- Email: [email protected]
PKB pathway and GLUT4
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2015 www.elsevier.com/locate/jbiosc
Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4 Yiming Wang, Jingfeng Wang, Yanlei Zhao, Shiwei Hu, Di Shi, and Changhu Xue* College of Food Science and Engineering, Ocean University of China, 5th Yushan Road, Qingdao, Shandong Province, 266003, China Received 25 January 2015; accepted 19 May 2015 Available online xxx
The present study investigated the anti-hyperglycemic properties and mechanisms of fucoidan, isolated from Cucumaria frondosa (Cf-FUC), in insulin resistant mice. Male C57BL/6J mice were fed regular diet or high-fat/high-sucrose diet for 19 weeks. Model animals were dietary administrated either rosiglitazone (RSG, 1 mg/kg$bw), fucoidan (Cf-FUC, 80 mg/kg$bw) or their combinations. Results showed that Cf-FUC significantly reduced fasting blood glucose and insulin levels, and enhanced glucose tolerance and insulin tolerance in insulin-resistant mice. Quantitative real-time PCR analysis showed that Cf-FUC increased the mRNA expressions of insulin receptors (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3 kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4). Western blot assays demonstrated that Cf-FUC showed no effect on total protein expression but nevertheless enhanced the phosphorylation of proteins listed above and increased translocation of GLUT4 to the cell membrane. Furthermore, Cf-FUC enhanced the effects of RSG. These results indicated that Cf-FUC exhibited significant anti-hyperglycemic effects via activating PI3K/ PKB pathway and GLUT4 in skeletal muscle and adipose tissue. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Sea cucumber fucoidan; Insulin resistance; Anti-hyperglycemic effects; Phosphatidylinositol 3 kinase pathway; Glucose transporter 4]
Diabetes mellitus is a serious, non-contagious disorder characterized by mellithemia due to an absolute or relative deficiency of insulin secretion or insulin resistance (1). In particular, type 2 diabetes is increasingly associated with morbidity and mortality world-wide (2). However, current therapies are often associated with inadequate glycemic control, adverse events and high secondary-failure rates (3). For instance, rosiglitazone (RSG) is an oral antidiabetic agent that can maintain glucose homeostasis and improve insulin resistance, but it also suffers from generally inadequate efficacy and a range of serious adverse effects (4,5). Therefore, seeking for safe and effective dietary regulations focused on ameliorating type 2 diabetes has been a hot spot of research. Hyperglycemia is the main pathological effect of type 2 diabetes and insulin resistance is the major risk factor for developing type 2 diabetes (6,7). Thus, alleviating insulin resistance is an important preventative step in halting the development of hyperglycemia and ultimately type 2 diabetes. In insulin resistance, target tissues respond poorly to the normal levels of the circulating hormone, which therefore fails to regulate the glucose homeostasis in skeletal muscle, liver, and adipose tissues (8). A fundamental mechanism for antagonizing insulin resistance is the rapid action of insulin to stimulate glucose uptake and metabolism in peripheral tissues which relied on the regular insulin signaling (9). Rains and Jain (10) confirmed that the insulin signal is amplified through the PI3K/PKB pathway. Skeletal
* Corresponding author. Tel.: þ86 532 82031948; fax: þ86 532 82032468. E-mail address: [email protected] (C. Xue).
muscle is considered the most important tissue in terms of glucose storage, because it accounts for approximately 75% of the total insulin-dependent glucose uptake in both human and rodents (11). Adipose tissue is another important destination and insulin insensitivity here plays a central role in the development of the overall insulin resistance, type 2 diabetes and cardiovascular diseases (12). Sea cucumber has been historically used in China because of its numerous health benefits. It contains various bioactive components such as collagen polypeptide, polysaccharide, saponins, and lipids (13e16). Fucoidan isolated from the sea cucumber (SC-FUC) is a sulfated polysaccharide containing a substantial percentage of L-fucose and sulfate ester groups (17). SC-FUC has been reported to have anticoagulant and antithrombotic properties (18), protection from gastric damage (19) and inhibition for osteoclastogenesis (20). In addition, multiple reports showed that fucoidan isolated from alga exhibited anti-hyperglycemia effects, for example, fucoidan extracted from Saccharina japonica had a pronounced hypoglycemic effect in alloxan-induced diabetic rats (21) and fucoidan derived from the Sporophyll of Undaria pinnatifida could regulate blood glucose homoeostasis in C57BL/KSJmþ/þdb and C57BL/KSJ db/db mice (22), while the literature on SC-FUC is scarce. We therefore isolated SC-FUC from Cucumaria frondosa (Cf-FUC) and demonstrated its anti-hyperglycemic potential in combination with RSG, which is a representative of classic hypoglycemic drugs. To investigate the mechanism of action, we determined the insulinmediated genes mRNA expression and protein phosphorylation levels of the classic PI3K/PKB pathway and translocation of GLUT4 protein in insulin resistant mice.
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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TABLE 2. Sequence of the primers used in the quantitative real-time PCR. Gene
Preparation of the Cf-FUC Dry sea cucumber C. frondosa was purchased from a marketplace (Qingdao, Shandong Province, China) and authenticated by Prof. Yulin Liao from Institute of Oceanology, Chinese Academy of Science. The preparation of Cf-FUC was performed according to the method described by Chang et al. (17). Briefly, the dry body wall of the sea cucumber was grinded and degreased with acetone. After hydrolysis with papain and precipitation with cetylpyridinium chloride, the crude polysaccharide was acquired and eluted in Sepharose Q Fast Flow column (GE Healthcare, Uppsala, Sweden) with linear gradient of NaCl solution. The eluent containing fucoidan was detected by chromatography (Aglient1100, Agilent Technologies, Santa Clara, CA, USA), then collected, dialyzed (10 kDa cutoff) and lyophilized. Pre-column derivatization high performance liquid chromatography and ion chromatography revealed the sulfate content of the Cf-FUC was 29.31% and the monosaccharide compositions were fucose, galactosamine, galactose and glucosamine, with a ratio of 1: 0.1: 0.3: 0.17. Animal experiment Male C57BL/6J mice, 4e5 weeks, were purchased from Vital River Laboratory Animal Center (Beijing, China; licensed ID: SCXK2007-0001) with free access to distilled water in a 12e12 h lightedark condition at 23 1 C. All animals experiments were conducted in accordance with internationally valid guidelines and experimental protocols with prior approval by the animal ethics committee according to the guidelines of the Standards for Laboratory Animals of China (GB 14922-94, GB 14923-94, and GB/T 14 925-94). The mice were randomly assigned to 6 groups of 10 animals each: normal control, model control, rosiglitazone group (RSG), Cf-FUC group, low-dose and highdose combination groups (RSGþ20Cf-FUC, RSGþ80Cf-FUC). Administration groups above were dietary supplied with rosiglitazone (1 mg/kg$bw), Cf-FUC (80 mg/ kg$bw) and combinations of low-dose or high-dose Cf-FUC (20 and 80 mg/kg$bw respectively) and rosiglitazone (1 mg/kg$bw). Mice in the normal control group were fed with a regular diet and the others were fed with high fat/sucrose diet (HFSD, according to the AIN-93 recipe). The dietary dose of Cf-FUC and rosiglitazone (Taji Group, Chongqing, China) as well as the forage compositions are shown in Table 1. During the experiment, the dietary dose of Cf-FUC and rosiglitazone was modified slightly according to the body weight and food intake to make a standardized comparison. The animals in each group were treated for 19 weeks. Body weight and food intake were measured during the experiment. Glucose tolerance and insulin tolerance tests were performed 10 days and 5 days before the last treatment respectively. After the last treatment, the hind limb skeletal muscle and epididymal adipose tissue were excised to analyze the PI3K/PKB pathway geneexpression levels, as well as GLUT4 translocation. Glucose tolerance tests Blood was collected from the tail veins of mice after an 8-h fasting period. Fasting blood glucose levels were determined using a commercial testing kit (Biosino Bio-technology and Science Inc., Beijing, China). Oral glucose tolerance test was conducted by measuring the blood glucose levels at 0, 0.5, 1 and 2 h after gavage of glucose at a dose of 2 g/kg$bw. The value was negatively estimated using area under curve (AUC), which was calculated as listed below. AUC ¼ 0.25 A þ 0.5 B þ 0.75 C þ 0.5 D where A, B, C and D represent glucose level at 0, 0.5, 1 and 2 h, respectively. Insulin tolerance tests After an 8-h fasting period, blood was collected through the tail vein and separated to determine fasting blood glucose levels. Insulin (0.75 U/kg$bw, Sigma-Aldrich, St. Louis, MO, USA) was administered by intraperitoneal injection. Blood glucose was determined at 0, 30, 60, and 120 min. Changes in glucose were plotted over time; AUC was also calculated as described before. Determination of insulin sensitivity Serum insulin level was assessed by insulin ELISA kit (R&D, Minneapolis, MN, USA). Homeostasis model assessment of insulin resistance index (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI) (23) were calculated as follows:
