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Growth Hormone
Chapter 20
Growth Hormone JENS OTTO LUNDE JORGENSEN
Introduction The GH Receptor Neuroregulation of GH Secretion GHRH Somatostatin Other Neurotransmitters Additional GH Secretagogues Feedback Inhibition of GH Secretion GH-Binding Proteins The Temporal Pattern of Circulating GH Sleep Stages Nutritional Status Exercise Chronological Age Body Composition Sex Steroids and Other Hormones Effects of GH on Cell Proliferation and Differentiation Effects of GH on Intermediary Fuel Metabolism
Principles of Medical Biology, Volume 10B Molecular and Cellular Endocrinology, pages 451-466. Copyright 9 1997by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-815-3 451
452 452 454 454 454 455 455 455 456 457 458 458 458 458 460 460 461 462
452
JENSOTTO LUNDE JORGENSEN INTRODUCTION
The gene locus of human pituitary growth hormone (GH) is on the long arm of chromosome 17. Apart from the normal gene (GH-N), a highly homologous variant gene (GH-V) has been identified, which is expressed in the human placenta. The predominant pituitary GH form is a single-chain pure peptide of 191 amino acids with two intramolecular disulfide bridges and a molecular mass of 22 kDa, designated 22K. The second most abundant pituitary GH is a 20-kDa variant (20K), which is a product of alternative splicing of GH-N mRNA. The 22K GH, which can be considered the GH prototype, constitutes approximately 75% of pituitary GH, and the 20K variant constitutes 5-10%. Additional heterogenous GH forms can be detected both within the pituitary and in the circulation. These include deamidated forms of 22K and oligomeric forms of both 22K and 20K, which represent native pituitary posttranslational modifications (Baumann, 1991). In the circulation, GH is associated with two specific binding proteins (GH-BP), as will be discussed below. The biological activity and role of 20K and the other heterogenous GH forms are unclear, wherefore this chapter will focus on the actions of monomeric pituitary 22K.
THE G H RECEPTOR Although the liver contains the highest concentration of GH binding sites, GH receptors have been identified in virtually all other mammalian tissues. The cDNA to the rabbit liver GH receptor has been purified, sequenced, and cloned, and the use of the rabbit cDNA as a probe has led to the cloning of a cDNA encoding the human receptor (Leung et al., 1987). The derived primary structure consists of 620 amino acids (aa), which can be subdivided into an extracellular domain of 246 aa, a single transmembrane region, and an intracellular domain of 350 aa. The isolated receptor has a molecular weight of 130,000, whereas the calculated molecular weight of the aa sequence is 70,000. This difference is only partly explained by glycosylation of the mature receptor. The GH receptor belongs to a large family of receptors which includes those for prolactin, erythropoietin, several interleukins, and the granulocyte macrophage-colony stimulating factor. The members of this so-called cytokine receptor family share homology in their extracellular domains, whereas the intracellular regions show little if any homology (Kelly et al., 1991). Little is known about the binding to and signal transduction of the GH receptor. It has been shown that GH has two sites for binding to the extracellular domain and that signal transduction requires a dimerization such that two receptors bind one GH molecule (Kelly et al., 1993). This dimer complex is thought to trigger tyrosine phosphorylation of cellular proteins through association with a tyrosine kinase, the precise nature of which remains to be identified (Silva et al., 1993).
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JENSOTTO LUNDE Jt~RGENSEN N E U R O R E G U L A T I O N OF G H SECRETION
The regulation of pituitary GH secretion is highly complex, being subject to modulation by both peripheral, hypothalamic and pituitary hormones, growth factors, and metabolites. The most important common path is the secretion of two hypothalamic hormones, one of which stimulates [GH-releasing hormone (GHRH)] and the other inhibits [somatotropin release-inhibiting hormone; somatostatin (SS)] GH secretion. Understanding of this control system is hampered by the fact that the underlying studies have involved a wide variety of species in which both the secretion and action of GH differ considerably.
GHRH Interestingly, GHRH was isolated from human GHRH-producing pancreatic islet cell adenomas that had caused acromegaly (i.e. the clinical syndrome of excess GH production and action, which usually is caused by pituitary GH-producing adenomas) (Rivier et al., 1982). Two GHRH forms have been identified in the human hypothalamus, of which the predominant form is a peptide of 44 aa [GHRH(144)NH2]. Neurons containing GHRH are located in the arcuate hypothalamic nucleus. The GHRH gene is located on chromosome 20. A single mRNA codes for a single preproGHRH, which is subsequently processed to GHRH(1-44)NH 2 or GHRH(1-40)OH. The latter has been shown to be biologically active and characterized by the absence of the four carboxy-terminal aa. The most important regulator of GHRH gene expression appears to be GH although data in humans are lacking. In rodents GH-deficiency is associated with increased GHRH mRNA levels, whereas GH treatment has the opposite effect. The structure of the GHRH receptor remains to be elucidated, whereas the second messenger is known to be cAMP (Frohman et al., 1992). Binding of GHRH to its receptor is associated with a rapid increase in intracellular Ca 2§ levels, but the interrelationship between Ca 2§ levels and cAMP (and other alleged second messengers) is uncertain. GHRH acts on the pituitary to secrete GH and long-term GHRH exposure is associated with enhanced GH gene transcription and GH mRNA levels, as well as hyperplasia of somatotrope cells. By radioimmunoassay GHRH has been measured in several parts of the gastrointestinal tract. The only action attributed to GHRH is stimulation of pituitary GH release.
Somatostatin The predominant form of somatostatin (SS) in the hypothalamus has 14 aa (SS-14) including 2 cysteine residues connected by a disulfide bond (Brazeau et al., 1973). It is notable that the structure of SS-14 is completely homologous in all mammalian species. Neurons containing SS are located in the anterior periventricular region. Additional SS-producing cells are widely distributed in the central
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nervous system and gastrointestinal tract. The SS mRNA codes for a large prepro SS precursor. Several amino terminally extended SS, in particular a 28 aa variant, are highly bioactive. The configuration of the pituitary SS receptor is not known but binding studies using iodinated SS indicate one single type of high affinity and low capacity. The inhibitory effect of SS on GH secretion involves inhibition of adenylyl cyclase activity and reduction in intracellular cAMP concentration (Frohman et al., 1992). On the other hand, SS has also been shown to inhibit GH release during conditions of high intracellular cAMP levels, and there is evidence to suggest that a coupled inhibition of Ca 2§ influx and stimulation of K § efflux is an important effect of SS. It thus seems that SS exerts many of its actions through inhibition of the effects of GHRH. In fact, SS seems to be able to block the actions of most, if not all GH secretagogues. Somatostatin has a broad spectrum of action apart from GH suppression, including inhibition of a large number of other peptide hormones (insulin, glucagon, thyrotropin, gastrointestinal hormones) and modulation of exocrine pancreatic function and intestinal motility and blood flow.
Other Neurotransmitters A large number of neuropeptides or hormones have been shown to either inhibit or stimulate the secretion of pituitary GH. It is generally believed that these substances act ultimately on the release of GHRH or SS. A number of secretagogues act through stimulation of a-adrenergic receptors (clonidine, dopamine), whereas 13-adrenergic agonists (salbutamol) inhibit GH release. Conversely, a-adrenergic blockers (phentolamine) and 13-adrenergic blockers (propranolol) have been shown to inhibit and stimulate GH secretion, respectively. Additional putative GH stimulating neurotransmitters include serotonin, acetylcholine, ?-aminobutyrate, and histamine. The anatomical site of action of these modulators is not fully known but seems to be at the hypothalamic level.
