The expression and function of a group VIA calcium-independent phospholipase A2 (iPLA2beta) in beta-cells

Many cells express a Group VIA phospholipase A2, designated iPLA2beta, that does not require calcium for activation, is stimulated by ATP, and is sensitive to inhibition by a bromoenol lactone suicide substrate (BEL). Studies in various cell systems have led to the suggestion that iPLA2beta has a role in phospholipid remodeling, signal transduction, cell proliferation, and apoptosis. We have found that pancreatic islets, beta-cells, and glucose-responsive insulinoma cells express an iPLA2beta that participates in glucose-stimulated insulin secretion but is not involved in membrane phospholipid remodeling. Additionally, recent studies reveal that iPLA2beta is involved in pathways that contribute to beta-cell proliferation and apoptosis, and that various phospholipid-derived mediators are involved in these processes. Detailed characterization of the enzyme suggests that the beta-cells express multiple isoforms of iPLA2beta, and we hypothesize that these participate in different cellular functions.     

Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.     

Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2 beta) and suppressed by inhibition of iPLA2 beta

The death of insulin-secreting beta-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive beta-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to beta-cell loss in diabetes. Agents that deplete ER calcium levels induce beta-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)beta). Overexpression of iPLA(2)beta 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)beta expression in various cell lines, and apoptosis is associated with stimulation of iPLA(2)beta activity, perinuclear accumulation of iPLA(2)beta protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA(2)beta 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)beta. These findings indicate that iPLA(2)beta participates in ER stress-induced apoptosis, a pathway that promotes beta-cell death in diabetes.     

Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma)

Although human group VIB calcium-independent phospholipase A(2) (iPLA(2)gamma) contains the lipase-consensus sequence Gly-Xaa-Ser-Xaa-Gly in the C-terminal half, its overall sequence exhibits a week similarity to those of other PLA(2)s, and thus no information on the catalytic site has been available. Here we show that the C-terminal region of human iPLA(2)gamma is responsible for the enzymatic activity. Comparison of this catalytic domain with those of the mouse homologue, human cytosolic PLA(2) (cPLA(2)), and the plant PLA(2) patatin reveals that an amino acid sequence of a short segment around Asp-627 of iPLA(2)gamma is conserved among these PLA(2)s, in addition to the Ser-483-containing lipase motif; the corresponding serine and aspartate in cPLA(2) and patatin are known to form a catalytic dyad. Since substitution of alanine for either Ser-483 or Asp-627 results in a loss of the PLA(2) activity, we propose that Ser-483 and Asp-627 of human iPLA(2)gamma constitute an active site similar to the Ser-Asp dyad in cPLA(2) and patatin.     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.     

Identification of calcium-independent phospholipase A2 (iPLA2) beta,and not iPLA2gamma,as the mediator of arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells. Enantioselective mechanism-based discrimination of mammalian iPLA2s

The agonist-stimulated release of arachidonic acid (AA) from cellular phospholipids in many cell types (e.g. myocytes, beta-cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A(2) (iPLA(2)s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA(2)s has been extended to include iPLA(2)gamma, which previously could not be pharmacologically distinguished from iPLA(2)beta. To determine whether iPLA(2)beta or iPLA(2)gamma (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA(2)beta and iPLA(2)gamma in intact cells. We hypothesized that the R- and S-enantiomers of BEL would possess different inhibitory potencies for iPLA(2)beta and iPLA(2)gamma. Accordingly, racemic BEL was separated into its enantiomeric constituents by chiral high pressure liquid chromatography. Remarkably, (S)-BEL was approximately an order of magnitude more selective for iPLA(2)beta in comparison to iPLA(2)gamma. Conversely, (R)-BEL was approximately an order of magnitude more selective for iPLA(2)gamma than iPLA(2)beta. The AVP-induced liberation of AA from A-10 cells was selectively inhibited by (S)-BEL (IC(50) approximately 2 microm) but not (R)-BEL, demonstrating that the overwhelming majority of AA release is because of iPLA(2)beta and not iPLA(2)gamma activity. Furthermore, pretreatment of A-10 cells with (S)-BEL did not prevent AVP-induced MAPK phosphorylation or protein kinase C translocation. Finally, two different cell-permeable protein kinase C activators (phorbol-12-myristate-13-acetate and 1,2-dioctanoyl-sn-glycerol) could not restore the ability of A-10 cells to release AA after exposure to (S)-BEL, thus supporting the downstream role of iPLA(2)beta in AVP-induced AA release.     

