Epigallocatechin-3-O-gallate (EGCG) protects the insulin sensitivity in rat L6 muscle cells exposed to dexamethasone condition.
Rat L6 muscle cells
The tea polyphenol epigallocatechin-3-O-gallate (EGCG) displays some antidiabetic effects; however the mechanisms are incompletely understood. In the present study, the investigation of the effects of EGCG on insulin resistance was performed in rat L6 cells treated with dexamethasone. We found that dexamethasone increased Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and reduced phosphorylation of AMPK and Akt. Furthermore, glucose uptake and glucose transporter (GLUT4) translocation were inhibited by dexamethasone. However, the treatment of EGCG improved insulin-stimulated glucose uptake by increasing GLUT4 translocation to plasma membrane. Furthermore, we also demonstrated these EGCG effects essentially depended on the AMPK and Akt activation. Together, our data suggested that EGCG inhibited dexamethasone-induced insulin resistance through AMPK and PI3K/Akt pathway.
[c] 2009 Elsevier GmbH. All rights reserved.
Insulin resistance is defined as the reduced ability of cells or tissues to respond to physiological levels of insulin. As we all known, glucocorticoids can induce insulin resistance when they are administered to human or experimental animals during excess condition (Gounarides et al. 2008; Klein et al. 2002). It is possible that glucocorticoids can act directly on peripheral tissues resulting in insulin resistance, or indirectly through changes in the blood levels of glucose, insulin or fatty acids (Gathercole et al. 2007; Lundgren et al. 2004). Although the understanding of glucocorticoids-induced insulin resistance has advanced considerably in recent years, effective therapeutic strategies to prevent or delay the development of this damage remain limited. Many researchers focus on searching for indigenous natural antidiabetic agents instead of synthetic drugs.
Green tea is a popular beverage worldwide; there has been a growing attention to its potential beneficial effects such as its anti-cancer and anti-oxidant properties (Hsu 2005; Wang and Bachrach 2002). Moreover, epidemiological study has shown a strong correlation between the consumption of tea and the prevention of diabetes (Iso et al. 2006; Mackenzie et al. 2007). In vivo animal and human studies indicate that insulin resistance could be alleviated by tea supplementation (Ryan et al. 2000; Wolfram et al. 2006). Particularly, oral supplementation with epigallocatechin-3-O-gallate (EGCG) (Fig. 1), the major polyphenol isolated from green tea, markedly enhances glucose tolerance in diabetic rodents (Wolfram et al. 2006) and improves glucose and lipid metabolism in hepatocytes (Zhen et al. 2006). In addition, EGCG also regulates the expression of genes encoding gluco-neogenesis enzymes, and suppresses hepatic glucose production (Collins et al. 2007; Csala et al. 2007).
Although EGCG is effective for antidiabetic activities in animal models, the effect of EGCG on skeletal muscle cells with insulin resistance under dexamethasone condition and its underlying mechanisms have not yet been elucidated. The findings from our study provide new insights into the underlying mechanisms by which EGCG exerts the antidiabetic effect in skeletal muscle cells.
Materials and methods
EGCG from green tea (E4143, [greater than or equal to]95% pure EGCG by HPLC), dexamethasone, insulin, [[.sup.3]H]-2-deoxyglucose, cytochalasin B were purchased from Sigma-Aldrich (St. Louis, MO). Rat L6 myoblast cell line was obtained from the ATCC (American Types Culture Collection, USA). High glucose-DMEM and fetal bovine serum (FBS) were purchased from G1BCO [TM]. Insulin receptor substrate-1 (IRS-1), phospho-IRS-1 ([Ser.sup.307]), AMP-activated protein kinase (AMPK), phospho-AMPK ([Thr.sup.172]), protein kinase B(Akt), phospho-A ([Ser.sup.473]) and HA-glucose transporter-4 (HA-GLUT4) antibodies were from Millipore (Bedford, MA, USA). [beta]-actin and second IgG HRP-linked antibodies were from Cell Signal Technology. Wortmannin was from Invitrogen (CA, USA) and compound C was from Merck (San Diego, CA).