Additional G H Secretagogues Several aa (in particular arginine), insulin-induced hypoglycemia, different endoand exotoxins and some arachidonic acid metabolites have been shown to stimulate GH secretion. Again, these secretagogues are most likely to act through modulation of GHRH or SS release.
Feedback Inhibition of GH Secretion The feedback regulation of GH secretion is complex and involves both GH itself, IGF-I, GHRH and SS (Frohman et al., 1992). GH is subject to auto-feedback inhibition, presumably through hypothalamic stimulation of SS and suppression of GHRH. IGF-I is also a potent inhibitor of GH secretion, which seems to involve both a direct pituitary mechanism in addition to stimulation of SS release. Circulating nutrients, in particular, non-esterified fatty acids (NEFA) and glucose are also
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potent inhibitors of GH secretion. Both hypothalamic and pituitary sites of action have been suggested for NEFA.
GH-BINDING
PROTEINS
It has previously been a dogma that peptide hormones circulate without being bound to specific binding proteins (Berson et al., 1968). Suggestive evidence of a specific binding protein for human GH was produced shortly after the introduction of a radio-immunoassay method (RIA) for GH (Hadden et al., 1964), but was long considered an experimental artefact. The existence of two specific GH-binding proteins (GHBPs) is now firmly established as predicted on the basis of the consistent and highly reproducible gel chromatographic profile of human plasma incubated with iodinated GH (Baumann et al., 1990) (Figure 2). The high-affinity GHBP corresponds to the extracellular region of the GH receptor. Indirect evidence suggesting this is that patients with Laron type dwarfism (LTD), which is known to be associated with absent GH receptors, also lack the high-affinity GHBP. It was also shown that GH receptor antibodies recognized the high-affinity GHBP. Ultimately the cloning of the GH receptor and sequencing of the GHBP led to identification of the latter as the extracellular domain of the GH receptor. In humans, only one GH receptor mRNA has been found, which indicates that the GHBP derives from shedding of the extracellular GH receptor domain. The liver is considered the main source of circulating high-affinity GHBP. It is a single-chain glycoprotein with a molecular weight of 61,000 (Baumann, 1991). A recombinant nonglycosylated GHBP has been shown to possess full biological activity. The binding stoichiometry of GH to GHBP is predicted to be 1"1, which is interesting if one considers that one molecule of GH binds to two GH receptors (see above). The regulation and functional role of the high-affinity GHBP is poorly understood. In humans, the high-affinity GHBP binds approximately 45% of circulating 22K GH. It has been shown to prolong the half-life of circulating GH. It is not subject to circadian fluctuation. The levels rise in early childhood whereafter they remain constant. Comparable levels are reported in males and females. Acromegaly and GH deficiency is not associated with abnormal GHBP levels of any substantial degree. Conditions in which the common denominator is high GH and low IGF-I levels (see below) are usually associated with low GHBP levels. Conversely, obesity, which is characterized by low GH and normal IGF-I levels (and activity), is accompanied by high GHBP levels. Both inhibitory and enhancing effects of the high-affinity GHBP on the actions of GH have been reported, depending on the experimental conditions. The low-affinity GHBP is less well characterized. It has a molecular weight of 100,000 and binds specifically 20K. Levels are normal in LTD. The physiological significance of this remains unclear (Baumann, 1991).
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THE TEMPORAL PATTERN OF CIRCULATING GH The circadian pattern of circulating GH is pulsatile and episodic. The most consistent feature is a large secretory burst shortly after the onset of sleep. Daytime secretion is less predictable but usually occurs a few hours postprandially. The elimination of GH follows first order kinetics with a serum half-time (tl/2) of 15-25 min (JCrgensen,1991). The metabolic clearance rate (MCR) and the distribution volume (DS) of GH are considered relatively constant in normal subjects, hence the changes in serum GH levels mainly reflect changes in the production rate of GH. Although the pharmacokinetic estimates are highly reproducible when measuring GH with conventional RIA, it has been shown that the different GH mass variants may differ in tl/2 (Baumann, 1991). As previously mentioned, secretion of GH is ultimately determined by the hypothalamic tone of GHRH and SS, but the outcome in terms of circadian GH levels is regulated by a number of physiological variables.
458
JENSOTTO LUNDE JORGENSEN Sleep Stages
A temporal association between nocturnal GH secretion and sleep patterns is well documented (Quabbe, 1977). One major nocturnal GH surge usually appears a few hours after onset of sleep which coincides with the first deep sleep stage [Stages 3 and 4, slow wave sleep (SWS)] as determined by electroencephalography (EEG). Artificial manipulation of the usual sleep-waking cycle indicates a causal relationship between SWS and GH secretion (Quabbe, 1977) but it is also evident that nocturnal secretion is subject to additional regulation, particularly by nutritional status.
Nutritional Status That nutritional status is an important determinant of GH secretion stems from two lines of studies. First, nutrients such as fatty acids and glucose suppress GH release, whereas some amino acids stimulate secretion. Second, and perhaps more importantly, is the impact of short-and long-term fasting (Figure 3). Frequent (20 min) blood sampling following one and five days of fasting has shown significant amplification of GH release. The increase was observed both in terms of peak and interpulse levels. It was further observed that fasting disclosed several regular cycles of GH secretion, but still with a major nocturnal component (Ho et al., 1988). The same study reported a significant increase in circulating lipid intermediates, and a significant reduction in blood glucose and serum IGF-I levels during fasting. Finally, the ability of exogenous GHRH to stimulate GH secretion was the same in the fed and fasted states. The underlying regulatory mechanisms responsible for fasting-induced amplification are unclear, but the decreased IGF-I level is one likely candidate. The metabolic role of GH during fasting is addressed on p. 463.
Exercise Both moderate and intensive exercise acutely stimulate GH secretion, whereas the short- and long-term responses to more prolonged exercise training are less well characterized (Howlett, 1987). The triggering events are unclear. Exercise-induced GH release is abolished or even suppressed if the normal rise in body temperature is circumvented. It is also noteworthy that the hormonal and metabolic changes observed during exercise bear many similarities to those observed during fasting and other catabolic states.
Chronological Age A substantial gradual decrease in GH secretion is a function of age (Rudman et al., 1981; Corpas et al., 1993). This is associated with a decline in circulating IGF-I levels. The secretory GH response to both arginine (an alleged inhibitor of SS secretion) and GHRH is blunted. Interestingly, repetitive administration of GHRH
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has been shown to partly restore GH responsiveness in elderly subjects. A possible causal relationship between the decline in GH secretion and the physiological senescent changes in body composition and organ function is presently the subject of intensive research.
Body Composition Obesity is associated with low-circulating GH levels mainly due to decreased production, and, in general, GH secretion correlates positively with lean body mass. GH secretion is restored in obese subjects following weight loss. Furthermore, IGF-I levels are usually normal in obesity. These findings imply that GH hyposecretion is an effect rather than a cause of obesity and underline the fact that nutrients are important stimulators of IGF-I production.
