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 stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct inh acellular compartments. We show that ablation of the endosomal system using 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. Introduction of a GST fusion protein encompassing the cytosolic tail of the V-SNARE cellubrevin inhibited GTP gamma S-stimulated GLUT4 translocation by similar to 40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTP gamma S-stimulated GLUT4 translocation but inhibited the insulin response by similar to 40%. GTP gamma S- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin (similar to 50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTP gamma S caused a marked accumulation of Rab4 and Rab5 at the cell surface, 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 using distinct sets of trafficking molecules.