Normal Physiology of ACTH and GH Release in the Hypothalamus and Anterior Pituitary in Man
Dr Bernard Khoo, and Prof Ashley B. Grossman
Updated]
Regulation of ACTH
Cells of origin

ACTH is released from corticotrophs in the human pituitary, constituting 15-20% of the cells of the anterior pituitary (see Asa). They are distributed in the median wedge, anteriorly and laterally, and posteriorly adjacent to the pars nervosa. These cells are characteristically identified from their basophil staining and PAS-positivity due to the high glycoprotein content of the N-terminal glycopeptide of pro-opiomelanocortin (vide infra), as well as ACTH immunopositivity. Scattered ACTH-positive cells are also present in the human homologue of the intermediate lobe. Some of these appear to extend into the posterior pituitary, so-called “basophilic invasion” (2).

ACTH/POMC

POMC gene structure

ACTH is derived from a 266 amino acid precursor, pro-opiomelanocortin (POMC]
The POMC promoter

The promoter of POMC has most extensively been studied in the rat. Common transcription elements such as a TATA box, a CCAAT box and an AP-1 site are found within the promoter. Corticotroph and melanotroph-specific transcription of POMC appears to be dependent on a CANNTG element motif synergistically binding corticotroph upstream transcription element-binding (CUTE) proteins (6). These include NeuroD (7), Ptx1 (8), and Tpit (9). Interestingly, Ikaros transcription factors, which had previously been characterized as being essential for B and T cell development, have recently been demonstrated to bind and regulate the POMC gene in mice. Moreover, Ikaros knockout mice demonstrate impaired corticotroph development in their pituitaries, as well as reduced circulating ACTH, MSH and corticosterone levels (10).

POMC transcription is positively regulated by corticotrophin releasing hormone (CRH]
POMC mRNA transcription is negatively regulated by glucocorticoids (15). The glucocorticoid effect appears, in the rat POMC promoter, to be dependent on a negative glucocorticoid response element partially overlapping the CCAAT box (16). The element binds the glucocorticoid receptor as a homodimer plus a monomer on the other side of the DNA helix (17). Glucocorticoid regulation of transcription may also be indirectly mediated via down-regulation of c-jun expression and direct protein-protein mediated inhibition of CRH-induced AP-1 binding (18), as well as inhibition of CRH receptor transcription (19).

Leukaemia Inhibitory Factor (LIF), a pro-inflammatory cytokine expressed in corticotrophs, has also been shown to stimulate POMC transcription, via activation of the Jak-STAT pathway (20, 21). This stimulation is synergistic with CRH. Deletional analysis of the POMC promoter has identified a LIF-responsive region from –407 to –301. It has been speculated that this pathway might form an interface between the immune system and regulation of the pituitary-adrenal axis (22).

Biogenesis of ACTH
Prohormone convertase enzymes PC1 and PC2 process POMC (Figure 1) at pairs of basic residues (lys-lys or lys-arg). This generates ACTH, the N-terminal glycopeptide, joining peptide, and beta-lipotropin (beta-LPH). ACTH can be further processed to generate alpha-melanocyte stimulating hormone (alpha-MSH) and corticotropin-like intermediate lobe peptide (CLIP), whereas beta-LPH can be processed to generate gamma-LPH and beta-endorphin (23). In corticotrophs, POMC is mainly processed to the N-terminal glycopeptide, joining peptide, ACTH and beta-LPH; smaller amounts of the other peptides are present (24). Other post-translational modifications include glycosylation of the N-terminal glycopeptide (25), C-terminal amidation of N-terminal glycopeptide, joining peptide and alpha-MSH (26, 27), and N-terminal acetylation of ACTH, alpha-MSH and beta-endorphin (28, 29).

Hypophysiotropic hormones affecting ACTH release
Corticotropin releasing hormone (CRH)
This 41 amino-acid neuropeptide (30) is derived from a 196-amino acid prohormone (31). CRH immunoreactivity is mainly found in the paraventricular (PV) nuclei of the hypothalamus, often co-localised with arginine vasopressin (32). CRH binds to a seven-transmembrane domain receptor (33) G-protein coupled to adenylate cyclase, stimulating cAMP synthesis and PK-A activity. Besides stimulating POMC transcription and ACTH biogenesis (vide supra), CRH stimulates the release of ACTH, leading to a biphasic response with the fast release of a pre-synthesised pool of ACTH, and the slower and sustained release of newly synthesised ACTH (34).