Sex Steroids and Other Hormones In contrast to rodents, the sexual dimorphism of GH secretion is less conspicuous in man. In general, there is a positive correlation between circulating levels of endogenous sex steroids and GH. Exogenous sex steroids enhance GH secretion, which is primarily characterized by a higher mass, but not frequency, of GH secretory pulses. In addition, there is a positive correlation between circulating IGF-I and endogenous levels of sex steroids in both sexes. In the case of estradiol, route of administration seems important: oral administration causes a decrease in IGF-I, whereas transdermally administered estradiol induces an increase in IGF-I. Both routes, however, stimulate GH secretion (Ho et al., 1992). The reason for this discrepancy is unsettled, but may involve a first-pass effect of estradiol on hepatic IGF-I and/or GHBP production. Most studies report GH secretion to be somewhat higher in women than in men, and GH secretion shows subtle changes during the menstrual cycle with high values during ovulation. The impact of corticosteroids on GH secretion is complex. There is evidence of both stimulation and inhibition of GH secretion. This discrepancy partly reflects differences in dosage and duration of corticosteroid administration, as well as differences in evaluation of GH secretion. The interaction between thyroid hormones and GH is also complex. In general, hypothyroidism is associated with low GH levels, whereas GH secretion is increased in hyperthyroidism. There is evidence to suggest that triiodothyronine may directly stimulate pituitary GH gene expression. Furthermore, in many in vitro models triiodothyronine plays a permissive role in the actions of GH including stimulation of IGF-I production. Growth hormone, on the other hand, seems to influence peripheral iodothyronine metabolism by enhancing extrathyroidal conversion of thyroxine to triiodothyronine. The latter is presumed to be the active hormone (JCrgensen et al., 1989).
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EFFECTS OF GH ON CELL PROLIFERATION A N D DIFFERENTIATION As mentioned earlier, little is known about the events that immediately follow activation of the receptor by GH binding. At the cellular or in vitro level a very large variety of mitogenic, growth-promoting and metabolic effects are induced. A few, but definitely not all, of these effects are due to GH-induced production of IGFs. The discovery of IGFs resulted from the observation that costal cartilage from hypophysectomized rats was unresponsive to in vitro addition of GH, whereas marked stimulation of matrix formation and mitogenesis were seen after addition of normal rat serum. Serum from hypophysectomized rats were completely ineffective, whereas serum from hypophysectomized rats treated with GH restored the effects seen with normal serum (Salmon et al., 1957). This ultimately led to the identification of IGF-I and II. It was originally thought that IGFs were solely produced by the liver and that they acted like classic hormones. It was, however, subsequently shown that IGFs are produced by a number of tissues and therefore also act as autocrine and paracrine agents. This local production of IGFs, in turn, makes it difficult to distinguish between the direct GH actions and those of IGF-mediated GH. This fundamental problem has been extensively investigated in relation to the stimulatory effect of GH on longitudinal bone growth both in vitro and in vivo (Isaksson et al., 1987). Administration of pituitary or biosynthetic GH to hypophysectomized rats significantly increases body weight and longitudinal bone growth. By comparison, administration of purified IGF-I induces a much weaker response despite the induction of comparable or higher levels of circulating IGF-I. Furthermore, local administration of GH into the epiphyseal growth plate of hypophysectomized rats has been shown to stimulate longitudinal bone growth on the injected side. Similar results were obtained with unilateral infusion of GH into the femoral artery. These findings imply that the growth-promoting effects of GH do not require hepatic production of IGF-I. Later it was shown that tibias from fetal rats secrete IGF-I into the medium. Furthermore, it is now known that administration of IGF-I antibodies diminishes the growth-promoting effects of locally administered GH on the growth plate. It is also known that GH and IGF-I exert different effects on the formation of epiphyseal chondrocyte cultures (Figure 4). This has been interpreted as indicating that GH directly stimulates the differentiation of prechondrocytes. During subsequent differentiation the cells gradually express the gene encoding for IGF-I leading to local IGF-I production. This local IGF-I, in turn, acts in an autocrine and paracrine manner leading to clonal expansion and maturation of the cells. This hypothesismknown as the dual effector theorymhas been suggested to apply to a variety of mesenchymal precursor cells. Although this hypothesis may appear simplistic, it is becoming evident that the effects of GH on cell proliferation at some stage involves the actions of IGFs.
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EFFECTS OF GH O N INTERMEDIARY FUEL METABOLISM Almost 60 years ago it was shown that hypophysectomized animals were hypersensitive to the actions of insulin and exhibited spontaneous hypoglycemia (Houssay, 1936). Subsequent studies involving the administration of purified pituitary GH in both normal subjects and GH-deficient patients unequivocally resulted in stimulation of lipolysis and hyperglycemia in spite of hyperinsulinemia. Similar results were obtained with biosynthetic GH. This has been taken to mean that the observed actions are due to the GH molecule and not to unidentified pituitary substances (JCrgensen, 1991). While the molecular basis of these actions are not understood, it is known that they do not involve interference with insulin binding to its receptor. In regard to lipolysis, there is evidence suggesting that cAMPdependent phosphorylation of intracellular lipases is one of the early steps ultimately leading to stimulation of the triacylglycerol/fatty acids substrate cycle. Moreover, inhibition of glucose uptake in adipocytes, and presumably striated muscle cells, is thought to involve inhibition of phosphatidylinositol phospholipase C, which is an alleged second messenger for insulin (Mr 1993). Initial studies of the metabolic actions of GH employed pharmacological amounts of GH. More recently, however, studies involving intravenous administration of small GH pulses (70-350 pg) in postabsorptive human subjects showed marked stimulation of lipolysis and suppression of glucose oxidation within 2-3 h (Mr 1993). This work supports the hypothesis that the primary event is the stimulation of lipolysis, and that the ensuing increased availability of fatty acids in tissues leads to substrate competition at the entry of the Krebs tricarboxylic acid cycle, and subsequently, inhibition of glucose oxidation (Randle et al., 1963). It is
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463
noteworthy that in these physiological experiments an effect of GH on hepatic glucose production was not observed. Since plasma glucose levels also remained unaltered, the inference drawn is that physiological GH exposure is also associated with an increase in nonoxidative glucose disposal. The site of this action is not fully known but it may involve glycogen synthesis in hepatic and other splanchnic tissues. High concentrations of GH, either experimentally or as seen in acromegaly, lead to an increase in hepatic glucose output, increased plasma glucose levels, and compensatory hyperinsulinemia (Mr 1993). To comprehend these metabolic actions of GH it is instructive to recall the physiological conditions in which GH is elevated. During daytime GH is usually elevated 2-4 h after food intake, i.e. in the immediate postabsorptive period. Sleep and more prolonged periods of fasting result in even more pronounced GH elevations. Exercise and other conditions of physical stress also elicit GH secretion. By contrast, GH is usually suppressed postprandially. Metabolic homeostasis in conditions of substrate depletion ultimately depends on lipid oxidation, inasmuch as adipose tissue constitutes the major source of energy. To minimize protein breakdown under these conditions also requires regulation (suppression) of glucose oxidation since amino acids provide the only pool for de novo glucose synthesis. Experimental studies of GH administration during short- and long-term fasting have indeed demonstrated both increased lipolysis and absolute or relative nitrogen retention (MOiler, 1993). A major challenge is to reconcile these metabolic GH actions, which seem to play a pivotal role during periods of fuel deprivation, with the previously described stimulating effects of GH on protein synthesis and cell proliferation, which seem to require relative fuel excess, and operate predominantly through induction of hepatic and peripheral IGF-I production. Some support can be obtained by recapitulating the observed changes in circulating IGF-I levels during different conditions. For example, fasting results in a progressive decline in IGF-I levels (Figure 5). It is likely that this is the main trigger of GH secretion by a positive feedback mechanism. Thus, fasting blocks IGF-I mediated GH effects and at the same time stimulates the direct lipolytic actions of GH. This open-loop regulation may also explain why the elevation in lipid intermediates does not suppress GH secretion during fasting as opposed to the well described suppressive effect of free fatty acid infusion on GH secretion during short-term experimental conditions. This model, however, does not address how GH acts to stimulate IGF-I production and action. It seems obvious that this axis of GH action requires both elevated GH levels and an adequate fuel supply. It will be remembered that in nonfasting subjects total serum IGF-I levels do not exhibit significant fluctuations (JCrgensen, 1991). The following hypothesis may now be offered: Meals of sufficient caloric content which are separated by periods without food intake ensure fuel supply and elicit late postprandial GH secretion. This
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Figure 5. Simplified diagram of IGF-I mediated versus direct effects of GH during conditions of adequate nutritional supply (A) and during fasting or other catabolic states (B).
