Inhibitory effect of ghrelin on insulin and pancreatic somatostatin secretion.

OBJECTIVE: Ghrelin is a 28 amino acid residue peptide identified in both human and rat stomach and which acts as an endogenous ligand for the GH secretagogue receptor (GHS-R) and stimulates GH release. GHS-Rs are expressed in a number of tissues, including the pancreas, and ghrelin-like immunoreactivity is present in peripheral plasma, where its levels increase during fasting and decrease after food intake. The relationship between nutritional status and circulating ghrelin concentrations prompted us to investigate the effect of this peptide on pancreatic hormone secretion. METHODS: The study was performed in the isolated rat pancreas perfused in situ. Insulin, glucagon and somatostatin were measured by radioimmunoassay. RESULTS: Addition of 10 nM ghrelin to the perfusate significantly reduced the insulin response to the secretagogues glucose, arginine and carbachol, which act on the B-cell via different mechanisms, as well as the somatostatin response to arginine. Ghrelin was without effect on the glucagon output induced by this amino acid. At a lower concentration (2 nM) ghrelin was also found to inhibit glucose-induced insulin release. CONCLUSION: These findings support the proposal that the inhibitory effect of ghrelin on insulin release constitutes a tonic regulation of the B-cell, contributing to restrain its secretory activity in the state of food deprivation. On the other hand, the inhibition of pancreatic somatostatin release by ghrelin suggests a blocking effect of this hormone on the widely distributed D-cell population.

Effect of leptin on insulin, glugacon and somatostatin secretion in the perfused rat pancreas.

We have investigated the effect of rat leptin as well as the 22-56 fragment of this molecule on pancreatic hormone secretion in the perfused rat pancreas. In pancreases from fed rats, leptin failed to alter the insulin secretion elicited by glucose, arginine or tolbutamide, but inhibited the insulin response to both CCK-8 and carbachol, secretagogues known to act on the B-cell by increasing phospholipid turnover. This insulinostatic effect was also observed with the 22-56 leptin fragment. In pancreases obtained from 24-hour fasted rats, no effect of leptin on carbachol-induced insulin output was found, perhaps as a consequence of depressed B-cell phospholipid metabolism. Leptin did not influence glucagon or somatostatin release. Our results do not support the concept of leptin as a major regulator of B-cell function. Leptin inhibition of carbachol-induced insulin output might reflect a restraining effect of this peptide on the cholinergic stimulation of insulin release.

Selective amylin inhibition of the glucagon response to arginine is extrinsic to the pancreas.

Amylin, a peptide hormone from pancreatic beta-cells, is reported to inhibit insulin secretion in vitro and in vivo and to inhibit nutrient-stimulated glucagon secretion in vivo. However, it has been reported not to affect arginine-stimulated glucagon secretion in vitro. To resolve if the latter resulted from inactive peptide (a problem in the early literature), those experiments were repeated here with well-characterized peptide and found to be valid. In isolated perfused rat pancreas preparations, coperfusion with 1 nM amylin had no effect on arginine-, carbachol-, or vasoactive intestinal peptide-stimulated glucagon secretion. Amylin also had no effect on glucagon output stimulated by decreasing glucose concentration from 11 to 3.2 mM or on glucagon suppression caused by increasing glucose from 3.2 to 7 mM. Amylin at 100 nM had no effect in isolated islets in which glucagon secretion was stimulated by exposure to 10 mM arginine, even though glucagon secretion in the same preparation was inhibited by somatostatin. In anesthetized rats, amylin coinfusion had no effect on glucagon secretion stimulated by insulin-induced hypoglycemia. To reconcile reports of glucagon inhibition with the absence of effect in the experiments just described, anesthetized rats coinfused with rat amylin or with saline were exposed sequentially to intravenous L-arginine (during a euglycemic clamp) and then to hypoglycemia. Amylin inhibited arginine-induced, but not hypoglycemia-induced, glucagon secretion in the same animal. In conclusion, we newly identify a selective glucagonostatic effect of amylin that appears to be extrinsic to the isolated pancreas and may be centrally mediated.

Effects of sodium tungstate on insulin and glucagon secretion in the perfused rat pancreas.

Both the direct effect of sodium tungstate on insulin and glucagon secretion in the perfused rat pancreas, and the insulin response to glucose and arginine in pancreases isolated from tungstate-pretreated rats were studied. Infusion of tungstate stimulated insulin output in a dose-dependent manner. The insulinotropic effect of tungstate was observed at normal (5.5 mM), and moderately high (9 mM) glucose concentrations, but not at a low glucose concentration (3.2 mM). Tungstate-induced insulin output was blocked by diazoxide, somatostatin, and amylin, suggesting several targets for tungstate at the B-cell secretory machinery. Glucagon release was not modified by tungstate. Pancreases from chronically tungstate-treated rats showed an enhanced response to glucose but not to arginine. Our results indicate that the reported reduction of glycemia caused by tungstate administration is, at least in part, due to its direct insulinotropic activity. Furthermore, chronic tungstate treatment may prime the B-cell, leading to over-response to a glucose stimulus.

