Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

Enterically derived high-density lipoprotein restrains liver injury through the portal vein.

The biogenesis of high-density lipoprotein (HDL) requires apoA1 and the cholesterol transporter ABCA1. Although the liver generates most of the HDL in the blood, HDL synthesis also occurs in the small intestine. Here, we show that intestine-derived HDL traverses the portal vein in the HDL3 subspecies form, in complex with lipopolysaccharide (LPS)-binding protein (LBP). HDL3, but not HDL2 or low-density lipoprotein, prevented LPS binding to and inflammatory activation of liver macrophages and instead supported extracellular inactivation of LPS. In mouse models involving surgical, dietary, or alcoholic intestinal insult, loss of intestine-derived HDL worsened liver injury, whereas outcomes were improved by therapeutics that elevated and depended upon raising intestinal HDL. Thus, protection of the liver from injury in response to gut-derived LPS is a major function of intestinally synthesized HDL.

Metabolic Effects of Selective Deletion of Group VIA Phospholipase A2 from Macrophages or Pancreatic Islet Beta-Cells.

To examine the role of group VIA phospholipase A2 (iPLA2ß) in specific cell lineages in insulin secretion and insulin action, we prepared mice with a selective iPLA2ß deficiency in cells of myelomonocytic lineage, including macrophages (MØ-iPLA2ß-KO), or in insulin-secreting ß-cells (ß-Cell-iPLA2ß-KO), respectively. MØ-iPLA2ß-KO mice exhibited normal glucose tolerance when fed standard chow and better glucose tolerance than floxed-iPLA2ß control mice after consuming a high-fat diet (HFD). MØ-iPLA2ß-KO mice exhibited normal glucose-stimulated insulin secretion (GSIS) in vivo and from isolated islets ex vivo compared to controls. Male MØ-iPLA2ß-KO mice exhibited enhanced insulin responsivity vs. controls after a prolonged HFD. In contrast, ß-cell-iPLA2ß-KO mice exhibited impaired glucose tolerance when fed standard chow, and glucose tolerance deteriorated further when introduced to a HFD. ß-Cell-iPLA2ß-KO mice exhibited impaired GSIS in vivo and from isolated islets ex vivo vs. controls. ß-Cell-iPLA2ß-KO mice also exhibited an enhanced insulin responsivity compared to controls. These findings suggest that MØ iPLA2ß participates in HFD-induced deterioration in glucose tolerance and that this mainly reflects an effect on insulin responsivity rather than on insulin secretion. In contrast, ß-cell iPLA2ß plays a role in GSIS and also appears to confer some protection against deterioration in ß-cell functions induced by a HFD.

Limited proliferation capacity of aortic intima resident macrophages requires monocyte recruitment for atherosclerotic plaque progression.

Early atherosclerosis depends upon responses by immune cells resident in the intimal aortic wall. Specifically, the healthy intima is thought to be populated by vascular dendritic cells (DCs) that, during hypercholesterolemia, initiate atherosclerosis by being the first to accumulate cholesterol. Whether these cells remain key players in later stages of disease is unknown. Using murine lineage-tracing models and gene expression profiling, we reveal that myeloid cells present in the intima of the aortic arch are not DCs but instead specialized aortic intima resident macrophages (MacAIR) that depend upon colony-stimulating factor 1 and are sustained by local proliferation. Although MacAIR comprise the earliest foam cells in plaques, their proliferation during plaque progression is limited. After months of hypercholesterolemia, their presence in plaques is overtaken by recruited monocytes, which induce MacAIR-defining genes. These data redefine the lineage of intimal phagocytes and suggest that proliferation is insufficient to sustain generations of macrophages during plaque progression.

B Cell-Mediated Antigen Presentation through MHC Class II Is Dispensable for Atherosclerosis Progression.

