Bovine serum albumin fraction V (BSA), o-phenylenediamine, hexadecyltrimethylammonium bromide (HTAB), 3-amino-1,2,4 triazole, as well as salts, buffers and reagents used in the biochemical analyses were purchased from Sigma-Aldrich (St. Louis, MO, USA). The dye reagent for protein titration was from Bio-Rad (Hercules, CA, USA). Polystyrene 96-wells ELISA MaxiSorp plates, with high affinity to proteins with mixed hydrophilic/hydrophobic domains, were purchased from Nunc (Roskilde, Denmark).

All experimental procedures involving animals were approved by “Comitato Etico-Scientifico per la Sperimentazione Animale” of the University of Naples Federico II and authorized by Italian Health Minister (260/2015-PR). This work complies with the animal ethics principles and regulations of the Italian Health Ministry. The authors ensured that all steps were taken to minimize the pain and suffering of the animals.

Male Sprague-Dawley rats (Charles River, Italy), of 30 (young) or 90 (adult) days of age were caged singly in a temperature-controlled room (23 ± 1°C) with a 12-h light/dark cycle (06.30–18.30). Young and adult rats were divided in three groups: the first one was euthanized at the beginning of the dietary treatment to measure initial body composition, while the remaining two groups were fed a fructose-rich or control diet for 2 weeks. The composition of the two diets is shown in Table 1. Rats were pair-fed for the whole experimental period, by giving them the same amount of diet, both as weight and as caloric content and each rat consumed the full portion of the diet. The amount of daily caloric intake of all groups of rats steadily increased during the experimental period. During the treatment, body weight and food intake were monitored daily. At the end of the experimental period, the rats were euthanized by decapitation, and blood and liver were collected, while the carcasses were used for body composition determination.

Oxygen consumption and carbon dioxide production of the rats were recorded over a period of 24 h with a four-chamber indirect open-circuit calorimeter (Panlab s.r.l., Barcelona, Spain). Measurements were performed every 15 min for 3 min in each cage. Urine was collected for the whole (24-h) period and urinary nitrogen levels were measured by an enzymatic colorimetric method (FAR Diagnostics s.r.l., Settimo di Pescantina, Italy). Urinary nitrogen levels were then used to calculate oxygen consumption and carbon dioxide production due to protein oxidation, and the obtained values were subtracted from total oxygen consumption and carbon dioxide production to calculate non-protein oxygen consumption and carbon dioxide production. These latter values were finally used to obtain the non-protein respiratory quotient (NPRQ) values in the different rat groups.

Glucose tolerance test was carried out the day before the euthanasia. To this end, rats were fasted for 6 h from 08.00 a.m. Basal, postabsorptive blood sample was obtained from a small tail clip and placed in EDTA–coated tubes and then glucose (2 g/kg body weight) was injected intraperitoneally. Blood samples were collected after 20, 40, 60, 90, and 120 min and placed in EDTA-coated tubes. The blood samples were centrifuged at 2000 × g for 15 min at 4°C. Plasma glucose concentration was measured by colorimetric enzymatic method (Pokler Italia, Genova, Italy), while plasma insulin concentration was measured using an ELISA kit (Mercodia AB, Uppsala, Sweden) in a single assay to remove inter-assay variations.

Guts were cleaned of undigested food and the carcasses were then autoclaved. After dilution in distilled water and subsequent homogenization of the carcasses with a Polytron homogeniser (Kinematica, Switzerland), duplicate samples of the homogenized carcass were analyzed for energy content by bomb calorimeter. The contribution of the liver to total body energy content was taken into account by measuring energy content of liver samples with the bomb calorimeter. Total body water content was determined by drying carcass samples in an oven at 60°C for 48 h. Total body lipids were measured by the Folch extraction method (Folch et al., 1957). The energy as lipid was calculated from body lipids by using the coefficient of 39.2 kJ/g, and was then subtracted from total body energy to obtain the energy as protein. Energy balance measurements were conducted by the comparative carcass technique over the experimental period, as previously detailed (Crescenzo et al., 2010). Briefly, daily food consumption was monitored, energy density of the diet was measured by a bomb calorimeter and gross energy intake was calculated. Energy content was measured in feces and urine and the values were subtracted from gross energy intake to obtain metabolizable energy (ME) intake. Body energy, lipid and protein gain were calculated as the difference between the final and initial content of body energy, fat and protein. 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.

