Hepatic Polyunsaturated Fatty Acids Research Articles

Maternal Gestational Diabetes Mellitus and High-Fat Diet Influenced Hepatic Polyunsaturated Fatty Acids Profile in the Offspring of C57BL/6J Mice.

This research examines the effects of maternal high-fat (HF) diet and gestational diabetes mellitus (GDM) on offspring lipid metabolism and polyunsaturated fatty acids (PUFA) profile. GDM is induced using the insulin receptor antagonist S961. Weaning offspring are categorized into HF-GDM, HF-CON, NC-GDM, and NC-CON groups based on maternal diet or GDM. Adult offspring are then grouped into NC-CON-NC, NC-CON-HF, NC-GDM-NC, NC-GDM-HF, HF-CON-NC, HF-CON-HF, HF-GDM-NC, and HF-GDM-HF according to dietary patterns. Gas chromatography determines PUFA composition. Western blot assesses PI3K/Akt signaling pathway-related protein expression. Feeding a normal chow diet until adulthood improves the distribution of hepatic PUFA during weaning across the four groups. PI3K expression is upregulated during weaning in HF-CON and HF-GDM, particularly in HF-CON-NC and HF-GDM-NC, compared to NC-CON-NC during adulthood. Akt expression increases in NC-GDM-NC after weaning with a normal diet. The hepatic PUFA profile in HF-CON-HF significantly distinguishes among the maternal generation health groups. Maternal HF diet exacerbates the combined impact of maternal GDM and offspring HF diet on hepatic PUFA and PI3K/Akt signaling pathway-related proteins during adulthood. Early exposure to HF diets and GDM affects hepatic PUFA profiles and PI3K/Akt signaling pathway protein expression in male offspring during weaning and adulthood.

Ketogenesis supports hepatic polyunsaturated fatty acid homeostasis via fatty acid elongation.

Therapeutic interventions targeting hepatic lipid metabolism in metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH) remain elusive. Using mass spectrometry-based stable isotope tracing and shotgun lipidomics, we established a novel link between ketogenesis and MASLD pathophysiology. Our findings show that mouse liver and primary hepatocytes consume ketone bodies to support fatty acid (FA) biosynthesis via both de novo lipogenesis (DNL) and FA elongation. Analysis of 13 C-labeled FAs in hepatocytes lacking mitochondrial D-β-hydroxybutyrate dehydrogenase (BDH1) revealed a partial reliance on mitochondrial conversion of D-βOHB to acetoacetate (AcAc) for cytoplasmic DNL contribution, whereas FA elongation from ketone bodies was fully dependent on cytosolic acetoacetyl-CoA synthetase (AACS). Ketone bodies were essential for polyunsaturated FA (PUFA) homeostasis in hepatocytes, as loss of AACS diminished both free and esterified PUFAs. Ketogenic insufficiency depleted liver PUFAs and increased triacylglycerols, mimicking human MASLD, suggesting that ketogenesis supports PUFA homeostasis, and may mitigate MASLD-MASH progression in humans.

Hepatic oxylipin profiles in mouse models of Wilson disease: New insights into early hepatic manifestations

Hepatic inflammation is commonly identified in Wilson disease (WD), a genetic disease of hepatic and brain copper accumulation. Copper accumulation is associated with increased oxidative stress and reactive oxygen species generation which may result in non-enzymatic oxidation of membrane-bound polyunsaturated fatty acids (PUFA). PUFA can be oxidized enzymatically via lipoxygenases (LOX), cyclooxygenases (COX), and cytochrome P450 monooxygenases (CYP). Products of PUFA oxidation are collectively known as oxylipins (OXL) and are bioactive lipids that modulate hepatic inflammation. We examined hepatic OXL profiles at early stages of WD in two mouse models, the toxic milk mouse from The Jackson Laboratory (tx-j) and the Atp7b knockout on a C57Bl/6 background (Atp7b−/−B6). Targeted lipidomic analysis performed by ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry showed that in both tx-j and Atp7b−/−B6 mice, hepatic OXL profiles were altered with higher thromboxane and prostaglandins levels. The levels of oxidative stress marker, 9-HETE were increased more markedly in tx-j mice. However, both genotypes showed upregulated transcript levels of many genes related to oxidative stress and inflammation. Both genotypes showed higher prostaglandins, thromboxin along with higher PUFA-derived alcohols, diols, and ketones with altered epoxides; the expression of Alox5 was upregulated and many CYP-related genes were dysregulated. Pathway analyses show dysregulation in arachidonic acid and linoleic acid metabolism characterizes mice with WD. Our findings indicate alterations in hepatic PUFA metabolism in early-stage WD and suggest the upregulation of both, non-enzymatic ROS-dependent and enzymatic PUFA oxidation, which could have implications for hepatic manifestations in WD and represent potential targets for future therapies.