Arginine vasopressin (AVP)
In the anterior pituitary, AVP principally binds to the seven-transmembrane domain V1b receptor, also known as the V3 receptor (35). The receptor is coupled to phospholipase C, phosphatidyl inositol generation and activation of protein kinase-C (36, 37) and not via adenylate cyclase and cAMP (11). AVP stimulates ACTH release weakly by itself, but synergises with the effects of CRH on ACTH release (38). Downregulation of protein kinase C by phorbol ester treatment abolishes the synergistic effect of AVP on ACTH release by CRH (39). AVP does not stimulate POMC transcription either by itself or in synergism with CRH (40).

Other influences on ACTH release]Oxytocin and AVP have been co-localised to the supraoptic and PV nuclei of the hypothalamus (41). Oxytocin controversially inhibits ACTH release in man (42-44) by competing for AVP receptor binding (45), although it may potentiate the effects of CRH on ACTH release (46).

Vasoactive intestinal peptide (VIP) and its relative, peptide histidine isoleucine (PHI), have been shown to activate ACTH secretion (47), a mechanism which may underlie the increase in ACTH after eating. This is most probably mediated indirectly via CRH (48).

Atrial natriuretic peptide (ANP) 1-28 has been localized to the supraoptic and PV nuclei (49). In healthy males, infusion of ANP 1-28 was reported to attenuate the ACTH release induced by CRH (50, 51), but this only occurs under highly specific conditions and is not readily reproducible. In physiological doses, ANP 1-28 does not appear to affect CRH-stimulated ACTH release (52).

Opiates and opioid peptides inhibit ACTH release (53). There does not seem to be a direct action at the pituitary level. It is likely that these act by modifying release of CRH at the hypothalamic level (54). Opiate receptor antagonists such as naloxone or naltrexone cause ACTH release by blocking tonic inhibition by endogenous opioid peptides (55).

The endocannabinoid system has recently appeared as a key player in regulating the baseline tone and stimulated peaks of ACTH release. The seven-transmembrane cannabinoid receptor type 1 (CB1) is found on corticotrophs, and the endocannabinoids anandamide and 2-arachidonoylglycerol can be detected in normal pituitaries (56). Antagonism of CB1 causes a dose-dependent rise in corticosterone levels in mice (57). CB1-/- knockout mice demonstrate higher corticosterone levels compared to wild-type CB1+/+ littermates, although the circadian rhythm is preserved. Treatment of the CB1-/- mice with low-dose dexamethasone did not significantly suppress their corticosterone levels and surprisingly caused a paradoxical rise in ACTH levels when compared to the wild-type, although high-dose dexamethasone suppressed corticosterone and ACTH to the same degree in both CB1-/- and CB1+/+ mice. These CB1-/- mice have]
Catecholamines act centrally via alpha1-adrenergic receptors to stimulate CRH release. Peripheral catecholamines do not affect ACTH release at the level of the pituitary in humans (60).

Ghrelin and the synthetic GH secretagogue hexarelin stimulate ACTH release, probably via stimulating predominantly AVP release with a much lesser effect on CRH (61-63).

GH releasing hormone (GHRH) has been shown to potentiate the ACTH and cortisol response to insulin-induced hypoglycaemia, but not to potentiate the ACTH and cortisol response after administration of CRH/AVP (64).

Angiotensin II (Ang II) is able to stimulate ACTH release in vitro from pituitary cells (65). Central Ang II is likely to stimulate CRH release via its receptors in the median eminence, as passive immunization with anti-CRH can abolish the effect of Ang II (66). Intraventricular Ang II can stimulate ACTH release in rats (67) and is able to stimulate the synthesis of CRH and POMC mRNA (68). There is some controversy as to whether peripheral Ang II can modulate ACTH secretion. It is likely that the ACTH rise seen after Ang II infusion into rats is mediated via circumventricular organ stimulation, as blockade of Ang II effects on the circumventricular organs with simultaneous infusion of saralasin blocks this rise (67).

In vitro studies have shown a possible inhibitory effect of somatostatin on ACTH release in pituitary cell lines. However, this effect appears to be dependent on the absence of glucocorticoids in the culture medium (69). Moreover, in vivo studies show no effect on basal or CRH-stimulated ACTH release (70), although somatostatin does decrease basal secretion in the context of Addison’s disease (71). It is unlikely, therefore, that somatostatin is an inhibitor of ACTH release in normal physiology.

Although there is some work to suggest that prepro-TRH 178-199 can inhibit both basal and CRH-stimulated ACTH release in AtT-20 cell lines and rat anterior pituitary cells (72, 73), other investigators have not been able to confirm this (74). The role of this peptide in ACTH release is currently in dispute.
Interleukins IL-1, IL-6 and possibly IL-2 appear to stimulate ACTH release, but most of the acute effects of these agents are almost certainly via the hypothalamus (75).

Leukaemia Inhibitory Factor is able to stimulate POMC synthesis, as noted above.

