Release Of Polyunsaturated Fatty Acids Research Articles

Infection, Inflammation, and Immunity in Sepsis.

Sepsis is triggered by microbial infection, injury, or even major surgery. Both innate and adaptive immune systems are involved in its pathogenesis. Cytoplasmic presence of DNA or RNA of the invading organisms or damaged nuclear material (in the form of micronucleus in the cytoplasm) in the host cell need to be eliminated by various nucleases; failure to do so leads to the triggering of inflammation by the cellular cGAS-STING system, which induces the release of IL-6, TNF-α, and IFNs. These cytokines activate phospholipase A2 (PLA2), leading to the release of polyunsaturated fatty acids (PUFAs), gamma-linolenic acid (GLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), which form precursors to various pro- and anti-inflammatory eicosanoids. On the other hand, corticosteroids inhibit PLA2 activity and, thus, suppress the release of GLA, AA, EPA, and DHA. PUFAs and their metabolites have a negative regulatory action on the cGAS-STING pathway and, thus, suppress the inflammatory process and initiate inflammation resolution. Pro-inflammatory cytokines and corticosteroids (corticosteroids > IL-6, TNF-α) suppress desaturases, which results in decreased formation of GLA, AA, and other PUFAs from the dietary essential fatty acids (EFAs). A deficiency of GLA, AA, EPA, and DHA results in decreased production of anti-inflammatory eicosanoids and failure to suppress the cGAS-STING system. This results in the continuation of the inflammatory process. Thus, altered concentrations of PUFAs and their metabolites, and failure to suppress the cGAS-STING system at an appropriate time, leads to the onset of sepsis. Similar abnormalities are also seen in radiation-induced inflammation. These results imply that timely administration of GLA, AA, EPA, and DHA, in combination with corticosteroids and anti-IL-6 and anti-TNF-α antibodies, may be of benefit in mitigating radiation-induced damage and sepsis.

Cannabidiol acts as molecular switch in innate immune cells to promote the biosynthesis of inflammation-resolving lipid mediators

Cannabinoids are phytochemicals from cannabis with anti-inflammatory actions in immune cells. Lipid mediators (LM), produced from polyunsaturated fatty acids (PUFA), are potent regulators of the immune response and impact all stages of inflammation. How cannabinoids influence LM biosynthetic networks is unknown. Here, we reveal cannabidiol (CBD) as a potent LM class-switching agent that stimulates the production of specialized pro-resolving mediators (SPMs) but suppresses pro-inflammatory eicosanoid biosynthesis. Detailed metabololipidomics analysis in human monocyte-derived macrophages showed that CBD (i)upregulates exotoxin-stimulated generation of SPMs, (ii) suppresses 5-lipoxygenase (LOX)-mediated leukotriene production, and (iii) strongly induces SPM and 12/15-LOX product formation in resting cells by stimulation of phospholipase A2-dependent PUFA release and through Ca2+-independent, allosteric 15-LOX-1 activation. Finally, in zymosan-induced murine peritonitis, CBD increased SPM and 12/15-LOX products and suppressed pro-inflammatory eicosanoid levels invivo. Switching eicosanoid to SPM production is a plausible mode of action of CBD and a promising inflammation-resolving strategy.

Development and characterization of omega-3-rich flaxseed oil microcapsules and evaluation of its stability and release behavior in probiotic millet yogurt

The study investigated the encapsulation potential of probiotics (PRO) and flaxseed oil (FS) using different wall materials, including sodium alginate-maltodextrin-inulin (SIN), skim milk (SK), whey protein (WP), and soy protein concentrate (SP) to produce oil-enriched millet yogurt. The microcapsules (SIN-FS-SK, SIN-FS-WP, and SIN-FS-SP) and co-microcapsules (SIN-FS-PRO-SK, SIN-FS-PRO-WP, and SIN-FS-PRO-SP) were characterized by encapsulation efficiency, lipid oxidation, FTIR, SEM, and GC–MS. Thus, the obtained emulsion droplets size was favorable (1.11 to 1.73 μm) for freeze-drying with an encapsulation rate above 95%, better oxidative stability at 4 °C, and microcapsule sizes between 800 and 1600 μm allowing the oil release during intestinal stage (51–65%). Thus, it was possible to obtain flaxseed oil enriched-millet yogurt with good oxidative stability. Adding co-microcapsules also improved the quantities of polyunsaturated fatty acids (PUFAs), particularly alpha-linolenic acid, in the various products, enabling a significant release of PUFAs during intestinal digestion, thus improving the omega 6/omega 3 ratio.

