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¹ Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 197 Ruijin Road II, Shanghai 200025, China² First College of Clinical Medicine, Medical Research Center, Nanjing University of Traditional Chinese Medicine, Nanjing 210029, China

(Correspondence should be addressed to M Chen; Email: mingdaochensh{at}yahoo.com)

^* (W Shang and Y Yang contributed equally to this work)

	Abstract

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A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb₁ has been shown to regulate peroxisome proliferator-activated receptor activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb₁ on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb₁ significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 µM and a time of 3 h. In adipocytes, Rb₁ promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb₁ increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb₁-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb₁ stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Declaration of interest References

 
Ginseng has been used as a tonic in oriental medicine to promote health for several millennia, and its constituents possess multiple pharmacological effects on the central nervous, cardiovascular, endocrine, and immune systems (Attele et al. 1999). Historical records in traditional Chinese medicine have shown that ginseng has been used to treat type 2 diabetes for a long time (Xie et al. 2005a). More recently, a series of clinical trials and animal experiments have demonstrated that ginseng and its major active components, ginsenosides, exert hypoglycemic and insulin-sensitizing action (Vuksan et al. 2000a,b, 2008, Attele et al. 2002, Yun et al. 2004, Liu et al. 2005, Xie et al. 2005b,c, Reay et al. 2006, Sievenpiper et al. 2006). These studies suggest that ginseng may be a useful therapy in the management of type 2 diabetes. However, cellular and molecular mechanisms of hypoglycemic action produced by ginseng and ginsenosides remain unclear.

Ginsenosides are the major active constituents responsible for pharmacological properties of ginseng. All ginsenosides have the same structure of four trans-ring rigid steroid skeleton with the difference in their number and site of attachment of hydroxyl groups (Gillis 1997, Attele et al. 1999). Ginsenoside Rb₁ is the most abundant one among more than 20 ginsenosides in ginseng root despite different sources and species (Attele et al. 2002, Washida & Kitanaka 2003, Lim et al. 2005). Our recent study showed that ginsenoside Rb₁, (Washida & Kitanaka 2003), facilitated adipogenesis of 3T3-L1 preadipocytes by enhancing peroxisome proliferator-activated receptor 2 (PPAR2) and CCAAT/enhancer-binding protein gene expression. In addition, Rb₁ binds to PPAR-ligand-binding domain as measured by surface plasmon resonance, which suggests that Rb₁ has the ability to activate PPAR (Shang et al. 2007b). Although treatment of differentiating adipocytes with Rb₁ lead to augmented basal and insulin-mediated glucose uptake, which reflects the degree of adipogenesis (Shang et al. 2007a), the direct effect of Rb₁ on glucose uptake in mature adipocytes remains unknown. We also found that Rb₁ could inhibit lipolysis in adipocytes (Shang et al. 2007b), which suggests that Rb₁ may possess insulin-like effect. Recent studies have reported that ginseng extract and ginsenoside Re stimulates glucose transport and inhibits lipolysis in adipocytes through activation of insulin signaling (Edens et al. 2001, Wang et al. 2006, Zhang et al. 2008). It has been indicated that the structural diversity of ginsenosides may lead to the different effects (Attele et al. 1999). Therefore, the effect of Rb₁ on glucose transport in insulin-sensitive cells and insulin signaling pathway in mature adipocytes merits further elucidation.

Insulin-mediated glucose transport in appropriate tissue is essential for maintenance of whole-body glucose homeostasis (Herman & Kahn 2006). Adipocytes and muscle cells are highly insulin-sensitive cells. In adipocytes, insulin stimulates glucose transport by enhancing the translocation of GLUT4 and, to a lesser extent, GLUT1 from the intracellular pool to the plasma membrane (PM) (Weiland et al. 1990, Watson et al. 2004). The classic pathway of insulin signaling involved in glucose transporter translocation to the cell surface is triggered by autophosphorylation of the insulin receptor on multiple tyrosine residues following insulin binding. This results in the tyrosine phosphorylation of a family of insulin receptor substrate (IRS) proteins and activation of a complex network of downstream molecules, including phosphatidylinositol 3-kinase (PI3K) and the serine/threonine kinase AKT (Khan & Pessin 2002, Watson et al. 2004). Muscle cells share the same signal cascade of regulating glucose transport (Zorzano et al. 2005).

