Abstract
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.
Highlights
Insulin stimulates glucose transport in muscles and adipose cells by promoting translocation of glucose transporter isoform, GLUT4, from intracellular compartmment(s) to the plasma membrane [1,2,3], the molecular mechanisms of the insulin action are still obscure
To elucidate the physiological significance of Rab4 in the insulin action, we incorporated into rat adipocytes a synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain and showed that the peptide inhibited glucose transport and GLUT4 translocation stimulated by insulin or GTP␥S1 [16], providing evidence that Rab4 plays a critical role in the insulin-induced GLUT4 translocation
We examined this possibility and demonstrated that guanine nucleotide exchange on Rab4 was stimulated by insulin in a wortmannin-sensitive manner, providing evidence for the first time that Rab4 lies downstream of the insulin-stimulated phosphatidyl inositol (PI) 3-kinase
Summary
Materials—125I-Labeled protein A and [35S]GTP␥S were from DuPont NEN. Wortmannin was obtained from Sigma and dissolved in dimethyl sulfoxide at 10 mM (stock solution). LY294002 was purchased from Calbiochem and dissolved in Me2SO at 5 mM (stock solution). Protein G-Sepharose was from Pharmacia Biotech Inc
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