Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.     

Propionate inhibits glucose-induced insulin secretion in isolated rat pancreatic islets

Dietary fibers, probably by generating short chain fatty acids (SCFA) through enterobacterial fermentation, have a beneficial effect on the control of glycemia in patients with peripheral insulin resistance. We studied the effect of propionate on glucose-induced insulin secretion in isolated rat pancreatic islets. Evidence is presented that propionate, one of the major SCFA produced in the gut, inhibits insulin secretion induced by high glucose concentrations (11.1 and 16.7 mM) in incubated and perfused pancreatic islets. This short chain fatty acid reduces [U-(14)C]-glucose decarboxylation and raises the conversion of glucose to lactate. Propionate causes a significant decrease of both [1-(14)C]- (84%) and [2-(14)C]-pyruvate (49%) decarboxylation. These findings indicate pyruvate dehydrogenase as the major site for the propionate effect. These observations led us to postulate that the reduction in glucose oxidation and the consequent decrease in the ATP/ADP ratio may be the major mechanism for the lower insulin secretion to glucose stimulus induced by propionate.     

Glucose stimulates the biosynthesis of rat I and II insulin to an equal extent in isolated pancreatic islets

The effects of glucose on insulin biosynthesis were studied by measuring the incorporation of radiolabelled amino acids into proinsulin/insulin in isolated rat islets. The islets were pulse labelled for 15 min with [3H]leucine (present in rat insulin I and II) or [35S]methionine (unique to rat insulin II) and then incubated for a 165 min post-label (chase) period during which the majority of labelled proinsulin was converted to insulin but under conditions whereby greater than 95% of radiolabelled proinsulin or insulin was retained in the islets. The newly synthesized, labelled, insulin was analyzed by high performance liquid chromatography. Rat I and II insulin biosynthesis was stimulated by 16.7 mM glucose to the same extent.     

Epoxyeicosatrienoic acids stimulate glucagon and insulin release from isolated rat pancreatic islets

Metapyrone and eicosatetraynoic acid but not indomethacin are effective inhibitors of the secretory response of isolated rat pancreatic islets to arginine and glucose. Epoxyeicosatrienoic acids, products of the cytochrome P-450-NADPH dependent arachidonic acid epoxygenase activity, are potent and selective mediators for the in vitro release of either insulin or glucagon from preparations of isolated rat pancreatic islets.     

Effect of prolonged fasting upon insulin and glucagon secretion from isolated rat pancreatic islets.

To study insulin-glucagon interrelationships in the regulation of pancreatic islet functions, glucose-mediated insulin and glucagon secretion have been studied in isolated pancreatic islets from fed and from 4 and 8-day fasted rats. At low glucose levels (50 mg %) a continuous decrease of insulin and increase of glucagon secretion were observed during prolonged fasting. High glucose concentrations 300 mg %) stimulated insulin and inhibited glucagon secretion until 4 days, but did not cause any effect after 8 days fasting. These results suggest that the secretory mechanisms of the two hormones may have a common basis.     

Human and rat amylin have no effects on insulin secretion in isolated rat pancreatic islets

Amylin, an islet amyloid peptide secreted by the pancreatic beta cell, has been proposed as a humoral regulator of islet insulin secretion. Four separate preparations of amylin were tested for effects on hormone secretion in both freshly isolated and cultured rat islets and in HIT-T15, hamster insulinoma cells. With all three experimental models, exposure to human amylin acid and human and rat amylin at concentrations as high as 100 nM had no significant effect on rates of insulin or glucagon secretion. These observations suggest that amylin, even at concentrations appreciably higher than those measured in peripheral plasma, is not a significant humoral regulator of islet hormone secretion.     