IR IRS-1 PI3K PKB GLUT4 b-Actin
Forward primer
Reverse primer
50 -CCTACTGCTATGGGCTTCG-30 50 -TTGCTTGGCACAATGTAGAA-30 5’-CCCATACAAGGTGTTAGCC-30 50 -CCAGATGGTAGCCAACAGT-30 50 -ACTAAGAGCACCGAGACCAA-30 50 -CAAGGCATTGCTGACAGGATG-30
30 -GTTCTGGTCTGGGCTTCTA-50 30 -GAGGATCGTCAATAGCGTAAC-5‘ 30 -ACTCTGACCTGGGATACCG-50 30 -GATAGAGTTTGAGGAGCCG-50 30 -CTGCCCGAAAGAGTCTAAAG-50 30 -GGTCGTCTACACCTAGTCGT-50
HOMA-IR ¼ fasting blood glucose level (mmol$L1) serum insulin level (mIU$ml1)/22.5 QUICKI ¼ 1/(lg(fasting blood glucose level (mmol$L1)) þ lg(serum insulin level (mIU$ml1))) Insulin stimulation and tissue processing The insulin stimulation was prepared according to the method of Wong et al. (24). At the end of 19 weeks, the animals were fasted overnight and injected intraperitoneally with 40 U of insulin (Sigma-Aldrich) per kg (n ¼ 4 per group) for phosphorylated protein analysis or equivalent volumes of saline (n ¼ 3 per group) for total protein levels and mRNA expression assays. After 5 min, the hind limb skeletal muscle and epididymal adipose tissue were excised and immediately frozen in liquid nitrogen and stored at 80 C. Skeletal muscle and adipose tissue were prepared for GLUT4 translocation assay in a similar manner as for protein phosphorylation, except that the fasted animals were injected with 0.5 U/kg insulin and sacrificed 30 min after injection. Skeletal muscle plasma membrane and adipose tissue plasma membrane were prepared according to the method as described previously (25,26). Briefly, 3 g of frozen skeletal muscle tissue was homogenized at low-speed in ice-cold lysis buffer. The homogenate was triple-centrifuged at 1200g, 9000g, and 190,000g successively to obtain pellets containing plasma membranes. The pellets were further separated by sucrose-gradient (25%, 32%, and 35% wt/wt) centrifugation at 150,000g for 16 h. Fractions containing membrane GLUT4 were collected from the fraction of 25% sucrose solution and subjected to 190,000g for 1 h and used for Western blot analysis. Adipose tissue (3 g) was homogenized in TES buffer at 4 C. The homogenate was centrifuged at 3000g to obtain the liquor and then centrifuged at 12,000g to obtain the pellets containing the adipose tissue plasma membranes. Quantitative real-time PCR analysis Total RNA was isolated from skeletal muscle and adipose tissue of mice using TRIzol reagent and the concentrations were detected by spectrophotometer. One microgram of RNA was converted to cDNA using M-MLV reverse transcriptase. Real-time PCR was conducted using the Bio-Rad iQ5 system (Bio-Rad, Hercules, CA, USA). Approximately 25 ml reaction volume was used for the quantitative real-time PCR assay which consisted of 12.5 ml Maxima SYBR Green qPCR Master mix, 10 mM primers (0.3 ml each of forward and reverse primer), 5.9 ml nuclease-free water and 6 ml template. The thermal procedure included an initial denaturation at 95 C for 10 min followed by 45 cycles of denaturation at 95 C for 15 s, annealing at 60 C for 20 s and extension at 72 C for 30 s. Data normalization was conducted using b-actin as the endogenous reference. The gene expression levels were analyzed by relative quantification using the standard curve method. The sequences of the primers (Sangon Biotech, Shanghai, China) are described in Table 2. Western blot analysis Approximately 50 mg of solubilized skeletal muscle protein was fractionated by electrophoresis on 1% SDS-PAGE gels. The fractionated proteins were transferred by electrophoresis to polyvinylidenefluoride membranes in transfer buffer. The membranes were blocked in 5% bovine serum albumin in TBST for 2 h at room temperature and then incubated overnight at 4 C with primary antibodies against b-actin IR-b, p-Tyr-IR-b, IRS-1, p-Tyr612-IRS-1, PI3K, p-p85-PI3K, PKB, p-Ser473-PKB or GLUT4 (Cell Signaling Technology,
TABLE 1. Ingredients of the mice forage (g/kg diet).a,b Composition Casein Cornstarch Sucrose Corn oil Lard Mineral mix Vitamin mix Cellulose Choline Bitartrate DL-Methionine Rosiglitazone Cf-FUC a b
Normal control
Model control
RSG
80Cf-FUC
RSG þ 20Cf- FUC
RSG þ 80Cf- FUC
200 650 0 50 0 35 10 50 3 2 e e
200 250 200 50 200 35 10 50 3 2 e e
200 249.925 200 50 200 35 10 50 3 2 0.075 e
200 248.8 200 50 200 35 10 50 3 2 e 1.2
200 249.625 200 50 200 35 10 50 3 2 0.075 0.3
200 248.725 200 50 200 35 10 50 3 2 0.075 1.2
RSG was fed in dose of 1 mg/kg$bw everyday. Mineral mix and vitamin mix were prepared according to AIN-93 recipe.
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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Danvers, MA, USA). The membranes were washed with TBST and incubated with horseradish peroxidase conjugated secondary antibodies for 2 h at room temperature. Membranes were washed again as above, and visualized by ECL detection kit (Applygen Technologies Inc., Beijing, China). The results were analyzed by Image J software and data were standardized using b-actin as the control. The process for adipose tissue was similar to skeletal muscle, except that the membranes were incubated with primary antibodies against IRS-1, p-Tyr612IRS-1, PI3K, p-p85-PI3K, PKB, p-Ser473-PKB or GLUT4. Statistical analysis Data were presented as mean SD. Data were analyzed statistically by one-way analysis of variance (ANOVA) followed by LSD test. Probability values were considered significant when P < 0.05.
RESULTS Cf-FUC decreased body weight gain As shown in Fig. 1 and Table 3, food intake was significantly lower in all HFSD groups than in the control group while there were no statistical differences in caloric intake between any of the groups. HFSD mice demonstrated significant weight gain compared to normal control group. Cf-FUC decreased the body weight gain by 25.77% in HFSD mice over the 19 weeks period. Compared to RSG group, high dose Cf-FUC combined with RSG significantly reduced the body weight gain in HFSD mice (P < 0.05). Cf-FUC reduced blood glucose Fasting blood glucose level and glucose tolerance were the major assessment criteria of diabetes mellitus. As shown in Table 4 and Fig. 2, the fasting blood glucose level and AUC in model control group were notably increased by HFSD. When mice were fed with Cf-FUC for 19 weeks, fasting blood glucose levels and AUC were significantly lowered by 17.53% and 18.87% respectively. The high dose Cf -FUC/RSG combination showed a superior effect compared to RSG group (P < 0.05). These results illustrated that Cf -FUC exhibited significant anti-hyperglycemic activity and favorable synergy with RSG in insulin resistant mice. Cf -FUC ameliorated insulin resistance As shown in Fig. 3, the AUC obtained with the blood glucose data during the insulin tolerance analysis was 1.32 fold higher in HFSD mice compared with that observed in control mice. Cf-FUC decreased the AUC by 7.91% in HFSD mice. Furthermore, high dose Cf-FUC combined with RSG decreased the AUC by 21.15% in HFSD mice and normalized the blood glucose levels. Cf -FUC enhanced insulin sensitivity Insulin resistance is usually accompanied by hyperinsulinemia, and typically evaluated by calculating the HOMA-IR and QUICKI indexes. As shown in Table 4, HFSD caused a significant increase in serum insulin level. Cf-FUC treatment relieved the hyperinsulinemia in insulin resistant animals, and additionally lowered the HOMA-IR and increased the QUICKI. Particularly, the high-dose Cf-FUC/RSG compound caused a remarkable reduction of in the HOMA-IR value compared to RSG group (P < 0.05), and increased the
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TABLE 3. Effects of Cf-FUC on food intake and body weight gain in insulin resistant mice.a,b
Normal control Model control RSG 80Cf-FUC RSG þ 20Cf-FUC RSG þ 80Cf-FUC
Food intake (g/d)
Calories intake (kcal/d)
Body weight gain (g)
4.10 0.08 3.49 0.24# 3.43 0.20 3.37 0.17 3.32 0.15 3.36 0.16
15.79 0.30 16.92 1.14 16.64 0.99 16.35 0.82 16.08 0.74 16.29 0.79
11.83 0.77 24.49 1.77## 18.43 0.86** 18.18 1.21** 18.35 1.66** 15.33 1.35**+
# P < 0.05, ##P < 0.01 vs normal control group; *P < 0.05, **P < 0.01 vs model control group; +P < 0.05 vs RSG group. a Data are presented as mean SD, n ¼ 10. b The normal control group was supplied with ordinary diet (3.85 kcal/g) and all the other groups were supplied with HFSD (4.85 kcal/g).
