Insulin recruits GLUT4 from specialized VAMP2-carrying vesicles as well asfrom the dynamic endosomal/trans-golgi network in rat adipocytes.

Insulin treatment of fat cells results in the translocation of the insulin- responsive glucose transporter type 4, GLUT4, from intracellular compartmen ts to the plasma membrane. However, the precise nature of these inh acellul ar GLUT4-carrying compartments is debated. To resolve the nature of these c ompartments, we have performed an extensive morphological analysis of GLUT4 -containing compartments, using a novel immunocytochemical technique enabli ng high labeling efficiency and 3-D resolution of cytoplasmic rims isolated from rat epididymal adipocytes. In basal cells, GLUT4 was localized to thr ee morphologically distinct intracellular structures: small vesicles, tubul es, and vacuoles. In response to insulin the increase of GLUT4 at the cell surface was compensated by a decrease in small vesicles, whereas the amount in tubules and vacuoles was unchanged. Under basal conditions, many small GLUT4 positive vesicles also contained IRAP (88%) and the V-SNARE, VAMP2 (5 7%) but not markers of sorting endosomes (EEA1), late endosomes, or lysosom es (lgp120). A largely distinct population of GLUT4 vesicles (56%) containe d the cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker prot ein that shuttles between endosomes and the trans-Golgi network (TGN). Ln r esponse to insulin, GLUT4 was recruited both from VAMP2 and CD-MPR positive vesicles. However, while the concentration of GLUT4 in the remaining VAMP2 -positive vesicles was unchanged, the concentration of GLUT4 in CD-MPR-posi tive vesicles decreased. Taken together, we provide morphological evidence indicating that, in response to insulin, GLUT4 is recruited to the plasma m embrane by fusion of preexisting VAMP2-carrying vesicles as well as by sort ing from the dynamic endosomal-TGN system.