S4031

Liver-specific PPARγ knockout (KO) mice were generated by breeding PPARγ-loxP mice (The Jackson Laboratory, Bar Harbor, ME) to transgenic Alb-Cre (B6.Cg-Tg (Alb-Cre) 21Mgn/J, The Jackson Laboratory) mice as previously described [25 (link)]. Two-month-old male KO or control (i.e., wildtype (WT) C57BL6/J; The Jackson Laboratory) mice were given ad lib access to sterile, irradiated low-fat (S4031, BioServ, Flemington, NJ) or high-fat (S3282, BioServ; 60% kcal from fat) diets, with or without 0.01% (w/w) pioglitazone hydrochloride (Pio, Sigma-Aldrich, St. Louis, MO) for 3 months (n = 4 − 5 mice/experimental group). Body weight was measured weekly and body composition analyzed at the experimental endpoint by ECHO MRI spectroscopy [26 (link)]; after which, mice were euthanized for tissue harvest and analysis. Blood glucose, serum insulin and free fatty acid levels, and liver histology and triglyceride content were determined as previously described [25 (link)–27 (link)]. All experiments were approved by the Washington University Institutional Animal Care and Use Committee (IACUC), and all animals received humane care in accordance with institutional guidelines and criteria in the “Guide for the Care and Use of Laboratory Animals” (8^th edition, 2011, https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf).

Obese wild-type C57BL/6 male mice fed a high-fat diet (HFD) and age- and sex-matched normal diet controls were purchased from the Jackson Laboratory. Foxp3-DTR-GFP reporter mouse founders (C57BL/6 background; first generated in the lab of Dr. Alexander Rudensky [61 (link)]) were also obtained from the Jackson Laboratory and bred in-house. Six-week-old male offspring were fed a high-fat diet (60 kcal% fat, 5.2 kcal/gram; Bio-Serv product # S3282). In all experiments, HFD was administered for at least 14 weeks to induce obesity. Normal weight controls were age- and sex-matched mice fed a conventional chow diet (10 kcal% fat, 3.8 kcal/gram, Bio-Serv product #S4031) in parallel. All mice were housed in a specific pathogen-free facility, and all procedures were approved by the Institutional Animal Care and Use Committee.