Ying Fan, Shan-xiao Zhang, Meng Ren, Li-feng Hong*, and Xiao-ni Yan

1Department of Cardiology, the Fifth Hospital of Wuhan & Institute of Cardiovascular Sciences of Jianghan University, Wuhan 430050, China

2Department of Cardiology, the First Hospital of Yangtze University, Jingzhou, Hubei 434000, China

ORIGINAL ARTICLE

Impact of 1, 25-(OH)2D3on Left Ventricular Hypertrophy in Type 2 Diabetic Rats△

Ying Fan1, Shan-xiao Zhang2, Meng Ren1, Li-feng Hong1*, and Xiao-ni Yan1

1Department of Cardiology, the Fifth Hospital of Wuhan & Institute of Cardiovascular Sciences of Jianghan University, Wuhan 430050, China

2Department of Cardiology, the First Hospital of Yangtze University, Jingzhou, Hubei 434000, China

1, 25-(OH)2D3; left ventricular hypertrophy; type 2 diabetes mellitus; rat

Objective To investigate the impact of 1, 25-(OH)2D3on left ventricular hypertrophy (LVH) in type 2 diabetic rats.

Methods Type 2 diabetic mellitus (DM) model rats were established by intraperitoneally injecting with 30 mg/kg streptozotocin. After 8 weeks, 19 male rats were identified as diabetic with left ventricular hypertrophy (LVH) by ultrasound examination, and randomly assigned into three groups：untreated (DM-LVH, n=7), treated with insulin (DM-LVH+INS, n=6), and treated with 1, 25-(OH)2D3(DM-LVH+VD, n=6). Healthy male rats were used as the controls group (n=6). The fasting blood glucose and the insulin level were determined weekly. The left ventricular mass index, myocardial collagen content, collagen volume fraction, and 1, 25-(OH)2D3-receptor level were determined by 4 weeks later.

Results In the DM-LVH model group, the insulin level was significantly decreased compared with the non-diabetic control group (p＜0.05), whereas the blood glucose, left ventricular mass index, myocardial collagen content, collagen volume fraction, and 1, 25-(OH)2D3-receptor expression were significantly increased (all p＜0.05). In the DM-LVH+INS and DM-LVH+VD groups, the insulin levels were significantly increased compared with the DM-LVH model group (p＜0.05), whereas the other parameters were significantly decreased (all p＜0.05).

Conclusion 1, 25-(OH)2D3could reverse LVH in diabetic rats and that the mechanism may involve stimulating insulin secretion and reducing blood glucose via direct up-regulation of 1, 25-(OH)2D3-receptor expression.

Chin Med Sci J 2015; 30(2)：114-120

TYPE 2 diabetes mellitus (DM) is considered to be a metabolic disorder syndrome resulting from the relative lack of insulin and/or reduced insulin biological activities, leading to high blood glucose. Consequently, it may involve bodily systems and multiple organs, having a particularly strong impact on the cardiovascular system. Diabetic cardiomyopathy is a disorder of the heart muscle in DM, and in particular, it is associated with three types of manifestations: coronary heart disease (CHD), cardiomyopathy, and cardiac autonomic neuropathy.1 Cardiomyopathy is a critical complication of DM with a mortality rate ranging from 70%–80%, and its importance has been considerably underestimated.2-3 Previous studies indicated that left ventricular hypertrophy (LVH) is an independent marker for cardiac events in type 2 DM patients, which include acute myocardial infarction, fatal arrhythmias, and sudden cardiac arrest.4-7 Therefore, reversal of LVH and reducing the cardiovascular events in diabetic patients is of vital importance.

Earlier studies reported that 1α, 25-dihydroxy vitamin D3 [1, 25-(OH)2D3] could exert some protective effects on islet β cells, including maintaining insulin secretion, ameliorating the inflammatory response and endothelial injury, and reducing the frequency of cardiovascular disease and its effects.8-10 However, whether 1, 25-(OH)2D3 can reverse LVH remained unknown. In this study, we investigated the impact of 1, 25-(OH)2D3 on myocardial hypertrophy in diabetic rats, in order to provide some new ideas for the prevention of diabetic cardiomyopathy.

