently been cloned and sequenced, encoding a 47-kDa protein of 423 amino acids [159]. GHRH has a selective action on GH release; it does not release other anterior pituitary or gut hormones. GHRH increases GH synthesis as well as secretion, and stimulates transcription of GH mRNA [64,160]. GHRH stimulates GH release from both stored and newly synthesized intracellular GH pools, with a greater effect on stored pools [161]. There are a number of heterogeneous GH pools, varying according to time of GH synthesis [162–164], molecular size [163], and response to provocative stimuli [163]. Somatostatin suppresses both basal and GHRHstimulated GH release, but does not affect GH biosynthesis [165]. It is thus apparent that GHRH stimulates both release and synthesis of GH independently of any effects of somatostatin. GHRH administered to normal adults elicits a prompt increase in serum GH levels, with higher levels occurring in female subjects [166]. Most acromegalic patients retain an intact GH response to administered GHRH [167]. SRIF

SRIF, a cyclic tetradecapeptide, is recognized as a heterogeneous group of molecules including quantitatively predominant, but less bioactive SRIF-14, more bioactive SRIF-28,

FIGURE 3.4. Amino acid structure of growth hormone-releasing hormone (GHRH) (1–44). GHRH (1–44) and GHRH (1–40) have similar GH-releasing potency in vivo. The tyrosine at the amino terminus confers biologic activity. Fragments as short as GHRH (1–29) exhibit full biologic activity. The carboxy terminal fragment (28–44) has no biologic activity.

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FIGURE 3.5. Amino acid structure of native somatostatin (SRIF). SRIF, a tetradecapeptide (14-amino acid peptide) designated SRIF-14, SRIF-28, which is extended at the amino terminal, and several larger forms range in size from 11.5 to 15.7 kDa.

sst

PHOSPHATASE

ADENYLCYCLASE K/Ca++ CHANNELS cAMP

Ca++

Calcineurin

Ser/Thr Phosphatase

– Exocytosis

INHIBITS SECRETION

FIGURE 3.6. SRIF receptor signaling pathway: somatostatin receptors mediate their responses via several cellular effectors such as adenylyl cyclase potassium and calcium channels and phosphatases. Adapted from Yogesh et al. [176a].

SRIF block the GH secretory response to insulin-induced hypoglycemia, exercise, arginine, morphine, levodopa, and sleep related GH release. Somatostatin exerts its biologic effects through specific membrane-bound high affinity receptors. At least five somatostatin receptor (SSTR) subtypes have been cloned [176], termed SSTRs1-5. Somatostatin receptors are coupled to guanine nucleotide protein (G), and have seven transmembrane domains. There is 42% to 60% amino acid homology among the five somatostatin receptor subtypes [177]. Somatostatin receptors mediate their responses via several cellular effectors such as adenylyl cyclase, protein phosphatases, Na+-H+ exchanger, cyclic GMP-dependent protein kinases, phospholipase C, potassium and calcium channels [176] (Fig. 3.6). The human pituitary gland expresses SSTR1, 2 and 5 [178,179], whereas human pituitary adenomas contain SSTR1, 2, 3 and [178–181]. Somatostatin analogs, octreotide and lanreotide, used to control GH hypersecretion in acromegaly, bind with high affinity to SSTR 2 and less efficiently to SSTR5 [182]. SSTR1-4 have highter affinity for SRIF-14, whereas SSTR5 bind more potently to SRIF-28 [176] (Table 3.2). GHRH and SRIF Interaction in Regulating GH Secretion

and still larger forms which range in molecular mass from 11.5 to 15.7-kDa [168] (Fig. 3.5). The SRIF precursor is a 116-amino acid prohormone consisting of a 24-amino acid signal peptide, a 64-amino acid connecting region, followed by SRIF-28 [169] which incorporates SRIF-14. Prosomatostatin is synthesized in neuronal perikarya of the anterior hypothalamic periventricular nuclei, and transported by axonal flow to the nerve terminals ending near the hypophyseal portal vessels. SRIF has also been isolated from pancreatic islets, gastrointestinal, neural and epithelial cells, and extrahypothalamic central nervous system neurons [170]. SRIF has a short plasma half-life of 2 to 3 minutes [168]. SRIF inhibits GH secretion, TSH release, TRH stimulation of TSH but not PRL [171], and pancreatic secretion of insulin and glucagon [172]. Dopamine [173], substance P, and neurotensin [174] stimulate SRIF release from the hypothalamus, while glucose inhibits SRIF secretion in cultured rat hypothalamus [174]. SRIF-28 binds to normal rat pituitary receptors with a threefold greater affinity than SRIF-14 [175]. Both SRIF14 and SRIF-28 block the effect of GHRH on GH release in vitro and in vivo in many different species. Infusions of

SRIF and GHRH secreted in independent waves from the hypothalamus interact to generate pulsatile GH release. SRIF inhibits GH secretion, while GHRH stimulates GH synthesis and secretion. GHRH stimulates pituitary GH synthesis and secretion, and also induces transcription of the GH gene [160]. SRIF suppresses GH secretion without altering GH mRNA levels [64]. GH secretion is further regulated by its target growth factor, IGF-I, which participates in a hypothalamic–pituitary–peripheral regulatory feedback system [183]. GH stimulates the liver and other peripheral tissues to produce IGF-I, which exerts a feedback effect on the hypothalamus and pituitary. IGF-I stimulates hypothalamic SRIF release [172] and inhibits pituitary GH gene transcription and GH secretion [51,184]. In vitro, IGF-I directly attenuates hGH gene expression [51]. Specific antibodies against GHRH and SRIF have been used to dissect the respective contributions of SRIF and GHRH in the generation of GH pulsatility in rats. Anti-SRIF antibody administration results in elevated baseline GH levels, with intact intervening GH pulses [185]. These studies imply that hypothalamic SRIF secretion generates GH troughs. AntiGHRH antibodies eliminate spontaneous GH surges. In humans, GH pulsatility persists when GHRH is tonically

Chapter 3 Table 3.2. receptors

Growth Hormone

87

Properties of the five cloned subtypes of human somatostatin

Properties

Somatostatin receptor Subtype 1

Subtype 2

Subtype 3

Subtype 4

Chromosomal location

14

17

22

20

16

G protein coupling

Yes

Yes

Yes

Yes

Yes

Effector system Adenylate cyclase activity Tryosine Phosphatase activity

Reduced Increased

Reduced Increased

Reduced ?

Reduced ?

Reduced ?

IC50 (nM) Somatostatin-14 Somatostatin-28 Octreotide Vapreotide (RC-160) Lanreotide (BIM-23014)

1.1 2.2 >1000 >1000 >1000

1.3 4.1 2.1 5.4 1.8

1.6 6.1 4.4–35 30.9 43

0.5 1.1 >1000 45 66

0.9 0.07 5.6 0.7 0.6

Brain, lungs, jejunum, kidneys, liver and pancreas

Brain and kidneys

Brain and pancreas

Brain and lungs

Brain, heart, adrenal glands placenta, pituitary, small intestine and skeletal muscle

Distribution in normal human tissue

Subtype 5

Adapted from Patel YC and Srikant CB [182].

elevated due to ectopic GHRH production by a tumor or during GHRH infusion [127,186], suggesting that hypothalamic SRIF is mainly responsible for GH pulsatility. The rat hypothalamus releases GHRH and SRIF 180° out of phase every 3 to 4 hours, resulting in pulsatile GH levels [185]. GHRH and SRIF also act synergistically, in that preexposure to SRIF has been shown to enhance somatotroph sensitivity to GHRH stimulation [187]. Hence, during a normal GH trough period, the high SRIF level probably primes the rat somatotroph to respond maximally to the subsequent GHRH pulse, thus optimizing GH release. In addition to exerting opposing and cooperative interactions on the pituitary somatotroph, SRIF exerts a central inhibitory effect on GHRH release via direct synaptic connections between SRIF-containing axons and GHRHcontaining perikarya in the rat hypothalamic arcuate nucleus [188]. GH Autoregulation

Chronic GHRH stimulation, either by continuous infusion or repeated bolus administration, eventually results in decreased GH release from cultured rat anterior pituitary cells [189] and in vivo [190] due to somatotroph desensitization. Loss of GH sensitivity to administered GHRH does not occur in acromegalic patients in vivo [191] or in somatotroph adenomas in vitro [192], possibly reflecting larger intracellular pools of GH or abnormal signaling. One possible mechanism of pituitary desensitization is depletion of a GHRH-sensitive pool of GH. GHRH pretreatment in

vitro also leads to a 50% decrease in somatotroph GHRH binding sites [193]. Feedback loops exist between GH and IGF-I and the release of SRIF and GHRH (Fig. 3.7). GH stimulates hypothalamic SRIF release in vitro [194], and in vivo, GH administration decreases GH responses to GHRH [195], most likely by increasing hypothalamic SRIF release [196]. GHRH and SRIF also autoregulate their own secretion. GHRH inhibits its own secretion but stimulates SRIF secretion in vitro [197], while SRIF inhibits its own secretion in vitro [198]. Growth Hormone Secretagogues (GHS) and Ghrelin

Small synthetic molecules termed growth hormone secretagogues (GHS) [199] stimulate and amplify pulsatile pituitary GH release [200], via a separate pathway distinct from GHRH/SRIF. A strategy of expression cloning was used to identify the GHS receptor, which is a heterotrimeric GTP-binding protein (G-protein)-coupled molecule [201]. This classic Gprotein coupled receptor has seven alpha helical membrane spanning domains and three intracellular and extracellular loops. The GHS receptor is most strongly expressed in the anterior pituitary and both the hypothalamic and nonhypothalamic regions of the brain. The ligand of the GHS receptor is a 28 amino acid peptide, Ghrelin isolated from stomach, with 3 serine residues and is n-octanoylated [202]. It releases GH both in vivo and in vitro and 0-n octanoylation appears to be essential for GH releasing activity. The

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FIGURE 3.7. Growth hormone (GH), insulin-like growth factor-I (IGF-I) Growth Hormone secretagogues GHS (GHRH and Ghrelin), somatostatin (SRIF) feedback loop in the control of somatic growth. A stimulatory effect of GHRH and Ghrelin and inhibitory effect of SRIF interact to result in GH secretion from the anterior pituitary. GH stimulates IGF-I production from the liver. This, in turn, has a negative feedback effect on pituitary GH secretion and a positive feedback effect on hypothalamic SRIF release. Pituitary GH secretion has a negative autoregulatory feedback effect on hypothalmic GHRH secretion. GH exerts an effect on somatic growth by a direct as well as indirectly via IGF-I.

gene encoding human Ghrelin has been isolated, and found to be expressed in the stomach and hypothalamus [202]. The isolation of Ghrelin implicates a control system in addition to GHRH/SRIF in the regulation of GH secretion. Ghrelin is analogous to GHRH/SRIF in that it is also synthesized in peripheral tissues (stomach) as well as centrally (hypothalamus). It would be anticipated that hypothalamic Ghrelin would control GH secretion, while peripheral sources may have other biological effects requiring further elucidation. Several artificial GH secretagogues (GHS) release GH in vitro [203]. GHSs, administered alone or in combination with GHRH are potent and reproducible GH releasers and are useful tools for the diagnosis of GH deficiency. GHSs act directly on the pituitary somatrophs [204], stimulating GH secretion by activating the cAmp-dependent protein kinase pathway [205]. GHRH and GHS thus act through distinct receptors, via different intracellular signaling pathways on somatotrope subpopulations. GHS increases the number of somatotropes releasing GH, without altering the amount of hormone released from each individual cell

[206]. Somatostatin too decreases the number of somatotropes that secrete GH, and it is postulated that GHS and SRIF may respectively depolarize and hyperpolarize the same somatotroph cell. GHRH, on the other hand, stimulates the amount of GH secreted per cell, as well as the number of cells secreting GH [206]. Evidence that the main action of GHS occurs at the hypothalamic level, and that the direct pituitary activity is ancillary, is supported by the fact that GHSs require the presence of a functional hypothalamus to exert their GH secreting effect. In patients with intact pituitaries, who have hypothalamic dysfunction leading to stalk section, GHSinduced GH release is blocked and the synergistic effect of GHRH and GHS is absent [207]. Current evidence suggests that the dual control of GH secretion postulated for GHRH/SRIF should be expanded to incorporate Ghrelin. If indeed Ghrelin acts at the hypothalamic level, its mechanism(s) of action could include release of endogenous GHRH. However available data seems to indicate that Ghrelin does not act via GHRH release in that the GH response to GHS is greater than that of GHRH alone [208];

Chapter 3

FIGURE 3.8. A new physiologic model of the regulation of GH secretion involving Ghrelin, GHRH and somatostatin (SRIF). From Diguez and Casanueva [218a].

GHSs potentiate GH release in response to a maximal stimulating dose of exogenous GHRH [209]. GHSs are unable to release GHRH in rat hypothalamic explants [210]; GHRH infusions block GHRH mediated GH release without altering the action of GHS [211,212]; after administration of a saturating dose of GHRh, a second dose of GHRH is ineffective, while GHS is fully effective [213]. The above evidence does not support a role of GHRH in the mechanism of action of GHS. GHSs do not seem to inhibit somatostatin release [212,214–216]. However, GHS mediated GH release is relatively insensitive to the inhibitory action of SRIF [217]. The presence of GHS in hypothalamic structures and the evidence that GHS-elicited GH secretion is not mediated by changes in endogenous GHRH or SRIF, but requires a functional hypothalamus, suggests that exogenous GHS may induce the release of another hypothalamic factor with GH-releasing capability [218] (Fig. 3.8). Release of another unknown hypothalamic factor with GH releasing capability is supposed by observations that GHSs effectively release GH when administered intravenously, subcutaneously, orally or intranasally [219]. Hypoglycemia increases GHS stimulated GH release [214] as does decreased circulating FFAs [220]. GHSs are resistant to wellknown inhibitors of GH secretion including increased FFA levels, hyperglycemia, SRIF infusion, atropine or drugs that enhance hypothalmic SRIF secretion [221]. GHS-mediated GH release is not reduced by a previous rise in GH [213], glucocorticoid treatment [222], or thyroid hormone abnormalities [223]. GHS-mediated GH release is more potent and reproducible than that elicited by GHRH alone [224].

Growth Hormone

89

GHRH-mediated GH release is more erratic and less reproducible, possibly due to the influence of metabolic and hormonal factors. Functional GHs receptors are detected in the human pituitary by the fifth week of gestation [225]. GHS mediated GH-release is demonstratable at birth [221], continues through infancy, increases at puberty and then decreases thereafter. Estrogen and testosterone increase GHS mediated GH release in childhood. When administered with GHRH, GHS has a synergistic action on GH release. As GH secretion elicited by combined GHRH and GHS administration is minimally altered by age, sex, or adiposity and is devoid of potential side effects (unlike insulin-induced hypogylcemia) this test has the potential to become a widely used diagnostic tool in the diagnosis of adult GHD. No side effects have been reported with the GHSs. Although highly specific for GH release, slight increases in prolactin and ACTH/cortisol have been reported with some GHSs [226], leading to the development of new more selective GHSs with no ACTH or PRLreleasing effects e.g. ipamorelin [227]. GHSs are being evaluated as potential therapeutic agents for enhancing circulating GH levels in patients with intact hypothalamic–pituitary axs, including children with GHD as well as adults with catabolic states, AIDS and heart failure. Long-term studies are required with a large number of subjects to determine dosage efficacy.

GH REGULATION

GH Transgenic Animals Transgenesis allows for the transfer of inheritable functional genes between animals of different species. GH transgene models of endocrine hyperfunction have also been useful in investigating regulatory factors required for tissue-specific gene targeting. When a rat or hGH reporter gene driven by a potent metallothionein-I (MT) promoter is expressed in a transgenic progeny, rat or human GH is produced by a number of tissues including the pituitary [228,229]. Circulating GH levels in these animals are several hundred times higher than normal with an increase in transgenic mouse size to twice that of normal littermates [230]. Circulating IGF-I levels are elevated two- to threefold, consistent with the notion that IGF-I mediates the allometric growth of these transgenic animals [228,229]. Interestingly, the effects of excess GH are only apparent after 3 weeks of age and no further growth occurs after 3 months. Immunocytochemical and ultrastructural study of transgenic pituitary glands revealed a marked reduction in both size and number of GH-secreting cells [229,231]. Pituitary lactotroph cells were also suppressed in male transgenic animals, but not in females. In contrast, both sexes exhibited corticotroph cell hyperplasia. Male transgenics had high levels of LH and highly active gonadotrophs, resembling “gonadectomy” pituitary cells. When ovariectomized female

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hGH transgenics were studied, adult female mice developed hypoprolactinemia, but had high levels of LH due to PRL-like activity of GH [232]. Metallothionein rat GH (MTrGH), metallothionein bovine GH (MTbGH) or metallothionein human GH (MThGH) female transgenics are sterile; however, metallothionein ovine GH (MToGH) female transgenics are fertile [233,234]. In MTrGH transgenic animals [235] both the liver and spleen undergo a threefold increase in size. Furthermore, MToGH male transgenics contain hepatic intranuclear inclusions whose presence correlates with high circulating levels of oGH [233]. The heart, lungs, kidneys, and thyroid glands of GH transgenics grew significantly larger when compared to nontransgenic control littermates. Interestingly, no increase was observed in brain or adrenal size. In contrast, methallothionein-driven IGF-I gene (MTIGF-1) transgenics [230] have an increased growth ratio of one to three times that of control littermates, which is only apparent from 6 to 8 weeks after birth. Spleen, pancreas, and kidneys increased in size in direct proportion to the increase in body weight both in IGF-I and GH transgenic models. Compared to GH transgenics, IGF-I transgenic mice show an increase in brain size but no enlargement of the liver or heart [230]. GH transgenics develop renal glomerular sclerosis, similar to abnormalities seen in diabetics, whereas this lesion is not present in IGF-I transgenics. GH transgenics also have elevated serum insulin and lipid levels [236]. Interestingly, mesangial renal proliferation followed by progressive glomerulosclerosis was also seen in transgenic GHRH animals [237], suggesting that the hypersomatotrophism associated with excess GHRH secretion is predominantly GH-mediated. While many of the sequelae of chronic GH overexpression such as increased growth and pituitary somatotroph atrophy were predictable, the abnormalities of hepatic and renal function, and the reduced fertility of female transgenics, were unexpected. These novel findings may provide useful models of human disease. Transgenesis is a useful tool to investigate ontogeny of gene expression, as well as a method of exposing animals to growth at a time when they would not usually be accessible to manipulation of factors responsible for both allometric as well as organ-specific growth. GH Binding Protein (GHBP)

Circulating GH is attached to specific binding proteins [238]. Two circulating GHBPs have been identified, one of high affinity and one of low affinity [239]. Although the site of GHBP synthesis is as yet undetermined, the liver has been postulated as the primary source [240]. The 60-kDa high-affinity BP corresponds to the extracellular domain of the hepatic GH receptor [241]. Under basal conditions, half of the circulating 22-kDa GH is bound to the highaffinity BP when GH levels are up to 10–15 ng/ml [242]. The 20-kDa GH binds preferentially to the low-affinity BP [243]. Binding to plasma GHBP prolongs GH plasma half-

life by decreasing GH metabolic clearance rate, as the GHbinding protein complex is too large for renal glomerular filtration [243]. The high-affinity BP also inhibits GH binding to surface receptors by competing for the ligand [244]. Thus GHBP dampens acute oscillations in serum GH levels caused by pulsatile pituitary GH secretion. Highaffinity BP levels are low in the fetus and neonate, rise most rapidly in the first 1 to 2 years after birth [245,246], and stay constant throughout adult life, with similar levels found in males and females [245]. GH itself does not regulate either type of BP, as evidenced by normal BP levels found in patients with either hypopituitarism or acromegaly [245]. GH resistance, as demonstrated in short stature, Laron dwarfism, and African pygmies, is characterized by decreased plasma levels of high-affinity BP [247,248]. Although these conditions provide insight into the role of GHBP in GH action, Laron dwarfs are also GH receptor-deficient, therefore, no conclusions can be drawn about GHBP function per se from these findings. In African pygmies, GHBP levels, although low, vary widely with considerable overlap with normal subjects.

