Mk 5000rq

Seven to eight days after the infusion of RFRP-3, indirect calorimetry was performed using an O₂/CO₂ metabolism-measuring system for small animals (MK-5000RQ; Muromachi Kikai, Tokyo, Japan). The system monitored VO₂ (mL/min) and VCO₂ (mL/min) at 3-min intervals and calculated the RQ ratio (VCO₂/VO₂). The locomotor activity was simultaneously measured using the SUPERMEX infrared ray passive sensor system (Muromachi Kikai). The measurements were collected hourly or every 15 min over a 24 h period (light period: 09:00 a.m.–21:00 p.m., dark period: 21:00 p.m.–09:00 a.m.) after 24 h of habituation for 7 d after the infusion of RFRP-3. Energy expenditure was calculated using Equation (1) [59 ]: $energy expenditure \left(\raisebox{1ex}{$cal$}\!\left/ \!\raisebox{-1ex}{$\raisebox{1ex}{$kg$}\!\left/ \!\raisebox{-1ex}{$h$}\right.$}\right.\right)=V{O}_{2 }\left(\raisebox{1ex}{$mL$}\!\left/ \!\raisebox{-1ex}{$\raisebox{1ex}{$kg$}\!\left/ \!\raisebox{-1ex}{$h$}\right.$}\right.\right) \times \left\{3.815+\left(1.232 \times RQ\right)\right\}.$

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.

Energy expenditure, VO₂ and VCO₂ were measured using a small-animal metabolic measurement system (MK-5000RQ, Muromachi Kikai, Tokyo, Japan). The respiratory quotient was obtained as the ratio of VCO₂ to VO₂.

Mouse daily activities and levels of O₂ consumption (VO₂, L/min) and CO₂ production (VCO₂, L/min) were measured using an indirect calorimetry system (MK-5000RQ, Muromachi Kikai Co., Ltd., Tokyo, Japan). VO₂ and VCO₂ were used to calculate the EE (kcal/day) using Weir’s equation in which EE = [(VO₂ × 3.941) + (VCO₂ × 1.11)] × 1.44, as previously reported [12 (link)].

After feeding mice the HFD or HJGE-mixed HFD for 4 weeks, they were separated and housed in individual metabolic cages to measure oxygen consumption (VO₂), carbon dioxide output (VCO₂), locomotor activity, and food intake (MK-5000RQ; Muromachi Kikai, Co., Ltd., Tokyo, Japan). The room temperature was maintained at 24°C throughout the experiments and measurements. HFD, HJGE-mixed HFD, and water were provided ad libitum. VO₂ and VCO₂ levels were corrected for the total body weight raised to the 0.75 power (Kon et al., 2019 (link)).

Twelve-week-old male GRf/f and GRmKO mice were analysed. Basal locomotor activity during 24 h was measured using ACTIMO-S food intake, drinking and locomotor activity monitoring system (Shintechno). Body temperature was measured using a rectal probe attached to a digital thermometer (Thermalert TH-5, Physitemp). Oxygen consumption (VO₂), carbon dioxide production (VCO₂) and respiratory exchange ratio (RER) were measured every 5 min over 24 h under resting condition using an O₂/CO₂ metabolism measuring system for small animals (MK-5000RQ, Muromachi Kikai). Energy expenditure was calculated from the gas exchange data [energy expenditure=(3.815+1.232 × RER) × VO₂].