Mouse treadmill exercise

Exercise capacity tests were carried out with the Exer 3/6 Rodent treadmill (Columbus Instrument, Columbus, OH). Prior to exercise capacity testing, the mice (12–15-week-old) were acclimated to the treadmill for three 10-min sessions on 3 consecutive days. The treadmill was set to 10° inclination and the speed was set to 0, 5, and 10 m/min for the first, second and third acclimation sessions, respectively. The electric shock grid at the rear end of the treadmill was turned on and set at stimulation intensity of 9 and frequency of 3 Hz. During exercise capacity testing, each mouse was placed into a lane of the treadmill. The genotype of the animals was blinded to the operator. The exercise protocol consisted of the following steps: (1) 10 m/min for 2 min for warm-up, (2) continuous acceleration from 15 m/min at a rate of 0.6 m/min² until the mouse was exhausted. Exhaustion was determined when the mouse stayed on the shock grid continuously for 5 s and was determined by the same observer for all experiments. Glucose and lactate were measured from a drop of blood from the tail tip before and immediately after exercise, using a OneTouch Ultra 2 glucometer (Lifescan, Inc) and a Lactate Plus lactate meter (Nova Biomedical), respectively.