Phospholipase A2 enzymes differently impact PUFA release and oxylipin formation ex vivo in rat hearts.

Phospholipase A2 (PLA2) enzymes cleave cell membrane phospholipids and release polyunsaturated fatty acids (PUFA), which can be converted into oxylipins. However, little is known about PLA2 preference for PUFA, and even less is known about how this further impacts oxylipin formation. Therefore, we investigated the role of different PLA2 groups in PUFA release and oxylipin formation in rat hearts. Sprague-Dawley rat heart homogenates were incubated without or with varespladib (VAR), methyl arachidonyl fluorophosphonate (MAFP) or EDTA. Free PUFA and oxylipins were determined by HPLC-MS/MS, and isoform expressions by RT-qPCR. Inhibition of sPLA2 IIA and/or V by VAR reduced the release of ARA and DHA, but only DHA oxylipins were inhibited. MAFP reduced the release of ARA, DHA, ALA, and EPA, and the formation of ARA, LA, DGLA, DHA, ALA, and EPA oxylipins. Interestingly, cyclooxygenase and 12-lipoxygenase oxylipins were not inhibited. mRNA expression levels of sPLA2 and iPLA2 isoforms were highest whereas levels of cPLA2 were low, consistent with activity. In conclusion, sPLA2 enzymes lead to the formation of DHA oxylipins, while iPLA2 is likely responsible for the formation of most other oxylipins in healthy rat hearts. Oxylipin formation cannot be implied from PUFA release, thus, both should be evaluated in PLA2 activity studies.

The fatty acid profiles of mixed fermented milk and its anti-inflammation properties in an LPS-induced RAW264.7 cell model.

Increasing knowledge of probiotics has shown that co-cultures of probiotics can achieve better fermentation and beneficial effects, and adding LAB to fermented milk fat products can increase the production of polyunsaturated fatty acids. In this study, the fatty acid profiles of milk fermented by L. reuteri DMSZ 8533, L. plantarum A3, and L. acidophilus CICC 6074 were investigated, and the strain difference on the polyunsaturated fatty acid profiles is also confirmed. The results found that L. plantarum A3 fermented milk with a mixed fermentation starter ratio of 1 : 1 : 2 could promote the hydrolysis of lipase to produce more polyunsaturated fatty acids. Furthermore, the milk fat complex hydrolyzed lipids can inhibit the expression of inflammatory factors TNF-α and IL-6 and promote the secretion of IL-10 through the MAPK-dependent NF-κB pathway. Furthermore, the anti-inflammation potential of the fatty acid profiles in the fermented milk products was also revealed in an LPS-induced RAW264.7 cell model. It is hoped that this study can shed light on the anti-inflammatory properties of fermented milk by the release of polyunsaturated fatty acids and the possibility to develop functional fermented milk with intestinal inflammation prevention properties.

