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动的机制，用腺病毒介导的基因转导系统在3T3-L1脂肪细胞中过表达了PI-3-K的表位标记的p110α亚单位。用抗标记表位抗体进行免疫印迹实验，证实p110α基因过表达。在基础状态下，p110α过表达诱导与其调节亚基相关的PI 3-激酶活性增加2.5倍，高于胰岛素刺激的对照细胞，而与磷酸酪氨酰蛋白相关的PI 3-K活性仅轻微升高。P110α的过表达诱导基础葡萄糖转运率增加约14倍，这也大于在刺激对照组观察到的。GLUT1和GLUT4蛋白在对照组和p110α过表达的3T3-L1脂肪细胞中的表达水平没有明显差异。亚细胞分级显示，在p110α过表达的基础细胞中，葡萄糖转运体从细胞内转移到质膜。用膜片法进一步证实了GLUT4蛋白向质膜的转位。这些结果表明，PI3K的p110α过表达导致PI3K活性增加，通过葡萄糖转运体的转位来刺激葡萄糖转运活性，即模仿胰岛素的作用。PI3-激酶激活对于胰岛素刺激的葡萄糖转运的重要性得到了使用PI3-激酶的显性负P85亚单位获得的结果的进一步支持。当P85的主要负性形式(野生型P85的SH间区域被HA标签取代)过表达到3T3-L1脂肪细胞中时，胰岛素刺激的葡萄糖转运明显受到抑制，GLUT4转位受到损害。

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.