Male Sprague-Dawley rats, aged 6 weeks, were adapted to room and cage environments for at least 5 days prior to the start of each experiment; they were caged singly in a temperature-controlled room (22 ± 1°C) with a 12-h light/dark cycle. They were maintained on a commercial pelleted chow diet (Mucedola 4RF21; Settimo Milanese, Milan, Italy) consisting, by energy, of 29.0% protein, 60.4% carbohydrates, and 10.6% fat, and had free access to tap water. The experiments were conducted after this period of adaptation in rats selected on the basis of body weight being within ±5 g of the mean body weight (200 g). This study was carried out in accordance with the recommendations of “Italian Health Ministry.” The protocol was approved by the “Comitato Etico-Scientifico per la Sperimentazione Animale” of the University “Federico II” of Naples.

Rats were food restricted on a daily basis for 14 days at approximately 50% of the spontaneous chow intake (calculated as mean of the food intake per day in the same rats before semistarvation). It has been previously shown that this degree of caloric restriction induces growth arrest, marked depletion of body fat stores, and leads to diminished energy expenditure (due to suppressed thermogenesis) that underlies a high efficiency of fat deposition during refeeding (Crescenzo et al., 2003). At the end of the semistarvation period, all the rats were separated into 3 groups (n = 8): one group was immediately euthanized by decapitation to calculate the body composition of restricted rats, while the other two groups were refed isocaloric amounts of two different high fat diets (58.2% by energy), rich in lard (mainly MUFA and SFA) or safflower/linseed oil (PUFA of omega-6 and omega-3 series). The amount of dietary energy provided to the refed animals corresponds to the metabolisable energy intake of spontaneously growing (non-restricted) weight-matched control animals fed on chow diet, as previously reported (Crescenzo et al., 2003). Furthermore, the level of fat in the high fat diet utilized here (i.e., 58% of energy intake) corresponds to dietary fat levels often utilized in rehabilitation (energy-dense) diets of malnourished infants and children in order to meet their high energy requirements for catch-up growth (Prentice and Paul, 2000). Details of diets composition and energy content are reported in Table 1. This experimental design, consisting of 2 weeks of controlled refeeding after 2 weeks of semistarvation, is similar to that previously described in establishing a rat model for studying adjustments in energy expenditure specific for accelerating fat deposition during refeeding (Dulloo and Girardier, 1992; Crescenzo et al., 2003).

At the end of refeeding period, all the rats were euthanized and blood was immediately collected for plasma preparation. Mesenteric and epididymal WAT (e-WAT) and interscapular BAT (i-BAT) were weighed and small aliquots of e-WAT and i-BAT were frozen for further measurements. Small aliquots of liver and hindleg skeletal muscle were also collected and used for measurements. The carcasses were finally used for body composition determination.

Twenty-four hours VO2 and VCO2 of the rats were recorded with a four-chamber indirect open-circuit calorimeter (Panlab s.r.l., Spain). Measurements were performed every 15 min for 3 min in each cage. During the above measurements, continuous recording of spontaneous activity was also carried out through the extensiometric weight transducers located beneath each cage. Urine was collected for the whole (24-h) period and urinary nitrogen levels were measured by an enzymatic colorimetric method (FAR S.r.l., Italy). Substrate oxidation rates were calculated from VO2, VCO2 and urinary nitrogen according to Even et al. (1994).

Rats were fasted for 6 h (8.00 a.m.–14.00 p.m.) and small blood samples were taken from the tail vein, placed in EDTA-coated tubes and transferred on ice. All the samples were then centrifuged at 1400 g_av for 15 min at 4°C, and then frozen. Plasma glucose concentration was measured by colorimetric enzymatic method (Pokler Italia, Italy), while plasma insulin concentration was measured using an ELISA kit (Mercodia AB, Sweden) in a single assay to remove inter-assay variations. Basal postabsorptive values of plasma glucose and insulin were used to calculate Homeostatic Model Assessment (HOMA) index as [Glucose (mg/dL) × Insulin (mU/L)]/405. HOMA index has been validated as an easy but accurate measure of insulin sensitivity in Wistar and Sprague-Dawley rats (Cacho et al., 2008). Plasma concentrations of triglycerides, cholesterol, non esterified fatty acids (NEFA), and alanine aminotransferase (ALT) were measured by colorimetric enzymatic method using commercial kits (SGM Italia, Italy and Randox Laboratories ltd., United Kingdom). Lipid peroxidation was determined according to Fernandes et al. (2006), by measuring thiobarbituric acid reactive substances (TBARS). Aliquots of plasma were added to 0.5 ml of ice-cold 40% trichloroacetic acid. Then, 1 ml of 0.67% of aqueous thiobarbituric acid containing 0.01% of 2,6-di-tert-butyl-p-cresol was added. The mixtures were heated at 90°C for 15 min, then cooled in ice for 10 min, and centrifuged at 850 g_av for 10 min. The supernatant fractions were collected and lipid peroxidation was estimated spectrophotometrically at 530 nm. The amount of TBARS formed was calculated using a molar extinction coefficient of 1.56 × 10⁵/M/cm and expressed as nmol TBARS/ml.