Myriocin Treatment Reverses Alcohol-Induced Alterations in Polyunsaturated Fatty Acid-Containing Phospholipid Expression in the Liver.

Chronic heavy alcohol exposure causes steatohepatitis manifested by abnormal intra-hepatocyte accumulation of lipid and parenchymal inflammation. Attendant alterations in polyunsaturated fatty acid (PUFA)-containing phospholipids could cause alcoholic liver disease (ALD) to progress by promoting oxidative stress, inflammation, and fibrogenesis. Previously we showed that myriocin, a serine palmitoyltransferase inhibitor, ameliorates experimental alcohol-induced steatohepatitis. However, the surprising overall therapeutic responses suggested that myriocin’s targets may go beyond sphingolipids. To this end, the present study examines the effects of myriocin on hepatic composition of docosahexaenoic acid (DHA)- and arachidonic acid (AA)-containing phospholipids in an experimental model of ALD. A chronic+binge ethanol exposure model was generated by feeding Long Evans rats with ethanol-containing diets (24% caloric content) for 8 weeks and simultaneously binge gavage administering 2 g/kg ethanol on Tuesdays, Thursdays and Saturdays during Weeks 6-8. Myriocin was administered by i.p. injection on Mondays, Wednesdays, and Fridays of Weeks 3-8. Control rats were studied in parallel. Upon euthanasia, the livers were harvested to examine ethanol- and/or myriocin-modulation of hepatic lipids using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). Results were analyzed statistically by two-way analysis of variance and depicted with data bar plots and heatmaps. Chronic+binge ethanol exposures significantly increased hepatic expression of AA-containing phospholipids including PE(36:4) (P = .005), PE(38:4) (P = .03), and PI(38:4) (P = .04) and reduced DHA-containing phospholipids including PS(40:6) (P = .03) and PE(40:6) (P = .04) relative to control. Myriocin partially reversed ethanol’s effects on hepatic PUFA expression by decreasing PE(36:4) (P = .004) and increasing PS(40:6) (P = .04) and PI(40:6) (P = .0003) relative to ethanol-exposed rats. Ethanol-mediated alterations in hepatic PUFA-containing phospholipids may contribute to hepatic oxidative and inflammatory injury by increasing AA and fibrogenesis by inhibiting DHA. The results suggest that Myriocin may help reduce or prevent long-term and progressive liver injury stemming from excessive chronic+binge ethanol consumption.

Hepatic Levels of DHA-Containing Phospholipids Instruct SREBP1-Mediated Synthesis and Systemic Delivery of Polyunsaturated Fatty Acids.

SummaryPolyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), play fundamental roles in mammalian physiology. Although PUFA imbalance causes various disorders, mechanisms of the regulation of their systemic levels are poorly understood. Here, we report that hepatic DHA-containing phospholipids (DHA-PLs) determine the systemic levels of PUFAs through the SREBP1-mediated transcriptional program. We demonstrated that liver-specific deletion of Agpat3 leads to a decrease of DHA-PLs and a compensatory increase of ARA-PLs not only in the liver but also in other tissues including the brain. Together with recent findings that plasma lysophosphatidylcholine (lysoPC) is the major source of brain DHA, our results indicate that hepatic AGPAT3 contributes to brain DHA accumulation by supplying DHA-PLs as precursors of DHA-lysoPC. Furthermore, dietary fish oil-mediated suppression of hepatic PUFA biosynthetic program was blunted in liver-specific Agpat3 deletion. Our findings highlight the central role of hepatic DHA-PLs as the molecular rheostat for systemic homeostasis of PUFAs.

Serum Cholesterol-Lowering Activity of β-Sitosterol Laurate Is Attributed to the Reduction of Both Cholesterol Absorption and Bile Acids Reabsorption in Hamsters.

The research was performed to delineate how β-sitosterol laurate (β-SLE) consumption influenced serum and hepatic lipids. The results showed that 220 mg/5 mL oil/kg body weight of β-SLE robustly reduced serum total triglyceride and cholesterol levels and the epididymal adipocyte size, and efficiently protected hepatic polyunsaturated fatty acids against lipid peroxidation through superoxide dismutase and glutathione transferase activity enhancement and malondialdehyde level reduction. Based on the changes of fecal cholesterol contents, fecal and hepatic bile acid (BAs) levels, and related protein expression, it was concluded that the mechanisms for lowering serum cholesterol by β-SLE involved (i) the enhanced excretion of fecal cholesterol via down-regulation of intestinal Niemann-Pick C1-like 1 protein; (ii) the increased conversion from cholesterol to primary BAs via up-regulation of cholesterol-7α-hydroxylase and sterol 27-hydroxylase, which was induced by the reduced BAs reabsorption through up-regulating ileal apical sodium-dependent bile acid transporter and ileal bile acid-binding protein.

