Hpyerglycemic Effect of a Mixture of Sea Cucumber and Cordyceps Sinensis in Streptozotocin-Induced Diabetic Rat

HU Shiwei, WANG Jingfeng, LI Zhaojie, FU Jia, WANG Yuming, and XUE Changhu

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, P. R. China

Hpyerglycemic Effect of a Mixture of Sea Cucumber and Cordyceps Sinensis in Streptozotocin-Induced Diabetic Rat

HU Shiwei, WANG Jingfeng*, LI Zhaojie, FU Jia, WANG Yuming, and XUE Changhu

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, P. R. China

Sea cucumber and cordyceps sinensis are used as both food and traditional medicines in Asia. This study was carried out in order to investigate the hpyerglycemic effect of a mixture of sea cucumber (Apostichopus japonicas) and cordyceps sinensis (Cordyceps militaris) (SCC) in diabetic rat and explore the mechanism underlining such an effect. The diabetic model rat was induced with intraperitoneal injection of streptozotocin (STZ). The diabetic model rats were randomly divided into control group (0.9% NaCl), low dose group (300 mg SCC·(kg body weight)−1) and high dose group (1200 mg SCC (kg body weight)−1). Sodium chloride and SCC were intragastrically administered once a day for 35 d. Changes in fasting serum glucose and serum insulin content, oral glucose tolerance and liver and muscle glycogen content were routinely evaluated. Pancreas tissue and β-cells of islets were observed under both optical and transmission electronic microscope, respectively. The abundance of glucose metabolism-relating genes in gastrocnemius and epididymal adipose tissue was determined with either reverse transcription PCR (RT-PCR) or western blotting. Results showed that SCC significantly decreased fasting serum glucose content, improved glucose tolerance and increased serum insulin and glycogen content; repaired STZ-injured β-cells of diabetic rat, and increased the expression of phosphatidylinositol 3 kinase (PI(3)K), protein kinase B (PKB) and glucose transporter 4 (Glut4) encoding protein in both gastrocnemius and adipose tissue, and Glut4 encoding gene in peripheral tissue. Our findings demonstrated that SCC exerted an anti-hyperglycemic effect by repairing β-cells and promoting insulin-mediated signal transduction pathway in insulin-sensitive gastrocnemius and adipose tissue.

sea cucumber; cordyceps sinensis; hpyerglycemic effect; β-cell; glucose transporter 4; phosphatidylinositol 3-kinase

1 Introduction

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia due to an absolute or partial deficiency of insulin secretion and/or insulin resistance. In developing and newly developed countries, DM is associated with severe metabolic disturbance, dysfunction and exhaustion of various organs (Alberti and Zimmet, 1998; Chopra et al., 2008). The macronutrient metabolism abnormality in DM is associated with deficient insulin activity due to insufficient secretion or diminished responsiveness of the insulin signaling pathway (Ramkumar et al., 2011). Although the occurrence of DM is continuously increasing, effective treatments remain unavailable. Considering the heterogeneity of diabetes and limitation of current pharmaceuticals such as insufficient regulated hyperglycemia, side effect and high secondary failure rate, it is crucial to identify new pharmacological approaches for effective prevention and treatment of this metabolic disorder.

As traditional food and medicine in Asia, sea cucumber and cordyceps sinensis have received an intensive attention in recent years. Early works found that sea cucumber exerts high anti-hyperglycemic effect via PI(3)K/PKB/ Glut4 pathway in DM therapy (Wang et al., 2011), and cordyceps sinensis improves glucose tolerance by repairing β-cells of islet in STZ-induced DM rat (Xu et al., 2011; Park et al., 2009; Fan, 2001). The philosophy of traditional Chinese medicine states that the effect of two tonics will be intensified once being combined. Unfortunately, no trial has been reported on the anti-diabetic effect of a combination of sea cucumber and cordyceps sinensis (SCC) to our knowledge. The present study was designed to evaluate the hpyerglycemic effect of SCC and determine the mechanism underlining such an effect.

2 Materials and Methods

2.1 Materials

Dry sea cucumber (Apostichopus japonicas) and cordyceps sinensis (Cordyceps militaris) were purchased from local market of Qingdao, Shandong Province, P. R. China. SCC superfine powder was prepared by grindingtheir mixture (2:1). SCC was stored at room temperature.

