Cooke D W, Lane M D
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3311, USA.
J Biol Chem. 1998 Mar 13;273(11):6210-7. doi: 10.1074/jbc.273.11.6210.
Prolonged treatment of 3T3-L1 adipocytes decreases expression of GLUT4, the insulin-responsive glucose transporter. Expression of promoter-reporter gene constructs that contained 2900 or 785 base pairs of 5'-flanking region of the murine GLUT4 gene was down-regulated by insulin (p < 0.0005), whereas expression of constructs that contained 641, 469, or 78 base pairs of 5'-flanking region was not. Nuclear extract from 3T3-L1 adipocytes protected the region from -707 to -681 in the GLUT4 5'-flanking region from DNase I digestion. Using an oligonucleotide probe that corresponded to this footprinted region, two major protein-DNA complexes were identified by a gel mobility shift assay. Southwestern analysis identified four protein bands with molecular masses from 38 to 46 kDa that bound to the insulin-responsive region probe. A reporter gene construct in which bases -706 to -676 were deleted was not repressed by insulin treatment, confirming that this sequence is necessary for the repression of the GLUT4 promoter by insulin in 3T3-L1 adipocytes. This sequence does not show homology to previously described insulin response elements and thus represents a distinct mechanism of gene regulation by insulin.
对3T3-L1脂肪细胞进行长时间处理会降低胰岛素反应性葡萄糖转运蛋白GLUT4的表达。含有小鼠GLUT4基因5'侧翼区2900或785个碱基对的启动子-报告基因构建体的表达受胰岛素下调（p<0.0005），而含有5'侧翼区641、469或78个碱基对的构建体的表达则不受影响。3T3-L1脂肪细胞的核提取物可保护GLUT4 5'侧翼区中从-707至-681的区域不被DNase I消化。使用与该足迹区域对应的寡核苷酸探针，通过凝胶迁移率变动分析鉴定出两种主要的蛋白质-DNA复合物。蛋白质印迹分析鉴定出四条分子量在38至46 kDa之间的蛋白质条带与胰岛素反应区域探针结合。一个缺失了-706至-676碱基的报告基因构建体在胰岛素处理后未被抑制，这证实了该序列对于3T3-L1脂肪细胞中胰岛素对GLUT4启动子的抑制作用是必需的。该序列与先前描述的胰岛素反应元件没有同源性，因此代表了胰岛素基因调控的一种独特机制。
