Curcumin and resveratrol inhibit nuclear factor-kappaB-mediated cytokine expression in adipocytes
© Gonzales and Orlando; licensee BioMed Central Ltd. 2008
Received: 27 February 2008
Accepted: 12 June 2008
Published: 12 June 2008
Adipocytes express inflammatory mediators that contribute to the low-level, chronic inflammation found in obese subjects and have been linked to the onset of cardiovascular disorders and insulin resistance associated with type 2 diabetes mellitus. A reduction in inflammatory gene expression in adipocytes would be expected to reverse this low-level, inflammatory state and improve cardiovascular function and insulin sensitivity. The natural products, curcumin and resveratrol, are established anti-inflammatory compounds that mediate their effects by inhibiting activation of NF-κB signaling. In the present study, we examined if these natural products can inhibit NF-κB activation in adipocytes and in doing so reduce cytokine expression.
Cytokine (TNF-α, IL-1β, IL-6) and COX-2 gene expression in 3T3-L1-derived adipocytes was measured by quantitative real-time PCR (qRT-PCR) with or without TNFα-stimulation. Cytokine protein and prostaglandin E2 (PGE2) expression were measured by ELISA. Effects of curcumin and resveratrol were evaluated by treating TNFα-stimulated adipocytes with each compound and 1) assessing the activation state of the NF-κB signaling pathway and 2) measuring inflammatory gene expression by qRT-PCR and ELISA.
Both preadipocytes and differentiated adipocytes express the genes for TNF-α, IL-6, and COX-2, key mediators of the inflammatory response. Preadipocytes were also found to express IL-1β; however, IL-1β expression was absent in differentiated adipocytes. TNF-α treatment activated NF-κB signaling in differentiated adipocytes by inducing IκB degradation and NF-κB translocation to the nucleus, and as a result increased IL-6 (6-fold) and COX-2 (2.5-fold) mRNA levels. TNF-α also activated IL-1β gene expression in differentiated adipocytes, but had no effect on endogenous TNF-α mRNA levels. No detectable TNFα or IL-1β was secreted by adipocytes. Curcumin and resveratrol treatment inhibited NF-κB activation and resulted in a reduction of TNF-α, IL-1β, IL-6, and COX-2 gene expression (IC50 = 2 μM) and a reduction of secreted IL-6 and PGE2 (IC50 ~ 20 μM).
Curcumin and resveratrol are able to inhibit TNFα-activated NF-κB signaling in adipocytes and as a result significantly reduce cytokine expression. These data suggest that curcumin and resveratrol may provide a novel and safe approach to reduce or inhibit the chronic inflammatory properties of adipose tissue.
Obesity is now known to play a causal role in the complex disease state of metabolic syndrome, as well as being a significant risk factor for cardiovascular disorders and diabetes [1, 2]. Although once thought to serve as a simple storage depot for excess fats, adipose tissue also regulates organismic metabolism through a variety of signaling mechanisms including autonomic nervous stimulation and secreted hormones [3, 4]. When in proper balance, these regulatory mechanisms effectively control energy preservation (lipogenesis) during the post-prandial period and energy mobilization (lipolysis) during times of increased energy expenditure.
In addition to these mechanisms of metabolic regulation, adipose tissue is also capable of producing proteins that are classical mediators of the inflammatory response. In the early 1990's, it was discovered that adipocytes synthesize and secrete the pro-inflammatory cytokine, Tumor Necrosis Factor-alpha (TNF-α) . Since then, it has been shown that a number of acute phase reactants and inflammatory mediators are made by adipocytes including plasminogen activator inhibitor-1, IL-1β, IL-6, IL-8, IL-10, IL-15, hepatocyte growth/scatter factor and prostaglandin E2 (PGE2) . In fact, enough of these factors are secreted by adipocytes that overall systemic levels are significantly elevated in obese subjects  and a number of studies have now identified a direct correlation between body mass index (BMI) and systemic levels of inflammatory proteins . These clinical observations provide key evidence linking obesity with cardiovascular disorders and begin to shed light on how low-level, chronic inflammation adversely affects cardiovascular function in obese subjects.
Recent evidence suggests that cytokine expression in adipose tissue is initiated by crosstalk occurring between adipocytes and macrophages [8–11]. Macrophages typically account for 5–10% of cells within adipose tissue obtained from non-obese donors; however, in diet-induced obesity, macrophage infiltration can account for up to 60% of all cells in adipose tissue . Cytokines secreted by macrophages, including TNFα, IL-1β and IL-6, are known to stimulate cytokine expression in adipocytes [13–15] and establish a paracrine loop between these two cell types . This paracrine stimulation in turn elevates systemic cytokine levels observed in obese individuals. In bone fide inflammatory cells, cytokine gene expression is activated following activation of the Nuclear Factor-kappaB (NF-κB) signal transduction pathway . Activation of the NF-κB pathway is mediated by a variety of signals including those initiated from the TNFα receptor and Toll-like receptor family. NF-κB itself is a heterodimeric transcription factor that is retained in the cytosol in its inactive state by complexing with a set of inhibitory proteins designated IκB. Upon receptor activation of NF-κB signaling the IκB complex is phosphorylated by IκB kinase (IKK). This in turn leads to its dissociation from NF-κB and rapid degradation by the proteosome. Free NF-κB is then able to translocate to the nucleus where it binds to specific promoter elements resulting in the activation of a battery of genes, including those encoding for inflammatory proteins.
