Quercetin attenuates inflammation in human macrophages ... - Nature

International Journal of Obesity (2011) 35, 1165–1172 & 2011 Macmillan Publishers Limited All rights reserved 0307-0565/11 www.nature.com/ijo

ORIGINAL ARTICLE Quercetin attenuates inflammation in human macrophages and adipocytes exposed to macrophageconditioned media A Overman, C-C Chuang and M McIntosh Department of Nutrition, University of North Carolina-Greensboro, Greensboro, NC, USA Background: Obesity is linked to chronic inflammation in white adipose tissue, which is exacerbated by infiltrating macrophages (MFs). We recently demonstrated that an extract from grape powder (GPE), which is abundant in quercetin (QUE), reduced inflammation in human MFs and prevented MF-mediated inflammation and insulin resistance in human adipocytes. However, we did not know how QUE individually affected these outcomes. Objective and design: We examined the extent to which QUE prevents inflammation in human MFs (that is, differentiated U937 cell line) and cross-talk with human adipocytes (that is, primary cultures of newly differentiated human adipocytes). Methods and results: Treatment of MFs with QUE attenuated the basal expression of inflammatory genes, such as tumor necrosis factor-a, interleukin (IL)-6, IL-8, IL-1b and interferon-g inducible protein-10, and cyclooxygenase-2, a marker of prostaglandin production. QUE also attenuated the abundance of phosphorylated c-Jun N-terminal kinase (JNK) and c-Jun, and IkBa degradation in MFs. Furthermore, conditioned media (CM) obtained from MFs treated with QUE decreased the capacity of this CM to inflame adipocytes and cause insulin resistance as evidenced by decreased: (1) inflammatory gene expression, (2) phosphorylation of JNK and c-Jun, (3) serine residue 307 phosphorylation of insulin receptor substrate (IRS)-1, 4) protein tyrosine phosphatase-1B gene expression and 5) suppression of insulin-stimulated glucose uptake. Conclusion: Taken together, these data suggest that QUE is one of the bioactive components of GPE that prevents inflammation in MFs and MF-mediated insulin resistance in adipocytes. International Journal of Obesity (2011) 35, 1165–1172; doi:10.1038/ijo.2010.272; published online 11 January 2011 Keywords: macrophages; adipocytes; quercetin; inflammation; insulin resistance

Introduction Obesity is a major health concern that is increasing worldwide.1,2 Obesity is characterized by low-grade, chronic inflammation that is linked to the metabolic syndrome (that is, atherosclerosis, type 2 diabetes, hypertension). One consistent feature of this chronic inflammatory state is macrophage (MF) infiltration into white adipose tissue (WAT).3,4 Activated MFs secrete an array of proinflammatory mediators, which contribute to the pathogenesis of these obesity-related diseases.5 For example, many obese individuals have increased levels of inflammatory markers, such as tumor necrosis factor (TNF)-a, interleukin (IL)-6 and

Correspondence: Dr MK McIntosh, Department of Nutrition, 318 Stone Building, PO Box 26170, University of North Carolina Greensboro, Greensboro, NC 27402-6170, USA. E-mail: [email protected] Received 10 September 2010; revised 17 November 2010; accepted 21 November 2010; published online 11 January 2011

monocyte chemoattractant protein-1 (see references 6, 7). Notably, several studies reported that MFs are the primary source for proinflammatory cytokine production in WAT and may be recruited to WAT by monocyte chemoattractant protein-1 (see references 8–12). In addition, numerous animal studies have shown the importance of MFs in inflammation-induced insulin resistance.11,13–15 Bioactive food components found in fruits and vegetables have the potential to prevent the obesity-related inflammation and insulin-resistance. Grapes are one of the most widely consumed fruits in the world.16 They contain high concentrations of polyphenols, which have been reported to have anti-inflammatory and anti-oxidant properties. For example, grape seed procyanidin modulated the inflammatory response in endotoxin-stimulated RAW264 MFs by inhibiting nuclear factor kappa B (NFkB).17 In addition, oligomerized grape seed polyphenols reduced NFkB transcriptional activity and activation of extracellular signalrelated kinase (ERK), a mitogen-activated protein kinase (MAPK), in a coculture of murine adipocytes and MFs.16