Peripheral GH Action GH Receptor

GH binds to its peripheral receptor and induces intracellular signaling by a phosphorylation cascade involving the JAK/STAT pathway. GH also acts indirectly by inducing synthesis of IGF-I, the potent growth and differentiation factor [249]. The GH receptor (GHR) is a 70 kd protein member of the class I cytokine/hematopoietin receptor superfamily [250]. GHR consists of an extracellular ligandbinding domain, a single membrane-spanning domain, and a cytoplasmic signaling component. The GH ligand complexes with two GHR components leading to receptor dimerization. This sequential ligand-receptor interaction and dimerization is critical for subsequent GH signaling. GHR dimerization is followed by rapid binding and activation of JAK2 tyrosine kinase, leading to phosphorylation of cytoplasmic signaling molecules, especially the signal transducing activators of transcription proteins (STAT) which comprise the critical signaling components for GH action. These cytoplasmic proteins are phosphorylated by JAK2 and directly translocated to the cell nucleus where they elicit GH-specific target gene expression by binding to nuclear DNA [250]. In addition, STAT1 and STAT5 may in fact interact directly with the GH receptor molecule [251]. Other target actions induced by GH include c-fos induction, IRS-1 phosphorylation, and insulin synthesis. As a differentiating and growth factor, IGF-1 is a critical protein induced by GH, and is likely responsible for most of the growth-promoting activities of GH [252]. IGF-I itself may also directly regulate GH [252] and GH receptor function [253]. The liver contains the highest abundance of GH receptors, and several peripheral tissues also express modest amounts of receptor, including muscle and fat [254].

Chapter 3

Mutations of the GH receptor may be associated with partial or complete GH insensitivity and growth failure. These syndromes are associated with normal or high circulating GH levels, decreased circulating GHBP levels and low levels of circulating IGF-I. Multiple homozygous or heterozygous exonic and intronic GHR mutations have been described, most of which occur in the extracellular ligandbinding domain of the receptor. Tissue responses to GH signaling may be determined by the pattern of GH secretion, rather than the absolute amount of circulating hormone. Thus, linear growth patterns, liver enzyme induction and STAT5b activity may be phenotypically distinct for male animals due to their higher rates of GH pulse frequency [255]. STAT5b is sensitive to repeated pulses of injected GH [256], unlike other GHinduced patterns which are desensitized by repeated GH pulsing. Mice harboring a disrupted STAT5b transgene exhibit impaired male pattern body growth [257] with IGFI and testosterone levels normally seen in female mice. Thus, the sexual dimorphic pattern of GH secretion and GH tissue targeting appears to be determined by STAT5b. The requirement for appropriate GH pulsatility to determine body growth also appears to be mediated by STAT5b [258]. In contrast, STAT5 does not appear to be critical for metabolic effects of GH on carbohydrate metabolism [259]. Unraveling STAT5 regulation in disease, especially those involving GH resistance, will provide novel mechanistic insights for dysregulated GH signaling in humans. Intracellular GH signaling is also abrogated by SOCS proteins, which disrupt the JAK/STAT pathway and thus exert a further level of control over the action of GH [260] (Fig. 3.9).

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91

IGF-I and IGF-II Structure and Synthesis

IGF-I and IGF-II are single-chain polypeptide molecules with three intrachain sulfide bridges (Figs 3.10 and 3.11). IGF-I, composed of 70 amino acids, and IGF-II, consisting of 67 amino acids, have a sequence homology of 62% [261]. The IGFs consist of B and A peptide domains (structurally homologous with the insulin B and A chains), a C domain analogous to the connecting (C) peptide of proinsulin, and a D domain. IGF-I and IGF-II are single distinct gene loci, localized on chromosome 12 (12q22–q24.1) and chromosome 11 (11p15), respectively [262,263]. The IGF-I gene primary transcript can be alternately spliced to different products resulting in IGF-Ia (exons 1,2,3,5) or IGF-Ib (exons 1,2,3,4). Several IGF-I mRNA species have been isolated from adult and fetal tissues. The liver is an important source of circulating IGF-I levels [264]. The IGF-I gene is expressed in human fetal conective tissues and cells of mesenchymal origin [265]. This ubiquitous localization of IGFs favors a paracrine/autocrine function as well as an endocrine function of IGF-I. GH is the major regulator of IGF-I gene expression in adult liver, heart, lung, and pancreas [266], and acts at the level of IGF-I transcription [267]. Fetal IGF-I production is GHindependent [268], and platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) also stimulate IGF-I production from human fibroblasts in vitro [269]. ACTH, TSH, LH, and FSH stimulate the paracrine production of IGF-I in their respective target tissues. Nutritional status is also an important regulator of IGF-I production at all ages [270]. As discussed below, IGF-I and IGF-II are bound to carrier proteins in the serum. IGFs are found in lymph [271], breast milk [272], saliva [273], and amniotic fluid [274]. IGF-I levels are low before birth, rise during childhood to high levels during puberty, and decline with age [275]. IGF-binding Proteins (IGFBPs)

FIGURE 3.9. Role of STAT proteins in growth hormone signaling. From Herrington, Smith, Schwartz, Carter-Su [260a].

IGF-I and IGF-II are complexed to six specific binding proteins in biological fluids [276] (Fig. 3.12 and Table 3.3). The IGFBPs are cysteine-rich proteins, with very similar amino acid sequences. They have a unique ability to bind IGF’s with high affinity. Recently several groups of cysteine-rich proteins have been described [277] with structural and functional similarities to the IGFBP’s, called IGFBP-related proteins (IGFBP-rPs). The IGFBP-rPs bind IGFs with low affinity. The physiological role of these IGFBP-rPs in the IGF system is not known. However some of these proteins unequivocally bind IGFs [277]. An IGFBP superfamily has been proposed, composed of the IGFBPs and the IGFBPrelated proteins (IGFBP-rPs) (Fig. 3.13). The major form of binding protein present in the human circulation is IGFBPIII, a glycoprotein associated with an IGF molecule and an 80-kDa acid-labile subunit (ALS) to form a 150- to 200kDa complex [278] (Fig. 3.14).

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FIGURE 3.10. Amino acid sequence of human insulin-like growth factor I (IGF-I). The black amino acids are identical to those in human insulin. The numbering corresponds to the numbering of residues in the proinsulin molecule. IGF-I consists of a 70-amino acid single peptide chain with A, B, C, and D, domains. A and B domains are structurally homologous to the A and B chains in the insulin molecule, and the C domain is equivalent to the connecting (C peptide in proinsulin). From Humbel [261a].

FIGURE 3.11. Primary structure of human insulin-like growth factor II (IGF-II). From Sara and Hall [261b].

Actions of the IGFBPs include modulation of IGF actions, storage of IGFs in extracellular matrices, and the carrier protein function of IGFBPs. In the serum most of the IGF circulates as a 150,000 dalton complex that consists of IGF-I or IGF-II plus IGFBP-III and a nonIGF binding component, the acid labile cubunit (ALS). Binding of IGF-I or II to IGFBP-III in the presence of ALS results in the formation of an IGF-IGFBP-ALS ternary complex, which is stabilized by IGF binding [279] 75% of circulating IGF-I and IGF-II is carried in this ternary 150-kDa complex. When associated with the 150-kDa complex, the IGFs do not readily leave the vascular compartment, and have prolonged half-lives of 12 to 15 hours [280] compared to the half-life of free IGF-I which is less than 10 minutes [281]. The exact function of the large store of IGF bound

to the 150-kDa ternary serum complex is unknown. It has been hypothesized to be a functional pool of material available for organisms to use during stress. The plasma concentrations of IGFBPs are hormonally regulated. Serum IGFBP-III levels correlate with IGF-I and II levels, double in patients with acromegaly and are reduced in hypopituitarism [282]. Malnutrition, insulin-dependent diabetes mellitus, and cirrhosis are associated with decreased IGF-I levels, as well as suppressed IGFBP-III levels [283]. IGFBP-I levels are high at birth and decline until puberty [284]. There is a diurnal variation with a nocturnal peak in serum IGFBP-I levels [285]. However, IGFBP-I levels are elevated in hypopituitarism [286] and decreased in acromegaly. IGFBP-I levels are regulated by insulin. The increased IGFBP-I levels associated with insulin-dependent

FIGURE 3.12. Simplified diagram of GH-IGF-I axis involving hypophysiotropic hormones controlling pituitary GH release, circulating GH-binding protein and its GH receptor source, IGF-I and its largely GHdependent binding proteins, and cellular responsiveness to GH and IGF-I interacting with their specific receptors. IGFR, IGF-I receptor; FFA, free fatty acids. From Rosenbloom [276a].

Table 3.3.

General characteristics of the human IGF binding protein No. of amino acids

Core molecular mass (kDa)

Chromosomal localization

IGF affinity

Modulation of IGF action

Source in biological fluids

IGFBP-I

234

25.3

7

I = II

Inhibition and/or potentiation

Amniotic fluid, serum, placenta, endometrium, milk, urine, synovial fluid, interstitial fluid and seminal fluid

IGFBP-II

289

31.4

2

II > I

Inhibition

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

IGFBP-III

264

28.7

7

I = II

Inhibition and/or potentiation

Serum, follicular fluid, milk, urine, CSF, amniotic fluid, synovial fluid, interstitial fluid, and seminal fluid

IGFBP-IV

237

25.9

17

I = II

Inhibition

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

IGFBP-V

252

28.5

5

II > I

Potentiation

Serum and CSF

IGFBP-VI

216

22.8

12

II > I

Inhibition

CSF, serum and amniotic fluid

Modified from Rajaram S et al. [278a].

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FIGURE 3.13. The IGF system. The components of this system include the peptides IGF-I and -II, IGFBP-1 to -6, IGFBP-rPs, IGFBP proteases, type I and type II IGF receptors, and potential IGFBP(s) and IGFBP-rP(s) receptors. M6P, Mannose-6-phosphate. From Hwa, Oh, Rosenfeld [277a].

diabetes mellitus [287] are normalized by insulin, and insulinoma is associated with suppressed IGFBP-I levels. Octreotide (a long-acting somatostatin analog) has been shown to increase IGFBP-I levels in acromegalic patients [288]. When normal subjects ingest glucose, the fall in IGFBP-I levels correlates inversely with the rise in insulin levels [289]. Hypophysectomy is associated with elevated IGFBP-II levels in rats, which fall with GH administration [283]. Insulin infused into normal rats increases IGFBP-II levels [283]. PARACRINE GH ACTION GH and GH-releasing factors are also produced in tissues outside the hypothalamic pituitary axis. The extrapituitary actions of GH are likely autocrine/paracrine and complement the classic endocrine action between the GHreleasing factors, GH and target tissues. GH gene expression is not restricted to the pituitary gland. GH immunoreactivity and GH mRNA have been localized to several extrapi-

tuitary tissues [290] including placenta, mammary gland muscle, spleen lymphocyte, suggesting an extrapituitary paracrine/autocrine action for GH. Classic endocrine actions of GH include GH promoting effects on bone and several metabolic actions.

Effect on Growth Whether GH exerts its growth-promoting action on the skeleton via a direct GH effect on the growth plate, or indirectly by stimulating hepatic or local IGF-I production, remains unclear. The somatomedin hypothesis proposed an indirect effect of GH on growth mediated through plasmatransported IGF-I that is synthesized in the liver [291]. The dual effector theory postulates a direct effect of GH by stimulating epiphyseal growth plate precursor cells to differentiate and become responsive to IGF-I, and to induce local IGF-I synthesis that in turn stimulates the multiplication of differentiating chondrocytes [292]. This proposes a dual role for GH and IGF-I in the control of tissue mass. IGF-1 has

Chapter 3

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95

which is rapidly restored by GH administration [299]. Hypophysectomized rats show a progressive decline in longitudinal bone growth rate [300], finally growing at only 0 to 10% of the normal rate. GH and IGF-I Effects on Bone in vitro

FIGURE 3.14. Relative distribution of various IGF pools in human serum. The distribution of IGFs between the 50-kDa, 150kDa, and the free pool as determined before and during continuous subcutaneous infusion of 30 mg/day of rhIGF-1 in healthy men. From Rajaram, Baylink, Mohan [278a].

a potent mitogenic action, especially on immature differentiating cells (adipocytes, chondrocytes), whereas GH is not a mitogen [293]. The dual effector theory proposes that although the overall effects of GH and IGF-I on growth at the tissue level appear to be the same, at the cellular level their effects are very different; GH has both a direct action and an indirect action mediated by IGF-I. The IGFBP regulatory hypothesis proposes that GH stimulates both IGF-I and IGFBP-III [294]. Circulating IGFBP-III regulates the bioavailability of IGF-I to the growth plate, and may inhibit IGF-I action. However, recently hepatic IGF-II production in the liver of mice was completely abolished using an IGF-I knockout mouse model, with complete inactivation of the IGF-I gene in the hepatocytes [295]. Serum IGF-I concentration was reduced by 75%. However the reduction in serum IGFI concentration had no discernible effect on postnatal body growth. Role of GH in Growth and Development

Congenital absence of the human pituitary gland does not result in abnormal birth or newborn weight [296], indicating that fetal and early postnatal development may occur independently of GH. Postpubertal mammalian longitudinal bone growth results from chondrocyte proliferation and differentiation in long bone epiphyseal growth plates. The proximal zone of the epiphyseal growth plate contains stem cells and is the layer of prechondrocyte origin [292]. These cells differentiate and enter the proliferative layer, where they undergo limited clonal expansion. After maturation, the cells become calcified and are incorporated into metaphyseal bone [297]. Hypophysectomy in the growing rat results in a marked reduction in tibial epiphyseal cartilage plate width [298],

Although earlier studies failed to demonstrate in vitro stimulatory effects of GH [301], subsequent studies have demonstrated significant direct effects of GH on chondrocyte and osteoblast cultures [302]. GH increases progenitor cell thymidine incorporation, chondrocyte differentiation, and osteoclast formation. The in vitro effect of IGF-I on chondrocytes has been more consistently demonstrated. IGF-I increases thymidine and sulfate incorporation into isolated chondrocytes [303], and this effect is additive with GH [302,303] occurring at different stages of maturation [304]. Cells from the proximal growth plate (prechondrocytes and young proliferating chondrocytes) form larger colonies in vitro, whereas chondrocytes from the more distal proliferative zone form smaller colonies [305]. Both GH and IGF-I induce chondrocyte colony formation [305] but exert differential effects on colonies of different sizes. GH preferentially potentiates formation of large size colonies, suggesting that GH stimulates prechondrocyte differentiation [304], while IGF-I induces smaller colonies, implicating IGF-I in epiphyseal chrondrocyte stimulation at a more mature stage of differentiation [304]. In vivo Effects of GH and IGF on Skeletal Growth

GH administered locally at the epiphyseal growth plate [306,307] or infused into a femoral artery [308] in hypophysectomized rats results in unilateral longitudinal bone growth and an increase in growth plate width. When IGFI antiserum is infused locally with GH this growth effect is eliminated [308], suggesting that GH exerts its growth promotion via local IGF-I. Local infusion of IGF-I alone into the epiphyseal growth plate results in longitudinal bone growth [307] and increased epiphyseal width. Local GH infusion at the growth plate additionally results in increased numbers of IGF-I-containing cells in the proliferative zone, as well as producing a simultaneous increase in plasma IGFI levels [309], suggesting that the in vivo GH effect may be mediated by locally produced IGF-I in cells that have been stimulated by GH. This may explain why GH-deficient children treated with GH grow significantly, even though their plasma IGF-I levels may not change appreciably [310]. The effects of systemic GH and IGF-I administration have been compared. IGF-I increases tibial epiphyseal width [311] and total body growth in hypophysectomized rats [312], but to a lesser degree than after replacement doses of GH. In mutant GH-deficient dwarf rats, hGH infusions had a more pronounced stimulatory effect on body weight and bone growth than hIGF-I administration, while hIGF-I caused a greater increase in kidney, adrenal, and spleen size than did hGH [313]. GH and IGF-I may also have

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differential tissue effects. Some of these observations suggest that GH acts directly on tibial plate cartilage via a locally produced paracrine or autocrine IGF-I action. The somatomedin hypothesis, however, suggests that plasma IGF-I levels are much higher than tissue levels, and that plasma IGF-I is indeed biologically active. The liver synthesizes more IGF-I in response to GH stimulation than does costal cartilage. Furthermore, IGF-I increases thymidine and sulfate uptake and incorporation in bone and cartilage cultures to a far greater degree than does GH. These findings favor the liver as the predominant site of IGF-I synthesis, with subsequent plasma transport of biologically active IGF-I to its target organ, bone, where it mediates growth. A model incorporating GH, IGF and IGFBP systems in the regulation of systemic growth integrates the dual effector theory and the somatomedin hypothesis (Fig. 3.15). GH stimulates hepatic/tissue IGF-I synthesis, and it is the endocrine action of this circulating IGF-I on the growth plate which regulates skeletal growth, by stimulating cell division. Furthermore, GH induces hepatic IGFBIII and ALS production, forming the circulating 150-kDa ternary

complex that binds IGF-I, thereby increasing its half-life. GH also directly induces prechondrocyte differentiation into chondrocytes at the growth plate, in addition to stimulating local IGF-I production, which acts in an autocrine/ paracrine fashion to further stimulate cell division. Thus the endocrine actions of circulating IGF-I largely contribute to cell division which results in intrauterine and post natal growth. This mechanism is augmented by the direct actions of GH stimulating prechondrocyte/chondrocyte differentiation at the growth plate as well as the local IGF-I stimulation by GH where it acts by an autocrine/ paracrine action to stimulate cell division.

GH and Metabolism In childhood, the multiple metabolic actions of GH contribute to linear growth. GH continues to be secreted in adulthood after growth cessation, implying that GH has important metabolic functions in adult life. The metabolic actions of GH are either acute insulin-like or chronic insulin antagonism. The metabolic actions of GH may be direct or indirectly mediated by IGF-I.

FIGURE 3.15. Integrated model of the GH-IGFBP-IGF axis in the growth process. Three mechanisms are proposed: (1) GH stimulates IGF-I production; circulating IGF-I (endocrine IGF-I) acts at the growth plate; (2) GH regulates hepatic production of IGFBP-III and ALS: IGF-I binds to IGFBP-III and thereafter with ALS, forming the 150 kDa ternary complex; proteases cleave IGFBP-III into fragments that release IGF-I in the intravascular space and at the growth plate; and (3) GH induces differentiation local IGF-I production, and IGF-I acts via an autocrine and paracrine mechanism to stimulate cell division.From Spagnol and Rosenfeld [313a].

Chapter 3 Table 3.4.

