Endurance Treadmill Test

To test exercise endurance capacity, mice of both sexes from selected crosses were run on a progressive treadmill running protocol. The mice were acclimatized to the motorized treadmill (Animal Treadmill Exer-3/6, Columbus Instruments, Columbus, OH, USA) for two days prior to the endurance test. Day 1 of acclimatization lasted 20 min consisting of 5 min on the treadmill at rest (0 m/min), 5 min at 8 m/min, 5 min at 10 m/min, followed by 5 min of rest (0 m/min). Day 2 also lasted 20 min and consisted of 5 min on the treadmill at rest (0 m/min), 5 min at 10 m/min, 5 min at 12 m/min, followed by 5 min of rest (0 m/min). On day 3, mice performed the endurance test to exhaustion, defined as when the mouse spent more than 5 sec on the shock grid (0.1 mA x163 V and 1 Hz) without re-engaging with the treadmill, as described previously⁵³. The mice ran at a speed of 10 m/min for the first 40 min, then the rate was increased at 1 m/min every 10 min until the speed reached 13 m/min, at which point the speed was increased at a rate of 1 m/min every 5 min until exhaustion. Mice were then removed from the treadmill and blood glucose and lactate were immediately measured from tail blood using hand-held analyzers for glucose (Bayer Contour Blood Glucose Meter, Ascensia Diabetes Care US, Inc., Parsippany, NJ, USA) and lactate (Nova Biomedical Lactate Plus Meter, Waltham, MA, USA). Mice were euthanized by decapitation, and muscles were rapidly dissected and frozen on dry ice. All tissues were stored at −80°C until used.