S. Destefanis

Ghrelin secretion is inhibited by glucose load and insulin-induced hypoglycaemia but unaffected by glucagon and arginine in humans.

objective  Circulating ghrelin levels are increased by fasting and decreased by feeding, glucose load, insulin and somatostatin. Whether hyperglycaemia and insulin directly inhibit ghrelin secretion still remains matter of debate. The aim of the present study was therefore to investigate further the regulatory effects of glucose and insulin on ghrelin secretion.

Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia–reperfusion injury in rat heart

AIMS The hypothalamic neuropeptide growth hormone-releasing hormone (GHRH) stimulates GH synthesis and release in the pituitary. GHRH also exerts proliferative effects in extrapituitary cells, whereas GHRH antagonists have been shown to suppress cancer cell proliferation. We investigated GHRH effects on cardiac myocyte cell survival and the underlying signalling mechanisms. METHODS AND RESULTS Reverse transcriptase-polymerase chain reaction analysis showed GHRH receptor (GHRH-R) mRNA in adult rat ventricular myocytes (ARVMs) and in rat heart H9c2 cells. In ARVMs, GHRH prevented cell death and caspase-3 activation induced by serum starvation and by the beta-adrenergic receptor agonist isoproterenol. The GHRH-R antagonist JV-1-36 abolished GHRH survival action under both experimental conditions. GHRH-induced cardiac cell protection required extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide-3 kinase (PI3K)/Akt activation and adenylyl cyclase/cAMP/protein kinase A signalling. Isoproterenol strongly upregulated the mRNA and protein of the pro-apoptotic inducible cAMP early repressor, whereas GHRH completely blocked this effect. Similar to ARVMs, in H9c2 cardiac cells, GHRH inhibited serum starvation- and isoproterenol-induced cell death and apoptosis through the same signalling pathways. Finally, GHRH improved left ventricular recovery during reperfusion and reduced infarct size in Langendorff-perfused rat hearts, subjected to ischaemia-reperfusion (I/R) injury. These effects involved PI3K/Akt signalling and were inhibited by JV-1-36. CONCLUSION Our findings suggest that GHRH promotes cardiac myocyte survival through multiple signalling mechanisms and protects against I/R injury in isolated rat heart, indicating a novel cardioprotective role of this hormone.

Proliferative and protective effects of growth hormone secretagogues on adult rat hippocampal progenitor cells.

Progenitor cells in the subgranular zone of the hippocampus may be of significance for functional recovery after various injuries because they have a regenerative potential to form new neuronal cells. The hippocampus has been shown to express the GH secretagogue (GHS) receptor 1a, and recent studies suggest GHS to both promote neurogenesis and have neuroprotective effects. The aim of the present study was to investigate whether GHS could stimulate cellular proliferation and exert cell protective effects in adult rat hippocampal progenitor (AHP) cells. Both hexarelin and ghrelin stimulated increased incorporation of (3)H-thymidine, indicating an increased cell proliferation. Furthermore, hexarelin, but not ghrelin, showed protection against growth factor deprivation-induced apoptosis, as measured by annexin V binding and caspase-3 activity and also against necrosis, as measured by lactate dehydrogenase release. Hexarelin activated the MAPK and the phosphatidylinositol 3-kinase/Akt pathways, whereas ghrelin activated only the MAPK pathway. AHP cells did not express the GHS receptor 1a, but binding studies could show specific binding of both hexarelin and ghrelin, suggesting effects to be mediated by an alternative GHS receptor subtype. In conclusion, our results suggest a differential effect of hexarelin and ghrelin in AHP cells. We have demonstrated stimulation of (3)H-thymidine incorporation with both hexarelin and ghrelin. Hexarelin, but not ghrelin, also showed a significant inhibition of apoptosis and necrosis. These results suggest a novel cell protective and proliferative role for GHS in the central nervous system.

Ghrelin and the endocrine pancreas.

