Ghrelin-induced GH secretion in domestic fowl in vivo and in vitro

Although avian and mammalian species differ significantly in their regulation of GH secretion, preliminary studies have demonstrated in vivo GH responses to ghrelin in chickens, as in mammals. However, the relative potency of ghrelin as a GH-releasing hormone (GHRH) in birds is uncertain, as is its site of action.The intravenous administration of human ghrelin to immature chickens promptly increased the circulating GH concentration (within 10 min), although this was transitory and was only maintained for 20 min. This GH response was dose-related with an EC50 of approximately 3.0 microg/kg, comparable with the reported potency of human GHRH in chickens. When incubated with dispersed pituitary cells, human ghrelin induced dose-dependent GH release over a range of 10(-6) to 10(-9) M, with an EC50 of 7.0 x 10(-8) M, comparable with that induced by human GHRH (EC50 6.0 x 10(-8) M), although it was less effective at doses of 10(-6) to 10(-8) M. This was due to direct effects on pituitary somatotrophs, since human ghrelin increased GH release (determined by the reverse hemolytic plaque assay) from individual pituitary cells. The incubation of these cells with human ghrelin induced a dose-dependent increase in the numbers of somatotrophs secreting GH and in the amount of GH released by each cell. In summary, these results demonstrated that ghrelin is a dose-related GH-releasing factor in chickens with a potency comparable with that induced by human GHRH. The GH-releasing action of ghrelin is due, at least in part, to stimulatory actions on the numbers of somatotrophs induced to release GH and upon the amount of GH released from individual somatotrophs.     

Effects of ghrelin on growth hormone secretion from cultured adenohypophysial cells in cattle

To clarify the direct effects of ghrelin on growth hormone (GH) release from anterior pituitary (AP) cells in cattle, GH-releasing effects of human ghrelin (hGhrelin) and rat ghrelin (rGhrelin) on bovine AP cells were compared with those of GH-releasing hormone (GHRH) in vitro. The AP cells were obtained from Holstein steers and were incubated for 2 h with the peptides after incubating in DMEM for 3 days. hGhrelin and rGhrelin significantly stimulated GH release from the cultured cells at doses from 10(-10) to 10(-7) M and from 10(-9) to 10(-7) M, respectively (P<0.05). The rates of increase in GH at 10(-10), 10(-9), 10(-8) and 10(-7) M hGhrelin were 26, 26, 59 and 100% compared with controls, respectively, and those of increase in GH at 10(-9), 10(-8) and 10(-7) M rGhrelin were 58, 74 and 106%, respectively. GHRH significantly increased GH concentrations in cultured media at a dose as low as 10(-13) M compared with the control (P<0.05). When hGhrelin (10(-8) M) and GHRH (10(-8) M) were added together, the release of GH induced by both peptides was significantly greater than that by hGhrelin alone (P<0.05), and tended to be greater than that by GHRH alone. Somatostatin (SS, 10(-7) M) significantly blunted GH release induced by hGhrelin (10(-8) M) and GHRH (10(-8) M) (P<0.05). In the presence of SS, the percent increase in GH released with hGhrelin plus GHRH was 42% and 14% greater than that by either hGhrelin or GHRH alone, respectively (P<0.05). These results show that ghrelin directly stimulates the release of GH from anterior pituitary cells, and that SS modifies ghrelin-stimulated GH release in cattle.     

Homologous and heterologous in vitro regulation of pituitary receptors for somatostatin, growth hormone (GH)-releasing hormone, and ghrelin in a nonhuman primate (Papio anubis)

