Human obesity is closely associated with a state of chronic low-grade inflammation, which also involves the adipose tissue with enhanced production of bioactive substances (adipokines). Calorie restriction (CR) reduces adipocytokine production and improves metabolic profile in rodents. Some of these effects are mediated through activation of the sirtuin 1 (Sirt1) enzyme, and in this study, we investigate whether the natural phytoalexin, resveratrol (RSV), which is a potent Sirt1 activator, has anti-inflammatory effects in human adipose tissue explants.
The effect of RSV on interleukin 1β (IL1β)-induced change of adipokine mRNA gene expression and secretion were measured in human adipose tissue explants.
Exposure of human adipose tissue in vitro to IL1β for 24 h increased secretion of the proinflammatory adipokines IL6, IL8 and monocyte chemoattractant protein 1 (MCP-1) 3–7.7-fold (P<0.05) and increased IL6, IL8, MCP-1, IL1β and PAI-1 mRNA expression 1.3–7.2-fold (P<0.05) accordingly. Concomitant incubations with RSV reversed the IL1β-stimulated secretion (16–36%) and gene expression (25–48%) of these adipokines. IL1β reduced adiponectin mRNA expression (40%), a decrement that was reversed by RSV treatment. Similar effects were observed in differentiated human preadipocytes in primary culture, indicating that human adipocytes are a potential target for RSV effects. Finally, the effects were neutralized by sirtinol, a Sirt1 inhibitor.
This study is the first to show anti-inflammatory effects of RSV on adipokine expression and secretion in human adipose tissue in vitro through the SIRT1 pathway. Thus, RSV is hypothesized to possess beneficial effects and might improve the metabolic profile in human obesity.
It is now well described that calorie restriction (CR) extends life span in organisms ranging from yeast to rodents.1, 2, 3 Already in 1934, Clive McCay and Mary Crowell showed how laboratory rodents fed 20–40% of ad libitum intake increased their max life span by up to 50%.4 Studies in humans also indicate that CR diet improves some markers of aging, that is levels of blood glucose, blood pressure and cholesterol.5, 6 The cellular pathways signalling the effects of CR are complex, but involve increased activity of sir2 in yeast and Sirt1 the mammal ortholog.2, 7 Food deprivation activates Sirt1 in mice, promoting fat mobilization and leanness.8
Large-scale screening of compounds capable of activating Sirt1 showed that resveratrol (RSV), which is a naturally occurring polyphenolic phytoalexin present in the skin of grape, berries and peanuts posseses strong Sirt1-activating properties.9
RSV treatment results in diminished fat accumulation. Some studies indicate increased lipolysis.8, 10, 11 Other studies found that increased Sirt1 activity in 3T3-L1 cells represses the pro-adipogenic nuclear receptor-γ.8 Finally, some studies indicate that some of the metabolic and anti-inflammatory effects of RSV may be mediated by other pathways such as interference with the estrogen receptors (ERs)12 and interaction with the nuclear factor-κB (NF-κB).13
Metabolic syndrome is characterized by obesity, insulin resistance, type 2 diabetes, cardiovascular diseases and premature death.14 It is now clear that obesity is associated with a low-grade chronic inflammation, characterized by abnormal levels of circulating proinflammatory factors and an abnormal production of bioactive factors/adipcytokines from the adipose tissue.15 These adipocytokines are suggested to have direct implications for the development of the metabolic syndrome.14, 16
We and others have provided clear evidence that the proinflammatory adipokines tumor necrosis factor (TNF-α), interleukin 6 (IL6) and IL8 are increased in obese subjects both with and without type 2 diabetes.17, 18 Both experimental and clinical findings suggest that IL1β is also associated with the pathology underlying these diseases.19 IL1β, IL6 and TNF-α are produced mainly by activated macrophages,20 but adipocytes and preadipocytes also produce these proinflammatory cytokines.21 A recent study indicated that IL1β induces insulin resistance in cultured murine and human adipocytes, and IL1β was upregulated in adipose tissue from an obese and insulin-resistant mouse model.22 IL1β has been shown earlier to decrease gene expression of adiponectin in human adipose tissue.23 Adiponectin is a protein uniquely produced by adipocytes24 and it has a positive influence on insulin sensitivity (anti-hyperglycemic effects).25 Circulating adiponectin is decreased in obese and insulin-resistant subjects, which is associated to obesity-related disease.26 Growing evidence now indicates that adiponectin mediates anti-atherogenic effects and increases insulin sensitivity in peripheral tissue such as liver and skeletal muscle, contributing to overall glucose homeostasis.27, 28
IL1β may also contribute to the increased production of monocyte chemoattractant protein 1 (MCP-1),29 which is elevated in obese subjects, and associated with recruitment of and macrophage infiltration in adipose tissue.30, 31 Adipose tissue macrophages are assumed to be an important source of inflammatory mediators such as TNF-α, IL8, IL6 and IL1β, interfering with adipocyte function by inhibiting insulin action.20 Thus, adipose tissue macrophages may be an essential link among chemokines between the low-grade chronic inflammation and insulin resistance.