Physiology of ACTH release

Pulsatility of ACTH release

Frequent sampling of ACTH with deconvolution analysis reveals that it is secreted in pulses from the corticotroph with 40 pulses +/- 1.5 measured per 24 hours, on analysis of 10 minute sampling data. These pulses temporally correlate with the pulsed secretion of cortisol, allowing for a 15 minute delay in secretion, and correlate in amplitude (76). Pulse concordance has been measured at 47% (ACTH to cortisol) and 60% (cortisol to ACTH) in one study (77), and 90% (ACTH to cortisol) in another (78). Although the pulsatility of ACTH secretion may result from pulsatile CRH release, there is evidence that isolated human pituitaries intrinsically release ACTH in a pulsatile fashion (79).

Circadian rhythm
In parallel with cortisol, ACTH levels vary in an endogenous circadian rhythm, reaching a peak between 0600-0900h, declining through the day to a nadir between 2300h-0200h, and beginning to rise again at about 0200-0300h. An increase in ACTH pulse amplitude rather than frequency is responsible for this rhythm (76).

The circadian rhythm is mediated via the supra-chiasmatic nucleus (SCN). An autoregulatory negative feedback system involving cyclical synthesis of period proteins PER1-3, CLOCK/BMAL1 and CRYPTOCHROME acts as the basic oscillator (80). Entrainment of the oscillator is achieved by light input from the retina, mediated via the retinohypothalamic tract. Light-activated transcription of immediate-early genes such as c-fos and JunB (81, 82) causes activation of PER1 gene transcription as well as modification of the acetylation pattern of histone tails. The latter are implicated in the control of chromatin structure and accessibility of genes to transcription (83).

Is a circadian rhythm in CRH secretion responsible for the ACTH rhythm? Although there is a report of a circadian rhythm in CRH secretion (84), other reports do not confirm this (85). Moreover, the circadian rhythm persists despite a continuous infusion of CRH, suggesting that other factors are responsible for the modulation of ACTH pulses (86). The most likely alternative candidate is AVP]
Stress

Stress, both physical and psychological, induces the release of ACTH, particularly via CRH and AVP (89, 90), and increases the turnover of these neurohypophysiotropic factors by increasing the transcription of CRH and AVP (91). The hypoglycaemia during the insulin tolerance test is one such stressor (Figure 2), as is venepuncture (92).

Interestingly, there is evidence that different stress paradigms have differential effects on CRH and AVP. In situ hybridization with intronic and exonic probes can be used to study the transcription of heterogenous nuclear RNA (hnRNA), followed by its processing (including splicing, capping and polyadenylation) to messenger RNA (mRNA) within 1-2 hours. CRH and AVP hnRNA levels in rats subjected to restraint show significant increases at 1 and 2 hours after the induction of stress, followed by significant increases in mRNA levels at 4 hours (93). In contrast, intraperitoneal hypertonic saline causes a rapid 8.6-fold increase in CRH hnRNA and mRNA within 15 minutes, returning to basal levels by 1 hour. AVP hnRNA responses are slower, peaking at 11.5-fold increase by 2 hours, followed by a prolonged elevation of AVP mRNA levels from 4 hours onwards (94).

Repetitive stress causes variable effects, enhancement or desensitization, on ACTH responses, depending on the stress paradigm involved. This appears to be positively correlated with changes in AVP binding to V1b receptors, reflecting changes in the number of binding sites and not their affinities. It is at present unclear whether this is due to changes in transcription of the V1b gene, alterations in mRNA stability, translational control or recruitment of receptors from intercellular pools (95).

Recent work has also characterised roles for endogenous nitric oxide (NO) and carbon monoxide (CO) in mediating the ACTH response to stress (96). Neuronal nitric oxide synthase co-localizes with AVP and to some extent CRH in paraventricular neurones (97, 98). Knockout mice lacking wild-type and neuronal nitric oxide synthase have much reduced quantities of POMC immunoreactivity in their arcuate nuclei and pituitaries compared to wild-type mice (97, 99). In general, inflammatory stressors appear to activate an endogenous inhibitory pathway whereby NO and CO attenuate the stimulated secretion of CRH and AVP. These effects can also be seen in terms of circulating AVP. However, the regulation of the pituitary-adrenal axis by other stressors may involve an activating role for these gaseous neurotransmitters.

Feedback regulation of ACTH release

Glucocorticoid feedback occurs at multiple levels]
inhibition of CRH and AVP synthesis and release in the PVN (100, 101).

inhibition of POMC transcription (as outlined above)

inhibition of ACTH release induced by CRH and AVP (102).

