Epigallocatechin Gallate (EGCG) Suppresses Lipopolysaccharide


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Epigallocatechin Gallate (EGCG) Suppresses LipopolysaccharideInduced Toll-like Receptor 4 (TLR4) Activity via 67kDa Laminin Receptor (67LR) in 3T3-L1 Adipocytes Suqing Bao, Yanli Cao, Haicheng Zhou, Xin Sun, Zhongyan Shan, and Weiping Teng J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 02 Mar 2015 Downloaded from http://pubs.acs.org on March 6, 2015

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Journal of Agricultural and Food Chemistry

Epigallocatechin Gallate (EGCG) Suppresses Lipopolysaccharide-Induced 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

Department of Endocrinology and Metabolism, the Institute of Endocrinology, Liaoning Provincial Key Laboratory of Endocrine Diseases, The First Affiliated Hospital of China Medical University, Shenyang, 110001, P.R. China

To whom correspondence should be addressed: Yanli Cao. 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, 110001 Shenyang, 110001, P.R. China, Tel.: 86-24-83282152; Fax: 86-24-83283294; Email: [email protected]

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Abstract

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Obesity-related insulin resistance is associated with chronic systemic low-grade inflammation, and

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toll-like receptor 4 (TLR4) regulates inflammation. We investigated the pathways involved in EGCG

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modulation of insulin and TLR4 signaling in adipocytes. Inflammation was induced in adipocytes by

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lipopolysaccharide (LPS). An antibody against the 67-kDa laminin receptor (67LR, to which EGCG

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exclusively binds) was used to examine EGCG’s effect on TLR4 signaling, and a TLR4/MD-2

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antibody was used to inhibit TLR4 activity and to determine the insulin sensitivity of differentiated

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3T3-L1 adipocytes. We found that EGCG dose-dependently inhibited LPS stimulation of adipocyte

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inflammation by reducing inflammatory mediator and cytokine levels (IKKβ, p-NF-κB, TNF-α and

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IL-6). Pretreatment with the 67LR antibody prevented EGCG inhibition of inflammatory cytokines,

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decreased GLUT4 expression and inhibited insulin-stimulated glucose uptake. TLR4 inhibition

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attenuated inflammatory cytokine levels and increased glucose uptake by reversing GLUT4 levels.

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These data suggest that EGCG suppresses TLR4 signaling in LPS-stimulated adipocytes via 67LR

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and attenuates insulin-stimulated glucose uptake associated with decreased GLUT4 expression.

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Keywords: adipocyte; epigallocatechin gallate; inflammation; insulin resistance; Toll-like receptor 4

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Journal of Agricultural and Food Chemistry

Introduction

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Accumulating evidence has demonstrated the importance of adipose tissue inflammation in

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disease occurrence, including obesity, type 2 diabetes and metabolic syndromes1, 2. One common

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feature of chronic adipose inflammation is increased cytokine production1-3. Adipocytes are integral

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cellular components throughout the whole body, and they induce the innate immune response,

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produce pro-inflammatory adipokines, such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6)

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and monocyte chemoattractant protein-1, and promote macrophage recruitment3. Furthermore,

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preadipocytes have the potential to efficiently and rapidly convert into macrophages under

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inflammatory conditions4. Therefore, there is a clear basis for the inflammatory response in adipose

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tissue.

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Toll-like receptors (TLRs) likely play a crucial role in obesity-related inflammation

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. TLR4

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was the first member to be characterized, and it has been the most-studied TLR in adipocytes5-7.

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Much of the research has shown that TLR4 expression is significantly higher in the adipose tissue of

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obese individuals, perhaps due to increased macrophage infiltration8-10. High levels of circulating

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free fatty acid (FFA) and lipopolysaccharide (LPS) activate TLR4 signaling in macrophages and

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adipocytes to induce the inflammatory response11-12. There are two primary signaling pathways

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initiated by TLR4 activation-one is modulated by myeloid differentiation primary response protein

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88 (MyD88), while the other is modulated by toll/IL-1-receptor-domain adaptor molecule. Both

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pathways can activate nuclear factor-κB (NF-κB) signaling and promote inflammatory cytokine

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secretion, which is a known cause of insulin resistance8-10.

