Time course of western diet (WD) induced nonalcoholic steatohepatitis (NASH) in female and male Ldlr-/- mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a global health problem. Identification of factors contributing to the onset and progression of NAFLD have the potential to direct novel strategies to combat NAFLD. METHODS: We examined the time course of western diet (WD)-induced NAFLD and its progression to nonalcoholic steatohepatitis (NASH) in age-matched female and male Ldlr-/- mice, with time-points at 1, 4, 8, 20 and 40 weeks on the WD. Controls included Ldlr-/- mice maintained on a purified low-fat diet (LFD) for 1 and 40 weeks. The approach included quantitation of anthropometric, plasma and liver markers of disease, plus hepatic histology, lipids, oxylipins, gene expression and selected metabolites. RESULTS: One week of feeding the WD caused a significant reduction in hepatic essential fatty acids (EFAs: 18:2, ω6, 18:3, ω3) which preceded the decline in many C20-22 ω3 and ω6 polyunsaturated fatty acids (PUFA) and PUFA-derived oxylipins after 4 weeks on the WD. In addition, expression of hepatic inflammation markers (CD40, CD44, Mcp1, Nlrp3, TLR2, TLR4, Trem2) increased significantly in both female & male mice after one week on the WD. These markers continued to increase over the 40-week WD feeding study. WD effects on hepatic EFA and inflammation preceded all significant WD-induced changes in body weight, insulin resistance (HOMA-IR), oxidative stress status (GSH/GSSG ratio) and histological and gene expression markers of macrosteatosis, extracellular matrix remodeling and fibrosis. CONCLUSIONS: Our findings establish that feeding Ldlr-/- mice the WD rapidly lowered hepatic EFAs and induced key inflammatory markers linked to NASH. Since EFAs have an established role in inflammation and hepatic inflammation plays a major role in NASH, we suggest that early clinical assessment of EFA status and correcting EFA deficiencies may be useful in reducing NASH severity.

Early transcriptome changes associated with western diet induced NASH in Ldlr-/- mice points to activation of hepatic macrophages and an acute phase response.

Background: Nonalcoholic fatty liver disease (NAFLD) is a global health problem. Identifying early gene indicators contributing to the onset and progression of NAFLD has the potential to develop novel targets for early therapeutic intervention. We report on the early and late transcriptomic signatures of western diet (WD)-induced nonalcoholic steatohepatitis (NASH) in female and male Ldlr-/- mice, with time-points at 1 week and 40 weeks on the WD. Control Ldlr-/- mice were maintained on a low-fat diet (LFD) for 1 and 40 weeks. Methods: The approach included quantitation of anthropometric and hepatic histology markers of disease as well as the hepatic transcriptome. Results: Only mice fed the WD for 40 weeks revealed evidence of NASH, i.e., hepatic steatosis and fibrosis. RNASeq transcriptome analysis, however, revealed multiple cell-specific changes in gene expression after 1 week that persisted to 40 weeks on the WD. These early markers of disease include induction of acute phase response (Saa1-2, Orm2), fibrosis (Col1A1, Col1A2, TGFß) and NASH associated macrophage (NAM, i.e., Trem2 high, Mmp12 low). We also noted the induction of transcripts associated with metabolic syndrome, including Mmp12, Trem2, Gpnmb, Lgals3 and Lpl. Finally, 1 week of WD feeding was sufficient to significantly induce TNFα, a cytokine involved in both hepatic and systemic inflammation. Conclusion: This study revealed early onset changes in the hepatic transcriptome that develop well before any anthropometric or histological evidence of NALFD or NASH and pointed to cell-specific targeting for the prevention of disease progression.

Nonalcoholic Fatty Liver Disease and Omega-3 Fatty Acids: Mechanisms and Clinical Use.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic fatty liver disease worldwide, particularly in obese and type 2 diabetic individuals. Currently, there are no therapies for NAFLD that have been approved by the US Food and Drug Administration. Herein, we examine the rationale for using ω3 polyunsaturated fatty acids (PUFAs) in NAFLD therapy. This focus is based on the finding that NAFLD severity is associated with a reduction of hepatic C20-22 ω3 PUFAs. Because C20-22 ω3 PUFAs are pleiotropic regulators of cell function, loss of C20-22 ω3 PUFAs has the potential to significantly impact hepatic function. We describe NAFLD prevalence and pathophysiology as well as current NAFLD therapies. We also present evidence from clinical and preclinical studies that evaluated the capacity of C20-22 ω3 PUFAs to treat NAFLD. Given the clinical and preclinical evidence, dietary C20-22 ω3 PUFA supplementation has the potential to decrease human NAFLD severity by reducing hepatosteatosis and liver injury.

A Lipidomic Analysis of Docosahexaenoic Acid (22:6, ω3) Mediated Attenuation of Western Diet Induced Nonalcoholic Steatohepatitis in Male Ldlr -/- Mice.

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.

Lipidomic and transcriptomic analysis of western diet-induced nonalcoholic steatohepatitis (NASH) in female Ldlr -/- mice.

BACKGROUND: Nonalcoholic 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. METHODS: Female 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. RESULTS: The WD induced all major hallmarks of NASH in female Ldlr -/- mice, including steatosis (SFA, MUFA, MUFA-containing di- and triacylglycerols), inflammation (TNFα), 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 ω6 PUFA-derived oxylipins (PGE2), as well as lipid markers of oxidative stress (8-iso-PGF2α). 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). CONCLUSION: WD-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.

Omega-3 fatty acids and nonalcoholic fatty liver disease in adults and children: where do we stand?

PURPOSE OF REVIEW: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic fatty liver disease worldwide. The incidence of NAFLD parallels the prevalence of obesity. Moreover, NAFLD can progress to nonalcoholic steatohepatitis (NASH), cirrhosis and primary hepatocellular cancer (HCC). As such, NAFLD has become a major public health concern. We discuss recent clinical trials and meta-analyses evaluating the efficacy of C20-22 ω3 polyunsaturated fatty acids (PUFA) to attenuate preexisting NAFLD in adults and children. RECENT FINDINGS: Humans with NAFLD and NASH; and preclinical mouse models of NASH, have a high abundance of hepatic saturated (SFA) and monounsaturated (MUFA) fat, but a low abundance of hepatic C20-22 ω3 PUFA. This change in hepatic fat type and abundance is associated with hepatic lipotoxicity, inflammation, oxidative stress and fibrosis. Recent meta-analyses and clinical trials evaluated the capacity of C20-22 ω3 PUFA dietary supplementation to improve health outcomes in adults and children with preexisting NAFLD. Diets supplemented with docosahexaenoic acid (DHA, 22 : 6,ω3) alone or with eicosapentaenoic acid (EPA, 20 : 5,ω3) are tolerated and effective at lowering liver fat in NAFLD patients. However, outcomes are mixed with respect to C20-22 ω3 PUFA attenuation of more severe NAFLD markers, such as hepatic injury, inflammation and fibrosis. SUMMARY: These studies suggest that dietary supplementation with C20-22 ω3 PUFA should be considered as a viable and effective option to lower liver fat in obese adults and children with NAFLD.