Immediately prior to study start, rats were block-randomized into four diet groups based on body weight (n = 10/group). Briefly, rats were sorted in order of ascending weight into blocks, and within each block, rats were then randomly assigned to receive either a control diet (Control), a high-fat diet (HFD), a high-fructose diet (HFr) or a high-fat/high-fructose/high-cholesterol-diet (NASH), (Research Diets, NJ, USA) for 16 weeks. The length of the study period was chosen to enhance the probability that NASH with concurrent fibrosis would be established, as has previously been described in Sprague–Dawley rats fed a high-fat diet . The exact nutritional composition of each diet is shown in Table 1. All diets were stored at − 20 °C throughout the study until use, and feed-remains were exchanged with fresh feed twice weekly to prevent purification. To obtain baseline values for all parameters assessed at the 16 week time point, animals were qCT-scanned, quantitative magnetic resonance (qMR)-scanned and blood sampled at study start. After 16 weeks, animals were euthanized by exsanguination under isoflurane anesthesia by incision of the abdominal aorta. Immediately following exsanguination, the liver, a 5 cm section of the proximal jejunum and the two epididymal fat depots were excised, weighed and freeze clamped for further analysis. Food intake and body weight was measured twice weekly throughout the study. Food intake was recorded by calculating the difference between the amount of administered food and remaining food in the cage. These data were used to calculate the accumulated energy intake after the 16 weeks. qCT- and qMR-scans was performed on a bi-monthly basis, and blood samples were collected at baseline and at study termination.Table 1Nutritional composition of dietsNutrient compositionControlNASHHFDHFrFat (%)10406010Carbohydrates (total) (%)70402070 Fructose (%)020060 Sucrose (%)01071 Corn starch (%)55009 Maltodextrin 10 (%)1510130Protein (%)20202020Cholesterol (%)–2––Metabolizable energy (kcal/g)3.854.495.243.85NASH NASH-diet, HFD high-fat diet, HFr high-fructose diet