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Green tea is a popular beverage worldwide, and its consumption has been associated with several

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health benefits, including protection against multiple diseases, including cancer, atherosclerosis and 3

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cardiovascular disorders13, 14. Epigallocatechin gallate (EGCG) is the primary active component of

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green tea, and it exclusively binds to 67-kDa laminin receptor (67LR), which is widely expressed in

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many cell types, including cancer cells, hepatocytes, and preadipocytes15-17. EGCG possesses a

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variety of biological activities14,

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differentiation and avoids diet-induced obesity19. EGCG attenuates TLR4 signaling8 and reduces

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hyperglycemia by promoting glucose transporter isoform 4 (GLUT4) translocation in rodents 8,20 and

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attenuates TNF-α-promoted ROS generation and increased glucose uptake ability in 3T3-L1

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adipocytes21.

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. It reduces adipose tissue mass by inhibiting adipocyte

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To date, the underlying molecular mechanism of EGCG-mediated suppression of TLR4 signaling

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and whether it involves insulin signaling remains unknown. Here, we investigated the pathways

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involved in EGCG modulation of insulin signaling and TLR4 signaling in LPS-stimulated

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adipocytes.

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Materials and Methods

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Reagents— —DMEM, fetal calf serum (FCS), fetal bovine serum (FBS) and serum-free medium were

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all purchased from Gibco (Life Technologies, Carlsbad, CA). Anti-TLR4 and anti-GLUT4 were

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from Abcam Biochemicals (Hong Kong, China); anti-PI-3K and p-NF-κB were obtained from Cell

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Signaling Technology (Shanghai, China); anti-TNF-α and anti-β-actin were obtained from Santa

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Cruz Technology (Carlsbad, CA). FITC-AffiniPure Donkey Anti-Rabbit IgG was from Jackson

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ImmunoResearch (West Grove, PA). The monoclonal antibody against mouse TLR4/MD-2

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(Affymetrix) was obtained from Affymetrix eBioscience (Carlsbad, CA). Mouse IL-6 enzyme-linked

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immunosorbent assay (ELISA) kits was obtained from Dakewe (Dakewe Bio -engineering Co., LTD. 4

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Beijing, China). Insulin (from bovine pancreas), 3-Isobutyl-1-methyl -xanthine (BioUltra, ≥ 99%),

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Dexamethasone (purity≥ 97%), Lipopolysaccharides from Escherichia coli 055:B5, EGCG (purity ≥

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95%, from green tea) and routine chemicals were all purchased from Sigma Chemical

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(Sigma-Aldrich, St. Louis, MO).

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Cell Culture and Differentiation— —3T3-L1 fibroblasts were obtained from the American Type

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Culture Collection (Manassas, VA, USA) and cultured at a 37°C, 5% CO2 and 95% humidity.

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Preadipocytes were induced to differentiate as follows 22. Cells were maintained in DMEM plus 10%

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heat-inactivated FCS and 0.5% penicillin- streptomycin (Invitrogen). After two days in culture when

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the cells reached confluence (day 0), they were induced to differentiate by adding medium containing

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10% FBS, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methyl-xanthine, and 5 µg/mL insulin for 48h.

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Fresh media containing only 5 µg/mL insulin and 10% FBS was added for an additional 48 h. Media

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was subsequently changed every 48 h. More than 90% of cells expressed the adipocyte phenotype

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between day 8-10 post-differentiation, and they were used for experiments.

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Oil Red O Staining of 3T3-L1 Adipocytes— —Conversion of 3T3-Ll fibroblasts to adipocytes was

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monitored by measurement of intracellular lipid accumulation using Oil red O staining. A 0.5% (w/v)

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solution of Oil Red-O was prepared in 60% isopropanol. After day 8, differentiated 3T3-L1

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preadipocytes were washed twice with PBS, fixed in 4% paraformaldehyde and then incubated with

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Oil Red-O working solution for 2 h at room temperature and rinsed to remove unbound dye. Staining

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was visualized with an OLYMPUS DP70 camera (OLYMPUS, Japan). As shown in Fig.1,

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differentiated 3T3-L1 cells were positive for fat droplets and lipid storage by Oil Red O. We used the 5

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cells for further experiments when more than 90% expressed the adipocyte phenotype between 8-10

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days post- differentiation.

