Evaluation of Methionine Content in a High-Fat and Choline-Deficient Diet on Body Weight Gain and the Development of Non-Alcoholic Steatohepatitis in Mice

<div><p>Aim</p><p>Non-alcoholic steatohepatitis (NASH) is a globally recognized liver disease. A methionine- and choline-deficient diet is used to induce NASH in mice; however, this diet also causes severe body weight loss. To resolve this issue, we examined the effects of methionine content in a high-fat and choline-deficient (HFCD) diet on body weight and the development of NASH in mice.</p><p>Methods</p><p>C57BL/6J mice (male, 10 weeks of age) were fed an L-amino acid rodent (control) diet, high-fat (HF) diet, or HFCD diet containing various amounts of methionine (0.1–0.6% (w/w)) for 12 weeks. Plasma lipid levels, hepatic lipid content and inflammatory marker gene expression were measured, and a pathological analysis was conducted to evaluate NASH.</p><p>Results</p><p>The 0.1% methionine in HFCD diet suppressed body weight gain, which was lower than that with control diet. On the other hand, the 0.2% methionine in HFCD diet yielded similar body weight gains as the control diet, while more than 0.4% methionine showed the same body weight gains as the HF diet. Liver weights and hepatic lipid contents were the greatest with 0.1% methionine and decreased in a methionine dose-dependent manner. Pathological analysis, NAFLD activity scores and gene expression levels in the liver revealed that 0.1% and 0.2% methionine for 12 weeks induced NASH, whereas 0.4% and 0.6% methionine attenuated the induction of NASH by HFCD diet. However, the 0.2% methionine in HFCD diet did not induce insulin resistance, despite the body weight gain.</p><p>Conclusions</p><p>The 0.2% methionine in HFCD diet for 12 weeks was able to induce NASH without weight loss.</p></div

NAFLD Activity Score (12 weeks).

<p>NAFLD Activity Score (12 weeks).</p

Pathological analysis.

<p>C57BL/6J mice (male, 10 weeks of age) were fed each experimental diet for 12 weeks. After overnight fasting, mice were killed and the liver was removed. (A) A pathological analysis with HE staining, serial red staining and F4/80 immuno-staining was conducted. Bar indicates 100 μm. Original magnification x200. (B) Fibrosis score was determined as the ratio of sirius red-positive area to the whole area in each section. Data are represented as means and SEM, n = 7 or 8 in each diet. ***<i>P</i> < 0.01 vs. control by one-way ANOVA with Bonferroni’s post-hoc test.</p

IPGTT was conducted in mice fed 0.2% methionine in HFCD diet for 12 weeks.

<p>C57BL/6J mice (male, 10 weeks of age) were fed a control diet or 0.2% methionine in HFCD diet for 12 weeks, and IPGTT was conducted after overnight fasting. (A) After injection of glucose (2 g/kg BW, i.p.), plasma glucose levels were measured at each time point. (B) AUC was calculated in (A). (C) Plasma insulin levels were measured using an ELISA kit. n = 6 in each diet.</p

Body Weight, Tissue Weights and Plasma Biomarkers (12 Weeks).

<p>Body Weight, Tissue Weights and Plasma Biomarkers (12 Weeks).</p

Hepatic lipid contents in each diet.

<p>C57BL/6J mice (male, 10 weeks of age) were fed a control diet, HF diet, or HFCD diet containing 0.1%, 0.2%, 0.4% or 0.6% methionine for 12 weeks. After overnight fasting, mice were killed and the liver was removed. Hepatic lipids were extracted according to Folch’s method, and TC, TG and NEFA levels were measured using enzymatic methods. *<i>P</i> < 0.05, ***<i>P</i> < 0.001 vs. control by a one-way ANOVA with Bonferroni’s post-hoc test.</p

NAFLD Activity Score (8 Weeks).

<p>NAFLD Activity Score (8 Weeks).</p

Data

File includes data for calculating intraclass correlation coefficients and Fleiss' kappa shown in the article entitled "Methods for estimating causal relationships of adverse events with dietary supplements"