Mechanisms of Islet Beta Cell Dysfunction in Diabetes

描述（由申请人提供）： 鞘脂神经酰胺[CER]已被证明是导致多种细胞反应（包括细胞凋亡）的信号转导过程的重要介质。尽管有令人信服的实验证据表明，CER依赖性信号传导机制可能是胰岛素分泌受损的体外和体内模型中细胞功能障碍的基础，但关于CER在导致胰岛细胞代谢失调的信号传导事件中的确切作用模式知之甚少。我们的初步研究结果表明，INS 832/13细胞和原代大鼠胰岛长期暴露于升高的葡萄糖和脂质促进冈田酸敏感性蛋白磷酸酶[CAPP]和吞噬NADPH氧化酶[NOX]的CER依赖性激活，导致线粒体失调。我们还提出了初步证据，表明这两种途径在Zucker糖尿病脂肪[ZDF]大鼠（一种广泛接受的2型糖尿病模型）的胰岛中加速。基于这些数据，我们假设，细胞内CER的积累，诱导分离的细胞长期暴露于葡萄糖和脂质，导致线粒体功能障碍，导致细胞死亡。所提出的研究的三个具体目的是：[I]证明糖脂毒性条件促进CER介导的CAPP的线粒体同种型的活化，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]证明糖脂毒性条件促进CER-介导的全酶组装和NOX的功能活化，导致ROS的产生和胰岛细胞线粒体功能障碍的相关发作;和[III]精确定义ZDF大鼠胰岛中CER合成抑制剂对线粒体缺陷和代谢功能障碍[根据目的I和II确定]的进展和预防。我们将采用一些生物化学，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。希望来自所提出的研究的数据将提供对特定CER敏感性信号传导步骤在线粒体功能障碍发作中的调节作用的新见解，线粒体功能障碍导致胰岛细胞在糖脂毒性条件的胁迫下死亡。我们的长期目标是开发特定的治疗方法，以防止这些细胞缺陷的建立和糖尿病的发生。我们提出的研究与VA研究和患者护理任务直接相关。现有数据清楚地表明，退伍军人比一般人群更容易患糖尿病，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，并且随着年龄的增长而增加。在接受退伍军人管理局医疗保健的退伍军人中，平均年龄高于一般人群，这一比例超过20%。根据退伍军人事务部的数据，通过该部门获得健康福利的750万退伍军人中有70%肥胖，五分之一患有糖尿病，这可能导致心脏病、高血压和截肢。我们设想，从拟议的研究中获得的数据将提供新的见解特定的CER敏感的信号步骤中的线粒体功能障碍的发病，导致死亡的细胞的胁迫下的glucolipotoxic条件下的监管作用。从我们的研究中获得的数据可能成为开发特定治疗方法的基础，以防止这些细胞缺陷的建立和糖尿病的发病。 公共卫生相关性： 最近越来越多的证据表明，胰岛细胞长期暴露于升高的葡萄糖和脂质[即，糖脂毒性]导致严重的代谢损伤和功能活性细胞质量的损失。这些代谢异常背后的精确分子和细胞机制仍然只是部分了解。该提案旨在确定神经酰胺（一种鞘脂）在线粒体缺陷发展中的新作用，线粒体缺陷最终导致细胞死亡，导致2型糖尿病发作。该项目的长期目标之一是开发特定的治疗方法，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.