Phospholipid-Derived Mediators and Insulin Secretion

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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.