Sulfasalazine and BAY 11-7082 Interfere with the Nuclear Factor-B and IB Kinase Pathway to Regulate the Release of Proinflammatory Cytokines
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内分泌学杂志 2005年第3期
Department of Obstetrics and Gynecology, University of Melbourne and Mercy Perinatal Research Center, Mercy Hospital for Women, East Melbourne, 3002 Victoria, Australia
Address all correspondence and requests for reprints to: Dr. Martha Lappas, Department of Obstetrics and Gynecology, University of Melbourne, Mercy Hospital for Women, 126 Clarendon Street, East Melbourne, 3002 Victoria, Australia. E-mail: mlappas@unimelb.edu.au.
Abstract
There is much evidence to indicate a role for adipocytokines in insulin resistance and/or type 2 diabetes mellitus. In experimental models, oral salicylates, through their ability to interfere with the nuclear factor-B (NF-B) transcription pathway, have been demonstrated to reverse insulin resistance. The aim of this study was to investigate whether NF-B regulates the release of adipocytokines in human adipose tissue and skeletal muscle. Human sc adipose tissue and skeletal muscle (obtained from normal pregnant women) were incubated in the absence (control) or presence of two NF-B inhibitors sulfasalazine (1.25, 2.5, and 5 mM) and BAY 11-7082 (25, 50, and 100 μM). After an 18-h incubation, the tissues were collected, and NF-B p65 DNA-binding activity and IB kinase (IKK-?) and insulin receptor-? protein expression were assessed by ELISA and Western blotting, respectively. The incubation medium was collected, and the release of TNF-, IL-6, IL-8, resistin, adiponectin, and leptin was quantified by ELISA. Treatment of adipose tissue and skeletal muscle with sulfasalazine and BAY 11-7082 significantly inhibited the release of IL-6, IL-8, and TNF-; NF-B p65 DNA-binding activity; and IKK-? protein expression (P < 0.05, by Newman-Keuls test). There was no effect of sulfasalazine and BAY 11-7082 on resistin, adiponectin, or leptin release. Both sulfasalazine and BAY 11-7082 increased the adipose tissue and skeletal muscle expression of insulin receptor-?. The data presented in this study demonstrate that the IKK-?/NF-B transcription pathway is a key regulator of IL-6, IL-8, and TNF- release from adipose tissue and skeletal muscle. Control of the IKK-?/NF-B pathway may therefore provide an alternative therapeutic strategy for regulating aberrant cytokine release and thereby alleviating insulin resistance in type 2 diabetes mellitus.
Introduction
INSULIN RESISTANCE IS defined as a failure of target tissues (including adipose tissue and skeletal muscle) to respond normally to insulin. Obesity is a major risk factor for insulin resistance and type 2 diabetes, and adipocytes secrete a number of cytokines, including TNF-, IL-6, IL-8, adiponectin, resistin, and leptin, all of which have been implicated in insulin resistance in type 2 DM (1, 2, 3, 4). TNF- and resistin appear to impair glucose tolerance, whereas leptin and adiponectin work to improve the metabolic status. We have recently demonstrated that in human gestational tissues, the nuclear factor-B (NF-B) signaling pathway is a regulator of TNF-, IL-6, and IL-8 release (5); therefore, the aim of this study was to determine whether NF-B also regulates cytokine release from adipose tissue and skeletal muscle, in particular TNF-, IL-6, IL-8, resistin, adiponectin, and leptin. Understanding how adipocytokines are regulated by drugs influencing insulin sensitivity will have major and important implications for the treatment of insulin resistance and obesity. The IB kinase (IKK-?)/NF-B pathway might impair insulin sensitivity at least in part via up- or down-regulation of adipocytokine synthesis. Pregnancy is a state of insulin resistance; therefore, the availability of adipose tissue and skeletal muscle from women undergoing elective cesarean section allows us the opportunity to study the regulation of cytokines in these tissues.
NF-B is a ubiquitous transcription factor that by regulating the expression of multiple inflammatory and immune genes plays a critical role in host defense and chronic inflammatory diseases (6). At least five genes belong to the NF-B family, with the most common NF-B dimer composed of the RelA (p65) and p50 subunits. NF-B dimers are usually sequestered in the cytoplasm bound to an inhibitory subunit, IB-. Upon stimulation, IB- is phosphorylated by the action of a specific IKK, ubiquitinated, and rapidly degraded resulting in the rapid translocation of NF-B to the nucleus, where it binds specific DNA elements (B motifs) in the promoter/enhancer region of target genes to initiate, enhance, or suppress the transcriptional process.
