[Abstract unavailable

The Ca 2+ -independent phospholipases A 2 (iPLA 2 s) are part of a diverse family of PLA 2 s that hydrolyze the sn -2 substituent from membrane phospholipids to release a free fatty acid and a lysolipid ( 1, 2 ). These enzymes are ubiquitously expressed, and in contrast to secretory PLA 2 s (sPLA 2 s) and cytosolic PLA 2 s (cPLA 2 s), do not require Ca 2+ for either translocation or activity. Some of the fi rst descriptions of iPLA 2 activity were in the mid-to late-1980s with the identifi cation of a plasmalogen-selective PLA 2 in Abstract Among the family of phospholipases A 2 (PLA 2 s) are the Ca 2+ -independent PLA 2 s (iPLA 2 s) and they are designated group VI iPLA 2 s. In relation to secretory and cytosolic PLA 2 s, the iPLA 2 s are more recently described and details of their expression and roles in biological functions are rapidly emerging. The iPLA 2 s or patatin-like phospholipases (PNPLAs) are intracellular enzymes that do not require Ca 2+ for activity, and contain lipase (GXSXG) and nucleotide-binding (GXGXXG) consensus sequences. Though nine PNPLAs have been recognized, PNPLA8 (membraneassociated iPLA 2 ␥ ) and PNPLA9 (cytosol-associated iPLA 2 ␤ ) are the most widely studied and understood. The iPLA 2 s manifest a variety of activities in addition to phospholipase, are ubiquitously expressed, and participate in a multitude of biological processes, including fat catabolism, cell differentiation, maintenance of mitochondrial integrity, phospholipid remodeling, cell proliferation, signal transduction, and cell death. As might be expected, increased or decreased expression of iPLA 2 s can have profound effects on the metabolic state, CNS function, cardiovascular performance, and cell survival; therefore, dysregulation of iPLA 2 s can be a critical factor in the development of many diseases. This review is aimed at providing a general framework of the current understanding of the iPLA 2 s and discussion of the potential mechanisms of action of the iPLA 2 s and related involved lipid mediators. -Ramanadham, S

The Group VIA Phospholipase A 2 (iPLA 2 ␤) is the first recognized cytosolic Ca 2؉ -independent PLA 2 and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes. To study iPLA 2 ␤ functions, we disrupted its gene by homologous recombination to generate mice that do not express iPLA 2 ␤. Heterozygous iPLA 2 ␤ ؉/؊ breeding pairs yield a Mendelian 1:2:1 ratio of iPLA 2 ␤ ؉/؉ , iPLA 2 ␤ ؉/؊ , and iPLA 2 ␤ ؊/؊ pups and a 1:1 male:female gender distribution of iPLA 2 ␤ ؊/؊ pups. Several tissues of wild-type mice express iPLA 2 ␤ mRNA, immunoreactive protein, and activity, and testes express the highest levels. Testes or other tissues of iPLA 2 ␤ ؊/؊ mice express no iPLA 2 ␤ mRNA or protein, but iPLA 2 ␤ ؊/؊ testes are not deficient in 20:4-containing glycerophosphocholine lipids, indicating that iPLA 2 ␤ does not play an obligatory role in formation of such lipids in that tissue. Spermatozoa from iPLA 2 ␤ ؊/؊ mice have reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo, and inhibiting iPLA 2 ␤ with a bromoenol lactone suicide substrate reduces motility of wild-type spermatozoa in a time-and concentration-dependent manner. Mating iPLA 2 ␤ ؊/؊ male mice with iPLA 2 ␤ ؉/؉ , iPLA 2 ␤ ؉/؊ , or iPLA 2 ␤ ؊/؊ female mice yields only about 7% of the number of pups produced by mating pairs with an iPLA 2 ␤ ؉/؉ or iPLA 2 ␤ ؉/؊ male, but iPLA 2 ␤ ؊/؊ female mice have nearly normal fertility. These findings indicate that iPLA 2 ␤ plays an important functional role in spermatozoa, suggest a target for developing male contraceptive drugs, and complement reports that disruption of the Group IVA PLA 2 (cPLA 2 ␣) gene impairs female reproductive ability.

The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.

The death of insulin-secreting β-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive β-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to β-cell loss in diabetes. Agents that deplete ER calcium levels induce β-cell apoptosis by a process that is independent of increases in [Ca 2+ ] i . Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A 2 (iPLA 2 β). Overexpression of iPLA 2 β amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA 2 β expression in various cell lines, and apoptosis is associated with stimulation of iPLA 2 β activity, perinuclear accumulation of iPLA 2 β protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA 2 β to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA 2 β. These findings indicate that iPLA 2 β participates in ER stress-induced apoptosis, a pathway that promotes β-cell death in diabetes.Diabetes mellitus (DM) 1 is the most prevalent human endocrine disease, and it results from loss and/or dysfunction of insulin-secreting β-cells in pancreatic islets. Type I DM is caused † This research was supported in part by grants from the National Institutes of Health (R37-DK34388, P41-RR00954, P01-HL57278, P60-DK20579, and P30-DK56341) and by an Award (to S.R.) from the American Diabetes Association.© 2004 American Chemical Society * To whom correspondence should be addressed: Department of Medicine, Washington University School of Medicine, Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110. Phone: (314) 362-8194. Fax: (314) 362-8188. sramanad@im.wustl.edu. 1 Abbreviations: AA, arachidonic acid; BEL, bromoenol lactone suicide inhibitor of iPLA 2 β; BME, β-mercaptoethanol; BSA, bovine serum albumin; CAD, collisionally activated dissociation; CM, ceramide; CNL, constant neutral loss; C3-I, caspase-3 inhibitor; cPLA 2 , group IV cytosolic phospholipase A2; ECL, enhanced chemiluminescence; EGFP, enhanced green fluorescence protein; ER, endoplasmic reticulum; ESI, electrospray ionization; FBS, fetal bovine serum; IF, immunocytofluorescence; iPLA 2 β, β-isoform of group VIA calcium-independent phospholipase A 2 ; IS, internal standard; MS, mass spectrometry; OE, iPLA 2 β-overexpressing cells; O/N, overnight; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PIC, protease inhibitor cocktail; PLA 2 , phospholipase A 2 ; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; SERCA, sarcoen-doplasmic reticulum Ca 2+ -ATPase; TBS-T, Tris-buffered saline-tween; TIC, total ion current; TLC, thin-layer chromatography;...