Depletion of B cells attenuates plaque development and modulates T cell responses in mouse models of atherosclerosis, suggesting that Ag presentation by B cells may promote disease progression. Thus, we set out to determine the role of B cell-mediated MHC class II (MHC II) Ag presentation during atherosclerotic plaque development. We developed murine conditional MHC II deletion and expression systems under control of the B cell-restricted CD19 promoter in an experimental model of atherosclerosis. Mice lacking MHC II expression only on B cells exhibited systemic shifts in germinal center and marginal zone B cell populations, leading to a reduced Ab response compared with littermate control animals. However, all populations were present and normal cholesterol uptake was detected in the plasma following high-fat diet treatment. In a second model, in which conditional expression of MHC II is limited only to B cells, showed similar overall cellularity characteristics compared with mice with complete MHC II deficiency. High-fat diet feeding showed no major changes in atherosclerotic plaque size or plaque cellular content in either conditional deletion or conditional expression approaches, compared with control animals. By testing the necessity and sufficiency of MHC II on B cells in the progression of atherosclerosis, we determine that MHC II on B cells does not directly regulate lesion development in murine models.

Polarization of Macrophages toward M2 Phenotype Is Favored by Reduction in iPLA2ß (Group VIA Phospholipase A2).

Macrophages are important in innate and adaptive immunity. Macrophage participation in inflammation or tissue repair is directed by various extracellular signals and mediated by multiple intracellular pathways. Activation of group VIA phospholipase A2 (iPLA2ß) causes accumulation of arachidonic acid, lysophospholipids, and eicosanoids that can promote inflammation and pathologic states. We examined the role of iPLA2ß in peritoneal macrophage immune function by comparing wild type (WT) and iPLA2ß-/- mouse macrophages. Compared with WT, iPLA2ß-/- macrophages exhibited reduced proinflammatory M1 markers when classically activated. In contrast, anti-inflammatory M2 markers were elevated under naïve conditions and induced to higher levels by alternative activation in iPLA2ß-/- macrophages compared with WT. Induction of eicosanoid (12-lipoxygenase (12-LO) and cyclooxygenase 2 (COX2))- and reactive oxygen species (NADPH oxidase 4 (NOX4))-generating enzymes by classical activation pathways was also blunted in iPLA2ß-/- macrophages compared with WT. The effects of inhibitors of iPLA2ß, COX2, or 12-LO to reduce M1 polarization were greater than those to enhance M2 polarization. Certain lipids (lysophosphatidylcholine, lysophosphatidic acid, and prostaglandin E2) recapitulated M1 phenotype in iPLA2ß-/- macrophages, but none tested promoted M2 phenotype. These findings suggest that (a) lipids generated by iPLA2ß and subsequently oxidized by cyclooxygenase and 12-LO favor macrophage inflammatory M1 polarization, and (b) the absence of iPLA2ß promotes macrophage M2 polarization. Reducing macrophage iPLA2ß activity and thereby attenuating macrophage M1 polarization might cause a shift from an inflammatory to a recovery/repair milieu.

Group VIA phospholipase A2 mitigates palmitate-induced ß-cell mitochondrial injury and apoptosis.

Palmitate (C16:0) induces apoptosis of insulin-secreting ß-cells by processes that involve generation of reactive oxygen species, and chronically elevated blood long chain free fatty acid levels are thought to contribute to ß-cell lipotoxicity and the development of diabetes mellitus. Group VIA phospholipase A2 (iPLA2ß) affects ß-cell sensitivity to apoptosis, and here we examined iPLA2ß effects on events that occur in ß-cells incubated with C16:0. Such events in INS-1 insulinoma cells were found to include activation of caspase-3, expression of stress response genes (C/EBP homologous protein and activating transcription factor 4), accumulation of ceramide, loss of mitochondrial membrane potential, and apoptosis. All of these responses were blunted in INS-1 cells that overexpress iPLA2ß, which has been proposed to facilitate repair of oxidized mitochondrial phospholipids, e.g. cardiolipin (CL), by excising oxidized polyunsaturated fatty acid residues, e.g. linoleate (C18:2), to yield lysophospholipids, e.g. monolysocardiolipin (MLCL), that can be reacylated to regenerate the native phospholipid structures. Here the MLCL content of mouse pancreatic islets was found to rise with increasing iPLA2ß expression, and recombinant iPLA2ß hydrolyzed CL to MLCL and released oxygenated C18:2 residues from oxidized CL in preference to native C18:2. C16:0 induced accumulation of oxidized CL species and of the oxidized phospholipid (C18:0/hydroxyeicosatetraenoic acid)-glycerophosphoethanolamine, and these effects were blunted in INS-1 cells that overexpress iPLA2ß, consistent with iPLA2ß-mediated removal of oxidized phospholipids. C16:0 also induced iPLA2ß association with INS-1 cell mitochondria, consistent with a role in mitochondrial repair. These findings indicate that iPLA2ß confers significant protection of ß-cells against C16:0-induced injury.

