INSULIN INDUCES A CHANGE IN RAB5 SUBCELLULAR-LOCALIZATION IN ADIPOCYTES INDEPENDENTLY OF PHOSPHATIDYLINOSITOL 3-KINASE ACTIVATION

We investigated whether Rab5, a small guanosine triphosphatase that re gulates early endocytic transport in different cell types is involved in the insulin-regulated endocytic pathways in adipocytes. Rab5 was de tected in freshly isolated adipocytes and 3T3-L1 adipocytes, but its e xpression level was not markedly increased with adipocyte differentiat ion. After subcellular fractionation of adipocytes incubated in the ab sence of insulin, Rab5 was found to be abundant in plasma membrane and cytosol, but was also present in high and low density microsomes. Thi s subcellular distribution was compatible with a role in early endocyt osis. When cells were incubated with insulin, the concentration of Rab 5 decreased by about 50% in the internal compartments. In contrast to Rab4, which also leaves the low density microsomes in response to insu lin, Rab5 was not found in Glut4-containing vesicles purified by immun oadsorption on antibodies to Glut4. When adipocytes were treated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, the effect of insulin on Rab5 movement was not affected, whereas the insulin-indu ced movements of Rab4 and Glut4 were abolished. In parallel, wortmanni n inhibited the increase in horseradish peroxidase uptake induced by i nsulin, an index of fluid phase endocytosis, but did not prevent the e ndocytosis of the glucose transporters. As a whole, our results sugges t that Rab5 is not involved in insulin-stimulated Glut4 exocytosis. Th ese results are compatible with the postulated role of Rab5 in the end ocytotic pathway, at a step that does not require phosphatidylinositol 3-kinase activation.