[FIGURE 1 OMITTED]
L6 cells were maintained in high glucose-DMEM supplemented with 10% FBS, 100 units/ml penicillin, 100 [micro]g/ml streptomycin, 2 mM L-glutamine, and kept at 37[degrees]C in a humidified atmosphere of 5% [CO.sub.2]. For differentiation, the cells were seeded in appropriate culture plates, and after sub-confluence (about 70%), the medium was changed to DMEM supplemented with 2% FBS. The medium was then changed every 2 days until the cells were fully differentiated. Cells were treated in the absence or presence of dexamethasone (l[micro]M) over a time course of 24 h. In set of experiments, cells were pretreated with different dose of EGCG for 3 h prior to dexamethasone stimulation.
2-Deoxyglucose uptake assay
The assay was initiated via the addition of [[.sup.3]H]-2-deoxyglucose (25 mM; 10 mCi/ml) to each of the wells for 10 min at 37[degrees]C. The assay was terminated via the addition and subsequent washing of the cells with ice-cold PBS. The cells were lysed in 10% SDS or 50 mM NaOH. Radioactivity was evaluated via scintillation counting of the lysates extracted in SDS, whereas total protein contents were determined in lysates extracted in NaOH via the Bradford procedure (Bio-Rad Laboratory, Richmond, CA, USA). The values of glucose uptake were corrected for non-carrier-mediated transport by measuring glucose uptake in the presence of 10 mM cytochalasin B.
GLUT4 translocation assay
HA-GLUT4 translocation to the plasma membrane was measured as previously described (Govers et al. 2004) with minor modifications. Briefly, at given time points, paraformaldehyde was added to the wells to a concentration of 3%. After 15 min, the paraformaldehyde was quenched by the addition of 50 mM glycine. The cells were washed extensively and incubated for 20 min with 5% normal goat serum (NGS) to analyze the amount of HA-GLUT4 at the PM or the total HA-GLUT4 content, respectively. Cells were incubated for 60 min with anti-HA or, as a control, a nonrelevant antibody (mouse 1gG1-MOPC21). Cells were extensively washed and incubated for 20 min in 5% NGS. Cells were then incubated with Cy3-conjugated goat-antimouse in PBS containing 2% NGS. Image acquisition was achieved using an Olympus FV500 fluorescent microscope and analyzed quantitatively by the HMIAS-2000 Imaging System, China. Ten randomly selected areas were taken from each cover lip to account for variations in the expression levels.
At the end of the experiments, cells were collected and stored at -80[degrees]C until further analysis. Cells were rinsed with PBS, scraped from the 100 mm plates, and collected in a total volume of 200 [micro]l of homogenizing buffer. Immediately after collection, cells were sonicated for 5 s prior to storage. Protein assays were conducted and 30 [micro]g of protein were used for immunoblotting. The blots were then visualized via ECL (Cell Signaling Technology, Beverly, MA).
Data are expressed as means [+ or -] S.D. Statistic analysis was conducted with one-way analysis of variance (ANOVA). [rho] value 0.05 was considered statistically significant.
EGCG increases insulin-induced glucose uptake in dexamethasone-treated L6 cells
To determine the roles of EGCG in glucose metabolism and insulin action, we assessed the co-treatment effects of EGCG with insulin on glucose uptake in dexamethasone-treated L6 cells. Our result showed that glucose uptake in insulin-stimulated control group was 3.0 times higher than that in normal control group; however, dexamethasone treatment alone inhibited glucose uptake by 30% compared to normal control group (Fig. 2A). In contrast to the insulin-stimulated control group, the dexamethasone treatment inhibited insulin-stimulated glucose uptake by over 60%. However, intervention with EGCG reversed the condition completely. Insulin-induced glucose uptake was increased by 80% and 105% respectively after intervention with 20 and 40 [micro]M EGCG for 24 h.
EGCG stimulates GLUT4 translocation in dexamethasone-treated L6 cells
We examined the effects of EGCG on the translocation of the glucose transporter GLUT4 to the plasma membrane in dexamethasone-treated L6 cells, because this is an essential step for inducible glucose uptake into muscle cells. The results showed that in contrast to the insulin-stimulated control group, dexamethasone inhibited GLUT4 translocation (Fig. 2B). In addition, compared with dexamethasone treatment as cytoplasmic vesicles, EGCG treatment induced obviously GLUT4 translocation into cell membrane.
