13-HODE Research Articles

Sexually Dimorphic Effects of CYP2B6 in the Development of Fasting-Mediated Steatosis in Mice: Role of the Oxylipin Products 9-HODE and 9-HOTrE

Background: Cytochrome P450 2B6 (CYP2B6) is a sexually dimorphic, anti-obesity CYP enzyme responsible for the metabolism of xeno- and endobiotics, including the metabolism of polyunsaturated fatty acids (PUFAs) into 9-hydroxyoctadecadienoic acid (9-HODE) and 9-hydroxyoctadecatrienoic acid (9-HOTrE). However, humanized CYP2B6 transgenic (hCYP2B6-Tg) mice are sensitive to diet-induced hepatic steatosis despite their resistance to obesity. The purpose of this study was to determine if 9-HODE, 9-HOTrE, or other factors contribute to the sexually dimorphic steatosis observed in hCYP2B6-Tg mice. Results: Cyp2b9/10/13-null (Cyp2b-null) mice were injected with either 9-HODE or 9-HOTrE for 2 days and were then subjected to a fasting period of 20 h to induce steatosis. Serum lipids were moderately increased, especially in females, after 9-HODE (triglycerides (TGs), very low-density lipoproteins (VLDLs)) and 9-HOTrE (high-density lipoproteins (HDLs), low-density lipoproteins (LDLs), cholesterol) treatment. No change in hepatic lipids and few changes in hepatic gene expression were observed in mice treated with either oxylipin, suggesting that these oxylipins had minimal to moderate effects. Therefore, to further investigate CYP2B6’s role in steatosis, hCYP2B6-Tg and Cyp2b-null mice were subjected to a 20 h fast and compared. Both male and female hCYP2B6-Tg mice exhibited increased steatosis compared to Cyp2b-null mice. Serum cholesterol, triglycerides, HDLs, and VLDLs were increased in hCYP2B6-Tg males. Serum triglycerides and VLDLs were decreased in hCYP2B6-Tg females, suggesting the greater hepatic retention of lipids in females. Hepatic oxylipin profiles revealed eight perturbed oxylipins in female hCYP2B6-Tg mice and only one in males when compared to Cyp2b-null mice. RNA-seq also demonstrated greater effects in females in terms of the number of genes and gene ontology (GO) terms perturbed. There were only a few overlapping GO terms between sexes, and lipid metabolic processes were enriched in hCYP2B6-Tg male mice but were repressed in hCYP2B6-Tg females compared to Cyp2b-nulls. Conclusions: hCYP2B6-Tg mice are sensitive to fasting-mediated steatosis in males and females, although the responses are different. In addition, the oxylipins 9-HODE and 9-HOTrE are unlikely to be the primary cause of CYP2B6’s pro-steatotic effects.

Severity-Dependent Long-Term Post-Traumatic Changes in the Circulating Oxylipin Profile.

Trauma causes the breakdown of membrane phospholipids and the subsequent degradation of the released polyunsaturated fatty acids (PUFAs) to partially bioactive oxylipins. Here, we screened for circulating PUFAs and oxylipins in patients (n = 34) differing from those of uninjured controls (n = 25) and analyzed their diagnostic potential. Patients were followed up for 1 to 240 h after minor/moderate, severe, and very severe injuries. Of the targeted oxylipins, 13 out of 80 (13/80) were detected in almost all patients and controls. Injury caused a long-term decrease in 9- and 13-hydroxyoctadecadienoic acids and in several dihydroxyeicosatetraenoic acids, the stable derivatives of bioactive anti-inflammatory epoxyeicosatrienoic acids, compared to controls. Frequently, these oxylipins correlated inversely to injury severity, days in the intensive care unit and hospital, and/or procalcitonin and pro-inflammatory cytokine levels 48 up to 240 h after trauma. Notably, 20/80 oxylipins were detected in some patients but not or less often in controls. Many of these oxylipins increased transiently immediately after injury. Their level is partly correlated with adverse clinical parameters at this early time point. The circulating oxylipidome was markedly affected by trauma. Several oxylipins showed injury-dependent alterations at different time points in the post-traumatic course.

Acute inflammation upregulates FAHFAs in adipose tissue and in differentiated adipocytes

Since the discovery of fatty acid hydroxy fatty acids (FAHFAs), significant progress has been made in understanding their regulation, biochemistry, and physiological activities. Here, we contribute to this understanding by revealing that inflammation induces the production of fatty acid hydroxy stearic acids (FAHSAs) and fatty acid hydroxyoctadecadienoic acids (FAHODEs) in white adipose tissue depots and in adipocytes co-cultured with macrophages. In LPS-induced co-culture systems, we confirm that adipose triglyceride lipase (ATGL) is required for inflammation-induced FAHFA generation and demonstrate that inflammation is necessary for producing hydroxy fatty acids. Chemically synthesized FAHODEs show anti-inflammatory activities in vivo, but only at supraphysiological concentrations. While endogenous FAHFAs are unlikely to be anti-inflammatory due to their low concentrations, conversion of pro-inflammatory hydroxy fatty acids into FAHFAs may modulate inflammation. We test this concept by showing the pro-inflammatory lipids—hydroxyeicosatetraenoic acids (HETEs) and leukotriene B4 (LTB4)—are converted into FAHFAs in cell culture, and that two LTB4-derived FAHFAs have are modestly anti- not pro-inflammatory. Further research is needed to establish whether these increased FAFHA levels have a role in inflammation or are simply markers of inflammation, but the discovery of significant increases in FAHFA upon acute inflammation advances our knowledge of FAHFAs.

