Clams system

Body composition was measured using a Bruker MiniSpec NMR. Whole-body VO₂, RER, and activity levels were measured using a CLAMS system (Columbus Instruments). Cold-tolerance testing was carried out in a 4 °C cold room. Prior to cold-tolerance testing, the mice were injected with a temperature-sensitive transponder (Bio Medic Data Systems, IPTT 300). One week after the injections, the mice were transferred to a 4 °C cold room for 6–8 h, and their core temperature was assessed using a Reader-Programmer (Bio Medic Data Systems, DAS 8007). The mice were single-housed in cages containing bedding with access to food and water throughout the cold-tolerance test. For long-term cold-exposure experiments, the mice were acclimated to the cold following a protocol used to acclimate UCP1 null mice to the cold [25 (link)]. Briefly, the mice were housed in a rodent incubator at 18°C for 2 weeks, after which the temperature was lowered to 6.5°C for 7 days. The mice were euthanized after a 4 h fast and their tissues were harvested. For glucose-tolerance testing, their blood glucose levels were measured following an IP injection of 20% glucose (5 μL/g body mass) at time points 0, 15, 30, 60, and 120 min.

Fat and lean mass was determined by echoMRI-100V. CT Scan was performed on Trifoil eXplore RS9 microCT system. Insulin tolerance test was performed on 6 h fasted mice and insulin (Humulin-R, Eli Lilly) was injected at 1.25 U/kg of body weight. Glucose tolerance test was performed on overnight fasted mice, glucose was injected at 1 mg/kg of body weight. Blood from mouse tail was taken and glucose levels were measured using Contour glucometer (Bayer). Metabolic cage experiments to measure physical activity were carried out on CLAMS System (Columbus). Liver TAG content was assessed using Triglyceride Colorimetric kit.

Indirect calorimetry in the CLAMS system (Columbus Instruments) was used to evaluate metabolic parameters and ambulatory locomotor activity during ad libitum, overnight fasted, or time and calorie restricted scheduled feeding (SF). All mice were on a 12:12 light-dark cycle with ad libitum access to food and water unless otherwise indicated. Lepr-cre; tdTomato mice were singly housed and acclimated to the CLAMS for 3 days prior to experiment start. The night before SF began, the SF cohort was fasted and cages were changed and thereafter were given 2–3 grams of food (PicoLab Rodent Diet 5053) at zeitgeber time (ZT) 6 for 10 days. The overnight fasted cohort had ad libitum food access until the night before sacrifice, when food was removed and cages were changed. We repeated this experiment a total of 3 times with 2 mice per condition each time (n = 6 mice / condition total).