This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. When the Perspective in Diabetes that is the subject of this retrospective was written, we had recently identified a phospholipase A2 (PLA2) activity in pancreatic islets that we believed might integrate several observations about glucose-induced phospholipid hydrolysis in pancreatic islets that had resulted from our work and that of R. Paul Robertson, Stewart Metz, Marjorie Dunlop, Claes Wollheim, and many others cited in the original Perspective (1). Such observations included the facts that exposure of islets to concentrations of D-glucose that stimulated insulin secretion resulted in hydrolysis of phospholipids, accumulation of nonesterified arachidonic acid, and generation of arachidonate oxygenation products. Glucose-induced phospholipid hydrolysis required that glucose be metabolized but was in part independent of Ca2+ influx, and the arachidonic acid that accumulated appeared to amplify the glucose-induced rise in -cell [Ca2+] by facilitating Ca2+ entry from the extracellular space and by inducing Ca2+ release from intracellular sequestration sites (1). Like a similar activity first recognized in myocardial cytosol but unlike other then recognized PLA2 enzymes, this islet PLA2 activity did not require Ca2+ for catalytic activity, was activated by ATP, and was sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate that did not inhibit other PLA2 enzymes at comparable concentrations. Moreover, BEL was found to suppress glucose-induced arachidonate release, insulin secretion, and the rise in -cell cytosolic [Ca2+], suggesting that this PLA2 might represent a component of the -cell fuel sensor apparatus (1). Subsequently, insulinoma cells were also found to express this PLA2 activity, and this facilitated its chromatographic analysis and purification (2). Such characterization of the -cell activity eventually resulted in the cloning from a rat islet cDNA library of an 84 kDa protein that contained a GXSXG serine lipase consensus sequence and 8 stretches of a repetitive motif similar to that in the integral membrane protein-binding domain of ankyrin (3). The recombinant protein exhibited PLA2 activity, and this enzyme is now classified as a group VIA PLA2 and given the trivial designation iPLA2 (4). Human islets were later found to express mRNA species encoding two distinct isoforms of iPLA2 that arise by an exon-skipping mechanism of alternative splicing (4), and pharmacologic inhibition studies with BEL in insulinoma cells supported a role for iPLA2 in phospholipid hydrolytic events in insulin secretion but failed to provide evidence that iPLA2 plays a housekeeping role in arachidonic acid incorporation into phospholipids (5) that had been suggested in other cells. Because BEL inhibits several enzymes in addition to iPLA2 , molecular biologic manipulations of iPLA2 activity provide an important complement to pharmacologic studies. Cultured insulinoma cell lines generated by stable transfection with retroviral vectors containing either iPLA2 cDNA (6) or small interfering RNA (7) express several-fold higher or lower levels of iPLA2 activity, respectively, than do cells transfected with empty vectors. Studies with such cell lines also support a role for iPLA2 in insulin secretion but not in -cell phospholipid remodeling (6, 7). Such cell lines also exhibit other properties that correlate with iPLA2 expression level, including increased proliferation rates in cells that overexpress iPLA2 (8) and reduced proliferation rates in cells in which iPLA2 expression is suppressed (7). Moreover, insulinoma cells that overexpress iPLA2 exhibit increased sensitivity to induction of apoptosis by agents that cause endoplasmic reticulum stress (9), suggesting that iPLA2 could participate in a complex variety of cell biologic processes that might differ among cells or within a given cell depending on specific circumstances. Consistent with that possibility, several iPLA2 isoforms that arise from alternative splicing of mRNA are now recognized (4), as are isoforms that arise from proteolytic processing (10). A predominant isoform in -cells arises from proteolytic removal of 14 kDa of the C-terminal amino acid sequence to yield a catalytically active 70 kDa protein involved in signaling (10). Protein-protein interactions and the subcellular location of iPLA2 could also affect the consequences of its action, and a major interacting protein in -cells is the Ca2+/calmodulin-dependent protein kinase II that plays important role in -cell Ca2+ signaling (11). Agents that elevate cAMP and amplify insulin secretion also induce subcellular redistribution of iPLA2 (4). Manipulation of iPLA2 expression level in whole animals can also provide insight into its potential biological functions. Early studies with transgenic mice that overexpress iPLA2 by several-fold specifically in -cells indicate that these mice have lower fasting and stimulated blood glucose levels and higher insulin levels than do wild-type mice (Figure 1). Characterization of the metabolic phenotype(s) of global knockout mice homozygous for an iPLA2 gene disrupted by homologous recombination is also ongoing. Male iPLA2 knockout mice have greatly reduced fertility and impaired motility of spermatozoa that might reflect impaired intracellular [Ca2+] regulation (12), and macrophages from such mice do not exhibit the normal response of increasing transcription of the inducible nitric oxide synthase gene when exposed to double-stranded RNA virus (13), suggesting that iPLA2 also plays a signaling role in that process. Early studies also suggest that iPLA2 knockout mice have lower mortality rates than wild-type mice, suggesting that iPLA2 might affect longevity (Figure 2), and additional roles for iPLA2 proposed by others have recently been reviewed (14, 15).
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