QUICKI nearly to the same score as control non-HFSD mice. These results suggested that Cf-FUC significantly alleviated insulin resistance in model animals and combined well with RSG. Cf -FUC activated the mRNA expression of PI3K/PKB pathway and GLUT4 The PI3K/PKB pathway is the main insulin signaling cascade that controls glycometabolism. GLUT4 is a key glucose transporter regulated by the PI3K/PKB pathway. Any deletion of these molecules may lead to insulin resistance. As shown in Fig. 4, mRNA expression levels of genes in PI3K/PKB pathway and GLUT4 were significantly decreased in the HFSD control group. Treatment with Cf-FUC normalized the expression of the above genes. It is important to note that the combination of CfFUC and RSG increased the mRNA expression of IRS-1, PKB and GLUT4 by 123.36%, 112.53% and 305.40% in skeletal muscle and 151.80%, 105.09% and 242.88% in adipose tissue respectively, resulting in genes expression comparable to that of the normal control group. Furthermore, the effect of high dose Cf-FUC combined with RSG was significantly more pronounced than in the RSG group (P < 0.05, P < 0.01). In conclusion, these data illustrated that Cf-FUC could enhance insulin signal transduction through the PI3K/ PKB pathway at the transcription level, and lead to improved glucose homeostasis if administered with RSG. Cf-FUC promoted GLUT4 protein translocation To test whether Cf-FUC reduced blood glucose level via promoting GLUT4 translocation, we analyzed GLUT4 and m-GLUT4 expression levels in skeletal muscle and adipose tissue by Western blotting and calculated relative expression of m-GLUT4/GLUT4. As shown in Figs. 5 and 6, significant decreases occurred in m-GLUT4/GLUT4 level of the HFSD control group in both skeletal muscle and adipose tissue. High-dose Cf-FUC/RSG combination significantly increased membrane GLUT4 protein expression of the insulin resistant mice compared to the RSG group (P < 0.05). This indicated that Cf-FUC stimulated the GLUT4 protein’s
TABLE 4. Effects of Cf-FUC on fasting blood glucose level, serum insulin level and HOMA-IR index and QUICKI in insulin resistant mice.a,b
Normal control Model control RSG 80Cf-FUC RSG þ 20Cf-FUC RSG þ 80Cf-FUC
FIG. 1. Effects of Cf-FUC on body weight in insulin resistant mice for 19 weeks.
Fasting blood glucose level (mmol$L1)
Fasting insulin level (mIU$L1)
HOMA-IR
QUICKI
9.28 1.75 13.12 0.82## 10.92 0.96* 10.82 1.41* 10.11 0.92* 9.72 1.24**
10.82 0.32 14.68 0.69## 13.36 0.14* 13.62 0.03* 13.01 0.19* 11.94 0.60**++
4.53 0.31 8.22 1.10## 5.86 0.25* 6.69 0.01* 5.91 0.11* 4.82 0.40**+
0.498 0.007 0.442 0.010## 0.472 0.004** 0.459 0.001** 0.471 0.002** 0.492 0.009**
# P < 0.05, ##P < 0.01 vs normal control group; *P < 0.05, **P < 0.01 vs model control group; +P < 0.05, ++P < 0.01 vs RSG group. a Data are presented as mean SD, n ¼ 10. b The normal control group was supplied with ordinary diet and all the other groups were supplied with HFSD.
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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FIG. 2. Effects of Cf-FUC on oral glucose tolerance in insulin resistant mice. Chart A is the serum glucose levels at 0, 0.5, 1, 2 h after the glucose gavage at the dose of 2 g$(kg$body weight)1. Chart B is the area under curve in OGTT. Data are presented as mean SD (n ¼ 10/group). ## P < 0.01 vs normal control group; * P < 0.05, ** P < 0.01 vs model control group; +P < 0.05 vs RSG group.
FIG. 3. Effects of Cf-FUC on insulin tolerance in insulin resistant mice. Chart A is the serum glucose levels at 0, 0.5, 1, 2 h after intraperitoneal injection of insulin at the dose of 0.75 U (kg$body weight)1. Chart B is the area under curve in insulin tolerance test. Data are presented as mean SD (n ¼ 10/group). ## P < 0.01 vs normal control group; * P < 0.05, ** P < 0.01 vs model control group.
translocation to the plasma membrane and the combination also led a superior efficacy. Cf-FUC stimulated PKB and PI3K phosphorylation As insulin-stimulated GLUT4 translocation was increased by Cf-FUC in both skeletal muscle and adipose tissue, we subsequently
determined the total protein expressions of PKB and PI3K as well as the level of their phosphorylation, which are the major upstream regulators of GLUT4 translocation. As shown in Figs. 5 and 6, no statistically significant differences were demonstrated with any of the proteins. However, the phosphorylations of p-
FIG. 4. Effects of Cf-FUC on the expression of PI3K/PKB genes in skeletal muscle and adipose tissue of insulin resistant mice. Chart A, B, C, D, and E are representative data of quantitative real-time-PCR for IR, IRS-1, PI3K, PKB, and GLUT4 mRNA relative expression levels respectively. Data are presented as mean SD (n ¼ 10/group). ## P < 0.01 vs normal control group. * P < 0.05, ** P < 0.01 vs model control group; +P < 0.05, ++P < 0.01 vs RSG group.
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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FIG. 5. Effects of Cf-FUC on insulin signaling pathway proteins in skeletal muscle of insulin resistant mice. Chart A, B, C, D and E are representative data of Western blot for the expression levels of p-Tyr-IR-b, IR-b, p-Ser307-IRS-1, IRS-1, p-p85-PI3K, PI3K, p-Ser473-PKB, PKB and m-GLUT4/GLUT4 proteins respectively. Results were normalized by b-actin, and the phosphorylation was normalized by their corresponding protein abundance. Data are presented as mean SD (n ¼ 10/group). # P < 0.05, ## P < 0.01 vs normal control group. * P < 0.05, ** P < 0.01 vs model control group; +P < 0.05, ++P < 0.01 vs RSG group.
p85-PI3K, p-Ser473-PKB proteins in skeletal muscle and adipose tissue were noteworthy. When compared with the normal control group, the phosphorylation levels of proteins listed above were significantly reduced in insulin resistant mice. The Cf-FUC treatment markedly accelerated the phosphorylation in various active sites of the genes in the PI3K/PKB pathway. Moreover, the high-dose combination exhibited a superior promoting effect compared with RSG group (P < 0.05, P < 0.01), which amplified the protein phosphorylation levels by several fold in insulin resistant animals. These results indicated that Cf-FUC facilitated the phosphorylation of PKB and PI3K, and had synergistic effects with RSG.
combination treatment further significantly influenced the change towards to normal range (P < 0.05, P < 0.01). These results indicated that Cf-FUC activated PKB and PI3K by increasing tyrosine phosphorylation of IR and IRS-1. We speculate that Cf-FUC promoted insulin secretion, and the increased insulin phosphorylated IR, which subsequently activated IRS-1, PI3K, PKB and finally enhanced GLUT4 translocation. As a result, glucose was transported from the cellular matrix to the cytoplasm and was then oxidized to produce ATP.
Cf-FUC enhanced IR-b and IRS-1 phosphorylation Since CfFUC enhanced the phosphorylation of PKB and PI3K, we also tested the total protein and tyrosine phosphorylation of IR-b and IRS-1, which are known to activate PI3K and PKB. As shown in Figs. 5 and 6, skeletal muscle IR-b, IRS-1 and adipose tissue IRS-1 total protein remained unchanged in normal control and HFSD control groups. Supplementation of Cf-FUC resulted in notable increases in skeletal muscle tyrosine phosphorylation of IR-b and IRS-1 and adipose tissue tyrosine phosphorylation of IRS-1, while the
In present study, we investigated the anti-hyperglycemic effects of fucoidan isolated from the sea cucumber C. frondosa. An animal model of type 2 diabetes model was developed by a classic method, namely feeding C57BL/6J mice with HFSD (27,28). The serum parameters associated with glucose metabolism were determined to observe the symptom of insulin resistant. Moreover, to detect the mechanism, gene expressions of the PI3K/PKB pathway and GLUT4 in skeletal muscle and adipose tissue were tested. Current data indicated that giving animals Cf-FUC dramatically reduced the signs
DISCUSSION
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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FIG. 6. Effects of Cf-FUC on insulin signaling pathway proteins in adipose tissue of insulin resistant mice. Chart A, B, C and D are representative data of Western blot for the expression levels of p-Ser307-IRS-1, IRS-1, p-p85-PI3K, PI3K, p-Ser473-PKB, PKB and m-GLUT4/GLUT4 proteins respectively. Results were normalized by b-actin, and the phosphorylation was normalized by their corresponding protein abundance. Data are presented as mean SD (n ¼ 10/group). # P < 0.05, ## P < 0.01 vs normal control group. * P < 0.05, ** P < 0.01 vs model control group; +P < 0.05 vs RSG group.
