Mechanisms of Islet Beta Cell Dysfunction in Diabetes

描述(由申请人提供)： 鞘磷脂神经酰胺[CER]已被证明是导致包括细胞凋亡在内的各种细胞反应的信号转导过程的重要中介。尽管有令人信服的实验证据表明，在胰岛素分泌受损的体外和体内模型中，CER依赖的信号机制可能是细胞功能障碍的基础，但对于CER在导致胰岛细胞代谢失调的信号事件中的确切作用模式，人们知之甚少。我们的初步发现表明，INS 832/13细胞和原代大鼠胰岛长期暴露于高糖和高脂促进CER依赖的冈田酸敏感蛋白磷酸酶[CAPP]和吞噬细胞NADPH-氧化酶[NOX]的激活，导致线粒体失调。我们还提供了初步证据表明，这两个途径在Zucker糖尿病肥胖[ZDF]大鼠的胰岛中加速，Zucker糖尿病肥胖[ZDF]大鼠是一种被广泛接受的2型糖尿病模型。基于这些数据，我们假设，在分离的细胞长期暴露于葡萄糖和脂肪后，细胞内CER的积累导致线粒体功能障碍，导致细胞死亡。拟议研究的三个具体目的是：[i]证明糖毒条件促进CER介导的CAPP线粒体异构体的激活，从而导致Bcl-2的去磷酸化和失活，最终导致胰岛细胞的线粒体功能障碍；[ii]证明糖毒条件促进CER介导的全酶组装和NOX的功能激活，从而导致ROS的产生和相关胰岛细胞线粒体功能障碍的发生；以及[iii]精确地确定CER合成抑制剂在ZDF大鼠胰岛[根据AIMS I和II鉴定]的进展和预防线粒体缺陷和代谢功能障碍。我们将使用许多生化、分子生物学、细胞生物学和免疫学方法来验证我们的假设，并在INS 832/13细胞、原代大鼠胰岛和整个动物中实现我们的目标。希望从这些拟议的研究中获得的数据将为特定的CER敏感信号步骤在线粒体功能障碍的发生中的调控作用提供新的见解，从而导致胰岛细胞在糖毒胁迫下的死亡。我们的长期目标是开发特定的治疗方式，以防止这些细胞缺陷的建立和糖尿病的发生。我们建议的研究与退伍军人管理局的研究和患者护理任务直接相关。现有数据清楚地表明，退伍军人比普通民众更有可能患有糖尿病，这是与肥胖有关的主要并发症之一。根据美国糖尿病协会的数据，超过7%的美国人口患有糖尿病，而且随着年龄的增长，这一比例会增加。在接受退伍军人医疗保健的退伍军人中，他们的平均年龄高于普通人群，这一比例超过20%。根据退伍军人管理局的数据，通过该部门领取医疗福利的750万退伍军人中，70%患有肥胖症，五分之一的退伍军人患有糖尿病，糖尿病可能导致心脏病、高血压和截肢。我们预计，从拟议的研究中获得的数据将为特定的CER敏感信号步骤在线粒体功能障碍的发生中的调控作用提供新的见解，从而导致细胞在糖毒条件下死亡。从我们的研究中获得的数据可能会成为开发特定治疗方式的基础，以防止这些细胞缺陷的建立和糖尿病的发生。 公共卫生相关性： 项目简介越来越多的最新证据表明，胰岛细胞长期暴露于高血糖和高血脂[即糖脂毒性]会导致严重的代谢损伤和功能活跃的细胞团的丧失。这些代谢异常背后的确切分子和细胞机制仍然只有部分了解。这项建议旨在定义神经酰胺，一种鞘磷脂，在线粒体缺陷的发展中所扮演的新角色，最终导致细胞死亡，导致2型糖尿病的发病。该项目的长期目标之一是开发特定的治疗方式，以防止这些细胞缺陷的建立和糖尿病的发病。

DESCRIPTION (provided by applicant): Project Summary The sphingolipid ceramide [CER] has been shown to be an important mediator of signal transduction processes leading to a variety of cellular responses, including apoptosis. Despite the compelling experimental evidence to suggest that CER-dependent signaling mechanisms might underlie -cell dysfunction in in vitro and in vivo models of impaired insulin secretion, very little is known with regard to the precise modes of action of CER in the signaling events leading to metabolic dysregulation of the islet -cell. Our preliminary findings suggest that long-term exposure of INS 832/13 cells and primary rat islets to elevated glucose and lipids promote CER-dependent activation of an okadaic acid-sensitive protein phosphatase [CAPP] and the phagocytic NADPH-oxidase [NOX] leading to mitochondrial dysregulation. We also present preliminary evidence to indicate that these two pathways are accelerated in islets from the Zucker Diabetic Fatty [ZDF] rat, a widely accepted model for type 2 diabetes. Based on these data we hypothesize that an accumulation of intracellular CER, induced following chronic exposure of isolated -cells to glucose and lipids, causes mitochondrial dysfunction leading to cell demise. The three Specific Aims of the proposed studies are: [I] to demonstrate that glucolipotoxic conditions promote CER-mediated activation of the mitochondrial isoform of CAPP leading to dephosphorylation and inactivation of Bcl-2 culminating in the mitochondrial dysfunction of the islet -cell; [II] to demonstrate that glucolipotoxic conditions promote CER-mediated holoenzyme assembly and functional activation of NOX to result in the generation of ROS and the associated onset of mitochondrial dysfunction of the islet -cell; and [III] to precisely define the progression, and prevention of mitochondrial defects and metabolic dysfunction [identified under Aims I and II] by CER synthesis inhibitors in the ZDF rat islet. We will employ a number of biochemical, molecular biological, cell biological and immunological approaches to validate our hypothesis and accomplish our goals in INS 832/13 cells, primary rat islets and whole animals. It is hoped that data derived from the proposed studies will provide fresh insights into the regulatory roles of specific CER-sensitive signaling steps in the onset of mitochondrial dysfunction leading to the demise of the islet -cell under the duress of glucolipotoxic conditions. Our long-term goal is to develop specific therapeutic modalities to prevent the establishment of these cell defects and the onset of diabetes. Our proposed studies have direct relevance to the VA research and patient care missions. Available data clearly suggest that veterans are more likely than the general population to have diabetes, one of the major complications associated with obesity. According to the American Diabetes Association, greater than 7% of the U.S. population has diabetes, and the rate increases with age. Among veterans receiving VA health care, who are on average older than the general population, the rate is greater than 20%. According to the VA, 70% of the 7.5 million veterans who receive health benefits through the department are obese, and one in five has diabetes, which can lead to heart disease, high blood pressure and amputations. We envision that data derived from the proposed studies will provide fresh insights into regulatory roles of specific CER-sensitive signaling steps in the onset of mitochondrial dysfunction leading to the demise of the -cell under the duress of glucolipotoxic conditions. The data accrued from our studies might form the basis for the development of specific therapeutic modalities to prevent the establishment of these -cell defects and the onset of diabetes. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE A growing body of recent evidence suggests that long-term exposure of islet -cells to elevated glucose and lipids [i.e., glucolipotoxicity] results in severe metabolic impairment and loss of functionally-active -cell mass. The precise molecular and cellular mechanisms underlying these metabolic abnormalities remain only partially understood. This proposal aims at defining novel roles for ceramide, a sphingolipid, in the development of mitochondrial defects culminating in the demise of the -cell leading to the onset of type 2 diabetes. One of the long-term goals of this project is to develop specific therapeutic modalities to prevent the establishment of these -cell defects and the onset of diabetes.