Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a.

Recent studies demonstrate widespread expression of ghrelin among tissues and have uncovered its pleiotropic nature. We have examined gene expression of ghrelin and its two receptor splice variants, growth hormone secretagogue receptors (GHS-R) 1a and 1b, in human bone biopsies and in the human pre-osteoblastic SV-HFO cell line during differentiation. Additionally, we examined proliferative effects of ghrelin and unacylated ghrelin (UAG) in differentiating and non-differentiating cells. We detected GHS-R1b mRNA in human bone and osteoblasts but not ghrelin's cognate receptor GHS-R1a, using two different real-time PCR assays and both total RNA and mRNA. In osteoblasts GHS-R1b mRNA expression remained low during the first 14 days of culture, but increased 300% in differentiating cells by day 21. Both human bone biopsies and osteoblasts expressed ghrelin mRNA, and osteoblasts were found to secrete ghrelin. Overall, ghrelin gene expression was greater in differentiating than non-differentiating osteoblasts, but was not increased during culture in either group. Ghrelin and UAG induced thymidine uptake dose-dependently, peaking at 1 and 10 nM respectively, at day 6 of culture in both non-differentiating and differentiating osteoblasts. The proliferative response to ghrelin and UAG declined with culture time and state of differentiation. The proliferative effects of ghrelin and UAG were suppressed by inhibitors of extracellular-signal-regulated kinase (ERK) and phosphoinositide-3 kinase, and both peptides rapidly induced ERK phosphorylation. Overall, our data suggest new roles for ghrelin and UAG in modulating human osteoblast proliferation via a novel signal transduction pathway.

Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans.

Ghrelin possesses strong GH-releasing activity but also other endocrine activities including stimulation of PRL and ACTH secretion, modulation of insulin secretion and glucose metabolism. It is assumed that the GH secretagogue (GHS) receptor (GHS-R) 1a mediates ghrelin actins provided its acylation in Serine 3; in fact, acylated ghrelin only is able to exert endocrine activities. Acylated ghrelin (AG) is present in serum at a 2.5 fold lower concentration than unacylated ghrelin (UAG). UAG, however, is not biologically inactive; it shares with AG some non-endocrine actions like cardiovascular effects, modulation of cell proliferation and even some influence on adipogenesis. Thus, these actions are likely to be mediated by GHS-R subtypes able to bind ghrelin independently of its acylation. In order to further clarify whether UAG is really devoid of any endocrine action, we studied the interaction of the combined administration of AG and UAG (1.0 microg/kg i.v.) in 6 normal young volunteers (age [mean +/- SE]: 25.4 +/- 1.2 yr; BMI: 22.3 +/- 1.0 kg/m2). As expected, AG induced marked increase (p < 0.01) in circulating GH, PRL, ACTH and cortisol levels. AG administration was also followed by a decrease in insulin levels (-285.4 +/- 64.8 mU*min/l; p < 0.05) and an increase in plasma glucose levels (1068.4 +/- 390.4 mg*min/dl; p < 0.01). UAG alone did not induce any change in these parameters. UAG also failed to modify the GH, PRL, ACTH and cortisol responses to AG. However, when UAG was co-administered together with AG, no significant change in insulin (-0.5 +/- 40.9 mU*min/l) and glucose levels (455.9 +/- 88.3 mg*min/dl) was recorded anymore, indicating that the insulin and glucose response to AG has been abolished by UAG. In conclusion, non-acylated ghrelin does not affect the GH, PRL, and ACTH response to acylated ghrelin but is able to antagonize the effects of acylated ghrelin on insulin secretion and glucose levels. These findings indicate that unacylated ghrelin is metabolically active and is likely to counterbalance the influence of acylated ghrelin on insulin secretion and glucose metabolism. As GHS-R1a is not bound by unacylated ghrelin, these findings suggest that GHS receptor subtypes mediate the metabolic actions of both acylated and unacylated ghrelin.

Effects of ghrelin on the insulin and glycemic responses to glucose, arginine, or free fatty acids load in humans.

