Daidzin decreases blood glucose and lipid in streptozotocin-induced diabetic mice

Purpose: To investigate the ameliorative effect of daidzin (DZ) on diabetes in streptozotocin (STZ)- induced diabetic Institute of Cancer Research (ICR) mice, with a view to determining its usefulness in the treatment of diabetes. Methods: The effect of DZ (100, 200 and 400 mg/kg) on blood glucose was investigated in both normal and STZ-induced diabetic mice with glibenclamide (3 mg/kg) and metformin (400 mg/kg) as positive control, respectively. Serum or hepatic levels of lipid, proinflammatory factors, malondialdehyde (MDA) and superoxide dismutase (SOD) were measured. Glucosidase activity assay and glucose uptake by C2C12 myotubes were performed in vitro and the expression of glucose transporter 4 (GLUT4) in C2C12 cells was determined by western blot. Results: DZ (200 and 400 mg/kg) did not decrease fasting blood glucose in normal mice but inhibited starch-induced postprandial glycemia. Oral administration of 400 mg/kg of DZ for 14 days significantly decreased mouse blood glucose (p < 0.01), as well as serum total cholesterol (TC, p < 0.01), triglycerides (TG, p < 0.01), low-density lipoprotein cholesterol (LDL-c, p < 0.01) levels in STZ-induced hyperglycemic mice and improved oral glucose tolerance. The serum and hepatic activity of SOD was enhanced (p < 0.01 and p < 0.001, respectively) while MDA level decreased (p < 0.001). Blood concentrations of interleukin-6 (IL-6, p < 0.001), tumor necrosis factor α (TNF-α, p < 0.01), monocyte chemotactic protein 1 (MCP-1, p < 0.01) were also significantly reduced. In vitro glucosidase activity results showed that DZ inhibited α-glucosidase with IC50 values of 82, 98 and 389 μg/mL for α- glucosidase from S. cerevisiae , Rhizopus sp. and rat intestines, respectively. It also stimulated glucose uptake and GLUT4 membrane translocation in C2C12 myotubes at 20 μM (p < 0.05). Conclusion: Oral administration of DZ is effective in alleviating diabetic hyperglycemia, dyslipidemia and inflammation. Inhibition of α-glucosidase and stimulation of glucose consumption by muscles may account for its inhibitory effect on blood glucose. Keywords: Daidzin, Diabetes, Inflammation, Superoxide dismutase (SOD), Malondialdehyde (MDA), Glucosidase, C2C12 myotubes, Glucose transporter


INTRODUCTION
Diabetes mellitus (DM) is becoming a most serious threat to human health with an incidence of 8.7 % (about 387 million patients) throughout the world, imposing a heavy burden on national healthcare systems, particularly in developing countries [1,2]. In the DM state, insulin activity is reduced, resulting in decreased insulinstimulated glucose uptake. Simultaneously, abnormal metabolism of fuel nutrients [3], disordered activities of antioxidant enzymes [4] and increased inflammation [5] lead to a pronounced oxidative stress, which is characterized by increased malondialdehyde (MDA) level and decreased activities of antioxidant enzymes such as superoxide dismutase (SOD). In the current circumstances, a search for an antidiabetic herbal product with fewer side effects has gained considerable importance in the medical field [6]. Daidzin (DZ) is a major isoflavone glycoside obtained from Puerariae radix, a well-known Chinese herbal medicine for the treatment of diabetes and cardiovascular diseases [7][8][9]. Various pharmacological activities of DZ have been reported including antioxidant [10], estrogenic [11] and anti-inflammatory [12]. The effect of DZ on blood glucose remains controversial. Choi et al [13] reported the inhibitive effect of DZ on yeast α-glucosidase, suggesting that DZ may be an alternative to acarbose for the treatment of diabetes. However, Meezan et al [14] reported that intraperitoneal injection of DZ impaired glucose tolerance in ob/ob diabetic mice. Recently, Zang et al [15] demonstrated that the mixture of DZ and glycitin can efficiently decrease blood glucose and serum HbA1c in hyperglycemic mice. Although the mixture of DZ and glycitin were effective in the treatment of DM, the exact role of DZ was not distinguished in their report.
In the present work, we investigated the effects of oral administration of DZ on diabetes-related hyperglycemia, dyslipidemia, perioxidative state, inflammation in streptozotocin (STZ)-induced, to demonstrate the potential utility of DZ in the prevention and treatment of diabetes.

