Role of ER Stress in Beta Cell Apoptosis

描述（由申请人提供）：本提案的总体目标是更好地了解1型胰岛素依赖型糖尿病（IDDM）中引起α细胞凋亡的机制。尽管IDDM的自身免疫病因学已得到很好的证实，但α-细胞死亡信号传导机制仍不完全清楚。内质网（ER）的Ca 2+稳态的破坏诱导的应激引起细胞凋亡的MIN6细胞和小鼠胰岛。ER应激和α-细胞死亡之间的分子信号尚不清楚。内质网应激在胰岛素依赖型糖尿病中的作用尚未明确。内质网应激诱导的细胞凋亡可能与半胱氨酸蛋白酶家族caspase的激活和转录因子CHOP（C/EBP同源蛋白，也称为GADD 153）基因表达增加有关。初步的实验表明，肌内质网钙ATP酶（SERCA），一个关键的调节ER Ca 2+稳态，是受损的胰岛非肥胖糖尿病（NOD）小鼠，胰岛素依赖型糖尿病模型。这提出了一个有趣的可能性，即糖尿病中功能性α细胞质量的丧失与ER应激有关。拟议的实验将：[1]鉴定α-细胞中ER应激诱导的分子信号;[2]确定细胞因子是否诱导β-细胞ER应激;[3]定义NOD小鼠中ER应激与功能性α-细胞质量损失之间的关系。将测试以下假设：[1] ER应激通过半胱天冬酶和CHOP依赖性途径激活β细胞凋亡;[2] ER应激诱导神经源性细胞死亡信号;[3]细胞毒性细胞因子扰乱β细胞ER Ca2+稳态，激活半胱天冬酶并诱导CHOP表达;[4] NOD小鼠中糖尿病的发展与ER应激相关。将研究MIN6细胞和小鼠胰岛细胞。实验程序将包括可视化的亚细胞Ca2+浓度梯度与遗传靶向Ca2+生物传感器，半胱天冬酶活性的测量，和应用激光捕获显微切割和实时PCR监测SERCA和CHOP在胰岛中的表达。拟议的研究将为调节β细胞活力的机制提供新的见解，并促进开发新的治疗策略，以保护和维持I型糖尿病患者的功能性β细胞群。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to provide better insight into mechanisms that cause a-cell apoptosis in Type 1, insulin-dependent diabetes mellitus (IDDM). Although the autoimmune etiology of IDDM is well established, a-cell death signaling mechanisms are incompletely understood. Endoplasmic reticulum (ER) stress induced by disruption of ER Ca2+ homeostasis causes apoptosis in MIN6 cells and in mouse islets of Langerhans. The molecular signals linking ER stress and a-cell death are unresolved. The role of ER stress in IDDM has not been defined. Apoptosis induced by ER stress may be related to activation of caspases, a family of cysteine proteases, and increased expression of the transcription factor CHOP (C/EBP homologous protein also known as GADD153) gene. Preliminary experiments suggest that sarcoendoplasmic reticulum calcium ATPase (SERCA), a key regulator of ER Ca2+ homeostasis, is impaired in islets from non-obese diabetic (NOD) mice, a model of IDDM. This raises an intriguing possibility that loss of functional a-cell mass in diabetes is related to ER stress. The proposed experiments will: [1] identify molecular signals induced by ER stress in a-cells; [2] determine whether cytokines induce beta-cell ER stress; and [3] define the relationship between ER stress and loss of functional a-cell mass in NOD mice. The following hypotheses will be tested: [1] ER stress activates beta-cell apoptosis via caspase- and CHOP-dependent pathways; [2] ER stress induces mitochondrially-derived cell death signals; [3] cytotoxic cytokines perturb beta-cell ER Ca2+ homeostasis, activate caspases and induce CHOP expression; and [4] development of diabetes in NOD mice is correlated with ER stress. MIN6 cells and mouse islet cells will be studied. Experimental procedures will include visualization of subcellular Ca2+ concentration gradients with genetically targeted Ca2+ biosensors, measurements of caspase activity, and application of Laser Capture Microdissection and real-time PCR to monitor SERCA and CHOP expression in islets. The proposed studies will provide new insights into mechanisms regulating beta-cell viability and facilitate development of novel therapeutic strategies to preserve and maintain functional beta-cell mass in patients with Type I diabetes.