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Mar 2, 2015 - Suqing Bao, Yanli Cao, Haicheng Zhou, Xin Sun, Zhongyan Shan, and Weiping Teng. Department of Endocrinology and Metabolism, Institute ...
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Epigallocatechin Gallate (EGCG) Suppresses LipopolysaccharideInduced Toll-like Receptor 4 (TLR4) Activity via 67 kDa Laminin Receptor (67LR) in 3T3-L1 Adipocytes Suqing Bao, Yanli Cao,* Haicheng Zhou, Xin Sun, Zhongyan Shan, and Weiping Teng

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Department of Endocrinology and Metabolism, Institute of Endocrinology, Liaoning Provincial Key Laboratory of Endocrine Diseases, The First Affiliated Hospital of China Medical University, No. 155, North Nanjing Street, Heping District, Shenyang, Liaoning 110001, People’s Republic of China ABSTRACT: Obesity-related insulin resistance is associated with chronic systemic low-grade inflammation, and toll-like receptor 4 (TLR4) regulates inflammation. We investigated the pathways involved in epigallocatechin gallate (EGCG) modulation of insulin and TLR4 signaling in adipocytes. Inflammation was induced in adipocytes by lipopolysaccharide (LPS). An antibody against the 67 kDa laminin receptor (67LR, to which EGCG exclusively binds) was used to examine the effect of EGCG on TLR4 signaling, and a TLR4/MD-2 antibody was used to inhibit TLR4 activity and to determine the insulin sensitivity of differentiated 3T3-L1 adipocytes. We found that EGCG dose-dependently inhibited LPS stimulation of adipocyte inflammation by reducing inflammatory mediator and cytokine levels (IKKβ, p-NF-κB, TNF-α, and IL-6). Pretreatment with the 67LR antibody prevented EGCG inhibition of inflammatory cytokines, decreased glucose transporter isoform 4 (GLUT4) expression, and inhibited insulin-stimulated glucose uptake. TLR4 inhibition attenuated inflammatory cytokine levels and increased glucose uptake by reversing GLUT4 levels. These data suggest that EGCG suppresses TLR4 signaling in LPSstimulated adipocytes via 67LR and attenuates insulin-stimulated glucose uptake associated with decreased GLUT4 expression. KEYWORDS: adipocyte, epigallocatechin gallate, inflammation, insulin resistance, toll-like receptor 4



INTRODUCTION Accumulating evidence has demonstrated the importance of adipose tissue inflammation in disease occurrence, including obesity, type 2 diabetes, and metabolic syndromes.1,2 One common feature of chronic adipose inflammation is increased cytokine production.1−3 Adipocytes are integral cellular components throughout the whole body, and they induce the innate immune response, produce pro-inflammatory adipokines, such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1, and promote macrophage recruitment.3 Furthermore, preadipocytes have the potential to efficiently and rapidly convert into macrophages under inflammatory conditions.4 Therefore, there is a clear basis for the inflammatory response in adipose tissue. Toll-like receptors (TLRs) likely play a crucial role in obesity-related inflammation.5,6 TLR4 was the first member to be characterized, and it has been the most-studied TLR in adipocytes.5−7 Much of the research has shown that TLR4 expression is significantly higher in the adipose tissue of obese individuals, perhaps because of increased macrophage infiltration.8−10 High levels of circulating free fatty acid (FFA) and lipopolysaccharide (LPS) activate TLR4 signaling in macrophages and adipocytes to induce the inflammatory response.11,12 There are two primary signaling pathways initiated by TLR4 activation: one is modulated by myeloid differentiation primary response protein 88 (MyD88), while the other is modulated by toll/IL-1-receptor-domain adaptor molecule. Both pathways can activate nuclear factor-κB (NFκB) signaling and promote inflammatory cytokine secretion, which is a known cause of insulin resistance.8−10 © 2015 American Chemical Society

Green tea is a popular beverage worldwide, and its consumption has been associated with several health benefits, including protection against multiple diseases, including cancer, atherosclerosis, and cardiovascular disorders.13,14 Epigallocatechin gallate (EGCG) is the primary active component of green tea, and it exclusively binds to 67 kDa laminin receptor (67LR), which is widely expressed in many cell types, including cancer cells, hepatocytes, and preadipocytes.15−17 EGCG possesses a variety of biological activities.14,18 It reduces adipose tissue mass by inhibiting adipocyte differentiation and avoids diet-induced obesity.19 EGCG attenuates TLR4 signaling,8 reduces hyperglycemia by promoting glucose transporter isoform 4 (GLUT4) translocation in rodents,8,20 and attenuates TNF-α-promoted reactive oxygen species (ROS) generation and increased glucose uptake ability in 3T3-L1 adipocytes.21 To date, the underlying molecular mechanism of EGCGmediated suppression of TLR4 signaling and whether it involves insulin signaling remain unknown. Here, we investigated the pathways involved in EGCG modulation of insulin signaling and TLR4 signaling in LPS-stimulated adipocytes.



