Advances in Metabolic Disorders: Volume 5

Advances in Metabolic Disorders, Volume 5 covers the developments in the study of metabolic disorders. The book discusses the hypothalamic control of the secretion of adenohypophysial hormones; the adrenocortical control of epinephrine synthesis in health and disease; and the role of the renin-angiotensin system in the physiological control of aldosterone secretion. The text also describes the general metabolic and systemic effects of progesterone and synthetic progestins, and the experimental and clinical studies of the role of the thymus glands in the development and expression of immune functions. The control of glucose metabolism in the human fetus and newborn infant, as well as the dynamics and regulation of the human adipose tissue are also considered. The book concludes by encompassing the possible association between several metabolic abnormalities and coronary artery disease. Endocrinologists and biochemists will find the text invaluable.

• Language: English
• Publisher: Academic Press
• Release date: Oct 22, 2013
• ISBN: 9781483215471

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Advances in Metabolic Disorders

Rachmiel Levine

City of Hope Medical Center, Duarte, California

Rolf Luft

Department of Endocrinology and Metabolism, Karolinska sjukhuset, Stockholm, Sweden

ISSN  0065-2903

Volume 5 • Number Suppl. (C) • 1971

Table of Contents

Cover image

Title page

Contributors to This Volume

Copyright page

Contributors

Contents of Previous Volumes

Hypothalamic Control of the Secretion of Adenohypophysial Hormones

Publisher Summary

I Introduction

II Chemistry of the Hypothalamic Releasing Factors

III Mechanisms of Action of the Hypothalamic Factors

IV Conclusions

Adrenocortical Control of Epinephrine Synthesis in Health and Disease

Publisher Summary

I Introduction

II Morphology of the Mammalian Adrenal Gland

III Catecholamine Biosynthesis in Neurons and Chromaffin Cells

IV Adrenocortical Control of Adrenomedullary Function

V PNMT Activity in the Brain

VI The Adrenal Cortex and Epinephrine Synthesis in Nonmammalian Vertebrates

VII Clinical Implications

Acknowledgments

The Relationship between Angiotensin and Aldosterone

Publisher Summary

I The Renin-Angiotensin System in the Physiological Control of Aldosterone Secretion

II Clinical Implications

III Conclusions

Acknowledgments

The Metabolic Influence of Progestins

Publisher Summary

I Introduction

II Electrolyte Metabolism

III Protein Metabolism

IV Growth Hormone Secretion

V Carbohydrate Metabolism

VI Fat Metabolism

VII Respiratory Effects

VIII The Influence of Synthetic Progestins on Electrolyte Metabolism

IX The Effect of Synthetic Progestins on Protein Metabolism

The Thymus Gland: Experimental and Clinical Studies of Its Role in the Development and Expression of Immune Functions

Publisher Summary

I Introduction

II Historical

III Phylogenetic and Embryonic Development of the Thymus Gland

IV Modern Concepts of Thymus Function Derived from Experimental Studies

V Thymus Malfunction and Clinical Disorders

VI Summary

Control of Glucose Metabolism in the Human Fetus and Newborn Infant

Publisher Summary

I Introduction

II Modification of Maternal Metabolism by the Conceptus

III Placental Transport of Nutrients and Hormones

IV Fetal Glucose Metabolism

V The Newborn Infant: the Effects of Birth

Human Adipose Tissue Dynamics and Regulation

Publisher Summary

I Introduction

II Sampling Techniques

III Morphology and Cell Composition

IV Human Adipose Tissue Metabolism

V Concluding Remarks

Coronary Disease and Carbohydrate and Fat Abnormalities

Publisher Summary

I Introduction

II Experimental Design

III Results

IV Discussion

V Summary

Author Index

Subject Index

Contributors to This Volume

Peter A.J. Adam, Per Björntorp, G.W. Boyd, Allan L. Goldstein, Richard Gorlin, Roger Guillemin, Michael V. Herman, Richard L. Landau, Jan Östman, W.S. Peart, L.A. Pohorecky, James T. Poulos, Abraham White and R.J. Wurtman

Copyright page

Copyright © 1971, by Academic Press, Inc.

all rights reserved

no part of this book may be reproduced in any form, by photostat, microfilm, retrieval system, or any other means, without written permission from the publishers.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by

ACADEMIC PRESS, INC. (LONDON) LTD.

Berkeley Square House, London W1X 6BA

Library of Congress Catalog Card Number: 64-14568

printed in the united states of america

Contributors

Peter A.J. Adam,     Department of Pediatrics, Case Western Reserve University School of Medicine at Cleveland Metropolitan General Hospital, Cleveland, Ohio (183)

Per Björntorp,     The First Medical Service, Sahlgrenska Sjukhuset, Göteborg, Sweden (277)

G.W. Boyd,     Medical Unit, St. Mary’s Hospital Medical School, London, England (77)

Allan L. Goldstein,     Department of Biochemistry, Albert Einstein Colloge of Medicine, Yeshiva University, Bronx, New York (149)

Richard Gorlin,     Cardiovascular Division, Peter Bent Brigham Hospital, and Harvard Medical School, Boston, Massachusetts (329)

Roger Guillemin,     The Salk Institute for Biological Studies, La Jolla, California (1)