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provides both fuel supply and GH elevations to stimulate synthesis of IGF-I and the major IGF binding protein IGFBP-3. During the late postprandial period both GH and insulin levels are elevated. Elevated insulin levels suppress the levels of IGFBP-1, another IGF-binding protein which binds a smaller fraction of IGF-I with a shorter half-life, thereby providing more free IGF-I to act on target tissues. In the postabsorptive state, IGF-I synthesis is blocked and low insulin levels ensure high IGFBP-1 levels, which inhibits IGF-I bioactivity. This in turn
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elicits GH secretion, which predominantly acts directly to promote lipolysis and decrease glucose oxidation. It should be stressed that this is only a hypothesis. Nonetheless, it is fascinating that there were qualitatively similar speculations 30 years ago. Knowledge then about IGFs and IGFBPs was limited. Rabinowitz and his colleagues (1963) write: We propose that, during a day, metabolism is dominated alternately by the action of insulin, or of human growth hormone, or of the combined effects of the two, in a three phased cycle determined by the intake of food. Exposure to insulin in the immediate postprandial period encourages storage of carbohydrate and fat, exposure to human growth hormone plus insulin in the delayed postprandial period encourages protein synthesis and exposure to human growth hormone in the remote postprandial phase encourages mobilization and peripheral oxidation of fat and retards translocation of glucose in muscle and adipose tissue.
REFERENCES Argente, J., Garcia-Sigura, L.M., Pozo, J., & Chowen, J.A. (1996). Growth hormone-releasing peptides. Hormone Res. 46, 155-159. Baumann, G., & Shaw, M.A. (1990). Plasma transport of the 20,000 dalton variant of human growth (20K):evidence for a 20K-specific binding site. J. Clin. Endocrinol. Metab. 71, 1339-1343. Baumann, G. (1991). Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr. Rev. 12, 424--449. Berson, S.A., & Yalow, R.S. (1968). Peptide hormones in plasma. Harvey Lecture 62, 107-163. Brazeau, P., Vale, W., & Burgus, R. (1973). Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 178, 77-79. Corpas, E., Harman, S.M., & Blackman, M.R. (1993). Human growth hormone and human aging. Endocr. Rev. 14, 20-39. Frohman, L.A., Downs, T.R., & Chomczynski, P. (1992). Regulation of growth hormone secretion. Front. Neuroendocrinol. 13, 344-405. Ho, K.Y., & Weissberger, A.J. (1992). Impact of short-term estrogen administration on growth hormone secretion and action: Distinct route-dependent effects on connective and bone tissue metabolism. J. Bone. Miner. Res.7, 821-827. Ho, K.Y., Veldhuis, J.D., Johnson, M.L., Furlanetto, R., Evans, W.S., Alberti, K.G.M.M., & Thorner, M.O. (1988). Fasting enhances growth hormone secretion and amplifies the complex rhythms of growth hormone secretion in man. J. Clin. Invest. 81,968-975. Howlett, T.A. (1987). Hormonal responses to exercise and training: a short review. Clin. Endocrinol. 26, 723-742. Hadden, D.R., & Prout, T.E. (1964). A growth hormone binding protein in normal human serum. Nature. 202, 1342-1343. Houssay, B.A. (1936). The hypophysis and metabolism. N. Engl. J. Med. 214, 961-986. Isaksson, O.G., Lindahl, A., Nilsson, A., & Isgaard, J. (1987). Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr. Rev. 8, 426-438. JCrgensen, J.O.L., Pedersen, S.A., Laurberg, P., Weeke, J., Skakkeb~ek, N.E., & Christiansen, J.S. (1989). Effects of growth hormone therapy on thyroid function of growth hormone-deficient adults with and without concomitant thyroxine-substituted central hypothyroidism. J. Clin. Endocrinol. Metab. 69, 1127-1132. JCrgensen, J.O.L. (1991). Human growth hormone replacement therapy: pharmacological and clinical aspects. Endocr. Rev. 12, 189-207.
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Kelly, EA., Djiane, J., Postel-Vinay, M-C., & Edery, M. ( 1991). The prolactin/growth hormone receptor family. Endocr. Rev. 12, 235-251. Kelly, EA., Ali, S., Rozakis, M., Goujon, L., Nagano, M., Pellegrini, I., Gould, D., Djiane J., Edery, M., Finidori, J., & Postel-Vinay, M.C. (1993). The growth hormone/prolactin receptor family. Rec. Progr. Horm. Res. 48, 123-164. Leung, D.W., Spencer, S.A., Cachianes, G., Hammonds, R.G., Collins, C., Henzel, W.J., Barnard, R., Waters, M.J., & Wood, W.I. (1987). Growth hormone receptor and serum binding protein:purification, cloning and expression. Nature (London) 330, 537-543. Mr N. (1993). In: Growth Hormone and Insulin-like Growth Factor I (Flyvbjerg, A., Orskov, H., & Alberti, K.G.M.M., Eds.), pp. 77-108, Wiley & Sons, New York. Rabinowitz, D., & Zierler, K.L. (1963). A metabolic regulating device based on the actions of human growth hormone and of insulin, singly and together, on the human forearm. Nature 199, 913-914. Randle, El., Garland, EB., Hales, C.N., & Newsholme, E.A. (1963). The glucose-fatty acid cycle:its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet i, 785-789. Rivier, J., Spiess, J., Thorner, M., & Vale, W.S. (1982). Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature (London) 300, 276-278. Ross, R.J.M., & Savage, M.O. (1996). Growth Hormone Resistance. Bailiere Tindall, London. Rudman, D., Vintner, M.H., Rogers, C.M., Lubin, M.E, Fleming, G.H., & Raymond, P.B. (1981). Impaired growth hormone secretion in the adult population. Relation to age and adiposity. J. Clin. Invest. 67, 1361-1369. Salmon, W.D., & Daughaday, W.H. (1957). A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J. Lab. Clin. Med. 49, 825-836. Scanlon, M.E, Issa, B.G., & Dieguez, C. (1996). Regulation of growth hormone secretion. Hormone Res. 46, 149-154. Silva, C.M., Weber, M.J., & Thorner, M.O. (1993). Stimulation of tyrosine phosphorylation in human cells by activation of the growth hormone receptor. Endocrinol. 132, 101-108. Quabbe, H.J. (1977). Chronobiology of growth hormone secretion. Chronobiologia. 4, 217-246.
RECOMMENDED READINGS Ross, R.J.M., & Savage, M.O. (1996). Growth Hormone Resistance. Bailliere Tindall, London. Scanlon, M.E, Issa, B.G., & Dieguez, C. (1996). Regulation of growth hormone secretion. Hormone Res. 46, 149-154. Spencer, E.M. (1991). Modern Concepts of Insulinlike Growth Factors. Elsevier, New York.
