Metformin

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.

C57BL/6JJcl mice (CLEA Japan, Tokyo, Japan) and B6.CB17-Prkdc^scid/SzJ (SCID) mice (The Jackson Laboratory, Bar Harbor, ME) were maintained with free access to chow and water. The mice were fed standard chow diet (STD) (MF, Oriental Yeast, Japan) or high fat-high sucrose diet (HFS: Protein: 16.4% kcal, Fat: 58% kcal, Carbohydrate: 25.5% kcal, Energy Density: 5.56 kcal/g) (D12331, Research Diets, New Brunswick, NJ).
For tumor growth assay, 4-week-old C57BL/6JJcl and B6.CB17-Prkdc^scid/SzJ male mice were fed with STD or HFS for 6 weeks and injected with 4 × 10⁵ B6 OVA-gene introduced B16 melanoma MO5 cells. Seven days after injection of MO5 cells, they were divided into 4 groups fed with (1) STD with water (STD), (2) STD with 5 mg/dl metformin (WAKO, Tokyo, Japan) water (STD + Met), (3) HFS with water (HFS), and (4) HFS with 5 mg/dl metformin water (HFS + Met) for 4 weeks. Mice that received metformin at 5 mg/ml achieved 1.70 μg/mL, which was consistent with plasma concentrations in patients (0.5–2 μg/mL)^{39 (link)}. Tumor infiltrating lymphocytes (TILs) were isolated at 48 h after the administration of metformin and subjected to cytokine assay.
4-week-old C57BL/6JJcl mice were fed with STD and HFS for 12 weeks and they were divided into STD, STD + Met, HFS, and HFS + Met for 2 weeks. Then, splenocytes were isolated and subjected to cytokine assay and Extracellular Flux Analyzer.

Recombinant human, rat, and mouse GK of liver and pancreas isoforms were produced by Teijin Pharma Limited (Tokyo, Japan). Human recombinant HK I, II, and III were purchased from ATgen (Seongnam-si, Korea). 2-[1,2-³H(N)]Deoxyglucose was obtaind from PerkinElmer (Boston, MA, USA). MIN6 cells [18 (link)] were obtained from Prof. Miyazaki (Graduate School of Medicine/Division of Medicine, Osaka University, Osaka, Japan). TMG-123 was synthesized by Teijin Pharma Limited. Metformin and Glibenclamide were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).