A Combination of Grape Seed-Derived Procyanidins and Gypenosides Alleviates Insulin Resistance in Mice and HepG2 Cells

Authors

  • H.-J. Zhang,

    1. Authors Zhang, Ji, Zhou, Luo, Yu, and Gao are with College of Food Science and Nutritional Engineering, China Agricultural Univ., Beijing, China. Author Chen is with The Key Laboratory of Food Science of MOE, Nanchang Univ., Nanchang, China. Author Zhang is with Ankang Inst. for Drug Control, Shanxi, China. Author Li is with Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China. Direct inquiries to authors Ji and Chen (E-mail: zn_jibp@163.com).
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  • B.-P. Ji,

    1. Authors Zhang, Ji, Zhou, Luo, Yu, and Gao are with College of Food Science and Nutritional Engineering, China Agricultural Univ., Beijing, China. Author Chen is with The Key Laboratory of Food Science of MOE, Nanchang Univ., Nanchang, China. Author Zhang is with Ankang Inst. for Drug Control, Shanxi, China. Author Li is with Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China. Direct inquiries to authors Ji and Chen (E-mail: zn_jibp@163.com).
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  • G. Chen,

    1. Authors Zhang, Ji, Zhou, Luo, Yu, and Gao are with College of Food Science and Nutritional Engineering, China Agricultural Univ., Beijing, China. Author Chen is with The Key Laboratory of Food Science of MOE, Nanchang Univ., Nanchang, China. Author Zhang is with Ankang Inst. for Drug Control, Shanxi, China. Author Li is with Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China. Direct inquiries to authors Ji and Chen (E-mail: zn_jibp@163.com).
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  • F. Zhou,

    1. Authors Zhang, Ji, Zhou, Luo, Yu, and Gao are with College of Food Science and Nutritional Engineering, China Agricultural Univ., Beijing, China. Author Chen is with The Key Laboratory of Food Science of MOE, Nanchang Univ., Nanchang, China. Author Zhang is with Ankang Inst. for Drug Control, Shanxi, China. Author Li is with Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China. Direct inquiries to authors Ji and Chen (E-mail: zn_jibp@163.com).
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  • Y.-C. Luo,

    1. Authors Zhang, Ji, Zhou, Luo, Yu, and Gao are with College of Food Science and Nutritional Engineering, China Agricultural Univ., Beijing, China. Author Chen is with The Key Laboratory of Food Science of MOE, Nanchang Univ., Nanchang, China. Author Zhang is with Ankang Inst. for Drug Control, Shanxi, China. Author Li is with Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China. Direct inquiries to authors Ji and Chen (E-mail: zn_jibp@163.com).
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  • H.-Q. Yu,

    1. Authors Zhang, Ji, Zhou, Luo, Yu, and Gao are with College of Food Science and Nutritional Engineering, China Agricultural Univ., Beijing, China. Author Chen is with The Key Laboratory of Food Science of MOE, Nanchang Univ., Nanchang, China. Author Zhang is with Ankang Inst. for Drug Control, Shanxi, China. Author Li is with Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China. Direct inquiries to authors Ji and Chen (E-mail: zn_jibp@163.com).
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  • F.-Y. Gao,

    1. Authors Zhang, Ji, Zhou, Luo, Yu, and Gao are with College of Food Science and Nutritional Engineering, China Agricultural Univ., Beijing, China. Author Chen is with The Key Laboratory of Food Science of MOE, Nanchang Univ., Nanchang, China. Author Zhang is with Ankang Inst. for Drug Control, Shanxi, China. Author Li is with Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China. Direct inquiries to authors Ji and Chen (E-mail: zn_jibp@163.com).
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  • Z.-P. Zhang,

    1. Authors Zhang, Ji, Zhou, Luo, Yu, and Gao are with College of Food Science and Nutritional Engineering, China Agricultural Univ., Beijing, China. Author Chen is with The Key Laboratory of Food Science of MOE, Nanchang Univ., Nanchang, China. Author Zhang is with Ankang Inst. for Drug Control, Shanxi, China. Author Li is with Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China. Direct inquiries to authors Ji and Chen (E-mail: zn_jibp@163.com).
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  • H.-Y. Li

    1. Authors Zhang, Ji, Zhou, Luo, Yu, and Gao are with College of Food Science and Nutritional Engineering, China Agricultural Univ., Beijing, China. Author Chen is with The Key Laboratory of Food Science of MOE, Nanchang Univ., Nanchang, China. Author Zhang is with Ankang Inst. for Drug Control, Shanxi, China. Author Li is with Tianjin Jianfeng Natural Product R&D Co., Ltd., Tianjin, China. Direct inquiries to authors Ji and Chen (E-mail: zn_jibp@163.com).
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Abstract

