Opt m3

Fat and lean body mass of each mouse were analyzed on an LF90 TD-NMR (Bruker Optics). For measuring metabolic parameters, mice were acclimated in the metabolic chambers (TSE Systems) for 2 days, and food and water intake, energy expenditure, and physical activities were measured for another 2 consecutive days. Constant airflow (0.2 l/min) was drawn through the chamber and the concentrations of oxygen and carbon dioxide were monitored at the inlet and outlet of the sealed chambers to calculate oxygen consumption. Each chamber was measured for 3.75 min at 30 min intervals. Physical activity was monitored by infrared technology (OPT-M3, Columbus Instruments), as the count of 3D beam breaking (X total, Y ambulatory, and Z) was measured.

Food intake, water consumption, feces outtake, and urine production were assessed by a metabolic cage (Tecniplast Metabolics Biological Monitoring, Italy). Briefly, 2-month-old male mice were individually housed in the metabolic cage 12 h light/dark cycle with ad libitum access to rodent chow diet and water. The data were collected by 12-h cycles for one month after a one-week adaption, and the data were normalized to the body weight.
Whole-body oxygen consumption (VO₂), carbon dioxide expiration (VCO₂), RER, and ambulatory activity were assessed by a Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments, USA). Briefly, 2-month-old male mice were acclimated to CLAMS cages individually with food and water ad libitum for 24 h. The data on oxygen consumption (VO₂) and carbon dioxide production (VCO₂) were recorded every 10 min for 3 d at 23°C following adaption. Infrared beams measured the ambulatory activity in a single horizontal plane (X-axis, Y-axis) and a vertical direction (Z-axis) (OPT-M3, Columbus Instruments). The RER was calculated as VCO₂/VO₂ automatically by the software.