Calcium-Independent PLA2Beta in Beta-Cell Apoptosis

Beta 细胞凋亡中的钙依赖性 PLA2Beta

• 批准号: 7258377
• 负责人: SASANKA RAMANADHAM
• 金额: $ 28.72万
• 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 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7258377
• 关键词: Apoptosis; Apoptotic; Arachidonic Acids; Attenuated; Beta Cell; Biochemical; Calcium; Caspase; Cell Survival; Cell membrane; Cell physiology; Cells; Ceramides; Chimeric Proteins; Cleaved cell; Complex; Condition; Consensus Sequence; Cytosol; Cytosolic Phospholipase A2; DNA; Diabetes Mellitus; Endoplasmic Reticulum; Functional disorder; Generations; Golgi Apparatus; HTATIP gene; Hydrolysis; Immunoblotting; In Situ Nick-End Labeling; Insulin-Dependent Diabetes Mellitus; Islet Cell; Islets of Langerhans; Link; Lipase; MAP Kinase Gene; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane; Monitor; Mus; Mutate; Mutation; Nature; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Numbers; Organelles; PLA2G4A gene; Pathway interactions; Phospholipase A2; Phospholipids; Phosphorylation; Polymerase Chain Reaction; Process; Protein Isoforms; Proteins; Protocols documentation; Rest; Role; Signal Transduction; Site; Spectrometry; Spectrometry, Mass, Electrospray Ionization; Stimulus; Stress; Testing; Time; caspase-3; human PLA2G4A protein; in vivo; insulinoma; islet; lipid mediator; prevent; type I and type II diabetes

DESCRIPTION (provided by applicant): Endoplasmic reticulum (ER) stress leads to beta-cell apoptosis that is suppressed by inhibition of the Group VIA Ca2+-independent phospholipase A2 (iPLA2beta), and our hypothesis is that iPLA2beta participates in ER stress-induced beta-cell apoptosis. In both type 1 and type 2 diabetes mellitus (DM), beta-cell apoptosis contributes to the loss of beta-cells and decreases in beta-cell function. It is therefore important to understand the mechanisms underlying beta-cell apoptosis if this process is to be prevented or delayed. The secretory nature of beta-cells endows them with a highly developed ER making them susceptible to ER stress. This process is suggested to promote beta-cell apoptosis and, in part, contribute to the decreases in beta-cell mass and beta-cell dysfunction in T2DM. The iPLA2beta catalyzes hydrolysis of arachidonic acid from membrane phospholipids and has a signal transduction role in beta-cells. We observed that ER stress-induced beta-cell apoptosis is related to the levels of iPLA2beta expression, is associated with perinuclear accumulation of a caspase-3-cleaved iPLA2beta isoform and increased generation of arachidonic acid and ceramides, and is suppressed by iPLA2beta inhibition. ER stress-induced apoptosis of pancreatic islets is also attenuated by inactivation of iPLA2beta and iPLA2beta -KO islets are less susceptible to ER stress. We propose to further examine the role of iPLA2a in a-cell apoptosis using pancreatic islets (wild type and iPLA2beta -KO) and INS-1 cells (parental, iPLA2beta over expressing, and iPLA2beta -KO) through the following Aims: Aim 1 is to characterize participation of iPLA2beta in beta-cell apoptosis using stimuli that induce both a-cell apoptosis and ER stress. Aim 2 is to examine the roles and subcellular localization of iPLA2beta isoforms in beta-cell apoptosis by expressing truncated and mutated iPLA2beta isoforms in a-cells. Aim 3 is to identify beta-cell subcellular phospholipid substrates for iPLA2beta during ER stress using ESI/MS/MS protocols. Aim 4 is to examine involvement of iPLA2beta phosphorylation and iPLA2beta -derived arachidonic acid in beta-cell apoptosis using LC/ESI/MS/MS and biochemical analyses.

描述（由申请人提供）：内质网（ER）应激导致β细胞凋亡，这种凋亡可通过抑制VIA组Ca2+独立磷脂酶A2（iPLA2beta）来抑制，我们的假设是iPLA2beta参与ER应激诱导的β细胞凋亡。在 1 型和 2 型糖尿病 (DM) 中，β 细胞凋亡会导致 β 细胞损失和 β 细胞功能下降。因此，如果要预防或延迟这一过程，了解β细胞凋亡的机制非常重要。 β 细胞的分泌性质赋予它们高度发达的内质网，使它们容易受到内质网应激的影响。该过程被认为可以促进 β 细胞凋亡，并在一定程度上导致 T2DM 中 β 细胞质量的减少和 β 细胞功能障碍。 iPLA2beta 催化膜磷脂水解花生四烯酸，并在 β 细胞中具有信号转导作用。我们观察到，内质网应激诱导的 β 细胞凋亡与 iPLA2β 表达水平有关，与 caspase-3 裂解的 iPLA2β 亚型的核周积累以及花生四烯酸和神经酰胺的生成增加有关，并且通过 iPLA2β 抑制而受到抑制。内质网应激诱导的胰岛细胞凋亡也会因 iPLA2β 的失活而减弱，并且 iPLA2β -KO 胰岛不易受内质网应激的影响。我们建议通过以下目标，使用胰岛（野生型和 iPLA2beta -KO）和 INS-1 细胞（亲本、iPLA2beta 过表达和 iPLA2beta -KO）进一步研究 iPLA2a 在 a 细胞凋亡中的作用： 目标 1 是使用诱导 iPLA2beta 和 iPLA2beta 的刺激来表征 iPLA2beta 在 β 细胞凋亡中的参与 a细胞凋亡和内质网应激。目标 2 是通过在 a 细胞中表达截短和突变的 iPLA2beta 同工型来检查 iPLA2beta 同工型在 β 细胞凋亡中的作用和亚细胞定位。目标 3 是使用 ESI/MS/MS 方案鉴定 ER 应激期间 iPLA2beta 的 β 细胞亚细胞磷脂底物。目标 4 是使用 LC/ESI/MS/MS 和生化分析检查 iPLA2beta 磷酸化和 iPLA2beta 衍生的花生四烯酸在 β 细胞凋亡中的参与情况。