Consuming a Linoleate-Rich Diet Increases Concentrations of Tetralinoleoyl Cardiolipin in Mouse Liver and Alters Hepatic Mitochondrial Respiration.

BACKGROUND: Hepatic mitochondrial dysfunction is a major cause of fat accumulation in the liver. Individuals with fatty liver conditions have hepatic mitochondrial structural abnormalities and a switch in the side chain composition of the mitochondrial phospholipid, cardiolipin, from poly- to monounsaturated fatty acids. Linoleic acid (LA), an essential dietary fatty acid, is required to remodel nascent cardiolipin (CL) to its tetralinoleoyl cardiolipin (L4CL, CL with 4 LA side chains) form, which is integral for mitochondrial membrane structure and function to promote fatty acid oxidation. It is unknown, however, whether increasing LA in the diet can increase hepatic L4CL concentrations and improve mitochondrial respiration in the liver compared with a diet rich in monounsaturated and saturated fatty acids. OBJECTIVES: The main aim of this study was to test the ability of a diet fortified with LA-rich safflower oil (SO), compared with the one fortified with lard (LD), to increase concentrations of L4CL and improve mitochondrial respiration in the livers of mice. METHODS: Twenty-four (9-wk-old) C57 BL/J6 male mice were fed either the SO or LD diets for ∼100 d, whereas food intake and body weight, fasting glucose, and glucose tolerance tests were performed to determine any changes in glycemic control. RESULTS: Livers from mice fed SO diet had higher relative concentrations of hepatic L4CL species compared with LD diet-fed mice (P value = 0.004). Uncoupled mitochondria of mice fed the SO diet, compared with LD diet, had an increased baseline oxygen consumption rate (OCR) and succinate-driven respiration (P values = 0.03 and 0.01). SO diet-fed mice had increased LA content in all phospholipid classes compared with LD-fed mice (P < 0.05). CONCLUSIONS: Our findings reveal that maintaining or increasing hepatic L4CL may result in increased OCR in uncoupled hepatic mitochondria in healthy mice whereas higher oleate content of CL reduced mitochondrial function shown by lower OCR in uncoupled mitochondria.

Different Structures-Similar Effect: Do Substituted 5-(4-Methoxyphenyl)-1H-indoles and 5-(4-Methoxyphenyl)-1H-imidazoles Represent a Common Pharmacophore for Substrate Selective Inhibition of Linoleate Oxygenase Activity of ALOX15?

Mammalian 15-lipoxygenases (ALOX15) are lipid peroxidizing enzymes that exhibit variable functionality in different cancer and inflammation models. The pathophysiological role of linoleic acid- and arachidonic acid-derived ALOX15 metabolites rendered this enzyme a target for pharmacological research. Several indole and imidazole derivatives inhibit the catalytic activity of rabbit ALOX15 in a substrate-specific manner, but the molecular basis for this allosteric inhibition remains unclear. Here, we attempt to define a common pharmacophore, which is critical for this allosteric inhibition. We found that substituted imidazoles induce weaker inhibitory effects when compared with the indole derivatives. In silico docking studies and molecular dynamics simulations using a dimeric allosteric enzyme model, in which the inhibitor occupies the substrate-binding pocket of one monomer, whereas the substrate fatty acid is bound at the catalytic center of another monomer within the ALOX15 dimer, indicated that chemical modification of the core pharmacophore alters the enzyme-inhibitor interactions, inducing a reduced inhibitory potency. In our dimeric ALOX15 model, the structural differences induced by inhibitor binding are translated to the hydrophobic dimerization cluster and affect the structures of enzyme-substrate complexes. These data are of particular importance since substrate-specific inhibition may contribute to elucidation of the putative roles of ALOX15 metabolites derived from different polyunsaturated fatty acids in mammalian pathophysiology.

Differential Lipid Accumulation on HepG2 Cells Triggered by Palmitic and Linoleic Fatty Acids Exposure.