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LPS and EGCG Treatment of Differentiated 3T3-L1 Adipocytes—Fully differentiated adipocytes

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incubated in serum-free medium and stimulated by adding different concentrations of LPS from

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Escherichia coli for 48 h to elicit an immune response in the absence or presence of 3 h pretreatment

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with EGCG. Cells were divided into eight groups, including one control group (blank), while the

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remaining seven groups were treated with 0.1 µg/ml - 1 µg/ml LPS, or 1 µg/ml LPS+10 µM - 100

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µM EGCG. After treatment, supernatants were collected for ELISA, and the adipocytes were

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collected for detection of inflammation-related proteins (p-NF-κB, IKKβ and TNF-α) and key insulin

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signaling protein (PI-3K and GLUT4) levels.

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Anti-67LR Treatment— —To evaluate the effect of EGCG on TLR4 signaling, we performed

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blocking experiments in adipocytes. Fully differentiated adipocytes were pretreated with 67LR

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antibody35 (10 µg/ml, MLuC5) for 30 minutes, followed by EGCG (100 µM) for 3 h and subsequent

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incubation with LPS (1 ug/ml) for 48 h. IL-6 levels in supernatants of treated and control cells were

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detected by ELISA, and inflammatory mediators and cytokines, such as TLR4, p-NF-κB p65 and

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TNF-α and the key insulin signaling protein GLUT4 were examined by western blotting.

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Anti-TLR4 Treatment—Cells were treated with a TLR4-specific antibody (anti-mouse TLR4/MD-2

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complex functional grade purified antibodies, MTS510)

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differentiated adipocytes were pretreated with MTS510 (5 µg/ml) or PBS for 30 min at 37°C and

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subsequently treated with LPS (1 µg/mL) for 48 h. IL-6 levels in supernatants and intracellular

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to inhibit their response to LPS. Fully

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p-NF-κB p65, TNF-α, and GLUT4 protein levels were determined by western blot.

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Cellular 3H-2-deoxy-D-glucose Uptake Measurements—3T3-L1 cells were seeded in 24-well

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plates (1*105 cells/well) and induced to differentiate as described above. On day 9, adipocytes were

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incubated for 2 h at 37°C in serum-free medium, and cells were washed twice with 37°C Krebs

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Ringer phosphate (KRP) buffer (pH 7.4) and placed in KRP buffer containing insulin (100 nM) for

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30 min, followed by the addition of 3H-2-deoxy-D -glucose (2 µCi/ml, Beijing Yuan Zi Gao Ke

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Corporation, China) for an additional 10 min at 37°C. Cells were then immediately washed three

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times in ice-cold PBS to terminate the reaction. Lastly, cells were solubilized in 0.5 M NaOH (0.4

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ml/well) for 2 h and subjected to scintillation counting for 3H radioactivity as disintegrations per min.

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The radioactivity of each sample was normalized to the protein concentration. Counts per minute and

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per mg of protein were measured to analyze experimental glucose uptake.

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IL-6 Measurements—IL-6 levels in culture supernatants were measured in duplicate by ELISA

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(Dakewe Biotech) kit according to the manufacturer’s instructions. The minimum detectable level of

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each kit was 15.6 pg/ml, the sensitivity was 8 pg/ml, and the coefficient of variation between the

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plates< 10%.

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GLUT4 Immunofluorescence—3T3-L1 cells were grown and induced to differentiate on glass

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coverslips and incubated for 2 h in serum-free culture medium, then stimulated with insulin (100 nM)

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before EGCG or LPS treatment. Cells were rinsed with PBS, fixed in 4% paraformaldehyde and

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0.05% glutaraldehyde for 30 min at room temperature and then blocked in bovine serum albumin and 7

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permeabilized with 0.3% Triton X-100 for 1 h, then incubated with GLUT4 antibody (5 µg/ml)

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overnight. Primary antibodies were detected with fluorescent secondary antibodies (FITC-AffiniPure

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Donkey Anti-Rabbit IgG, 1:200) and imaged with a Leica TCS SP5 X camera (Germany).

