All animal experiments were carried out with the Guide for the Care and Use of Laboratory Animals published by the National Academy Press (26). The protocol was also approved by the Animal Care and Research Committee of Kanazawa Medical University on the Ethics of Animal Experiments. OLETF rats, a well-known animal model for type 2 diabetes mellitus and obesity, were procured from Otsuka Pharmaceuticals (Tokushima, Japan). OLETF rats spontaneously develop obesity, hyperglycemia, hyperinsulinemia and insulin resistance during their lifespan. OLETF rats depict insulin resistance at about the age of 10–15 wks and type 2 diabetes mellitus at the age of 25–30 wks (22). Otsuka Long-Evans Tokushima (OLET) rats, which do not develop diabetes mellitus spontaneously, were used as controls. Five-week-old male OLETF and OLET rats were housed in stainless-steel wire mesh cages at 23 ± 2°C with a relative humidity of 50 ± 10% in 12-h light-dark cycles and 10–15 air changes per hour. All animals had access to sterile drinking water ad libitum throughout the experiment. Both OLETF and OLET rats were sacrificed at the age of 10, 20 and 30 wks to evaluate any histopathological changes, auto-induction of CYP2E1 through aging and related oxidative stress.
Western Blotting for CYP2E1
The effect of aging on autoinduction of CYP2E1 in OLETF and OLET rats were evaluated by Western blotting and immunohistochemical staining for CYP2E1. About 100 mg liver tissue was homogenized in 1 mL ice-cold 50 mmol/L Tris-HCl buffer (pH 8) containing 150 mmol/L NaCl, 1 mmol/L EDTA and 1% Triton X-100. The protease inhibitors 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), 5 µg/mL aprotinin and 1 µg/mL pepstatin were added to the buffer just before use. The homogenate was centrifuged at 10,000g for 10 min at 4°C, and the supernatant was collected. The protein concentration in the supernatant was determined by using the Coomassie plus protein assay (Pierce Biotechnology, Rockford, IL, USA), and the samples were stored at −20°C until assayed. The proteins were denatured and resolved on 4–20% sodium dodecyl sulfate-polyacrylamide gradient gel (Bio-Rad, Hercules, CA, USA) and electroblotted to an activated polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Nonspecific binding sites were blocked with 5% nonfat dry milk, and the membrane was incubated overnight on a rocker at 4°C with CYP2E1 polyclonal antibody raised in rabbit against rat full-length native protein as immunogen (#ab28146; Abcam, Cambridge, MA, USA). After incubation, the membrane was washed three times and treated with horseradish peroxidase-conjugated rabbit second antibody (Biomeda, Foster City, CA, USA) at room temperature for 2 h. The membrane was washed again, treated with enhanced chemiluminescence reagent (GE Healthcare, Piscataway, NJ, USA), exposed to Kodak autoradiography film (BioMax XAR, New Haven, CT, USA) and developed. The membranes were reprobed by using Western ReProbe buffer (G-Biosciences, St. Louis, MO, USA) with a monoclonal antibody to GAPDH (Novus Biologicals, Littleton, CO, USA) that served as a control protein. The Western blotting images were quantified by using Gel-Pro analyzer software (Media Cybernetics, Silver Spring, MD, USA).
Administration of Alcohol
Thirty-week-old male OLETF rats (620 ± 15 g) were administered 10 mL of 10% ethanol or water by using an intragavage tube for 5, 3, 2 and 1 d/wk for 3 consecutive weeks on a fixed time schedule. As the treated control, 30-wk-old male OLET rats (460 ± 10 g) were administered the same amount of ethanol or water concurrently. All the animals were sacrificed on d 21 from the beginning of exposure under ether anesthesia and blood, and livers were collected immediately. The liver tissue was instantly weighed, and a small portion of the tissue from the center of the lobe (about 100 mg) was cut into small pieces and fixed in RNAlater solution (Life Technologies, Tokyo, Japan) and stored at −20°C for polymerase chain reaction (PCR) studies. The median lobe of the liver tissue was cut into 3-mm pieces and instantly fixed in 10% phosphate-buffered formalin for histopathological and immunohistochemical studies. The remaining liver tissue was flash-frozen in liquid nitrogen and stored at −80°C for protein analysis.
Measurement of ALT, AST, γ-GTP Total Cholesterol, Triglycerides, Glucose, Adiponectin and Insulin in Serum
Blood was allowed to clot for 3–5 h at 37°C, and the serum was separated in the conventional method. Serum alanine transaminase (ALT) and aspartate transaminase (AST) and glucose were measured using an auto-analyzer. Serum γ-glutamyl transpeptidase (γ-GTP) was determined by using L-γ-glutamyl-3-carboxy-4-nitroanilide according the method of Theodorsen and Stromme (27). Total cholesterol present in the serum was measured following the enzymatic method of Kayamori et al. (28) using β-NAD. Serum triglycerides were determined by using a commercial kit (Sigma-Aldrich, St. Louis, MO, USA) according to the method of Bucolo and David (29). Adiponectin levels in the serum was determined using an ELISA kit (cat. no. K1002-1; B-Bridge International, Otsuka Pharmaceuticals, Otsuka, Japan) as per the manufacturer’s protocol. Serum insulin was measured by using a chemiluminescence insulin assay kit (Diagnostic Automation, Calabasas, CA, USA).