Cancer-related gene expression is associated with disease severity and modifiable lifestyle factors in non-alcoholic fatty liver disease.

The aim of this study was to determine whether hepatic gene expression related to hepatocellular carcinoma (HCC) is associated with disease severity and modifiable lifestyle factors in non-alcoholic fatty liver disease (NAFLD). In a cross-sectional study, the associations between hepatic gene expression and liver histology, insulin resistance, anthropometrics, diet, and physical activity were assessed in patients with non-alcoholic steatohepatitis (NASH; n = 19) or simple steatosis (SS; n = 20). In a group of patients with NASH, we then conducted a 1-y, single-arm, pilot study using ω-3 polyunsaturated fatty acid (PUFA) supplementation to determine whether changes in hepatic PUFA content would have a modulating effect on hepatic gene expression and would affect liver histology. In the cross-sectional study, histological features of disease severity correlated with AKR1B10, ANXA2, PEG10, SPP1, STMN2, MT1A, and MT1B in NASH and with EEF1A2, PEG10, and SPP1 in SS. In addition, PEG10, SPP1, ANXA2, and STMN2 expression correlated positively with insulin resistance in NASH. SPP1 and UBD correlated strongly with body mass index in SS. Associations between ENPP2, AKR1B10, SPP1, UBD, and waist circumference depended on sex and diagnosis. Several genes correlated with protein, fat, or carbohydrate intake. PEG10 correlated positively with physical activity in NASH and inversely with plasma vitamin C in both groups. Despite increased erythrocyte and hepatic ω-3 PUFA, supplementation did not alter hepatic gene expression and liver histology. HCC-related gene expression was associated with liver histology, body mass index, waist circumference, diet, and physical activity but was not affected by ω-3 PUFA supplementation.

Changes in PUFA Levels and Inflammation in a Cell Model of Hepatic Endoplasmic Reticulum Stress

Background The presence of endoplasmic reticulum (ER) stress is as an important contributing factor in various liver diseases, including alcoholic liver disease, nonalcoholic steatohepatitis, drug-induced liver injury, acute-on-chronic liver failure and hepatocellular carcinoma. Thus, investigating the ER stress response in cell cultures can help to understand the pathology of these diseases. This study aimed to determine hepatic polyunsaturated fatty acids (PUFAs) and inflammatory response in a cell model of ER stress. Materials and Methods Liver THLE-3 cells were treated with tunicamycin (TM) to induce ER stress and tauroursodeoxycholic acid (TUDCA) was administered in advance to decrease cytotoxic effects. Cell viability was determined via MTT assay. ER stress was confirmed by immunofluorescence and western blot analysis of C/EBP-homologous protein (CHOP) and 78-kDa glucose-regulated protein (GRP78). Arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) in THLE-3 cells were determined by LC-MS/MS. Phospholipase A2 (PLA2), cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in cells via ELISA. Results Hepatic ER stress was accomplished by TM and was alleviated by TUDCA. Tunicamycin treatment significantly decreased PUFAs in THLE-3 cells compared to controls. Activity of PLA2, COX and PGE2 levels were significantly increased in TM treated THLE-3 cells compared to controls. Tauroursodeoxycholic acid lead to a partial restoration of PUFA levels and decreased PLA2, COX and PGE2. Discussion In summary, we report that TM treatment results in significantly decreased PUFA levels and leads to significantly increased activity of cPLA2, COX and PGE2 levels in human hepatocytes. We also show that TUDCA increases PUFA levels and alleviates cPLA2, COX and PGE2 levels in TM treated liver cells. Conclusion To our best knowledge, this is the first study reporting decreased PUFA levels in ER stress and supports the use of omega-3 fatty acids in liver diseases demonstrating ER stress.

Effect of tauroursodeoxycholic acid on PUFA levels and inflammation in an animal and cell model of hepatic endoplasmic reticulum stress.