MATERIALS AND METHODS

Reagents. Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), fetal bovine serum (FBS), and serum-free medium were all purchased from Gibco (Life Technologies, Carlsbad, CA).

Received: Revised: Accepted: Published: 2811

November 19, 2014 March 2, 2015 March 2, 2015 March 2, 2015 DOI: 10.1021/jf505531w J. Agric. Food Chem. 2015, 63, 2811−2819

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Journal of Agricultural and Food Chemistry

Figure 1. 3T3-L1 cell differentiation detected by Oil Red O staining. Panels A and B show pre-Oil Red O staining, and panels C and D show postOil Red O staining. Panels A and C represent undifferentiated cells, and panels B and D represent differentiated cells (magnification, 100×; bar, 200 μm). Anti-TLR4 and anti-GLUT4 were from Abcam Biochemicals (Hong Kong). Anti-PI-3K and p-NF-κB were obtained from Cell Signaling Technology (Shanghai, China). Anti-TNF-α and anti-β-actin were obtained from Santa Cruz Technology (Carlsbad, CA). Fluorescein (FITC)-AffiniPure donkey anti-rabbit immunoglobulin G (IgG) was from Jackson ImmunoResearch (West Grove, PA). The monoclonal antibody against mouse TLR4/MD-2 (Affymetrix) was obtained from Affymetrix eBioscience (Carlsbad, CA). Mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kits were obtained from Dakewe (Dakewe Biotech Co., Ltd., Beijing, China). Insulin (from bovine pancreas), 3-isobutyl-1-methylxanthine (BioUltra, ≥99%), dexamethasone (purity of ≥97%), lipopolysaccharides from Escherichia coli 055:B5, EGCG (purity of ≥95%, from green tea), and routine chemicals were all purchased from Sigma Chemical (Sigma-Aldrich, St. Louis, MO). Cell Culture and Differentiation. 3T3-L1 fibroblasts were obtained from the American Type Culture Collection (Manassas, VA) and cultured at a 37 °C, 5% CO2, and 95% humidity. Preadipocytes were induced to differentiate as follows.22 Cells were maintained in DMEM plus 10% heat-inactivated FCS and 0.5% penicillin−streptomycin (Invitrogen). After 2 days in culture when the cells reached confluence (day 0), they were induced to differentiate by adding medium containing 10% FBS, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 5 μg/mL insulin for 48 h. Fresh media containing only 5 μg/mL insulin and 10% FBS was added for an additional 48 h. Media was subsequently changed every 48 h. More than 90% of cells expressed the adipocyte phenotype between days 8 and 10 post-differentiation, and they were used for experiments. Oil Red O Staining of 3T3-L1 Adipocytes. Conversion of 3T3Ll fibroblasts to adipocytes was monitored by measurement of intracellular lipid accumulation using Oil Red O staining. A 0.5% (w/ v) solution of Oil Red O was prepared in 60% isopropanol. After day 8, differentiated 3T3-L1 preadipocytes were washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde, then incubated with Oil Red O working solution for 2 h at room

temperature, and rinsed to remove unbound dye. Staining was visualized with an Olympus DP70 camera (Olympus, Japan). As shown in Figure 1, differentiated 3T3-L1 cells were positive for fat droplets and lipid storage by Oil Red O. We used the cells for further experiments when more than 90% expressed the adipocyte phenotype between 8 and 10 days post-differentiation. LPS and EGCG Treatment of Differentiated 3T3-L1 Adipocytes. Fully differentiated adipocytes incubated in serum-free medium and stimulated by adding different concentrations of LPS from E. coli for 48 h to elicit an immune response in the absence or presence of 3 h of pretreatment with EGCG. Cells were divided into eight groups, including one control group (blank), while the remaining seven groups were treated with 0.1−1 μg/mL LPS or 1 μg/mL LPS + 10−100 μM EGCG. After treatment, supernatants were collected for ELISA and the adipocytes were collected for detection of inflammation-related proteins (p-NF-κB, IKKβ, and TNF-α) and key insulin signaling protein (PI-3K and GLUT4) levels. Anti-67LR Treatment. To evaluate the effect of EGCG on TLR4 signaling, we performed blocking experiments in adipocytes. Fully differentiated adipocytes were pretreated with 67LR antibody35 (10 μg/mL, MLuC5) for 30 min, followed by EGCG (100 μM) for 3 h, and subsequent incubation with LPS (1 μg/mL) for 48 h. IL-6 levels in supernatants of treated and control cells were detected by ELISA, and inflammatory mediators and cytokines, such as TLR4, p-NF-κB p65, and TNF-α, and the key insulin signaling protein GLUT4 were examined by western blotting. Anti-TLR4 Treatment. Cells were treated with a TLR4-specific antibody (anti-mouse TLR4/MD-2 complex functional-grade purified antibodies, MTS510)43 to inhibit their response to LPS. Fully differentiated adipocytes were pretreated with MTS510 (5 μg/mL) or PBS for 30 min at 37 °C and subsequently treated with LPS (1 μg/ mL) for 48 h. IL-6 levels in supernatants and intracellular p-NF-κB p65, TNF-α, and GLUT4 protein levels were determined by western blotting. 2812