In adipocytes, both expression and activity of NF-κB increase during differentiation  suggesting that it is a key player in mediating adipose-specific cytokine expression. Moreover, excessive NF-κB activity has been associated with the development of type 2 diabetes as obese individuals have high circulating levels of TNF-α, IL-1β and IL-6 that, like cardiovascular risk, directly correlate with insulin resistance [7, 19, 20]. Collectively, these observations suggest that therapeutic targeting of the NF-κB signaling pathway in adipose tissue represents a logical pursuit to reduce systemic cytokine levels and reverse their negative influence on cardiovascular function and diabetic progression.
There are no shortages of reported inhibitors of NF-κB activation as they now number in the hundreds . Unfortunately, the targets for many of these inhibitors are not necessarily restricted to the NF-κB pathway raising concerns of potential side effects due to off-target inhibition. As an alternative, we have investigated the effectiveness of certain natural products to inhibit the NF-κB signaling pathway . Many natural products have been shown to possess low level toxicity and potent anti-inflammatory properties by targeting similar pathways as non-steroidal anti-inflammatory drugs (NSAIDs). Two natural polyphenols of particular interest are curcumin and resveratrol. These natural products are modest inhibitors of NF-κB activation and inflammatory gene expression [22–30] and have proven safe in human clinical trials [31–33]. In the present study, we examined if curcumin and resveratrol are also able to inhibit NF-κB activation in adipocytes and in doing so inhibit cytokine expression in these cells. We believe that using natural products to inhibit the chronic inflammatory response of adipose tissue may provide a novel approach to reduce systemic cytokine levels which in turn is expected to improve cardiovascular health and insulin sensitivity.
TNFα was obtained by R&D Systems, Minneapolis, MN and was used in all experiments at a final concentration of 20 ng/ml. Lipopolysaccharide (LPS) was purchased from Sigma, St. Louis, MO and used to activate BV-2 murine macrophages at a final concentration of 20 μg/ml. Curcumin was synthesized in the lab  and resveratrol was purchased from A.G. Scientific Inc., San Diego, CA.
Cell culture and adipocyte differentiation
For our studies, we utilize an in vitro cell culture system that has been extensively characterized for adipocyte differentiation, namely mouse 3T3-L1 fibroblasts . Following induction into the differentiation pathway, 3T3-L1 cells undergo growth arrest, become spherical, and form large intracellular lipid droplets. Subcutaneous implantation of these cells in mice results in tissue masses that are histologically indistinguishable from white adipose tissue [36, 37]. 3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal calf serum (Irvine Scientific, Santa Ana, CA), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin. Cells were cultured at 37°C with 10% CO2 and passaged twice weekly. To differentiate 3T3-L1 cells into adipocytes, cells were incubated with 250 nM dexamethasone, 450 μM 3-isobutyl-1-methylxanthine, and 167 nM insulin for 2 days, followed by 167 nM insulin for an additional 3 days.
BV-2 murine macrophages (a gift from Dr. Paul Stemmer, Wayne State University, Detroit, MI) were grown in RPMI-1640 (Hyclone®, Logan, UT) supplemented with 10% (v/v) fetal calf serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin. Cells were cultured at 37°C with 5% CO2 and passaged twice weekly.
qRT-PCR and RT-PCR analysis
Gene and primer information used in this study.
5' primer – top sequence
3' primer – bottom sequence
Base pairs amplified
qRT-PCR was performed using ABsolute QPCR SYBR Green Mix (Fisher Scientific, Atlanta, GA) with the following cycling parameters: 1 cycle, 95°C, 15 min; 40 cycles, 95°C, 15 sec, 63°C, 1 min. Changes in gene expression were determined by the Comparative CT method. Since β-actin gene expression is unaffected by TNFα treatment, β-actin mRNA levels were quantified in each sample using identical cycling conditions and used to normalize values obtained for COX-2, IL-1β, IL-6, and TNFα expression. Amplified products were separated on 3% agarose gels and stained with Gel Star® (Cambrex, Rockland, ME).
Cell lysates were prepared using 1× Laemmli sample buffer (Sigma-Aldrich). After heating samples at 95°C for 10 min, they were vortexed on high for 20 s to shear DNA and reduce viscosity. Proteins were then separated by SDS-PAGE and transferred to PVDF membrane (0.2 μm, BioRad, Hercules, CA) using a wet tank transfer system (BioRad). Membranes were blocked with 20 mM Tris, pH 7.4, 150 mM NaCl (TBS) containing 0.1% (v/v) Tween-20, 5% (v/v) calf serum for 30 minutes at 23°C and incubated with either anti-IκB monoclonal antibody (2 μg/ml, Imgenex, San Diego, CA) or anti-β-actin monoclonal antibody (1:500, no. A-4700, Sigma-Aldrich) for 24 h at 23°C. Membranes were washed three times (10 min each) with TBS, 0.1% (v/v) Tween-20, and bound antibodies were detected with goat anti-mouse HRP-conjugated secondary antibody (1:3000, BioRad) followed by chemiluminescence detection with Immobilon™ Western according to the manufacturer's instructions (Millipore, Billerica, MA). Images were captured using a Syngene GeneGnome system equipped with a Peltier-cooled 16-bit CCD camera and saturation detection. Densitometry was performed using ImageJ software (version 1.37; National Institutes of Health, http://rsb.info.nih.gov/ij/).