The present study sought to explore the effects of Rb₁ on glucose transport in the differentiated 3T3-L1 adipocytes and myotubes, as well as elucidation of its mechanism in adipocytes. Our results presented here show that Rb₁ stimulates glucose transport in adipocytes by inducing the translocations of GLUT1 and GLUT4 through activating insulin-like signaling pathways. These findings may help to understand the hypoglycemic and anti-diabetic effects of ginseng and ginsenosides.

	Materials and Methods

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Reagents

Dulbecco modified Eagle medium (DMEM) and horse serum were purchased from Gibco. Fetal calf serum (FCS) was obtained from PAA Laboratories (Pasching, Austria). Ginsenoside Rb₁, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich. Recombinant human insulin (Ins) was purchased from Lily (Fegersheim, France). 2-Deoxy-D-[1-³H] glucose (2-DOG) and -³²P ATP were purchased from Amersham. Lipofectamine 2000 was from Invitrogen. BCA protein assay kit and West Pico chemiluminescent substrate were purchased from Pierce (Rockford, IL, USA). Unless otherwise specified, all other reagents were analytic grade.

Antibodies and chemical inhibitors

GLUT1 rabbit polyclonal antibody was purchased from Chemicon (Temecula, CA, USA) and GLUT4 monoclonal antibody was obtained from R&D (Minneapolis, MN, USA). Polyclonal IRS-1 (phosphor Y612) and mouse monoclonal clathrin heavy chain antibody were purchased from Abcam (Cambridge, UK). Anti-p85 subunit of PI3K antibody was obtained from Upstate (Temecula, CA, USA). Phospho-insulin receptor antibody (Tyr1135/1136), phospho-AKT antibody (Ser473 and Thr308), AKT antibody, horseradish peroxidase-linked anti-rabbit immunoglobulin G (IgG) and anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Wortmannin was obtained from Biomol International LP (Plymouth Meeting, PA, USA). LY294002 and hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester (HNMPA-(AM)₃) were purchased from Calbiochem (La Jolla, CA, USA).

Cell culture

Mouse 3T3-L1 fibroblast cells and C2C12 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). 3T3-L1 fibroblast cells were cultured and differentiated into adipocytes as described previously with some modifications (Perrini et al. 2004). Briefly, the fibroblasts were grown in DMEM containing 10% FCS, 25 mM glucose, 2 mM glutamine, 3.4 mg/ml NaHCO₃, 3.6 mg/ml HEPES, 64 µg/ml penicillin, and 100 µg/ml streptomycin (growth medium) at 37 °C in 7% CO₂ atmosphere. After confluence, the fibroblasts were maintained for another 2 days. Differentiation was induced by treating the cells with standard differentiation inducers (DMEM containing 0.5 mM IBMX, 1 µM DEX, 1.7 µM Ins, and 10% FCS) for 48 h. The cells were re-fed with DMEM supplemented with 1.7 µM Ins and 10% FCS for the following 48 h, then the medium was replaced with growth medium, changed every 2 days. More than 90% of the cells expressed the adipocyte phenotype between 8 and 10 days after the initiation of differentiation and were used for the experiments. C2C12 cells were maintained in growth medium. When the cells achieved 70% confluence, they were differentiated in DMEM containing 2% horse serum for 4 days. The differentiated C2C12 cells were fused into myotubes and were used for the experiments. The cells used for the experiments were serum starved overnight in DMEM containing 0.2% BSA (Roche Applied Science). Ginsenosides and all inhibitors were dissolved in DMSO as a 1000-fold stock, and added to 0.2% BSA–DMEM at various concentrations as shown in each figure. DMSO was present in the control culture at a same concentration less than 0.2% (v/v).

