High fat diet 32

Male B6 mice (14 weeks old) were purchased from the Hebei Medical University, Center for Animal Experimentation (Shijiazhuang, China) and were maintained in the animal facilities of Hebei Medical University with standard animal care procedures based on the institutional guidelines. The mice were fed a normal laboratory diet (22.3% protein, 6.2% fat, 3.0% fiber, 6.5% ash and 47.8% complex carbohydrate) with free access to water, and were housed with a regular 12-h light/dark cycle according to the Hebei Medical University Guidelines for the Care and Use of Laboratory Animals. Following acclimatization for two weeks, B6 mice were fed a high-fat chow (High-Fat Diet 32; CLEA Japan, Inc., Tokyo, Japan) for four weeks, then the mice were divided into two groups (n=7/group); those fed the high-fat chow for four weeks (HF group), and those fed the high-fat chow with 0.0064% wt/wt ezetimibe (5 mg/kg/day) for four weeks (HF+EZ group). After 16-h fasting, all mice were sacrificed under anaesthesia by intraperitoneal administration of pentobarbital (60 mg/kg body weight; Nembutal; Dainippon Sumimoto Pharma Co., Ltd., Osaka Japan) and medetomidine (0.3 mg/kg body weight; Domitor; Meiji Seika Kaisha, Ltd., Tokyo, Japan). The ezetimibe was provided by Merck Sharp & Dohme (Whitehouse Station, NJ, USA).

Hif1α-floxed (Hif1αF/F) mice containing loxP sites flanking exons 13–15 of the Hif1α gene [29] (link) were crossed with mice harboring the Cre recombinase under the control of the aP2 promoter (aP2-Cre mice; a gift from Ronald M. Evans, Salk Institute for Biological Studies, CA) and under the control of the lysozyme M promoter (LysM-Cre mice; a gift from Irmgard Förster, Institut für Umweltmedizinische Forschung, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany [30] (link)), generating adipocyte-specific HIF-1α knockout (ahKO) mice and macrophage-specific HIF-1α knockout (mhKO) mice, respectively. All mice were on the C57BL/6 background and only male mice were used for experiments. The mice were maintained under temperature- and light-controlled environmental settings with free access to water and dissection was performed in the morning. Six-week-old mice were fed either a normal diet (ND) (10% kcal consisting of fat; MF (Oriental Yeast)) or a high fat diet (HFD) (57% kcal consisting of fat; high fat diet 32 (CLEA Japan)) for another 20 weeks.

All procedures were carried out in accordance with relevant guidelines and regulations as approved by the Animal Care Committees of The University of Tokyo and the Institute of Medical Science, Asahi Life Foundation, and complied with the standards stated in the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health, revised 2011). The study is reported in accordance with ARRIVE guidelines. Male mice were 6–55 weeks of age at the time of the study. They were housed in cages and maintained on a 12 h light–dark cycle with access to chow and water ad libitum. In these experiments, we used normal chow diet consisting of 24.9% (wt/wt) proteins, 4.6% fibers, 7.1% ashes, 49.5% carbohydrates, 4.8% fat and 9.1% water (CE-2, CLEA Japan Inc.) or high-fat diet consisting of 25.5% (wt/wt) protein, 2.9% fibers, 4.0% ashes, 29.4% carbohydrates, 32.0% fat and 6.2% water (High Fat Diet 32, CLEA Japan Inc.). High-fat diet was fed to male mice from 6 weeks old onwards. C57Bl/6 mice were purchased from Charles River Laboratories Japan, Inc. and Japan SLC, Inc. Akita mice were purchased from Japan SLC, Inc.