Calcium-Independent PLA2Beta in Beta-Cell Apoptosis

描述（由申请人提供）：内质网（ER）应激导致β细胞凋亡，通过抑制VIA组钙离子非依赖性磷脂酶A2（iPLA 2 β）抑制β细胞凋亡，我们的假设是iPLA 2 β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细胞凋亡导致β细胞丢失和β细胞功能降低。因此，重要的是要了解β细胞凋亡的机制，如果这个过程是要预防或延迟。β细胞的分泌性质赋予它们高度发达的ER，使它们对ER应激敏感。这一过程被认为促进β细胞凋亡，并在一定程度上导致T2 DM中β细胞质量和β细胞功能障碍的减少。iPLA 2 β催化花生四烯酸从膜磷脂中水解，并在β细胞中具有信号转导作用。我们观察到ER应激诱导的β细胞凋亡与iPLA 2 β表达水平相关，与caspase-3切割的iPLA 2 β亚型的核周积累以及花生四烯酸和神经酰胺的产生增加相关，并被iPLA 2 β抑制剂抑制。ER应激诱导的胰岛细胞凋亡也通过iPLA 2 β和iPLA 2 β的失活而减弱-KO胰岛对ER应激较不敏感。我们建议通过以下目的使用胰岛（野生型和iPLA 2 β-KO）和INS-1细胞（亲本、iPLA 2 β过表达和iPLA 2 β-KO）进一步检查iPLA 2 α在α-细胞凋亡中的作用：目的1是使用诱导α-细胞凋亡和ER应激的刺激物表征iPLA 2 β参与β-细胞凋亡。目的2是通过在α细胞中表达截短和突变的iPLA 2 β亚型来检查iPLA 2 β亚型在β细胞凋亡中的作用和亚细胞定位。目的3是使用ESI/MS/MS方案鉴定ER应激期间iPLA 2 β的β细胞亚细胞磷脂底物。目的4是使用LC/ESI/MS/MS和生物化学分析来检查iPLA 2 β磷酸化和iPLA 2 β衍生的花生四烯酸在β细胞凋亡中的参与。

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.