DOI: 10.1021/jf505531w J. Agric. Food Chem. 2015, 63, 2811−2819

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Journal of Agricultural and Food Chemistry

Figure 2. EGCG decreases inflammation-related protein levels and increases the expression of key insulin signaling proteins in adipocytes. Various doses of LPS (0.1, 0.5, and 1 μg/mL) stimulated inflammatory cytokine expression in mature 3T3-L1 adipocytes. Mature 3T3-L1 adipocytes pretreated with EGCG (10, 30, 50, and 100 μM) decreased LPS-stimulated inflammatory-related factor expression (IKKβ, p-NF-κB, and TNF-α) to different degrees. EGCG (10, 30, 50, and 100 μM) increased key insulin signaling protein expression (PI-3K and GLUT4). Results are expressed as the mean ± SD (n = 6). (∗) p < 0.05 versus the 0 ng/mL LPS + 0 μM EGCG group. (#) p < 0.05 versus the 1 μg/mL LPS group. Cellular 3H-2-Deoxy-D-glucose Uptake Measurements. 3T3L1 cells were seeded in 24-well plates (1 × 105 cells/well) and induced to differentiate, as described above. On day 9, adipocytes were incubated for 2 h at 37 °C in serum-free medium, and cells were washed twice with 37 °C Krebs Ringer phosphate (KRP) buffer (pH 7.4) and placed in KRP buffer containing insulin (100 nM) for 30 min, followed by the addition of 3H-2-deoxy-D-glucose (2 μCi/mL, Beijing Yuan Zi Gao Ke Corporation, China) for an additional 10 min at 37 °C. Cells were then immediately washed 3 times in ice-cold PBS to terminate the reaction. Lastly, cells were solubilized in 0.5 M NaOH (0.4 mL/well) for 2 h and subjected to scintillation counting for 3H radioactivity as disintegrations per minute. The radioactivity of each sample was normalized to the protein concentration. Counts per minute and per milligram of protein were measured to analyze experimental glucose uptake. IL-6 Measurements. IL-6 levels in culture supernatants were measured in duplicate by the ELISA (Dakewe Biotech) kit according to the instructions of the manufacturer. The minimum detectable level of each kit was 15.6 pg/mL; the sensitivity was 8 pg/mL; and the coefficient of variation between the plates was 0.05). Furthermore, GLUT4 levels were reduced by approximately 72.4% in the 67LR antibody group compared to the EGCG group (Figure 4E; p < 0.05). These data suggest that 67LR antibody blocks the effect of EGCG on LPS-induced TLR4 signaling, resulting in a subsequent series of changes, such as increased inflammatory cytokine expression and decreased GLUT4 levels. However, we did not detect any obvious differences in 67LR expression among the control (blank), EGCG (100 μM EGCG plus 1 μg/mL LPS), and LPS

21.9, and 14.0%, respectively) expression and increased PI-3K (approximately by 1.4-, 2.0-, 2.0-, and 1.8-fold, respectively) and GLUT4 (approximately by 1.9-, 2.1-, 2.9-, and 3.1-fold, respectively) levels compared to treatment with 1 μg/mL LPS alone (Figure 2; p < 0.05 for each). These data suggest that EGCG decreases LPS-stimulated inflammatory cytokine and insulin signaling protein expression in adipocytes. EGCG Reverses Intracellular GLUT4 Expression in LPSStimulated Adipocytes. We used immunofluorescence labeling to investigate cellular GLUT4 expression. GLUT4 fluorescence intensity demonstrated that LPS treatment decreased GLUT4 levels. As shown in Figures 2 and 3A, various doses of LPS (0.1, 0.5, and 1 μg/mL) dramatically decreased the GLUT4 signal. In contrast, EGCG treatment (100 μM) increased GLUT4 expression compared to LPS (1 μg/mL) treatment alone (Figures 2 and 3B). 67LR Antibody Blocks the Effect of EGCG on LPSInduced TLR4 Signaling. Upon treatment with the 67LR 2815

