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

Metabolic data during exhaustive exercise prior to the three diet interventions.

<p>Day 1.</p

Concentrations of metabolites and hormones in fasted condition the day after exhaustive exercise and diet interventions, and during and after the performance test.

<p>Data are mean and error bars represents SEM. C: p<0.05 compared to CHO; W: p<0.05 compared to CHO+PROT (whey); P: p<0.05 compared to placebo. N = 8 for all data points.</p

Plasma concentrations of amino acids during the first 2 h of recovery when provided CHO, CHO+PROT or PLA.

<p>Data for 18 amino acids are presented; no data are presented on cysteine and aspartate. Data are mean and error bars represents SEM. C: p<0.05 compared to CHO; W: p<0.05 compared to CHO+PROT (whey); P: p<0.05 compared to placebo. N = 8 for all data points.</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

Performance test after 18 h recovery receiving CHO, CHO+PROT or PLA.

<p>The performance test was time to exhaustion cycling at W_72% (237±6 W). Data are mean and error bars represents SEM. N = 8 at all tests. a: p<0.05 compared to CHO+PROT; b: p<0.05 compared to CHO.</p

Concentrations of amino acids in fasted condition the day after exhaustive exercise and diet interventions, and during and after the performance test.

<p>Data are mean and error bars represents SEM. C: p<0.05 compared to CHO; W: p<0.05 compared to CHO+PROT (whey); P: p<0.05 compared to placebo. N = 8 for all data points.</p

Plasma concentrations of lactate, glucose, insulin, FFA, glycerol and glucagon during the first 2 h recovery when provided CHO, CHO+PROT or PLA.

<p>Data are mean and error bars represents SEM. C: p<0.05 compared to CHO; W: p<0.05 compared to CHO+PROT (whey); P: p<0.05 compared to placebo. N = 8 for all data points.</p

Plasma concentrations of uric acid, ornithine, citrulline and 1-methyl-histidine during the first 2 h of recovery when provided CHO, CHO+PROT or PLA.

<p>Data are mean and error bars represents SEM. C: p<0.05 compared to CHO; W: p<0.05 compared to CHO+PROT (whey); P: p<0.05 compared to placebo. N = 8 for all data points.</p

Nitrogen balance and urine excretion of urea and creatinine. a: p<0.05 compared to CHO+PROT; b: p<0.05 compared to CHO.

<p>Data are mean and error bars represents SEM. C: p<0.05 compared to CHO; W: p<0.05 compared to CHO+PROT (whey); P: p<0.05 compared to placebo. N = 8 for all data points.</p