Fish protein was isolated from sardine fillets obtained from a fishery (Oran, Algeria). The heads, internal organs and bones of the sardines were removed. The proteins of the muscle tissue were then solubilized in 10 volumes of water with NaOH (Sigma-Aldrich, St. Louis, MO, USA) added to obtain pH 10.5 according to the method outlined by Undeland et al (20). The mixture was centrifuged (18,000 x g, 20 min, 4°C), causing the light oil fraction to rise to the top of the suspension, then the muscle proteins were precipitated and collected. The crude protein content was determined by the Kjeldahl method (21). The crude fat content was measured by the Soxhlet method (22). The moisture content was calculated as the loss in weight following drying at 105°C for 24 h. The ash amount was analyzed by direct ignition at 550°C for 24 h. The amino acid composition of the sardine protein, as determined through analysis by a commercial service (Institute of Chemistry, Center for Technical and Scientific Research in Physical-Chemical Analysis, Algiers, Algeria), is presented in Table I.

A total of 24 male Wistar rats obtained from Iffa-Credo (l'Arbresle, France) weighing 190–200 g at the beginning of the experiment were used for the current study. The rats were kept in a laboratory animal house with a 12 h light:dark cycle (07:00–19:00). Throughout the experiment, the temperature of the animal room was maintained at 24°C and humidity at 60%. Rats were assigned to four equal-weight groups and fed the following diets for 2 months: Group 1 (C-HF), diet containing 20% casein (C) and 64% fructose (Prolabo, Paris, France); group 2 (S-HF), 20% sardine protein and 64% fructose; group 3 (C), 20% casein; group 4 (S), 20% sardine protein. The compositions of the experimental diets are presented in Table II. Diets were isoenergetic (16.28 MJ/kg) and contained identical amounts of lipids, vitamins (UAR 200; UAR, Villemoisson-sur-Orge, France), minerals (UAR 205 B; UAR) and fiber. Food and water were provided ad libitum. The body weights of the animals were recorded every week and food intake was measured daily. The food efficiency ratio was calculated as follows: Weight gain (g) / food intake (g), where weight gain was calculated as the difference between the final weight and the initial weight of each rat during the 60-day experiment and food intake was determined as the difference between the rest amount of food and the amount of food administered for each rat during the 60-day experiment. The glycemia was measured weekly using a handheld glucometer (Accu-Chek Aviva; Roche Diagnostics, Basel, Switzerland) with blood obtained from the caudal vein. The general guidelines for the Care and Use of Laboratory Animals as set forth by the Council of European Communities were followed (23).

At the end of the experimental period, the rats were sacrificed by anesthesia using intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight; Abbott Laboratories, North Chicago, IL, USA) following overnight starvation. Blood samples were collected from the abdominal aorta in citric acid tubes, and the plasma was separated by centrifugation (3,000 × g, 15 min, 4°C) and stored at -70°C until required for chemical analysis. Liver, kidney, heart and gastrocnemius muscle tissues were harvested, washed with ice-cold 150 mmol/l NaCl (Sigma-Aldrich), weighed and immediately frozen at -70°C until required for analysis. Urine samples were collected on day 59 of the experiment in the four groups.

Plasma glucose was analyzed by the method previously described by Bergmeyer et al (24). Insulin in plasma was measured by radioimmunoassay according to Leclercq-Meyer et al (25). These measurements were used to calculate the homeostasis model assessment (HOMA) index (mmol/l/22.5): [The product of the plasma insulin concentration (mmol/l) × plasma D-glucose concentration (mmol/l)]. GLP-1 level was measured in plasma using a GLP-1 (Active) ELISA kit (BioVendor Research and Diagnostic Products, Karasek, Czech Republic). Plasma fructose levels were determined enzymatically using a BioSentec Glucose/Fructose/Sucrose kit (BioSentec, Toulouse, France). Protein concentrations were measured according to the method of Lowry et al (26) using bovine serum albumin (Sigma-Aldrich) as a standard. Plasma and urine creatinine, uric acid and albumin levels were determined using an enzymatic Spinreact Colorimetric Kinetic Jaffé kit (cat. no. 1001110), a Uricase-POD Spinreact Enzymatic Colorimetric kit (cat. no. 1001011), and a Bromocresol Green Spinreact Colorimetric kit (cat. no. 1001020; Spinreact, Girona, Spain).