Secretion of GH by pituitary somatotrophs is primarily stimulated by GHRH and ghrelin and inhibited by somatostatin through the activation of specific receptors [GHRH receptor (GHRH-R), GH secretagogue receptor (GHS-R) and somatostatin receptors (sst1-5), respectively]. However, we have shown that somatostatin, at low doses, can also stimulate GH release, directly and specifically, in primary pituitary cultures from a nonhuman primate (baboons, Papio anubis) and pigs. To determine whether somatostatin, GHRH, and ghrelin can also regulate the expression of their receptors in primates, pituitary cultures from baboons were treated for 4 h with GHRH or ghrelin (10(-8) m) or with high (10(-7) m) and low (10(-15) m) doses of somatostatin, and GH release and expression levels of all receptors were measured. GHRH/ghrelin decreased the expression of their respective receptors (GHRH-R and GHS-R). Both peptides increased sst1, only GHRH decreased sst5 expression, whereas sst2 expression remained unchanged. The effects of GHRH/ghrelin were completely mimicked by forskolin (adenylate cyclase activator) and phorbol 12-myristate 13-acetate (protein kinase C activator), respectively, indicating the regulation of receptor subtype levels by GHRH and ghrelin involved distinct signaling pathways. In contrast, high-dose somatostatin did not alter GH release but increased sst1, sst2, and sst5 expression, whereas GHRH-R and GHS-R expression were unaffected. Interestingly, low-dose somatostatin increased GH release and sst1 mRNA but decreased sst5 and GHRH-R expression, similar to that observed for GHRH. Altogether, our data show for the first time in a primate model that the primary regulators of somatotroph function (GHRH/ghrelin/somatostatin) exert both homologous and heterologous regulation of receptor synthesis which is dose and subtype dependent and involves distinct signaling pathways.     

Leptin stimulates growth hormone secretion via a direct pituitary effect combined with a decreased somatostatin tone in a median eminence-pituitary perifusion study

The aim of this study was to examine the effect of recombinant human leptin on growth hormone (GH) secretion in perifused anterior pituitary slices from adult pigs. Anterior pituitary slices from sows were perifused and treated with recombinant human leptin (10 nM) and GH-releasing hormone (GHRH; 1 nM). In some experiments, pituitary slices were coincubated with stalk median eminence (SME). In a subset of the coincubation experiments, immunoneutralization of endogenous GHRH and somatostatin (SRIH) release was performed with antisera to GHRH and SRIH. Leptin increased GH secretion in pituitary slices alone (up to 100% vs. control at 40 min) as well as in pituitary slices coincubated with SME (up to 122% vs. control at 40 min). A significant difference was observed in GH secretion from pituitary slices when the tissue was coincubated with leptin and GHRH at a low concentration (0.1 nM), but not when GHRH was used at 1 and 10 nM. Furthermore, anti-SRIH antiserum increased GH release from pituitary slices in coincubation experiments with SME. Finally, SRIH secretion was significantly reduced by leptin (down by 35% vs. control from 0 to 30 min of treatment) in cultured SME. These data show that leptin is effective in stimulating GH secretion by acting at two different levels: (1) it stimulates GH secretion directly from pituitary slices, and (2) it reduces SRIH tone from the median eminence and, indirectly, increases GH secretion from the pituitary. These results support the hypothesis that leptin may be an interesting hormonal mediator of growth and related metabolic effects by acting directly on the hypothalamic-pituitary axis.     

Novel ghrelin analogs with improved affinity for the GH secretagogue receptor stimulate GH and prolactin release from human pituitary cells

OBJECTIVE: Ghrelin, a recently identified 28-amino acid peptide is a potent GH secretagogue (GHS) produced predominantly by the stomach. Ghrelin stimulates GH secretion through binding to the GHS receptor in the hypothalamus and pituitary. In addition to the GH-releasing action, ghrelin has been found to be a powerful orexigenic factor. To assess the direct in vitro effects of ghrelin on human pituitary hormone secretion we have produced a panel of novel ghrelin analogs (molecular weight, 3323-3384; human native ghrelin, 3371) with enhanced affinity for the human GHS receptor (IC(50) 0.38-1.09 nM; human ghrelin, 1.2-2.2 nM). METHODS: The peptidic analogs were tested for their effect on GH secretion using dispersed human fetal pituitaries (21 to 23 weeks of gestation) and cultured GH- and prolactin (PRL)-secreting adenomas. The expression of the GHS receptor in normal (fetal and adult) human pituitary tissues, GH- and PRL-cell adenomas was established using RT-PCR. RESULTS: The effects of ghrelin, its analogs and GH-releasing hormone (GHRH) alone or in combination on GH and PRL secretion were compared at various concentrations. The ghrelin analogs stimulated GH release by 35-60% from human fetal pituitary cells (1-10 nM; P<0.05) and by 50-75% from cultured pituitary adenomas (10 nM; P<0.05). This releasing effect was dose-dependent, achieving maximal stimulation with analog concentrations at 100 nM. Human ghrelin was less potent as compared with its analogs in stimulating human GH, in keeping with the improved binding affinity of the analogs for the GHS-1a receptor. The ghrelin analogs and GHRH had comparable effects on GH secretion from both normal and adenomatous cells, and in combination produced an additive stimulatory effect on GH (150%; P<0.0001). In contrast, ghrelin and its analogs induced a comparable increase in PRL release ranging between 25 and 40% (P<0.05) from fetal cells and 30 and 70% (P<0.001) from cultured PRL-cell and mixed GH-PRL adenomas. CONCLUSIONS: Our results have demonstrated for the first time that ghrelin analogs with enhanced affinity for the GHS receptor are potent stimulators of GH secretion from human pituitary cells, and thus may possess potential clinical therapeutic benefits.     