Possible role of phosphatidylinositol turnover in insulin release from isolated rat pancreatic islets

We have previously reported occurrence of Ca2+-activated, phospholipid-dependent protein kinase (referred as protein kinase C) in the rat pancreatic islets. It has been suggested that unsaturated diacylglycerol which results from hydrolysis of phosphatidylinositol by phospholipase C-like enzyme activates protein kinase C. Therefore, we studied the effect of exogenous phospholipase C on insulin release from isolated islets of rat pancreas. Bacterial phospholipase C enhanced insulin release induced by glucose in a dose dependent manner. The effect, however, was decreased in the islets pretreated with colchicine. Both phospholipase C and glucose caused an increase in 32p incorporation into phosphatidylinositol. These results indicate that phospholipid metabolism is linked to the insulin release mechanism.     

Blockade of IRS1 in isolated rat pancreatic islets improves glucose-induced insulin secretion

Several neural, hormonal and biochemical inputs actively participate in the balance of insulin secretion induced by blood glucose fluctuations. The exact role of insulin as an autocrine and paracrine participant in the control of its own secretion remains to be determined, mostly due to insufficient knowledge about the molecular phenomena that govern insulin signaling in pancreatic islets. In the present experiments we demonstrate that higher insulin receptor and insulin receptor substrates-1 and -2 (IRS1 and IRS2) concentrations are predominantly encountered in cells of the periphery of rat pancreatic islets, as compared to centrally located cells, and that partial blockade of IRS1 protein expression by antisense oligonucleotide treatment leads to improved insulin secretion induced by glucose overload, which is accompanied by lower steady-state glucagon secretion and blunted glucose-induced glucagon fall. These data reinforce the inhibitory role of insulin upon its own secretion in isolated, undisrupted pancreatic islets.     

Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion from isolated rat pancreatic islets

Islet amyloid polypeptide has 37 amino acids and is a major component of amyloid deposition in pancreatic islets of patients with type 2 diabetes mellitus. To determine whether the peptide is involved in the impaired insulin secretion in this type of diabetes mellitus, we synthesized islet amyloid polypeptide and its fragments and examined its effect on insulin secretion. Islet amyloid polypeptide inhibited the glucose-stimulated insulin secretion from isolated rat pancreatic islets, as calcitonin gene-related peptide did, but the fragments failed to inhibit the secretion. Thus, we propose that amyloid deposition may be an important factor in the impairment of insulin secretion in type 2 diabetes mellitus.     

Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.     

Effect of chronic psychological stress on insulin release from rat isolated pancreatic islets

Despite documented studies, the exact role of stress on diabetes is still unclear. The present study investigates the effect of chronic psychological stress on insulin release from isolated rat pancreatic islets. Male Wistar rats were divided into two groups of control and stressed (n=8/group). The animals of the stressed group were exposed to restraint stressors (1 h twice daily) for 15 or 30 consecutive days. At the beginning and end of the experimental periods, the animals were weighed and blood samples taken to determine the fasting plasma levels of glucose, insulin and corticosterone. On the following day the pancreatic islets of 5/group of the above animals were isolated and the static release of insulin in the presence of different glucose concentrations (2.8, 5.6, 8.3, 16.7 mM) was assessed. The results showed that in the stressed group, fasting plasma glucose levels were increased significantly on the 15th day as compared to the control group. However there was no significant increase on the 30th day. Fasting plasma insulin was significantly decreased on the 15th and 30th days of the experiment in the stressed group. Stressed rats showed significantly higher fasting plasma corticosterone levels, only on the 15th day, as compared to the control rats. In response to increasing concentrations of glucose, insulin release from islets of the stressed group was increased significantly on the 30th day of the experiment as compared to the control group. We conclude that chronic psychological stress could increase responsiveness of pancreatic beta cells to glucose, in vitro, and thus, low insulin levels of the stressed animals, in vivo, may be due to reason(s) other than the reduction of insulin releasing capacity of pancreatic beta cells.     

Mechanism of 3-phenylpyruvate-induced insulin release from isolated pancreatic islets.

3-Phenylpyruvate evoked a monophasic insulin release from perifused mouse islets. L-Phenylalanine was not an insulin secretagogue and was oxidized by islets at a very low rate, suggesting that 3-phenylpyruvate does not trigger insulin release by enhancing production of reducing equivalents. Moreover, allosteric activation of glutamate dehydrogenase does not play a role in 3-phenylpyruvate-induced insulin secretion.