2.2 Animal Treatments

Male Sprague-Dawley rats (220–250 g in body weight) were purchased from Vital River Laboratory Animal Center (Beijing, China; Licensed ID: SCXK2007- 0001) and cultured according to the Standards for Laboratory Animals of China (GB 14923-94, GB 14922-94, and GB/T 14925-94). The rats were housed at 23℃ ± 1℃ with a rhythm of 12 h light and 12 h dark and free accession to distilled water and a standard sterile diet.

The animals were randomly assigned to a normal group (10 rats) and an experimental group. A freshly prepared solution of streptozotocin (STZ, 50 mg (kg body weight)−1, Sigma, USA) in 0.1 mol L−1cold citrate buffer (pH 4.3) was injected into the tail vein of overnight-fasted rats and reinforced (10 mg (kg body weight)−1) next day. After STZ inducing for one week, animals with fasting blood glucose content higher than 16.7 mmol L−1were defined as successful diabetic model rats and selected for further experiments. The diabetic rats were divided into 3 groups (10 rats each): the model control, low-dose group (SCC at 300 mg (kg body weight)−1) and high-dose group (SCC at 1200 mg (kg body weight)−1). The rats in model control and normal control groups were given 0.9% NaCl at 10 mL (kg body weight)−1. The dosage of SCC was determined by referring to the recommnende to human body nutrient intake and our early findings. The rats were intragastrically given once a day for 35 d. After the last administration, the blood was collected to assay fasting serum glucose and insulin content. The liver, gastrocnemius and epididymal adipose tissue were separated to measure glycogen content and analyze the expression of glucose metabolism-related genes. The tail of pancreas was cut off carefully to observe the histological change of islet of langerhans.

2.3 Serum Glucose Content and Oral Glucose Tolerance After Fasting

Blood was collected from the tail vein each rats after a 5-h fasting. Serum glucose content was determined using a glucose test kit. An oral glucose tolerance test was conducted at 0, 0.5, and 2 h after intragastrically giving glucose (2 g (kg body weight)−1). The area under curve (AUC) was calculated as follows:

where A, B and C stand for serum glucose content measured at 0, 0.5, and 2 h, respectively.

2.4 Serum Insulin Content

After 10 h fasting, blood of the rats was collected. Serum insulin content was assessed using insulin ELISA kit (R&D Systems, USA), and the insulin sensitivity index was presented as the ratio of serum insulin content to fasting serum glucose content.

2.5 Glycogen Content

The liver and gastrocnemius (80–100 mg) were homogenized with lye and heated in boiling water for 30 min. After cooling in an ice bath and centrifuged at 8500 r min−1for 30 min. The precipitate was suspended in 1.5 mL distilled water with glycogen concentration measured with a glycogen test kit (Zhongsheng Beikong, China).

2.6 Microscopic and Submicroscopic Structure of Islet of Langerhans and β-cells

Parts of the selected pancreas were fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (HE). The microscopic structure of islet of langerhans was imaged under optical microscope (BH-2, Olympus, Japan). The other parts were fixed in 4% glutaraldehyde and 1% osmic acid, embedded in epon, sectioned into ultrathin pieces and stained with uranyl acetate and lead citrate. The submicroscopic structure of β-cells was imaged under a transmission electron microscope (TEM, H-7000, Hitachi, Japan).

2.7 Glucose Metabolism-related Genes Expression Analysis by RT-PCR

Table 1 Primers and other information for determining the abundance of glucose metabolism related gene transcripts

The abundance of glucose metabolism-related gene transcripts such as that of PI(3)K, PBK and Glut4 encoding genes were determined with reverse transcription polymerase chain reaction (RT-PCR). The housekeeping β-actin gene was used as control. The total RAN from gastrocnemius or adipose tissue was extracted using TRIzol reagent, and 1 μg RNA was reversely transcribed to cDNA with M-MLVPCR was carried out in 30 μL system cantaining 0.5 μg cDNA on a MJ Research thermocycler (TC-96/G/H(b)A; Hangzhou, China). The thermocycling was performed by denaturing at 94℃ for 45 s, annealing at the temperature desirable to each primer pair for 45 s and extending at 72℃ for 45 s for cycling number set for each primer pair followed by am extra extending at 72℃ for 10 min. The primers, product length, annealing temperature and the number of cycles used for the amplification of each gene are listed in Table 1. PCR products were separated in 1% agarose gel buffered with tris-acetate-EDTA and visualized with ethidium bromide staining. Bands were quantitated using the Image J program (JS-780; NIH, USA). The abundance of gene transcripts was expressed as the ratio of signal intensity of gene transcripts to that of β-actin gene transcript.