Fast feedback occurs within seconds to minutes and involves inhibition of ACTH release by the corticosteroids. In vitro this appears to involve inhibition of stimulated ACTH and CRH release, but basal secretion is not affected. Protein synthesis is not required, implying that the glucocorticoid effect is non-genomic, e.g. by inhibition of second-messenger systems (103, 104). Recent evidence implicates the endocannabinoids in mediating this fast feedback inhibition (105). Intermediate feedback occurs within 4 hours time frame and involves inhibition of CRH synthesis and release, but does not affect ACTH synthesis (104). Slow feedback occurs over longer timeframes and involves inhibition of POMC transcription (104).

There is evidence that ACTH can inhibit CRH synthesis in the context of elevated CRH levels due to Addison’s disease or hypopituitarism, although not in the context of normal human subjects (106). Immunohistochemical studies of the paraventricular nuclei in adrenalectomised or hypophysectomised rats show a reduction of CRH and AVP positive cells when these rats are given ACTH infusions (107).

Regulation of GH release

Somatotroph development and differentiation

Somatotrophs make up approximately 50% of the cell population of the anterior pituitary (see Asa). These cells are characteristically acidophilic, polyhedral and immunopositive for GH and Pit-1. A smaller number of such cells are mammosomatotrophs, i.e. immunopositive for GH and prolactin (108).

During the process of cytodifferentiation in the Rathke’s pouch primordium, a cascade of transcription factors is activated to specify anterior pituitary cell types. The two factors particularly involved in differentiation of the lactotroph, somatotroph and thyrotroph lineages are Prop-1 (Prophet of Pit-1) and Pit-1. Prop-1 is a paired-like homeodomain transcription factor; mutations in this gene cause combined GH, prolactin and TSH deficiency. Mutations of Prop-1 will also give abnormalities of gonadotroph function and, occasionally, corticotroph reserve. Interestingly, these deficiciencies are often progressive over time.

Pit-1, also known as GHF-1, is part of the POU homeodomain family of transcription factors that includes unc-86, Oct-1 and Oct-2 (109). Pit-1 is a key transcription factor that activates GH gene transcription in the somatotroph (vide infra).

Growth hormone
This is a 191 amino-acid single chain polypeptide hormone that occurs in various modified forms in the circulation. During spontaneous pulses of secretion the majority full-length isoform of 22 kDa makes up 73%, the alternatively spliced 20 kDa isoform contributes 16%, while the ‘acidic’ desamido and N-alpha acylated isoforms make up 10%. During basal secretion between pulses other forms (30 kDa, 16 kDa and 12 kDa) can also be identified which consist of immunoreactive fragments of GH (133-135).

Higher molecular weight forms of GH exist in the circulation, representing GH bound to binding proteins or GHBP (136). The high-affinity GHBP consists of the extracellular domain of the hepatic GH receptor, and this binds the 22 kDa GH isoform preferentially (137). The low-affinity GHBP binds the 20 kDa isoform preferentially (138). Binding of GH to GHBP prolongs the circulation time of GH as the complex is not filtered through the glomeruli (134). GH/GHBP interactions may also compete for GH binding to its surface receptors (139).

Hypophysiotropic hormones affecting GH secretion

GHRH

GHRH was originally isolated from a pancreatic tumour taken from a patient that presented with acromegaly and somatotroph hyperplasia (140). GHRH is derived from a 108 amino-acid prepro-hormone to give GHRH(1-40) and (1-44) (Figure 4), which are both found in the human hypothalamus (141, 142). The C-terminal 30-44 residues appear to be dispensable, as residues 1-29 show full bioactivity. GHRH binds to a seven-transmembrane domain G-protein coupled receptor that activates adenylate cyclase (143), which stimulates transcription of the GH gene as well as release of GH from intracellular pools (144, 145). No other hormone is released by GHRH, although GHRH has homology to other neuropeptides such as PHI, glucagon, secretin and GIP (146).

Somatostatin

Somatostatin (a.k.a. somatotropin release inhibitory factor or SRIF) is derived from a 116 amino-acid prohormone to give rise to two principal forms, somatostatin-28 and -14 (147). Both of these are cyclic peptides due to an intramolecular disulphide bond (Figure 4). Somatostatin has multiple effects on anterior pituitary as well as pancreatic, liver and gastrointestinal function]
It inhibits GH secretion directly from somatotrophs (148, 149) and antagonizes the GH secretagogue activity of ghrelin (150).

It inhibits GH secretion indirectly via antagonizing GHRH secretion (see Lechan and Toni).

It inhibits GH secretion indirectly via inhibiting the secretion of ghrelin from the stomach (151-153).

It inhibits secretion of TSH, and TRH stimulation of TSH secretion from the pituitary (154, 155).

