Chinese Medical Journal 2014;127(7):1298-1303:10.3760/cma.j.issn.0366-6999.20132950
Gliquidone versus metformin: differential effects on aorta in streptozotocin induced diabetic rats

Tan Zhongju, Xu Zherong, Gui Qifeng, Wu Weizhen and Yang Yunmei

Keywords
diabetes mellitus, type 2; atherosclerosis; endothelial function; metformin; gliquidone
Abstract
Background Diabetic cardiovascular complication is a major cause of mortality in type 2 diabetic patients. Hyperglycemia markedly increases the risk of cardiovascular disease. Endothelial dysfunction is common in type 2 diabetes mellitus (DM) and is an early indicator of diabetic vascular disease. Therefore, it is necessary to identify the effect of different hypoglycemic agents on vascular endothelium. The aim of the study was to examine and compare the effects of metformin and gliquidone on atherosclerotic lesions in streptozotocin-induced diabetic rats.
Methods Forty male Sprague-Dawley rats (age, 8 weeks; weight, 180–200 g) were included in this study and fed with a normal chow diet for 1 week. Rats (n=10) served as the normal control group (NC group) were fed with a normal chow for another 2 weeks and received an injection of saline. The rest 30 rats fed with a high-fat diet for 2 weeks and injected streptozotocin were randomly assigned to three groups (n=10 rats per group) as follow: type 2 DM group (DM group), DM + gliquidone group (GLI group) and DM + metformin group (MET group). Five weeks later, all rats were fasted overnight and taken tail blood samples for biochemical determinations. Then rats in the NC and DM groups were administrated with normal saline, while rats in the MET and GLI groups were administrated with metformin (100 mg/kg) or gliquidone (10 mg/kg), respectively. All medicines were given via intragastric administration for 8 weeks. After 16 weeks, plasma triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) were measured. The aortic arch was isolated from diabetic rats and was assessed by pathological sectioning using H&E staining.
Results Metformin treatment prevented weight gain ((315.80±52.16) g vs. (318.70±68.48) g, P=0.773), improved plasma TG, HDL-C and LDL-C levels (P=0.006, 0.003, 0.001, respectively, all P <0.05). However, gliquidone showed no significant effects on plasma TG and TC levels (P=0.819, 0.053, respectively). LDL-C and HDL-C in the GLI group changed ((0.46±0.10) mmol/L vs. (0.36±0.14) mmol/L, P=0.007; (0.99±0.27) mmol/L vs. (1.11±0.18) mmol/L, P=0.049). Both metformin and gliquidone treatment lowered blood glucose levels (P=0.001, 0.004, respectively, P <0.05). Under light microscopy, no changes were observed in the aortic wall structure of each layer; the intima was smooth and the membrane elastic fibers were normal in the NC group. In the DM group, the aortic wall structure was unclear, the intima was thickened with irregular intima, and membrane elastic fibers collapsed. The aortic intima in the MET and GLI groups was smoother compared with the DM group, but the endothelial structure of the MET group was closer to that of the NC group.
Conclusions Both metformin and gliquidone have anti-atherosclerotic effects. But the endothelial structure of the MET group was closer to that of the NC group. Metformin and gliquidone therapy can reduce serum level of LDL-C and increase level of HDL-C, whereas gliquidone therapy did not lose weight and decrease serum level of TG. These data may have important implications for the treatment of patients with type 2 DM.
With the population aging, urbanization, and an increasing prevalence of obesity and physical inactivity, the prevalence of diabetes mellitus (DM) is increasing. The total number of people with DM was approximately 171 million in 2000, and by the year 2030 the number is projected to 366 million.1Patients with type 2 DM (T2DM) have an increased risk for various diseases, including vascular diseases, premature deaths, blindness, renal failure, amputation, fracture, depression and impairment of cognitive function.2Epidemiological studies have demonstrated that those diseases are directly associated with blood glucose levels.