MATERIALS AND METHODS

Model establishment

Forty-six Wistar rats weighing 196 ± 18 g were purchased from the Laboratory Animal Center of Zhongnan Hospital of Wuhan University. We attempted to establish diabetes in 46 male Wistar rats. Streptozotocin (STZ, Sigma-Aldrich, San Francisco, CA, USA) was dissolved in 0.1 mol/L sodium citrate buffer solution (pH 4.4) to obtain the final concentration of 1% solution, and injected intraperitoneally at 30 mg/kg body weight after rats were fasted for 10 hours.

A tail vein was pricked 5 days later to obtain blood to determine the blood glucose using the One Touch blood glucose meter. Rats with a blood glucose level ≥16.7 mmol/L were considered to have type 2 diabetes, resulting in 37 eligible rats for the in vivo studies. Then rats were housed 5 per cage, the water was changed regularly and a high-fat high-sugar diet was provided. When changing the bedding, we monitored the general condition of the rats, including their weight and water consumption as well as the cage-bottom bedding wetness.

Ultrasonography

The heart section was monitored by ultrasonography and the standard measurements in cardiac hypertrophy in the rats were defined previously.11-12Rats were anesthetized using 1% pentobarbital sodium intraperitoneally. The rats were fixed on a board in the supine position, the skin of the left chest was prepared, and a high-frequency linear-array probe of 7-15 MHz was applied to detect the heart. The left ventricular long-axis was also viewed from the two-dimensional ultrasound-guided M-curve. Ultrasound indicators determined included left ventricular posterior wall thickness diastolic (LVPWTd), interventricular septum thickness diastolic (IVSTd), and left ventricular end diastolic diameter (LVDd), from which we calculated the left ventricular mass (LVM)=1.04×[(LVDd+LVPWTd+IVSTd)3-LVDd3]-13.6.13

The 37 diabetic rats were screened for myocardial hypertrophy by ultrasonography during the first 8 weeks. Defining myocardial hypertrophy in diabetes as an LVM＞985.55 mg, a total of 19 diabetic rats could be considered as fulfilling this criterion for a diabetic cardiac hypertrophy model.

Treatment groups

Nineteen model rats for diabetic myocardial hypertrophy were randomly divided into three groups: (1) DM-LVH group: 7 successful model rats for diabetic myocardial hypertrophy with no treatment; (2) DM-LVH+INS group: 6 successful model rats given regular subcutaneous 25 U/kg insulin injection for 4 weeks; (3) DM-LVH+VD group: 6 successful model rats given daily gavage of 2 μg/kg·d 1, 25-(OH)2D3(Sigma-Aldrich) for 4 weeks. Meanwhile，healthy male rats were used as the controls group (n=6).

Monitoring fasting plasma glucose and insulin

The rats were fasted for 8 hours each time prior to determination of the fasting blood glucose level. The blood glucose test was performed using the peripheral blood glucose tester SureStepPlus (Lifescan Inc., USA) on blood obtained by cutting a vein in the tail tip weekly.

The insulin level was determined weekly following 8 hours of fasting using an insulin radioimmunoassay kit.

Determination of LVM index

After 4 weeks of treatment, all of the rats weresacrificed after weighing and the heart removed. We separated the left and right ventricles, and determined the LVM using an electronic balance. We calculated the LVM index (the left ventricular weight to body weight ratio). In addition, we confirmed the presence of LVH in the model rats.

Semi-quantitative analysis of the myocardial collagen morphology

A portion of the left ventricle muscle tissue was taken, fixed in 4% formalin, and embedded in paraffin, from which 4-μm slices were cut. We stained the slices using Van-Gieson (VG) staining for collagen (under light microscopy the myocardial cells were yellow and collagen red), then analyzed the slices by automatic image analyzer. We selected one slice from each specimen to determine the left ventricular collagen volume fraction (CVF=left ventricular collagen area/visual-field area measured) from 6 randomly selected views.

Determination of collagen content

The collagen content was determined from the hydroxyproline content of the myocardial tissue using the hydroxyproline kit according to the manufacturer’s instructions (Nanjing Jiancheng Bio-Engineering Company, China). Because the mean hydroxyproline content of interstitial collagen is 13.4%, the collagen content was calculated by multiplying the hydroxyproline content by 7.46, giving a value in mg/g tissue.

Western blot analysis of vitamin D-receptor (VDR) levels

We obtained 200 mg of myocardial cells from each group for lysis. From the lysate, 20 μg of total protein extract were separated by 5%-8% discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was incubated with rabbit anti-rat VDR monoclonal antibody (diluted 1:500, Santa Cruz Inc., CA, USA) followed by horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (Santa Cruz Inc.), the color developed using 3,3'-diaminobenzidine, using phosphate-buffered saline to terminate the reaction, and photographed. Lab Work 3.0 UVP software was applied to analyze the band density.