The Influence of Phospholipase A 2 (PLA 2 ) Enzymes on Fatty Acid Release and Oxylipin Formation in Rat Hearts

Polyunsaturated fatty acids (PUFA) released by PLA2 are converted into diverse bioactive lipid mediators called oxylipins. However, little is known about the preference of PLA2 isoforms for PUFA release, and even less is known about how this further impacts the formation of oxylipins. Further, oxylipin levels in tissues do not always mimic the levels of their precursor PUFA, so conclusions about oxylipins cannot be drawn from PUFA content. Therefore, we aimed to determine the impact of PLA2 isoforms on PUFA release and oxylipin formation. We hypothesized that different PLA2 isoforms would impact PUFA release and oxylipin formation differently, and that oxylipin formation would not necessarily reflect changes seen in PUFA. Rat heart homogenates were incubated at 37C for 20 min with or without inhibitors, as the increase of both free PUFA and oxylipins was linear at 20 min incubation. Free PUFA and oxylipins were analyzed by HPLC-MS/MS. Two inhibitors were used: Varespladib (VAR) targeting secreted (sPLA2) isoforms IIA, V and X, and methyl arachidonyl fluorophosphonate (MAFP) targeting cytosolic (cPLA2) and calcium-independent (iPLA2) isoforms. Arachidonic Acid (ARA) release was similarly inhibited by both VAR and MAFP, however, while 15 out of 29 ARA oxylipins were inhibited with MAFP, only 3 out of 29 were inhibited with VAR. Docosahexaenoic acid (DHA) release was more strongly inhibited by VAR than by MAFP, which corresponded with 7 out of 12 DHA oxylipins being more strongly inhibited by VAR than by MAFP. Both alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA) were only inhibited by MAFP, which also inhibited 3 out of 5 ALA and 1 out of 3 EPA oxylipins. Further, 9 out of 10 LA oxylipins were inhibited by MAFP while none were affected by VAR. In conclusion, in the rat heart ARA is released by sPLA2 IIA, V, and/or X as well as by cPLA2 and/or iPLA2, DHA is released more by sPLA2 IIA, V, and/or X than by cPLA2 and/or iPLA2, and EPA and ALA are released primarily by cPLA2 and/or iPLA2. Effects on oxylipin formation largely reflected those on precursor PUFA release, with an exception, as inhibition of sPLA2 IIA, V and X by VAR mostly did not affect ARA oxylipin formation even though it inhibited ARA release. Therefore, careful evaluation of the effects on the oxylipin profile is needed when considering PLA2 inhibitors for potential treatments of diseases.

"Cell Membrane Theory of Senescence" and the Role of Bioactive Lipids in Aging, and Aging Associated Diseases and Their Therapeutic Implications.

Lipids are an essential constituent of the cell membrane of which polyunsaturated fatty acids (PUFAs) are the most important component. Activation of phospholipase A2 (PLA2) induces the release of PUFAs from the cell membrane that form precursors to both pro- and ant-inflammatory bioactive lipids that participate in several cellular processes. PUFAs GLA (gamma-linolenic acid), DGLA (dihomo-GLA), AA (arachidonic acid), EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) are derived from dietary linoleic acid (LA) and alpha-linolenic acid (ALA) by the action of desaturases whose activity declines with age. Consequently, aged cells are deficient in GLA, DGLA, AA, AA, EPA and DHA and their metabolites. LA, ALA, AA, EPA and DHA can also be obtained direct from diet and their deficiency (fatty acids) may indicate malnutrition and deficiency of several minerals, trace elements and vitamins some of which are also much needed co-factors for the normal activity of desaturases. In many instances (patients) the plasma and tissue levels of GLA, DGLA, AA, EPA and DHA are low (as seen in patients with hypertension, type 2 diabetes mellitus) but they do not have deficiency of other nutrients. Hence, it is reasonable to consider that the deficiency of GLA, DGLA, AA, EPA and DHA noted in these conditions are due to the decreased activity of desaturases and elongases. PUFAs stimulate SIRT1 through protein kinase A-dependent activation of SIRT1-PGC1α complex and thus, increase rates of fatty acid oxidation and prevent lipid dysregulation associated with aging. SIRT1 activation prevents aging. Of all the SIRTs, SIRT6 is critical for intermediary metabolism and genomic stability. SIRT6-deficient mice show shortened lifespan, defects in DNA repair and have a high incidence of cancer due to oncogene activation. SIRT6 overexpression lowers LDL and triglyceride level, improves glucose tolerance, and increases lifespan of mice in addition to its anti-inflammatory effects at the transcriptional level. PUFAs and their anti-inflammatory metabolites influence the activity of SIRT6 and other SIRTs and thus, bring about their actions on metabolism, inflammation, and genome maintenance. GLA, DGLA, AA, EPA and DHA and prostaglandin E2 (PGE2), lipoxin A4 (LXA4) (pro- and anti-inflammatory metabolites of AA respectively) activate/suppress various SIRTs (SIRt1 SIRT2, SIRT3, SIRT4, SIRT5, SIRT6), PPAR-γ, PARP, p53, SREBP1, intracellular cAMP content, PKA activity and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α). This implies that changes in the metabolism of bioactive lipids as a result of altered activities of desaturases, COX-2 and 5-, 12-, 15-LOX (cyclo-oxygenase and lipoxygenases respectively) may have a critical role in determining cell age and development of several aging associated diseases and genomic stability and gene and oncogene activation. Thus, methods designed to maintain homeostasis of bioactive lipids (GLA, DGLA, AA, EPA, DHA, PGE2, LXA4) may arrest aging process and associated metabolic abnormalities.

