A06071314

Male C57BL/6J mice were obtained from Shanghai SLAC Laboratory and carefully housed under pathogen-free conditions. Mice were fed with a NASH diet containing 40% fat (of which 18% was trans-fat), 22% fructose, and 2% cholesterol (D09100301, Research Diets Inc., New Brunswick, NJ, USA) or a normal diet for 16 or 20 weeks. Mice were fed with a choline-deficient, l-amino acid-defined, high-fat diet with a 0.1% methionine (CDAHFD) diet (A06071302, Research Diets Inc.) or a control diet (A06071314, Research Diets Inc.) for 6 weeks. All animal-related experimental procedures were performed in accordance with the National Institutes of Health guidelines and approved by the Laboratory Animal Ethical Committee of Fudan University. After being fed separately, the mice were sacrificed for analysis. Just before sacrifice, blood was drawn directly from the heart, and the serum was separated and stored at −80°C. Liver tissues were rapidly harvested and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) or snap-frozen in liquid nitrogen and stored at −80°C for later assessment.

Female HNKO and WT mice (10–16 weeks of age) were randomized into four groups based on lean mass. Mice were either fed an L-amino-acid defined, low MCD (A06071302; Research Diets) (39 (link)) or a matched, purified, L-amino acid defined low-fat PD (A06071314; Research Diets) for 3 weeks. Body composition was determined before and after the diet intervention using an NMR scanner (EchoMRI 4-1; EchoMRI). Before sacrifice, mice were fasted for 4 h from ZT = 3. Mice were sedated with pentobarbital as previously described (16 (link)) and killed by cardiac puncture with a heparin-coated needle. Blood was transferred to tubes with EDTA and was centrifuged at 6000g at 4 °C for 6 min. Plasma was transferred to a new tube and stored at −80 °C. Part of the hepatic median lobe was fixed in 4% paraformaldehyde (PFA) for histology. The remaining liver was snap frozen and stored at −80 °C. The frozen liver was crushed in liquid nitrogen before further analysis.

Three-week-old male C57BL/6J mice were purchased from CLEA Japan Inc. and acclimated for 3 weeks prior to be using in experiments. After acclimation, six-week-old mice were divided into two groups: SD group fed with standard diet (SD; A06071314, Research Diets Inc.) and CDAHFD group fed with choline-deficient, L-amino acid-defined, high-fat diet with 0.1% methionine (CDAHFD; A06071302, Research Diets Inc.). The mice were housed on 12-h light/dark cycle and had free access to food and tap water. All animal experiments in this study were conducted after obtaining approval from the Institutional Animal Care and Use Committee of the National University Corporation at Hokkaido University in accordance with Hokkaido University Regulations of Animal Experimentation. This study was also carried out in compliance with the ARRIVE guidelines.