Increasing evidence underline the role of inflammation in the behavioral, emotional and cognitive dysregulations displayed in anorexia nervosa (AN). Among the inflammatory mediators acting at both peripheral and central levels, growing attention receives a class of lipids derived from arachidonic acid (AA), called eicosanoids (eiCs), which exert a complex, multifaceted role in a wide range of neuroinflammatory processes, peripheral inflammation, and generally in immune system function. To date, little is known about their possible involvement in the neurobiological underpinnings of AN. The present study evaluated whether the activity-based model of AN (ABA) may alter AA-metabolic pathways by changing the levels of AA-derived eiCs in specific brain areas implicated in the development of the typical anorexic-like phenotype, i.e. in prefrontal cortex, cerebral cortex, nucleus accumbens, caudate putamen, amygdala, hippocampus, hypothalamus and cerebellum. Our results point to brain region-specific alterations of the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 epoxygenase (CYP) metabolic pathways rendering altered levels of AA-derived eiCs (i.e. prostaglandins, thromboxanes and hydroxyeicosatetraenoic acids) in response to induction of and recovery from the ABA condition. These changes, supported by altered messenger RNA (mRNA) levels of genes coding for enzymes involved in eiCs-related methabolic pathways (i.e., PLA2, COX-2, 5-LOX and 15-LOX), underlie a widespread brain dysregulation of pro- and anti-inflammatory eiC-mediated processes in the ABA model of AN. These data suggest the importance of eiCs signaling within corticolimbic areas in regulating key neurobehavioral functions and highlight eiCs as biomarker candidates for monitoring the onset and development of AN, and/or as possible targets for pharmacological management.

The biogenesis of peroxisomes in relation to the trafficking of proteins to peroxisomes has been extensively examined. However, the supply of phospholipids, which is needed to generate peroxisomal membranes in mammals, remains unclear. Therefore, we herein investigated metabolic alterations induced by clofibric acid, a peroxisome proliferator, in the synthesis of phospholipids, particularly phosphatidylethanolamine (PE) molecular species, and their relationship with the biogenesis of peroxisomal membranes. The subcutaneous administration of clofibric acid to rats at a relatively low dose (130 mg/kg) once a day time-dependently and gradually increased the integrated perimeter of peroxisomes per 100 μm2 hepatocyte cytoplasm (PA). A strong correlation was observed between the content (μmol/mg DNA) of PE containing arachidonic acid (20:4) and PA (r2 = 0.9168). Moreover, the content of PE containing octadecenoic acid (18:1) positively correlated with PA (r2 = 0.8094). The treatment with clofibric acid markedly accelerated the formation of 16:0-20:4 PE by increasing the production of 20:4 and the activity of acyl chain remodeling of pre-existing PE molecular species. Increases in the acyl chain remodeling of PE by clofibric acid were mainly linked to the up-regulated expression of the Lpcat3 gene. On the other hand, clofibric acid markedly increased the formation of palmitic acid (16:0)-18:1 PE through de novo synthesis. These results suggest that the enhanced formation of particular PE molecular species is related to increases in the mass of peroxisomal membranes in peroxisome proliferation in the liver.

The oleaginous microalga Lobosphaera incisa (Trebouxiophyceae, Chlorophyta) contains arachidonic acid (ARA, 20:4 n-6) in all membrane glycerolipids and in the storage lipid triacylglycerol. The optimal growth temperature of the wild-type (WT) strain is 25°C; chilling temperatures (≤15°C) slow its growth. This effect is more pronounced in the delta-5-desaturase ARA-deficient mutant P127, in which ARA is replaced with dihomo-γ-linolenic acid (DGLA, 20:3 n-6). In nutrient-replete cells grown at 25°C, the major chloroplast lipid monogalactosylglycerol (MGDG) was dominated by C18/C16 species in both strains. Yet ARA constituted over 10% of the total fatty acids in the WT MGDG as a component of C20/C18 and C20/C20 species, whereas DGLA was only a minor component of MGDG in P127. Both strains increased the percentage of 18:3 n-3 in membrane lipids under chilling temperatures. The temperature downshift led to a dramatic increase in triacylglycerol at the expense of chloroplast lipids. WT and P127 showed a similarly high photochemical quantum yield of photosystem II, whereas non-photochemical quenching (NPQ) and violaxanthin de-epoxidation were drastically higher in P127, especially at 15°C. Fluorescence anisotropy measurements indicated that ARA-containing MGDG might contribute to sustaining chloroplast membrane fluidity upon dropping to the chilling temperature. We hypothesize that conformational changes in chloroplast membranes and increased rigidity of the ARA-deficient MGDG of P127 at chilling temperatures are not compensated by trienoic fatty acids. This might 'lock' violaxanthin de-epoxidase in the activated state causing high constitutive NPQ and alleviate the risk of photodamage under chilling conditions in the mutant.