Inflammation has been implicated as a major pathogenic mediator of type 2 diabetes mellitus and/or insulin resistance. Elevated levels of acute phase reactant and inflammatory mediators circulate in patients with type 2 diabetes mellitus (7), and inflammatory cytokines have been shown to attenuate insulin signaling in experimental models (3). Furthermore, the ability of salicylates, including sulfasalazine, aspirin, and sodium salicylate, to lower blood sugar has been recognized since the late 19th century (8). Recently, Juan et al. (9) demonstrated that high doses of salicylates reverse high blood sugar, high insulin, and high blood fat levels in obese rodents by inhibiting the NF-B and its upstream activator, IKK-?. In recent studies we have demonstrated that the antiinflammatory effects of sulfasalazine could also be attributed to its ability to inhibit NF-B DNA-binding activity (5). However, the effects of salicylates on cytokine release and insulin signaling from human adipose tissue and skeletal muscle are not known and may have important implications for the treatment of type 2 diabetes.
The hypothesis to be tested is that inhibition of NF-B DNA-binding activity suppresses the release of TNF-, IL-6, IL-8, resistin, adiponectin, and leptin from adipose tissue and skeletal muscle. Tissues were incubated in the presence of sulfasalazine (1.25, 2.5, and 5 mM) and BAY 11-7082 (an inhibitor of IB kinase (10); (E)-3-[4-methylphenylsulphonyl]2-propenenitrile); NF-B p65 DNA-binding activity in nuclear extracts was analyzed by a ELISA; IKK-?, and insulin receptor-? (IR-?) tissue protein expression were analyzed by Western blotting; and the release of TNF-, IL-6, and IL-8 into the incubation medium was quantified by ELISA.
Materials and Methods
Reagents
All chemicals were purchased from BDH Chemicals Australia (Melbourne, Victoria, Australia) unless stated otherwise. DMEM was obtained from Invitrogen Life Technologies, Inc. (Grand Island, NY). BSA (RIA grade), dithiothreitol, EDTA, leupeptin, lipopolysaccharides (from Escherichia coli 026:B6), ?-NADH (disodium salt), 3,3',5,5'-tetramethylbenzidine, pyruvic acid (dimer free), and sulfasalazine were supplied by Sigma-Aldrich Corp. (St. Louis, MO). BAY 11-7082 was purchased from Sapphire Bioscience (Crows Nest, Australia). Pefabloc SC [4-(2-amnoethyl)-benzenesulfonyl fluoride] was purchased from Roche (Mannheim, Germany). Streptavidin-horseradish peroxidase conjugate and the IL-6, IL-8, TNF-, and leptin kits were supplied by BioSource International (Camarillo, CA). The resistin and adiponectin ELISA kits were supplied by CytoLab (Rehovot, Israel) and R&D Systems (Minneapolis, MN), respectively. NF-B p65 ELISA was purchased from BD Clontech (Palo Alto, CA). Rabbit polyclonal IKK-? polyclonal antibody, rabbit polyclonal IR-? polyclonal antibody, and horseradish peroxidase-conjugated goat antirabbit immunoglobulin G were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Tissue collection and preparation
Subcutaneous adipose tissue (from the anterior abdominal wall) and skeletal muscle (pyramidalis) where obtained from 10 pregnant women who delivered healthy, singleton infants at term (37 wk gestation) after undergoing elective cesarean section (indications for cesarean section were breech presentation and/or previous cesarean section). The clinical characteristics of the subjects are collated in Table 1. Approval for this study was obtained from the Mercy Hospital for Women’s research and ethics committee, and informed consent was obtained from all participating subjects.
TABLE 1. Characteristics of the study group
Tissues were obtained within 10 min of delivery, and dissected fragments were placed in ice-cold PBS. Tissue were dissected into small fragments visibly free of connective tissue and blood. Tissue fragments were placed in DMEM at 37 C in a humidified atmosphere of carbogen gas (95% O2 and 5% CO2) for 1 h. Explants were blotted dry on sterile filter paper and transferred to 24-well tissue culture plates (100–150 mg wet weight/well). The explants were incubated in duplicate in 2 ml DMEM containing penicillin G (100 U/ml) and streptomycin (100 μg/ml). Tissues were incubated in the absence (control) or presence of sulfasalazine (1.25, 2.5, and 5 mM) or BAY 11-7082 (25, 50, and 100 μM). The concentrations used in this study were chosen according to our previous studies (5).