Growth Hormone JENS OTTO LUNDE JORGENSEN
Introduction The GH Receptor Neuroregulation of GH Secretion GHRH Somatostatin Other Neurotransmitters Additional GH Secretagogues Feedback Inhibition of GH Secretion GH-Binding Proteins The Temporal Pattern of Circulating GH Sleep Stages Nutritional Status Exercise Chronological Age Body Composition Sex Steroids and Other Hormones Effects of GH on Cell Proliferation and Differentiation Effects of GH on Intermediary Fuel Metabolism
Principles of Medical Biology, Volume 10B Molecular and Cellular Endocrinology, pages 451-466. Copyright 9 1997by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-815-3 451
452 452 454 454 454 455 455 455 456 457 458 458 458 458 460 460 461 462
452
JENSOTTO LUNDE JORGENSEN INTRODUCTION
The gene locus of human pituitary growth hormone (GH) is on the long arm of chromosome 17. Apart from the normal gene (GH-N), a highly homologous variant gene (GH-V) has been identified, which is expressed in the human placenta. The predominant pituitary GH form is a single-chain pure peptide of 191 amino acids with two intramolecular disulfide bridges and a molecular mass of 22 kDa, designated 22K. The second most abundant pituitary GH is a 20-kDa variant (20K), which is a product of alternative splicing of GH-N mRNA. The 22K GH, which can be considered the GH prototype, constitutes approximately 75% of pituitary GH, and the 20K variant constitutes 5-10%. Additional heterogenous GH forms can be detected both within the pituitary and in the circulation. These include deamidated forms of 22K and oligomeric forms of both 22K and 20K, which represent native pituitary posttranslational modifications (Baumann, 1991). In the circulation, GH is associated with two specific binding proteins (GH-BP), as will be discussed below. The biological activity and role of 20K and the other heterogenous GH forms are unclear, wherefore this chapter will focus on the actions of monomeric pituitary 22K.
THE G H RECEPTOR Although the liver contains the highest concentration of GH binding sites, GH receptors have been identified in virtually all other mammalian tissues. The cDNA to the rabbit liver GH receptor has been purified, sequenced, and cloned, and the use of the rabbit cDNA as a probe has led to the cloning of a cDNA encoding the human receptor (Leung et al., 1987). The derived primary structure consists of 620 amino acids (aa), which can be subdivided into an extracellular domain of 246 aa, a single transmembrane region, and an intracellular domain of 350 aa. The isolated receptor has a molecular weight of 130,000, whereas the calculated molecular weight of the aa sequence is 70,000. This difference is only partly explained by glycosylation of the mature receptor. The GH receptor belongs to a large family of receptors which includes those for prolactin, erythropoietin, several interleukins, and the granulocyte macrophage-colony stimulating factor. The members of this so-called cytokine receptor family share homology in their extracellular domains, whereas the intracellular regions show little if any homology (Kelly et al., 1991). Little is known about the binding to and signal transduction of the GH receptor. It has been shown that GH has two sites for binding to the extracellular domain and that signal transduction requires a dimerization such that two receptors bind one GH molecule (Kelly et al., 1993). This dimer complex is thought to trigger tyrosine phosphorylation of cellular proteins through association with a tyrosine kinase, the precise nature of which remains to be identified (Silva et al., 1993).
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JENSOTTO LUNDE Jt~RGENSEN N E U R O R E G U L A T I O N OF G H SECRETION
The regulation of pituitary GH secretion is highly complex, being subject to modulation by both peripheral, hypothalamic and pituitary hormones, growth factors, and metabolites. The most important common path is the secretion of two hypothalamic hormones, one of which stimulates [GH-releasing hormone (GHRH)] and the other inhibits [somatotropin release-inhibiting hormone; somatostatin (SS)] GH secretion. Understanding of this control system is hampered by the fact that the underlying studies have involved a wide variety of species in which both the secretion and action of GH differ considerably.
GHRH Interestingly, GHRH was isolated from human GHRH-producing pancreatic islet cell adenomas that had caused acromegaly (i.e. the clinical syndrome of excess GH production and action, which usually is caused by pituitary GH-producing adenomas) (Rivier et al., 1982). Two GHRH forms have been identified in the human hypothalamus, of which the predominant form is a peptide of 44 aa [GHRH(144)NH2]. Neurons containing GHRH are located in the arcuate hypothalamic nucleus. The GHRH gene is located on chromosome 20. A single mRNA codes for a single preproGHRH, which is subsequently processed to GHRH(1-44)NH 2 or GHRH(1-40)OH. The latter has been shown to be biologically active and characterized by the absence of the four carboxy-terminal aa. The most important regulator of GHRH gene expression appears to be GH although data in humans are lacking. In rodents GH-deficiency is associated with increased GHRH mRNA levels, whereas GH treatment has the opposite effect. The structure of the GHRH receptor remains to be elucidated, whereas the second messenger is known to be cAMP (Frohman et al., 1992). Binding of GHRH to its receptor is associated with a rapid increase in intracellular Ca 2§ levels, but the interrelationship between Ca 2§ levels and cAMP (and other alleged second messengers) is uncertain. GHRH acts on the pituitary to secrete GH and long-term GHRH exposure is associated with enhanced GH gene transcription and GH mRNA levels, as well as hyperplasia of somatotrope cells. By radioimmunoassay GHRH has been measured in several parts of the gastrointestinal tract. The only action attributed to GHRH is stimulation of pituitary GH release.
Somatostatin The predominant form of somatostatin (SS) in the hypothalamus has 14 aa (SS-14) including 2 cysteine residues connected by a disulfide bond (Brazeau et al., 1973). It is notable that the structure of SS-14 is completely homologous in all mammalian species. Neurons containing SS are located in the anterior periventricular region. Additional SS-producing cells are widely distributed in the central
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nervous system and gastrointestinal tract. The SS mRNA codes for a large prepro SS precursor. Several amino terminally extended SS, in particular a 28 aa variant, are highly bioactive. The configuration of the pituitary SS receptor is not known but binding studies using iodinated SS indicate one single type of high affinity and low capacity. The inhibitory effect of SS on GH secretion involves inhibition of adenylyl cyclase activity and reduction in intracellular cAMP concentration (Frohman et al., 1992). On the other hand, SS has also been shown to inhibit GH release during conditions of high intracellular cAMP levels, and there is evidence to suggest that a coupled inhibition of Ca 2§ influx and stimulation of K § efflux is an important effect of SS. It thus seems that SS exerts many of its actions through inhibition of the effects of GHRH. In fact, SS seems to be able to block the actions of most, if not all GH secretagogues. Somatostatin has a broad spectrum of action apart from GH suppression, including inhibition of a large number of other peptide hormones (insulin, glucagon, thyrotropin, gastrointestinal hormones) and modulation of exocrine pancreatic function and intestinal motility and blood flow.
Other Neurotransmitters A large number of neuropeptides or hormones have been shown to either inhibit or stimulate the secretion of pituitary GH. It is generally believed that these substances act ultimately on the release of GHRH or SS. A number of secretagogues act through stimulation of a-adrenergic receptors (clonidine, dopamine), whereas 13-adrenergic agonists (salbutamol) inhibit GH release. Conversely, a-adrenergic blockers (phentolamine) and 13-adrenergic blockers (propranolol) have been shown to inhibit and stimulate GH secretion, respectively. Additional putative GH stimulating neurotransmitters include serotonin, acetylcholine, ?-aminobutyrate, and histamine. The anatomical site of action of these modulators is not fully known but seems to be at the hypothalamic level.