Role of ghrelin in the control of growth hormone secretion in prepubertal rats: interactions with excitatory amino acids

Ghrelin is a 28-amino-acid peptide, with an essential n-octanoyl modification at Ser3, that elicits growth-hormone (GH) secretion in rats and humans. At present, the mechanisms of ghrelin action and its interactions with other systems controlling GH secretion remain poorly characterized. In this context, the present study was undertaken to obtain information about ontogeny and possible gender differences in the GH-releasing activity of ghrelin, and to delineate its primary site(s) of action at the hypothalamus and/or pituitary. In addition, the interactions between ghrelin and other relevant signals in the control of GH secretion, such as excitatory amino acids (EAAs), nitric oxide (NO) and serotonin, were assessed. Experiments were carried out in infantile-prepubertal animals, when GH pulsatility is not yet established. Systemic administration of ghrelin (25 nmol/rat, i.p.) to 5-, 10- and 23-day-old male and female rats increased plasma GH levels from day 10 onwards. This action was NO dependent, since it disappeared in 23-day-old males after pretreatment with an inhibitor of NO synthase (NAME). Similarly, central infusion of ghrelin (3 nmol/rat, i.c.v.) elicited GH responses in 10- and 23-day-old animals significantly higher than after systemic administration. By contrast, in vitro challenge of pituitary tissue with increasing doses of ghrelin (10(-9)-10(-7) M) failed to enhance GH release into the incubation medium, whereas stimulation with GH-releasing hormone (GHRH; 10(-7) M) or GHRP-6 (10(-7) M) was effective. Finally, effects of ghrelin were blocked by pretreatment with MK-801 and NBQX antagonists of EAA ionotropic receptors and after manipulation of endogenous serotoninergic tone. In addition, the potent releasing activity of EAA agonists NMDA and AMPA was blunted by pretreatment with D-Lys3-GHRP-6, a selective antagonist of the cognate ghrelin receptor, i.e. the GH-secretagogue receptor. In conclusion, our results demonstrate that GH-releasing activity of ghrelin appears early in the infantile period, is NO dependent and involves a primary hypothalamic site of action. The data also demonstrate for the first time the existence of a cross-talk between ghrelin and other neurotransmitter systems, such as EAAs and serotonin, in precise control of GH secretion.     

Pituitary vasoactive intestinal peptide regulates prolactin secretion in the hypothyroid rat

In this study, we demonstrated that the cell content and basal secretion of vasoactive intestinal peptide (VIP) in primary rat pituitary cell cultures were increased in hypothyroidism. VIP release from hypothyroid pituitary cells in vitro was stimulated by thyrotropin releasing hormone (TRH 10(-8) to 10(-6) M) and growth hormone (GH)-releasing hormone (GHRH 10(-9) to 10(-8) M) but not by corticotropin-releasing hormone or luteinizing hormone-releasing hormone in concentrations up to 10(-6) M. In the presence of anti-VIP antisera, there was a significant decrease in basal prolactin secretion from cultured hypothyroid pituitary cells (p less than 0.005) indicating that VIP exerts a tonic stimulatory effect on prolactin (PRL) secretion. The increment in PRL secretion following TRH was not affected by exposure to anti-VIP indicating that PRL release after TRH is not mediated by VIP at the pituitary level. In contrast to changes in PRL, exposure to anti-VIP had no effect on basal GH secretion, indicating that the PRL changes are hormone specific. Similarly, GHRH-induced GH release was unaffected by VIP immunoneutralization.     

