Energy expenditure by indirect calorimetry

After 7 weeks of the assigned diet, the rats were fasted for 6 h before the beginning of the dark phase and energy expenditure was measured. Energy expenditure was assessed by indirect calorimetry measuring average oxygen consumption (VO₂) and average carbon dioxide production (VCO₂): a rat was placed in the metabolic chambers (airflow = 800 ml/min) with a computer-controlled O₂ and CO₂ measurement system (Biopac Systems Inc., Goleta, CA) for 30 min. The respiratory quotient (RQ) and resting energy expenditure were calculated using the equations described by Niwa et al. [19]. After the experiment, data were averaged over 1 min intervals and VO₂ and VCO₂ values were corrected for metabolic body size (kg^0.75) [20]. The amounts of carbohydrate and fat oxidation were calculated from non-protein oxygen consumption as were their relative oxidative proportions and the amount of oxygen consumed per gram of substrate oxidized [20].