Lipid metabolism pathways such as ß-oxidation, lipolysis and, lipogenesis, are mainly associated with normal liver function. However, steatosis is a growing pathology caused by the accumulation of lipids in hepatic cells due to increased lipogenesis, dysregulated lipid metabolism, and/or reduced lipolysis. Accordingly, this investigation hypothesizes a selective in vitro accumulation of palmitic and linoleic fatty acids on hepatocytes. After assessing the metabolic inhibition, apoptotic effect, and reactive oxygen species (ROS) generation by linoleic (LA) and palmitic (PA) fatty acids, HepG2 cells were exposed to different ratios of LA and PA to study the lipid accumulation using the lipophilic dye Oil Red O. Lipidomic studies were also carried out after lipid isolation. Results revealed that LA was highly accumulated and induced ROS production when compared to PA. Lipid profile modifications were observed after LA:PA 1:1 (v/v) exposure, which led to a four-fold increase in triglycerides (TGs) (mainly in linoleic acid-containing species), as well as a increase in cholesterol and polyunsaturated fatty acids (PUFA) content when compared to the control cells. The present work highlights the importance of balancing both PA and LA fatty acids concentrations in HepG2 cells to maintain normal levels of free fatty acids (FFAs), cholesterol, and TGs and to minimize some of the observed in vitro effects (i.e., apoptosis, ROS generation and lipid accumulation) caused by these fatty acids.

Of fruits and fats: high-sugar diets restore fatty acid profiles in the white adipose tissue of captive dwarf lemurs.

Fat-storing hibernators rely on fatty acids from white adipose tissue (WAT) as an energy source to sustain hibernation. Whereas arctic and temperate hibernators preferentially recruit dietary polyunsaturated fatty acids (PUFAs), tropical hibernators can rely on monounsaturated fatty acids that produce fewer lipid peroxides during oxidation. Nevertheless, compositional data on WAT from tropical hibernators are scant and questions remain regarding fat recruitment and metabolism under different environmental conditions. We analyse fatty acid profiles from the WAT of captive dwarf lemurs (Cheirogaleus medius) subjected to high-sugar or high-fat diets during fattening and cold or warm conditions during hibernation. Dwarf lemurs fed high-sugar (compared to high-fat) diets displayed WAT profiles more comparable to wild lemurs that fatten on fruits and better depleted their fat reserves during hibernation. One PUFA, linoleic acid, remained elevated before hibernation, potentially lingering from the diets provisioned prior to fattening. That dwarf lemurs preferentially recruit the PUFA linoleic acid from diets that are naturally low in availability could explain the discrepancy between captive and wild lemurs' WAT. While demonstrating that minor dietary changes can produce major changes in seasonal fat deposition and depletion, our results highlight the complex role for PUFA metabolism in the ecology of tropical hibernators.

Effect of chelated source of additional zinc and selenium on performance, yolk fatty acid composition, and oxidative stability in laying hens fed with oxidised oil.

1. The following study determined whether the effects of the combined addition of zinc amino acid complex (ZA) and selenomethionine (SM) was superior to their single addition in controlling the oxidative stress induced by dietary oxidised fat in laying hens.2. Two hundred and forty 32-week-old laying hens were divided into the following dietary treatments (each consisting of six replicates of eight birds): 1) a fresh soy oil (FSO) diet; 2) an oxidised soy oil (OSO) diet; 3) an OSO diet plus 20 mg zinc as ZA/kg (OSO+ZA); 4) an OSO diet plus 0.2 mg selenium as SM/kg (OSO+SM); and 5) an OSO diet plus ZA and SM (OSO+ZA+SM).3. After 10 weeks of feeding hens, feed intake, egg production, and egg mass in the OSO+ZA+SM group were similar to the FSO group but better (P < 0.05) than those in the OSO group. Shell thickness and shell breaking strength were significantly improved by the OSO+ZA and OSO+ZA+SM treatments.4. Increases in the yolk concentrations of palmitic acid and total saturated fatty acids (SFA), and decreases in yolk linoleic acid, n-6 polyunsaturated fatty acids (PUFA), total PUFA, and PUFA/SFA ratio were induced by dietary oxidised fat which were normalised (P < 0.05) by OSO+SM and OSO+ZA+SM.5. An increase (P < 0.05) in malondialdehyde and a decrease in 2,2-diphenyl-picrylhydrazyl radical scavenging activity in the yolk, induced by dietary oxidised fat, was significantly improved by all dietary supplementations, but only birds fed the OSO+ZA+SM diet exhibited similar values to those fed FSO.6. In conclusion, the simultaneous inclusion of organic zinc plus selenium in the oxidised fat diets was beneficial for improving egg-laying performance, yolk fatty acid profile, and oxidative stability, but not for internal egg quality, compared with either zinc or selenium alone in laying hens.

UPLC-MS based plasma metabolomics and lipidomics reveal alterations associated with IgG4-related disease.