of diabetes and also normalized the PI3K/PKB pathway and GLUT4 translocation. The insulin-stimulated glucose uptake is mainly controlled by the PI3K/PKB pathway, which is a crucial signaling cascade triggered by insulin and other growth factors. As the prior receptor tyrosine kinase of this pathway, the IR is a heterotetramer consisting of two extracellular a-subunits and two transmembrane bsubunits held together by disulfide bonds (29). Insulin binding to the a-subunit leads to derepression of the kinase activity in the bsubunit followed by transphosphorylation of the b-subunits and a conformational change that further increases kinase activity (11). The activated IR then catalyzes the phosphorylation at the tyrosine residues of several intracellular substrates, including the IRS proteins (30). In insulin resistant models, the phosphorylation of IR and IRS is markedly inhibited (31,32). When treated with Cf-FUC, the gene expression levels together with the tyrosine phosphorylation of IR and IRS-1 were both increased in skeletal muscle and adipose tissue of insulin resistant mice. These indicated that Cf-FUC could activate IR/IRS-1 signaling cascades. Phosphorylation of IRS will subsequently activate PI3K and PKB, both of which are crucial for insulin signal transduction and stimulating glucose transport in target tissues. As an essential role in glucose uptake and GLUT4 translocation, inhibition of PI3K with pharmacological inhibitors or transfection with dominant-negative constructs will block almost all metabolic actions of insulin (33). PI3K consists of a p110 catalytic subunit and a p85 regulatory subunit that possesses two SH2 domains that interact with tyrosine-phosphorylated pYMXM and pYXXM motifs in IRS proteins (34). Upon phosphorylation, IRS activates PI3K to produce PIP3, which subsequently activates PKB (35). PKB phosphorylated in serine 473 is one of the key downstream mediators of PI3K. The main mechanism relating to glucose uptake of the phosphorylated
PKB is activating the phosphoinositide-3-phosphate-5-kinase to subsequently stimulate the translocation of GLUT4 vesicles to the plasma membrane (9). In addition, a study by Kim et al. (36) showed that high carbohydrate diet has no effect on the resultant total protein expression of the PI3K/PKB pathway. However, a study by Hu et al. (26) showed that hyper glycemia and insulin resistance could be promoted via phosphorylation PI3K/PKB signaling cascade in skeletal muscle of insulin resistant mice without any effects on total protein expressions. In the present study, Cf-FUC provided significant increase in PI3K and PKB gene expression levels of insulin resistant mice. Meanwhile, Cf-FUC enhanced phosphorylation of PI3K in p85 and of PKB in serine473. These results suggest that Cf-FUC acts via phosphorylating the p85 regulatory subunit of PI3K leading to the activation of PKB and consequently stimulating glucose uptake. The principal glucose transporter protein that mediates glucose uptake is GLUT4, which plays a key role in regulating whole body glucose homeostasis (37). Insulin promotes glucose uptake in cells by mobilizing the GLUT4 storage vesicles and stimulating the translocation of GLUT4 from the vesicles to the plasma membrane (38). During the development of type 2 diabetes, this action is compromised. The biogenesis of the insulin responsive vesicles is inhibited and GLUT4 slowly recycles between the plasma membrane and the intracellular vesicles (39). A number of previous reports have shown that a marked reduction in the transcription of GLUT4 could be detected in animal models of insulin resistance and type 2 diabetes (40e42). Furthermore, multiple studies demonstrated that these effects can be attenuated through upregulating the numbers of membrane GLUT4 transporters. Gandhi et al. (43) found that embelin isolated from Embelia ribes promoted the translocation of GLUT4 in STZ-induced insulin resistant rats. Wong et al. (24) discovered that Des-aspartate-angiotensin-I and
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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angiotensin IV improved glucose tolerance and membrane GLUT4 concentrations in diet-induced insulin resistant mice. In our study, Cf-FUC dramatically augmented GLUT4 gene expression level and membrane GLUT4 abundance in skeletal muscles of diet-induced insulin resistant mice. All of these studies demonstrated that the transcription and translocation of GLUT4 gene correlated with antihyperglycemic effects of the experimental compounds. The present study demonstrated that Cf-FUC possessed significant anti-hyperglycemic activity. The underlying mechanisms of these effects involved the activation of PI3K/PKB insulin signaling cascade which led to the translocation of GLUT4. In addition, Cf-FUC exhibited well synergistic effects with RSG. Thus, if nutritional supplementation with Cf-FUC for human is validated, Cf-FUC may be used as a complementary therapy for diet-induced type 2 diabetes. ACKNOWLEDGMENTS We acknowledged supports from National Natural Science Foundation of China (No. 31371876, No. 31471684 and No. U1406402). References 1. Alberti, K. G. and Zimmet, P. Z.: Definition, diagnosis and classification of diabetes mellitus and its complications. Part1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation, Diabet. Med., 15, 539e553 (1998). 2. Lloyd, D. J., Wheeler, M. C., and Gekakis, N.: A point mutation in Sec61a1 leads to diabetes and hepatosteatosis in mice, Diabetes, 59, 460e470 (2010). 3. Gao, R., Wang, Y., Wu, Z., Ming, J., and Zhao, G.: Interaction of barley b-glucan and tea polyphenols on glucose metabolism in streptozotocin-induced diabetic rats, J. Food Sci., 77, 128e134 (2012). 4. Bailey, C. J.: Drugs on the horizon for diabesity, Curr. Diab. Rep., 5, 353e359 (2005). 5. Hemmeryckx, B., Hoylaerts, M. F., Gallacher, D. J., Lu, H. R., Himmelreich, U., D’hooge, J., Swinnen, M., and Lijnen, H. R.: Does rosiglitazone affect adiposity and cardiac function in genetic diabetic mice, Eur. J. Pharmacol., 700, 23e31 (2013). 6. Donath, M. Y. and Shoelson, S. E.: Type 2 diabetes as an inflammatory disease, Nat. Immunol., 11, 98e107 (2011). 7. Alsahli, M. and Gerich, J. E.: Hypoglycemia, Endocrinol. Metab. Clin. North Am., 42, 657e676 (2013). 8. Shulman, G. I.: Cellular mechanisms of insulin resistance, J. Clin. Invest., 106, 171e176 (2000). 9. Choi, K. and Kim, Y. B.: Molecular mechanism of insulin resistance in obesity and type 2 diabetes, Korean J. Intern. Med., 25, 119e129 (2010). 10. Rains, J. L. and Jain, S. K.: Oxidative stress, insulin signaling, and diabetes, Free Radic. Biol. Med., 50, 567e575 (2011). 11. Saltiel, A. R. and Kahn, C. R.: Insulin signaling and the regulation of glucose and lipid metabolism, Nature, 414, 799e806 (2001). 12. Romacho, T., Elsen, M., Rohrborn, D., and Eckel, J.: Adipose tissue and its role in organ crosstalk, Acta Physiol., 210, 733e753 (2014). 13. Xu, J., Wang, Y. M., Feng, T. Y., Zhang, B., Sugawara, T., and Xue, C. H.: Isolation and anti-fatty liver activity of a novel cerebroside from the sea cucumber Acaudina molpadioides, Biosci. Biotechnol. Biochem., 75, 1466e1471 (2011). 14. Bordbar, S., Anwar, F., and Saari, N.: High-value components and bioactives from sea cucumbers for functional foodsda review, Mar. Drugs, 9, 1761e1805 (2011). 15. Hu, X., Wang, Y., Wang, J., Xue, Y., Li, Z., Nagao, K., Yanagita, T., and Xue, C.: Dietary saponins of sea cucumber alleviate orotic acid-induced fatty liver in rats via PPARa and SREBP-1c signaling, Lipids Health Dis., 9, 25 (2010). 16. Yu, L., Ge, L., Xue, C., Chang, Y., Zhang, C., Xu, X., and Wang, Y.: Structural study of fucoidan from sea cucumber Acaudina molpadioides: a fucoidan containing novel tetrafucose repeating unit, Food Chem., 142, 197e200 (2014). 17. Chang, Y., Xue, C., Tang, Q., Li, D., Wu, X., and Wang, J.: Isolation and characterization of a sea cucumber fucoidan-utilizing marine bacterium, Lett. Appl. Microbiol., 50, 301e307 (2010). 18. Chen, S., Hu, Y., Ye, X., Li, G., Yu, G., Xue, C., and Chai, W.: Sequence determination and anticoagulant and antithrombotic activities of a novel sulfated fucan isolated from the sea cucumber Isostichopus badionotus, Biochim. Biophys. Acta, 1820, 989e1000 (2012). 19. Wang, Y., Su, W., Zhang, C., Xue, C., Chang, Y., Wu, X., Tang, Q., and Wang, J.: Protective effect of sea cucumber Acaudina molpadioides fucoidan against ethanol-induced gastric damage, Food Chem., 133, 1414e1419 (2012).
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S., Walton, F. S., Saunders, R. J., and Stýblo, M.: Characterization of the impaired glucose homeostasis produced in C57BL/6J mice by chronic exposure to arsenic and high-fat diet, Environ. Health Perspect., 119, 1104 (2011). 29. Belfiore, A., Frasca, F., Pandini, G., Sciacca, L., and Vigneri, R.: Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease, Endocr. Rev., 30, 586e623 (2009). 30. Saltiel, A. R. and Pessin, J. E.: Insulin signaling pathways in time and space, Trends Cell Biol., 12, 65e71 (2002). 31. Mima, A., Ohshiro, Y., Kitada, M., Matsumoto, M., Geraldes, P., Li, C., Li, Q., White, G. S., Cahill, C., Madsen, C. R., and King, G. L.: Glomerular-specific protein kinase C-b-induced insulin receptor substrate-1 dysfunction and insulin resistance in rat models of diabetes and obesity, Kidney Int., 79, 883e896 (2011). 32. Whelan, S. A., Dias, W. B., Thiruneelakantapillai, L., Lane, M. D., and Hart, G. W.: Regulation of insulin receptor substrate 1 (IRS-1)/AKT kinasemediated insulin signaling by O-Linked b-N-acetylglucosamine in 3T3-L1 adipocytes, J. Biol. Chem., 285, 5204e5211 (2010). 33. Taniguchi, C. M., Emanuelli, B., and Kahn, C. R.: Critical nodes in signalling pathways: insights into insulin action, Nat. Rev. Mol. Cell Biol., 7, 85e96 (2006). 34. Myers, M. G., Backer, J. M., Sun, X. J., Shoelson, S., Hu, P., Schlessinger, J., Yoakim, M., Schaffhausen, B., and White, M. F.: IRS-1 activates phosphatidylinositol 3’-kinase by associating with src homology 2 domains of p85, Proc. Natl. Acad. Sci. USA, 89, 10350e10354 (1992). 35. Tsatsoulis, A., Mantzaris, M. D., Bellou, S., and Andrikoula, M.: Insulin resistance: an adaptive mechanism becomes maladaptive in the current environmentdan evolutionary perspective, Metabolism, 62, 622e633 (2012). 36. Kim, S., Jwa, H., Yanagawa, Y., and Park, T.: Extract from Dioscorea batatas ameliorates insulin resistance in mice fed a high-fat diet, J. Med. Food, 15, 527e534 (2012). 37. Huang, S. and Czech, M. P.: The GLUT4 glucose transporter, Cell Metab., 5, 237e252 (2007). 38. Rubin, B. R. and Bogan, J. S.: Intracellular retention and insulin-stimulated mobilization of GLUT4 glucose transporters, Vitam. Horm., 80, 155e192 (2009). 39. Bogan, J. S. and Kandror, K. V.: Biogenesis and regulation of insulin-responsive vesicles containing GLUT4, Curr. Opin. Cell Biol., 22, 506e512 (2010). 40. Kumar, R., Balaji, S., Uma, T. S., and Sehgal, P. K.: Fruit extracts of Momordica charantia potentiate glucose uptake and up-regulate Glut-4, PPARg and PI3K, J. Ethnopharmacol., 126, 533e537 (2009). 41. Wang, Z. Q., Zhang, X. H., Yu, Y., Poulev, A., Ribnicky, D., Floyd, E., and Cefalu, W. T.: Bioactives from bitter melon enhance insulin signaling and modulate acyl carnitine content in skeletal muscle in high-fat diet-fed mice, J. Nutr. Biochem., 22, 1064e1073 (2011). 42. Koren, G. M., Aviram, M., and Hayek, T.: Paraoxonase 1 (PON1) reduces insulin resistance in mice fed a high-fat diet, and promotes GLUT4 overexpression in myocytes, via the IRS-1/Akt pathway, Atherosclerosis, 229, 71e78 (2013). 43. Gandhi, G. R., Stalin, A., Balakrishna, K., Ignacimuthu, S., Paulraj, M. G., and Vishal, R.: Insulin sensitization via partial agonism of PPARg and glucose uptake through translocation and activation of GLUT4 in PI3K/p-Akt signaling pathway by embelin in type 2 diabetic rats, Biochim. Biophys. Acta, 1830, 2243e2255 (2012).
