Effects of neuromedin B on insulin and glucagon release from the isolated perfused rat pancreas

The effect of neuromedin B (NMB) on insulin and glucagon release was studied in isolated perfused rat pancreas. Infusion of NMB (10 nM, 100 nM and 1 microM) did not affect the insulin release under the perusate conditions of 5.5 mM glucose plus 10 mM arginine and 11 mM glucose plus 10 mM arginine, although 10 nM NMB tended to slightly suppress it under the perfusate condition of 5.5 mM glucose alone. The degree of stimulation of insulin release provoked by the addition of 5.5 mM glucose to the perfusate was not affected by the presence of 10 nM NMB. The glucagon release was slightly stimulated by the infusion of 100 nM and 1 microM NMB but not by 10 nM NMB under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. The effect of C-terminal decapeptide of gastrin releasing peptide (GRP-10) was also examined and similar results were obtained; 10 nM and 100 nM GRP-10 did not affect insulin release and 100 nM GRP-10 stimulated glucagon release under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. The present results concerning glucagon release are consistent with the previous results obtained with isolated perfused canine and porcine pancreas. However, the results regarding insulin release are not. Species differences in insulin release are also evident with other neuropeptides such as substance P and the mechanism of such differences remains for be clarified.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

Insulin release from the perfused rat pancreas. Mode of action of tolbutamide

Dextran-linked tolbutamide causes first-phase insulin release when perfused through the isolated rat pancreas. The linked sulphonylurea is also able to stimulate the membranal adenylate cyclase present in mouse islets. These facts make it highly likely that sulphonylureas exert their pharmacological action by interaction with the plasma membrane. Their action may be mediated via the adenylate cyclase enzyme.     

Failure of parathormone to affect insulin and glucagon release from the perfused rat pancreas.

Parathormone (0.15 U/ml) failed to affect the rate of glucagon and insulin release by the perfused rat pancreas exposed to glucose in either low (3.3 mM) or high (8.3 mM) concentration. Parathormone also failed to interfere with the suppressive effect of glucose (16.6mM) upon glucagon release and its stimulatory action upon insulin secretion. Likewise, the biphasic release of both glucagon and insulin evoked by arginine (10.0 mM) in the presence of glucose (8.3 mM) was unaffected by parathormone. These findings suggest that the endocrine pancreas may not be a target organ for any direct and immediate action of parathormone.     

Effect of exercise training on insulin and glucagon release from perfused rat pancreas

The effect of physical training on insulin and glucagon release in perfused rat pancreas was examined in the spontaneously exercised group running in a wheel cage an average of 1.4 km/day for 3 weeks and in the sedentary control group kept in the cage whose rotatory wheel was fixed on purpose. Pancreatic immunoreactive insulin (IRI) responses to glucose and arginine were reduced by 28% and 47.8% respectively in trained rats compared with untrained rats, while IRI content of the pancreas was similar in these two groups. The demonstrated decrease in insulin secretion of the beta-cell of the trained rats, in response to the glucose and arginine stimulations, may be functional in nature. On the other hand, neither pancreatic glucagon immunoreactivity (GI) response to glucose and arginine nor GI content of the pancreas was modified by exercise training. These results demonstrate that exercise training reduces IRI responses to glucose as well as to arginine stimulations, but does not modify any secretory response of pancreatic GI.     

Effects of 3-hydroxybutyrate and hyperosmolarity on glucagon release from isolated perfused canine pancreas

The effects of 3-hydroxybutyrate (3-OHB) and hyperosmolarity on glucagon secretion were examined in the isolated perfused canine pancreas. When 3-OHB was infused for 15 min into the pancreas perfused with 2.8 mM glucose, 5 and 20 mM sodium 3-OHB inhibited it after a transient stimulation, whereas a similar transient stimulation was observed also by the infusion of 20 mM NaCl in a control experiment. The above inhibition was not observed under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. When the isolated canine pancreas was perfused under the perfusate condition of acidosis (pH 7.1), ketoacidosis (pH 7.1 and 20 mM 3-OHB) or hyperosmolarity (+60 mOsm/kg with sucrose) throughout the experiment, the glucagon concentrations produced by 2.8 mM glucose under the ketoacidotic and hyperosmolar conditions, were less than half of those obtained under the standard condition. The insulin level was not influenced by the above perfusate conditions. These results suggest that 3-OHB inhibits glucagon secretion stimulated by glucopenia, but does not inhibit it stimulated by amino acids, and that hyperosmolarity inhibits glucagon secretion but does not inhibit insulin secretion. The pathophysiological significance of these results must be slight, considering the presence of hyperglucagonemia during prolonged starvation or diabetic ketoacidosis.     

Inhibition of the glucose induced insulin release by somatostatin in the isolated perfused rat pancreas. Action of cyclic AMP, glucagon and glibenclamide.