Glucose uptake measurement

The assay was performed essentially as described previously (Perrini et al. 2004). The cells were washed thrice with PBS, then incubated in Krebs–Ringer phosphate buffer (KRP, 1.32 mM NaCl, 4.71 mM KCl₂, 47 mM CaCl₂, 1.24 mM MgSO₄, 2.48 mM Na₃PO₄, 10 mM HEPES (pH 7.4)) for 10 min at 37 °C, then 0.5 µCi/ml 2-DOG as the final concentration was added to the cells. After 10-min incubation, the medium was aspirated and the cells were washed thrice with ice-cold KRP containing 10 mM glucose to terminate the reaction. The cells were lysed with 0.1 M NaOH and the radioactivity taken up by the cells was determined using a scintillation counter (Beckman Instruments, Fullerton, CA, USA). The d.p.m. value was corrected by protein content in each well which was measured using a BCA protein assay kit.

Extract of total cell protein and subcellular membrane fractions

For extracting total protein from adipocytes, the cells were washed twice with ice-cold PBS and harvested in a lysis buffer (radioimmunoprecipitation assay (RIPA), 50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulphonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 mM sodium orthovanadate (pH 7.4)). After sonication and centrifugation at 13 000 g for 30 min at 4 °C, the protein content of the resulting supernatant was determined using the BCA protein assay kit.

Subcellular fractions were prepared from 3T3-L1 adipocytes as previously described with minor modifications (Piper et al. 1991). After washing with PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO4, 8 mM Na₂HPO₄ (pH 7.4)), adipocytes were scraped in ice-cold buffer A (20 mM HEPES, 250 mM sucrose, 1 mM EDTA (pH 7.4)) containing protease inhibitor cocktail (10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF), and subsequently homogenized with 30 strokes in a glass Dounce homogenizer at 4 °C. All subsequent procedures were performed at 4 °C. After centrifugation at 1000 g for 10 min to remove large cell debris and unbroken cells, the supernatant was then centrifuged at 8000 g for 20 min to yield a pellet of total cellular membranes and a supernatant representing the cytosolic fraction. The pellet was resuspended (30 strokes in a glass Dounce homogenizer) in buffer B (20 mM HEPES, 250 mM sucrose, 1 mM EDTA (pH 7.4)) containing protease inhibitor cocktail and layered on 1.12 M sucrose cushion. Following 1 h centrifugation at 100 000 g, the PM layer at the interface was collected by centrifugation, and resuspended in buffer B. The 16 000 g supernatant was centrifuged at 41 000 g for 20 min; the resulting supernatant was centrifuged for 75 min at 180 000 g to pellet the low-density membrane (LDM) fraction, which was resuspended in buffer B. The protein concentrations of PM and LDM were determined with BCA protein assay kit.

Western blot analysis

Protein samples were incubated in Laemmli buffer for 5 min at 95 °C, and then samples were separated by 6 or 8% SDS-PAGE, as appropriate, and subjected to immunoblotting with the appropriate antibodies, as described previously (Perrini et al. 2004). Immunoblots were developed using the ECL kit. The immunoblotting signals were quantitated using a Bio-Rad GS-710 densitometer with QuantityOne 4.5 software.

Immunoprecipitation and assay of PI3-kinase activity

PI3-kinase activity was measured as described previously (Imamura et al. 1999). The 3T3-L1 adipocytes were washed twice with ice-cold buffer A (137 mM NaCl; 20 mM Tris–HCl (pH 7.4) 1 mM CaCl₂; 1 mM MgCl₂, and 0.1 mM sodium orthovanadate) and were lysed with ice-cold lysis buffer (buffer A containing 1% NP-40 and 1 mM PMSF). The samples were centrifuged for 10 min at 13 000 g at 4 °C to sediment insoluble material; the supernatant fractions were subjected to immunoprecipitation with 4 µg anti-P85 antibody overnight at 4 °C. Immunocomplexes were then precipitated from the supernatant with protein A/G PLUS-agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and washed. The washed immunocomplexes were incubated with phosphatidylinositol (Sigma) and -³²P ATP for 10 min at 37 °C. After reactions were stopped by 6 M HCl, the radiolabeled lipid was extracted with a mixture of CHCl₃:MeOH (1:1), and separated by thin layer chromatography. The phosphorylated products were then visualized by autoradiography.