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [14C]ethanolamine, and revealed that overexpressed AC lowered the levels of 14C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.

A high intake in polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) (C20:5 n-3), is cardioprotective. Dietary PUFAs incorporate into membrane phospholipids, which may modify the function of membrane proteins. We investigated the consequences of the membrane incorporation of several PUFAs on the key antiatherogenic ABCA1-mediated cholesterol efflux pathway. Human THP-1 macrophages were incubated with EPA, arachidonic acid (AA) (C20:4 n-6) or docosahexaenoic acid (DHA) (C22:6 n-3) for a long time to mimic a chronic exposure. EPA 70 μM, but not AA 50 μM or DHA 15 μM, increased ABCA1-mediated cholesterol efflux to apolipoprotein (apo) AI by 28% without altering aqueous diffusion. No variation in ABCA1 expression or localization was observed after EPA treatment. EPA incorporation did not affect the phenotype of THP-1 macrophages. The membrane phospholipids composition of EPA cells displayed higher levels of both EPA and its elongation product docosapentaenoic acid, which was associated with drastic lower levels of AA. Treatment by EPA increased the ATPase activity of the transporter, likely through a PKA-dependent mechanism. Eicosanoids were not involved in the stimulated ABCA1-mediated cholesterol efflux from EPA-enriched macrophages. In addition, EPA supplementation increased the apo AI binding capacity from macrophages by 38%. Moreover, the increased apo AI binding in EPA-enriched macrophages can be competed. In conclusion, EPA membrane incorporation increased ABCA1 functionality in cholesterol-normal human THP-1 macrophages, likely through a combination of different mechanisms. This beneficial in vitro effect may partly contribute to the cardioprotective effect of a diet enriched with EPA highlighted by several recent clinical trials.

Cystic fibrosis (CF) is the most common genetic disease in Caucasians. CF is manifested by abnormal accumulation of mucus in the lungs, which serves as fertile ground for the growth of microorganisms leading to recurrent infections and ultimately, lung failure. Mucus in CF patients consists of DNA from dead neutrophils as well as mucins produced by goblet cells. MUC5AC mucin leads to pathological plugging of the airways whereas MUC5B has a protective role against bacterial infection. Therefore, decreasing the level of MUC5AC while maintaining MUC5B intact would in principle be a desirable mucoregulatory treatment outcome. Fenretinide prevented the lipopolysaccharide-induced increase of MUC5AC gene expression, without affecting the level of MUC5B, in a lung goblet cell line. Additionally, fenretinide treatment reversed the pro-inflammatory imbalance of fatty acids by increasing docosahexaenoic acid and decreasing the levels of arachidonic acid in a lung epithelial cell line and primary leukocytes derived from CF patients. Furthermore, for the first time we also demonstrate the effect of fenretinide on multiple unsaturated fatty acids, as well as differential effects on the levels of long- compared to very-long-chain saturated fatty acids which are important substrates of complex phospholipids. Finally, we demonstrate that pre-treating mice with fenretinide in a chronic model of P. aeruginosa lung infection efficiently decreases the accumulation of mucus. These findings suggest that fenretinide may offer a new approach to therapeutic modulation of pathological mucus production in CF.