Genetic modulation of islet ß-cell iPLA2ß expression provides evidence for its impact on ß-cell apoptosis and autophagy.

ß-cell apoptosis is a significant contributor to ß-cell dysfunction in diabetes and ER stress is among the factors that contributes to ß-cell death. We previously identified that the Ca²âº-independent phospholipase A2ß (iPLA2ß), which in islets is localized in ß-cells, participates in ER stress-induced ß-cell apoptosis. Here, direct assessment of iPLA2ß role was made using ß-cell-specific iPLA2ß overexpressing (RIP-iPLA2ß-Tg) and globally iPLA2ß-deficient (iPLA2ß-KO) mice. Islets from Tg, but not KO, express higher islet iPLA2ß and neutral sphingomyelinase, decrease in sphingomyelins, and increase in ceramides, relative to WT group. ER stress induces iPLA2ß, ER stress factors, loss of mitochondrial membrane potential (∆Ψ), caspase-3 activation, and ß-cell apoptosis in the WT and these are all amplified in the Tg group. Surprisingly, ß-cells apoptosis while reduced in the KO is higher than in the WT group. This, however, was not accompanied by greater caspase-3 activation but with larger loss of ∆Ψ, suggesting that iPLA2ß deficiency impacts mitochondrial membrane integrity and causes apoptosis by a caspase-independent manner. Further, autophagy, as reflected by LC3-II accumulation, is increased in Tg and decreased in KO, relative to WT. Our findings suggest that (1) iPLA2ß impacts upstream (UPR) and downstream (ceramide generation and mitochondrial) pathways in ß-cells and (2) both over- or under-expression of iPLA2ß is deleterious to the ß-cells. Further, we present for the first time evidence for potential regulation of autophagy by iPLA2ß in islet ß-cells. These findings support the hypothesis that iPLA2ß induction under stress, as in diabetes, is a key component to amplifying ß-cell death processes.

Group VIB Phospholipase A(2) promotes proliferation of INS-1 insulinoma cells and attenuates lipid peroxidation and apoptosis induced by inflammatory cytokines and oxidant agents.

Group VIB Phospholipase A(2) (iPLA(2)γ) is distributed in membranous organelles in which ß-oxidation occurs, that is, mitochondria and peroxisomes, and is expressed by insulin-secreting pancreatic islet ß-cells and INS-1 insulinoma cells, which can be injured by inflammatory cytokines, for example, IL-1ß and IFN-γ, and by oxidants, for example, streptozotocin (STZ) or t-butyl-hydroperoxide (TBHP), via processes pertinent to mechanisms of ß-cell loss in types 1 and 2 diabetes mellitus. We find that incubating INS-1 cells with IL-1ß and IFN-γ, with STZ, or with TBHP causes increased expression of iPLA(2)γ mRNA and protein. We prepared INS-1 knockdown (KD) cell lines with reduced iPLA(2)γ expression, and they proliferate more slowly than control INS-1 cells and undergo increased membrane peroxidation in response to cytokines or oxidants. Accumulation of oxidized phospholipid molecular species in STZ-treated INS-1 cells was demonstrated by LC/MS/MS scanning, and the levels in iPLA(2)γ-KD cells exceeded those in control cells. iPLA(2)γ-KD INS-1 cells also exhibited higher levels of apoptosis than control cells when incubated with STZ or with IL-1ß and IFN-γ. These findings suggest that iPLA(2)γ promotes ß-cell proliferation and that its expression is increased during inflammation or oxidative stress as a mechanism to mitigate membrane injury that may enhance ß-cell survival.

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A(2)-VIA (iPLA(2)ß)-knockout mice.