8638 NEGR1 is a Mediator of Exercise-Induced Neuritogenesis in Subcutaneous White Adipose Tissue, Implications for Improved Systemic Metabolism

Abstract Disclosure: P. Nigro: None. N.P. Carbone: None. T. Caputo: None. M. Vamvini: None. M.M. Columbus: None. M.F. Hirshman: None. R. Middelbeek: None. L.J. Goodyear: None. Emerging research shows that exercise training results in robust and beneficial adaptions to subcutaneous white adipose tissue (scWAT), leading to improved systemic metabolism. The mechanisms for these benefits of exercise on are not fully understood, but we have recently demonstrated that training in mouse scWAT refines nerve connections in scWAT promoting neurite outgrowth (neuritogenesis). Our multi-omics and advanced microscopy data identifies Neuronal Growth Regulator 1 (NEGR1) as a potential player in this process. Here, we determine the role of NEGR1 as a mediator of scWAT neuritogenesis and regulator of systemic metabolism. Exercise training in mice for 11 days significantly increased NEGR1 protein expression by 35% (p<0.05). Next, we directly injected NEGR1 AAV into the inguinal scWAT of untrained male mice for three weeks resulting in a 43% increase in NEGR1 protein (p<0.05). Overexpression of NEGR1 led to an increase in the innervation markers tyrosine hydroxylase (+38%, p<0.01) and synaptophysin (+39%, p<0.01), and remarkably, a 16% decrease in adipocyte cell size (p<0.05), and a 15% reduction in fasting blood glucose levels (p<0.05). This suggests that NEGR1 plays a fundamental role in regulating scWAT morphology and function, adaptations that improve systemic glucose homeostasis. To determine mechanisms for exercise training-induced increases in NEGR1 expression, we used in silico and cell culture experiments and found that NEGR1 is under the regulation of the Peroxisome Proliferator-Activated Receptor gamma (PPARg) transcription factor. Given that numerous natural and synthetic PPARg ligands are lipids, we used targeted lipidomics to identify exercise-induced lipids capable of binding and inducing PPARg expression in inguinal scWAT. We identified 13-oxo-octadecadienoic acid (13-oxo-ODE), an oxylipin derived from linoleic acid, as a potent agonist and inducer of PPARg expression. Exercise training increased 13-oxo-ODE by 3-fold (p<0.05), and 13-hydroxyoctadecadienoic acid (13-HODE), the 13-oxo-ODE precursor was also increased by 3-fold (p<0.01). To validate that 13-oxo-ODE increases NEGR1, adipocytes were incubated with 2μM of 13-oxo-ODE for 48 hours. 13-oxo-ODE increased NEGR1 expression by 27-fold (p<0.0001) and NEGR1 protein by 2-fold (p<0.01). In summary, NEGR1 is a novel exercise-induced molecule that regulates neuronal refinement in scWAT. Our data also suggest that the synthesis of 13-oxo-ODE leads to PPARγ activation that in turn promotes NEGR1 expression in scWAT. NEGR1 has beneficial effects on scWAT that are associated with improved systemic metabolism, making NEGR1 a novel target for treatment of obesity and cardiometabolic diseases. Presentation: 6/1/2024

Mass spectrometry unveils heat-induced changes in yolk oxylipins and key lipid molecules during home cooking

IntroductionOxylipins, as a widespread class of metabolic markers following oxidative stress, and several studies have reported dietary regulation of lipid metabolism. However, there is a lack of investigation of dietary oxylipins, especially cooking-induced changes in food lipid oxidation. ObjectivesInvestigated the effects of cooking methods and lipid profiles on polyunsaturated fatty acids derived oxylipins generation within egg yolks. MethodsThe lipid profile of egg yolk was determined by UPLC-QTOF-MS/MS, oxylipins were detected by HPLC-QTRAP-MS/MS, while the total fatty acid content was quantified by GC-FID. Random Forest (RF) and Partial Least Squares (PLS) regression models were employed to explore the association between oxidized lipids and key lipid species. ResultsHeating reduced egg yolk docosahexaenoic acid (DHA) content, and no consistent trends for arachidonic acid (AA), linoleic acid (LA), and linolenic acid (ALA). Yolk lipid composition affected triacylglycerol (TG), phosphatidylethanolamine (PE), and LA-monoepoxide contents after cooking. 9- and 13-HODE (hydroxyoctadecadienoic acid), 9,10,13-TriHOME (trihydroxyoctadecenoic acid), 9,10- and 12,13-EpOME (epoxyoctadecenoic acid), 9,10- and 12,13-DiHOME (dihydroxyoctadecenoic acid), 5-HETE (hydroxyeicosatetraenoic acid), and 4-HDHA (hydroxydocosahexaenoic acid) were the prevalent oxylipins with high concentrations, accounting for 1.08 %–29.58 % of the total content of 29 oxylipins. Steaming resulted in a 1.9-fold increase in oxylipin concentrations in yolks compared to raw yolks, and boiling with or without shells (poaching) resulted in a 1.30- to 1.76-fold increase in oxylipin concentrations. In contrast, pan-fried yolks exhibited the lowest and least variable levels of total oxylipins, while still retaining some epoxides, including epoxyeicosatrienoic acid (EET) and EpOME. Utilizing big data analysis, we mapped the oxylipin network in both ordinary and DHA-enriched egg yolks, revealing a strong correlation between cooking-induced oxylipin production and variations in 24 lipid species. ConclusionRevealed the potential mechanisms and key lipid molecules for heating-induced oxylipin production of yolk through lipidomics and big data analysis.

