Dataset Information

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

ABSTRACT:

Background

Nonalcoholic 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.

Methods

We 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.

Results

Mice 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.

Conclusion

These 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.

SUBMITTER: Lytle KA

PROVIDER: S-EPMC4711955 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:BackgroundNonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, particularly in obese and type 2 diabetic individuals. NAFLD ranges in severity from benign steatosis to nonalcoholic steatohepatitis (NASH); and NASH can progress to cirrhosis, primary hepatocellular carcinoma (HCC) and liver failure. As such, NAFLD has emerged as a major public health concern. Herein, we used a lipidomic and transcriptomic approach to identify lipid markers associated with western diet (WD) induced NASH in female mice.MethodsFemale mice (low-density lipoprotein receptor null (Ldlr -/-) were fed a reference or WD diet for 38 and 46 weeks. Transcriptomic and lipidomic approaches, coupled with statistical analyses, were used to identify associations between major NASH markers and transcriptomic & lipidomic markers.ResultsThe WD induced all major hallmarks of NASH in female Ldlr -/- mice, including steatosis (SFA, MUFA, MUFA-containing di- and triacylglycerols), inflammation (TNF?), oxidative stress (Ncf2), and fibrosis (Col1A). The WD also increased transcripts associated with membrane remodeling (LpCat), apoptosis & autophagy (Casp1, CtsS), hedgehog (Taz) & notch signaling (Hey1), epithelial-mesenchymal transition (S1004A) and cancer (Gpc3). WD feeding, however, suppressed the expression of the hedgehog inhibitory protein (Hhip), and enzymes involved in triglyceride catabolism (Tgh/Ces3, Ces1g), as well as the hepatic abundance of C18-22 PUFA-containing phosphoglycerolipids (GpCho, GpEtn, GpSer, GpIns). WD feeding also increased hepatic cyclooxygenase (Cox1 & 2) expression and pro-inflammatory ?6 PUFA-derived oxylipins (PGE2), as well as lipid markers of oxidative stress (8-iso-PGF2?). The WD suppressed the hepatic abundance of reparative oxylipins (19, 20-DiHDPA) as well as the expression of enzymes involved in fatty epoxide metabolism (Cyp2C, Ephx).ConclusionWD-induced NASH in female Ldlr -/- mice was characterized by a massive increase in hepatic neutral and membrane lipids containing SFA and MUFA and a loss of C18-22 PUFA-containing membrane lipids. Moreover, the WD increased hepatic pro-inflammatory oxylipins and suppressed the hepatic abundance of reparative oxylipins. Such global changes in the type and abundance of hepatic lipids likely contributes to tissue remodeling and NASH severity.

Project description:BackgroundNonalcoholic 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.MethodsUsing livers from our previous study, we carried out a global non-targeted metabolomic approach to quantify diet-induced changes in hepatic metabolism.ResultsLivers 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 C20-22 n-3 PUFA-containing phosphoglycerolipids, C20-22 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 C20-22 PUFA content. Hepatic C20-22 n-3 PUFA content was inversely associated with hepatic ?-tocopherol and ascorbate content and positively associated with urinary F2- and F3-isoprostanes, revealing diet effects on whole body oxidative stress.ConclusionDHA 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.

Project description:Nonalcoholic fatty liver disease (NAFLD) is a major public health problem worldwide. NAFLD ranges in severity from benign steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and primary hepatocellular cancer (HCC). Obesity and type 2 diabetes mellitus (T2DM) are strongly associated with NAFLD, and the western diet (WD) is a major contributor to the onset and progression of these chronic diseases. Our aim was to use a lipidomic approach to identify potential lipid mediators of diet-induced NASH. We previously used a preclinical mouse (low density lipoprotein receptor null mouse, Ldlr -/-) model to assess transcriptomic mechanisms linked to WD-induced NASH and docosahexaenoic acid (DHA, 22:6, ω3)-mediated remission of NASH. This report used livers from the previous study to carry out ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography coupled with dynamic multi-reaction monitoring (HPLC-dMRM) to assess the impact of the WD and DHA on hepatic membrane lipid and oxylipin composition, respectively. Feeding mice the WD increased hepatic saturated and monounsaturated fatty acids and arachidonic acid (ARA, 20:4, ω6) in membrane lipids and suppressed ω3 polyunsaturated fatty acids (PUFA) in membrane lipids and ω3 PUFA-derived anti-inflammatory oxylipins. Supplementing the WD with DHA lowered hepatic ARA in membrane lipids and ARA-derived oxylipins and significantly increased hepatic DHA and its metabolites in membrane lipids, as well as C20-22 ω3 PUFA-derived oxylipins. NASH markers of inflammation and fibrosis were inversely associated with hepatic C20-22 ω3 PUFA-derived Cyp2C- and Cyp2J-generated anti-inflammatory oxylipins (false discovery rate adjusted p-value; q ≤ 0.026). Our findings suggest that dietary DHA promoted partial remission of WD-induced NASH, at least in part, by lowering hepatic pro-inflammatory oxylipins derived from ARA and increasing hepatic anti-inflammatory oxylipins derived from C20-22 ω3 PUFA.