Diabetic complications, especially vascular complication, are the main etiology for a great percentage of morbidity and mortality in patients with T2DM. Patients with DM present complex vascular changes, such as an accelerated atherosclerotic process and hypercoagulability, and an increased risk for future cardiovascular events. The risk of dying from a cardiovascular cause is increased by about 3-folds in patients with T2DM, compared with non-diabetic individuals.3Atherosclerosis (AS) is a major factor that accelerates diabetic macrovascular complications. T2DM, as an independent risk factor, can accelerate the process of AS.4Moreover, AS causes coronary heart disease, which accounts for 75% of mortality in both type 1 and type 2 diabetic patients.5Endothelial dysfunction is considered to be the earliest detectable abnormality in the process of AS. And impaired endothelial function has been demonstrated in patients with T2DM.6
Hyperglycemia is generally considered as a detrimental factor in vascular dysfunction, and the focus has been on tight blood glucose control as a treatment for diabetic patients. However, clinical trials, aimed at improving cardiovascular outcomes with intensive glycemic control, do not support the assumption that strict glucose control reduces cardiovascular risk in complicated T2DM, questioning the role of hyperglycemia in poor cardiovascular outcomes.7-9Nevertheless, intensive glycemic control is especially important in patients with T2DM. Therefore, oral hypoglycemic agents are needed in patients with T2DM. Oral hypoglycemic agents have been widely used for 60 years which improve insulin sensitivity, vascular function and carotid intimal media thickness compared with insulin.10Clinical studies have also reported the cardiovascular effects of oral antidiabetic agents,11,12but the different vascular protective effects between metformin and sulfonylureas remains controversial, especially for pathological changes. Metformin (a biguanide) appears to reduce bodyweight,13and has beneficial effects on plasma lipoproteins14and vascular function.15In the United Kingdom Prospective Diabetes Study (UKPDS),16intensive glycemic control in overweight patients treated with metformin was associated with a lower risk of DM-related end points, all-cause deaths and myocardial infarction. There is debate that sulfonylureas increase the risk of adverse cardiovascular events. And there are very few data concerning the vascular effects of gliquidone.17There is not enough evidence about which one should be preferred in order to improve cardiovascular prognosis. Thus, in the present study, we compared the effects of metformin and gliquidone on vascular (aorta artery) changes in diabetic Sprague-Dawley rats.
METHODS
Animal groups and diabetic model
A total of 40 male Sprague-Dawley rats (Vital River Laboratory Animal Technology Co., Ltd, Beijing, China) weighing approximately 180–200 g, were housed four per cage in a room with a 12/12-hour light/dark cycle and appropriate temperature and humidity. Rats were given with food and waterad libitumfor 1 week. Then, the normal control rats (NC group;n=10) were fed with a normal chow diet and while the other rats were fed the high-fat diet (contained 16.2% fat, 12.6% carbohydrate and 18.8% protein, Shanghai Slac Laboratory Animal Co., Ltd, Shanghai, China) for 2 weeks. After 2 weeks on either diet, animals fed with the high-fat diet were intraperitoneally injected with streptozotocin (STZ, Sigma, USA). STZ (35 mg/kg) in freshly prepared citrate buffer (pH=4.4) after an overnight fast. Rats in the NC group were intraperitoneally injected with vehicle citrate buffer (PH=4.4) in a dose volume of 1 ml/kg. Tail blood samples were taken to test fasting glucose levels 72 hours after injections. Rats whose fasting glucose was ≥7.8 mmol/L twice were considered diabetic. At the same time, the rats that proved to be diabetic were randomly assigned to three groups as follows: DM group (T2DM), GLI group (DM + gliquidone) and MET group (DM + metformin), 10 rats per each group. Following blood samples, all rats received food and waterad libitumfor another 5 weeks. Animals were fasted over night and blood samples were obtained from tails for biochemical determinations. Then the rats were administrated with medications as follows. DM group served as a diabetic group without drug administration. In GLI group, rats received gliquidone (10 mg/kg per day, gliquidone (30 mg) tablets, Beijing Wanhui Double-Crane Pharmaceutical Co. Ltd, Beijing, China). In MET group, rats received metformin (100 mg/kg per day, metformin hydrochloride enteric-coated (250 mg) tablets, Beijing Liling Hengtai Pharmaceutical Co. Ltd, Beijing, China). It will last for a period of 8 weeks, with food and waterad libitum. The total experimental duration was 16 weeks. All animal experiments were performed according to the Zhejiang University guidelines for animal care and approved by the Animal Ethics Review Committee of Zhejiang University.
