Wortmannin inhibits insulin secretion in pancreatic islets and beta-TC3 cells independent of its inhibition of phosphatidylinositol 3-kinase

Glucose is the primary stimulus for insulin secretion by pancreatic beta-cells, and it triggers membrane depolarization and influx of extracellular Ca2+, Cholinergic agonists amplify insulin release by several pathways, including activation of phospholipase C, which hydrolyzes membrane polyphosphoinositides. A novel phospholipid, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P-3], a product of phosphatidylinositol 3-kinase (PI 3-kinase), has recently been found in various cell types, We demonstrate by immunoblotting that PI 3-kinase is present in both cytosolic and membrane fractions of insulin-secreting beta-TC3 cells and in rat islets, The catalytic activity of PI 3-kinase in immunoprecipitates of islets and beta-TC3 cells was measured by the production of radioactive phosphatidylinositol 3-monophosphate from phosphatidylinositol (PtdIns) in the presence of [gamma-P-32]ATP, Wortmannin, a fungal metabolite, dose dependently inhibited PI 3-kinase activity of both islets and beta-TC3 cells, with an IC50 of 1 nmol/l and a maximally effective concentration of 100 nmol/l, when it was added directly to the kinase assay, However, if intact islets were incubated with wortmannin and PI 3-kinase subsequently was determined in islet immunoprecipitates, similar to 50% inhibition of PI 3-kinase activity (but no inhibition of glucose- and carbachol-stimulated insulin secretion) from intact islets was obtained at wortmannin concentrations of 100 nmol/l, Wortmannin, at higher concentrations (1 and 10 mu mol/l), inhibited glucose- and carbachol-induced insulin secretion of intact rat islets by 58 and 92%, respectively, Wortmannin had no effect on the basal insulin release from rat islets, A similar dose curve of inhibition of glucose- and carbachol-induced insulin secretion by wortmannin was obtained when beta-TC3 cells were used, Cellular metabolism was not changed by any wortmannin concentrations tested (0.01-10 mu mol/l). Both basal cytosolic [Ca2+](i) and carbamyl choline-induced increases of [Ca2+](i) were unaffected be wortmannin in the presence of 2.5 mmol/l Ca2+, while Ca2+ mobilization from intracellular stores was partially decreased by wortmannin. Together, these data suggest that wortmannin at concentrations that inhibit PI 3-kinase does not affect insulin secretion, PI 3-kinase is unlikely to have a major role in insulin secretion induced by glucose and carbachol.