Full composition-wide association study identifies the chemical markers to distinguish different processed camellia oils: Integrating multi-targets with chemometrics

To identify chemical-markers from hot-pressed, cold-pressed, organic-solvent, aqueous-enzymatic and water extracted camellia oils (HPO, CPO, OSO, AEO, WEO). We report a full composition-wide association study based on GC–MS, LC-MS and 1HNMR. Squalene, β-amyrin and lupeol were potential-markers in distinguishing different oils through GC–MS. Naringenin, FA 18:1 + 10, undecanedioic acid and tridecanedioic acid exhibited were up-regulated in HPO. 16-Hydroxyhexadecanoic acid, octadecanoic acid and 9-hydroxyoctadecadienoic acid were potential-metabolites in CPO. Characteristic-markers in WEO were hydroquinidine and undecanedioic acid. Gallic acid, hydroquinidine, lichesterylic acid and 7,4′-dihydroxyflavone were biomarkers in AEO. Oleic acid, linoleic acid and triacylglycerols may be potential key markers to distinguish AEO from others via 1HNMR. Finally, Naringenin, gallic acid, kaempferol, 7,4′-dihydroxyflavone, (Z)-5,8,11-trihydroxyoctadec-9-enoic acid and β-amyrin were screened and validate through integration of nonglyceride minor components and trace metabolites. Results provided understanding of chemical diversity for different processed-camellia oils, and proposed a complementary strategy to distinguish different camellia oils for multidimensional perspective.

Lipid metabolites and nitric oxide production in the cerebrospinal fluid and plasma of dogs with meningoencephalitis of unknown origin and idiopathic epilepsy: a pilot study.

Idiopathic epilepsy (IE) and meningoencephalomyelitis of unknown origin (MUO) are common causes of brain diseases leading to seizures in dogs. In this study, the concentrations of 196 lipid metabolites and nitrogen oxide (NO) production in the cerebrospinal fluid (CSF) and plasma of dogs with MUO or IE were measured using a LC-MS/MS and a NOx analyzer, respectively. Nine clinically healthy dogs and 11 and 12 dogs with IE and MUO, respectively, were included in the study. Lipid analysis revealed variations in the levels of four and six lipid metabolites in CSF and plasma, respectively, between the groups. The levels of 6-keto-prostaglandin (PG) F1α (PGF1α), 20-carboxy arachidonic acid (20-carboxy-AA), 9-hydroxyoctadecadienoic acid, and lyso-platelet-activating factor were high in the CSF of dogs with MUO. In addition, the plasma levels of 11,12-dihydroxyeicosatrienoic acid, 20-carboxy-AA, and oleoylethanolamide were high in dogs with IE, and those of PGF1α were high in dogs with MUO. NO production levels were high in CSF but not in plasma in dogs with MUO or IE. It remains unknown whether these changes represent the cause or effect of diseases of the central nervous system; however, lipid metabolites and NO production in CSF and plasma may be used as diagnostic biomarkers and could be exploited for treating idiopathic or inflammatory epilepsy in dogs.

Biomarkers for ideal protein: rabbit diet metabolomics varying key amino acids

With the main aim of identifying biomarkers that contribute to defining the concept of ideal protein in growing rabbits under the most diverse conditions possible this work describes two different experiments. Experiment 1: 24 growing rabbits are included at 56 days of age. The rabbits are fed ad libitum one of the two experimental diets only differing in lysine levels. Experiment 2: 53 growing rabbits are included at 46 days of age, under a fasting and eating one of the five experimental diets, with identical chemical composition except for the three typically limiting amino acids (being fed commercial diets ad libitum in both experiments). Blood samples are taken for targeted and untargeted metabolomics analysis. Here we show that the metabolic phenotype undergoes alterations when animals experience a rapid dietary shift in the amino acid levels. While some of the differential metabolites can be attributed directly to changes in specific amino acids, creatinine, urea, hydroxypropionic acid and hydroxyoctadecadienoic acid are suggested as a biomarker of amino acid imbalances in growing rabbits’ diets, since its changes are not attributable to a single amino acid. The fluctuations in their levels suggest intricate amino acid interactions. Consequently, we propose these metabolites as promising biomarkers for further research into the concept of the ideal protein using rabbit as a model.