Long-chain acyl-coenzyme A synthetases (ACSLs) are a family of enzymes that convert free long-chain fatty acids into their acyl-coenzyme A (CoA) forms. ACSL4, belonging to the ACSL family, shows a preferential use of arachidonic acid (AA) as its substrate and plays a role in the remodeling of AA-containing phospholipids by incorporating free AA. However, little is known about the roles of ACSL4 in inflammatory responses. Here, we assessed the roles of ACSL4 on the effector functions of bone marrow-derived macrophages (BMDMs) obtained from mice lacking ACSL4. Liquid chromatography-tandem mass spectrometry analysis revealed that various highly unsaturated fatty acid (HUFA)-derived fatty acyl-CoA species were markedly decreased in the BMDMs obtained from ACSL4-deficient mice compared with those in the BMDMs obtained from wild-type mice. BMDMs from ACSL4-deficient mice also showed a reduced incorporation of HUFA into phosphatidylcholines. The stimulation of BMDMs with lipopolysaccharide (LPS) elicited the release of prostaglandins (PGs), such as PGE2, PGD2 and PGF2α, and the production of these mediators was significantly enhanced by ACSL4 deficiency. In contrast, neither the LPS-induced release of cytokines, such as IL-6 and IL-10, nor the endocytosis of zymosan or dextran was affected by ACSL4 deficiency. These results suggest that ACSL4 has a crucial role in the maintenance of HUFA composition of certain phospholipid species and in the incorporation of free AA into the phospholipids in LPS-stimulated macrophages. ACSL4 dysfunction may facilitate inflammatory responses by an enhanced eicosanoid storm.

Epilepsy is a highly common chronic neurological disorder, manifested in many different types, affecting ~1% of the worldwide human population. The molecular mechanisms of epileptogenesis have not yet been clarified, and pharmacoresistance exhibited by 30-40% of epilepsy patients remains a major obstacle in medical care. Growing evidence indicates a role of lipid signalling pathways in epileptogenesis, thus lipid signals emerge as potential biomarkers for the onset and evolving course of the epileptic disorder, as well as potential therapeutic agents and targets. For this purpose, we applied a lipidomic strategy to unravel lipid alterations in brain regions, periphery tissues and plasma that are specific for acute epileptic seizures in mice at 1h after seizure induction by systemic kainic acid injection as compared to vehicle controls. Specifically, levels of (i) selected phospholipids and sphingomyelins, (ii) the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), and the endocannabinoid-related compounds oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), (iii) arachidonic acid (AA), (iv) selected eicosanoids, and (v) fatty acyl content of lipidome were determined in pulverized tissues from six brain regions of kainic acid induced epileptic seizure models and vehicle controls: hypothalamus, hippocampus, thalamus, striatum, cerebellum and cerebral cortex, and from peripheral organs, such as heart and lungs, and in plasma. Alterations in lipid levels after acute epileptic seizures as compared to non-seizure controls were found to be brain region- and periphery tissue-specific, including specific plasma lipid correlates, highlighting their value as marker candidates in translational research studies, and/or drug discovery and response monitoring.

Bone is a dynamic tissue that is constantly remodelled by bone resorbing osteoclasts and bone forming osteoblasts, respectively. A breakdown in the remodelling process underlies several bone diseases such as osteoporosis. Unsaturated fatty acids (UFAs) have been shown to have beneficial effects on bone health. However, the mechanism of action of UFAs in bone remains unclear. Free fatty acid receptor 4 (FFAR4) is expressed in bone cells and preferentially binds ω-3 and ω-7 UFAs. Therefore, we sought to determine if FFAR4 influenced the action of different classes of UFAs in bone cells. FFAR4 and potential signalling pathways, β-arrestin 2 (βarr2) and Gαq, were silenced in RAW264.7 murine macrophages (pre-osteoclasts) and MC3T3-E1 murine pre-osteoblasts. Cell differentiation, activation of signalling pathways and expression of regulatory genes were evaluated. The ω-3 UFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and the ω-7 UFA, palmitoleic acid (PLA), were shown to require the FFAR4/βarr2 signalling pathway to inhibit osteoclast differentiation in RAW264.7 murine macrophages. The ω-6 UFA, arachidonic acid, and the ω-9 UFA, oleic acid (OA), were shown to inhibit osteoclast formation but did not use FFAR4. DHA, EPA, PLA and OA enhanced osteoblast signalling through the FFAR4/βarr2 signalling axis. This study reveals that FFAR4/βarr2 signalling may mediate the bone protective effects of different classes of UFAs in osteoclasts and osteoblasts.

