Obesity is linked to chronic inflammation in white adipose tissue, which is exacerbated by infiltrating macrophages (MΦs). We recently demonstrated that an extract from grape powder (GPE), which is abundant in quercetin (QUE), reduced inflammation in human MΦs and prevented MΦ-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 MΦs (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 MΦs with QUE attenuated the basal expression of inflammatory genes, such as tumor necrosis factor-α, interleukin (IL)-6, IL-8, IL-1β and interferon-γ 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 IκBα degradation in MΦs. Furthermore, conditioned media (CM) obtained from MΦs 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.
Taken together, these data suggest that QUE is one of the bioactive components of GPE that prevents inflammation in MΦs and MΦ-mediated insulin resistance in adipocytes.
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 (MΦ) infiltration into white adipose tissue (WAT).3, 4 Activated MΦs 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)-α, interleukin (IL)-6 and monocyte chemoattractant protein-1 (see references 6, 7). Notably, several studies reported that MΦs are the primary source for proinflammatory cytokine production in WAT and may be recruited to WAT by monocyte chemoattractant protein-1 (see references 8, 9, 10, 11, 12). In addition, numerous animal studies have shown the importance of MΦs in inflammation-induced insulin resistance.11, 13, 14, 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 MΦs by inhibiting nuclear factor kappa B (NFκB).17 In addition, oligomerized grape seed polyphenols reduced NFκB transcriptional activity and activation of extracellular signal-related kinase (ERK), a mitogen-activated protein kinase (MAPK), in a coculture of murine adipocytes and MΦs.16 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 NFκB and activator protein (AP)-1 in human MΦs, 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 MΦs, and block inflammation and insulin resistance in human adipocytes treated with MΦ-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 NFκB, and their subsequent induction of inflammatory genes in human MΦs. Furthermore, we postulated that QUE pretreatment of MΦs would decrease inflammation and insulin resistance in primary cultures of human adipocytes incubated with MΦ-CM.
Materials and methods
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 NH2-terminal 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 MΦs
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 MΦs, including irregular shape, lobed nuclei, and an intense phagocytic activity. In addition, differentiated U937 cells adhere to each other and to a material surface, a characteristic feature of mature MΦs. In vivo, mature MΦs 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 μg l−1 phorbol 12-myristate (PMA) for 24 h in RPMI-1640 (containing 10% fetal bovine serum, 60 U ml−1 penicillin, 60 U l−1 streptomycin and 25 μg ml−1 amphotericin B). Media was then changed to PMA-free RPMI and 24 h later the experiments were initiated with the MΦ monolayers. Cultures were incubated at 37 °C 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 <32.0 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 μM isobutylmethylxanthine and 1 μM thiazolidinediones rosiglitazone (BRL 49653; a gift from Dr Per Sauerberg, Novo Nordisk A/S, Copenhagen, Denmark) for 3 days.20 Mφ-free cultures containing ∼50% preadipocytes and ∼50% 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 μg 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 was conducted as previously described.20 Briefly, total cellular protein was harvested using phosphate-buffered saline (pH 7.5) lysis buffer containing 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, 30 μgml−1 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, and stored at −80 °C. Protein concentration was determined using the BCA assay (Thermo Scientific, Rockford, IL, USA). Total cellular protein, 20 μg, was separated by electrophoresis on 4–12% SDS-polyacrylamide gradient gels (NuPAGE mini-gel system; Invitrogen), transferred to a polyvinylidene difluoride membrane using a wet transfer module (Trans-Blot 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).
MΦ-CM experiments in primary human adipocytes
CM was collected from differentiated U937 cultures treated with 0, 0.3, 3, 10, or 30 μM QUE for 5 h. The 5-h incubation time was chosen based on the results of a pilot time-course study (data not shown). MΦ-CM obtained from each experiment was pooled and stored at −80 °C 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) MΦ-CM, or (4) MΦ-CM treated with QUE. The amount and duration of MΦ-CM treatment varied depending on the outcome measured.
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 MΦ treated with 0, 3, 10, or 30 μM 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 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, P<0.05. Data are expressed as means±s.e.m.
QUE decreases inflammatory gene expression in human MΦs
To determine the extent to which QUE attenuated markers of inflammation, MΦs were treated for 5 h with QUE (0, 3, 10, or 30 μM) 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-γ inducible protein-10, IL-1β, TNF-α 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)γ and ABCA1 gene expression in human MΦs
Given the important role MΦs have in lipid metabolism and inflammation, we examined the impact of QUE on the expression of PPARγ and ABCA1, two regulators involved in lipid metabolism and inflammation.22 MΦs were treated for 5 h with QUE (0, 0.3, 3, 10, or 30 μM) and the expression of PPARγ and ABCA1 was measured by qPCR. Whereas 0.3 μM QUE increased PPARγ and ABCA1 gene expression, the expression levels of these lipogenic genes treated with 3, 10 and 30 μM QUE were similar to the controls (Figure 2).
