Phospholipid-Derived Mediators and Insulin Secretion

磷脂衍生介质和胰岛素分泌

基本信息

批准号：
8010459
负责人：
JOHN W TURK
金额：
$ 9.82万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-02-04 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8010459
关键词：
6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one Acetylglucosamine Affect Alleles Apoptosis Cell Line Cell Nucleus Cell physiology Cell secretion Cells Ceramides Complex Consensus Diabetes Mellitus Electrospray Ionization Engineering Enzymes Ethers Exocytosis Gene Structure Generations Genes Glucose Green Fluorescent Proteins Homeostasis Human Immunoblotting In Vitro Islet Cell Islets of Langerhans Islets of Langerhans Transplantation Isoenzymes Ligands Lipids Location Measures Mediating Mediator of activation protein Membrane Membrane Fusion Messenger RNA Modification Molecular Movement Mus Organ Donor Pathologic Peroxisome Proliferator-Activated Receptors Phosphatidic Acid Phosphatidylinositol 4,5-Diphosphate Phospholipase A2 Phospholipids Phosphorylation Plasmalogens Post-Translational Protein Processing Process Production Protein Kinase Proteins Recombinants Role Signal Transduction Site Site-Directed Mutagenesis Source System Tissues Transgenic Mice arachidonate base caspase-3 embryonic stem cell homologous recombination in vivo insulin secretion insulinoma islet overexpression peroxisome protein protein interaction response success suicide substrates yeast two hybrid system

项目摘要

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.

我们在上一个项目期间的假设是胰岛Ca2+独立的磷脂酶A2 （IPLA2P）在用促分泌物刺激后激活，其产物参与P细胞信号传导。现在，我们已经从胰岛mRNA克隆IPLA2P，并确定了人IPLA2基因结构和染色体位置。重组IPLA2P被溴苯酚内酯（BEL）自杀底物抑制还抑制葡萄糖诱导的胰岛素分泌，IPLA2P过表达会放大胰岛素瘤细胞分泌和扩散。我们还发现，参与膜融合和胞吐作用在P细胞中很丰富，这些醚脂质是由过氧化物酶体 - 派生的中间体。靶向过氧化物酶体的IPLA2Y同工酶也在胰岛中表达，可能参与调节复杂脂质合成。过氧化物酶体失调可能导致病理学组织脂质在糖尿病中的积累。人类胰岛移植的最新成功和有限的供体器官的可用性强调了识别影响P细胞分泌的基因及其产品的需求和生存以促进工程P细胞线的构建，这可能是可移植的P细胞。在即将到来的项目期间，我们建议进一步表征IPLA2同工酶的角色， P细胞功能中的复杂脂质和过氧化物酶体，并随着改变的转基因小鼠发展体内研究的IPLA2P表达。目的1是表征分泌，增殖和其他反应 IPLA2P表达通过分子生物学手段操纵的胰岛素瘤细胞和胰岛。 AIM 2是表征复杂脂质在P细胞函数和IPLA2同酶和过氧化物酶体中的作用形成。目的3是表征IPLA2P蛋白的调节后翻译后修饰。目标4是在细胞室之间进行IPLA2P易位的细胞生物学研究，并与其他蛋白质。 AIM 5是开发带有IPLA2P表达的转基因小鼠的体内研究。我们已经准备了小鼠胚胎干细胞，其中ipla2p等位基因已被破坏同源重组是产生不表达酶的小鼠的步骤。