It inhibits the secretion of CCK, glucagon, gastrin, secretin, GIP, insulin and VIP from the pancreas (156).

Somatostatin binds to specific seven-transmembrane domain G-protein coupled receptors, of which there are at least 5 subtypes. Subtypes 2 and 5 are the most abundant in the pituitary (157). The somatostatin receptors couple to various 2nd messenger systems such as adenylate cyclase, protein phosphatases, phospholipase C, cGMP dependent protein kinases, potassium, and calcium ion channels (158).

Ghrelin

Ghrelin is the most recently discovered GH regulatory factor and was isolated from stomach as the endogenous ligand of the GH secretagogue receptor (GHS-R), another member of the seven-transmembrane receptor family G-protein coupled to the phospholipase C-phosphoinositide pathway (159, 160). Ghrelin is derived from preproghrelin, a 117 amino-acid peptide, by cleavage and n-octanoylation at the third residue to give a 28 amino-acid active peptide (Figure 4). The majority of circulating ghrelin exists as the des-octanoylated (des-acyl) form]
In the circulation, ghrelin appears to be bound to a subfraction of HDL particles containing clusterin and the A-esterase paraoxonase. It has been suggested that paraoxonase may be responsible for catalyzing the conversion of ghrelin to des-acyl ghrelin (163). However, inhibition of paraoxonase in human serum does not inhibit the de-acylation of ghrelin, and there is a negative correlation in these sera between the paraoxonase activity and ghrelin degradation. Instead, it is more likely that butyrylcholinesterase and other B-esterases are responsible for this activity (164).

Ghrelin is present in the arcuate nucleus of the hypothalamus and in the anterior pituitary (165). Immunofluoresence studies show that ghrelin is localized in somatotrophs, thyrotrophs and lactotrophs but not in corticotrophs and gonadotrophs, suggesting that ghrelin may be acting in a paracrine fashion in the anterior pituitary (166). It stimulates GH release in vitro directly from somatotrophs (159) and also when infused in vivo, although the latter action appears to require the participation of an intact GHRH system (150). Ghrelin stimulates GH secretion in a synergistic fashion when co-infused with GHRH (63). Besides its GH releasing activity, ghrelin has orexigenic activity (167, 168), stimulates insulin secretion (169), ACTH and prolactin release (170). Knocking out the ghrelin gene in mice does not seem to affect their size, growth rate, food intake, body composition and reproduction, indicating that ghrelin is not dominantly and critically involved in mouse viability, appetite regulation and fertility (171). Ghrelin null mice show an increased utilization of fat as an energy substrate when placed on a high-fat diet, which may indicate that ghrelin is involved in modulating the use of metabolic substrates (172). GHS-R knockout mice have the same food intake and body composition as their wild-type littermates, although their body weight is decreased in comparison. However, treatment of GHS-R null mice with ghrelin does not stimulate GH release or food intake, confirming that these properties of ghrelin are mediated through the GHS-R (173).

This is not to say, however, that des-acyl ghrelin does not have any biological effects. It has been shown to inhibit apoptosis and cell death in primary cardiomyocyte and endothelial cell cultures (174), to have varying effects on the proliferation of various prostate carcinoma cell lines (175), to inhibit isoproterenol-induced lipolysis in rat adipocyte cultures (176), and to induce hypotension and bradycardia when injected into the nucleus tractus solitarii of rats (177). More controversially, intracerebroventricular or peripherally adminstered des-acyl ghrelin causes a decrease in food consumption in fasted mice and inhibits gastric emptying. Des-acyl ghrelin overexpression in transgenic mice causes a decrease in body weight, food intake, fat pad mass weight and decreased linear growth compared to normal littermates (178). These observations were not replicated by other researchers, who found no effect of des-acyl ghrelin on feeding (179). The effects of des-acyl ghrelin appear not to be mediated via the type 1a or 1b GHS-R (174-176). The effects of peripherally administered des-acyl ghrelin on stomach motility can be inhibited by intracerebrovascular CRH receptor type 2 antagonists, suggesting that CRH receptor type 2 is involved, but there is no direct evidence that des-acyl ghrelin binds this receptor (180)

As noted above, the actions of ghrelin in vivo seem to require an intact GHRH system, as immunoneutralisation of GHRH blocks GH secretion induced by ghrelin (150). The actions of GH secretagogues are blocked by hypothalamo-pituitary disconnection, which suggests that in vivo ghrelin’s stimulatory actions are indirect and mediated by GHRH (181). However, GHRH cannot be the sole mediator of ghrelin’s actions as the GH response to ghrelin is greater than that to GHRH (182), and, as noted above, ghrelin synergistically potentiates GH release by a maximal dose of GHRH (63). There is no evidence to suggest that ghrelin decreases somatostatinergic tone as immunoneutralisation of somatostatin does not block ghrelin’s ability to release GH (150). There may therefore be another mediator, the so-called ‘U’ factor, released by ghrelin, which causes GH secretion (183).