9-HODE and 9-HOTrE alter mitochondrial metabolism, increase triglycerides, and perturb fatty acid uptake and synthesis associated gene expression in HepG2 cells

Non-Alcoholic Fatty Liver Disease (NAFLD) prevalence is rising and can lead to detrimental health outcomes such as Non-Alcoholic Steatohepatitis (NASH), cirrhosis, and cancer. Recent studies have indicated that Cytochrome P450 2B6 (CYP2B6) is an anti-obesity CYP in humans and mice. Cyp2b-null mice are diet-induced obese, and human CYP2B6-transgenic (hCYP2B6-Tg) mice reverse the obesity or diabetes progression, but with increased liver triglyceride accumulation in association with an increase of several oxylipins. Notably, 9-hydroxyoctadecadienoic acid (9-HODE) produced from linoleic acid (LA, 18:2, ω-6) is the most prominent of these and 9-hydroxyoctadecatrienoic acid (9-HOTrE) from alpha-linolenic acid (ALA, 18:3, ω-3) is the most preferentially produced when controlling for substrate concentrations in vitro. Transactivation assays indicate that 9-HODE and 9-HOTrE activate PPARα and PPARγ. In Seahorse assays performed in HepG2 cells, 9-HOTrE increased spare respiratory capacity, slightly decreased palmitate metabolism, and increased non-glycolytic acidification in a manner consistent with slightly increased glutamine utilization; however, 9-HODE exhibited no effect on metabolism. Both compounds increased triglyceride and pyruvate concentrations, most strongly by 9-HOTrE, consistent with increased spare respiratory capacity. qPCR analysis revealed several perturbations in fatty acid uptake and metabolism gene expression. 9-HODE increased expression of CD36, FASN, PPARγ, and FoxA2 that are involved in lipid uptake and production. 9-HOTrE decreased ANGPTL4 expression and increased FASN expression consistent with increased fatty acid uptake, fatty acid production, and AMPK activation. Our findings support the hypothesis that 9-HODE and 9-HOTrE promote steatosis, but through different mechanisms as 9-HODE is directly involved in fatty acid uptake and synthesis; 9-HOTrE weakly inhibits mitochondrial fatty acid metabolism while increasing glutamine use.

Calprotectin Is Associated with HETE and HODE Acids in Inflammatory Bowel Diseases.

Intestinal diseases are identified as autoimmune phenomena attributed to a specific virus that binds to the mucosal epithelium. The importance of precise diagnostic processes and identification is emphasized, but the multifaceted and complex etiological factors pose challenges for effective treatment. A recent supplementary study suggested a linkage between the secretion of calprotectin, a protein associated with inflammatory processes, and increased levels of hydroxyeicosatrienoic acids (HETE) and hydroxyoctadecadienoic (HODE) compounds. Sixty-two patients (average age: 14.06 ± 2.93 years) suffering from inflammatory bowel diseases were included in this study. Comparative analyses were performed to assess the concentrations of calprotectin against the levels of arachidonic acid derivatives. The calprotectin concentration was determined using the enzyme-linked immunosorbent assay (ELISA) method. The derivatives of HETE and HODE were identified through liquid chromatography. Patients with Crohn's disease (CD) displayed higher average concentrations of fatty acid metabolites; however, no correlation with calprotectin was observed. A dependency of 12S HETE concentration relative to age was noted in the CD group, and a similar trend was also identified in ulcerative colitis (UC), with the significant metabolites being 15 HETE and 5 oxoETE. In UC patients, a positive correlation was established between the calprotectin concentration and the acids 5-HETE and 12-HETE. These findings may be instrumental for monitoring the inflammatory states of patients and indicating a pathway for intervention. The metabolite 16RS HETE is associated with UC activity, and 15-HETE is related to the disease's duration. A relatively more significant role of HETE acids in the progression of the disease was observed in UC.

Genetic Regulation of Carnitine Metabolism Controls Lipid Damage Repair Mechanisms and Hemolytic Propensity of Human Red Blood Cells during Aging In Vivo and in Vitro