Michael V. Herman,     Department of Medicine, Peter Bent Brigham Hospital, and Harvard Medical School, Boston, Massachusetts (329)

Richard L. Landau,     The Fisher Endocrinology Laboratories, Department of Medicine, The University of Chicago, Chicago, Illinois (119)

Jan Östman,     Department of Endocrinology and Metabolism, Karolinska Sjukhuset, Stockholm, Sweden

W.S. Peart,     Medical Unit, St. Mary’s Hospital Medical School, London, England (77)

L.A. Pohorecky,     Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts (53)

James T. Poulos,     The Fisher Endocrinology Laboratories, Department of Medicine, The University of Chicago, Chicago, Illinois (119)

Abraham White,     Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York (149)

R.J. Wurtman,     Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts (53)

Numbers in parentheses indicate the pages on which the authors’ contribution begin.

Contents of Previous Volumes

Volume 1

Glycogen Storage Disease

H. G. Hers

The Parathyroids

G. D. Aurbach and John T. Potts, Jr.

Mitochondrial Respiratory Control: Biochemical, Physiological and Pathological Aspects

Lars Ernster and Rolf Luft

Osteoporosis

B. E. C. Nordin

Basal Metabolic Rate and Thyroid Hormones

J. R. Tata

Insulin Antagonists and Inhibitors

J. Vallance-Owen

Aldosterone: Its Biochemistry and Physiology

John H. Laragh and William G. Kelley

Folic Acid Deficiency in Man and its Interrelationship with Vitamin B12 Metabolism

A. Leonard Luhby and Jack M. Cooperman

author index—subject index

Volume 2

Gout

James B. Wyngaarden

Nitrogen-Retaining Steroids and Their Application in Disease

A. Querido and A. A. H. Kassenaar

Macroglobulinemia

Jan Waldenstrom

Testing the Functional Capacity of the Tryptophan-Niacin Pathway in Man by Analysis of Urinary Metabolites

J. M. Price, R. R. Brown, and Norma Yess

The Syndrome of Testicular Feminization

A. Louis Southren

author index—subject index

Volume 3

Ketogenesis and Its Regulation

O. Wieland

Spontaneous Diabetes and/or Obesity in Laboratory Rodents

Albert E. Renold

Ethanol-Induced Hypoglycemia

Leonard L. Madison

Hormonal Syndromes Associated with Neoplasia

Mortimer B. Lipsett

Metabolic Disorders of the Ovary

Bruno Lunenfeld, Aliza Eshkol, Vaclav Insler, and Zaki Kraiem

The Nature and Significance of the Long-Acting Thyroid Stimulator

Joseph P. Kriss

Thyroid Autoantibodies in Thyroid Diseases

Noel R. Rose and Ernest Witebsky

Erythropoietin

Clifford W. Gurney

author index—subject index

Volume 4

Intestinal Factors in the Regulation of Insulin Secretion

Vincent Marks and Ellis Samols

Antidiuretic Hormone Synthesis, Release, and Action under Normal and Pathological Circumstances

Niels A. Thorn

Disaccharidase Deficiency

David H. Alpers and Kurt J. Isselbacher

Hormonal Control of Fetal Development and Metabolism

Alfred Jost and Luc Picon

The Metabolism of the Placenta

Andrew J. Szabo and Richard D. Grimaldi

Metabolic Aspects of Obesity

E. S. Gordon

The Use of Liquid Formula Diets in Metabolic Studies: 15 Years’ Experience

E. H. Ahrens, Jr.

author index—subject index

Hypothalamic Control of the Secretion of Adenohypophysial Hormones†

Roger Guillemin*,     *The Salk Institute for Biological Studies, La Jolla, California

Publisher Summary

This chapter explains the mechanisms involved in the fine regulations and control of the secretions of the hormones of the anterior lobe of the pituitary gland that reside in the central nervous system (CNS). There are various areas of the CNS that participate in the fine regulation of the secretion of all adenohypophysial hormones. The ultimate integrator of this information of CNS origin is the hypothalamus. The various areas of the limbic system and those nuclei of the hypothalamus that are involved in control of the secretion of the adenohypophysial hormones are sensitive to, and see their electrical activity modified by, hormones of the peripheral glands that are acted upon by the adenohypophysis through the secretion of each one of its tropic hormones. The final information from the hypothalamus to the adenohypophysis is not in the form of nerve impulses with eventual liberation of one of the classical transmitters but as specific hypothalamic hypophysiotropic substances.

I Introduction

A Hypothalamo-hypophysial Relationships

B Neuroendocrime Integrations. Telencephalic Influences upon the Hypothalamus. Hormonal Feedback on Hypothalamic and Extra-hypothalamic Centers

C Origin of the Releasing Factors

D Chemical Nature of the Hypothalamic Releasing Factors

E Assays for Hypothalamic Releasing Factors

F Hypothalamic Releasing Factors Present in Portal Blood and Injected in Portal Vessels

II Chemistry of the Hypothalamic Releasing Factors

A Thyrotropin Releasing Factor (TRF)

B Luteinizing Hormone (LH) Releasing Factor (LRF)

C Follicle Stimulating Hormone (FSH) Releasing Factor (FRF)

D Growth Hormone Releasing Factor (GRF) and Growth Hormone Release Inhibiting Factor (GIF)