2.8 Analysis of Glut4 Protein Content by Western Blotting

Gastrocnemius or adipose tissue was lysed with lysis buffer (20 mmol L−1Tris-HCl, pH 8.0), 2 mmol L−1EDTA, 137 mmol L−1NaCl, 1 mmol L−1Na3VO4, 10% glycerol, 1% Triton X-100 and 1 mmol L−1phenylmethylsulfonyl fluoride), centrifuged at 12000 r min−1for 5 min) and fractionated with 10% SDS-PAGE. Protein was transferred to PVDF membrane and incubated with rabbit anti-Glut4 primary antibody and then with goat-anti rabbit secondary antibody (Promega, USA). GAPDH was used as an internal control for normalization. Antigen reactivity was detected using ECL detection kit (Applygen Technologies Inc., China). The Glut4 protein relative content was quantitated as the ratio of target protein band to that of GAPDH with the Image J program (JS-780; NIH, USA).

2.9 Statistical Analysis

Data were presented as mean ± SD. Statistical comparison was done with one-way analysis of variance (ANOVA). A P value less than 0.05 was considered statistically significant.

Table 2 Effect of SCC on the content of fasting serum glucose, serum insulin and glycogen and insulin secretion index in liver and muscle of diabetic rats

3 Results

3.1 SCC Reduced Serum Glucose Content After Fasting and Improved Oral Glucose Tolerance

Fasting serum glucose content and glucose tolerance were the major assessment criteria of DM. Glucose tolerance was negatively related to AUC. After being STZ inducted, fasting serum glucose content and AUC in model control group were increased by 366.87% and 260.11%, respectively, of the normal control, indicating that the diabetic model was established successfully. The fasting serum glucose content and AUC in SCC-treated group were significantly decreased compared with those in the model group (Table 2 and Fig.1). These findings showed that SCC exerted a significant anti-hyperglycemic activity in diabetic rats.

Fig.1 Effect of SCC on oral glucose tolerance in diabetic rats. Mean ± S.D., n = 10; ##, extremely significantly different (P < 0.01) from normal control; *, significantly (P < 0.05), and **, extremely significantly (P < 0.01) different from model control.

3.2 SCC Increased Serum Insulin Content

When being treated with SCC for 35 days, serum insulin content was significantly increased in diabetic rats. Serum insulin content in low- and high-dosage SCC groups was increased to 126.54% and 133.23% (P < 0.05) in diabetic animals, respectively (Table 2). As an important reference in diagnosis, treatment and prevention to diabetic, insulin secretion index is typically used to assess the insulin secretory function of β-cells. Insulin secretion index of rats in low and high dosage SCC-treated groups was also 35.74% and 36.81% higher than that of model control, respectively, suggesting that SCC significantly ameliorated the insulin secretory function of β-cells.

3.3 SCC Increased Glycogen Content

When being treated with SCC for 35 d, the glycogen content in liver and in gastrocnemius of high-dose SCC group was increased remarkably in diabetic rats (Table 2), showing SCC facilitated the anabolism of glycogen in liver and gastrocnemius.

3.4 SCC Repaired β-Cells of Islets

STZ induced the diabetic development in rats because of the oxidation injury of most β-cells in islet of langerhans. Various pathologic manifestations such as atrophy of islet of langerhans, cell necrosis and substantial inflammatory infiltration were found in the model control group. However, these histological changes in SCC groups were alleviated in comparison with those in model control group (Fig.2). In model control group, β-cells were found to contain irregularity nuclei, compacted chromatin, less condensed mitochondria and partly damaged cristae in mitochondria, density reduced granules, and dilatant granular endoplasmic reticulum. In contrast, β-cells in high-dose SCC group were less changed in these characteristics (Fig.3), being similar to those of normal control. SSC repaired damaged βcells.

Fig.2 Effect of SCC on microstructure of islets of langerhans in diabetes rats. × 400; arrow points to islets of langerhans; A, normal control; B: model control; C: low-dose SCC; D: high-dose SCC.