Additional G H Secretagogues Several aa (in particular arginine), insulin-induced hypoglycemia, different endoand exotoxins and some arachidonic acid metabolites have been shown to stimulate GH secretion. Again, these secretagogues are most likely to act through modulation of GHRH or SS release.
Feedback Inhibition of GH Secretion The feedback regulation of GH secretion is complex and involves both GH itself, IGF-I, GHRH and SS (Frohman et al., 1992). GH is subject to auto-feedback inhibition, presumably through hypothalamic stimulation of SS and suppression of GHRH. IGF-I is also a potent inhibitor of GH secretion, which seems to involve both a direct pituitary mechanism in addition to stimulation of SS release. Circulating nutrients, in particular, non-esterified fatty acids (NEFA) and glucose are also
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potent inhibitors of GH secretion. Both hypothalamic and pituitary sites of action have been suggested for NEFA.
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It has previously been a dogma that peptide hormones circulate without being bound to specific binding proteins (Berson et al., 1968). Suggestive evidence of a specific binding protein for human GH was produced shortly after the introduction of a radio-immunoassay method (RIA) for GH (Hadden et al., 1964), but was long considered an experimental artefact. The existence of two specific GH-binding proteins (GHBPs) is now firmly established as predicted on the basis of the consistent and highly reproducible gel chromatographic profile of human plasma incubated with iodinated GH (Baumann et al., 1990) (Figure 2). The high-affinity GHBP corresponds to the extracellular region of the GH receptor. Indirect evidence suggesting this is that patients with Laron type dwarfism (LTD), which is known to be associated with absent GH receptors, also lack the high-affinity GHBP. It was also shown that GH receptor antibodies recognized the high-affinity GHBP. Ultimately the cloning of the GH receptor and sequencing of the GHBP led to identification of the latter as the extracellular domain of the GH receptor. In humans, only one GH receptor mRNA has been found, which indicates that the GHBP derives from shedding of the extracellular GH receptor domain. The liver is considered the main source of circulating high-affinity GHBP. It is a single-chain glycoprotein with a molecular weight of 61,000 (Baumann, 1991). A recombinant nonglycosylated GHBP has been shown to possess full biological activity. The binding stoichiometry of GH to GHBP is predicted to be 1"1, which is interesting if one considers that one molecule of GH binds to two GH receptors (see above). The regulation and functional role of the high-affinity GHBP is poorly understood. In humans, the high-affinity GHBP binds approximately 45% of circulating 22K GH. It has been shown to prolong the half-life of circulating GH. It is not subject to circadian fluctuation. The levels rise in early childhood whereafter they remain constant. Comparable levels are reported in males and females. Acromegaly and GH deficiency is not associated with abnormal GHBP levels of any substantial degree. Conditions in which the common denominator is high GH and low IGF-I levels (see below) are usually associated with low GHBP levels. Conversely, obesity, which is characterized by low GH and normal IGF-I levels (and activity), is accompanied by high GHBP levels. Both inhibitory and enhancing effects of the high-affinity GHBP on the actions of GH have been reported, depending on the experimental conditions. The low-affinity GHBP is less well characterized. It has a molecular weight of 100,000 and binds specifically 20K. Levels are normal in LTD. The physiological significance of this remains unclear (Baumann, 1991).
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THE TEMPORAL PATTERN OF CIRCULATING GH The circadian pattern of circulating GH is pulsatile and episodic. The most consistent feature is a large secretory burst shortly after the onset of sleep. Daytime secretion is less predictable but usually occurs a few hours postprandially. The elimination of GH follows first order kinetics with a serum half-time (tl/2) of 15-25 min (JCrgensen,1991). The metabolic clearance rate (MCR) and the distribution volume (DS) of GH are considered relatively constant in normal subjects, hence the changes in serum GH levels mainly reflect changes in the production rate of GH. Although the pharmacokinetic estimates are highly reproducible when measuring GH with conventional RIA, it has been shown that the different GH mass variants may differ in tl/2 (Baumann, 1991). As previously mentioned, secretion of GH is ultimately determined by the hypothalamic tone of GHRH and SS, but the outcome in terms of circadian GH levels is regulated by a number of physiological variables.
458
JENSOTTO LUNDE JORGENSEN Sleep Stages
A temporal association between nocturnal GH secretion and sleep patterns is well documented (Quabbe, 1977). One major nocturnal GH surge usually appears a few hours after onset of sleep which coincides with the first deep sleep stage [Stages 3 and 4, slow wave sleep (SWS)] as determined by electroencephalography (EEG). Artificial manipulation of the usual sleep-waking cycle indicates a causal relationship between SWS and GH secretion (Quabbe, 1977) but it is also evident that nocturnal secretion is subject to additional regulation, particularly by nutritional status.
Nutritional Status That nutritional status is an important determinant of GH secretion stems from two lines of studies. First, nutrients such as fatty acids and glucose suppress GH release, whereas some amino acids stimulate secretion. Second, and perhaps more importantly, is the impact of short-and long-term fasting (Figure 3). Frequent (20 min) blood sampling following one and five days of fasting has shown significant amplification of GH release. The increase was observed both in terms of peak and interpulse levels. It was further observed that fasting disclosed several regular cycles of GH secretion, but still with a major nocturnal component (Ho et al., 1988). The same study reported a significant increase in circulating lipid intermediates, and a significant reduction in blood glucose and serum IGF-I levels during fasting. Finally, the ability of exogenous GHRH to stimulate GH secretion was the same in the fed and fasted states. The underlying regulatory mechanisms responsible for fasting-induced amplification are unclear, but the decreased IGF-I level is one likely candidate. The metabolic role of GH during fasting is addressed on p. 463.
Exercise Both moderate and intensive exercise acutely stimulate GH secretion, whereas the short- and long-term responses to more prolonged exercise training are less well characterized (Howlett, 1987). The triggering events are unclear. Exercise-induced GH release is abolished or even suppressed if the normal rise in body temperature is circumvented. It is also noteworthy that the hormonal and metabolic changes observed during exercise bear many similarities to those observed during fasting and other catabolic states.
Chronological Age A substantial gradual decrease in GH secretion is a function of age (Rudman et al., 1981; Corpas et al., 1993). This is associated with a decline in circulating IGF-I levels. The secretory GH response to both arginine (an alleged inhibitor of SS secretion) and GHRH is blunted. Interestingly, repetitive administration of GHRH
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has been shown to partly restore GH responsiveness in elderly subjects. A possible causal relationship between the decline in GH secretion and the physiological senescent changes in body composition and organ function is presently the subject of intensive research.
Body Composition Obesity is associated with low-circulating GH levels mainly due to decreased production, and, in general, GH secretion correlates positively with lean body mass. GH secretion is restored in obese subjects following weight loss. Furthermore, IGF-I levels are usually normal in obesity. These findings imply that GH hyposecretion is an effect rather than a cause of obesity and underline the fact that nutrients are important stimulators of IGF-I production.
