Phospholipid-Derived Mediators and Insulin Secretion

我们在前一个项目期间的假设是，胰岛Ca2+非依赖性磷脂酶A2 （iPLA2p）在用促分泌素刺激时被激活，并且其产物参与p细胞信号传导。 我们现在已经从胰岛mRNA中克隆了iPLA2p，并确定了人iPLA2基因结构， 染色体定位重组iPLA2p被溴烯醇内酯（BEL）自杀底物抑制， 也抑制葡萄糖诱导的胰岛素分泌，iPLA2p过表达放大胰岛素瘤细胞 分泌和增殖。我们还发现，含有花生四烯酸的缩醛磷脂， 膜融合和胞吐，在p细胞中是丰富的，这些醚脂是由过氧化物酶体产生的， 衍生中间体。靶向过氧化物酶体的iPLA 2 γ同工酶也在胰岛中表达， 参与调节复合脂质合成。过氧化物酶体失调可能导致病理性 糖尿病中的组织脂质积聚。近年来人类胰岛移植的成功和有限的 供体器官的可用性突出了识别影响β细胞分泌的基因及其产物的必要性 和存活，以促进工程化的p细胞系的构建，所述工程化的p细胞系可用作 可移植的P细胞。在接下来的项目期间，我们建议进一步表征iPLA 2同工酶的作用， 复杂的脂质和过氧化物酶体在p细胞功能，并开发基因修饰的小鼠与改变 用于体内研究的iPLA2p表达。目的1是表征分泌，增殖和其他反应， 胰岛素瘤细胞和胰岛，其中通过分子生物学手段操纵iPLA 2p表达。目标二是 表征复合脂质在p细胞功能中的作用以及脂质中iPLA 2同工酶和过氧化物酶体的作用， 阵目的3是表征iPLA2p蛋白的调节性翻译后修饰。目标4是 进行iPLA2p在细胞区室之间易位的细胞生物学研究以及与 其他蛋白质。目的5是开发具有改变的iPLA 2p表达的遗传修饰小鼠，用于体内实验。 问题研究我们已经制备了小鼠胚胎干细胞，其中iPLA2p等位基因已经被破坏， 同源重组作为产生不表达酶的小鼠的步骤。

Our hypothesis in the previous project period was that a pancreatic islet Ca2+-independent phospholipase A2 (iPLA2p) is activated upon stimulation with secretagogues and that its products participate in p-cell signaling. We have now cloned iPLA2p from islet mRNA and determined the human iPLA2 gene structure and chromosomal location. Recombinant iPLA2p is inhibited by a bromoenol lactone (BEL)suicide substrate that also suppresses glucose-induced insulin secretion, and iPLA2p overexpression amplifies insulinoma cell secretion and proliferation. We have also found that arachidonate-containingplasmalogens, which participate in membrane fusion and exocytosis, are abundant in p-cells, and these ether lipids are produced from peroxisome- derived intermediates. An iPLA2y isozyme targeted to peroxisomes is also expressed in islets and may participate in regulating complex lipid synthesis. Peroxisomal dysregulation could contribute to pathologic tissue lipid accumulation in diabetes. The recent success of human islet transplantation and the limited availability of donor organs highlights the need to identify genes and their products that affect p-cell secretion and survival to facilitate construction of engineered p-cell lines that might serve as an alternate source of transplantable p-cells. In the coming project period, we propose to further characterize roles of iPLA2 isozymes, complex lipids, and peroxisomes in p-cell function and to develop genetically modified mice with altered iPLA2p expression for in vivo studies. Aim 1 is to characterize secretion, proliferation, and other responses of insulinoma cells and islets in which iPLA2p expression is manipulated by molecular biologic means. Aim 2 is to characterize roles of complex lipids in p-cell function and of iPLA2 isozymes and peroxisomes in lipid formation. Aim 3 is to characterize regulatory post-translational modifications of the iPLA2p protein. Aim 4 is to conduct cell biologic studies of iPLA2p translocation among cellular compartments and interactions with other proteins. Aim 5 is to develop genetically modified mice with altered iPLA2p expression for in vivo studies. We have prepared mouse embryonic stem cells in which an iPLA2p allele has been disrupted by homologous recombination as a step to generate mice that do not express the enzyme.