Acute metabolic effects of growth hormone

Enhanced lipolysis and lipid oxidation ÆMobilization of stored triglyceride Protein synthesis stimulation Antagonism of insulin action Water and sodium retention

Acute, transient, insulin-like effects of GH can be demonstrated on carbohydrate, protein, and lipid metabolism (Table 3.4). The insulin-like effects are more easily demonstrated in tissues that have not recently been exposed to GH (for example, those derived from hypophysectomized animals, or GH-deficient humans) than in normal tissues [314]; casting doubt on the physiologic relevance of the insulin-like effects of GH, because GH is secreted endogenously in frequent bursts, resulting in significant changes in ambient GH levels. The chronic antiinsulin effects of GH result in decreased glucose utilization, increased lipolysis, and tissue refractoriness to the insulin-like effects of GH. Antiinsulin effects occur at both hepatic and peripheral sites [315]. Effects on Carbohydrate Metabolism

GH-deficient children have decreased fasting glucose levels [316,317], decreased insulin secretion [316], contradictory impairment of glucose tolerance [318–321], and increased insulin sensitivity due to increased glucose utilization and blunted hepatic glucose release. GH replacement increases fasting glucose levels [317,318], insulin levels [318], and hepatic glucose production [317]. Endogenous physiologic GH secretion antagonizes insulin action [322]. Normally, GH secretion increases 3 to 5 hours after oral glucose ingestion, resulting in decreased disposal of a second oral glucose challenge, associated with hyperinsulinemia occurring 2 hours after GH levels peak. Both intravenous and oral glucose tolerance tests are impaired if performed during periods of increased GH secretion, such as sleep onset. GH-deficient adults have elevated fasting insulin levels and a positive correlation between fasting plasma insulin and both fat mass and waist girth [323], suggesting insulin resistance, which is confirmed by hyperinsulinemic euglycemic clamp [324]. GH replacement initially further increases insulin resistance, in the first 1 to 6 weeks of therapy, but subsequently, although hyperinsulinemia persists, carbohydrate metabolism returns to baseline after 3 months of GH treatment [325]. Acromegaly is the experiment of nature for effects of excess GH secretion on carbohydrate metabolism. Several stages of impaired glucose tolerance occur in acromegaly, possibly indicative of a stepwise deterioration in carbohydrate metabolism as the disease progresses [326]. Glucose tolerance after both intravenous [326] and oral glucose administration is impaired in association with hyperinsu-

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linemia [327]. When acromegalic patients eventually manifest overt diabetes mellitus, postglucose insulin levels are depressed [327]. Effects on Protein Metabolism

Somatic growth is under the primary control of GH and, as such, GH is an anabolic hormone. GH causes urinary nitrogen retention and decreased plasma urea levels in GH deficient children [328], and in normal [329] and obese [330] adults. Furthermore, GH-deficient children increase their muscle mass following GH treatment [331]. Both insulin and IGF-I have been implicated in the anabolic effects of GH on protein metabolism, especially as insulin is a protein anabolic hormone and GH stimulates circulating insulin levels. However, GH-stimulated protein synthesis precedes the GH stimulated increase in insulin levels. Physiologically, GH may promote nitrogen retention during starvation, as GH levels rise during fasting.

Effects on Lipid Metabolism GH Effect on Adipose Tissue

GH plays an important role in fat metabolism. GHdeficient children are mildly obese [331,332], with a decreased total number of fat cells [333,334] that are larger in size, with a greater lipid content than normal. GH replacement therapy leads to a decrease in body fat and, eventually, a decrease in the size and lipid content of subcutaneous adipocytes. GH deficient adults have altered body composition, with increased fat mass and decreased lean body mass. Initial acute effects of GH on lipid metabolism are antilipolytic (insulin-like) and subsequently, GH exerts a lipolytic (antiinsulin) effect. Lipolysis

GH increases fat mobilization and decreases fat deposition, thereby decreasing body fat. GH activates hormonesensitive lipase [335], resulting in increased hydrolysis of triglycerides to free fatty acids and glycerol (lipolysis). GH also decreases fatty acid reesterification [336]. The lipolytic actions of GH can be counteracted by insulin and are additive with glucocorticoids [337], which decrease the antilipolytic effects of insulin [338]. In vivo Effects on Lipids and Lipoproteins

Physiologic doses of GH have multiple, inconsistently demonstrated effects on free fatty acids and ketones [339]. Lipolytic and ketogenic effects of GH are easily demonstrable in normal subjects with suppressed insulin levels [318] and in GH-deficient insulin-dependent diabetic patients infused with GH [340,341]. GH deficient adults have elevated total cholesterol, low density lipoprotein cholesterol (LDL) and apolipoprotein B (ApoB) [342], with low high density lipoprotein levels (HDL) and high triglyceride levels. This lipid profile is

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associated with premature atherosclerosis and cardiovascular disease. GH replacement treatment, decreases total cholesterol (342) LDL cholesterol and ApoB and increases HDL. Long term surveillance will be required to determine whether GH replacement therapy reverses premature atheroclerosis and reduces cardiovascular morbidity and mortality in GH-deficient adults.

the abdomen [323]. The reduction in abdominal fat mass is mainly due to reduced visceral fat mass [343,344]. GH therapy increases total body water [343] especially extracellular water within 3 to 5 days [345,351]. Total blood volume increases after 3 months of treatment [346]. A direct action of GH and/or IGF-I on renal tubular sodium reabsorption, as well as activation of the renin angiotensin system [345,352], contributes to the antinatiuretic GH effects.

GH Effects on Body Composition

The anabolic, lipolytic and antinatiuretic actions of GH impact body composition affecting fat mass, lean body mass and fluid volume in GH-deficient adults. Lean body mass (LBM) is reduced [323,343], and fat mass is increased in GH-deficient adults compared to predicted values for age, sex, and height matched normal controls in GH-deficient adults [323]. Excess fat accumulates mostly in the visceral component in a central distribution, mainly abdominal [343,344]. Total body water is reduced in adults with GH deficiency. Reduced plasma volume [345] and total blood volume [346], contribute to the reduced extracellular water [347,345]. GH replacement therapy reverses these effects on body composition by increasing LBM (Fig. 3.16) [343,344, 348–350]. There is a significant increase in skeletal muscle, the most important component of LBM [348]. GH replacement also reduces fat mass by 4–6 kg in GH deficient adults [343,344,348–350] with the most significant reduction in

GH Effects on Bone Metabolism and Bone Mineral Density (BMD)

GH-deficient adults have reduced bone mass, [353] measured by bone mineral density (BMD), resulting in a 2 to 3 times increased fracture rate [354]. The pathogenesis of the bone loss is unclear, with studies demonstrating unchanged [355], increased, [356] or reduced [357] bone formation as well as unchanged bone resorption [358] and delayed mineralization [359]. With GH replacement therapy there is predominant bone resorption in the first 3 to 6 months, resulting in initial reduced BMD after 6 to 12 months [360] with subsequent increased bone formation leading to a net gain in bone mass after 18 to 24 months of treatment [361]. GH Effects on Muscle Strength and Exercise Performance

GH deficiency is associated with reduced muscle strength, due to altered body composition [362]. Reduction in muscle cross-sectional area, as well as lack of conditioning and training, may contribute to the weakness. Prolonged GH replacement therapy for at least 12 months is required to significantly increase muscle strength [362] and for at least 3 years to normalize the muscle strength [363]. GH Effects on Cardiovascular Function

FIGURE 3.16. Effect of GH replacement on (a) lean body mass (LBM) and (b) fat mass in 24 adults with GHD. Reproduced with permission from Salomon et al. [347a].

Hypopituitary adults have increased vascular mortality [364] mostly attributable to cerebrovascular and cardiovascular disease [365]. GH deficiency has been implicated [366] based on the hypothesis that adult GH deficiency predisposes to the development of premature atherosclerosis, as evidenced by increased carotid artery intima-medial thickening and plaque formation [367], increased triglycerides, plasminogen activator inhibitor-1 activity and fibrinogen [368]. The role of GH in regulating cardiac structure and function has not been established. In GH-sufficient adults with dilated cardiomyopathy and congestive heart failure, GH has been shown to improve cardiac function and increase ventricular mass [369] by some workers and to have no beneficial effect by others. GH deficient adults have reduced left ventricular mass and impaired systolic function. Short term GH treatment for 6 months improves the deficits in cardiac function and structure, with a sustained effect on cardiac performance for up to 3 years of GH treatment [370]. Effects of GH excess in acromegaly manifest as left ventricular hypertrophy, hypertension, cardiac failure and increased mortality due to cardiovascular causes [371]. The long term effects of GH

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replacement on cardiac structure and function are unknown. GH replacement should aim to restore somatic deficiency, including cardiac size and function. In view of the deleterious cardiac effects of excessive GH, supraphysiological GH doses should be avoided.

surements to be consistently more reproducible and sensitive than repeated pharmacologic stimulation tests [377]. These conflicting findings may be attributable to differences in control groups in the discrepant studies.

TESTS OF GH SECRETION

An alternative approach to be assessment of spontaneous GH secretion is the measurement of GH concentrations in urine [378]. As urinary GH concentrations are very low, assays require high affinity antibodies [379]. Several different immunoassay methods for urinary GH measurement have been developed [380], none of which have the ability to reflect pharmacological GH testing or to adequately discriminate between normal and abnormal GH secretion [381–383]. Clinical utility of urinary GH measurements in the diagnosis of GH deficiency will require attainment of adequate-age-and-sex-matched controls and a decision as to whether GH excretion should be expressed relative to body weight or creatinine excretion [384]. 24-hour urinary collections have been assayed for GH in an attempt to diagnose acromegaly [385,386]. Overnight urinary GH measurements were, however, not useful in differentiating acromegalic patients from normal controls [385]. However, others have found 24-hour urine collections for GH measurement to be useful in diagnosis of acromegaly [385].

Urinary GH Measurement

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

Measurement of Spontaneous GH Secretion Twenty-four-hour Integrated Concentration of GH

Pituitary GH secretion occurs episodically during waking hours, as well as during sleep, necessitating continuous measurement of integrated secretion over 24 hours [372] rather than over shorter periods. Constant blood collection over a 24-hour period allows the determination of a true mean or IC-GH. Measurement of the 24-hour IC-GH concentration ideally requires a nonthrombogenic continuous withdrawal pump [373] or patent indwelling catheter from subjects whose food intake and physical activity are not limited. Sampling intervals of 20 minutes are most widely used [373], but 5-minute and 30-second sampling frequencies detect significantly more pulses per hour [374]. Samples from collection periods may be pooled, producing a combined aliquot in which the ICGH concentration is measured. The 24-hour IC-GH reflects the average GH concentration over a 24-hour period, eliminating peak or trough levels that might otherwise be obtained by single random sampling of GH when the latter is released in a pulsatile manner. The discriminating power of continuous GH measurement in the diagnosis of GH deficiency has been disputed [375,376]. Measuring spontaneous GH secretion in prepubertal short children was found to be an insensitive test, with no clear diagnostic advantage over GH stimulation tests and considerable overlap between values obtained in normal, short children and children with GHD [375]. As young normal controls have IC-GH levels which overlap with those of organic hypopituitary patients [377] measurement of spontaneous GH secretion has limitations in the diagnosis of organic GH deficiency in adults. As measurement of spontaneous GH secretion has limitations in the diagnosis of adult GH deficiency, 12-hour overnight spontaneous GH secretion has been measured. Night-to-night variation in results occurred when the test was repeated on consecutive nights, as compared with GH stimulation tests which proved to be more reproducible [376]. However, others have found 24-hour IC–GH mea-

Evaluation of GH Hypersecretion Increased serum IGF-I levels are a consistent finding in acromegaly [387]. Integrated 24-hour serum GH levels are elevated and show a log (dose) response correlation with serum IGF-I levels [388]. The currently accepted diagnostic test of GH hypersecretion is failure of GH levels to be suppressed to less than 1 ng/mL within 2 hours following a 75 g oral glucose load using an IRMA (two site immunoradiometric assay) or chemiluminescent assay [389]. In normal subjects receiving oral glucose loading, serum GH levels initially fall and then subsequently increase as plasma glucose declines. However, in acromegalic patients, oral glucose fails to suppress GH to the normal range [390]. GH levels may increase in response to an oral glucose load in acromegaly (in approximately 28%) [391], remain unchanged (in approximately 36%), or fall (in approximately 36% of patients). As GH secretion is pulsatile, random GH measurements may be misleading.

Evaluation of GH Deficiency Random GH and 1GF-I Measurements

Random GH measurements are not diagnostic of GH deficiency, as GH secretion is pulsatile and daytime levels are often low in normal subjects. Low IGF-I levels are suggestive of GH deficiency, but are also found in malnutrition [392], acute illness, celiac disease, poorly controlled diabetes mellitus, liver disease, and estrogen ingestion [393]. Fifteen

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percent of children diagnosed to be GH deficient by stimulation tests may have normal IGF-I levels [394]. IGF-I levels are normally very low before 3 years of age and highest in adolescence. Normal and GH-deficient children may have IGF-I levels which overlap with those observed in infancy [395]. Furthermore, both normal and low IGFI levels are present in children with growth delay and genetic short stature [396]. IGF-I levels do not always correlate with GH levels after provocative GH stimulation [397]. Low IGFBP-III levels are also encountered in patients with GH deficiency [398]. The diagnosis of adult GHD is more reliably established by provocative testing of GH secretion, as a single ageadjusted IGF-I level has limited diagnostic value [377]. Whereas, in acromegaly there is an excellent correlation between IC-GH and IGF-I [388], in adult GH deficiency, age-adjusted IGF-I should be regarded as a simple, low-cost screening test, because of the limitations outlined above. IGFBP-III is the major serum carrier protein for the IGF peptides, and is GH-dependent [282]. IGFBP-III assays have several advantages over IGF-I assays in that (i) they are technically simple, not requiring separation of IGF peptides from BPs; (ii) circulate in the plasma in high concentrations, so that assay sensitivity is not compromised; (iii) IGFBP-III plasma concentrations are less influenced by nutritional factors than IGF-I levels; (iv) plasma levels vary minimally with age, so that identification of abnormal concentrations in infancy or puberty is clear-cut [384,399]. IGFBP-III is a sensitive test for diagnosis of severe childhood GHD [518], with superior reproducibility to GH stimulation tests [400]. Provocative Tests

Dynamic testing of GH reserve involves stimulation of the somatotrophs to secrete GH in response to a pharmacologic stimulus. Several GH stimulatory agents have been utilized, including insulin, clonidine, arginine, L-dopa, GHRH, and propranolol. Insulin-induced Hypoglycemia (Insulin Tolerance Test, ITT)

This reliable stimulus for GH secretion is the gold standard provocative test [401]. The ITT is the best validated GH stimulation test and is recommended by the Growth Hormone Research Society as the test of choice [401]. Regular insulin 0.1 IU/kg is administered intravenously to decrease the basal glucose level by 50% to a value below 40 mg/dI. Maximal GH secretion peaks at 30 to 60 minutes. Patients may experience symptoms of hypoglycemia, including light headedness, anxiety, tremulousness, sweating, tachycardia seizures and rarely, unconsciousness. Insulin induced hypoglycemia is contraindicated in patients with a history of seizure disorder, coronary artery disease, or over the age of 55 years. The test should be performed under close supervision, and intravenous glucose (50%) should be readily at hand for rapid administration. The risk of inducing profound hypoglycemia is greater in GH-deficient patients because of their increased sensitivity to insulin.

Clonidine

This alpha-adrenergic agonist stimulates GH release via a central action [402]. Clonidine (0.15 mg/m2) is administered orally, with a maximum GH secretory peak occurring after 60 to 90 minutes. Patients may experience some drowsiness, with a decrease in systolic blood pressure in sodium depleted GH deficient adults at doses required to release GH (0.25–0.30 mg orally.) Clonidine, frequently used as a stimulus for GH release in children, is not reliable in adults [403]. L-dopa/propranolol

l-dopa, the immediate metabolic precursor of dopamine, stimulates GH release by stimulating hypothalamic dopaminergic receptors. Adrenergic blockade (propranolol) enhances the GH response to l-dopa [404]. l-dopa is administered orally according to the patient’s weight (125 mg if weighing 30 kg) together with propranolol 0.75 mg/kg (maximum dose 40 mg) after an overnight fast. Maximum GH secretion occurs after 60 to 90 minutes. l-dopa is very effective in stimulating GH release and rarely results in adverse effects [255]. Arginine/GHRH

Recently it has been shown that arginine potentiates maximal somatotroph responsiveness to GHRH [405]. After an overnight fast, GHRH 1 ug/kg is administered as an intravenous bolus at 0 minutes with Arginine 30 g in 100 ml infused from 0 to 30 minutes, with subsequent blood sampling for GH performed every 15 minutes for 90 minutes. Combined Arginine/GHRH responses are ageindependent and is a highly reproducible GH provocative test [405], at least as sensitive as insulin induced hypoglycemia [406]. GHRH/GHRP-6

GHRH 1 ug/kg plus GHRP-6 1 ug/kg is given intravenously at 0 minutes and blood is drawn for GH sampling at 0 and 120 minutes [407]. GH releasing peptide-6 (GHRP-6) is an artificial hexapeptide [203] that activates a hypothalmic and pituitary receptor whose natural ligand Ghrelin was recently characterized [202]. Combined administration of GHRP-6 and GHRH is the most potent stimulus to GH release, with excellent reproducibility and no side effects [203]. GHRH/GHRP-6 is a good alternative to the ITT in patients with organic pituitary disease, however there is some overlap between GH levels attained in the control group and severely GH-deficient patients. Since GHRH and GHRP act directly on the pituitary, it is possible that their administration restored GH secretion in patients who had a deficiency of these secretagogues because of hypothalamic disease [408]. Pitfalls of Provocative Testing

Provocative testing has several limitations. The diagnosis of childhood onset GH deficiency is determined by an inad-

Chapter 3

equate GH response to two distinct provocative tests. The definition of an adequate response is arbitrary. The availability of recombinant human GH in the mid-1980s resulted in a loosening of the diagnostic cut-off from 5–7 ng/ml to 10 ng/ml. Sequential tests on the same day have been attempted [409], but discordant individual results are obtained in approximately 30% of subjects. By altering the temporal relationship between the administration of provocative agents, the amount of GH released can be decreased or increased [410–412]. A combination of two provocative agents administered simultaneously has been tried, but this approach failed to identify some patients who demonstrated GH deficiency following separate testing with each agent individually [413]. The reproducibility of provocative tests has been questioned, as only a moderate correlation was found between pharmacologic tests carried out on two separate occasions, 6 weeks apart [414]. Many physical states alter stimulated GH levels. Increased GH response to various provocative stimuli during puberty [415] have been attributed to increased estrogens and androgens [416]. Sex steroid priming with either estrogens or testosterone 3 to 5 days prior to provocative testing in prepubertal children has therefore been recommended by some. Mildly impaired GH release in response to provocative stimuli in hypothyroid patients is a frequent finding [417]. Patients should thus be euthyroid at the time of provacative GH testing. Furthermore, 67% of patients with “idiopathic” GHD diagnosed in childhood normalize their GH secretion, when reevaluated after completion of treatment [418].