Sex Steroids and Other Hormones In contrast to rodents, the sexual dimorphism of GH secretion is less conspicuous in man. In general, there is a positive correlation between circulating levels of endogenous sex steroids and GH. Exogenous sex steroids enhance GH secretion, which is primarily characterized by a higher mass, but not frequency, of GH secretory pulses. In addition, there is a positive correlation between circulating IGF-I and endogenous levels of sex steroids in both sexes. In the case of estradiol, route of administration seems important: oral administration causes a decrease in IGF-I, whereas transdermally administered estradiol induces an increase in IGF-I. Both routes, however, stimulate GH secretion (Ho et al., 1992). The reason for this discrepancy is unsettled, but may involve a first-pass effect of estradiol on hepatic IGF-I and/or GHBP production. Most studies report GH secretion to be somewhat higher in women than in men, and GH secretion shows subtle changes during the menstrual cycle with high values during ovulation. The impact of corticosteroids on GH secretion is complex. There is evidence of both stimulation and inhibition of GH secretion. This discrepancy partly reflects differences in dosage and duration of corticosteroid administration, as well as differences in evaluation of GH secretion. The interaction between thyroid hormones and GH is also complex. In general, hypothyroidism is associated with low GH levels, whereas GH secretion is increased in hyperthyroidism. There is evidence to suggest that triiodothyronine may directly stimulate pituitary GH gene expression. Furthermore, in many in vitro models triiodothyronine plays a permissive role in the actions of GH including stimulation of IGF-I production. Growth hormone, on the other hand, seems to influence peripheral iodothyronine metabolism by enhancing extrathyroidal conversion of thyroxine to triiodothyronine. The latter is presumed to be the active hormone (JCrgensen et al., 1989).
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EFFECTS OF GH ON CELL PROLIFERATION A N D DIFFERENTIATION As mentioned earlier, little is known about the events that immediately follow activation of the receptor by GH binding. At the cellular or in vitro level a very large variety of mitogenic, growth-promoting and metabolic effects are induced. A few, but definitely not all, of these effects are due to GH-induced production of IGFs. The discovery of IGFs resulted from the observation that costal cartilage from hypophysectomized rats was unresponsive to in vitro addition of GH, whereas marked stimulation of matrix formation and mitogenesis were seen after addition of normal rat serum. Serum from hypophysectomized rats were completely ineffective, whereas serum from hypophysectomized rats treated with GH restored the effects seen with normal serum (Salmon et al., 1957). This ultimately led to the identification of IGF-I and II. It was originally thought that IGFs were solely produced by the liver and that they acted like classic hormones. It was, however, subsequently shown that IGFs are produced by a number of tissues and therefore also act as autocrine and paracrine agents. This local production of IGFs, in turn, makes it difficult to distinguish between the direct GH actions and those of IGF-mediated GH. This fundamental problem has been extensively investigated in relation to the stimulatory effect of GH on longitudinal bone growth both in vitro and in vivo (Isaksson et al., 1987). Administration of pituitary or biosynthetic GH to hypophysectomized rats significantly increases body weight and longitudinal bone growth. By comparison, administration of purified IGF-I induces a much weaker response despite the induction of comparable or higher levels of circulating IGF-I. Furthermore, local administration of GH into the epiphyseal growth plate of hypophysectomized rats has been shown to stimulate longitudinal bone growth on the injected side. Similar results were obtained with unilateral infusion of GH into the femoral artery. These findings imply that the growth-promoting effects of GH do not require hepatic production of IGF-I. Later it was shown that tibias from fetal rats secrete IGF-I into the medium. Furthermore, it is now known that administration of IGF-I antibodies diminishes the growth-promoting effects of locally administered GH on the growth plate. It is also known that GH and IGF-I exert different effects on the formation of epiphyseal chondrocyte cultures (Figure 4). This has been interpreted as indicating that GH directly stimulates the differentiation of prechondrocytes. During subsequent differentiation the cells gradually express the gene encoding for IGF-I leading to local IGF-I production. This local IGF-I, in turn, acts in an autocrine and paracrine manner leading to clonal expansion and maturation of the cells. This hypothesismknown as the dual effector theorymhas been suggested to apply to a variety of mesenchymal precursor cells. Although this hypothesis may appear simplistic, it is becoming evident that the effects of GH on cell proliferation at some stage involves the actions of IGFs.
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EFFECTS OF GH O N INTERMEDIARY FUEL METABOLISM Almost 60 years ago it was shown that hypophysectomized animals were hypersensitive to the actions of insulin and exhibited spontaneous hypoglycemia (Houssay, 1936). Subsequent studies involving the administration of purified pituitary GH in both normal subjects and GH-deficient patients unequivocally resulted in stimulation of lipolysis and hyperglycemia in spite of hyperinsulinemia. Similar results were obtained with biosynthetic GH. This has been taken to mean that the observed actions are due to the GH molecule and not to unidentified pituitary substances (JCrgensen, 1991). While the molecular basis of these actions are not understood, it is known that they do not involve interference with insulin binding to its receptor. In regard to lipolysis, there is evidence suggesting that cAMPdependent phosphorylation of intracellular lipases is one of the early steps ultimately leading to stimulation of the triacylglycerol/fatty acids substrate cycle. Moreover, inhibition of glucose uptake in adipocytes, and presumably striated muscle cells, is thought to involve inhibition of phosphatidylinositol phospholipase C, which is an alleged second messenger for insulin (Mr 1993). Initial studies of the metabolic actions of GH employed pharmacological amounts of GH. More recently, however, studies involving intravenous administration of small GH pulses (70-350 pg) in postabsorptive human subjects showed marked stimulation of lipolysis and suppression of glucose oxidation within 2-3 h (Mr 1993). This work supports the hypothesis that the primary event is the stimulation of lipolysis, and that the ensuing increased availability of fatty acids in tissues leads to substrate competition at the entry of the Krebs tricarboxylic acid cycle, and subsequently, inhibition of glucose oxidation (Randle et al., 1963). It is
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noteworthy that in these physiological experiments an effect of GH on hepatic glucose production was not observed. Since plasma glucose levels also remained unaltered, the inference drawn is that physiological GH exposure is also associated with an increase in nonoxidative glucose disposal. The site of this action is not fully known but it may involve glycogen synthesis in hepatic and other splanchnic tissues. High concentrations of GH, either experimentally or as seen in acromegaly, lead to an increase in hepatic glucose output, increased plasma glucose levels, and compensatory hyperinsulinemia (Mr 1993). To comprehend these metabolic actions of GH it is instructive to recall the physiological conditions in which GH is elevated. During daytime GH is usually elevated 2-4 h after food intake, i.e. in the immediate postabsorptive period. Sleep and more prolonged periods of fasting result in even more pronounced GH elevations. Exercise and other conditions of physical stress also elicit GH secretion. By contrast, GH is usually suppressed postprandially. Metabolic homeostasis in conditions of substrate depletion ultimately depends on lipid oxidation, inasmuch as adipose tissue constitutes the major source of energy. To minimize protein breakdown under these conditions also requires regulation (suppression) of glucose oxidation since amino acids provide the only pool for de novo glucose synthesis. Experimental studies of GH administration during short- and long-term fasting have indeed demonstrated both increased lipolysis and absolute or relative nitrogen retention (MOiler, 1993). A major challenge is to reconcile these metabolic GH actions, which seem to play a pivotal role during periods of fuel deprivation, with the previously described stimulating effects of GH on protein synthesis and cell proliferation, which seem to require relative fuel excess, and operate predominantly through induction of hepatic and peripheral IGF-I production. Some support can be obtained by recapitulating the observed changes in circulating IGF-I levels during different conditions. For example, fasting results in a progressive decline in IGF-I levels (Figure 5). It is likely that this is the main trigger of GH secretion by a positive feedback mechanism. Thus, fasting blocks IGF-I mediated GH effects and at the same time stimulates the direct lipolytic actions of GH. This open-loop regulation may also explain why the elevation in lipid intermediates does not suppress GH secretion during fasting as opposed to the well described suppressive effect of free fatty acid infusion on GH secretion during short-term experimental conditions. This model, however, does not address how GH acts to stimulate IGF-I production and action. It seems obvious that this axis of GH action requires both elevated GH levels and an adequate fuel supply. It will be remembered that in nonfasting subjects total serum IGF-I levels do not exhibit significant fluctuations (JCrgensen, 1991). The following hypothesis may now be offered: Meals of sufficient caloric content which are separated by periods without food intake ensure fuel supply and elicit late postprandial GH secretion. This
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A GH, Lipolysis
Fuelsupply
~ Glucoseoxidation IGF-I
Protein~anabolism
Protein sparing
B Ib=
GH
, •Lipolysis ~ Glucoseoxidation s?