Ghrelin possesses central and peripheral endocrine actions including influence on the endocrine pancreatic function. To clarify this latter ghrelin action, in seven normal young subjects [age (mean +/- SEM), 28.3 +/- 3.1 yr; body mass index, 21.9 +/- 0.9 kg/m(2)), we studied insulin and glucose levels after acute ghrelin administration (1.0 microg/kg i.v.) alone or combined with glucose [oral glucose tolerance test (OGTT), 100 g orally], arginine (ARG, 0.5 g/kg i.v.) or free fatty acid (FFA, Intralipid 10%, 250 ml). Ghrelin inhibited (P < 0.05) insulin and increased (P < 0.05) glucose levels. OGTT increased (P < 0.01) glucose and insulin levels. FFA increased (P < 0.05) glucose but did not modify insulin levels. ARG increased (P < 0.05) both insulin and glucose levels. Ghrelin did not modify both glucose and insulin responses to OGTT as well as the FFA-induced increase in glucose levels; however, ghrelin administration was followed by transient insulin decrease also during FFA. Ghrelin blunted (P < 0.05) the insulin response to ARG and enhanced (P < 0.05) the ARG-induced increase in glucose levels. In all, ghrelin induces transient decrease of spontaneous insulin secretion and selectively blunts the insulin response to ARG but not to oral glucose load. On the other hand, ghrelin raises basal glucose levels and enhances the hyperglycemic effect of ARG but not that of OGTT. These findings support the hypothesis that ghrelin exerts modulatory action of insulin secretion and glucose metabolism in humans.

Concomitant impairment of growth hormone secretion and peripheral sensitivity in obese patients with obstructive sleep apnea syndrome.

To clarify the impairment of the GH/IGF-I axis in obstructive sleep apnea syndrome (OSAS), in 13 adult male patients with OSAS (OSA) as well as 15 weight-matched patients with simple obesity (OB) and 10 normal lean male subjects (NS), we studied: 1) the GH response to GHRH (1 micro g/kg iv) plus arginine (30 g iv); and 2) the IGF-I and IGF binding protein-3 responses to a very low dose recombinant human (rh)GH treatment (5.0 microg/kg sc per day for 4 d). The GH response to arginine plus GHRH in OSA was lower than in OB (P < 0.05), which in turn was lower than in NS (P < 0.001). Basal IGF-I levels in OSA were lower than in OB (P < 0.05), which in turn were lower than in NS (P < 0.03). As opposed to OB and NS, in OSA a very low rhGH dose did not affect IGF-I. Adjusting for age and basal values, rhGH-induced IGF-I rise in OSA was lower than in OB (P < 0.01). IGF binding protein-3, glucose, and insulin levels in the three groups were not modified by rhGH. OSA show a more marked impairment of the maximal secretory capacity of somatotroph cells together with reduced IGF-I sensitivity to rhGH stimulation. These findings suggest that OSAS is connoted by a concomitant impairment of GH secretion and sensitivity.

Neuroendocrine and metabolic effects of acute ghrelin administration in human obesity.

Ghrelin stimulates appetite and plays a role in the neuroendocrine response to energy balance variations. Ghrelin levels are inversely associated with body mass index (BMI), increased by fasting and decreased by food intake, glucose load, insulin, and somatostatin. Ghrelin levels are reduced in obesity, a condition of hyperinsulinism, reduced GH secretion, and hypothalamus-pituitary-adrenal axis hyperactivity. We studied the endocrine and metabolic response to acute ghrelin administration (1.0 microg/kg i.v.) in nine obese women [OB; BMI (mean +/- SD) 36.3 +/- 2.3 kg/m(2)] and seven normal women (NW; BMI 20.3 +/- 1.7 kg/m(2)). Basal ghrelin levels in NW were higher than in OB (P < 0.05). In NW, ghrelin increased (P < 0.05) GH, prolactin (PRL), ACTH, cortisol, and glucose levels but did not modify insulin. In OB, ghrelin increased (P < 0.01) GH, PRL, ACTH, and cortisol levels. The GH response to ghrelin in OB was 55% lower (P < 0.02) than in NW, whereas the PRL, ACTH, and cortisol responses were similar. In OB, ghrelin increased glucose and reduced insulin (P < 0.05). Thus, obesity shows remarkable reduction of the somatotroph responsiveness to ghrelin, suggesting that ghrelin hyposecretion unlikely explains the impairment of somatotroph function in obesity. On the other hand, in obesity ghrelin shows preserved influence on PRL, ACTH, and insulin secretion as well as in glucose levels.

Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity.

We investigated the metabolic actions of ghrelin in humans by examining the effects of acute administration of acylated ghrelin, unacylated ghrelin, and the combination in eight adult-onset GH-deficient patients. We followed glucose, insulin, and free fatty acid concentrations before and after lunch and with or without the presence of GH in the circulation. We found that acylated ghrelin, which is rapidly cleared from the circulation, induced a rapid rise in glucose and insulin levels. Unacylated ghrelin, however, prevented the acylated ghrelin-induced rise in insulin and glucose when it was coadministered with acylated ghrelin. Surprisingly, the injection of acylated ghrelin induced an acute increase in unacylated ghrelin and therefore total ghrelin levels. Finally, acylated ghrelin decreased insulin sensitivity up to the end of a period of 6 h after administration. This decrease in insulin sensitivity was prevented by coinjection of unacylated ghrelin. This combined administration of acylated and unacylated ghrelin even significantly improved insulin sensitivity, compared with placebo, for at least 6 h, which warrants studies to investigate the long-term efficacy of this combination in the treatment of disorders with disturbed insulin sensitivity.