Variability of GH Assays The comparisons of results of various GH assays obtained in different laboratories is difficult because of differences between almost all aspects of the immunoassays. Plasma GH is usually measured by radioimmunoassay (polyclonal or monoclonal; RIA) or by immunoradiometric assay (dual monoclonal; IRMA). Comparative GH measurements are obtained using 11 commercial immunoassays varied by a factor of three [419]. As previously mentioned, there are several different circulating forms of GH, most of which are not measured in GH assays because reagents to detect all these forms do not exist. Because monomeric 22 k is the only GH form available as a standard in sufficient purity and quantity, and because monomeric 22 k is also the most abundant circulating form, it is used as the basis for GH measurement: however it accounts for only about 25% of circulating immunoreactivity [420]. The other GH forms are recognized to varying and largely unknown degrees. Thus an assay result is an averaged look at a variety of GH forms, and different antibodies or assay designs yield different answers. Polyclonal antibodies were used in the early RIAs, inducing higher estimates of GH, because they recognized several molecular forms of GH compared to newer immunometric

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assays employing highly specific monoclonal antibodies [421,422]. GH standards also affect comparison of GH values in different laboratories. In 1994, the first international standard for somatotropin, IRP 88/624 [423], was prepared by the WHO, using recombinant technology in contrast to the previous standards prepared from pituitary extracts. Use of an international standard enables uniformity of calibration between different GH kits. GH-binding proteins (GHBPs) may influence GH estimates by interfering in some of the GH assays. In the circulation, approximately 50% of GH is complexed to GHBP, which corresponds to the extracellular domain of the GH receptor. Both high affinity and low-affinity BPs are present in the serum. As the low-affinity GHBP has a greater affinity for the 20-KD GH molecule, it presumably does not interfere in GH estimates in the new GH assays specific for the 22-KD GH molecule. The influence of GHBP in noncompetitive immunometric assays could lead to too low estimates of GH if the GHBP competes with one or both antibodies. In competitive assays like the RIAs, the error in the GH estimates will depend on whether the GHBPbound 125 I-GH is in the antibody-bound or in the free fraction. In RIAs using polyethylene glycol or double antibody for separation, the GHBP-complexed GH will end up in the free fraction if the antibodies are directed against epitopes in the GHBP-binding area and thus give spuriously high values [424]. Another factor contributing to the heterogeneity of results from GH immunoassays is the antibody used. As different antibodies employed in immunoassays bind to a different spectrum of GH isoforms, GH concentrations measured by immunoassay are likely to depend on the particular antibody used. Furthermore, as the distribution of the different GH isoforms varies between individuals, results from different GH immunoassays cannot be compared. The heterogeneity of GH immunoassay results poses a major problem in the definition of standards for the diagnosis of GHD. Consensus statements including cut-off recommendations refer to the polyclonal-antibody-based RIA. However RIA is now used infrequently, therefore clinicians should be aware of the GH assay used by their laboratory and how it compares to polyconal RIA. Recently, three new GH assays have been developed, two of which, ESTA and IFA, measure GH bioactivity rather than immunoactivity as measured by convential RIA. A third, GHEA, measures the different GH isoforms in the circulation [425]. ESTA or IFA bioassays could replace RIA if they become commercially available, as they more closely reflect biologically active GH than that measured by RIA. CLINICAL USE OF GH

Recombinant hGH (rhGH) The application of recombinant DNA technology has made available a sufficient quantity of hGH for treatment of GH

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deficiency [426]. Early trials investigating the effects of recombinant human GH compared to pituitary-derived hGH in adults showed comparable kinetics in the two preparations, as well as similar ability to stimulate IGF-I levels. Both compounds resulted in a similar decrease in serum blood-urea nitrogen (BUN) and cholesterol, and increased serum triglyceride levels [427] and clinical growth-promoting effects.

GH Therapy in Childhood Classic GH Deficiency

Childhood GH deficiency ranges from complete absence of GH associated with severe growth retardation to partial GH deficiency resulting in short stature. Diagnosis is based on decreased height (more than 2.5 standard deviations (SD) below the mean height for age-matched normal children), poor growth velocity (less than the twenty-fifth percentile), delayed bone age and a predicted adult height below mean parental height [428]. GH deficiency (GHD) is usually confirmed by inadequate pituitary GH responses to standard provocative stimuli (peak serum GH value of less then 10 mg/L) [429], with values less than 5 mg/L in severe GH deficiency. Combined clinical evaluation and provocative testing are used in the assessment of childhood GH deficiency. Concomitant endocrine deficiencies, especially hypothyroidism, should be corrected to maximize the growth-promoting benefits of hGH. GH replacement should be started as early as possible before height drops below the third percentile, as total height gain is inversely proportional to the pretreatment chronologic and bone age [430], as well as severity of GH deficiency. The most pronounced acceleration in linear growth rate occurs during the first two years of treatment [431]. Dose and frequency of administration of hGH both influence height velocity [432]. Currently, the recommended weekly dose is 0.18 mg/kg (0.54 iu/kg) divided into six or seven daily subcutaneous injections or as three injections/ weekly administered on alternate days. The maximal replacement weekly dosage is 0.3 mg/kg (0.9 iu/kg) divided into equal doses given on three alternate days. Idiopathic Short Stature

Idiopathic short stature describes otherwise normal children who are at or below the fifth percentile for height, with normal growth hormone responses to provocative stimuli. In the past, scarcity of human pituitary GH limited its therapeutic uses to children with severe GH deficiency diagnosed by a decreased growth rate and inadequate GH response to two provocative stimuli. As short stature may affect psychological development, trials of GH in “slow” growing children have been initiated to determine whether GH treatment can increase final adult height in normal children without complications of treatment. GH treatment in these children is controversial. Long-term studies in short

children treated for up to ten years have shown conflicting results, some showing no significant difference in the SD score for final height [433] while others report final heights exceeding the predicted height [434]. Approximately onethird of all children on GH treatment in the USA are nonGH deficient with idiopathic short stature [435]. In view of the contradictory data on the benefical effect of GH therapy on final height, non-GH-deficient children and their families should have realistic expectations. In children with no identifiable cause of growth failure GH therapy is indicated if growth is retarded more than 2.5 SD below the mean for age and very slow growth velocity (less than the twenty-fifth percentile).

GH Neurosecretory Dysfunction (GHND) A subgroup of GH-deficient children has been described who have normal responses to GH provocative tests, low serum IGF-I levels and abnormal 24-hour GH secretory patterns [436]. This syndrome, termed “growth hormone neurosecretory dysfunction,” is characterized by height below the first percentile, bone age delayed by 2 or more years, and a growth velocity of 4 cm/year or less, in combination with the serum abnormalities described above. Classical GH deficiency, GHND, and normal children can be distinguished by assessing the number of GH pulses during a 24-hour period, mean peak GH pulse amplitude, highest nocturnal peak, and mean 24-hour GH concentration. Patients with GHND may secrete less GH than patients with classical GH deficiency, and the nocturnal GH surge is maintained but with a lower magnitude [436]. GHND has also been associated with Turner’s and Noonan’s syndrome, empty sella syndrome, and precocious puberty [437]. hGH was administered intramuscularly three times weekly for at least 6 months to children with GHND [436]. This treatment resulted in a doubling of growth velocity, comparable to that seen in children with GH deficiency receiving GH. Turner’s Syndrome

Patients with Turner’s syndrome manifest dysmorphic body features, ovarian failure, and reduced growth rate, starting during intrauterine life and continuing through childhood and puberty, resulting in reduced final adult height [438]. Earlier studies evaluating the use of hGH in Turner’s syndrome were inconclusive, possibly due to inadequate dosage regimens and short-term follow-up [439]. Recently, GH therapy in girls with Turner’s syndrome has been shown to significantly increase predicted height, with a greater increase in height in girls treated with combined GH and oxandrolone (an anabolic steroid) [440]. Turner’s syndrome is an approved FDA indication for GH use. Low Birth Weight

Children with low birth weight generally have reduced adult height [441]. An early study investigating the use of

Chapter 3

hGH in the treatment of short children with low birth weight yielded inconclusive results, probably due to the inadequate dosage regimen. Subsequent studies using higher, more frequent hGH dosages [442,443] have shown an increased growth rate for these children, but no increase in final height [444]. Chronic Diseases

hGH has been used in an attempt to increase height in children with short stature associated with a variety of chronic nonendocrine disorders. Growth retardation may be due to specific effects of the disease of may be secondary to poor nutrition or infection. Increased height velocity has been demonstrated after hGH treatment in prepubertal children and infants with chronic renal failure and following renal transplantation [445,446]. Chronic renal insufficiency prior

Table 3.5. deficiency

Causes of adult growth hormone (GH)

Presenting in childhood Congenital Idiopathic Genetic GHRH receptor defect Transcription factor defect Embryologic defects GH resistance Laron dwarfism Pygmy Neurosecretory defects Radiation for juvenile brain tumors, leukemia Head trauma Perinatal birth injury Child abuse Accidental Inflammatory diseases Viral encephalitis Meningitis, bacterial, fungal, tuberculosis Acquired in adulthood Postpituitary surgery Pituitary mass-adenoma, craniophyringioma Hematologic disorders Hemochromatosis Sickle cell disease Thalassemia Autoimmunity Lymphocytic hypophysitis Irradiation: central nervous system radiation for tumors Histiocytosis Granulomas Sarcoidosis Idiopathic Infection Tuberculosis Syphilis Vascular

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to transplantation is an FDA approved indication for GH therapy. Achondroplasia and Hypochondroplasia

Achondroplasia and hypochondroplasia result in short stature, with normal GH responses to provocative tests. GH therapy increases growth in both conditions, however there is worsening of body disproportion [447]. GH Treatment in Adults

As part of the normal aging process, adults experience loss of lean body mass, atrophy of skeletal muscle and organs, and increased adipose tissue [448]. Skeletal muscle atrophy is associated with a loss of muscle strength. These changes have been attributed to GH deficiency, as GH and IGF-I levels decline with age in healthy adults. With the increased availability of hGH, studies have been performed to evaluate the effects of rhGH on body composition in normal elderly individuals [449–451]. These studies have reported an increase in lean body mass, decreased adipose tissue content and increased retention of sodium, calcium, and nitrogen. None of these parameters translate into a measurable improvement in muscle size or strength. Adults may be truly GH-deficient due to isolated GH deficiency or to panhypopituitarism, from a number of causes (Table 3.5). GH-deficient adults have altered body composition with increased fat and decreased muscle volume and strength, lower psychosocial achievement, altered glucose and lipid metabolism, decreased bone mineral density and possibly increased mortality due to cardiovascular disease (Table 3.6). The same pharmacologic stimulating tests, used in children, have been used in adults. However, the definition of an adequate response differs. IGF-I, IGFBP-III, cholesterol and LDL levels can also be determined (Table 3.7). Mean 24-hour GH secretion sampled every 20 minutes, insulin-induced hypoglycemia and IGF-I levels have been compared to normals in the diagnosis of adult GHD [452]. IGF-I is a poor indicator of adult GHD as levels may overlap with normal. The diagnosis of adult GH deficiency is confirmed by provocative testing of GH secretion. Other hormonal deficits should be adequately replaced prior to GH provocative testing. The

Table 3.6. Physical findings in the adult growth hormone deficiency syndrome. Adapted from Cuneo et al. [451a] Truncal adiposity Waist/hip ratio increased Thin, dry, cool skin Exercise performance reduced Muscle strength reduced Reduced bone density Mood depressed Psychosocial impairment

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Table 3.7. Diagnosis of adult GH deficiency. Adapted from Cuneo et al. [451a] Stimulated GH 9% Leu) is associated with onset and transmission of autosomal dominant neurohypophyseal diabetes insipidus. J Clin Endocrinol Metab 1994;79:421–427. 49 Yuasa H, Ito M, Nagasaki H et al. Glu-47, which forms a salt bridge between neurophysin-II and arginine vasopressin, is deleted in patients with familial central diabetes insipidus. J Clin Endocrinol Metab 1993;77:600–604. 50 Ito M, Mori Y, Oiso Y, Saito H. A single base substitution in the coding region for neurophysin II associated with familial central diabetes insipidus. J Clin Invest 1991;87:725–728. 51 Nagasaki H, Ito M, Yuasa H et al. Two novel mutations in the coding region for neurophysin-II associated with familial central diabetes insipidus. J Clin Endocrinol Metab 1995;80:1352–1356.

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Chapter 7 215 Swift RG, Sadler DB, Swift M. Psychiatric findings in Wolfram syndrome homozygotes. Lancet 1990;336:667–669. 216 Inoue H, Tanizawa Y, Wasson J et al. A gene encoding a transmembrane protein is mutated in patients with diabetes mellitus and optic atrophy (Wolfram syndrome). Nature Genet 1998;20:143–148. 217 Strom TM, Hortnagel K, Hofmann S et al. Diabetes insipidus, diabetes mellitus, optic atrophy and deafness (DIDMOAD) caused by mutations in a novel gene (wolframin) coding for a predicted transmembrane protein. Hum Mol Genet 1998;7:2021–2028. 218 Hardy C, Khanim F, Torres R et al. Clinical and molecular genetic analysis of 19 Wolfram syndrome kindreds demonstrating a wide spectrum of mutations in WFS1. Am J Hum Genet 1999;65:1279–1290. 219 Howard RL, Bichet DG, Schrier RW. Hypernatremic and polyuric states. In: Seldin DW, Giebisch G, eds. The kidney: physiology and pathophysiology 2nd ed., New York: Raven Press, 1992:1753–1778. 220 Seibold A, Brabet P. Rosenthal W et al. Structure and chromosomal localization of the human antidiuretic hormone receptor gene. Am J Hum Genet 1992;51:1078–1083. 221 Tamarappoo BK, Verkman AS, Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones. J Clin Invest 1998;101:2257–2267. 222 Morello JP, Salahpour A, Laperrière A et al. Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. J Clin Invest 2000;105:887–895. 223 Oksche A, Rosenthal W. The molecular basis of nephrogenic diabetes insipidus. J Mol Med 1998;76:326–337. 224 Nomura Y, Onigata K, Nagashima T et al. Detection of skewed X-inactivation in two female carriers of vasopressin type 2 receptor gene mutation. J Clin Endocrinol Metab 1997;82:3434–3437. 225 Crawford JD, Bode HH. Disorders of the posterior pituitary in children. In: Gardner LI, eds. Endocrine and genetic diseases of childhood and adolescence 2nd ed., Philadelphia: WB Saunders, 1975:126–158. 226 Forssman H. On the mode of hereditary transmission in diabetes insipidus. Nordisk Medicine 1942:16:3211–3213. 227 Waring AG, Kajdi L, Tappan V. Congenital defect of water metabolism. Am J Dis Child 1945;69:323–325. 228 Williams RM, Henry C. Nephrogenic diabetes insipidus transmitted by females and appearing during infancy in males. Ann Int Med 1947;27: 84–95. 229 Niaudet P, Dechaux M, Trivin C et al. Nephrogenic diabetes insipidus: clinical and pathophysiological aspects. Adv Nephrol Necker Hosp 1984;13:247–260. 230 McIlraith CH. Notes on some cases of diabetes insipidus with marked family and hereditary tendencies. Lancet 1892;2:767–768. 231 Reeves WB, Andreoli TE. Nephrogenic diabetes insipidus. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disease 7th ed., New York: McGraw-Hill, 1995:3045–3071. 232 Streitz JMJ, Streitz JM. Polyuric urinary tract dilatation with renal damage. J Urol 1988;139:784–785. 233 Boyd SD, Raz S, Ehrlich RM. Diabetes insipidus and nonobstructive dilatation of urinary tract. Urology 1980;16:266–269. 234 Gautier B, Thieblot P, Steg A. Mégauretère, Mégavessie et diabète insipide familial. Sem Hop 1981;57:60–61. 235 Arthus M-F, Lonergan M, Crumley MJ et al. Report of 33 novel AVPR2 mutations and analysis of 117 families with X-linked nephrogenic diabetes insipidus. J Am Soc Nephrol 2000;11:1044–1054. 236 Bode HH, Crawford JD. Nephrogenic diabetes insipidus in North America: the Hopewell hypothesis. N Engl J Med 1969;280:750–754. 237 Bichet DG, Hendy GN, Lonergan M et al. X-linked nephrogenic diabetes insipidus: from the ship Hopewell to restriction fragment length polymorphism studies. Am J Hum Genet 1992;51:1089–1102. 238 Bichet DG, Arthus M-F, Lonergan M et al. X-linked nephrogenic diabetes insipidus mutations in North America and the Hopewell hypothesis. J Clin Invest 1993;92:1262–1268. 239 Holtzman EJ, Kolakowski LF, O’Brien D et al. A null mutation in the vasopressin V2 receptor gene (AVPR2) associated with nephrogenic diabetes insipidus in the Hopewell kindred. Hum Mol Genet 1993;2:1201–1204. 240 Bichet DG, Birnbaumer M, Lonergan M et al. Nature and recurrence of AVPR2 mutations in X-linked nephrogenic diabetes insipidus. Am J Hum Genet 1994;55:278–286. 241 Fujiwara TM, Morgan K, Bichet DG. Molecular analysis of X-linked nephrogenic diabetes insipidus. Eur J Endocrinol 1996;134:675–677. 242 Hobbs HH, Russell DW, Brown MS, Goldstein JL. The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet 1990;24:133–170.

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243 Schöneberg T, Yun J, Wenkert D, Wess J. Functional rescue of mutant V2 vasopressin receptors causing nephrogenic diabetes insipidus by a coexpressed receptor polypeptide. EMBO J 1996;15:1283–1291. 244 Schöneberg T, Sandig V, Wess J et al. Reconstitution of mutant V2 vasopressin receptors by adenovirus-mediated gene transfer. J Clin Invest 1997;100: 1547–1556. 245 Oksche A, Schulein R, Rutz C et al. Vasopressin V2 receptor mutants that cause X-linked nephrogenic diabetes insipidus: analysis of expression, processing, and function. Mol Pharmacol 1996;50:820–828. 246 Tsukaguchi H, Matsubara H, Taketani S et al. Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked nephrogenic diabetes insipidus. J Clin Invest 1995;96: 2043–2050. 247 Wenkert D, Schoneberg T, Merendino JJ Jr et al. Functional characterization of five V2 vasopressin receptor gene mutations. Mol Cell Endocrinol 1996;124: 43–50. 248 Schulz A, Grosse R, Schultz G et al. Structural implication for receptor oligomerization from functional reconstitution studies of mutant V2 vasopressin receptors. J Biol Chem 2000;275:2381–2389. 249 Vargas-Poussou R, Forestier L, Dautzenberg MD et al. Mutations in the vasopressin V2 receptor and aquaporin-2 genes in 12 families with congenital nephrogenic diabetes insipidus. J Am Soc Nephrol 1997;8:1855–1862. 250 Sadeghi H, Robertson GL, Bichet DG et al. Biochemical basis of partial NDI phenotypes. Mol Endocrinol 1997;11:1806–1813. 251 Ala Y, Morin D, Mouillac B et al. Functional studies of twelve mutant V2 vasopressin receptors related to nephrogenic diabetes insipidus: molecular basis of a mild clinical phenotype. J Am Soc Nephrol 1998;9:1861–1872. 252 Knoers N, Monnens LA. A variant of nephrogenic diabetes insipidus: V2 receptor abnormality restricted to the kidney. Eur J Pediatr 1991;150:370–373. 253 Langley JM, Balfe JW, Selander T et al. Autosomal recessive inheritance of vasopressin-resistant diabetes insipidus. Am J Med Genet 1991;38:90–94. 254 Lonergan M, Birnbaumer M, Arthus M-F et al. Non-X-linked nephrogenic diabetes insipidus: phenotype and genotype features. J Am Soc Nephrol 1993;4:264A. 255 Bichet DG, Razi M, Lonergan M et al. Hemodynamic and coagulation responses to 1-desamino[8-D-arginine]vasopressin (dDAVP) infusion in patients with congenital nephrogenic diabetes insipidus. N Engl J Med 1988;318: 881–887. 256 Bichet DG, Razi M, Arthus M-F et al. Epinephrine and dDAVP administration in patients with congenital nephrogenic diabetes insipidus. Evidence for a pre-cyclic AMP V2 receptor defective mechanism. Kidney Int 1989;36:859–866. 257 Brenner B, Seligsohn U, Hochberg Z. Normal response of factor VIII and von Willebrand factor to 1-deamino-8D-arginine vasopressin in nephrogenic diabetes insipidus. J Clin Endocrinol Metab 1988;67:191–193. 258 Fushimi K, Uchida S, Hara Y et al. Cloning and expression of apical membrane water channel of rat kidney collecting tubule. Nature 1993;361: 549–552. 259 Uchida S, Sasaki S, Furukawa T et al. Molecular cloning of a chloride channel that is regulated by dehydration and expressed predominantly in kidney medulla [published erratum appears in J Biol Chem 1994 Jul 22;269(29): 19192]. J Biol Chem 1993;268:3821–3824. 260 Deen PMT, Verdijk MAJ, Knoers NVAM et al. Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. Science 1994;264:92–95. 261 Sasaki S, Fushimi K, Saito H et al. Cloning, characterization, and chromosomal mapping of human aquaporin of collecting duct. J Clin Invest 1994;93: 1250–1256. 262 van Lieburg AF, Verdijk MAJ, Knoers NVAM et al. Patients with autosomal nephrogenic diabetes insipidus homozygous for mutations in the aquaporin 2 water-channel gene. Am J Hum Genet 1994;55:648–652. 263 Hochberg Z, van Lieburg A, Even L et al. Autosomal recessive nephrogenic diabetes insipidus caused by an aquaporin-2 mutation. J Clin Endocrinol Metab 1997;82:686–689. 264 Mulders SB, Knoers NVAM, van Lieburg AF et al. New mutations in the AQP2 gene in nephrogenic diabetes insipidus resulting in functional but misrouted water channels. J Am Soc Nephrol 1997;8:242–248. 265 Canfield MC, Tamarappoo BK, Moses AM et al. Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response. Hum Mol Genet 1997;6:1865–1871. 266 Mulders SM, Bichet DG, Rijss JPL et al. An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the golgi complex. J Clin Invest 1998;102:57–66.