Recently, studies have revealed that RSV inhibits the production of the proinflammatory adipokines TNF-α, MCP-1, IL6, PAI-1 and increases adiponectin production in 3T3-L1 cells.32, 33 Earlier studies with RSV and adipose tissue have mainly been performed in rodents or in cell cultures. However, we recently showed that Sirt1 is expressed in human adipose tissue, and Sirt1 expression in human adipose tissue was also upregulated by severe CR.10 In addition, we found that RSV in vitro increased the adrenergically-induced lipolysis similar to the findings in rodents. Thus, RSV may have direct effects in human adipose tissue on inflammation, insulin resistance and metabolic syndrome through some of these mediators.
In this study, we therefore investigated whether RSV can influence the production of various adipokines, also in human adipose tissue. Our hypothesis is that RSV possesses beneficial effects in human adipose tissue that may help fight the metabolic syndrome by changing the local production of adipokines released from human adipose tissue.
Subcutaneous abdominal adipose tissue was obtained from eight normal to overweight subjects (seven women, one man, age 43.6±11.9 years, body mass index 25.3±3.9 kg m−2). The subjects were all Caucasian, healthy, and did not receive any medication known to influence adipose tissue metabolism. Adipose tissue was obtained by liposuction performed in general anaesthesia (propophol, ramifentanil and fentanyl) at a cosmetic surgery clinic. The adipose tissue was mixed with isotonic saline and transported to the laboratory in a sterile container within 30 min after removal. The study was approved by the Local Ethics Committee at the institution and all subjects gave written informed consent.
RSV was purchased from Cayman Chemical, Ann Arbor, MI, USA and dissolved in dimethyl sulfoxide (DMSO) (final DMSO concentration in incubations=0.05%). ER antagonist (ICI 182780), Tocris Bioscience Ellisville, MO, USA. Fetal calf serum, bovine serum albumin, amphotericin B and penicillin/streptomycin, Invitrogen A/S, Thistedgade 6, 2630 Tåstrup, Denmark. All other reagents were purchased from Sigma Aldrich, Saint Louis, MO, USA, unless otherwise stated.
Adipose tissue incubation
All subsequent procedures were carried out under a laminar airflow hood and under sterile conditions. The adipose tissue was placed in Medium 199, cut into smaller fragments (∼10–20 mg each), rinsed in NaCl. Then, 500 mg adipose tissue per 5 ml incubation medium was used per tube. All incubations were performed in duplicate. After preincubation for 24 h, the medium was replaced with incubation medium containing IL1β and RSV at the indicated concentrations. After incubating for 24 h, the adipose tissue and medium were harvested, snap frozen in liquid nitrogen and kept at −80 °C. The medium was used for later measurement of adipokine secretion and the tissue was stored for RNA isolation.