OBJECTIVE: The pathogenesis of IgG4-related disease (IgG4-RD) remains unclear. Metabolomic profiling of IgG4-RD patients offers an opportunity to identify novel pathophysiological targets and biomarkers. This study aims to identify potential plasma biomarkers associated with IgG4-RD. METHODS: Thirty newly diagnosed IgG4-RD patients, age-matched healthy controls and post-treated IgG4-RD patients were enrolled. Patients' clinical data, laboratory parameters and plasma were collected. Plasma was measured for ultraperformance liquid chromatography-tandem mass spectrometry based metabolomics and lipidomics profiling. Multivariate and univariate statistical analyses were conducted to identify potential biomarkers. The receiver operating characteristic and the correlations between biomarkers and clinical parameters were investigated. RESULTS: The plasma metabolites are altered among healthy controls, newly diagnosed IgG4-RD and post-treated IgG4-RD groups. Of the identified features, eight metabolites were significantly perturbed in the IgG4-RD group, including glyceric acid 1,3-biphosphate (1,3-BPG), uridine triphosphate (UTP), uridine diphosphate glucose (UDP-Glc) or uridine diphosphate galactose (UDP-Gal), lysophospholipids, linoleic acid derivatives and ceramides. Receiver operating characteristic analysis indicated that UTP, UDP-Glc/UDP-Gal and LysoPC (18:1) had high sensitivity and specificity in diagnosis of IgG4-RD. A Pearson correlation analysis showed that 1,3-BPG and UTP were strongly correlated with clinical parameters. CONCLUSION: IgG4-RD patients have a unique plasma metabolomic profile compared with healthy controls. Our study suggested that metabolomic profiling may provide important insights into pathophysiology and testable biomarkers for diagnosis of IgG4-RD.

Biochemical Characterization of 13-Lipoxygenases of Arabidopsis thaliana.

13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (Vmax app) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s-1·mg protein-1), with a Km value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg2+, Ca2+, Cu2+ and Cd2+, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.

Impact of a high-fat diet on the fatty acid composition of the retina.

Structure and function of the retina mainly rely on its fatty acid (FA) composition. Evidence from epidemiological studies and from animal experiments indicates that FA composition of the retina is influenced by the diet. Mice under chronic high-fat diet (HFD) develop metabolic syndrome, a risk factor for diabetes that is associated with structural and functional alterations of the retina. Here, we studied the impact of chronic exposure of mice to HFD on retinal FA composition. C57BL/6 J male mice were fed either a chow diet or a HFD for 11 weeks. As expected, HFD induced weight gain, adiposity, hyperglycemia and dyslipidemia. The retinal FA composition was determined by gas chromatography coupled to flame ionization detection. No significant change in the relative abundance of total saturated FAs (SFAs), total monounsaturated FAs (MUFAs) or total polyunsaturated FAs (PUFAs) was observed. However, retinas of HFD-fed mice displayed decreased amounts of C24:0 (p = 0.0231), C16:1n-7 (p < 0.0001), C18:1n-7 (p < 0.0001), C20:3n-9 (p = 0.0425) and C20:3n-6 (p = 0.0008), and an increased amount of C20:2n-6 (p < 0.0001). In addition, the ratio of linoleic acid (C18:2n-6) to alpha-linolenic acid (C18:3n-3) was increased in the retinas of HFD-fed mice (15.0 ± 0.8 versus 11.8 ± 0.6 in HFD and CD, respectively, p = 0.0045). No modification in the contents of arachidonic acid (C20:4n-6, AA) and docosahexaenoic acid (C22:6n-3, DHA) were observed. Analysis of dimethylacetals (DMA), which are residues of plasmalogens (Pls), revealed that the amount of Pls containing octadecanal-aldehydes (DMA C18:0) was significantly increased in HFD-fed mice (p = 0.0447). This increase was, at least in part, balanced by a decrease in Pls containing 7-octadecanal-aldehydes (DMA C18:1n-7) (p = 0.0007). In conclusion, HFD had an impact on the relative proportion of essential dietary fatty acids linoleic acid and alpha-linolenic acid that are incorporated in the retina. However, this imbalance in PUFA precursors did not alter the content of the two major retinal long-chain PUFAs, AA and DHA. HFD consumption also led to alterations in the retinal SFAs, MUFAs and Pls profiles.