Quercetin attenuates inflammation in human macrophages and adipocytes A Overman et al

1166 Consistent with these data, we recently found that a grape powder extract (GPE) made from table grapes from the California Table Grape Commission attenuated inflammatory signaling, such as MAPKs and transcription factors NFkB and activator protein (AP)-1 in human MFs, and cross-talk with adipocytes.18 Furthermore, we found that this GPE had relatively high levels of quercetin (QUE). However, the ability of QUE alone to prevent inflammation in human MFs, and block inflammation and insulin resistance in human adipocytes treated with MF-conditioned media (CM) is unknown. Furthermore, the potential anti-inflammatory mechanisms of action of QUE are unknown. We hypothesized, on the basis of these data, that QUE would attenuate the activation of inflammatory MAPKs, AP-1 and NFkB, and their subsequent induction of inflammatory genes in human MFs. Furthermore, we postulated that QUE pretreatment of MFs would decrease inflammation and insulin resistance in primary cultures of human adipocytes incubated with MF-CM.

Materials and methods Materials All cell culture-ware were purchased from Fisher Scientific (Norcross, GA, USA). Fetal bovine serum was purchased from Hyclone (Logan, UT, USA). RPMI-1640 was purchased from ATCC (Manassas,VA, USA). Tri-Reagent was purchased from Molecular Research Center (Cincinnati, OH, USA). Gene-specific primers were purchased from Applied Biosystems (Foster City, CA, USA). A polyclonal antibody for anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho (Thr183/Tyr185) and total c-jun NH2terminal kinase (JNK), anti-phospho (Ser63) and total c-Jun antibodies, and anti-phospho (Ser307) and total insulin receptor substrate (IRS)-1 were purchased from Cell Signaling Technologies (Beverly, MA, USA). Immunoblotting buffers and precast gels were purchased from Invitrogen (Carlsbad, CA, USA). Western Lightning chemiluminescence substrate was purchased from Perkin Elmer Life Sciences (Boston, MA, USA). All other reagents and chemicals were purchased from Sigma-Aldrich unless otherwise stated.

Culturing of human MFs Human U937 monocytes were purchased from ATCC. The U937 cell line was originally derived from a patient with diffuse histiocytic lymphoma.19 These cells can be stimulated to differentiate using 12-otetradecanoylphorbol 13-acetate (PMA), which rapidly activates protein kinase C, initiating a cascade of differentiation signals. Once differentiated, U937 cells develop the characteristics of mature MFs, including irregular shape, lobed nuclei, and an intense phagocytic activity. In addition, differentiated U937 cells adhere to each other and to a material surface, International Journal of Obesity

a characteristic feature of mature MFs. In vivo, mature MFs adhere to tissues and secrete inflammatory mediators that contribute to obesity-associated inflammatory disease. Cells were seeded in 35-mm dishes at 0.75 106 cells per 35 mm or 1.25 106 cells per 60 mm dish and differentiated with 30 mg l1 phorbol 12-myristate (PMA) for 24 h in RPMI1640 (containing 10% fetal bovine serum, 60 U ml1 penicillin, 60 U l1 streptomycin and 25 mg ml1 amphotericin B). Media was then changed to PMA-free RPMI and 24 h later the experiments were initiated with the MF monolayers. Cultures were incubated at 37 1C in a humidified O2:CO2 (95:5%) atmosphere.