Primary culture of human preadipocytes
The tissue sample was rinsed in phosphate-buffered saline to remove adhering blood. Adipose tissue was digested using 1 mg ml−1 collagenase in phosphate-buffered saline containing 20 mg ml−1 bovine serum albumin, pH 7.4, for 50 min under intermittent shaking. Stromal cells were inoculated in DME/Ham's F-12 medium (1:1 vol/vol) supplemented with 10% fetal calf serum, 15 nM NaHCO3, 15 mM Hepes and antibiotics. After 16–20 h, the medium was removed, and the cells were repeatedly washed. Then, the cells were cultured in serum-free DME/Ham's F-12 medium supplemented with 33 mM biotin, 17 mM pantothenate, 10 mg ml−1 human transferrin and to induce adipose differentiation 100 nM cortisol, 100 nM insulin, 200 pM triiodothyronine and, for the first 3 days, 0.2 mM isobutylmethylxanthine. The medium was changed every 2–3 days. At day 6, the cells started to accumulate visible lipid droplets, and at day 12 >80% were filled with lipid droplets. At this time, the cells were stimulated with the indicated agents (IL1β, RSV and Sirtinol).
Isolation of RNA
Total RNA was isolated from the biopsies and cells using the TriZol reagent (Gibco BRL, Life Technologies, Roskilde, Denmark); RNA was quantified by measuring absorbance at 260 and 280 nm, and the ratio should be 1.8. Finally, the integrity of the RNA was checked by visual inspection of the two ribosomal RNAs, 18S and 28S, on an agarose gel.
Real-time reverse transcriptase polymerase chain reaction
For real-time reverse transcriptase polymerase chain reaction (PCR), complementary DNA was constructed using random hexamer primers as described by the manufacturer (GeneAmp RNA PCR Kit from PerkinElmer Cetus, Norwalk, CT, USA). PCR mastermix, containing the specific primers, Hot star Taq DNA polymerase and SYBR-Green PCR buffer were then added. The following primers were designed using the primer analysis software Oligo version 6.64 and synthesis performed by DNA Technology A/S Voldbjergvej 16, 8240 Risskov, Denmark: adiponectin: sense primer 5′-CATGACCAGGAAACCACGACT-3′ and antisense primer 5′-TGAATGCTGAGCGGTAT-3′, MCP-1: sense primer 5′-GACATCCTGGAACTGCCCTACC-3′ and antisense primer 5′-ACTGTGCCGCTCTCGTTCAC-3′, IL1β: sense primer 5-ATGGCAGAAGTACC TGAGCTC-3′ and antisense primer 5′-TTAGGAAGACACAAATTGCAT-3′. IL6: sense primer 5′-AAATGCCAGCCTGCTGACGAAC-3′ and antisense primer 5′-AACAACAATCTGAGGTGCCCATGCTAC, IL8: sense primer 5′-TTGGCAGCCT TCCTGATT-TC-3′, and antisense primer 5′-AACTTCTCCACAACCCTCTG-3′, PAI-1: sense primer 5′-CGACATCCTGGAACTGCCCTACC-3′ and antisense primer 5′-CACTGTGCCGCTCTCGTTC-AC-3′, β-Actin was used as house-keeping gene: sense primer 5′-ACGGGGTCACCCAC-ACTGTGC-3′ and antisense primer 5′-CTAGAAGCATTTGCGGTGGACGATG-3′. Real-time quantification of genes was performed by SYBR-green real-time reverse transcription PCR assay (Qiagen, Valencia, CA, USA) using an ICycler from Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA). Gene mRNA was amplified in separate tubes and the increase in fluorescence was measured in real time. The threshold cycle was calculated, and the relative gene expression was calculated essentially as described in the User Bulletin no. 2, 1997, from Perkin-Elmer. All samples were amplified in duplicate. A similar set-up was used for negative controls, except that the reverse transcriptase was omitted, and no PCR products were detected under these conditions.
ELISA measurement of MCP-1, IL8 and IL6
Cytokine protein concentration in culture medium was measured using a commercial human ELISA assay (MCP-1, IL8 and IL6 DuoSet; R&D Systems Europe Ltd, Abingdon, UK) with an intraassay coefficient of variation of 4.4% (n=20). The MCP-1, IL8 and IL6 samples were diluted 1:100, 1:800 and 1:200, respectively, to be within the assay range (31.2–1000 ng l−1).
Differences between group means were determined using one-way analysis of variance with Bonferroni post hoc test. The level of significance was 0.05. Data represent mean±s.e.m. All analyses were performed with SigmaStat (Systat software, Inc., Richmond, CA, USA) statistical software.