Effects of High and Low Molecular Weight Hyaluronic Acids on the Omega-3 and Omega-6 Fatty Acid Release upon Activation of the Toll-Like Receptors in Astrocytes

Hyaluronic acid (HA) is one of the key polymer components of the extracellular matrix; it can exert different effects on intracellular signaling depending on its molecular weight. We compared the effects of low (LMW) and high molecular weight (HMW) HA polymers on the ability of primary astrocytes, glial brain cells, to release into the extracellular medium omega-3 unsaturated fatty acids, docosahexaenoic (DHA) and eicosapentaenoic (EPA), as well as omega-6 unsaturated arachidonic acid (AA), which were determined by HPLC-MS/MS. It was shown that upon the addition of LMW HA for 4.5 h the concentration of AA reduced. HMW HA did not affect the release of AA but increased the release of DHA and EPA. Adaptation of the cells for 48 h to the action of the HA polymers of different molecular weights led to a decrease in the release of omega-3 and omega-6 unsaturated fatty acids by astrocytes. A comparison of the ability of HA to modulate astrocyte responses to stimulation by Toll-like receptor (TLR) agonists showed that HA affected TLR4-stimulated induction of IL-1β proinflammatory marker gene after 0.5-h exposure to LMW HA and HMW HA followed by further stimulation with TLR4 agonist for 4 h. Thus, the release of polyunsaturated fatty acids in astrocytes was sensitive to HA and depended on the molecular weight of the polymers; long (48 h) exposure to HA led to adaptation of astrocytes; HA differently affected the release of omega-6 (AA) and omega-3 (DHA) unsaturated fatty acids, indicating a difference in the mechanisms of their release. As DHA is believed to perform anti-inflammatory and protective functions in the brain, these findings open up prospects for appliance of HA polymers as modulators of inflammatory responses of astrocytes.

Particle formation and characterization of mackerel reaction oil by gas saturated solution process.

Most of the health benefits of fish oil can be attributed to the presence of omega-3 fatty acids like Docosahexenoic acid (DHA) and Eicosapentaenoic acid (EPA). There are few dietary sources of EPA and DHA other than oily fish. EPA and DHA have great potential effect on human health. In this research, Supercritical carbon dioxide (scCO2) extracted mackerel oil was reacted by enzyme at different systems to improve the EPA and DHA. Different types of immobilize enzyme TL-IM, RM-IM, Novozyme 435 were assessed for improving PUFAs. Best result was found at non-pressurized system using TL-IM. Reacted oil particle were obtained with polyethylene glycol by gas saturated solution process (PGSS). Different parameters like temperature, pressure, agitation speed and nozzle size effect on particle formulation were observed. SEM and PSA analysis showed, small size non spherical particles were obtained. It was found that after particle formation poly unsaturated fatty acids (PUFAs) were present in particle as same in oil. PUFAs release from particle was almost linear against constant time duration. Oil quality in particle not change significantly, in this contrast this study will be helpful for food and pharmaceutical industry to provide high EPA and DHA containing powder.