Fig.3 Effect of SCC on ultrastructure of β-cells in diabetes rats. × 5,000; Cn, cell nucleus; Mt: mitochondrion; Er: granular endoplasmic reticulum; Dg: dense granules; A, normal control; B, model control; C, low-dose SCC; D, high-dose SCC.

3.5 SCC Up-Regulated the Expression of PI(3)K, PKB and Glut4 Gene in Gastrocnemius and Adipose Tissues

PI(3)K/PKB/Glut4 pathway mediates the glucose transport and uptake, which is stimulated by insulin. Loss of any signal molecule or link may result in diabetes. To evaluate the effect of SCC on the expression of major genes in the pathway, the abundance of PI(3)K, PKB and Glut4 gene transcripts in insulin-sensitive gastrocnemius and adipose tissues were determined. The abundance of PI(3)K and PKB gene transcripts of model control was significantly decreased in gastrocnemius and in adipose tissues (Figs.4 and 5) relative to those of the normal control. After being treated with SCC, the expression of these two genes was significantly higher than that of model control. Such a up regulation was more pronounced in high-dose SCC group, where the abundance of PI(3)K and PKB gene transcripts increased by 137.74%, 33.19% in gastrocnemius (Fig.4) and 42.86%, 27.78% in adipose tissues (Fig.5), respectively. The abundance of Glut4 gene transcripts in model control group was significantly decreased by 33.98% in gastro-cnemius and 26.67% in adipose tissues in comparison with that of normal control group. Different dosages of SCC caused the abundance of Glut4 gene transcripts increases by 30.06% and 29.58% in average in these two tissues of diabetic rats, respectively, illustrating that SCC functions on the PI(3)K/ PKB/Glut4 insulin signaling pathway at the level of transcription.

3.6 SCC Improved Glut4 Synthesis in Gastrocnemius and Adipose Tissues

Glut4 protein content significantly decreased by 37.32% and 93.68% (P < 0.01) of in gastrocnemius and adipose tissues of model control group, respectively in comparison with that of normal control group (Fig.6). Once different doses of SCC were supplemented, the average content of Glut4 protein increased by 21.64% in gastrocnemius and 255.78% in adipose tissues in comparisonwith that of model control group, indicating that SCC up-regulated the Glut4 protein synthesis.

Fig.4 Effect of SCC on the abundance of PI(3)K (A), PKB (B) and Glut4 (C) gene transcript in gastrocnemius of diabetic rats (mean ± S.D., n = 10; # and ## indicate significant (P < 0.05) and extremely significant (P < 0.01) difference from normal control group; * and ** indicate significant (P < 0.05) and extremely significant (P < 0.01) difference from model control).

Fig.5 Effect of SCC on the abundance of PI(3)K (A), PKB (B) and Glut4 (C) gene transcripts in epididymal adipose tissues of diabetic rats (mean ± S.D., n = 10; # and ## indicate significant (P < 0.05) and extremely significant (P < 0.01) difference from normal control group; * and ** indicate significant (P < 0.05) and extremely significant (P < 0.01) difference from model control).

Fig.6 Effect of SCC on Glut4 synthesis in gastrocnemius (A) and epididymal adipose tissues (B) of diabetic rats (mean ± S.D. n = 10; # and ## indicate significant (P < 0.05) and extremely significant (P < 0.01) difference from normal control group; * and ** indicate significant (P < 0.05) and extremely significant (P < 0.01) difference from model control).

4 Discussion

The STZ-induced diabetic rat is an ideal model of studying mechanisms of rising serum glucose content and impaired glucose tolerance which may aid to developing novel DM therapeutic interventions (Jung et al., 2005). We evaluated the hpyerglycemic effect of SCC in STZ-induced DM rats, and found that SCC decreased fasting serum glucose content, improved glucose tolerance, increased serum insulin and glycogen content and repaired β-cells within 35 d of SCC-treatment. SCC exhibited a significant anti-hyperglycemic activity in diabetic rats.

Either absolute absence or low insulin secretion can lead to an aberrant glucose metabolism and onset of DM. In STZ-diabetic animals, insulin is markedly depleted because of partial oxidative damage of β-cells in islets of langerhans (Tomlinson et al., 1992). In our study, SCC was found to promote the secretion of insulin, a key factor of regulation the signaling transduction pathway of PI(3)K/PKB/Glut4, and repair β-cells in islets of langerhans. These findings suggested that SCC improved β cells and subsequently increased insulin secretion and played a important role in the signaling pathway of PI(3)K/PKB/ Glut4 and anti-hyperglycemic activity.

