Carlotta Gauna

Unacylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin releasing factor 2 receptor

Both unacylated ghrelin (UAG) and acylated ghrelin (AG) exert metabolic effects. To investigate the interactions between AG and UAG on ghrelin receptors we evaluated the effects of AG and UAG on INS-1E rat insulinoma cells, using insulin secretion after 30min static incubation as a read-out. A possible involvement of the growth hormone secretagogue receptor type 1a (GHS-R1a) or the corticotropin-releasing factor 2 (CRF2) receptor (CRF2R), as a putative receptor for UAG, was also studied determining their mRNA expression and the functional effects of receptor antagonists on insulin release. Both UAG and AG stimulated insulin release dose-dependently in the nanomolar range. The AG-induced insulin output was antagonized by two GHS-R1a antagonists ([d-Lys(3)]GHRP-6 and BIM28163), which did not block UAG actions. These effects occurred in the presence of low levels of GHS-R1a mRNA. Neither CRF2R expression nor effects of the CRF2R antagonist (astressin(2)B) on insulin output were observed. In conclusion, we provide a sensitive and reproducible assay for specific effects of UAG, which in this study is responsible for insulin release by INS-1E cells. Our data support the existence of a specific receptor for UAG, other than the CRF2R and GHS-R1a. The stimulatory effect on insulin secretion by AG in this cell line is mediated by the GHS-R1a.

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.

Unacylated ghrelin and obestatin increase islet cell mass and prevent diabetes in streptozotocin-treated newborn rats

The ghrelin gene products, namely acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob), were shown to prevent pancreatic beta-cell death and to improve beta-cell function under treatment with cytokines, which are major cause of beta-cell destruction in diabetes. Moreover, AG had been described previously to prevent streptozotocin (STZ)-induced diabetes in rats; however, the effect of either UAG or Ob has never been examined in this context. In the present study, we investigated the potential of UAG and Ob to increase islet beta-cell mass and to reduce diabetes at adult age in STZ-treated neonatal rats. One-day-old rats were injected with STZ and subsequently administered with either AG, UAG or Ob for 7 days. On day 70, plasma glucose levels, plasma and pancreatic insulin levels, pancreatic islet area and number, insulin and pancreatic/duodenal homeobox-1 (Pdx1) gene expression, and antiapoptotic BCL2 protein expression were determined. Similarly to AG, both UAG and Ob counteracted STZ-induced high glucose levels and improved plasma and pancreatic insulin levels, which were reduced by the diabetogenic compound. UAG and Ob increased islet area, islet number, and beta-cell mass with respect to STZ treatment alone. Finally, in STZ-treated animals, UAG and Ob up-regulated insulin and Pdx1 mRNA and increased the expression of BCL2 similarly to AG. Taken together, our results suggest that in STZ-treated newborn rats, UAG and Ob improve glucose metabolism and preserve islet cell mass, granting a therapeutic potential in medical conditions associated with impaired beta-cell function.

Unacylated ghrelin is not a functional antagonist but a full agonist of the type 1a growth hormone secretagogue receptor (GHS-R)

Recent findings demonstrate that the effects of ghrelin can be abrogated by co-administered unacylated ghrelin (UAG). Since the general consensus is that UAG does not interact with the type 1a growth hormone secretagogue receptor (GHS-R), a possible mechanism of action for this antagonistic effect is via another receptor. However, functional antagonism of the GHS-R by UAG has not been explored extensively. In this study we used human GHS-R and aequorin expressing CHO-K1 cells to measure [Ca(2+)](i) following treatment with UAG. UAG at up to 10(-5)M did not antagonize ghrelin induced [Ca(2+)](i). However, UAG was found to be a full agonist of the GHS-R with an EC(50) of between 1.6 and 2 microM using this in vitro system. Correspondingly, UAG displaced radio-labeled ghrelin from the GHS-R with an IC(50) of 13 microM. In addition, GHS-R antagonists were found to block UAG induced [Ca(2+)](i) with approximately similar potency to their effect on ghrelin activation of the GHS-R, suggesting a similar mode of action. These findings demonstrate in a defined system that UAG does not antagonize activation of the GHS-R by ghrelin. But our findings also emphasize the importance of assessing the concentration of UAG used in both in vitro and in vivo experimental systems that are aimed at examining GHS-R independent effects. Where local concentrations of UAG may reach the high nanomolar to micromolar range, assignment of GHS-R independent effects should be made with caution.

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.

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.