E Corticotropin Releasing Factors (CRFs)

F Prolactin Release Inhibiting Factor (PIF) and Prolactin Releasing Factor (PRF)

G Melanocyte Stimulating Hormone Release Inhibiting Factor (MIF)and Releasing Factor (MRF)

H Mechanisms of Inactivation of the Releasing Factors

III Mechanisms of Action of the Hypothalamic Releasing Factors

A Effects of Electrolytes on the Hypophysiotropic Activity of Releasing Factors. Effects of Elevated [K+]

B Source of Energy for the Activity of the Releasing Factors in Stimulating Secretion of Pituitary Hormones. Role of Cyclic AMP

C Effects of Releasing Factors on Biosynthesis of Pituitary Hormones

IV Conclusions

References

I Introduction

A Hypothalamo-hypophysial Relationships

There is now ample evidence from observations and experimental data obtained in both clinical medicine and experimental physiology that the mechanisms involved in the fine regulations and control of the secretions of the hormones of the anterior lobe of the pituitary gland reside in the central nervous system. Integration of the secretory activity of the adenohypophysis by the central nervous system involves widely separated and diverse structures of the mesencephalon, and certain cortical areas; the final common pathway of the afferents from these various parts of the central nervous system is to be found, however, in a small area of the diencephalon, the ventral hypothalamus (see Figs. 1 and 2). The area more particularly involved is located around the third ventricle and expands from the suprachiasmatic region to the mamillary bodies; it has been termed the hypothalamic hypophysiotropic area. Also well established is the concept that this hypothalamic involvement in the control of the secretion of adenohypophysial hormones does not make use of a direct innervation of adenohypophysial secretory cells by hypothalamic nerve fibers but involves a neurohumoral mechanism in which substances of hypothalamic origin are transported to the adenohypophysial parenchyma by a peculiar system of capillary vessels (Fig. 1a-f). It was the merit of G. W. Harris to recognize early the importance of this hypothalamo-hypophysial portal system of capillaries as a possible means of conveyance of the then hypothetical hypothalamic substances now referred to as hypothalamic releasing factors or hypothalamic hypophysiotropic hormones. The last twenty years have witnessed a prolific literature describing the search for these hypothalamic releasing factors, efforts to establish their chemical characterization as well as studies aimed at elucidating their physiological role and significance.

Fig. 1a-f Hypothalamo-hypophysial relationship, (la) Diagram of the areas of the ventral hypothalamus, which upon electrocoagulation leads to changes in the secretion of adenohypophysial hormones as shown, (lb) Diagram of the relationship between anterior hypothalamus (SON, supra-optic nucleus), PVN, paraventricular nucleus, and posterior pituitary; the axonic fibers depicted constitute the hypothalamo-hypophysial tract of Cajal. (lc) Diagram of the hypothalamo-hypophysial portal vessels connecting the median eminence area to the adenohypophysis. (Id) Photomicrograph of the capillary vessels depicted in (lc) visualized in the rabbit brain after injection of india ink. RI, infundibular recess; Cap., capillaries of the primary plexus; AH, adenohypophysis. (Courtesy Professor H. Duvernoy, College of Medicine, Besançon, France.) (1e, 1f) Diagrammatic representation of the hypophysiotropic area of the ventral hypothalamus, in which some hypothalamic elements synthesize releasing factors and release them in the hypothalamo-hypo-physial portal vessels.

Fig. 2.(a) Hypothalamic afferents. Diagrammatic representation of neural connections between hypothalamus, telencephalon, and midbrain; this simple diagram attempts to show some of the most important of these connections and has no pretense at anatomical exactness.

The well-established observation by both experimenters and clinicians of what has been termed the feedback system between peripheral hormones (thyroid, adrenal cortex, gonads) and the corresponding pituitary hormones has now been shown to be for the most part, mediated by effects of these hormones on various centers of the central nervous system, and particularly the hypothalamus. This modern version of the neuroendocrine feedback system, will be discussed in some detail below.

Few fields of modern physiology during the same time have generated or suffered as many controversies as have been encountered in this new discipline now referred to as neuroendocrinology. This has been due essentially to the fact that all studies on these hypophysiotropic releasing factors have had perforce to be conducted using bioassays, many of which have been of doubtful specificity, reliability, or even significance (see below pp. p11"–15).

This chapter in the series of monographs on Advances in Metabolic Disorders will be a critical review of what I consider to be solid information on some aspects of this field of research while pointing out what I consider to be still of doubtful significance or validity in what sometimes is a deceivingly elegant and attractive series of physiological constructs.

B Neuroendocrine Integrations. Telencephalic Influences upon the Hypothalamus. Hormonal Feedback on Hypothalamic and Extra-hypothalamic Centers

We have said above that the integration of the adenohypophysial secretion is in the hypothalamus. The hypothalamus, however, is not to be construed as some sort of a semi-isolated structure, only in contact with the adenohypophysis. Quite to the contrary, anatomical studies reveal that the hypothalamus receives a large number of afferent fibers mainly from the oldest parts of the cortex and the basal ganglia (see Fig. 2a). These structures comprise the hippocampus, the cortex of the pyriform lobe, the cortex of the cingular gyrus, the anterior insula and the posterior orbito-frontal region, also the amygdala and globus pallidus. All these structures with the exception of the globus pallidus are incorporated in what is now called the limbic system.