Effects of short-term administration of low-dose rhGH on IGF-I levels in obesity and Cushing's syndrome: indirect evaluation of sensitivity to GH.

OBJECTIVE: To verify the hypothesis of an increased sensitivity to GH in obesity (OB) and Cushing's syndrome (CS). DESIGN: We studied the effects of short-term administration of low-dose rhGH on circulating IGF-I levels in patients with simple OB or CS and in normal subjects (NS). METHODS: Nineteen women with abdominal OB aged (mean +/- s.e.m.) 38.2+/-3.1 years, body mass index 40.7+/-2.5 kg/m(2), waist to hip ratio 0.86+/-0.02, ten with CS (50.4+/-4.2 years, 29.7 +/- 3.3 kg/m(2)) and 11 NS (35.0+/-3.6 years, 20.5+/-0.5 kg/m(2)) underwent s.c. administration of 5 microg/kg per day rhGH at 2200 h for four days. Serum IGF-I, IGF-binding protein-3 (IGFBP-3), GH-binding protein (GHBP), insulin and glucose levels were determined at baseline and 12 h after the first and the last rhGH administration. RESULTS: Basal IGF-I levels in NS (239.3+/-22.9 microg/l) were similar to those in OB (181.5+/-13.7 microg/l) and CS (229.0+/-29.1 microg/l). Basal IGFBP-3, GHBP and glucose levels in NS, OB and CS were similar while insulin levels in NS were lower (P<0.01) than those in OB and CS. In NS, the low rhGH dose induced a sustained rise of IGF-I levels (279.0+/-19.5 microg/l, P<0.001), a non-significant IGFBP-3 increase and no change in GHBP, insulin and glucose levels. In OB and CS, the IGF-I response to rhGH showed progressive increase (246.2+/-17.2 and 311.0+/-30.4 microg/l respectively, P<0.01 vs baseline). Adjusting by ANCOVA for basal values, rhGH-induced IGF-I levels in CS (299.4 microg/l) were higher than in OB (279.1 microg/l, P<0.01), which, in turn, were higher (P<0.05) than in NS (257.7 microg/l). In OB, but not in CS, IGFBP-3 and insulin levels showed slight but significant (P<0.05) increases during rhGH treatment, which did not modify glucose levels in any group; thus, in the OB patient group a significant fall in glucose/insulin ratio was observed. CONCLUSIONS: Short-term treatment with low-dose rhGH has enhanced stimulatory effect on IGF-I levels in OB and, particularly, in hypercortisolemic patients. These findings support the hypothesis that hyperinsulinism and hypercortisolism enhance the sensitivity to GH in humans.

Cortistatin-17 and -14 exert the same endocrine activities as somatostatin in humans.

Cortistatin (CST) is a neuropeptide, which binds with high affinity all somatostatin (SS) receptor subtypes and shows high structural homology with SS itself. A receptor specific for CST only, i.e., not recognized by SS, has been recently described in agreement with data reporting that not all CST actions are shared by SS. Interestingly, CST but not SS also binds ghrelin receptor (GHS-R1a) in vitro, suggesting a potential interplay between CST and ghrelin system. The aim of this study was to investigate in humans the endocrine and metabolic activities of human CST-17 in comparison with rat CST-14 that has previously been shown to exert the same endocrine actions of SS in healthy volunteers. To this aim, in six healthy male volunteers (age [median, 3rd-97th centiles]: 28.5; 23.6-34.3 years; Body Mass Index: 23.5; 21.0-25.1 kg/m(2)), we studied the effects of human CST-17 (2.0 microg/kg/h iv over 120 min), rat CST-14 (2.0 microg/kg/h iv over 120 min) and SS-14 (2.0 microg/kg/h iv over 120 min) on: (a) spontaneous GH, ACTH, PRL, cortisol, insulin and glucose levels; (b) the GH responses to GHRH (1.0 microg/kg iv at 0 min); (c) the GH, PRL, ACTH, cortisol, insulin and glucose responses to ghrelin (1.0 microg/kg iv at 0 min). CST-17 inhibited (p < 0.01) basal GH secretion to the same extent of CST-14 and SS-14. Spontaneous PRL, ACTH and cortisol secretion were not significantly modified by CST-17, CST-14 or SS-14. CST-17 as well as CST-14 and SS-14 also inhibited (p < 0.05) spontaneous insulin secretion to a similar extent. None of these peptides modified glucose levels. The GH response to GHRH was inhibited to the same extent by CST-17 (p < 0.01), CST-14 (p < 0.01) and SS-14 (p < 0.05 ). The ghrelin-induced GH response was higher than that elicited by GHRH (p < 0.01) and inhibited by CST-17 (p < 0.05) as well as by CST-14 (p < 0.05) and SS-14 (p < 0.01). The PRL, ACTH and cortisol responses to ghrelin were unaffected by CST-17, CST-14 or SS-14. On the other hand, the inhibitory effect of ghrelin on insulin levels was abolished by CST-17, CST-14 or SS-14 (p < 0.05) that, in turn, did not modify the ghrelin-induced increase in glucose levels. In conclusion, this study demonstrates that human CST-17 and rat CST-14 exert the same endocrine activities of SS in humans. The endocrine actions of human and rat CST therefore are likely to reflect activation of classical SS receptors.

