Regulated expression of 5'-deleted mouse GLUT4 minigenes

GLUT 4是胰岛素反应性葡萄糖转运蛋白，介导骨骼肌和脂肪组织中葡萄糖代谢的限速步骤。运动训练上调和高脂喂养下调GLUT 4 mRNA。通过使用5 '-缺失的小鼠GLUT 4小基因转基因小鼠，我们先前已经证明小鼠GLUT 4的运动和高脂肪反应元件位于相对于转录起始的-1000和-442之间的558 bp处。为了缩小这一区域，制备了-701、-551和-423 GLUT 4小基因转基因小鼠，运动和高脂反应元件的作用位于-551 <$-442和-701 <$-551。采用凝胶位移分析法检测了肌肉细胞核提取物与GLUT 4 -1：-556-527，GLUT 4 -2：-531-502，GLUT 4 -3：-506-470，GLUT 4 -4：-476-438的结合活性。与GLUT 4 -3的结合在运动小鼠的核提取物中比在非运动小鼠的核提取物中更大。此外，使用-751的552的DNA酶I足迹分析显示，来自高碳水化合物和高脂肪喂养的小鼠的WAT核提取物保护了-706至-683和-674至-652的区域免受DNA酶I消化。因此，使用WF 1：-712-688，WF 2：-690-650，WF 3：-655-616，WF 4：-620-581，WF 5：-585-546进行凝胶位移分析，以检测来自两种饲料喂养小鼠的WAT核提取物的结合活性。与喂食高碳水化合物饮食的小鼠相比，喂食高脂肪饮食的小鼠与WF 4和WF 5的结合减少更多。这些结果表明，运动和高脂喂养分别导致GLUT 4 -3结合因子的增加和WF 4、WF 5结合因子的减少，可能是GLUT 4上调和下调的原因。

GLUT4, the insulin responsive-glucose transporter, mediates the rate limiting step of glucose metabolism in skeletal muscles and adipose tissues. Exercise training up-regulates and High-fat feeding down-regulates GLUT4 mRNA. By using 5'-deleted mouse GLUT4 minigene transgenic mice, we have previously demonstrated that exercise and high-fat responsive elements of mouse GLUT4 are located 558 bp between -1000 and -442 relative to transcription initiation. To narrowing this area, -701, -551 and -423 GLUT4 minigene transgenic mice were made and the effects of exercise and high fat responsive elements are located -551〜-442 and -701〜-551. Furthermore, denervation responsive element is located downstream of -423 and different to exercise responsive element.Gel shift analysis was performed to test the binding activities of muscle nuclear extracts to GLUT4-1:-556〜-527, GLUT4-2:-531〜-502, GLUT4-3:-506〜-470, GLUT4-4:-476〜-438. The binding to GLUT4-3 was greater in nuclear extracts from exercised mice than those of non-exercised mice. Moreover, DNase I footprinting analysis using -751〜-552 revealed that WAT nuclear extracts from high-carbohydrate and high-fat fed mice protected the region from -706 to -683 and from -674 to -652 from DNase I digestion. Therefore, gel shift analysis was performed to test the binding activities of WAT nuclear extracts from both diet fed mice using WF1:-712〜-688, WF2:-690〜-650, WF3:-655〜-616, WF4:-620〜-581, WF5:-585〜-546. The binding to WF4 and WF5 was more decreased in mice fed the high-fat diet than those fed the high-carbohydrate diet. These results demonstrated that increased binding factor to GLUT4-3 and decreased binding factor to WF4, WF5 may be responsible for up- and down-regulation of GLUT4 by exercise and high-fat feeding, respectively.