NF-κB nuclear localization assay
BV-2 murine macrophages were cultured in the absence or presence of LPS (20 μg/ml) for 24 h. 3T3-L1-derived adipocytes were cultured in the absence or presence of TNFα (20 ng/ml) or incubated with TNFα together with curcumin or resveratrol or vehicle alone (dimethylsulfoxide at 0.1% final concentration) for 62 h. Nuclear localized NF-κB was quantified using a Transcription Factor ELISA Kit to detect activated p65 subunit of NF-κB (Panomics, Fremont, CA). All reagents required for preparing nuclear extracts and performing ELISA assays were included and their use was described by the manufacturer.
Cells were grown in 96-well plates to 80–90% confluency. Media was replaced with fresh complete media containing the indicated concentrations of curcumin or resveratrol, or vehicle alone (dimethylsulfoxide at 0.1% final concentration). After a 24 h incubation, WST-1 (Roche Molecular Biochemicals, Indianapolis, IN) was added to the cultures to a final concentration of 10% (vol/vol). Following an additional incubation at 37°C for 60 min, absorbance was recorded for each well (450 nm; reference wavelength, 690 nm).
Cytokine and PGE2 ELISA
Quantitation of cytokine protein levels from cell culture supernatants was done by ELISA Ready-SET-Go! kit (eBioscience, San Diego, CA) per manufacturer's instructions. Parameter™ PGE2 competitive binding ELISA kit (R&D systems, Minneapolis, MN) was used to measure PGE2 levels.
All experimental protocols were done in at least triplicate points and error bars represent standard deviations of mean values. Student's t-test was performed on some figures using data sets composed of a minimum of triplicate values. Comparison of data sets resulting in p values < 0.05 were considered statistically significant.
Cytokine expression profile in 3T3-L1 preadipocytes and differentiated adipocytes
With this criteria met, we next examined the cytokine expression profile of 3T3-L1 preadipocytes as well as cytokine expression after differentiation to adipocytes. Specifically, we assessed TNFα, IL-1β, IL-6, and COX-2 expression using RT-PCR analysis. We included COX-2 in our list of inflammatory genes to examine since its expression, like the other inflammatory mediators, has also been associated with obesity . As shown in Figure 1B, both 3T3-L1 preadipocytes and differentiated adipocytes express several key mediators of the inflammatory response: TNFα, IL-6, and COX-2. Notably, preadipocytes were found to express IL-1β; however, IL-1β expression was absent in fully differentiated adipocytes.
TNFα treatment activates IL-1β expression in differentiated adipocytes and increases expression of IL-6 and COX-2
Activation of NF-κB in adipocytes
Curcumin and resveratrol show very little cytotoxicity to adipocytes
Curcumin and resveratrol inhibit IκB degradation and NF-κB translocation to the nucleus in adipocytes
Curcumin and resveratrol reduce cytokine and COX-2 gene expression in adipocytes
Secreted cytokine protein and PGE2 levels are reduced by curcumin and resveratrol
Increased adiposity is now a well established risk factor for developing complications related to metabolic syndrome and type II diabetes mellitus. Mounting evidence indicates that low level, chronic inflammation resulting from cytokines secreted by adipose tissue may play a significant role in causing, or at the very least aggravating, the inflammatory component of cardiovascular disease and in desensitizing cells to insulin leading to high circulating glucose levels. These observations suggest a hypothesis that reducing or preventing the inflammatory properties of adipose tissue represents a novel and promising therapeutic approach to curb the progression of cardiovascular disease and to restore insulin sensitivity in type II diabetics.
Macrophage infiltration has recently been postulated to be a primary stimulus for fueling the inflammatory properties of adipose tissue . Monocyte chemoattractants [9, 11], such as monocyte chemoattractant protein-1 (MCP-1) which is synthesized and secreted by adipocytes , are thought to mediate macrophage infiltration and intensify macrophage expression of TNFα . TNFα has pleiotropic effects on adipocyte physiology including an induction of lipolysis to increase the mobilization of free fatty acids [46, 47], activating cytokine expression  and promoting insulin resistance [5, 48]. Observations such as these provide sufficient evidence suggesting that TNFα is the predominant factor that mediates the crosstalk between macrophages and adipocytes and that elevated TNFα levels found in obese individuals establishes a paracrine loop in adipose tissue  that is responsible for the elevated systemic levels of cytokines seen in obesity.