Regulation of somatotroph differentiation and growth hormone (GH) secretion by corticosterone and GH-releasing hormone during embryonic development

The role of extracellular factors in the regulation of anterior pituitary cell differentiation and GH secretion during embryonic development was investigated. Previously, we reported that somatotrophs become a significant population by embryonic day (e-) 16 of the chick and that corticosterone is the active compound responsible for the observed GH cell-differentiating activity of e-16 serum. More recently, the influence of hormone interactions on somatotroph differentiation and GH secretion during mid- to late embryogenesis was evaluated. Anterior pituitary cells from e-12, -14, and -17 chicks were cultured for 2, 3, and 6 days with corticosterone (10(-9) M) and GH-releasing hormone (GHRH; 10(-10)-10(-7) M) alone and in combination. Medium samples were analyzed for GH concentrations, and recovered cells were subjected to GH reverse hemolytic plaque assay for determination of somatotroph percentages and the relative amount of GH secretion from individual somatotrophs. GHRH significantly (P < 0.05) increased GH secretion from e-17, but not e-12 and e-14, pituitary cells during 2 and 3 days of culture. Corticosterone alone failed to increase GH secretion from e-12, -14, and -17 pituitary cells; however, corticosterone in combination with GHRH increased GH secretion from cells of all three ages. Culture with GHRH decreased percentages of e-17 GH-secreting cells in a concentration-dependent manner (from basal levels of 12.3 +/- 2.4% to 3.2 +/- 0.7% by 2 days), but did not affect percentages of e-12 and e-14 somatotrophs. Conversely, corticosterone increased percentages of e-12 and e-14 GH-secreting cells (by as much as 14- and 3-fold above basal levels, respectively), but did not alter the proportions of e-17 GH cells. Corticosterone in combination with GHRH was more effective than either hormone alone for increasing percentages of e-12 GH-secreting cells (from 9.6 +/- 0.8% with corticosterone to 15.9 +/- 1.5% with corticosterone plus GHRH), but this synergistic effect was not apparent until after 3 days of culture. Exposure to corticosterone in culture for 2, 3, and 6 days increased subsequent GH release from e-12 and e-14 pituitary cells during reverse hemolytic plaque assay. Combined treatment with corticosterone and GHRH further increased subsequent GH release from e-12 and e-14 cells. We conclude that glucocorticoids induce GH cell differentiation and that corticosterone and GHRH can interact at specific stages of embryonic development to regulate somatotroph differentiation and GH secretion.     

Cortistatin mimics somatostatin by inducing a dual, dose-dependent stimulatory and inhibitory effect on growth hormone secretion in somatotropes

Cortistatin is a recently discovered neuropeptide that is structurally related to somatostatin, the classic inhibitor of growth hormone (GH) release. Cortistatin binds with high affinity to all five somatostatin receptors (sst1-5), and, like somatostatin, cortistatin inhibits in vivo GH release in man and rats. In this report, we compared the in vitro actions of cortistatin and somatostatin using primary pig pituitary cell cultures. In this species, we have previously reported that somatostatin not only inhibits GH-releasing hormone (GHRH)-stimulated GH release at high doses, but also stimulates basal GH release at low (pM) doses, a dual response that is markedly dependent on the subpopulation of pituitary somatotropes examined. Results reported herein demonstrate that cortistatin closely mimics the dose-dependent inhibitory and stimulatory effects of somatostatin on GH secretion. As cortistatin, unlike somatostatin, binds to the human receptor for ghrelin/GH secretagogs (GHS-R), we also investigated whether cortistatin stimulates GH release through this receptor by using a synthetic, short form of cortistatin, cortistatin-8 (CST8), which lacks the sst-binding capacity of full-length cortistatin but retains its GHS-R-binding capacity. Interestingly, CST8 stimulated GH release only at low doses (10(-15) M), and did not reduce GH secretion stimulated by GHRH, ghrelin, or low-dose, full-length cortistatin, yet it counteracted that induced by a nonpeptidyl GHS, L-163 255. Taken together, our results indicate that the dual, inhibitory and stimulatory effects of cortistatin on GH release closely parallel those of somatostatin and are probably mediated by the same receptor(s) and signaling pathway(s) for both peptides. Furthermore, they suggest that the pathway(s) activated by cortistatin (and somatostatin) to stimulate GH release are not initiated by GHS-R activation.     