Endocannabinoids, such as anandamide (ANA) and 2-arachidonoylglycerol (2AG), are lipid-signaling molecules that can be oxidized by lipid-peroxidizing enzymes, and this oxidation alters the bioactivity of these lipid mediators. Here, under strictly comparable experimental conditions, we explored whether ANA and 2AG function as substrates for four human (ALOX15, ALOX15B, ALOX12, ALOX5) and three mice Alox isoforms (Alox15, Alox12, Alox5) and compared the rates of product formation with those of arachidonic acid oxygenation. Except for ALOX5, the two endocannabinoids were more efficiently oxygenated than arachidonic acid by human ALOX isoforms. Mice Alox15 oxygenated ANA more efficiently than arachidonic acid, but the other mice Alox isoforms exhibited reduced reaction rates for endocannabinoid conversion. Like its human ortholog, mice Alox5 did not oxygenate ANA, but the formation of 5-HETE-containing 2AG derivatives was observed for this enzyme. 1AG and 2AG were similarly effective substrates for human ALOX isoforms. Molecular docking studies, the pattern of oxygenation products, and site-directed mutagenesis experiments suggested a similar substrate alignment of arachidonic acid and endocannabinoids at the active site of ALOX15 orthologs. The product specificity of arachidonic acid oxygenation was conserved for endocannabinoid metabolization, and the triad concept describing the molecular basis for the reaction specificity of ALOX15 orthologs is applicable for endocannabinoid oxygenation. Taken together, these data indicate that, except for ALOX5 orthologs, endocannabinoids are suitable substrates for most mammalian ALOX isoforms.

Secreted LOX from Pseudomonas aeruginosa (PA-LOX) has previously been identified as arachidonic acid 15S-lipoxygenating enzyme. Here we report that the substitution of Ala420Gly in PA-LOX leads to an enzyme variant with pronounced dual specificity favoring arachidonic acid 11R-oxygenation. When compared with other LOX-isoforms the molecular oxygen affinity of wild-type PA-LOX is 1-2 orders of magnitude lower (Km O2 of 0.4mM) but Ala420Gly exchange improved the molecular oxygen affinity (Km O2 of 0.2mM). Experiments with stereo-specifically deuterated linoleic acid indicated that the formation of both 13S- and 9R-HpODE involves abstraction of the proS-hydrogen from C11 of the fatty acid backbone. To explore the structural basis for the observed functional changes (altered specificity, improved molecular oxygen affinity) we solved the crystal structure of the Ala420Gly mutant of PA-LOX at 1.8Å resolution and compared it with the wild-type enzyme. Modeling of fatty acid alignment at the catalytic center suggested that in the wild-type enzyme dioxygen is directed to C15 of arachidonic acid by a protein tunnel, which interconnects the catalytic center with the protein surface. Ala420Gly exchange redirects intra-enzyme O2 diffusion by bifurcating this tunnel so that C11 of arachidonic acid also becomes accessible for O2 insertion.