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267 Deen PMT, Croes H, van Aubel RAMH et al. Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing. J Clin Invest 1995;95:2291–2296. 268 van Os CH, Deen PM. Aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus. Proc Assoc Am Physicians 1998;110:395–400. 269 Kamsteeg EJ, Wormhoudt TA, Rijss JP et al. An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus. Embo J 1999;18: 2394 –2400. 270 Boton R, Gaviria M, Batlle DC. Prevalence, pathogenesis, and treatment of renal dysfunction associated with chronic lithium therapy. Am J Kidney Dis 1987;10:329–345. 271 Christensen S, Kusano E, Yusufi AN et al. Pathogenesis of nephrogenic diabetes insipidus due to chronic administration of lithium in rats. J Clin Invest 1985;75:1869–1879. 272 Cogan E, Svoboda M, Abramow M. Mechanisms of lithium-vasopressin interaction in rabbit cortical collecting tubule. Am J Physiol 1987;252: F1080–1087. 273 Goldberg H, Clayman P, Skorecki K. Mechanism of Li inhibition of vasopressin-sensitive adenylate cyclase in cultured renal epithelial cells. Am J Physiol 1988;255:F995–1002. 274 Marples D, Christensen S, Christensen EI et al. Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla. J Clin Invest 1995;95:1838–1845. 275 Marples D, Frokiaer J, Dorup J et al. Hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex. J Clin Invest 1996;97:1960–1968. 276 Frokiaer J, Marples D, Knepper MA et al. Bilateral ureteral obstruction downregulates expression of vasopressin-sensitive AQP-2 water channel in rat kidney. Am J Physiol 1996;270:F657–F668. 277 Sands JM, Flores FX, Kato A et al. Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats. Am J Physiol 1998;274:F978–985. 278 Kanno K, Sasaki S, Hirata Y et al. Urinary excretion of aquaporin-2 in patients with diabetes insipidus [see comments]. N Engl J Med 1985;332: 1540–1545. 279 Batlle DC, von Riotte AB, Gaviria M et al. Amelioration of polyuria by amiloride in patients receiving long-term lithium therapy. N Engl J Med 1985;312:408–414. 280 Barlow ED, de Wardener HE. Compulsive water drinking. Q J Med New Series 1959;28:235–258. 281 Moses AM, Scheinman SJ, Oppenheim A. Marked hypotonic polyuria resulting from nephrogenic diabetes insipidus with partial sensitivity to vasopressin. J Clin Endocrinol Metab 1984;59:1044–1049. 282 Amico JA. Diabetes insipidus and pregnancy. In: Czernichow P, Robinson AG, eds. Frontiers of hormone research, vol 13, Diabetes insipidus in man. Basel: Switzerland, Karger, 1985:266–277. 283 Barron WM, Cohen LH, Ulland LA et al. Transient vasopressin-resistant diabetes insipidus of pregnancy. N Engl J Med 1984;310:442–444. 284 Durr JA, Hoggard JG, Hunt JM, Schrier RW. Diabetes insipidus in pregnancy associated with abnormally high circulating vasopressinase activity. N Engl J Med 1987;316:1070–1074. 285 Iwasaki Y, Oiso Y, Kondo K et al. Aggravation of subclinical diabetes insipidus during pregnancy [see comments]. N Engl J Med 1991;324:522–526. 286 Robertson GL. Dipsogenic diabetes insipidus: a newly recognized syndrome caused by a selective defect in the osmoregulation of thirst. Trans Assoc Am Physicians 1987;100:241–249. 287 Leaf A. Neurogenic diabetes insipidus. Kidney Int 1979;15:572–580. 288 Robertson GL. Diagnosis of diabetes insipidus. In: Czernichow P, Robinson AG, eds. Frontiers of hormone research, vol 13, Diabetes insipidus in man. Basel: Karger, 1985:176–189. 289 Miller M, Dalakos T, Moses AM et al. Recognition of partial defects in antidiuretic hormone secretion. Ann Intern Med 1970;73:721–729. 290 Zerbe RL, Robertson GL. A comparison of plasma vasopressin measurements with a standard indirect test in the differential diagnosis of polyuria. N Engl J Med 1981;305:1539–1546. 291 Stern P, Valtin H. Verney was right, but . . . [editorial]. N Engl J Med 1981;305:1581–1582. 292 Maas AH, Siggaard-Andersen O, Weisberg HF, Zijlstra WG. Ion-selective electrodes for sodium and potassium: a new problem of what is measured and what should be reported. Clin Chem 1985;31:482–485. 293 Redetzki HM, Hughes JR, Redetzki JE. Differences between serum and plasma osmolalities and their relationship to lactic acid values. Proc Soc Exp Biol Med 1972;139:315–318.

294 Thompson CJ, Baylis PH. Thirst in diabetes insipidus: clinical relevance of quantitative assessment. Q J Med 1987;65:853–862. 295 Debelak L, Pollak R, Reckard C. Arginine vasopressin versus desmopressin for the treatment of diabetes insipidus in the brain dead organ donor. Transplant Proc 1990;22:351–352. 296 Knoers N, Monnens LA. Nephrogenic diabetes insipidus: clinical symptoms, pathogenesis, genetics and treatment. Pediatr Nephrol 1992;6:476–482. 297 Bichet DG, Oksche A, Rosenthal W. Congenital nephrogenic diabetes insipidus. J Am Soc Nephrol 1997;8:1951–1958. 298 Bichet DG, Fujiwara TM. Nephrogenic diabetes insipidus. In: Scriver CR, Beaudet AL, Sly WS et al., eds. The metabolic and molecular bases of inherited disease 8th ed., New York: McGraw-Hill, 2001. 299 Anderson RJ. Hospital-associated hyponatremia [clinical conference]. Kidney Int 1986;29:1237–1247. 300 Anderson RJ, Chung HM, Kluge R, Schrier RW. Hyponatremia: a prospective analysis of its epidemiology and the pathogenetic role of vasopressin. Ann Intern Med 1985;102:164–168. 301 Verbalis JG. Hyponatremia. In: Alberti KG, Besser GM, Bierich JR et al., eds. Baillière’s Clinical Endocrinology and Metabolism. vol 3, Water and Salt Homeostasis in Health and Disease. London: Baillière Tindall, 1989:499–530. 302 Berl T, Anderson RJ, McDonald KM, Schrier RW. Clinical disorders of water metabolism. Kidney Int 1976;10:117–132. 303 Bichet DG, Kluge R, Howard RL, Schrier RW. Hyponatremic states. In: Seldin DW, Giebisch G, eds. The kidney: physiology and pathophysiology 2nd ed., New York: Raven Press, 1992:1727–1751. 304 Berl T. Psychosis and water balance [editorial]. N Engl J Med 1988;318: 441–442. 305 Goldman MB, Luchins DJ, Robertson GL. Mechanisms of altered water metabolism in psychotic patients with polydipsia and hyponatremia. N Engl J Med 1988;318:397–403. 306 Verbalis JG. An experimental model of syndrome of inappropriate antidiuretic hormone secretion in the rat. Am J Physiol 1984;247:E540–E553. 307 Verbalis JG, Drutarosky MD. Adaptation to chronic hypoosmolality in rats. Kidney Int 1988;34:351–360. 308 Verbalis JG, Drutarosky MD, Ertel RJ, Vollmer RR. Adaptive responses to sustained volume expansion in hyponatraemic rats. J Endocrinol 1989;122: 421–431. 309 Verbalis JG. The syndrome of inappropriate antidiuretic hormone secretion and other hypoosmolar disorders. In: Schrier RW, Gottschalk CW, eds. Diseases of the kidney 6th ed., vol III. Boston: Little, Brown and Company, 1997: 2393–2427. 310 Schwartz WB, Bennett W, Curelop S, Bartter FC. A syndrome of renal sodium loss and hyponatremia probably resulting from inappropriate secretion of antidiuretic hormone. Am J Med 1957;23:529–542. 311 Berl T, Kumar S. Disorders of water metabolism. In: Johnson RJ, Feehally J, eds. Comprehensive Clinical Nephrology. London: Mosby, 2000:9.1–9.20. 312 Smitz S, Legros JJ, Franchimont P, le Maire M. Identification of vasopressinlike peptides in the plasma of a patient with the syndrome of inappropriate secretion of antidiuretic hormone and an oat cell carcinoma. Acta Endocrinol (Copenh.) 1988;119:567–574. 313 Verbalis JG. Tumoral hyponatremia [editorial]. Arch Intern Med 1986;146: 1686–1687. 314 Cullen MJ, Cusack DA, O’Briain DS et al. Neurosecretion of arginine vasopressin by an olfactory neuroblastoma causing reversible syndrome of antidiuresis. Am J Med 1986;81:911–916. 315 Osterman J, Calhoun A, Dunham M et al. Chronic syndrome of inappropriate antidiuretic hormone secretion and hypertension in a patient with olfactory neuroblastoma. Evidence of ectopic production of arginine vasopressin by the tumor. Arch Intern Med 1986;146:1731–1735. 316 Schwaab G, Micheau C, Le Guillou C et al. Olfactory esthesioneuroma: a report of 40 cases. Laryngoscope 1988;98:872–876. 317 Singh W, Ramage C, Best P, Angus B. Nasal neuroblastoma secreting vasopressin. A case report. Cancer 1980;45:961–966. 318 Srigley JR, Dayal VS, Gregor RT et al. Hyponatremia secondary to olfactory neuroblastoma. Arch Otolaryngol 1983;109:559–562. 319 Houde I, René de Cotret P, Zingg H, Bichet DG. Messenger RNA (mRNA) for arginine-vasopressin (AVP) in an olfactory neuroblastoma, a rare tumor associated with the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Kidney Int 1990;37:266A. 320 Nolph KD, Schrier RW. Sodium, potassium and water metabolism in the syndrome of inappropriate antidiuretic hormone secretion. Am J Med 1970;49:534–545. 321 Beck LH. Hypouricemia in the syndrome of inappropriate secretion of antidiuretic hormone. N Engl J Med 1979;301:528–530.

Chapter 7 322 Falardeau P, Proulx J, Nawar T et al. Clinical and biochemical profiles of inappropriate secretion of antidiuretic hormone. Proceedings of the Seventh International Congress of Nephrology 1978:D-32. 323 Decaux G, Genette F, Mockel J. Hypouremia in the syndrome of inappropriate secretion of antidiuretic hormone. Ann Intern Med 1980;93:716–717. 324 Cogan E, Debieve MF, Pepersack T, Abramow M. Natriuresis and atrial natriuretic factor secretion during inappropriate antidiuresis. Am J Med 1988;84:409–418. 325 Manoogian C, Pandian M, Ehrlich L et al. Plasma atrial natriuretic hormone levels in patients with the syndrome of inappropriate antidiuretic hormone secretion. J Clin Endocrinol Metab 1988;67:571–575. 326 Kamoi K, Ebe T, Kobayashi O et al. Atrial natriuretic peptide in patients with the syndrome of inappropriate antidiuretic hormone secretion and with diabetes insipidus. J Clin Endocrinol Metab 1990;70:1385–1390. 327 Dillingham MA, Anderson RJ. Inhibition of vasopressin action by atrial natriuretic factor. Science 1986;231:1572–1573. 328 Nonoguchi H, Sands JM, Knepper MA. Atrial natriuretic factor inhibits vasopressin-stimulated osmotic water permeability in rat inner medullary collecting duct. J Clin Invest 1988;82:1383–1390. 329 Zerbe R, Stropes L, Robertson G. Vasopressin function in the syndrome of inappropriate antidiuresis. Annu Rev Med 1980;31:315–327. 330 DeFronzo RA, Goldberg M, Agus ZS. Normal diluting capacity in hyponatremic patients. Reset osmostat or a variant of the syndrome of inappropriate antidiuretic hormone secretion. Ann Intern Med 1976;84: 538–542. 331 Michelis MF, Fusco RD, Bragdon RW, Davis BB. Reset of osmoreceptors in association with normovolemic hyponatremia. Am J Med Sci 1974;267: 267–273. 332 Penney MD, Murphy D, Walters G. Resetting of osmoreceptor response as cause of hyponatraemia in acute idiopathic polyneuritis. Br Med J 1979;2:1474–1476. 333 Skowsky WR, Kikuchi TA. The role of vasopressin in the impaired water excretion of myxedema. Am J Med 1978;64:613–621. 334 Arieff AI, Llach F, Massry SG. Neurological manifestations and morbidity of hyponatremia: correlation with brain water and electrolytes. Medicine (Baltimore) 1976;55:121–129. 335 Arieff AI. Hyponatremia associated with permanent brain damage. Adv Intern Med 1987;32:325–344. 336 Arieff AI. Osmotic failure: physiology and strategies for treatment. Hosp Pract (Off Ed) 1988;23:173–178, 183–174, 187–179 passim. 337 Sterns RH. Severe symptomatic hyponatremia: treatment and outcome: a study of 64 cases. Ann Intern Med 1987;107:656–664. 338 Berl T. Treating hyponatremia: damned if we do and damned if we don’t [clinical conference]. Kidney Int 1990;37:1006–1018. 339 Kleinschmidt-DeMasters BK, Norenberg MD. Rapid correction of hyponatremia causes demyelination: relation to central pontine myelinolysis. Science 1981;211:1068–1070. 340 Norenberg MD, Leslie KO, Robertson AS. Association between rise in serum sodium and central pontine myelinolysis. Ann Neurol 1982;11: 128–135. 341 Sterns RH. Unsafe at any speed? Am Kidney Fund Lett 1989;6:1–10. 342 Sterns RH, Riggs JE, Schochet SS Jr. Osmotic demyelination syndrome following correction of hyponatremia. N Engl J Med 1986;314: 1535–1542. 343 Antonarakis S. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat 1998;11:1–3. 344 Baylis PH. Vasopressin and its neurophysin. In: DeGroot LJ, Besser JM, Cahill GFJ et al., eds. Endocrinology. 2nd ed, vol 1. Philadelphia: WB Saunders, 1989:213–229. 345 Robertson GL. Diseases of the posterior pituitary. In: Felig D, Baxter JD, Broadus AE, Frohman LA, eds. Endocrinology and Metabolism, New York: McGraw-Hill, 1981:251–277. 346 Robertson GL. The physiopathology of ADH secretion. In: Tolis G, Labrie F, Martin JB et al., eds. Clinical neuroendocrinology: A pathophysiological approach. New York: Raven Press, 1979:247–260. 347 Richter D, Schmale H. Molecular aspects of the expression of the vasopressin gene. In: Czernichow P, Robinson AG, eds. Frontiers of hormone research, vol 13, Diabetes insipidus in man. Basel: S. Karger, 1985:37–41. 348 Zerbe RL, Robertson GL. Disorders of ADH. Med North America 1984;13: 1570–1574. 349 Vokes T, Robertson GL. Physiology of secretion of vasopressin. In: Czernichow P, Robinson AG, eds. Diabetes insipidus in man, Basel: S. Karger, 1985.

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350 Robertson GL. Regulation of vasopressin secretion. In: Seldin DW, Giebisch G, eds. The kidney: physiology and pathophysiology, New York: Raven Press, 1985:869–884. 351 Chini B, Mouillac B, Ala Y et al. Tyr115 is the key residue for determining agonist selectivity in the V1a vasopressin receptor. Embo J 1995;14: 2176–2182. 352 van Lieburg AF, Verdijk MAJ, Schoute F et al. Clinical phenotype of nephrogenic diabetes insipidus in females heterozygous for a vasopressin type 2 receptor mutation. Hum Genet 1995;96:70–78. 353 Jinnouchi H, Araki E, Miyamura N et al. Analysis of vasopressin receptor type II (V2R) gene in three Japanese pedigrees with congenital nephrogenic diabetes insipidus: identification of a family with complete deletion of the V2R gene. Eur J Endocrinol 1996;134:689–698. 354 Sharif M, Hanley M. Stepping up the pressure. Nature 1992;357: 279–280. 355 Cheong HI, Park HW, Ha IS et al. Six novel mutations in the vasopressin V2 receptor gene causing nephrogenic diabetes insipidus. Nephron 1997;75: 431–437. 356 Wildin RS, Antush MJ, Bennett RL et al. Heterogeneous AVPR2 gene mutations in congenital nephrogenic diabetes insipidus. Am J Hum Genet 1994;55:266–277. 357 Holtzman EJ, Kolakowski LFJ, Geifman-Holtzman O et al. Mutations in the vasopressin V2 receptor gene in two families with nephrogenic diabetes insipidus. J Am Soc Nephrol 1994;5:169–176. 358 Knoers NV, van den Ouweland AM, Verdijk M et al. Inheritance of mutations in the V2 receptor gene in thirteen families with nephrogenic diabetes insipidus. Kidney Int 1994;46:170–176. 359 Oksche A, Dickson J, Schülein R et al. Two novel mutations in the vasopressin V2 receptor gene in patients with congenital nephrogenic diabetes insipidus. Biophys Biochem Res Com 1994;205:552–557. 360 Yuasa H, Ito M, Oiso Y et al. Novel mutations in the V2 vasopressin receptor gene in two pedigrees with congenital nephrogenic diabetes insipidus. J Clin Endocrinol Metab 1994;79:361–365. 361 Holtzman EJ, Harris HWJ, Kolakowski LFJ et al. Brief report: a molecular defect in the vasopressin V2-receptor gene causing nephrogenic diabetes insipidus. N Engl J Med 1993;328:1534–1537. 362 Wenkert D, Merendino JJJ, Shenker A et al. Novel mutations in the V2 vasopressin receptor gene of patients with X-linked nephrogenic diabetes insipidus. Hum Mol Genet 1994;3:1429–1430. 363 Pan Y, Metzenberg A, Das S et al. Mutations in the V2 vasopressin receptor gene are associated with X-linked nephrogenic diabetes insipidus. Nat Genet 1992;2:103–106. 364 Faa V, Ventruto ML, Loche S et al. Mutations in the vasopressin V2-receptor gene in three families of Italian descent with nephrogenic diabetes insipidus. Hum Mol Genet 1994;3:1685–1686. 365 Rosenthal W, Seibold A, Antaramian A et al. Molecular identification of the gene responsible for congenital nephrogenic diabetes insipidus. Nature 1992;359:233–235. 366 Shoji Y, Takahashi T, Suzuki Y et al. Mutational analyses of AVPR2 gene in three Japanese families with X-linked nephrogenic diabetes insipidus: two recurrent mutations, R137H and delta V278, caused by the hypermutability at CpG dinucleotides. Hum Mutat 1998;Suppl 1:S278–S283. 367 Tsukaguchi H, Matsubara H, Aritaki S et al. Two novel mutations in the vasopressin V2 receptor gene in unrelated Japanese kindreds with nephrogenic diabetes insipidus. Biochem Biophys Res Commun 1993;197: 1000–1010. 368 van den Ouweland AM, Dreesen JC, Verdijk M et al. Mutations in the vasopressin type 2 receptor gene (AVPR2) associated with nephrogenic diabetes insipidus. Nat Genet 1992;2:99–102. 369 Tsukaguchi H, Matsubara H, Inada M. Expression studies of two vasopressin V2 receptor gene mutations, R202C and 804insG, in nephrogenic diabetes insipidus. Kidney Int 1995;48:554–562. 370 Yokoyama K, Yamauchi A, Izumi M et al. A low-affinity vasopressin V2receptor gene in a kindred with X-linked nephrogenic diabetes insipidus. J Am Soc Nephrol 1996;7:410–414. 371 Merendino JJJ, Speigel AM, Crawford JD et al. Brief report: a mutation in the vasopressin V2-receptor gene in a kindred with X-linked nephrogenic diabetes insipidus. N Engl J Med 1993;328:1538–1541. 372 Tajima T, Nakae J, Takekoshi Y et al. Three novel AVPR2 mutations in three Japanese families with X-linked nephrogenic diabetes insipidus. Pediatr Res 1996;39:522–526. 373 Pan Y, Wilson P, Gitschier J. The effect of eight V2 vasopressin receptor mutations on stimulation of adenylyl cyclase and binding to vasopressin. J Biol Chem 1994;269:31933–31937.