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Immunoblotting Analyses—Mature 3T3-L1 adipocytes were washed three times in PBS and

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lysed (KGP 250 kit, Keygen Biotech, Nanjing, China) on ice. All steps were carried out according to

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the manufacturers’ instructions. Bound antibodies were detected with horseradish peroxidase-

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conjugated anti-IgG with an enhanced chemiluminescence kit (Pierce) protocol and visualized with

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the Chem Doc XRS with Quantity One software (Bio-Rad). Blots were repeated at least six times.

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The band intensities were quantified using Image J analysis software (National Institutes of Health,

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USA).

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Statistical Analyses—All values were expressed as mean ± SD. Differences between groups

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were tested for statistical significance using one-way analysis of variance (ANOVA), and when the

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F-value indicated significance, least significant difference (LSD) was used to correct for multiple

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comparisons. All analyses were performed using SPSS statistics software, version 17.0 (SPSS Inc.,

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Chicago, IL, USA) on a computer with a Windows operating system. A p value less than 0.05 was

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considered statistically significant.

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Results

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EGCG decreases inflammation-related protein levels and increases insulin signaling protein

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expression in adipocytes—We treated mature 3T3-L1 adipocytes with various doses of LPS (0, 0.1 8

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µg/ml, 0.5 µg/ml, 1 µg/ml) for 48 h to induce inflammation. Compared to the control group, LPS

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treatment increased inflammation-related protein levels to different degrees. IKKβ (approximately

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1.1-fold, 1.6-fold and 1.7-fold, respectively), p-NF-κB (approximately 3.9-fold, 5.0-fold and 5.8-fold,

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respectively) and TNF-α (approximately 1.5-fold, 2.0-fold and 2.5-fold, respectively) protein levels

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significantly increased (p<0.05 vs. baseline), while the expression of insulin signaling proteins, such

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as PI-3K (approximately 89.9%, 64.9% and 40.5%, respectively,) and GLUT4 (approximately 84.7%,

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73.7% and 29.6%, respectively) decreased (p<0.05, Fig. 2).

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Various doses of EGCG (10 µM, 30 µM, 50 µM, 100 µM) decreased LPS-induced IKKβ,

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p-NF-κB and TNF-α expression. EGCG reduced IKKβ (approximately 64.5%, 66.8%, 31.9% and

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10.4%, respectively), p-NF-κB (approximately 89.5%, 85.8%, 67.5% and 41.2%, respectively), and

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TNF-α (approximately 71.6%, 45.7%, 21.9% and 14.0%, respectively) expression, and increased

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PI-3K (approximately by 1.4-fold, 2.0-fold, 2.0-fold and 1.8-fold, respectively) and GLUT4

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(approximately by 1.9-fold, 2.1-fold, 2.9-fold and 3.1-fold respectively) levels, compared to

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treatment with 1 µg/ml LPS alone (Fig. 2, p<0.05 for each). These data suggest that EGCG decreases

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LPS-stimulated inflammatory cytokine and insulin signaling protein expression in adipocytes.

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EGCG reverses intracellular GLUT4 expression in LPS-stimulated adipocytes—We used

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immunofluorescence labeling to investigate cellular GLUT4 expression. GLUT4 fluorescence

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intensity demonstrated that LPS treatment decreased GLUT4 levels. As shown in Fig. 2 and 3A,

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various doses of LPS (0.1 µg/ml, 0.5 µg/ml and 1 µg/ml) dramatically decreased the GLUT4 signal.

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In contrast, EGCG treatment (100 µM) increased GLUT4 expression compared to LPS (1 µg/ml) 9

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treatment alone (Fig. 2 and 3B).

