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 proteins, has been shown to be associated with GLUT4-containing vesicles and implicated in the insulin action on glucose transport in rat adipocytes. In the present study, we investigated the insulin effects on the guanine nucleotide exchange on Rab4. In electrically permeabilized rat adipocytes, the amount of 35S]guanosine 5'-O-(3-thiotrisphosphate) (GTPgammaS) bound to Rab4 increased in a time-dependent manner during 45 min of the incubation period. Addition of insulin resulted in about a 2-fold stimulation of the binding of 35S]GTPgammaS to Rab4, indicating that insulin stimulated the guanine nucleotide exchange on the GTPase. Pretreatment of the cells with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, completely abolished the stimulatory effect of insulin on 35S]GTPgammaS binding to Rab4. Wortmannin also attenuated the nucleotide binding to Rab4 in the basal cells, suggesting that phosphatidylinositol 3-kinase activity may be essential for regulation of guanine nucleotide exchange on the GTPase and insulin may up-regulate the exchange activity by stimulating the lipid kinase. Insulin-induced subcellular redistribution of Rab4 from the microsomal fraction to the soluble fraction was also inhibited by wortmannin. These results suggest that insulin stimulates the guanine nucleotide exchange on Rab4 via a phosphatidylinositol 3-kinase-dependent signaling pathway and that Rab4 is one of possible targets of insulin action on intracellular vesicle traffic in rat adipocytes.