Large scale genomics studies and vein-to-vein databases have started to reveal that donor biology and genetics influence red blood cell (RBC) storability and transfusion outcomes. To further delve into this concept, here we performed metabolomics analyses of 13,091 packed RBC units from donors enrolled in the Recipient Epidemiology and Donor Evaluation (REDS) RBC Omics study. End of storage (day 42) units were tested for metabolomics and hemolytic propensity. Donors ranking in the 5 th and 95 th percentile were contacted again and invited to donate a second unit of blood, which was stored again for 42 days (n=643). Correlation of end of storage metabolomics measurements for the first (index) and second (recalled) donation identified a core of metabolites involved in carnitine synthesis and acyl-carnitine metabolism as the most reproducible within the same donor across multiple donations ( Figure 1.A). Carnitine and its precursors, methyl- and trimethyl-lysine were the most significantly reproducible of all the metabolites tested in this study ( Figure 1.A). Association of L-carnitine measurements to genomics data (i.e., 879,000 single nucleotide polymorphisms that were assayed via a precision transfusion medicine array developed for this study - Figure 1.B) identified non-synonymous coding polymorphisms in the carnitine transporter SLC22A16 as a critical genetic factor contributing to inter-donor heterogeneity in end of storage carnitine levels ( Figure 1.B). Donors carrying two alleles of this SNP were characterized by the lowest L-carnitine levels, and associated depletion of the whole carnitine pool. Functionally, stored RBCs with the lowest levels of carnitine pools were characterized by significant elevation in in vitro hemolysis and the highest degree of vesiculation, in parallel to increases in lipid peroxidation markers (hydroxy-eicosatetraenoic - HETEs and hydroxy-octadecadienoic acids - HODEs). We leveraged a novel CFDA-SE staining protocol, which allows flow cytometry sorting of end of storage RBCs in two highly enriched preparations: a morphologically-altered subpopulation (CFDA-SE high) and a morphologically-normal RBC subpopulation (CFDA-SE low). The morphologically-altered subpopulations contains mostly storage-induced micro-erythrocytes (SMEs), i.e., RBCs with decreased surface/volume ratio upon storage-induced vesiculation. This RBC sub-population has increased susceptibility to extravascular hemolysis via splenic sequestration upon transfusion in vivo. Metabolic characterization of SMEs (i.e., CFDA-SE low) vs morphologically-normal end of storage RBCs (i.e., CFDA-SE low) showed that the former are characterized by a complete depletion of acyl-carnitine pools compared to the latter or to fresh RBCs. We thus propose a model where storage promotes lipid peroxidation, which is in turn counteracted by activation of the Lands cycle, a pathway that relies on acyl-carnitine pools to repair lysophospholipids secondary to oxidation of fatty acids in membrane lipids. To determine the translational relevance of this mechanism to RBCs as they age in the bloodstream in vivo, we separated packed RBCs via Percoll density gradients and determined total carnitine pools in each fraction. Our results indicate that the oldest circulating RBCs are not only the smallest, but are also characterized by the lowest levels of carnitine pools. Incubation of the youngest, oldest and intermediate RBC populations with radio-labeled palmitate showed that incorporation of labeled hexadecenoic acid into phosphatidylethanolamines is constrained by carnitine pools in old and average age RBCs, compared to the youngest population. Altogether, our results suggest an important role for L-carnitine levels during RBC aging in vivo and in vitro. As such, carnitine supplementation may represent a viable strategy to boost RBC storage quality and modulate hemolytic propensity, especially in RBCs from donors harboring genetic polymorphism associated with partial ablation of the carnitine transport system. Figure 1 - Levels of carnitine metabolites are reproducible across multiple donations from the 643 REDS RBC Omics donors across two consecutive donations (index and recalled in A), a phenomenon in part explained by genetic polymorphisms to the carnitine transporter SLC22A16 as gleaned by L-carnitine measurements in end of storage units from 13,091 blood donors ( B).

Eicosapentaenoic Acid Influences the Lipid Profile of an In Vitro Psoriatic Skin Model Produced with T Cells.

Psoriasis is a skin disease characterized by epidermal hyperplasia and an inappropriate activation of the adaptive immunity. A dysregulation of the skin's lipid mediators is reported in the disease with a predominance of the inflammatory cascade derived from n-6 polyunsaturated fatty acids (n-6 PUFAs). Bioactive lipid mediators derived from arachidonic acid (AA) are involved in the inflammatory functions of T cells in psoriasis, whereas n-3 PUFAs' derivatives are anti-inflammatory metabolites. Here, we sought to evaluate the influence of a supplementation of the culture media with eicosapentaenoic acid (EPA) on the lipid profile of a psoriatic skin model produced with polarized T cells. Healthy and psoriatic skin substitutes were produced following the auto-assembly technique. Psoriatic skin substitutes produced with or without T cells presented increased epidermal and dermal linolenic acid (LA) and AA levels. N-6 PUFA lipid mediators were strongly measured in psoriatic substitutes, namely, 13-hydroxyoctadecadienoic acid (13-HODE), prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE). The added EPA elevated the amounts of EPA, n-3 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in the epidermal and dermal phospholipids. The EPA supplementation balanced the production of epidermal lipid mediators, with an increase in prostaglandin E3 (PGE3), 12-hydroxyeicosapentaenoic acid (12-HEPE) and N-eicosapentaenoyl-ethanolamine (EPEA) levels. These findings show that EPA modulates the lipid composition of psoriatic skin substitutes by encouraging the return to a cutaneous homeostatic state.

