| Project/Area Number |
08457266
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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 |
内分泌・代謝学
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| Research Institution | Yamaguchi University |
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Principal Investigator |
OKA Yoshitomo Yamaguchi University School of Medicine, Professor, 医学部, 教授 (70175256)
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| Co-Investigator(Kenkyū-buntansha) |
NOMIYAMA Jun Yamaguchi University School of Medicine Hospital, instructor, 医学部・附属病院, 医員
TAKEUCHI Yasuo Yamaguchi University School of Medicine Hospital, instructor, 医学部・附属病院, 医員
TAKEUCHI Hideo Yamaguchi University School of Medicine Hospital, instructor, 医学部・附属病院, 医員
UEDA Kouhei Yamaguchi University School of Medicine Hospital, Instructor, 医学部・附属病院, 医員
EMOTO Masahiro Yamaguchi University School of Medicine Hospital, Assistant professor, 医学部・附属病院, 助手 (50294640)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥8,600,000 (Direct Cost: ¥8,600,000)
Fiscal Year 1997: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1996: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Keywords | insuliln / glucose transport / PI3-kinase / gene transduction / GLUT4 / adenovirus vector / アデノウイルスベクター / グルコキナーゼ |
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| Research Abstract |
To elucidate the mechanisms whereby phosphatidylinositol (PI) 3-kinase is involved in insulin-stimulated glucose transport activity, the epitope-tagged p110alpha subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Overexpression of p110alpha was confirmed by immunoblot using anti-tagged epitope antibody. p110alpha overexpression induced an 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, which was greater than that observed in maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated. Overexpression of p110alpha induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control. No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110alpha-overexpressing 3T3-L1 adipocytes. Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110alpha-overexpressing cells. The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay. These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110alpha of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin. The importance of PI3-kinase activation for insulin-stimulated glucose transport was further supported by the results obtained using the dominant negative p85 subunit of PI3-kinase. When the dominant negative form of p85, of which inter-SH region of the wild type p85 was replaced by HA tag, was overexpressed into 3T3-L1 adipocytes, insulin-stimulated glucose transport was markedly inhibited with impairment of GLUT4 translocation.
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