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374 Friedman E, Bale AE, Carson E et al. Nephrogenic diabetes insipidus: an X chromosome-linked dominant inheritance pattern with a vasopressin type 2 receptor gene that is structurally normal. Proc Natl Acad Sci USA 1994;91: 8457–8461. 375 Oksche A, Moller A, Dickson J et al. Two novel mutations in the aquaporin-2 and the vasopressin V2 receptor genes in patients with congenital nephrogenic diabetes insipidus. Hum Genet 1996;98:587–589. 376 Fushimi K, Sasaki S, Marumo F. Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel. J Biol Chem 1997;272:14800–14804. 377 Katsura T, Gustafson C, Ausiello D, Brown D. Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells. Am J Physiol 1997;272:F817–F822. 378 Cheng A, van Hoek AN, Yeager M et al. Three-dimensional organization of a human water channel. Nature 1997;387:627–630.

379 Burbach JP, Luckman SM, Murphy D, Gainer H. Gene regulation in the magnocellular hypothalamo-neurohypophysial system. Physiol Rev 2001;81: 1179–1267. 380 Abbes AP, Bruggerman B, van Den Akker EL. Identification of two distinct mutations at the same nucleotide position, concomitantly with a novel (polymorphism in the vasopressin-neurophysin II gene (AVP-NP II) in two dutch families with familiarl neurohypophyseal (diabetes insipidus Clin Chem 2000;46:1699–1702. 381 Maghnie M, Cosi G, Genovese E et al. Central diabetes insipidus in children and young adults. N Engl J Med 2000;343:998–1007. 382 Kuwahara M, Iwai K, Ooeda T et al. Three families with autosomal dominant nephrogenic diabetes insipidus cansed by aquaporin-2 mutations in the C-terminus. Am J Hum Genet 2001;69:738–748.

S e c t i o n 2

Hypothalamic–Pituitary Dysfunction

C h a p t e r

8 The Hypothalamus Glenn D. Braunstein

ANATOMY The hypothalamus is one of the major portions of the diencephalon, and is situated at the base of the brain below the thalamus and above the pituitary (Figs 8.1 and 8.2). The anterior margin of the optic chiasm forms the anterior boundary of the hypothalamus, while the posterior margins of the mamillary bodies delineate the posterior boundary. The lateral borders are less well defined and vary at different levels. They are composed of the optic tracts, internal capsule, pes pedunculi, globus pallidus, and ansa lenticularis [1]. Between the chiasm and the mamillary bodies on the ventral surface is the tuber cinereum from which the pituitary stalk arises. The third ventricle lies in the center of the hypothalamus, and is connected to the lateral ventricles through the foramen of Monro, and to the fourth ventricle by the aqueduct of Sylvius. The overall dimensions of the hypothalamus are aproximately 1.5 (top-to-bottom) ¥ 1.5 (front-to-back) ¥ 1.3 (side-to-side) cm, and the weight is about 2.5 g [2]. This relatively small structure is packed with groups of nerve cell bodies which form distinct nuclei (see Figs 8.1 and 8.2). These nuclei can be divided into three zones (periventricular, medial, lateral) or four regions moving anterior to posterior (preoptic, supraoptic, tuberal, and mamillary) (Table 8.1) [3–5]. In addition to the nuclei, numerous afferent and efferent fibers connect the hypothalamus to the cerebral cortex and the brain stem.

HYPOTHALAMIC FUNCTIONS A number of functions have been ascribed to the hypothalamus based upon animal studies, clinical observations of disease states involving the hypothalamus, and electrical stimulation or destruction of hypothalamic regions in

humans. Because of the close association of hypothalamic nuclei to afferent and efferent tracts from cortical, thalamic, limbic, midbrain, and spinal regions, it has been difficult to localize precisely specific functions to specific nuclei in the hypothalamus. A lesion in a nucleus may also damage or interrupt transmissions from adjacent nerve fibers. Indeed, the nucleus may only serve as a synaptic junction for neural transmissions that begin and terminate elsewhere. In addition, many nuclei appear to subserve multiple functions, and more than one pair of nuclei may be involved with the same function. For example, the ventromedial nucleus is involved in appetite control, emotional expression, and short-term memory retention. The following sections summarize the current concepts regarding the normal functions of the hypothalamus. The reader is referred to several publications which review the clinical and experimental evidence upon which these summaries are based [3,6–9].

Water Metabolism Antidiuretic hormone (ADH; arginine vasopressin) is synthesized in the nerve cell bodies of the magnocellular neurons of the supraoptic and paraventricular nuclei. The hormone is packaged in secretory granules with a specific neurophysin and transported through axoplasmic streaming down long axons that terminate in the pituitary stalk and posterior pituitary. ADH is released into the blood when serum osmolarity increases or vascular volume decreases. Blood volume status is monitored by stretch receptors present in the left atrium and large pulmonary veins, while serum osmolarity changes are detected by peripheral and hypothalamic osmoreceptors. Increased serum osmolarity is the dominant stimulus for ADH release, and this is mediated primarily through the hypothalamic osmoreceptors located in the medial preoptic anterior hypothalamic region. 317

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Table 8.1.

Hypothalamic–Pituitary Dysfunction

Major hypothalamic nuclei Zone

Region

Periventricular

Medial

Lateral

Preoptic

Medial preoptic nucleus

Lateral preoptic nucleus

Mamillary

Posterior hypothalamic nucleus

Anterior hypothalamic nucleus Medial portion of supraoptic nucleus Dorsomedial hypothalamic nucleus Ventromedial hypothalamic nucleus Premamillary nucleus Medial mamillary nucleus

Lateral portion of supraoptic nucleus

Tuberal

Preoptic periventricular nucleus Anterior periventricular nucleus Suprachiasmatic nucleus Paraventricular nucleus Arcuate (infundibular) nucleus

Supraoptic

Lateral hypothalamic nucleus Lateral mamillary nucleus Intercalatus nucleus

Modified from [3–5].

their water permeability, allowing water to be reabsorbed from the urine into the hypertonic renal medullary interstitial region, from which it reenters the bloodstream. This reabsorbed water along with the water ingested in response to activation of the thirst mechanism reestablishes volume and decreases osmolarity, closing the feedback loop. Other factors that stimulate the release of ADH include hypotension, nausea, vomiting, nicotine, hypoglycemia, hypoxia, barbiturates, b-adrenergic drugs, morphine, tricyclic antidepressants, cholinergic drugs, and angiotensin II infusions. ADH release is inhibited by ethanol, atropine, aadrenergic drugs, diphenylhydantoin, and chlorpromazine [10].

Temperature Regulation

FIGURE 8.1 Schematic representation of lateral brain section demonstrating hypothalamic nuclei. Dashed lines represent the frontal (coronal) section planes illustrated in Figs. 8.2 and 8.3. Key to numbers: 1, preoptic nucleus; 2, paraventricular nucleus; 3, anterior hypothalamic area; 4, supraoptic nucleus; 5, arcuate nucleus; 6, dorsal hypothalamic area; 7, dorsomedial nucleus; 8, ventromedial nucleus; 9, posterior hypothalamic area; 10, mamillary body; 11, optic chiasm; 12, optic nerve.

The preoptic anterior hypothalamus harbors receptors for warmth (“warm receptors”), as well as “cold receptors” that respond to cold. When peripheral warm receptors are stimulated by a rise in ambient temperature, and the hypothalamic adrenergic warm receptors are activated by an increase in the temperature of the blood, efferent signals are transmitted to the lateral portion of the posterior hypothalamus via the median forebrain bundle. This leads to activation of the heat dissipating responses of vasodilatation and sweating. In contrast, activation of peripheral cold receptors through a decrease in environmental temperature, or activation of the serotonergic hypothalamic cold receptors, leads to medially placed neurons in the posterior hypothalamus activating the heat production and conservation mechanisms of shivering and vasoconstriction [6,7].

Appetite Control Osmoreceptors located in the lateral preoptic anterior hypothalamic region stimulate thirst in response to increased serum osmolarity [6]. Hypovolemia and hypotension also stimulate thirst. Under the influence of the secreted ADH, the distal tubules and collecting ducts of the kidneys increase

The physiology of caloric homeostasis is poorly understood. Feeding behavior involves cerebral cortical, limbic, and hypothalamic input. Animal studies have defined the ventromedial medial nucleus as the “satiety center”, which inhibits feeding when stimulated, and leads to hyperphagia

FIGURE 8.2 Frontal (coronal) sections of the hypothalamic regions. (a) represents the preoptic region (frontal section plane 1 in Fig. 8.1); (b) represents the supraoptic region (frontal section plane 2 in Fig. 8.1); (c) represents the tuberal region (frontal section plane 3 in Fig. 8.1); (d) represents the mamillary region (frontal section plane 4 in Fig. 8.1)

Chapter 8 The Hypothalamus

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when destroyed. In addition, a “feeding center” is present in the lateral hypothalamus, whereby stimulation leads to hyperphagia and destruction to hypophagia [11,12]. The mechanisms by which the body monitors caloric balance are unknown. Peripheral lipid sensors, intestinal mechanoreceptor, hepatic glucoreceptors, and hypothalamic glucoreceptors have been proposed [7]. The hypothalamus does contain glucoreceptors, and hypoglycemia can stimulate them to increase feeding behavior, but these receptors do not appear to be of physiologic importance [7].

Sleep–wake Cycle and Circadian Rhythm Control The most important area governing wakefulness is the reticular activating system of the brain stem. Lesions in this area result in coma, a state in which the individual cannot be aroused, even with noxious stimuli. The anterior hypothalamus contains a “sleep center,” stimulation of which leads to inhibition of the reticular activating system and sleep, from which, in contrast to coma, the animal or individual can be aroused. Stimulation of the posterior hypothalamus (“wakefulness center”) leads to wakefulness and arousal. The normal sleep–wake cycle may reflect the integrative activities of these two centers; alternatively, the cycle may be entrained in other regions such as the brain stem [6,7]. The suprachiasmatic nuclei control the circadian rhythms in anterior pituitary hermone release, as well as other physiologic rhythms [13,14]. Many of these rhythms are entrained through the visual system via the retinohypothalamic tract [15].

the medial tuberal region [3], increased motility activity of the gastrointestinal tract with stimulation of the preoptic anterior hypothalamus and posterior dorsolateral regions, and reduced bowel motility with ventromedial hypothalamic stimulation [3,7,16]. Gastric juice volume, acidity and pepsin content are increased with stimulation of the anteromedial hypothalamus, as well as the tegmentum of the brain stem [6,17].

Emotional Expression and Behavior Through the use of electrode stimulation or production of lesions in various hypothalamic regions of animals, as well as clinical observations on humans with hypothalamic diseases, the ventromedial nucleus has been found to play an important role in integrating cortical input with regard to behavior. Lesions in this area lead to rage with aggressive, often violent behavior, associated with activation of the sympathetic nervous system [7]. This behavior is referred to as “sham rage” to distinguish it from voluntary or cortical rage. The autonomic response is probably mediated through activation of the posterior hypothalamic sympathetic area. In man, electrical stimulation of the medial or posterior hypothalamus results in the sensations of fear or horror, while apathy and reduced activity is found with destructive lesions in these areas [8,18,19]. Lesions in the limbic system in the region of the caudal hypothalamus have been associated with aggressive, hypersexual behavior [20]. A “pleasure center” located in the medial forebrain bundle in the lateral hypothalamus of rats has been described [6], as has a “nourishing region” around the septal area, stimulation of which leads to lapping, licking, and chewing [3].

Regulation of Visceral (Autonomic) Functions Integration of sympathetic and parasympathetic autonomic nervous system activity is an important function of the hypothalamus. Stimulation of the “sympathetic region” in the posteromedial hypothalamus results in activation of the thoracolumbar autonomic response and a “fight-or-flight” reaction with pupillary dilatation, a rise in blood pressure, tachycardia, increased cardiac output, tachypnea, piloerection, vasoconstriction of the a-adrenergic receptor visceral vascular beds, and vasodilatation of the b-adrenergic responsive blood vessels in skeletal muscle [8]. Stimulation of the “parasympathetic region” in the preoptic anterior hypothalamus leads to increased vagal and sacral autonomic response with pupillary constriction, bradycardia, hypotension, increased blood flow in the visceral vascular bed, and decreased flow in the muscle blood vessels [7,8,16]. Because of the multitude of autonomic fibers running through the hypothalamus, stimulation of one area may result in a sympathetic response, while a parasympathetic type of response may be found with stimulation of an adjacent area. Other types of autonomic function that have been described in nonhuman animal studies include stimulation of micturition and defecation with electrical stimulation of

Memory Memory is a complex process that requires an intact brain stem reticular formation, limbic system, and hypothalamus. Short-term or recent memory requires intact ventromedial nuclei and hippocampus [6,7]. The role of the mamillary nuclei and dorsal medial nucleus in short-term memory is presently unclear [21].

Control of Anterior Pituitary Function The hypothalamus synthesizes and secretes several hypophysiotropic releasing and inhibitory hormones that regulate anterior pituitary function. The physiologic and pharmacologic factors that control the hypothalamic– pituitary–target organ axes are described in detail elsewhere in this volume. Several immunohistochemical studies have localized the various factors in the hypothalamus. Although the nerve cell bodies in which the factors are synthesized are widely distributed throughout the hypothalamus, the axons converge at the median eminence (neurovascular zone) as part of the tuberoinfundibular system and terminate on or near the hypothalamohypophysial portal vessels in

Chapter 8

which they discharge the hypophysiotropic substances under appropriate stimulation [22]. The highest concentrations of nerve cell bodies for gonadotropin-releasing hormone (GnRH) are located in the medial basal hypothalamus and preoptic areas [23]. Thyrotropin-releasing hormone (TRH) neurons are found in the suprachiasmatic, preoptic medial, and paraventricular nuclei [24], while corticotropin-releasing hormone (CRH) has been localized to the paraventricular nucleus [25]. Growth hormone-releasing hormone (GHRH)-containing neurons are found in the arcuate nucleus [26], as are neurons synthesizing somatostatin [27]. Dopaminergic neurons, which presumably inhibit prolactin secretion through dopamine release into the hypothalamohypophysial portal vessels, are found primarily in the arcuate nucleus, with smaller amounts found in the dorsomedial, ventromedial, periventricular, paraventricular nuclei, and median forebrain bundle [28]. In addition to the peptides and amines with established physiologic pituitary regulatory functions, the hypothalamus is replete with a large number of biologically active substances, many of which are located in the same neurons that harbor the hypophysiotropic factors (Table 8.2). PATHOPHYSIOLOGICAL PRINCIPLES Considering the large number of important physiologic functions that depend upon the integrity of the hypothalamus, the close proximity of the nuclei and tracts, and the small overall size of the structure, one would anticipate that diseases involving the hypothalamus would give rise to a plethora of clinical syndromes. Indeed, this is the case, but despite the diversity of findings from patient to patient, several general principles regarding the pathophysiology of signs and symptoms of hypothalamic dysfunction have been established through careful clinical observation [6,7, 29–33]. 1. The spectrum of diseases that can affect the hypothalamus is large, and different lesions may produce identical signs and symptoms of hypothalamic damage. Multiple pathologic processes in each of the major disease categories can involve the hypothalamus (Table 8.3). Bauer reviewed 60 patients with hypothalamic involvement by a variety of diseases documented by autopsy [29,30]. Despite the diversity of pathologic abnormalities, 78% had neuroophthalmologic abnormalities (in 13% these were the first manifestations), 75% developed pyramidal tract or sensory nerve involvement, 65% had headaches, 62% showed extrapyramidal cerebellar signs, and 40% exhibited recurrent vomiting. Findings more specific to the hypothalamus included precocious puberty in 40% (undoubtedly reflecting a selection bias due to the types of case reports in which autopsies were performed), diabetes insipidus in 35%, hypogonadism in 32%, somnolence in 30%, dysthermia in 28%, and obesity

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Table 8.2. Biologically active substances present in paraventricular and arcuate nucleus neurons Paraventricular nucleus Magnocellular division Angiotensin II Cholecystokinin Glucagon Oxytocin Peptide 7B2 Proenkephalin B (dynorphin, rimorphin, a-neoendorphin) Vasopressin Parvocellular division Angiotensin II Atrial natriuretic factor Cholecystokinin Corticotropin-releasing hormone Dopamine Follicle-stimulating hormone-releasing factor g-Aminobutyric acid Galanin Glucagon Neuropeptide Y Neurotensin Peptide 7B2 Proenkephalin A (methionine enkephalin, leucine enkephalin, BAM 22P, metorphamide, [Met]enkephalin-Arg6-Phe7-Leu8, [Met]enkephalin-Arg6Gly7-Leu8) Somatostatin Thyrotropin-releasing hormone Vasopressin Vasoactive intestinal polypeptide/Peptide histidine isoleucine Arcuate nucleus Acetylcholine Dopamine Galanin g-Aminobutyric acid Growth hormone-releasing hormone Neuropeptide Y Neurotensin Pancreatic polypeptide Proenkephalin A Prolactin Proopiomelanocortin (adrenocorticotropic hormone, b-lipotropin, g-melanocyte-stimulating hormone, b-endorphin) Somatostatin Substance P From Lechan [22].

or emaciation in 25%. Although most of the different hypothalamic syndromes can result from a large proportion of the disease listed in Table 8.3, some pathologic processes result in a restricted number of syndromes. For instance, the gliosis of the supraoptic and paraventricular nuclei that occurs in familial or idiopathic diabetes insipidus has diabetes insipidus as its only hypothalamic

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Table 8.3.