Neuroendocrine and metabolic determinants of the adaptation of GH/IGF-I axis to obesity.

Obese patients show marked impairment in spontaneous secretion as well as in the somatotroph responsiveness to all provocative stimuli. GH insufficiency in obese patients has been reported reversible after long-term diet and marked weight loss but somatotroph secretion is not restored by fasting. Among potential neuroendocrine causes, GHRH hypoactivity has been shown but it is likely that alterations in the influence of ghrelin, the gastric-derived natural ligand of the GHS-R, and or of the NPY/leptin interplay could have a role. Among metabolic alterations, the chronic elevation of FFA levels and hyperinsulinism probably have a key role in causing GH insufficiency in obesity. Despite marked GH insufficiency, total IGF-I levels are basically preserved while free IGF-I levels are even increased thus questioning real hypoactivity of GH/IGF-I axis in obesity. Peripheral GH hypersensitivity due to increased GH receptor status, hyperinsulinism and reduced IGFBP-I levels likely explain almost normal total IGF-I and increased free IGF-I levels which, in turn, probably exert an increased negative feedback action on somatotroph cells.

Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway.

Ghrelin is expressed in normal human adrenocortical cells and induces their proliferation through growth hormone secretagogue receptor 1a (GHS-R1a). Consequently, it was of interest to us to determine whether acylated ghrelin and its predominant serum isoform, unacylated ghrelin, also act as factors for adrenocortical carcinoma cell growth. To examine a potential ghrelin-regulated system in adrenocortical tumors, we measured proliferative effects of acylated and unacylated ghrelin in the adrenocortical carcinoma cell lines SW-13 and NCI-H295R. We also examined the expression of ghrelin, GHS-R1a, and corticotrophin-releasing factor receptor 2 (CRF-R2). Acylated and unacylated ghrelin in the nanomolar range dose-dependently induced adrenocortical cell growth up to 200% of untreated controls, as measured by thymidine uptake and WST1 assay. The proliferative effects of acylated and unacylated ghrelin in SW-13 cells was blocked by [D-Lys(3)]growth hormone-releasing peptide 6 (GHRP6), but a CRF-R2 antagonist had no effect on unacylated ghrelin growth stimulation. Cell cycle analysis suggests that acylated and unacylated ghrelin suppress the sub-G(0)/apoptotic fraction by up to 50%. Measurement of DNA fragmentation and caspase-3 and -7 activity in SW-13 cells confirmed that acylated and unacylated ghrelin suppress apoptotic rate. SW-13 cells express preproghrelin mRNA and secrete ghrelin, and [D-Lys(3)]GHRP6 suppresses their basal proliferation rate, strongly suggesting that ghrelin could act as an auto/paracrine growth factor. Acylated and unacylated ghrelin are potential auto/paracrine factors acting through an antiapoptotic pathway to stimulate adrenocortical tumor cell growth. Unacylated ghrelin-stimulated growth is suppressed by an antagonist of GHS-R1a, suggesting either that unacylated ghrelin is acylated before its action or that ghrelin, unacylated ghrelin, and [D-Lys(3)]GHRP-6 bind to a novel receptor in these cells.

Somatostatin, cortistatin, ghrelin and glucose metabolism.