Calcium-independent phospholipase A(2) group VIA (iPLA(2)ß) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA(2)ß gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA(2)ß in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA(2)ß(+/+)) and knockout (iPLA(2)ß(-/-)) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA(2), cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA(2)ß(+/+) mice, iPLA(2)ß(-/-) mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA(2)ß(-/-) mice, brain levels of iPLA(2)ß mRNA, protein, and activity were decreased, as was the iPLA(2)γ (Group VIB PLA(2)) mRNA level, while levels of secretory sPLA(2)-V mRNA, protein, and activity and cytosolic cPLA(2)-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA(2)ß deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.

Group VIA PLA2 (iPLA2ß) is activated upstream of p38 mitogen-activated protein kinase (MAPK) in pancreatic islet ß-cell signaling.

Group VIA phospholipase A(2) (iPLA(2)ß) in pancreatic islet ß-cells participates in glucose-stimulated insulin secretion and sarco(endo)plasmic reticulum ATPase (SERCA) inhibitor-induced apoptosis, and both are attenuated by pharmacologic or genetic reductions in iPLA(2)ß activity and amplified by iPLA(2)ß overexpression. While exploring signaling events that occur downstream of iPLA(2)ß activation, we found that p38 MAPK is activated by phosphorylation in INS-1 insulinoma cells and mouse pancreatic islets, that this increases with iPLA(2)ß expression level, and that it is stimulated by the iPLA(2)ß reaction product arachidonic acid. The insulin secretagogue D-glucose also stimulates ß-cell p38 MAPK phosphorylation, and this is prevented by the iPLA(2)ß inhibitor bromoenol lactone. Insulin secretion induced by d-glucose and forskolin is amplified by overexpressing iPLA(2)ß in INS-1 cells and in mouse islets, and the p38 MAPK inhibitor PD169316 prevents both responses. The SERCA inhibitor thapsigargin also stimulates phosphorylation of both ß-cell MAPK kinase isoforms and p38 MAPK, and bromoenol lactone prevents both events. Others have reported that iPLA(2)ß products activate Rho family G-proteins that promote MAPK kinase activation via a mechanism inhibited by Clostridium difficile toxin B, which we find to inhibit thapsigargin-induced ß-cell p38 MAPK phosphorylation. Thapsigargin-induced ß-cell apoptosis and ceramide generation are also prevented by the p38 MAPK inhibitor PD169316. These observations indicate that p38 MAPK is activated downstream of iPLA(2)ß in ß-cells incubated with insulin secretagogues or thapsigargin, that this requires prior iPLA(2)ß activation, and that p38 MAPK is involved in the ß-cell functional responses of insulin secretion and apoptosis in which iPLA(2)ß participates.

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA(2)ß (VIA)-deficient mice.

Ca(2+)-independent phospholipase A(2)ß (iPLA(2)ß) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA(2)ß-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M(1,3,5) receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA(2)ß(-/-), iPLA(2)ß(+/-), and iPLA(2)ß(+/+) mice, and [1-(14)C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J(in), representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA(2)ß(-/-) or iPLA(2)ß(+/-) compared with iPLA(2)ß(+/+) mice showed widespread and significant baseline reductions in k* and J(in) for DHA. Arecoline increased both parameters in brain regions of iPLA(2)ß(+/+) mice but quantitatively less so in iPLA(2)ß(-/-) and iPLA(2)ß(+/-) mice. Consistent with iPLA(2)ß's reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA(2)ß deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M(1,3,5) receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.

Effects of endoplasmic reticulum stress on group VIA phospholipase A2 in beta cells include tyrosine phosphorylation and increased association with calnexin.