Other influences on GH secretion

Glucocorticoids

Glucocorticoid treatment has a biphasic effect on GH secretion]
Leptin

Leptin is a 167 amino-acid peptide primarily produced by white adipose tissue (187), which regulates body fat mass (188) by feedback inhibition of the appetite centres of the hypothalamus (189). Leptin and its receptor has been detected both by RT-PCR and immunohistochemistry in surgical pituitary adenoma specimens and in normal pituitary tissue (190, 191). However, pituitary adenoma cells in culture do not secrete GH in response to leptin treatment (191, 192).

In rats, immunoneutralisation with leptin antisera decreases GH secretion. Intracerebroventricular leptin administration reverses the inhibitory effect of fasting on GH levels in rats. However, intracerebroventricular leptin by itself does not significantly influence GH secretion (193). These observations, however, may not be extendable to humans, as the physiology of GH in humans appears to be very different from rats, e.g. GH levels in humans are increased by fasting in contrast to suppression in rats (vide infra).

Catecholamines

In general, alpha-adrenergic pathways stimulate GH secretion, by stimulation of GHRH release and inhibition of somatostatinergic tone, while beta-adrenergic pathways inhibit secretion by increasing somatostatin release (194, 195). The alpha2-adrenoceptor agonist clonidine can therefore be used as a provocative test of GH secretion (196, 197) although clinical experience suggests that this is an unreliable stimulatory test for GH secretion in practice. L-dopa stimulates GH secretion; however, this action does not appear to be mediated via dopamine receptors as specific blockade of these receptors with pimozide does not alter the GH response to L-dopa (198). Instead, L-dopa’s effects appear to depend on conversion to noradrenaline or adrenaline, as a-adrenoceptor blockade with phentolamine disrupts the GH response to L-dopa (199).

Acetylcholine

Muscarinic pathways are known to stimulate GH secretion, probably by modulating somatostatinergic tone (200). Pyridostigmine, an indirect agonist which blocks acetylcholinesterase, increases the 24 hour secretion of GH by selectively increasing GH pulse mass (201). On the other hand, the muscarinic antagonist atropine is able to blunt the GH release associated with slow wave sleep (202) and that associated with GHRH administration (203). Passive immunization with anti-somatostatin antibodies abolishes the pyridostigmine induced rise in GH in rats, but not immunization with anti-GHRH antibodies, supporting the central role of somatostatinergic tone in mediating this response (204).

Endogenous opioids

Endorphins and enkephalins are able to stimulate GH secretion in man (205), and blockade with opiate antagonists can attenuate the GH response to exercise (206). Passive immunization against GHRH in rats inhibits GH release in response to an enkephalin analogue, which argues for stimulation of GHRH in response to these compounds (207). This cannot be the only mechanism, however, as the met-enkephalin analogue DAMME is able to increase GH release over and above the levels released during maximal stimulation by a GHRH analogue (208). It is possible that the actions of endogenous opioids occur via an interaction with the GHS-R, as the original GH secretagogues characterised were derived from the enkephalins (162).

Endocannabinoids

As with ACTH/cortisol, the endocannabinoids may also influence the release of GH. Somatotroph cells bear the CB1 receptor (56). The administration of THC for 14 days suppresses the GH secretion in response to hypoglycaemia in healthy human subjects (59). Oddly enough, THC and anandamide appear to have opposed effects on GH levels in ovariectomized rats]
Other neuropeptides

Many neuropeptides, including the ones in the following paragraphs, have been shown to influence GH secretion in various contexts. For the most part, however, their physiological role in man is not well characterised.

Infusion of galanin, a 29 amino-acid peptide originally isolated from the small intestine, causes stimulation of GH secretion when infused alone and also enhances GHRH-stimulated GH secretion (212).

Calcitonin, the 32 amino-acid peptide secreted from the C cells of the thyroid gland, appears to inhibit the stimulated secretion of GH by arginine and insulin-induced hypoglycaemia (213, 214).

Neuropeptide Y (NPY) is an orexigenic peptide that has been shown to inhibit GH secretion in rats (215-217), from human somatroph tumour cells in culture (218), and from rat hypothalamic explants (219). When infused into patients with prolactin-secreting pituitary adenomas, 9 out of 15 patients showed a paradoxical rise in GH levels (220). However, when infused into healthy young men overnight, NPY did not have any significant effect on GH secretion (221).