Ghrelin is a 28-amino-acid peptide predominantly produced by the stomach, while substantially lower amounts derive from other tissues including the pancreas. It is a natural ligand of the GH secretagogue (GHS) receptor (GHS-R1a) and strongly stimulates GH secretion, but acylation in serine 3 is needed for its activity. Ghrelin also possesses other endocrine and nonendocrine actions reflecting central and peripheral GHS-R distribution including the pancreas. The wide spectrum of ghrelin activities includes orexigenic effect, control of energy expenditure, and peripheral gastroenteropancreatic actions. Circulating ghrelin levels mostly reflect gastric secretion as indicated by evidence that they are reduced by 80% after gastrectomy and even after gastric by-pass surgery. Ghrelin secretion is increased in anorexia and cachexia but reduced in obesity, a notable exception being Prader-Willi syndrome. The negative association between ghrelin secretion and body weight is emphasized by evidence that weight increase and decrease reduces and augments circulating ghrelin levels in anorexia and obesity, respectively, and agrees with the clear negative association between ghrelin and insulin levels. In fact, ghrelin secretion is increased by fasting whereas it is decreased by glucose load as well as during euglycemic clamp but not after arginine or free fatty acid load in normal subjects; in physiological conditions, however, the most remarkable inhibitory input on ghrelin secretion is represented by somatostatin as well as by its natural analog cortistatin that concomitantly reduce β-cell secretion. This evidence indicates that the endocrine pancreas plays a role in directly or indirectly modulating ghrelin secretion. As anticipated, ghrelin, in turn, is expressed within the endocrine pancreas, although it is still matter of debate if it is expressed by β-, α-, or non-α/non-β cells. Moreover, GHS-R1a expression in the pancreas has been demonstrated by many authors. Some impact of synthetic GHS on insulin secretion and glucose metabolism had been reported in both animal and human studies. Depending on dose and experimental conditions ghrelin has been shown able to inhibit or stimulate insulin secretion in animals. In humans, ghrelin administration is followed by transient inhibition of insulin levels that surprisingly follows persistent increase in plasma glucose levels suggesting that ghrelin would also directly or indirectly activate glycogenolisis. Current studies indicate that ghrelin also blunts the insulin response to arginine but not that to oral glucose load in humans. These acute effects of ghrelin are independent of any cholinergic mediation and are not shared by synthetic, peptidyl GHS indicating they are likely mediated by a non-GHS-R1a receptor. These acute effects of ghrelin on insulin secretion would be short-lasting, and it has to be remembered that long-term treatment with synthetic non peptidyl GHS in healthy elderly subjects was followed by insulin resistance. In all, it is already clear that ghrelin has remarkable impact in modulating insulin secretion and glucose metabolism. Insulin and ghrelin secretions seem linked by a negative functional relationship that strengthens the hypothesized role of ghrelin in participating in the management of the neuroendocrine and metabolic response to variations in energy balance.

Unacylated as well as acylated ghrelin promotes cell survival and inhibit apoptosis in HIT-T15 pancreatic β -cells

Ghrelin is mainly produced by the stomach, although it is expressed in other tissues, including the pancreas. Among its pleiotropic actions, ghrelin prevents the development of diabetes in rats and exerts mitogenic and antiapoptotic effects in different cell types. In addition, a ghrelin -producing ε-cell population has been demonstrated in rodent islets, suggesting a direct role in the control of islet cell survival. In this study, we investigated the effect of acylated ghrelin (AG) and unacylated ghrelin (UAG) on cell survival of HIT-T15 pancreatic β cells. We show that both AG and UAG equally prevented β cell death induced by serum withdrawal. In addition, both peptides inhibited serum starvation-induced apoptosis. These findings indicate that UAG and AG prevent cell death and apoptosis of pancreatic β cells. Since only AG, but not UAG, binds the GRLN receptor, a different and as yet unknown receptor is likely involved in these survival mechanisms.

Alprazolam (a benzodiazepine activating GABA receptor) reduces the neuroendocrine responses to insulin‐induced hypoglycaemia in humans

objective Alprazolam (ALP), a benzodiazepine‐activating GABAergic receptor, possesses clear centrally mediated inhibitory effects on ACTH and cortisol secretion that could reflect an inhibitory influence on CRH‐ and/or AVP‐secreting neurones. An inhibitory effect of ALP on catecholamine release has also been shown while its effect on GH secretion is unclear. To further clarify the neuroendocrine actions of ALP, we studied the ALP effects on the neurohormonal responses to hypoglycaemia in a group of normal subjects.