Leptin augments alveolar macrophage leukotriene synthesis by increasing phospholipase activity and enhancing group IVC iPLA2 (cPLA2gamma) protein expression

Leptin is a hormone secreted by adipocytes in correlation with total body fat mass. In addition to regulating energy homeostasis, leptin modulates immune functions such as macrophage phagocytosis and cytokine synthesis. Previously, we reported defective leukotriene synthesis in macrophages from leptin-deficient mice that could be restored with exogenous leptin. In the present study, we utilized macrophages from normal rodents to explore the mechanism by which leptin could enhance cellular leukotriene synthesis. Leptin pretreatment of either rat alveolar or murine peritoneal macrophages for 16 h dose dependently increased the synthesis of leukotriene B4 and cysteinyl leukotrienes in response to calcium ionophore or the particulate zymosan. Leptin also enhanced calcium ionophore-stimulated release of free arachidonic acid. Calcium-dependent and -independent arachidonoyl-selective phospholipase activities in macrophage lysates were likewise increased following leptin treatment. Immunoblot analysis of leptin-treated cells revealed that group IVC iPLA2 (cPLA2gamma) protein expression increased approximately 80%. These data demonstrate for the first time that phospholipase A2 activity and cPLA2gamma protein levels in alveolar macrophages represent targets for upregulation by leptin and provide previously unrecognized mechanisms by which this hormone can promote inflammatory responses.     

Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid (TDGA) on glucose and fatty acid oxidation in isolated rat soleus muscle

1. The effect of 2-tetradecylglycidic acid (TDGA), a potent, specific inhibitor of long-chain fatty acid oxidation, on fatty acid and glucose oxidation by isolated rat soleus muscle was studied. 2. TDGA inhibited [1-14C]palmitate oxidation by soleus muscle in a concentration-dependent manner. 3. TDGA inhibited the activity of soleus muscle mitochondrial carnitine palmitoyltransferase A (CPT-A). 4. Added palmitate (0.5 mM) significantly inhibited D-[U-14C]glucose oxidation and, under conditions where TDGA inhibited palmitate oxidation, the oxidation of D-[U-14C]glucose by isolated soleus muscle was significantly stimulated. 5. TDGA stimulation of glucose oxidation was reversed by octanoate, a medium-chain fatty acid whose oxidation is not inhibited by TDGA. 6. When nondiabetic rats were treated with TDGA (10 mg/kg p.o./day x 3 days), fasting plasma glucose was significantly lowered and the ability of isolated contralateral soleus muscles to oxidize palmitate was inhibited while glucose oxidation was significantly stimulated.     

Cloning and recombinant expression of human group IIF-secreted phospholipase A(2)

Mammalian-secreted phospholipases A(2) (sPLA(2)) form a diverse family of at least nine enzymes that hydrolyze phospholipids to release free fatty acids and lysophospholipids. We report here the cloning and characterization of human group IIF sPLA(2) (hGIIF sPLA(2)). The full-length cDNA codes for a signal peptide of 20 amino acid followed by a mature protein of 148 amino acids containing all of the structural features of catalytically active group II sPLA(2)s. hGIIF sPLA(2) gene is located on chromosome 1 and lies within a sPLA(2) gene cluster of about 300 kbp that also contains the genes for group IIA, IIC, IID, IIE, and V sPLA(2)s. In adult tissues, hGIIF is highly expressed in placenta, testis, thymus, liver, and kidney. Finally, recombinant expression of hGIIF sPLA(2) in Escherichia coli shows that the enzyme is Ca(2+)-dependent, maximally active at pH 7-8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference.     

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 muM) palmitate oxidation was decreased by 95% (P<0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P<0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.     

