Zhongmin Ma

Studies of the Role of Group VI Phospholipase A2 in Fatty Acid Incorporation, Phospholipid Remodeling, Lysophosphatidylcholine Generation, and Secretagogue-induced Arachidonic Acid Release in Pancreatic Islets and Insulinoma Cells

An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet β-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet β-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells.

Pancreatic islets express a Ca2+-independent phospholipase A2 enzyme that contains a repeated structural motif homologous to the integral membrane protein binding domain of ankyrin.

Pancreatic islets express a Ca2+-independent phospholipase A2 (CaI-PLA2) activity that is sensitive to inhibition by a haloenol lactone suicide substrate that also attenuates glucose-induced hydrolysis of arachidonic acid from islet phospholipids and insulin secretion. A cDNA has been cloned from a rat islet cDNA library that encodes a protein with a deduced amino acid sequence of 751 residues that is homologous to a CaI-PLA2 enzyme recently cloned from Chinese hamster ovary cells. Transient transfection of both COS-7 cells and Chinese hamster ovary cells with the cloned islet CaI-PLA2 cDNA resulted in an increase in cellular CaI-PLA2 activity, and this activity was susceptible to inhibition by haloenol lactone suicide substrate. The domain of the islet CaI-PLA2 from amino acid residues 150-414 is composed of eight stretches of a repeating sequence motif of approximately 33-amino acid residues in length that is highly homologous to domains of ankyrin that bind both tubulin and integral membrane proteins, including several proteins that regulate ionic fluxes across membranes. These findings complement previous pharmacologic observations that suggest that CaI-PLA2 may participate in regulating transmembrane ion flux in glucose-stimulated β-cells.

Human Pancreatic Islets Express mRNA Species Encoding Two Distinct Catalytically Active Isoforms of Group VI Phospholipase A2 (iPLA2) That Arise from an Exon-skipping Mechanism of Alternative Splicing of the Transcript from the iPLA2 Gene on Chromosome 22q13.1

An 85-kDa Group VI phospholipase A2 enzyme (iPLA2) that does not require Ca2+ for catalysis has recently been cloned from three rodent species. A homologous 88-kDa enzyme has been cloned from human B-lymphocyte lines that contains a 54-amino acid insert not present in the rodent enzymes, but human cells have not previously been observed to express catalytically active iPLA2 isoforms other than the 88-kDa protein. We have cloned cDNA species that encode two distinct iPLA2 isoforms from human pancreatic islet RNA and a human insulinoma cDNA library. One isoform is an 85-kDa protein (short isoform of human iPLA2 (SH-iPLA2)) and the other an 88-kDa protein (long isoform of human iPLA2(LH-iPLA2)). Transcripts encoding both isoforms are also observed in human promonocytic U937 cells. Recombinant SH-iPLA2 and LH-iPLA2 are both catalytically active in the absence of Ca2+ and inhibited by a bromoenol lactone suicide substrate, but LH-iPLA2 is activated by ATP, whereas SH-iPLA2 is not. The human iPLA2gene has been found to reside on chromosome 22 in region q13.1 and to contain 16 exons represented in the LH-iPLA2 transcript. Exon 8 is not represented in the SH-iPLA2 transcript, indicating that it arises by an exon-skipping mechanism of alternative splicing. The amino acid sequence encoded by exon 8 of the human iPLA2 gene is proline-rich and shares a consensus motif of PX 5PX 8HHPX 12NX 4Q with the proline-rich middle linker domains of the Smad proteins DAF-3 and Smad4. Expression of mRNA species encoding two active iPLA2 isoforms with distinguishable catalytic properties in two different types of human cells demonstrated here may have regulatory or functional implications about the roles of products of the iPLA2 gene in cell biologic processes.

The molecular biology of the group VIA Ca2+-independent phospholipase A2.

