Diabetes-prone male Otsuka Long-Evans Tokushima fatty (OLETF) rats (4 weeks old) and non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats were obtained from the Otsuka Pharmaceutical Company (Tokushima, Japan) and maintained in the animal facility at Gyeongsang National University (Republic of Korea). All experiments were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. The University Animal Care Committee for Animal Research of Gyeongsang National University approved the study protocol. LETO and OLETF rats were housed individually with an alternating 12-h light/dark cycle. OLETF rats (aged 12 weeks) were randomly separated into two groups (n = 9–10 per group) and were fed standard chow with or without ALA (200 mg/kg/day, Bukwang Pharmaceutical Company, Seoul, South Korea) for 16 weeks. LETO rats were fed standard chow without ALA. All rats were weighed immediately before sacrifice at 28 weeks of age.
Tissue collection and sample preparation
For tissue analysis, rats were anesthetized with Zoletil (5 mg/kg, Virbac Laboratories, Carros, France) and then perfused transcardially with heparinized saline followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS). The hearts were fixed with the same reagent for 12 h at 4°C. The samples were then processed for paraffin embedding, and 5 μm-thick sections were cut. Sections were stained with hematoxylin and eosin (H&E). The sections were visualized under a BX51 light microscope (Olympus, Tokyo, Japan), and digital images were captured and documented.
Sirius red staining
Sirius red staining is commonly used to identify collagens. To determine cardiac collagen accumulation, deparaffinized heart sections were stained with Weigert’s hematoxylin (Sigma-Aldrich, MO, USA) for 8 min, washed, and restained with picro-sirius red (Sigma) for 1 h and washed. Sections were dehydrated through graded alcohols, cleared in xylene, covered with a coverslip, and sealed with Permount (Sigma).
Sircol collagen assay
The Sircol collagen assay is a dye-binding method designed for the analysis of acid and pepsin-soluble collagens, which are newly synthesized during inflammation and wound healing. The heart tissues were frozen in liquid nitrogen and stored at -80°C prior to the assay. The collagen concentration was analysed using a Sircol assay kit (Bioclor Ltd., Northern Ireland, UK) according to the instructions provided by the manufacturer. A standard curve was derived and the collagen content of the sample was calculated.
Deparaffinized heart sections were placed in a solution of 0.3% H2O2 for 10 min. After washing, sections were treated with diluted blocking goat serum for 20 min. Slides were incubated overnight at 4°C in a humidified chamber with anti-mouse-Cu/Zn-superoxide dismutase (SOD) (1:100, Santa Cruz Biotechnology, USA) diluted in blocking serum. After washing three times with 0.1 M PBS, sections were incubated for 1 h at room temperature with a secondary antibody (1:200). After washing, sections were incubated in avidin-biotin-peroxidase complex solution (ABC solution, Vector Laboratories, Burlingame, CA, USA). Sections were developed with 0.05% diaminobenzidine (DAB, Sigma) containing 0.05% H2O2 and were dehydrated through graded alcohols, cleared in xylene, covered with a coverslip, and sealed with Permount (Sigma). Sections were visualized under a BX51 light microscope (Olympus). For immunostaining of collagen tissue growth factor (CTGF), heart sections were incubated with the rabbit anti-rat CTGF (1:500, Abcam, Cambridge, MA, USA) overnight at 4°C. Sections were incubated with AlexaFluor 594-conjugated donkey anti-rabbit antibody (1:1,000, Invitrogen, Carlsbad, CA, USA). Fluorescence was visualized under a confocal microscope (FV-1000, Olympus).
Cytosolic and nuclear fraction
For cytosolic and nuclear fractions, the hearts were promptly excised and placed in ice-cold PBS. After chopping in ice-cold lysis buffer (10 mM HEPES-KOH [pH7.9], 1.5 mM MgCl2, 10 mM KCl, 1 μg/ml aprotinin, 3 μg/ml pepstatin, 0.5 μg/ml leupeptin, 0.2 mM PMSF, 0.5 mM DTT), the hearts were homogenized. The fractions of heart were prepared according to Andrews and Faller .
The hearts were promptly excised and placed in ice-cold PBS. After chopping in ice-cold hypertonic lysis buffer (10 mM Tris, 10 mM NaCl, 3 mM MgCl2, 1 mM sodium vanadate, 5 μg/ml aprotinin, 3 μg/ml pepstatin, 5 μg/ml leupeptin, 1 mM EDTA, 1mM DTT), the hearts were homogenized. Homogenates were centrifuged at 12,500 × g for 15 min. The resulting pellet were resuspended in 1% Triton lysis buffer and centrifuged at 12,500 × g for 15 min.
Western blot analysis
For total heart extracts, frozen hearts were homogenized in a T-PER tissue protein extraction reagent (Thermo scientitic, IL, USA) containing Halt protease inhibitor cocktail (Thermo scientitic). The following antibodies were used: LKB1 (Wako Pure Chemical Company, Osaka, Japan); phospho-AMPK, AMPK, phospho-acetyl-CoA carboxylase (ACC), ACC, Sterol regulatory element-binding protein-1 (SREBP-1, BD Biosciences, CA, USA), glucose transporter 4 (GLUT4, Cell Signaling Technology, Danvers, MA, USA), receptor for advanced glycosylation end products (RAGE), heme oxygenase-1 (HO-1), Cu/Zn-SOD, and transforming growth factor-β1 (TGF-β1) (all from Santa Cruz Biotechnology). The membranes were probed with each antibody or α-tubulin antibody (Sigma) and visualized using an enhanced chemiluminescence substrate (Pierce, Rockford, IL, USA). The Multi-Gauge V 3.0 image analysis program (Fujifilm, Tokyo, Japan) was used to measure band density.
Differences between LETO, OLETF, and OLETF rats following ALA administration were determined with one-way ANOVA, followed by Bonferroni post-hoc analysis. Values are expressed as the mean ± standard error of the mean (SEM). A p value < 0.05 was considered statistically significant.