Hypothalamic–Pituitary Dysfunction

Etiologies of hypothalamic dysfunction

Congenital Acquired Developmental malformations Anencephaly Porencephaly Agenesis of the corpus callosum Septooptic dysplasia Suprasellar arachnoid cyst Colloid cyst of the third ventricle Hamartoma Aqueductal stenosis Trauma Intraventricular hemorrhage Genetic (Familial or sporadic cases) Hypothalamic hypopituitarism Familial diabetes insipidus Prader–Willi syndrome Bardet–Biedl syndrome Wolfram’s syndrome Pallister–Hall syndrome Tumors Primary intracranial tumors Angioma of the third ventricle Craniopharyngioma Ependymoma Ganglioneuroma Germ cell tumors Glioblastoma multiforme Glioma Hamartoma Hemangioma Lipoma Lymphoma Medulloblastoma Meningioma Neuroblastoma Pinealomas Pituitary tumors Plasmacytoma Sarcoma Metastatic tumors Infiltrative Histiocytosis Leukemia Sarcoidosis Immunologic Idiopathic diabetes insipidus Paraneoplastic syndrome

Nutritional/metabolic Anorexia nervosa Kernicterus Wernicke–Korsakoff syndrome Weight loss Degenerative Glial scarring Parkinson’s Infectious Bacterial Meningitis Mycobacterial Tubrculosis Spirochetal Syphilis Viral Cytomegalovirus Encephalitis Jakob–Creutzfeldt Kuru Poliomyelitis Varicella Vascular Aneurysm Arteriovenous malformation Pituitary apoplexy Subarachnoid hemorrhage Vasculitis Trauma Birth injury Head injury Postneurosurgical Functional Diencephalic epilepsy Drugs Hayek–Peake syndrome Idiopathic SIADH Kleine–Levin syndrome Periodic syndrome of Wolff Psychosocial deprivation syndrome Other Radiation Porphyria Toluene exposure

manifestation. Similarly, hamartomas have precocious puberty and galastic seizures as their primary manifestations, due to their endocrine activity and/or their specific location in the tuber cinereum. Many of the pathologic processes have characteristic appearances on

magnetic resonance imaging that are helpful diagnostically [34]. 2. As a general rule, patients with systemic illnesses such as sarcoidosis, histiocytosis, and infections that involve the hypothalamus usually, but not uniformly, have nonhypothalamic manifestations of the disease process. Isolated sarcoid lesions may be found in the hypothalamus, but more commonly ophthalmologic and extracranial disease coexists. Unifocal eosinophilic granulomas have been described in the hypothalamus, but usually such involvement reflects disseminated histiocytosis and bony lesions generally are present also. Tuberculous meningitis, neurosyphilis, and viral illnesses are rarely confined to the hypothalamus, although hypothalamic symptoms may be early manifestations of the disease. 3. The site of a lesion causing a dysfunction does not necessarily correspond to the site from which the function emanates. As noted above, the hypothalamic nuclei are closely packed and interspersed among various fiber tracts whose origins or destinations may be the cerebral cortex, midbrain, thalamus, limbic system, spinal cord, or even other nuclei within the hypothalamus. Since disease processes involving the hypothalamus tend to be rather large in relation to the size of the hypothalamus, it is rare to find a lesion involving only one nucleus or a single tract. Therefore, it is not surprising that in Bauer’s series most patients had mixtures of neurologic or neuroophthalmologic signs and symptoms in addition to endocrine abnormalities [29,30]. 4. The clinical manifestations depend in part upon the rate of progression of the disease process. Patients with small, rapidly progressive lesions often develop symptoms early, while slowly progressive lesions may remain asymptomatic for long periods, allowing some to obtain relatively immense size before clinical evidence of the disease becomes apparent. Presumably in the latter instance, the slow growth allows for the other areas of the hypothalamus or extrahypothalamic regions to compensate for the deficits induced by the lesion. Acute insults, such as vascular accidents or trauma, tend to result in decreased consciousness, hyperthermia, and diabetes insipidus which may be transient if the patient survives the initial injury. Chronic lesions tend to alter cognitive ability and endocrine function, and are not reversible. 5. Although lesions that involve a single, unilateral area of the hypothalamus may result in symptoms, most lesions resulting in chronic hypothalamic syndromes are bilateral, though not necessarily symmetrical. Since most of the hypothalamic functions are controlled by one or more pairs of nuclei, destruction of a single nucleus usually is not sufficient to result in a clinical syndrome. From a pathophysiologic standpoint, this implies that pathologic processes that are multiple (i.e., metastatic

Chapter 8

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tumors, granulomatous diseases), arise in or around the third ventricle (colloid cysts), cause enlargement of the third ventricle (pinealomas, germ cell tumors, midbrain gliomas, aqueductal stenosis), or impinge upon or invade the floor of the hypothalamus (craniopharyngiomas, optic gliomas, pituitary adenomas) will be more likely to result in clinical signs and symptoms of hypothalamic disease than will diseases that affect the lateral portions of the hypothalamus. 6. Lesions involving hypothalamic nuclei may give different syndromes depending upon whether the lesion results in stimulation or destruction of the nuclei. Thus, stimulatory lesions in the tuberal area may result in precocious puberty, while destructive lesions may lead to hypogonadism. Hyperthermia is associated with stimulation of the preoptic region, while hypothermia is the clinical consequence of destruction of the same area. 7. The clinical manifestations of hypothalamic disease depends upon the age of onset. As a rule, the hypothalamus of neonates is quite immature, and diseases afflicting the neonatal or infant hypothalamus present different symptoms than the same disease affecting the same region in an older child or an adult. The diencephalic syndrome of infancy due to a glioma involving the anterior hypothalamus is an example of this phenomenon. The affected infants eat seemingly adequate quantities of food, yet lose weight. They tend to be hyperactive and euphoric. After the age of two years, the surviving infants undergo a dramatic change by gaining weight, becoming obese, and displaying irritable behavior [7]. Another type of age-related disease manifestation is the effect of hypopituitarism due to hypothalamic abnormalities. Gonadotropin deficiency that occurs before puberty will result in a lack of pubertal changes with maintenance of the sexually infantile state. Acquired hypothalamic hypogonadism that has its onset in an adult may lead to some regression of secondary sexual characteristics, but such individuals do not appear sexually infantile. GH deficiency due to hypothalamic disease in a prepubertal individual is associated with short stature, while a similar deficiency in an adult is clinically inapparent.

Manifestations of Hypothalamic Disease Keeping the above general principles in mind, and based upon careful pathologic studies of patients with hypothalamic diseases, a topographic map of the hypothalamus which correlates clinical findings with anatomic sites of lesions can be constructed [35–44] (Fig. 8.3).

Disorders of Water Metabolism Central Diabetes Insipidus

This condition results from a partial or complete absence of ADH. Without sufficient ADH, the distal tubules and

FIGURE 8.3 Clinical findings associated with hypothalamic lesions located at various anatomical sites. Clinicopathologic correlation based upon multiple studies [31–40]. (a) corresponds to region depicted in Fig. 8.2a; (b) corresponds to region depicted in Fig. 8.2c; (c) corresponds to section depicted in Fig. 8.2d.

collecting ducts of the kidneys are unable to adequately reabsorb water, leaving the urine inappropriately hypotonic relative to the plasma osmolarity. The persistent diuresis leads to polyuria (up to 10–12 l/day) and nocturia, which in turn stimulates the thirst mechanism to bring about water-seeking behavior and polydipsia. If the osmoreceptor mechanism is intact and the patient is conscious and has

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access to fluids, the plasma osmolarity may be maintained within the normal range. However, if the osmoreceptors of the thirst center are damaged, or if the patient is unable to ingest adequate quantities of water, hypernatremic dehydration may occur and result in rapid deterioration of the sensorium from lethargy to stupor to coma. Patients with lesser deficiencies of ADH may release enough of the hormone to maintain adequate water balance under basal conditions. In contrast to patients with complete diabetes insipidus, patients with partial diabetes insipidus may increase their urine osmolarity to a level above their plasma osmolarity during dehydration. However, in both conditions, the administration of exogenous vasopressin to a dehydrated patient will result in a further increase in urine osmolarity, while dehydrated normal individuals will show little or no further increase in urine osmolarity after a standard dehydration test. Diabetes insipidus results from lesions involving the magnocellular neurons of the supraoptic and paraventricular nuclei or that interrupt the supraopticohypophysial tracts that terminate in the pituitary stalk or posterior pituitary. Such lesions are commonly found in patients with hypothalamic disorders. Transient diabetes insipidus may be found in individuals with posterior pituitary or low pituitary stalk lesions, or in patients with acute, reversible hypothalamic lesions. In Bauer’s series of anatomically proven chronic hypothalamic lesions, 21 (35%) of the patients had diabetes insipidus at some time during the course of their illness [29,30]. Although it was the second-most frequent manifestation of hypothalamic disease, in only 3% of cases was diabetes insipidus the initial manifestation. Diabetes insipidus is often associated with hypogonadism and obesity, reflecting the anterior, medial hypothalamic localization of lesions affecting the supraoptic and paraventricular nuclei. The spectrum of pathologic lesions accounting for diabetes insipidus from two large series is shown in Table 8.4. Idiopathic diabetes insipidus comprises the largest single category of causes and over a third of these patients have circulating vasopressin cell antibodies suggesting an autoimmune etiology [48]. In this condition, loss of magnocellular nerve cell bodies and gliosis is found in the supraoptic and paraventricular nuclei [49]. The same pathologic findings are present in patients with familial central diabetes insipidus but these patients do not have anti-ADH antibodies. Rather, mutations in the vasopressin precursor molecule gene have been found in some families [50]. Both sex-linked recessive and autosomal dominant forms of this latter condition have been described. An autosomal recessive form (Wolfram’s syndrome) exists, composed of central diabetes insipidus, insulin-dependent diabetes mellitus, primary optic atrophy, bilateral sensorineural deafness, and in some families, autonomic neurogenic bladder and ataxia [51]. Degeneration of the paraventricular and, to a lesser extent, the supraoptic nuclei have been noted in this syndrome, as has atrophy of the posterior pituitary [52].

Table 8.4.

Etiologies of diabetes insipidus

Etiology

Number of patients

Percentage

Idiopathic/familial Neoplasm Primary intracranial Metastatic Lymphoma Leukemia Trauma Histiocytosis Infectious Neurosyphilis Meningitis Postencephalitic Sarcoidosis Other*

138

42

71 15 3 3 18 23

21 5 1 1 5 7

9 3 5 4 40

3 1 2 1 11

Total

332

100

* Cerebral atherosclerosis, birth injury, postvaccinal, giant cell granuloma, systemic illness, postirradiation, congenital malformation, and postoperative. Adapted from [45–47].

Deficiency of ADH is frequently seen with hypothalamic involvement by suprasellar germinomas (85%) [53,54], pineal germinomas (40%) [53,55], the chronic disseminated form of histiocytosis (50%) [56], and sarcoidosis (58%) [57–60]. Diabetes insipidus may also be found in patients with septooptic dysplasia (23%) [61–64], pinealomas (18%) [55], hypothalamic gliomas (17%) [65,66], and craniopharyngiomas (14%) [53]. Adipsic or Essential Hypernatremia (Cerebral Salt Retention Syndrome)

Damage to the osmoreceptors in the anterior medial and anterior lateral preoptic regions of the hypothalamus may bring about essential hypernatremia which is characterized by chronic, fluctuating elevations of serum sodium (and chloride), often to dangerously high levels, despite the spontaneous ingestion of amounts of fluid (1–2 l/day) that are capable of maintaining appropriate plasma osmolarity in otherwise normal adults. Affected individuals have an impaired thirst mechanism, demonstrating hypodipsia or adipsia despite the marked elevations in serum sodium. Nevertheless, these patients have a normal volume of extracellular fluid and are not dehydrated, and, therefore, maintain a normal blood pressure, pulse rate, blood–urea nitrogen, serum creatinine, and creatinine clearance. Since vascular volume status also regulates ADH release, these patients can release ADH and concentrate their urine with volume depletion. However, even while hypernatremic, an oral or intravenous intake of a large volume of water only results in inhibition of ADH release due to increased volume, culminating in the excretion of a dilute urine. Most

Chapter 8

of these patients do have partial diabetes insipidus, as their urine osmolarity does increase with exogenous administration of ADH [7,29,30,32,67,68]. Clinically, few symptoms reflecting hypernatremia are found with serum sodium concentrations below 160 mmol/l. Above this level, patients develop fatigue, lethargy, weakness, muscle tenderness and cramps, anorexia, depression, and irritability. Stupor and frank coma may be found with sodium concentrations greater than 180 mmol/l. Although the pituitary gland at autopsy is normal, anterior pituitary hormone deficiencies are found in 71% of patients, reflecting the hypothalamic etiology of the hypopituitarism [67]. Obesity has been noted also in 43% of the patients. Additionally, hypertriglyceridemia has been found in five of six subjects (83%) in whom this measurement has been reported [67]. The pathologic processes that have been associated with essential hypernatremia include suprasellar germinomas, histiocytosis, sarcoidosis, craniopharyngiomas, ruptured aneurysms, optic nerve gliomas, pineal tumors, trauma, hydrocephalus, cysts, inflammatory conditions, and toluene exposure [69]. Recently a few children have been described with essential hypernatremia but without a structural hypothalamic defect being found (Hayek–Peake syndrome). They demonstrate recurrent hypernatremia, hypodipsia, obesity, hyperprolactinemia, hypothyroidism, hyperlipidemia, lethargy, increased perspiration, and in some cases, central hypoventilation [20,21]. The findings suggest a functional derangement in the anterior medial hypothalamic region with involvement of the osmoreceptors and the ventral medial nucleus. Syndrome of Inappropriate Secretion of ADH (SIADH)

This condition is characterized by serum hypoosmolarity and hyponatremia, an inappropriately concentrated urine for the low serum osmolarity, continued urinary excretion of sodium despite the low serum sodium, and hypouricemia in a patient with normal renal, adrenal, and thyroid function, and who does not exhibit findings of extracellular fluid volume expansion (i.e., no evidence of congestive heart failure, cirrhosis, or other edematous states). This condition may be due to drugs, activation of peripheral volume receptors, peripheral neuropathies, ectopic production of ADH from neoplasms, or intracranial processes. In some individuals the syndrome is due to a decrease in the setpoint for the serum osmolarity release of ADH. Indeed, SIADH may occur, usually transiently, following head trauma, intracranial bleeds, meningitis, encephalitis, transsphenoidal pituitary surgery, and other neurosurgical procedures. It has also been noted in some patients with hydrocephalus, craniopharyngiomas, germinomas, pinealomas, central pontine myelinolysis, and acute intermittent porphyria [53]. In the latter situation it is not clear whether this is a reflection of a hypothalamic lesion or the result of a peripheral neuropathy [7,32]. An idiopathic, cyclic form of the syndrome has been found in young women with menstrual irregu-

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larities, and enlarged lateral ventricles. No hypothalamic pathology has been identified in patients with the idiopathic variety [10]. Besides symptoms from the underlying disease, these patients demonstrate the clinical findings of water intoxication. Mild decrements in serum sodium, between 130 and 120 mmol/l, generally result in weight gain without edema, anorexia, nausea, vomiting, headache, weakness, withdrawal, and lethargy. Mental confusion is common at concentrations below 120 mmol/l, and seizures and coma may also develop, especially if the decrease in sodium occurs rapidly.

Dysthermia Hyperthermia

Acute injury to the anterior hypothalamic and preoptic areas from intracranial bleeds, neurosurgical procedures in the region of the floor of the third ventricle, or trauma may result in temperature elevations up to 41°C, tachycardia, and unconsciousness that generally lasts for less than 2 weeks if the patient survives. With such lesions, heat production continues, while the heat-dissipating mechanisms fail to respond appropriately. The pulse rate in patients with hyperthermia due to hypothalamic lesions is not increased to the same extent for a given elevation in temperature as is the pulse rate in patients with fever from infections or inflammatory processes [3,6,7,29,30]. Acute hyperthermia to 41°C or greater is a characteristic of the neuroleptic malignant syndrome. This syndrome develops in susceptible individuals over 24 to 72 hours following exposure to phenothiazines, butyrophenones, thioxanthenes, or ioxapine. The potential for development of the syndrome roughly parallels the antidopaminergic D2 receptor potency of the neuroleptic drug. It has been hypothesized that the syndrome results from basal ganglia dopamine D2 receptor blockade which activates heat generation through muscle contraction, impairment of heat dissipation through hypothalamic injury, and inhibition of diaphoresis through a peripheral anticholinergic effect of the neuroleptics [72]. Autopsy studies have shown injury in the preoptic medial and tuberal nuclei [72]. Other clinical characteristics of the syndrome include hypertonicity of the skeletal muscles with “lead-pipe” type of rigidity, fluctuating consciousness varying from agitation to stupor to coma, and instability of the autonomic nervous system reflected by pallor, diaphoresis, wide swings in blood pressure, tachycardia and arrhythmias, tremors, and akinesis. Leukocytosis, elevations of serum creatine phosphokinase, and nonspecific encephalopathic findings on electroencephalography (EEG) are also found [73]. The syndrome lasts 5 to 10 days and currently carries a 20–30% mortality rate [74]. Sustained or chronic hyperthermia is found with lesions in the tuberoinfundibular region. Ten percent of the patients in Bauer’s series exhibited chronic hyperthermia

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[29,30], which may result from loss of heat-dissipating mechanisms, stimulation of the heat-conservation mechanisms, or elevation of the setpoint for activation of the heat dissipation [7]. Patients with chronic hypothalamic hyperthermia do not exhibit the generalized malaise that accompanies elevated temperatures due to infections, and also have paradoxical peripheral vasoconstriction with cold, clammy extremities. The hyperthermia may respond to sedatives or anticonvulsant medications, but not to salicylates [29,30]. Spontaneous paroxysmal hyperthermia of probable hypothalamic origin but without pathologic lesions in the hypothalamus has been described in a few patients. In most individuals, the episodes occur sporadically and are characterized by shaking chills, fever, hypertension, vomiting, and peripheral vasoconstriction. Resolution over minutes to hours is accompanied by vasodilatation and diaphoresis. A similar syndrome occurring at regular 3-week intervals was described by Wolff, and may represent a form of diencephalic epilepsy [7,75,76]. Hypothermia

Chronic hypothermia with temperatures below 32°C was present in 12% of the patients described by Bauer [29,30], and this finding was usually associated with large lesions involving the anterior and/or posterior hypothalamus. Destruction of the thermoregulatory mechanisms by such lesions results in an inability to generate heat through shivering and vasoconstriction. Hypothermia has been noted in third-ventricular and large hypothalamic neoplasms, poliomyelitis, neurosyphilis, sarcoidosis, multiple sclerosis, gliosis of the anterior hypothalamus, posterior hypothalamic neuronal pyknosis in Parkinson’s disease, and with the periventricular and mamillary body destruction seen with Wernicke’s encephalopathy [57,76–81]. Episodic or paroxysmal hypothermia, also known as diencephalic autonomic epilepsy, is a distinct syndrome in which body temperatures abruptly decrease, often to 32°C or lower, over a period of minutes to days, associated with a variety of signs and symptoms of autonomic nervous system dysfunction [7,35,82–88]. The frequency of attacks varies from daily to decades apart. Patients experience flushing, diaphoresis, fatigue, hypotension, bradycardia, salivation, lacrimation, pupillary dilatation, Cheyne–Stokes respirations, nausea, vomiting, asterixis, ataxia, and obtundation. Thus, during the episodes heat generation is impaired and heat loss is increased due to the vasodilatation and sweating. EEG slowing occurs during the episodes. Recovery occurs spontaneously over hours to days, and is associated with heat generation through shivering and vasoconstriction. Attacks often begin in the teenage years and the frequency and duration of attacks may increase as the patient ages. Some degree of thermal regulation is maintained during the episodes, since experimentally lowering the temperature further results in shivering and vasoconstriction, suggesting that there is a resetting of the thermostat during the episodes

[87]. This syndrome has been found in some patients with tumors involving the floor and lower walls of the third ventricle [35,82]. In others, gliosis and loss of the arcuate nucleus and the premamillary area have been noted at autopsy. In addition, approximately half of the patients with episodic hypothermia have an agenesis of the corpus callosum, a combination given the eponym “Shapiro’s syndrome” [83,84]. Such patients may also have hypogonadism, precocious puberty, diabetes insipidus, reset osmostat, and GH deficiency [83,86,87]. Poikilothermia

This condition – the tendency of the individual to assume the ambient temperature – results from loss of both heat conservation and heat-loss homeostatic mechanisms. Wide fluctuations of temperature are seen, and affected patients do not experience thermal discomfort, nor attempt to alter their environment to maintain their core body temperature. This condition, noted in 1.7% of the patients in Bauer’s series, is found with large lesions involving the posterior hypothalamus and rostral mesencephalon, as well as in patients with both anterior and posterior hypothalamic destruction [7,29,30]. Poikilothermia may also be found in Wernicke’s encephalopathy [7].