Biochemical determinations
At the time of sacrifice, the rats were fasted for 12–16 hours, then weighed and anesthetized with 4% chloral hydrate (7 ml/kg, intraperitoneally). Serum samples were collected from inferior vena cava for biochemical determination; plasma was separated by centrifuge at 4 000 r/min for 6 minutes. Fasting plasma glucose (FPG), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were measured using automatic biochemical analyzer according to the instruction.
Pathological sectioning
After blood collection, the thoracic aorta was dissected carefully, cleaned of fat and adherent connective tissues, cut into segments of 4 mm in length, and dissolved in 15 ml centrifugal tubes with 10% neutral formaldehyde solution containing: formaldehyde 100 ml, sodium dihydrogen phosphate 4 g, disodium hydrogen phosphate 6.5 g and distilled water 900 ml. The thoracic segments were assessed by pathological sectioning using hematoxylin and eosin staining and light microscopy.
Statistical analysis
Data are presented as mean± standard deviation (SD). The paired Student’st-test was used in order to assess changes from before-treatment to after-treatment within the GLI and MET groups. Differences in body weight, blood glucose, TG, TC, LDL-C, HDL-C at baseline among groups were analyzed with one-way analysis of variance (ANOVA) following least-significant difference (LSD). All the data analysis was performed with the Statistical Package for the Social Sciences (SPSS) 19.0 software (SPSS Inc., USA). Significance was defined asP<0.05.
RESULTS
Biochemical results
As shown in Figure 1, before treatment, DM, GLI and MET groups had significantly elevated glucose, compared with the NC group (P=0.000, 0.000, 0.001, respectively). Weight in the DM, GLI, MET groups decreased significantly, compared with the NC groups ((318.60±79.08) g vs. (398.28±61.00) g,P=0.012; (278.16±73.55) g vs. (398.28±61.00) g,P=0.000; (315.80±52.16) g vs. (398.28±61.00) g,P=0.009). Moreover, there were significant differences in TG in the DM group, GLI group and MET group, compared with NC group ((0.57±0.21) mmol/L vs. (0.38±0.09) mmol/L,P=0.043; (0.67±0.28) mmol/L vs. (0.38±0.09) mmol/L,P=0.003; (0.65±0.17) mmol/L vs. (0.38±0.09) mmol/L,P=0.005, respectively). Before treatment, there were no significant differences in TG, TC, HDL-C, LDL-C, FPG and weight among the DM group, GLI group and MET group (allP>0.05) (Table 1).
Glycemic responses were similar after 8 weeks of treatment: FPG was (10.64±2.36) mmol/L in the GLI group vs. (11.30±3.29) mmol/L in the MET group (P=0.621). FPG significantly reduced in the GLI and MET group since rats were administrated with gliquidone and metformin. FPG was (18.48±4.22) mmol/L in the DM group vs. (10.64±2.36) mmol/L in the GLI group (P=0.000), (18.48±4.22) mmol/L in the DM group vs. (11.30±3.29) mmol/L in the MET group (P=0.000).