ABSTRACT:  This study investigated the effects of grape seed-derived procyanidins (GSP), gypenosides (GPE), and combination procyanidins/gypenosides on insulin resistance in mice and HepG2 cells. ICR mice were randomly divided into 2 control and 4 treatment groups. The control mice were to receive either normal diet (ND) or high-fat diet (HFD), and the treatment groups were fed high-fat diet with either 80 mg/kg of GSP (GSP80), GPE (GPE80), GSP + GPE (1: 1, GSP40 + GPE40), or 500 mg/kg of metformin for a 6-wk period. All the groups of mice except the normal control were on high-fat diet along with fructose (15%) administered in drinking water throughout the period of treatment. An insulin-resistant HepG2 cell model was developed after 24 h of 5 × 10−7 mol/L insulin incubation. The treatment of GPE80 could significantly reduce the index of insulin resistance (HOMA-IR) and increase hepatic glycogen concentration, compared with HFD group (P < 0.05). When GSP and GPE were administered simultaneously, synergic effects were observed in decreasing the HOMA-IR index and serum total cholesterol (TC) level and enhancing glucose tolerance. All treatment groups showed considerable raise of hepatic glucokinase activity (P < 0.05 compared with HFD group). GSP application increased the consumption of extracellular glucose in HepG2 cells. Our data suggest that the combination of GSP and GPE may have functional efficacy in consumers with insulin resistance.

Introduction

The metabolic syndrome/insulin resistance disorder has become increasingly common around the world. Insulin resistance is a central pathophysiological feature of type 2 diabetes and is a common metabolic abnormality seen associated with obesity, hypertension, dyslipidemia, and coronary artery disease (Cordain and others 2003). Current nonpharmaceutical risk-reducing therapies include regular physical activity and body weight reduction. Intervention by nutraceuticals aimed at improvement of insulin resistance and correction of hyperlipidemia could be another promising option.

Procyanidins, a class of flavonoids, are oligomeric forms of catechins that are abundant in red wine, grapes, cocoa, and apples (Scalbert and Williamson 2000). It has been shown that they were highly bioavailable and provided protection against free radicals and free radical-induced lipid peroxidation and DNA damage (Bagchi and others 1998). Previous study had reported antihyperglycemic property of procyanidins. Acute treatment with grape seed procyanidin extract reduced the blood glucose concentration in streptozotozin-induced diabetic rats. Such effect could be explained in part by the insulinomimetic activity of procyanidins on insulin-sensitive cell lines (Pinent and others 2004). Al-Awwadi and others (2004) also showed the antihyperglycemic role of red wine polyphenol extract.

Gynostemma pentaphyllum Makino (family Cucurbitaceae) is a perennial liana growing wild in the mountainous regions of China, Japan, and many other Asian countries. It has been used in Chinese folk medicine for its heat clearing, detoxification, cough relieving, as well as antitussive and antibronchitis. Phytochemical studies of this plant had identified more than 100 dammarane-type glycosides, mainly named gypenosides (Cui and others 1999), which had a variety of pharmacological properties, such as anti-inflammatory, antihyperlipidemic, and anticardiovascular properties (Aktan and others 2003; Circosta and others 2005; Megalli and others 2005). G. pentaphyllum could improve glucose tolerance in obese Zucker rats but not lean rats suggested that it might improve insulin receptor sensitivity (Megalli and others 2006). Moreover, a novel insulin-releasing substance, isolated from ethanol extracts of G. pentaphyllum, was a potent initiator and potentiator of insulin secretion both in vitro and in vivo in the rat (Norberg and others 2004; Hoa and others 2007).

Therefore, due to their individual therapeutic effects from different perspectives, the combination of grape seed-derived procyanidins and gypenosides might be more appropriate to target on the complexity and redundancy of the pathological mechanisms of insulin resistance. In the present study, we sought to determine whether there was a synergic effect between grape seed-derived procyanidins and gypenosides on hypoglycemic, hypolipidemic effects in ICR mice fed a high-fat diet along with fructose administered in drinking water. HepG2 cells were used in the present study due to their common physiological function in glucose metabolism similar to normal hepatic cells (Xu and others 2003).