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Figure 4. (A−E) 67LR antibody blocks the effects of EGCG on TLR4 signaling and GLUT4 expression in LPS-stimulated adipocytes. (F) EGCG did not affect 67LR expression between the control (blank group), EGCG (100 μM EGCG plus 1 μg/mL LPS), and LPS groups (1 μg/mL LPS). (A−F) Protein levels were determined by western blotting. Bars represent the mean ± SD, and both are expressed as the ratio to the control group (the level of the control group was normalized to 1). (∗) p < 0.05, for the LPS group versus the control group; (#) p < 0.05, for the 67LR blocker group versus the EGCG group. ns = not significant when the EGCG group was compared to the LPS or control group.

Figure 5. TLR4 mediates LPS-induced inflammation activation and insulin resistance, and EGCG has similar effects to the anti-TLR4 antibody. (A− D) Protein levels determined by western blotting. Bars represent the mean ± SD, expressed as the ratio to the control group (the level of the control group was normalized to 1; n = 6 for each group). (∗) p < 0.05, for the LPS group versus the control group. (#) p < 0.05, for the LPS group versus the EGCG group and TLR4-inhibited group.

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Figure 6. EGCG and the anti-TLR4 antibody inhibit IL-6 production, and an inhibitor of 67LR blocks EGCG effects. IL-6 levels in culture supernatants measured by ELISA. (A and B) IL-6 levels in cell culture supernatants were measured in duplicate. (∗) p < 0.05. ns = not significant between the two groups. Panel A showed that EGCG inhibits IL-6 production in the context of LPS and does not work effectively with an inhibitor of 67LR. Panel B showed that IL-6 production was blocked with the anti-TLR4 antibody and has similar effect to EGCG.

groups (1 μg/mL LPS) (Figure 4F; p > 0.05). We hypothesize that EGCG performs its anti-inflammatory role by regulating 67LR activity or translocation. TLR4 Mediates LPS-Induced Inflammatory Activation and Insulin Resistance. As shown in Figure 5, the specific TLR4/MD2 inhibitor MTS510 (5 μg/mL) treatment significantly reduced p-NF-κB (67.9% decrease; p < 0.05; Figure 5B), TNF-α (21.5% decrease; p < 0.05; Figure 5C), and IL-6 (45.5% decrease; p < 0.05; Figure 6B) levels after LPS stimulation, although IL-6 levels were still higher than the blank group, perhaps because of the effects of LPS. Meanwhile, MTS510 treatment increased GLUT4 expression by 1.3-fold (p < 0.05; Figure 5D) compared to LPS-induced control cells. The above data suggest that TLR4/MD2 inhibition decreased NF-κB activation and upregulated GLUT4 expression and EGCG had similar effects to the anti-TLR4 antibody. EGCG Upregulates Cellular 3H-2-Deoxy-D-glucose Uptake by Decreasing TLR4 Expression. To determine the effects of TLR4 on glucose transport in inflammation, cellular 3H-2-deoxy-D-glucose uptake contents were measured (Figure 7). We found that insulin-stimulated glucose influxes were significantly greater than untreated control cells (p < 0.05). Pretreatment with EGCG (100 μM) for 3 h before 48 h of LPS exposure (1 μg/mL) significantly increased glucose

uptake compared to LPS stimulation alone (∼1.2-fold; p < 0.05), while anti-67LR Ab reversed the trend (about 84.4% of the EGCG group; p < 0.05). Anti-TLR4 Ab increased glucose uptake even in the absence of EGCG (about 1.2-fold higher than the LPS group; p < 0.05). Our data suggest that TLR4 attenuates insulin-stimulated glucose uptake and EGCG blocks these changes by decreasing TLR4 signaling.



