Oxylet pro

O₂ consumption, CO₂ production and energy expenditure were measured by using the OxyletPro indirect calorimetry system (Panlab, Barcelona, Spain). We defined the value immediately before the delivery of caffeine or control as basal value. The parameters were normalized by lean body mass (lbm), which was measured by using a Minispec LF50 body composition analyzer (Bruker, Rheinstetten, Germany). For wheel-running locomotor activity, mice were housed in cages with steel wheels (diameter=11.5 cm), and were accustomed to the cages and running wheels for 3 days. Reagents were administered at 11:00 AM and the revolution number was recorded by a digital counter connected to a magnet sensor.

To measure the basal oxygen consumption (VO₂), carbon dioxide production (VCO₂), and respiratory exchange rate (RER) in mice, a gas analyzer (LE 405 Gas Analyzer; Panlab, Cornellà de Llobregat, Spain) and Metabolism software (Panlab) were used. The mice were placed in metabolic cages (Oxylet Pro; Panlab) for 24 h, and VO₂ and energy expenditure were measured after 25 h. The mice were maintained under a 12 h light/dark cycle with lights on at 6 AM and off at 6 PM and were allowed free access to food and water.

The metabolic performance was analysed individually in metabolic cages by indirect calorimetry with an oxyletpro System (Panlab Harvard Apparatus, Barcelona, Spain) according to the manufacturer’s instructions and data analysis were performed with Metabolism V 3.0 software (Panlab, Harvard Apparatus, Barcelona, Spain) (oxyletpro-system-physiocage-1.html">https://www.harvardapparatus.com/oxyletpro-system-physiocage-1.html). For the analysis, 2 rats under AL, HCT, and NCT were used and metabolic parameters were measured every 20 min for 3 days following 2 days of habituation. Photoperiod and feeding conditions were kept the same as in the home cages. As a marker of nutritional status, total protein was measured in serum by the Bradford method. Lactate dehydrogenase activity was determined by an enzyme-coupled reaction to the reduction of NAD⁺ to NADH using the conversion of lactate to pyruvate measured at λ = 340 nm.

Exercise tolerance in mice (Wt and G93A*SOD1) were assessed by a graded maximal exercise test, as described previously [52 (link),53 (link)]. Briefly, mice were acclimated to the treadmill (OxyletPro, Panlab; Harvard Apparatus, Holliston, MA, USA) in three training sessions and allowed to rest for one week before the experiment. For acclimation, mice were placed in the motionless treadmill, followed by activation of the shock grid (1.5 mA). Subsequently, the treadmill was set up to a walking speed of 6 m/minute (m/min) for 5 min and progressively increased the speed to 12 m/min for 12 min. After a week of rest following acclimation, mice were placed on the treadmill at 0⁰ incline and activated the shock grid. The mice were allowed to run on the treadmill with progressively increasing speeds and inclinations until exhaustion [52 (link),53 (link)]. Mice spending more than 5 s on the shock grid or getting more than 10 shocks indicate exhaustion time. The data were analyzed using Metabolism version 3.0 software (Harvard Apparatus).