Identification of wortmannin-sensitive targets in 3T3-L1 adipocytes - Dissociation of insulin-stimulated glucose uptake and GLUT4 translocation

The current studies investigated the contribution of phosphatidylinositol 3 -kinase (PI3-kinase) isoforms to insulin-stimulated glucose uptake and gluc ose transporter 4 (GLUT4) translocation, Experiments involving the microinj ection of antibodies specific for the p110 catalytic subunit of class I PI3 -kinases demonstrated an absolute requirement for this form of the enzyme i n GLUT4 translocation. This finding was confirmed by the demonstration that the PI3-kinase antagonist wortmannin inhibits GLUT4 and insulin-responsive aminopeptidase translocation with a dose response identical to that requir ed to inhibit another class I PI3-kinase-dependent event, activation of pp7 0 SG-kinase, Interestingly, wortmannin inhibited insulin-stimulated glucose uptake at much lower doses, suggesting the existence of a second, higher a ffinity target of the drug. Subsequent removal of wortmannin from the media shifted this dose-response curve to one resembling that for GLUT4 transloc ation and pp70 SG-kinase, This is consistent with the lower affinity target being p110, which is irreversibly inhibited by wortmannin, Wortmannin did not reduce glucose uptake in cells stably expressing Myr-Akt, which constit utively induced GLUT4 translocation to the plasma membrane; this demonstrat es that wortmannin does not inhibit the transporters directly. In addition to elucidating a second wortmannin-sensitive pathway in 3T3-L1 adipocytes, these studies suggest that the presence of GLUT4 on the plasma membrane is not sufficient for activation of glucose uptake.