Materials and Methods

Materials

Grape seed-derived procyanidins extract (GSP) was provided by Jianfeng, Inc. (Tianjin, China). The total polyphenol content of this GSP extract was 95.0% evaluated colorimetrically using the Folin–Ciocalteau reagent (Singleton and Rossi 1965). G. pentaphyllum extract containing more than 95% gypenosides (GPE) was purchased from Xi' An Zhongxin Biotechnology Co. Ltd. (Xi' An, China). Quantitative analysis of gypenosides was performed using the method described by Chang and others (2005), and total gypenosides content was 96.8%. The standards of catechin, gallic acid, and epicatechin were purchased from Natl. Inst. for the Control of Pharmaceutical and Biological Products (Beijing, China). The standard of procyanidin B2 was provided by Jianfeng, Inc. (Tianjin, China). The standards of Gypenoside A (Gypenoside XLIII) were provided by Ankang Pharmaceutical Inst. of the Beijing Univ. (Shanxi, China). HepG2 cells were from Cell Culture Center of Peking Union Medical Science (Beijing, China). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), and bovine insulin were obtained from Sigma (St. Louis, Mo., U.S.A.). Metformin was supplied by Beijing Medicinal Co. (Beijing, China). Final concentrations of Dimethyl Sulphoxide (DMSO) used for dissolving drugs in medium were below 0.05% (v/v).

GSP and GPE analysis using high-performance liquid chromatography (HPLC)

The HPLC system consisted of a Shimadzu HPLC (Model LC-10ATvp two Pumps and DGU-12A Degasser) equipped with a diode array detector (Model SPD-M10Avp) (Shimadzu, Kyoto, Japan). The separation of GSP was performed on a ProdigyTM ODS(3) column (250 × 4.6 mm I.D., particle size 5 μm) (Phenomenex, Torrance, Calif., U.S.A.). For HPLC analysis, a 10-μL sample was injected into the columns and eluted at room temperature with a constant flow rate of 1.0 mL/min. Acetonitrile: water: acetic acid (80: 19.6: 0.4, v/v) (solvent A) and acetic acid (2%, v/v) (solvent B) were used. A gradient elution used was 0 to 3 min, 0% A; 3 to 6 min, 0% to 4% A; 6 to 15 min, 4% to 10% A; 15 to 30 min, 10% to 15% A; 30 to 50 min, 15% to 23% A; 50 to 60 min, 23% to 25% A; 60 to 66 min, 25% to 30% A; 66 to 80 min, 30% to 50% A; 80 to 83 min, 50% to 80% A; 83 to 85 min, 80% to 0% A; 85 to 105 min, 0% A. The detection wavelength was set to 280 nm. Catechin, gallic acid, epicatechin, and procyanidin B2 in GSP were identified by comparison of their retention times with those obtained from the chromatograms of mixed standards. The quantification was done by external standard calibration, based on peak areas.

The separation of GPE was performed on a Shim-Pack VP-ODS column (150 × 4.6 mm I.D., particle size 5 μm) with a guard column (Shim-pack G VP-ODS, 10 × 4.6 mm I.D., particle size 5 μm) (Shimadzu, Kyoto, Japan). The mobile phase consisted of acetonitrile (solvent A) and water (solvent B) at a flow rate of 1.0 mL/min. To achieve better separation gradient elution was used starting with 20% of A, and then followed by gradient to obtain 30% of A at 10 min, 39% of A at 45 min and held for 10 min, 40% of A at 65 min and held for 5 min, 41% of A at 75 min held for 10 min, 44% of A at 95 min, 62% of A at 100 min, 67% of A at 120 min, 95% of A at 130 min and held for 5 min, 20% of A at 140 min. The detection wavelength was set to 203 nm. The concentration of GPE was 2.5 mg/mL, and each injection volume was 20 μL. The quantification of Gypenoside A was done by external standard calibration, based on peak areas.

Animals and treatments

Healthy, 6-wk-old male CD–1 (ICR) mice were obtained from the Beijing Vital River Laboratory Animal Center [Certificated Nr SCXK (Beijing) 2007–0001] and were acclimatized for 1 wk. Mice were housed in an animal room with 12-h light: dark cycle and controlled for temperature and humidity. All animal procedures were conducted in accordance with Natl. Inst. of Health guidelines for animal care (NRC 1985). During the acclimatization period, each animal was raised on regular diet freely. At 7-wk old, the ICR mice were randomly divided into 6 groups; 2 control and 4 treatment groups. The normal diet (ND) control group and the high-fat diet (HFD) control group received a gavage of vehicle (tap water). The 3rd, 4th, and 5th groups of mice received 80 mg/kg of GSP (GSP80), GPE (GPE80), and GSP + GPE (1: 1, GSP40 + GPE40), respectively, once daily, orally for 6 wk. As a positive control, metformin was administrated at the dose of 500 mg/kg. All the groups of mice except the normal control were on high-fat diet along with fructose (15%) administered in drinking water throughout the period of treatment. The high-fat diet comprised the following ingredients: lard 10 g, cholic acid 0.2 g, cholesterol 1 g, and standard animal chow to 100 g (Experiment Animal Center of Beijing, China). Body weights were measured weekly. At the end of the experiment period, blood was collected from the orbital plexus, after 13 h of food deprivation, and then the animals were sacrificed by cervical dislocation. The serum was isolated by centrifugation at 1500 g, 4 °C for 10 min, and the liver and abdominal adipose tissue were excised and weighed.