The carcasses were incised and the gut cleaned of undigested food, then the whole carcasses were homogenized in distilled water, aliquoted, and frozen. Samples of the homogenized carcass were dried and analyzed for energy content by bomb calorimetry (Parr Instruments, IL, USA) and for fat content by the Folch extraction method (Folch et al., 1957). Body protein was determined from a general formula relating energy derived from fat, total energy value of the carcass, and energy derived from protein (Dulloo and Girardier, 1992); the caloric values for body fat and protein were taken as 38.6 and 22.7 kJ/g, respectively. Water, fat, and fat free dry mass were then calculated.

Energy balance measurements were conducted during refeeding by the comparative carcass technique over 2 weeks, during which metabolisable energy (ME) intake was monitored, as detailed previously (Iossa et al., 1997). Energy expenditure was determined as the difference between energy gain and ME intake, and the energetic efficiency was calculated as the percentage of total energy gain per ME intake.

Fatty acid synthase (FAS) activity was measured according to the protocol described by Penicaud et al. (1991) on protein extracts from liver, e-WAT and i-BAT. FAS activity was assessed in e-WAT since previous results have shown a downregulation of its activity in this specific fat pad in rats refed with SFA-MUFA diet (Marcelino et al., 2013).

Tissue triglycerides were measured by colorimetric enzymatic method using commercial kits (SGM Italia, Italy). Tissue glycogen content was assessed by direct enzymatic procedure (Roehrig and Allred, 1974).

Lipid peroxidation was determined in liver and skeletal muscle by using the same procedure used for plasma samples. The amount of TBARS formed was expressed as nmol TBARS/g tissue.

The determination of MPO activity can be used as a surrogate marker of inflammation, since it has been shown that the activity of MPO solubilised from the inflamed tissue is directly proportional to the number of neutrophils seen in histologic sections (Krawisz et al., 1984). MPO activity was therefore assessed in liver samples as reported by Kim et al. (2012). Briefly, tissue samples (100 mg) were homogenized in 1 ml of hexadecyltrimethylammonium bromide (HTAB) buffer (0.5% HTAB in 50 mM phosphate buffer, pH 6.0) and centrifuged at 13,400 × g_av for 6 min at 4°C. MPO activity was measured spectrophotometrically: 10 μl of supernatant were combined with 200 μl of 50 mM phosphate buffer, pH 6.0, containing 0.167 mg/ml 0-dianisidine hydrochloride and 1.25% hydrogen peroxide. The change in absorbance at 450 nm was measured and one unit of MPO activity was defined as that degrading 1 μmol of peroxide per minute at 25°C.

TNF-α concentrations in protein extracts from liver were determined using a rat specific ELISA (R&D Systems, MN, USA) according to manufacturer's instruction. Briefly, the wells of a microtitre plate were coated with 100 μl of mouse anti-rat TNF-α (4 μg/ml) in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4), and incubated overnight at room temperature. The antibody excess was then removed by washing with Wash Buffer (containing 0.05% (v/v) Tween 20 in PBS, pH 7.4), and the remaining sites on the plate were blocked with reagent diluent (PBS containing 1% BSA) (1 h, room temperature). After extensive washing, samples were added to the wells and incubated for 2 h at room temperature. After further washing, the wells were incubated with biotinylated goat anti-rat TNF-α (225 ng/ml in reagent diluent) followed by treatment with Streptavidin-HRP (1:200 dilution; 1 h, room temperature). Peroxidase-catalyzed color development from o-Phenylenediamine was measured at 492 nm.

Isolation of mitochondria from liver and skeletal muscle and measurements of respiration were carried out as previously reported (Iossa et al., 2003) and outlined below.

Tissue fragments from fresh liver were gently homogenized with a medium containing 220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 1 mM EDTA, and 0.1% (w/v) fatty acid free bovine serum albumin (BSA), pH 7.4, in a Potter Elvehjem homogenizer set at 500 rpm (4 strokes/min). After withdrawn of aliquots for further assays, the homogenate was then centrifuged at 1,000 g_av for 10 min and the resulting supernatant was again centrifuged at 3,000 g_av for 10 min. The mitochondrial pellet was washed twice and finally resuspended in a medium containing 80 mM KCl, 50 mM HEPES, 5 mM Tris-PO₄, 1 mM EGTA, 0.1% (w/v) fatty acid-free BSA, pH 7.0.

Freshly isolated hind leg muscles were freed of excess connective tissue, finely minced, washed in a medium containing 100 mM KCl, 50 mM Tris, pH 7.5, 5 mM MgCl₂, 1 mM EDTA, 5 mM EGTA, and 0.1% (w/v) fatty acid free BSA, and treated with protease nagarse (1 mg/g) for 4 min. Tissue fragments were then homogenized with the above medium (1:8, w/v) at 500 rpm (4 strokes/min). The homogenate was centrifuged at 3,000 g for 10 min, the resulting supernatant was rapidly discarded and the pellet was resuspended and centrifuged at 500 g for 10 min. The supernatant was then centrifuged at 3,000 g for 10 min, the pellet was washed once and resuspended in a suspension medium (250 mM sucrose, 50 mM Tris, pH 7.5 and 0.1% fatty acid free BSA). Control experiments of enzymatic and electron microscopy characterization have shown that our isolation procedure (centrifugation at 3,000 g_av for 10 min) results in a cellular fraction, which is essentially constituted by mitochondria.