Statistical analysis

Statistical analyses were performed using SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). Data were expressed as means ± standard deviation. The means of multiple samples were compared using a one-way analysis of variance (ANOVA). P＜0.05 was considered statistically significance.

RESULTS

Comparison of fasting blood glucose and insulin levels

After 4 weeks of treatment, insulin intervention decreased the fasting blood glucose level to 9.72 ± 0.71 mmol/L, which was significantly lower compared with the DM-LVH model group (P=0.025, Fig. 1A). 1,25-(OH)2D3intervention also reduced the fasting blood glucose after 4 weeks to 11.19 ± 0.83 mmol/L, which was significantly lower than that in the DM-LVH model group (P=0.015). There was no significant difference between the insulinand 1, 25-(OH)2D3- intervention groups (Fig. 1A).

Before initiation of treatment there were no significant differences in the fasting insulin among the diabetic model groups, being less than the 24.43 ± 2.53 μIU/ml of the control group (P=0.031, Fig. 1B). The fasting insulin level gradually increased in the insulin-intervention group to 11.78 ± 1.93 μIU/mL after 4 weeks, which was significantly higher than that in the untreated model group (P=0.022). Similarly, in the 1, 25-(OH)2D3-intervention group, the fasting insulin level gradually increased over 4 weeks to the end of the experiment to 15.32 ± 1.81 μIU/ml, which again was significantly higher compared with the untreated model group (P=0.018). There was no significant difference between the 1, 25-(OH)2D3- and insulinintervention groups (Fig. 1B).

LVM and LVM index

The LVM and LVM index in the DM-LVH model group were significantly higher than those in the control group (P=0.005), as well as those in the 1, 25-(OH)2D3- and insulin-intervention groups (P=0.034 and P=0.027, Table 1). There were no significant differences between the insulin- and 1, 25-(OH)2D3-intervention groups.

VG collagen staining results

In the control group, the heart tissue was normal with little fibrous tissue and the cell gap was wider than that in the DM-LVH group, where the myocardial cells were arranged more closely and small blood vessels were present around the heart (Fig. 2). The myocardial interstitial collagen deposition and CVF were significantly higher in the DM-LVH group than those in the control group (P=0.008, Table 2). In the insulin- and 1, 25-(OH)2D3-intervention groups, the myocardial collagen deposition and CVF were significantly lower than those in the DM-LVHgroup (P=0.003), whereas there were no significant differences between the insulin- and 1, 25-(OH)2D3-intervention groups (Fig. 2, Table 2).

Collagen-content

The myocardial tissue collagen content in the DM-LVH group was significantly higher than that in the control group (P=0.003). In the insulin- and 1, 25-(OH)2D3-intervention groups, the collagen content was significantly lower than that in the DM-LVH group (P=0.005). There was no statistical significance between the insulin- and 1, 25-(OH)2D3-intervention groups. (Table 2)

VDR expression

Expression of VDR in the myocardial tissue was significantly higher in the diabetic myocardial hypertrophy group compared with the control group (P=0.017, Fig. 3). The insulin- and vitamin D-intervention groups expressed significantly lower VDR compared with the DM-LVH group (P=0.041). There was no statistical difference in the VDR expression between the vitamin D- and insulin-intervention groups (Fig. 3).

Figure 1. Comparisons of fasting blood glucose (A) and insulin levels (B) in the four groups.Control group: non-diabetic Wistar rats (n=6); DM-LVH group: left ventricular hypertrophy in type 2 diabetic rats without treatment (n=7); DM-LVH+INS group: left ventricular hypertrophy in type 2 diabetic rats with insulin intervention (n=6); DM-LVH+VD group: left ventricular hypertrophy in type 2 diabetic rats with 1, 25-(OH)2D3intervention (n=6).Data were expressed as means ± standard deviation.*P＜0.05 compared with the DM-LVH group.

Table 1. Comparisons of the left ventricular mass and left ventricular mass index after 4 weeks of treatment§

Figure 2．Van-Gieson staining of myocardial tissue in rat. (×500)

Table 2. Comparisons of the total CVF and collagen content after 4 weeks of treatment§

Figure 3. Results of Western blot analysis of myocardial vitamin D-receptor (VDR) expression in the four groups.Data were expressed as means ± standard deviation.*P＜0.01 compared with the control group;#P＜0.05 compared with the DM-LVH group.

