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素刺激胰岛素分泌。的 本提案中概述的研究将扩大我们的观察， 线粒体甘油磷酸脱氢酶（mGPD），参与 通过磷酸甘油穿梭在由 糖酵解，在胰腺β细胞中比其他细胞中更丰富 组织中这表明磷酸甘油穿梭是重要的 胰岛素分泌。支持这一观点的是最近的报告， 在非胰岛素的啮齿动物模型中，胰岛中的mGPD活性较低 依赖性糖尿病和几个患有这种疾病的人。我们 最近克隆了大鼠mGPD cDNA，发现推导的序列 含有与以下的钙结合位点同源的区域： 钙调素这可以解释mGPD的钙激活， 这表明钙直接与mGPD相互作用，以激活它， 葡萄糖刺激我们分离了大鼠胰岛和人mGPD cDNA， 人类基因组克隆，并已产生有效的mGPD抗体，以 研究mGPD在胰岛素释放和糖尿病中的作用。由于没有 已知有效的mGPD特异性抑制剂，我们将尝试获得直接的 磷酸甘油穿梭对胰岛素很重要的证据 通过设计用核酶降低mGPD水平的实验和 β细胞系中的反义策略。转基因小鼠 β细胞mGPD将通过原核注射胰岛素产生 启动子-mGPD-核酶构建体。将通过以下方法产生mGPD无效小鼠： 定向基因破坏由葡萄糖诱导的胰岛素分泌将 与不需要穿梭活性的化合物诱导的相比。 mGPD酶活性、蛋白质和mRNA水平以及甘油磷酸 将测量航天飞机活动。实验描述了克隆和 表征mGPD基因，包括定位内含子-外显子剪接点 并寻找组织（胰岛）特异性cDNA。mGPD基因增加 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，其中mGPD是非常 甲状腺激素丰富，则无影响。启动子研究将调查 胰岛中这种明显的高水平组成型表达， 与肝脏中的甲状腺激素反应性一样。的重要因素 启动子和5'侧翼结构域将被鉴定， 将研究与这些区域相互作用的机制。研究还将 通过体外研究mGPD蛋白中的功能位点， 突变的FAD位点，钙结合位点和推定的 甘油磷酸结合域。mGPD的合作研究， 非胰岛素依赖型糖尿病家庭和动物模型 疾病将继续。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理解。 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.