Effect of telmisartan on the expression of adiponectin receptors and nicotinamide adenine dinucleotide phosphate oxidase in the heart and aorta in type 2 diabetic rats

Thirty-six male Wistar rats weighing 140-180g, purchased from Physiological Laboratory of Shanxi Medical University (Taiyuan, Shanxi, China), were used in the study. All rats were housed in a temperature-controlled room (22-24^oC) and kept on a 12 hour light/dark cycle. All animals received humane care in accordance with the principles of the Chinese Council on Animal Care. After two week’s adaptation, all rats were randomly divided into 2 groups: control (C, n = 10) and diabetic (n = 26). Control rats were fed with standard rat chow. Diabetic rats were fed with high-fat chow (ingredients: 10% refined lard, 20% sucrose, 2% cholesterol, 1% sodium cholate and 67% common food), which were provided by Animal Experimental Centre of Shanxi Medical University. Four weeks later, diabetic rats were given the peritoneal injection of a low dose of streptozotocin (30 mg/kg body weight; Sigma, St. Louis, MO, USA) [21], while the control group was given equivalent volume of citric acid buffer. After one week of STZ injection, fasting plasma glucose (FPG) was tested and the rats with FPG ≥7.8 mmol/L and insulin resistance were considered to be diabetic (n = 20) [21]. Diabetic rats were again randomly divided into diabetic (D, n = 10) and diabetic treated (DT, n = 10). Telmisartan (5mg/kg/d, Boehringer Ingelheim pharmaceutical company, Germany) [22] was administrated to diabetic treated rats by gavage for 12 weeks. The equivalent volume of normal sodium was administrated to control and diabetic rats by gavage for 12 weeks. Subsequently, blood samples were collected every 2 weeks. Blood was centrifuged at 10,000 g for 45 minutes, plasma was collected and plasma glucose levels were measured. At termination, 1 day before the experiments were finished, all animals were fasted for 12-14 h and then were anesthetized with an intraperitoneal injection of 10% chloral hydrate (0.3mL/100g body weight). The maximum descent-speed of pressure in isovolumetric relaxation period in the left ventricle (-dp/dtmax) and the maximum ascendent-speed of pressure in isovolumic contraction period in the left ventricle (+dp/dtmax) were measured by carotid artery cannula. The rats were sacrificed after blood sample had been withdrawn from abdominal cardinal vein. The blood sample was centrifuged and plasma aliquots were stored at –80°C until assays were done. The heart was immediately taken out of thoracic cavity and the abdominal aorta was immediately taken out of abdominal cavity after the rat was sacrificed. The heart and the abdominal aorta were rinsed with normal sodium and dried by filter-paper and then the heart was weighed. The ratio of heart weight to body weight was calculated. The apex of heart and a part of abdominal aorta were fixed in 10% neutral buffered formalin and processed for histological analysis. The rest part of cardiac ventricle and abdominal aorta were immediately thrown into the liquid nitrogen and then stored in the -70°C refrigerator until analyses were carried out.

Following anesthesia with an intraperitoneal injection of 10% chloral hydrate (0.3ml/100g), the neck skin was cut open and the right common carotid artery was fully exposed. A micromanometer-tipped catheter was inserted into the left ventricle through the right common carotid artery for measurement of left ventricular pressure. Left ventricular end-diastolic pressure (LVEDP) and the maximal rate of rise and decline of ventricular pressure (±dp/dt[max]) were obtained by BL-410 Bio-signal analysis system (Chengdu TME Technology Co., Ltd, Sichuan, China).

The plasma glucose level was measured by the glucose oxidase method using an autoanalyzer (Beckman Instruments, USA). Plasma cholesterol, triglyceride and free fatty acid were measured colorimetrically by using commercially available kit (SiRuiKe Biotechnology Co., Ltd, Shanghai, China). Plasma adiponectin was measured by using a commercially available ELISA kit (Westang Biotechnology Co., Ltd, Shanghai, China). Plasma insulin was measured by using a commercially available radioimmunoassay kit (China Institute of Atomic Energy, Beijing, China). Insulin sensitivity index (ISI) was calculated as the following formula: ISI = In 1 FPG × FINS.