At first sight, the title is confusing as it seems to try to merge four unrelated topics into a single presentation. Somatostatin, cortistatin (CST) and ghrelin display broad biological activities, including metabolic effects. However, although apparently unrelated, these peptides entities have more in common than it might be expected and their reciprocal interactions give a new perspective to the hormonal regulation of glucose metabolism. Let's analyze the ghrelin receptor subtype GH secretagogue (GHS)-receptor 1a (R1a). Taking into account the GHS-R1a as receptor of reference, acylated ghrelin is one of its natural ligands. Interestingly, it has been demonstrated that also CST, a neuropeptide, binds with high affinity to the GHS-R1a in human hypothalamus and pituitary tissues. CST is a recently described neuropeptide showing high structural homology with somatostatin that binds to all somatostatin receptor subtypes (SSTRs) with an affinity (1-2 nM). In fact, CST and somatostatin exhibit the same endocrine activities. The existence of specific receptors which selectively bind somatostatin or CST has been hypothesized, based on evidence that CST possesses an action profile different from somatostatin and that CST and somatostatin are often co-expressed in the same neurons but are regulated by different stimuli. Given these findings, the ability of CST to bind the GHS-R1a is of particular relevance because somatostatin and its fragments do not bind the same receptor. Interestingly, the classical synthetic somatostatin analogs, i.e. octreotide, lanreotide and vapreotide bind the GHS-R1a with an affinity lower than that of CST. These findings have generated the hypothesis that CST, because of its ability to bind both SSTRs and GHSRs, would represent the link between ghrelin and "somatostatin/CST" system that had not previously been demonstrated. On the other hand, the GHS-R1a is unlikely to be the only GHS-R. It has been already demonstrated that a GHS-R subtype able to bind non-acylated as well as acylated ghrelin exists and likely mediates biological activities. Another GHS-R subtype likely mediates the influence of unacylated ghrelin on glucose metabolism, since it does not bind nor activates the GHS-R1a. Given this complexity, it is clear that further studies are required to clarify whether ghrelin is the sole ligand or one of a number of ligands activating the GHS-R 1a and whether that receptor used for ghrelin isolation is the sole receptor or one of a group of receptors for such ligands.

Pituitary function during severe and life-threatening illnesses.

The catabolic state of prolonged critical illness is associated with a low activity of anterior pituitary functions. Before considering endocrine intervention in these conditions, a detailed understanding of the neuroendocrinology of the stress response is warranted. It is now clear that the acute phase and the later phase of critical illness behave differently from an endocrinological point of view. When the disease process becomes prolonged, there is a uniformly-reduced pulsatile secretion of anterior pituitary hormones with proportionally reduced concentrations of peripheral anabolic hormones. Apparently, there is a constant interaction between neuroendocrine and internal immunoregulatory mechanisms that assures the fine tuning of both the neuro-endocrine and the immune system, so that both are able to preserve homeostasis of patients during severe and life-threatening illnesses.

Glucagon administration elicits blunted GH but exaggerated ACTH response in obesity.

Reduction in both spontaneous and stimulated GH secretion in obesity has been clearly demonstrated. Mild hyperactivity of hypothalamus-pituitary-adrenal (HPA) axis has been also reported. Glucagon, at least after im administration, induces clear increase in either GH or ACTH and F levels but its effect on somatotroph and corticotroph secretion in obesity has never been studied. In 7 patients with abdominal obesity (OB, aged 24-42 yr, BMI: 29.1-43.9 kg/m2, waist/hip ratio [WHR]: 0.86-1.00) we studied the GH, ACTH and F responses to the im administration of glucagon (0.017 mg/kg at 0 min). The results in OB were compared with those in a group of 6 age-matched controls normal subjects (Ns aged 26-32 yr, BMI 19.7-22.5 kg/m2). In Ns glucagon administration induced clear increase in GH (peak vs baseline, mean+/-SE: 11.6+/-3.4 vs 3.3+/-0.7 microg/l, p<0.02), and ACTH (52.9+/-15.2 vs 19.0+/-1.5 pg/ml, p<0.02) levels which peaked at +150 and +165 min, respectively. Increase in F levels (222.3+/-23.8 vs 158.3+/-7.0 ng/ml, p<0.05) was also recorded but peaked at +180 min. In OB glucagon administration induced GH response (7.4+/-2.3 vs 0.8+/-0.6 microg/l) lower (p<0.05) than that recorded in Ns; when the GH responses were evaluated by co-variance analysis, a significant difference between the 2 groups was recorded in term of peaks but not of AUCs. On the other hand, the ACTH response to glucagon in OB was higher than that in Ns (11452.6+/-2447.7 vs 4892.2+/-719.4 pg/ml x min, p<0.05). The F response to glucagon in OB and Ns was, however, similar (24057.9+/-4109.1 vs 29835.9+/-1566.0 ng/ml x min). In conclusion, this study demonstrates that in obese patients the im administration of glucagon elicits blunted GH response but exaggerated ACTH increase which is uncoupled with the adrenal response. These findings agree with the existence of concomitant GH insufficiency and altered corticotroph function in obesity.

