All animal procedures were performed under a project licence (PPL P94B395E0) approved by the U.K. Home Office under the Animals (Scientific Procedures) Act 1986 and the University of Aberdeen ethics review board. Studies were performed following the recommendations in the ARRIVE guidelines under guidance by the Veterinary Surgeon and Animal Care and Welfare Officers of the institutional animal research facility. Thus, all methods were performed in accordance with the relevant guidelines and regulations. Male LDLR−/− mice, aged 4–6 weeks, were purchased from The Jackson Laboratory (supplied by Charles River UK Ltd), male and female ApoE−/− mice were bred in-house (University of Aberdeen). All mice were fed chow diet until 12 weeks of age then placed into three groups and fed the following diets (all Research Diets Inc.) to induce atherogenesis and NAFLD for 14 weeks: control (10% kCal fat D14121001) or high-fat/high-cholesterol diet (HFD, 40% kCal fat from cocoa butter and soybean oil, 34.5% kcal and 5.5% kcal respectively, plus 1.25% cholesterol, Clinton/Cybulsky D12108C) + /- 0.04% Fenretinide (FEN-HFD, D18061502,16 (link),27 (link)–29 (link)). Mice were maintained at 22–24 °C on 12-h light/dark cycle with free access to food/water. At week 14, mice were fasted for 5 h and injected intraperitoneally with either saline or insulin (10 mU/g body weight) for 10 min prior to CO2-induced anaesthesia followed by cervical dislocation. Heart and aortic tissues were collected for histological analysis. Peripheral metabolic tissues (liver, muscle and white adipose tissue (WAT)) were frozen in liquid nitrogen and stored at − 80 °C until subsequent analysis.
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