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，而内质网应激通过钙非依赖性磷脂酶A2(iPLA2β)抑制内质网(ER)诱导的β细胞凋亡，我们的假设是iPLA2β参与内质网应激诱导的β细胞凋亡。在1型和2型糖尿病(DM)中，β细胞的凋亡都会导致β细胞的丧失和β细胞功能的降低。因此，如果要阻止或推迟这一过程，了解β细胞凋亡的潜在机制是很重要的。β细胞的分泌特性赋予它们高度发达的内质网，使它们容易受到内质网压力的影响。这一过程被认为促进了β细胞的凋亡，并在一定程度上导致了T2 DM患者β细胞质量和β细胞功能障碍的减少。IPLA2β催化膜磷脂中花生四烯酸的水解，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的表达水平有关，与caspase-3裂解的iPLA2β亚型在核周聚集以及花生四烯酸和神经酰胺的生成增加有关，并被iPLA2β抑制。IPLA2β失活也可减弱内质网应激诱导的胰岛细胞凋亡，iPLA2β-KO胰岛对内质网应激较不敏感。我们建议通过以下目的进一步研究iPLA2a在胰岛(野生型和iPLA2β-KO)和INS-1细胞(亲本，iPLA2β过表达和iPLA2β-KO)中的作用：目的1利用诱导a细胞凋亡和内质网应激的刺激，表征iPLA2β在β细胞凋亡中的参与。目的2通过在a细胞中表达截短和突变的iPLA2β亚型，研究iPLA2β亚型在β细胞凋亡中的作用和亚细胞定位。目的3是利用ESI/MS/MS方法鉴定内质网应激过程中iPLA2β的β细胞亚细胞磷脂底物。目的4通过LC/ESI/MS/MS和生化分析，研究iPLA2β磷酸化及其衍生的花生四烯酸在β细胞凋亡中的作用。

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.