The Group VIA phospholipase A(2) (iPLA(2)ß) hydrolyzes glycerophospholipids at the sn-2-position to yield a free fatty acid and a 2-lysophospholipid, and iPLA(2)ß has been reported to participate in apoptosis, phospholipid remodeling, insulin secretion, transcriptional regulation, and other processes. Induction of endoplasmic reticulum (ER) stress in ß-cells and vascular myocytes with SERCA inhibitors activates iPLA(2)ß, resulting in hydrolysis of arachidonic acid from membrane phospholipids, by a mechanism that is not well understood. Regulatory proteins interact with iPLA(2)ß, including the Ca(2+)/calmodulin-dependent protein kinase IIß, and we have characterized the iPLA(2)ß interactome further using affinity capture and LC/electrospray ionization/MS/MS. An iPLA(2)ß-FLAG fusion protein was expressed in an INS-1 insulinoma cell line and then adsorbed to an anti-FLAG matrix after cell lysis. iPLA(2)ß and any associated proteins were then displaced with FLAG peptide and analyzed by SDS-PAGE. Gel sections were digested with trypsin, and the resultant peptide mixtures were analyzed by LC/MS/MS with database searching. This identified 37 proteins that associate with iPLA(2)ß, and nearly half of them reside in ER or mitochondria. They include the ER chaperone calnexin, whose association with iPLA(2)ß increases upon induction of ER stress. Phosphorylation of iPLA(2)ß at Tyr(616) also occurs upon induction of ER stress, and the phosphoprotein associates with calnexin. The activity of iPLA(2)ß in vitro increases upon co-incubation with calnexin, and overexpression of calnexin in INS-1 cells results in augmentation of ER stress-induced, iPLA(2)ß-catalyzed hydrolysis of arachidonic acid from membrane phospholipids, reflecting the functional significance of the interaction. Similar results were obtained with mouse pancreatic islets.

Mice deficient in group VIB phospholipase A2 (iPLA2gamma) exhibit relative resistance to obesity and metabolic abnormalities induced by a Western diet.

Phospholipases A(2) (PLA(2)) play important roles in metabolic processes, and the Group VI PLA(2) family is comprised of intracellular enzymes that do not require Ca(2+) for catalysis. Mice deficient in Group VIA PLA(2) (iPLA(2)beta) develop more severe glucose intolerance than wild-type (WT) mice in response to dietary stress. Group VIB PLA(2) (iPLA(2)gamma) is a related enzyme distributed in membranous organelles, including mitochondria, and iPLA(2)gamma knockout (KO) mice exhibit altered mitochondrial morphology and function. We have compared metabolic responses of iPLA(2)gamma-KO and WT mice fed a Western diet (WD) with a high fat content. We find that KO mice are resistant to WD-induced increases in body weight and adiposity and in blood levels of cholesterol, glucose, and insulin, even though WT and KO mice exhibit similar food consumption and dietary fat digestion and absorption. KO mice are also relatively resistant to WD-induced insulin resistance, glucose intolerance, and altered patterns of fat vs. carbohydrate fuel utilization. KO skeletal muscle exhibits impaired mitochondrial beta-oxidation of fatty acids, as reflected by accumulation of larger amounts of long-chain acylcarnitine (LCAC) species in KO muscle and liver compared with WT in response to WD feeding. This is associated with increased urinary excretion of LCAC and much reduced deposition of triacylglycerols in liver by WD-fed KO compared with WT mice. The iPLA(2)gamma-deficient genotype thus results in a phenotype characterized by impaired mitochondrial oxidation of fatty acids and relative resistance to the metabolic abnormalities induced by WD.

Age-related changes in bone morphology are accelerated in group VIA phospholipase A2 (iPLA2beta)-null mice.

Phospholipases A(2) (PLA(2)) hydrolyze the sn-2 fatty acid substituent, such as arachidonic acid, from phospholipids, and arachidonate metabolites are recognized mediators of bone modeling. We have previously generated knockout (KO) mice lacking the group VIA PLA(2) (iPLA(2)beta), which participates in a variety of signaling events; iPLA(2)beta mRNA is expressed in bones of wild-type (WT) but not KO mice. Cortical bone size, trabecular bone volume, bone mineralizing surfaces, and bone strength are similar in WT and KO mice at 3 months and decline with age in both groups, but the decreases are more pronounced in KO mice. The lower bone mass phenotype observed in KO mice is not associated with an increase in osteoclast abundance/activity or a decrease in osteoblast density, but is accompanied by an increase in bone marrow fat. Relative to WT mice, undifferentiated bone marrow stromal cells (BMSCs) from KO mice express higher levels of PPAR-gamma and lower levels of Runx2 mRNA, and this correlates with increased adipogenesis and decreased osteogenesis in BMSCs from these mice. In summary, our studies indicate that age-related losses in bone mass and strength are accelerated in iPLA(2)beta-null mice. Because adipocytes and osteoblasts share a common mesenchymal stem cell origin, our findings suggest that absence of iPLA(2)beta causes abnormalities in osteoblast function and BMSC differentiation and identify a previously unrecognized role of iPLA(2)beta in bone formation.