Transduction of GLUT4-green fluorescence protein (GFP) in CHO cells

Chinese Hamster Ovary cells (CHO, obtained from ATCC) were maintained in 10% FCS–DMEM and plated. On the next day, the 95% confluent cells were transfected with GLUT4-eGFP plasmid (kindly provided by Alan R Saltiel) (Inoue et al. 2003) using Lipofectamine 2000 according to the manufacturers' protocol. At 48 h post-transfection, the cells were serum starved for 8 h and treated with Rb₁ for 3 h or insulin for 30 min. The translocation of GLUT4 was observed under fluorescence microscopy (Leica, Wetzlar, Germany).

Statistical analyses

Quantitative data were presented as the mean±S.E.M. Statistical analysis was performed using Student's t-test or ANOVA with SPSS software (Chicago, IL, USA). The significance of differences was set at P<0.05.

	Results

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Ginsenoside Rb₁ stimulates basal and insulin-mediated glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes

To observe the effect of Rb₁ on glucose transport, 3T3-L1 cells were induced to mature adipocytes and then incubated with various concentrations of Rb₁ for different lengths of time, after which the rate of glucose uptake was measured. Rb₁ induced a time- and dose-dependent increase in glucose uptake rates in 3T3-L1 adipocytes. Rb₁ exerted an initial significant effect at 0.01 µM (168% of Con; P<0.05) and maximal effect at 1 µM in glucose uptake (185% of Con; P<0.05; Fig. 1A). However, the effect of Rb₁ was less potent than that of insulin (P<0.05, Fig. 1C). With 1 µM Rb₁, glucose uptake was augmented maximally at 3 h of treatment with Rb₁ (P<0.05; Fig. 1B). Furthermore, after 3 h of incubation with 1 µM and 10 µM Rb₁, 100 nM insulin-mediated glucose uptake was increased significantly (119 and 118% of Con with insulin, P<0.05; Fig. 1C).

View larger version (14K): [in this window] [in a new window]   Figure 1 Ginsenoside Rb1 stimulated basal and insulin-mediated glucose uptake in 3T3-L1 adipocytes. (A) Adipocytes were incubated with the indicated concentrations of Rb1 for 3 h or (B) with 1 µM Rb1 for the indicated times before 2-DOG uptake measurements. (C) After incubation with the indicated concentrations of Rb1 for 3 h, 2-DOG uptake in adipocytes were measured in the absence or presence of 100 nM insulin (Ins) for 30 min. Data represent mean±S.E.M. of three to four independent experiments. *P<0.05 vs 0 µM or 0 h; #P<0.05 vs 0 µM with insulin.

View larger version (12K): [in this window] [in a new window]   Figure 2 Ginsenoside Rb1 stimulated basal and insulin-mediated glucose uptake in C2C12 myotubes. (A) Myotubes were incubated with the indicated concentrations of Rb1 for 3 h or with 100 nM insulin (Ins) for 30 min or (B) with 1 µM Rb1 for the indicated times before 2-DOG uptake measurements. (C) After incubation with 1 µM Rb1 for 3 h, 2-DOG uptake rates in myotubes were measured in the absence or presence of 100 nM insulin (Ins) for 30 min. Data represent mean±S.E.M. of two independent experiments. *P<0.05 vs 0 µM Rb1 or 0 h; #P<0.05 vs 0 µM with insulin.

