1995 Fiscal Year Final Research Report Summary
Mechanism of insulin action on pancreatic Alpha cells-gene analysis of insulin receptor-
| Project/Area Number |
06671043
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
内分泌・代謝学
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
KISHIKAWA Hideki Kumamoto University School of Medicine, Lecturer, 医学部, 講師 (30161441)
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| Co-Investigator(Kenkyū-buntansha) |
ARAKI Eiich Kumamoto University School of Medicine, Research Associate, 医学部, 助手 (10253733)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Pancreatic Alpha cell / In-R1-G9 cells / alpha TC clone 6 / Insulin receptor / Glucagon secretion |
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| Research Abstract |
In pancreatic Alpha cells, the existence and function of insulin receptor has not been fully studied yet. In this study, to confirm the expression of functional insulin receotor in pancreatic Alpha cells, we perfomed 1)insulin receptor binding assay, 2)Northern blot analysis and RT-PCR (reverse transcription-polymerase chain reaction) amplification of insulin recetor mRNA,3)immunocytochemical staining, 4)biosynthetical labelling of insulin receptor protein using [^<35>S] methionine, 5)the analysis of insulin-stimulated autophosphorylation of the insulin receptor in glucagon secreting cell lines ; In-R1-G9 and alpha TC clone 6 cells.
The glucagon secretion decreased by the addition of insulin in both cells. The receptor binding studies using[125I-Tyr-A14] insulin revealed that both cells possessed the significant number of insulin receptors (In-R1-G9 : K_1=2.1x10^9mol/l, K_2=6.2x10_7mol/l, R_1=0.27x10^4, R_2=1.86x10^4sites/cell ; alpha TC clone 6 : K_1=2.1x10^9mol/l, K_2=7.3x10^7mol/l, R_1=0.27x10^4, R_2=1.95x10^4sites/cell). Northern blot analysis as well as RT-PCR amplification showed the mRNA specific for insulin receptor in both cells. By immunocytochemical staining using antiinsulin receptor a subunit antibody, positive immunostainings for insulin receptor were observed in both cells. [^<35>S] methionine labelling of both cells followed by immunoprecipitation using antiinsulin receptor antibody showed correct size of insulin receptor protein. The insulin receptor expressed in these cells underwent autophosphorylation by insulin stimulation.
It is concluded that functional insulin receptors are properly expressed in In-R1-G9 and alpha TC clone 6 cells.
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