The continuous infusion of acylated ghrelin enhances growth hormone secretion and worsens glucose metabolism in humans

Context: Acylated ghrelin (AG) has been discovered as a natural ligand of the GH secretagogue receptor type 1a and is now recognized as an important orexigenic factor. Besides stimulation of GH secretion and appetite, it exerts other central and peripheral actions including modulation of insulin secretion, glucose and lipid metabolism. Objective: To define the effects of the continuous iv infusion of AG in humans with particular attention to metabolic parameters. Materials and methods: We studied the effects of 16h (from 21:00 to 13:00 h) infusion of AG (0.5 μg/kg/h) or saline in 8 young volunteers who were provided with isocaloric balanced meals. GH, cortisol, insulin, glucose, free fatty acid (FFA), and ghrelin levels were assayed every 20 min. Results: AG infusion increased circulating total ghrelin to a steady state that was maintained over 16 h infusion of the peptide. With respect to saline, AG infusion significantly modified GH, Cortisol, insulin, and glucose profiles and decreased FFA area under the curve (p<0.01). AG increased GH pulse frequency and approximate entropy (p<0.05). AG enhanced the glucose response to both dinner (p<0.02) and breakfast (p<0.03). AG infusion blunted the early insulin response to dinner (p<0.03) but enhanced the second-phase insulin response to dinner and breakfast (p<0.05). Conclusions: The continuous exposure to AG in humans enhances somatotroph secretion but also worsens glucose metabolism, although it inhibits lipolysis. These findings in normal young volunteers are consistent with data from studies in animals and suggest that acylated ghrelin is likely to play a negative role in glucose metabolism.

GH/IGF-I axis in anorexia nervosa

Patients with anorexia nervosa (AN) may develop multiple endocrine abnormalities, including amenorrhea, hyperactivity of the hypothalamus-pituitary-adrenal axis, hypothyroidism and particular changes in the activity of the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. Exaggerated GH secretion and reduced IGF-I levels are usually found in AN, as well as in conditions of malnutrition and malabsorption, insulin-dependent diabetes mellitus, liver cirrhosis and catabolic states. In AN, GH hypersecretion at least partially reflects malnutrition-induced peripheral GH resistance, which leads to reduced IGF-I synthesis and release; this implies an impairment of the negative IGF-I feedback action on GH secretion. On the other hand, primary alterations in the neural control of GH secretion cannot be ruled out. The neuroendocrine alterations include enhanced somatotroph responsiveness to growth hormone releasing hormone (GHRH) and impaired GH response to most central nervous system-mediated stimuli. Particular resistance to cholinergic manipulation has also been demonstrated, thus suggesting a somewhat specific alteration in the somatostatin (SS)-mediated cholinergic influence on GH secretion. Moreover, paradoxical GH responses to glucose load, thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH) have also been reported. The effect of reduced leptin levels on GH hypersecretion in AN is still unclear, but ghrelin (the gastric hormone that is a natural ligand of the GH secretagogue receptor and strongly stimulates somatotroph secretion) is thought to play a major role. Regardless of the supposed central and peripheral alterations, it has to be emphasised that the activity of the GH/IGF-I axis in AN is generally restored by nutritional and stable weight gain. It therefore reflects an impaired nutritional state and cannot be considered a primary hallmark of the disease.

ALPRAZOLAM, A BENZODIAZEPINE, DOES NOT MODIFY THE ACTH AND CORTISOL RESPONSE TO HCRH AND AVP, BUT BLUNTS THE CORTISOL RESPONSE TO ACTH IN HUMANS

