Regulated expression of 5'-deleted mouse GLUT4 minigenes

GLUT4是胰岛素反应葡萄糖转运体，在骨骼肌和脂肪组织中介导葡萄糖代谢的限速步骤。运动训练上调GLUT4基因表达，高脂饮食下调GLUT4基因表达。通过使用5‘端缺失的小鼠GLUT4微型基因转基因小鼠，我们先前已经证明了小鼠GLUT4的运动和高脂反应元件位于相对于转录启动的558bp的-1000到-442之间。为了缩小这一区域，制备了-701、-551和-423 GLUT4转基因小鼠，运动和高脂反应元件的影响位于-551~-442和-701~-551。此外，去神经反应元件位于-423的下游，与运动反应元件不同。通过凝胶位移分析检测了肌核提取物与GLUT4-1：-556~-527、GLUT4-2：-531~-502、GLUT4-3：-506~-470、GLUT4-4：-476~-438的结合活性。运动小鼠的核提取液与GLUT4-3的结合比未运动小鼠强。此外，使用-751~-552进行的DNase I足迹分析表明，高碳水化合物和高脂肪喂养的小鼠的Wat核提取物保护-706到-683和-674到-652区域不被DNase I消化。因此，使用WF1：-712~-688，WF2：-690~-650，WF3：-655~-616，WF4：-620~-581，WF5：-585~-546，对两种饲料喂养小鼠的WAT核提取物进行凝胶位移分析。高脂饮食的小鼠与WF4和WF5的结合比高碳水化合物饮食的小鼠更低。这些结果表明，运动和高脂饮食分别上调和下调GLUT4-3和WF4、WF5的结合因子可能与GLUT4-3结合因子增加和WF4结合因子减少有关。

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.