All formations of this system, through various routes, send fibers into the medial forebrain bundle of the hypothalamus in addition to more specific projections upon various hypothalamic nuclei. The hypothalamus has also important connections both afferent and efferent, with the midbrain and particularly, with the reticular activating substance. Electrophysiological studies as well as ablation studies show that these various hypothalamic afferents constitute the basis for extensive reverberating circuits and feedback mechanisms through which, eventually, all hypothalamic efferents, including the hypophysiotropic hypothalamic efferents (the hypothalamic releasing factors) are affected.

There is good experimental evidence that the integrity of all these extra-hypothalamic systems is necessary for a harmonious function of the neuroendocrine mechanisms involved in maintaining homeostasis. The limbic system appears to influence the hypophysiotropic functions of the hypothalamus, acting as some sort of a modulator of functional patterns which seem to be eventually integrated at the level of hypothalamic formations. For instance, certain lesions of the amygdala, the hippocampus or the midbrain will inhibit, prevent, or facilitate the release of ACTH, the gonadotropins, TSH or growth hormone (see Fig. 2b) as though some of these higher centers exerted stimulatory as well as inhibitory influences on the neuroendocrine functions of the hypothalamus—eventually resulting in corresponding modifications of secretion of the adenohypophysis. The same appears to be true for lesions or electrical stimulation in various areas of the reticular formation of the midbrain (reviews in Dell and Dumont, 1961; Mason, 1968; Beyer and Sawyer, 1969). It is of considerable physiological interest that these same extrahypothalamic systems influence the classical electrical activity of the hypothalamic centers in the same directions (excitatory or inhibitory), as they modify neuroendocrine activities of the hypothalamic centers. In other words, we see the neuroendocrine activity of the hypothalamus to be modified and modulated by the same neurophysiological components which modify and modulate the other autonomic functions of the hypothalamic centers (Dell and Dumont, 1961).

Fig. 2.(b) On the same diagram of anatomical formations as in (a), simplified locations of eventual activation or inhibition of some of the adenohypophysial functions are shown, again in a simplified manner. A, amygdala; A6, area 6 of frontal lobe; L, limbic system; Fo, fornix; ST, stria terminalis; H, hippocampus; 0, orbito-frontal cortex; MFB, medial forebrain bundle; P.O., preoptic nucleus; P.V., paraventricular nucleus; 3d.V., 3d. ventricle; Ma., mamillary bodies; Arc, arcuate nucleus; O.C., optic chiasm; P.P., posterior pituitary; A.P., anterior pituitary.

It has also been shown that lesions of various areas of the limbic system or of the midbrain will inhibit the circadian or diurnal variations of the secretion of pituitary hormones such as ACTH or TSH, and will modify the rapid cycles of secretion of growth hormone as it normally relates to sleep patterns. Equally significant in their physiological and clinical implications are the observations that peripheral hormones, such as corticoids and gonadal steroids, can alter in profound fashion the electrical activity of several formations of the central nervous system, particularly the hypothalamus and the various hypothalamic afferents mentioned above. Studies with steroid implantations have shown that these hormones can act directly on nerve cells in the brain as if they reacted as chemoreceptors, the steroids modifying the threshold for activation or the inherent firing frequencies or amplitude in numerous studies using microelectrode recordings. The various areas of the hypothalamic centers as well as those of the hypothalamic afferents which seem to be more apt to localize gonadal steroids or corticosteroids appear to be the same areas which, following electrical stimulation or electrocoagulation, modify the secretion of gonadotropins or ACTH (Beyer and Sawyer, 1969; Davidson, 1969).

It is thus tempting to consider the presence of the gonadal or corticosteroids preferentially in those areas of the central nervous system involved in the final control of the secretion of the pertinent pituitary hormones, i.e., the gonadotropins and ACTH, as part of the feedback mechanisms discussed above.

Clear evidence for localization of gonadal steroids feedback receptors relates to testosterone; a basomedial hypothalamic area for the testosterone inhibitory feedback mechanism appears to be well established (Davidson, 1969); estrogens, on the other hand, may have a primary action on the basomedial hypothalamus with also probably other effects at the anterior pituitary level; it is difficult here to know what is truly physiological from what may be pharmacological and relating to experimental conditions. There is good evidence for progesterone inhibitory feedback systems at the level of the basomedial hypothalamus, as well as at the level of other areas of the limbic system, the steroids modifying the electrical activity of these various centers as usually affected by stimulation of the reticular activating formation, as in the elegant experiments of Barraclough and Cross (1963). These observations appear of considerable interest when it is realized that most of the synthetic steroids used as antifertility agents in humans, such as norethindrone (Norlutin) and norethynodrel, elevate differentially the EEG-afterreaction threshold and block the ovulation induced by copulation without inhibiting the sexual behavior or modifying the EEG-arousal threshold in a series of studies in the rabbits reported by Sawyer (1967). Thus, these antifertility steroids appear to block the pituitary secretion of ovulating hormone by preventing the limbic-hypothalamic stimuli from reaching the area of median eminence, thereby preventing release of the hypothalamic LRF, hence the release of LH. There are indeed similar evidences that the glucocorticoids do modify the neuronal electrical activity of various areas of the midbrain, the limbic system and the hypothalamus, all areas known from electrical stimulation or ablation studies to be involved in the control of ACTH secretion. The mechanisms of the feedback between secretion of the thyroid gland and of TSH are somewhat different and will be discussed below in some detail.