Bolus administration of obestatin does not change glucose and insulin levels neither in the systemic nor in the portal circulation of the rat

Obestatin is a second peptide derived from the preproghrelin polypeptide. It was originally thought to have anorexigenic effects, thereby functioning as an antagonist of ghrelin. However, this has been a subject of debate ever since. Since acylated ghrelin strongly induces insulin resistance, it could be hypothesized that obestatin plays a role in glucose homeostasis as well. In the present study we evaluated the effect of obestatin on glucose and insulin metabolism in the systemic and portal circulation. Obestatin 200 nmol/kg was administered systemically as a single intravenous bolus injection to fasted pentobarbital anesthetized adult male Wistar rats. Up to 50 min after administration, blood samples were taken to measure glucose and insulin concentrations, both in the portal and in the systemic circulation. The effect of obestatin was evaluated in fasted and in glucose-stimulated conditions (IVGTT) and compared to control groups treated with saline or IVGTT, respectively. Intravenous administration of obestatin did not have any effect on glucose and insulin concentrations, neither systemic nor portal, when compared to the control groups. Only the glucose peak 1 min after administration of IVGTT was slightly higher in the obestatin treated rats: 605.8+/-106.3% vs. 522.2+/-47.1% in the portal circulation, respectively (NS), and 800.7+/-78.7% vs. 549.6+/-37.0% in the systemic circulation, respectively (P<0.02), but it can be debated whether this has any clinical relevance. In the present study, we demonstrated that intravenously administered obestatin does not influence glucose and insulin concentrations, neither in the portal nor in the systemic circulation.

Effects of cortistatin-14 and somatostatin-14 on the endocrine response to hexarelin in humans.

Cortistatin (CST)-14, a neuropeptide with high structural homology with somatostatine (SS)-14, binds all SS receptor subtypes but also shows activities not shared by SS. CST and SS are often co-expressed in the same neurons but are regulated by different stimuli. Moreover, CST, but not SS, also binds the GH secretagogue (GHS) receptor. We compared the effects of CST-14 and SS-14 (2.0 μg/kg/h iv from -30 to +90 min) on the endocrine response to hexarelin (HEX, 1.0 μg/kg iv at 0 min), a synthetic GHS, in 6 normal volunteers [age (mean±SEM): 28.7±2.9 yr; body mass index: 23.4±0.8 kg/m2]. GH, PRL, ACTH, cortisol, insulin and glucose levels were measured at each time point. CST-14 inhibited spontaneous GH secretion [Δ-areas under curves (-AUC): −83.57±44.8 vs 2.3±2.7 μg/l/h, p<0.01] to the same extent of SS-14 (−186.1±162.9 μg/l/h, p<0.01). CST-14 as well as SS-14 also inhibited insulin secretion (p<0.05). The GH response to HEX was similarly inhibited by either CST-14 (AUC: 3814.1±924.2 vs 1212.9±379.8 μg/l/h, p<0.05) or SS-14 (720.9±158.6 μg/l/h, p<0.05). HEX significantly increased PRL, ACTH and cortisol levels but these responses were not modified by either CST-14 or SS-14. The effects of CST-14 and SS-14 on insulin and glucose levels were not modified by HEX. In conclusion, this study shows that CST-14 inhibits the GH response to HEX to the same extent of SS-14. Like SS-14, CST-14 also inhibits insulin secretion but both do not modify the stimulatory effects of HEX on lactotroph and corticotroph secretion. Thus, CST-14 exerts full SS-14 activity in humans.

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.

Unsaturated fatty acids prevent desensitization of the human growth hormone secretagogue receptor by blocking its internalization

The composition of the plasma membrane affects the responsiveness of cells to metabolically important hormones such as insulin and vasoactive intestinal peptide. Ghrelin is a metabolically regulated hormone that activates the G protein-coupled receptor GH secretagogue receptor type 1a (GHSR) not only in the pituitary gland but also in peripheral tissues such as the pancreas, stomach, and T cells in the circulation. We have investigated the effects of lipids and altered plasma membrane composition on GHSR activation. Oligounsaturated fatty acids (OFAs) disrupt the structure of membranes and make them more fluid. Prolonged (96 h), but not acute, treatment of the GHSR cells with the 18C OFAs oleic and linoleic acid caused a significant increase in sensitivity of the receptor to ghrelin (EC(50) reduced by a factor of 2.4 and 2.9 at 60 and 120 microM OFAs, respectively). OFAs were found to block the inhibitory effects of ghrelin pretreatment on subsequent ghrelin responsiveness, suggesting that OFAs suppress desensitization of GHSR. Radioligand displacement studies did not show a significant shift in receptor binding after incubation with OFAs. However, it was found that OFA treatment suppressed GHSR internalization, likely explaining OFA-induced refractoriness to ligand-induced desensitization. The involvement of lipid rafts in this process was indicated by the altered responsiveness of GHSR under conditions that alter membrane cholesterol. In conclusion, our findings demonstrate the importance of membrane composition for GHSR activation and desensitization and indicate at least part of the mechanism through which OFAs and cholesterol could affect ghrelins activity in vivo.