~ IGF-I
Protein sparing
Figure 5. Simplified diagram of IGF-I mediated versus direct effects of GH during conditions of adequate nutritional supply (A) and during fasting or other catabolic states (B).
2.
3.
provides both fuel supply and GH elevations to stimulate synthesis of IGF-I and the major IGF binding protein IGFBP-3. During the late postprandial period both GH and insulin levels are elevated. Elevated insulin levels suppress the levels of IGFBP-1, another IGF-binding protein which binds a smaller fraction of IGF-I with a shorter half-life, thereby providing more free IGF-I to act on target tissues. In the postabsorptive state, IGF-I synthesis is blocked and low insulin levels ensure high IGFBP-1 levels, which inhibits IGF-I bioactivity. This in turn
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elicits GH secretion, which predominantly acts directly to promote lipolysis and decrease glucose oxidation. It should be stressed that this is only a hypothesis. Nonetheless, it is fascinating that there were qualitatively similar speculations 30 years ago. Knowledge then about IGFs and IGFBPs was limited. Rabinowitz and his colleagues (1963) write: We propose that, during a day, metabolism is dominated alternately by the action of insulin, or of human growth hormone, or of the combined effects of the two, in a three phased cycle determined by the intake of food. Exposure to insulin in the immediate postprandial period encourages storage of carbohydrate and fat, exposure to human growth hormone plus insulin in the delayed postprandial period encourages protein synthesis and exposure to human growth hormone in the remote postprandial phase encourages mobilization and peripheral oxidation of fat and retards translocation of glucose in muscle and adipose tissue.
REFERENCES Argente, J., Garcia-Sigura, L.M., Pozo, J., & Chowen, J.A. (1996). Growth hormone-releasing peptides. Hormone Res. 46, 155-159. Baumann, G., & Shaw, M.A. (1990). Plasma transport of the 20,000 dalton variant of human growth (20K):evidence for a 20K-specific binding site. J. Clin. Endocrinol. Metab. 71, 1339-1343. Baumann, G. (1991). Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr. Rev. 12, 424--449. Berson, S.A., & Yalow, R.S. (1968). Peptide hormones in plasma. Harvey Lecture 62, 107-163. Brazeau, P., Vale, W., & Burgus, R. (1973). Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 178, 77-79. Corpas, E., Harman, S.M., & Blackman, M.R. (1993). Human growth hormone and human aging. Endocr. Rev. 14, 20-39. Frohman, L.A., Downs, T.R., & Chomczynski, P. (1992). Regulation of growth hormone secretion. Front. Neuroendocrinol. 13, 344-405. Ho, K.Y., & Weissberger, A.J. (1992). Impact of short-term estrogen administration on growth hormone secretion and action: Distinct route-dependent effects on connective and bone tissue metabolism. J. Bone. Miner. Res.7, 821-827. Ho, K.Y., Veldhuis, J.D., Johnson, M.L., Furlanetto, R., Evans, W.S., Alberti, K.G.M.M., & Thorner, M.O. (1988). Fasting enhances growth hormone secretion and amplifies the complex rhythms of growth hormone secretion in man. J. Clin. Invest. 81,968-975. Howlett, T.A. (1987). Hormonal responses to exercise and training: a short review. Clin. Endocrinol. 26, 723-742. Hadden, D.R., & Prout, T.E. (1964). A growth hormone binding protein in normal human serum. Nature. 202, 1342-1343. Houssay, B.A. (1936). The hypophysis and metabolism. N. Engl. J. Med. 214, 961-986. Isaksson, O.G., Lindahl, A., Nilsson, A., & Isgaard, J. (1987). Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr. Rev. 8, 426-438. JCrgensen, J.O.L., Pedersen, S.A., Laurberg, P., Weeke, J., Skakkeb~ek, N.E., & Christiansen, J.S. (1989). Effects of growth hormone therapy on thyroid function of growth hormone-deficient adults with and without concomitant thyroxine-substituted central hypothyroidism. J. Clin. Endocrinol. Metab. 69, 1127-1132. JCrgensen, J.O.L. (1991). Human growth hormone replacement therapy: pharmacological and clinical aspects. Endocr. Rev. 12, 189-207.
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Kelly, EA., Djiane, J., Postel-Vinay, M-C., & Edery, M. ( 1991). The prolactin/growth hormone receptor family. Endocr. Rev. 12, 235-251. Kelly, EA., Ali, S., Rozakis, M., Goujon, L., Nagano, M., Pellegrini, I., Gould, D., Djiane J., Edery, M., Finidori, J., & Postel-Vinay, M.C. (1993). The growth hormone/prolactin receptor family. Rec. Progr. Horm. Res. 48, 123-164. Leung, D.W., Spencer, S.A., Cachianes, G., Hammonds, R.G., Collins, C., Henzel, W.J., Barnard, R., Waters, M.J., & Wood, W.I. (1987). Growth hormone receptor and serum binding protein:purification, cloning and expression. Nature (London) 330, 537-543. Mr N. (1993). In: Growth Hormone and Insulin-like Growth Factor I (Flyvbjerg, A., Orskov, H., & Alberti, K.G.M.M., Eds.), pp. 77-108, Wiley & Sons, New York. Rabinowitz, D., & Zierler, K.L. (1963). A metabolic regulating device based on the actions of human growth hormone and of insulin, singly and together, on the human forearm. Nature 199, 913-914. Randle, El., Garland, EB., Hales, C.N., & Newsholme, E.A. (1963). The glucose-fatty acid cycle:its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet i, 785-789. Rivier, J., Spiess, J., Thorner, M., & Vale, W.S. (1982). Characterization of a growth hormone-releasing factor from a human pancreatic islet tumour. Nature (London) 300, 276-278. Ross, R.J.M., & Savage, M.O. (1996). Growth Hormone Resistance. Bailiere Tindall, London. Rudman, D., Vintner, M.H., Rogers, C.M., Lubin, M.E, Fleming, G.H., & Raymond, P.B. (1981). Impaired growth hormone secretion in the adult population. Relation to age and adiposity. J. Clin. Invest. 67, 1361-1369. Salmon, W.D., & Daughaday, W.H. (1957). A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J. Lab. Clin. Med. 49, 825-836. Scanlon, M.E, Issa, B.G., & Dieguez, C. (1996). Regulation of growth hormone secretion. Hormone Res. 46, 149-154. Silva, C.M., Weber, M.J., & Thorner, M.O. (1993). Stimulation of tyrosine phosphorylation in human cells by activation of the growth hormone receptor. Endocrinol. 132, 101-108. Quabbe, H.J. (1977). Chronobiology of growth hormone secretion. Chronobiologia. 4, 217-246.
RECOMMENDED READINGS Ross, R.J.M., & Savage, M.O. (1996). Growth Hormone Resistance. Bailliere Tindall, London. Scanlon, M.E, Issa, B.G., & Dieguez, C. (1996). Regulation of growth hormone secretion. Hormone Res. 46, 149-154. Spencer, E.M. (1991). Modern Concepts of Insulinlike Growth Factors. Elsevier, New York.