C Origin of the Releasing Factors

Biological activities associated with releasing factors and highly purified materials with hypophysiotropic activities have been obtained from crude extracts of the hypothalamus of all mammalian species studied so far. There is good evidence for the existence of a TRF (TSH releasing factor), LRF (LH releasing factor), CRF (corticotropin releasing factor), GRF (growth hormone releasing factor); there are also biological activities which have been related to an FRF (FSH releasing factor), to a PIF (prolactin release inhibiting factor), MIF (MSH release inhibiting factor). All these are discussed in some detail in the following pages of this review.

There is no unquestionable evidence for the exclusive localization of these releasing factors in specific nuclei of the hypothalamus. To the contrary, they can be extracted from hypothalamic tissues with no exact correlation with what morphologists have classically described as specific nuclei of the hypothalamus. Indeed, equivalent weight of fragments of the hypothalamus dissected from the supra-optic area to the median eminence and eventually to the mamillary bodies appear to yield equivalent quantities of TRF activity, (for instance), per unit weight (Guillemin, 1965). Statements claiming specific localization in the hypothalamus for LRF (McCann, 1962) must be viewed with caution in terms of the relative reliability of the bioassays involved in these studies. As this review is written, the specific origin of the releasing factors, in terms of the type of hypothalamic cell which would be involved in their synthesis (neurons, glial cells or other cells specifically endowed with neuroendocrine activity) is totally unknown.

At this time, nothing is known concerning the biosynthesis of any of the hypothalamic releasing factors. In view of the recently established structure of TRF as a simple tripeptide derivative, its biosynthesis might be considered to take place via a nonribosomal enzyme system; or it could be cleaved from a larger prohormone or precursor protein of plasma or cytoplasmic origin (cf. angiotensin); in the latter case, the releasing factors would be more closely related to tissue or plasma kinins than to true hormonal secretions. Both of these working hypotheses require experimental evidence to ascertain their possible validity.

D Chemical Nature of the Hypothalamic Releasing Factors

The earlier reports in 1955 which brought good evidence for the existence of hypothalamic releasing factors put forward the hypothesis that these substances would probably prove to be polypeptides of relatively small molecular weight, since other areas of the hypothalamus were known to secrete vasopressin and oxytocin, both octapeptides. These early reports had also eliminated the classical transmitters of the nervous system (acetylcholine, catecholamines, histamine, etc.) as possibly being the releasing factors. Indeed, for the next ten years, attempts at purifying hypothalamic releasing factors were conducted by methods classically used for purification of polypeptides. Because of peculiarities, which we will discuss below, that were encountered when dealing with purified TRF, we suggested in 1966 that TRF (at least) might not be a simple homomeric polypeptide, even though we clearly accepted the possibility that an unusual amino acid sequence might still explain the observed resistance of the biological activity to proteolytic enzymes as well as other peculiarities of the physical characteristics of the purified material obtained at that time (Guillemin et al., 1966). Schally et al. (1966a, 1969a) concurred later with these conclusions on the basis of their own observations and went so far as to state that the non-peptidic moiety which constitutes the largest part of the molecular weight of TRF was necessary for biological activity. At about the same time, White, Schally, and collaborators (White et al., 1968; Schally et al., 1968) concluded on the basis of a series of bioassays based on the depletion of pituitary content of FSH, that the FSH releasing activity in extracts of porcine hypothalamus could be entirely accounted for in terms of its content in polyamines such as putrescine, histamine, spermine, and spermidine—another evidence against the polypeptidic nature of the releasing factors. New information has severely questioned the physiological significance of the earlier results reported by White et al. using the FSH pituitary depletion assay (see below, section on FRF). Furthermore, recent observations from our laboratory have now shown ovine TRF to be a tripeptide derivative and a similar conclusion has recently been reached by Schally et al. regarding TRF of porcine origin. There is also recent evidence that the LH releasing activity in highly purified fractions of ovine hypothalamic origin is associated with a polypeptide or polypeptide derivative with a PCA-N-terminus from studies with a highly specific pyrrolidone-carboxylyl peptidase (Amoss et al., 1970).

The hypothalamic releasing factors can thus be reasonably considered to be polypeptides or polypeptide derivatives.

E Assays for Hypothalamic Releasing Factors

All studies on hypothalamic releasing factors must make use of some method of bioassay to ascertain the presence of the (hypophysiotropic) factor under study, its localization in the effluent of some chromatographic" column used in its purification, or to study variations of its concentrations in the extract of hypothalamic tissues or portal blood obtained in one experimental condition or another. Though the recent elucidation of the chemical structure of TRF may allow, in the future, the devising of methods to measure TRF based on radioimmunoassays or isotope dilution, at the moment, there is no method available other than bioassays to study specifically and quantitatively any of the releasing factors. It is thus obvious that any of these studies will not be any better than the bioassay method they are conducted with. I consider that the readers of this review whose primary interest is, presumably, in fields other than those of hypothalamo-pituitary relationships, should be presented with some critical comments about the bioassays presently utilized by investigators working on hypothalamo-pituitary relationships.

