| Project/Area Number |
18390271
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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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 | Hiroshima University |
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Principal Investigator |
ASANO Tomoichiro Hiroshima University, Graduate School of Biomedical Sciences, Professor (70242063)
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| Co-Investigator(Kenkyū-buntansha) |
SAKODA Hideyuki University of Tokyo, University Hospital, Assistant professor (50376464)
FUJISHIRO Midori University of Tokyo, University Hospital, Assistant professor (50420211)
UCHIJIMA Yasunobu University of Tokyo, Garduate School of Medicine, Research Associate (90272426)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥17,400,000 (Direct Cost: ¥15,600,000、Indirect Cost: ¥1,800,000)
Fiscal Year 2007: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000)
Fiscal Year 2006: ¥9,600,000 (Direct Cost: ¥9,600,000)
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| Keywords | insulin / Diabetes mellitus / proteomics / glucose transporter / Pin1 / IRS-1 / insulin resistance / シグナル伝達 / 糖取り込み |
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| Research Abstract |
IRS-proteins are phosphorylated on multiple tyrosine residues by the activated insulin receptor. We identified Pin1 to be associated with IRS-1 by screening a cDNA library using 32P-ATP labeled IRS-1 probe and identifying Pin1 in the immunoprecipitates of overexpressed IRS-1.with myc and FLAG tags in mouse livers. Pin1 reportedly binds to pSer/Pro or pThr/Pro containing motif and converts the cis-trans conformational change of praline residue, which induce the conformational change of target protein. The association of Pin1 with IRS-1 was observed in not only overexpression experiments using Sf-9 and HepG2 cells, but also endogenously in the mouse liver. The association between Pin1 and IRS-1 in HepG2 cells was significantly increased by the treatment of okadaic acid. IRS-1 produced in Sf-9 cells was pulldowned by GST-pin1 but not GST alone, while IRS-1 treated with alkaline phosphatase was not. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed that WW d
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omain located in the N-terminus of Pin1 and Ser 434 in the SAIN domain of IRS-1 is involved in their association.
Next, we investigated the role of Pin1 on IRS-1 mediating insulin signaling. The overexpression of Pin1 in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events; PI 3-kinase binding with IRS-1, Akt phosphorylation and GSK3beta phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 inhibitor Juglone suppressed them. In addition, overxpression of Pin1 in ob/ob mouse liver elevated IRS-1 mediating insulin signaling, while Pin1 KO mice exhibited insulin resistance and glucose intolerance.
Finally, the regulation of Pin1 expression in the mouse liver and muscle was investigated. After 2 weeks high-fat diet feeding, in both liver and muscle, Pin1 protein expression and IRS-1 associated with Pin1 was increased by several folds. It was also shown that Pin1 expression is low in the fasted condition, and increased by re-feeding. Taken all results together, we conclude that Pin1 expression is regulated by the nutrient conditions, and play an important role on enhancing IRS-1 mediating insulin actions in accordance with the nutrient conditions. Less
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