Targeting of the hydrophobic metabolome by pathogens.

The hydrophobic molecules of the metabolome – also named the lipidome – constitute a major part of the entire metabolome. Novel technologies show the existence of a staggering number of individual lipid species, the biological functions of which are, with the exception of only a few lipid species, unknown. Much can be learned from pathogens that have evolved to take advantage of the complexity of the lipidome to escape the immune system of the host organism and to allow their survival and replication. Different types of pathogens target different lipids as shown in interaction maps, allowing visualization of differences between different types of pathogens. Bacterial and viral pathogens target predominantly structural and signaling lipids to alter the cellular phenotype of the host cell. Fungal and parasitic pathogens have complex lipidomes themselves and target predominantly the release of polyunsaturated fatty acids from the host cell lipidome, resulting in the generation of eicosanoids by either the host cell or the pathogen. Thus, whereas viruses and bacteria induce predominantly alterations in lipid metabolites at the host cell level, eukaryotic pathogens focus on interference with lipid metabolites affecting systemic inflammatory reactions that are part of the immune system. A better understanding of the interplay between host–pathogen interactions will not only help elucidate the fundamental role of lipid species in cellular physiology, but will also aid in the generation of novel therapeutic drugs.

Involvement of GPR40, a long-chain free fatty acid receptor, in the production of central post-stroke pain after global cerebral ischemia

Central post-stroke pain (CPSP), one of the complications of cerebral ischemia and neuropathic pain syndrome, is associated with specific somatosensory abnormalities. Although CPSP is a serious problem, detailed underlying mechanisms and standard treatments for CPSP are not well established. In this study, we assessed the role of GPR40, a long-chain fatty acid receptor, showing anti-nociceptive effects, in CPSP. We also examined the role of astrocytes in CPSP due to their effects in mediating the release of polyunsaturated fatty acids, which act as potential GPR40 ligands. The aim of this study was to determine the interactions between CPSP and astrocyte/GPR40 signaling. Male ddY mice were subjected to 30min of bilateral carotid artery occlusion (BCAO). The development of hind paw mechanical hyperalgesia was measured after BCAO using the von Frey test. Neuronal damage was estimated by histological analysis on day 3 after BCAO. The thresholds for hind paw mechanical hyperalgesia were significantly decreased on days 1–28 after BCAO when compared with those of pre-BCAO assessments. BCAO-induced mechanical hyperalgesia was significantly decreased by intracerebroventricular injection of docosahexaenoic acid or GW9508, a GPR40 agonist; furthermore, these effects were reversed by GW1100, a GPR40 antagonist. The expression levels of glial fibrillary acidic protein, an astrocytic marker, and some free fatty acids were significantly decreased 5h after BCAO, although no effects of BCAO were noted on hypothalamic GPR40 protein expression. Our data show that BCAO-induced mechanical hyperalgesia is possible to be regulated by astrocyte activation and stimulation of GPR40 signaling.

Plasmalogens in the retina: from occurrence in retinal cell membranes to potential involvement in pathophysiology of retinal diseases.

Plasmalogens (Pls) represent a specific subclass of glycerophospholipids characterized by the presence of a vinyl-ether bond at the sn-1 position of glycerol. Pls are quantitatively important in membranes of neuronal tissues, including the brain and the retina, where they can represent until almost two-third of ethanolamine glycerophospholipids. They are considered as reservoirs of polyunsaturated fatty acids as several studies have shown that arachidonic and docosahexaenoic acids are preferentially esterified on Pls when compared to other glycerophospholipids. Reduced levels of Pls were observed in a number of neurodegenerative disorders such as glaucoma, the second leading cause of blindness worldwide. In a mouse model of Pls deficiency, "glaucoma-like" optic nerve abnormalities were observed as well as developmental defects in the eye. These included microphthalmia, dysgenesis of the anterior segment of the eye, and abnormalities in retinal vessel architecture. Several data from animal and invitro studies suggest that Pls may be involved in the regulation of retinal vascular development through the release of polyunsaturated fatty acids by a calcium-independent phospholipase A2.