FPG decreased significantly in the GLI group ((16.97±5.90) mmol/L before treatment vs. (10.64±2.36) mmol/L after treatment,P=0.004) and MET group ((18.52±6.78) mmol/L before treatment vs. (11.30±3.29) mmol/L after treatment,P=0.001, respectively). Serum TG and LDL-C decreased in the MET group ((0.65±0.17) mmol/L before treatment vs. (0.47±0.16) mmol/L after treatment,P=0.006; (0.44±0.06) mmol/L before treatment vs. (0.30±0.08) mmol/L after treatment,P=0.001, respectively) (Table 2). LDL-C decreased in the GLI group ((0.46±0.10) mmol/L before treatment vs. (0.36±0.14) mmol/L after treatment,P=0.007). Serum HDL-C was increased in the GLI group and MET group ((0.99±0.27) mmol/L before treatment vs. (1.11±0.18) mmol/L after treatment,P=0.049); ((0.83±0.16) mmol/L before treatment vs. (1.01±0.15) mmol/L after treatment,P=0.003, respectively). Weight in the MET group kept ((315.80±52.16) g vs. (318.70±68.48) g,P=0.773), while weight in the GLI group increased ((278.16±73.55) g vs. (307.46±88.74) g,P=0.001). There were no significant changes of TG, TC in the GLI group (P=0.819, 0.053, allP>0.05). Weight in the NC group increased significantly ((398.28±61.00) g vs. (441.68±77.74) g,P=0.000). There were no significant changes of FPG, TG, TC, LDL-C and HDL-C in the NC group after 8 weeks.
Pathological results
As shown in Figure 2, in the NC group, the aortic intimal was smooth, endothelial cell integrity was preserved, the single layer was close to the elastic plate and medial thickness was uniform. However, in the DM group, the typical three-layer structure of the aortic wall was not clear, and intimal thickness increased with irregular intima. In the MET and GLI group, the aortic intima was smooth and intimal thickening was less compared with the DM group. Progression of AS was attenuated by metformin and gliquidone therapy, whereas the endothelial structure of the MET group was similar to that of the NC group.
DISCUSSION
DM is a prevalent disease in the elderly population. Its cardiovascular complications are major threaten in diabetic patients. Although there were studies demonstrated glycemic control decreased the risk of microvascular disease, it was uncertain which hypoglycemic strategy and hypoglycemic agents were beneficial, at least did not increase the risk of cardiovascular diseases. Metformin and gliquidone have great difference in their mode of action and metabolic effects. Gliquidone is a second generation sulfonylurea that improves glycemic control via stimulating insulin secretion by promoting closure of adenosine triphosphate-sensitive potassium channels in pancreatic β-cells. Metformin lowers blood glucose predominantly by reducing hepatic glucose output and can also increase peripheral glucose utilisation. In our study, the rats were administrated with metformin, 100 mg/kg per day18and gliquidone, 10 mg/kg per day.19Because the dose not only played drug efficacy, but had fewer side effects. There was no significance of weight, serum TG, TC, LDL-C, HDL-C and FPG among the DM group, the GLI group, the MET group.
T2DM is related to metabolic syndrome such as obesity, hypertension and dyslipidemia. It is known that 70%–90% of T2DM patients are overweight or obese. Obese or overweight is common comorbidity in DM. These concomitant factors such as obesity and physical inactivity affect the development of cardiovascular disease and DM and contribute to insulin resistance and impaired glucose tolerance. Body mass has resulted in increased cardiovascular disease in some studies. Abdominal adiposity, physical inactivity and abnormal glucose metabolism are associated with carotid intimal-medial wall thickness, suggesting these factors contribute to atherogenesis.20In our study, weight in the DM group did not change significantly. Perhaps it was the cause of DM itself. There was a significant difference in treatment-induced changes in weight between the two groups: gliquidone gained while metformin neither gained nor losed weight. Although weight in the gliquidone group compared with that in other two groups was lighter at baseline, there was no significance among these groups. Speculatively, the effect of metformin may be responsible for ameliorated vascular endothelial dysfunction. UKPDS also demonstrated the standpoint. Weight in the NC group elevated significantly, while vascular did not change. Because obese is just a risk for cardiovascular diseases, and rats in the NC group are relatively healthy. Another reason was our short duration.
