| Project/Area Number |
11671122
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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 | Ehime University School of Medicine |
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Principal Investigator |
MAKINO Hideichi Ehime University School of Medicine, Department of Laboratory Medicine Professor, 医学部, 教授 (50009578)
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| Co-Investigator(Kenkyū-buntansha) |
ONUMA Hiroshi Ehime University School of Medicine, Department of Laboratory Medicine Instructor, 医学部, 助手 (00294794)
OSAWA Haruhiko Ehime University School of Medicine, Department of Laboratory Medicine Associate Professor, 医学部, 助教授 (90294800)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Insulin / PDE3B / adenovirus / Akt / PI 3-K / PKB / PKCλ |
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| Research Abstract |
Phosphodiesterase (PDE) 3B is a key enzyme involved in anti-lipolytic action of insulin. PDE3B is phosphorylated and activated by insulin, which results in a reduced output of free fatty acids (FFA) from adipocytes. We have established cell-free assay system for PDE kinase, an unknown kinase which phosphorylates PDE3B.This system enabled us to find this kinase is activated by insulin at the downstream of phosphatidylinositol 3-kinase (PI3-kinase). Akt is located at the downstream of PI3-kinase in some of important insulin signaling pathways such as ones leading to protein synthesis, thus, could be a molecule which activates PDE kinase. To examine a role of Akt in PDE kinase activation by insulin, we established gene transfer system into 3T3-L1 adipocytes using adnovirus vectors. Adenovirus vectors containing dominant negatives for Akt or PI3-K, and β-galactosidase (β-gal) as controls were provided by Dr.Ogawa at Kobe University. Last year, we infected 293 cells with these adenovirus vectors and prepared high titer virus reagents. This year, we transfected these adenovirus vectors into 3T3-L1 adipocytes. We determined optimal titers for each virus reagents by checking either introduced β-gal, Akt, or PI 3-K gene expression in 3T3-L1 adipocytes by Western blotting. We are now examining the effect of overexpression of dominant negatives for Akt or PI 3-K on insulin-induced PDE3B activation and PDE3B phosphorylation by PDE kinase. These experiments will clarify roles of Akt and PI 3-K in insulin-induced PDE kinase activation.
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