Serum measurements

Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), and serum glucose concentrations were assessed enzymatically with conventional diagnostic kits (Biosino Bio-technology and Science Incorporation, Beijing, China). The serum level of glucose was analyzed by the glucose oxidase method, and corresponding diagnostic kits (Nanjing Jiancheng Bioengineering Inst., Nanjing, China) were used according to the manufacturer's instructions. Insulin concentration was measured with a radioimmunological assay kit (Insulin RIA kit, Atom HighTech Co. Ltd., Beijing, China).

Oral glucose tolerance test

An oral glucose tolerance test (OGTT) was performed at the end of the treatment. After 13 h of food deprivation, glucose (2 g/kg) was loaded by gavage. Blood samples were taken from the tail at 0, 30, 60, and 120 min after glucose administration. Blood glucose concentrations were measured using a calibrated OneTouch Ultra® glucometer for each time point. Total area under the curve (AUC) was calculated as millimoles per liter per minute using trapezoidal method. The R-value of homeostasis model (HOMA-IR) of Matthews and others (1985) was expressed as an index of insulin resistance. HOMA-IR index was calculated by the formula: fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5.

Glucokinase activity and hepatic glycogen content

The glucokinase activity was determined using a spectrophotometric continuous assay as described by Davidson and Arion (1987). One unit of glucokinase was defined as the enzyme activity resulting in the formation of 1 μmol of glucose-6-phosphate per minute per milligram of protein. The glycogen concentration was determined by the method of Seifter and others (1950) and corresponding diagnostic kits (Nanjing Jiancheng Bioengineering Inst.) were used according to the manufacturer's instructions.

Insulin-resistant HepG2 cell model

The HepG2 cells were seeded into 96 multi-well plates in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were cultured in a humidified incubator (5% CO2) at 37 °C, and were allowed to attach for 24 h. Insulin-resistant cell model was induced according to the previous method (Xie and others 2006) with a slight modification. In brief, HepG2 cells were incubated with fresh medium containing 1% FBS and 5 × 10−7 mol/L bovine insulin for 24 h. Subsequently, the medium was exchanged with medium containing 10−9 mol/L insulin and GSP, GPE, GSP + GPE, or metformin, incubation was conducted for 24 h.

Extracellular glucose in HepG2 cells

After treatment, glucose was assayed in 10 μL of medium by enzymatic methods with diagnostic kits (Nanjing Jiancheng Bioengineering Inst.). Data were expressed as consumption of extracellular glucose content (μmol/mg protein) =[extracellular glucose content (μmol)0  h− extracellular glucose content (μmol) 24  h]/mg cell protein (Xie and others 2006). Protein content was measured using a BCA protein assay (Pierce, Rockford, Ill., U.S.A.).

Cytotoxicity assay

The HepG2 cells were seeded on 96 multi-well plates at 104 cells/well and cultured for 24 h. The cells were washed with DMEM once, and then incubated with GSP, GPE, GSP + GPE, or metformin in DMEM for 24 h. Subsequently, the medium was removed, cells were washed with DMEM once, and 100 μL of MTT (0.5 mg/mL in DMEM) were added to each well and incubated for 4 h. The MTT medium was subsequently removed and 150 μL of DMSO were added to dissolve the formazan formed. After shaking, the optical densities (OD) at 570 nm were measured using a Labsystems Multiskan MK3 (Thermo Labsystems, Helsinki, Finland) (Hansen and others 1989; Van de Loosdrecht and others 1991).

Statistical analysis

All data were presented as mean ± SEM. Statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, Ill., U.S.A.). The statistical significance comparing data between groups was assessed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. A P value less than 0.05 was considered to be statistical significant, while P value less than 0.01 was considered very significant.

Results

HPLC analysis of GSP and GPE

Chromatograms of GSP and GPE were shown in Figure 1. As shown in Figure 1A, a separation of catechin, gallic acid, epicatechin, and procyanidin B2 was obtained. The content of catechin, gallic acid, epicatechin, and procyanidin B2 in GSP was 8.50%, 0.04%, 5.02%, and 1.92%, respectively. The content of Gypenoside A in GPE was 16.27% (Figure 1B).

Figure 1—.