Dyslipidemia is a major risk factor for atherosclerosis. Reducing high levels of cholesterol, particularly low-density lipoprotein cholesterol, decreases the risks for developing atherosclerosis. While higher concentrations of HDL-C contributed to lower the risk for atherosclerosis. Reed et al. showed that high-fat-induced diabetic rats had higher free fatty acids (FFAs) and triglyceride levels compared with normal rats.21Compared with the DM, GLI and MET groups, lower serum TG level in the NC group indicates DM might cause dyslipidemia. In our study, gliquidone treatment did not induce any significant changes in TG, TC levels, but induced changes in LDL-C and HDL-C levels. This was not totally consistent with early studies. It has been demonstrated that SUs reduced plasma total cholesterol, total triglyceride, very low-density lipoprotein-cholesterol, and LDL-cholesterol and apolipoprotein B.22,23Some studies24,25showed that patients treated with SUs had a lower HDL-cholesterol level compared with patients treated with insulin, despite comparable glycemic control. In our study, metformin decreased TG, LDL-C and increased HDL-C in agreement with published data. Metformin has favorable effects on circulating lipids26and FFAs.27Metformin appears to have beneficial effects on blood lipoproteins by reducing LDL-C13,14and may increase HDL-C levels.14The changes of serum lipid levels may be a reason to explain why the vascular lesion in the metformin group was slighter than that in the gliquidone.
DM is an independent risk for cardiovascular diseases in both men and women.28DM also is associated with severe carotid atherosclerosis.20In our study, both gliquidone and metformin reduced the level of FPG. Consequently, the endothelial lesion in the MET and GLI groups was slighter than that in the DM group.
Impaired endothelial function is present in the preclinical stages of atherosclerosis. In our study, vascular endothelial structure impaired in the DM group. Our study showed that metformin and gliquidone both delays the progression of endothelial dysfunction despite metformin treatment is more effective. The UKPDS sub-study with metformin16demonstrated that metformin had vascular protective effect compared with conventionally treated patients, suggesting metformin enhanced vascular endothelial function and exerted anti-atherogenic effects. Vascular effects of sulfonylureas have not been extensively studied except few studies suggest a neutral effect of the agents on vascular function.29The Steno-2 study showed that multifactorial treatment, based on a SU, reduced the risk of micro- and macrovascular complications in high-risk patients.30
We did not study the mechanism of the vascular endothelial protective effect. Gliquidone has potent anti-aggregatory activity31and diabetic vascular complications are often related to altered platelet function. However, Tugba et al19showed that gliquidone had some oxidant effects on aorta as it increased LPO and decreased GSH levels. Metformin improves insulin resistance.32Metformin therapy may ameliorate endothelial dysfunction by improving insulin resistance. And metformin contributed to weight loss, and improved endothelial dysfunction, the fibrinolytic system, inflammation and oxide stress.33-35Metformin has been shown to exert anti-oxidative effects on the vascular beds.36,37The therapeutic effects of metformin may be mediated by its activation of AMP-activated protein kinase (AMPK).38,39AMPK signaling in endothelial cells is essential for angiogenesis; its activation stimulates endothelial nitric oxide synthesis (eNOS) and nitric oxide production.40eNOS exerts a vasodilatory effect and is cardiovascularly protective. Metformin, either as a monotherapy or in combination with a SU, ameliorated hyperglycemia and lead to a decrease in several cardiovascular disease risk factors in patients with T2DM.41
The present results, using H&E staining, provided additional evidence that metformin and gliquidone exert anti-atherosclerotic effects, despite metformin was somewhat better than gliquidone. There are several limitations of the current study. The first limitation is the relative short-term duration of the study. The second limitation derives from certain differences between humans and rodents. In humans with T2DM, several risk factors for cardiovascular diseases, such as obesity, hypertension, hyperinsulinemia, dyslipidemia and procoagulant state, are often present in patients with T2DM. The clustering of these risk factors is termed as the metabolic syndrome. The presence of each additional risk factor raises the risk for cardiovascular diseases. So it is important to remember that glycemic control is just one aspect of the overall management of AS. And patients require an individualized therapy that takes into account other cardiovascular disease risk factors in the selection of an appropriate antidiabetic therapy, such as hypertension and dyslipidemia. Thirdly, just using H&E staining to test endothelial lesion is not convincing. In our later study, we would adopt a more advanced detection means, such as measuring carotid intimal-medial wall thickness by B-mode ultrasonography. Additionally, cellular alterations involved in diabetic endothelial dysfunction have not been determined in this study. However, given that favorable changes were observed, we may be able to partly explain the improvement in endothelial function. Moreover, vascular endothelial function may vary depending on the different site of vascular beds, and the various stages, severity and duration of DM.