The aim of this study was to evaluate hepatic polyunsaturated fatty acids (PUFAs) and inflammatory response in an animal and cell model of endoplasmic reticulum (ER) stress. Rats were divided into control, tunicamycin (TM)-treated, and TM + tauroursodeoxycholic acid (TUDCA)-treated groups. Hepatic ER stress was induced by TM and the ER stress inhibitor TUDCA was injected 30 min before induction of ER stress. Liver THLE-3 cells were treated with TM and TUDCA was administered in advance to decrease cytotoxic effects. Necroinflammation was evaluated in liver sections, while cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kit. ER stress was confirmed by immunofluorescence and Western blot analysis of C/EBP-homologous protein and 78-kDa glucose-regulated protein. Arachidonic acid (C20:4n-6), dihomo-γ-linolenic acid (C20:3n-6), eicosapentaenoic acid (C20:5n-3), and docosahexaenoic acid (C22:6n-3) in liver tissue and THLE-3 cells were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Phospholipase A2 (PLA2), cyclooxygenase (COX), and prostaglandin E2 (PGE2) were measured in tissue and cell samples. Hepatic ER stress was accomplished by TM and was alleviated by TUDCA. TM treatment significantly decreased PUFAs in both liver and THLE-3 cells compared to controls. PLA2, COX, and PGE2 levels were significantly increased in TM-treated rats and THLE-3 cells compared to controls. TUDCA leads to a partial restoration of liver PUFA levels and decreased PLA2, COX, and PGE2. This study reports decreased PUFA levels in ER stress and supports the use of omega-3 fatty acids in liver diseases demonstrating ER stress.

Docosahexaenoic acid blocks progression of western diet-induced nonalcoholic steatohepatitis in obese Ldlr-/- mice.

BackgroundNonalcoholic fatty liver disease (NAFLD) is a major public health concern in western societies. Nonalcoholic steatohepatitis (NASH), the progressive form of NAFLD, is characterized by hepatic steatosis, inflammation, oxidative stress and fibrosis. NASH is a risk factor for cirrhosis and hepatocellular carcinoma. NASH is predicted to be the leading cause of liver transplants by 2020. Despite this growing public health concern, there remain no Food and Drug Administration (FDA) approved NASH treatments. Using Ldlr -/- mice as a preclinical model of western diet (WD)-induced NASH, we previously established that dietary supplementation with docosahexaenoic acid (DHA, 22:6,ω3) attenuated WD-induced NASH in a prevention study. Herein, we evaluated the capacity of DHA supplementation of the WD and a low fat diet to fully reverse NASH in mice with pre-existing disease.MethodsLdlr -/- mice fed the WD for 22 wks developed metabolic syndrome (MetS) and a severe NASH phenotype, including obesity, dyslipidemia, hyperglycemia, hepatic steatosis, inflammation, fibrosis and low hepatic polyunsaturated fatty acid (PUFA) content. These mice were randomized to 5 groups: a baseline group (WDB, sacrificed at 22 wks) and 4 treatments: 1) WD + olive oil (WDO); 2) WD + DHA (WDD); 3) returned to chow + olive oil (WDChO); or 4) returned to chow + DHA (WDChD). The four treatment groups were maintained on their respective diets for 8 wks. An additional group was maintained on standard laboratory chow (Reference Diet, RD) for the 30-wk duration of the study.ResultsWhen compared to the WDB group, the WDO group displayed increased hepatic expression of genes linked to inflammation (Opn, Il1rn, Gdf15), hepatic fibrosis (collagen staining, Col1A1, Thbs2, Lox) reflecting disease progression. Mice in the WDD group, in contrast, had increased hepatic C20-22 ω3 PUFA and no evidence of NASH progression. MetS and NASH markers in the WDChO or WDChD groups were significantly attenuated and marginally different from the RD group, reflecting disease remission.ConclusionWhile these studies establish that DHA supplementation of the WD blocks WD-induced NASH progression, DHA alone does not promote full remission of diet-induced MetS or NASH.

92 - Decreased Hepatic Polyunsaturated Fatty Acids and Inflammatory Response in a Rat Model of Endoplasmic Reticulum Stress

In this study, we sought to evaluate the effect of endoplasmic reticulum (ER) stress on hepatic polyunsaturated fatty acids (PUFAs) and inflammatory response through cyclooxygenase (COX)-mediated pathway. Male Wistar rats which were allowed free access to standard rat chow were randomly divided into control, tunicamycin (TM) treated and TM + tauroursodeoxycholic acid (TUDCA) treated groups. Hepatic ER stress was induced by intraperitoneal injection of tunicamycin and the ER stress inhibitor TUDCA was injected 30 minutes before hepatic induction of ER stress. The presence of ER stress was confirmed by increased intracellular levels of C/EBP-homologous protein (CHOP) and 78-kDa glucose-regulated protein (GRP78). Necroinflammation was evaluated in liver sections stained with hematoxylin-eosin using the Ishak-modified hepatic activity index. Levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) in liver tissue were determined by an optimized multiple reaction monitoring method using ultra fast-liquid chromatography coupled with tandem mass spectrometry. Phospholipase A2 (PLA2), COX and prostaglandin E2 (PGE2) were measured in tissue samples to evaluate changes in the inflammatory pathways. Tunicamycin treatment significantly decreased all measured PUFAs and increased AA/EPA ratio in liver tissue compared to controls. Tissue activity of PLA2, COX and PGE2 levels were significantly increased in liver tissue of TM treated rats compared to controls. Tauroursodeoxycholic acid lead to a partial restoration of liver PUFA levels and significantly decreased PLA2, COX and PGE2 levels compared to TM treated rats. The results of this study reveal the presence of a proinflammatory state in hepatic ER stress as shown by significantly increased AA/EPA ratio. To our best knowledge, this is the first study reporting altered PUFA levels in ER stress and supports the use of omega-3 fatty acids as adjuvant treatment in liver diseases demonstrating ER stress. Acknowledgement This study was supported by a grant from TUBITAK; # 214S223.