Chromatograms of grape seed-derived procyanidins extract (A) and gypenosides (B). HPLC conditions were described in the Materials and Methods section.

Effects of grape seed-derived procyanidins and gypenosides on the adipose tissue and body weight

Body weight was determined once a week. Weight gains in ND and HFD groups during the 6-wk period were 9.8 ± 0.6 g (43% increase over the initial body weight) and 13.2 ± 1.5 g (58% increase over the initial body weight), respectively (Table 1). Animals fed GSP80, GPE80, or GSP40 + GPE40 showed a gradual increase in body weight, but the increase was significantly less than that of HFD control in spite of continued and prolonged access to the high-fat diet. The mice in HFD group had higher adipose tissue weight and relative adipose tissue weight than those in ND group did (P < 0.05). GSP40 + GPE40 treated group showed a significant decrease in relative adipose weight relative to the HFD group (P < 0.05).

Table 1—.  Effect of grape seed-derived procyanidins and gypenosides on body and tissue weight.
GroupsBody weight (g)Weight gain (g)Adipose tissue weight (g)aRelative adipose tissue weightb
InitialFinal
  1. Values represent the mean ± SE (n= 10).

  2. P < 0.05 compared with ND.

  3. *P < 0.05 compared with HFD.

  4. aAdipose tissue: epididymal fat pad and abdominal adipose tissue weight.

  5. bRelative adipose weight was expressed as g adipose tissue × 100/g body weight.

  6. ND = normal diet; HFD = high-fat diet; GSP = grape seed-derived procyanidins; GPE = gypenosides.

ND22.7 ± 0.4 32.6 ± 0.5  9.8 ± 0.6 0.5 ± 0.11.5 ± 0.3
HFD22.8 ± 0.436.1 ± 1.6†13.2 ± 1.5†  1.1 ± 0.3† 2.9 ± 0.6†
GSP8022.4 ± 0.231.7 ± 0.8*9.3 ± 0.9*0.7 ± 0.12.2 ± 0.2
GPE8022.3 ± 0.231.3 ± 0.7*9.0 ± 0.8*0.6 ± 0.12.0 ± 0.2
GSP40 + GPE4022.7 ± 0.5 32.5 ± 0.4 9.8 ± 0.6*0.5 ± 0.1 1.5 ± 0.2*
Metformin22.8 ± 0.4 32.9 ± 1.0 10.1 ± 0.6  0.7 ± 0.12.1 ± 0.5

Serum glucose, insulin, and insulin resistance index

Fasting serum glucose and insulin levels were measured at the end of the treatment. A modest but significant hyperglycemia was developed in the HFD group compared with the ND group for 6-wk trial (Table 2). GPE, GSP + GPE administered mice showed significant decrease in serum glucose levels relative to those in HFD group. As a result of increased serum glucose and insulin levels, homeostatic model assessment values for insulin resistance (HOMA-IR), of HFD group was 1.6 times higher than that of the ND group. The insulin resistance indices of GPE80 (P < 0.05) and GSP40 + GPE40 (P < 0.01) groups were significantly reduced compared with the HFD group. Amelioration of insulin resistance in GSP40 + GPE40 treated group was comparable to the metformin-treated group.

Table 2—.  Effect of grape seed-derived procyanidins and gypenosides on serum glucose, insulin, and homeostasis model assessment for insulin resistance (HOMA-IR).
GroupsSerum glucose (mmol/L)Insulin (μU/mL)HOMA-IR
  1. Values represent the mean ± SE (n= 10) homeostasis model assessment was used to calculate an index of insulin resistance as glucose (mmol/L) × insulin (μU/mL)/22.5.

  2. P < 0.05.

  3. ††P < 0.01 compared with ND.

  4. *P < 0.05.

  5. **P < 0.01 compared with HFD.

  6. ND = normal diet; HFD = high-fat diet; GSP = grape seed-derived procyanidins; GPE = gypenosides.

ND6.9 ± 0.26.6 ± 0.52.0 ± 0.2
HFD8.9 ± 0.3††8.3 ± 0.8†3.3 ± 0.4††
GSP807.8 ± 0.47.8 ± 1.12.4 ± 0.4
GPE807.5 ± 0.4*6.9 ± 0.52.3 ± 0.2*
GSP40 + GPE406.8 ± 0.2**6.3 ± 0.6*1.9 ± 0.2**
Metformin7.4 ± 0.5*5.9 ± 0.9*2.0 ± 0.4*

Oral glucose tolerance test (OGTT)

After 6-wk administration of GSP and GPE, OGTT was performed (Table 3). Among the groups, blood glucose levels at 0 time were significantly different. Glucose challenge dramatically increased the blood glucose levels in HFD group compared with those in ND group, while GSP40 + GPE40 administration significantly prevented the blood glucose levels from rising, especially at 30 min time points. When the area under the curve (AUC) was compared between groups, GPE80 and GSP40 + GPE40 administered groups, respectively, showed 5% and 13% reduction in the AUC compared with the HFD group, and the reduction in GSP40 + GPE40 group was comparable to that in metformin-treated group. But there was no significant difference for the AUC between GSP80-treated group and HFD group. The effect of reduction in the AUC was intensified by the combination of GSP and GPE treatment, showing very significant difference from that in HFD group (P < 0.01).

