CALCIUM-INDEPENDENT PLA2BETA IN BETA-CELL APOPTOSIS

β 细胞凋亡中钙独立的 PLA2BETA

• 批准号: 7783955
• 负责人: SASANKA RAMANADHAM
• 金额: $ 3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7783955
• 关键词: Address; Affect; Apoptosis; Apoptotic; Autoimmune Diabetes; Autoimmune Process; Autoimmunity; Beta Cell; Biological Assay; Biology; Calcium; Cell Death; Cell physiology; Cells; Ceramides; Cleaved cell; Confocal Microscopy; Dependence; Development; Diabetes Mellitus; Enzymes; Event; Evolution; Exhibits; Flow Cytometry; Functional disorder; Future; Generations; Genetic; Glucose; Goals; Health; Human; Hydrolysis; Hyperglycemia; Immunoblotting; Inbred NOD Mice; Insulin-Dependent Diabetes Mellitus; Knockout Mice; Knowledge; Laboratories; Lead; Lipids; Mass Spectrum Analysis; Mediating; Mitochondria; Modeling; Molecular Biology; Mus; Nitric Oxide; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Organelles; Outcome; Pathway interactions; Phospholipase A2; Predisposition; Process; Proteins; Protocols documentation; Radiolabeled; Resistance; Role; SRE-1 binding protein; Spectrometry, Mass, Electrospray Ionization; Sphingomyelinase; Sphingomyelins; Staining method; Stains; Stimulus; Stress; Testing; Therapeutic Intervention; Transgenic Organisms; Work; caspase-3; cytokine; design; diabetic; in vivo; islet; lipid mediator; mouse model; prevent; public health relevance; radiotracer; response; tool

DESCRIPTION (provided by applicant): ¿-cell apoptosis contributes to loss of ¿-cells and decreases in ¿-cell function in both types 1 and 2 diabetes mellitus. It is therefore important to understand the mechanisms underlying ¿-cell apoptosis if this process is to be prevented or delayed. Our hypothesis in the 1st project period (10/04-7/09) was that the group VIA Ca2+-independent phospholipase A2 (iPLA2¿) participates in ER stress-induced ¿-cell apoptosis. We observed that (a) ER stress promotes accumulation and activity of iPLA2¿ in the ER and mitochondria, (b) iPLA2¿ activation during ER stress increases ceramide generation via neutral sphingomyelinase (NSMase)-catalyzed hydrolysis of sphingomyelins triggering the mitochondrial apoptotic pathway and ¿-cell apoptosis, (c) these outcomes are suppressed by inhibition of iPLA2¿ or NSMase, (d) iPLA2¿-null islets are less and iPLA2¿-transgenic (Tg) islets more sensitive to ER stress, (e) NSMase expression is unaffected in iPLA2¿-null islets and amplified in iPLA2¿-Tg islets, and (f) ¿-cell iPLA2¿ and NSMase messages are higher in the Akita mouse model of spontaneous ¿-cell ER stress that leads to diabetes. We also find that iPLA2¿ participates in ¿-cell apoptosis induced by hyperglycemia and cytokines and that islet iPLA2¿ and NSMase messages are elevated in pre-diabetic NOD mice. We hypothesize that iPLA2¿-mediated ceramide generation and triggering of mitochondrial abnormalities are critical contributory events to ¿-cell apoptosis. The studies proposed herein are designed to elucidate the precise role of iPLA2¿ in this process and whether this pathway, activated by ER stress, is also elicited by hyperglycemia and cytokines. Our Aims will address the following: Aim 1, role of iPLA2¿ activation and iPLA2¿-derived lipid mediators. Hyperglycemia and cytokines promote ¿-cell apoptosis, in part, by inducing ER stress and the role of iPLA2¿ and generation of iPLA2¿-derived lipid mediators in this process will be assessed; Aim 2, induction of iPLA2¿ and ceramide-generating pathway. SREBP-1 and CaMKII¿ are known to affect iPLA2¿ and are activated during ER stress. Their roles in iPLA2¿ induction and the requirement of iPLA2¿ activity for ceramide generation will be examined; Aim 3, requirement of iPLA2¿ mobilization and activation to trigger mitochondrial abnormalities. The affects of genetic modulation, mobilization, and organelle-specific expression of iPLA2¿ on the mitochondrial apoptotic pathway will be examined; Aim 4, sensitivity to ER stress following in vivo modulation of iPLA2¿. A role of iPLA2¿ in modulating ER stress in ¿-cells will be tested by crossing iPLA2¿-null with ER stress-sensitive Akita mice and iPLA2¿-Tg with ER stress-resistant CHOP-null mice; and Aim 5, contribution of iPLA2¿ to ¿-cell apoptosis during the evolution of autoimmune DM. The dependence of cytokine-induced ¿-cell apoptosis on an iPLA2¿/nitric oxide (NO)-dependent pathway and the contribution of iPLA2¿ to ¿-cell apoptosis in NOD mice will be assessed. These Aims will be addressed using apoptosis, flow cytometry, immunoblotting, qRT-PCR, enzymatic activity assay, confocal microscopy, molecular biology, and mass spectrometry protocols. Findings from our studies will lead to increased understanding of iPLA2¿ biology in the ¿-cell. The long-range goal of our laboratory is to assess the role of iPLA2¿ in ¿-cell apoptosis during the development and progression of diabetes mellitus. PUBLIC HEALTH RELEVANCE: ¿-cell apoptosis contributes to decreases in ¿-cell mass and ¿-cell dysfunction during the evolution of diabetes mellitus and is therefore important to understand the mechanisms underlying ¿-cell apoptosis if this process is to be prevented or delayed. Observations in the PI's laboratory indicate involvement of iPLA2¿ in the ¿-cell apoptotic pathway and we propose to examine in greater detail the role of iPLA2¿, as it relates to its expression, activation, and localization, and of iPLA2¿-derived lipid mediators in ¿-cell apoptosis due to stimuli that contribute to ¿-cell death during the evolution of diabetes mellitus. Findings from these studies will extend our knowledge of factors that adversely affect ¿-cell health and identify targets for future therapeutic interventions to prevent ¿-cell death.