Culturing of human primary adipocytes Abdominal WAT was obtained from non-diabetic Caucasian and African American females, between the ages of 20–50 years old with a body mass index o32.0 following abdominoplasty. Approval was obtained from the Institutional Review Board at the University of North Carolina at Greensboro and the Moses Cone Memorial Hospital in Greensboro, NC, USA. Tissue was digested using collagenase and stromal vascular cells were isolated, proliferated and induced to differentiate in adipocyte medium-1 plus 250 mM isobutylmethylxanthine and 1 mM thiazolidinediones rosiglitazone (BRL 49653; a gift from Dr Per Sauerberg, Novo Nordisk A/S, Copenhagen, Denmark) for 3 days.20 Mf-free cultures containing B50% preadipocytes and B50% adipocytes, based on visual observations and previous analyses,21 were treated between day 6–12 of differentiation. Each experiment was repeated at least twice at different times using a mixture of cells from 2–3 subjects unless otherwise indicated.

RNA isolation and real-time quantitative PCR (qPCR) Following treatment, cells were harvested and total RNA was isolated using Tri-Reagent according to the manufacturer’s protocol. For real-time qPCR, 2.0 mg total RNA was converted into first strand complementary DNA using Applied Biosystems High-Capacity cDNA Archive Kit. The qPCR was performed in an Applied Biosystems 7500 FAST Real-Time PCR System using Taqman Gene Expression Assays. To account for possible variation related to complementary DNA input or the presence of PCR inhibitors, the endogenous reference gene, GAPDH, was simultaneously quantified for each sample, and data were normalized accordingly.

Immunoblotting Immunoblotting was conducted as previously described.20 Briefly, total cellular protein was harvested using phosphatebuffered saline (pH 7.5) lysis buffer containing 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, 30 mgml1 aprotinin, 1 mM phenylmethylsulfonyl fluoride and 1 mM sodium orthovanadate. The samples were incubated on ice with frequent vortexing and centrifuged for 20 min at 15 000 g,


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2-[3H]deoxy-glucose (2-DOG) uptake Primary human adipocytes were incubated with low glucose (5 mM) and insulin (20 pM) containing media for 24 h. Cultures were then treated with CM from MF treated with 0, 3, 10, or 30 mM QUE for 24 h. The 24-h incubation time was chosen on the basis of the results of a pilot time-course study (data not shown). Basal and insulin-stimulated 2-DOG were measured as described previously.20

Statistical analysis Statistical analyses were performed using a one-way analysis of variance (JMP Version 6.03, SAS Institute, Cary, NC, USA). Student’s t-tests were used to compute individual pairwise comparisons of least square means, Po0.05. Data are expressed as means±s.e.m.

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MF-CM experiments in primary human adipocytes CM was collected from differentiated U937 cultures treated with 0, 0.3, 3, 10, or 30 mM QUE for 5 h. The 5-h incubation time was chosen based on the results of a pilot time-course study (data not shown). MF-CM obtained from each experiment was pooled and stored at 80 1C until used. Distinct pools were used for each experiment. For RNA isolation and qPCR experiments, primary human adipocytes were seeded in 35-mm dishes at 0.5 106 per dish and allowed to differentiate for 6 days. On day 6, media was changed and cells were incubated in 1 ml of adipocyte medium-1. After 24 h, the following were added to the cultures: (1) fresh adipocyte medium-1, (2) fresh RPMI, (3) MF-CM, or (4) MF-CM treated with QUE. The amount and duration of MF-CM treatment varied depending on the outcome measured.

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and stored at 80 1C. Protein concentration was determined using the BCA assay (Thermo Scientific, Rockford, IL, USA). Total cellular protein, 20 mg, was separated by electrophoresis on 4–12% SDS-polyacrylamide gradient gels (NuPAGE minigel system; Invitrogen), transferred to a polyvinylidene difluoride membrane using a wet transfer module (TransBlot Module; Bio-Rad Inc., Hercules, CA, USA), and prepared for immunodetection. Following primary and secondary antibody exposure, each protein was detected using Western Lightning (Perkin Elmer Life Sciences) chemiluminescence substrate. Chemiluminescence was visualized following exposure of the membrane to X-ray film (X-OMAT; Eastman Kodak Co., Rochester, NY, USA).

