NEW ANIMAL MODEL FOR EARLY ONSET NONIMMUNE DIABETES

长期目标是评估钙的变化 胰腺β细胞内的稳态导致 非免疫性糖尿病的早期发病。 转基因 已经产生了小鼠，其中大鼠胰岛素II基因启动子 与鸡的钙调素小基因相连 表达 这种小基因对产生胰岛素的β细胞是特异性的， 导致钙调素含量增加。 钙调素是一种 钙结合蛋白，以高浓度存在于 β细胞，并用于调节参与 葡萄糖介导的胰岛素分泌。 增加5倍， 钙调蛋白在胚胎β细胞发育早期出现， 导致糖尿病，在几个小时后变得明显 出生 这种疾病的特征是血糖升高， 胰腺和血清中的胰岛素减少， 消耗β细胞。 除了胰岛素含量降低， 晚期胚胎的β细胞表现正常，而2 日龄新生儿明显异常。 结果表明，过量 钙调素干扰β细胞功能， 糖尿病 糖尿病状态和过量的钙调素参与 β细胞毒性 我们建议完成对两者的分析 胎儿和新生儿胰腺中存在的转基因品系。 分析将包括电子显微镜检查和 钙调素和胰岛激素的免疫金定位。 将分离胰岛并评价胰岛素 葡萄糖反应分泌，葡萄糖代谢，葡萄糖 敏感性K+通道、Ca ++电导和 胞内Ca ++。 第二个目标是确定是否 糖尿病特别是由于钙调素升高或增加 Ca ++缓冲能力 我们将生产新的 转基因小鼠，其中转基因将是突变体 一种钙调素分子，通常与钙结合，但不能与 或激活钙调素依赖酶。 血糖状态 将通过测量血糖来评估这些小鼠的 和胰岛素。 如果这些也是糖尿病患者，类似的实验， 将对当前的转基因系进行所述的方法。 最后一个目标是解决正常水平的重要性， 钙调素对内分泌胰腺发育的影响， β细胞的增殖和分化功能。 这 问题将利用转基因小鼠进行评估， 钙调蛋白反义RNA或钙调蛋白结合蛋白 在β细胞中。 目的是降低钙调蛋白水平， 质疑这种减少的功能意义。 这些目标的完成将提供对分子生物学的深入了解。 可能导致糖尿病的特征类似于 与人的胰岛素依赖型糖尿病和非胰岛素依赖型糖尿病中存在的蛋白质结合。

The long term objective is to evaluate how changes in calcium homeostasis in the beta cell of the pancreas results in development of very early onset non-immune diabetes. Transgenic mice have been produced in which the rat insulin II gene promotor has been ligated to a chicken calmodulin minigene. Expression of this minigene is specific to the insulin-producing beta cells and results in an increased calmodulin content. Calmodulin is a calcium binding protein that is present at high concentration in beta cells and serves to regulate several enzymes involved in glucose-mediated insulin secretion. The 5X increase in calmodulin occurs early in embryonic beta cell development and results in diabetes that becomes evident within a few hours after birth. The disease is characterized by increased blood glucose, decreased insulin in both pancreas and serum as well as gradual depletion of beta cells. Except for reduced insulin content, beta cells in the late embryo appear normal whereas those of 2 day neonates are markedly abnormal. Results suggest that excess calmodulin interferes with beta cell function leading to diabetes. The diabetic state and excess calmodulin are involved in beta cell toxicity. We propose to complete analysis of both fetal and neonatal pancreas in the existing transgenic lines. Analysis will include electron microscopic examination and immunogold localization of calmodulin and the islet hormones. Pancreatic islets will be isolated and evaluated for insulin secretion in response to glucose, glucose metabolism, glucose sensitive K+ channels, Ca++ conductance and changes in intracellular Ca++. The second aim is to determine whether diabetes is specifically due to elevated calmodulin or increased Ca++ buffering capacity. We will generate new lines of transgenic mice in which the transgene will be a mutant calmodulin molecule that binds calcium normally but cannot bind to or activate calmodulin-dependent enzymes. The glycemic state of these mice will be evaluated by measurement of blood glucose and insulin. If these are also diabetic, similar experiments as described for the current transgenic lines will be performed. The final aim is to address the importance of normal levels of calmodulin on development of the endocrine pancreas and proliferation and differentiated function of beta cells. This problem will be evaluated utilizing transgenic mice that express either a calmodulin antisense RNA or a calmodulin binding protein in beta cells. The purpose is to decrease calmodulin levels and question the functional significance of this reduction. Completion of these aims will provide insight into molecular events that could produce diabetes with characteristics similar to those present in both IDDM and NIDDM in humans.