Comparative Effects of Glutathione Peroxidase‐1 Overexpression and Cu/Zn‐superoxide Dismutase Knockout on Murine Hepatic Fatty Acid Metabolism

Glutathione peroxidase‐1 (GPX1) and Cu/Zn‐superoxide dismutase (SOD1) are two major antioxidant enzymes, while polyunsaturated fatty acids (PUFA) including eicosapentaenoic acid (20:5n‐3, EPA) and docosahexaenoic acid (22:6n‐3, DHA) are health‐promoting nutrients. This study was conducted to determine the effects of GPX1 overexpression and SOD1 knockout on hepatic synthesis, accumulation, and oxidation of PUFA. GPX1 overexpressing (OE, 6–7 mo), and SOD1 knockout (SKO, 2 mo) mice and their respective wild‐type controls were fed a Torula yeast basal diet supplemented with 0 or 5% defatted microalgae (Nannochloropsis oceanica, 16% EPA) for 8 wk (n = 5 to 7 per group). Overexpression of GPX1 decreased (P < 0.01) linoleic acid (18:2n‐6) concentration and raised (P < 0.05) ω‐3/ω‐6 fatty acid ratio in the liver of mice regardless of diets. In comparison, knockout of SOD1 decreased (P < 0.05) concentrations of PUFA, in particular eicosatrienoic acid (20:3n‐3, precursor of EPA) and DHA, in mice fed both diets. The knockout of SOD1 also resulted in lower (P < 0.01) hepatic ω‐3/ω‐6 fatty acid ratio than the wild‐type mice when both genotypes were fed the algae diet. Inclusion of 5% algae in the diet elevated (P < 0.05) the hepatic DHA concentration in only wild‐type mice of SOD1. Results of qPCR analysis showed that knockout of SOD1 down‐regulated (P < 0.05) hepatic mRNA levels of elongase 2 and 5, the major enzymes involved in long chain PUFA synthesis, compared with the wild‐type controls. Meanwhile, the OE mice had lower (P < 0.05) hepatic mRNA levels of PUFA oxidation‐related genes including cyclooxygenase 1 (COX1), COX2, and arachidonate 5‐lipoxygenase than their wild‐type controls. In addition, feeding the algae diet decreased (P < 0.01) non‐esterified fatty acid concentrations in both plasma and liver of the OE mice, and concentration of triglycerides in the liver of the SOD1 wild‐type mice. In conclusion, overexpression of GPX1 and knockout of SOD1 exerted differential effects on synthesis, accumulation and oxidation of hepatic PUFA in mice. Further study requires to measure oxidized metabolites of PUFA and to reveal molecular mechanisms by which GPX1 and SOD1 regulate PUFA metabolism.Support or Funding InformationSupported in part by a NIH grant DK53018 and a USDA/DOE Biomass R&D Initiative Grant

Is Western Diet-Induced Nonalcoholic Steatohepatitis in Ldlr-/- Mice Reversible?