Plasma concentrations of alanine aminotransferase (ALT), triglycerides, and non esterified fatty acids (NEFA) were measured by colorimetric enzymatic method using commercial kits (SGM Italia, Rome, Italy for ALT and triglycerides and Roche Diagnostics, Mannheim, Germany for NEFA). Lipid peroxidation was determined according to Fernandes et al. (2006), by measuring thiobarbituric acid reactive substances (TBARS), using the thiobarbituric acid assay.

Liver triglycerides were measured by colorimetric enzymatic method using commercial kits (SGM Italia, Rome, Italy). Hepatic lipid content data were also confirmed by histological analysis. Frozen 10 μm-thick sections were obtained after embedding and freezing liver fragments in Killik (Bio Optica, Milan, Italy). Sections were then stained with hematoxylin-eosin and mounted in PBS/glycerol (9:1, v/v) (Tussellino et al., 2012; Alwahsh et al., 2014). Sections were photographed with a Leica CTR 6500 UV microscope (Leica Microsystems CMS gmbH, Wetzlar, Germany) and degree of steatosis was evaluated by ImageJ software.

Hepatic glycogen content was assessed by direct enzymatic procedure (Roehrig and Allred, 1974).

Liver ceramide content was evaluated by ELISA using 96-well Polysorp plates (Nunc, Rochester, NY, USA). Briefly, hepatic lipids extracted with the Folch method (Folch et al., 1957) were adsorbed to well bottoms overnight at 4°C. Plates were blocked with 10 mM phosphate buffered saline (PBS), 140 mM NaCl, 0.1% Tween, pH 7.4 supplemented with 1% bovine serum albumin for 1 h at 37°C. The plates were then washed three times with washing buffer containing 10 mM PBS, 140 mM NaCl, 0.05% Tween, pH 7.4 and incubated with monoclonal anti-ceramide antibody (2 μg/ml) for 1 h at 37°C. After three washings, peroxidase-conjugated goat anti-mouse IgM (1:5000 dilution) was incubated for 1 h at 37°C. After four washings, the wells were incubated with 100 μl of a color development solution (20 mg of o-Phenylenediamine dihydrochloride in 50 ml of 70 mM Na₂HPO₄, 30 mM citric acid, pH 5, supplemented with 120 μl of 3% H₂O₂). After 15 min at 37°C, the reaction was stopped by the addition of 50 μl of 2.5 M H₂SO₄ and the absorbance was measured at 492 nm. All tests were done in triplicate. Immunoreactivity was normalized to starting tissue weight. Negative control reactions were performed by omitting the addition of primary antibody.

As plasma marker of inflammation, the concentration of the acute phase protein haptoglobin (Hpt) was measured by ELISA as previously reported (Spagnuolo et al., 2014). Individual samples were diluted (1:10,000–1:60,000) with coating buffer (7 mM Na₂CO₃, 17 mM NaHCO₃, 1.5 mM NaN₃, pH 9.6), and incubated in the wells of a microtitre plate (Immuno MaxiSorp; overnight, 4°C). After four washes by Tween-Tris buffered saline (T-TBS) (130 mM NaCl, 20 mM Tris-HCl, 0.05% Tween 20, pH 7.4) and four washes by high salt TBS (500 mM NaCl in 20 mM Tris-HCl at pH 7.4), the wells were blocked with TBS (130 mM NaCl, 20 mM Tris-HCl, pH 7.4) containing 0.5% BSA (1 h, 37°C). After washing, the wells were incubated (1 h, 37°C) with 60 μl of rabbit anti-Hpt IgG (ICL Lab, 1:1000 dilution) followed by goat anti-rabbit peroxidase-conjugated secondary antibody (GAR-HRP IgG; 1:8,000 dilution; Sigma-Aldrich, MO, USA) for Hpt detection. Peroxidase-catalyzed color development from o-phenylenediamine was measured at 492 nm.

Tumor necrosis factor (TNF)-alpha concentrations in protein extracts from liver were determined using a rat specific enzyme linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA) according to manufacturer's instruction.

