Metformin (metformin hydrochloride) was purchased from Wako Pure Chemical Industries, Ltd., Tokyo, Japan.

Eight-week-old male C57BL/6N mice were purchased from CLEA Japan Inc. Mice were housed for 15 weeks on a 12-h light/dark cycle, and food and water were accessible ad libitum. Mice were fed either a methionine- and choline-deficient (MCD) diet (Oriental Yeast, Tokyo, Japan) or a normal diet. Mice were divided into three experimental groups and fed for 15 weeks. Group 1 was given a methionine- and choline-deficient (MCD) diet (MCD, n=10). Group 2 was fed an MCD diet with 2.4 mg/day metformin (Wako Pure Chemical Industries) (MCD + metformin, 2.4 mg/day, n=10). Group 3 was fed normal chow (NC, n=7). Group 2 was fed with an MCD diet and treated with 2.4 mg/day metformin given in the drinking water. The dose of metformin was calculated at 2.4 mg/mouse/day and corresponds to 4,800 mg/60 kg in a human. Group 3 was fed a standard diet and received untreated drinking water ad libitum. The mice were fed for 15 weeks to recreate the advanced stages of steatohepatitis. After 15 weeks on each diet, the mice were euthanized and the liver and body weight were measured. Livers were fixed in 10% formalin or flash frozen in liquid nitrogen for histological analysis. The samples were stored at −80°C until further analysis. All animal procedures were performed in accordance with the guidelines of the Committee on Experimental Animals of Kagawa University, Kagawa, Japan.

Blood samples were obtained from the right ventricle, and the levels of AST, ALT and ALP were measured by an autoanalyzer (TBA-200FR NEO; Toshiba Medical Systems Corp., Tokyo, Japan).

To determine whether metformin decreased MCD-induced steatosis and fibrosis in the liver, Oil red-O and Azan staining was performed, respectively. In all the experimental groups, 5-μm sections of formalin-fixed and paraffin-embedded liver samples were processed for Azan staining. Oil red-O staining was also performed with all the liver samples to estimate the degree of hepatic steatosis. Areas of the digital photomicrographs were quantified with a computerized image analysis system (ImageJ).

The total RNA of each liver sample was extracted using a miRNeasy Mini Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA was measured using an RNA 6000 Nano kit (Agilent Technologies, Tokyo, Japan). The samples were labeled using a miRCURY Hy3/Hy5 Power Labeling kit and were hybridized on a mouse miRNA Oligo chip (v. 17.0; Toray Industries, Inc., Tokyo, Japan). Scanning was conducted with a 3D-Gene Scanner 3000 (Toray Industries). 3D-Gene extraction version 1.2 software (Toray Industries) was used to read the raw intensity of the image. To determine the change in miRNA expression between the metformin-treated and control samples, the raw data were analyzed via GeneSpring GX v10.0 (Agilent Technologies). The samples were first normalized relative to the 28S RNA level and then baseline-corrected to the median of all the samples.

Replicate data were consolidated into two groups, including those from metformin-treated animals and those from control animals. The data were organized using the hierarchical clustering and analysis of variance (ANOVA) functions in the GeneSpring software. Hierarchical clustering was completed using the clustering function (condition tree) and a Euclidean correlation as a distance metric. A two-way ANOVA and asymptotic P-value computation without any error correction was performed on the samples to search for the miRNAs that varied most prominently across the different groups. The P-value cut-off was set to 0.05. Only changes >50% for at least one of the time-points within each sample were considered significant. All the analyzed data were scaled by global normalization. The statistical significance of differentially expressed miRNAs was analyzed by the Student’s t-test. Our microarray data in the present study were submitted as a complete data set to the NCBI Gene Expression Omnibus (GEO) no.: GSE55593; control vs. MCD (http:ncbi.nlm.nih.gov/=GSE55593) and no.: GSE55523; MCD vs. MCD + metformin (http:ncbi.nlm.nih.gov/=GSE55523).

To show alterations in the expression level of nine miRNAs, we created a heatmap in which each cell represents the expression level of each miRNA for four individual subjects from the MCD-fed mice that were treated with and without metformin. The heatmap was color-coded according to a log 2-transformed expression level. The center level of the color code was set as the median value for all the values used to create the heatmap. Thus, white was considered the mean number; red, an increase and blue, a decrease in the heat map.

Data are shown as the means ± standard deviation (SD). Statistical significance was defined as P<0.05 for the unpaired t-test and as P<0.025 for the Bonferroni corrections.

To determine the effect of metformin on the development of NASH, mice were fed an MCD or normal diet. In MCD-fed mice, the levels of plasma ALT, AST and ALP were higher than those for mice fed a normal diet. Treatment of MCD-fed mice with metformin significantly attenuated this increase (P<0.025; significant differences were confirmed with a Bonferroni correction) (Fig. 1A). In the present study, the liver/body weight ratio was not significantly different between MCD-fed mice treated with and without metformin (Fig. 1B). To determine whether metformin decreased MCD-induced liver steatosis, Oil red-O staining was completed. Consistent with the results from the plasma ALT, AST and ALP levels, MCD-fed mice treated with 2.4 mg metformin showed significantly suppressed development of MCD-induced liver steatosis by 75% (9.91±2.03 vs. 2.51±0.70%, P<0.001) (Fig. 2). These results suggested that metformin suppressed the inflammation and steatosis induced by MCD.

Histological analysis of the liver was performed to determine whether the liver fibrosis induced in MCD-fed mice was inhibited by metformin. Perivenular fibrosis was detected around the central vein in MCD-fed mice. By contrast, metformin treatment significantly downregulated the extent of perivenular fibrosis (Fig. 3).

To elucidate the miRNA profiles during the development of NASH, we analyzed the expression levels of 1,135 mouse miRNA probes using liver tissue from control and MCD-fed mice. As shown in Table IA, 71 miRNAs were significantly upregulated, and 60 miRNAs were downregulated in MCD-fed mice. Unsupervised hierarchical clustering analysis using a Pearson’s correlation showed that MCD-fed mice clustered separately from the control group (Fig. 4). These subsets of 131 miRNAs exhibited significant alterations in their expression levels between the MCD-fed and control mice.

miRNA profiles were examined in MCD-fed mice after metformin treatment. As shown in Table IB, 23 miRNAs were significantly upregulated, and 25 miRNAs were downregulated in MCD-fed mice treated with metformin. Unsupervised hierarchical clustering analysis using a Pearson’s correlation showed that MCD-fed mice without metformin clustered separately from the MCD-fed mice treated with metformin (Fig. 5). These subsets of 48 miRNAs exhibited significant alterations in their expression levels between the MCD-fed mice with or without metformin. Notably, miR-122, miR-194, miRNA-101b, and miRNA-705 were upregulated and miRNA-376a, miRNA-127, miRNA-34a, miRNA-300 and miRNA-342-3p were downregulated in the liver tissue of MCD-fed mice treated with or without metformin (Table IB and Fig. 6). The four upregulated miRNAs, i.e., miR-122, miR-194, miRNA-101b and miRNA-705, in mice treated with or without metformin were consistent with four of the 60 downregulated miRNAs from the control group and MCD-fed mice. The five downregulated miRNAs i.e., miRNA-376a, miRNA-127, miRNA-34a, miRNA-300 and miRNA-342-3p, were identical to five of the 71 upregulated miRNAs in control and MCD-fed mice.