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4 Yiming Wang, Jingfeng Wang, Yanlei Zhao, Shiwei Hu, Di Shi, and Changhu Xue* College of Food Science and Engineering, Ocean University of China, 5th Yushan Road, Qingdao, Shandong Province, 266003, China Received 25 January 2015; accepted 19 May 2015 Available online xxx
The present study investigated the anti-hyperglycemic properties and mechanisms of fucoidan, isolated from Cucumaria frondosa (Cf-FUC), in insulin resistant mice. Male C57BL/6J mice were fed regular diet or high-fat/high-sucrose diet for 19 weeks. Model animals were dietary administrated either rosiglitazone (RSG, 1 mg/kg$bw), fucoidan (Cf-FUC, 80 mg/kg$bw) or their combinations. Results showed that Cf-FUC significantly reduced fasting blood glucose and insulin levels, and enhanced glucose tolerance and insulin tolerance in insulin-resistant mice. Quantitative real-time PCR analysis showed that Cf-FUC increased the mRNA expressions of insulin receptors (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3 kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4). Western blot assays demonstrated that Cf-FUC showed no effect on total protein expression but nevertheless enhanced the phosphorylation of proteins listed above and increased translocation of GLUT4 to the cell membrane. Furthermore, Cf-FUC enhanced the effects of RSG. These results indicated that Cf-FUC exhibited significant anti-hyperglycemic effects via activating PI3K/ PKB pathway and GLUT4 in skeletal muscle and adipose tissue. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Sea cucumber fucoidan; Insulin resistance; Anti-hyperglycemic effects; Phosphatidylinositol 3 kinase pathway; Glucose transporter 4]
Diabetes mellitus is a serious, non-contagious disorder characterized by mellithemia due to an absolute or relative deficiency of insulin secretion or insulin resistance (1). In particular, type 2 diabetes is increasingly associated with morbidity and mortality world-wide (2). However, current therapies are often associated with inadequate glycemic control, adverse events and high secondary-failure rates (3). For instance, rosiglitazone (RSG) is an oral antidiabetic agent that can maintain glucose homeostasis and improve insulin resistance, but it also suffers from generally inadequate efficacy and a range of serious adverse effects (4,5). Therefore, seeking for safe and effective dietary regulations focused on ameliorating type 2 diabetes has been a hot spot of research. Hyperglycemia is the main pathological effect of type 2 diabetes and insulin resistance is the major risk factor for developing type 2 diabetes (6,7). Thus, alleviating insulin resistance is an important preventative step in halting the development of hyperglycemia and ultimately type 2 diabetes. In insulin resistance, target tissues respond poorly to the normal levels of the circulating hormone, which therefore fails to regulate the glucose homeostasis in skeletal muscle, liver, and adipose tissues (8). A fundamental mechanism for antagonizing insulin resistance is the rapid action of insulin to stimulate glucose uptake and metabolism in peripheral tissues which relied on the regular insulin signaling (9). Rains and Jain (10) confirmed that the insulin signal is amplified through the PI3K/PKB pathway. Skeletal
* Corresponding author. Tel.: þ86 532 82031948; fax: þ86 532 82032468. E-mail address: [email protected] (C. Xue).
muscle is considered the most important tissue in terms of glucose storage, because it accounts for approximately 75% of the total insulin-dependent glucose uptake in both human and rodents (11). Adipose tissue is another important destination and insulin insensitivity here plays a central role in the development of the overall insulin resistance, type 2 diabetes and cardiovascular diseases (12). Sea cucumber has been historically used in China because of its numerous health benefits. It contains various bioactive components such as collagen polypeptide, polysaccharide, saponins, and lipids (13e16). Fucoidan isolated from the sea cucumber (SC-FUC) is a sulfated polysaccharide containing a substantial percentage of L-fucose and sulfate ester groups (17). SC-FUC has been reported to have anticoagulant and antithrombotic properties (18), protection from gastric damage (19) and inhibition for osteoclastogenesis (20). In addition, multiple reports showed that fucoidan isolated from alga exhibited anti-hyperglycemia effects, for example, fucoidan extracted from Saccharina japonica had a pronounced hypoglycemic effect in alloxan-induced diabetic rats (21) and fucoidan derived from the Sporophyll of Undaria pinnatifida could regulate blood glucose homoeostasis in C57BL/KSJmþ/þdb and C57BL/KSJ db/db mice (22), while the literature on SC-FUC is scarce. We therefore isolated SC-FUC from Cucumaria frondosa (Cf-FUC) and demonstrated its anti-hyperglycemic potential in combination with RSG, which is a representative of classic hypoglycemic drugs. To investigate the mechanism of action, we determined the insulinmediated genes mRNA expression and protein phosphorylation levels of the classic PI3K/PKB pathway and translocation of GLUT4 protein in insulin resistant mice.
