PATHOBIOLOGY OF BETA CELL DYSFUNCTION AND APOPTOSIS IN DIABETES MELLITUS

这个项目的总体目标是定义分子缺陷。 与糖尿病中的β细胞功能障碍相关的技术 分子生物学与整体器官和细胞生理学相结合。 将对隔离的灌流胰腺进行研究，以确定 药物对胰岛素振荡分泌的调节作用 它们影响胰岛素分泌途径中的特定部位，包括 三磷酸腺苷敏感钾通道和L钙通道。结果将是 与氨基酸诱导的胰岛素分泌相比。建筑的改动 分泌振荡及其对这些促分泌剂的反应将是 由自身免疫引起的糖尿病动物的胰腺中检测到的 β细胞破坏(DP BB/WOR大鼠)或β细胞质量减少(GK 大鼠)和大量输注葡萄糖以诱导β细胞的动物 功能障碍。这些实验将使我们能够检验这一假设 β细胞功能障碍的状态与低波幅有关， 不规则的分泌振荡。此外，我们建议研究 编码葡萄糖激酶的基因表达的变化，以及 在细胞内钙离子调节中起关键作用的两种蛋白质 信号转导，即电压依赖性钙通道和IP3受体。 结果将与GLUT2表达的变化进行比较 吉恩。这些研究将使我们能够检验糖尿病的假设 导致GLUT2、葡萄糖激酶或ATP敏感K+的异常 电压依赖性钙通道和IP3相对备用的通道 受体。最后，将进行实验以确定是否 正常胰岛存在振荡分泌胰岛素的模式持续存在 在糖尿病大鼠分离的胰岛中，分散的胰岛和胰岛素 分泌细胞系。作为模型β细胞系，BetaTC3细胞系具有 已被证明可以增强其对胰岛素的反应性 与高糖相比，在低糖中孵化。分子 高糖的这种影响的机制将在下面进行研究 这些细胞在高糖和低糖中孵育。电压的作用 依赖的钙、钾通道与IP3受体对心肌细胞的调节 该细胞系胰岛素分泌的研究将通过将正常 以及这些基因的突变形式和对胰岛素的影响 分泌性输出。这些研究将增加我们对正常的理解 IDDM的β细胞生理学和病理生理学，将促进 开发新的药理和其他治疗方法 最大限度地提高我们保存和维持贝塔细胞功能的能力 早期IDDM患者和胰岛细胞移植受者。

The overall goal of this project is to define the molecular defects associated with the beta cell dysfunction in diabetes using techniques of molecular biology combined with whole organ and cellular physiology. Studies will be conducted with the isolated perfused pancreas to define the regulation of oscillatory insulin secretion using pharmacologic agents which affect specific sites in the insulin secretory pathway including the ATP-sensitive K+ channel and the L-type Ca2+ channel. The results will be compared with amino acid induced insulin secretion. Alterations in the secretory oscillations and their response to these secretagogues will be determined in pancreata from animals with diabetes due either to autoimmune beta cell destruction (DP BB/Wor rat) or reduced beta cell mass (the GK rat) and animals infused with large amounts of glucose to induce beta cell dysfunction. These experiments will enable us to test the hypothesis that states of beta cell dysfunction are associated with low amplitude, irregular secretory oscillations. In addition we propose to examine alterations in expression of the genes which encode for glucokinase, and two proteins which play a key role in the regulation of intracellular Ca2+ signalling, i.e., the voltage dependent Ca2+-channel and the IP3 receptor. The results will be compared with alterations in expression of the GLUT2 gene. These studies will allow us to test the hypothesis that diabetes causes abnormalities in either GLUT2, glucokinase or the ATP-sensitive K+ channel with relative sparing of the voltage dependent Ca2+ channel and IP3 receptor. Finally, experiments will be performed to determine if the pattern of oscillatory insulin secretion present in normal islets persists in islets isolated from diabetic rats, dispersed islets and in insulin secreting cell lines. A model beta cell line, the betaTC3 cell line has been demonstrated to increase its responsiveness to insulin after incubation in low glucose when compared to high glucose. The molecular mechanisms underlying this effect of high glucose will be studied following incubation of these cells in high and low glucose. The role of voltage dependent Ca2+ and K+ channels and the IP3 receptor in the regulation of insulin secretion in this cell line will be studied by transfecting normal and mutant forms of these genes and characterizing the effects on insulin secretory output. These studies will increase our understanding of normal beta cell physiology and pathophysiology in IDDM and will facilitate the development of novel pharmacologic and other therapeutic approaches to maximize our ability to preserve and maintain beta cell function in patients with early IDDM and in islet cell transplant recipients.