A Screening System for Development of Novel Insulin Secretagogues

开发新型胰岛素分泌剂对糖尿病的治疗具有重要意义。磺脲类药物广泛用于治疗2型糖尿病。磺脲类药物通过抑制胰岛β细胞中atp敏感的K^+通道来刺激胰岛素分泌。我们最近发现β细胞的K_<ATP>通道由向内整流K^+通道亚基Kir6.2和磺酰脲受体亚基SURI组成，该亚基对磺酰脲类格列本脲具有高亲和力。我们还克隆了一个SUR1的异构体，称为SUR2A，它对格列本脲的亲和力较低。为了建立新型磺酰脲类药物及其衍生物的有效性筛选体系，我们确定了1)β细胞K_<ATP>通道的亚基化学计量学，2)磺酰脲类药物在SUR1中的结合位点；3)产生Kir6.2缺陷小鼠。利用SUR1与Kir6.2的融合蛋白，我们发现当SUR1亚基与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^+通道被认为是同源或异源四聚体，这表明β细胞K_<ATP>通道是由四个Kir6.2亚基和四个SUR1亚基组成的异源八聚体。在SUR1和SUR2A之间制备了多种嵌合体。我们检测了^3H标记的格列本脲与转染了这些嵌合体的COS-1细胞的结合，以及格列本脲对转染了每种嵌合体和Kir6.2的COS-1细胞^< 36b> Rb外排的影响。我们发现格列本脲的高亲和力结合位点位于SUR1.3跨膜段的第15和第16段之间。我们通过遗传破坏Kir6.2产生K_<ATP>通道缺陷小鼠，该通道形成K^+离子选择孔。纯合子小鼠（Kir6.2^<-/->）缺乏K_<ATP>通道活性。在K_<ATP>通道缺陷小鼠（Kir6.2^<-/->）中，通过胰岛的灌注和批量培养评估，没有发现葡萄糖或磺酰脲甲磺丁酰胺对胰岛素分泌的反应。我们的研究将为胰岛素分泌新药的开发提供有用的信息。少

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