Measurement of Hepatic Triglycerides
Hepatic triglyceride content in the frozen liver tissue was determined as described before (30). Triglycerides present in the rat liver tissue were quantified by using a triglyceride reagent (#T2449; Sigma-Aldrich), which contains lipase for hydrolysis of triglycerides to glycerol. About 200 mg frozen liver tissue in a microfuge tube was treated with 350 µL ethanolic KOH (two parts ethanol:one part 30% KOH) and incubated overnight at 55°C. The tubes were vortexed well until the tissue was digested completely. The volume was brought to 1 mL with ethanol:H2O (1:1), mixed well and centrifuged at 1,500 × g for 5 min. Exactly 200 µL supernatant was transferred to a new microfuge tube, followed by 200 µL 1 mol/L MgCl2. It was vortexed well, incubated for 10 min on ice and centrifuged at 1,500 × g for 5 min. The resultant supernatant was transferred to a new microfuge tube. A total of 1 mL triglyceride reagent (Sigma-Aldrich) was diluted with 5 mL free glycerol reagent (#F6428; Sigma-Aldrich). Exactly, 1 mL of diluted triglyceride reagent was added to a 5-mL plastic tube followed by 30 µL of the sample prepared above. Different concentrations of glycerol standard (#G7793; Sigma-Aldrich) were made up to 30 µL with water, and 30 µL water was used as the blank. All tubes were vortexed and allowed to stand at room temperature for 15 min. The resultant color was read at 540 nm in a spectrophotometer. The hepatic triglyceride content was calculated as per determination of serum triglyceride concentration by using Sigma-Aldrich glycerol standard and represented as milligrams per gram fresh liver tissue.
Histopathological Evaluation of the Liver Tissue
The formalin-fixed liver tissues were processed in an automatic tissue processor optimized for liver tissue, embedded in paraffin blocks and cut into sections of thickness. The sections were stained with hematoxylin and eosin (H&E) as per the standard protocol. The stained sections were examined under an Olympus BX50 microscope attached with a DP 71 digital camera (Olympus Corporation, Tokyo, Japan) and photographed.
Liver histology in the different animal groups was assessed for NASH activity by a hepatopathologist for steatosis, lobular inflammation and hepatocellular ballooning by using the NASH Clinical Research Network scoring system (31,32). Scores for steatosis (score 0–3), lobular inflammation (score 0–3) and ballooning (score 0–3) were summed to produce the NAFLD activity score (NAS), thus ranging from 0 to 9. A liver tissue with a NAS >5 was considered as NASH.
Immunohistochemical Staining for CYP2E1 and 4-HNE in Rat Liver
Immunohistochemical staining for CYP2E1 and 4-hydroxy-nonenal (4-HNE) was carried out on paraffin liver sections to examine the upregulation of CYP2E1 and increased production of reactive oxygen species (ROS) during ethanol treatment on OLETF and OLET rats. The liver sections were deparaffinized by using xylene and alcohol and hydrated to water. Immunohistochemistry was performed using a broad-spectrum histostain kit (Invitrogen, Carlsbad, CA, USA). After blocking, the liver sections were treated with CYP2E1 (a gift from Jerome M. Lasker, CYP450-GP, Vista, CA, USA) and 4-HNE (Nikken Seil, Shizuoka, Japan) primary antibodies and incubated in a moisturized slide chamber (Evergreen Scientific, Los Angeles, CA, USA) at 4°C overnight. The sections were then washed three times in cold phosphate-buffered saline and incubated with broad-spectrum biotinylated secondary antibody for 2 h at room temperature. The slides were washed again and treated with strepta-vidin-peroxidase conjugate and incubated for another 1 h at room temperature. The final stain was developed by using 3% 3-amino-9-ethylcarbazole (AEC) in N,N-dimethylformamide. The stained sections were washed and counterstained with Mayer’s hematoxylin for 2 min and mounted by using aqueous-based mounting medium. The slides were examined under a microscope (Olympus BX50, Tokyo, Japan) attached with a digital camera (Olympus DP71) and photographed. The staining intensity in 10 randomly selected microscopic fields were quantified by using WinRoof image analyzing software (Mitani, Fukui, Japan). Data were presented as percentage square microns, where the sample with maximum staining intensity (square microns) was considered as 100%.
Quantitative Real-Time PCR
Quantitative real-time reverse transcription (RT)-PCR was carried out to evaluate the rate of expression of tumor necrosis factor (TNF)-α and peroxisome proliferator-activated receptor-γ (PPAR-γ) in the rat hepatic tissue during chronic alcohol administration in OLETF and OLET rats. Total cellular RNA was isolated from rat liver by using an RNeasy lipid tissue Mini Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. The purity of the isolated RNA was evaluated by using ultraviolet spectrometry, and the A260:A280 ratio was always >1.8. About 1–2 µg pure isolated RNA was reversely transcribed into complementary DNA (cDNA) by using Sprint RT eight-well strips (Clontech, Mountain View, CA, USA) in a total volume of 20 µL in RNAse free H2O at 42°C for 60 min. The primer set for TNF-α (TaqMan Rn00562055_m1) (NM_012675.3) and PPAR-γ (TaqMan Rn00440945_m1) (NM_001145366.1) was procured from Applied Biosystems (Carlsbad, CA, USA). The expression rate of TNF-α and PPAR-γ mRNA was quantified by using TaqMan real-time RT-PCR (7900HT Real-Time PCR system; Applied Biosystems). Each reaction was multiplexed with β-actin (Taq-Man Rn00667869_m1) (#NM_031144.2) as a housekeeping gene, and all data were normalized based on the expression levels of β-actin. All samples were run in triplicate. The quantitative PCR was performed as follows: the samples were denatured at 95°C for 20 s (1 cycle), amplification of 1 s at 95°C for denature and 20 s at 60°C for annealing (40 cycles), a final melting curve at 50°C for 1 min (1 cycle) and cooling to 25°C (1 cycle).
Arithmetic mean and standard deviation (SD) were calculated for all the data and presented as mean ± SD. All data were analyzed and compared by using analysis of variance or Student t test. A value of P < 0.05 was considered statistically significant.