Insulin release in the perfused isolated rat pancreas was measured after stimulation with 16.5 mM glucose with and without somatostatin (cycle form, 100 ng/ml) in the medium. A complete blockage of the typical biphasic pattern of insulin release ocurred with somatostatin in the medium. Such blockage was abolished when cAMP (2.5 mM) and a 0.5 ml solution of glucagon (1 mg/ml) were continuously perfused for 20-minute periods and for 30-second periods correspondently. It did not take place when glibenclamide (HB-419) was perfused for a 20-minute period at a rate of 10 mug/ml. The results suggest that the adenylcyclase dependent mechanisms of glucose-induced insulin release are involved in the inhibition of the glucose-induced insulin secretion by somatostatin.     

Effects of glucagon-like peptide 1 (7-36) amide and glucagon on amylin release from perfused rat pancreas.

The effects of glucagon-like peptide 1 (7-36) amide [GLP-1 (7-36) amide] and glucagon on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas were studied. In the presence of 5.6 mM glucose, GLP-1 (7-36) amide and glucagon stimulated the release of amylin from the perfused pancreas. The infusion of GLP-1 (7-36) amide at a concentration of 10(-9) M elicited a biphasic release of amylin similar to that of insulin. The cumulative output of amylin induced by 10(-9)M GLP-1 (7-36) amide was significantly higher than that by 10(-9)M glucagon (p less than 0.01). The amylin/insulin molar ratios induced by GLP-1 (7-36) amide and glucagon were about 1% and did not differ significantly. These findings suggest that GLP-1 (7-36) amide and glucagon stimulate the release of amylin from the pancreas and that the concomitant secretion of amylin and insulin might contribute to glucose homeostasis.     

Somatostatin, insulin and glucagon secretion by the perfused pancreas from the cysteamine-treated rat

In rats, administration of a single dose of cysteamine (300 mg/kg, intragastrically) induces a depletion of pancreatic somatostatin content (approximately 60%) without modifying pancreatic insulin or glucagon content. In perfused pancreases from cysteamine-treated rats, there was a lack of somatostatin response to glucose, arginine or tolbutamide. In the absence of stimulated somatostatin release, the secretory responses of insulin and glucagon to glucose, to arginine, and to tolbutamide were not significantly different from those observed in pancreases from control rats. Our data do not support the concept that pancreatic somatostatin plays a major role in the control of insulin and glucagon release.     

Effects of pancreastatin on insulin, glucagon and somatostatin secretion by the perfused rat pancreas

Pancreastatin is a novel peptide, isolated from porcine pancreatic extracts, which has been shown to inhibit glucose-induced insulin release "in vitro". To achieve further insight into the influence of pancreastatin on pancreatic hormone secretion, we have studied the effects of this peptide on unstimulated insulin, glucagon and somatostatin output, as well as on the responses of these hormones to glucose and to tolbutamide in the perfused rat pancreas. Pancreastatin strongly inhibited unstimulated insulin release as well as the insulin responses to glucose and to tolbutamide. It did not significantly affect glucagon or somatostatin output under any of the above-mentioned conditions. These findings suggest that pancreastatin inhibits B-cell secretory activity directly, and not through an A-cell or D-cell paracrine effect.     

Homologous pancreastatin inhibits insulin secretion without affecting glucagon and somatostatin release in the perfused rat pancreas.

The identification of pancreastatin in pancreatic extracts prompted the investigation of its effects on islet cell function. However, in most of the investigations to date, pig pancreastatin was tested in heterologous species. Since there is great interspecies variability in the amino acid sequence of pancreastatin, we have investigated the influence of rat pancreastatin on insulin, glucagon and somatostatin secretion in a homologous animal model, namely the perfused rat pancreas. During 5.5 mM glucose infusion, pancreastatin (40 nM) inhibited insulin secretion (ca. 40%, P less than 0.025) as well as the insulin responses to 10 mM arginine (ca. 50%, P less than 0.025) and to 1 nM vasoactive intestinal polypeptide (ca. 50%; P less than 0.05). Pancreastatin failed to significantly modify glucagon or somatostatin release under any of the above experimental conditions. In addition, a lower pancreastatin concentration (15.7 nM) markedly suppressed the insulin release evoked by 11 mM glucose (ca. 85%, P less than 0.05). Our present observations reinforce the concept that pancreastatin is an effective inhibitor of insulin secretion, influencing the B-cell function directly and not through an A-cell or D-cell paracrine effect.     