TNFα mediates its affects on adipocytes by activating the NF-κB signaling pathway [11, 18]; a signaling event that has been studied extensively in the innate immune response. In conventional immune cells, activation of the NF-κB signaling pathway requires relocation of the NF-κB heterodimer from the cytoplasm to the nucleus where it functions as part of a multi-protein transcription complex controlling the expression of most inflammatory mediators. In adipose tissue, low level NF-κB activation has been identified in vivo  suggesting that, like in conventional immune cells, NF-κB is largely responsible for cytokine gene expression in adipocytes. Only recently has the role of NF-κB in adipose function come under scrutiny. Berg, et al., examined NF-κB expression and activity during adipocyte differentiation and found both parameters to be elevated in fully differentiated adipocytes . Consistent with their findings, we were able to activate NF-κB signaling in differentiated adipocytes with TNFα treatment and in doing so demonstrate an increase in NF-κB nuclear translocation. However, to extend these observations we also examined the upstream signaling event that is directly responsible for NF-κB activation, namely IκB degradation. We found that IκB was rapidly degraded in adipocytes following TNFα treatment and with kinetics similar to those measured for true immune cells . These data provide compelling evidence that NF-κB signaling in adipocytes shares a similar time course of activation as inflammatory cells.
Because the NF-κB signaling pathway is such a pleiotropic pro-inflammatory and pro-survival factor in a wide range of disorders, it has been an attractive target for small-molecule inhibitor development. Thus far almost 800 compounds have been reported to inhibit NF-κB activation . A large fraction of these inhibitors include natural products that are capable of targeting multiple checkpoints in the NF-κB activation pathway. Of particular interest are the polyphenolic natural compounds, curcumin and resveratrol. Curcumin is derived from the spice turmeric, which comes from the root of Curcuma longa of the ginger family. It is an established inhibitor of NF-κB activation  and has recently been shown to specifically target IKK . Inhibitors targeting IKK have so far proven to be the most effective compounds for preventing the activation of NF-κB [50–54] by directly preventing the phosphorylation of IκB, and as a consequence, block NF-κB translocation to the nucleus. Important for clinical drug development, curcumin has also been found safe in six human trials at oral doses up to 8 g/day administered for 3 months [31, 32]. The other natural product that has been a focus of our laboratory is resveratrol . A product of red grapes, resveratrol possesses multiple biological activities including anti-oxidant and anti-cancer activities, and like curcumin, is an inhibitor of NF-κB activation  through targeted inhibition of IKK . In addition, although the extent of its bioavailability is still under investigation [57, 58], resveratrol has been shown to be quite safe in preclinical trials . In the present study, we examined if curcumin and resveratrol might represent promising therapeutics to combat the chronic inflammatory properties of adipose tissue by exploring their effects on NF-κB activation and inflammatory cytokine expression in adipocytes. We first identified that curcumin and resveratrol are able to inhibit NF-κB translocation to the nucleus in TNFα-stimulated adipocytes. Moreover, we also found that both natural products are able to prevent IκB degradation. These data establish that curcumin and resveratrol carry out their inhibitory functions either at the level of IκB phosphorylation by IKK or upstream from this checkpoint in the NF-κB activation pathway. We next examined the effects of curcumin and resveratrol on downstream gene regulation in adipocytes since NF-κB activation is largely responsible for mediating inflammatory gene expression in immune cells. Indeed, treatment of TNFα-stimulated adipocytes with curcumin or resveratrol resulted in a significant reduction in TNFα, IL-1β, IL-6, and COX-2 gene expression. The IC50 values measured for inhibition of IL-1β, IL-6, and COX-2 gene expression by either compound were found to be < 2 μM; for TNFα gene expression, the IC50 value was ~8 μM.
During the course of identifying inhibitors for NF-κB signaling, many studies will limit their analysis to measuring the effects of inhibitors on the transcriptional status of cytokine genes. Although these studies provide a wealth of data regarding the direct control of cytokine gene expression by the state of NF-κB activity, they fall short of identifying additional mechanisms of regulation at post-transcriptional levels. Limiting inhibitor identification to effects on transcriptional levels in bona fide immune cells may be acceptable since the NF-κB signaling pathway that mediates these immunological responses has been well studied . However, because much less is known about potential multi-level regulatory elements in non-immune cells that may affect NF-κB signaling, cytokine expression analyses should include a quantitative assessment of secreted cytokines to identify possible post-transcriptional control of cytokine expression. By extending our analysis to measuring levels of secreted cytokines, we have identified unique expression patterns that may have significant impact on our understanding of adipocyte contributions to systemic inflammation. First, although adipocytes express TNFα mRNA, we were unable to measure any secreted TNFα by ELISA. This observation suggests that the major source of circulating TNFα found in obese subjects arises from adipose-infiltrating macrophages rather than adipocytes. A similar observation was made by Fain and colleagues when comparing isolated adipocytes to stromal vascular cells obtained from human adipose explants . In this study the authors found significant amounts of TNFα secreted by stromal vascular cells, with little or no detectable TNFα secreted by adipocytes. One caveat of this study stems from the fact that the adipocytes were removed from the in vivo environment where they are exposed to macrophage-derived TNFα. Removal of TNFα-stimulation from the isolated adipocytes would discontinue signaling events that arguably might be necessary to sustain TNFα secretion by adipocytes. Our study clearly addresses this concern by demonstrating the lack of TNFα secretion in TNFα-stimulated adipocytes.
