C57BL/6JJcl mice (CLEA Japan, Tokyo, Japan) and B6.CB17-Prkdcscid/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-Prkdcscid/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.
For tumor growth assay, 4-week-old C57BL/6JJcl and B6.CB17-Prkdcscid/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.
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