Our preliminary studies suggest the hypothesis that an arachidonate 12-lipoxygenase product (e.g. 12-HPETE) participates in insulin secretion since glucose stimulates synthesis of 12-lipoxygenase products by isolated pancreatic islets and 12-lipoxygenase inhibitors suppress glucose-induced insulin secretion. This hypothesis will be further evaluated by examining 1) the time course of rat islet 12-HETE and prostaglandin biosynthesis compared to the biphasic secretion of insulin after glucose stimulation; 2) the insulin secretagogue properties and islet biosynthesis of recently described metabolites of 12-HPETE; 3) the influence islet insulin secretion in response to exogenous 12-lipoxygenase products); 4) the influence of lipoxygenase inhibitors on glucose-induced insulin secretion in isolated human pancreatic islets and 5) the biosynthesis of 12-HETE by human islets in response to glucose. Exogenous addition of the 12-lipoxygenase product 12-HETE only partially reverses the suppression of glucose-induced insulin secretion from isolated islets by lipoxygenase inhibitors. I will examine the possibilities that stimulation of insulin secretion can be more readily demonstrated with: 1) enantiopure rather than racemic 12-HETE and 12-HPETE and 2) carboxylic acid esters of 12-HETE and 12-HPETE rather than the free carboxylate anions (which may not readily enter cells). To explore a possible mechanism for effects of 12-lipoxygenase products, the influence of these compounds on calcium flux from digitonin- permeabilized islets will also be examined. Arachidonate for 12-HPETE synthesis may be derived in part from phosphatidyl inositol-4,5-biphosphate with attendant generation of diacyclglycerol (DG) and inositol-1,4, 5-tris- phosphate (IP3). To evaluate the hypothesis that the coordinated release and degradation of free arachidonate, its metabolites, and of DG and IP3 regulate the phasic nature of glucose-induced insulin secretion, mass spectrometric methods will be developed for quantitation of AA, DG, and IP3 which are similar to those already developed for measurement of 12-HETE. These methods will then be applied to study the time course of 12-HETE, AA, DG, and IP3 appearance in relationship to insulin secretion after glucose stimulation.
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