Frozen heart tissue was pulverized and homogenized at 4°C in cold buffer (20 mM Tris-HCl, pH 7.5, 50 mM 2-mercaptoethanol, 5 mM EGTA, 2 mM EDTA, 1 mM PMSF, 10 mM NaF, 25 μg/ml leupeptin, 2 μg/ml aprotinin) and then centrifuged at 1500 g for 5 minutes at 4°C. The supernatant was collected and stored at –80°C until analyses were conducted. The protein content of the samples was measured using the Bradford protein assay [23] with the use of bovine serum albumin as a standard. Cardiac adiponectin was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Westang Biotechnology Co., Ltd, Shanghai, China).

Tissues fixed in 10% buffered formalin were embedded in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin (HE) and Masson’s trichrome for light microscopic morphologic study.

Ventricular samples and abdominal aorta were immediately fixed in 10% neutral buffered formalin overnight and embedded in paraffin. Paraffin embedded tissue blocks were sectioned at 3 μm and sections were mounted on positively charged slides. The slides were deparaffinized, rehydrated, blocked with 3% hydrogen peroxide [to block endogenous peroxidase activity, washed with phosphate-buffered saline (PBS)], and blocked with 5% normal goat serum in PBS for 30 min. The slides were subsequently incubated with primary rabbit polyclonal adiponectin receptor type 1 (1:200) and type 2 (1:400) antibody (Beijing Biosynthesis Biotechnology Co., Ltd, Beijing, China), connective tissue growth factor antibody (1:100, Wuhan Boster Biological Engineering Company Limited, Hubei, China ), MCP-1 antibody (1:100,Beijing Biosynthesis Biotechnology Co., Ltd, Beijing, China) and NF-κB antibody(1:50,Zhongshan Goldenbridge Biotechnology Co., Ltd, Beijing, China)in PBS containing 1% normal goat serum overnight at 4°C. The primary antibody was rinsed off with PBS, and the sections were incubated with biotin labeling goat anti-rabbit secondary antibodies (1:100, Zhongshan Goldenbridge Biotechnology Co., Ltd, Beijing, China) for 30 min. After three washing steps in PBS were completed, the sections were stained using horseradish enzyme labeling strepto-avidin solution (1:100, Zhongshan Goldenbridge Biotechnology Co., Ltd, Beijing, China) for 10 min, washed with PBS, coloured with 3, 3′-diaminobenzidine (DAB), and washed with distilled water. The sections were counterstained using hematoxylin, washed in running water, dehydrated in increasing grades of alcohol, and cleared in xylene before being mounted in resinous mounting medium with coverslips. Some sections incubated with nonspecific rabbit immunoglobulins (IgG) served as negative controls. Quantification was performed with the observer blinded to details. With the use of high power microscope, all slides were observed and photographed. The average of five fields under microscope (×400) in each slice were randomly selected, and the positive stained intensity or the average value of gray scale which had an inverse proportion to the positive stained intensity was quantified by using BI-2000 color image processing system (Chengdu TME Technology Co., Ltd, Sichuan, China).

Total RNA extraction was performed using Trizol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s recommendations. Primer sequences were described in Table 1. The PCR reaction was initiated by 4-minute incubation at 94°C, 32 cycles for denaturation at 94°C for 20 seconds, annealing at 54-56°C for 30 seconds, and extension at 72°C for 30 seconds, terminated after a 10-minute extension at 72°C. Ten microliters of each PCR reaction mixture were electrophoresed in a 1.5% agarose gel and bands were visualized by ethidium bromide staining. The density of the DNA bands of the PCR products was analyzed by using the software (Tianjin DiDe Technology Co., Ltd, Tianjin, China).

