Mms 2

Mice were individually placed in air-tight 15 × 15 × 15 cm plexi glass cages, and oxygen consumption was measured for 24 h by indirect calorimetry with MK-5000RQ and MMS-2 software (Muromachi Kikai). Spontaneous locomotor activity was measured in SUPERMEX apparatus and Compact AMS3 software (Muromachi Kikai). Mice were acclimated to monitoring for 1 h once a day for 3 days before 24-h recording.

Insulin sensitivity was assessed by the ipITT after 4 h of fasting. Blood glucose concentrations were measured before and 15, 30, 60, and 120 min after an intraperitoneal injection of 0.75 U/kg recombinant human insulin (Humulin R; Eli Lilly Japan K.K., Japan). Glucose tolerance was assessed by the OGTT after 6 h of fasting. Blood glucose concentrations were measured before and 15, 30, 60 and 120 min after oral administration of 2.0 g/kg glucose^{29 (link)}. HOMA-IR was calculated as an index for insulin resistance as follows: fasting serum glucose (mmol/L) × fasting serum insulin (pmol/L)/22.5. The serum concentration of insulin was determined using the Mouse Insulin ELISA kit (Mercodia) according to the manufacturers’ instructions. Oxygen consumption was measured with an O₂/CO₂ metabolism measuring system for small animals (MK-5000RQ; Muromachi Kikai, Tokyo, Japan), and the results were analyzed using MMS-2 software (Muromachi Kikai). Each mouse was placed in an airtight chamber maintained at 25 °C with 0.50 L/min of air flow for more than 24 h, and the oxygen consumption was normalized by kilogram^0.75 body weight.