Animal experiments

This study was approved by the institutional review board at Keio University. All experiments were performed in accordance with the Keio University animal experimentation guidelines. All animal studies were carried out in compliance with the ARRIVE guidelines. Five-week-old male C57BL/6J mice (Japan SLC, Inc., Shizuoka, Japan) were acclimated for 1 week. At 6 weeks of age, their diets were changed to an open source control or HF diet with or without iron supplementation. Body weight and food intake were measured weekly. The open source control diet (D12450B) contained 10 kcal% fat, 20 kcal% protein, and 70 kcal% carbohydrates, whereas the HF diet (D12492) contained 60 kcal% fat, 20 kcal% protein, and 20 kcal% carbohydrates (Research Diets, Inc., New Brunswick, NJ, USA). Both the open source control diet and HF diet originally contained iron (see Supplementary Table ^S3 online). Iron supplemented mice were fed with 0.023% (w/w) SFC (Komatsuya, Osaka, Japan) simultaneously as the HF diet. These food compositions are shown in the Supplementary Tables ^S2 and ^S3 online. Mice always had free access to food and water. After 7 weeks of each diet, body temperature of the rectum was measured using a thermo-register for mice (AD-1687) (A&D, Tokyo, Japan). After 8 weeks of each diet, glucose parameters were measured. After 15 weeks of each diet, mice were fasted for 4 h and anesthetized with isoflurane, and tissues were collected. Each tissue was weighed and then rapidly frozen in liquid nitrogen. It was then used for RNA isolation, lipid, iron, and ATP measurements, and histological analysis. The facility where all the mice were housed had a 12-h light (from 8 a.m. to 8 p.m.)-dark (from 8 p.m. to 8 a.m.) cycle and temperature control at 23–25 ℃.