DISCUSSION

The pathophysiology of type 2 diabetes is based on a relative absolute decline in insulin, thus the role of insulin is decreased. Furthermore, insulin resistance and decreased insulin sensitivity cause the liver to produce excessive glucose (glucose increases gluconeogenesis, glycogenolysis, and glycogen synthesis to reduce this increase), while reducing glucose uptake by the surrounding tissues and its utilization and processing, thus giving rise to high blood sugar, which is characterized by chronic progressive β-cell failure. In type 2 diabetes and CHD, LVH and cardiac fibrosis are closely associated with the cardiovascular disease process, which can lead to cardiac hypertrophy.14

The mechanism of diabetes-induced cardiac hypertrophy is currently not understood. It may, among various factors, be related to high blood sugar, high cholesterol, renin-angiotensin-aldosterone system activation, cytokine secretion, and abnormal collagen degradation.15-18In the present study, we established a cardiac hypertrophy model in diabetic rats confirmed initially by ultrasound screening. We found that the LVH determined by ultrasound was consistent with the autopsy results. Once the cardiac hypertrophy model in the diabetic rats was successfully established after 8 weeks, the fasting blood glucose, LVM index, and collagen content were found to be significantly higher than those in the control group, which indicated that cardiac hypertrophy was present in the diabetic rats. Insulin intervention significantly improved all of the indicators. This suggests that insulin has a therapeutic effect on myocardial hypertrophy in STZ-induced diabetes, and that this effect may be related to insulin regulation of glucose and lipid metabolism.

Vitamin D is an essential vitamin, and is a member of the steroidal hormone family. The main active metabolic product in the body is 1, 25-dihydroxy bile calciferol [1, 25-(OH)2D3]. It has numerous activities, including regulation of renal and intestinal calcium, phosphate metabolism, and tissue growth and differentiation. In the present study, we found that 1, 25-(OH)2D3significantly reduced blood glucose levels in type 2 diabetic rats and increased blood insulin levels. Studies have shown that vitamin D is the physiological glucose-stimulated insulin secretion under the conditions and the material necessary to maintain normal glucose tolerance.19Vitamin-D deficiency can lead to the inhibition of insulin secretion.20When vitamin D was given to type 2 diabetic patients, insulin secretion significantly improved.21Several clinical studies reported that serum vitamin-D levels and the incidence of type 2 diabetes were inversely related.22-23Inthe present study, we found that 1, 25-(OH)2D3stimulated insulin secretion in diabetic rats and reduced their blood glucose levels. This treatment may reverse cardiac hypertrophy in diabetic rats.

In addition, we found that 1, 25-(OH)2D3reduced type 2 diabetes-induced myocardial interstitial collagen production, thus reversing the cardiac hypertrophy. The mechanism may involve a reversal in the decline in VDR expression. Vitamin D and mild hypercalcemia were reported to reverse neonatal rat cardiac hypertrophy,24-26whereas VDR knockout in the rat may lead to cardiac hypertrophy.27Bodyak et al28found that the vitamin-D analog paricalcitol reversed cardiac hypertrophy with no change in blood flow. Several clinical studies reported that a lack of vitamin D in childhood was associated with a significantly increased incidence of heart disease and heart failure.29The blood vitamin-D level were significantly reduced in renal-failure patients with myocardial hypertrophy.30Myocardial cells and cardiac fibroblasts in cardiac hypertrophy were found to increase VDR expression.31On vitamin-D treatment, VDR expression decreased, and the myocardial-hypertrophy phenotype was prevented. This effect was direct, and was not a hemodynamic effect.31

The current experiment suggests that 1, 25-(OH)2D3can reverse cardiac hypertrophy in type 2 diabetic rats. The mechanism is very complex, and may be related to direct reversal of cardiac hypertrophy via the VDR and/or indirectly by regulating blood sugar levels and stimulating insulin secretion. Therefore, vitamin D can not only improve insulin resistance and thus type 2 DM, but may also reverse diabetic cardiac hypertrophy, which is of clinical significance in the prevention and treatment of cardiomyopathy in type 2 DM.

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for publication February 12, 2014.

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△Supported by the Research Fund for Public Health of the Health and Family Planning Commission of Wuhan Municipality (WG13B12).