Further investigation of the mechanism involved in SCC protection against DM was focused on the changes in glucose metabolism-related signaling pathway. PI(3)K has a pivotal role in glucose uptake and Glut4 trans-location in glucose metabolism-related signaling pathway. Inhibition of this enzyme with pharmachological inhibitors such as wortmannin completely blocks the stimulation of glucose uptake by insulin (Okada T et al., 1994). Subsequently, several studies have demonstrated that impaired insulin-stimulated Glut4 translocation and glucose uptake in gastrocnemius and adipose tissues are associated with defective PI(3)K activation (Tremblay et al., 2003; Su et al., 2006). As a central node in signaling downstream of PI(3)K, serine/threonine protein kinase PKB is an important and versatile protein kinase. One of the most important physiological functions of PKB is its active stimulation of glucose uptake in response to insulin (Manning and Newgard, 2007). The activation of PKB causes Glut4 translocation to the cell membrane (Berwick et al., 2004). Zhen et al. have shown that inhibition of PKB activation by PKB siRNA has a significantly negative effect on insulin-stimulated glucose transport in 3T3-L1 adipocytes (Jiang et al., 2003). In present study, SCC caused significant increases of the abundance of PI(3)K and PKB gene transcripts in gastrocnemius and adipose tissues. These findings suggested that SCC upregulated and activated PI(3)K/PKB signal pathway.

As the major insulin-dependent transporter, Glut4 is predominantly expressed in muscle and adipose tissues and mediates whole-body glucose homeostasis (Watson and Pessin, 2001). Recent data have suggested that Glut4 abundance determines the maximal effect of insulin on glucose transport (Furuta et al., 2002). Reduction of Glut4 content decreases glucose uptake and therefore contributes to the increased blood glucose content in diabetic condition. A marked reduction of Glut4 abundance in muscle and adipose tissues of STZ-diabetic rats was observed in many subsequent experiments (Wang et al., 2005; Mohammad et al., 2006). In our study, SCC dramatically enhanced Glut4 gene transcription and Glut4 synthesis in both gastrocnemius and adipose tissues of diabetic rats in a dose-dependent way. We proposed that PI(3)K/PKB gene transcription, enhancement of signaling transmission and promotion of Glut4 gene transcription and protein synthesis may be achieved by SCC induction of glucose transmembrane transport. These effects decrease hyperglycemia through facilitating glucose utilization in skeletal muscle and adipose tissues.

Early works have found that both Apostichopus japonicas and Cordyceps militaris increase fasting serum glucose and insulin content and improve glucose tolerance (Long et al., 2012; Lo et al., 2006; Guo et al., 2011); however, the substance exerting hpyerglycemic activity remained unclear. The proposed included collagen polypeptide, polysaccharide, triterpene glycosides, cerebroside, gangliosides in sea cucumber (Xu et al., 2011; Bordbar et al., 2011; Liu et al., 2010; Zhang et al., 2010) and cordycepin, adenosine, ergosterol, and polysaccharides in cordyceps sinensis (Yu et al., 2009; Kim and Yun, 2005; Chang et al., 2008; Won and Park, 2008; Panda and Swain, 2011). We have isolated and purified some bioactive substances in sea cucumber, which included fucosylated chondroitin sulfate, fucoidan, saponin, cerebroside and among others (Hu et al., 2010). Further studies on the identification of the substance exerting hpyerglycemic activity are in progress.

4 Conclusions

SCC exhibited a significant anti-hyperglycemic activity by repairing β-cells in the islets of langerhans and strengthening insulin-mediated PI(3)K/PKB/Glut4 signaling transduction pathway in STZ-induced diabetic rats. Depth investigations are appreciated in order to decipher the mechanism underlining such functions and identify the components of both sea cucumber and cordyceps sinensis exerting hpyerglycemic activity.

Acknowledgements

The research was supported by National Marine Public Welfare Scientific Research Project of China (No. 2011 05029), the National Key Technology S&D Program (No. 2012BAD33B07), and Program for Changjiang Scholars and Innovative Research Team in University (IRT1188).

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(Edited by Qiu Yantao)

(Received June 13, 2012; revised July 30, 2012; accepted April 20, 2013)

© Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2014

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E-mail: jfwang@ouc.edu.cn