*2.2. Animals*

UCP3-ablated mice were derived from those that were described by Gong et al. [3] and they were backcrossed to the C57BL/6 strain for ten generations.

Male wild type (WT) and UCP3 knockout mice (KO) were used in the present study. Mice were housed in thermoneutrality (30 ± 1 ◦C) with a 12/12 h light-dark cycle and free access to food and water. This study was carried out in accordance with recommendations in the EU Directive 2010/63/ for the Care and Use of Laboratory Animals. The Committee on the Ethics of Animal Experiments of the University of Napoli Federico II (Italy) and the Italian Minister of Health approved all of the animal protocols. Every effort was made to minimize animal pain and suffering. At the end of the treatments described below, the mice were anesthetized with Zoletil (40 mg/100 g bw) and sacrificed by decapitation.

To detect metabolic parameters and mitochondrial functionality, four–five month-old mice of each genotype acclimated to 30 ◦C ± 1 for 2–3 weeks and fed a standard diet were used to detect resting metabolic rate (RMR), respiratory quotient (RQ), energy expenditure (EE), and mitochondrial functional parameters. Other groups of WT and KO mice were kept one per cage and were housed at 30 ◦C ± 1 for 80 days since weaning and fed with a standard diet or a high fat diet (HFD) for 80 days in order to detect body composition and energy gains as well as to perform histological analysis of mice under different "lipid loads''. Standard/low fat diet (STD) consisted of 10% lipids, 20% proteins, 70% carbohydrates with a gross energy density of 15.5 kJ/g wet food. HFD consisted of 45% lipid, 20% proteins, and 35% carbohydrates with a gross energy density of 19.2 kJ/g wet food. Both of the diets were from Mucedola (Milano Italy).

## *2.3. Metabolic Parameters*

Oxygen consumption (VO2) and carbon dioxide production (CO2) measurements were made using a four-chamber, indirect open-circuit calorimeter (Columbus Instrument), with one mouse per chamber at a room temperature of 30 ◦C to evaluate basal metabolic parameters. Measurements were performed between 1100 and 1600 h. After a 1-h period of adaptation to the metabolic chamber, VO2 and VCO2 were measured when the mice were not moving for at least 10 min. The system settings included a flow rate of 0.5 L/min., a sample line-purge time of 2 min., and a measurement period of 30 s every 12 min. Mice were placed in separate 2.5-L calorimetry chamber with ad libitum access to water. Values of VO2 and VCO2 were obtained by means of three different consecutive measurements during which the mice were not moving. These data were used to calculate the respiratory quotient (RQ; VCO2/VO2) and the resting energy expenditure (REE) ([3.815 + 1.232 RQ] VO2). The contribution of fatty acid oxidation to REE was calculated, as described using the following equation: percentage of fat contribution = [468.6 (1 − RQ)]/[5.047 (RQ − 0.707) + 4.686 (1 − RQ)] [23].

#### *2.4. SkM Mitochondrial Respiration*

As soon as euthanasia was performed, SkM were excised and all visible contaminating tissues were removed. The tissues were either immediately processed for mitochondria isolation or frozen in liquid nitrogen and then stored at −80 ◦C for later analysis. Mitochondria from SkM were isolated by differential centrifugation, as reported by Lombardi et al. [24]. The mitochondrial respiration and fatty acid oxidation rate were assessed by the polarographic method while using a Clark-type electrode at 37 ◦C by using different respiratory substrates. SkM mitochondrial respiration was detected in a final volume of a 0.5 mL respiration medium consisting of 80 mM KCl, 50 mM HEPES (pH 7.0), 1 mM EGTA, 5 mM K2HPO4, and 0.5% fatty acid-free BSA (w/v). The mitochondria were incubated for three min in the respiratory medium, and the respiration was initiated by the addition of succinate (5 mM) in the presence of rotenone (4 μM) or pyruvate (10 mM) in the presence of malate (2 mM), or palmitoyl-carnitine (40 μM) in the presence of malate (2 mM). Once State 2 of respiration was reached, ADP (300 μM) was added to the incubation medium to induce State 3 respiration; when ADP was exhausted, State 4 was reached.

#### *2.5. Separation of Respiratory Complexes by Blue-Native Page (BN-PAGE) and Histochemical Staining for in-Gel Activity*

Solubilisation of mitochondrial membranes by detergents, BN-PAGE, staining, and densitometric quantification of oxidative phosphorylation complexes were performed, as described in Scagger et al. [25] and Lombardi et al. [26], with some minor variations. Mitochondria enriched fraction was suspended in a low-salt buffer (50 mM NaCl, 50 mM imidazole, pH 7.0) and solubilised with 10% (w/v) dodecyl-maltoside to solubilise the individual respiratory chain complexes. The electrophoretic run was carried out on 4–13% gradient polyacrylamide gels and enzymatic colorimetric reactions were performed essentially as reported by Zerbetto et al. [27]. The activity of complex I activity was evaluated by incubating the gel slices with 2 mM Tris–HCl, pH 7.4, 0.1 mg/mL NADH, and 2.5 mg/mL nitro blue tetrazolium (NTB) at room temperature. To detect complex II activity, gel slices were incubated at room temperature in a 100 mM Tris/glycine buffer at pH 7.4 containing 1 mg/mL NTB and 1 mM sodium succinate. Complex IV activity was assessed by incubating BN-PAGE gels with 5 mg 3,3-diaminobenzidine tetrahydrochloride (DAB) that was dissolved in a 9 mL phosphate buffer (0.05 M, pH 7.4), 1 mL catalase (20 μg/mL), 10 mg cytochrome c, and 750 mg sucrose. The original colour of the complex I, II, or IV-reacting bands was preserved by fixing the gels in 50% methanol and 10% acetic acid. In parallel, another electrophoretic run was performed to stain the gels with Coomassie Blue G to obtain the total band pattern of the respiratory complexes. After gel scanning, the areas of the bands were expressed as absolute values (arbitrary units).

#### *2.6. Determination of Glycerol Release from White Adipose Tissue*

100 mg of epididymal white adipose tissue samples were removed. Samples were cut into 20 mg sections to evaluate the glycerol diffusion from tissue to the medium better; 100 mg of tissue were incubated at 37 ◦C in 500 μL of Krebs Ringer buffer (KRB; 12 mM HEPES, 121 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 0.33 mM CaCl2) containing 2% FA-free bovine serum albumin (BSA) and 0.1% glucose in the presence or absence of 10 μM isoproterenol (Sigma). Tissue was incubated for 1 h at 37 ◦C in a shaking bath and then gassed with 95% O2-5% CO2. At the end of the incubation period, an aliquot of the medium was used for the analysis of glycerol. A commercially available absorbance-based enzyme assay for glycerol (Free Glycerol Reagent; Sigma) was converted to fluorescence-based detection by the inclusion of the hydrogen peroxide-sensitive dye Amplex UltraRed, as reported by Clark et al. [28].