Initially, a concentration–response curve was performed, and a concentration of RSV at 50 μM was found to be most effective in reducing the IL1β-induced MCP-1 expression in human adipose tissue fragments (Figure 1). Therefore, 50 μM RSV was chosen in all subsequent experiments. This concentration level is well in line with other studies using different cell types, where 50 μM RSV is most often used.33, 34 In addition, a time-course analysis was performed (6–72 h), showing that using 24-h incubations were optimal in showing effects of RSV (data not shown).
We have tested several different housekeeping genes, and β-actin was stabily expressed in the different study groups, and therefore, it was chosen as a reference gene in this study.
Effects of RSV on mRNA levels of adipokines in human adipose tissue explants
Effects on IL6, IL8, IL1β, MCP-1 and PAI-1
Stimulation with IL1β (2 ng ml−1) for 24 h increased adipose tissue IL6 mRNA expression 2.6-fold (P<0.05; Figure 2a). Concomitant incubation with RSV (50 μM) reduced this increase with 35% (P<0.05). Similarly, adipose tissue IL6 secretion to the media was increased after IL1β stimulation threefold (P<0.05), and concomitant incubation with RSV (50 μM) reduced this increase by 16% (P<0.05; Figure 3a).
Very similar changes were obtained concerning IL8, MCP-1 and PAI-1: stimulation with IL1β increased IL8, MCP-1 and PAI-1 mRNA expression by 7.2 (P<0.05), 1.8 (P<0.05) and 1.3-fold (P<0.05), respectively. In all cases, this was reduced significantly by RSV as shown in Figure 2. Secretion of IL8 and MCP-1 to the media was increased by IL1β stimulation 7.7 (P<0.05) and 2.6-fold (P<0.05), respectively, and reduced by RSV, though not significantly for MCP-1. Finally, incubation with IL1β was able to upregulate its own gene expression 7.7-fold (P<0.05), and RSV was able to inhibit this stimulation of IL1β by 37% (P<0.05; Figure 2c).
Effects on adiponectin
Well in line with the earlier findings showing that IL1β induced an increase in adipose tissue cytokine production, we found IL1β to inhibit adipose tissue production of the anti-inflammatory adipokine; adiponectin. Incubation with IL1β (2 ng ml−1) for 24 h decreased adipose tissue adiponectin mRNA expression by 40% (P<0.05), which could be normalized by concomitant RSV stimulation (P<0.05; Figure 2f).
Effect of RSV on MCP-1 gene expression in differentiated human preadipocyte in primary culture
As human adipose tissue explants contain many different cell types, we isolated and differentiated human preadipocyte to study whether mature adipocytes were affected by RSV. Stimulation with IL1β for 24 h increased MCP-1 mRNA expression 40-fold (P<0.05; Figure 4). Similar findings were observed for IL6 and IL8 (data not shown) Concomitant incubation with RSV (50 μM) reduced this increase by 50% (P<0.05; Figure 4), indicating that RSV affected adipocyte adipokine production.
Effects of Sirt1 and ER antagonist
Parallel incubation with Sirtinol 10 μM (an inhibitor of the Sirt1 enzyme activity) was able to diminish the anti-inflammatory effect of RSV (P<0.05; Figure 4), indicating that the anti-inflammatory effects of RSV, at least in part, are mediated through the Sirt1 enzyme. Similarly, incubating with 1 μM ER antagonist (ICI 182780), showed no significant effect on the ant-inflammatory effects of RSV.
Testing for hypoxia
We have measured LDH, pH and lactate in the medium and found no indication of hypoxia or cell death during our incubations (data not shown).