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Figure 1 Quercetin (QUE) decreases inflammatory gene expression in human MFs. MFs were treated with 0, 3, 10, or 30 mM QUE for 5 h. The mRNA levels were measured by quantitative PCR (qPCR). Data are representative of three independent experiments. Means (±s.e.m.; n ¼ 3) without a common letter (a–c) differ (Po0.05).

or 30 mM) and the expression of several inflammatory genes was measured by qPCR. The 5-h incubation time was chosen on the basis of the results of a pilot time-course study (data not shown). QUE decreased the mRNA levels IL-6, IL-8, interferon-g inducible protein-10, IL-1b, TNF-a and cyclooxygenase-2 (Figure 1). No visual signs of QUE cytotoxicity were observed (for example, no floating cells, no visual differences in the number of adherent cells or protein concentrations, no abnormal changes in cell morphology).

QUE increases peroxisome proliferator activated receptor (PPAR)g and ABCA1 gene expression in human MFs Given the important role MFs have in lipid metabolism and inflammation, we examined the impact of QUE on the expression of PPARg and ABCA1, two regulators involved in lipid metabolism and inflammation.22 MFs were treated for 5 h with QUE (0, 0.3, 3, 10, or 30 mM) and the expression of PPARg and ABCA1 was measured by qPCR. Whereas 0.3 mM QUE increased PPARg and ABCA1 gene expression, the expression levels of these lipogenic genes treated with 3, 10 and 30 mM QUE were similar to the controls (Figure 2).

Results QUE decreases inflammatory gene expression in human MFs To determine the extent to which QUE attenuated markers of inflammation, MFs were treated for 5 h with QUE (0, 3, 10,

QUE decreases the degradation of IkBa and the activation of c-Jun and JNK in human MFs Given the important roles of MAPKs, AP-1 and NFkB in inducing the transcription of inflammatory genes, we International Journal of Obesity


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QUE (M) Figure 4 QUE treatment of MFs decreases MF-CM-mediated inflammatory

Figure 3 QUE decreases the degradation of IkBa and the activation of c-Jun and JNK human MFs. MFs were treated with 0, 3, 10, or 30 mM QUE for 2.5 h. Proteins were immunoblotted and probed with antibodies for IkBa, GAPDH, and phospho-(P) or non-P specific c-Jun and JNK. Data are representative at least three independent experiments.

examined the effects of QUE on IkBa degradation, and JNK and c-Jun activation in MFs treated for 2.5 h with 0, 3, 10, or 30 mM QUE. QUE attenuated the degradation of IkBa and the phosphorylation of c-Jun and JNK in a dose-dependent manner (Figure 3). In contrast, QUE had no effect on the phosphorylation status of ERK or p38, (data not shown).

QUE treatment of MFs decreases MF-CM-mediated inflammation and insulin resistance in human adipocytes Several studies have reported cross-talk between murine MFs and adipocytes; that is, activated MFs can inflame adipocytes and vice-versa.18,23 Therefore, we hypothesized that QUE treatment of MFs would prevent MF-CM-mediated International Journal of Obesity

gene expression in human adipocytes. MFs were treated with 0, 3, 10, or 30 mM QUE for 5 h. MF-CM was collected and used to treat adipocytes for 3 h at a 1:1 ratio in adipocyte medium-1 (AM-1). Adipocytes were treated with MF-CM for 3 h and then harvested for qPCR analysis. Means (±s.e.m.; n ¼ 3) without a common letter (a–e) differ (Po0.05). Data are representative of at least three independent experiments. NT, adipocytes treated with 1:1 ratio of RPMI and AM-1.

inflammation and insulin resistance in human adipocytes. Consistent with our hypothesis, QUE decreased MF-mediated induction of inflammatory gene expression (Figure 4) and the phosphorylation of c-Jun and JNK (Figure 5) in human adipocytes. In contrast, QUE did not attenuate MF-mediated phosphorylation of ERK or IkBa degradation (data not shown). Lastly, QUE decreased MF-mediated phosphorylation of serine residue 307 on IRS-1 (Ser307-IRS-1) (Figure 6a) and protein tyrosine phosphatase-1B gene expression (Figure 6b), which are negative regulators of insulin sensitivity, and increased insulin-stimulated 2-DOG uptake (Figure 6c) in human adipocytes.