Effects of alprazolam, a benzodiazepine, on the ACTH-, GH- and PRL-releasing activity of hexarelin, a synthetic peptidyl GH secretagogue (GHS), in patients with simple obesity and in patients with Cushing's disease.

GH secretagogues (GHS) possess potent GH-releasing activity but also stimulate PRL, ACTH and cortisol (F) secretion. To further clarify the endocrine activities of GHS, in 9 obese patients, 9 patients with Cushing's disease and 14 controls we studied the ACTH, F, GH and PRL responses to hexarelin (HEX, 2.0 micrograms/kg i.v.), a peptidyl GHS, alone and preceeded by alprazolam (ALP, 0.02 mg/kg p.o.), a benzodiazepine. The HEX-induced ACTH response in controls was similar to that in obese patients (delta peak: 9.9 +/- 1.9 and 24.7 +/- 7.6 ng/L, respectively) and both were lower (p < 0.002) than that in Cushing's patients (peak: 210.7 +/- 58.4 ng/L). The GH response to HEX in controls (peak: 58.1 +/- 10.3 x g/L) was higher (p < 0.001) than those in obese and Cushing's patients (18.2 +/- 3.8 and 12.6 +/- 5.4 x g/L, respectively) which, in turn, were similar. The PRL responses to HEX in controls, obese and Cushing's patients (peak: 11.9 +/- 1.6, 18.0 +/- 4.5 and 12.4 +/- 1.4 x g/L, respectively) were similar. In controls the HEX-induced ACTH response was abolished by ALP (peak: 8.6 +/- 2.4 vs 28.0 +/- 6.7 ng/L, p < 0.03) which, on the other hand, only blunted that in obese (peak: 12.7 +/- 2.1 vs 42.4 +/- 8.4 ng/L, p < 0.02) and did not modify that in Cushing's patients (205.6 +/- 55.4 vs 175.9 +/- 47.6 ng/L). ALP blunted the GH response to HEX in controls (peak: 31.0 +/- 7.1 x g/L, p < 0.03) while did not modify those in obese and in Cushing's patients (14.5 +/- 5.3 and 13.3 +/- 11.1 x g/L, respectively). ALP did not modify the HEX-induced PRL response in controls, obese and Cushing's patients (peak: 13.8 +/- 0.9, 16.3 +/- 2.4 and 19.2 +/- 1.1 x g/L, respectively). In conclusion, alprazolam inhibits the ACTH response to hexarelin in normal and obese subjects while fails to modify the exaggerated ACTH response in Cushing's Disease suggesting that GHS activate the HPA axis via the hypothalamus in normal and obese subjects but not in patients with Cushing's disease. Alprazolam is also able to blunt the GH-releasing activity of hexarelin in normal subjects but not the low GH response to the hexapeptide in obese and Cushing's patients. The PRL-releasing activity of hexarelin in controls, obese and hypercortisolemic patients is similar and is not modified by alprazolam pretreatment.

The GH/IGF-I axis in obesity: influence of neuro-endocrine and metabolic factors.

In this review we propose an integrated neuro-endocrine-metabolic point of view on the alterations (adaptations?) of GH/IGF-1 axis in obesity, summarizing the evidence from the literature, particularly focusing the data on humans and adding where possible results from our studies in this field. It is well-known that GH secretion is deeply impaired in overweight patients: we reviewed the multiple mechanisms underlying this issue, considering either central (CNS-related, such as impairment of GHRH tone or increased somatostatin release) or peripheral (ie metabolic: insulin, free fatty acids, glucose) factors. A central point of the debate about GH insufficiency in obesity is if it represents a simple adaptive phenomenon or reflects a true impairment of the axis activity. Evaluation of IGF-I levels and generation in obesity was the mean used to address this question: a bulk of evidence on IGF-I balance in human obesity has been provided, but the matter is still uncertain and unsolved.

Alprazolam, a benzodiazepine, blunts but does not abolish the ACTH and cortisol response to hexarelin, a GHRP, in obese patients.