The determination of myeloperoxidase (MPO) activity can be used as a surrogate marker of inflammation, since it has been shown that the activity of MPO solubilized 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 HTAB buffer (0.5% HTAB in 50 mM phosphate buffer, pH 6.0) and centrifuged at 13,400 × g for 6 min at 4°C. Then, 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.

For Real-Time RT-PCR analysis of inflammation markers in liver, total RNA was extracted from single liver for sample randomly selected for each treatment using Tri-Reagent (Sigma, St. Louis, MO, USA) according to the manufacturer's recommendations. cDNAs were obtained, from 1 μg of total RNA, using the Super Script VILO cDNA synthesis kit (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Real-time PCR was performed using Power SYBR Green Master Mix kits (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) using the 96-well optical reaction plate in 20 μL total reaction volume. The reaction solution and amplification reactions were carried out as reported in De Marco et al. (2017) and Tussellino et al. (2016), respectively. β-actin (Alwahsh et al., 2014) was designed as endogenous reference (housekeeping gene). cDNA from samples of adult rat were used as controls. Primers used to detect Monocyte Chemoattractant Protein 1 (MCP1) (Sheldon et al., 2015), Arginase (Arg) (Li et al., 2012), F4/80 (Sheldon et al., 2015), and Interleukin 1 beta (IL-1β) (Sheldon et al., 2015) are shown in Table 2.

Isolation of liver mitochondria and measurement of state 3 respiration were carried out as previously reported (Crescenzo et al., 2012). Briefly, liver tissue fragments 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 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 for 10 min and the resulting supernatant was again centrifuged at 3000 g for 10 min. The mitochondrial pellet was washed twice and finally resuspended 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. Oxygen consumption rate was measured polarographically in liver homogenates and isolated mitochondria with a Clark-type electrode (Yellow Springs Instruments, Yellow Springs, 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. All samples were allowed to oxidize their endogenous substrates for 3 min and then 10 mM glutamate + 2.5 mM malate, 10 mM succinate + 3.8 μM rotenone, or 40 μM palmitoyl-carnitine + 2.5 mM malate were added as substrate. State 3 oxygen consumption was measured in the presence of 0.3 mM ADP. Control experiments of enzymatic and electron microscopy characterization have shown that our isolation procedure (centrifugation at 3, 000 × g for 10 min) results in a cellular fraction, which is constituted essentially by mitochondria.

De novo lipogenesis in liver was evaluated by assessing fatty acid synthase (FAS) activity, according to the protocol described by Pénicaud et al. (1991) on hepatic homogenates.

Lipid peroxidation was determined in liver homogenates and isolated mitochondria by using the same procedure used for plasma samples as reported above.

As marker of protein oxidative modifications, the level of N-Tyr in liver homogenates and isolated mitochondria was quantified by ELISA. Samples were diluted (1:500, 1:1,500, 1:3,000, and 1:6,000) with coating buffer, and aliquots (50 μl) were then incubated in the wells of a microtitre plate (overnight, 4°C). After four washes by T-TBS and four washes by high salt TBS, the wells were blocked with TBS containing 0.5% BSA (1 h, 37°C). After washing, the wells were incubated (2 h, 37°C) with 50 μl of rabbit anti-N-Tyr (Covalab, purchased by Vinci Biochem, Vinci, Italy; 1: 800 dilution in T-TBS containing 0.25% BSA) followed by 60 μl of GAR-HRP IgG (1:4,000 dilution; 1 h, 37°C). Peroxidase-catalyzed color development from o-phenylenediamine was measured at 492 nm. Data were reported as OD per mg of proteins.

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

Proteins were extracted from livers by homogenizing frozen tissues (−80°C) in 10 volumes (w/v) of 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, St. Louis, MO, USA) using a Potter homogeniser, shaken for 2 h at 4°C. Homogenates were centrifuged at 14,000 g_av for 20 min at 4°C and the supernatants were collected. Then, aliquots (65 μg) were denatured in Laemmli's buffer (60.0 mM Tris pH 6.8, 10% sucrose, 2% SDS, 4% β-mercaptoethanol) and fractionated by electrophoresis under denaturing and reducing conditions on 10% (for p-IRS and p-Akt) or 12% (for LCN-2) polyacrylamide gel. After the run in electrode buffer (25 mM Tris, pH 8.3, 192 mM glycine, 0.1% SDS), the gels were transferred onto polyvinylidene difluoride membranes (PVDF, Immobilon-P, Merck Millipore, Darmstadt, Germany) at 0.8 mA/cm² for 90 min.