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lation of JNK and c-Jun in human adipocytes. MFs were treated with 0, 0.3, 3, 10, or 30 mM QUE for 5 h. MF-CM was collected and used to treat adipocytes for 30 min at a 1:1 ratio in AM-1. Proteins were immunoblotted and probed with antibodies for GAPDH and phospho (P) or non-P specific JNK and c-Jun. Data are representative of three independent experiments.

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In this study, we showed that 3–30 mM QUE, a tetrahydroxyflavonol found in grapes and grape products like GPE,18 attenuated basal inflammatory gene expression (that is, IL-6, IL-8, IL-1b, interferon-g inducible protein-10, TNF-a and cyclooxygenase-2) (Figure 1) and increased PPARg and ABCA1 gene expression (Figure 2), two key players involved in MF lipid metabolism.22 QUE also attenuated the basal activation of NFkB, c-Jun and JNK (Figure 3). Moreover, we demonstrated that QUE decreased: (1) induction of inflammatory genes (that is, interferon-g inducible protein-10, monocyte chemoattractant protein-1 and TNF-a) (Figure 4), (2) activation of JNK and c-Jun (Figure 5), (3) phosphorylation of Ser307-IRS-1 (Figure 6a), (4) induction of protein tyrosine phosphatase-1B gene expression (Figure 6b) and (5) suppression of insulin-stimulated 2-DOG uptake (Figure 6c) in human adipocytes treated with MF-CM. Taken together, these findings demonstrate that QUE inhibits the basal activation of inflammatory transcription factors and MAPKs that induce inflammatory gene expression in cultures of human MFs. On the basis of these data, we speculate that QUE directly attenuates the basal activation of NFkB, c-Jun and JNK in MFs, thereby preventing the induction and secretion of inflammatory cytokines and chemokines MFs and their cross-talk with adipocytes. However, it should be noted that the effects of QUE were lost at higher doses with regards to PPARg and ABCA1 gene expression in MFs. This U-shaped dose response suggests that lower doses of QUE may be more effective than higher doses with regards to improving lipid metabolism in MFs. Consistent with our data, QUE has been reported to have anti-inflammatory properties in vivo and in vitro. For example, QUE reduced systolic blood pressure and plasma

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Figure 6 QUE treatment of MFs decreases MF-CM-mediated insulin resistance in human adipocytes. MFs were treated with 0, 0.3, 3, 10, or 30 mM QUE for 5 h. MF-CM was collected and used to treat adipocytes at a 1:1 ratio in AM-1 for 30 min to determine the protein level of serine residue 307 phosphorylation of insulin receptor substrate (IRS)-1 (p-Ser307-IRS-1) by immunoblotting (a), for 3 h to determine the mRNA level of protein tyrosine phosphatase (PTP)-1B by qPCR (b), or for 24 h to determine insulin-stimulated 2-[3H]deoxy-glucose (2-DOG) uptake (c). Data are representative of two (a) or three (b, c) independent experiments. (b, c) Means (±s.e.m.; n ¼ 3–4) without a common letter (a–d) differ (Po0.05).

oxidized low density lipoprotein concentrations in overweight subjects with a high cardiovascular disease risk phenotype.24 QUE supplementation of high-fat fed mice25 or obese Zucker rats26 reduced circulating markers of inflammation. QUE also attenuated atherosclerotic lesion size, decreased markers of inflammation, improved nitric oxide bioavailability, and increased heme oxygenase-1 protein expression in ApoE gene knockout mice.27 In vitro, QUE reduced the levels of inflammatory markers in lipopolysaccharide (LPS)-treated U937-derived MFs28 and suppressed the degradation of IkBa and the phosphorylation International Journal of Obesity


