Effects of insulin on intracellular GLUT4 vesicles in adipocytes: evidencefor a secretory mode of regulation

The facilitative glucose transporter, GLUT4 undergoes insulin-dependent mov ement to the cell surface in adipocytes, The magnitude of the insulin effec t is much greater for CLUT4 than other recycling proteins such as the CD-MP R, In the present study we have studied the colocalisation of these protein s in adipocytes in an effort to explain this selective insulin-dependent re cruitment of CLUT4, Using immunofluorescence microscopy or immuno-EM on 3T3 -L1 adipocytes we find that there is considerable colocalisation between th ese proteins particularly within the area of the TGN, However, the distribu tion of CD-MPR was not significantly effected by insulin. The insulin-depen dent recruitment of GLUT4 was concomitant with a selective decrease in GLUT 4 labelling of cytoplasmic vesicles whereas the amount of CLUT4 in the TGN region (approx. 50% of total GLUT4) was relatively unaffected. To explore t he possibility that the cytoplasmic GLUT4(+) vesicles represent an intracel lular insulin-responsive storage compartment we performed quantitative immu no-EM on whole mounts of intracellular vesicles isolated from basal and ins ulin-stimulated adipocytes. These studies revealed that: (1) GLUT4 and CD-M PR were concentrated in small (30-200 nm) vesicles at a labelling density o f 1-20+ gold particles/vesicle; (2) there was significant overlap between b oth proteins in that 70% of the total GLUT4 pool colocalised with CD-MPR; ( 3) a significant amount of GLUT4 (approx, 50% of total) was found in a subp opulation of vesicles that contained as little as 5% of the total CD-MPR po ol; (4) the GLUT4(+)/CD-MPR(-) vesicles were highly insulin-responsive, and (5) the total number of GLUT4(+) vesicles, but not CD-MPR(+) vesicles, dec reased by approx. 30% in response to insulin treatment. These data are cons istent with a model in which GLUT4 is selectively sorted into a vesicular c ompartment in adipocytes that is recruited to the plasma membrane by insuli n stimulation.