QUE decreases the degradation of IκBα and the activation of c-Jun and JNK in human MΦs
Given the important roles of MAPKs, AP-1 and NFκB in inducing the transcription of inflammatory genes, we examined the effects of QUE on IκBα degradation, and JNK and c-Jun activation in MΦs treated for 2.5 h with 0, 3, 10, or 30 μM QUE. QUE attenuated the degradation of IκBα 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 MΦs decreases MΦ-CM-mediated inflammation and insulin resistance in human adipocytes
Several studies have reported cross-talk between murine MΦs and adipocytes; that is, activated MΦs can inflame adipocytes and vice-versa.18, 23 Therefore, we hypothesized that QUE treatment of MΦs would prevent MΦ-CM-mediated inflammation and insulin resistance in human adipocytes. Consistent with our hypothesis, QUE decreased MΦ-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 MΦ-mediated phosphorylation of ERK or IκBα degradation (data not shown). Lastly, QUE decreased MΦ-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.
In this study, we showed that 3–30 μM QUE, a tetrahydroxyflavonol found in grapes and grape products like GPE,18 attenuated basal inflammatory gene expression (that is, IL-6, IL-8, IL-1β, interferon-γ inducible protein-10, TNF-α and cyclooxygenase-2) (Figure 1) and increased PPARγ and ABCA1 gene expression (Figure 2), two key players involved in MΦ lipid metabolism.22 QUE also attenuated the basal activation of NFκB, c-Jun and JNK (Figure 3). Moreover, we demonstrated that QUE decreased: (1) induction of inflammatory genes (that is, interferon-γ inducible protein-10, monocyte chemoattractant protein-1 and TNF-α) (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 MΦ-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 MΦs. On the basis of these data, we speculate that QUE directly attenuates the basal activation of NFκB, c-Jun and JNK in MΦs, thereby preventing the induction and secretion of inflammatory cytokines and chemokines MΦs and their cross-talk with adipocytes. However, it should be noted that the effects of QUE were lost at higher doses with regards to PPARγ and ABCA1 gene expression in MΦs. 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 MΦs.
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 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 MΦs28 and suppressed the degradation of IκBα and the phosphorylation of p38 and Akt in LPS-stimulated bone marrow-derived MΦs.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 NFκB in murine skin epidermal (JB6 P+) cells.31 Also, QUE impaired inflammatory cytokine and chemokine production and decreased activation of ERK, JNK, Akt and NFκB 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 MΦs.33 Consistent with these data, luteolin, a tetrahydroxyflavone found in celery and green peppers, reduced JNK phosphorylation and AP-1 activation in microganglia34 and NFκB, and AP-1 activation in alveolar MΦs.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 NFκB 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 MΦs in WAT. MΦ-secreted factors have been shown to increase inflammation and decrease insulin-stimulated glucose uptake in adipocytes.13, 38, 39, 40 Consistent with these findings, we previously reported that LPS increased the activation of MAPK, NFκB and AP-1 in human MΦs, increasing their capacity to cause inflammation and insulin resistance in primary human adipocytes.18 Pretreatment of human MΦs with GPE, which is rich in QUE, attenuated inflammation in MΦs and adipocytes, and MΦ-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 NFκB or AP-1 (see reference 41). Concerning this last mechanism, QUE may block inflammation by increasing PPARγ expression or activation. Notably, we found that PPARγ expression was increased by the lowest level of QUE (Figure 2). However, we did not measure PPARγ protein levels or activity. PPARγ has been reported as a negative regulator of MΦ activation, and has been implicated in improving lipid homeostasis and insulin sensitivity and preventing inflammation. PPARγ agonists such as thiazolidinediones reduce inflammatory gene expression in MΦs.42 When administered before the onset of inflammation, TZDs exhibit beneficial effects on experimental models of inflammation, such as colitis,43, 44, 45, 46, 47, 48 atherosclerosis,49, 50, 51, 52 asthma,53, 54, 55 psoriasis,56 myocarditis57, 58 and allergic encephalomyelitis.59, 60 Consistent with these data, anthocyanins such as cyanidin-3-O-β-glucoside have also been shown to enhance the expression and transcriptional activities of PPARγ in MΦs.61, 62 Although the underlying mechanisms are not fully elucidated, it has been suggested that PPARγ 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 PPARγ 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 PPARγ and ABAC1 gene expression, possibly by suppressing the activation of NFκB, c-Jun and JNK. Furthermore, QUE decreased the inflammatory capacity of MΦ-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 MΦ-mediated inflammation and insulin resistance.
We thank the North Carolina Agricultural Research Service (NCARS 02288) for providing financial support for these studies.