Validation of explant cultures and viability
To determine the effects of sulfasalazine and BAY 11-7082 treatment on cell membrane integrity, the release of the intracellular enzyme lactate dehydrogenase (LDH) into incubation medium was determined as described previously (5). LDH release was investigated over the 18-h time course of tissue explants. Explants were incubated in either medium alone or medium containing sulfasalazine (1.25, 2.5, and 5 mM) or BAY 11-7082 (25, 50, and 100 μM). Neither in vitro incubation nor experimental treatment with sulfasalazine or BAY 11-7082 significantly affected LDH activity in the incubation medium (data not shown). These data indicate that the concentrations used in this study did not affect cell viability.
Experimental assays
After an 18-h incubation, the explant incubation medium was collected, and the release of IL-6, IL-8, TNF-, and leptin was determined by sandwich ELISA according to the manufacturer’s instructions (BioSource International, Camarillo, CA). The limits of detection of the IL-6, IL-8, TNF-, and leptin assays (defined as 2 SD from the zero standard) were 3, 2.8, 7.2, and 7.2 pg/ml, respectively. The release of resistin (CytoLab, Rehovot, Israel) and adiponectin (R&D Systems) in the explant incubation medium was determined by sandwich ELISA according to the manufacturer’s instructions. The intra- and interassay coefficients of variation for the resistin ELISA were 3.4% and 4.5%, respectively, and the minimum detectable limit of the assay was 16 pg/ml. The intra- and interassay coefficients of variation for the adiponectin ELISA were 4.2% and 4.7%, respectively, and the minimum detectable limit of the assay was 32 pg/ml. All data were corrected for total protein and expressed as picograms per milligram of protein. The protein content of tissue homogenates was determined using the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL) using BSA as a reference standard, as previously described (5).
Assessment of NF-B p65 DNA-binding activity by ELISA
After the 18-h incubation, tissues were collected, and protein was extracted as previously described (5). NF-B DNA binding was assessed using a commercially available NF-B p65 ELISA according to manufacturer’s instructions (BD Clontech) in which TNF--stimulated HeLa nuclear protein extract was used as a positive control for NF-B activation, and the specificity of NF-B binding was assessed using wild-type and mutated consensus oligonucleotides. A Benchmark Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA) was used to read the sample absorbance, and data are expressed as absorbance at 655 nm.
Assessment of IKK-? and IR-? protein expression by Western blotting
Assessment of IKK-? and IR-? protein expression in tissues was analyzed by Western blotting. Forty micrograms of tissue protein extracts were separated on a 10% polyacrylamide gel and transferred to nitrocellulose as previously described (11). Protein expression of IKK-? or IR-? was identified by comigration with a positive control and by comparison with the mobility of protein standard.
Statistical analysis
Statistical analyses were performed using a commercially available statistical software package (STSC, Statgraphics, Rockville, MD). The homogeneity of data was assessed by Bartlett’s test, and when significant, data were logarithmically transformed before additional analysis. Comparisons between groups were performed using Newman-Keuls multiple range tests. Statistical difference was indicated by P < 0.05. Data are expressed as the mean ± SEM and represent replication with independent tissue explants obtained from at least three women.
Results
Effects of sulfasalazine and BAY 11-7082 on NF-B-binding activity
The binding ability of NF-B p65 to DNA consensus sequences was measured using a commercially available kit. An NF-B wild-type consensus oligonucleotide was used to monitor the specificity of the assay. The wild-type oligonucleotide, by competing for NF-B binding to the probe immobilized on the plate, acted as an effective competitor for NF-B p65 binding (data not shown). The specificity of binding was also demonstrated using wells coated with mutated consensus oligonucleotide. In these experiments no binding was detected in the presence of the positive control (data not shown). Incubation of adipose tissue and skeletal muscle extracts with sulfasalazine at 2.5 and 5 mM significantly inhibited NF-B p65 DNA-binding activity (Fig. 1A; n = 3). Incubation of adipose tissue with BAY 11-7082 at all concentrations tested significantly inhibited NF-B p65 DNA-binding activity (Fig. 1B; n = 3), whereas in skeletal muscle, BAY 11-7082 at 50 and 100 μM significantly inhibited NF-B p65 DNA-binding activity (Fig. 1B; n = 3).
