Calo cages

Mouse body composition, energy expenditure, activity, and food intake were measured at the Animal Metabolism Phenotyping Core at UNC under the supervision of Dr Kunjie Hua. Body composition was determined by MRI at 11 weeks of age (EchoMRI, Houston, TX) [60 (link)]. Energy expenditure, activity, and food intake were measured at 13–14 weeks of age using CaloCages (PhenoMaster, TSE systems, Chesterfield, MO). Data were collected for 2 days; the first day was considered the acclimation period and was excluded from data analysis [61 (link)]. Mice had unlimited access to food and water for the entire duration in metabolic chambers.

VO₂ and VCO₂ was measured in open-circuit indirect calorimetry cages as described previously [21] (link). In short, the mice were housed in CaloCages (Phenomaster, TSE Systems), equipped with infrared light-beam frames (ActiMot2). VO₂ and VCO₂ was measured for each cage, i.e., each mouse, for 1.9 min once every 30 min, while light-beam breaks were measured continuously. Measurements were performed for a total of 72 h while all groups were fed the LF diet and consecutively for 72 h on the respective HF diets. For each 72 h period of measurements the first 24 h were regarded as an adaptation period and only the subsequent 48 h were used for analyses; Based on two consecutive light (06.00–17.30 h) and dark (18.00–05.30 h) phases respiratory exchange ratio (RER) was calculated from VO₂ and VCO₂ and spontaneous locomotor activity was defined as total counts of light-beam breaks. Energy expenditure (EE) was calculated as follows; 16.3 kJ/L × L VO₂ +4.6 kJ/L × L VCO₂[37] (link).

After treatment week, rats of all the groups were individually housed in Calo-cages of a TSE PhenoMaster system (TSE, Germany) at 25°C. The animals were left undisturbed for 24 hours before making the calorimetry measurements to allow for acclimatization to the individual housing conditions and to minimize the so-called novelty effect [21 (link)]. After gas calibration and calibration of the food sensors, the experimental protocol was executed for three days. The volumes of respiratory gases oxygen (VO₂) and carbon dioxide (VCO₂) were measured based on open-circuit indirect calorimetry. In addition, the RQ and EE per hour and per kg of body wt (Kcal/h/kg) were measured. FI was automatically recorded using a calibrated sensor at a sensitivity of 0.01 g. The measurements were taken and recorded every 15 min and those of the first six hours were omitted to ensure the stability of the recording process. Data used for the analyses included VO₂, VCO₂, RQ, TEE, FI, and body wt [22 (link)].