Low-grade inflammation in the adipose tissue has recently been implicated in the pathogenesis of the metabolic syndrome14, 35 and is probably linked to high local production of cytokines in the adipose tissue.14 RSV, which is a polyphenolic phytoestrogen and a potent Sirt1 activator, has received increased interest, due to beneficial metabolic effects in animal models.11, 36 A recent study showed how mice on high-fat diet treated with RSV were protected against obesity and insulin resistance, both characteristic features of the metabolic syndrome.11 Some of these effects of RSV might be mediated in a Sirt1-dependent manner.36 Recently, we found that Sirt1 is present in human adipose tissue and is upregulated in response to severe CR.10 New results showed how RSV effectively inhibits TNF-α-induced secretion of MCP-1, IL6 and PAI, while restoring the decreased adiponectin secretion, in 3T3-L1 adipocytes.32, 33 In this respect, Sirt1 activation posed anti-inflammatory effect, by decreasing TNF-α-induced NF-κB activity in 3T3-L1 adipocytes.33 In addition, recent findings illustrate how Sirt1 in a non-adipose human cell line, modulates NF-κB transcription, by deacetylating the Re1/p65 subunit of NF-κB at lysine 310 subunit.37 Independent of Sirt1 activation, RSV itself, might also have direct antagonistic effect on TNF-α-induced NF-κB activity, improving the inflammatory profile in adipose tissue.33 Finally, the ERs α and β are present in human adipocytes,38 and some very new findings indicate that some of the positive metabolic effects of RSV on glucose metabolism in rodents are mediated through binding to especially ERα by RSV.12 Thus, the exact molecular pathways involved in our findings of reduced cytokine production in human adipose tissue caused by RSV stimulation are still not known.
In this study, we showed that RSV in vitro regulated the gene expression and secretion of several adipokines from human adipose tissue in organ culture. The general pattern was that RSV changed the adipokine expression and secretion in a beneficial direction. We used IL1β stimulation to increase the adipokine production and in parallel tubes studied the effects of added RSV. In all situations, RS diminished the gene expression of the inflammatory adipokines like IL6, IL8, MCP-1, IL1β and PAI-1, and increased adiponectin gene expression. When studying the secreted adipokines to the incubation medium, a similar pattern was observed that is IL1β stimulation increases the secretion of IL8, IL6 and MCP-1, whereas concomitant RSV stimulation decreases the secreted adipokines (although not significantly for MCP-1 secretion). In this study, the IL1β-induced mRNA response is relatively small, whereas protein response is considerable larger, suggesting different time dependencies. However, in earlier studies using the same technique, we have been able to show a robust increase in the inflammatory response in human adipose tissue fragments, cultured under similar circumstances.39 As human tissue fragments consist of several different cell types, the observed positive response to RSV could be mediated in each of these cell types. Therefore, we used differentiated human preadipocytes, to study whether adipocytes can be targeted directly by RSV. These studies clearly showed that the adipocyte inflammatory response could be controlled directly by RSV in differentiated human adipocytes in primary culture.
Finally, we found that sirtinol, a Sirt1 inhibitor, significantly neutralized RSV-induced decrement of IL1β-induced MCP-1 gene expression, indicating that the effects of RSV are, at least to some extent, mediated through the Sirt1 enzyme in human adipocytes. We also studied whether an ER antagonist could modify the RSV effects, but ER blockage did not change the effects of RSV in human adipocytes.
In conclusion our results show that RSV ameliorates the proinflammatory response in human adipose tissue and increases adiponectin expression changes, which is hypothetically beneficial, as adipose tissue from obese individuals with metabolic syndrome, expresses lower adiponectin levels and higher levels of proinflammatory adipokines.40 We are aware that it is difficult to compare in vitro incubations and in vivo conditions, and especially it is difficult to know whether the concentrations used in our study are a meaningful in a clinical setting. It is known that orally administrated RSV is rapidly absorbed and then quickly metabolized, but the local concentration of RSV in different tissue compartments are unknown and it is also not known if glucuronidated RSV can be locally de-glucuronidated and thus reach a higher local concentration.41 However, the concentration used in our in vitro experiments (50 μM) is similar to most other reports on RSV effects in different cell types.32, 42
In conclusion, this study is the first to show anti-inflammatory effects of RSV on adipokine expression and secretion in human adipose tissue in vitro. Small interfering molecules such as RSV are in this matter hypothesized to possess beneficial effects and might improve the metabolic profile in human obesity. The present in vitro study must therefore be followed by in vivo studies where the safety and efficacy of Sirt1 activators can be determined for the treatment of the metabolic syndrome.
The expert technical assistance of Lenette Pedersen and Pia Hornbek is appreciated. The study was part of the DanORC consortium. DanOrc is supported by the Danish Council for Strategic Research. The study was supported by grants from NovoNordisk Foundation and the Danish Medical Research Council.