GH secretagogues (GHS) act on specific receptors at the pituitary and hypothalamic level and possess potent GH-releasing activity but also stimulate prolactin (PRL), ACTH and cortisol (F) secretion. However, hyperactivity of the HPA axis in obesity has been reported. The objective of this study was to clarify the endocrine activity of GHS in obesity. In nine obese patients (obese OB), 9 F, age, (34.8 +/- 3.7 y, body mass index (BMI), 35.0 +/- 2.2 kg/m2; WHR, 0.9 +/- 0.02), 14 controls (normal subjects, NS), 14 F, 30.4 +/- 0.9 y, 20.0 +/- 0.4 kg/m2), we studied the ACTH, F and GH responses to hexarelin (HEX, 2.0 microg/kg), a peptidyl GHS, alone and preceded by alprazolam (ALP, 0.02 mg/kg), and a benzodiazepine which has an inhibitory effect on corticotroph secretion. The HEX-induced ACTH response in OB was higher than that in n.s., but this difference did not attain statistical significance. In n.s. the HEX-induced ACTH response was abolished by ALP (P < 0.03) which, however, only blunted that in OB (P < 0.02). The GH response to HEX in OB was lower (P < 0.02) than that in n.s.. ALP blunted the GH response to HEX in n.s. (P < 0.03) while it did not modify that in OB. The GABAergic activation by alprazolam abolishes the ACTH response to hexarelin in normal subjects, while it only blunts that in obese subjects. Moreover, alprazolam blunts the GH response to hexarelin in normal but not in obese subjects. Thus, obese patients show partial refractoriness to the inhibitory effect of alprazolam on both corticotroph and somatotroph function.

The activity of GH/IGF-I axis in anorexia nervosa and in obesity: a comparison with normal subjects and patients with hypopituitarism or critical illness.

GH/IGF-I axis activity changes have been reported both in anorexia nervosa (AN) and in obesity (OB). AN is characterized by GH hypersecretion and very low IGF-I levels as a result of undernutrition and acquired peripheral GH resistance. On the other hand OB is a GH hyposecretory state but IGF-I levels are generally preserved. The activity of GH/IGF-I axis in AN and OB has never been directly compared with that of other pathophysiological conditions such as hypopituitarism and critical illness in which a reduction of both GH and IGF-I secretion has been demonstrated. To this aim, we evaluated IGF-I levels and both basal and GHRH (1 microgram/kg) IV-induced GH secretion in 20 female patients with anorexia nervosa (mean age: 19.1 +/- 0.8 years) and in 15 female and 5 male patients with simple obesity (mean age: 39.0 +/- 3.0 years). We then compared the results with those of hypopituitaric patients with severe GH deficiency (GHD), including 10 female and 10 patients (mean age: 32.0 +/- 2.1 years), and with 4 female and 7 male patients with critical illness (CRI) following multiple trauma 72 hours after ICU admission (mean age: 59.2 +/- 1.2 years). Twenty-six normal subjects (NS) including 14 female and 12 male patients (mean age: 37.8 +/- 3.7 years) were studied as controls. Basal IGF-I levels in AN patients (93.5 +/- 11 micrograms/L) were lower (p < 0.001) than in the NS (201.7 +/- 13.5 micrograms/L) and OB (194.5 +/- 28.6 micrograms/L), which, in turn, were similar. IGF-I levels in AN patients were lower than in CRI patients (162.8 +/- 17.4 micrograms/L) and higher than in GHD patients (76.7 +/- 13.5 micrograms/L) but these differences did not attain statistical significance. Basal GH levels in AN patients (7.6 +/- 2.5 micrograms/L) were higher (p < 0.001) than in NS (1.8 +/- 0.3 micrograms/L), OB patients (1.1 +/- 0.5 micrograms/L), CRI patients (1.8 +/- 0.5 micrograms/L) and GHD patients (0.3 +/- 0.1 microgram/L), which were the lowest (p < 0.01). The GHRH-induced GH rise in AN patients (AUC: 2032.9 +/- 253.5 micrograms/L/h) was three fold higher (p < 0.001) than in NS (662.1 +/- 80.3 micrograms/L). On the other hand in OB (332.4 +/- 74.7 micrograms/L/h) the GH response to GHRH was similar to that in CRI (199.6 +/- 98.8 micrograms/L/h); both were clearly higher (p < 0.01) than in GHD patients (25.1 +/- 5.2 micrograms/L/h) but lower (p < 0.01) than in NS. These findings demonstrate that in AN patients, in spite of a clear increase of both basal and GHRH-induced GH secretion, IGF-I synthesis and release are as markedly impaired as in patients with panhypopituitarism and severe GHD. On the other hand in OB and in CRI, IGF-I synthesis and release are preserved despite marked impairment to GHRH-induced GH secretion. These results reinforce the major role of nutrition in conditioning the activity of GH/IGF-I axis in different patho-physiological states.

Short-term fasting abolishes the sex-related difference in GH and leptin secretion in humans.