EGCG inhibits dexamethasone-induced IRS-1 [Ser.sup.307] phosphorylation in L6 cells
Since phosphorylation precedes IRS-1 degradation and [Ser.sup.307] phosphorylation of IRS-1 has become a molecular indicator of insulin resistance, therefore we investigated the effect of EGCG on IRS-1 [Ser.sup.307] phosphorylation with phosphorylation-specific IRS-1 [Ser.sup.307] antibody by immunoblotting in dexamethasone-treated L6 cells. We found that dexamethasone (1 [micro]M, 24 h) induced IRS-1 [Ser.sup.307] phosphorylation (Fig. 3); however, exposure with EGCG (20 or 40 [micro]M) reduced IRS-1 [Ser.sup.307] phosphorylation in a dose-dependent manner.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
EGCG induces AMPK phosphorylation in dexamethasone-treated L6 cells
AMPK activation is thought to be a key proximal event in the cellular energy balance response, and AMPK phosphorylation level in [Thr.sup.172] is currently accepted as a marker of AMPK activity. AMPK has been proved to be required for antidiabetic effects of some clinical drugs in insulin-resistant rat L6 cells. In order to find the factor mediating IRS-1 phosphorylation. we examined the phosphorylation of AMPK in L6 cells cotreated with EGCG and dexamethasone. As shown in Fig. 4A, insulin significantly stimulated AMPK phosphorylation compared to normal control group. There is a basal level of phosphorylated AMPK in L6 cells under excessive dexamethasone condition; however, these levels were increased by coincubation with EGCG in a dose-dependent manner. Interestingly, the addition of compound C, an AMPK specific inhibitor, could diminish the EGCG-induced IRS-1 [Ser.sup.307] phosphorylation inhibition (Fig. 4B). As the observations, it has raised the possibility that EGCG increased insulin sensitivity in dexamethasone-treated L6 cells might be attribute to the regulation of AMPK activation.
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
Effects of EGCG on insulin-induced Akt phosphorylation under dexamethasone
Akt is the key molecular which mediates the metabolic effects of insulin signaling. It lays downstream of PI-3K and facilitates glucose uptake in muscle cells. Dexamethasone treatment clearly blocked the insulin-induced [Ser.sup.473] phosphorylation of Akt in muscle cells (Fig. 5A). The inhibition of Akt [Ser.sup.473] phosphorylation can be reversed by treatment with EGCG in a dose-dependent way. Notably, we also observed that wortmannin, a PI-3K specific inhibitor, inhibited the EGCG-induced phosphorylation of Akt, which suggests that the EGCG action may go through upstream of PI-3K/Akt signaling pathway (Fig. 5B). This result confirmed that the action targets of EGCG should be placed upstream of Akt molecules.
The cell model of dexamethasone-induced insulin resistance in skeletal muscle was established in the present study. Emerging evidences are in favor of the concept that glucocorticoids can induce insulin resistance in vivo and in vitro (Gounarides et al. 2008; Kern et al. 1999). The usage of L6 cells in culture provides a means to focus on the effect of glucocorticoids directly. In the present study we found that dexamethasone treatment for 24 h downregulated the insulin-stimulated glucose uptake significantly, which was consistent with previous studies in cell culture (Li et al. 2005).
A recent search of the literature revealed that the natural product EGCG is the major polyphenolic constituent found in green tea [dried fresh leaves of the plant Camellia sinensis L. Ktze. (Theaceae)] (Demeule et al. 2002). More than 50% of the mass of all catechin combination in green tea is composed of EGCG and a vast body of scientific research suggests that EGCG is responsible for the majority of the potential health benefits attributed to green tea consumption (Nagle et al. 2006). Dozens of studies have demonstrated that non-physiologically relevant high concentrations of EGCG can potentially interfere with many disease-related biochemical processes in vitro (Collins et al. 2007; Zhang and Zhuo 2006). Although EGCG has previously been shown to display antidiabetic property (Babu et al. 2007), molecular mechanism underlying the effect is still unclear. The present study demonstrated an inhibitory effect of EGCG on insulin resistance induced by dexamethasone in L6 cells. We also found that EGCG intervention for 24 h could attenuate the effect of dexamethasone on glucose uptake and increase glucose uptake in a dose-dependent manner in differentiated L6 cells. Additionally, the movement of the insulin responsive GLUT4 to cell surface is an essential step for insulin-responsive glucose transport in muscle tissue, which becomes defective in dexamethasone-induced insulin resistance (Coster et al. 2004). To further confirm the antidiabetic property of EGCG, we also determined GLUT4 translocation in dexamethasone treated L6 cells. Our data showed that EGCG exhibited an effect on stimulating GLUT4 translocation by several folds in L6 cells compared to that in dexamethasone treatment. These results indicate that EGCG is highly potent and efficacious on stimulating GLUT4 translocation in insulin responsive cells.