Bioactive Oxylipins in Infants and Children With Congenital Heart Disease Undergoing Pediatric Cardiopulmonary Bypass.

OBJECTIVES: To determine the production of 9-hydroxyoctadecadienoic acid and 13-hydroxyoctadecadienoic acid during cardiopulmonary bypass in infants and children undergoing cardiac surgery, evaluate their relationship with increase in cell-free plasma hemoglobin, provide evidence of bioactivity through markers of inflammation and vasoactivity (WBC count, milrinone use, vasoactive-inotropic score), and examine their association with overall clinical burden (ICU/hospital length of stay and mechanical ventilation duration). DESIGN: Prospective observational study. SETTING: Twelve-bed cardiac ICU in a university-affiliated children's hospital. PATIENTS: Children were prospectively enrolled during their preoperative clinic appointments with the following criteria: greater than 1 month to less than 18 years old, procedures requiring cardiopulmonary bypass INTERVENTIONS:: None. MEASUREMENTS AND MAIN RESULTS: Plasma was collected at the start and end of cardiopulmonary bypass in 34 patients. 9-hydroxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, plasma hemoglobin, and WBC increased. 9:13-hydroxyoctadecadienoic acid at the start of cardiopulmonary bypass was associated with vasoactive-inotropic score at 2-24 hours postcardiopulmonary bypass (R = 0.25; p < 0.01), milrinone use (R = 0.17; p < 0.05), and WBC (R = 0.12; p < 0.05). 9:13-hydroxyoctadecadienoic acid at the end of cardiopulmonary bypass was associated with vasoactive-inotropic score at 2-24 hours (R = 0.17; p < 0.05), 24-48 hours postcardiopulmonary bypass (R = 0.12; p < 0.05), and milrinone use (R = 0.19; p < 0.05). 9:13-hydroxyoctadecadienoic acid at the start and end of cardiopulmonary bypass were associated with the changes in plasma hemoglobin (R = 0.21 and R = 0.23; p < 0.01). The changes in plasma hemoglobin was associated with milrinone use (R = 0.36; p < 0.001) and vasoactive-inotropic score less than 2 hours (R = 0.22; p < 0.01), 2-24 hours (R = 0.24; p < 0.01), and 24-48 hours (R = 0.48; p < 0.001) postcardiopulmonary bypass. Cardiopulmonary bypass duration, 9:13-hydroxyoctadecadienoic acid at start of cardiopulmonary bypass, and plasma hemoglobin may be risk factors for high vasoactive-inotropic score. Cardiopulmonary bypass duration, changes in plasma hemoglobin, 9:13-hydroxyoctadecadienoic acid, and vasoactive-inotropic score correlate with ICU and hospital length of stay and/mechanical ventilation days. CONCLUSIONS: In low-risk pediatric patients undergoing cardiopulmonary bypass, 9:13-hydroxyoctadecadienoic acid was associated with changes in plasma hemoglobin, vasoactive-inotropic score, and WBC count, and may be a risk factor for high vasoactive-inotropic score, indicating possible inflammatory and vasoactive effects. Further studies are warranted to delineate the role of hydroxyoctadecadienoic acids and plasma hemoglobin in cardiopulmonary bypass-related dysfunction and to explore hydroxyoctadecadienoic acid production as a potential therapeutic target.

Lipolysis modulates the biosynthesis of inflammatory lipid mediators derived from linoleic acid in adipose tissue of periparturient dairy cows.

