Study on the mechanism of insulin stimulation of glucose transport

在本研究中，我们研究了胰岛素刺激大鼠脂肪细胞上的鸟嘌呤核苷酸交换。这种胰岛素作用可被磷脂酰肌醇(PI)3-激酶抑制剂Wortmannin或LY294002显著减弱，提示P13-K可能是激活Rab4胰岛素的重要信号成分。此外，我们还研究了Rab4与Synaxin4的相互作用，Synaxin4是一种t-SNARE蛋白，与胰岛素诱导的含有GLUT4的囊泡与质膜的胞外融合有关。用免疫共沉淀法从大鼠脂肪细胞匀浆中分离出Rab4和Synaxin4。GTP结合蛋白调节大鼠脂肪细胞含GLUT4囊泡的胞吐和胞内步骤。此外，我们还利用CHO细胞中表达的突变型Dynamin阐明了Dynamin是调节GLUT4内吞作用的GTPase。

In the present study, we calrified that insulin stimulated guanine nucleotide exchange on Rab4 in rat adipocytes. This insulin action was markedly attenuated with phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin or LY294002, suggesting that Pl 3-kinase may be an essential signaling component for insulin activation of Rab4. Additionally, we investigated the interaction of Rab4 with syntaxin4, a t-SNARE protein implicated in the insulin-induced exocytic fusion of the GLUT4-containing vesicle with the plasma membrane. Rab4 and syntaxin4 were co-immunoprecipitated from the homogenate of rat adipocytes. The interaction of the two proteins were attenuated by pretreatment of the cells with insulin but enhanced with GTPgammaS.GTP-binding proteins regulate both exocytotic and endocytotic steps of GLUT4-containing vesicles in rat adipocytes. Furthermore, we demonstrated that protein kinase Cgamma may be the upstream regulator of the insulin action on Rab4.We also clarified that dynamin is a regulatory GTPase of GLUT4 endocytosis by using mutant dynamin expressed in CHO cells.