Stimulatory effect of exogenous diadenosine tetraphosphate on insulin and glucagon secretion in the perfused rat pancreas.

1. Diadenosine triphosphate (AP3A) and diadenosine tetraphosphate (AP4A) are released by various cells (e.g. platelets and chromaffin cells), and may act as extracellular messengers. In pancreatic B-cells, AP3A and AP4A are inhibitors of the ATP-regulated K+ channels, and glucose increases intracellular levels of both substances. 2. We have studied the effect of exogenous AP3A and AP4A on insulin and glucagon secretion by the perfused rat pancreas. 3. AP3A did not significantly modify insulin or glucagon release, whereas AP4A induced a prompt, short-lived insulin response ( approximately 4 fold higher than basal value; P<0.05) in pancreases perfused at different glucose concentrations (3.2, 5.5 or 9 mM). AP4A-induced insulin release was abolished by somatostatin and by diazoxide. These two substances share the capacity to activate ATP-dependent K+ channels, suggesting that these channels are a potential target for AP4A in the B-cell. 4. AP4A stimulated glucagon release at both 3.2 and 5.5 mM glucose. This effect was abolished by somatostatin. 5. The results suggest that extracellular AP4A may play a physiological role in the control of insulin and glucagon secretion.

Inhibitory effect of enterostatin on the beta cell response to digestive insulinotropic peptides.

OBJECTIVE AND DESIGN: We have investigated the effect of enterostatin on insulin release by the perfused rat pancreas: (1) under conditions of prolonged fasting and (2) under beta cell stimulation by digestive insulinotropic piptides. RESULTS: In pancreases from 24-h starved rats, the insulin response to glucose was reduced (approximately 75%) as compared to that observed in fed rats. This minimal response was abolished by 100 nM enterostatin. In fed rats, 100 nM enterostatin blocked the insulin output evoked by gastric inhibitory peptide (GIP) (approximately 75%) and by glucagon-like peptide-1 (GLP-1) (approximately 80%). Since both peptides exert their insulinotropic activity by activating the adenylate cyclase/cyclic AMP system, the interference of this pathway by enterostatin may be considered. Enterostatin (100 nM) did not modify the insulin responses to 26-33 fragment of cholecystokinin (CCK-8) and carbachol, substances which activate phosphoinositol turnover within the beta cell. The inhibitory effect of 100 nM enterostatin on glucose-induced insulin release was also observed at 50 nM and 10 nM enterostatin. Finally, des-Arg-enterostatin (100 nM) did not modify glucose-induced insulin output. CONCLUSION: Enterostatin inhibits the insulin response to glucose--both under fed and fasted conditions, and to the main digestive insulinotropic peptides--GIP and GLP-I. The entire enterostatic molecule seems to be necessary for it to exert its beta cell blocking effect. White the physiological role of enterostatin has not as yet been established, our observations allow speculation as to whether this peptide is implicated in the entero-insular axis as an antiincretin agent.

Influence of glucose concentration on the inhibitory effect of amylin on insulin secretion. Study in the perfused rat pancreas.

The effect of amylin on insulin secretion is a matter of controversy. Short-term experiments have shown that amylin, at 75 pmol/l, inhibits the insulin release elicited by a modest increase in the perfusate glucose concentration (from 5.5 mmol/l to 9 mmol/l). The present work was undertaken to further investigate the effect of amylin on glucose-induced insulin release at different glucose concentrations. The study was performed in the isolated perfused rat pancreas. Amylin, at 75 pmol/l, markedly blocked the insulin response when the perfusate glucose concentration was increased from 3.2 mmol/l to 7 mmol/l (by 90%; P < 0.01) or from 5.5 mmol/l to 9 mmol/l (by 80%; P < 0.01). At the same amylin concentration, no significant inhibition of insulin output was observed when the perfusate glucose level was augmented from 5.5 mmol/l to 16.6 mmol/l, from 7 mmol/l to 11 mmol/l or from 9 mmol/l to 13 mmol/l. At a higher concentration (750 pmol/l), amylin also failed to inhibit the insulin response induced by increasing glucose levels from 5.5 mmol/l to 16.6 mmol/l or from 9 to 13 mmol/l. These findings indicate that, in the rat pancreas, amylin only inhibits insulin release when evoked by elevations of glucose levels comparable to those occurring in normal subjects under physiological conditions.

