1997 Fiscal Year Final Research Report Summary
Analysis of Mechanizm of Glucose Responsive Human Insulin Gene Expression.
| Project/Area Number |
08671174
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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 |
内分泌・代謝学
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
SHIROTANI Tetsuya KUMAMOTO UNIVERSITY HOSPITAL,RESEARCH ASSOCIATE, 医学部・附属病院, 助手 (30274715)
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| Co-Investigator(Kenkyū-buntansha) |
MIYAMURA Nobuhiro KUMAMOTO UNIVERSITY HOSPITAL,RESEARCH ASSOCIATE, 医学部・附属病院, 助手 (40274716)
ARAKI Eiichi KUMAMOTO UNIVERSITY HOSPITAL,LECTURER, 医学部・附属病院, 講師 (10253733)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Insulin gene promoter / Pancreatic and duodenal homeobox gene-1 (PDX-1) / A3 element / protein kinase C / trans acting factor / Electrophoretic mobility shift assay (EMSA) |
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| Research Abstract |
The results are summarized as follows.
1.Analysis of glucose responsive element in human insulin gene promoter : In CAT assay using deletion mutants of the human insulin gene promoter, the region -230 to -201, which contained A3 element, revealed to be important for the flucose responsive expression.
2.Analysis of trans-acting factor which bound to glucose responsive element : EMSA revealed that two nuclear proteins from MIN6 cells bound to A3 element in a glucose dependent manner. The molecular weights of the nuclear proeins were 36 and 48 kDa, respecively, and gel supershift analysis confirmed that both of them bound to anti-PDX-1 antiserum. These results indicated that the 48 kDa protein was PDX-1, and the 36 kDa protein might be degradated PDX-1 or its homologue.
Analysis of PDX-1 : The binding activity of PDX-1 reduced significantly by potato acid phosphatase treatment, suggesting that DNA binding activity of PDX-1 was affected by its phosphorylation status. Western blot analysis using anti-phosphopeptide antibodies demonstrated that phosphorylation of threonine in PDX-1 was increased with high glucose concentration. In in vitro experiment, PDX-1 was phosphorylated by PKC.
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