9-Hydroxyoctadecadienoic acid plays a crucial role in human skin photoaging

Human skin is regularly exposed to ultraviolet (UV) rays from sunlight, leading to photoaging, which differs from intrinsic aging. Although the acute effects of UV exposure have been extensively studied, limited research has addressed the long-term consequences of chronic UV exposure. This study aimed to investigate the underlying causes of chronic photoaging. A questionnaire-based assessment of sunlight exposure was conducted among volunteers in their 20s and 50s, and the stratum corneum of their skin was analyzed for bioactive lipid content. Volunteers were categorized into low and high UV exposure groups based on the questionnaire scores. The analysis results revealed a significant increase in 9-hydroxyoctadecadienoic acid (9-HODE) levels in the skin of individuals in their 50s with high UV exposure. However, UV exposure did not affect 9-HODE levels in the skin of individuals in their 20s. In vitro experiments further indicated that 9-HODE contributes to chronic inflammation, pigmentary changes, and extracellular matrix alterations during photoaging. Specifically, 9-HODE stimulated cytokine production [interleukin-6 (IL6), IL8, and granulocyte-macrophage colony-stimulating factor (GM-CSF)] and reduced dickkopf-1 (DKK1) production in keratinocytes. In fibroblasts, 9-HODE stimulated matrix metalloproteinase-1 (MMP1) and MMP3 production while reducing collagen I (COL1) production. The expression of G2A, the receptor for 9-HODE, was also confirmed in fibroblasts, suggesting that 9-HODE exerts its effects via G2A, as observed in keratinocytes. Overall, these findings indicate that 9-HODE is a mediator of chronic photoaging and highlight its potential significance in photoaging prevention.

CES1 Releases Oxylipins from Oxidized Triacylglycerol (oxTAG) and Regulates Macrophage oxTAG/TAG Accumulation and PGE2/IL-1β Production.

Triacylglycerols (TAGs) are storage forms of fat, primarily found in cytoplasmic lipid droplets in cells. TAGs are broken down to their component free fatty acids by lipolytic enzymes when fuel reserves are required. However, polyunsaturated fatty acid (PUFA)-containing TAGs are susceptible to nonenzymatic oxidation reactions, leading to the formation of oxylipins that are esterified to the glycerol backbone (termed oxTAGs). Human carboxylesterase 1 (CES1) is a member of the serine hydrolase superfamily and defined by its ability to catalyze the hydrolysis of carboxyl ester bonds in both toxicants and lipids. CES1 is a bona fide TAG hydrolase, but it is unclear which specific fatty acids are preferentially released during lipolysis. To better understand the biochemical function of CES1 in immune cells, such as macrophages, its substrate selectivity when it encounters oxidized PUFAs in TAG lipid droplets requires study. We sought to identify those esterified oxidized fatty acids liberated from oxTAGs by CES1 because their release can activate signaling pathways that enforce the development of lipid-driven inflammation. Gaining this knowledge will help fill data gaps that exist between CES1 and the lipid-sensing nuclear receptors, PPARγ and LXRα, which are important drivers of lipid metabolism and inflammation in macrophages. Oxidized forms of triarachidonoylglycerol (oxTAG20:4) or trilinoleoylglycerol (oxTAG18:2), which contain physiologically relevant levels of oxidized PUFAs (<5 mol %), were incubated with recombinant CES1 to release oxylipins and nonoxidized arachidonic acid (AA) or linoleic acid (LA). CES1 hydrolyzed each oxTAG, yielding regioisomers of hydroxyeicosatetraenoic acids (5-, 11-, 12-, and 15-HETE) and hydroxyoctadecadienoic acids (9- and 13-HODE). Furthermore, human THP-1 macrophages with deficient CES1 levels exhibited a differential response to extracellular stimuli (oxTAGs, lipopolysaccharide, and 15-HETE) as compared to those with normal CES1 levels, including enhanced oxTAG/TAG lipid accumulation and altered cytokine and prostaglandin E2 profiles. This study suggests that CES1 can metabolize oxTAG lipids to release oxylipins and PUFAs, and it further specifies the substrate selectivity of CES1 in the metabolism of bioactive lipid mediators. We suggest that the accumulation of oxTAGs/TAGs within lipid droplets that arise due to CES1 deficiency enforces an inflammatory phenotype in macrophages.

Quorum Sensing-Mediated Lipid Oxidation Further Regulating the Environmental Adaptability of Aspergillus ochraceus.

Quorum sensing (QS) is a cellular strategy of communication between intra- and inter-specific microorganisms, characterized by the release of quorum sensing molecules (QSMs) that achieve coordination to adaptation to the environment. In Aspergillus, lipids carry population density-mediated stresses, and their oxidative metabolite oxylipins act as signaling to transmit information inside cells to regulate fungal development in a synchronized way. In this study, we investigated the regulation of density-dependent lipid metabolism in the toxigenic fungi Aspergillus ochraceus by the oxidative lipid metabolomics in conjunction with transcriptomics. In addition to proven hydroxyoctadecadienoic acids (HODEs), prostaglandins (PGs) also appear to have the properties of QSM. As a class of signaling molecule, oxylipins regulate the fungal morphology, secondary metabolism, and host infection through the G protein signaling pathway. The results of combined omics lay a foundation for further verification of oxylipin function, which is expected to elucidate the complex adaptability mechanism in Aspergillus and realize fungal utilization and damage control.

Characterization of gut microbial and metabolite alterations in faeces of Goto Kakizaki rats using metagenomic and untargeted metabolomic approach.