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67LR antibody blocks EGCG’s effect on LPS-induced TLR4 signaling—Upon treatment with the

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67LR antibody (10 µg/ml), TLR4 protein levels increased by more than 1.2-fold, compared to the

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EGCG group (Fig. 4B, p<0.05). As expected, NF-κB p65 phosphorylation significantly increased

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(1.4-fold, Fig. 4C, p<0.05), as did the downstream protein TNF-α (1.6-fold, Fig.4D, p<0.05). The

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level of IL-6 increased in the supernatant upon 67LR antibody treatment compared to the EGCG

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group (1.8-fold, Fig. 6A, p<0.05), and there was no difference between the +/-EGCG treatment

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groups in the presence of the 67LR antibody (p>0.05). Furthermore, GLUT4 levels were reduced by

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approximately 72.4% in the 67LR antibody group, compared to the EGCG group (Fig. 4E, p<0.05).

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These data suggest that 67LR antibody blocks EGCG’s effect on LPS-induced TLR4 signaling,

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resulting in a subsequent series of changes, such as increased inflammatory cytokine expression and

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decreased GLUT4 levels. However, we did not detect any obvious differences in 67LR expression

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among the control (blank), EGCG (100 µM EGCG plus 1 µg/ml LPS) and LPS groups (1 µg/ml LPS)

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(Fig. 4F, p>0.05). We hypothesize that EGCG performs its anti-inflammatory role by regulating

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67LR activity or translocation.

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TLR4 mediates LPS-induced inflammatory activation and insulin resistance—As shown in Fig 5,

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the specific TLR4/MD2-inhibitor MTS510 (5 µg/ml) treatment significantly reduced p-NF-κB

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(67.9% decrease, p<0.05, Fig. 5B), TNF-α (21.5% decrease, p<0.05, Fig. 5C) and IL-6 (45.5%

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decrease, p<0.05, Fig. 6B) levels after LPS stimulation, though IL-6 levels were still higher than the

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blank group, perhaps due to the effects of LPS. Meanwhile, MTS510 treatment increased GLUT4 10

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expression by 1.3-fold (p<0.05, Fig. 5D) compared to LPS-induced control cells. The above data

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suggest that TLR4/MD2 inhibition decreased NF-κB activation and upregulated GLUT4 expression,

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and EGCG had similar effects to the anti-TLR4 antibody.

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EGCG upregulates cellular 3H-2-deoxy-D-glucose uptake by decreasing TLR4 expression — To

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determine the effects of TLR4 on glucose transport in inflammation, cellular 3H-2-deoxy-D-glucose

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uptake contents were measured (Fig. 7). We found that insulin-stimulated glucose influxes were

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significantly greater than untreated control cells (p<0.05). Pretreatment with EGCG (100 µM) for 3 h

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before 48 h LPS exposure (1 µg/ml) significantly increased glucose uptake compared to LPS stimulation

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alone (~1.2-fold, p<0.05), while anti-67LR Ab reversed the trend (about 84.4% of EGCG group, p<0.05).

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Anti-TLR4 Ab increased glucose uptake even in the absence of EGCG (about 1.2-fold higher than the

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LPS group, p<0.05). Our data suggest that TLR4 attenuates insulin-stimulated glucose uptake, and

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EGCG blocks these changes by decreasing TLR4 signaling.

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Discussion

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Here, we showed that EGCG suppresses the TLR4 signaling pathway in adipocytes via 67LR.

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Upon 67LR inhibition, EGCG’s suppressive effect on TLR4 activity was blocked, inflammatory

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cytokine levels such as TNF-α and IL-6 increased, and GLUT4 expression decreased. These effects

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led to attenuated insulin signal transduction and decreased glucose uptake. In contrast, TLR4

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inhibition inactivated the inflammation pathway, phenocopying EGCG’s effect and increasing

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expression of the key insulin signaling protein GLUT4 in LPS-stimulated adipocytes, thus promoting

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insulin-stimulated glucose uptake. These results suggest that EGCG improves insulin signaling by

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suppressing TLR4 activity in adipocytes. 11

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Type 2 diabetes and obesity are characterized by low-grade inflammation with increased

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inflammatory cytokine levels and changes in the gut microbiota23. As an important pathogen

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recognition receptor, high TLR4 levels impair insulin action and negatively regulate insulin