For quantification of p-IRS, the membranes were blocked in TBS containing 3% BSA and 0.1% Tween for 1 h and then incubated overnight at 4°C with rabbit anti-pIRS1 (Tyr612) (Thermo Fisher Scientific, Waltham, MA, USA, 1:500 dilution in blocking buffer). Membranes were washed 3 times 12 min in TBS/0.1% Tween 20 and 3 times 12 min in TBS and then incubated with GAR-HRP IgG (1: 7,000 dilution in blocking buffer; 1 h, 37°C) (Sigma-Aldrich, St. Louis, MO, USA). The membranes were washed as above described, rinsed in distilled water and the immunocomplexes were detected by the ECL detection system (Immobilon Western Chemiluminescent HRP kit, distributed by Microtech, Naples, Italy). After p-IRS detection, the membrane was extensively washed with TBS/0.1% Tween 20, and submerged in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7; 45 min, 50°C). Total IRS 1 was revealed by incubation (overnight, 4°C) with rabbit anti-IRS1 (Cell Signaling, Danvers, MA, USA, 1:500 dilution in blocking buffer) followed by GAR-HRP IgG (1: 20,000 dilution in blocking buffer; 1 h, 37°C).

For quantification of p-Akt, the membranes were blocked in blocking buffer (TBS; 5% milk powder; 0.1% Tween 20) for 1 h and then incubated overnight at 4°C with rabbit anti-p-Akt (Cell Signaling, Danvers, MA, USA, diluted 1:2,000 in blocking buffer). Membranes were washed 3 times 15 min in TBS/0.1% Tween 20 and 3 times 15 min in TBS and then incubated 1 h at room temperature with an anti-rabbit alkaline phosphatase-conjugated secondary antibody (GAR-AP IgG; diluted 1:5,000 in blocking buffer; 1 h, 37°C) (Promega, Madison, WI, USA). The membranes were washed as above described, rinsed in distilled water and incubated at room temperature with a chemiluminescent substrate, CDP-Star (Sigma-Aldrich, St Louis, MO, USA). After p-Akt detection, the membrane was stripped as above and incubated overnight at 4°C with rabbit anti-Akt (Cell Signaling, Danvers, MA, USA, 1:2000 dilution in blocking buffer), followed by an anti-rabbit alkaline phosphatase-conjugated secondary antibody (GAR-AP IgG; diluted 1:5,000 in blocking buffer; 1 h, 37°C) (Promega, Madison, WI, USA).

For quantification of LCN-2, the membranes were blocked in blocking buffer (TBS; 5% milk powder; 0.1% Tween 20) for 1 h, 37°C and then incubated overnight at 4°C with goat anti- LCN-2 (Thermo Fischer Scientific, Waltham, MA, USA; 1:300 dilution in blocking buffer containing 0.5% milk powder). Membranes were washed 3 times 15 min in TBS/0.1% Tween 20 and 3 times 15 min in TBS and then incubated 1 h at room temperature with a rabbit anti-goat peroxidase-conjugated secondary antibody (RAG-HRP IgG, 1:130,000 dilution in blocking buffer; 1 h, 37°C) (Sigma-Aldrich, St. Louis, MO, USA). The membranes were washed as above described, rinsed in distilled water and incubated at room temperature with the Excellent Chemiluminescent detection Kit (ElabScience, distributed by Microtech, Naples, Italy).

Rat plasma samples (75 μg protein) were denatured at 100°C for 5 min in Laemmli's buffer and fractionated by electrophoresis on 12% polyacrylamide gel, under denaturing and reducing conditions. After protein transfer onto PVDF as above described, the membranes were incubated with the same dilution of goat anti-LCN-2 and GAR-HRP IgG used for liver homogenates.