Involvement of plasmalogens in post-natal retinal vascular development.

ObjectiveProper development of retinal blood vessels is essential to ensure sufficient oxygen and nutrient supplies to the retina. It was shown that polyunsaturated fatty acids (PUFAs) could modulate factors involved in tissue vascularization. A congenital deficiency in ether-phospholipids, also termed “plasmalogens”, was shown to lead to abnormal ocular vascularization. Because plasmalogens are considered to be reservoirs of PUFAs, we wished to improve our understanding of the mechanisms by which plasmalogens regulate retinal vascular development and whether the release of PUFAs by calcium-independent phospholipase A2 (iPLA2) could be involved.Methods and ResultsBy characterizing the cellular and molecular steps of retinal vascular development in a mouse model of plasmalogen deficiency, we demonstrated that plasmalogens modulate angiogenic processes during the early phases of retinal vascularization. They influence glial activity and primary astrocyte template formation, endothelial cell proliferation and retinal vessel outgrowth, and impact the expression of the genes involved in angiogenesis in the retina. These early defects led to a disorganized and dysfunctional retinal vascular network at adult age. By comparing these data to those obtained on a mouse model of retinal iPLA2 inhibition, we suggest that these processes may be mediated by PUFAs released from plasmalogens and further signalling through the angiopoietin/tie pathways.ConclusionsThese data suggest that plasmalogens play a crucial role in retinal vascularization processes.

Structure, Activity, and Substrate Selectivity of the Orf6 Thioesterase from Photobacterium profundum

Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

Deficiency of carboxylesterase 1/esterase-x results in obesity, hepatic steatosis, and hyperlipidemia

Increased lipogenesis, together with hyperlipidemia and increased fat deposition, contribute to obesity and associated metabolic disorders including nonalcoholic fatty liver disease. Here we show that carboxylesterase 1/esterase-x (Ces1/Es-x) plays a regulatory role in hepatic fat metabolism in the mouse. We demonstrate that Ces1/Es-x knockout mice present with increased hepatic lipogenesis and with oversecretion of apolipoprotein B (apoB)-containing lipoproteins (hepatic very-low density lipoproteins), which leads to hyperlipidemia and increased fat deposition in peripheral tissues. Consequently, Ces1/Es-x knockout mice develop obesity, fatty liver, hyperinsulinemia, and insulin insensitivity on chow diet without change in food intake and present with decreased energy expenditure. Ces1/Es-x deficiency prevents the release of polyunsaturated fatty acids from triacylglycerol stores, leading to an up-regulation of sterol regulatory element binding protein 1c-mediated lipogenesis, which can be reversed with dietary ω-3 fatty acids. These studies support a role for Ces1/Es-x in the partitioning of regulatory fatty acids and concomitant control of hepatic lipid biosynthesis, secretion, and deposition.

Comparative study of post‐natal retinal vascular development in mice models of iPLA2 inhibition and plasmalogen deficiency

AbstractPurpose Plasmalogens are particular phospholipids characterized by the presence of a polyunsaturated fatty acid (PUFA) at sn‐2 position of glycerol. Plasmalogen deficiency in mouse leads to developmental abnormalities in retinal vasculature. We propose that liberation of PUFA by the specific calcium‐independent phospholipase A2 (iPLA2), is involved in the mechanism by which plasmalogens control retinal vascular development. To confirm this hypothesis, we performed a comparative study of retinal vascular development in a mouse model of retinal iPLA2 inhibition and a model of plasmalogen deficiency.Methods Vessel and astrocyte networks were visualized on flat‐mounted retinas through immunostaining methods.Results Similar abnormalities were observed in retina of both mouse models. They consisted in an increased number of vessel ramifications at PN14, and in an abnormal glial cells migration from the optic nerve, at PN14 and at PN21. An activation of microglial cells was also observed at adult age.Conclusion These results confirm the implication of plasmalogen in the control of retinal vessel development through PUFA release from their sn‐2 position.