We studied growth hormone (GH) and leptin secretion in eight male (age 29.3 +/- 1.2 yr, body mass index 22.2 +/- 0.5 kg/m(2)) and seven female normal subjects (28.0 +/- 0.8 yr, 20.1 +/- 0.7 kg/m(2)) before and after 36 h of fasting. In the fed state, 8-h mean GH and leptin concentrations were higher in females (P < 0.05 and P < 0. 0001, respectively). Fasting increased GH and decreased leptin in both sexes. There was significant interaction between gender and fasting (P < 0.05 for GH and P < 0.005 for leptin). Females showed a slighter increase in GH but a more marked decrease in leptin, so that there was no significant gender-related difference in GH and leptin after fasting. Fasting did not modify insulin-like growth factor (IGF) I, IGF binding protein (IGFBP)-3, acid-labile subunit, or GH binding protein; increased IGFBP-1 and free fatty acids (P < 0.0001) but decreased glucose (P < 0.001) and insulin levels (P < 0.05). In males, insulin levels were higher (P < 0.05) in the fed state and underwent deeper reduction after fasting (interaction P < 0.03). In conclusion, GH and leptin secretions are higher in women than in men in the fed but not in the fasting condition, which abolishes these gender-related differences in humans.

Assessment of GH/IGF-I axis in obesity by evaluation of IGF-I levels and the GH response to GHRH+arginine test.

The GH response to provocative stimuli in obese is often as low as in panhypopituitaric patients with severe GHD; however, IGF-I levels are normal or slightly reduced. In 53 patients with simple obesity (11 M and 42 F, age: 40.3+/-1.6 yr, BMI: 39.1+/-1.0 Kg/m2), we evaluated the GH response to GHRH (1 microg/kg iv)+arginine (ARG, 0.5 g/kg iv), and total IGF-I levels. The mean (+/-SE) GH peak after GHRH+ARG was markedly lower (74% reduction, p<0.0001) in obese (16.8+/-2.0 microg/l) than in normal subjects (62.7+/-4.3 microg/l). IGF-I levels in obese patients (134.0+/-7.6 microg/l) were lower (33% reduction, p<0.001) than in normal subjects (200.8+/-5.7 microg/l). Taking into account the 3rd centile limit of normal response, the GH response to GHRH+ARG was reduced in 62.3% (33/53) of the obese patients, and 21.2% (7/33) of them had low IGF-I levels. Assuming the 1st centile limit, it was reduced in 33.9% (18/53) obese subjects, and 22% (4/18) of them had low IGF-I levels. Considering 3.0 microg/L as arbitrary cut-off, the GH response was reduced in 5.7% (3/53) of the obese patients, and still one of them had low IGF-I levels. Our findings: a) confirm that the secretory capacity of somatotroph cells is often deeply impaired in obesity; b) demonstrate that IGF-I assay generally rules out severe impairment of GH/IGF-I axis in obese patients with marked reduction of the GH secretion; c) indicate that the percentage of obese patients with concomitant reduction of GH secretion and IGF-I levels is not negligible. Thus, IGF-I assay should be routinely performed in obese patients; those presenting with low IGF-I levels should undergo further evaluation of their hypothalamo-pituitary function and morphology, particularly in the presence of empty sella.

Dehydroepiandrosterone sulfate levels in women. Relationships with age, body mass index and insulin levels.

Sex and age are the major determinants of serum levels of dehydroepiandrosterone sulfate (DHEA-S): they are about twice in men than in women and show a progressive reduction from the end of the puberty to aging in both sexes. It has been reported that DHEA-S levels are also negatively influenced by insulin. Moreover, DHEA-S levels reduction has been associated to increased risk for cardiovascular disease, which connotes hyperinsulinemic states, such as obesity. We have evaluated serum levels of DHEA-S and insulin as function of age and body mass index (BMI) in 376 adult women (age 18.1-89.6 yrs, median 42.2; BMI 15.7-57.8 kg/m2, median 32.7) by multiple regression and piecewise regression analysis. Insulin levels positively associated to BMI (p=0.000002) and DHEA-S levels negatively associated with age (p=0.000001). Considering the whole population, DHEA-S levels were related positively with BMI (p=0.0013) independently of age. DHEA-S were also directly related to insulin levels independently of age (p=0.042), but this association disappeared after correction for BMI. Piecewise regression analysis did not reveal a threshold level for the increase of BMI (p=0.0004). Interestingly, DHEA-S levels and BMI were positively associated before but not after menopause. Taking into account only obese population, (no.=143, age 18.7-67.3 yrs, mean 39.0, median 39.4) DHEA-S levels were again related negatively with age and positively with BMI, while were unrelated with waist to hip ratio (p=0.391). Our data show that increasing body mass and insulin secretion is not associated to DHEA-S reduction in women. This evidence suggests that DHEA-S is unlikely implicated in the pathogenesis of cardiovascular disease in obese women.