Data detection was carried out by exposing autoradiography films (Eastman Kodak Company, Long Island city, NY, USA) to the membranes and quantification of signals was carried out by Un-Scan-It gel software (Silk Scientific, Orem, UT, USA).

Data were expressed as mean values ± SEM. The program GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) was used to verify that raw data have normal distribution and to perform two-tailed, two-way ANOVA followed by the Tukey post-hoc test. A probability of < 5% (P < 0.05) was considered statistically significant in all analyses.

After 2 weeks of dietary treatment, body composition was evaluated. Even after this short term dietary treatment, the isoenergetic intake of a fructose-rich diet elicited a significant increase in body energy, body lipids, body energy gain, body lipid gain in adult rats, but not in young rats (Table 3). A trend toward increase was also found in young rats, but the relative variations were less evident and did not reach statistical significance. At the end of the dietary treatment, indirect calorimetry measurements were performed and values of oxygen consumption, carbon dioxide production, and urinary nitrogen were used to calculate energy expenditure per lean body mass, RQ and NPRQ. No significant difference in energy expenditure/g body protein was evident at the end of the dietary treatment and between age groups. On the other hand, a significant increase in RQ and NPRQ was found in adult but not in young rats after high fructose feeding (Figures 1A,B), thus indicating that after 2 weeks of high fructose intake the utilization of carbohydrates as metabolic fuels is increased. Part of this increase is due to the activation of de novo lipogenesis, as evidenced by the finding of increased FAS activity in the livers of adult rats (Figure 1C). No significant increase in FAS activity was found in young rats.

Metabolic characterization of the glucose homeostatic system was obtained by carrying out glucose tolerance test the day before the euthanasia (Figures 2A,B). As shown in Figure 2C, area under the curve of plasma glucose was significantly higher in fructose-fed adult rats and tended to be higher in fructose-fed young ones. In addition, area under the curve of plasma insulin was significantly higher in adult than in young rats whatever the dietary treatment and was significantly higher in both young and adult rats fed a fructose-rich diet compared to controls (Figure 2D). Insulin signaling pathway in the liver was assessed through the degree of activation (by phosphorylation) of downstream targets, namely IRS1 and Akt. Significantly lower phosphorylation of both proteins was found in young rats after 2 weeks of fructose feeding (Figures 3A,B), while no significant effect was detected in the liver of adult rats (Figures 3A,B).

Plasma metabolites were assessed in young and adult rats fed a control or fructose-rich diet. A significant increase in plasma triglycerides (Figure 4A) and NEFA (Figure 4B) independent of age was elicited by fructose-rich diet, while no variation was found in plasma TBARS (Figure 4C) and ALT (Figure 4D). Liver glycogen content increased with fructose-rich diet in young and adult rats (Figure 5A). Fructose-rich diet significantly increased liver ceramide content only in young rats (Figure 5B), and liver content of triglycerides only in adult rats (Figures 5C,D).

As markers of inflammation we measured both Hpt and LCN-2 in plasma samples. The level of Hpt was found significantly higher in young and adult rats fed a fructose-rich diet (Figure 6A), while no variation was detected in plasma levels of LCN-2 (Figure 6B). Further, TNF-alpha protein levels and MPO activity were higher in rats fed fructose-rich diet regardless of age (Figures 6C,D). On the other hand, the amount of hepatic LCN-2 was found significantly increased by the fructose-rich diet only in young but not in adults (Figure 6E).

Liver gene expression profiling of inflammatory molecules, namely marker of macrophages (F4/80), of classical (MCP-1, IL-1), and alternative (Arg) macrophage activation revealed a marked upregulation of all the above markers in adult rats fed a fructose-rich diet (Figures 7A–D), while in young rats only slight changes (upregulation in MCP-1 and downregulation in Arg) were evident (Figure 7).

Oxidative damage to hepatic lipids was higher both in the whole tissue and at the mitochondrial level in fructose-fed rats (Figures 8A,B), while N-Tyr level, used as marker of protein oxidative modification, was found significantly increased only in fructose fed young rats both in the whole tissue and at the mitochondrial level (Figures 8C,D). No variation was found in mitochondrial oxidative capacities (Figures 8E,F) and in Mn-SOD specific activity in both groups of rats (data not shown).

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.