Oxidized linoleic acid metabolites (OXLAM) are products of adipocyte lipolysis with the potential to modulate adipose tissue (AT) lipid metabolism and inflammation. In periparturient cows, linoleic acid is preferentially mobilized from AT during lipolysis by hormone-sensitive lipase (HSL) compared with other polyunsaturated fatty acids. Enzymatic and nonenzymatic reactions generate OXLAM from linoleic acid. Among OXLAM, 9-, 10-, and 12-hydroxy-octadecadienoic acids (HODE) are associated with pro-inflammatory responses, whereas 9- and 13-oxo-octadecadienoic acids (oxoODE) and 13-HODE can facilitate inflammation resolution and promote lipogenesis. This study evaluated the effect of HSL activity on OXLAM biosynthesis using subcutaneous AT explants collected from multiparous dairy cows at 10 d before and again at 10 and 24 d after calving. Explants were treated for 3 h without or with the ß-adrenergic agonist isoproterenol (ISO; 1 µM; MilliporeSigma, Burlington, MA) to induce HSL activity. The contribution of HSL to OXLAM biosynthesis was determined by inhibiting its activity with CAY10499 (2 µM; Cayman Chemical, Ann Arbor, MI). After treatments, media and explants were collected for lipidomic analysis using HPLC-tandem mass spectroscopy. Results indicated that ISO increased the biosynthesis of 9-, 12-, and 13-HODE and 9-oxoODE, and this effect was reduced at 24 d after calving. Inhibiting HSL activity partially reversed ISO effects on HODE and 9-oxoODE. Our ex vivo model demonstrated for the first time a direct effect of HSL activity on the biosynthesis of OXLAM in AT, especially at 10 d before and 10 d after calving. The biosynthesis of anti-inflammatory OXLAM is limited during the first weeks after parturition and may promote AT inflammation and lipolytic responses to negative energy balance. These results indicate that HSL activity releases linoleic acid for OXLAM biosynthesis in concentrations of a magnitude that may bypass the need for the activation of phospholipases linked with the inflammatory cascade and thus supports, in part, lipolysis-driven inflammation within AT of periparturient cows.

Influence of Lipoxygenase Inhibition on Glioblastoma Cell Biology.

BACKGROUND: The relationship between glioblastoma (GBM) and fatty acid metabolism could be the key to elucidate more effective therapeutic targets. 15-lipoxygenase-1 (15-LOX), a linolenic acid and arachidonic acid metabolizing enzyme, induces both pro- and antitumorigenic effects in different cancer types. Its role in glioma activity has not yet been clearly described. The objective of this study was to identify the influence of 15-LOX and its metabolites on glioblastoma cell activity. METHODS: GBM cell lines were examined using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to identify 15-LOX metabolites. GBM cells treated with 15-LOX metabolites, 13-hydroxyoctadecadeinoic acid (HODE) and 9-HODE, and two 15-LOX inhibitors (luteolin and nordihydroguaiaretic acid) were also examined. Dose response/viability curves, RT-PCRs, flow cytometry, migration assays, and zymograms were performed to analyze GBM growth, migration, and invasion. RESULTS: Higher quantities of 13-HODE were observed in five GBM cell lines compared to other lipids analyzed. Both 13-HODE and 9-HODE increased cell count in U87MG. 15-LOX inhibition decreased migration and increased cell cycle arrest in the G2/M phase. CONCLUSION: 15-LOX and its linoleic acid (LA)-derived metabolites exercise a protumorigenic influence on GBM cells in vitro. Elevated endogenous levels of 13-HODE called attention to the relationship between linoleic acid metabolism and GBM cell activity.

Determining the Cytotoxicity of Oxidized Lipids in Cultured Caco-2 Cells Using Bioimaging Techniques.

Fish lipids are comprised of considerable quantities of polyunsaturated acids and are prone to oxidation, producing reactive oxygen species and hydroperoxides. This study aimed to evaluate the biochemical and structural alterations in Caco-2 cells following exposure to 100 µg/mL methyl linoleate or fish oil, and then radiated for 24, 48 or 72 h. Electron spin resonance spectroscopy detected free radicals in the lipid membrane, Raman microscopy observed biochemical alterations and atomic force microscopy identified changes in morphology, such as the breakdown of DNA bonds. The study showed that bioimaging and biochemical techniques can be effective at detecting and diagnosing cellular injuries incurred by lipid peroxidation.

Eicosanoids in Nonalcoholic Fatty Liver Disease (NAFLD) Progression. Do Serum Eicosanoids Profile Correspond with Liver Eicosanoids Content during NAFLD Development and Progression?

Nonalcoholic fatty liver disease (NAFLD) is becoming a major public health problem worldwide. The study aimed to evaluate the concentration of eicosanoids in serum and liver tissue during steatosis progression and to assess whether eicosanoid change scores may predict liver tissue remodeling. Thirty six eight-week-old male Sprague Dawley rats were enrolled and sacrificed at different stages of NAFLD. Eicosanoid concentrations, namely lipoxin A4, hydroxyeicosatetraenoic acids (HETE), hydroxyloctadecadienoic acids (HODE), protectin DX, Maresine1, leucotriene B4, prostaglandin E2, and resolvin D1 measurement in serum and liver tissue with Agilent Technologies 1260 liquid chromatography were evaluated. For the liver and serum concentrations of 9-HODE and 13-HODE, the correlations were found to be strong and positive (r > 0.7, p < 0.05). Along with NAFLD progression, HODE concentration significantly increased, and change scores were more abundant in the liver. The moderate positive correlation between liver and serum (r = 0.52, p < 0.05) was also observed for resolvin E1. The eicosanoid concentration decreased during NAFLD progression, but mostly in serum. There were significant correlations between HETE concentrations in liver and serum, but their associations were relatively low and changes the most in liver tissue. Eicosanoids profile, predominantly 9-HODE and 13-HODE, may serve as a potential biomarker for NAFLD development.

Sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity during the transition to endothermy in an altricial bird.

Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane pump critical to muscle calcium cycling during contraction, and SERCA has also been proposed as the basis for a non-shivering thermogenesis mechanism in birds. Despite its potential importance to both shivering and non-shivering thermogenesis, the activity of this transporter has rarely been studied in altricial birds, and never during the developmental transition from ectothermy to endothermy. Here, we describe SERCA activity in the pectoralis muscle and heart ventricle of red-winged blackbird (Agelaius phoeniceus) nestlings, fledglings and adults. Additionally, using a diet manipulation, we tested the hypothesis that muscle SERCA activity is affected by dietary fatty acid composition, as has been shown in some previous studies. In blackbird hearts, SERCA activity increased throughout development and into adulthood, conspicuously jumping higher just prior to fledging. In pectoralis muscle, SERCA activity increased throughout the nestling period, but then declined after fledging, an effect we attribute to remodeling of the muscle from a primarily heat-generating organ to a primarily force-generating organ. SERCA activity of the pectoralis muscle was correlated with the proportion of linoleic acid in muscle phospholipids when including all ages in the control group. However, in diet-manipulated birds, there was no consistent relationship between SERCA activity and muscle membrane fatty acid composition at any tested age (5-9 days old). It is unclear whether SERCA might be affected by developmental changes in fatty acid composition at younger ages.

Targeting CYP2J to reduce paclitaxel-induced peripheral neuropathic pain.

Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a severe dose- and therapy-limiting side effect of widely used cytostatics that is particularly difficult to treat. Here, we report increased expression of the cytochrome-P450-epoxygenase CYP2J6 and increased concentrations of its linoleic acid metabolite 9,10-EpOME (9,10-epoxy-12Z-octadecenoic acid) in dorsal root ganglia (DRGs) of paclitaxel-treated mice as a model of CIPNP. The lipid sensitizes TRPV1 ion channels in primary sensory neurons and causes increased frequency of spontaneous excitatory postsynaptic currents in spinal cord nociceptive neurons, increased CGRP release from sciatic nerves and DRGs, and a reduction in mechanical and thermal pain hypersensitivity. In a drug repurposing screen targeting CYP2J2, the human ortholog of murine CYP2J6, we identified telmisartan, a widely used angiotensin II receptor antagonist, as a potent inhibitor. In a translational approach, administration of telmisartan reduces EpOME concentrations in DRGs and in plasma and reverses mechanical hypersensitivity in paclitaxel-treated mice. We therefore suggest inhibition of CYP2J isoforms with telmisartan as a treatment option for paclitaxel-induced neuropathic pain.

Changes in Gene Expression and Metabolite Profiles in Platanus acerifolia Leaves in Response to Feeding Damage Caused by Corythucha ciliata.

The sycamore lace bug, Corythucha ciliata (Say) is a highly invasive pest insect that feeds on sycamore trees (Platanus spp.) worldwide. The interaction between Platanus species and this insect pest has not yet been studied at the molecular level. Therefore, a recent study was conducted to compare the gene expression and metabolite profiles of Platanus acerifolia leaves in response to C. ciliata feeding damage after 24 and 48 h. We employed high throughput RNA sequencing (RNA- seq) to identify a total of 2,828 significantly differentially expressed genes (DEGs) after C. ciliata feeding. In addition, 303 unigenes were found to be up-regulated at both time points. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that monoterpenoid biosynthesis, the linoleic acid metabolism pathway, and alpha- linolenic acid metabolism were the most prominent pathways among the DEGs. Further analysis of the metabolite profiles showed that nine metabolites were significantly different before and after C. ciliata damage. In addition, we analyzed DEGs detected in the P. acerifolia and C. ciliata interaction using Mapman. The terpene synthase gene family was also identified. We suggest that the results obtained from DEGs and metabolite analysis can provide important information for the identification of genes involved in the P. acerifolia-C. ciliata interaction, which might be necessary for controlling C. ciliata efficiently.