BackgroundNonalcoholic fatty liver disease (NAFLD) is a major public health burden in western societies. The progressive form of NAFLD, nonalcoholic steatohepatitis (NASH), is characterized by hepatosteatosis, inflammation, oxidative stress, and hepatic damage that can progress to fibrosis and cirrhosis; risk factors for hepatocellular carcinoma. Given the scope of NASH, validating treatment protocols (i.e., low fat diets and weight loss) is imperative.MethodsWe evaluated the efficacy of two diets, a non-purified chow (NP) and purified (low-fat low-cholesterol, LFLC) diet to reverse western diet (WD)-induced NASH and fibrosis in Ldlr-/- mice.ResultsMice fed WD for 22–24 weeks developed robust hepatosteatosis with mild fibrosis, while mice maintained on the WD an additional 7–8 weeks developed NASH with moderate fibrosis. Returning WD-fed mice to the NP or LFLC diets significantly reduced body weight and plasma markers of metabolic syndrome (dyslipidemia, hyperglycemia) and hepatic gene expression markers of inflammation (Mcp1), oxidative stress (Nox2), fibrosis (Col1A, LoxL2, Timp1) and collagen crosslinking (hydroxyproline). Time course analyses established that plasma triglycerides and hepatic Col1A1 mRNA were rapidly reduced following the switch from the WD to the LFLC diet. However, hepatic triglyceride content and fibrosis did not return to normal levels 8 weeks after the change to the LFLC diet. Time course studies further revealed a strong association (r2 ≥ 0.52) between plasma markers of inflammation (TLR2 activators) and hepatic fibrosis markers (Col1A, Timp1, LoxL2). Inflammation and fibrosis markers were inversely associated (r2 ≥ 0.32) with diet-induced changes in hepatic ω3 and ω6 polyunsaturated fatty acids (PUFA) content.ConclusionThese studies establish a temporal link between plasma markers of inflammation and hepatic PUFA and fibrosis. Low-fat low-cholesterol diets promote reversal of many, but not all, features associated with WD-induced NASH and fibrosis in Ldlr-/- mice.

Changes of hepatic lipid mediators associated with intake of high-fat diet for 12 weeks in endotoxemic rats using LC-ESI-MS/MS

It has recently been reported that anti-inflammatory lipid mediators are increased in the late phase of acute inflammation, whereas proinflammatory lipid mediators are regulated at the initiation of inflammation. The purpose of this study was to evaluate changes of hepatic lipid mediators due to high-fat diet (HFD) feeding in endotoxemic rats. Male Wistar rats were fed either HFD or control diet for 12 weeks, and were then killed 0, 1.5, and 6 h after lipopolysaccharide (LPS) injection. Analyses included lipidomics assessment of mediators using liquid chromatography-electrospray ionization/multi-stage mass spectrometry; measuring expression of hepatic polyunsaturated fatty acid (PUFA)-oxidizing enzyme, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and inducible nitric oxide synthase mRNA levels; blood biochemical tests; and liver histology. HFD feeding worsened liver injury, increased expression of TNF-α and IL-6 mRNA, and increased oxidative stress after LPS injection. PUFA-oxidizing enzymes were higher in HFD-fed rats after LPS injection. The proinflammatory prostaglandin (PG)E2 and thromboxane B2 were increased 1.5 h after LPS injection, and had decreased by 6 h in HFD-fed rats. In contrast, potent pro-resolving resolvins derived from eicosapentaenoic acid and docosahexaenoic acid were not detected, but anti-inflammatory epoxyeicosatrienoic acids, lipoxin A4, and 15-deoxy-PGJ2 were increased after LPS injection in HFD-fed rats. HFD feeding for 12 weeks enhanced proinflammatory lipid mediators 1.5 h after LPS injection suggesting relation to liver injury.

Maternal fat intake in rats alters 20:4n-6 and 22:6n-3 status and the epigenetic regulation of Fads2 in offspring liver

Poor prenatal nutrition, acting through epigenetic processes, induces persistent changes in offspring phenotype. We investigated the effect of maternal fat intake on polyunsaturated fatty acid (PUFA) status and on the epigenetic regulation of Fads2, encoding Δ6 desaturase (rate limiting in PUFA synthesis), in the adult offspring. Rats (n=6 per dietary group) were fed either 3.5% (w/w), 7% (w/w) or 21% (w/w) butter or fish oil (FO) from 14days preconception until weaning. Offspring (n=6 males and females per dietary group) were fed 4% (w/w) soybean oil until postnatal day 77. 20:4n-6 and 22:6n-3 levels were lower in liver phosphatidylcholine (PC) and phosphatidylethanolamine and plasma PC (all P<.0001) in offspring of dams fed 21% than 3.5% or 7% fat regardless of type. Hepatic Fads2 expression related inversely to maternal dietary fat. Fads2 messenger RNA expression correlated negatively with methylation of CpGs at −623, −394, −84 and −76 bases relative to the transcription start site (all P<.005). Methylation of these CpGs was higher in offspring of dams fed 21% than 3.5% or 7% fat; FO higher than butter. Feeding adult female rats 7% fat reduced 20:4n-6 status in liver PC and Fads2 expression and increased methylation of CpGs −623, −394, −84 and −76 that reversed in animals switched from 7% to 4% fat diets. These findings suggest that fat exposure during development induces persistent changes, while adults exhibit a transient response, in hepatic PUFA status in offspring through epigenetic regulation of Fads2. Thus, epigenetic regulation of Fads2 may contribute to short- and long-term regulation of PUFA synthesis.

