Hepatic Methionine Homeostasis Is Conserved in C57BL/6N Mice on High-Fat Diet Despite Major Changes in Hepatic One-Carbon Metabolism

<div><p>Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine β-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments <i>in vitro</i>, using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARα) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-α-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of <i>de novo</i> DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARα-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine.</p> </div

Influence of HF diet on hepatic Dnmt gene expression and global DNA methylation.

<p>(<b>A–C</b>) Quantification of Dnmt gene expression after 12 weeks of feeding (n = 5–6). Control mice showed stronger expression for Dnmt1 (Ct = 25.13±0.06) and <i>de novo</i> Dnmt3a (Ct = 28.10±0.15) than for <i>de novo</i> Dnmt3b (Ct = 30.55±0.31). Upon HF feeding, Dnmt1 mRNA expression was unaltered (Ct = 25.03±0.11), but gene expression of Dnmt3a (Ct = 28.28±0.18) and Dnmt3b (Ct = 31.37±0.14) decreased, respectively. (<b>D</b>) Analysis of hepatic global DNA methylation of control and HF animals (n = 6). DNA methylation was calculated from the (<i>Hpa</i>II/<i>Msp</i>I) ratio, whereby a ratio of 1 indicates 0% methylation and a ratio approaching 0 corresponds to 100% DNA methylation at the investigated sites. Data are presented as mean ± SEM. Open and grey bars represent control and HF animals, respectively. Asterisk indicates statistical significance (p<0.05).</p

Impact of HF diet on selected hepatic metabolite concentrations after 12 weeks of feeding.

<p>Data are presented as box and whisker plot. (<b>A</b>) Selected data for taurine, L-glutamine, L-α-amino-n-butyrate, L-citrulline, L-ornithine, hydroxyproline and L-methionine (n = 9–11). (<b>B</b>) Analysis of S-adenosyl-methionine, S-adenosyl-homocysteine, L-homocysteine, cystathionine (n = 5–6) and choline, betaine and dimethylglycine (n = 7–9). (<b>C</b>) Selected ratios between measured hepatic metabolite concentrations. Open and grey bars represent control and HF mice, respectively. Asterisk indicates statistical significance (p<0.05).</p

Schematic presentation of analyzed changes in hepatic C1-metabolism after HF feeding in C57BL/6N mice.

<p>Observed changes of mRNAs (Bhmt, Cbs, Csad, Got, Gss, PPARα), proteins (BHMT and CBS) and measured metabolites (taurine, homocysteine, methionine, betaine, DMG) are depicted. Dotted lines represent inhibitory effects of insulin (reported by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057387#pone.0057387-Ratnam1" target="_blank">[48]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057387#pone.0057387-Ratnam2" target="_blank">[49]</a>) and PPARα (this study) on the regulation of transcription. Cross means disrupted inhibitory effect of insulin reported in hyperglycemic mice (40–43).</p