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. 2017 Dec;38(12):1601-1610.
doi: 10.1038/aps.2017.127. Epub 2017 Sep 21.

Omega-3 PUFA ameliorates hyperhomocysteinemia-induced hepatic steatosis in mice by inhibiting hepatic ceramide synthesis

Yong-Qiang Dong  1 , Xing-Zhong Zhang  1 , Lu-Lu Sun  1 , Song-Yang Zhang  1 , Bo Liu  1 , Hui-Ying Liu  1 , Xian Wang  1 , Chang-Tao Jiang  1
Affiliations

Omega-3 PUFA ameliorates hyperhomocysteinemia-induced hepatic steatosis in mice by inhibiting hepatic ceramide synthesis

Yong-Qiang Dong et al. Acta Pharmacol Sin. 2017 Dec.

Abstract

Hyperhomocysteinemia (HHcy) is a key risk factor in hepatic steatosis. In this study, we applied a metabolomic approach to investigate the changes in the metabolite profile due to HHcy-induced hepatic steatosis and the effects of omega-3 PUFA (polyunsaturated fatty acid) supplementation in mice. HHcy was induced in mice by giving DL-Hcy (1.8 g/L) in drinking water for 6 weeks, then the mice were sacrificed, and the metabolic profiles of the liver and plasma were analyzed through UPLC-ESI-QTOFMS-based lipidomics. Hepatic triglycerides and cholesterol were further assayed. The expression of ceramide metabolism-related genes was measured by quantitative PCR. Compared with control mice, HHcy mice exhibited hepatic steatosis with a notable increase in ceramide-related metabolites and subsequent upregulation of ceramide synthesis genes such as Sptlc3, Degs2, Cer4 and Smpd4. Omega-3 PUFA was simultaneously administered in HHcy mice through chow diet containing 3.3% omega-3 PUFA supplement for 6 weeks, which significantly ameliorated Hcy-induced hepatic steatosis. The decrease in hepatic lipid accumulation was mainly due to reduced hepatic levels of ceramides, which was partly the result of the lower expression of ceramide synthesis genes, Sptlc3 and Degs2. Similar beneficial effects of DHA were observed in Hcy-stimulated primary hepatocytes in vitro. In summary, Hcy-induced ceramide elevation in hepatocytes might contribute to the development of hepatic steatosis. Furthermore, downregulation of ceramide levels through omega-3 PUFA supplementation ameliorates hepatic lipid accumulation. Thus, ceramide is a potential therapeutic target for the treatment of hepatic steatosis.

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Figures

Figure 1
Figure 1
Effects of HHcy on lipid accumulation in vivo. (A) ORO staining of lipids in representative liver sections. (B) Liver weight. (C) Ratios of liver weight/body weight. (D) Hepatic TG content. (E) Hepatic TC content. (F) The mRNA levels of genes involved in hepatic lipogenesis, FFA oxidation, TG secretion and FFA uptake. (A-F) Male C57BL/6 mice (6 weeks old) were treated with vehicle or Hcy (1.8 g/L) for 6 weeks (n=6/group). All data are presented as the mean±SEM. Two-tailed Student's t-test: *P<0.05 compared with vehicle.
Figure 2
Figure 2
Metabolic profiling of HHcy mice and control mice. (A) Heat map of lipid metabolites in liver. (B) Score scatter plot of the partial least squares discriminant analysis (PLS-DA) model of lipid metabolites in the liver; the red plot represents the vehicle group, and the green plot represents the HHcy group. (C) Variable importance in projection (VIP) plot generated from the PLS-DA, displaying the top 10 most important metabolite features in the liver. (D) The levels of saturated ceramides in the liver. (A-D) Male C57BL/6 mice (6 weeks old) were treated with vehicle or Hcy (1.8 g/L) for 6 weeks (n=4/group). (D) The data are presented as the mean±SEM. Two-tailed Student's t-test: *P<0.05 compared to vehicle.
Figure 3
Figure 3
Hcy induces ceramide synthases production both in vivo and in vitro. (A) The mRNA levels of ceramide synthases in the liver. (B) The mRNA levels of ceramide hydrolases in the liver. (A, B) Male C57BL/6 mice (6 weeks old) were treated with vehicle or Hcy (1.8 g/L) for 6 weeks (n=6/group). (C) The mRNA levels of ceramide synthases in primary hepatocytes. (D) The mRNA levels of ceramide hydrolases in primary hepatocytes. (C, D) Primary hepatocytes were treated with Hcy (100 μmol/L) for 24 h (n=5). All data are presented as the mean±SEM. Two-tailed Student's t-test: *P<0.05 compared with vehicle or control as appropriate.
Figure 4
Figure 4
Administration of omega-3 PUFA attenuates the development of HHcy-induced hepatic steatosis. (A) ORO staining of lipids in representative liver sections. (B) Liver weight. (C) Ratios of liver weight/body weight. (D) Hepatic TG content. (E) Hepatic TC content. (F) The mRNA levels of genes involved in hepatic lipogenesis, FFA oxidation, TG secretion and FFA uptake. Hcy-treated mice were fed a standard chow diet or a chow diet supplemented with omega-3 PUFA (33 mg/g) for 6 weeks and were compared with mice administered vehicle for 6 weeks (n=6/group). All data are presented as the mean±SEM. (B-F) One-way ANOVA with Tukey's correction: *P<0.05 compared with vehicle; #P<0.05 compared to HHcy.
Figure 5
Figure 5
Omega-3 PUFA treatment ameliorates the levels of ceramides in the livers of HHcy mice. (A) Heat map of lipid metabolites in the liver. (B) Score scatter plot of the Principal Component Analysis (PCA) model of lipid metabolites in the liver; the red plot represents the vehicle group, the green plot represents the HHcy group and the purple represents the HHcy plus omega-3 group. (C) The saturated ceramide levels in the liver. Hcy-treated mice were fed either a standard chow diet or a chow diet supplemented with omega-3 PUFA (33 mg/g) for 6 weeks and were compared with mice administered vehicle 6 weeks (n=4/group). All data are presented as the means±SEM. One-way ANOVA with Tukey's correction: *P<0.05 compared with vehicle; #P<0.05 compared with HHcy.
Figure 6
Figure 6
Omega-3 PUFA decreases the expression of ceramide synthases induced by Hcy in hepatocytes. (A) The mRNA levels of ceramide synthases in the liver. (B) The mRNA levels of ceramide hydrolases in the liver. Hcy-treated mice were fed either a standard chow diet or a chow diet supplemented with omega-3 PUFA (33 mg/g) for 6 weeks and were compared with mice administered vehicle for 6 weeks (n=6/group). (C) The mRNA levels of ceramide synthases in primary hepatocytes. (D) The mRNA levels of ceramide hydrolases in primary hepatocytes. Primary hepatocytes were pretreated with DHA (20 μmol/L) for 1 h before Hcy administration (100 μmol/L) for 24 h (n=5). (A-D) All data are presented as the mean±SEM. (A-D) One-way ANOVA with Tukey's correction: *P<0.05 compared with vehicle or control as appropriate; #P<0.05 compared to HHcy or Hcy as appropriate.
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