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.
, Heywon Lee, Hui Su Lee, Jong Sik Ha, Ji Hee Lee Src family tyrosine kinases(TK) have been found to be involved in LPS induction of signal cascades. Furthermore Lipopolysaccharide(LPS) or Tumor necrosis factor alpha (TNF-α) activate nuclear transcription factor κB(NF-κB) by inducing serine or tyrosine phosphorylation of the inhibitory subunit of NF-κ B(I κ B-α). In this study, it is our purpose to search the role of Src TK in LPS induced activation of NF-κ B and NF-κ B dependent induced inflammatory factors.
Nuclear extracts were prepared from RAW 264.7 cells pretreated with damnacanthal or PP1 and then stimulate with LPS. After that, we figured out the dffects of inhibition of Src family kinases on LPS-induced activation of NF-κB by EMSA. We investigated effects of damnacanthal of PP1 on the production of NO by Griess assay and LPS-induced serine phosphorylation and degradation of Iκ B-α by Western blots in LPS-stimulated RAW263.7 cells.
Inhibition of Src TK with damnacanthal or PP1 blocked LPS-induced NF-κB activation at the range of nanomolar concentrations. Substantial inhibition in LPS-induced production of NO was also observed in cells treated with damnacanthal or PP1. These kinase inhibitors blocked LPS-induced the serine phosphorylation, and the degradation of Iκ B-α.
we investigated the role of Src TK in NF-κ B activation and production of nitric oxide (NO) in LPS stimulated RAW 264.7 macrophages and the underlying mechanism by which Src TK play a role in LPS-induction of the possible pathways leading to NF-κ B activation. Src kinase specific inhibitors, damnacanthal and PP1 blocked LPS induced activating NF-κ B and producing Nitric Oxide in Raw 264.7 machrophages. Moreover, Damnacanthal and PP1 inhibited LPS induced serine phosphorylation and degradation of Iκ B-α.
, Young Hyun Go, Ji Hee Lee 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 Gαh, 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 Gαh, 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, Gαh is not involved in the GLUT4 translocation process.
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