In recent years, the incidence of type 2 diabetes (T2DM) has shown a rapid growth trend. Goto Kakizaki (GK) rats are a valuable model for the study of T2DM and share common glucose metabolism features with human T2DM patients. A series of studies have indicated that T2DM is associated with the gut microbiota composition and gut metabolites. We aimed to systematically characterize the faecal gut microbes and metabolites of GK rats and analyse the relationship between glucose and insulin resistance. To evaluate the gut microbial and metabolite alterations in GK rat faeces based on metagenomics and untargeted metabolomics. Ten GK rats (model group) and Wistar rats (control group) were observed for 10 wk, and various glucose-related indexes, mainly including weight, fasting blood glucose (FBG) and insulin levels, homeostasis model assessment of insulin resistance (HOMA-IR) and homeostasis model assessment of β cell (HOMA-β) were assessed. The faecal gut microbiota was sequenced by metagenomics, and faecal metabolites were analysed by untargeted metabolomics. Multiple metabolic pathways were evaluated based on the differential metabolites identified, and the correlations between blood glucose and the gut microbiota and metabolites were analysed. The model group displayed significant differences in weight, FBG and insulin levels, HOMA-IR and HOMA-β indexes (P < 0.05, P < 0.01) and a shift in the gut microbiota structure compared with the control group. The results demonstrated significantly decreased abundances of Prevotella sp. CAG:604 and Lactobacillus murinus (P < 0.05) and a significantly increased abundance of Allobaculum stercoricanis (P < 0.01) in the model group. A correlation analysis indicated that FBG and HOMA-IR were positively correlated with Allobaculum stercoricanis and negatively correlated with Lactobacillus murinus. An orthogonal partial least squares discriminant analysis suggested that the faecal metabolic profiles differed between the model and control groups. Fourteen potential metabolic biomarkers, including glycochenodeoxycholic acid, uric acid, 13(S)-hydroxyoctadecadienoic acid (HODE), N-acetylaspartate, β-sitostenone, sphinganine, 4-pyridoxic acid, and linoleic acid, were identified. Moreover, FBG and HOMA-IR were found to be positively correlated with glutathione, 13(S)-HODE, uric acid, 4-pyridoxic acid and allantoic acid and ne-gatively correlated with 3-α, 7-α, chenodeoxycholic acid glycine conjugate and 26-trihydroxy-5-β-cholestane (P < 0.05, P < 0.01). Allobaculum stercoricanis was positively correlated with linoleic acid and sphinganine (P < 0.01), and 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate was negatively associated with Prevotella sp. CAG:604 (P < 0.01). The metabolic pathways showing the largest differences were arginine biosynthesis; primary bile acid biosynthesis; purine metabolism; linoleic acid metabolism; alanine, aspartate and glutamate metabolism; and nitrogen metabolism. Metagenomics and untargeted metabolomics indicated that disordered compositions of gut microbes and metabolites may be common defects in GK rats.

Oxidized linoleic acid metabolites regulate neuronal morphogenesis in vitro.

Linoleic acid (LA, 18:2n-6) is an essential nutrient for optimal infant growth and brain development. The effects of LA in the brain are thought to be mediated by oxygenated metabolites of LA known as oxidized LA metabolites (OXLAMs), but evidence is lacking to directly support this hypothesis. This study investigated whether OXLAMs modulate key neurodevelopmental processes including axon outgrowth, dendritic arborization, cell viability and synaptic connectivity. Primary cortical neuron-glia co-cultures from postnatal day 0-1 male and female rats were exposed for 48h to the following OXLAMs: 1) 13-hydroxyoctadecadienoic acid (13-HODE); 2) 9-hydroxyoctadecadienoic acid (9-HODE); 3) 9,10-dihydroxyoctadecenoic acid (9,10-DiHOME); 4) 12(13)-epoxyoctadecenoic acid (12(13)-EpOME); 5) 9,10,13-trihydroxyoctadecenoic acid (9,10,13-TriHOME); 6) 9-oxo-octadecadienoic acid (9-OxoODE); and 7) 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME). Axonal outgrowth, evaluated by Tau-1 immunostaining, was increased by 9-HODE, but decreased by 12,13-DiHOME in male but not female neurons. Dendrite arborization, evaluated by MAP2B-eGFP expression, was affected by 9-HODE, 9-OxoODE, and 12(13)-EpOME in male neurons and, by 12(13)-EpOME in female neurons. Neither cell viability nor synaptic connectivity were significantly altered by OXLAMs. Overall, this study shows select OXLAMs modulate neuron morphology in a sex-dependent manner, with male neurons being more susceptible.