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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J. BIOSCI. BIOENG., MATERIALS AND METHODS
TABLE 2. Sequence of the primers used in the quantitative real-time PCR. Gene
Preparation of the Cf-FUC Dry sea cucumber C. frondosa was purchased from a marketplace (Qingdao, Shandong Province, China) and authenticated by Prof. Yulin Liao from Institute of Oceanology, Chinese Academy of Science. The preparation of Cf-FUC was performed according to the method described by Chang et al. (17). Briefly, the dry body wall of the sea cucumber was grinded and degreased with acetone. After hydrolysis with papain and precipitation with cetylpyridinium chloride, the crude polysaccharide was acquired and eluted in Sepharose Q Fast Flow column (GE Healthcare, Uppsala, Sweden) with linear gradient of NaCl solution. The eluent containing fucoidan was detected by chromatography (Aglient1100, Agilent Technologies, Santa Clara, CA, USA), then collected, dialyzed (10 kDa cutoff) and lyophilized. Pre-column derivatization high performance liquid chromatography and ion chromatography revealed the sulfate content of the Cf-FUC was 29.31% and the monosaccharide compositions were fucose, galactosamine, galactose and glucosamine, with a ratio of 1: 0.1: 0.3: 0.17. Animal experiment Male C57BL/6J mice, 4e5 weeks, were purchased from Vital River Laboratory Animal Center (Beijing, China; licensed ID: SCXK2007-0001) with free access to distilled water in a 12e12 h lightedark condition at 23 1 C. All animals experiments were conducted in accordance with internationally valid guidelines and experimental protocols with prior approval by the animal ethics committee according to the guidelines of the Standards for Laboratory Animals of China (GB 14922-94, GB 14923-94, and GB/T 14 925-94). The mice were randomly assigned to 6 groups of 10 animals each: normal control, model control, rosiglitazone group (RSG), Cf-FUC group, low-dose and highdose combination groups (RSGþ20Cf-FUC, RSGþ80Cf-FUC). Administration groups above were dietary supplied with rosiglitazone (1 mg/kg$bw), Cf-FUC (80 mg/ kg$bw) and combinations of low-dose or high-dose Cf-FUC (20 and 80 mg/kg$bw respectively) and rosiglitazone (1 mg/kg$bw). Mice in the normal control group were fed with a regular diet and the others were fed with high fat/sucrose diet (HFSD, according to the AIN-93 recipe). The dietary dose of Cf-FUC and rosiglitazone (Taji Group, Chongqing, China) as well as the forage compositions are shown in Table 1. During the experiment, the dietary dose of Cf-FUC and rosiglitazone was modified slightly according to the body weight and food intake to make a standardized comparison. The animals in each group were treated for 19 weeks. Body weight and food intake were measured during the experiment. Glucose tolerance and insulin tolerance tests were performed 10 days and 5 days before the last treatment respectively. After the last treatment, the hind limb skeletal muscle and epididymal adipose tissue were excised to analyze the PI3K/PKB pathway geneexpression levels, as well as GLUT4 translocation. Glucose tolerance tests Blood was collected from the tail veins of mice after an 8-h fasting period. Fasting blood glucose levels were determined using a commercial testing kit (Biosino Bio-technology and Science Inc., Beijing, China). Oral glucose tolerance test was conducted by measuring the blood glucose levels at 0, 0.5, 1 and 2 h after gavage of glucose at a dose of 2 g/kg$bw. The value was negatively estimated using area under curve (AUC), which was calculated as listed below. AUC ¼ 0.25 A þ 0.5 B þ 0.75 C þ 0.5 D where A, B, C and D represent glucose level at 0, 0.5, 1 and 2 h, respectively. Insulin tolerance tests After an 8-h fasting period, blood was collected through the tail vein and separated to determine fasting blood glucose levels. Insulin (0.75 U/kg$bw, Sigma-Aldrich, St. Louis, MO, USA) was administered by intraperitoneal injection. Blood glucose was determined at 0, 30, 60, and 120 min. Changes in glucose were plotted over time; AUC was also calculated as described before. Determination of insulin sensitivity Serum insulin level was assessed by insulin ELISA kit (R&D, Minneapolis, MN, USA). Homeostasis model assessment of insulin resistance index (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI) (23) were calculated as follows:
IR IRS-1 PI3K PKB GLUT4 b-Actin
Forward primer
Reverse primer
50 -CCTACTGCTATGGGCTTCG-30 50 -TTGCTTGGCACAATGTAGAA-30 5’-CCCATACAAGGTGTTAGCC-30 50 -CCAGATGGTAGCCAACAGT-30 50 -ACTAAGAGCACCGAGACCAA-30 50 -CAAGGCATTGCTGACAGGATG-30
30 -GTTCTGGTCTGGGCTTCTA-50 30 -GAGGATCGTCAATAGCGTAAC-5‘ 30 -ACTCTGACCTGGGATACCG-50 30 -GATAGAGTTTGAGGAGCCG-50 30 -CTGCCCGAAAGAGTCTAAAG-50 30 -GGTCGTCTACACCTAGTCGT-50
HOMA-IR ¼ fasting blood glucose level (mmol$L1) serum insulin level (mIU$ml1)/22.5 QUICKI ¼ 1/(lg(fasting blood glucose level (mmol$L1)) þ lg(serum insulin level (mIU$ml1))) Insulin stimulation and tissue processing The insulin stimulation was prepared according to the method of Wong et al. (24). At the end of 19 weeks, the animals were fasted overnight and injected intraperitoneally with 40 U of insulin (Sigma-Aldrich) per kg (n ¼ 4 per group) for phosphorylated protein analysis or equivalent volumes of saline (n ¼ 3 per group) for total protein levels and mRNA expression assays. After 5 min, the hind limb skeletal muscle and epididymal adipose tissue were excised and immediately frozen in liquid nitrogen and stored at 80 C. Skeletal muscle and adipose tissue were prepared for GLUT4 translocation assay in a similar manner as for protein phosphorylation, except that the fasted animals were injected with 0.5 U/kg insulin and sacrificed 30 min after injection. Skeletal muscle plasma membrane and adipose tissue plasma membrane were prepared according to the method as described previously (25,26). Briefly, 3 g of frozen skeletal muscle tissue was homogenized at low-speed in ice-cold lysis buffer. The homogenate was triple-centrifuged at 1200g, 9000g, and 190,000g successively to obtain pellets containing plasma membranes. The pellets were further separated by sucrose-gradient (25%, 32%, and 35% wt/wt) centrifugation at 150,000g for 16 h. Fractions containing membrane GLUT4 were collected from the fraction of 25% sucrose solution and subjected to 190,000g for 1 h and used for Western blot analysis. Adipose tissue (3 g) was homogenized in TES buffer at 4 C. The homogenate was centrifuged at 3000g to obtain the liquor and then centrifuged at 12,000g to obtain the pellets containing the adipose tissue plasma membranes. Quantitative real-time PCR analysis Total RNA was isolated from skeletal muscle and adipose tissue of mice using TRIzol reagent and the concentrations were detected by spectrophotometer. One microgram of RNA was converted to cDNA using M-MLV reverse transcriptase. Real-time PCR was conducted using the Bio-Rad iQ5 system (Bio-Rad, Hercules, CA, USA). Approximately 25 ml reaction volume was used for the quantitative real-time PCR assay which consisted of 12.5 ml Maxima SYBR Green qPCR Master mix, 10 mM primers (0.3 ml each of forward and reverse primer), 5.9 ml nuclease-free water and 6 ml template. The thermal procedure included an initial denaturation at 95 C for 10 min followed by 45 cycles of denaturation at 95 C for 15 s, annealing at 60 C for 20 s and extension at 72 C for 30 s. Data normalization was conducted using b-actin as the endogenous reference. The gene expression levels were analyzed by relative quantification using the standard curve method. The sequences of the primers (Sangon Biotech, Shanghai, China) are described in Table 2. Western blot analysis Approximately 50 mg of solubilized skeletal muscle protein was fractionated by electrophoresis on 1% SDS-PAGE gels. The fractionated proteins were transferred by electrophoresis to polyvinylidenefluoride membranes in transfer buffer. The membranes were blocked in 5% bovine serum albumin in TBST for 2 h at room temperature and then incubated overnight at 4 C with primary antibodies against b-actin IR-b, p-Tyr-IR-b, IRS-1, p-Tyr612-IRS-1, PI3K, p-p85-PI3K, PKB, p-Ser473-PKB or GLUT4 (Cell Signaling Technology,
TABLE 1. Ingredients of the mice forage (g/kg diet).a,b Composition Casein Cornstarch Sucrose Corn oil Lard Mineral mix Vitamin mix Cellulose Choline Bitartrate DL-Methionine Rosiglitazone Cf-FUC a b
Normal control
Model control
RSG
80Cf-FUC
RSG þ 20Cf- FUC
RSG þ 80Cf- FUC
200 650 0 50 0 35 10 50 3 2 e e
200 250 200 50 200 35 10 50 3 2 e e
200 249.925 200 50 200 35 10 50 3 2 0.075 e
200 248.8 200 50 200 35 10 50 3 2 e 1.2
200 249.625 200 50 200 35 10 50 3 2 0.075 0.3
200 248.725 200 50 200 35 10 50 3 2 0.075 1.2
RSG was fed in dose of 1 mg/kg$bw everyday. Mineral mix and vitamin mix were prepared according to AIN-93 recipe.
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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Danvers, MA, USA). The membranes were washed with TBST and incubated with horseradish peroxidase conjugated secondary antibodies for 2 h at room temperature. Membranes were washed again as above, and visualized by ECL detection kit (Applygen Technologies Inc., Beijing, China). The results were analyzed by Image J software and data were standardized using b-actin as the control. The process for adipose tissue was similar to skeletal muscle, except that the membranes were incubated with primary antibodies against IRS-1, p-Tyr612IRS-1, PI3K, p-p85-PI3K, PKB, p-Ser473-PKB or GLUT4. Statistical analysis Data were presented as mean SD. Data were analyzed statistically by one-way analysis of variance (ANOVA) followed by LSD test. Probability values were considered significant when P < 0.05.
RESULTS Cf-FUC decreased body weight gain As shown in Fig. 1 and Table 3, food intake was significantly lower in all HFSD groups than in the control group while there were no statistical differences in caloric intake between any of the groups. HFSD mice demonstrated significant weight gain compared to normal control group. Cf-FUC decreased the body weight gain by 25.77% in HFSD mice over the 19 weeks period. Compared to RSG group, high dose Cf-FUC combined with RSG significantly reduced the body weight gain in HFSD mice (P < 0.05). Cf-FUC reduced blood glucose Fasting blood glucose level and glucose tolerance were the major assessment criteria of diabetes mellitus. As shown in Table 4 and Fig. 2, the fasting blood glucose level and AUC in model control group were notably increased by HFSD. When mice were fed with Cf-FUC for 19 weeks, fasting blood glucose levels and AUC were significantly lowered by 17.53% and 18.87% respectively. The high dose Cf -FUC/RSG combination showed a superior effect compared to RSG group (P < 0.05). These results illustrated that Cf -FUC exhibited significant anti-hyperglycemic activity and favorable synergy with RSG in insulin resistant mice. Cf -FUC ameliorated insulin resistance As shown in Fig. 3, the AUC obtained with the blood glucose data during the insulin tolerance analysis was 1.32 fold higher in HFSD mice compared with that observed in control mice. Cf-FUC decreased the AUC by 7.91% in HFSD mice. Furthermore, high dose Cf-FUC combined with RSG decreased the AUC by 21.15% in HFSD mice and normalized the blood glucose levels. Cf -FUC enhanced insulin sensitivity Insulin resistance is usually accompanied by hyperinsulinemia, and typically evaluated by calculating the HOMA-IR and QUICKI indexes. As shown in Table 4, HFSD caused a significant increase in serum insulin level. Cf-FUC treatment relieved the hyperinsulinemia in insulin resistant animals, and additionally lowered the HOMA-IR and increased the QUICKI. Particularly, the high-dose Cf-FUC/RSG compound caused a remarkable reduction of in the HOMA-IR value compared to RSG group (P < 0.05), and increased the
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TABLE 3. Effects of Cf-FUC on food intake and body weight gain in insulin resistant mice.a,b
Normal control Model control RSG 80Cf-FUC RSG þ 20Cf-FUC RSG þ 80Cf-FUC
Food intake (g/d)
Calories intake (kcal/d)
Body weight gain (g)
4.10 0.08 3.49 0.24# 3.43 0.20 3.37 0.17 3.32 0.15 3.36 0.16
15.79 0.30 16.92 1.14 16.64 0.99 16.35 0.82 16.08 0.74 16.29 0.79
11.83 0.77 24.49 1.77## 18.43 0.86** 18.18 1.21** 18.35 1.66** 15.33 1.35**+
# P < 0.05, ##P < 0.01 vs normal control group; *P < 0.05, **P < 0.01 vs model control group; +P < 0.05 vs RSG group. a Data are presented as mean SD, n ¼ 10. b The normal control group was supplied with ordinary diet (3.85 kcal/g) and all the other groups were supplied with HFSD (4.85 kcal/g).