Evidence of the stimulation of the release of a pituitary hormone (by some hypothalamic factor) can be evidenced by criteria of two orders: (a) criteria of the first order will be those measuring directly, in peripheral plasma or in the incubation medium of some in vitro incubation method, changes in the (peripheral) concentration or rate of secretion of the pituitary hormone under study; this can be done by bioassays or radioimmunoassays; (b) criteria of the second order are those measuring indirectly (presumed) changes in the secretion of a given pituitary hormone, by assessing changes of some peripheral effects of that hormone or one or another of its peripheral targets—changes in the plasma or adrenal concentration of corticoids as evidence of adrenal stimulation, hence ACTH secretion; changes in the ovarian ascorbic acid content in specially prepared animals—as evidence of endogenous release of LH, since it is known that injection of exogenous LH will modify ovarian ascorbic acid content, etc.

Whatever the method chosen to assess the secretion of the pituitary hormone under study, it must be highly specific for that hormone; it must be relatively sensitive, i.e., be compatible with the use of reasonably small amounts of tissues or body fluid or samples; it should have a good index of discrimination, i.e., be able to assess with the proper statistical analysis relatively small changes in the secretion of the pituitary hormone under study, thus allowing quantitative measurements and studies with their pertinent mathematical and statistical evaluation; it must be reliable, i.e., based on (biological) metameters and (analytical) methodology well enough understood and established to give reproducible results, in similar conditions, in any laboratory and at any time.

To these somewhat obvious requirements, must be added that of the proof that one is dealing with an immediate or direct pituitary activation (by the presumed releasing factor), i.e., that one has established that the effect observed is not due to activation of the hypothalamus of the assay animal, which in turn would stimulate its pituitary, the result observed being, in fact, due to an indirect (i.e., transhypothalamic) stimulation of pituitary secretion (this is particularly true and important for study of the control of secretions of ACTH, TSH, STH, prolactin, which we now know are stimulated by what is called nonspecific stress, to use Selye’s terminology).

However surprising to the investigator foreign to this field, let me say that few of the methods currently used in studying the hypothalamic control of pituitary secretion meet these elementary and obvious criteria; this laxity, which appears to be in direct proportion with the number of publications emanating from some working in this field, has contributed much, if not all, of the confusion and sometimes doubt, extant in much of the literature on these hypothalamic releasing factors.

Criteria of the first order, as defined above, have been almost impossible to achieve until recent availability of radioimmunoassays for pituitary hormones, with the only exception of plasma TSH, which, in rat blood is present in large enough concentrations so that it could be measured reliably by a bioassay. Reliable assays for hypophysiotropic factors have recently been or are presently devised, based on direct measurements in otherwise normal animals, of plasma concentrations of ACTH, FSH, LH, growth hormones, prolactin by radioimmunoassays (Amoss and Guillemin, 1969; Daughaday et al., 1970). With candid knowledge of the limitations of the immunological methods, these assays appear to be the approach of choice.

As part of the criteria of the first order, are also to be mentioned in vitro methods in which fragments of adenohypophysis survive in short-term incubation or in classical organ culture or tissue cultures, in all cases the end point being the measurement by bioassays or recently radioimmunoassays of the amount of one pituitary hormone or another released in the incubation medium. These in vitro methods are powerful tools in the characterization of hypothalamic releasing factors since they contribute the most exclusive proof that a substance of hypothalamic origin act directly at the level of the adenohypophysial tissue. These methods, however, have often been used indiscriminately by several investigators ignoring their many limitations and requirements for obtaining meaningful and specific information (Guillemin and Schally, 1959; Guillemin and Vale, 1970).