Nutritional Modulation of Guinea Pig Skin Hyperproliferation by Essential Fatty Acid Deficiency is Associated with Selective Down Regulation of Protein Kinase C-β

In a previous study we demonstrated that 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns 4,5-P2) and released as 13-HODE-containing-diacylglycerol (13-HODE-DAG). In vitro, 13-HODE-DAG was shown to selectively inhibit epidermal total protein kinase C (PKC-β) activity. To determine whether these observations are relevant in vivo, guinea pigs were made essential fatty acid deficient (EFAD) by feeding them a basal diet supplemented with 4% hydrogenated coconut oil for 8 wk. Tissue levels of putative 13-HODE-DAG, protein kinase C (PKC) isozymes and tissue hyperproliferation were determined in the epidermal preparations from skin of control safflower oil-fed guinea pigs, those fed EFAD diet and those fed EFAD diet followed by the control diet for 2 wk. Our data revealed that cutaneous 13-HODE and 13-HODE-DAG were significantly lower in EFAD animals than in safflower-fed controls. These reductions were associated with both elevated epidermal hyperproliferation and elevated expressions and activities of PKC-α and β-isozymes. Refeeding the animals with safflower oil for 2 wk replenished tissue levels of 13-HODE-DAG, which inversely correlated with the selective down regulation of PKC-β expression and activity and the reversal of hyperproliferation. In contrast, although the expression and activity of PKC-α was elevated in the epidermis of the EFAD guinea pigs, this elevated PKC-α expression was not down regulated after refeeding the safflower oil diet to the animals. These results suggest that the epidermal level of 13-HODE plays a role in vivo in modulating cutaneous hyperproliferation via the generation of 13-HODE-DAG via signal transduction and its selective suppression of PKC-β isozyme.

PRDX6 Promotes Fatty Acid Oxidation via PLA2-Dependent PPARα Activation in Rats Fed High-Fat Diet.

Aims: Nonalcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease globally, which is defined as an excess accumulation of fat caused by the imbalance of lipogenesis and lipid catabolism. Recently, increasing evidence suggests that peroxiredoxin 6 (PRDX6) is involved in the pathogenesis and progression of NAFLD. However, little is known regarding its role in liver lipid catabolism. Results: We found that PRDX6 level was significantly increased in liver tissues after high-fat diet (HFD) treatment. PRDX6 knockout (KO) exacerbated HFD-induced hepatic steatosis. PRDX6 KO did not affect messenger RNA (mRNA) and protein levels of peroxisome proliferator-activated receptor alpha (PPARα). However, PRDX6 KO decreased the mRNA and protein levels of carnitine palmitoyltransferase-1alpha (CPT-1α) and acyl-CoA oxidase 1 (ACOX1), the target genes of PPARα. PRDX6 KO also did not activate AMP-activated protein kinase (AMPK)α/proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), the upstream signal of PPARα. However, PRDX6 KO reduces the levels of PPARα activators, the oxidized fatty acids (9- and 13-hydroxyoctadecadienoic acid) in HFD rats. More interestingly, PRDX6 promoted the production of oxidized fatty acids by hydrolyzing oxidized low-density lipoprotein (Ox-LDL), which depends on its phospholipase A2 (PLA2) activity. PRDX6 mutation on its PLA2 and its competitive phospholipase inhibitor inhibited the production of the oxidized fatty acids as well as the activation of PPARα. Furthermore, PRDX6 overexpression enhanced the transcriptional activation of PPARα. Innovation and Conclusion: This study elucidates for the first time the role of PLA2 enzyme activity of PRDX6 in fatty acid oxidation and reveals a novel mechanism of PRDX6 involved in liver steatosis. Antioxid. Redox Signal. 38, 1184-1200.

Lyciumbarbarum polysaccharides ameliorate canine acute liver injury by reducing oxidative stress, protecting mitochondrial function, and regulating metabolic pathways.

The development of acute liver injury can result in liver cirrhosis, liver failure, and even liver cancer, yet there is currently no effective therapy for it. The purpose of this study was to investigate the protective effect and therapeutic mechanism of Lyciumbarbarum polysaccharides (LBPs) on acute liver injury induced by carbon tetrachloride (CCl4). To create a model of acute liver injury, experimental canines received an intraperitoneal injection of 1 mL/kg of CCl4 solution. The experimental canines in the therapy group were then fed LBPs (20 mg/kg). CCl4-induced liver structural damage, excessive fibrosis, and reduced mitochondrial density were all improved by LBPs, according to microstructure data. By suppressing Kelch-like epichlorohydrin (ECH)-associated protein 1 (Keap1), promoting the production of sequestosome 1 (SQSTM1)/p62, nuclear factor erythroid 2-related factor 2 (Nrf2), and phase II detoxification genes and proteins downstream of Nrf2, and restoring the activity of anti-oxidant enzymes like catalase (CAT), LBPs can restore and increase the antioxidant capacity of liver. To lessen mitochondrial damage, LBPs can also enhance mitochondrial respiration, raise tissue adenosine triphosphate (ATP) levels, and reactivate the respiratory chain complexes I‒V. According to serum metabolomics, the therapeutic impact of LBPs on acute liver damage is accomplished mostly by controlling the pathways to lipid metabolism. 9-Hydroxyoctadecadienoic acid (9-HODE), lysophosphatidylcholine (LysoPC/LPC), and phosphatidylethanolamine (PE) may be potential indicators of acute liver injury. This study confirmed that LBPs, an effective hepatoprotective drug, may cure acute liver injury by lowering oxidative stress, repairing mitochondrial damage, and regulating metabolic pathways.