INSULIN STIMULATES GUANINE-NUCLEOTIDE EXCHANGE ON RAB4 VIA A WORTMANNIN-SENSITIVE SIGNALING PATHWAY IN RAT ADIPOCYTES

Rab4, a member of the Rab family of Ras-related small GTP-binding prot eins, has been shown to be associated with GLUT4-containing vesicles a nd implicated in the insulin action on glucose transport in rat adipoc ytes. In the present study, we investigated the insulin effects on the guanine nucleotide exchange on Rab4. In electrically permeabilized ra t adipocytes, the amount of [S-35]guanosine 5'-O-(3-thiotrisphosphate) (GTP gamma S) bound to Rab4 increased in a time-dependent manner duri ng 45 min of the incubation period. Addition of insulin resulted in ab out a 2-fold stimulation of the binding of [S-35]GTP gamma S to Rab4, indicating that insulin stimulated the guanine nucleotide exchange on the GTPase. Pretreatment of the cells with wortmannin, a specific inhi bitor of phosphatidylinositol 3-kinase, completely abolished the stimu latory effect of insulin on [S-35]GTP gamma S binding to Rab4. Wortman nin also attenuated the nucleotide binding to Rab4 in the basal cells, Suggesting that phosphatidylinositol 3-kinase activity may be essenti al for regulation of guanine nucleotide exchange on the GTPase and ins ulin may up-regulate the exchange activity by stimulating the lipid ki nase. Insulin-induced subcellular redistribution of Rab4 from the micr osomal fraction to the soluble fraction was also inhibited by wortmann in. These results suggest that insulin stimulates the guanine nucleoti de exchange on Rab4 via a phosphatidylinositol 3-kinase-dependent sign aling pathway and that Rab4 is one of possible targets of insulin acti on on intracellular vesicle traffic in rat adipocytes.