Promethion metabolic cage system

The fat mass and lean mass of the mice were analyzed once a month through NMR (Niumag QMR23-060H-I, Suzhou, China) following the instruction of the manufacturer. At 16–17 weeks of age, indirect calorimetry measurements were performed using the Promethion Metabolic Cage System (Sable Systems International, Las Vegas, NV, United States). Mice were acclimatized for 24 h in the Promethion system before the measurement was started. Instrument control and data acquisition were performed according to the instructions of the manufacturer. Raw data were processed using ExeData software (Sable Systems).

After FMT, mice were transferred to MIGAL-Galilee Research Institute, and their metabolic phenotype including body weight gain, body composition, total 24-h food (energy) intake, total 24-h energy expenditure, and respiratory quotient was measured over the following 10 days [17 (link)]. Indirect calorimetry measurements were performed using an 8-cage multi-plex system equipped with food hoppers connected to load cells for food intake monitoring (Promethion Metabolic Cage System, Sable Systems, Las Vegas). Mice were placed with bedding in the calorimetry unit for 10 days, and measurements were recorded after a 2-day habituation [17 (link)]. Raw data were collected for each mouse for 0.5 min periods, at 5-min intervals and processed by ExpeData v.1.8.2 (Sable Systems), including all steps of data correction and transformation. The system sampled CO₂, O₂, and H₂O levels in the cage air, and food consumption was monitored by weight of the food dispenser. Respiratory quotient (RQ) was calculated as the volume of carbon dioxide released, and the volume of oxygen absorbed during respiration.

Fat and lean mass were measured using the EchoMRI-900 Body Composition Analyser (EchoMRI Corporation Pte Ltd, Singapore) in accordance with the manufacturer’s instructions. Whole-body energy expenditure, the respiratory exchange ratio, food intake, and locomotor activity were assessed using a 16 chamber Promethion Metabolic Cage System (Sable Systems International). Studies were commenced after 8 h of acclimation to the metabolic chamber, and metabolic parameters assessed at 30 min intervals for 48 h.

Fat mass and lean mass were measured in isoflurane-anesthetized mice using a Lunar PIXImus II DEXA Densitometer. For metabolic determinations, we utilized a Promethion metabolic cage system (Sable Systems). A standard 12-h light/dark cycle was maintained throughout the calorimetry studies and all animals had ad libitum access to standard rodent chow and water throughout the study. Prior to data collection, animals were acclimated to the cages for 24 h. The calorimetry system consisted of 16 metabolic cages (identical to home cages with bedding) equipped with water bottles, food hoppers, and home shelters connected to load cells for food and water intake as well as body weight monitoring throughout the study. All cages contained running wheels (4.5″ (11.5 cm) diameter, MiniMitter) wired to record revolutions/second continuously using a magnetic reed switch and standard XYZ beambreak assembly to monitor cage activity. Data were acquired with Metascreen v2.3.15.11 and analyzed using Expedata v1.9.27 (Sable Systems) as previously described (DeMambro et al., 2015 (link)). ANCOVA analysis between body composition and energy expenditure parameters was performed. No significant effects of body composition were found between energy expenditure and any of the variables. Thus, energy expenditure is reported in kcal/h.

Indirect calorimetry assessments were performed using the Promethion metabolic cage system (Sable Systems, NV, USA). Mice were single-housed in Promethion cages for 72–96 h with ad libitum access to food and water. Monitoring was performed for 48 h following an initial 24-h acclimatization period. RQ was calculated as the ratio of VCO₂/VO₂.