Body weight, plasma and hepatic biochemical levels.

<p>(A) Body weight of the three experimental groups. Eight-week-old male STAM mice were divided into three experimental groups and fed for 4 weeks as follows: 1) high-fat diet (HFD) (control group); 2) HFD mixed with 0.28% L-carnitine (L-carnitine group); and 3) HFD mixed with 0.01% α-tocopherol (α-tocopherol group). After 4 weeks, mice were weighed. (B) Fasting blood glucose levels of the three experimental groups. (C) Plasma biochemical findings of experimental groups. (D) Hepatic triglyceride levels of experimental groups. Data are expressed as mean ± standard deviation (SD).</p

Quantitative real-time PCR results and Western blotting results for expression of hepatic mitochondrial pathway-related genes.

<p>(A) mRNA levels of L-carnitine transport-related gene OCTN-2 and long chain fatty acid transport-related genes Cpt1a and Cpt2 were analyzed. (B) mRNA levels of mitochondrial β-oxidation-related gene MCAD were measured. (C) mRNA levels of antioxidant system-related genes Sod2, CAT, and Gpx1 were analyzed. Data are expressed as means ± SD. (D) Western blotting was performed with the following antibodies directed to Cpt1a, Cpt2, Sod2, CAT and Gpx4. β-actin was used as a loading control. *P<0.05. OCTN, organic cation/carnitine transporter; Cpt, carnitine palmitoyltransferase; MCAD, medium chain acyl CoA dehydrogenase; Sod, superoxide dismutase; CAT, catalase; Gpx, glutathione peroxidase.</p

Intestinal microbiome analysis.

<p>(A) The 100% stacked column chart of microbiome from intestinal feces. (B) Representative percentages of bacterial species in experimental groups. *P<0.05.</p

Liver histological findings.

<p>(A) Representative H&E-stained liver sections are shown. (B) Non-alcoholic fatty liver disease activity scores (NAS) for mouse liver specimens of the three experimental groups. Steatosis, inflammation, and hepatocyte ballooning were categorized, and the sum of these scores was designated as NAS. Data are expressed as means ± SD. *P<0.05.</p

Iron metabolism related pathway analysis.

<p>(A) Representative immunohistochemical staining for ferritin of STAM mouse liver tissue. Intensity of ferritin was calculated by computerized image analysis using Olympus cellSens imaging software. (B) mRNA levels of iron uptake-related hepcidin coding gene Hamp and iron transport-related gene DMT-1 were analyzed. Data are expressed as means ± SD. (C) Liver extracted proteins were analyzed with Western blotting with antibodies directed to DMT-1 and Hamp (hepcidine). *P<0.05. DMT, divalent metal transporter; Hamp, hepcidin coding gene.</p

Assessment of oxidative stress and inflammation in the liver.

<p>(A) Concentrations of 8-OHdG in the liver. (B) Representative immunohistochemical staining for 4-HNE in STAM mouse liver tissue. The intensity of 4-HNE was calculated by computerized image analysis with Olympus cellSens imaging software. (C) Results of quantitative real-time PCR assay to detect TNF-α and IL-1β mRNA levels are shown. (D) Western blotting analysis of the hepatic extracts was performed with each antibodies. Data are expressed as means ± SD. *P<0.05. 8-OHdG, 8-hydroxy-deoxyguanosine; 4-HNE, 4-hydroxynonenal; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin-1β.</p

Metabolomic analysis (L-carnitine/Control, α-tocopherol/Control, L-carnitine/α-tocopherol).

¶<p>; ratio of left divided by right.</p>||<p>; p-value: ***p<0.001, **p<0.01, *p<0.05.</p

Effects of drugs on preventing hepatocarcinogenesis.

<p>(A) Control group developed numerous tumors on the liver surface, while groups receiving drug treatments developed fewer tumors. (B) Histological findings showed that the tumors are hepatocellular carcinoma. (C) Number of tumors was significantly lower in the L-carnitine group. In the α-tocopherol group, the average tumor number and size were not reduced. Data are expressed as means ± SD. *P<0.05.</p