We also found that preadipocytes express the gene for IL-1β, yet differentiated adipocytes show no mRNA expression. Interestingly, TNFα treatment was able to re-activate IL-1β mRNA expression in differentiated adipocytes; however, in spite of this re-activation we were unable to detect any secreted IL-1β from treated adipocytes indicating that post-transcriptional mechanisms are in place to prevent expression of IL-1β protein. These observations may be interpreted based on the effects of long-term treatment of adipocytes with IL-1β. Such treatment has been shown to inhibit insulin receptor substrate -1 (IRS-1) expression  and activation  thereby inducing insulin resistance. By repressing IL-1β transcription during adipocyte differentiation, insulin responsiveness can be maintained for proper glucose homeostasis. Furthermore, because expansion of adipose tissue is accompanied by accelerated macrophage infiltration providing a substantial source of secreted TNFα, which we show can activate IL-1β gene expression, additional levels of regulation become necessary to prevent secretion of IL-1β protein by adipocytes. Collectively, these observations indicate that multiple regulatory checkpoints are in place to prevent IL-1β expression and ensure proper insulin responsiveness by adipocytes.
In contrast to the results obtained for measurements of secreted TNFα and IL-1β, TNFα-stimulation of adipocytes did have a pronounced effect on secreted levels of IL-6 and PGE2. We found little or no IL-6 secreted by unstimulated, fully differentiated adipocytes; however, when stimulated with TNFα, a significant level of secreted IL-6 was measured. In spite of a lack of secreted IL-6, we found that the IL-6 gene is expressed in unstimulated adipocytes and is responsive to TNFα stimulation as mRNA levels increased by 6-fold. These data indicate that TNFα stimulation of adipocytes not only increases transcriptional activity of the IL-6 gene, but also activates post-transcriptional events to produce secreted IL-6. Secreted PGE2 levels were also measured as a direct assessment of COX-2 activity. We found that TNFα-stimulation modestly increased COX-2 gene expression by 2-fold and increased secreted PGE2 by 3-fold over basal levels found in unstimulated adipocytes. Notably, both curcumin and resveratrol treatment of TNFα-stimulated adipocytes significantly reduced secreted levels of IL-6 and PGE2 in a dose-dependent manner. IC50 values for curcumin and resveratrol inhibition of IL-6 are estimated to be ~20 μM. By contrast, IC50 values for inhibition of PGE2 differ for each compound; ~2 μM for curcumin and > 20 μM for resveratrol. These IC50 values determined for secreted levels of IL-6 and PGE2 are noticeably higher than what was measured for inhibition of IL-6 and COX-2 gene expression. These differences are most likely due to previously unidentified effects of curcumin and resveratrol on post-transcriptional events and highlight the importance of measuring the final product in addition to transcriptional levels when identifying the quantitative effects of potential inhibitory compounds.
Whenever a compound is being developed as a potential therapeutic, issues involving in vivo bioavailability must be addressed. In this regard, data thus far presented on the pharmacokinetics of curcumin [62, 63] and resveratrol  have been confusing and often times contradictory. Both polyphenols have relatively short half-lives in vivo as they are rapidly metabolized to their glucuronide and sulfated forms. These metabolites, readily found in the circulation, typically demonstrate very low cell permeability and questionable bioactivity when compared to their unmetabolized forms. In spite of these hurdles, the in vivo efficacies of curcumin and resveratrol have been reproducibly shown by numerous investigators. Many challenges lie ahead in order to systematically and quantitatively address the pharmacokinetics of these natural products. Immediate questions that need to be addressed to improve on in vivo efficacy include, 1) do the metabolites of curcumin and resveratrol have comparable bioactivity with the parent compounds, 2) does the circulating pool of metabolites represent a source of inhibitor that can be modified to their more active forms, and 3) can chemical substitutions be made to the base structures of curcumin and resveratrol making them more active and less susceptible to conjugation.
Most importantly for our hypothesis, the results presented here provide proof-of-principle evidence that use of curcumin and resveratrol represents a promising new therapeutic approach to reduce both local and systemic inflammatory contributions by adipose tissue. At present, we believe that the low μM IC50 values of curcumin and resveratrol together with their positive in vivo effects make these natural products excellent lead compounds to guide the development of more potent inhibitors of NF-κB activation and inflammatory gene expression. Toward this goal, we have recently developed chemical libraries of synthetic analogs based on the chemical structures of curcumin  and resveratrol . Studies are currently underway to identify if these novel structural analogs improve upon the inhibitory properties of the parent compounds while also critically addressing the challenges of bioavailability and in vivo metabolism.
We thank Drs. Lorraine Deck, Robert Royer and David Vander Jagt for contributing the curcumin and resveratrol used in this study. We thank Dr. Paul Stemmer (Wayne State University, Detroit, MI) for providing the BV-2 cell line for our studies. This work was supported by an American Heart Association Grant-in-aid no. 0555647Z (to R.A.O.).