DISCUSSION Here, we showed that EGCG suppresses the TLR4 signaling pathway in adipocytes via 67LR. Upon 67LR inhibition, The suppressive effect of EGCG on TLR4 activity was blocked; inflammatory cytokine levels, such as TNF-α and IL-6, increased; and GLUT4 expression decreased. These effects led to attenuated insulin signal transduction and decreased glucose uptake. In contrast, TLR4 inhibition inactivated the inflammation pathway, phenocopying the effect of EGCG and increasing expression of the key insulin signaling protein GLUT4 in LPS-stimulated adipocytes, thus promoting insulinstimulated glucose uptake. These results suggest that EGCG improves insulin signaling by suppressing TLR4 activity in adipocytes. Type 2 diabetes and obesity are characterized by low-grade inflammation with increased inflammatory cytokine levels and changes in the gut microbiota.23 As an important pathogen recognition receptor, high TLR4 levels impair insulin action and negatively regulate insulin signaling.36,37 Increased TLR4 levels underlie non-infectious chronic inflammation and dysfunction of adipose tissue in obesity.5,24,25 In our previous study, we found that a high-fat diet increases the TLR4mediated inflammatory response in the adipose tissue of obese rats.8 TLR4-deficient mice are partially protected from inflammation and insulin resistance,26 and inhibition of TLR4 signaling protects against type 2 diabetes in mice.27,38,39 Studies have shown that the TLR4 ligand LPS activates NF-κB and increases inflammatory biomediator release in 3T3-L1 adipocytes, a process which may be blocked by a TLR4 inhibitor.12 Importantly, these biomediators also work on insulin target cells and insulin-producing cells, such as adipocytes, pancreatic islet cells, and the hypothalamus, further impairing insulin sensitivity and secretion.25,28−30 Consistently, our work suggests that LPS activates the TLR4 signaling

Figure 7. Cellular 3H-2-deoxy-D-glucose uptake. (∗) p < 0.05. 2817

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pathway, a specific TLR4/MD2 inhibitor partially weakens inflammatory cytokines, such as TNF-α and IL-6, and upregulates insulin signaling protein GLUT4 expression, and TLR4 inhibition has similar effects to EGCG treatment in 3T3L1 adipocytes. EGCG is the most abundant polyphenol in tea and has many biological properties.13−15,17−19 Its metabolites can exist in methylated, glucuronide, and sulfate forms, and the phenolic hydroxyls in their structure determine their antibacterial, anticardiovascular disease, and antitumor activities.31 Studies have shown that EGCG exclusively binds to 67LR and influences downstream insulin signaling,17,32,33 and treatment with 67LR antiserum blocks extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation while increasing insulin receptor-β and insulin receptor substrates 1 and 2 (IRS1 and IRS2) phosphorylation in preadipocytes.17,32 Our work demonstrated that pretreatment of adipocytes with a 67LR blocking antibody blocked the suppression of the TLR4 signaling pathway of EGCG, activated the NF-κB signal pathway, induced inflammatory cytokine transcription, and decreased GLUT4 expression, leading to a further decrease in cellular 3H-2-deoxy-D-glucose uptake, which is associated with insulin signaling transduction. Our work demonstrated that 67LR expression did not change upon EGCG treatment. It has yet to be determined whether 67LR membrane localization changes upon EGCG treatment. However, previous research33 stated that EGCG increases 67LR efficiency by inducing its translocation from the cytoplasm to the membrane. EGCG downregulation of inflammatory signals may be induced by Tollip, which is associated with decreased TLR signaling-mediated NF-κB activation.34,35 Therefore, it is crucial to further delineate the mechanisms involved to support our findings. Additionally, TLRs and nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are very remarkable pathogen recognition receptors in obesity and type 2 diabetes characterized by low-grade inflammation.23,40,41 Both TLRs and NLRs mediate NF-κB activation, leading to inflammatory cytokine production and insulin resistance.40−42 Therefore, we speculate that EGCG may simultaneously decrease inflammation through the NLR signaling pathway. Therefore, further studies are necessary to target TLR- or NLR-regulated pathways in obesity-related metabolic complications. Taken together, these data suggest that EGCG affects the TLR4 signaling pathway, reduces chronic low-grade inflammation, and further influences insulin signaling protein expression, while the entire process is blunted by 67LR inhibition, which further strengthens the link between the TLR4-induced inflammatory response and insulin resistance in adipocytes. Our work also provides insight into better prevention of insulin resistance by revealing the possible molecular mechanism and developing targeted therapies to protect against it.



The authors declare no competing financial interest.



ABBREVIATIONS USED EGCG, epigallocatechin gallate; TLR4, toll-like receptor 4; LPS, lipopolysaccharide; GLUT4, glucose transporter type 4; 67LR, 67 laminin receptor; FFA, free fatty acid



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AUTHOR INFORMATION

Corresponding Author

*Telephone: 86-24-83282152. Fax: 86-24-83283294. E-mail: vanilla421@163.com. Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant 81000327), the Chinese Society of Endocrinology (12030470347), and the General Scientific Research Fund of Liaoning Provincial Education Department (L2013300). 2818

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Journal of Agricultural and Food Chemistry

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DOI: 10.1021/jf505531w J. Agric. Food Chem. 2015, 63, 2811−2819