Phenomaster

After 4 weeks of CR, the mice were placed in a metabolic cage (TSE PhenoMaster, Germany) for 24 h to assess heat production and oxygen consumption.

At W13, rats were housed in the metabolic chamber individually. The whole-body metabolic phenotyping of rats was recorded through an indirect calorimetry and locomotor activity monitoring system (TSE Phenomaster, TSE, Germany), including metabolic rates (oxygen consumption (VO₂), carbon dioxide production (VCO₂)) and heat production [29 (link)]. We placed rats in metabolic chambers for 72 h for acclimation and then collected data from them every 15 min for an additional 24 h. The parameter values obtained during the final 24 h were used for statistics, and then the ratio of VCO₂ versus VO₂ was calculated as the respiratory exchange ratio (RER). Three rats were selected randomly from each group for detection. Water and food were freely available to all rats in each metabolic chamber.

Mice were acclimatized to indirect calorimetry cages (PhenoMaster, TSE, Germany) for 3 days, before expired carbon dioxide (VCO2) and inhaled oxygen (VO2) were measured over another 3 days to determine respiratory exchange ratio (RER). Total activity was measured with laser beam breaks and in a subset of mice food intake was measured.

At the age of 6 weeks, this cohort of animals was divided into two groups and fed HFD + Met or HFD (n = 16 each) for 12 weeks. At the age of 18 weeks, mice were kept for 48 h (starting at 10 am) in an open circuit measurement system (PhenoMaster, TSE Systems GmbH, Bad Homburg, Germany) after having been acquainted to the new environment. Light phase was from 7:30 PM until 6:30 AM. The following parameters were obtained: CO₂ production, O₂ consumption, home cage activity, food intake, water intake and feeding events, and values for energy expenditure (EE) and respiratory exchange ratio (RER; CO₂ production/O₂ consumption) were derived from these measurements.

Eight-week old C57BL6/J healthy mice (n = 60, 22 ± 2 g) were randomly divided in two groups (n = 30 each) and fed for 6 weeks, either with a standard diet (SD) (A04, SAFE Augy, France, 2900 kcal/kg), or a high-saturated-fat (60%) diet HFD (260HF, SAFE Augy, France, 5505 kcal/kg). After 6 weeks of diet diversification, mice (n = 20 HFD, n = 20 SD) fed ad libitum, or 16 h fasting, were subjected to noninvasive MRI acquisitions (n = 10 HFD, n = 10 SD), or phenotyping evaluation (n = 10 HFD, n = 10 SD) in an automatically monitored cage system (Phenomaster, TSE Systems GmbH, Germany) (Supplementary Fig. 1).
Animals were euthanized with a high-power microwave fixation system focused in the brain (TMW-4012C 5 kW, Muromachi Kikai Co. Ltd., Japan). Fixed brains were then isolated from the skull, the hypothalamus (Hyp), hippocampus (Hipc), and temporal cortex (Ctx) dissected, and the samples kept at −80 °C for subsequent HRMAS acquisitions. Design and reporting adhered to the ARRIVE guidelines [29 (link)].

Indirect calorimetry analyses were carried out using a 16-chamber TSE PhenoMaster monitoring system (TSE Systems GmbH, Bad Homburg, Germany). Alb-R26^Met and R26^stopMet mice were placed in acclimatization cages similar to experimental cages, with one mouse per cage. Acclimation starts the Friday before the experiment, with the experiment running for 5 consecutive days. Mice were on a 12 h light–dark cycle, and room temperature was maintained at 22 ± 2 °C. Food and water were provided ad libitum in appropriated devices that allow to measure cumulative food intake and drink consumption. The determination of different parameters was carried out over a period of 84 h. The respiratory exchange ratio (RER) was estimated by calculating the ratio of VCO₂/VO₂. Energy expenditure (H(1)) was calculated as follows: H(1) = (3.185 + 1.232 × RER) × VO₂. Total locomotor activity (ambulatory and fine) was measured simultaneously along the x and y axes using an infrared photocell beam grid and represented as the average of the total number of beam breaks along the x and y axes during light and dark periods.

An automated phenotyping homecage system (TSE PhenoMaster) was used to perform behavioral and metabolic tests in single-caged mice. The specificity of the TG construct was tested in bTG and WT mice by recording daily liquid intake of water or 5mM aspartame and 600ppm sucralose in 3 subsequent, alternate days.