Alpha-lipoic acid attenuates cardiac fibrosis in Otsuka Long-Evans Tokushima Fatty rats

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.

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 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).

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).

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 [17].

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.

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.

OLETF rats were fed ALA for 16 weeks. Without ALA treatment, the body weight of OLETF rats at 28 weeks was significantly higher than that of LETO rats (p < 0.05) (Table 1). However, ALA caused a significant reduction in the body weight of OLETF rats (p < 0.05). Whole heart weights were measured at the time of sacrifice, and the heart/body weight ratio was calculated for each group. Although OLETF heart weights were higher than those of LETO rats, the heart/body weight ratio was significantly lower in OLETF rats without ALA treatment than in OLETF rats with ALA treatment (p < 0.05).

To determine the effect of ALA on cardiac LKB1 expression, Western blot analysis was performed (Figure 1A). Levels of cardiac LKB1 expression were significantly lower in OLETF rats than in LETO rats (p < 0.05). However, ALA significantly increased LKB1 expression in OLETF rats (p < 0.05). The effects of ALA on the phosphorylation of AMPK and ACC expression were then evaluated (Figure 1B). Western blot analysis showed that the levels of cardiac phospho (p)-AMPK and p-ACC in OLETF rats were lower than in LETO rats and that they increased after ALA administration (p < 0.05). To investigate the dynamics of the downstream AMPK signalling pathways in the heart, Western blot analysis of SREBP1 and GLUT4 expression was performed (Figure 1C and D). Compared with LETO rats, western blot revealed that there is an increase of precursor segment of SREBP-1 expression in both total and cytosolic lysates OLETF rats approximately to 1.42 and 4.51 times, respectively (Figure 1C). Also, mature segment of SREBP-1 was increased in OLETF rats compared with LETO rats. However, ALA treatment attenuated SREBP-1 expression in total and nuclear lysates from the heart of OLETF rats (1.24 and 2.76 times, respectively, p < 0.05). In the heart tissues, the GLUT4 levels in all the lysates were decreased in OLETF rats compared with LETO rats (Figure 1D). ALA treatment increased GLUT4 translocation from intracellular sites to the plasma membrane.

The effect of ALA on cardiac RAGE expression was evaluated in OLETF rats by Western blot analysis (Figure 2A). Cardiac RAGE expression was significantly higher in OLETF rats than in LETO rats, and ALA significantly decreased RAGE expression in OLETF rats (p < 0.05) (Figure 2B).

To investigate the effect of ALA on antioxidant enzyme activity in response to oxidative stress in OLETF rat hearts, Western blot analysis of HO-1 and Cu/Zn-SOD expression and immunohistochemistry of Cu/Zn-SOD were performed (Figure 3). Western blot analysis revealed that cardiac HO-1 and Cu/Zn-SOD expression levels were significantly lower in OLETF rats than in LETO rats (Figure 3A and B). However, the expression of both proteins was significantly increased in OLETF rats by ALA treatment (p < 0.05). Immunohistochemistry showed that Cu/Zn-SOD-positive cells were distributed throughout the cardiomyocytes of LETO rats and OLETF rats with ALA treatment (Figure 3C). However, without ALA treatment, Cu/Zn-SOD-positive cells were stained more weakly in OLETF rats.

To examine the effects of ALA on cardiac morphology in 28-week-old OLETF rats, H&E staining was performed (Figure 4A). No significant morphological changes were observed between LETO and OLETF rats. Sirius red-stained collagen deposits were observed in the left ventricles (LV) of OLETF rat hearts (Figure 4B and C). However, ALA treatment reduced Sirius red-stained collagen deposition. The effect of ALA on collagen synthesis in OLETF rat hearts was confirmed using the Sircol collagen assay (Figure 4D). As observed with Sirius red staining, OLETF rats had significantly more soluble collagen than LETO rats (p < 0.05). After ALA administration, a significant decrease in the quantity of collagen was observed (p < 0.05).

The effect of ALA on cardiac TGF-β1 and CTGF expression was evaluated in OLETF rats by Western blot and immunofluorescence analyses, respectively (Figure 5). Cardiac TGF-β1 expression was significantly higher in OLETF rats than in LETO rats (p < 0.05), and ALA treatment significantly decreased TGF-β1 expression in OLETF rats (p < 0.05) (Figure 5A and B). CTGF-positive cells were distributed throughout the cardiomyocytes in OLETF rats (Figure 5C). However, CTGF staining was weak in LETO rats and OLETF rats treated with ALA.

