Experimental setup

Eight‐ to nine‐week‐old LKO and Lox/Lox littermate mice were randomly divided into two groups, receiving either a high‐fat high‐fructose (HFF) diet (D09100304: 20% protein, 40% carbohydrates and 40% fat) where 50% of the calories were derived from fructose (Research Diets, Inc., New Brunswick, NJ) or a matched control diet (CON) (D09100304: 20% protein, 70% carbohydrates and 10% fat) ad libitum (Research Diets, Inc.). Fructose was used to induce more severe liver steatosis as previously reported (Trevaskis et al. 2012). Body weight and food intake were registered weekly throughout the intervention period. In addition, all mice were MR scanned (Echo MRI, Echo Medical Systems) at intervention start, 8 weeks into the intervention and 2 days prior to euthanization.

After receiving the respective diet for 9 weeks, the HFF group was further divided into a HFF untrained (HFF UT) and a HFF exercise trained (HFF ExT) group. The HFF ExT group was adapted to the treadmill (TSE Systems GmbH, Bad Homburg, Germany) for 10 min 2 times per day for 4 days, before starting the exercise training protocol, consisting of 1 h of treadmill running (TSE Systems GmbH, Bad Homburg, Germany) at 15 m/min with 10^o incline, once a day, 6 days/week for 5 weeks.

By the end of the 14th week, the CON UT, HFF UT and HFF ExT groups were each divided into sedentary (CON UT, HFF UT, HFF ExT) and acute exercise groups (CON Ex, HFF UT Ex, HFF ExT Ex) resulting in 6 groups in total. The acute exercise bout consisted of 1 h of treadmill running (TSE Systems GmbH, Bad Homburg, Germany) at 15 m/min with 10^o incline and the mice were euthanized 2 h after the acute exercise running bout, together with the sedentary mice. The 2 h time point was chosen based on the previous findings that UPR markers were regulated at this time point (Kristensen et al. 2018). Exercise trained mice performed the last exercise bout 24 h prior to euthanization to prevent acute effects of exercise. All mice were euthanized by cervical dislocation at ~12 am–2 pm, trunk blood was quickly collected, and liver was removed and snap‐frozen in liquid nitrogen. Plasma was obtained by centrifugation of the blood at 2600 g and 4°C for 15 min. Liver and plasma samples were stored at −80°C until further analyses. To ensure homogeneity of the liver tissue, liver samples were crushed in liquid nitrogen before analyses.