2,2′-Azino-bis-(3-ethylbenzthioazoline-6-sulfonic acid) (ABTS) was purchased from VWR (Fontenay sous Bois, France), amyloglucosidase (AMGD) from Roche Diagnostic (Meylan, France), glucose and phosphate-buffered saline (PBS) from Fisher Scientific (Illkirch, France), eosin, Harris hematoxylin, paraffin, ethanol and toluene from Labonord (Templemars, France). The (+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and all other products were purchased from Sigma-Aldrich (St Quentin Fallavier, France).
The study was performed in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH publication No. 85–23, revised 1996), and the present protocol was approved by the local ethics committee (Comité Régional d’Ethique en Matière d’Expérimentation Animale CREMEAS, approval AL/02/11/05/12). All efforts were made to minimize animal suffering and reduce the number of animals used.
Animals and induction of diabetes
Sixty-five male Wistar rats (8 weeks old; 204 ± 1 g), supplied by Depré (Saint Doulchard, France), were housed in a temperature-controlled room, in a 12-h-light/dark cycle environment with ad libitum access to water and food. At the beginning of the study, 5 rats were sacrificed (Ctr-rats, M0). After 2 weeks, the rats (312 ± 2 g) were randomly divided into four groups of 15 rats each. The first group had free access to a standard diet “Normal Diet” (ND) from SAFE (Augy, France), with the following macronutrient composition: 3.1 % fat, 16.1 % protein, 3.9 % fibre, and 5.1 % ash (minerals). The second group “High Fructose” (HF) had the same normal diet, but with an additional 25 % of fructose (Sigma, France) in water. The third group, “High Fat Diet” (HFD), received a purified laboratory hypercaloric rodent diet “WESTERN RD” (SDS, Special Diets Services, Saint Gratien, France) containing 21.4 % fat, 17.5 % protein, 50 % carbohydrate, 3.5 % fibre, and 4.1 % ash. The fourth group, “High Fat High Fructose” (HFHF), had both the enriched diet and fructose in water. Both groups had free access to water. The body weight and calorie intake of each animal was recorded once a week. 5 rats were sacrificed at the beginning of the study (M0), and then 5 rats of all groups were sacrificed at 2 and 8 months (M) after starting administration of each diet.
Before anaesthesia, body weight was recorded, capillary glucose levels were measured, and tail vein blood samples were taken to estimate metabolic parameters. After anaesthesia with an intraperitoneal injection of 50 mg/kg pentobarbital (Centravet, France), blood was drawn from the abdominal aorta, and plasma and serum were frozen in liquid nitrogen and stored at −80 °C after centrifugation (4 °C, 2 min, 10,000 × g) for later biochemical analysis. Liver tissue was cleaned, weighed and embedded in Tissue-Tek® OCT (Optimal Cutting Temperature compound, Leica Microsystem SAS, Nanterre, France) or directly frozen in liquid nitrogen and stored at −80 °C. The main superior mesenteric artery was excised and bathed in Krebs bicarbonate solution (119 mM NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4, 1.25 mM CaCl2, 25 mM NaHCO3, and 11 mM d-glucose, pH 7.4, 37 °C) for dissection.
Biochemical plasmatic analysis
Plasmatic metabolic parameters
Glucose tolerance was evaluated by measuring intraperitoneal glucose tolerance (IpGTT) of fasting rats. Capillary glycaemia at baseline and 15, 30, 60, and 120 min after an intraperitoneal (IP)-injection of 2 g/kg glucose (20 % solution) was measured with a glucometer (Accu-Chek Performa®, Roche Diagnostic, France). Blood samples were collected from the tail vein at 0 and 60 min after injection, in order to measure blood glucose (glucose RTU®, Biomérieux, France) and C-peptide levels (Elisa C-peptide kit, Mercodia, Uppsala, Sweden) to evaluate insulin sensitivity. Measuring C-peptide was preferred to measuring insulin for evaluating insulinemia, because it is more stable in blood and is not affected by haemolysis . Results were expressed in g/L for plasma glucose and in pmol/L for plasma C-peptide. Fasting leptin was measured by ELISA (Elisa Leptin kit, Linco Research Inc., St Louis, MO, USA) as An index of fat mass . Plasmatic cholesterol was quantified by a colorimetric method Cholesterol RTU™ (BioMérieux, Lyon, France) using a cholesterol calibrator. Insulin resistance was evaluated using the homeostasis model assessment (HOMA2). HOMA2-IR was calculated for fasting plasma glucose and fasting C-peptide using the HOMA2 model calculator (http://www.dtu.ox.ac.uk/homacalculator). All parameters were measured once a month.
