A Metabolomic Analysis of Omega-3 Fatty Acid-Mediated Attenuation of Western Diet-Induced Nonalcoholic Steatohepatitis in <i>LDLR</i>^<i>-/-</i> Mice

<div><p>Background</p><p>Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease and a risk factor for cirrhosis, hepatocellular carcinoma and liver failure. Previously, we reported that dietary docosahexaenoic acid (DHA, 22:6,n-3) was more effective than eicosapentaenoic acid (EPA, 20:5,n-3) at reversing western diet (WD) induced NASH in LDLR^-/- mice.</p> <p>Methods</p><p>Using livers from our previous study, we carried out a global non-targeted metabolomic approach to quantify diet-induced changes in hepatic metabolism. </p> <p>Results</p><p>Livers from WD + olive oil (WD + O)-fed mice displayed histological and gene expression features consistent with NASH. The metabolomic analysis of 320 metabolites established that the WD and n-3 polyunsaturated fatty acid (PUFA) supplementation had broad effects on all major metabolic pathways. Livers from WD + O-fed mice were enriched in saturated (SFA) and monounsaturated fatty acids (MUFA), palmitoyl-sphingomyelin, cholesterol, n-6 PUFA, n-6 PUFA-containing phosphoglycerolipids, n-6 PUFA-derived oxidized lipids (12-HETE) and depleted of C_20-22 n-3 PUFA-containing phosphoglycerolipids, C_20-22 n-3 PUFA-derived oxidized lipids (18-HEPE, 17,18-DiHETE) and S-lactoylglutathione, a methylglyoxal detoxification product. WD + DHA was more effective than WD + EPA at attenuating WD + O-induced changes in NASH gene expression markers, n-6 PUFA and oxidized lipids, citrate and S-lactosyl glutathione. Diet-induced changes in hepatic MUFA and sphingolipid content were associated with changes in expression of enzymes involved in MUFA and sphingolipid synthesis. Changes in hepatic oxidized fatty acids and S-lactoylglutathione, however, correlated with hepatic n-3 and n-6 C_20-22 PUFA content. Hepatic C_20-22 n-3 PUFA content was inversely associated with hepatic α-tocopherol and ascorbate content and positively associated with urinary F2- and F3-isoprostanes, revealing diet effects on whole body oxidative stress. </p> <p>Conclusion</p><p>DHA regulation of hepatic SFA, MUFA, PUFA, sphingomyelin, PUFA-derived oxidized lipids and S-lactoylglutathione may explain the protective effects of DHA against WD-induced NASH in LDLR^-/- mice. </p> </div

Diet effects on hepatic one-carbon, choline and glutathione metabolism.

<p>Panel A: Pathways for one-carbon, choline, glutathione and sphingomyelin metabolism. Panel B: Metabolites quantified by the metabolomic analysis were expressed as Metabolite-Fold Change and represented as mean ± SD, n=8 per group; *, <i>p</i> ≤ 0.05 versus chow; #, <i>p</i> ≤ 0.05 versus WD + O. </p

Diet effects on urinary isoprostanes and hepatic α-tocopherol, ascorbate and Nrf2.

<p>Panel A: Levels of 24-Hour urinary F2- and F3-IsoPs were quantified as described [17]. F2-IsoPs are derived from arachidonic acid (20:4 n-6) and F3-IsoPs are derived from eicosapentaenoic acid (20:5 n-3) (Fig. 14). Results are represented as Urinary Isoprostanes (ng/mg creatinine) mean <u>+</u> SD, n=3; urine from 2 pools of mice (3 mice/pool) from each diet group were assayed. Panel B: Hepatic α-tocopherol (vitamin E) and ascorbate (vitamin C) were quantified by the metabolomic analysis (Methods) and represented as Metabolite-Fold Change, relative to chow-fed mice; mean ± SD; n=8/group. Panel C: Hepatic nuclear abundance of Nrf2. Hepatic nuclear extracts were assayed for Nrf2 and the loading control protein, TATA-binding protein (TBP) using methods previously described [17]. Nrf2 nuclear abundance was normalized to TBP for each sample. Results are represented as Nrf2 Nuclear Abundance-Fold Change, mean <u>+</u> SD, n=8 group; *, <i>P</i> ≤ 0.05 versus chow; #, <i>P</i> ≤ 0.05 versus WD + O.</p

Heat map of diet effects on liver.