FIG. 1. Effects of 1.25, 2.5, and 5 mM sulfasalazine (A; n = 5) and 25, 50, and 100 μM BAY 11-7082 (B; n = 3) on NF-B p65 DNA-binding activity in human adipose tissue () and skeletal muscle (). *, P < 0.05 vs. basal adipose tissue NF-B p65 DNA-binding activity; , P < 0.05 vs. basal skeletal muscle NF-B p65 DNA-binding activity.
Effects of sulfasalazine and BAY 141-7082 on IKK-?
A band corresponding to approximately 85 kDa was identified as IKK-? and was present in all samples of adipose tissue and skeletal muscle. Both sulfasalazine (Fig. 2A; n = 3) and BAY 11-7082 (Fig. 2B; n = 3) at the highest concentrations reduced IKK-? protein in adipose tissue and skeletal muscle.
FIG. 2. Effects of 1.25, 2.5, and 5 mM sulfasalazine (A) and 25, 50, and 100 μM BAY 11-7082 (B) on IKK-? protein expression in human adipose tissue () and skeletal muscle (). *, P < 0.05 vs. basal adipose tissue IKK-? protein expression; , P < 0.05 vs. basal skeletal muscle IKK-? protein expression. A representative Western blot is also shown, demonstrating the effects of sulfasalazine and BAY 11–7082 on IKK-? protein.
Effect of sulfasalazine on cytokine release
The release of TNF- from adipose tissue and skeletal muscle was detectable in only three of the six adipose tissue samples and three of the seven skeletal muscle samples tested. In these tissues, TNF- release (Fig. 3A) was significantly inhibited at all sulfasalazine concentrations tested. Treatment of adipose tissue with sulfasalazine completely suppressed TNF- release, whereas in skeletal muscle, complete suppression was observed at 5 mM sulfasalazine. IL-6 and IL-8 release was detected from all adipose and skeletal muscle tissue samples. Sulfasalazine concentrations greater than 2.5 mM significantly suppressed both IL-6 (Fig. 3B) and IL-8 (Fig. 3C) from adipose tissue and skeletal muscle. At the highest sulfasalazine concentration of 5 mM, there were 8- and 3-fold reductions in IL-6 release and 22- and 6-fold reductions in IL-8 release from adipose tissue and skeletal muscle, respectively. Treatment of adipose tissue and skeletal muscle with sulfasalazine had no effect on the release of adiponectin, resistin, and leptin (Table 2).
FIG. 3. Effects of 1.25, 2.5, and 5 mM sulfasalazine on TNF- (A), IL-6 (B), and IL-8 (C) release from human adipose tissue (; TNF-, n = 3; IL-6 and IL-8, n = 7) and skeletal muscle (; TNF-, n = 3; IL-6 and IL-8, n = 6). *, P < 0.05 vs. basal adipose tissue cytokine release; , P < 0.05 vs. basal skeletal muscle cytokine release.
TABLE 2. Effect of sulfasalazine on leptin, resistin, and adiponectin release from human adipose tissue and skeletal muscle
Effect of BAY 11-7082 on proinflammatory cytokine release
BAY 11-7082 at 100 μM significantly decreased the release of TNF- from adipose tissue (Fig. 4A), whereas the release of IL-6 (Fig. 4B) and IL-8 (Fig. 4C) was significantly inhibited at all concentrations of BAY 11-7082 tested. Treatment of skeletal muscle with BAY 11-7082 at concentrations greater than 50 μM significantly decreased the release of TNF- (Fig. 4A), IL-6 (Fig. 4B), and IL-8 (Fig. 4C). Treatment of adipose tissue and skeletal muscle with BAY 11-7082 had no effect on the release of adiponectin, resistin, and leptin (Table 3).
FIG. 4. Effects of 25, 50, and 100 μM BAY 11-7082 on TNF- (A), IL-6 (B), and IL-8 (C) release from human adipose tissue (; n = 3) and skeletal muscle (; n = 3). *, P < 0.05 vs. basal adipose tissue cytokine release; , P < 0.05 vs. basal skeletal muscle cytokine release.
