NEW ANIMAL MODEL FOR EARLY ONSET NONIMMUNE DIABETES

长期目标是评估钙离子的变化 胰腺β细胞的动态平衡导致 出现起病极早的非免疫性糖尿病。转基因 已经产生了大鼠胰岛素II基因启动子的小鼠 已经连接到鸡钙调素迷你基因上。的表达 这种微型基因是产生胰岛素的β细胞所特有的， 导致钙调蛋白含量增加。钙调蛋白是一种 钙结合蛋白，存在于高浓度的 β细胞，并调节几种酶参与 葡萄糖介导的胰岛素分泌。年增长5倍 钙调蛋白出现在胚胎β细胞发育的早期， 导致糖尿病，在几个小时内变得明显 出生。这种疾病的特点是血糖升高， 胰腺和血清中胰岛素的降低以及渐进性 β细胞耗尽。除了胰岛素含量降低外， 晚期胚胎中的β细胞看起来正常，而2 日间新生儿明显异常。结果表明，过量 钙调蛋白干扰β细胞功能导致 糖尿病。糖尿病状态和过量的钙调蛋白有关 在β细胞毒性方面。我们建议完成对这两个问题的分析 现有转基因品系中的胎儿和新生儿胰腺。 分析将包括电子显微镜检查和 钙调蛋白和胰岛激素的免疫金标定位。 将分离胰岛并对其进行胰岛素检测 分泌对葡萄糖的反应，葡萄糖代谢，葡萄糖 敏感的K+通道、钙离子电导和血管内皮细胞的变化 细胞内钙离子。第二个目标是确定是否 糖尿病的具体原因是钙调蛋白升高或增加 CA++缓冲能力。我们将生成新的系列 转基因小鼠中的转基因基因将是一个突变体 正常结合钙但不能结合的钙调蛋白分子 激活钙调蛋白依赖的酶。血糖升高状态 将通过测量血糖来评估这些小鼠的 还有胰岛素。如果这些也是糖尿病患者，类似的实验 将对目前的转基因品系进行描述。 最终目标是解决正常水平的重要性 钙调蛋白对内分泌胰腺发育的影响 β细胞的增殖和分化功能。这 将利用转基因小鼠来评估问题，转基因小鼠表达 钙调蛋白反义RNA或钙调蛋白结合蛋白 在贝塔细胞中。其目的是降低钙调蛋白水平和 质疑这一削减的功能意义。 完成这些目标将提供对分子的洞察 可能导致具有相似特征的糖尿病的事件 在人类的IDDM和NIDDM中都存在。

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.