| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
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.
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