- Grundy SM: Obesity, metabolic syndrome, and coronary atherosclerosis. Circulation 2002, 105: 2696-2698. 10.1161/01.CIR.0000020650.86137.84View ArticleGoogle Scholar
- Krauss RM, Winston M, Fletcher RN, Grundy SM: Obesity: impact of cardiovascular disease. Circulation 1998, 98: 1472-1476.View ArticleGoogle Scholar
- Attele AS, Shi ZQ, Yuan CS: Leptin, gut, and food intake. Biochemical Pharmacology 2002, 63: 1579-1583. 10.1016/S0006-2952(02)00883-3View ArticleGoogle Scholar
- Mohamed-Ali V, Pinkney JH, Coppack SW: Adipose tissue as an endocrine and paracrine organ. Int J Obes Relat Metab Disord 1998,22(12):1145-1158. 10.1038/sj/ijo/0800770View ArticleGoogle Scholar
- Hotamisligil GS, Shargill NS, Spiegelman BM: Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993, 259: 87-91. 10.1126/science.7678183View ArticleGoogle Scholar
- Fain JN, Madan AK, Hiler ML, Cheema P, Bahouth SW: Comparison of the release of adipokines by adipose tissue, adipose tissue matrix, and adipocytes from visceral and subcutaneous abdominal adipose tissues of obese humans. Endocrinology 2004, 145: 2273-2282. 10.1210/en.2003-1336View ArticleGoogle Scholar
- Cottam DR, Mattar SG, Barinas-Mitchell E, Eid G, Kuller L, Kelley DE, Schauer PR: The chronic inflammatory hypothesis for the morbidity associated with morbid obesity: implications and effects of weight loss. Obes Surg 2004, 14: 589-600. 10.1381/096089204323093345View ArticleGoogle Scholar
- Berg AH, Scherer PE: Adipose tissue, inflammation, and cardiovascular disease. Circ Res 2005, 96: 939-949. 10.1161/01.RES.0000163635.62927.34View ArticleGoogle Scholar
- Fantuzzi G: Adipose tissue, adipokines, and inflammation. J Allergy Clin Immunol 2005, 115: 911-919. quiz 920 10.1016/j.jaci.2005.02.023View ArticleGoogle Scholar
- Tilg H, Moschen AR: Adipocytokines: mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol 2006, 6: 772-783. 10.1038/nri1937View ArticleGoogle Scholar
- Wellen KE, Hotamisligil GS: Obesity-induced inflammatory changes in adipose tissue. J Clin Invest 2003, 112: 1785-1788.View ArticleGoogle Scholar
- Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr: Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003, 112: 1796-1808.View ArticleGoogle Scholar
- Permana PA, Menge C, Reaven PD: Macrophage-secreted factors induce adipocyte inflammation and insulin resistance. Biochem Biophys Res Commun 2006, 341: 507-514. 10.1016/j.bbrc.2006.01.012View ArticleGoogle Scholar
- Lin Y, Lee H, Berg AH, Lisanti MP, Shapiro L, Scherer PE: The lipopolysaccharide-activated toll-like receptor (TLR)-4 induces synthesis of the closely related receptor TLR-2 in adipocytes. J Biol Chem 2000, 275: 24255-24263. 10.1074/jbc.M002137200View ArticleGoogle Scholar
- Rajala MW, Scherer PE: Minireview: The adipocyte – at the crossroads of energy homeostasis, inflammation, and atherosclerosis. Endocrinology 2003, 144: 3765-3773. 10.1210/en.2003-0580View ArticleGoogle Scholar
- Suganami T, Nishida J, Ogawa Y: A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor alpha. Arterioscler Thromb Vasc Biol 2005, 25: 2062-2068. 10.1161/01.ATV.0000183883.72263.13View ArticleGoogle Scholar
- Andreakos E, Sacre S, Foxwell BM, Feldmann M: The toll-like receptor-nuclear factor kappaB pathway in rheumatoid arthritis. Front Biosci 2005, 10: 2478-2488. 10.2741/1712View ArticleGoogle Scholar
- Berg AH, Lin Y, Lisanti MP, Scherer PE: Adipocyte differentiation induces dynamic changes in NF-kappaB expression and activity. Am J Physiol Endocrinol Metab 2004, 287: E1178-1188. 10.1152/ajpendo.00002.2004View ArticleGoogle Scholar
- Grimble RF: Inflammatory status and insulin resistance. Curr Opin Clin Nutr Metab Care 2002, 5: 551-559. 10.1097/00075197-200209000-00015View ArticleGoogle Scholar
- Pickup JC, Crook MA: Is type II diabetes mellitus a disease of the innate immune system? Diabetologia 1998, 41: 1241-1248. 10.1007/s001250051058View ArticleGoogle Scholar
- Gilmore TD, Herscovitch M: Inhibitors of NF-kappaB signaling: 785 and counting. Oncogene 2006, 25: 6887-6899. 10.1038/sj.onc.1209982View ArticleGoogle Scholar
- Heynekamp J, Weber W, Hunsaker L, Gonzales A, Orlando R, Deck L, Jagt DV: Substituted trans-Stilbenes, Including Analogs of the Natural Product Resveratrol, Inhibit the TNFα-induced Activation of Transcription Factor NF-B. J Med Chem 2006, 49: 7182-7189. 10.1021/jm060630xView ArticleGoogle Scholar
