John W. Turk, MD, PhD

Professor of Medicine

Washington University in St. Louis (WU)

Our hypothesis that a pancreatic islet Ca2+-independent phospholipase A2 (iPLA2ß) is activated by secretagogues and that its products participate in ß-cell signaling will be pursued with tools developed in this project period, including insulinoma cells with stably altered iPLA2ß expression that exhibit secretory and proliferative properties and sensitivities to Endoplasmic Reticulum (ER) stress that correlate with iPLA2ß expression level. New ESI/MS/MS methods for lipid analyses reveal that iPLA2ß overexpression enhances ER stress-induced ceramide accumulation and increases lysophosphatdiylcholine (LPC) levels. New MS proteomics tools reveal that ß-cells proteolytically remove an iPLA2ß inhibitory domain and that iPLA2ß hydrolyzes its inhibitor BEL to an intermediate that alkylates cysteine thiols. New iPLA2ß fluorescent constructs reveal that iPLA2ß translocates to perinuclear membranes in stimulated ß-cells, and it interacts with a Ca2+/calmodulin-dependent protein kinase Iiß isoform expressed in ß-cells. Our iPLA2ß-null mice have reduced male fertility and impaired ability to increase macrophage inducible NO synthase expression and become more glucose intolerant than wild-type mice on a high-fat diet, but they are resistant to diet-induced increases in adiposity and mortality. Our emphasis will now shift to in vivo studies with genetically modified mice, but we will continue our Aims to: 1.) Characterize insulinoma cells with altered iPLA2ß or ? expression with respect to secretion, proliferation, ER stress, and ion channel function; 2.) Characterize diet-induced changes in ß-cell and tissue fatty acid desaturases and complex lipids e.g., lysophosphatidic acid, cardiolipin, and NO2-fatty acids; 3.) Characterize iPLA2ß protein modifications, e.g., proteolysis, oxidation, and phosphorylation; 4.) Conduct cell biologic studies of iPLA2ß isoform-specific organelle association and protein interactions, e.g., with MSS4 and cytochrome c oxidase; and 5.) Characterize genetically modified mice with altered iPLA2 expression. With iPLA2ß-null and transgenic mice, we will examine insulin sensitivity and secretion in vivo; dietary effects on glucose-tolerance, body composition, and mortality; macrophage sensitivity to cholesterol-induced apoptosis; and atherogenesis in apoE-/-/iPLA2ß-/- double knockout mice. Goals also include preparing mice with tissue-specific iPLA2ß knockouts or altered iPLA2? expression. The public health relevance is that manipulating iPLA2 expression might be useful in genetic engineering of ß-cell lines for Type I diabetes mellitus (DM) cellular replacement therapy, and mechanisms to expand ß-cell mass and improve insulin secretion in Type II DM could also be identified. Studies with iPLA2ß-null mice could also provide evidence that iPLA2ß pharmacologic inhibitors might have beneficial therapeutic effects, such as reducing diet-induced obesity and atherogenesis, and insight into inhibitor design could result from iPLA2ß inactivation studies.