Atropine blockade of growth hormone (GH)-releasing hormone-induced GH secretion in man is not exerted at pituitary level

The role of acetylcholine (Ach) in the regulation of human GH secretion was assessed using atropine, which selectively blocks cholinergic muscarinic receptors. Paired tests were performed in seven normal subjects using GH-releasing hormone (GHRH) 1-44 (1 microgram/kg iv), with and without atropine pretreatment (1 mg im). The GHRH 1-44-induced GH secretory peak [20.7 +/- 4.5 (SEM) ng/ml] was completely blocked by atropine administration (2.3 +/- 0.6 ng/ml) (P less than 0.01). To determine whether this atropine blockade was at the pituitary level, a series of in vitro studies were conducted using monolayer cultures of cells from bovine anterior pituitary glands. GHRH 1-44 (10(-8) M) stimulated bovine GH release (11.1 +/- 1.5 micrograms/ml) as compared to control values (5.1 +/- 0.4 microgram/ml) (P less than 0.01). This response was not altered by 10(-6) M atropine (14.9 +/- 0.9 microgram/ml). Similar results were obtained with GHRH, 10(-9) M, with or without atropine, 10(-7) M. Addition of 10(-6) M Ach to the incubation medium significantly increased bovine GH release (12.7 +/- 1.2 microgram/ml) and the effect of 10(-6) M Ach and 10(-8) M GHRH was additive (20.9 +/- 2.1 micrograms/ml) (P less than 0.01). Similar results were obtained with Ach, 10(-5) M, and GHRH, 10(-9) M. Atropine or eserine alone did not alter basal GH secretion, and atropine blocked Ach-stimulating activity. In conclusion, atropine blockade of GHRH-induced GH secretion appears to be exerted at a site other than pituitary.     

Short-chain fatty acids inhibit the release and content of growth hormone in anterior pituitary cells of the goat

The effects of short-chain fatty acids (SCFA: acetate, propionate, and butyrate) on growth hormone (GH)-releasing hormone (GHRH)-induced GH secretion from pituitary somatotrophs were assessed on isolated anterior pituitary cells of goats. Cells were cultured in Dulbecco's modified Eagle's medium for 3 days, either in the presence (1, 3, or 10 mM) or in the absence of each SCFA, and then stimulated with GHRH (10(-12) to 10(-7) M) for 30 min, again in the presence of and at the concentration of SCFA used over the previous 3 days. In the cells cultured in the absence of SCFA, the addition of SCFA to the medium during the GHRH stimulation period did not significantly change GHRH-induced GH release. However, in cells cultured in the presence of either propionate (3 or 10 mM) or butyrate (1, 3, or 10 mM), the addition of SCFA to the medium during GHRH stimulation significantly reduced the GHRH-induced GH release. The inhibitory effects of SCFA were dependent on the concentrations of SCFA and were greater for butyrate than for propionate. In the cells cultured in the presence of butyrate, but not in the absence, the total GH production (the sum of the released GH and the remaining GH after stimulation) was also significantly reduced. The GHmRNA expression was reduced in the cells cultured with 10 mM butyrate, whereas it was enhanced by the stimulation with 10(-7) M GHRH. These findings suggest that propionate and butyrate may inhibit GHRH-induced GH release and GH production by caprine anterior pituitary cells.     

Cortistatin inhibits growth hormone release from human fetal and adenoma pituitary cells and prolactin secretion from cultured prolactinomas

CONTEXT: Cortistatin (CST) is a neuropeptide that shares high homology with somatostatin and binds with high affinity to all somatostatin receptor (SSTR) subtypes. Many of its endocrine and biological activities overlap with those of somatostatin. OBJECTIVE/DESIGN: The objective of the study was to assess the direct in vitro effects of CST on human pituitary hormone secretion. SETTING: This study was performed in the endocrine laboratory of a tertiary academic medical center. MATERIALS: Primary cell cultures of human fetal (21-25 wk gestation) pituitary tissues and cultured hormone-secreting adenoma cells were used in this study. INTERVENTIONS: Cell cultures were incubated with CST-14 or CST-17, somatostatin, GHRH, SSTR analogs, and ghrelin analogs, and hormone secretion was analyzed. OUTCOME MEASURES: GH and prolactin (PRL) medium concentrations were tested by hormone assay, and SSTR mRNA was tested by RT-PCR. RESULTS: CST-14 (10 nm) inhibited GH secretion by up to 65% in all fetal pituitary specimens after 4-h incubation (P < 0.05). CST-14 or CST-17 (10 nm) inhibited basal GH secretion in six of the 13 GH-cell adenomas and two of the three GH-PRL mixed adenomas. CST-17 (100 nm) suppressed the GH response to GHRH and ghrelin analog (10 nm each) by 30-50% in adenomas (P < 0.05). Three PRL-adenomas treated with CST-17 (10 nm) showed a 20-40% inhibition of PRL release (P < 0.05), whereas in three others no suppression or mild response was achieved at this concentration. A comparable inhibition of PRL secretion was obtained with SSTR5-selective analog but significantly less with SSTR2-preferential compounds. RT-PCR revealed the expression of both SSTR2 and SSTR5 in all GH-cell and mixed adenomas studied and all PRL-secreting adenomas studied, except for two of the CST-resistant prolactinomas, in which SSTR5 was absent. CONCLUSIONS: This is the first report of in vitro CST suppression of human GH and PRL in cultured pituitary tissues. The regulation of PRL release from cultured adenomas appears to be primarily mediated by SSTR5.     

