GLUCOSE/SECRETAGOGUE METABOLISM IN PANCREATIC ISLETS

这个项目的长期目标是探索新陈代谢是如何 葡萄糖和其他促分泌剂刺激胰岛素分泌。这个 这项提案中概述的研究将扩大我们的观察范围 线粒体甘油磷酸脱氢酶(MGPD)参与 通过甘油磷酸穿梭在NADH的再氧化中产生 糖酵解，在胰腺的β细胞中比在其他细胞中更丰富 纸巾。这表明磷酸甘油穿梭是重要的。 用于胰岛素分泌。支持这一观点的是最近的报道， 非胰岛素啮齿动物模型胰岛MGPD活性低 依赖型糖尿病和几名患有这种疾病的人。我们 最近克隆了大鼠MGPD的cDNA，并发现推导出的序列 包含一个与钙结合位点同源的区域 钙调素。这可能解释了众所周知的MGPD和MGPD的钙激活 提示钙直接与MGPD相互作用以激活它 葡萄糖刺激。我们已经分离了大鼠胰岛和人MGPD的cDNA和 人类基因组克隆，并产生了一种有效的MGPD抗体，以便 研究MGPD在胰岛素释放和糖尿病中的作用。因为没有 已知的有效的MGPD特异性抑制剂，我们将尝试直接获得 有证据表明磷酸甘油穿梭对胰岛素很重要 用核酶降低MGPD水平的实验分泌 β细胞系中的反义策略。转基因小鼠的体重减少 胰岛素原核注射将产生β细胞MGPD 启动子-MGPD-核酶构建。将通过以下方式生成MGPD空鼠标 有针对性的基因破坏。葡萄糖诱导的胰岛素分泌将是 与不需要穿梭活性的化合物相比。 MGPD酶活性、蛋白质和信使核糖核酸水平以及甘油磷酸 航天飞机的活动将被测量。描述了克隆和克隆的实验 MGPD基因的特征，包括定位内含子-外显子剪接连接 并寻找组织(胰岛)特异的cDNA。MGPD基因增加 通过甲状腺激素在其活性较低的组织中，如肝脏， 而在组织中，如胰岛，MGPD非常 充足时，甲状腺激素不起作用。启动子研究将调查 在胰岛中这种明显高水平的结构性表达也是如此 作为甲状腺激素在肝脏中的反应性。的重要元素 启动子和5‘侧翼结构域将被确定和交易因子 将对与这些区域相互作用的区域进行研究。研究也将是 对MGPD蛋白的功能位点进行体外研究 FAD位点、钙结合位点和推定基因的突变 甘油磷酸结合结构域。MGPD在中国的合作研究 非胰岛素依赖型糖尿病家系及其动物模型研究 疾病将会继续下去。从这些研究中获得的信息将 增加对葡萄糖刺激的胰岛素释放和非胰岛素释放的认识 胰岛素依赖型糖尿病。

The long term objective of this project is to explore how the metabolism of glucose and other secretagogues stimulates insulin secretion. The studies outlined in this proposal will expand on our observation that mitochondrial glycerol phosphate dehydrogenase (mGPD), which participates via the glycerol phosphate shuttle in the reoxidation of NADH produced by glycolysis, is more abundant in the pancreatic beta cell than in other tissues. This suggests that the glycerol phosphate shuttle is important for insulin secretion. In support of this idea are recent reports that mGPD activity is low in pancreatic islets in rodent models of non-insulin dependent diabetes mellitus and in several humans with this disease. We recently cloned the rat mGPD cDNA and found that the deduced sequence contains a region with homology to the calcium-binding sites of calmodulin. This may explain the long-known calcium activation of mGPD and suggests that calcium interacts directly with mGPD to activate it during glucose stimulation. We have isolated rat islet and human mGPD cDNAs and human genomic clones, and have produced a potent mGPD antibody in order to examine the role of mGPD in insulin release and diabetes. As there are no known potent specific inhibitors of mGPD, we will attempt to obtain direct evidence that the glycerol phosphate shuttle is important for insulin secretion by experiments designed to reduce mGPD levels with ribozyme and antisense strategies in beta cell lines. Transgenic mice with decreased beta cell mGPD will be produced by pronuclear injection of an insulin promoter-mGPD-ribozyme construct. An mGPD null mouse will be produced by targeted gene disruption. Insulin secretion induced by glucose will be compared with that induced by compounds not requiring shuttle activity. mGPD enzyme activity, protein, and mRNA levels, and glycerol phosphate shuttle activity will be measured. Experiments are described to clone and characterize the mGPD gene, including mapping intron-exon splice junctions and looking for tissue (islet) specific cDNAs. The mGPD gene is increased by thyroid hormone in tissues where its activity is low, such as liver, whereas in tissues, such as the pancreatic islet, where mGPD is extremely abundant, thyroid hormone has no effect. Promoter studies will investigate this apparent high level of constitutive expression in the islet, as well as the thyroid hormone responsiveness in liver. Important elements in the promoter and 5' flanking domain will be identified and transacting factors that interact with these regions will be studied. Research will also be undertaken to study the functional sites in the mGPD protein by in vitro mutagenesis of the FAD site, the calcium binding site and the putative glycerol phosphate binding domain. Collaborative studies of mGPD in families with non-insulin dependent diabetes and in animal models of this disease will be continued. Information gained from these studies will increase the understanding of glucose-stimulated insulin release and non- insulin dependent diabetes.