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)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 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-32P]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, approximately 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 micromol/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 micromol/l). Both basal cytosolic [Ca2+]i and carbamyl choline-induced increases of [Ca2]i 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.
Gao
Z
Z
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA.
Konrad
R J
RJ
Collins
H
H
Matschinsky
F M
FM
Rothenberg
P L
PL
Wolf
B A
BA
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D007328:Insulin; D002217:Carbachol; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D002122:Calcium Chloride; D002118:Calcium; D000077191:Wortmannin