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，这是通过抑制组VIA Ca2+独立磷脂酶A2 （ipla2 β）来抑制的，我们的假设是ipla2 β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细胞凋亡导致β细胞损失和β细胞功能下降。因此，了解β细胞凋亡的机制对于预防或延缓这一过程至关重要。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。这一过程被认为促进了β细胞凋亡，并在一定程度上导致了T2DM中β细胞质量的减少和β细胞功能障碍。ipla2 β可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ipla2 β的表达水平有关，与caspase-3-cleaved ipla2 β亚型的核周积累和花生四烯酸和神经酰胺的生成增加有关，并被ipla2 β抑制所抑制。内质网应激诱导的胰岛凋亡也可通过ipla2 β的失活而减弱，ipla2 β -KO胰岛对内质网应激的敏感性较低。我们建议进一步研究iPLA2a在a细胞凋亡中的作用，使用胰岛（野生型和ipla2 β -KO）和in -1细胞（亲代、ipla2 β过表达和ipla2 β -KO），通过以下目的：目的1是通过诱导a细胞凋亡和内质网应激的刺激来表征ipla2 β参与β细胞凋亡。目的2是通过在a细胞中表达截断和突变的ipla2 β亚型，研究ipla2 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协议鉴定内质网应激下β细胞亚细胞磷脂底物ipla2 β。目的4是通过LC/ESI/MS/MS和生化分析来检测ipla2 β磷酸化和ipla2 β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.