Molecular approach for mechanisms of glucose transport and insulin action

为了阐明磷脂酰肌醇（PI） 3-激酶参与胰岛素刺激的葡萄糖转运活性的机制，我们利用腺病毒介导的基因转导系统在3T3-L1脂肪细胞中过表达PI 3-激酶表位标记的p110 α亚基。使用抗标记表位抗体免疫印迹法证实p110alpha过表达。p110alpha过表达诱导基础状态下与其调控亚基相关的PI 3-激酶活性增加2.5倍，这比在胰岛素刺激最大的对照细胞中观察到的要高，而与磷酸酪氨酸蛋白相关的PI 3-激酶活性仅适度升高。p110 α的过表达诱导了基础葡萄糖转运率大约14倍的增加，这也比在受刺激的对照组中观察到的要大。在对照组和过表达p110α的3T3-L1脂肪细胞中，GLUT1或GLUT4蛋白的细胞表达水平均无明显差异。亚细胞分离揭示了葡萄糖转运体在基底p110 α过表达细胞中从胞内转运到质膜的易位。利用膜片试验进一步证实了GLUT4蛋白在质膜上的易位。这些发现表明，PI 3激酶的p110 α过表达诱导PI 3激酶活性的增加，通过葡萄糖转运体的易位刺激葡萄糖转运活性，即模仿胰岛素的作用。利用pi3激酶的显性负p85亚基获得的结果进一步支持了pi3激酶激活对胰岛素刺激的葡萄糖运输的重要性。当p85的显性阴性形式（野生型p85的sh间区被HA标签取代）在3T3-L1脂肪细胞中过表达时，胰岛素刺激的葡萄糖转运被明显抑制，GLUT4易位受损。

To elucidate the mechanisms whereby phosphatidylinositol (PI) 3-kinase is involved in insulin-stimulated glucose transport activity, the epitope-tagged p110alpha subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Overexpression of p110alpha was confirmed by immunoblot using anti-tagged epitope antibody. p110alpha overexpression induced an 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, which was greater than that observed in maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated. Overexpression of p110alpha induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control. No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110alpha-overexpressing 3T3-L1 adipocytes. Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110alpha-overexpressing cells. The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay. These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110alpha of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin. The importance of PI3-kinase activation for insulin-stimulated glucose transport was further supported by the results obtained using the dominant negative p85 subunit of PI3-kinase. When the dominant negative form of p85, of which inter-SH region of the wild type p85 was replaced by HA tag, was overexpressed into 3T3-L1 adipocytes, insulin-stimulated glucose transport was markedly inhibited with impairment of GLUT4 translocation.