The role of EDS1 (enhanced disease susceptibility) during singlet oxygen‐mediated stress responses of Arabidopsis

Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2) that is restricted to the plastid compartment. Distinct sets of genes are activated that are different from those induced by hydrogen peroxide/superoxide. One of the genes that is rapidly upregulated is EDS1 (enhanced disease susceptibility). The EDS1 protein has been shown to be required for the resistance to biotrophic pathogens and the accumulation of salicylic acid (SA) that enhances the defenses of a plant by inducing the synthesis of pathogen-related (PR) proteins. Because of the similarity of its N-terminal portion to the catalytic site of lipases, EDS1 has also been implicated with the release of polyunsaturated fatty acids and the subsequent formation of various oxylipins. The release of singlet oxygen in the flu mutant triggers a drastic increase in the concentration of free SA and activates the expression of PR1 and PR5 genes. These changes depend on the activity of EDS1 and are suppressed in flu/eds1 double mutants. Soon after the beginning of singlet oxygen production, the synthesis of oxylipins such as jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) also start and plants stop growing and induce a cell-death response. The inactivation of EDS1 does not affect oxylipin synthesis, growth inhibition and the initiation of cell death, but it does allow plants to recover much faster from singlet oxygen-mediated growth inhibition and it also suppresses the spread of necrotic lesions in leaves. Hence, singlet oxygen activates a complex stress-response program with EDS1 playing a key role in initiating and modulating several steps of it. This program includes not only responses to oxidative stress, but also responses known to be activated during plant-pathogen interactions and wounding.

Docosahexaenoic acid and arachidonic acid release in rat brain astrocytes is mediated by two separate isoforms of phospholipase A2 and is differently regulated by cyclic AMP and Ca2+.

1. Docosahexaenoic acid (DHA) and arachidonic acid (AA), polyunsaturated fatty acids (PUFAs), are important for central nervous system function during development and in various pathological states. Astrocytes are involved in the biosynthesis of PUFAs in neuronal tissue. Here, we investigated the mechanism of DHA and AA release in cultured rat brain astrocytes. 2. Primary astrocytes were cultured under standard conditions and prelabeled with [(14)C]DHA or with [(3)H]AA. Adenosine 5'-triphosphate (ATP) (20 micro M applied for 15 min), the P2Y receptor agonist, stimulates release of both DHA (289% of control) and AA (266% of control) from astrocytes. DHA release stimulated by ATP is mediated by Ca(2+)-independent phospholipase A(2) (iPLA(2)), since it is blocked by the selective iPLA(2) inhibitor 4-bromoenol lactone (BEL, 5 micro M) and is not affected either by removal of Ca(2+) from extracellular medium or by suppression of intracellular Ca(2+) release through PLC inhibitor (U73122, 5 micro M). 3. AA release, on the other hand, which is stimulated by ATP, is attributed to Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)). AA release is abolished by U73122 and, by removal of extracellular Ca(2+), is insensitive to BEL and can be selectively suppressed by methyl arachidonyl fluorophosphonate (3 micro M), a general inhibitor of intracellular PLA(2) s. 4. Western blot analysis confirms the presence in rat brain astrocytes of 85 kDa cPLA(2) and 40 kDa protein reactive to iPLA(2) antibodies. 5. The influence of cAMP on regulation of PUFA release was investigated. Release of DHA is strongly amplified by the adenylyl cyclase activator forskolin (10 micro M), and by the protein kinase A (PKA) activator dibutyryl-cAMP (1 mM). In contrast, release of AA is not affected by forskolin or dibutyryl-cAMP, but is almost completely blocked by 2,3-dideoxyadenosine (20 micro M) and inhibited by 34% by H89 (10 micro M), inhibitors of adenylyl cyclase and PKA, respectively. 6. Other neuromediators, such as bradykinin, glutamate and thrombin, stimulate release of DHA and AA, which is comparable to the release stimulated by ATP. 7. Different sensitivities of iPLA(2) and cPLA(2) to Ca(2+) and cAMP reveal new pathways for the regulation of fatty acid release and reflect the significance of astrocytes in control of DHA and AA metabolism under normal and pathological conditions in brain.

