The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide, concurrent with increased obesity. Thus, there is urgent need for research that can lead to effective NAFLD prevention/treatment strategies. Omega-3 polyunsaturated fatty acids (n-3 PUFAs), including eicosapentaenoic acid (EPA), improve inflammation- and dyslipidemia-related metabolic disorders; however, mechanisms mediating the benefits of n-3 PUFAs in NAFLD treatment are less understood. We previously reported that EPA reversed obesity-induced hepatic steatosis in high-fat (HF)-fed B6 mice. Utilizing a combination of biochemical analyses of liver tissues from HF and HF-EPA-fed mice and a series of in vitro studies in tumor necrosis factor-alpha (TNF-α)-stimulated HepG2 cells, we dissect the mechanistic effects of EPA in reducing hepatic steatosis, including the role of EPA-targeted microRNAs (miRNA). With EPA, hepatic lipid metabolism was improved in HF-EPA mice, as indicated by decreased protein and messenger RNA (mRNA) levels of fatty acid synthase (FASN) and acetyl-CoA carboxylase (Acaca) gene, and increased mRNA levels for the peroxisome proliferator activated receptor-α (Pparα), and carnitine palmitoyltransferase (Cpt) 1a and 2 genes in the HF-EPA mice. Additionally, inflammation was reduced, as shown by decreased tumor necrosis factor-alpha (Tnfα) gene expression. Accordingly, EPA also significantly reduced FASN and ACACA mRNAs in human HepG2 cells. Glycolysis, estimated by extracellular acidification rate, was significantly reduced in HepG2 cells treated with EPA vs. vehicle. Furthermore, we identified several miRNAs that are regulated by EPA in mouse liver, including miR-19b-3p, miR-21a-5p, and others, which target lipid metabolism and inflammatory pathways. In conclusion, our findings provide novel mechanistic evidence for beneficial effects of EPA in NAFLD, through the identification of specific genes and miRNAs, which may be further exploited as future NAFLD therapies.

Visceral obesity may be a driving factor in nonalcoholic fatty liver disease (NAFLD) development. Previous studies have shown that the omega-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA), ameliorates obesity in high-fat (HF) fed male, C57Bl/6 mice at thermoneutral conditions, independent of uncoupling protein 1 (UCP1). Our goals herein were to investigate sex-dependent mechanisms of EPA in the livers of wild type (WT) and UCP1 knockout (KO) male and female mice fed a HF diet (45% kcal fat; WT-HF, KO-HF) with or without supplementation of 36 g/kg EPA (WT-EPA, KO-EPA). KO significantly increased body weight in males, with no significant reductions with EPA in the WT or KO groups. In females, there were no significant differences in body weight among KO groups and no effects of EPA. In males, liver TGs were significantly higher in the KO-HF group and reduced with EPA, which was not observed in females. Accordingly, gene and protein markers of mitochondrial oxidation, peroxisomal biogenesis and oxidation, as well as metabolic futile cycles were sex-dependently impacted by KO and EPA supplementation. These findings suggest a genotypic difference in response to dietary EPA supplementation on the livers of male and female mice with diet-induced obesity and housed at thermoneutrality.

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Hepatic steatosis is characterized by excessive accumulation of triglyceride (TG). Mitochondrial membrane uncoupling proteins (UCPs) are potential targets for obesity treatment, given their ability to dissipate energy as heat. Previous studies in our lab have indicated that the omega‐3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA), reduced obesity, inflammation and hepatic steatosis, and upregulated brown adipose tissue UCP1 protein in high fat (HF) fed mice housed at ambient temperature (22–23°C). However, EPA‐mediated improvements were attenuated and were independent of UCP1 when mice were housed in a thermoneutral environment (28–30°C). Thus, the objective of this work is to determine mechanisms of EPA‐mediated improvements in liver steatosis in UCP1 Knockout (KO) mice. Wild type (WT) and UCP1‐KO B6 mice were fed a HF diet with 36 g/kg AlaskOmega, 800mg/g fish oil of EPA (WT‐EPA, KO‐EPA) or without EPA (WT‐HF, KO‐HF) for 14 weeks. Mice were metabolically phenotyped during the feeding period, and histology and molecular analyses were performed in the liver after euthanasia. The KO‐HF group were highest in body weight, while KO‐HF, WT‐HF and WT‐EPA were comparable. Surprisingly, EPA attenuated weight gain in the KO‐EPA group compared to KO‐HF. Further, liver TG levels were significantly reduced in the KO‐EPA group compared to the KO‐HF. Consistent with this finding, EPA upregulated fatty acid beta‐oxidation markers only in the KO‐EPA group, indicated by significant increases in peroxisome proliferator activated receptor‐alpha (Ppara) and carnitine palmitoyltransferase (Cpt) 1a compared to KO‐HF. In conclusion, EPA rescued the detrimental effects of UCP1 deficiency in the livers of HF fed mice in a thermoneutral environment. Further studies are ongoing to dissect mechanistic basis for UCP1‐independent beneficial effects of EPA in liver steatosis. Our promising findings warrant further EPA supplementation studies in humans with obesity, given the limited activity of brown adipose tissue and downregulation of UCP1 in this population. Support or Funding Information Funded by NIH/NCCIH grant #R15AT008879‐01A1 (N.M.M.)