QUICKI nearly to the same score as control non-HFSD mice. These results suggested that Cf-FUC significantly alleviated insulin resistance in model animals and combined well with RSG. Cf -FUC activated the mRNA expression of PI3K/PKB pathway and GLUT4 The PI3K/PKB pathway is the main insulin signaling cascade that controls glycometabolism. GLUT4 is a key glucose transporter regulated by the PI3K/PKB pathway. Any deletion of these molecules may lead to insulin resistance. As shown in Fig. 4, mRNA expression levels of genes in PI3K/PKB pathway and GLUT4 were significantly decreased in the HFSD control group. Treatment with Cf-FUC normalized the expression of the above genes. It is important to note that the combination of CfFUC and RSG increased the mRNA expression of IRS-1, PKB and GLUT4 by 123.36%, 112.53% and 305.40% in skeletal muscle and 151.80%, 105.09% and 242.88% in adipose tissue respectively, resulting in genes expression comparable to that of the normal control group. Furthermore, the effect of high dose Cf-FUC combined with RSG was significantly more pronounced than in the RSG group (P < 0.05, P < 0.01). In conclusion, these data illustrated that Cf-FUC could enhance insulin signal transduction through the PI3K/ PKB pathway at the transcription level, and lead to improved glucose homeostasis if administered with RSG. Cf-FUC promoted GLUT4 protein translocation To test whether Cf-FUC reduced blood glucose level via promoting GLUT4 translocation, we analyzed GLUT4 and m-GLUT4 expression levels in skeletal muscle and adipose tissue by Western blotting and calculated relative expression of m-GLUT4/GLUT4. As shown in Figs. 5 and 6, significant decreases occurred in m-GLUT4/GLUT4 level of the HFSD control group in both skeletal muscle and adipose tissue. High-dose Cf-FUC/RSG combination significantly increased membrane GLUT4 protein expression of the insulin resistant mice compared to the RSG group (P < 0.05). This indicated that Cf-FUC stimulated the GLUT4 protein’s
TABLE 4. Effects of Cf-FUC on fasting blood glucose level, serum insulin level and HOMA-IR index and QUICKI in insulin resistant mice.a,b
Normal control Model control RSG 80Cf-FUC RSG þ 20Cf-FUC RSG þ 80Cf-FUC
FIG. 1. Effects of Cf-FUC on body weight in insulin resistant mice for 19 weeks.
Fasting blood glucose level (mmol$L1)
Fasting insulin level (mIU$L1)
HOMA-IR
QUICKI
9.28 1.75 13.12 0.82## 10.92 0.96* 10.82 1.41* 10.11 0.92* 9.72 1.24**
10.82 0.32 14.68 0.69## 13.36 0.14* 13.62 0.03* 13.01 0.19* 11.94 0.60**++
4.53 0.31 8.22 1.10## 5.86 0.25* 6.69 0.01* 5.91 0.11* 4.82 0.40**+
0.498 0.007 0.442 0.010## 0.472 0.004** 0.459 0.001** 0.471 0.002** 0.492 0.009**
# P < 0.05, ##P < 0.01 vs normal control group; *P < 0.05, **P < 0.01 vs model control group; +P < 0.05, ++P < 0.01 vs RSG group. a Data are presented as mean SD, n ¼ 10. b The normal control group was supplied with ordinary diet and all the other groups were supplied with HFSD.
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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FIG. 2. Effects of Cf-FUC on oral glucose tolerance in insulin resistant mice. Chart A is the serum glucose levels at 0, 0.5, 1, 2 h after the glucose gavage at the dose of 2 g$(kg$body weight)1. Chart B is the area under curve in OGTT. Data are presented as mean SD (n ¼ 10/group). ## P < 0.01 vs normal control group; * P < 0.05, ** P < 0.01 vs model control group; +P < 0.05 vs RSG group.
FIG. 3. Effects of Cf-FUC on insulin tolerance in insulin resistant mice. Chart A is the serum glucose levels at 0, 0.5, 1, 2 h after intraperitoneal injection of insulin at the dose of 0.75 U (kg$body weight)1. Chart B is the area under curve in insulin tolerance test. Data are presented as mean SD (n ¼ 10/group). ## P < 0.01 vs normal control group; * P < 0.05, ** P < 0.01 vs model control group.
translocation to the plasma membrane and the combination also led a superior efficacy. Cf-FUC stimulated PKB and PI3K phosphorylation As insulin-stimulated GLUT4 translocation was increased by Cf-FUC in both skeletal muscle and adipose tissue, we subsequently
determined the total protein expressions of PKB and PI3K as well as the level of their phosphorylation, which are the major upstream regulators of GLUT4 translocation. As shown in Figs. 5 and 6, no statistically significant differences were demonstrated with any of the proteins. However, the phosphorylations of p-
FIG. 4. Effects of Cf-FUC on the expression of PI3K/PKB genes in skeletal muscle and adipose tissue of insulin resistant mice. Chart A, B, C, D, and E are representative data of quantitative real-time-PCR for IR, IRS-1, PI3K, PKB, and GLUT4 mRNA relative expression levels respectively. Data are presented as mean SD (n ¼ 10/group). ## P < 0.01 vs normal control group. * P < 0.05, ** P < 0.01 vs model control group; +P < 0.05, ++P < 0.01 vs RSG group.
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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FIG. 5. Effects of Cf-FUC on insulin signaling pathway proteins in skeletal muscle of insulin resistant mice. Chart A, B, C, D and E are representative data of Western blot for the expression levels of p-Tyr-IR-b, IR-b, p-Ser307-IRS-1, IRS-1, p-p85-PI3K, PI3K, p-Ser473-PKB, PKB and m-GLUT4/GLUT4 proteins respectively. Results were normalized by b-actin, and the phosphorylation was normalized by their corresponding protein abundance. Data are presented as mean SD (n ¼ 10/group). # P < 0.05, ## P < 0.01 vs normal control group. * P < 0.05, ** P < 0.01 vs model control group; +P < 0.05, ++P < 0.01 vs RSG group.
p85-PI3K, p-Ser473-PKB proteins in skeletal muscle and adipose tissue were noteworthy. When compared with the normal control group, the phosphorylation levels of proteins listed above were significantly reduced in insulin resistant mice. The Cf-FUC treatment markedly accelerated the phosphorylation in various active sites of the genes in the PI3K/PKB pathway. Moreover, the high-dose combination exhibited a superior promoting effect compared with RSG group (P < 0.05, P < 0.01), which amplified the protein phosphorylation levels by several fold in insulin resistant animals. These results indicated that Cf-FUC facilitated the phosphorylation of PKB and PI3K, and had synergistic effects with RSG.
combination treatment further significantly influenced the change towards to normal range (P < 0.05, P < 0.01). These results indicated that Cf-FUC activated PKB and PI3K by increasing tyrosine phosphorylation of IR and IRS-1. We speculate that Cf-FUC promoted insulin secretion, and the increased insulin phosphorylated IR, which subsequently activated IRS-1, PI3K, PKB and finally enhanced GLUT4 translocation. As a result, glucose was transported from the cellular matrix to the cytoplasm and was then oxidized to produce ATP.