Ghrelin main action on the regulation of growth hormone release is exerted at hypothalamic level

Ghrelin, a recently isolated hormone, seems to participate in the physiological regulation of GH secretion. Exogenously administered ghrelin stimulates GH discharge in all species so far tested including man, but whether this action is exerted at pituitary or alternatively at hypothalamic level is not known at present. To understand the point of ghrelin action a group of patients with organic lesion mainly in the hypothalamic area and matched controls were studied. Patients showed a severe GH deficiency after hypothalamic stimulation (ITT), but partial response after GHRH administration. Cases and controls were tested on three separate days by either ghrelin; GHRH; and ghrelin plus GHRH; always at 1 micro g/Kg iv. The mean GH peak after stimulation in the patients were: 0.4 +/- 0.1 micro g/L by ITT; 3.1 +/- 0.5 micro g/L after GHRH; 2.0 +/- 0.8 micro g/L after ghrelin; and 9.6 +/- 2.9 micro g/L after the combination of GHRH plus ghrelin. In the controls GHRH induced a GH peak of 21.2 +/- 7.5 micro g/L, and 75.1 +/- 16.0 micro g/L after ghrelin with a peak after GHRH + ghrelin of 103.5 +/- 26.4 micro g/L. These data indicate that when hypothalamic structures are not operative ghrelin, either alone or in combination with GHRH, is not able to significantly release GH. In addition to postulating a hypothalamic point of action for the ghrelin-induced GH secretion, these results suggests that ghrelin will not have significant clinical utility in patients with GH deficiency due to organic lesion.     

Interrelationship between the novel peptide ghrelin and somatostatin/growth hormone-releasing hormone in regulation of pulsatile growth hormone secretion

GH is an anabolic hormone that is essential for normal linear growth and has important metabolic effects throughout life. The ultradian rhythm of GH secretion is generated by the intricate patterned release of two hypothalamic hormones, somatostatin (SRIF) and GHRH, acting both at the level of the pituitary gland and within the central nervous system. The recent discovery of ghrelin, a novel GH-releasing peptide identified as the endogenous ligand for the GH secretagogue receptor and shown to induce a positive energy balance, suggests the existence of an additional neuroendocrine pathway for GH control. To further understand how ghrelin interacts with the classical GHRH/SRIF neuronal system in GH regulation, we used a combined physiological and histochemical approach. Our physiological studies of the effects of ghrelin on spontaneous pulsatile GH secretion in conscious, free-moving male rats demonstrate that 1) ghrelin, administered either systemically or centrally, exerts potent, time-dependent GH-releasing activity under physiological conditions; 2) ghrelin is a functional antagonist of SRIF, but its GH-releasing activity at the pituitary level is not dependent on inhibiting endogenous SRIF release; 3) SRIF antagonizes the action of ghrelin at the level of the pituitary gland; and 4) the GH response to ghrelin in vivo requires an intact endogenous GHRH system. Our dual chromogenic and autoradiographic in situ hybridization experiments provide anatomical evidence that ghrelin may directly modulate GHRH mRNA- and neuropeptide Y mRNA-containing neurons in the hypothalamic arcuate nucleus, but that SRIF mRNA-expressing cells are not major direct targets for ghrelin. Together, these findings support the idea that ghrelin may be a critical hormonal signal of nutritional status to the GH neuroendocrine axis serving to integrate energy balance and the growth process.     