Oxygen consumption rate of isolated mitochondria was measured polarographically with a Clark-type electrode (Yellow Springs Instruments, OH, USA) in a 3 mL-glass cell, at a temperature of 30°C in a medium containing 80 mM KCl, 50 mM HEPES, 5 mM K₂HPO₄, 1 mM EGTA, 0.1% (w/v) fatty acid free BSA, pH 7.0, for liver mitochondria or 30 mM KCl, 6 mM MgCl₂, 75 mM sucrose, 1 mM EDTA, 20 mM KH₂PO₄, pH 7.0, and 0.1% (w/v) fatty acid-free BSA, pH 7.0 for skeletal muscle mitochondria. The substrates used were 10 mM succinate + 3.75 μM rotenone, 10 mM glutamate + 2.5 mM malate, 40 μM palmitoyl-carnitine + 2.5 mM malate. All samples were allowed to oxidize their endogenous substrates for 3 min and then 10 mM succinate + 3.75 μM rotenone, 40 μM palmitoyl-carnitine + 2.5 mM malate, or 10 mM glutamate + 2.5 mM malate were added as substrate. State 3 oxygen consumption was measured in the presence of 0.3 mM ADP.

Active aconitase specific activity was measured spectrophotometrically by following the formation of NADPH (340 nm) at 25°C in a mixture containing 0.2 mM NADP⁺, 5 mM sodium citrate, 0.6 mM MnCl₂, 1 U/mL concentration of isocitric dehydrogenase, 50 mM Tris-HCl, pH 7.4 (Gardner, 2002). Aconitase inhibited by ROS in vivo was reactivated so that total activity could be measured by incubating mitochondrial extracts in a medium containing 50 mM dithiothreitol, 0.2 mM Na₂S, and 0.2 mM ferrous ammonium sulfate.

Lipid peroxidation was determined in isolated mitochondria by using the same procedure described before and was expressed as nmol TBARS/mg protein.

SOD specific activity was measured in a medium containing 0.1 mM EDTA, 2 mM KCN, 50 mM KH₂PO₄ pH 7.8, 20 mM cythocrome c, 0.1 mM xanthyne, and 0.01 units of xanthyne oxidase. Determinations were carried out spectrophotometrically (550 nm) at 25°C, by monitoring the decrease in the reduction rate of cythocrome c by superoxide radicals, generated by the xanthine-xanthine oxidase system (Flohè and Otting, 1974). One unit of SOD activity is defined as the concentration of enzyme that inhibits cythocrome c reduction by 50% in the presence of xanthine + xanthine oxidase.

Tissue samples were homogenized in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2.7 mM KCl, 5% (v/v) glycerol, 1% (v/v) Triton X-100, and 50 μL/g tissue of protease inhibitor cocktail (all from Sigma-Aldrich, MO, USA) using a Potter homogeniser, shaken for 2 h at 4°C, and centrifuged at 14,000 g_av for 20 min at 4°C. The supernatants were collected, aliquots were denatured in a buffer (60.0 mM Tris pH 6.8, 10% sucrose, 2% SDS, 4% β-mercaptoethanol) and loaded onto a 12% SDS-Polyacrylamide gel. After the run in electrode buffer (50 mM Tris, pH 8.3, 384 mM glycine, 0.1% SDS), the gels were transferred onto polyvinylidene difluoride membranes (Immobilon-P, Merck Millipore, Germany) at 0.8 mA/cm² for 90 min. The membranes were preblocked in blocking buffer (PBS; 5% milk powder; 0.5% Tween 20) for 1 h and then incubated overnight at 4°C with a rabbit antibody for UCP1 (Alpha Diagnostic International, TX, USA, at 1 μg/mL dilution in blocking buffer). The membranes were washed and then incubated for 1 h at room temperature with an anti-rabbit alkaline phosphatase-conjugated secondary antibody (Promega, WI, USA). The membranes were finally washed, rinsed in distilled water and incubated at room temperature with a chemiluminescent substrate, CDP-Star (Sigma-Aldrich, MO, USA). Data detection was carried out by exposing autoradiography films (Eastman Kodak Company, NY, USA) to the membranes. Quantification of signals was carried out by Un-Scan-It gel software (Silk Scientific, UT, USA). Actin was detected with polyclonal antibody (Sigma-Aldrich, MO, USA diluted 1:250 in blocking buffer) and used to normalize the UCP1 signal.

Data are given as means with their standard errors. Statistical analyses were performed by two-tailed, unpaired, Student's t-test. Probability values less than 0.05 were considered to indicate a significant difference. All analyses were performed using GraphPad Prism 6 (GraphPad Software, CA, USA).

All chemicals used were of analytical grade and were purchased from Sigma-Aldrich (MO, USA).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.