Figure 1: Study Design.

C57Bl/6J mice consumed either a low-fat diet (LFD) or high-fat diet (HFD) for 12 weeks. The percentage of total kilocalories from fat/carbohydrate/protein for LFD was 10/70/20 and for HFD was 60/20/20. At week 4 of the diet intervention, mice either remained sedentary (SED) or began graded aerobic exercise training (EX-Tr). Aerobic exercise training was performed on a motorized treadmill for 50 minutes per day, 5 days per week. Mice were acclimated to the motorized treadmill (Panlab, Harvard Apparatus, Holliston, MA, USA) for one week with low speed (0-12m/min) for 15 minutes. Exercise then gradually progressed in intensity and duration from 10m/min at 0% incline for 30 minutes to 17m/min for 50 minutes at 10% incline which comprised the last 12 days of exercise training. A graded exercise test was performed after acclimation and at the end of the training period to assess exercise capacity. The belt speed started at 6m/min and 0% incline for 5 minutes then increased by 3m/min and 5% incline every 2 minutes until 18m/min and 15% incline was reached. Speed was then increased by 1-2 m/min every minute until exhaustion. Mice were removed from the treadmill when they refused to run despite sitting on the shock grid continuously for 5 seconds. In-cage metabolic assessment (Promethion, Sable Systems Int.) was performed at week 10 along with body composition measures using dual-energy x-ray absorptiometry (GE Lunar, PIXImus2). Following an acclimation period of 24 hours, in-cage substrate oxidation during fed and fasted conditions were assessed in 12-hour cycles and following a 3-5 hour fast, respectively, using indirect calorimetry (see Fat Oxidation, below, for calculations). At week 12, mice were anesthetized by sodium pentobarbital overdose following a 4 hour fast, 36 hours after their last bout of exercise or remaining sedentary. Tissues were collected, flash frozen in liquid nitrogen, and stored at −80°C until further analysis (n=10 mice/condition).