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signaling36, 37. Increased TLR4 levels underlie non-infectious chronic inflammation and dysfunction

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of adipose tissue in obesity5, 24, 25. In our previous study, we found that a high-fat diet increases the

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TLR4-mediated inflammatory response in the adipose tissue of obese rats8. TLR4-deficient mice are

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partially protected from inflammation and insulin resistance26, and inhibition of TLR4 signaling

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protects against type 2 diabetes in mice27, 38, 39. Studies have shown that the TLR4 ligand LPS

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activates NF-κB and increases inflammatory bio-mediator release in 3T3-L1 adipocytes, a process

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which may be blocked by a TLR4 inhibitor12. Importantly, these bio-mediators also work on insulin

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target cells and insulin-producing cells, such as adipocytes, pancreatic islet cells and hypothalamus,

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further impairing insulin sensitivity and secretion25, 28-30. Consistently, our work suggests that LPS

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activates the TLR4 signaling pathway, and a specific TLR4/MD2 inhibitor partially weakens

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inflammatory cytokines, such as TNF-α and IL-6, and up-regulates insulin signaling protein GLUT4

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expression, and TLR4 inhibition has similar effects to EGCG treatment in 3T3-L1 adipocytes.

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EGCG is the most abundant polyphenol in tea and has many biological properties13-15, 17-19. Its

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metabolites can exist in methylated, glucuronide and sulfate forms, and the phenolic hydroxyls in

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their structure determine their anti-bacterial, anti-cardiovascular disease and anti-tumor activities31.

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Studies have shown that EGCG exclusively binds to 67LR and influences downstream insulin

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signaling17, 32, 33, and treatment with 67LR antiserum blocks extracellular signal regulated kinase 1/2

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(ERK1/2) phosphorylation while increasing insulin receptor-beta and insulin receptor substrates 1

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and 2 (IRS1 and IRS2) phosphorylation in preadipocytes`17,

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. Our work demonstrated that

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pretreatment of adipocytes with a 67LR blocking antibody blocked EGCG’s suppression of the

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TLR4 signaling pathway, activated the NF-κB signal pathway, induced inflammatory cytokine

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transcription, and decreased GLUT4 expression, leading to a further decrease in cellular

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H-2-deoxy-D-glucose uptake, which is associated with insulin signaling transduction.

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Our work demonstrated that 67LR expression did not change upon EGCG treatment. It has yet

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to be determined whether 67LR membrane localization changes upon EGCG treatment. However,

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previous research33 stated that EGCG increases 67LR efficiency by inducing its translocation from

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the cytoplasm to the membrane. EGCG down-regulation of inflammatory signals may be induced by

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Tollip, which is associated with decreased TLR signaling-mediated NF-κB activation34, 35. Therefore,

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it is crucial to further delineate the mechanisms involved to support our findings.

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Additionally, TLRs and nucleotide-binding and oligomerization domain (NOD)-like receptors

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(NLRs) are very remarkable pathogen recognition receptors in obesity and type 2 diabetes

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characterized by low-grade inflammation23, 40, 41. Both TLRs and NLRs mediate NF-κB activation,

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leading to inflammatory cytokine production and insulin resistance40, 41, 42. Therefore, we speculate

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that EGCG may simultaneously decrease inflammation through the NLR signaling pathway.

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Therefore, further studies are necessary to target TLR- or NLR- regulated pathways in obesity related

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metabolic complications.

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Taken together, these data suggest that EGCG affects the TLR4 signaling pathway, reduces

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chronic low-grade inflammation, and further influences insulin signaling protein expression, while

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the entire process is blunted by 67LR inhibition, which further strengthens the link between the

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TLR4-induced inflammatory response and insulin resistance in adipocytes. Our work also provides

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insight into better prevention of insulin resistance by revealing the possible molecular mechanism 13

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and developing targeted therapies to protect against them.