Extracted RNA and synthesized cDNA were described above. Real-time quantitative polymerase chain reaction was performed with 50 μl reaction volumes containing 25μl 2× FastStart Universal SYBR Green Master Mix (Roche Ltd., Basel, Switzerland), 1μl 0.3 μM of each primer, 5 μl of cDNA template, and 19 μl deionized water. Primer sequences are described in Table 2. PCR amplifications were done on a real-time fluorescence quantitative PCR detection system (Xi’an Tianlong Science and Technology Co., Ltd,Xi’an, China) using the following parameters: initial denaturation of 95°C for 10 min, followed by 40 cycles for denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 45 seconds. Relative gene expression levels were quantified using the comparative ΔCt (cycle threshold) method. This method normalized Ct values of the detected gene to the average of that of the housekeeping genes and calculated the relative expression values as fold changes of the control. Ct values were extracted using the software. Data analysis was performed using the 2^-ΔΔCt method [24].

Data are expressed as mean ± S.E.M. Statistical analysis was performed by one –way ANOVA followed by Tukey’s post hoc test. P <0.05 was considered statistically significant.

Plasma glucose levels were significantly increased in diabetic rats compared to controls. Telmisartan treatment slightly but significantly reduced the plasma glucose level in diabetic rats. The levels of plasma insulin, total cholesterol, triglycerides and free fatty acid were significantly increased in diabetic rats compared to controls and telmisartan treatment prevented these changes in diabetic rats. Insulin sensitivity index (ISI) were significantly reduced in diabetic rats compared to controls and telmisartan treatment significantly increased the insulin sensitivity index in diabetic rats. The ratio of heart weight to body weight, an index of cardiac hypertrophy, was significantly increased in diabetic rats compared to controls and telmisartan treatment significantly decreased the ratio of heart weight to body weight in diabetic rats (See table one in reference [6], a paper we previously published. The samples we used in this paper were the same as in reference [6]).

The results of the myocardium stained by Masson’s trichrome and observed through the light microscope were as following: The myocardial cells lined up in order, the spherical or oval cellular nucleus was in the same size and was stained dark and in uniformity, and the cytoplasm was stained red and in uniformity in control rats. The myocardial cells lined up in disorder, the cellular nucleus was in irregular size, breakdown and lost, the myocardial fiber was broken, the boundary between cells was unclear, and collagen fiber was increased in diabetic rats. The pathological changes were obviously improved in diabetic rats treated with telmisartan. The myocardial collagen content was increased in diabetic rats and was decreased in diabetic rats treated with telmisartan (Figure 1).

The results of the aorta stained by hematoxylin and eosin (HE) and observed through the light microscope were as following: the intima of aorta was smooth, all level cells lined up in order, endothelial cells were applanate and clung to the smooth and straight internal elastic lamina, and medial elastic lamina lined up in parallel with smooth muscle cell orderly and alternately in control rats. Endothelial cells were broken off and the shallow smooth muscle cells in medial elastic lamina were proliferated and lined up in disorder in diabetic rats. The pathological changes of aorta were obviously improved in diabetic rats treated with telmisartan (Figure 2).

Compared to controls, +dp/dtmax and -dp/dtmax were significantly reduced and LVEDP was significantly increased in diabetic rats, indicating that the heart function was significantly decreased in diabetic rats. Telmisartan treatment attenuated these changes in diabetic rats (See table two in reference [6], a paper we previously published. The samples we used in this paper were the same as in reference [6]).

The yellow positive staining of adipoR2 was mainly located in myocardial cellular member and cytoplasm. The adipoR2 expression was quantified using the average value of gray scale which had an inverse proportion to the positive stained intensity. The average value of gray scale of adipoR2 was significantly increased in the heart in diabetic rats compared to controls, indicating that the positive stained intensity of adipoR2 was significantly decreased in the heart in diabetic rats compared to controls. Telmisartan treatment significantly decreased the average value of gray scale of adiponectin receptor 2 in the heart in diabetic rats, indicating that telmisartan treatment significantly increased the positive stained intensity of adipoR2 in the heart in diabetic rats (Figure 3).

The yellow positive staining of CTGF was mainly located in myocardial cellular cytoplasm. The integrated optical density(IOD)of positive staining of CTGF was significantly increased in the heart in diabetic rats compared to controls. Telmisartan treatment significantly decreased the integrated optical density (IOD) of positive staining of CTGF in the heart in diabetic rats (Figure 4).