TCDD-Elicited Effects on Liver, Serum, and Adipose Lipid Composition in C57BL/6 Mice

The aryl hydrocarbon receptor (AhR) mediates alterations in hepatic lipid composition elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to further investigate the effects of TCDD, liver, serum, and gonadal white adipose tissue (gWAT) fatty acid methyl esters (FAMEs) and lipids were examined in fasted 4-week-old female mice orally gavaged with 30 µg/kg TCDD at 24, 72, and 168 h postdose. Mean hepatic FAME levels increased (236.7 µmol/g in controls compared with 392.2 µmol/g in TCDD treated) with minimal changes in gWAT and serum. In the liver, TCDD decreased saturated fatty acids (SFAs 16:0, 18:0, 20:0, and 22:0) and increased monounsaturated fatty acids (MUFAs 16:1n7, 18:1n9, and 20:1n9). Hepatic polyunsaturated fatty acids (PUFAs) 20:2n6, 20:3n6, 18:3n3, and 22:5n3 also increased, whereas 20:4n6 and 22:6n3 levels decreased. gWAT PUFAs 20:2n6 and 20:3n6 exhibited modest increases, whereas serum 18:0 decreased and 18:1n9 increased. Serum analyses also identified a ~25% decrease in total cholesterol (CHOL), low-density lipoprotein (LDL), and high-density lipoprotein following TCDD treatment. The decrease in serum CHOL was consistent with the induction of hepatic reverse CHOL transport genes Lcat (2.0-fold), Apoa1 (1.7-fold), and Ldlr (3.6-fold), and the repression of CHOL biosynthesis genes Hmgcs1 (-2.1-fold) and Hmgcr (-2.3-fold). In addition, TCDD decreased serum Apob100 (4.4-fold) and Apob48 (2.2-fold) protein levels, suggesting serum lipid clearance and decreased hepatic efflux. Collectively, the TCDD-elicited decreases in serum lipid levels are consistent with AhR-mediated enhancement of dietary fat distribution to the liver.

CYP4V2 in Bietti's Crystalline Dystrophy: Ocular Localization, Metabolism of ω-3-Polyunsaturated Fatty Acids, and Functional Deficit of the p.H331P Variant

Bietti's crystalline corneoretinal dystrophy (BCD) is a recessive degenerative eye disease caused by germline mutations in the CYP4V2 gene. More than 80% of mutant alleles consist of three mutations, that is, two splice-site alterations and one missense mutation, c.992C>A, which translates to p.H331P. In the present study, we analyzed the expression of CYP4 family members in human tissues and conducted functional studies with the wild-type and p.H331P enzymes, to elucidate the link between CYP4V2 activity and BCD. Expression analysis of 17 CYP1 to CYP4 genes showed CYP4V2 to be a major cytochrome P450 in ARPE-19 cells (a human cell line spontaneously generated from normal human retinal pigmented epithelium) and the only detectable CYP4 transcript. Immunohistochemical analyses demonstrated that CYP4V2 protein was present in epithelial cells of the retina and cornea and the enzyme was localized to endoplasmic reticulum. Recombinant reconstituted CYP4V2 protein metabolized eicosapentaenoic acid and docosahexaenoic acid (an important constituent of the retina) to their respective ω-hydroxylated products at rates similar to those observed with purified CYP4F2, which is an established hepatic polyunsaturated fatty acid (PUFA) hydroxylase. The disease-associated p.H331P variant was undetectable in Western blot analyses of HepG2 cells stably transduced with lentiviral expression vectors. Finally, overexpression of functional CYP4V2 in HepG2 cells altered lipid homeostasis. We demonstrated that CYP4V2 protein is expressed at high levels in ocular target tissues of BCD, that the enzyme is metabolically active toward PUFAs, and that the functional deficit among patients with BCD who carry the H331P variant is most likely a consequence of the instability of the mutant protein.

Non-Alcoholic Fatty Liver Disease in HIV Infection Associated with Altered Hepatic Fatty Acid Composition