For assessing whether the increased glucose transport induced by Rb₁ in 3T3-L1 adipocytes was due to the translocations of GLUT1 and GLUT4, which are two predominant informs of glucose transporters expressed in 3T3-L1 adipocytes, the amounts of glucose transporter protein in LDM and PM fractions in the basal state, and after treatment with Rb₁ or insulin were measured. Rb₁ induced a significant increase of GLUT1 and GLUT4 protein in the PM content (P<0.05, respectively, Fig. 3A and B). In addition, a significant decrease of GLUT4 protein and a modest but no significant decrease of GLUT1 in the LDM fraction was observed after insulin treatment (P<0.05; P>0.05 respectively, Fig. 3A and B). However, the levels of GLUT1 and GLUT4 in the LDM fraction did not show significant changes in response to Rb₁ (Fig. 3A and B). The experiment in CHO cells transfected with GLUT4-eGFP demonstrated that Rb₁ induced translocation of GLUT4 to the cell surface (Fig. 3C).

View larger version (50K): [in this window] [in a new window]   Figure 3 Ginsenoside Rb1 promoted the translocations of GLUT1 and GLUT4 in 3T3-L1 adipocytes. (A) Adipocytes were incubated with 1 µM Rb1 for 3 h or 100 nM insulin for 30 min, and then subcellular membrane fractions were extracted and subjected to immunoblot analysis. (B) The immunoblotting signals of GLUT1 and GLUT4 in LDM and PM fractions from five independent experiments were quantified using densitometer. Data represent mean±S.E.M. *P<0.05 versus LDM of Con, #P<0.05 versus PM of Con. (C) GLUT4-eGFP transfected CHO cells were treated with 1 µM Rb1 for 3 h or 100 nM insulin for 30 min, and then the translocation of GLUT4 was photographed under fluorescence microscopy. The arrowhead points to GLUT4-eGFP accumulated at cell membrane.

To observe the effects of Rb₁ on the insulin signaling pathway, the activations of IR, IRS-1, PI3K, and AKT were measured. As expected, insulin stimulation resulted in augmentation of IR-β (Tyr1150/1151), IRS-1 (Y612) tyrosine phosphorylation, and AKT (Ser473) serine and threonine (Thr308) phosphorylation (Fig. 4A and B) and increased the activity of PI3K (Fig. 4C). Rb₁ also significantly enhanced IRS-1 (Y612), AKT (Thr308) phosphorylation (P<0.05 respectively; Fig. 4A and B) and stimulated the activity of PI3K (Fig. 4C). However, Rb₁ treatment did not produce detectable change in tyrosine phosphorylation of IR-β (Fig. 4A). Thus, we concluded that Rb₁ activates IRS-1 in the absence of the activation of IR, while activating PI3K and AKT.

View larger version (56K): [in this window] [in a new window]   Figure 4 Effects of ginsenoside Rb1 on the insulin signaling pathway in 3T3-L1 adipocytes. (A) Adipocytes were treated with 1 µM Rb1 for 3 h or 100 nM insulin for 30 min; total protein was extracted and subjected to immunoblot analysis with the indicated antibodies. (B) The immunoblotting signals of the indicated proteins for Rb1 and insulin from three independent experiments were quantified using densitometer. Data represent mean±S.E.M. *P<0.05 versus Con. (C) Adipocytes were treated with 1 µM Rb1 for 3 h or 100 nM insulin for 30 min, and then total protein was extracted for assay of PI3K activity and immunoblotting with anti-p85 antibody.

Whether enhancing glucose transport induced by Rb₁ is associated with the activation of PI3K was determined next. Pretreatment with HNMPA-(AM)3 (specific insulin receptor tyrosine kinase inhibitor), and wortmannin and LY294004 (specific PI3K inhibitors) markedly inhibit insulin-mediated glucose uptake in adipocytes (P<0.05 versus insulin control, Fig. 5), as previously reported (Li et al. 2005). Increased glucose uptake induced by Rb₁ was inhibited by both wortmannin and LY294004 (P<0.05 versus Rb₁ control, Fig. 5); however, this was not significantly affected by HNMPA-(AM)3. This result was consistent with that of Rb₁-activated IRS-1 independent of IR (Fig. 4). To assess the association of Rb₁-induced translocations of GLUT1 and GLUT4 with the enhanced activity of PI3K, GLUT1, and GLUT4 in the LDM and PM were measured in adipocytes pretreated with wortmannin. Wortmannin prevented the translocation of GLUT4 from the LDM to PM fractions induced by Rb₁ or insulin (P<0.05 or 0.01 respectively; Fig. 6A and C). GLUT1 in PM also showed a tendency to decline in PM in the presence of wortmannin, but it was not statistically significant (Fig. 6A and B). Therefore, we concluded that the activation of the insulin signaling cascade, including IRS-1, PI3K, and AKT, is required for Rb₁-induced glucose uptake and translocations of GLUT4, and to some extent, of GLUT1.