The yellow positive staining of NF-κB was mainly located in cellular nucleus and the yellow positive staining of MCP-1 was mainly located in cellular cytoplasm in abdominal aorta. The integrated optical density (IOD) of positive staining of NF-κB and MCP-1 was significantly increased in aorta in diabetic rats compared to controls. Telmisartan treatment significantly decreased the integrated optical density (IOD) of positive staining of NF-κB and MCP-1 in aorta in diabetic rats (Figures 5 and 6).

Plasma and myocardial adiponectin levels were significantly decreased in diabetic rats compared to controls. Telmisartan treatment significantly increased plasma and myocardial adiponectin levels in diabetic rats (See Table two in reference [6], a paper we previously published. The samples we used in this paper were the same as in reference [6]).

The mRNA expression of myocardial adiponectin receptor 2 and GLUT4 was significantly reduced in diabetic rats compared to controls. Telmisartan treatment significantly increased the mRNA expression of myocardial adiponectin receptor 2 and GLUT4 in diabetic rats (Figures 7 and 8).

The mRNA expression of myocardial MCP-1, NOX4 and p22phox was significantly increased in diabetic rats compared to controls. Telmisartan treatment significantly reduced the mRNA expression of myocardial MCP-1, NOX4 and p22phox in diabetic rats (Figures 9, 10 and 11).

The mRNA expression of adiponectin receptor 1 in abdominal aorta was significantly reduced in diabetic rats compared to controls. Telmisartan treatment significantly increased the mRNA expression of adiponectin receptor 1 in abdominal aorta in diabetic rats (Figure 12).

Adiponectin exerts its effect through adiponectin receptor 1 and 2, which are both expressed in the heart in vivo [5] and in the cardiomyocyte in vitro [4]. Adiponectin receptors were also expressed in atherosclerotic lesions, macrophages and vascular endothelial cells [5, 25]. Overweight patients with coronary artery disease had decreased surface expression of adiponectin receptors in peripheral monocytes [26]. Although the protein expression of adiponectin receptor 1 in coronary arterioles and aortas was similar between control and diabetic mice, the protein expression of adiponectin receptor 2 was significantly reduced in type 2 diabetic mice [27]. Adenovirus-mediated overexpression of AdipoR1 and 2 in vascular endothelial cells significantly enhanced the antiinflammatory effect of adiponectin [28]. Our study showed that the expression of adipoR2 in the heart and the expression of adipoR1 in aorta were decreased in diabetic rats. The decreased adiponectin receptors may be due to hyperinsulinemia since it has been reported that insulin deficiency increased, but insulin replenishment decreased the expression of adipoR1/2 in animals in vivo [29]. The decreased adiponectin and its receptors may lead to adiponectin resistance, which may limit adiponectin to produce its biological effects, especially its protective effect on diabetic heart and aorta. The decreased adiponectin and its receptors may together play a role in the occurrence and progression of cardiomyopathy and atherosclerosis in type 2 diabetic rats. Adiponectin receptors could be modulated by agonists of the nuclear receptors PPARalpha, PPARgamma, and LXR [25]. Our study showed that telmisartan, a unique angiotensin II receptor antagonist with selective PPARgamma-modulating activity [16, 17], increased the mRNA expression of AdipoR2 in the heart and the mRNA expression of AdipoR1 in the aorta in diabetic rats, indicating that angiotensin II could down-regulate, while PPARgamma agonist could up-regulate the expression of adiponectin receptors. The results of our study are in agreement with the results of studies [25, 30] showing that adiponectin receptors were expressed in adult ventricular cardiomyocytes, atherosclerotic lesions and macrophages, and upregulated by activation of peroxisome proliferator-activated receptor gamma.