In conclusion, our study demonstrates that in rats with T2DM metformin treatment for 8 weeks more effectively improves endothelial function and circulating lipis despite similarly improved glycemic control, compared with gliquidone. Further research is warranted to assess whether the difference in the vascular effects of these widely used substances may affect the selection of antidiabetic agents for the treatment of T2DM.
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(Received November 20, 2013)
Edited by Guo Lishao

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Figure 1.Blood glucose in rats of NC, DM, GLI and MET groups.
Table 1.Baseline laboratory measurements in the GLI and MET groups
Groups n Weight (g) FPG (mmol/L) TG (mmol/L) TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L)
NC 10 398.28±61.00 4.75±1.07 0.38±0.09 1.55±0.43 0.98±0.27 0.39±0.14
DM 10 318.60±79.08 19.03±5.00 0.57±0.21 1.43±0.18 0.86±0.12 0.36±0.10
GLI 10 278.16±73.55 16.97±5.90 0.67±0.28 1.57±0.37 0.99±0.27 0.46±0.10
MET 10 315.80±52.16 18.52±6.78 0.65±0.17 1.41±0.25 0.83±0.16 0.46±0.09
Data are presented as mean±SD.
Table 2.Laboratory measurements in NC, DM, GLI and MET groups of before-treatment and after-treatment
Groups
n
Weight (g)
FPG (mmol/L)
TG (mmol/L)
TC (mmol/L)
HDL-C (mmol/L)
LDL-C (mmol/L)
Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post
NC 10 398.28±61.00 441.68±77.74* 4.75±1.07 5.68±1.22 0.40±0.11 0.35±0.13 1.55±0.43 1.46±0.35 0.98±0.27 1.01±0.25 0.39±0.14 0.40±0.11
DM 10 318.60±79.08 322.93±78.71 19.03±5.00 18.48±4.22 0.57±0.21 0.50±0.13 1.43±0.18 1.37±0.12 0.86±0.12 0.87±0.11 0.41±0.09 0.36±0.10
GLI 10 278.16±73.55 307.46±88.74* 16.97±5.90 10.64±2.36* 0.67±0.28 0.65±0.26 1.57±0.37 1.43±0.26 0.99±0.27 1.11±0.18* 0.46±0.10 0.36±0.14*
MET 10 315.80±52.16 318.70±68.48 18.52±6.78 11.30±3.29* 0.65±0.17 0.47±0.16* 1.41±0.25 1.38±0.22 0.83±0.16 1.01±0.15* 0.44±0.06 0.30±0.08*
Data are presented as mean±SD.*P<0.05, Pre vs. Post.Pvalues are shown for within group comparisons (before treatment compare with post treatment within each group, pairedttest).

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Figure 2. Vascular changes from normal control group (A), DM group (B), metformin group (C) and liquidone group (D) (Hematoxylin and eosin staining, original magnification ×40).
  1. a grant from Major Science and Technology Projects of Zhejiang Province (No. 2011c13032-2).