Several groups of investigators over the years have been using a bioassay for studies on releasing factor for FSH, TSH, and growth hormone particularly, based on the premises that if large enough amounts of any of these pituitary hormones are released rapidly under the action of an acutely administered releasing factor, the pituitary content in that hormone measured a few minutes after administration of the hypothalamic extract, crude or purified, will show a considerable decrease when compared with the proper control, i.e., a depletion of the pituitary content in that particular hormone—which depletion can be considered as proof of acute secretion (David et al., 1965). A considerable number of publications have recently appeared over the last few years using these pituitary depletion assays in physiological studies dealing with releasing factors or in biochemical studies aimed at the purification and eventually isolation of these releasing factors. I have always been of the opinion that the premises on which these pituitary depletion assays are based were highly questionable: The content in a given hormone of an actively secreting gland is obviously, at any given time, the value of a complicated function with multiple variables including the available pool at time zero, rate of synthesis of that hormone—modified by availability of the proper precursors, etc., rate of release of that hormone, etc., to mention only a few of the important metameters. Thus, knowledge of a numerical value for only one of these variables and for obviously one single time only, does not and cannot define the overall function characterizing the secretory rate of the pituitary gland. Furthermore the adenohypophysis is a storage gland which contains, at all times, quantities of any pituitary hormone many thousandfold the amount of that hormone present in the remainder of the body outside of the pituitary pool. Thus, even in case of maximum stimulation by a large dose of a releasing factor, one can anticipate that only a small amount of that particular hormone will be released after the injection of a releasing factor even though the peripheral plasma levels of that hormone may have increased 10- or 20-fold. Indeed, we have evidence to support this statement in the case of TSH in a series of experiments in which a large dose of TRF was injected in a series of normal rats, the plasma TSH levels being measured individually after injection of TRF at the peak of the peripheral plasma TSH concentration; a few seconds later, each animal was decapitated, its pituitary removed, and its TSH content measured individually in 4-point assays. Even though plasma TSH levels increased from 5- to 10-fold in each of the TRF injected animals, under no circumstance could we demonstrate statistically valid difference in the pituitary TSH content of the animals receiving TRF, when compared to the control animals receiving saline (see Fig. 3). There are further complications regarding the pituitary depletion assays: White et al. (1968) reported recently that injection of a series of polyamines, such as putrescine, cadaverine, methylhistamine, histamine, from nanogram to microgram doses in experimental animals would deplete (down to 20% of the control values) the pituitary FSH content of these animals a few minutes after administration, the pituitary FSH content being measured by the Steelman and Pohley bioassay method. A large number of papers appeared from this and other groups reporting physiological studies on the release of FSH as assessed by this method as well as studies on the isolation and characterization of a hypothalamic FSH releasing factor. The amount of FSH that would have been released by the polyamines had the assay data truly reflected depletion of the pituitary FSH content would have been so high as to be easily measured by the bioassay in the plasma of these animals; such plasma levels of FSH were never observed either by the bioassay or by recently performed radioimmunoassay for FSH. Furthermore, the polyamines were shown to be unable to release FSH when added to pituitaries incubated in vitro, at doses corresponding to those which had been active in vivo in the so-called pituitary FSH depletion assay. Schally et al. (1970b) have thus recently recognized that the polyamines do not represent true releasing factors for FSH. If we accept the bioassay data observed by these various investigators in their test for pituitary FSH when attempting to measure FSH content, no satisfactory hypothesis is available to explain their assay data in terms of release of FSH. Similar observations are being made regarding the pituitary growth hormone content depletion assay as a test for physiological studies as well as chemical purifications of a hypothetical hypothalamic growth hormone releasing factor. Thus, considerable caution should be exercised when reading any of these publications either on the physiology or on the purification of one releasing factor or another based on these pituitary depletion assays (see discussions in Meites, 1970a, pp. 57–59, 161–170).

Fig. 3 Injection of a large dose of TRF which elevates plasma TSH levels 5- to 10-fold when compared to control levels does not produce a statistically valid depletion of pituitary TSH content (a) measured in the same animals at the time of the peak TSH concentration in plasma (b) (From Takemura et al., 1970.)

In the various following chapters on each of the releasing factors, we will also call attention to what we consider limitation of the specific assay methods employed and reported in the literature.

F Hypothalamic Releasing Factors in Portal Blood and Injected in Portal Vessels

Two important requirements of the theory proposing a role for the hypothalamo-hypophysial portal capillaries in the concept of a neuro-humoral hypothalamic control of pituitary function would be that portal blood collected from the primary plexus of this system should contain hypophysiotropic activities in greater amounts than peripheral blood, that these hypophysiotropic activities should vary with physiological or experimental conditions and that injections of purified hypothalamic substances (releasing factors) in the collecting veins downstream toward the secondary plexus of the system should indeed stimulate the secretion of pituitary hormones. There is now very good evidence for both of these requirements. Porter and collaborators (1970a) have demonstrated the existence in blood collected from the primary plexus of the hypothalamo-hypophysial portal system of biological activities best explained by the presence of TRF as well as LRF in blood sampled in various experimental conditions—electrical stimulation of the preoptic area would increase TRF activity of portal blood; perfusion of the third ventricle with dopamine will increase LRF activity in portal blood. Fink et al. (1967) and Harris and Ruf (1970) have reached similar conclusions. Recently Porter, in collaboration with our laboratories, has shown that microperfusion of pituitary stalk vessels leading to the secondary plexus of the portal system is followed by considerable stimulation of the secretion of TSH or LH when measured in peripheral plasma following perfusion of TRF or highly purified LRF (Porter et al., 1970b).

These two important tenets for the theory of a neurohumoral hypothalamic control of pituitary function are now well established.

II Chemistry of the Hypothalamic Releasing Factors

A Thyrotropin Releasing Factor (TRF)