- Weber WM, Hunsaker LA, Roybal CN, Bobrovnikova-Marjon EV, Abcouwer SF, Royer RE, Deck LM, Jagt DL: Activation of NFkappaB is inhibited by curcumin and related enones. Bioorg Med Chem 2006, 14: 2450-2461. 10.1016/j.bmc.2005.11.035View ArticleGoogle Scholar
- Aggarwal S, Ichikawa H, Takada Y, Sandur SK, Shishodia S, Aggarwal BB: Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IkappaBalpha kinase and Akt activation. Mol Pharmacol 2006, 69: 195-206.Google Scholar
- Brennan P, O'Neill LA: Inhibition of nuclear factor kappaB by direct modification in whole cells – mechanism of action of nordihydroguaiaritic acid, curcumin and thiol modifiers. Biochem Pharmacol 1998, 55: 965-973. 10.1016/S0006-2952(97)00535-2View ArticleGoogle Scholar
- Manna SK, Mukhopadhyay A, Aggarwal BB: Resveratrol suppresses TNF-induced activation of nuclear transcription factors NF-kappa B, activator protein-1, and apoptosis: potential role of reactive oxygen intermediates and lipid peroxidation. J Immunol 2000, 164: 6509-6519.View ArticleGoogle Scholar
- Nam NH: Naturally occurring NF-kappaB inhibitors. Mini Rev Med Chem 2006, 6: 945-951. 10.2174/138955706777934937View ArticleGoogle Scholar
- Singh S, Aggarwal BB: Activation of transcription factor NF-kappa B is suppressed by curcumin (diferuloylmethane) [corrected]. J Biol Chem 1995, 270: 24995-25000. 10.1074/jbc.270.25.14867View ArticleGoogle Scholar
- Singh S, Khar A: Biological effects of curcumin and its role in cancer chemoprevention and therapy. Anticancer Agents Med Chem 2006, 6: 259-270. 10.2174/187152006776930918View ArticleGoogle Scholar
- Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS: Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation. Mutat Res 2001, 480–481: 243-268.View ArticleGoogle Scholar
- Chainani-Wu N: Safety and anti-inflammatory activity of curcumin: a component of tumeric (Curcuma longa). J Altern Complement Med 2003, 9: 161-168. 10.1089/107555303321223035View ArticleGoogle Scholar
- Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, Ko JY, Lin JT, Lin BR, Ming-Shiang W, Yu HS, Jee SH, Chen GS, Chen TM, Chen CA, Lai MK, Pu YS, Pan MH, Wang YJ, Tsai CC, Hsieh CY: Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res 2001, 21: 2895-2900.Google Scholar
- Aggarwal BB, Bhardwaj A, Aggarwal RS, Seeram NP, Shishodia S, Takada Y: Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res 2004, 24: 2783-2840.Google Scholar
- Weber WM, Hunsaker LA, Gonzales AM, Heynekamp JJ, Orlando RA, Deck LM, Jagt DL: TPA-induced up-regulation of activator protein-1 can be inhibited or enhanced by analogs of the natural product curcumin. Biochem Pharmacol 2006, 72: 928-940. 10.1016/j.bcp.2006.07.007View ArticleGoogle Scholar
- Green H, Meuth M: An established pre-adipose cell line and its differentiation in culture. Cell 1974, 3: 127-133. 10.1016/0092-8674(74)90116-0View ArticleGoogle Scholar
- Green H, Kehinde O: Formation of normally differentiated subcutaneous fat pads by an established preadipose cell line. J Cell Physiol 1979, 101: 169-171. 10.1002/jcp.1041010119View ArticleGoogle Scholar
- Vannier C, Gaillard D, Grimaldi P, Amri EZ, Djian P, Cermolacce C, Forest C, Etienne J, Negrel R, Ailhaud G: Adipose conversion of ob17 cells and hormone-related events. Int J Obes 1985, 1: 41-53.Google Scholar
- Cousin B, Munoz O, Andre M, Fontanilles AM, Dani C, Cousin JL, Laharrague P, Casteilla L, Penicaud L: A role for preadipocytes as macrophage-like cells. FASEB J 1999,13(2):305-312.Google Scholar
- Fain JN, Ballou LR, Bahouth SW: Obesity is induced in mice heterozygous for cyclooxygenase-2. Prostaglandins Other Lipid Mediat 2001, 65: 199-209. 10.1016/S0090-6980(01)00136-8View ArticleGoogle Scholar
- Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ, Sole J, Nichols A, Ross JS, Tartaglia LA, Chen H: Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 2003, 112: 1821-1830.View ArticleGoogle Scholar
- Li Q, Verma IM: NF-kappaB regulation in the immune system. Nat Rev Immunol 2002, 2: 725-734. 10.1038/nri910View ArticleGoogle Scholar
- Aggarwal BB, Shishodia S: Molecular targets of dietary agents for prevention and therapy of cancer. Biochem Pharmacol 2006, 71: 1397-1421. 10.1016/j.bcp.2006.02.009View ArticleGoogle Scholar
- Bremner P, Heinrich M: Natural products as targeted modulators of the nuclear factor-kappaB pathway. J Pharm Pharmacol 2002, 54: 453-472. 10.1211/0022357021778637View ArticleGoogle Scholar