Effect of enterostatin on insulin, glucagon, and somatostatin secretion in the perfused rat pancreas.

Enterostatin is a pentapeptide generated by trypic digestion of procolipase in the small intestine. Both peripheral and central administration of this peptide to rats has been shown to reduce food intake, this reduction being due to specific suppression of fat intake. In perifused pancreatic rat islets, enterostatin has been shown to inhibit the insulin response to a high glucose concentration. In the present study, we have investigated the effect of exogenous enterostatin on insulin, glucagon, and somatostatin secretion by the isolated perfused rat pancreas. Enterostatin, at 100 mmol/l, inhibited the insulin response to 9 mmol/l glucose (by 70%), 0.1 mmol/l tolbutamide (by 40%), and 5 mmol/l arginine (by 70%). Enterostatin had no effect on glucagon and somatostatin release at a maintained glucose level (5.5 mmol/l) or in response to 5 mmol/l arginine. Finally, preinfusion of the rat pancreas with a high enterostatin concentration (500 nmol/l) did not alter the insulin response to glucose, an observation that would rule out a toxic effect of this peptide on the beta-cell. In summary, in the perfused rat pancreas, enterostatin, at putatively physiological concentrations, inhibits insulin secretion without affecting glucagon or somatostatin output, thus pointing to a direct effect of enterostatin on the beta-cell and not through an alpha-cell or delta-cell paracrine effect. Because enterostatin is generated in the small intestine after feeding, it might play a role in the enteroinsular axis as an anti-incretin agent.

Effect of (8-32) salmon calcitonin, an amylin antagonist, on insulin, glucagon and somatostatin release: study in the perfused pancreas of the rat.

1. The 8-32 fragment of salmon calcitonin ((8-32) sCT) has been proposed as a highly selective amylin receptor antagonist. 2. In the present study, we have studied the influence of (8-32) sCT on the inhibitory effect of both amylin and its structural congener, calcitonin gene-related peptide (CGRP), on insulin secretion in the rat perfused pancreas. 3. Both amylin and CGRP, at 75 pM, clearly inhibited glucose-induced insulin release (by 80% and by 70%, respectively). Simultaneous infusion of 10 microM (8-32) sCT reversed the inhibitory effect of amylin (by 80%; P < 0.05 vs. amylin experiments) but did not significantly affect the inhibition of glucose-induced insulin output elicited by CGRP. Furthermore, at the same concentration (10 microM), (8-32) sCT alone potentiated the insulin response to 7 mM glucose (2.5 fold; P < 0.05) whilst it did not affect glucagon or somatostatin secretion. 4. The observation that infusion of an amylin antagonist into the rat pancreas potentiates the insulin response to glucose, favours the concept of endogenous amylin as an inhibitor of insulin release. 5. Finally, as an amylin antagonist at the level of the beta-cell, (8-32) sCT might be considered of potential interest in experimental and clinical pharmacology.

Inhibitory effect of amylin (islet amyloid polypeptide) on insulin response to non-glucose stimuli. Study in perfused rat pancreas.

Amylin, also called islet amyloid polypeptide (IAPP), can inhibit the glucose-induced insulin secretion in perfused rat pancreas at 75 pmol/l, a concentration comparable to that found in the effluent of this experimental model. To further explore the influence of amylin on insulin release, we investigated the effect of synthetic rat amylin (75 pmol/l) on insulin response to non-glucose secretagogues. These agents stimulate B-cell secretion via different mechanisms, such as a dihydropyridine derivative (BAY K 8644, 10 mmol/l) which activates Ca(2+)-channels, a sulfonylurea (tolbutamide, 0.2 mmol/l) which blocks ATP-dependent K(+)-channels, KCL (11 mmol/l) which depolarizes B cells and the 26-33 fragment of cholecystokinin (8-CCK, 1 nmol/l) which increases phospholipid turnover. The study was performed in perfused rat pancreas. Amylin significantly inhibited insulin response to BAY K 8644 (65%), KCI (60%) and 8-CCK (80%) as well as the early phase of tolbutamide-induced insulin output (70%). Thus, amylin can inhibit insulin release induced by secretagogues that interact at different levels of B-cell stimulus-secretion coupling. This inhibition may be due to a multifarious influence of amylin on the B-cell secretory mechanism and/or a disturbing effect on a distal, crucial step in the insulin-releasing mechanism, e.g. by affecting exocytosis of the secretory granule or by inhibiting an essential metabolic pathway within the B cell.