Previous indirect studies have suggested that activation of phospholipase A2 (PLA2) is a pivotal step in hormone release, in particular that of insulin. However, there has been no specific demonstration of the presence of a PLA2 in intact islets and its activation by relevant islet stimuli such as D-glucose. Furthermore, most studies have focused on one hydrolytic by-product of PLA2 action, arachidonic acid (AA) and the contribution of lipoxygenase-derived metabolites of AA such as 12-hydroperoxyeicosatetraenoic acid (12-HPETE) to insulin (I) release; the role of the second phospholipid moiety generated by PLA2 action (lysophospholipids; lyso-PLs) remains virtually unexplored in any endocrine cell system. Our preliminary data strongly implicate lyso-PLs in the initiation of glucose-induced I release, with the co-generation of AA metabolites serving to modulate this response. This protocol, therefore, seeks to measure specific PLA2 activity (and its response to islet agonists, such as glucose; inhibitors, such as epinephrine; or pathophysiologic perturbations such as starvation or chemically-induced diabetes) in intact adult rat islets by the simultaneous determination of lyso-PL and 12-HPETE accumulation using high performance liquid chromatographic analysis of membrane extracts or media from islets. Islets will be pre-labelled with 3H-AA, 14C-stearate, (32P)-Pi and/or 3H-glycerol in order to label, respectively, the sn-2 carbon, the sn-1-carbon, the polar head groups, and the 3-carbon backbone of phospholipids. The accumulation of various phospholipids and lysophospholipids will be monitored by HPLC after exposure of the islets to D-glucose, exogenous PLA2 and other test compounds in the presence or absence of p-hydroxymercuribenzoate (to impede the removal of any lyso-PL generated). I secretion is assessed, in parallel, in static incubations of intact rat islets, and in perifusions of intact islets and of monolayer-cultured, dispersed neonatal islet cells. We will examine the time- and dose-correspondence between I release and lyso-PL formation in response to various perturbations of islet secretion, and will assess the stereo- and anomeric specificity, Ca++ dependence and dependence on fuel flux of the phospholipid response to glucose. In turn, the mechanism of lyso-PL stimulation of I release will be assessed by examining its response to relatively specific pharmacologic perturbations and through measurements of relevant parameters such as cyclic AMP or intracellular Ca++ accumulation. We will assess whether exogenous lyso-PLs or exogenous PLA2 can reverse the blockade of glucose-induced I release caused by phospholipase inhibitors, fasting, chemically-induced diabetes, epinephrine or somatostatin. These studies should define the presence of, and basic mechanisms involved in, regulating islet PLA2 activity. These studies may provide insights into stimulus-secretion coupling in general and specifically into the insulin secretory defect characteristic of human diabetes mellitus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK037312-02
Application #
3236153
Study Section
Metabolism Study Section (MET)
Project Start
1986-07-01
Project End
1991-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
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
Metz, S A; Meredith, M; Vadakekalam, J et al. (1999) A defect late in stimulus-secretion coupling impairs insulin secretion in Goto-Kakizaki diabetic rats. Diabetes 48:1754-62
Li, G; Segu, V B; Rabaglia, M E et al. (1998) Prolonged depletion of guanosine triphosphate induces death of insulin-secreting cells by apoptosis. Endocrinology 139:3752-62
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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