Animal studies
Male Institute of Cancer Research (ICR) mice (20 -22 g each) were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing, China. The study was carried out according to the "Principles of Laboratory Animal Care" [16] and approved by Animal Ethics Committee of Zhengzhou University (no. 2014090210). A standard pellet diet and water were given ad libitum. Animals were housed under a constant 12-h light and dark cycle and in an environment of 21 -23 °C.
To test the effect of DZ in normal mice, the animals were randomly divided into five groups with eight mice in each group. Mice were daily treated with glibenclamide (3 mg/kg) or DZ (as indicated) orally for 14 days. Control group was given equal volume of distilled water. At the end the experiment, animals were fasted for 4 hours and the fasting blood glucose levels were evaluated. For postprandial glycemia test, animals were fasted for 4 h then treated with the corresponding agents orally. After another 15 min, each mouse was given 4 g/kg of starch (p.o.) [17]. Blood glucose levels at 0, 1 and 2 h after starch administration were determined by a blood glucose meter (Roche, ACCU-CHEK Active). Area under the curve was calculated as previously reported [17].
Next, male ICR mice were rendered diabetic by intraperitoneal injection of STZ (80 mg/kg) [18] in citrate buffer, pH 4.5. Five days later, the animals were fasted for 8 h, blood samples taken from their tail vein and the plasma glucose concentration was measured. Animals with blood glucose concentration above 12 mM were selected for the study.
STZ-induced diabetic mice were divided into groups randomly with 8 mice in each group. Animals were daily treated with metformin (400 mg/kg) [19] or DZ (100, 200 and 400 mg/kg) orally for 14 days. Control group was given an equal volume of distilled water. At the end of the experiments and after fasted for 4 h, animals were euthanized and the blood and liver samples was obtained for the estimation of various biochemical parameters by specific kits following the manufacturer's instructions.
For oral glucose tolerance test (OGTT), the animals were fasted for 4 h then treated with corresponding agents orally. After another 2 h, each mouse was given 4 g/kg of glucose (p.o.) [20]. Blood glucose levels were determined at 0, 0.5, 1, 1.5 and 2 h after glucose administration by a blood glucose meter.

Cell culture
C2C12 cells, originating from the American Type Culture Collection (ATCC) (Manassas, VA, USA), were obtained from the Peking Union Medical College. C2C12 myoblasts were cultured in DMEM supplemented with 10 % FBS at 37 o C and 5 % CO 2 . To induce differentiation, media was replaced with DMEM containing 2 % horse serum (Sigma-Aldrich, Inc., St Louis, USA) whenever the cells reached confluence. Experiments were performed in differentiated C2C12 myotubes 5 days after differentiation was induced.

Glucose uptake assay
Glucose uptake assay was performed as previously reported [21]. Briefly, differentiated C2C12 myotubes were incubated with the serum-free DMEM containing the fluorescent glucose analog 2-NBDG (10 μM) and DZ (1, 5 or 10 μM) in the presence or absence of insulin (100 nM). After incubation for 12 h, medium was removed and cells were washed with phosphatebuffered saline (PBS) twice. Cells were scraped out in 1 mL of PBS and transferred into 5 mL polystyrene round-bottom tubes (BD Falcon) and kept at 4 °C. The amount of 2-NBDG taken up by the cells was measured by determining the fluorescence intensities at Ex/Em=475 nm/550 nm using a Tecan Infinite M1000Pro Microplate Reader (TECAN Group Ltd, Shanghai, China).

Western blot
GLUT4 membrane translocation in C2C12 myotubes was analyzed by western blot. Cell membrane preparation and western blot procedure were performed as previously reported [6]. GAPDH was used as internal reference. The immunoreactive bands were quantified using ImageJ 4.1 software (NIH, Bethesda, USA).

α-Glucosidase inhibition assay
Inhibition of α-glucosidase and α-amylase activities were performed according to the chromogenic method described previously [22], using PNPG as substrate. Acarbose was used as positive control. The inhibitory activity was calculated as in Eq 1. (1) where Ac and At are the absorbance of control and test samples, respectively.

Statistical analysis
The results are expressed as mean ± standard error of the mean (SEM). The data were analyzed by one-way analysis of variance (ANOVA) using SPSS 13.0 software. Significant difference was set at p < 0.05.

Effect of daidzin in normal mice
The effect of daidzin (DZ) on blood glucose was first assessed in normal mice. As shown in Figure 1A, DZ did not decrease fasting blood glucose levels in normal mice. This indicates that DZ may not stimulate insulin secretion and hence has a low risk of inducing hypoglycemia. In contrast, treatment with DZ (200 and 400 mg/kg) significantly inhibited the increase of serum glucose levels after oral administration of starch (p < 0.05) ( Figure 1B and 1C), suggesting that DZ is effective in preventing postprandial glycemia.

Effect of daidzin in diabetic mice
We further evaluated the antihyperglycemic effect of DZ in streptozotocin (STZ)-induced diabetic mice. Oral administration of DZ (200 and 400 mg/kg) decreased fasting blood glucose (p < 0.05 and p < 0.01, respectively) ( Figure 2A) and improved glucose tolerance in oral glucose tolerance test ( Figure 2B and C). The potency of DZ at 400 mg/kg was significant but weaker than metformin (400 mg/kg), a popular antidiabetic medicine. These results suggested that DZ can decrease blood glucose levels and improve glucose tolerance in diabetic animals.

Daidzin effect on blood lipid of diabetic mice
As shown in Figure 3, treatment with DZ dosedependently reduced the levels of serum TC, TG and LDL-c. At the optimal dose (400 mg/kg), DZ decreased serum concentration of TC, TG and LDL-c by 23.81, 29.84 and 21.43 %, respectively, which is lesser than the effect of metformin (400 mg/kg). The level of serum HDL-c was elevated by metformin but not affected by DZ (Figure 3).

