PATHOBIOLOGY OF BETA CELL DYSFUNCTION AND APOPTOSIS IN DIABETES MELLITUS

这个项目的总体目标是确定分子缺陷 与糖尿病中β细胞功能障碍相关的研究， 分子生物学结合整体器官和细胞生理学。 将用离体灌注胰腺进行研究，以确定 使用药理学试剂调节振荡胰岛素分泌 其影响胰岛素分泌途径中的特定位点， ATP敏感性K+通道和L型Ca 2+通道。 结果将 与氨基酸诱导的胰岛素分泌相比。 的改变 分泌振荡和它们对这些促分泌素的反应将是 在患有糖尿病的动物的胰腺中测定，糖尿病是由于自身免疫性疾病 β细胞破坏（DP BB/Wor大鼠）或β细胞群减少（GK 大鼠）和输注大量葡萄糖以诱导β细胞的动物 功能障碍 这些实验将使我们能够验证这样一个假设， β细胞功能障碍的状态与低幅度有关， 不规则分泌振荡 此外，我们建议研究 编码葡糖激酶的基因表达的改变，和 这两种蛋白质在调节细胞内Ca 2+中起关键作用 信令，即，电压依赖性钙通道和IP 3受体。 将结果与GLUT 2表达的改变进行比较。 基因 这些研究将使我们能够验证糖尿病 导致GLUT 2、葡萄糖激酶或ATP敏感性K+ 相对保留电压依赖性Ca 2+通道和IP 3通道 受体的 最后，将进行实验以确定 正常胰岛中存在的振荡胰岛素分泌模式持续存在 在从糖尿病大鼠分离的胰岛、分散的胰岛和胰岛素中， 分泌细胞系。 一种模型β细胞系，β TC 3细胞系具有 已被证明可以增加其对胰岛素的反应性， 当与高葡萄糖相比时，在低葡萄糖中孵育。 分子 下面将研究高葡萄糖这种效应的潜在机制 将这些细胞在高和低葡萄糖中孵育。 电压的作用 依赖性Ca ~（2+）和K ~+通道及IP 3受体在调节 将通过检测正常人的胰岛素分泌来研究该细胞系中的胰岛素分泌。 以及这些基因的突变形式，并表征对胰岛素的影响， 分泌量 这些研究将增加我们对正常 β细胞生理学和病理生理学，并将促进 开发新的药理学和其他治疗方法， 最大限度地保护和维持β细胞功能， 早期胰岛素依赖型糖尿病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.