1170 of p38 and Akt in LPS-stimulated bone marrow-derived MFs.29 Similarly, QUE attenuated differentiation and markers of inflammation in murine 3T3-L1 adipocytes30 and suppressed tissue plasminogen activator-mediated activation of MEK/ERK, AP-1 and NFkB in murine skin epidermal (JB6 P þ ) cells.31 Also, QUE impaired inflammatory cytokine and chemokine production and decreased activation of ERK, JNK, Akt and NFkB in LPS-stimulated dendritic cell.32 In addition, a semi-synthetic acetyl QUE derivative inhibited LPS-induced nitric oxide production and iNOS expression in J774A.1 MFs.33 Consistent with these data, luteolin, a tetrahydroxyflavone found in celery and green peppers, reduced JNK phosphorylation and AP-1 activation in microganglia34 and NFkB, and AP-1 activation in alveolar MFs.35 Thus, QUE has the capacity to reduce markers of inflammation in vivo and in vitro, possibly by attenuating inflammatory MAPKs, such as ERK and JNK and transcription factors, such as NFkB and AP-1 that induce inflammatory gene expression and also the expression of protein tyrosine phosphatase-1B, a negative regulator of insulin signaling that dephosphorylates tyrosine residues on IRS-1.36,37 In obesity, the increased production of these inflammatory mediators originates mainly from infiltrating MFs in WAT. MF-secreted factors have been shown to increase inflammation and decrease insulin-stimulated glucose uptake in adipocytes.13,38–40 Consistent with these findings, we previously reported that LPS increased the activation of MAPK, NFkB and AP-1 in human MFs, increasing their capacity to cause inflammation and insulin resistance in primary human adipocytes.18 Pretreatment of human MFs with GPE, which is rich in QUE, attenuated inflammation in MFs and adipocytes, and MF-mediated insulin resistance in adipocytes.18 Mechanisms by which polyphenols like QUE have been reported to reduce inflammation include: (1) serving as an antioxidant or inducing the expression of antioxidant genes, (2) interfering with inflammatory signaling pathways, (3) blocking inflammatory gene expression by preventing histone acetylation, or (4) increasing the activation of transcription factors that antagonize NFkB or AP-1 (see reference 41). Concerning this last mechanism, QUE may block inflammation by increasing PPARg expression or activation. Notably, we found that PPARg expression was increased by the lowest level of QUE (Figure 2). However, we did not measure PPARg protein levels or activity. PPARg has been reported as a negative regulator of MF activation, and has been implicated in improving lipid homeostasis and insulin sensitivity and preventing inflammation. PPARg agonists such as thiazolidinediones reduce inflammatory gene expression in MFs.42 When administered before the onset of inflammation, TZDs exhibit beneficial effects on experimental models of inflammation, such as colitis,43–48 atherosclerosis,49–52 asthma,53–55 psoriasis,56 myocarditis57,58 and allergic encephalomyelitis.59,60 Consistent with these data, anthocyanins such as cyanidin-3-O-b-glucoside have also been shown to enhance the expression and transcripInternational Journal of Obesity

tional activities of PPARg in MFs.61,62 Although the underlying mechanisms are not fully elucidated, it has been suggested that PPARg activation exerts its anti-inflammatory function by transrepressing the NFB and MAPK pathways.63,64 Thus, QUE may be attenuating activation of NFB and MAPKs by acting as a PPARg agonist. Studies are needed to test this hypothesis. Taken together, these data demonstrate that relatively low levels of QUE, which is abundantly found in GPE, attenuates inflammatory gene expression and increases PPARg and ABAC1 gene expression, possibly by suppressing the activation of NFkB, c-Jun and JNK. Furthermore, QUE decreased the inflammatory capacity of MF-CM, attenuating its ability to cause inflammation and insulin resistance in primary human adipocytes. Limitations of these in vitro studies include the high doses of QUE used. In vivo studies are needed to confirm these in vitro effects of QUE on MF-mediated inflammation and insulin resistance.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements We thank the North Carolina Agricultural Research Service (NCARS 02288) for providing financial support for these studies.

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