Wortmannin Inhibits Insulin Secretion in Pancreatic Islets and β-TC3 Cells Independent of Its Inhibition of Phosphatidylinositol 3-Kinase

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Abstract
Glucose is the primary stimulus for insulin secretion by pancreatic β-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)P3[, 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 β-TC3 cells and in rat islets. The catalytic activity of PI 3-kinase in immunoprecipitates of islets and β-TC3 cells was measured by the production of radioactive phosphatidylinositol 3-monophosphate from phosphatidylinositol (Ptdlns) in the presence of [γ-32P[ATP. Wortmannin, a fungal metabolite, dose dependency inhibited PI 3-kinase activity of both islets and P-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, ∼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 µmol/l. Wortmannin, at higher concentrations (1 and 10 µmol/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 β-TC3 cells were used. Cellu-lar metabolism was not changed by any wortmannin concentrations tested (0.01–10 µmol/l). Both basal cytosolic [Ca2+]1 and carbamyl choline-induced increases of [Ca2+]1 were unaffected by 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.Diabetes 45:854–862, 1996

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