Calcium-independent phospholipase A2 (iPLA2 beta)-mediated ceramide generation plays a key role in the cross-talk between the endoplasmic reticulum (ER) and mitochondria during ER stress-induced insulin-secreting cell apoptosis

Endoplasmic reticulum (ER) stress induces INS-1 cell apoptosis by a pathway involving Ca(2+)-independent phospholipase A(2) (iPLA(2)beta)-mediated ceramide generation, but the mechanism by which iPLA(2)beta and ceramides contribute to apoptosis is not well understood. We report here that both caspase-12 and caspase-3 are activated in INS-1 cells following induction of ER stress with thapsigargin, but only caspase-3 cleavage is amplified in iPLA(2)beta overexpressing INS-1 cells (OE), relative to empty vector-transfected cells, and is suppressed by iPLA(2)beta inhibition. ER stress also led to the release of cytochrome c and Smac and, unexpectedly, their accumulation in the cytosol is amplified in OE cells. These findings raise the likelihood that iPLA(2)beta participates in ER stress-induced apoptosis by activating the intrinsic apoptotic pathway. Consistent with this possibility, we find that ER stress promotes iPLA(2)beta accumulation in the mitochondria, opening of mitochondrial permeability transition pore, and loss in mitochondrial membrane potential (Delta Psi) in INS-1 cells and that these changes are amplified in OE cells. ER stress also led to greater ceramide generation in ER and mitochondria fractions of OE cells. Exposure to ceramide alone induces loss in Delta Psi and apoptosis and these are suppressed by forskolin. ER stress-induced mitochondrial dysfunction and apoptosis are also inhibited by forskolin, as well as by inactivation of iPLA(2)beta or NSMase, suggesting that iPLA(2)beta-mediated generation of ceramides via sphingomyelin hydrolysis during ER stress affect the mitochondria. In support, inhibition of iPLA(2)beta or NSMase prevents cytochrome c release. Collectively, our findings indicate that the iPLA(2)beta-ceramide axis plays a critical role in activating the mitochondrial apoptotic pathway in insulin-secreting cells during ER stress.     

Role of group VIA calcium-independent phospholipase A2 in arachidonic acid release, phospholipid fatty acid incorporation, and apoptosis in U937 cells responding to hydrogen peroxide

Group VIA calcium-independent phospholipase A2 (iPLA2) has been shown to play a major role in regulating basal phospholipid deacylation reactions in certain cell types. More recently, roles for this enzyme have also been suggested in the destruction of membrane phospholipid during apoptosis and after oxidant injury. Proposed iPLA2 roles have rested heavily on the use of bromoenol lactone as an iPLA2-specific inhibitor, but this compound actually inhibits other enzymes and lipid pathways unrelated to PLA2, which makes it difficult to define the contribution of iPLA2 to specific functions. In previous work, we pioneered the use of antisense technology to decrease cellular iPLA2 activity as an alternative approach to study iPLA2 functions. In the present study, we followed the opposite strategy and prepared U937 cells that exhibited enhanced iPLA activity by stably expressing a plasmid containing iPLA2 cDNA. Compared with control cells, the iPLA2 -overexpressing U937 cells showed elevated responses to hydrogen peroxide with regard to both arachidonic acid mobilization and incorporation of the fatty acid into phospholipids, thus providing additional evidence for the key role that iPLA2 plays in these events. Long-term exposure of the cells to hydrogen peroxide resulted in cell death by apoptosis, and this process was accelerated in the iPLA2-overexpressing cells. Increased phospholipid hydrolysis and fatty acid release also occurred in these cells. Unexpectedly, however, abrogation of U937 cell iPLA2 activity by either methyl arachidonyl fluorophosphonate or an antisense oligonucleotide did not delay or decrease the extent of apoptosis induced by hydrogen peroxide. These results indicate that, although iPLA2-mediated phospholipid hydrolysis occurs during apoptosis, iPLA2 may actually be dispensable for the apoptotic process to occur. Thus, beyond a mere destructive role, iPLA2 may play other roles during apoptosis.     

Glycogen synthase kinase-3beta negatively regulates group IIA phospholipase A2 expression in human aortic smooth muscle and HepG2 hepatoma cells

The present study shows that the IFN-gamma-mediated upregulation of secretory phospholipase A2 of group IIA (sPLA2-IIA) in HASMC and HepG2 cells is synergistically increased after simultaneous inhibition of glycogen synthase kinase-3beta (GSK-3beta) by indirubin-3'-monoxime, 5-iodo or AR-A014418. The effect of GSK-3beta inhibition was dose- and time-dependent and can be further augmented by its concomitant incubation with Clostridium difficile toxin B, an inhibitor of small Rho proteins, or H-1152, an inhibitor of Rho-associated kinase. Using AG-490 and caffeic acid phenethyl ester (CAPE), it is further demonstrated that the effect of GSK-3beta inhibition on sPLA2-IIA expression depends on Janus kinase-2 and NF-kappaB-signaling.     

