Glucose-induced insulin secretion is defective in type II diabetes mellitus, and the molecular events coupling glucose recognition to insulin secretion by pancreatic islets beta-cells are incompletely understood. Glucose induces hydrolysis of islet membrane phospholipids and accumulation of nonesterified arachidonic acid and its 12- lipoxygenase products by a process that requires glucose metabolism and is mediated in part by an ATP-stimulated, Ca2+-independent phospholipase A2 enzyme (ASCl-PLA2), which may be a component of the beta-cell fuel sensor. ASCl-PLA2 blockade with a specific suicide substrate suppresses arachidonate release, the rise in beta-cell [(Ca2+], and insulin secretion induced by glucose. Further studies of the role of this enzyme in beta-cells will include: A.) Characterizing beta-cell ASCl-PLA2 activity in islets from 3 species and insulinoma cells. Activation by ATP, modulation of this response (by factors including ADP and glycolytic intermediates which influence ASCl-PLA2 chromatographic behavior), ASCl- PLA2 membrane translocation, and ASCl-PLA2 expression in models of perturbed islet function will be examined. B.) Isolation of beta-cell ASCl-PLA2 and its distinct catalytic and regulatory proteins will be achieved by ATP-affinity chromatography, gel filtration, and FPLC. Relationships between catalytic and regulatory proteins from islets and insulinoma cells will be studied by cross-complementation. ASCl-PLA2 cellular and subcellular distribution and changes enzyme mass in perturbed-islet models will be studied with antibodies to the purified proteins. C.) Molecular characterization of islet phospholipids by HPLC and GC/MS will determine the islet content of plasmalogen substrates preferred by ASCl-PLA2; the phospholipid composition of islet subcellular membranes; the influence of insulin secretagogues on phospholipid arachidonate mass; and the depletion of critical beta-cell arachdionate pools in glucose-desensitized islets. D.) The influence of arachidonate on Ca2+ entry into beta-cells will be examined in studies of the effects of arachidonate on beta-cell voltage-operated Ca2+ channels (VOCC) and of effects of arachidonate and omega3 polyunsaturated fatty acids on the rise in beta-cell [Ca2+] induced by glucose and depolarization and the participation of omega-conotoxin-sensitive VOCC in these events. The major islet arachidonate-metabolizing enzyme is a 12-lipoxygenase (12-LO) selectively expressed by beta-cells which is not found in other islet cells or in a pancreatic acini, and 12-LO blockade suppresses insulin secretion. E.) The role of 12-LO in beta-cells will be studied by examining: 12-LO membrane tran-location in glucose-stimulated islets; islet conversion of the primary 12-LO product 12-HPETE and of linoleate to active products; effects of 12-LO products on beta-cell membrane potential and K+ permeability; and esterification of 12-LO products in islet plasma membrane and secretory granule phospholipids.
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