权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular approach for mechanisms of glucose transport and insulin action

葡萄糖转运和胰岛素作用机制的分子方法

基本信息

批准号：
08457266
负责人：
OKA Yoshitomo
金额：
$ 5.5万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

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.

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，使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-激酶的表位标记的 p110α 亚基。使用抗标记表位抗体通过免疫印迹证实了p110α的过度表达。 p110α过表达诱导基础状态下与其调节亚基相关的PI 3激酶活性增加2.5倍，这比在最大胰岛素刺激的对照细胞中观察到的要高，而与磷酸酪氨酰蛋白相关的PI 3激酶活性仅适度升高。 p110α 的过度表达导致基础葡萄糖转运率增加约 14 倍，这也高于刺激对照中观察到的水平。在对照和 p110α 过表达 3T3-L1 脂肪细胞之间，没有观察到 GLUT1 或 GLUT4 蛋白的细胞表达水平存在明显差异。亚细胞分级分离揭示了基础 p110α 过表达细胞中葡萄糖转运蛋白从细胞内易位到质膜。使用膜片测定进一步证实了 GLUT4 蛋白易位至质膜。这些发现表明，PI 3-激酶的 p110α 过度表达诱导的 PI 3-激酶活性增加通过葡萄糖转运蛋白的易位刺激葡萄糖转运活性，即模拟胰岛素的作用。使用 PI3 激酶的显性负性 p85 亚基获得的结果进一步支持了 PI3 激酶激活对于胰岛素刺激的葡萄糖转运的重要性。当 p85 的显性失活形式（其中野生型 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受到显着抑制，同时 GLUT4 易位受损。