The role of pituitary ghrelin in growth hormone (GH) secretion: GH-releasing hormone-dependent regulation of pituitary ghrelin gene expression and peptide content

Ghrelin is a GH-releasing peptide originally purified from the rat stomach. It has been demonstrated that ghrelin expression, within the gastroenteric system, is regulated by both the metabolic and GH milieu. Our laboratory and others have previously reported that ghrelin is also produced in the pituitary. Given that the receptor for ghrelin [GH secretagogue receptor (GHS-R)] is also expressed by the pituitary, the possibility exists that locally produced ghrelin plays an autocrine/paracrine role in regulating GH release. Because we have previously reported that GHRH infusion increases pituitary levels of ghrelin mRNA, we hypothesized that GHRH could be a key regulator of pituitary ghrelin expression. In this report, we demonstrate that 4-h GHRH infusion increased pituitary ghrelin peptide content. Interestingly, under experimental conditions in which hypothalamic GHRH expression is increased, e.g. GH deficiency due to GH gene mutation, glucocorticoid deficiency, and hypothyroidism, we observed that pituitary ghrelin expression (mRNA levels and peptide content) was also increased. Consistent with this positive correlation between GHRH and ghrelin, pituitary ghrelin expression (mRNA levels and peptide content) was found to be decreased in conditions in which hypothalamic GHRH expression is decreased, e.g. GH treatment, glucocorticoid excess, hyperthyroid state, and food deprivation. Collectively, these results suggest that pituitary ghrelin expression is GHRH dependent. We also conducted functional studies to examine whether the pituitary ghrelin/GHS-R system contributes to GH release after GHRH stimulation, by challenging pituitary cell cultures with GHRH in the presence of a GHS-R-specific inhibitor ([d-Lys-3]-GHRP-6). The GHS-R inhibitor did not affect GH release in the absence of GHRH, but significantly reduced GHRH-mediated GH release. This is the first report demonstrating that endogenous pituitary ghrelin can play a physiological role in GH release, by optimizing somatotroph responsiveness to GHRH.     

Endocrine activities of ghrelin, a natural growth hormone secretagogue (GHS), in humans: comparison and interactions with hexarelin, a nonnatural peptidyl GHS, and GH-releasing hormone

An endogenous ligand for the GH secretagogue-receptor (GHS-receptor) has recently been isolated, from both the rat and the human stomach, and named ghrelin. It is a 28-amino-acid peptide showing a unique structure with an n-octanoyl ester at its third serine residue, which is essential for its potent stimulatory activity on somatotroph secretion. In fact, it has been demonstrated that ghrelin specifically stimulates GH secretion from both rat pituitary cells in culture and rats in vivo. The aim of the present study was to test the GH-releasing activity of ghrelin in humans and to compare it with that of GHRH and hexarelin (HEX), a nonnatural peptidyl GHS, which possesses strong GH-releasing activity but also significantly stimulates PRL, ACTH, and cortisol secretion. To clarify the mechanisms of action underlying the GH-releasing activity of ghrelin in humans, its interaction with GHRH and HEX was also studied. Seven normal young volunteers (7 men; 24-32 yr old; body mass index, 20-24 kg/m(2)) were studied. All subjects underwent the administration of ghrelin, HEX, and GHRH-29 (1.0 microg/kg i.v. at 0 min) as well as placebo (2 mL isotonic saline i.v. at 0 min). Six subjects also underwent the combined administration of ghrelin and GHRH or HEX. Blood samples were taken every 15 min from -15 up to +180 min. GH levels were assayed at each time point in all sessions; PRL, ACTH, cortisol, and aldosterone levels were also assayed after administration of ghrelin and/or HEX. Ghrelin administration induced a prompt and marked increase in circulating GH levels (Cmax, mean +/- SEM, 92.1 +/- 16.7 microg/L; area under the curve, 1894.9 +/- 347.8 microg/L.h). The GH response to ghrelin was clearly higher (P < 0.01) than the one recorded after GHRH (26.7 +/- 8.7 microg/L; 619.6 +/- 174.4 microg/L.h) and even significantly higher (P < 0.05) than after HEX (68.4 +/- 14.7 microg/L; 1546.9 +/- 380.0 microg/L x h). Ghrelin administration also induced an increase in PRL, ACTH, and cortisol levels; these responses were higher (P < 0.05) than those elicited by HEX. A significant increase in aldosterone levels was recorded after ghrelin but not after HEX. The endocrine responses to ghrelin were not modified by the coadministration of HEX. On the other hand, the coadministration of ghrelin and GHRH had a real synergistical effect (P < 0.05) on GH secretion (133.6 +/- 22.5 microg/L; 3374.3 +/- 617.3 microg/L x h). In conclusion, ghrelin, a natural ligand of GHS-receptor, exerts a strong stimulatory effect on GH secretion in humans, releasing more GH than GHRH and even more than a nonnatural GHS such as HEX. Ghrelin, as well as HEX, also stimulates lactotroph and corticotroph secretion. Ghrelin shows no interaction with HEX, whereas it has a synergistical effect with GHRH on GH secretion. Thus, ghrelin is a new hormone playing a major role in the control of somatotroph secretion in humans, and its effects are imitated by nonnatural GHS.     

