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