Differential regulation of secretory compartments containing the insulin-responsive glucose transporter 4 in 3T3-L1 adipocytes

Insulin and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) both stimulat e glucose transport and translocation of the insulin-responsive glucose tra nsporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies s uggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by di stinct trafficking mechanisms, possibly involving mobilization of distinct inh acellular compartments. We show that ablation of the endosomal system u sing transferrin-HRP causes a modest inhibition (similar to 30%) of insulin -stimulated GLUT4 translocation. In contrast, the GTP gamma S response was significantly attenuated (similar to 85%) under the same conditions. Introd uction of a GST fusion protein encompassing the cytosolic tail of the V-SNA RE cellubrevin inhibited GTP gamma S-stimulated GLUT4 translocation by simi lar to 40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane prot ein-2 had no significant effect on GTP gamma S-stimulated GLUT4 translocati on but inhibited the insulin response by similar to 40%. GTP gamma S- and i nsulin-stimulated GLUT1 translocation were both partially inhibited by GST- cellubrevin (similar to 50%) but not by GST-vesicle-associated membrane pro tein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with G TP gamma S caused a marked accumulation of Rab4 and Rab5 at the cell surfac e, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently usin g distinct sets of trafficking molecules.