POTENTIAL ROLE OF RAB4 IN THE REGULATION OF SUBCELLULAR-LOCALIZATION OF GLUT4 IN ADIPOCYTES

A role for Rab4 in the translocation of the glucose transporter Glut4 induced by insulin has been recently proposed, To study more directly the role of this small GTPase, freshly isolated adipocytes were transi ently transfected with the cDNAs of both an epitope-tagged Glut4-myc a nd Rab4, a system which allows direct measurement of the concentration of Glut4 molecules at the cell surface, When cells were cotransfected ,vith Glut4-myc and Rab4, the concentration of Glut4-myc at the cell s urface decreased in parallel with the increased expression of Rab4, su ggesting that Rab4 participates in the intracellular retention of Glut 4, In parallel, the amount of Rab4 associated with the Glut4-containin g vesicles increased, When Rab4 was moderately overexpressed, the numb er of Glut4-myc molecules recruited to the cell surface in response to insulin was similar to that observed in mock-transfected cells, and t hus the insulin efficiency was increased, When Rab4 was expressed at a higher level, the amount of Glut4-myc present at the cell surface in response to insulin decreased, Since the overexpressed protein was pre dominantly cytosolic, this suggests that the cytosolic Rab4 might comp lex some factor(s) necessary for insulin action, This hypothesis was s trengthened by the fact that Rab4 Delta CT, a Rab-l mutant lacking the geranylgeranylation sites, inhibited insulin-induced recruitement of Glut4-myc to the cell surface, even when moderately overexpressed, Rab 3D was without effect on Glut4-mye subcellular distribution in basal o r insulin-stimulated conditions, While two mutated proteins unable to bind GTP did not decrease the number of Glut4-myc molecules in basal o r insulin-stimulated conditions at the plasma membrane, the behavior o f a mutated Rab4 protein without GTPase activity was similar to that o f the wild-type Rab4 protein, indicating that GTP binding but not its hydrolysis was required for the observed effects, Altogether, our resu lts suggest that Rab4, but not Rab3D, participates in the molecular me chanism involved in the subcellular distribution of the Glut4 molecule s both in basal and in insulin-stimulated conditions in adipocytes.