描述(申请人提供)：在1型和2型糖尿病中，细胞凋亡都会导致细胞丢失和细胞功能下降。因此，如果要阻止或推迟这一过程，了解细胞凋亡的潜在机制是很重要的。我们在第一个项目期间(10/04-7/09)的假设是，该组通过钙非依赖性磷脂酶A2(IPLA2)参与了内质网应激诱导的细胞凋亡。我们观察到：(A)内质网应激促进内质网和线粒体中iPLA2的积累和活性，(B)内质网应激时iPLA2的激活通过中性鞘磷脂酶(NSMase)催化的神经鞘蛋白水解酶(NSMase)催化神经酰胺的产生，从而触发线粒体凋亡途径和细胞凋亡，(C)这些结果被iPLA2或NSMase抑制，(D)iPLA2缺失的胰岛较少，iPLA2转基因(TG)胰岛对ER应激更敏感，(E)iPLA2缺失的胰岛中NSMA2的表达不受影响，iPLA2-TG胰岛中的NSMA2表达被放大和(F)自发性细胞内质网应激导致糖尿病的秋田小鼠模型中，细胞iPLA2和NSMase的信息更高。我们还发现iPLA2参与了高血糖和细胞因子诱导的细胞凋亡，糖尿病前期NOD小鼠的胰岛iPLA2和NSMase信息增加。我们假设iPLA2？介导的神经酰胺的产生和线粒体异常的触发是导致 --细胞凋亡。本文提出的研究旨在阐明iPLA2在这一过程中的确切作用，以及这一由内质网应激激活的途径是否也被高血糖和细胞因子所诱导。我们的目标将解决以下问题：目标1，iPLA2激活和iPLA2衍生的脂质介质的作用。高血糖和细胞因子部分通过诱导内质网应激促进细胞凋亡，将评估iPLA2在此过程中的作用和iPLA2衍生的脂质介质的产生；目的2，诱导iPLA2和神经酰胺的产生途径。已知SREBP-1和CaMKII会影响iPLA2，并在内质网应激时被激活。将研究它们在iPLA2诱导中的作用以及iPLA2活性对神经酰胺生成的要求；目标3，iPLA2动员和激活以触发线粒体异常的要求。本课程将探讨iPLA2的遗传调控、动员和细胞器特异性表达对线粒体凋亡途径的影响；目标4，体内iPLA2调控后对内质网应激的敏感性。IPLA2？在调节ER应激中的作用将通过与ER应激敏感的秋田小鼠和iPLA2？-TG与ER应激抗性CHOP缺失的小鼠杂交来测试；以及目标5，iPLA2在自身免疫性糖尿病进化过程中对？细胞凋亡的贡献。将评估细胞因子诱导NOD小鼠细胞凋亡对iPLA2/一氧化氮(NO)依赖途径的依赖性以及iPLA2在细胞凋亡中的作用。这些目标将使用细胞凋亡、流式细胞术、免疫印迹、qRT-PCR、酶活性分析、共聚焦显微镜、分子生物学和质谱学方法来解决。我们的研究结果将增加对细胞内iPLA2生物学的理解。我们实验室的长期目标是评估iPLA2在糖尿病发生发展过程中细胞凋亡中的作用。 公共卫生相关性：在糖尿病的发展过程中，细胞凋亡有助于减少细胞质量和细胞功能障碍，因此，如果要防止或推迟这一过程，了解细胞凋亡的机制是很重要的。PI实验室的观察表明，iPLA2参与了细胞凋亡途径，我们建议更详细地研究iPLA2的作用，因为它与其表达、激活和定位有关，以及iPLA2衍生的脂质介质在糖尿病演变过程中导致细胞死亡的刺激所致的细胞凋亡中的作用。这些研究的结果将扩大我们对不利影响细胞健康的因素的了解，并为未来预防细胞死亡的治疗干预确定目标。