The group VIA PLA2 is a member of the PLA2 superfamily. This enzyme, which is cytosolic and Ca2+-independent, has been designated iPLA2beta to distinguish it from another recently cloned Ca2+-independent PLA2. Features of iPLA2beta molecular structure offer some insight into possible cellular functions of the enzyme. At least two catalytically active iPLA2beta isoforms and additionalsplicing variants are derived from a single gene that consists of at least 17 exons located on human chromosome 22q13.1. Potential tumor suppressor genes also reside at or near this locus. Structural analyses reveal that iPLA2beta contains unique structural features that include a serine lipase consensus motif (GXSXG), a putative ATP-binding domain, an ankyrin-repeat domain, a caspase-3 cleavage motif DVTD138Y/N, a bipartite nuclear localization signal sequence, and a proline-rich region in the human long isoform. iPLA2beta is widely expressed among mammalian tissues, with highest expression in testis and brain. iPLA2beta prefers to hydrolyze fatty acid at the sn-2 fatty acid substituent but also exhibits phospholipase A1, lysophospholipase, PAF acetylhydrolase, and transacylase activities. iPLA2beta may participate in signaling, apoptosis, membrane phospholipid remodeling, membrane homeostasis, arachidonate release, and exocytotic membrane fusion. Structural features and the existence of multiple splicing variants of iPLA2beta suggest that iPLA2beta may be subject to complex regulatory mechanisms that differ among cell types. Further study of its regulation and interaction with other proteins may yield insight into how its structural features are related to its function.

Cloning and expression of a group IV cytosolic Ca2+-dependent phospholipase A2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca2+-independent phospholipase A2

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may play signaling or effector roles in insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet beta-cells express low molecular weight secretory phospholipases A2 (PLA2) and a Group VI, Ca2+-independent PLA2 (iPLA2). Previous studies indicate that islets also express a protein recognized by antibodies against a Group IV, cytosolic, Ca2+-dependent PLA2 (cPLA2). To further examine the possible expression of cPLA2 by islets, we screened a rat islet cDNA library with a probe that recognizes cPLA2 sequence, and isolated a full-length cPLA2 cDNA. The rat islet cPLA2-deduced amino acid sequence is 96% identical to those of human and mouse cPLA2. Transfection of COS-7 cells with cPLA2 cDNA in an expression vector induced expression of Ca2+-dependent PLA2 activity and of a protein recognized by anti-cPLA2 antibody. Comparison of recombinant islet cPLA2 and iPLA2 activities expressed in transfected COS-7 cells indicated that iPLA2 but not cPLA2 is stimulated by ATP. Both activities are similarly sensitive to inhibition by arachidonyltrifluoromethyl ketone, but iPLA2 is more effectively inhibited by a haloenol lactone suicide substrate than cPLA2. RT-PCR experiments with RNA from purified islet beta-cells and from an alpha-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA2 mRNA is more abundant in the beta-cell population. Immunoblotting analyses indicate that islets express cPLA2-immunoreactive protein, and that interleukin-1 does not affect its expression. The cPLA2 is thus one of at least three classes of PLA2 enzymes with distinct properties expressed in beta-cells.

Interleukin-1 Enhances Pancreatic Islet Arachidonic Acid 12-Lipoxygenase Product Generation by Increasing Substrate Availability through a Nitric Oxide-dependent Mechanism

Interleukin-1 (IL-1) impairs insulin secretion from pancreatic islets and may contribute to the pathogenesis of insulin-dependent diabetes mellitus. IL-1 increases islet expression of nitric oxide (NO) synthase, and the resultant overproduction of NO participates in inhibition of insulin secretion because NO synthase inhibitors, e.g.NG-monomethyl-arginine (NMMA), prevent this inhibition. While exploring effects of IL-1 on islet arachidonic acid metabolism, we found that IL-1 increases islet production of the 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid 12-(HETE). This effect requires NO production and is prevented by NMMA. Exploration of the mechanism of this effect indicates that it involves increased availabilty of the substrate arachidonic acid rather than enhanced expression of 12-lipoxygenase. Evidence supporting this conclusion includes the facts that IL-1 does not increase islet 12-lipoxygenase protein or mRNA levels and does not enhance islet conversion of exogenous arachidonate to 12-HETE. Mass spectrometric stereochemical analyses nonetheless indicate that 12-HETE produced by IL-1-treated islets consists only of the S-enantiomer and thus arises from enzyme action. IL-1 does enhance release of nonesterified arachidonate from islets, as measured by isotope dilution mass spectrometry, and this effect is suppressed by NMMA and mimicked by the NO-releasing compound 3-morpholinosydnonimine. Although IL-1 increases neither islet phospholipase A2 (PLA2) activities nor mRNA levels for cytosolic or secretory PLA2, a suicide substrate which inhibits an islet Ca2+-independent PLA2 prevents enhancement of islet arachidonate release by IL-1. IL-1 also impairs esterification of [3H8]arachidonate into islet phospholipids, and this effect is prevented by NMMA and mimicked by the mitochondrial ATP-synthase inhibitor oligomycin. Experiments with exogenous substrates indicate that NMMA does not inhibit and that the NO-releasing compound does not activate islet 12-lipoxygenase or PLA2 activities. These results indicate that a novel action of NO is to increase levels of nonesterified arachidonic acid in islets.