Plasmatic inflammatory and oxidative parameters
TNFα was assessed on plasma according to the manufacturer’s instructions (Rat TNF-α ELISA Kit, Millipore, Fontenay sous Bois, France). Total antioxidant capacity (TAOC) with the radical cation ABTS•+ was performed by a trolox equivalent antioxidant capacity method as previously described . Lipid peroxidation as a consequence of OS was estimated by measuring TBARS using a kit (OxiSelect™ TBARS Assay Kit-MDA Quantitation, Cell Biolabs Inc., San Diego, CA, USA) according to the manufacturer’s instructions, and expressed in μmol/L TBARS.
Histological and functional studies
Morphological analysis and immunohistochemistry
The degree of hepatic histological changes was assessed by eosin/hematoxylin coloration, Oil Red O (steatosis), and Masson’s Trichrome (fibrosis) staining on 10-μm cryosections fixed with 4 % paraformaldehyde. Steatosis was evaluated according to the standard Kleiner Classification  of grading and staging. Degree of steatosis was scored as the percentage of hepatocytes per lipid droplet: 0 (less than 5 %), 1 (between 5 and 33 %), 2 (between 33 and 66 %) and 3 (higher than 66 %), complicated or not by fibrosis.
In situ liver macrophages
As previously described by Dal S et al.  frozen-embedded liver sections (10 μm) were fixed and incubated with rabbit anti-Iba-1 (Rat, 1:1000, Wako Chemicals GmbH, Germany). Macrophage density was expressed as the percentage of brown pixels per field in comparison to control values (100 %). Six slides were prepared for each animal, and five fields were analysed per slide at a magnification of × 20.
Hepatic triglycerides and glycogen quantification
Extraction of hepatic triglyceride content was performed on piece of fresh liver (100 mg) mixed with a high-speed homogeniser (Polytron PT MR2100, Kinematica AG, Luzern, Switzerland) in a chloroform and methanol buffer (CHCl3/Methanol/H2O, v/v: 2/1/0,6), and centrifuged (1000 × g, 10 min, ambient temperature). The clot was mixed with a fresh solution of chloroform-Triton (X100, 2 %), evaporated (55 °C), and diluted in milli-Q water. Triglycerides were determined using the Triglycerides Quantification Kit (Abcam, Paris, France) according to the manufacturer’s instructions. Samples were measured at 550 nm and concentrations were expressed in nmol/mg of liver.
Hepatic glycogen content extraction was performed on piece of fresh liver (100 mg) according to the method described previously  and expressed in mg of glycogen/mg of liver.
Tissue oxidative stress
The oxidative fluorescent dye dihydroethidine (DHE) was used to evaluate in situ formation of ROS according to a method described by Dal-Ros et al. . Unfixed liver and mesenteric artery were cut into 10-μm-thick sections, treated with DHE (2.5 μM), and incubated in a light-protected humidified chamber at 37 °C for 30 min. The level of ROS was determined using microscopy and whole fluorescence of tissue was quantified with the microscope assistant (NIS-Elements BR, Nikon, France), and expressed as a percentage of that in age-matched ND rats.
As previously described , liver tissue (5 mg) from experimental rats was homogenized using NP-40 buffer (NaCl 150 mmol/L, 1.0 % Triton X-100, Tris 50 mmol/L, pH 8) with a protease/phosphatase inhibitor cocktail (Roche Diagnostics, Meylan, France) using an ULTRA-TURRAX. Supernatants were collected and protein contents measured by the Bradford method  SOD and catalase activities were performed (50 mg of proteins) according to the manufacturer’s instructions (Superoxide dismutase assay kit and Catalase Assay Kit, Abcam, Paris, France) and expressed respectively in % of inhibition rate and (μmol/L). Lipid peroxidation was estimated by measuring TBARS using a kit (OxiSelect™ TBARS Assay Kit-MDA Quantitation, Cell Biolabs Inc., San Diego, CA, USA) according to the manufacturer’s instructions, and expressed in μmol/L TBARS/mg of proteins.
Vascular reactivity studies
Mesenteric artery rings were suspended in organ baths for the determination of changes in isometric tension, as described previously . The NO-mediated component of relaxation was determined in the presence of indomethacin (10 μM) and charybdotoxin (CTX) plus apamin (APA) (100 nM each) to rule out the formation of vasoactive prostanoids and EDHF, respectively. The EDHF-mediated component of relaxation was determined in the presence of indomethacin (10−5M) and NѠ-nitro-l-arginine (L-NA, 10−4M) to rule out the formation of vasoactive prostanoids and NO, respectively. Levcromakalim- (an ATP-sensitive K+channel opener; 0.1 nM–10 μM) induced relaxations were examined in endothelium-denuded rings of mesenteric artery to test the vascular smooth muscle cells relaxations without EDHF production by endothelial cells.
Values are expressed as means ± SEM, and n indicates the number of rats. Statistical analysis was performed with Student’s t-test for unpaired data or ANOVA followed by Tukey’s protected least-significant difference test, where appropriate (Statistica®, StatSoft, France). p < 0.05 was considered to be statistically significant.