Insulin-regulated sorting of glucose transporters in 3T3-L1 adipocytes

Two glucose transporters (GLUT-4 and GLUT-1) move from within the cell to the plasma membrane (PM) when 3T3-L1 adipocytes are stimulated with insulin. To study the sorting of these two molecules, vesicles containing GLUT-4 and GLUT-1 were immunoadsorbed from basal and insulin-treated cells. Two different vesicle populations were isolated as follows: 1) a compartment that contained the majority of intracellular GLUT-4 and GLUT-1 and 2) a subpopulation of vesicles containing 43% of the intracellular GLUT-4 that was highly insulin regulatable and that contained relatively low levels of GLUT-1. After incubation at 19 degrees C, basal glucose transport was slightly increased, whereas insulin-dependent transport was blocked. Consistent with these observations, cell surface GLUT-1 levels were increased in the basal state, whereas insulin-dependent translocation of GLUT-4 to the PM was blocked at 19 degrees C. However, insulin-dependent sorting of GLUT-4 within the intracellular compartment was still evident at 19 degrees C. These data indicate that GLUT-1 and GLUT-4 are heterogeneously distributed throughout the same intracellular compartment in 3T3-L1 adipocytes. Furthermore, we have uncoupled two distinct steps in the insulin-dependent movement of GLUT-4 to the cell surface. These include movement of GLUT-4 out of its storage compartment and accumulation of GLUT-4 at the cell surface. Only the former step occurs in cells preincubated at 19 degrees C.