Agouti-related peptide,neuropeptide Y,and somatostatin-producing neurons are targets for ghrelin actions in the rat hypothalamus

Ghrelin, the endogenous ligand of the GH secretagogue receptor, acts at central level to elicit GH release and regulate food intake. To elucidate the neural circuit that exerts its effects, we measured the expression of hypothalamic neuropeptides involved in weight regulation and GH secretion after ghrelin administration. Adult male rats, fed or fasted for 72 h, were treated centrally (intracerebroventicularly) with a single dose of ghrelin (5 micro g). After 2, 4, and 6 or 8 h, agouti-related peptide, melanin-concentrating hormone, neuropeptide Y, prepro-orexin, GHRH, and somatostatin mRNA levels were measured by in situ hybridization. We found that ghrelin increased agouti-related peptide and neuropeptide Y expression in the arcuate nucleus of the hypothalamus of fed and fasted rats. In contrast, no change was demonstrated in the mRNA levels of the other neuropeptides studied at any time evaluated. Finally, we examined the effect of ghrelin on GHRH and somatostatin mRNA levels in GH-deficient (dwarf) rats. Our results show that ghrelin increases somatostatin mRNA levels in the hypothalamus of these rats. This study furthers our understanding of the molecular basis and mechanisms involved in the effect of ghrelin on food intake and GH secretion.     

Homologous and heterologous in vitro regulation of pig pituitary somatostatin receptor subtypes, sst1, sst2 and sst5 mRNA

Somatostatin (SRIF) is commonly regarded as an inhibitor of GH release in rodents and humans. However, in pigs, SRIF can stimulate the release of GH at low (picomolar) doses, while inhibiting GHRH-stimulated GH release at high (nanomolar) doses in primary pituitary cell cultures. One possible mechanism by which pig cells respond differently to the actions of SRIF is by differential expression and regulation of SRIF receptor subtypes. As no information is available on the homologous regulation of SRIF receptors in pigs, we examined the acute (4 h) in vitro effects of SRIF on mRNA levels of SRIF receptors sst1, sst2 and sst5 by multiplex RT-PCR. These particular sst subtypes were selected because all three have been implicated in the inhibitory effects of SRIF on GH release in both rodents and humans. At a high dose (10(-7) M), SRIF stimulated the expression of sst1, sst2 and sst5 in pig pituitary cell cultures. At a low dose (10(-13) M), SRIF markedly increased sst1, without affecting sst2 or sst5. Given that our laboratory has shown SRIF at high and low doses stimulates cAMP production in a subpopulation of pig somatotropes, we sought to determine if this signaling pathway may be responsible for the stimulatory effect of SRIF on its own receptor expression. The receptor-independent cAMP activator forskolin elevated sst1 and sst2 mRNA levels but did not affect sst5 expression, suggesting the stimulatory actions of high- and low-dose SRIF on sst1 and high-dose SRIF on sst2 mRNA levels can be mediated by activation of cAMP, whereas the stimulatory effect of high-dose SRIF on sst5 mRNA is elicited by a cAMP-independent pathway. Interestingly, both GHRH (10(-8) M) and ghrelin (10(-6) M), which release GH in pig pituitary cell cultures via cAMP-dependent mechanisms, decreased sst5 without altering sst1 or sst2 mRNA levels. Since the actions of GHRH and ghrelin on sst expression markedly contrasted with that observed for SRIF and forskolin these results clearly indicate GHRH and ghrelin are regulating sst5 mRNA levels by a cAMP-independent signaling pathway. Taken together, our results demonstrate that expression of pig SRIF receptors is under a complex, receptor subtype-selective regulation, wherein the concerted actions of key regulators of somatotrope function would play divergent and dose-dependent effects.