Disrupted membrane homeostasis and accumulation of ubiquitinated proteins in a mouse model of infantile neuroaxonal dystrophy caused by PLA2G6 mutations.

Mutations in the PLA2G6 gene, which encodes group VIA calcium-independent phospholipase A2 (iPLA(2)beta), were recently identified in patients with infantile neuroaxonal dystrophy (INAD) and neurodegeneration with brain iron accumulation. A pathological hallmark of these childhood neurodegenerative diseases is the presence of distinctive spheroids in distal axons that contain accumulated membranes. We used iPLA(2)beta-KO mice generated by homologous recombination to investigate neurodegenerative consequences of PLA2G6 mutations. iPLA(2)beta-KO mice developed age-dependent neurological impairment that was evident in rotarod, balance, and climbing tests by 13 months of age. The primary abnormality underlying this neurological impairment was the formation of spheroids containing tubulovesicular membranes remarkably similar to human INAD. Spheroids were strongly labeled with anti-ubiquitin antibodies. Accumulation of ubiquitinated protein in spheroids was evident in some brain regions as early as 4 months of age, and the onset of motor impairment correlated with a dramatic increase in ubiquitin-positive spheroids throughout the neuropil in nearly all brain regions. Furthermore accumulating ubiquitinated proteins were observed primarily in insoluble fractions of brain tissue, implicating protein aggregation in this pathogenic process. These results indicate that loss of iPLA(2)beta causes age-dependent impairment of axonal membrane homeostasis and protein degradation pathways, leading to age-dependent neurological impairment. iPLA(2)beta-KO mice will be useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration with brain iron accumulation.

Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2beta in pancreatic beta-cells and in iPLA2beta-null mice.

Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A(2) (iPLA(2)beta) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA(2)beta, for example, exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic (TG) mice in which the rat insulin 1 promoter (RIP) drives iPLA(2)beta overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA(2)beta expression is increased severalfold, as reflected by quantitative PCR of iPLA(2)beta mRNA, immunoblotting of iPLA(2)beta protein, and iPLA(2)beta enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA(2)beta overexpression in RIP-iPLA(2)beta-TG islet beta-cells without obviously perturbed islet morphology. Male RIP-iPLA(2)beta-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA(2)beta-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA(2)beta-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA(2)beta-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca(2+) concentration that suggest a molecular mechanism for the physiological role of iPLA(2)beta to amplify insulin secretion.

Attenuated free cholesterol loading-induced apoptosis but preserved phospholipid composition of peritoneal macrophages from mice that do not express group VIA phospholipase A2.

Mouse macrophages undergo ER stress and apoptosis upon free cholesterol loading (FCL). We recently generated iPLA(2)beta-null mice, and here we demonstrate that iPLA(2)beta-null macrophages have reduced sensitivity to FCL-induced apoptosis, although they and wild-type (WT) cells exhibit similar increases in the transcriptional regulator CHOP. iPLA(2)beta-null macrophages are also less sensitive to apoptosis induced by the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin and the scavenger receptor A ligand fucoidan, and restoring iPLA(2)betaexpression with recombinant adenovirus increases apoptosis toward WT levels. WT and iPLA(2)beta-null macrophages incorporate [(3)H]arachidonic acid ([(3)H]AA]) into glycerophosphocholine lipids equally rapidly and exhibit identical zymosan-induced, cPLA(2)alpha-catalyzed [(3)H]AA release. In contrast, although WT macrophages exhibit robust [(3)H]AA release upon FCL, this is attenuated in iPLA(2)beta-null macrophages and increases toward WT levels upon restoring iPLA(2)beta expression. Recent reports indicate that iPLA(2)beta modulates mitochondrial cytochrome c release, and we find that thapsigargin and fucoidan induce mitochondrial phospholipid loss and cytochrome c release into WT macrophage cytosol and that these events are blunted in iPLA(2)beta-null cells. Immunoblotting studies indicate that iPLA(2)beta associates with mitochondria in macrophages subjected to ER stress. AA incorporation into glycerophosphocholine lipids is unimpaired in iPLA(2)beta-null macrophages upon electrospray ionization-tandem mass spectrometry analyses, and their complex lipid composition is similar to WT cells. These findings suggest that iPLA(2)beta participates in ER stress-induced macrophage apoptosis caused by FCL or thapsigargin but that deletion of iPLA(2)beta does not impair macrophage arachidonate incorporation or phospholipid composition.