TABLE 3. Effect of BAY 11-7082 on leptin, resistin, and adiponectin release from human adipose tissue and skeletal muscle
Effect of sulfasalazine and BAY 141-7082 on IR-?
A band corresponding to approximately 95 kDa was identified as IR-? and was present in all samples of adipose tissue and skeletal muscle. Sulfasalazine at 5 mM (Fig. 5A) and BAY 11-7082 at 100 μM (Fig. 5B) increased IR-? protein in both adipose tissue and skeletal muscle.
FIG. 5. A representative Western blot demonstrating the effect of 5 mM sulfasalazine and 100 μM BAY 11-7082 on IR-? protein expression in human adipose tissue and skeletal muscle. Both sulfasalazine and BAY 11-7082 increased IR-? protein expression in adipose tissue and skeletal muscle. Similar results were obtained in three experiments.
Discussion
Current dogma suggests that there is a link between type 2 diabetes mellitus and inflammation (7, 12). Although early studies have demonstrated that antiinflammatory agents, including aspirin, improve glucose metabolism in patients with type 2 diabetes mellitus (8), it has only been recently that the mechanisms by which aspirin works is being uncovered. Salicylates inhibit the activity of IKK-?, an upstream activator of the NF-B pathway (13, 14). In this study adipose tissue and skeletal muscle obtained from normal pregnant women were used to determine the effects of two inhibitors of the IKK-?/NF-B pathway, sulfasalazine and BAY 11-7082. The data presented demonstrate that suppression of the IKK-?/NF-B signaling pathway results in inhibition of proinflammatory cytokine release from human adipose tissue and skeletal muscle. However, there was no effect of NF-B inhibition on adiponectin, resistin, and leptin release from either adipose tissue or skeletal muscle.
Sulfasalazine, at concentrations of at least 2.5 mM, suppressed NF-B p65 DNA-binding activity in human adipose tissue and skeletal muscle. This inhibition was associated with a significant and concomitant inhibition of IL-6, IL-8, and TNF- release. Although similar findings in both gestational and nongestational tissues have revealed that salicylate suppression of NF-B is associated with a reduction in cytokine expression and/or release (5, 14, 15, 16, 17), this is the first study to demonstrate that the NF-B signaling pathway coordinates cytokine release in adipose tissue and skeletal muscle and may therefore have important implications in the treatment of insulin resistance and type 2 diabetes mellitus. It has been demonstrated that sulfasalazine and other salicylates prevent nuclear translocation of NF-B p65 due to the inhibition of IKK-? or another upstream signal (13, 14). In this study we have demonstrated that sulfasalazine inhibits IKK-? protein expression in both adipose tissue and skeletal muscle.
There is now increasing evidence demonstrating salicylates as therapeutic agents for type 2 diabetes mellitus. Yuan et al. (9) demonstrated that treatment of insulin-resistant Zucker rats with aspirin and salicylate increased IR tyrosine phosphorylation in liver and skeletal muscle. Furthermore, treatment with aspirin reverses the TNF--induced reduction in IR-? and IRS-1 tyrosine phosphorylation in cultured 3T3-L1 adipocytes, and IRS-2 tyrosine phosphorylation in Fao hepatoma cells. Kim et al. (18) reported that high dose salicylate prevented fat-induced insulin resistance in skeletal muscle by blocking the activity of IKK-? and subsequent phosphorylation of IRS-1 by IKK-? and decreased activation of IRS-1-associated phosphatidylinositol 3-kinase. Similarly, in this study we have demonstrated that sulfasalazine and BAY 11-7082 increased IR-? protein expression. Taken together, these studies demonstrate the potential importance of IKK-? in mediating insulin resistance and the ability of high dose salicylate to inhibit IKK-? activity. The importance of IKK-? in the treatment of type 2 diabetes mellitus was also recently demonstrated by Hundal et al. (19), who reported improvements in glucose metabolism in type 2 diabetes mellitus subjects treated with high dose aspirin.
BAY 11-7082 is a selective inhibitor of the actions of IKK-?, thus preventing the translocation of free NF-B to the nucleus (10), and was used in this study to confirm the results obtained using sulfasalazine. BAY 11-7082 treatment inhibited both IKK-? protein expression and NF-B p65 DNA-binding activity from adipose tissue and skeletal muscle, which was associated with a concomitant decrease in the release of TNF-, IL-6, and IL-8.