Proinflammatory treatment of astrocytes with lipopolysaccharide results in augmented Ca2+ signaling through increased expression of via phospholipase A2 (iPLA2)

Many Ca(2+)-regulated intracellular processes are involved in the development of neuroinflammation. However, the changes of Ca(2+) signaling in the brain under inflammatory conditions were hardly studied. ATP-induced Ca(2+) signaling is a central event of signal transmission in astrocytic networks. We investigated primary astrocytes after proinflammatory stimulation with lipopolysaccharide (LPS; 100 ng/ml) for 6-24 h. We reveal that Ca(2+) responses to purinergic ATP stimulation are significantly increased in amplitude and duration after stimulation with LPS. We detected that increased amplitudes of Ca(2+) responses to ATP in LPS-treated astrocytes can be explained by substantial increase of Ca(2+) load in stores in endoplasmic reticulum. The mechanism implies enhanced Ca(2+) store refilling due to the amplification of capacitative Ca(2+) entry. The reason for the increased duration of Ca(2+) responses in LPS-treated cells is also the amplified capacitative Ca(2+) entry. Next, we established that the molecular mechanism for the LPS-induced amplification of Ca(2+) responses in astrocytes is increased expression and activity of VIA phospholipase A(2) (VIA iPLA(2)). Indeed, both gene silencing with specific small interfering RNA and pharmacological inhibition of VIA iPLA(2) with S-bromoenol lactone reduced the load of the Ca(2+) stores and caused a decrease in the amplitudes of Ca(2+) responses in LPS-treated astrocytes to values, which were comparable with those in untreated cells. Our findings highlight a novel regulatory role of VIA iPLA(2) in development of inflammation in brain. We suggest that this enzyme might be a possible target for treatment of pathologies related to brain inflammation.     

Fatty acid oxidation and myocardial phospholipase A2 activity

Summary Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A₂ activity (PLA₂) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca²⁺-dependent PLA₂ was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA, activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC₅₀ for arachidonic acid was approx 65 M. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37°C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA₂ inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA₂s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury.     

Skeletal muscle fatty acid handling in insulin resistant men

Disturbances in skeletal muscle lipid metabolism may precede or contribute to the development of whole body insulin resistance. In this study, we examined fasting and postprandial skeletal muscle fatty acid (FA) handling in insulin resistant (IR) men. Thirty men with the metabolic syndrome (MetS) (National Cholesterol Education Program-Adult Treatment Panel III) were included in this sub-study to the LIPGENE study, and divided in two groups (IR and control) based on the median of insulin sensitivity (S(I) = 2.06 (mU/l(-1))·min(-1)·10(-4)). Fasting and postprandial skeletal muscle FA handling were examined by combining the forearm balance technique with stable isotopes of palmitate. [(2)H(2)]-palmitate was infused intravenously to label endogenous triacylglycerol (TAG) and free FAs (FFAs) in the circulation and [U-(13)C]-palmitate was incorporated in a high-fat mixed meal (2.6 MJ, 61 E% fat) to label chylomicron-TAG. Muscle biopsies were taken to determine muscle TAG, diacylglycerol (DAG), FFA, and phospholipid (PL) content, their fractional synthetic rates (FSRs) and degree of saturation, as well as messenger RNA (mRNA) expression of genes involved in lipid metabolism. In the first 2 h after meal consumption, forearm muscle [(2)H(2)]-labeled TAG extraction was higher in IR vs. control (P = 0.05). Fasting percentage saturation of muscle DAG was higher in IR vs. control (P = 0.016). No differences were observed for intramuscular TAG, DAG, FFA, and PL content, FSR, and muscle mRNA expression. In conclusion, increased muscle (hepatically derived) TAG extraction during postprandial conditions and increased saturation of intramuscular DAG are associated with insulin resistance, suggesting that disturbances in skeletal muscle FA handling could play a role in the development of whole body insulin resistance and type 2 diabetes.