Platelets are collected for transfusion to patients with different haematological disorders, and for logistical reasons, platelets are stored as concentrates. Despite carefully controlled conditions, platelets become activated during storage, and platelet concentrates (PlaCs) may cause adverse inflammatory reactions in recipients. The time-dependent changes in the lipidome of clinical PlaCs, platelets isolated from PlaCs, and extracellular vesicles (EVs) thereof were examined by mass spectrometry. The relative amount of arachidonic acid containing glycerophospholipids, especially those in the phosphatidylethanolamine and phosphatidylserine classes during storage, but the relative amount of other polyunsaturated fatty acid containing glycerophospholipids remained stable in all sample types. These changes were not directly translated to lipid mediator (LM) profile since the levels of arachidonic acid-derived proinflammatory LMs were not specifically elevated. Instead, several monohydroxy pathway markers and functionally relevant LMs, both proinflammatory and proresolving, were detected in the PlaCs and the EVs, and some representatives of both kind clearly accumulated during storage. By Western blot, the key enzymes of these pathways were shown to be present in platelets, and in many cases, EVs. Since the EVs were enriched in the fatty acid precursors of LMs in their (phospholipid) membranes, harboured LM-producing enzymes, contained the related monohydroxy pathway markers, and secreted the final LM products, PlaC-derived EVs could participate in the regulation of inflammation and healing, and thereby aid the platelets in exerting their essential physiological functions.

Microvascular dysfunction is a key contributor to vascular hypertension, one of the most common chronic diseases in the world. Microvascular dysfunction leads to the loss of nitric oxide-mediated endothelial dilation and the subsequent compensatory function of endothelium-derived hyperpolarizing (EDH) factors in the regulation of vascular tone. Previously, we showed that lactone metabolite derived from arachidonic acid induces endothelial-dependent vasodilation in isolated human microvessels. Based on structural similarities, we hypothesize that additional lactone metabolites formed from eicosapentaenoic fatty acid (EPA) may bear EDH properties.

A high consumption of polyunsaturated fatty acids (PUFAs), particularly n-3 PUFAs, is atheroprotective. PUFAs incorporation into membrane phospholipids alters the functionality of membrane proteins. We studied the consequences of the in vitro supplementation of several PUFAs on the FA profiles and on ABCA1-dependent cholesterol efflux capacities from cholesterol-loaded macrophages. Arachidonic acid (AA, C20:4 n-6) and, to a lesser extent, eicosapentaenoic acid (EPA, C20:5 n-3), dose-dependently impaired cholesterol efflux from cholesterol-loaded J774 mouse macrophages without alterations in ABCA1 expression, whereas docosahexaenoic acid (DHA, C22:6 n-3) had no impact. AA cells exhibited higher proportions of arachidonic acid and adrenic acid (C22:4 n-6), its elongation product. EPA cells exhibited slightly higher proportions of EPA associated with much higher proportions of docosapentaenoic acid (C22:5 n-3), its elongation product and with lower proportions of AA. Conversely, both EPA and DHA and, to a lesser extent, AA decreased cholesterol efflux from cholesterol-loaded primary human macrophages (HMDM). The differences observed in FA profiles after PUFA supplementations were different from those observed for the J774 cells. In conclusion, we are the first to report that AA and EPA, but not DHA, have deleterious effects on the cardioprotective ABCA1 cholesterol efflux pathway from J774 foam cells. Moreover, the membrane incorporation of PUFAs does not have the same impact on cholesterol efflux from murine (J774) or human (HMDM) cholesterol-loaded macrophages. This finding emphasizes the key role of the cellular model in cholesterol efflux studies and may partly explain the heterogeneous literature data on the impact of PUFAs on cholesterol efflux.

Arachidonic acid lipoxygenases (ALOXs) are lipid-metabolizing enzymes that have been implicated in cell differentiation, but also in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Most mammalian genomes involve six or seven functional ALOX genes and among the corresponding ALOX-isoforms the ALOX15 orthologs are somewhat unique since they exhibit variable reaction specificity using arachidonic acid as substrate. The Evolutionary Hypothesis of mammalian ALOX15 reaction specificity (Prog. Lipid Res. 72, 55, 2018) suggests that ALOX15 orthologs of primates ranked higher in evolution than gibbons are 15-lipoxygenating enzymes. In contrast, mammals ranking lower than gibbons express dominantly 12-lipoxygenating lipoxygenases and gibbon ALOX15 constitutes a transition enzyme with pronounced dual reaction specificity. Here we predicted the reaction specificity of 95 different prototherian, metatherian and eutherian ALOX15 orthologs on the basis of their primary structures and characterized experimentally the reaction specificity of ten novel metatherian/eutherian enzymes representing different stages of mammalian evolution (gorilla, opossum, cape golden mole, dog, horseshoe bat, hedgehog, Sunda flying lemur, pika, chinchilla, kangaroo rat). We found that 97% of the currently sequenced mammalian ALOX15 including the enzymes of living and extinct hominids follow the Evolutionary Hypothesis. However, the ALOX15 orthologs of rabbits and of the Ord's kangaroo rat violate this mechanistic concept. Taken together, this data confirms the Evolutionary Hypothesis of ALOX15 reaction specificity and puts this concept on a more reliable experimental basis.