- Cancello R, Clement K: Is obesity an inflammatory illness? Role of low-grade inflammation and macrophage infiltration in human white adipose tissue. Bjog 2006, 113: 1141-1147.View ArticleGoogle Scholar
- Christiansen T, Richelsen B, Bruun JM: Monocyte chemoattractant protein-1 is produced in isolated adipocytes, associated with adiposity and reduced after weight loss in morbid obese subjects. Int J Obes (Lond) 2005, 29: 146-150. 10.1038/sj.ijo.0802839View ArticleGoogle Scholar
- Ryden M, Dicker A, van Harmelen V, Hauner H, Brunnberg M, Perbeck L, Lonnqvist F, Arner P: Mapping of early signaling events in tumor necrosis factor-alpha – mediated lipolysis in human fat cells. J Biol Chem 2002, 277: 1085-1091. 10.1074/jbc.M109498200View ArticleGoogle Scholar
- Souza SC, Palmer HJ, Kang YH, Yamamoto MT, Muliro KV, Paulson KE, Greenberg AS: TNF-alpha induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3-L1 adipocytes. J Cell Biochem 2003, 89: 1077-1086. 10.1002/jcb.10565View ArticleGoogle Scholar
- Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM: Tumor necrosis factor alpha inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA 1994, 91: 4854-4858. 10.1073/pnas.91.11.4854View ArticleGoogle Scholar
- Carlsen H, Moskaug JO, Fromm SH, Blomhoff R: In vivo imaging of NF-kappa B activity. J Immunol 2002, 168: 1441-1446.View ArticleGoogle Scholar
- Braun T, Carvalho G, Coquelle A, Vozenin MC, Lepelley P, Hirsch F, Kiladjian JJ, Ribrag V, Fenaux P, Kroemer G: NF-kappaB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome. Blood 2006, 107: 1156-1165. 10.1182/blood-2005-05-1989View ArticleGoogle Scholar
- Braun T, Carvalho G, Fabre C, Grosjean J, Fenaux P, Kroemer G: Targeting NF-kappaB in hematologic malignancies. Cell Death Differ 2006, 13: 748-758. 10.1038/sj.cdd.4401874View ArticleGoogle Scholar
- Haefner B: Targeting NF-kappaB in anticancer adjunctive chemotherapy. Cancer Treat Res 2006, 130: 219-245.View ArticleGoogle Scholar
- Magne N, Toillon RA, Bottero V, Didelot C, Houtte PV, Gerard JP, Peyron JF: NF-kappaB modulation and ionizing radiation: mechanisms and future directions for cancer treatment. Cancer Lett 2006, 231: 158-168. 10.1016/j.canlet.2005.01.022View ArticleGoogle Scholar
- Redell MS, Tweardy DJ: Targeting transcription factors for cancer therapy. Curr Pharm Des 2005, 11: 2873-2887. 10.2174/1381612054546699View ArticleGoogle Scholar
- Kundu JK, Surh YJ: Molecular basis of chemoprevention by resveratrol: NF-kappaB and AP-1 as potential targets. Mutat Res 2004, 555: 65-80.View ArticleGoogle Scholar
- Kundu JK, Shin YK, Kim SH, Surh YJ: Resveratrol inhibits phorbol ester-induced expression of COX-2 and activation of NF-kappaB in mouse skin by blocking IkappaB kinase activity. Carcinogenesis 2006, 27: 1465-1474. 10.1093/carcin/bgi349View ArticleGoogle Scholar
- Walle T, Hsieh F, DeLegge MH, Oatis JE Jr, Walle UK: High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos 2004, 32: 1377-1382. 10.1124/dmd.104.000885View ArticleGoogle Scholar
- Yu C, Shin YG, Chow A, Li Y, Kosmeder JW, Lee YS, Hirschelman WH, Pezzuto JM, Mehta RG, van Breemen RB: Human, rat, and mouse metabolism of resveratrol. Pharm Res 2002, 19: 1907-1914. 10.1023/A:1021414129280View ArticleGoogle Scholar
- Carmody RJ, Chen YH: Nuclear factor-kappaB: activation and regulation during toll-like receptor signaling. Cell Mol Immunol 2007, 4: 31-41.Google Scholar
- Jager J, Gremeaux T, Cormont M, Le Marchand-Brustel Y, Tanti JF: Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression. Endocrinology 2007, 148: 241-251. 10.1210/en.2006-0692View ArticleGoogle Scholar
- Lagathu C, Yvan-Charvet L, Bastard JP, Maachi M, Quignard-Boulange A, Capeau J, Caron M: Long-term treatment with interleukin-1beta induces insulin resistance in murine and human adipocytes. Diabetologia 2006, 49: 2162-2173. 10.1007/s00125-006-0335-zView ArticleGoogle Scholar
- Hsu CH, Cheng AL: Clinical studies with curcumin. Adv Exp Med Biol 2007, 595: 471-480.View ArticleGoogle Scholar
- Sharma RA, Steward WP, Gescher AJ: Pharmacokinetics and pharmacodynamics of curcumin. Adv Exp Med Biol 2007, 595: 453-470.View ArticleGoogle Scholar
- Baur JA, Sinclair DA: Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov 2006, 5: 493-506. 10.1038/nrd2060View ArticleGoogle Scholar
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