Despite earlier claims based on questionable methodology (see reviews in Davis et al., 1967; Guillemin, 1967; Schally et al., 1968), the first incontrovertible evidence for the existence and early purification of a hypothalamic TSH releasing factor was reported by Guillemin et al. (1962b): A 2 N acetic acid extract of several hundred fragments of hypothalamic tissues from sheep brain was filtered on Sephadex G-25; in an animal in vivo bioassay designed to demonstrate exogenously injected or endogenously secreted TSH (Yamazaki et al., 1963), two zones of biological activity were found in the effluent (Fig. 4). Nonretarded materials were active also in hypophysectomized animals thus showing that the substance involved in this zone of the effluent had TSH-like activity; the second zone of activity in the bioassay was strongly retarded (elution volume similar to that of a α-MSH just preceding that of Arg-8-vasopressin), was active in the assay animals with an intact pituitary but not in hypophysectomized animals; its biological activity was inhibited by pretreatment of the assay animals by thyroxine and it stimulated release of TSH from pituitaries incubated in vitro. The conclusion was reached that the materials so purified corresponded to the then hypothetical TSH releasing factor (TRF) of hypothalamic origin. The biological activity (stimulation of the release of TSH) of this preparation was resistant to heating and to incubation with trypsin but was destroyed by 6 N HC1 hydrolysis (110°C, 24 hours). The material with TRF activity obtained by filtration on G-25 considered to be polypeptidic in nature was further purified by ion-exchange chromatography on CM-cellulose (Jutisz et al., 1963). These early observations have been amply confirmed using extracts of the hypothalamus of sheep, pig, beef, rats, guinea pigs, mice, and rabbits and also fragments of human brain origin (reviews in Guillemin, 1967; Schally et al., 1968; Burgus and Guillemin, 1970a). Guillemin et al. (1965) reported purification of ovine TRF from 2 N acetic acid extracts of acetone powder from approximately 80,000 sheep hypothalamic fragments by gel filtration, countercurrent distribution, ion-exchange chromatography on IRC-50 and finally thin-layer chromatography (TLC), obtaining 400 μg of material active at approximately 0.1 μg/dose in vivo. TRF activity was found in a ninhydrin-positive, Pauly-positive zone on TLC; amino acid composition after 6 N HC1 hydrolysis was Lys, His 4, Thr, Ser, Glu 3, Pro 3, Gly, Ala, Met, Leu, Thr. No definitive analysis was claimed nor evidence of homogeneity of the sample prepared, because of the small quantities of material available which simply precluded reliable conclusions with the methodology available.

Fig. 4 Hormonal and releasing factor activities in the effluent of a filtration on Sephadex G-25 of a crude extract of hypothalamic tissues. This is a composite diagram incorporating the results of many laboratories. Showing numbers of exclusion volumes on the abscissa makes the diagram valid for any size column of Sephadex G-25, equilibrated in 0.1 N acetic acid or pyridine-acetate buffer. Only TRF and Arg-8-vasopressin (Amoss, Blackwell, Burgus, and Guillemin, unpublished) have actually been isolated and characterized from these hypothalamic extracts. The other zones and curves shown here correspond thus to biological activities. The question mark after FRF and PIF indicates that some authors claim to have separated these two activities from LRF even at the G-25 filtration stage.

Working with material of porcine origin, Schally et al. (1966a) reported later purification of TRF from the acetic acid extract of 20,000 hypothalamic fragments following gel filtration, phenol extraction, CM-cellulose chromatography, countercurrent distribution and free flow electrophoresis. In all the chromatographic systems, the TRF of porcine origin showed properties similar or identical to those obtained for TRF of bovine or ovine origin. From these studies approximately 900 μg were obtained of a TRF reported to be active at 10 ng in vivo which, upon acid hydrolysis, yielded the amino acids Gly 1, His 5, Pro 5, Thr 6 (later corrected to Glu 6, see Guillemin, 1967), Leu 0.5, Ser 0.6, and Lys 0.4.

From such experiments, it became obvious that huge quantities of hypothalamic tissues would have to be processed to obtain a sufficient amount of material to perform meaningful chemical studies (Guillemin, 1964, 1967). During the next three to four years, two laboratories accepted this challenge, Guillemin and co-workers working with ovine TRF, and Schally and co-workers working primarily with porcine TRF. A number of preliminary reports appeared from both laboratories reporting progress and vicissitudes in the development of various purification schemes and observation on the chemical nature of TRF. They derived from the processing of about 750,000 fragments of ovine hypothalamic tissues and approximately 250,000 fragments of porcine tissues. The final schemes utilized by the two laboratories for the purification of TRF were essentially identical in several purification steps and very similar in others. Schally et al. (1966a) obtained from 100,000 porcine hypothalamic fragments 2.8 mg of TRF claimed to be homogeneous by TLC and thin-layer electrophoresis, and reported to be active at doses smaller than 1 ng in vivo. This material was reported to contain His, Glu, Pro in essentially equimolar ratios accounting for 30% of the weight of the preparation. More recently, the same material was subjected to additional states of partition chromatography, absorption chromatography on charcoal (Burgus and Guillemin, 1970a, p. 505), analytical gel filtration and finally, paper chromatography (Schally et al., 1969a). No changes in the amino acid content or specific biological activity were reported. Another batch of 165,000 porcine hypothalamic fragments purified by the same sequence produced essentially an identical material with about the same amino acid content (33.6%) for a total yield of about 7 mg of a material reported to be homogeneous from 265,000 hypothalamic fragments.

In 1967, we proposed that TRF might not be a simple (homomeric) polypeptide based on the observations that purified preparations of TRF appeared to contain no more than 5-8% of amino acids following HC1 hydrolysis and the observation that pepsin, trypsin, pronase, carboxypep-tidases A and B, or leucine aminopeptidase failed to inactivate the biological activity of TRF (Burgus et al., 1966a). Dansylation followed by two-dimensional chromatoelectrophoresis gave no evidence of a free N-terminus and biological activity was found to be present in a ninhydrin negative zone on TLC or TLE but in a zone that always gave a color reaction to Pauly reagent in all chromatographic and electrophoretic systems tested. We pointed out, however, that infrared and high resolution nuclear magnetic resonance (NMR) spectra of