Table 3—.  Plasma glucose and the area under the curve responses to an oral glucose challenge (2 g/kg) after 13 h of food deprivation at the end of the treatment.
Groups0 (mmol/L)30 (mmol/L)60 (mmol/L)120 (mmol/L)AUC (mmol/L × min)
  1. Values represent the mean ± SE (n= 10).

  2. P < 0.05.

  3. ††P < 0.01 compared with ND.

  4. *P < 0.05.

  5. **P < 0.01 compared with HFD.

  6. ND = normal diet; HFD = high-fat diet; GSP = grape seed-derived procyanidins; GPE = gypenosides.

ND7.0 ± 0.2 16.3 ± 0.711.6 ± 0.57.7 ± 0.21346.1 ± 38.7  
HFD 9.0 ± 0.3††  19.1 ± 0.7†12.8 ± 0.5 8.8 ± 0.4†1544.6 ± 29.5††
GSP808.0 ± 0.4 17.0 ± 1.013.1 ± 0.79.3 ± 0.51500.0 ± 73.7  
GPE807.8 ± 0.2* 16.4 ± 1.213.2 ± 0.48.4 ± 0.31456.7 ± 28.1* 
GSP40 + GPE406.8 ± 0.2*   15.8 ± 0.7**11.8 ± 0.6 7.5 ± 0.3* 1332.2 ± 53.8**
Metformin7.5 ± 0.5*  17.0 ± 0.7*11.4 ± 0.4 7.2 ± 0.3* 1351.8 ± 37.9**

Serum lipid levels

The effects of GSP and GPE on serum lipid levels were examined at the end of the treatment (Table 4). In HFD group, TC increased by 2.1-fold (6.81 to 14.21 mmol/L), and LDL-C increased by 1.6-fold (0.37 to 0.58 mmol/L) compared with those in the ND group. Simultaneous treatment with GSP and GPE, serum TC decreased 23% compared with HFD group, which was comparable to that of metformin-treated group. The HDL-C level in all treatment groups was elevated significantly (P < 0.05 or 0.01) than that in the ND group, showing no significant difference with that in HFD group.

Table 4—.  Effects of grape seed-derived procyanidins and gypenosides on serum lipid levels.
GroupsLDL-C (mmol/L)HDL-C (mmol/L)TC (mmol/L)TG (mmol/L)
  1. Values represent the mean ± SE (n= 10).

  2. P < 0.05.

  3. ††P < 0.01 compared with ND.

  4. *P < 0.05 compared with HFD.

  5. ND = normal diet; HFD = high-fat diet; GSP = grape seed-derived procyanidins; GPE = gypenosides.

ND0.37 ± 0.022.71 ± 0.17    6.81 ± 0.38 1.33 ± 1.09
HFD  0.59 ± 0.05††3.59 ± 0.45   14.21 ± 0.87††1.48 ± 0.92
GSP800.55 ± 0.054.45 ± 0.42††12.17 ± 1.02 1.15 ± 0.16
GPE800.54 ± 0.073.83 ± 0.41† 11.79 ± 1.03 1.60 ± 0.20
GSP40 + GPE400.53 ± 0.063.99 ± 0.49††10.89 ± 1.20* 1.27 ± 0.31
Metformin0.62 ± 0.043.97 ± 0.28††10.79 ± 0.69* 1.23 ± 0.92

Hepatic glucokinase activity and glycogen level

The hepatic glucokinase activity was significantly lowered in HFD group than in ND group (Figure 2A). The supplementation of GSP or GPE alone significantly elevated hepatic glucokinase activity compared with the HFD group by 60% and 75%, respectively (P < 0.05 or 0.01). GSP40 + GPE40 treated group also showed considerable raise of hepatic glucokinase activity (P < 0.05 compared with HFD). The HFD group showed 29% decrease in hepatic glycogen content compared with ND group (Figure 2B). However, treatment with GPE reversed hepatic glycogen level. The elevation of glycogen level in GPE80 group was comparable to that in metformin-treated group. The glycogen level of GSP80 and GSP40 + GPE40 group was not significantly different from that of HFD group.