Hexadecenoic Fatty Acid Positional Isomers and De Novo PUFA Synthesis in Colon Cancer Cells.

Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9cis-16:1; n-7 series) and sapienic acid (6cis-16:1; n-10 series), respectively. The corresponding biological consequences and lipidomic research on these positional monounsaturated fatty acid (MUFA) isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid (5cis,8cis-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 µM) and provide evidence of the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodelling was influenced by the type of fatty acid and positional isomer, with an increase of 8cis-18:1, n-10 PUFA and a decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in cases with sapienic acid. Sapienic acid was the less toxic among the tested fatty acids, showing the highest EC50s and inducing death only in 75% of cells at the highest concentration tested. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.

P-MAPA and Interleukin-12 Reduce Cell Migration/Invasion and Attenuate the Toll-Like Receptor-Mediated Inflammatory Response in Ovarian Cancer SKOV-3 Cells: A Preliminary Study.

Immunotherapies have emerged as promising complementary treatments for ovarian cancer (OC), but its effective and direct role on OC cells is unclear. This study examined the combinatory effects of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride, known as P-MAPA, and the human recombinant interleukin-12 (hrIL-12) on cell migration/invasion, apoptosis, toll-like receptor (TLR)-mediated inflammation, and cytokine/chemokine profile in human OC cell line SKOV-3. P-MAPA and IL-12 showed cancer cell toxicity under low doses after 48 h. Although apoptosis/necrosis and the cell cycle were unchanged by the treatments, P-MAPA enhanced the sensitivity to paclitaxel (PTX) and P-MAPA associated with IL-12 significantly reduced the migratory potential and invasion capacity of SKOV-3 cells. P-MAPA therapy reduced TLR2 immunostaining and the myeloid differentiation factor 88 (MyD88), but not the TLR4 levels. Moreover, the combination of P-MAPA with IL-12 attenuated the levels of MyD88, interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kB p65). The IL-12 levels were increased and P-MAPA stimulated the secretion of cytokines IL-3, IL-9, IL-10, and chemokines MDC/CCL22 and, regulated on activation, normal T cells expressed and secreted (RANTES)/CCL5. Conversely, combination therapy reduced the levels of IL-3, IL-9, IL-10, MDC/CCL22, and RANTES/CCL5. Collectively, P-MAPA and IL-12 reduce cell dynamics and effectively target the TLR-related downstream molecules, eliciting a protective effect against chemoresistance. P-MAPA also stimulates the secretion of anti-inflammatory molecules, possibly having an immune response in the OC microenvironment.

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis.

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the "hydroperoxide-binding domain" (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.

Dietary Linoleic Acid and Its Oxidized Metabolites Exacerbate Liver Injury Caused by Ethanol via Induction of Hepatic Proinflammatory Response in Mice.

Alcoholic liver disease is a major human health problem leading to significant morbidity and mortality in the United States and worldwide. Dietary fat plays an important role in alcoholic liver disease pathogenesis. Herein, we tested the hypothesis that a combination of ethanol and a diet rich in linoleic acid (LA) leads to the increased production of oxidized LA metabolites (OXLAMs), specifically 9- and 13-hydroxyoctadecadienoic acids (HODEs), which contribute to a hepatic proinflammatory response exacerbating liver injury. Mice were fed unsaturated (with a high LA content) or saturated fat diets (USF and SF, respectively) with or without ethanol for 10 days, followed by a single binge of ethanol. Compared to SF+ethanol, mice fed USF+ethanol had elevated plasma alanine transaminase levels, enhanced hepatic steatosis, oxidative stress, and inflammation. Plasma and liver levels of 9- and 13-HODEs were increased in response to USF+ethanol feeding. We demonstrated that primarily 9-HODE, but not 13-HODE, induced the expression of several proinflammatory cytokines in vitro in RAW264.7 macrophages. Finally, deficiency of arachidonate 15-lipoxygenase, a major enzyme involved in LA oxidation and OXLAM production, attenuated liver injury and inflammation caused by USF+ethanol feeding but had no effect on hepatic steatosis. This study demonstrates that OXLAM-mediated induction of a proinflammatory response in macrophages is one of the potential mechanisms underlying the progression from alcohol-induced steatosis to alcoholic steatohepatitis.