Daidzin improves perioxidative states in diabetic mice
Treatment with DZ (100, 200 and 400 mg/kg) dose-dependently increased SOD activity and decreased the concentration of MDA in serum and liver (Figure 4). The antioxidative effect of DZ at 400 mg/kg was more pronounced than that of metformin (400 mg/kg).

Daidzin stimulates glucose uptake and increases GLUT4 translocation in C2C12 myotubes
We also found that DZ activated glucose consumption in a dose-dependent manner in C2C12 myotubes. The effect of DZ on glucose uptake was assessed by NBD-glucose (2-NBDG) uptake assay. Treatment with DZ for 12 h increased 2-NBDG uptake by C2C12 myotubes in a dose-dependent manner ( Figure 6A), suggesting a potent activity of DZ in stimulating glucose uptake by myotubes. In accordance with this finding, DZ largely increased GLUT4 membrane translocation without significantly enhancing its total expression level ( Figure 6B and 6C).

Daidzin inhibits the activity of α-glucosidase in vitro
We further investigated the inhibitive effect of DZ on the activity of α-glucosidase in vitro. As shown in Figure 7, DZ inhibited the activity of αglucosidase in a dose-dependent manner. The IC 50 values of DZ on α-glucosidase from S. cerevisiae, Rhizopus sp. and rat intestines were 82 μg/mL, 98 μg/mL and 389 μg/mL, respectively. The in vitro effect of DZ on glucosidase was in good accordance to its inhibitive action on postprandial glycaemia in ICR mice ( Figure 1B and C). These results suggest that DZ is an effective inhibitor of α-glucosidase in vitro and in vivo.

DISCUSSION
Daidzin (DZ) is a major isoflavone glycoside from Puerariae radix, a herb widely used in Chinese medicinal formulae for the treatment of diabetes [7][8][9]. Many pharmacological studies have been made on DZ but its effect on blood glucose remains controversial. In the present work, we demonstrated that oral administration of DZ is adequate in alleviating diabetes mellitus in STZinduced hyperglycemic mice.
Treatment with DZ did not decrease fasting blood glucose in normal mice, suggesting that it has minimal effect on insulin secretion and thus possesses less risk for hypoglycemia. However, oral administration of DZ significantly decreased blood glucose and improved oral glucose tolerance in diabetic mice, showing an adequate effect on hyperglycemia. It also significantly decreased serum levels of TC, TG and LDL-c and slightly increased HDL-c levels. Simultaneously, diabetes-related inflammation and perioxidative state were largely alleviated by DZ. These results demonstrated that oral administration of DZ is beneficial for the treatment of hyperglycemia and diabetes-related syndromes.
Meezan et al [14] found that intraperitoneal injection (i.p.) of DZ was detrimental for glucose homeostasis. This contradiction may be due to the different route by which DZ is administrated. As is well-known that isoflavones including DZ will undergo intensive metabolism in the gut of animals by gut microbiota after oral administration. Therefore, the actual bioactive compounds after DZ oral administration may likely be its metabolites other than DZ itself which will require further investigation. A recent study by Zang et al [15] demonstrated the positive role of the mixture of DZ and glycitin in decreasing blood glucose and serum HbA1c, which partly supported our findings.
There are many approaches to efficiently decrease blood glucose level such as promoting insulin secretion [24], inhibiting glucose absorption [25], stimulating glucose uptake by tissues [26] and increasing insulin sensitivity [27]. Insulinotropic agents such as glibenclamide, stimulate insulin secretion and dramatically decrease blood glucose level in both normal and diabetic animals. Treatment with DZ showed no effects on blood glucose in normal mice. It is thus unlikely to promote insulin secretion. However, it significantly inhibited postprandial glycemia induced by starch, implying that DZ may be effective in inhibiting α-glycosidases and slowing glucose ingestion.
α-Glucosidase data showed that DZ inhibited the activities of α-glucosidase of various origins in a dose-dependent manner, which was in good accordance with its in vivo action on postprandial glycaemia, suggesting that DZ may inhibit hyperglycemia through at least in part inhibition of α-glucosidase. Promoting glucose uptake by muscles is another effective way to decrease blood glucose [8], in which GLUT4 plays a key role. Glucose uptake assay in C2C12 myotubes showed that DZ significantly promoted glucose consumption by myotubes and enhanced GLUT4 membrane translocation as well, indicating that stimulating glucose uptake by muscles and enhancing GLUT4 membrane translocation may involve in the blood glucose-lowering action of DZ.

CONCLUSION
The findings of this work demonstrate that daidzin (DZ) can effectively decrease blood glucose and lipid, improve peroxidation and alleviate inflammation in diabetic animals. Stimulation of glucose consumption and inhibition of glucosidase may be involved in the blood glucose modulating effect of DZ. These results suggest that DZ is an effective regulator of glucose consumption in mice and may find application in the treatment of type 2 diabetes in human.