Indirect calorimetry study

Mice (4 months old) were individually housed 14 days before and presented with training bottle and acclimated to the chambers for 48 hours before experimental measurements. For the experiment, they were fed 7 days with ND and then 7 days with HFD. They were analyzed for whole energy expenditure, oxygen consumption and carbon dioxide production, RQ (VCO₂/VO₂), food intake, and locomotor activity (beam breaks per hour) using a calorimetric device (LabMaster, TSE Systems GmbH). Data were recorded every 15 min for each animal during the entire experiment. Data analysis was performed on excel XP using extracted raw value of VO₂ consumed, VCO₂ production (expressed in milliliter per hour), and energy expenditure (kilocalories per hour). Subsequently, each value was normalized to the whole lean body mass extracted from the EchoMRI (Whole Body Composition Analyzers, EchoMRI, Houston, TX, USA) analysis as previously described (45). No practical method of estimation of the basal metabolism is available presently. However, an estimation could be made from windows of time during which animals should have no access to food, their previous intake had been far enough in time to discard the thermic effect of food, and the spontaneous activity had been the lowest possible within the past 30 min. Data points of energy expenditure were considered as the best estimation of basal metabolism when the spontaneous activity was lower, within the last 30 min, than 5% of the highest daily value and when no intake was recorded within the last 60 min. Fatty acid oxidation was calculated from the following equation: fat ox (kcal/hour) = energy expenditure (kcal/hour) × (1-RER/0,3) according to (46).