Abstract

• As much as 29.3% of GLUT4, 45.1% of GLUT1, 58.5% of clathrin, and 17.2% of insulin receptor imrnunoreactivities in rat adipocyte plasma membranes(PM) were found to be insoluble upon 1% Triton extraction at basal state. By insulin treatment the distributions of insoluble fraction were changed by 16.2% of GLUT4, 48.5% of GLUT1, 65.3% of clathrin and 31.0% of insulin receptor, respectively. SDS-PAGE revealed that the Triton-insoluble PM fraction contains a number of protein species including 110,80,70,50 30-33 and 15 KDa polyeptides. When PM was prewashed with alkaline buffer and 0.5M Tris buffer, which removed most of extrinsic membrane proteins including clathrin and AP-2 from PM, virtually all of the GLUT4 and GLUT1 in PM became soluble in 1% Triton. Subcellular fractionation fo11owed by Western blot indicated that AP-2 distribute to 4.8% at PM/NM, 25.7% at HDM, 38.9% at LDM and 30.6% at cytosol, respectively. An insulin treatment which increased GLUT4 content in PM by 1.5 fo1d increased the AP-2 content in PM nearly 2.3 fold with a concomitant decrease in cytosol AP-2 contents. These findings suggest that subpopulations of GLUT4 and insulin receptor in the plasma membrane of adipocytes are in specific association with extrinsic proteins, possibly clathrin and/or AP-2, and this association may play a key role in the translocation mechanism of GLUT4 in rat epididymal adipocytes.