The level of lipid peroxidation in the liver, kidney, heart and muscle tissues was studied by measuring thiobarbituric acid reactive substances (TBARS) in tissue homogenates using the method of Quintanilha et al (27). For TBARS measurement, the tissue homogenates were deproteinized with 10% trichloroacetic acid (TCA) (Sigma-Aldrich) and the precipitate was treated with thiobarbituric acid (Sigma-Aldrich) at 90°C for 1 h. The pink color formed gave a measure of the TBARS. The concentration was expressed as µmol/g tissue. Additionally, liver, kidney, heart and muscle hydroperoxides were assayed using the method described by Eymard & Genot (28). The color developed was read at 560 nm using a Beckman Coulter DU 640 spectrophotometer (Beckman Coulter, Inc., Cridersville, OH, USA). The concentration was expressed as µmol/g tissue. The level of protein carbonyl was measured by the method of Levine et al (29). The tissue was homogenized in 10 mM HEPES buffer (Sigma-Aldrich) containing 137 mM NaCl, 4.0 mM potassium chloride, 1.0 mM potassium dihydrogen phosphate and 0.6 mM magnesium sulfate (Sigma-Aldrich). The homogenate was centrifuged at 40,000 × g for 20 min at 25°C. The supernatant was mixed with dinitrophenyl hydrazine in 2M hydrochloric acid and allowed to stand at room tempera ture for 1 h. The protein hydrazone derivative was precipitated with TCA and the precipitate was washed three times with ethanol ethylacetate (1:1) (Sigma-Aldrich). The color in the supernatant was read at 390 nm using a Beckman Coulter DU 640 spectrophotometer. The concentrations were expressed as nmol/g tissue. Tissue nitric oxide (NO) was assessed using the Griess reagent (sulfanilamide and N-naphthyl ethylenediamine) (30). Tissue homogenates were clarified by zinc sulfate solution (Sigma-Aldrich), NO₃ (Sigma-Aldrich) was then reduced to NO₂ by cadmium (Sigma-Aldrich) overnight at 20°C under agitation. Samples were added to the Griess reagent and incubated for 20 min at room temperature. The absorbance of these solutions was measured at 540 nm using a Beckman Coulter DU 640 spectrophotometer. Sodium nitrite (Sigma-Aldrich) was used for the standard curve. The data were expressed as µmol/g tissue.

Liver, kidney, heart and muscle tissue homogenates prepared on ice at a ratio of 1 g wet tissue to 9 ml 150 mmol/l KCl using a POLYTRON^® PT 2100 homogenizer (Kinematica AG, Lucerne, Switzerland), were used for superoxide dismutase (SOD; EC 1.15.1.1), glutathione peroxidase (GSH-Px; EC 1.11.1.9) and catalase (CAT; EC 1.11.1.6) determinations. Tissue SOD activity was determined using a SOD and GSH-Px Reagent Assay Cayman Chemical kit (Cayman Chemical Company, Ann Arbor, MI, USA). Briefly, the method uses xanthine and xanthine oxidase to generate superoxide radicals, which react with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride to form a formazan dye. The SOD activity was measured by the degree of inhibition of the reaction, using a spectrophotometer. The results were expressed as U/mg of protein. CAT activity was determined according to the method described by Aebi (31) and the results were expressed as nmol/mg of protein. Tissue GPH-Px activity was measured using an enzymatic method with a kit from Cayman Chemical Company. The data were expressed in nmol/min/mg of protein.

Liver α-tocopherol levels were determined by the method as described by Baker et al (32). The level of α-tocopherol was estimated by the reduction of ferric ions to ferrous ions by α-tocopherol and the formation of a red-colored complex with 2,2-dipyridyl was measured at 520 nm using a Beckman Coulter DU 640 spectrophotometer. Ascorbic acid concentrations were measured using a LiChrospher 100 RP18 high performance liquid chromatography instrument (EMD Millipore, Billerica, MA, USA).

Values are presented as the mean ± standard deviation of six rats per group. Statistical analysis of the data was conducted with Statistica software, version 6 (Dell Software, Aliso Viejo, CA, USA). Data were analyzed using two way analysis of variance with the type of protein and fructose as independent variables. When the interaction was significant, Fisher's protected least significant difference test was performed. P<0.05 was considered to indicate a statisti cally significant difference.

