The subcellular distribution of glucose transporter in rat hepatocytes was studied in the absence and presence of insulin by measuring glucose-sensitive cytochalasin B binding sites and immunoreactivities to antibodies specific to hepatocyte glucose transporter. Total hepatocyte membranes bound cytochalasin B at a class of glucose-sensitive sites with a K_d of l.6×l0^-6M. and a B_t of 6.8 pmol/mg protein. The glucose-sensitive cytochalasin B binding sites were found in various subcellular membrane fractions with a relative abundance of 47% in a plasma membrane-nuclei-mitochondria-enriched fraction(PM/NM). 29% in a lysosome-enriched fraction(LYSO). 16% in a Golgi-enriched. high density microsomal fraction(HDM) and 8% in the low density microsome fraction(LDM). Relative abundances of two well known plasma membrane markers. 5'-nucleotidase and cell surface carbohydrate label, on the other hand, were found to he 48~50% in PM/NM. 41~43% in LYSO. 6~8% in HDM and l~2% in LDM.

Insulin treatment of intact hepatocytes did not induce any significant changes in the subcellular distributions of the glucose-sensitive. cytochalasin B binding activities, the immunoreactivities to the transporter specific antibodies, or the two cell surface membrane markers. These findings indicate that as much as 15% of the total hepatocyte glucose transporters occur in organelle(s) other than the pasma membrane, most likely representing an intracellular storage pool. which is not decreased by insulin. It is concluded that the rat hepatocyte lacks the insulin-mediated, glucose transporter translocation mechanism, thus would be a valuable experimental system in which one can study the celluar and molecular basis of this deficiency.

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.

Insulin stimulates glucose transport in muscle cell and adipocyte via the rapid redistribution of GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. The mechanism that insulin treggers the translocation of glucose transporters in not known yet whether it is due to the structural differences among glucose transporters or there is cell specific targetting/translocation apparatus insulin-sensitive cells.

This study was planned to examine this question by strdying insulin effect on the glucose transport rate at adipocyte and hepatocyte fused with GLUT1 vesicle, respecitively.

The results showed that treatment of 37nM insulin increased the transport rate of 3-0-methylglucose by 3.8-fold at adipocyte fused with GLUT1 but increased lnly by 1.2-fold at hepatocyte fused with GLUT1.

Therefore, it is suggested that insulin sensitive cell has a cell-specific targetting/translocation machinery which is triggered by insulin-insulin receptor interaction but insulin sensitivity may not dependent on isoform(structural)-specific manner.