Fig. 8. Study design.

Normal-weight and DIO mice followed same study procedure. At day -9, mice were weighted and MR-scanned and allocated into groups matched on weight and body composition. At day -7 mice were transferred into a closed temperature controlled indirect calometry system from SABLE Systems International (NV, USA). Mice were single housed with bedding but without hide and nesting material. The temperature was either set to 22, 25, 27.5, or 30 °C. After a week of acclimatization (day -7 to day 0, no handling of animals), data was collected for four consecutive days (day 0–4, data presented in Figs. 1, 2, 5). Hereafter, the mice housed at 25, 27.5, and 30 °C was left at unaltered conditions until day 17. Meanwhile, the temperature in the 22 °C group was increased in 2 °C intervals every other day, adjusting temperature at start of light cycle (0600 h) (data are presented in Fig. 1). At day 15, temperature was lowered to 22 °C and data was collected for two days to provide a baseline data for following procedures. At day 17, a hide was added to all mice, and nesting material was added at day 20 (Fig. 5). At day 23, mice were weighted and MR-scanned and were thereafter left undisturbed for 24 h. At day 24, mice were fasted from start of light cycle (0600 h) and were subjected to an OGTT (2 g/kg) at 1200 h (6–7 h fasted). Mice were thereafter returned to their respective housing conditions in the SABLE system and were euthanized the following day (day 25).