<p>The heat map represents the fold-change for each metabolite relative to control chow-fed versus WD-fed mice. The WD was supplemented with olive (O), EPA (E), DHA (D) or EPA and DHA (E + D). Results are sorted by fold-change within each pathway. </p

Diet effects on plasma endotoxin.

<p>Plasma endotoxin was quantified as described in Materials and Methods. Results are presented as Plasma Endotoxin, Fold Change. Mean ± SD relative to chow fed mice; *, <i>P</i> ≤ 0.05 versus chow. </p

Diet effects on glucose metabolism and <i>de</i><i>novo</i> MUFA synthesis.

<p>Panel A: Glucose conversion to saturated and monounsaturated fatty acids. Panel B: Metabolites involved in glucose metabolism were quantified by the metabolomic analysis and expressed as Metabolite-Fold Change; mean ± SD, n=8 per group. Panel C: Metabolites involved in <i>de </i><i>novo</i> MUFA synthesis were quantified by the metabolomic analysis and expressed as Metabolite-Fold Change; mean ± SD, n=8 per group; *, <i>p</i> ≤ 0.05 versus chow; #, <i>p</i>≤ 0.05 versus WD + O.</p

Pathways for the formation of oxidized lipids from arachidonic acid (20:4,n-6) [Panel A] and eicosapentaenoic acid (20:5,n-3)[Panel B].

<p>See text for explanation.</p

Diet effects on hepatic oxidized lipids.

<p>N-6 and n-3 PUFA and oxidized fatty acids were quantified by the metabolomic analysis (Methods). Results are expressed and Metabolite-Fold Change relative to the chow-fed group, mean ± SD, n=8/group; *, <i>p</i> ≤ 0.05 versus chow; #, <i>p</i> ≤ 0.05 versus WD + O.</p

Diet effects on hepatic markers of inflammation, SFA, MUFA and damage.

<p>Linear regression analysis of hepatic palmitoyl-sphingomyelin (Fold Change relative to chow) versus hepatic MCP1 mRNA expression (fold change relative to chow) (Panel A); hepatic total MUFA content (µmoles total MUFA/mg protein) (Panel B); hepatic palmitate (16:0) (µmoles /mg protein) (Panel C); and plasma AST (units (U)/ml of plasma) (Panel D). Palmitoyl-sphingomyelin was quantified in the metabolomic analysis while hepatic MCP1, MUFA, palmitate, and plasma AST were quantified and reported previously [8]. Each data point in Panels A-D represents the relative abundance of palmitoyl-sphingomyelin and hepatic MCP1 mRNA, MUFA, 16:0 or plasma AST for each animal. The groups are colored-coded to facilitate visualization of the distribution in each group.</p

Diet effects on S-lactoylglutathione and metabolites from carbohydrate and lipid oxidation.

<p>Panel A: Pathway of methylglyoxal formation and detoxification. Panel B: Hepatic abundance of S-lactoylglutathione was quantified by the metabolomic analysis (Methods). Results are represented as S-Lactoylglutathione-Fold Change, relative to chow-fed mice; mean ± SD, n=8/group. Panel C: Expression of enzymes involved in S-lactoylglutathione formation and degradation, i.e., glyoxalase 1 (Glo 1) and glyoxalase 2 (Glo 2). Results are represented as mRNA Abundance-Fold Change, relative to chow-fed mice; mean <u>+</u> SD, n=8/group. </p