Cf-FUC enhanced IR-b and IRS-1 phosphorylation Since CfFUC enhanced the phosphorylation of PKB and PI3K, we also tested the total protein and tyrosine phosphorylation of IR-b and IRS-1, which are known to activate PI3K and PKB. As shown in Figs. 5 and 6, skeletal muscle IR-b, IRS-1 and adipose tissue IRS-1 total protein remained unchanged in normal control and HFSD control groups. Supplementation of Cf-FUC resulted in notable increases in skeletal muscle tyrosine phosphorylation of IR-b and IRS-1 and adipose tissue tyrosine phosphorylation of IRS-1, while the
In present study, we investigated the anti-hyperglycemic effects of fucoidan isolated from the sea cucumber C. frondosa. An animal model of type 2 diabetes model was developed by a classic method, namely feeding C57BL/6J mice with HFSD (27,28). The serum parameters associated with glucose metabolism were determined to observe the symptom of insulin resistant. Moreover, to detect the mechanism, gene expressions of the PI3K/PKB pathway and GLUT4 in skeletal muscle and adipose tissue were tested. Current data indicated that giving animals Cf-FUC dramatically reduced the signs
DISCUSSION
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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FIG. 6. Effects of Cf-FUC on insulin signaling pathway proteins in adipose tissue of insulin resistant mice. Chart A, B, C and D are representative data of Western blot for the expression levels of p-Ser307-IRS-1, IRS-1, p-p85-PI3K, PI3K, p-Ser473-PKB, PKB and m-GLUT4/GLUT4 proteins respectively. Results were normalized by b-actin, and the phosphorylation was normalized by their corresponding protein abundance. Data are presented as mean SD (n ¼ 10/group). # P < 0.05, ## P < 0.01 vs normal control group. * P < 0.05, ** P < 0.01 vs model control group; +P < 0.05 vs RSG group.
of diabetes and also normalized the PI3K/PKB pathway and GLUT4 translocation. The insulin-stimulated glucose uptake is mainly controlled by the PI3K/PKB pathway, which is a crucial signaling cascade triggered by insulin and other growth factors. As the prior receptor tyrosine kinase of this pathway, the IR is a heterotetramer consisting of two extracellular a-subunits and two transmembrane bsubunits held together by disulfide bonds (29). Insulin binding to the a-subunit leads to derepression of the kinase activity in the bsubunit followed by transphosphorylation of the b-subunits and a conformational change that further increases kinase activity (11). The activated IR then catalyzes the phosphorylation at the tyrosine residues of several intracellular substrates, including the IRS proteins (30). In insulin resistant models, the phosphorylation of IR and IRS is markedly inhibited (31,32). When treated with Cf-FUC, the gene expression levels together with the tyrosine phosphorylation of IR and IRS-1 were both increased in skeletal muscle and adipose tissue of insulin resistant mice. These indicated that Cf-FUC could activate IR/IRS-1 signaling cascades. Phosphorylation of IRS will subsequently activate PI3K and PKB, both of which are crucial for insulin signal transduction and stimulating glucose transport in target tissues. As an essential role in glucose uptake and GLUT4 translocation, inhibition of PI3K with pharmacological inhibitors or transfection with dominant-negative constructs will block almost all metabolic actions of insulin (33). PI3K consists of a p110 catalytic subunit and a p85 regulatory subunit that possesses two SH2 domains that interact with tyrosine-phosphorylated pYMXM and pYXXM motifs in IRS proteins (34). Upon phosphorylation, IRS activates PI3K to produce PIP3, which subsequently activates PKB (35). PKB phosphorylated in serine 473 is one of the key downstream mediators of PI3K. The main mechanism relating to glucose uptake of the phosphorylated
PKB is activating the phosphoinositide-3-phosphate-5-kinase to subsequently stimulate the translocation of GLUT4 vesicles to the plasma membrane (9). In addition, a study by Kim et al. (36) showed that high carbohydrate diet has no effect on the resultant total protein expression of the PI3K/PKB pathway. However, a study by Hu et al. (26) showed that hyper glycemia and insulin resistance could be promoted via phosphorylation PI3K/PKB signaling cascade in skeletal muscle of insulin resistant mice without any effects on total protein expressions. In the present study, Cf-FUC provided significant increase in PI3K and PKB gene expression levels of insulin resistant mice. Meanwhile, Cf-FUC enhanced phosphorylation of PI3K in p85 and of PKB in serine473. These results suggest that Cf-FUC acts via phosphorylating the p85 regulatory subunit of PI3K leading to the activation of PKB and consequently stimulating glucose uptake. The principal glucose transporter protein that mediates glucose uptake is GLUT4, which plays a key role in regulating whole body glucose homeostasis (37). Insulin promotes glucose uptake in cells by mobilizing the GLUT4 storage vesicles and stimulating the translocation of GLUT4 from the vesicles to the plasma membrane (38). During the development of type 2 diabetes, this action is compromised. The biogenesis of the insulin responsive vesicles is inhibited and GLUT4 slowly recycles between the plasma membrane and the intracellular vesicles (39). A number of previous reports have shown that a marked reduction in the transcription of GLUT4 could be detected in animal models of insulin resistance and type 2 diabetes (40e42). Furthermore, multiple studies demonstrated that these effects can be attenuated through upregulating the numbers of membrane GLUT4 transporters. Gandhi et al. (43) found that embelin isolated from Embelia ribes promoted the translocation of GLUT4 in STZ-induced insulin resistant rats. Wong et al. (24) discovered that Des-aspartate-angiotensin-I and
Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012
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angiotensin IV improved glucose tolerance and membrane GLUT4 concentrations in diet-induced insulin resistant mice. In our study, Cf-FUC dramatically augmented GLUT4 gene expression level and membrane GLUT4 abundance in skeletal muscles of diet-induced insulin resistant mice. All of these studies demonstrated that the transcription and translocation of GLUT4 gene correlated with antihyperglycemic effects of the experimental compounds. The present study demonstrated that Cf-FUC possessed significant anti-hyperglycemic activity. The underlying mechanisms of these effects involved the activation of PI3K/PKB insulin signaling cascade which led to the translocation of GLUT4. In addition, Cf-FUC exhibited well synergistic effects with RSG. Thus, if nutritional supplementation with Cf-FUC for human is validated, Cf-FUC may be used as a complementary therapy for diet-induced type 2 diabetes. ACKNOWLEDGMENTS We acknowledged supports from National Natural Science Foundation of China (No. 31371876, No. 31471684 and No. U1406402). References 1. Alberti, K. G. and Zimmet, P. Z.: Definition, diagnosis and classification of diabetes mellitus and its complications. Part1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation, Diabet. Med., 15, 539e553 (1998). 2. Lloyd, D. J., Wheeler, M. C., and Gekakis, N.: A point mutation in Sec61a1 leads to diabetes and hepatosteatosis in mice, Diabetes, 59, 460e470 (2010). 3. Gao, R., Wang, Y., Wu, Z., Ming, J., and Zhao, G.: Interaction of barley b-glucan and tea polyphenols on glucose metabolism in streptozotocin-induced diabetic rats, J. Food Sci., 77, 128e134 (2012). 4. Bailey, C. J.: Drugs on the horizon for diabesity, Curr. Diab. Rep., 5, 353e359 (2005). 5. Hemmeryckx, B., Hoylaerts, M. F., Gallacher, D. J., Lu, H. R., Himmelreich, U., D’hooge, J., Swinnen, M., and Lijnen, H. R.: Does rosiglitazone affect adiposity and cardiac function in genetic diabetic mice, Eur. J. Pharmacol., 700, 23e31 (2013). 6. Donath, M. Y. and Shoelson, S. E.: Type 2 diabetes as an inflammatory disease, Nat. Immunol., 11, 98e107 (2011). 7. Alsahli, M. and Gerich, J. E.: Hypoglycemia, Endocrinol. Metab. Clin. North Am., 42, 657e676 (2013). 8. Shulman, G. I.: Cellular mechanisms of insulin resistance, J. Clin. Invest., 106, 171e176 (2000). 9. Choi, K. and Kim, Y. B.: Molecular mechanism of insulin resistance in obesity and type 2 diabetes, Korean J. Intern. Med., 25, 119e129 (2010). 10. Rains, J. L. and Jain, S. K.: Oxidative stress, insulin signaling, and diabetes, Free Radic. Biol. Med., 50, 567e575 (2011). 11. Saltiel, A. R. and Kahn, C. R.: Insulin signaling and the regulation of glucose and lipid metabolism, Nature, 414, 799e806 (2001). 12. Romacho, T., Elsen, M., Rohrborn, D., and Eckel, J.: Adipose tissue and its role in organ crosstalk, Acta Physiol., 210, 733e753 (2014). 13. Xu, J., Wang, Y. M., Feng, T. Y., Zhang, B., Sugawara, T., and Xue, C. H.: Isolation and anti-fatty liver activity of a novel cerebroside from the sea cucumber Acaudina molpadioides, Biosci. Biotechnol. Biochem., 75, 1466e1471 (2011). 14. Bordbar, S., Anwar, F., and Saari, N.: High-value components and bioactives from sea cucumbers for functional foodsda review, Mar. Drugs, 9, 1761e1805 (2011). 15. Hu, X., Wang, Y., Wang, J., Xue, Y., Li, Z., Nagao, K., Yanagita, T., and Xue, C.: Dietary saponins of sea cucumber alleviate orotic acid-induced fatty liver in rats via PPARa and SREBP-1c signaling, Lipids Health Dis., 9, 25 (2010). 16. Yu, L., Ge, L., Xue, C., Chang, Y., Zhang, C., Xu, X., and Wang, Y.: Structural study of fucoidan from sea cucumber Acaudina molpadioides: a fucoidan containing novel tetrafucose repeating unit, Food Chem., 142, 197e200 (2014). 17. Chang, Y., Xue, C., Tang, Q., Li, D., Wu, X., and Wang, J.: Isolation and characterization of a sea cucumber fucoidan-utilizing marine bacterium, Lett. Appl. Microbiol., 50, 301e307 (2010). 18. Chen, S., Hu, Y., Ye, X., Li, G., Yu, G., Xue, C., and Chai, W.: Sequence determination and anticoagulant and antithrombotic activities of a novel sulfated fucan isolated from the sea cucumber Isostichopus badionotus, Biochim. Biophys. Acta, 1820, 989e1000 (2012). 19. Wang, Y., Su, W., Zhang, C., Xue, C., Chang, Y., Wu, X., Tang, Q., and Wang, J.: Protective effect of sea cucumber Acaudina molpadioides fucoidan against ethanol-induced gastric damage, Food Chem., 133, 1414e1419 (2012).
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Please cite this article in press as: Wang, Y., et al., Fucoidan from sea cucumber Cucumaria frondosa exhibits anti-hyperglycemic effects in insulin resistant mice via activating the PI3K/PKB pathway and GLUT4, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.05.012