Dietary supplementation with methyl donors reduces fatty liver and modifies the fatty acid synthase DNA methylation profile in rats fed an obesogenic diet

Abstract

Non-alcoholic fatty liver disease (NAFLD) is one of the first hepatic manifestations of metabolic syndrome, whose progression can lead to cirrhosis and hepatic carcinoma. Interestingly, methyl donor supplementation could improve obesogenic diet-induced hepatic triglyceride accumulation. The aim of this research is to describe methyl donor effects on a high-fat-sucrose (HFS) diet in both sexes and epigenetic changes induced on fatty acid synthase (FASN) promoter methylation pattern as well as gene expression of NAFLD key metabolic genes. Twenty-four male and 28 female Wistar rats were assigned to three dietary groups: control, HFS, and HFS supplemented with methyl donors (choline, betaine, vitamin B12, and folic acid). After 8 weeks of treatment, somatic, biochemical, mRNA, and epigenetic measurements were performed. Rats fed the HFS diet presented an overweight phenotype and alterations in plasma biochemical measurements. Methyl donor supplementation reverted the HFS-diet-induced hepatic triglyceride accumulation. Analysis of FASN promoter cytosine methylation showed changes in both sexes due to the obesogenic diet at -1,096, -780, -778, and -774 CpG sites with respect to the transcriptional start site. Methyl donor supplementation modified DNA methylation at -852, -833, -829, -743, and -733 CpGs depending on the sex. RT-PCR analysis confirmed that FASN expression tended to be altered in males. Our findings reinforce the hypothesis that methyl donor supplementation can prevent hepatic triglyceride accumulation induced by obesogenic diets in both sexes. Changes in liver gene expression profile and epigenetic-mediated mechanisms related to FASN DNA hypermethylation could be involved in methyl donor-induced NAFLD improvement.

Fig. 1

Hepatic triglyceride content (in μmol of triglycerides per gram of tissue) in (a) male and (b) female Wistar rats. Different letters indicate significant differences between groups of at least p < 0.05 (c control group—white, HFS high-fat-sucrose group—grey, HFSsupp high-fat-sucrose supplemented with methyl donors group—black)

Fig. 2

Quantitative DNA methylation analysis using MassARRAY system for FASN promoter in liver tissue from (a) male and (b) female Wistar rats (TSS transcriptional start site). The results are shown as percentages of methylation. All results are expressed as mean ± SD. Different letters indicate significant differences between groups of at least p < 0.05 (Control group, white; high-fat-sucrose group, grey; high-fat-sucrose supplemented with methyl donors group; black; **p < 0.01, ***p < 0.001)

Fig. 3

Expression levels (mRNA) of SREBPF1, CACA, FASN, MGLL, CCL2 and FABP4 measured by RT-PCR in liver from C, HFS and HFSsupp groups in male Wistar rats. Data are presented as mean ± SE measured in arbitrary units after normalization with ACTB, GAPDH and PPIA. Different letters in the same gene indicate statistically significant differences between groups of at least p < 0.05. (control group, white; high-fat-sucrose group, grey; high-fat-sucrose supplemented with methyl donors group, black; *p < 0.05, **p < 0.01)