The tree shrew (Tupaia belangeri) is a rat-sized mammal, which is more closely related to humans than mice and rats. However, the use of tree shrew to explore the patho-mechanisms of human inflammatory disorders has been limited since nothing is known about eicosanoid metabolism in this mammalian species. Eicosanoids are important lipid mediators exhibiting pro- and anti-inflammatory activities, which are biosynthesized via lipoxygenase and cyclooxygenase pathways. When we searched the tree shrew genome for the presence of cyclooxygenase and lipoxygenase isoforms we found copies of functional COX1, COX2 and LOX genes. Interestingly, we identified four copies of ALOX15 genes, which encode for four structurally distinct ALOX15 orthologs (tupALOX15a-d). To explore the catalytic properties of these enzymes we expressed tupALOX15a and tupALOX15c as catalytically active proteins and characterized their enzymatic properties. As predicted by the Evolutionary Hypothesis of ALOX15 specificity we found that the two enzymes converted arachidonic acid predominantly to 12S-HETE and they also exhibited membrane oxygenase activities. However, their reaction kinetic properties (KM for arachidonic acid and oxygen, T- and pH-dependence) and their substrate specificities were remarkably different. In contrast to mice and humans, tree shrew ALOX15 isoforms are highly expressed in the brain suggesting a role of these enzymes in cerebral function. The genomic multiplicity and the tissue expression patterns of tree shrew ALOX15 isoforms need to be considered when the results of in vivo inflammation studies obtained in this animal are translated into the human situation.

Lysophospholipid acyltransferases (LPLATs) incorporate a fatty acid into the hydroxyl group of lysophospholipids (LPLs) and are critical for determining the fatty acid composition of phospholipids. Previous studies have focused mainly on their molecular identification and their substrate specificity regarding the polar head groups and acyl-CoAs. However, little is known about the positional specificity of the hydroxyl group of the glycerol backbone (sn-2 or sn-1) at which LPLATs introduce a fatty acid. This is mainly due to the instability of LPLs used as an acceptor, especially for LPLs with a fatty acid at the sn-2 position of the glycerol backbone (sn-2-LPLs), which are essential for the enzymatic assay to determine the positional specificity. In this study, we established a method to determine the positional specificity of LPLAT by preparing stable sn-2-LPLs in combination with PLA2 digestion, and applied the method for determining the positional specificity of several LPLATs including LPCAT1, LYCAT and LPCAT3. We found that LPCAT1 introduced palmitic acid both at the sn-1 and sn-2 positions of palmitoyl-LPC, while LYCAT and LPCAT3 specifically introduced stearic acid at the sn-1 position of LPG and arachidonic acid at the sn-2 position of LPC, respectively. The present method for evaluating the positional specificity could also be used for biochemical characterization of other LPLATs.

