1998 Fiscal Year Final Research Report Summary
A Screening System for Development of Novel Insulin Secretagogues
| Project/Area Number |
09557075
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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 | 展開研究 |
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| Research Field |
内分泌・代謝学
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| Research Institution | Chiba University Graduate School of Medicine |
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Principal Investigator |
SEINO Susumu Chiba University, Graduate School of Medicine, Professor, 大学院・医学研究科, 教授 (80236067)
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| Co-Investigator(Kenkyū-buntansha) |
WATANABE Toshifumi Takeda Chemical Industries, LTD.Pharmaceutical Research Division, Research Fello, 創薬研究本部, 主任研究員
GONOI Tohru Chiba University, Research Center for Pathogenic Fungi, and Microbial Toxicoses,, 真菌医学研究センター, 助手 (30134365)
MIKI Takashi Chiba University, Graduate School of Medicine, Assistant, 大学院・医学研究科, 助手 (50302568)
YANO Hideki Chiba University, Graduate School of Medicine, Associato Professor, 大学院・医学研究科, 教授 (30288576)
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| Project Period (FY) |
1997 – 1998
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| Keywords | ATP-sensitive K^+ channels / Insulin / sulfonvlurea receptors / inward rectfier / glibenclamide / diabetes mellitus |
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| Research Abstract |
Development of novel insulin secretagogues is important for the treatment of diabetes mellitus. Sulfonylureas are widely used in the treatment of type 2 diabetes. Sulfonylureas stimulate insulin secretion by inhibiting ATP-sensitive K^+ channels in pancreatic beta-cells. We have recently shown that the beta-cell K_<ATP> channel comprises the inward rectfier K^+ channel subunit Kir6.2 and the sulfonylurea receptor subunit SURI which shows high affinity for the sulfonylurea glibenclamide. We also cloned an isoform of SUR1, called SUR2A which shows low affinity for glibenclamide. To develop screening system of the effectiveness of novel sufonylureas and their derivatives, we have determined 1) the subunit stoichiometry of the beta-cell K_<ATP> channel, 2) sulfonylurea binding sites in the SUR1 ; and 3) we generated Kir6.2 deficient mice.
1). By using fusion proteins of SUR1 and Kir6.2, we found that the activity of K_<ATP> channels is optimized when the the SUR1 subunit and the Kir6.2 subu
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nit are coexpressed with a molar ratio of 1 : 1. Since inward rectifier K^+ channels are thought to function as homo- or hetero-tetramers, this suggests that the beta-cell K_<ATP> channel functions as a hetero- octamer composed of four Kir6.2 subunits and four SUR1 subunits.
2). Various chimeras between SUR1 and SUR2A were prepared. We examined ^3H labeled glibenclamide binding to COS-1 cells transfected with these chimeras and also the effect of glibenclamide on ^<36>Rb efflux from COS-1 cells transfected with each chimera and Kir6.2. We found that high affinity binding site for glibenclamide locates between 15 th and 16th transmembrane segement of SUR1.
3). We generated K_<ATP> channel-deficient mice by genetic disruption of Kir6.2, which forms the K^+ ion-selective pore of the channel. The homozygous mice (Kir6.2^<-/->) lack K_<ATP> channel activity. No significant insulin secretion in response to either glucose or the sulfonylurea tolbutamide was found in K_<ATP> channel-deficient mice (Kir6.2^<-/->), as assessed by perifusion and batch incubation of pancreatic islets. Our studies should provide useful information for the development of new drugs for insulin secretion. Less
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