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a hypothalamic C-terminally amidated 38 residue peptide hormone originally characterised on the basis of its ability to stimulate cAMP accumulation from anterior pituitary cells (222). In rats, PACAP stimulates GH release from pituitary cell lines and also when infused in vivo (223-225). When infused into human volunteers, however, GH levels do not appear to be affected (226).

Feedback loops in GH secretion

Multiple negative feedback loops exist to autoregulate the GH axis (Figure 5)]
Somatostatin auto-inhibits its own secretion (227).

GHRH auto-inhibits its own secretion by stimulating somatostatin release (228).

GH auto-regulates its own secretion by stimulating somatostatin release and inhibiting GHRH-stimulated GH release (229-231). There is also a negative feedback on stomach ghrelin release by GH (232).

IGF-I, whose production is stimulated by GH, inhibits GH release in a biphasic manner]
Physiology of GH secretion
Pulsatility of GH secretion

Circulating GH levels are pulsatile, with high peaks separated by valleys where the GH is undetectable by conventional RIAs or IRMAs (Figure 6). The recent development of sensitive chemiluminescent assays for GH with high frequency sampling and deconvolution analysis has allowed the detailed study of GH secretion. This shows that there are detectable levels of basal GH secretion in the ‘valleys’ (235). On average, there are 10 pulses of GH secretion per day lasting on average 96.4 mins with 128 mins between each pulse (236).

There is a dynamic interplay of pulsatile GHRH and somatostatin secretion]
Via crosstalk]
Via synergistic actions on somatotrophs]
However, continuous GHRH administration does not affect the pulsatility of GH secretion (238). Moreover, patients with an inactivating mutation of the GHRH receptor continue to show pulsatile GH secretion, suggesting that somatostatin pulsatility is sufficient to determine GH pulsatility (239).

GH and sexual dimorphism

The technical developments in sensitive detection of GH referred to above have elucidated differences in secretion between men and women. Women have higher mean GH levels throughout the day than men due to higher incremental and maximal GH peak amplitudes (Figure 6), but show no significant difference in GH half-life, interpulse times or pulse frequency (240). The higher basal GH levels may underlie the higher nadir GH levels seen in normal women after GH suppression with oral glucose (241).

Differences in GH secretion patterns between the sexes, with male ‘pulsatile’ secretion versus female ‘continuous’ secretion, can cause different patterns of gene activation in target tissues, e.g. induction of linear growth patterns, gain of body weight, induction of liver enzymes and STAT 5b signalling pathway activity (242).

Sleep
The secretion rate of GH shows a circadian pattern, with peak rates measured during sleep. These are approximately triple the daytime rate (243). GH secretion is especially associated with slow wave sleep (SWS – stages 3 and 4) (244). The decline in GH secretion during aging is paralleled by the decreasing proportion of time spent in SWS, although it is unclear which is cause and which is effect (245). In early data from a clinical trial, GH deficient patients have increased sleep fragmentation and decreased total sleep time, and it is conjectured that such alterations in sleep patterns may be responsible for excessive daytime sleepiness in such patients (246).

Sleep deprivation, in the laboratory or due to travel causing ‘jet lag’, causes two alterations in the GH secretory pattern]
Adminstration of a GHRH antagonist reduces the nocturnal GH pulsatility by 75% (248). Normal subjects remain sensitive to GHRH boluses during the night, however, and the lowering of somatostatinergic tone during the night may be responsible for the increase in GH secretion rate (249). Recent work, however, has also demonstrated that ghrelin levels rise through the night in lean men (250). It is likely, therefore, that a combination of increased GHRH, decreased somatostatin and increased ghrelin levels underlie the circadian variation in GH secretion.

Adminstration of GHRH augments the increased nocturnal GH release and promotes SWS. Somatostatin does not change nocturnal GH release, does not affect the proportion of SWS but may increase rapid eye movement (REM) sleep density (251). Ghrelin has been shown to promote slow wave sleep at the expense of REM sleep, accompanied by an increase in GH and prolactin release when administered exogenously (252).

Exercise

Exercise is a powerful stimulus to secretion of GH (253), which occurs by about 15 min from the start of exercise (254). The kinetics may vary between subjects, an effect which is likely to be related to differences in age, sex and body composition (255). Ten minutes of high-intensity exercise is required to stimulate a significant rise in GH (256). Anaerobic exercise causes a larger release of GH than aerobic exercise of the same duration (257). Acetylcholine, adrenaline, noradrenaline and endogenous opioids have been implicated in exercise-induced GH release (200). However, ghrelin levels do not rise in acute exercise, indicating that ghrelin may not have a role to play in exercise-induced GH release (258).