A polymorphism of TM6SF2 associates with hepatic lipid accumulation and reduction of triacylglycerol (TAG) secretion, but the function of the encoded protein has remained enigmatic. We studied the effect of stable TM6SF2 knock-down on the lipid content and composition, mitochondrial fatty acid oxidation and organelle structure of HuH7 hepatoma cells. Knock-down of TM6SF2 resulted in intracellular accumulation of TAGs, cholesterol esters, phosphatidylcholine (PC) and phosphatidylethanolamine. In all of these lipid classes, polyunsaturated lipid species were significantly reduced while saturated and monounsaturated species increased their proportions. The PCs encountered relative and absolute arachidonic acid (AA, 20:4n-6) depletion, and AA was also reduced in the total cellular fatty acid pool. Synthesis and turnover of the hepatocellular glycerolipids was enhanced. The TM6SF2 knock-down cells secreted lipoprotein-like particles with a smaller diameter than in the controls, and more lysosome/endosome structures appeared in the knock-down cells. The mitochondrial capacity for palmitate oxidation was significantly reduced. These observations provide novel clues to TM6SF2 function and raise altered mebrane lipid composition and dynamics among the mechanism(s) by which the protein deficiency disturbs hepatic TAG secretion.

Pseudomonas aeruginosa is a gram-negative pathogen, which causes life-threatening infections in immunocompromized patients. These bacteria express a secreted lipoxygenase (PA-LOX), which oxygenates free arachidonic acid to 15S-hydro(pero)xyeicosatetraenoic acid. It binds phospholipids at its active site and physically interacts with lipid vesicles. When incubated with red blood cells membrane lipids are oxidized and hemolysis is induced but the structures of the oxygenated membrane lipids have not been determined. Using a lipidomic approach, we analyzed the formation of oxidized phospholipids generated during the in vitro incubation of recombinant PA-LOX with human erythrocytes and cultured human lung epithelial cells. Precursor scanning of lipid extracts prepared from these cells followed by multiple reaction monitoring and MS/MS analysis revealed a complex mixture of oxidation products. For human red blood cells this mixture comprised forty different phosphatidylethanolamine and phosphatidylcholine species carrying oxidized fatty acid residues, such as hydroxy-octadecadienoic acids, hydroxy- and keto-eicosatetraenoic acid, hydroxy-docosahexaenoic acid as well as oxygenated derivatives of less frequently occurring polyenoic fatty acids. Similar oxygenation products were also detected when cultured lung epithelial cells were employed but here the amounts of oxygenated lipids were smaller and under identical experimental conditions we did not detect major signs of cell lysis. However, live imaging indicated an impaired capacity for trypan blue exclusion and an augmented mitosis rate. Taken together these data indicate that PA-LOX can oxidize the membrane lipids of eukaryotic cells and that the functional consequences of this reaction strongly depend on the cell type.

The interest in understanding the capacity of aquatic invertebrates to biosynthesise omega-3 (ω3) long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) has increased in recent years. Using the common octopus Octopus vulgaris as a model species, we previously characterised a ∆5 desaturase and two elongases (i.e. Elovl2/5 and Elovl4) involved in the biosynthesis of LC-PUFA in molluscs. The aim of this study was to characterise both molecularly and functionally, two methyl-end (or ωx) desaturases that have been long regarded to be absent in most animals. O. vulgaris possess two ωx desaturase genes encoding enzymes with ∆12 and ω3 regioselectivities enabling the de novo biosynthesis of the C18 PUFA 18:2ω6 (LA, linoleic acid) and 18:3ω3 (ALA, α-linolenic acid), generally regarded as dietary essential for animals. The O. vulgaris ∆12 desaturase ("ωx2") mediates the conversion of 18:1ω9 (oleic acid) into LA, and subsequently, the ω3 desaturase ("ωx1") catalyses the ∆15 desaturation from LA to ALA. Additionally, the O. vulgaris ω3 desaturase has ∆17 capacity towards a variety of C20 ω6 PUFA that are converted to their ω3 PUFA products. Particularly relevant was the affinity of the ω3 desaturase towards 20:4ω6 (ARA, arachidonic acid) to produce 20:5ω3 (EPA, eicosapentaenoic acid), as supported by yeast heterologous expression, and enzymatic activity exhibited in vivo when paralarvae were incubated in the presence of [1-14C]20:4ω6. These results confirmed that several routes enabling EPA biosynthesis are operative in O. vulgaris whereas ARA and docosahexaenoic acid (DHA, 22:6ω3) should be considered essential fatty acids since endogenous production appears to be limited.