Human serum paraoxonase 1 decreases macrophage cholesterol biosynthesis: possible role for its phospholipase-A2-like activity and lysophosphatidylcholine formation.

Human serum paraoxonase 1 (PON1) activity is inversely related to the risk of developing an atherosclerotic lesion, which contains cholesterol-loaded macrophage foam cells. To assess a possible mechanism for this relationship, we analyzed the effect of PON1 on cellular cholesterol biosynthesis. Mouse peritoneal macrophages (MPMs) were harvested from PON1-deficient mice (PON1o and PON1o/Eo mice on the genetic background of C57BL/6J and Eo mice, respectively). PON1o/Eo mice exhibited a significantly 51% increased atherosclerotic lesion area and 35% increased macrophage cholesterol content compared with control E degrees mice. In parallel, macrophage cholesterol biosynthesis rates were increased in PON1-deficient mice MPMs by 50% compared with their controls. Incubation of macrophages with human PON1 revealed a dose-dependent inhibitory effect (up to 84%) on macrophage cholesterol biosynthesis. We demonstrated a PON1 phospholipase-A2-like activity on MPMs, evidenced by release of polyunsaturated fatty acids and formation of lysophosphatidylcholine. On incubation of macrophages with lysophosphatidylcholine, a dose-dependent inhibition (up to 40%) of cellular cholesterol biosynthesis was noted. The inhibitory effect of PON1 on macrophage cholesterol biosynthesis was shown to be downstream to mevalonate, probably at the lanosterol metabolic point. PON1 inhibits macrophage cholesterol biosynthesis and atherogenesis probably through its phospholipase-A2-like activity.

Role of polyunsaturated fatty acids in the inhibitory effect of human adipocytes on osteoblastic proliferation

As previously reported, the association of bone loss with an increase in bone marrow adipose volume may be related to the inhibition of human osteoblastic cell proliferation in the presence of human adipocytes. In the osteoblastic supernatant, fatty acid composition varied after coculture with mature adipocytes, with a marked increase in the proportion of docosahexaenoic acid (22:6 n-3; DHA) (+90 ± 8%). This suggests that polyunsaturated fatty acids (PUFA) may contribute to the inhibitory effect of adipocytes on osteoblastic cell proliferation. The purpose of the present study was to evaluate the effects of two PUFA, DHA and arachidonic acid (20:4 n-6; AA), on the proliferation of primary human osteoblastic (hOB) cells and human osteosarcoma cell line, MG-63. The effects of cholesterol and oleic acid, a monounsaturated FA (18:1 n-9; OA), both being present in adipocyte lipidic vacuoles, were also investigated. At between 10 and 50 μmol/L, DHA and AA induced a significant dose-dependent decrease in hOB cell proliferation (p < 0.0001 and p < 0.006 for DHA and AA, respectively) when compared with control hOB cells exposed to the vehicle (bovine serum albumin). This inhibition reached −50% with 50 μmol/L of DHA or 20 μmol/L of AA. This effect was not related to cell apoptosis, as shown by terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling (TUNEL) and Hoechst dye staining. In contrast, OA and cholesterol had no effect on hOB cell proliferation, even at a high concentration (200 μmol/L). Similar results were observed with regard to MG-63 cell proliferation. In addition, flow cytometric analysis showed that the number of hOB cells in the S phase of the cycle was twofold lower when treated with 50 μmol/L of DHA or AA. In vitro results indicate that mature adipocytes may contribute to age-related bone loss through the release of polyunsaturated fatty acids, which impair osteoblastic proliferation.