The prostaglandin system and insulin release. Studies with the isolated perfused rat pancreas

Using the isolated perfused rat pancreas PGE₂ (1 μM and 10 μM) had no effect on basal or glucose (10 and 20 mM)-induced insulin release (IR). PGF_2α stimulated basal IR at 1 μM and inhibited IR at 10 μM. The glucose-induced IR was unaffected by this PG. Furosemide (5 and 10 mM) led to a monophasic IR at low glucose (glu) and to a potentiation of IR at high glu. Only high indomethacin (Indo) (50 μg/ml) inhibited glu-induced IR. The stimulatory effect of furosemide on IR could not be inhibited by indomethacin. However mepacrine (0.1 mM) abolished the furosemide effect. Also glu-induced IR was inhibited by mepacrine. Acetylsalicylic acid (30 mg/100 ml) had no significant influence on glu-induced IR.These findings provide evidence that phospholipase activation rather than increased PG synthesis might primarily be involved in the secretory process of insulin.     

Pituitary adenylate cyclase activating polypeptide stimulates insulin release from the isolated perfused rat pancreas.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide structurally related to vasoactive intestinal peptide (VIP) and glucagon like peptide-1(7-36) amide (tGLP-1) in its N-terminal portion. Therefore, their levels of insulinotropic potency were compared using an isolated rat pancreas perfusion. It was found that 0.1 nM PACAP (1-27) amide (PACAP27) significantly stimulated insulin release under a perfusate glucose concentration of 5.5 mM, whereas 1 nM PACAP27 did not under a perfusate glucose concentration of 2.8 mM. The potency was evaluated as tGLP-1 greater than PACAP27 greater than VIP. These results indicate that PACAP is a glucagon superfamily peptide which stimulates insulin release in a glucose dependent manner.     

The effects of prostaglandins on secretion of glucagon and insulin by the perfused rat pancreas.

The secretion of both glucagon and insulin by the isolated perfused rat pancreas was significantly stimulated by 10(-7) M PGH2. Experiments to show that the stimulated secretion was mediated by conversion of PGH2 to TXA2 or TXB2 revealed no correlation between the amount of secretion and the amount of thromboxane formed. Conversion of PGH2 with a crude platelet thromboxane synthase preparation caused a progressive loss of ability to secret insulin, whereas the capacity to stimulate release of glucagon remained at about one-half the maximal level. This relatively stable and selective secretagogue action on the alpha-cells appeared to be due to the formation of PGD2 by the platelet preparation. Direct administration of PGD2 confirmed this interpretation and showed clearly that this prostaglandin is a potent secretagogue for glucagon with little activity in stimulating the release of insulin. Our results have shown high and relatively equal stimulation of secretion by alpha- and beta-cells with exogenous PGE2, PGF2 alpha, and PGH2, little or no secretion by either cell type with TXA2, TXB2, or PGI2, and a unique selective stimulatory action of PGD2 upon the alpha-cell.     

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.     

Jejunal factor stimulating insulin release in the isolated perfused canine pancreas and jejunum.

In the present study using an isolated perfused preparation of canine jejunum and pancreas, an insulin-releasing factor was found in the venous effluent of the jejunum. Insulin secretion by the pancreas rose twofold after 10% glucose was infused in the lumen of the jejunum and remained at a high level even after the stimulus was discontinued. No modification of the exocrine pancreatic secretion occurred during the insulin release, and therefore it seems unlikely that gastrin, secretin or cholecystokinin-pancreozymin were released by the jejunal mucosa. In control experiments the values of hyperglycaemia observed previously and intraluminal hyperosmolarity were tested: at these levels, they did not affect insulin secretion. The nature of this intestinal insulin-releasing factor remains unknown however, but may be identifiable when intestinal hormones in blood can be assayed reliably.     

The effect of ventromedial hypothalamic nuclei destruction on somatostatin and insulin release from the isolated perfused rat pancreas

The effect of electrolytic lesions in the ventromedial hypothalamic nuclei (VMH) on somatostatin and insulin release was studied using the isolated perfused rat pancreas. Obesity gradually developed in the rats after placement of the VMH lesions, and fasting insulin levels determined immediately before the isolation of the pancreas were significantly higher than those in sham-operated controls. In the presence of 4.4 mM glucose, both perfusate somatostatin and insulin responses to arginine were significantly greater than in the controls, suggesting that VMH lesions cause not only hypersecretion of insulin but hypersecretion of somatostatin as well.     

Amino acid--induced insulin release from the perfused rat pancreas. The influence of phenylalanine and tyrosine

The effects of L or D phenylalanine and L tyrosine on insulin release from the perfused rat pancreas were investigated. It was found that in the presence of D-glucose, all three amino-acids stimulate insulin secretion. After L-Phe had been removed from perfusate in the presence or absence of L-Tyr, the secondary rise of insulin release (an "off response") was noticed. This phenomenon did not follow to either D-Phe or L-Tyr.     

