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A Screening System for Development of Novel Insulin Secretagogues

新型胰岛素促分泌剂开发的筛选系统

基本信息

批准号：
09557075
负责人：
SEINO Susumu
金额：
$ 7.62万
依托单位：
Chiba University Graduate School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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 … More 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

新型胰岛素促分泌剂的开发对于糖尿病的治疗具有重要意义。磺酰脲类药物广泛用于治疗2型糖尿病。磺酰脲类药物通过抑制胰腺 β 细胞中 ATP 敏感的 K^+ 通道来刺激胰岛素分泌。我们最近表明，β细胞K_<ATP>通道包含内向整流K^+通道亚基Kir6.2和磺酰脲类受体亚基SURI，其对磺酰脲类格列本脲表现出高亲和力。我们还克隆了 SUR1 的同种型，称为 SUR2A，它对格列本脲显示出低亲和力。为了开发新型磺酰脲类药物及其衍生物的有效性筛选系统，我们确定了 1) β 细胞 K_<ATP> 通道的亚基化学计量，2) SUR1 中的磺酰脲类结合位点； 3) 我们生成了 Kir6.2 缺陷小鼠。1)。通过使用 SUR1 和 Kir6.2 的融合蛋白，我们发现当 SUR1 亚基和 Kir6.2 亚基以 1:1 的摩尔比共表达时，K_<ATP> 通道的活性得到优化。由于内向整流 K^+ 通道被认为作为同源或异源四聚体发挥作用，这表明 β 细胞 K_<ATP> 通道的功能为由四个 Kir6.2 亚基和四个 SUR1 亚基组成的异八聚体。2)。制备了SUR1和SUR2A之间的各种嵌合体。我们检查了 3 H标记的格列本脲与用这些嵌合体转染的COS-1细胞的结合，以及格列本脲对来自用每种嵌合体和Kir6.2转染的COS-1细胞的 36 Rb流出的影响。我们发现格列本脲的高亲和力结合位点位于SUR1.3的第15和第16跨膜片段之间。我们通过对 Kir6.2 进行基因破坏，产生了 K_<ATP> 通道缺陷小鼠，Kir6.2 形成了通道的 K^+ 离子选择性孔。纯合小鼠(Kir6.2^-/->)缺乏K_<ATP>通道活性。通过胰岛的灌注和分批培养进行评估，在K_<ATP>通道缺陷型小鼠(Kir6.2^-/->)中没有发现响应于葡萄糖或磺酰脲类甲苯磺丁脲的显着胰岛素分泌。我们的研究应该为胰岛素分泌新药的开发提供有用的信息。较少的