Hepatic fatty acid (FA) composition, especially a reduction in n-3 polyunsaturated FA (PUFA) may contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD), which is common in HIV-infection.. In a cross-sectional study we compared hepatic FA composition between 20 HIV-infected men with NAFLD (HIV/NAFLD), 21 HIV-negative men with NAFLD (NAFLD), and 7 healthy controls. Within HIV/NAFLD we compared simple steatosis (HIV/SS) to steatohepatitis (HIV/NASH). FA composition in liver and erythrocytes, oxidative stress, diet, and exercise were assessed. Major findings (P<0.05) were: 1) higher hepatic n-6/n-3 ratio in HIV/NAFLD [median (range)] [8.08 (1.08-21.52)] compared to controls [5.83 (3.58-6.93)] and NAFLD [5.97 (1.46-10.40)], with higher n-6 PUFA in HIV/NAFLD compared to NAFLD; 2) lower n-3 PUFA in erythrocytes (mol%), a marker for dietary intake, in HIV/NAFLD [5.26 (1.04-11.75)] compared to controls [8.92 (4.79-12.67)]; 3) the ratios of long-chain PUFA products to essential FA precursors of the n-6 and n-3 series were lower in HIV/NAFLD and NAFLD compared to controls. In contrast, the ratio of oleic/stearic acid was higher in HIV/NAFLD compared to the other groups. These ratios are indirect markers of enzymatic FA desaturation and elongation. Hepatic PUFA, especially biologically active long-chain PUFA, were also lower in HIV/NASH compared to HIV/SS. Oxidative stress was not different among the groups. We conclude that HIV/NAFLD is associated with altered hepatic FA composition. Changes may be due to impaired FA metabolism or suboptimal n-3 PUFA intake. The potential role of n-3 PUFA (e.g. fish oil) to treat or prevent HIV/NAFLD warrants further investigation.

Effects of weight loss on liver and erythrocyte polyunsaturated fatty acid pattern and oxidative stress status in obese patients with non-alcoholic fatty liver disease.

Our aim was to study the influence of weight loss on the fatty acid (FA) composition of liver and erythrocyte phospholipids and oxidative stress status in obese, non-alcoholic, fatty liver disease (NAFLD) patients. Seven obese NAFLD patients who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in roux and Y were studied immediately and 3 months after surgery. Seven non-obese patients who underwent anti-reflux surgery constituted the control group. Serum F2-isoprostane levels were measured by GS/NICI-MS/MS and FA composition was determined by GC. At the time of surgery, controls and obese patients exhibited a hepatic polyunsaturated fatty acid (PUFA) pattern that correlated with that of erythrocytes. Three months after surgery, NAFLD patients lost 21% of initial body weight; serum F2-isoprostane levels decreased by 76%; total PUFA, long-chain PUFA (LCPUFA), n-3 PUFA, and n-3 LCPUFA increased by 22, 29, 81, and 93%, respectively; n-6/n-3 LCPUFA ratio decreased by 51%; docosahexaenoic acid/docosapentaenoic acid ratio increased by 19-fold; and the n-3 product/precursor ratio (20: 5 + 22: 5 + 22: 6)/18: 3 increased by 164% (p<0.05). It is concluded that weight loss improves the n-3 LCPUFA status of obese patients in association with significant amelioration in the biomarkers of oxidative stress, membrane FA insaturation, and n-3 LCPUFA biosynthesis capacity, thus representing a central therapeutic issue in the improvement of obesity-related metabolic alterations involved in the mechanism of hepatic steatosis.

Effects of weight loss on liver and erythrocyte polyunsaturated fatty acid pattern and oxidative stress status in obese patients with non-alcoholic fatty liver disease

Our aim was to study the influence of weight loss on the fatty acid (FA) composition of liver and erythrocyte phospholipids and oxidative stress status in obese, non-alcoholic, fatty liver disease (NAFLD) patients. Seven obese NAFLD patients who underwent subtotal gastrectomy with a gastro-jejunal anastomosis in roux and Y were studied immediately and 3 months after surgery. Seven non-obese patients who underwent anti-reflux surgery constituted the control group. Serum F2-isoprostane levels were measured by GS/NICI-MS/MS and FA composition was determined by GC. At the time of surgery, controls and obese patients exhibited a hepatic polyunsaturated fatty acid (PUFA) pattern that correlated with that of erythrocytes. Three months after surgery, NAFLD patients lost 21% of initial body weight; serum F2-isoprostane levels decreased by 76%; total PUFA, long-chain PUFA (LCPUFA), n-3 PUFA, and n-3 LCPUFA increased by 22, 29, 81, and 93%, respectively; n-6/n-3 LCPUFA ratio decreased by 51%; docosahexaenoic acid/docosapentaenoic acid ratio increased by 19-fold; and the n-3 product/precursor ratio (20: 5 + 22: 5 + 22: 6)/18: 3 increased by 164% (p<0.05). It is concluded that weight loss improves the n-3 LCPUFA status of obese patients in association with significant amelioration in the biomarkers of oxidative stress, membrane FA insaturation, and n-3 LCPUFA biosynthesis capacity, thus representing a central therapeutic issue in the improvement of obesity-related metabolic alterations involved in the mechanism of hepatic steatosis.