Regulated expression of 5'-deleted mouse GLUT4 minigenes

Research Project

Project/Area Number	10671088
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Metabolomics
Research Institution	National Institute of Health and Nutrition
Principal Investigator	EZAKI Osamu National Institute of Health and Nutrition, Division of Clinical Nutrition, Division Director, 臨床栄養部, 部長 (90191923)
Project Period (FY)	1998 – 1999
Project Status	Completed (Fiscal Year 1999)
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)
Keywords	GLUT4 / Cis-element / Exercise / High-fat diet / Denervation / Transgenic mice
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.