Disorders of Caloric Balance Hypothalamic Obesity

Obesity is a common finding in patients with hypothalamic diseases, occurring in approximately 25% of individuals with anatomically proven lesions, although rarely is it the initial manifestation of hypothalamic dysfunction [29,30]. Most patients with hypothalamic obesity have large lesions or extensive involvement of multiple areas of the hypothalamus. Nevertheless, based upon careful study of the few patients with well-described, discrete lesions, it is clear that bilateral destruction of the ventromedial nucleus results in obesity in man, as it does in experimental studies in animals [3,11,29,30,37,38,42]. In patients with documented structural involvement, close to 90% have a neoplasm, most often a craniopharyngioma (approximately 60%) [11]. Approximately 6% are the result of inflammatory or granulomatous processes including sarcoidosis, tuberculosis, arachnoiditis, and encephalitis, 5% are posttraumatic, and 2% are due to leukemic infiltration [11]. As would be anticipated from the location of the lesions that lead to obesity, other clinical findings are commonly present. In a series of 69 patients analyzed by Bray and Gallagher, 72% had headaches, 72% had decreased vision or visual field abnormalities, 56% exhibited reproductive dysfunction such as amenorrhea, impotence, or diminished libido, 35% had disordered water metabolism with diabetes insipidus, polyuria, and/or polydipsia, 40% were somnolent, 20% had behavioral abnormalities, and 7% had seizures [11]. The association between obesity and hypogonadism has long

Chapter 8

been noted since Froehlich described his patient, who was subsequently found to have a craniopharyngioma, with “dystrophic adiposogenitalis.” The affective disorders that coexist with hypothalamic obesity vary from antisocial behavior to sham rage [38]. The obesity is clearly the result of hyperphagia. In many instances the abnormality appears to reflect a resetting of the satiety setpoint. This is best seen in patients with obesity that develops following trauma. Most affected individuals gain weight for approximately 6 months following the trauma, followed by a period of stabilization as the energy expenditure equals the caloric content of the ingested food, with a subsequent gradual decrease in food intake and a loss of weight [11]. Similarly, patients with tumor destruction of the ventromedial nuclei may develop hyperphagia and a rapid gain in weight, followed by a plateau, and then a further weight gain as the neoplasm grows [7]. Some patients display an indiscriminate food intake and even will ingest left-over scraps destined for the garbage, while others will show a finickiness that closely resembles that seen in rats with bilateral lesions in the ventromedial nuclei [11]. These patients have hyperinsulinemia to a greater extent than do patients with essential obesity, and it has been proposed that this is due to enhanced insulin secretion through stimulation of the vagus nerve, as increased vagal firing rate has been noted in animals with ventromedial nucleus lesions [12]. Hypothalamic Cachexia in Adults

In Bauer’s series, 18% of the patients exhibited substantial weight loss, 7% had anorexia, and 8% were bulimic [29,30]. Destruction of both the ventromedial nuclei and the lateral hypothalamus leads to anorexia and emaciation, as do lesions isolated to the lateral hypothalamus [37,38,41]. The features of the lateral hypothalamic syndrome include rapid weight loss, muscle wasting, decreased activity, and hypophagia leading to cachexia and death. The most common lesions accounting for this syndrome are neoplasms, although cysts, and malignant multiple sclerosis also have been described as causes [37,41]. Diencephalic Syndrome of Infancy

In 1951, Russell described an unusual syndrome in infants with hypothalamic tumors of severe emaciation despite an apparently good food intake, associated with an alert appearance and euphoric affect, and nystagmoid eye movements [89]. The majority (80%) of these infants have been found to have low-grade hypothalamic or optic nerve gliomas that destroy the ventromedial nuclei [6,7,90]. Rarely ependymomas, gangliogliomas, and dysgerminomas give rise to the syndrome [90]. The infants appear normal at birth and demonstrate normal feeding and developmental parameters during the first 3–12 months. Towards the end of the first year of life, the infants begin to lose weight and subcutaneous fat, show signs of hyperactivity and a cheerful, happy

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The Hypothalamus

affect, but continue to grow normally. They exhibit an alert appearance secondary to eyelid retraction (Collier sign) [90]. Other findings including nystagmus, pallor, vomiting, tremor, and optic atrophy may be present (Table 8.5). Endocrine evaluation is generally normal, although absent diurnal variation in plasma cortisol concentrations, and elevated basal serum GH levels with a paradoxical rise following a glucose load, have been found [32]. The elevated GH levels are not specific to these patients, since other illnesses associated with weight loss, such as anorexia nervosa, also are accompanied by such elevations. Usually the infants succumb to the tumor and emaciation by the age of 2 years. Paradoxically, infants who survive beyond age 2, either due to spontaneous stabilization or therapy, often maintain their good appetite, gain weight, and become obese. In addition, their pleasant personality is replaced by irritability and rage, and they may develop somnolence and precocious puberty [6,90,91]. This syndrome nicely illustrates the fact that the manifestations of hypothalamic disease are related in part to the age of patient and maturity of the hypothalamus. Anorexia Nervosa

The typical patient with anorexia nervosa is a young, white female from a middle to upper socioeconomic background who inappropriately views herself as obese, and, therefore, severely restricts her food intake, exercises excessively, and may engage in bulimic binges with self-induced vomiting, and diuretic and cathartic abuse. The typical age of onset is less than 25 years, the patients lose more than 15% of their weight, and are usually 25% below their ideal body weight.

Table 8.5. Clinical findings in 67 patients with diencephalic syndrome of infancy Feature Emaciation Alert appearance Increased vigor and/or hyperkinesis Vomiting Euphoria Pallor Hydrocephalus Nystagmus Irritability Optic atrophy Tremor Sweating Large hands/feet Large genitalia Polyuria Papilledema Endocrine abnormalities From Burr et al. [90].

Percentage 100 87 72 68 59 55 33 55 32 23 23 15 150

Surgery Radiotherapy Bromocriptine Octreotide

PRL normalized GH remained elevated

353

Acidophilic stem cell adenoma

F

151/2

179 (>95th %ile)

71

8700

Surgery Cabergoline Octreotide LAR

PRL, GH, and IGF normalized

135

Mammosomatotroph adenoma positive for GH > PRL

M

8

140.5 (+2.5 SD)

28

120

Surgery Bromocriptine

Patient doing well

362

Pituitary GH adenoma

F

2

+4.4 SD

135

370

Surgery

GH normalized

358

McCune–Albright syndrome

M

6

149.7 (+5.9 SD)

1316 mIU

Surgery Octreotide

Growth started after surgery, but stopped on octreotide

363

GRFoma from foregut with metastases liver

F

15

n/a

>120

Surgery GRFoma resected Octreotide

GH and GRF suppressed Growth stopped 21/2 months after SMS start

359

Unknown (no surgery)

M

12

+4.2 SD

15

52

Bromocriptine Octreotide

Normalized GH, IGF-I, PRL and growth velocity

354

Unknown (no surgery)

M

159.9 (+4 SD)

17–40

>60

Bromocriptine Radiotherapy

GH near normal

360

Pathology

Ht (cm)

GH (ng/ml)

Outcome

Author*

Unclear tumor type

21

Mixed GH and PRL cells

11

Other

71/2

44.9 340–450

* Adapted from Maheshwari et al. [133]. Abbreviations: GH: growth hormone, PRL: prolactin, IGF-I: insulin-like growth factor-1, n/a: not available.

428

SECTION 3

Pituitary Tumors

rheumatologic disorders (Table 11.6). In a study in the UK, only 13% of 256 acromegalic patients diagnosed during a 20-year period, presented with primary symptoms of altered facial appearance or enlargement of extremities [136]. In a review of several hundred patients worldwide, 98% were reported with acral enlargement, while hyperhidrosis was prominent in 70% [140]. Morever, the time between onset of symptoms and diagnosis of acromegaly ranges from 6.6 to 10.2 years, with a mean delay of almost 9 years [8,135]. Generalized visceromegaly occurs with enlargement of the tongue, bones, salivary glands, thyroid, heart, liver, and spleen. Clinically apparent hepatosplenomegaly, however, is rare. Patients have characteristic facial features, large fleshy nose, spade-like hands, and frontal bossing. Some patients, if presenting early, may have subtle facial and peripheral features. Serial review of old photographs often accentuates the progress of these subtle physical changes [141]. Increase in shoe, ring, or hat size is commonly reported. Progressive

Table 11.5.

Clinical features of acromegaly

Local tumor effects Pituitary enlargement Visual field defects Cranial nerve palsy Headache Somatic Acral enlargement Thickening of soft tissue of hands and feet Musculoskeletal Prognathism Malocclusion Arthralgias Carpal tunnel syndrome Acroparesthesia Proximal myopathy Hypertrophy of frontal bones Skin Hyperhidrosis Oily Skin tags Colon Polyps Cardiovascular Left ventricular hypertrophy Asymmetric septal hypertrophy Hypertension Congestive heart failure Sleep disturbances Sleep apnea Narcolepsy

Visceromegaly Tongue Thyroid Salivary gland Liver Spleen Kidney Endocrine-metabolic Reproduction Menstrual abnormalities Galactorrhea Decreased libido, impotence, low sex hormone-binding globulin Multiple endocrine neoplasia (1) Hyperparathyroidism Pancreatic islet cell tumors Carbohydrate Impaired glucose tolerance Insulin resistance and hyperinsulinemia Diabetes mellitus Lipids Hypertriglyceridemia Mineral Hypercalciuria, increased 1,25 (OH)2 vitamin D3 Urinary hydroxyproline Electrolyte Low renin Increased aldosterone Thyroid Low thyroxine-binding globulin

* The mean delay to diagnosis from onset of symptoms is 8.7 years.

acral changes will, if untreated, lead to severe facial and skeletal disfigurement especially if the excess GH secretion begins prior to closure of the epiphyses (Fig. 11.4) [142–144]. Skeletal Changes

Periosteal new bone formation in response to IGF-I [145] results in skeletal overgrowth leading to mandibular overgrowth with prognathism, maxillary widening, teeth separation, frontal bossing, jaw malocclusion and overbite, and nasal bone hypertrophy [149]. Characteristic voice deepening with a sonorous resonance occurs because of laryngeal hypertrophy and enlarged paranasal sinuses. Arthropathy occurs in about 70% of patients with acromegaly, most of whom exhibit objective signs of joint swelling, hypermobility and cartilaginous thickening [5,147]. Up to half of patients experience joint symptoms severe enough to limit or impair daily activities [148–153]. Severe joint pain unusually signifies irreversible joint degeneration. Knees, hips, shoulders, lumbosacral joints, elbows, and ankles are affected in decreasing order of frequency. Joint involvement may be mono- or polyarticular, and although crepitus, stiffness, tenderness, and hypermobility are common, joint effusions are rarely encountered [144,152]. Local periarticular fibrous tissue thickening may cause subsequent joint stiffening, deformities, and nerve entrapment. Neural enlargement, local fluid retention and swelling of wrist soft tissues may lead to carpal tunnel syndrome, a painful entrapment median neuropathy, which occurs in up to half of all patients. This condition generally resolves early

Table 11.6.

Presentation of acromegaly*

Presenting chief complaint

Frequency (%)

Menstrual disturbance Change in appearance/acral growth Headaches Paresthesias/carpal tunnel syndrome Diabetes mellitus/impaired glucose tolerance Heart disease Visual impairment Decreased libido/impotence Arthopathy Thyroid disorder Hypertension Gigantism Fatigue Hyperhidrosis Somnolence Other Chance (detected by unrelated physical or dental examination or X-ray)

13 11 8 6 5 3 3 3 3 2 1 1 0.3 0.3 0.3 5 40

* From Molitch [143], based on 310 patients.

Chapter 11

Acromegaly

429

(c) (a),(b)

(d)

(f) (e) FIGURE 11.3. Clinical signs of hypersomatotrophism. (a) Original figure depicting earliest illustration of clinical features of acromegaly by Minkowski in 1887. Note acromegalic facies, fleshy fingers and toes, and frontal bossing. (b) Acromegaly in a young male with active perspiration, oily skin, acne, and widened tooth gap. (c) Prominent skin tags may be associated with the presence of colon polyps. (d) Jaw overbite and widening of spaces between incisors due to mandibular growth in acromegaly. (e) X-ray image of bony “tufting” seen at ends of terminal phalanges indicates bony overgrowth. (f) Increased heal pad thickness. (d) and (f) from Melmed and Braunstein [139].

after treatment. Spinal involvement including osteophytosis, disc space widening, and increased anteroposterior vertebral length may lead to dorsal kyphosis [151]. Pathology of Arthropathy

Proliferation of chondrocytes with subsequent increased joint space occurs early in response to increased GH and IGF-I levels. Presumably, induction of local IGF-I by GH stimulates uneven chondrocyte growth. Ulcerations and fissures present on the weight-bearing areas of new cartilage are often accompanied by new bone formation. This process eventually results in debilitating osteoarthritis associated

with bone remodeling, osteophyte formation, subchondral cysts, narrowed joint spaces, and lax periarticular ligaments. Osteophytes are commonly seen at the tufts of the phalanges and over the anterior aspects of spinal vertebrae. Ossification of ligaments and periarticular calcium pyrophosphate deposition are also found [147]. Although the duration of hypersomatotrophism appears to directly correlate with the severity of the joint changes, it is unclear whether higher GH levels are associated with increased severity of articular disease [151]. Responses to therapy (see below) will usually depend upon the degree of irreversible cartilage degeneration already in place.

430

SECTION 3

Pituitary Tumors

FIGURE 11.4. Severe skeletal disfigurement in three patients with growth hormone-secreting pituitary tumors. From Whitehead et al. [142].

Skin Changes

Hyperhidrosis and oily skin with an unpleasant odor are common early signs, occurring in up to 70% of patients. Patients often relate the need to increase their use of deodorant or cosmetic powders. Facial wrinkles, nasolabial folds and heel pads are increased in thickness, and body hair may become coarsened [154,155]. These effects may correlate with IGF-1 levels, and improve after treatment. Thickening of the skin has been attributed to glycosaminoglycan deposition [156], while connective tissue collagen production is also increased [160]. Skin tags are common and these may be important markers for the concomitant presence of adenomatous colonic polyps [158]. Raynaud’s phenomenon may also be present in up to one-third of patients. Cardiovascular Complications

Cardiovascular disease is a major cause of morbidity and mortality [159–169] with symptomatic cardiac disease present in about 20% of patients. Arterial hypertension, accelerated atherosclerosis, sodium and fluid retention leading to expanded extracellular fluid volume, and cardiac arrhythmias are the common cardiovascular manifestations. About half of patients with active acromegaly have hypertension, and 50% of these have evidence of left ventricular dysfunction [169]. Interestingly, left ventricular hypertrophy is also reported in about half of normotensive acromegalic patients. Although asymmetric septal hypertrophy is common, cardiac failure with early or mild cardiomegaly may occur in the absence of obvious causes of myocardial failure. Patients may exhibit subclinical left ventricular diastolic dysfunction, consistent with unique pathologic findings including myocardial hypertrophy, interstitial fibrosis, and lymphocytic myocardial infiltrates. Electrocardiograms are abnormal in about 50% of patients, with S-T segments,T-wave abnormalities, conduction defects and arrhythmias accounting for most changes. Plasma renin

levels are suppressed and endogenous plasma digitalis-like activity with chronic volume expansion has been identified [170]. Coexisting hypertension and coronary artery disease account for most of the cardiovascular disease encountered in patients with acromegaly, while a GH- or IGF-1mediated specific cardiomyopathy may also be present [164]. Cardiovascular disease is the most important cause of mortality in acromegaly, accounting for approximately 60% of deaths [159]. The presence of cardiovascular disease at the time of diagnosis is associated with high mortality rates, and effective control of GH and IGF-I levels results in improved cardiac function [172]. Respiratory Complications

Prognathism, thick lips, macroglossia, and hypertrophied nasal structures may result in significant airway obstructions [173–175]. Additional clinical features of acromegaly contribute to impaired upper respiratory function. Irregular hypertrophy of laryngeal mucosa and cartilage may lead to unilateral or bilateral vocal cord fixation or laryngeal stenosis with accompanying voice changes [173]. Tracheal calcification and cricoarytenoid joint arthropathy may also be present. These obstructive features may necessitate tracheostomy either to maintain adequate baseline airway function, or especially at the time of surgical anesthesia. Difficulty in tracheal intubation is often encountered in patients undergoing anesthesia. Central respiratory center depression as well as upper airways obstruction may contribute to the development of paroxysmal daytime sleep (narcolepsy), sleep apnea and habitual excessive snoring [173]. The obstructive sleep apnea syndrome, characterized by excessive daytime sleepiness with at least five episodes of obstructive apnea per hour, is an important cause of daytime somnolence in men with acromegaly. These patients may also have a ventilation perfusion defect with hypoxemia. The sleep apnea of acromegaly may be due to either obstruction of the respi-

Chapter 11

ratory tract, or central in origin. Interestingly, the central form of sleep apnea is associated with higher GH and IGFI levels, possibly reflecting a loss of central somatostatin tone accounting for the disorder [176–178]. Neuromuscular Changes

Peripheral acroparesthesias occur in almost half of all patients. Synovial edema and hyperplastic wrist ligaments and tendons contribute to painful median nerve compression with the resultant carpal tunnel syndrome [180]. A true symmetrical peripheral neuropathy has also been described. This rare mixed motor and sensory impairment should be distinguished from characteristic diabetic neuropathy, which may occur secondarily to acromegaly. The pathologic features of median neuropathy have been ascribed to increased edema, rather than extrinsic compression [181]. About half of all patients develop proximal myopathy which may be accompanied by myalgias and cramps and nonspecific electromyogram (EMG) myopathic changes. Histologic examination reveals hypertrophy and necrosis of skeletal muscle fiber in patients with proximal muscle weakness and elevated creatine phosphokinase (CPK) levels [182]. Although bony overgrowth of frontal bones may mask eye changes, true exophthalmos may be present. Open angle glaucoma may also result from impaired aqueous filtration through hypertrophied tissue surrounding the canal of Schlemm. Psychologic Changes

Self-esteem may diminish with progressive facial and bodily disfigurement. It is unclear whether reported depression, mood swings and apathy result from these physical effects or whether they are intrinsic central effects of high GH levels. There is no clear evidence for an increased incidence of psychologic disorders in acromegaly [183,184]. Morbidity and Mortality in Acromegaly

The impact of the disease on mortality outcome has recently become apparent, and a significant increased (approximately threefold) mortality has been reported in several studies [8,9,159,160,185–190]. In a retrospective study reported in 1966, 50% of patients died before the age of 50, with cardiovascular disease being the most common cause of death (Table 11.7). In 194 patients with acromegaly, a reduced life expectancy was found, with cardiovascular disorders accounting for 24% of deaths followed by respiratory (18%), and cerebrovascular disease (14%). Diabetes mellitus, found in 20% of patients, was associated with 2.5 times the predicted risk of death, while hypertension had been present in 45% of patients with acromegaly [8,9,159]. Analysis of more recent reports indicates that cardiovascular disease, respiratory disorders, diabetes and malignancy account for mortality in acromegaly. The most significant determinants of mortality appear to be the GH level and the presence of coexisting cardiac disease [159]. Moreover, control of GH levels to