Our recent studies indicated that a phospholipase A2 (PLA2) and a phospholipase D (PLD) are present in pancreatic islets; furthermore, the phospholipid hydrolysis products which they generate (arachidonic acid, lysophospholipids, and phosphatidic acid) are insulinotropic. However, the physiologic regulation enzymes in intact islet cells is largely unexplored. We propose to clarify their regulation and the mechanisms whereby their lipid byproducts modulate exocytosis. The major preparation is adult rat islets; dispersed, neonatal rat islet cells will be employed in circumstances when a single cell preparation or increased cell mass is needed. In general, intact cells will be used so that lipid biochemistry and insulin secretion can be studied in virtually identical preparation in parallel; for selected questions, permeabilized cells and broken cell preparations will be studied. Phospholipid turnover is assessed using thin layer chromatographic or high performance liquid chromatographic analysis of organic and aqueous cell extracts of prelabelled islets; both isotopic and confirmatory mass measurements of phospholipids are made. Emphasis is placed upon the regulation of PLA2 and PLD by changes in nutrient availability, [Ca++i], protein kinase activity, G protein activation, pH, and upon the effects of physiological modulators of islet function (e.g., alpha 2 agonists, somatostatin). In addition to phospholipid hydrolysis, a second, potentially rate-limiting step (the removal of phospholipid byproducts by re-esterification or resynthesis) will be systematically assessed. We will use unambiguous lipid markers (such as lysophospholipid accumulation of PLA2, and phosphatidylethanol generation for PLD). The latter reflects the transphosphatidylation activity inherent in PLD--that is, in the presence of nucleophilic acceptors such as ethanol (glycerol), PLD transfers a phosphatidyl group (principally from phosphatidylcholine) to form phosphatidic acid. To examine the mechanism of action of lipid mediators, lysophosphatidylcholine and phosphatidate will be generated endogenously (as well as provided exogenously); we will also attempt to inhibit or deplete islet phospholipase activity and then reverse any effects on secretion by """"""""repleting"""""""" enzyme activity or its specific lipid byproducts. Conversely, the generation of phosphatidate by PLD can be """"""""ablated"""""""" via providing acceptors which support transphosphatidylation. We will also continue to probe the mechanism whereby lipoxygenase-mediated metabolism of arachidonic acid promotes secretion, by providing both exogenous (lipoxygenase) enzyme and authentic lipoxygenase-derived metabolites to permeabilized islets. The effects of relevant lipid species on cell signalling systems and insulin release can then be related. These studies are unique in delineating a definitive fashion the biochemical nature and, simultaneously, the physiologic function of the phospholipase A2 and D of the pancreatic islet. Such findings may have applicability to the understanding of endocrine secretion in general and, ultimately, to hypersecretory states such as diabetes mellitus, specifically.

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National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Metabolism Study Section (MET)
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Laughlin, Maren R
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Pacific Northwest Research Institute
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Huo, Jianxin; Luo, Rui-Hua; Metz, Stewart A et al. (2002) Activation of caspase-2 mediates the apoptosis induced by GTP-depletion in insulin-secreting (HIT-T15) cells. Endocrinology 143:1695-704
Metz, S; Holland, S; Johnson, L et al. (2001) Inosine-5'-monophosphate dehydrogenase is required for mitogenic competence of transformed pancreatic beta cells. Endocrinology 142:193-204
Li, G D; Luo, R H; Metz, S A (2000) Effects of inhibitors of guanine nucleotide synthesis on membrane potential and cytosolic free Ca2+ levels in insulin-secreting cells. Biochem Pharmacol 59:545-56
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Segu, V B; Li, G; Metz, S A (1998) Use of a soluble tetrazolium compound to assay metabolic activation of intact beta cells. Metabolism 47:824-30
Vadakekalam, J; Metz, S (1998) Isotopic efflux studies as indices of phospholipase activation. Methods Mol Biol 105:175-85
Kowluru, A; Metz, S A (1998) Purine nucleotide- and sugar phosphate-induced inhibition of the carboxyl methylation and catalysis of protein phosphatase-2A in insulin-secreting cells: protection by divalent cations. Biosci Rep 18:171-86
Vadakekalam, J; Rabaglia, M E; Metz, S A (1997) Roles of GTP and phospholipase C in the potentiation of Ca(2+)-induced insulin secretion by glucose in rat pancreatic islets. J Endocrinol 153:61-71
Kowluru, A; Metz, S A (1997) Ceramide-activated protein phosphatase-2A activity in insulin-secreting cells. FEBS Lett 418:179-82

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