| Project/Area Number |
12671104
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Metabolomics
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| Research Institution | Yamanashi Medical University |
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Principal Investigator |
YOKOMORI Norihiko Yamanashi Medical University, assistant, 医学部, 助手 (20293475)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMURA Hiroki Yamanashi Medical University, Medical Staff, 医学部, 医員 (40303416)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | GLUT4 / 3T3-L1 cell / Leptin / Transcription factor / Methylation / Epigenetic / 脂肪細胞 / 転写抑制因子 |
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| Research Abstract |
We recently showed that methylation of specific CpG sites of the GLUT4 gene, as well as a inethylation sensitive transcription factor (55kDa), contribute to GLUT4 gene regulation during 3T3-L1 differentiation. Similar mechanisms may also regulate the expression of other genes induced during adipocyte differentiation. The mouse leptin gene, a major hormonal regulator of appetite and fat cell mass, expresses during the differentiation of 3T3-L1 preadipocytes to adipocytes. To determine if DNA methylation is involved in regulating leptin gene expression, we examined the level of methylation and methylation-sensitive transcription factors during 3T3-L1 differentiation. Transient transfection of reporter constructs with the leptin promoter showed that preadipocytes that do not transcribe the leptin gene show enough transactivation, suggesting the presence of an additional regulatory mechanism. We identified eight CpG sites in the promoter up to nt -161, all of which were highly methylated (
… More
>92 %) in preadipocytes. Seven of these sites showed varying degree of demethylation during differentiation, while the site at.nt -54 remained methylated. In electrophoretic mobility-shift assays, DNA fragments from nt -115 to nt -70 generated a methylation-sensitive band with nuclear extracts from preadipocytes when the CpG sites were methylated. Southwestern analysis identified a 52 kDa protein that binds strongly to the methylated probes, and also identified this 52 kDa protein is same as a 55 kDa protein that binds to the methylated GLUT4 gene. Promoter activity was reduced by methylation of the CpG sites up to nt -115, but not up to nt -70. These results suggest that methylation of specific CpG sites and the same methylation-sensitive protein may contribute to GLUT4 and leptin gene expression during adipocyte differentiation in 3T3-L1 cells. We are now trying the cloning this methylation-sensitive protein, and sequencing the obtained positive clone. We will analyze this clone in future study. Less
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