Expedata v 1

A standard 12 h light/dark cycle was maintained throughout the indirect calorimetry studies. Mice (10-week-old) were acclimated to the cages for 48 h before data collection. Energy expenditure and food intake were measured using a computer controlled indirect calorimetry system (Promethion, Sable Systems, North Las Vegas, NV, USA). Food and water were provided ad libitum, and data were collected for three days after acclimation. Oxygen consumption (VO₂) and carbon dioxide production (VCO₂) were measured for each mouse at 10-min intervals. Incurrent air reference values were determined after the analysis of the mice in all cages. The respiratory quotient (RQ) was calculated as the ratio of CO₂ production to O₂ consumption. Data acquisition and instrument control were performed using MetaScreen v. 1.6.2 software, and the obtained raw data were processed using ExpeData v. 1.4.3 (Sable Systems) with an analysis script detailing all aspects of data transformation.

The Promethion High-Definition Room Calorimetry System was used for the indirect calorimetry studies (GA3, Sable Systems, Las Vegas, NV). Prior to acquisition eight-12-week-old male mice (4 P2rx7^+/+ and 4 P2rx7^−/−) were acclimated to cages for 48 h. A standard 14 h light- 10 h dark cycle was maintained and animals had access ad libitium to standard chow and water. For the experiments, mice had ad libitium access to standard chow and water. Data acquisition and instrument control were coordinated by MetaScreen v. 1.6.2 and the obtained raw data was processed using Expe-Data v. 1.4.3 (Sable Systems, Las Vegas, NV) using an analysis script detailing all aspects of data transformation. For this study, we focused on food uptake, animal locomotion and energy expenditure (EE). Results for food uptake (gram), locomotion (meter) and EE (kcal/hr) were presented as the mean ± SEM in function of the circadian cycle.

The Promethion High-Definition Room Calorimetry System was used for the indirect calorimetry studies (GA3, Sable Systems. Las Vegas, NV). Data acquisition and instrument control were coordinated by MetaScreen v. 1.6.2 and the obtained raw data was processed using ExpeData v. 1.4.3 (Sable Systems, Las Vegas, NV) using an analysis script detailing all aspects of data transformation. A standard 12 h light/dark cycle (6:00–12:00) was maintained throughout the calorimetry studies. Prior to data collection, all animals were acclimated to cages for 7 days. Mice were subsequently placed in metabolic cages at thermoneutrality. Mice were prior subjected to a 48 h measurement at 30 °C following a switch at 10 °C for 24 h. At the end of the procedure, mice were returned to a 10 °C environment for 10 days before a second round of measurement at 10 °C.

The Promethion High-Definition Room Calorimetry System was used for indirect calorimetry studies (GA3, Sable Systems. Las Vegas, NV). Data acquisition and instrument control were coordinated by MetaScreen v. 1.6.2 and the obtained raw data was processed using ExpeData v. 1.4.3 (Sable Systems, Las Vegas, NV) using an analysis script detailing all aspects of data transformation. A standard 12 h light/dark cycle (6:00–18:00) was maintained throughout the calorimetry studies. Prior to data collection, all animals were acclimated to cages for 3 days followed by 4 days of data acquisition^{26 (link)}. The derived Weir’s equations^{27 (link)} revised by the non-protein assumption^{28 (link)} were used to estimate mouse oxidative rates for carbohydrates (4.585 CO₂–3.226 VO₂ (mg/min/kg body weight)) and fat (1.695 VO₂–1.701 CO₂ (mg/min/kg body weight)).

For indirect calorimetry studies, C57Bl/6 mice were acclimated to metabolic cages after which energy expenditure was measured using a computer-controlled indirect calorimetry system (Promethion, Sable Systems, Las Vegas, NV) as described (Kaiyala et al., 2015 (link); Kaiyala et al., 2012 (link); Kaiyala et al., 2016 (link)) with support from the Nutrition Obesity Research Center Energy Balance Core. For each animal, O₂ consumption and CO₂ production were measured for 1 min at 5 min (acute studies) or 10 min (chronic studies) intervals. RQ was calculated as the ratio of CO₂ production to O₂ consumption. Energy expenditure was calculated from VO₂ and VCO₂ data using the Weir equation (Weir, 1949 (link)). Ambulatory activity was measured continuously with consecutive adjacent infrared beam breaks in the x-, y-, and z-axes were scored as an activity count that was recorded every 5 or 10 min. Data acquisition and instrument control were coordinated by MetaScreen v.1.6.2 and raw data was processed using ExpeData v.1.4.3 (Sable Systems) using an analysis script documenting all aspects of data transformation.

Energy expenditure was measured using a computer-controlled indirect calorimetry system (Promethion^®, Sable Systems, Las Vegas, NV, USA) as described previously [22 (link)]. O₂ consumption and CO₂ production were measured at 10-min intervals. Respiratory quotient (RQ) was calculated as the ratio of CO₂ production to O₂ consumption. Energy expenditure was calculated using the Weir equation [23 (link)]. Ambulatory activity was determined simultaneously with the collection of calorimetry data. Consecutive adjacent infrared beam breaks in the x-, y-, and z-axes were scored as an activity count, and a tally recorded every 10 min. Data acquisition and instrument control were coordinated by MetaScreen v.1.6.2 and raw data was processed using ExpeData v.1.4.3 (Sable Systems) using an analysis script documenting all aspects of data transformation. Mice were evaluated over three consecutive 24-hour periods, with standard alternating 12-hour light and dark cycles.

Energy expenditure was measured using a Promethion M indirect calorimetry system using MetaScreen v. 1.6.2 software (Sable Systems, Las Vegas, NV, U.S.A.). After 48 h of acclimation, energy expenditure was measured for 72 h, and mice had free access to food and water. A flow rate of 2 000 mL·min⁻¹ was used. O₂ consumption and CO₂ production were measured for each mouse at 5-min intervals. The Weir equation (kcal per h = 60*(0.003 941*VO₂ + 0.001 106*VCO₂) was used to calculate energy expenditure, and the results were normalized to lean body mass. Raw data were processed using ExpeData v.1.4.3 (Sable Systems).