Insulin secretory responses and phospholipid composition of pancreatic islets from mice that do not express Group VIA phospholipase A2 and effects of metabolic stress on glucose homeostasis.

Studies involving pharmacologic or molecular biologic manipulation of Group VIA phospholipase A(2) (iPLA(2)beta) activity in pancreatic islets and insulinoma cells suggest that iPLA(2)beta participates in insulin secretion. It has also been suggested that iPLA(2)beta is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels and arachidonate incorporation into phosphatidylcholine (PC). We have generated iPLA(2)beta-null mice by homologous recombination and have reported that they exhibit reduced male fertility and defective motility of spermatozoa. Here we report that pancreatic islets from iPLA(2)beta-null mice have impaired insulin secretory responses to D-glucose and forskolin. Electrospray ionization mass spectrometric analyses indicate that the abundance of arachidonate-containing PC species of islets, brain, and other tissues from iPLA(2)beta-null mice is virtually identical to that of wild-type mice, and no iPLA(2)beta mRNA was observed in any tissue from iPLA(2)beta-null mice at any age. Despite the insulin secretory abnormalities of isolated islets, fasting and fed blood glucose concentrations of iPLA(2)beta-null and wild-type mice are essentially identical under normal circumstances, but iPLA(2)beta-null mice develop more severe hyperglycemia than wild-type mice after administration of multiple low doses of the beta-cell toxin streptozotocin, suggesting an impaired islet secretory reserve. A high fat diet also induces more severe glucose intolerance in iPLA(2)beta-null mice than in wild-type mice, but PLA(2)beta-null mice have greater responsiveness to exogenous insulin than do wild-type mice fed a high fat diet. These and previous findings thus indicate that iPLA(2)beta-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages.

Genetic and pharmacologic evidence that calcium-independent phospholipase A2beta regulates virus-induced inducible nitric-oxide synthase expression by macrophages.

Recent evidence supports a regulatory role for the calcium-independent phospholipase A2 (iPLA2) in the antiviral response of inducible nitric-oxide synthase (iNOS) expression by macrophages. Because two mammalian isoforms of iPLA2 (iPLA2beta and iPLA2gamma) have been cloned and characterized, the aim of this study was to identify the specific isoform(s) in macrophages that regulates the expression of iNOS in response to virus infection. Bromoenol lactone (BEL), a suicide substrate inhibitor of iPLA2, inhibits the activity of both isoforms at low micromolar concentrations. However, the R- and S-enantiomers of BEL display approximately 10-fold greater potency for inhibition of the enzymatic activity of iPLA2gamma and iPLA2beta, respectively. In this study, we show that the iPLA2beta-selective (S)-BEL inhibits encephalomyocarditis virus (EMCV)-induced iNOS expression, nitric oxide production, and iPLA2 enzymatic activity in macrophages in a concentration-related manner that closely resembles the inhibitory properties of racemic BEL. cAMP response element-binding protein (CREB) is one downstream target of iPLA2 that is required for the transcriptional activation of iNOS in response to virus infection, and consistent with the effects of BEL enantiomers on iNOS expression, (S)-BEL more effectively inhibits EMCV-induced CREB phosphorylation than (R)-BEL in macrophages. Using macrophages isolated from iPLA2beta-null mice, virus infection fails to stimulate iNOS mRNA accumulation and protein expression, thus providing genetic evidence that iPLA2beta is required for EMCV-induced iNOS expression. These findings provide evidence for a signaling role for iPLA2beta in virus-induced iNOS expression by macrophages.