Energy expenditure was evaluated via indirect calorimetry (Oxymax System, Columbus Instruments) over periods of 24 hour. Briefly, energy expenditure was calculated by multiplying oxygen consumption (VO2) by the calorific value (CV = 3.815 + 1.232 × respiratory exchange ratio) and corrected by lean mass. Locomotor activity and food intake were also measured simultaneously. For glucose and insulin tolerance tests, mice were subjected to intraperitoneal bolus injection of glucose (1mg/g of body weight) or insulin (1.5mU/g) after fasting for overnight (14–16 hours) or 6 hours with water ad libitum, respectively. Blood glucose levels were measured at the indicated intervals. For histological analysis, tissues were fixed in 4% paraformaldehyde and embedded in paraffin. 5 μm sections were stained with hematoxylin and eosin (H&E). Irx3 expression in brain and lung was examined using Irx3lacZ knockin mice with beta-galactosidase staining.