View larger version (17K): [in this window] [in a new window]   Figure 5 Inhibition of PI3K abrogated glucose uptake induced by Rb1 in 3T3-L1 adipocytes. Adipocytes were preincubated for 30 min with 50 µM HNMPA-(AM)3 (HNMPA), 100 nM wortmannin (Wort), and 50 µM LY294002, and then treated with 1 µM Rb1 for 3 h or 100 nM insulin for 30 min before 2-DOG uptake measurements. Data represent mean±S.E.M. of three independent experiments. *P<0.05 versus Con with DMSO; #P<0.05 versus Rb1 with DMSO; P<0.05 versus insulin with DMSO.

View larger version (36K): [in this window] [in a new window]   Figure 6 Effects of wortmannin on the translocations of GLUT1 and GLUT4 induced by Rb1 in 3T3-L1 adipocytes. (A) Adipocytes were preincubated with 100 nM wortmannin (Wort) for 30 min, and then treated with 1 µM Rb1 for 3 h or 100 nM insulin for 30 min. Subcellular membrane fractions were extracted and subjected to immunoblot analysis. (B and C) The immunoblotting signals of GLUT1 and GLUT4 in LDM and PM fractions from three independent experiments were quantified using densitometer. Data represent mean±S.E.M. *P<0.05 versus LDM of Con, #P<0.05 versus PM of Con, P<0.01 versus PM of Rb1, P<0.05 versus PM of insulin.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Declaration of interest References

To understand the mechanism underlying the stimulation of glucose transport by Rb₁, we examined the effect of this compound on GLUT1 and GLUT4 redistribution and the insulin signaling pathway in 3T3-L1 adipocytes. Both GLUT1 and GLUT4 are essential transporters of glucose uptake in adipocytes (Herman & Kahn 2006, Liao et al. 2006). In the present study, we showed that Rb₁was able to stimulate basal glucose uptake in adipocytes simultaneously accompanied by promoted translocations of the GLUT1 and GLUT4 protein. Previous literature has documented that the PI3K pathway plays an important role in the insulin signaling cascade leading to glucose transport translocation (Khan & Pessin 2002, Watson et al. 2004). The inhibition of PI3K activity blocks the translocation of both GLUT1 and GLUT4 in adipocytes (Clarke et al. 1994, Perrini et al. 2004). In our study, GLUT1 and GLUT4 translocations induced by Rb₁was associated with the activation of PI3K. Pretreatment of adipocytes with a specific PI3K inhibitor, wortmannin, abrogated Rb₁-induced GLUT1 and GLUT4 translocations as well as glucose uptake. Therefore, glucose transport and transporter translocation induced by Rb₁ is dependent on the involvement of PI3K. A recent study found that ginsenoside Re, which is another kind of ginsenosides, protopanaxatriol, also activated insulin signaling pathway independent of the insulin receptor and stimulated glucose uptake in adipocytes (Zhang et al. 2008). These results may indicate that insulin-like property is the underlying mechanism of action of ginsenosides.