The biosynthesis of glucagon in perfused rat pancreas

The biosynthesis of glucagon was studied by using the recirculated, isolated perfused rat pancreas. [³H]Tryptophan was initially incorporated into acid–ethanol-extractable protein, which on gel filtration was eluted with a molecular weight of about 9000 and contained a small amount of glucagon immunoreactivity. With longer incubation [³H]tryptophan incorporation into a second peak was obtained in an identical position with that of the majority of rat glucagon immunoreactivity. This peak of labelled protein exhibited migration characteristics on polyacrylamide-gel electrophoresis identical with those of rat glucagon and was identified as newly synthesized glucagon by demonstration of specific binding and dissociation behaviour with glucagon antibodies. The incorporation of [³H]tryptophan into acid–ethanol-extractable protein was inhibited by cycloheximide. High concentrations of glucose increased [³H]tryptophan incorporation into high-molecular-weight protein but decreased incorporation into proteins smaller than cytochrome c. The pattern of [³H]leucine incorporation into protein was similar to that of [³H]tryptophan.     

Adrenergic modulation of insulin and glucagon secretion from the isolated perfused rat pancreas

In order to observe the effect of the adrenergic system on pancreatic glucagon secretion in the isolated perfused rat pancreas, phenylephrine, an alpha-adrenergic agonist, and isoproterenol, a beta-adrenergic agonist, were added to the perfused solution. 1.2 microM phenylephrine suppressed glucagon secretion at 2.8 mM glucose, and it also decreased insulin secretion at 11.1 mM glucose. 240 nM isoproterenol enhanced glucagon secretion not only at 2.8 mM glucose, but also at 11.1 mM glucose, as well as insulin secretion at 11.1 mM. In order to study the role of intra-islet noradrenalin, phentolamine, an alpha-adrenergic antagonist, and propranolol, a beta-adrenergic antagonist, were infused with the perfused solution. 10 and 100 microM phentolamine caused an increase in insulin secretion, and 25 microM propranolol decreased insulin secretion, while they did not cause any change in glucagon secretion. From these results, it can be concluded that alpha-stimulation suppresses not only insulin but also glucagon secretion, while beta-stimulation stimulates glucagon secretion, as well as insulin secretion. Intra-islet catecholamine may have some effect on the B cell, whereas it seems to have no influence on the A cell.     

Inhibitory effect of rat amylin on the insulin responses to glucose and arginine in the perfused rat pancreas

Amylin, a 37-amino acid polypeptide, is the main component of amyloid deposits in the islets of Langerhans, and has been identified in the B-cell secretory granules. We have investigated the effect of rat amylin on the insulin and glucagon release by the isolated, perfused rat pancreas. Amylin infusion at 750 nM, markedly reduced unstimulated insulin release (ca. 50%, P less than 0.025), whereas it did not modify glucagon output. At the same concentration, amylin also blocked the insulin response to 9 mM glucose (ca. 80%, P less than 0.025) without affecting the suppressor effect of glucose on glucagon release. The inhibitory effect of amylin on glucose-induced insulin secretion was confirmed by lowering the amylin concentration (500 nM) and increasing the glucose stimulus (11 mM); again, no effect of amylin on glucagon release was observed. Finally, amylin, at 500 nM, reduced the insulin response to 3.5 mM arginine (ca. 40%, P less than 0.025) without modifying the secretion of glucagon elicited by this amino acid. It can be concluded that, in the rat pancreas, the inhibitory effect of homologous amylin on unstimulated insulin secretion, as well as on the insulin responses to metabolic substrates (glucose and arginine), favours the concept of this novel peptide as a potential diabetogenic agent.     

Bombesin stimulates the release of insulin and glucagon, but not pancreatic somatostatin, from the isolated perfused dog pancreas.

Synthetic bombesin, perfused in the isolated canine pancreas at a rate of 340-380 ng/min for 10 min, elicited a 4-fold rise in insulin to a peak at 2 min; a rapid decline followed discontinuation of bombesin. Glucagon rose by 50% to a peak at 6 min, but remained elevated after discontinuation of the bombesin. Somatostatin-like immunoreactivity was not significantly affected by perfusion with bombesin.     

Effects of salicylate on insulin and glucagon secretion by the isolated and perfused rat pancreas

The effects of sodium salicylate, a prostaglandin synthesis inhibitor, on glucose-induced secretion of insulin and glucagon by the isolated perfused rat pancreas have been studied. Sodium salicylate inhibited both basal (2.8 mM glucose) and stimulated (16.7 mM glucose) insulin release in a dose dependent manner (1, 5 and 10 mM). This inhibition is not interpretable in terms of a simple inhibition of cyclooxygenase by sodium salicylate. Basal glucagon release was not changed by 1 mM sodium salicylate but the latter partially blocked its inhibition by 16.7 mM glucose. Higher doses of sodium salicylate (5 and 10 mM) inhibited basal glucagon secretion without affecting its response to 16.7 mM glucose. These findings suggest a predominant stimulatory action of endogenous prostaglandins on glucagon release.