Hypoglycaemia

Insulin-induced hypoglycaemia is another powerful stimulus to GH secretion (Figure 7) (259, 260). The peak GH levels achieved during insulin stress testing correlate well with those achieved during slow wave sleep (261). The hypoglycaemic response is mediated by a2-adrenergic receptors (262) to cause inhibition of somatostatin release (200), although other evidence argues for a role of stimulated GHRH release, as a GHRH receptor antagonist significantly suppressed hypoglycaemic GH release (263). Ghrelin is unlikely to be involved in the GH response to insulin-induced hypoglycaemia as ghrelin levels are suppressed by the insulin bolus (264).

Other stressors
Other physical stresses such as hypovolaemic shock (265) and elective surgery (266) cause increased GH release. a-adrenergic dependent mechanisms are thought to underly this, as blockade with phentolamine inhibits the response (266).

Hyperglycaemia
In contrast to hypoglycaemia, ingestion of an oral glucose load causes an initial suppression of plasma GH levels for 1-3 hours (Figure 8), followed by a rise in GH concentrations at 3-5 hours (267). The initial suppression could be mediated by increased somatostatin release as pyridostigmine, a postulated inhibitor of somatostatin release, blocks this suppression (268). Circulating ghrelin levels also fall following ingestion of glucose (269). The GH response to ghrelin and GHRH infusions is blunted by oral glucose, an effect that is probably mediated by somatostatin (270). The later rise in GH levels is postulated to be due to a decline in somatostatinergic tone plus a reciprocal increase in GHRH, leading to a ‘rebound’ rise (200).

In type I diabetes mellitus, GH dynamics are disordered, with elevated 24 hour release of GH (271). Deconvolution analysis shows that GH pulse frequencies and maximal amplitudes are increased. The latter is accounted for by higher ‘valley’ levels (272). Better glycaemic control appears to normalize these disordered dynamics (273). The pathophysiological mechanism appears to involve reduced somatostatinergic tone (200).

There is conflicting evidence for increased, decreased or normal GH dynamics in type II diabetics. It is likely that this reflects two factors acting in opposite directions]
Obesity and malnutrition
Chronic malnutrition states such as marasmus and kwashiorkor cause a rise in GH levels (274). A voluntary 5-day fast also leads to significant increases in discrete GH pulse frequency, 24 hour integrated GH concentration and maximal pulse amplitude (275). On the other hand, obesity is known to be associated with lower GH levels, partially due to decreased levels of GH binding protein and partially due to decreased frequency of GH pulses (276). Visceral adiposity, as assessed by CT scanning and dual energy X-ray absorptiometry, seems to be especially important, and correlates negatively with mean 24 hour GH concentrations (277).

The mechanism of decreased GH release in obesity has been ascribed to increased somatostatinergic tone, as pyridostigmine is able to reverse this, to some extent, by suppressing somatostatin release (278-280). However, this cannot be the full explanation, as pyridostigmine is not able to fully reverse the hyposomatotropinism of obesity, even when combined with GHRH and the GH secretagogue GHRP-6 (281).

Although leptin has been shown to be influential on GH secretion in rats (193), this may not be so in humans. Leptin-deficient subjects have been compared with obese non-deficient control subjects in their GH responses when stimulated with GHRH plus GHRP-6. Both these groups have decreased GH peaks compared to non-obese control subjects, as expected. There was no significant difference in mean GH peaks between leptin-deficient and leptin-replete controls, suggesting that leptin does not play a significant role in the GH suppression seen in obese humans, and that the lower GH secretion of obesity is mediated via other mechanisms (282).

Another candidate for the mechanism linking obesity to GH secretion is ghrelin. Its levels correlate negatively with body fat content (283). A comparative study between 5 lean and 5 obese men employed rapid sampling and pulse analysis of ghrelin levels over 24 hours. Ghrelin levels increased at night in the lean controls but did not in the obese group (250). Weight loss caused circulating ghrelin levels to rise in two studies (284, 285). Contradicting this, however, Lindeman and colleagues found that ghrelin levels paradoxically correlated positively with visceral fat area, in contrast with 24 hour GH secretion, which correlated negatively. Moreover, in their study, weight loss increased GH secretion but did not affect ghrelin levels (286). The role of ghrelin in linking nutritional status to GH secretion is therefore at present unclear.

Amino acids
GH release is stimulated by a protein meal (287). L-arginine, an essential amino acid, can be used as a provocative test for GH secretion (288). Evidence that L-arginine acts through inhibition of somatostatin release includes the observation that L-arginine can still enhance the GH response to GHRH despite the use of maximal doses of GHRH (289). However, a specific GHRH antagonist blunted the GH response to L-arginine, an observation that supports the notion that L-arginine also acts through stimulation of GHRH secretion (263). Unlike oral glucose, L-arginine does not modify the GH response to ghrelin infusion (270).