In the insulin pathway, the activation of PI3K results from the autophosphorylation of intracellular β subunit tyrosine residues of the insulin receptor, subsequent activation of downstream signaling molecules, including IRS-1, and then leads to phosphorylation of AKT which is downstream from PI3K (Khan & Pessin 2002, Watson et al. 2004). To determine the engagement of other proteins in the insulin signaling cascade, we examined several upstream and downstream elements of PI3K. In 3T3-L1 adipocytes, Rb₁ was found to enhance the phosphorylation of IRS-1 at position 612 and AKT at position 308 without a detectable activation of IR-β. Although we did not directly measure the IRS-1-associated PI3K activity, because the tyrosine at position 612 is important for IRS-1 to activate PI3K and mediate the translocation of GLUT4 (Esposito et al. 2001), our results suggest that Rb₁ stimulates PI3K by a similar mechanism involving IRS-1 as that in insulin action. However, unlike insulin action, Rb₁ stimulated IRS-1 independent of insulin receptor activation. This conclusion was confirmed by the fact that the inhibition of IR with specific IR inhibitor did not significantly affect Rb₁-induced glucose transport. It is not clear how Rb₁ initiate the activation of the IRS/PI3K/AKT pathway. A distinct mechanism may be involved in the activation of insulin signaling cascade induced by Rb₁. Although the activation of IR is necessary for insulin to stimulate the insulin signaling pathway (Khan & Pessin 2002), actually insulin's action could be mimicked independent of insulin receptor activation in some cases. For example, dehydroepiandrosterone (DHEA), one of the steroid hormone, activated IRS/PI3K to result in the translocations of GLUT1 and GLUT4 and the augmentation of glucose transport in adipocytes in the absence of the activated insulin receptor. The authors suggested that DHEA might bind to specific cell-surface G-protein receptors to activate the IRS/PI3K pathway. Another report indicated that selenium promoted glucose transport and anti-lipolysis in an IRS-1/PI3K-dependent manner with no activation of the insulin receptor (Heart & Sung 2003). JAK2, as a growth hormone (GH) receptor-associated, GH-activated tyrosine kinase has been shown to interact with IRS-1 and stimulate binding of IRS-1 to the 85 kDa regulatory subunit of PI3K, which provides a biochemical basis for the insulin-like metabolic effects of GH (Argetsinger et al. 1995). Owing to their structural features of steroid skeleton and lipid-soluble ability, ginsenosides traverse the PM and initiate genomic effects; on the other hand, they might interact with some membrane receptor (Attele et al. 1999). Therefore, we speculate that Rb₁ may initiate insulin signaling pathway at IRS-1 responsible for the translocation of glucose transporters by other membrane receptors, which remain further clarified.

Our previous studies have shown that Rb₁ could bind to PPAR and regulate its expression to promote adipogenesis in adipocytes, suggesting that Rb₁ might be a PPAR ligand (Shang et al. 2007a,b). In this study, we found that besides promoting adipocytes differentiation, Rb₁ is also able to stimulate glucose transport in mature adipocytes through activating some elements of the insulin signaling pathway. It has been shown that troglitazone acutely stimulated glucose transport by activating PI3K in skeletal muscle (Kausch et al. 2001) and promoting GLUT4 translocation in adipocytes and myotubes (Shintani et al. 2001, Yonemitsu et al. 2001). These results imply that independent of activating PPAR, ginsenoside Rb₁ has a direct effect on insulin signaling pathway, similar to the effect pattern of troglitazone on adipose tissue.

In conclusion, we have demonstrated that ginsenoside Rb₁ stimulates basal and insulin-mediated glucose uptake in adipocytes and muscle cells. Rb₁ increases basal glucose transport and the translocations of GLUT1 and GLUT4 in adipocytes through an insulin-like signaling pathway. These results help to clarify the glucose-lowering action and anti-diabetic properties of ginseng and ginsenosides and further support their therapeutic importance in the treatment of diabetes.

	Declaration of interest

Top Abstract Introduction Materials and Methods Results Discussion Declaration of interest References

 
The authors state no conflict of interest.

	Funding

 
This study was supported by a fund for key laboratory of endocrinology and metabolic diseases from the Chinese Ministry of Education (Y0204) and a grant from the cultivation program of Shanghai excellence young medical talent fund (2004). This study was also supported by the grants from the Shanghai-Unilever Research and Development Fund (2004).

	Acknowledgements

 
We thank Alan R Saltiel for the gift of pCDNA3-GLUT4-eGFP.

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Received in final form 21 May 2008
Accepted 11 June 2008
Made available online as an Accepted Preprint 11 June 2008

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