Alprazolam (AL), a benzodiazepine which activates gamma-amino butyrric acid (GA-BA)-ergic receptors, exerts a clear inhibitory effect on the activity of the hypothalamo-pitu-itary-adrenal (HPA) axis and is able to markedly reduce the ACTH response to metyrapone-induced inhibition of glucocorticoid feedback. It has been suggested that its inhibitory action could be regulated by CRH or AVP mediated mechanisms. However, the effect of benzodi-azepines on the HPA response to CRH or AVP is contradictory. It has been shown that benzodi-azepines have specific receptors on the adrenal gland but it is unclear if they mediate biological effects in humans. In order to further clarify the mechanisms underlying the inhibitory effect of benzodiazepine on HPA axis in humans, we studied the effect of AL (0.02 mg/kg po at −90 min) or placebo in 7 healthy young volunteers (7 female, age: 26–34 yr; wt: 50–58 kg, BMI 20–22 kg/m2) on: 1) the ACTH and cortisol responses to hCRH (2.0 μg/kg iv at 0 min) or AVP (0.17 U/kg im at 0 min); 2) the cortisol, aldosterone and DHEA responses to ACTH 1–24 (0.06 and 250 μg iv at 0 and 60 min, respectively). After placebo, the ACTH and cortisol responses to hCRH (peaks, mean±SE: 29.8± 4.4 pg/ml and 199.3±19.6 μg/l) were similar to those recorded after AVP (31.7±6.5 pg/ml and 164.8±18.0 μg/l); the cortisol response to 0.06 μg ACTH (190.4±11.8 μg/l) was similar to that recorded after hCRH and AVP but lower (p<0.01) than that after 250 μg ACTH (260.6±17.4 μg/l). AL did not modify the ACTH response to both hCRH (42.5±7.1 pg/ml) and AVP (33.3±2.7 pg/ml), which even showed a trend toward increase. AL also failed to significantly modify the cortisol response to both hCRH (156.3±12.7 μg/l) and AVP (119.4±14.5 μg/l), which, on the other hand, showed a trend toward decrease. The cortisol peaks after 0.06 μg ACTH were significantly reduced (p<0.02) by AL pre-treatment (115.0±7.7 μg/l) which, in turn, did not modify the cortisol response to the subsequent ACTH bolus (214.7±16.6 μg/l). The DHEA and aldos-terone responses to all the ACTH doses were not significantly modified by AL. In conclusion, these data show that the HPA response to AVP as well as to hCRH is refractory to the inhibitory effect of AL which, in turn, blunts the cortisol response to low ACTH dose. These findings suggest that both CRH- and AVP-mediated mechanisms could underlie the CNS-mediated inhibitory effect of AL on HPA axis; in the meantime, these results suggest that benzodiazepines could also act on adrenal gland by blunting the sensitivity of the fasciculata zone to ACTH.

Standard light breakfast inhibits circulating ghrelin level to the same extent of oral glucose load in humans, despite different impact on glucose and insulin levels.

Ghrelin levels are increased by fasting and energy restriction, decreased by food intake, glucose load and insulin but not by lipids and amino acids. Accordingly, ghrelin levels are elevated in anorexia and cachexia and reduced in obesity. Herein we compared the effects of a standardized light breakfast (SLB) on morning circulating ghrelin levels with those of oral glucose load (OGTT) in normal subjects. Specifically, 8 young adult volunteers [age (mean±SEM): 28.0±2.0 yr; body mass index (BMI): 22.4±0.6 kg/m2] underwent the following testing sessions: a) OGTT (100 g po at 0 min, about 400 kcal); b) SLB (about 400 kcal, 45% carbohydrates, 13% proteins and 42% lipids at 0 min) on three different days; c) placebo (100 ml water po). In all sessions, at baseline, blood samples were withdrawn twice at 5-min interval to characterize the inter- and intra-individual reproducibility of the variables assayed. After placebo and OGTT, blood samples were withdrawn every 15 min up to +120 min. After SLB, blood samples were taken at 60 min only. Ghrelin, insulin and glucose levels were assayed at each time point in all sessions. Similarly to insulin and glucose levels, at baseline, ghrelin showed remarkable intra-subject reproducibility both in the same sessions and among the different sessions. Placebo did not significantly modify ghrelin, insulin and glucose. OGTT increased (p<0.01) glucose (baseline vs peak: 80.0±3.6 vs 140.5±6.3 mg/dl) and insulin (20.2±6.2 vs 115.3±10.3 mU/l) levels. SLB increased (p<0.05) both insulin (16.3±1.8 vs 48.3±6.3 mU/l) and glucose (74.5±3.7 vs 82.9±3.1 mg/dl) levels. Notably both the insulin and glucose increases after OGTT were significantly higher (p<0.01) than that induced by SLB. After OGTT, ghrelin levels underwent a significant reduction (baseline vs nadir: 355.7±150.8 vs 243.3±98.8 pg/ml; p<0.05) reaching the nadir at time +60 min. Similarly, ghrelin levels 60 min after SLB (264.8±44.8 pg/ml) were significantly (p<0.01) lower than at baseline (341.4±54.9 pg/ml). No significant differences in the reduction of ghrelin levels after OGTT and SLB were observed. In conclusion, these findings show that light breakfast inhibits ghrelin secretion to the same extent of OGTT in adults despite lower variations in glucose and insulin levels.