Figure 2—.

Effect of grape seed-derived procyanidins and gypenosides on the hepatic glucokinase (A) and glycogen (B) content in normal and experimental animals. Values represent the mean ± SE (n= 10). ††P < 0.01 compared with ND. *P < 0.05, **P < 0.01 compared with HFD. ND = normal diet; HFD = high-fat diet; GSP = grape seed-derived procyanidins; GPE = gypenosides.

Effect of GSP and GPE on sensitivity to exogenous insulin in insulin-resistant HepG2 cells

The cytotoxicity of grape seed-derived procyanidins and gypenosides on HepG2 cells was examined by MTT assay. There was no significant difference of the MTT OD value between treatment groups and control group (Figure 3). So, the compounds at these concentration ranges did not influence cellular bioactivity. Following 10−9 mol/L insulin incubation, of HepG2 cell there was a significant decrease in the consumption of extracellular glucose in control with 5 × 10−7 mol/L insulin pretreatment (P < 0.05) compared with blank control without insulin pretreatment (Table 5). Insulin at the final concentration of 10−9 mol/L, combined with metformin (16.5 μg/mL), GSP (6.25, 12.5 μg/mL), or GSP + GPE (6.25 μg/mL + 6.25 μg/mL), respectively, significantly increased consumption of extracellular glucose in HepG2 cells pretreated with 5×10−7 mol/L insulin. Treatment with 6.25, 12.5 μg/mL GSP, or GSP + GPE (6.25 μg/mL + 6.25 μg/mL) showed 52%, 48%, or 45% increase in consumption of extracellular glucose, respectively, compared with control group.

Figure 3—.

Effect of grape seed-derived procyanidins and gypenosides on MTT in HepG2 cells. Values represent the mean ± SE (n= 6). GSP = grape seed-derived procyanidins; GPE = gypenosides.

Table 5—.  Effect of grape seed-derived procyanidins and gypenosides on sensitivity to exogenous insulin in insulin-resistance HepG2 cells pretreated with 5 × 10−7 mol/L insulin.
GroupsDosage (μg/mL)Consumption of extracellular glucose (μmol/mg cell protein)
  1. Values represent the mean ± SE (n= 6).

  2. P < 0.05.

  3. ††P < 0.01 compared with blank control without 5 × 10−7 mol/L insulin pretreatment.

  4. **P < 0.01 compared with control with 5 × 10−7 mol/L insulin pretreatment.

  5. GSP = grape seed-derived procyanidins; GPE = gypenosides.

Blank control 42.38 ± 1.56
Control  36.78 ± 1.66†
GSP6.25   55.92 ± 2.33**††
12.5   54.63 ± 1.30**††
GPE12.536.44 ± 2.17
2535.70 ± 2.34
GSP + GPE6.25 + 6.25  53.38 ± 3.15**†
12.5 + 12.536.17 ± 2.54
Metformin16.5  45.29 ± 1.55**

Discussion

It is well known that obesity leads to a high incidence of type 2 diabetes (Colditz and others 1990). Increasing ingestion of high-caloric food, which matters in relation to development of overweight, was considered to be the major cause of insulin resistance and type 2 diabetes (Friedman 2003). The obesity in mice was developed by feeding HFD, and the obese mice had the characters of hyperglycemia and insulin resistance (Yun and others 2004). In the present study, we used a simple protocol of supplying mice with high-fat food and high fructose dinking water to elevate caloric consumption. This diet-induced mouse model showed mild obesity, hyperglycemia, and insulin resistance, and was appropriate to develop the preventive agent for type 2 diabetes.

In the present study, as a result of high-fat diet combined with fructose administration for 6 wk, mice in HFD group developed a prediabetic state associated with overweight, hyperglycemia, insulin resistance, and dyslipidemia. GSP failed to decrease fasting blood glucose and HOMA-IR index significantly. In OGTT, GSP did not improve glucose tolerance. Pinent and others (2004) firstly reported antihyperglycemic effect of GSP, they showed that GSP could mimic the role of insulin in an insulin-deficient animal model. El-Alfy and others (2005) reported that GSP reduced the increase in serum glucose concentration induced by alloxan. Another study had shown that wine flavonoids improved oxidative status of diabetic situation, but had no effect on glycemia (Landrault and others 2003). The difference between our results and those obtained by others might be due to the different protocols, including the dosage of procyanidins and animal models. In the present experiment, the insulin resistance animal induced by high-fat diet and fructose was not insulin-deficient, but mild hyperinsulinemia.