Adiponectin increases insulin sensitivity by increasing fatty acid oxidation, resulting in reduced circulating fatty acid levels and reduced triglyceride (TG) content in muscle [25]. Energy homeostasis is vital for continuous cardiac pumping activity, and adiponectin controls energy homeostasis by modifying through glucose uptake [26]. In our previous studies, serum adiponectin was shown to be expressed at lower levels in OLETF rats than in LETO rats, and ALA increased adiponectin levels in OLETF rats. [27]. AMPK is phosphorylated and activated by its upstream kinase, LKB1, and both are conserved serine/threonine kinases that regulate metabolism [28]. In this study, diabetes-prone OLETF rats had low cardiac LKB1 expression, which was increased by ALA administration. This result is consistent with the report that obese insulin-resistant Zucker rats have decreased LKB1 content in muscle [29]. Moreover, the lower expression of LKB1 in the heart correlated closely with lower AMPK/ACC signalling pathway activity. These results support a role for ALA in promoting the effects of SIRT1 activation and LKB1-AMPK signalling on insulin sensitivity [30, 31]. SREBP1, which is negatively regulated by AMPK, is a major regulator of fatty acid synthesis [32]. Consistent with the observation that AMPK inhibits lipogenesis by reducing SREBP1 expression and by activating glucose uptake via GLUT4 upregulation [27, 33], ALA reversed the increase in the levels of SREBP1 and decreased the levels of GLUT4 in OLETF rat hearts. In our previous study, we also confirmed the effect of ALA on SREBP1 and GLUT4 expression in non-alcoholic fatty liver disease of OLETF rats [27]. SREBP1 expression is significantly higher in nonalcoholic fatty liver disease than in control animals [34]. ALA reduces circulating free fatty acids (FFA) and TG levels by reducing lipid accumulation in non-adipose tissue as well as in adipose tissue [27, 35]. In addition, our study confirms that ALA may contribute to inhibit the proteolytic cleavage and nuclear translocation of SREBP-1 in the heart of diabetic OLETF rats. This finding is in agreement with the results reported by Hao et al. [36] that high glucose increase lipogenesis by increasing precursor and mature (cleaved form) segment of SREBP-1 in renal tubular cells and HKC cells. The roles of cardiac glucose uptake and insulin action have been demonstrated in mice with cardiac-specific ablation of GLUT4, which developed cardiac hypertrophy resembling that of the diabetic heart [37]. In OLETF rats, caloric restriction improves insulin resistance in association with increased adipocyte-specific GLUT4 expression. It has been reported that impairment of glucose uptake in obesity is closely associated with the reduction of cellular GLUT4 content and translocation into plasma membrane [38, 39]. Our study shows that the protein expression of both total lysates and plasma membrane is decreased, indicating that glucose metabolism would be reduced in the heart of OLETF rats. However, ALA enhanced cellular GLUT4 contents and translocation. This finding is in agreement with the results reported by Park et al. [38] and Guo et al. [33] that caloric restriction or telmisartan reduces insulin resistance by improving GLUT4 gene expression and GLUT4 translocation to the plasma membrane. Penumathsa et al. [40] also demonstrated that the antioxidant resveratrol enhances GLUT4 translocation in the STZ-induced diabetic heart. This suggests that obesity-induced cardiac dysfunction may be attributable to chronic alterations in cardiac glucose and lipid metabolism and in the levels of circulating adipokines, including adiponectin.

In addition to cardiac dysfunction caused by energy disturbances and oxidative stress, an association between the deleterious effects of AGEs and diabetic vascular complications has been suggested in many human studies [41]. Kuhla et al. [42] suggested that targeting the AGE/RAGE interaction with an inhibitor of RAGE may be of therapeutic value in oxidative stress-induced hepatic inflammation. Our results showed that ALA inhibited increased cardiac RAGE expression in OLETF rats. These data indicate that the oxidative stress-dependent AGE/RAGE interaction may be regulated by the antioxidant ALA. In this study, ALA increased the antioxidant activity in OLETF rats. Ogborne et al. [43] first reported that ALA increases HO-1 expression in human monocytic THP-1 cells. In Zucker diabetic fat rats, upregulation of HO-1 activity induced by protoporphyrin increased adiponectin levels and improved insulin sensitivity by increasing AMPK phosphorylation, and decreased adipose tissue volumes [44]. In addition to HO-1 expression, Cu/Zn-SOD expression, which was reduced in OLETF rat hearts, was increased by ALA treatment. The downregulation of antioxidant enzymes, including HO-1 and Cu/Zn-SOD, under conditions of chronic obesity or insulin resistance-induced oxidative stress, may promote the progression of diabetic cardiomyopathy.

Diabetes-induced cardiac fibrosis is a major risk factor for the progression of diabetic cardiomyopathy, which can result in cardiac cell death, fibrosis, and endothelial dysfunction [45, 46]. Guo et al. [6] demonstrated that decreased plasma adiponectin levels may contribute to myocardial hypertrophy in STZ-induced diabetic rats. Furthermore, adiponectin supplementation improved concentric cardiac hypertrophy in adiponectin-deficient mice [9]. Recent study showed that recombinant human granulocyte-colony stimulating factor (G-CSF) ameliorates cardiac diastolic dysfunction and fibrosis in OLETF rats [47]. Although cardiac hypertrophy was not detected in the OLETF rat model used in this study, recent study showed that ALA (100 mg/kg/day) reversed impairment of systolic function in STZ-treated diabetic rats compared to controls [21]. Indeed, diabetic heart disease is associated with increased interstitial fibrosis, which is caused by collagen accumulation via an increase in the level of type III collagen [48]. Consistent with the observation that ALA ameliorates cardiac fibrosis in STZ-induced diabetes [20], Sirius red staining also showed that ALA inhibited collagen accumulation in marginal regions between the right and left ventricles in OLETF rat hearts. Thus this data suggest that comparing STZ-induced diabetic rats, diabetes prone-OLETF rats induce mild diabetic cardiomyopathy. TGF-β1 is a key factor in the formation of fibrosis, which results from collagen deposition. During cardiac pathology, TGF-β1 is expressed at high levels in the heart [49]. CTGF, which is a potent profibrotic factor, induces the accumulation of collagen by stimulating cardiac fibroblasts in response to TGF-β1 [50]. Western blot analysis of TGF-β1 expression and immunofluorescence analysis of CTGF expression showed that CTGF-positive cell number was reduced by ALA treatment. Our results support the hypothesis that hyperglycemia induces changes in cardiac structure via the generation of AGEs and ROS, and via TGF-β1 stimulation [51].