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Abbreviations Used EGCG, Epigallocatechin Gallate; TLR4, Toll-like receptor 4; LPS, Lipopolysaccharide; GLUT4,

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Glucose transporter type 4; 67LR, 67 laminin receptor; FFA, free fatty acid

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Acknowledgments

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*This work was supported by grants from the National Natural Science Foundation of China

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(Grant No. 81000327), the Chinese Society of Endocrinology (No.12030470347) and the General

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Scientific Research Fund of Liaoning Provincial Education Department (L2013300).

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Figure captions

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Figure 1. 3T3-L1 cell differentiation detected by Oil-Red O staining. Top panels show

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pre-Oil-Red-O staining (Fig 1A and 1B), bottom panels show after-Oil-Red O staining (Fig 1C and

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1D). Left panels represent undifferentiated cells, and right panels represent differentiated cells

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(Magnification = 100X, Bar: 200µm).

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Figure 2. EGCG decreases inflammation-related protein levels and increases the expression of key

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insulin signaling proteins in adipocytes. Various doses of LPS (0.1 µg/ml, 0.5 µg/ml, 1 µg/ml)

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dose-dependently stimulated inflammatory cytokine expression in mature 3T3-L1 adipocytes.

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Mature 3T3-L1 adipocytes pretreated with EGCG (10 µM, 30 µM, 50 µM, 100 µM) decreased

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LPS-stimulated inflammatory related factor expression (IKKβ, p-NF-κB and TNF-α) in a

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dose-dependent manner. EGCG (10 µM, 30 µM, 50 µM, 100 µM) increased key insulin signaling

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protein expression (PI-3K, GLUT4). Results are expressed as mean±SD (n=6), *p<0.05 vs. (0 ng/ml

408

LPS + 0 µM EGCG) group; #p<0.05 vs. (1 µg/ml LPS) group.

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Figure 3. EGCG reversed intracellular GLUT4 expression in LPS-stimulated adipocytes. GLUT4

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(FITC, green) and nuclear (DAPI, blue) immunofluorescence. Fig 3A-a. control group; 3A-b. 0.1

411

µg/ml LPS group; 3A-c. 0.5 µg/ml LPS group; 3A-d. 1 µg/ml LPS group; 3B-a. control group. 3B-b.

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1 µg/ml LPS group; 3B-c. (100 µM EGCG + 1 µg/ml LPS) group. (Magnification = 400X).

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Figure 4. 67LR antibody blocks EGCG’s effects on TLR4 signaling and GLUT4 expression in

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LPS-stimulated adipocytes. 4f: EGCG did not affect 67LR expression between the control (blank

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group), EGCG (100 µM EGCG plus 1 µg/ml LPS) and LPS groups (1 µg/ml LPS). 4a-4f: Protein

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levels determined by Western blot. Bars represent mean ± SD, and both are expressed as the ratio to

417

control group (The level of control group was normalized to 1). *: p< 0.05, LPS group vs. Control 20

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group; #: p< 0.05, 67LR blocker group vs. EGCG group; n.s.: not significant when the EGCG group

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was compared to the LPS or control group.

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Figure 5. TLR4 mediates LPS-induced inflammation activation and insulin resistance, and EGCG

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has similar effects to the anti-TLR4 antibody. a-d: protein levels determined by Western blot. Bars

422

represent mean ± SD, expressed as the ratio to the control group (The level of control group was

423

normalized to 1, n = 6 for each group). *: p< 0.05, LPS group vs. Control group; #: p< 0.05, LPS

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group vs. EGCG group & TLR4 inhibited group.

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Figure 6. EGCG and the anti-TLR4 antibody inhibit IL-6 production and an inhibitor of 67LR

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blocks EGCG effects. IL-6 levels in culture supernatants measured by ELISA. 6A-6B: IL-6 levels in

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cell culture supernatants were measured in duplicate. *: p< 0.05, n.s.: not significant between the two

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groups. Figure 6A showed that EGCG inhibits IL-6 production in the context of LPS, and EGCG

429

does not work effectively that with an inhibitor of 67LR.

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was blocked with the anti-TLR4 antibody, and it has similar effects to the EGCG.

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Figure 7. Cellular 3H-2-deoxy-D-glucose uptake. *: p< 0.05.

Figure 6B showed that IL-6 production

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