To investigate the mechanism responsible for the GLUT4 translocation stimulated by EGCG, we examined the actions of EGCG on cellular signaling pathways that are known to mediate this translocation process in dexamethasone-treated L6 cells. AMPK pathway is a major regulator of GLUT4 translocation in response to some antidiabetic agents such as AICAR and metformin (Ju et al. 2005; Russell et al. 1999), and hence we investigated whether EGCG activated AMPK. Our study confirmed that EGCG could increase the phosphorylation of AMPK, suggesting that the AMPK signaling pathway is likely responsible for the stimulation of GLUT4 translocation by EGCG.
To be further, we also found that a predominant signal transduction pathway by which EGCG improved insulin resistance was through PI-3K/Akt pathway in L6 cells. Our data demonstrated that EGCG exhibited an enhancement on insulin-mediated Akt phosphorylation in dexamethasone-treated L6 cells, which was inhibited by wortmannin, an inhibitor of PI-3K. Indeed, previous reports indicate that EGCG alone does not affect Akt phosphorylation, but it does augment insulin signaling pathway by activating AMPK, eventually results in Akt activation (Lin and Lin 2008). Reports concerning interactions between PI-3K/Akt and AMPK are controversial. AMPK has been reported to stimulate GLUT4 translocation independently of PI-3K/Akt. However, it is also proposed that AMPK has a potential role in regulation of insulin action, thus increasing the complexity of understanding the interconnection of these two signaling pathways. Taken together, the interaction between these two signaling pathways appears to be cell type-dependent and text-dependent. Further studies are required to elucidate the precise mechanisms interconnecting these two signaling pathway.
The search for the modulation of IRS activities has been suggested to focus on its phosphorylation state. Most notably, serine/threonine phosphorylation has been shown to modulate both positive and negative signaling transmission via IRS (Kumar and Dey 2002). In this study, we demonstrated that the exposure to dexamethasone resulted in [Ser.sup.307] phosphorylation of IRS-1 which was consistent with the previous study (Lin and Lin 2008). This indicates that increasing IRS-1 [Ser.sup.307] phosphorylation can affect insulin action. Moreover, our study also displayed that the EGCG treatment reversed these dexamethasone effects significantly via inhibiting IRS-1 [Ser.sup.307] phosphorylation.
An interesting finding was that AMPK mediated the EGCG inhibition of IRS-1 serine phosphorylation. In this study we confirmed that EGCG was indeed inhibitory to [Ser.sup.307] phosphorylation in IRS-1. However, this effect could be abolished by the AMPK inhibitor compound C. Interestingly, both this study and previous study have found that EGCG could activate AMPK, and the activation of AMPK was associated with EGCG-induced effect (Lin and Lin 2008). Our work showed that EGCG insulin-enhancing effect in dexamethasone-treated L6 cells was required for the activation of AMPK and the consequent inhibition of IRS-1 serine phosphorylation.
In summary, using L6 cells, we investigated how dexamethasone preferentially impaired insulin down-signaling and what the role EGCG played to alleviate this insulin resistance state. Our work found that EGCG inhibited dexamethasone-induced insulin resistance through AMPK and PI-3K/Akt pathway. Further research should aim to analyze the mechanism of EGCG which may lead to identification of molecular targets for the therapeutic agents of insulin resistance disease.
This work was supported by the foundation (No. 2006BAD27B01) from the Ministry of Science and Technology of the People's Republic of China.
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Z.F. Zhang, Q. Li, J. Liang, X.Q. Dai, Y. Ding, J.B. Wang, Y. Li *
Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing 100191, PR China
Abbreviations: Akt, Protein kinase B; AMPK, AMP-activated protein kinase; EGCG, Epigallotatechin-3-O-gallate; FBS, Fetal bovine serum; GLUT4, Glucose transporter-4; IRS-1, Insulin receptor substrate-1; NGS, Normal goat serum; P1-3K, Phosphatidylinositol 3 kinase
* Corresponding author. Tel./fax: +86 10 82801177.
E-mail address: email@example.com (Y. Li).
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