Ca2+-independent phospholipase A2 is required for agonist-induced Ca2+ sensitization of contraction in vascular smooth muscle.

Excitatory agonists can induce significant smooth muscle contraction under constant free Ca2+ through a mechanism called Ca2+ sensitization. Considerable evidence suggests that free arachidonic acid plays an important role in mediating agonist-induced Ca2+-sensitization; however, the molecular mechanisms responsible for maintaining and regulating free arachidonic acid level are not completely understood. In the current study, we demonstrated that Ca2+-independent phospholipase A2 (iPLA2) is expressed in vascular smooth muscle tissues. Inhibition of the endogenous iPLA2 activity by bromoenol lactone (BEL) decreases basal free arachidonic acid levels and reduces the final free arachidonic acid level after phenylephrine stimulation, without significant effect on the net increase in free arachidonic acid stimulated by phenylephrine. Importantly, BEL treatment diminishes agonist-induced Ca2+ sensitization of contraction from 49 ± 3.6 to 12 ± 1.0% (p< 0.01). In contrast, BEL does not affect agonist-induced diacylglycerol production or contraction induced by Ca2+, phorbol 12,13-dibutyrate (a protein kinase C activator), or exogenous arachidonic acid. Further, we demonstrate that adenovirus-mediated overexpression of exogenous iPLA2 in mouse portal vein tissue significantly potentiates serotonin-induced contraction. Our data provide the first evidence that iPLA2 is required for maintaining basal free arachidonic acid levels and thus is essential for agonist-induced Ca2+-sensitization of contraction in vascular smooth muscle.

Studies of phospholipid metabolism, proliferation, and secretion of stably transfected insulinoma cells that overexpress group VIA phospholipase A2

A cytosolic 84 kDa Group VIA phospholipase A2 (iPLA2β) that does not require Ca2+ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet β-cells, and other sources. Proposed iPLA2β functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (IPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet β-cells. To further examine iPLA2β functions in β-cells, we prepared stably transfected INS-1 insulinoma cell lines that overexpress iPLA2β activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPIA2β cDNA. The iPLA2β-overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [3H] choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA2β-overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [3H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA2β. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA2β-overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA2β-overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA2β plays a signaling role in β-cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.

Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion.

Insulin secretion by pancreatic islet beta-cells is impaired in diabetes mellitus, and normal beta-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on beta-cell phospholipid composition and secretory responses could be involved in changes in beta-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.

Type IB secretory phospholipase A2 is contained in insulin secretory granules of pancreatic islet β-cells and is co-secreted with insulin from glucose-stimulated islets

Stimulation of pancreatic islets with d-glucose induces insulin secretion from secretory granules contained within the islet beta-cells. Accumulating evidence suggests that secretory phospholipases A2 (sPLA2) may play a role in the distal events of secretory processes in many different cell types. Since intact pancreatic islets have been reported to contain sPLA2, it was of interest to determine the cellular and subcellular localization of the sPLA2 enzymes in pancreatic islets. Our findings indicate that rat pancreatic islets express mRNA for both types IB and IIA sPLA2 enzymes and mRNA for an sPLA2 membrane receptor. Immunoblotting analyses with antibodies directed against type IB sPLA2 or against type IIA sPLA2 indicate that the type IB isoform is much more abundant than the type IIA isoform in islets. Studies with purified populations of islet beta-cells prepared from dispersed islet cells by fluorescence-activated cell sorting indicate that both sPLA2 activity and type IB sPLA2 immunoreactive protein are substantially more abundant in beta-cells than in non-beta-cells. Subcellular fractionation studies indicate that sPLA2 activity and type IB sPLA2 immunoreactive protein are contained in insulin secretory granules. Stimulation of intact islets with insulin secretagogues results in the co-secretion of insulin and of sPLA2 activity and type IB sPLA2 immunoreactive protein into the incubation medium. These findings raise the possibility that type IB sPLA2 participates in the secretory process of pancreatic islet beta-cells.