However, GPE was helpful to maintain glucose homeostasis and clear the postprandial glucose load (Table 3). These results were consistent with previous observation in obese Zucker rats (Megalli and others 2006). Simultaneous treatment with GSP and GPE inhibited the subsequent development of obesity, hyperglycemia, and insulin resistance in HFD fed mice. The combination of GSP and GPE resulted in improvement of insulin resistance by decreasing blood glucose and insulin levels at a fasting state. It should be mentioned that the dosage of GSP/GPE in GSP40 + GPE40 group was only half of that in GSP80/GPE80 group, respectively. Meanwhile, when GSP and GPE were given individually at a daily dose of 40 mg/kg, no changes in the glucose tolerance were observed after 6-wk treatment compared with HFD group in our preliminary studies (data not shown). In contrast, when GSP and GPE were administered simultaneously, the HOMA-IR index was decreased and glucose intolerance was improved, reflecting a synergic effect. Moreover, the same phenomenon was found in serum TC level. To the best of our knowledge, this is the 1st finding on the occurrence of a synergic effect of GSP and GPE to alleviate insulin resistance and decrease serum TC level in HFD mice.

The effect of ameliorating insulin resistance of combined GSP/GPE was comparable to that of metformin, a biguanide agent that reduces hyperinsulinemia and improves hepatic insulin resistance. Activity of the hepatic glucose-phosphorylating enzyme, glucokinase, was reduced by 42% in HFD group compared with ND group in the present study. This was consistent with the observation that liver glucokinase was decreased by high-fat diet in rat (Gustafson and others 2002). Since the glucokinase gene was induced by insulin (Granner and Pilkis 1990), the decreased glucokinase activity was in keeping with the fact that HFD feeding induced liver insulin resistance. Insulin decreased the hepatic glucose output by activating glycogen synthesis and glycolysis, and by inhibiting gluconeogesis (McGarry 1992). An increased glucokinase activity enhanced glucose utilization and glucose uptake in the liver (Jung and others 2008), and the increase in glucokinase activity in the GSP and GPE-supplemented HFD fed mice probably induced improvement of insulin resistance. The blood glucose-lowering activity of other flavonoids had been defined for hesperidin and naringin (Jung and others 2004), which could increase glucokinase activity in C57BL/KsJ-db/db mice. The elevation of hepatic glucokinase activity could increase the utilization of blood glucose for glycogen storage in the liver (Iynedjian and others 1988). In the present study, GPE not only markedly elevated the hepatic glucokinase activity but also significantly increased hepatic glycogen concentration compared with HFD group.

The study in vivo suggested that GSP and GPE could elevate the hepatic glucokinase activity, which would be related to improvement of hepatic insulin resistance. In vitro, we study the effect of GSP and GPE on sensitivity to exogenous insulin in insulin-resistant HepG2 cells, to verify whether the improvement of insulin sensitivity in vivo was associated with enhancement of insulin action in hepatic cells. Therefore, an insulin-resistant HepG2 cell model was developed after 24 h of 5 × 10−7 mol/L insulin incubation. Here, consumption of extracellular glucose content in hepatic cells was adopted to evaluate the disposal of them to extracellular glucose. High-concentration insulin induced insulin resistance in HepG2 cells and showed an insensitive response to low-concentration insulin. GSP significantly improved insulin action in HepG2 cells, which was consistent with the results in insulin-sensitive cell line (Pinent and others 2004). Grape seed-derived procyanidins has an insulin-like effect on glucose uptake in 2 insulin-sensitive cell lines: 3T3-L1 adipocytes and L6E9 myotubes. There was a synergic effect between procyanidins and insulin in 3T3-L1 adipocytes on stimulation of glucose uptake (Pinent and others 2004). In the current study, GPE had no effect on insulin action in insulin-resistant HepG2 cells, and high dose of GPE counteracted the effects of GSP. However, GPE and GSP + GPE could elevate hepatic glucokinase activity in HFD fed mice. These indicated that results in vitro would not necessarily consist with those in vivo or GPE might be converted into more active form in vivo. The precise mechanisms require further investigation.

Conclusions

The present study suggested that supplementation with GPE improved insulin resistance in HFD mice by, at least in part, increasing glucose utilization, which seemingly was mediated via elevating glucokinase activity and hepatic glycogen concentration. GSP could improve insulin action in insulin-resistant HepG2 cells. In particular, there was a synergic effect between GSP and GPE on improvement of insulin resistance and decrease of serum TC level in HFD fed mice. Thus, combination of GSP and GPE was hopeful to develop into an adjuvant for handling of diabetic patients with clinically manifested insulin resistance in the coming future.

Acknowledgments

The authors thank ZhiWei Yang, Tao Li, and Jun Zhao for their excellent technical assistance.

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