Phosphatidylinositol 3-Phosphate [PtdIns(3)P] Is Generated at thePlasma Membrane by an Inositol Polyphosphate 5-Phosphatase: Endogenous PtdIns(3)P Can Promote GLUT4 Translocation to the Plasma Membrane

Exogenousdelivery of carrier-linked phosphatidylinositol 3-phosphate[PtdIns(3)P] to adipocytes promotes the trafficking, but not theinsertion, of the glucose transporter GLUT4 into the plasma membrane.However, it is yet to be demonstrated if endogenous PtdIns(3)Pregulates GLUT4 trafficking and, in addition, the metabolic pathwaysmediating plasma membrane PtdIns(3)P synthesis are uncharacterized. Inunstimulated 3T3-L1 adipocytes, conditions under whichPtdIns(3,4,5)P3 was not synthesized, ectopic expression ofwild-type, but not catalytically inactive 72-kDa inositol polyphosphate5-phosphatase (72-5ptase), generated PtdIns(3)P at the plasma membrane.Immunoprecipitated 72-5ptase from adipocytes hydrolyzedPtdIns(3,5)P2 , forming PtdIns(3)P. Overexpression of the72-5ptase was used to functionally dissect the role of endogenousPtdIns(3)P in GLUT4 translocation and/or plasma membrane insertion. Inunstimulated adipocytes wild type, but not catalytically inactive,72-5ptase, promoted GLUT4 translocation and insertion into the plasmamembrane but not glucose uptake. Overexpression of FLAG-2xFYVE/Hrs,which binds and sequesters PtdIns(3)P, blocked 72-5ptase-induced GLUT4translocation. Actin monomer binding, using latrunculin A treatment,also blocked 72-5ptase-stimulated GLUT4 translocation.72-5ptase expression promoted GLUT4 trafficking via a Rab11-dependentpathway but not by Rab5-mediated endocytosis. Therefore, endogenousPtdIns(3)P at the plasma membrane promotes GLUT4translocation.