Adiponectin can improve both glucose metabolism and insulin resistance [31]. Glucose enters the heart via the facilitative glucose transporters GLUT1 and GLUT4 [32]. Glucose transporter expression in the heart is altered in various pathological states. Our study showed that the mRNA expression of GLUT4 was decreased, indicating that glucose metabolism would be reduced in diabetic rat hearts. It was reported that changes in glucose transporter expression contributed to myocardial dysfunction in diabetes [33]. In addition, GLUT4-deficient mice developed striking cardiac hypertrophy [34]. Normalization of glucose homeostasis by transgenic re-expression of GLUT4 in the skeletal muscle resulted in a reversal of the cardiac pathology in mice heterozygous for GLUT4 ablation [35]. Hence, we propose that the decreased cardiac GLUT4 observed in this study may contribute to the deterioration in heart function and to the cardiac hypertrophy seen in diabetic rats. Our study showed that telmisartan treatment reduces cardiac hypertrophy, improved the heart function, and increased myocardial expression of GLUT4 in diabetic rats. The increased adiponectin and its receptors may partly explain the increased GLUT4, which may contribute to the ameliorated heart function in diabetic rats treated with telmisartan.

Heart failure, diabetes, and obesity are recognized as states of chronic inflammation. Inflammatory cytokines may play a role in all three of these conditions [36, 37]. Monocyte chemotactic protein-1 (MCP-1) has been shown to be produced and released by the heart and to be increased in failing heart [38, 39]. Increased levels of MCP-1 are associated with previously unknown abnormal glucose regulation in patients with acute ST-elevation myocardial infarction [40]. Our study showed that cardiac expression of MCP-1 was increased in diabetic rats. Telmisartan treatment decreased the cardiac expression of MCP-1 in diabetic rats. It was reported that adiponectin suppressed MCP-1 production in lipopolysaccharide-treated 3T3-L1 adipocytes [41]. Oxidative stress increased the level of MCP-1[42]. Therefore, the elevated cardiac MCP-1 may be due to the decreased adiponectin and the increased oxidative stress, and may be involved in the diabetic cardiac deterioration. The increased adiponectin and decreased NADPH oxidase by telmisartan treatment may lead to the decreased inflammation factor MCP-1 in the heart, which may result in the improved heart function in diabetic rats.

This study showed that the expression of MCP-1 and NF-κB was increased in aorta in diabetic rats. Telmisartan treatment significantly reduced the expression of MCP-1 and NF-κB and alleviated the pathological alteration in aorta in diabetic rats, suggesting that telmisartan may lessen the atherosclerotic degree by downregulating the expression of MCP-1 and NF-κB in aorta in diabetic rats. It was reported that adiponectin reduced aortic NF-kappaB expression in ApoE knockout mice and inhibited IκBα phosphorylation and nuclear factor κB protein expression in aorta of type 2 diabetic mice [27, 43]. Adenovirus-mediated overexpression of AdipoR significantly enhanced the suppressive effect of adiponectin on NF-kappaB activation in vascular endothelial cells [28]. Therefore, the increased expression of MCP-1 and NF-κB in aorta may be associated with the reduced expression of adiponectin and its receptors.

Oxidative stress has been suggested to be involved in the development and progression of diabetes-induced cardiomyopathy (7). NADPH oxidase has emerged as the main source of ROS in the cardiovascular tissues [44]. It was reported that the expression of NADPH oxidase subunits, p22phox and Nox4 was significantly increased in the heart of diabetic mice and rats [13, 14, 45, 46], indicating that Nox4 was an important source of ROS in the left ventricle and Nox4-derived ROS contribute to cardiomyopathy at early stages of type 1 diabetes [45]. Consistent with these studies, the present study also showed that the expression of myocardial p22phox and Nox4 was significantly increased in type 2 diabetic rats, suggesting that NADPH oxidase might play a crucial role in the development of diabetic cardiomyopathy. It was reported that telmisartan treatment reduced the protein expression of renal and vascular NADPH oxidase subunits in diabetic mice and cardiac NADPH oxidase activity in diabetic rats [47, 48]. Our study showed that telmisartan treatment decreased the myocardial mRNA expression of NADPH oxidase subunits, p22phox and Nox4, which may result in the reduced oxidative stress and the improved heart function, in diabetic rats.