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.     

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.     

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.     

Effect of the alpha 2-blocker DG-5128 on insulin and somatostatin release from the isolated perfused rat pancreas

2[2-(4.5-Dihydro-1H-imidazol-2-yl)-1-phenylethyl] pyridine dihydrochloride sesquihydrate (DG-5128) is an alpha 2-specific-adrenergic antagonist. We have studied the effect of DG-5128 on insulin and somatostatin release from the isolated perfused rat pancrease. DG-5128 stimulated somatostatin and insulin release not only at a low glucose concentration but also at a high glucose concentration. These findings suggest that an alpha 2-adrenergic receptor plays an important role in the regulation of insulin and somatostatin secretion.     

Biotin enhances glucose-stimulated insulin secretion in the isolated perfused pancreas of the rat

The effects of biotin on insulin secretion in pair-fed control rats and biotin-deficient rats were investigated using the method of isolated pancreas perfusion. Isolated pancreas perfusion was performed using 20 mM glucose, 10 mM arginine, and 20 mM glucose plus various concentrations of biotin (20 mM glucose + biotin solution) as stimulants of insulin secretion. The insulin response to 20 mM glucose in biotin-deficient rats was approximately 22% of that seen in control rats. The level of the insulin response to 10 mM arginine was also significantly lower in biotin-deficient rats than in control rats. These results indicate that insulin release from the pancreas was disturbed in biotin-deficient rats. The insulin responses to 20 mM glucose + 1 mM biotin in biotin-deficient and control rats increased to 165% and 185%, respectively, of that to 20 mM glucose. These biotin-induced increases in glucose-stimulated insulin release were evident within the first few minutes of the infusion. An enhancement of the arginine-induced insulin response in control rats was not found when arginine and biotin was administered. These results suggest that biotin may play an important role in the mechanism by which glucose stimulates insulin secretion from the beta cells of the pancreatic islets.     

Effect of cholecystokinin, secretin, and gastric inhibitory polypeptide on insulin release from the isolated perfused rat pancreas

The actions of gastric inhibitory polypeptide (GIP) on insulin release from the isolated perfused rat pancreas were compared with those of pure secretin and cholecystokinin (CCK). At dose levels physiologically achievable for GIP (1 ng/mL perfusate), infusions of CCK stimulated significant insulin release both on a weight (1 ng/mL) and a molar (770 pg/mL) basis. Although 50% as potent as GIP on a weight basis and 43% as potent on a molar basis, the insulin response to CCK was multiphasic and sustained for the duration of the infusion. The action of CCK, like that of GIP, was glucose dependent yielding no significant insulin release at a low perfusate glucose concentration (80 mg/dL). Irrespective of perfusate glucose concentration or dose (1 or 5 ng/mL), secretin failed to stimulate significant release of insulin from the perfused pancreas. It was concluded that secretin is ineffective as an incretin and that a physiological role for CCK in an enteroinsular axis awaits accurate measurement of circulating levels of immunoreactive CCK.     

The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta-cells in the isolated perfused rat pancreas.

The isolated perfused rat pancreas was used to test the hypothesis that total cellular ATP or the ratio of ATP/free ADP plays the primary role in coupling intermediary metabolism to the biophysical events that are the basis of glucose-stimulated insulin release. The pancreas was preperfused for 20 min with 4.0 mM of a physiological mixture of 20 amino acids plus 4.2 mM glucose, and insulin release was then stimulated for 150 s by suddenly increasing the glucose to 8.3 mM. The pancreas was sampled at 24, 48, 72, and 150 s after the switch. The content of total ATP, ADP, AMP, Pi, phosphocreatine, and creatine were measured in beta-cell enriched cores of pancreatic islets microdissected from freeze-dried pancreas cryostat sections. Metabolites were measured by quantitative histochemical enzymatic cycling techniques. Modeling studies were carried out to assess the impact of biochemical analytical results on the membrane potential of the beta-cells. The level of free ADP was calculated using the creatine kinase equilibrium reaction and an intracellular pH of 7.2. First phase insulin release was stimulated at least 10-fold with the maximum reached 45 s after adding high glucose. The biochemical analytical data demonstrate that the total cellular level of the putative coupling factor ATP and of the ratios ATP/free ADP and ATP/free ADP x Pi are not significantly influenced by a glucose level change that causes a more than 10-fold surge of insulin release. The strength and limitations of the present experimental strategy and the implications of the results for our understanding of metabolic coupling in glucose-stimulated insulin release are discussed.