Following 8 weeks of feeding, the HF-fed rats were significantly heavier than the control rats, despite a low cumulative energy intake over the 8 weeks of study (P<0.05; Table III). Consequently, food efficiency was significantly higher (P<0.05) in HF-fed animals as compared with control animals. Consistent with this, the weights of the liver, kidney and heart were greater in rats on the HF diets compared with control rats. The constituents (g/100 g) of the protein obtained by these operations were 93 g protein, 0.9 g lipids, 2.5 g ash and 3.6 g moisture. Rats on sardine protein diets gained less weight and had reduced food and energy intakes than those fed casein diets. The liver weight was significantly reduced with S and S-HF diets, while heart, kidney and skeletal muscle weights were not significantly different among HF rats with any of the protein sources.

The results of the time course of glycemia assessed in the rats is presented in Fig. 1. Rats receiving HF diets exhibited significantly higher blooD-glucose levels at 7, 14, 21, 28, 35, 42, 48 and 60-days compared with control diets. In S-HF rats, the glycemia was significantly reduced at 35, 42, 48 and 60-days of the experiment compared with C-HF rats. In addition, the values recorded in the S group were significantly reduced at days 48 and 60 compared with the C group. However, the results of blooD-glucose were closely comparable between the two protein groups on days 7, 14, 21 and 28 of the experimental period.

Following 8 weeks on the HF diet, it was observed that plasma glucose was significantly increased as demonstrated by the data presented in Table IV. Plasma insulin levels were increased only in S-HF-fed rats as compared with S fed rats. The HOMA-insulin resistance (HOMA-IR) index was observed to be higher in the HF groups compared with the control groups. Likewise, plasma GLP-1 levels were significantly reduced in the HF group compared with control rats. In addition, rats on HF diets had higher plasma fructose levels than those fed control diets. Plasma and urine creatinine and uric acid, and urinary albumin concentration were significantly higher (P<0.05) in rats fed the HF diet than in those on the C-diet. In the rats fed the HF diet, it was observed that the S-HF diet, compared with the C-HF diet, attenuated the rise in plasma glucose (21%), insulin (35%), HOMA-IR (42%) and fructose (17%), and increased the GLP-1 (29%) level. Consumption of the S diet significantly reduced plasma insulin, fructose and HOMA-IR compared with the C-diet. Rats fed the S-HF diet exhibited reduced plasma levels of creatinine (37%) and uric acid (22%), and urine levels of creatinine (34%) and albumin concentration (16%) compared with rats fed the C-HF diet. In addition, the S diet reduced plasma levels of creatinine (25%) and uric acid (21%), and urinary levels of uric acid (29%) and albumin concentration (29%) compared with the C-diet.

Table V presents the status of oxidative stress parameters in the tissues of experimental and control rats. Addition of fructose to the protein diets resulted in oxidative stress that was demonstrated by the increase in TBARS in the heart and liver tissues compared with control diets. In the kidneys, the concentration of TBARS was significantly increased in rats fed the C-HF diet compared with rats fed the C-diet. Higher hydroperoxide and protein carbonyl levels, however reduced NO contents in all tissues were observed in all HF-fed rats compared with control rats. Additionally, following the treatment with sardine protein (S-HF group), hydroperoxide concentrations in the liver (31%), heart (16%), kidney (11%) and muscle (19%) tissues were significantly reduced compared with the C-HF group. In addition, the S group rats presented with low hydroperoxide (25%) levels in the liver tissue. Protein carbonyl and NO contents were significantly reduced in the liver, kidney and heart tissues of S-HF-fed rats compared with C-HF-fed rats. Feeding rats the S diet led to reduced carbonyls in the heart and reduced NO concentrations in the kidney and heart compared with the C-diet.

Following 8 weeks of feeding, the diets supplemented with high dietary fructose induced a significant reduction in the activity of SOD, CAT and GSH-Px in all tissues in comparison with the control diets (Fig. 2) (33). Feeding rats the S diet resulted in an increase in liver SOD activity (21%) and in the CAT activity of the liver (13%) and muscle (28%) compared with the C-diet. The administration of the S-HF diet to rats increased SOD activity in liver (31%) and kidney (14%) tissues compared with the C-HF diet. Furthermore, liver and muscle CAT activity was increased in S-HF-fed rats by 28 and 48%, respectively, when compared with the C-HF-fed rats. The activity of GSH-Px, which serves a role in peroxide removal, was significantly higher in the liver (31%), heart (51%) and muscle (24%) homogenates of S-HF-fed rats than C-HF-fed rats.

A reduction in liver ascorbic acid and α-tocopherol levels was observed in the HF-fed rats compared with the control diets (Table VI). Administration of sardine protein to rats with or without fructose significantly increased levels of liver ascorbic acid relative to casein fed rats. In addition, the levels of liver α-tocopherol were higher in the S group than in the C group.