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.

Insulin stimulation of glucose transport in adipocytes results from the translocation of vesicles containing the GLUT4 glucose transporter from an intracellular pool to the plasma membrane. In mammalian cells a family of GTP-binding proteins has been implicated in the control of cellular trffic. Thus this study was planned to see whether G-proteins such as Rab, a small molecular mass G-protein and Gα_h, a large molecular mass G-protein are involved in insulin induced GLUT4 translocation process.

Diabetic rats(Spraque-Dauley, 200-250g) were prepared by injection of streptozotocin(60mg/kg,IP) and treated with or without insulin(20U/rat) for 4 weeks. The purpose of the study is to elucidate a possible functional relationship between G-protein and the insulin-responsive GLUT4 translocation by immunoblotting method from the subcellular fractions of adipocytes of epididymal tissues.

As results Rab4 protein was coexisted in the membrane of GLUT4 immunoprecipitates of adipocyte total homogenates in normal rats, however Gα_h, could not be detected. The amount of GLUT4 at plasma membrane(PM) obtained from insulin treated rats were increased by 21. 35% compared to that of streptozotocin diabetic rats. The increase of Rab4 at the same plasma membrane was negligible. On the other hand, the amounts of GLUT4 and Rab4 at low density microsome(LDM) were decreased by 7.82% and 9.25%, respectively.

These results show that Rab4 is co-localized with GLUT4 in an insulin-responsive intracellular compartment and Rab4 protein plays role in the action of insulin on the GLUT4 translocation but a large molecular G-protein, Gα_h is not involved in the GLUT4 translocation process.