There is an apparent conflict between the doses of sulfasalazine and BAY 11-7082 required to inhibit TNF- and those that inhibit IKK-? and NF-B p65. However, this is most likely due to these inhibitors acting on more than one of the NF-B dimers and/or IKK isoforms. In mammals, the NF-B dimers arise from five polypeptides: p50, p52, p65, c-Rel, and RelB, with the p50/p65 heterodimer and the p50 homodimer being the most abundant. Some, but not all, of the other possible dimers have been shown to exist in cells. The homodimers of p50 and p52 and the p50/p52 heterodimer function as transcriptional repressors because they do not contain a distinct activation domain. The remaining combinations of dimeric NF-B proteins, containing at least one monomer of p65, c-Rel, or RelB, act as activators. Furthermore, IKK is an unusual kinase, in that in most cells IKK contains (at least) three distinct subunits: IKK-, IKK-?, and IKK-.
IL-6, IL-8, and/or TNF- protein and mRNA transcripts have been identified in both adipose tissue and skeletal muscle (7, 20, 21). In this study we have found the IKK-?/NF-B signaling pathway to be important for the formation of proinflammatory cytokines in these tissues. This has important implications because there is much evidence to indicate a role for cytokines, in particular TNF-, in insulin resistance in type 2 diabetes mellitus (reviewed in Ref. 3). TNF- down-regulates insulin-stimulated glucose uptake via effects on IR autophosphorylation and IRS-1. In adipose tissue and skeletal muscle, TNF- decreases both IR phosphorylation and tyrosine phosphorylation of IRS-1 (22, 23). In adipocytes, TNF- down-regulates the insulin-sensitive glucose transporter GLUT-4, resulting in decreased glucose uptake (24), whereas in skeletal muscle, glucose uptake is increased through the up-regulation of GLUT-1 (23). There is a direct correlation between insulin resistance and circulating IL-6 levels (25), and TNF- increases the release of IL-8 from adipose tissue (21).
Activation of the IKK-?/NF-B signaling pathway can initiate both extracellular and intracellular regulatory events that result in autoregulation of the inflammatory cascade (reviewed in Ref. 7). IKK-? activation may initiate NF-B-mediated transcription, which in certain cells would enhance the production of TNF-, which is capable of activating NF-B (26). This positive feedback loop could amplify and perpetuate a vicious cycle of low level inflammatory signaling, leading to insulin resistance.
In this study the IKK-?/NF-B pathway regulated TNF-, IL-6, and IL-8 release from adipose tissue and skeletal muscle; therefore, we hypothesized that this pathway may also regulate other adipocytokines that are known to influence insulin sensitivity and glucose metabolism, namely, adiponectin, resistin, and leptin. However, in this study we demonstrated that the IKK-?/NF-B pathway does not regulate adiponectin, resistin, or leptin synthesis from adipose tissue and skeletal muscle.
In this study we have established that in adipose tissue and skeletal muscle, the IKK-?/NF-B signaling pathway is a key regulator of the proinflammatory cytokines, IL-6, IL-8, and TNF-, as well as IR-?. By identifying the pathway by which obesity, fatty diet, and sedentary lifestyle cause insulin resistance, and because the IKK-?/NF-B pathway has already been shown to be a "drugable" target, efforts to identify better new drug treatments for type 2 diabetes mellitus can now be pursued.
Acknowledgments
We gratefully acknowledge the assistance of the clinical research midwives, Angie Denning, Val Bryant, Sarah Mitchell, Melissa Ryan, and Ellen Smith, and the obstetrics and midwifery staff of the Mercy Hospital for Women for their cooperation.
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Address all correspondence and requests for reprints to: Dr. Martha Lappas, Department of Obstetrics and Gynecology, University of Melbourne, Mercy Hospital for Women, 126 Clarendon Street, East Melbourne, 3002 Victoria, Australia. E-mail: mlappas@unimelb.edu.au.
Abstract
There is much evidence to indicate a role for adipocytokines in insulin resistance and/or type 2 diabetes mellitus. In experimental models, oral salicylates, through their ability to interfere with the nuclear factor-B (NF-B) transcription pathway, have been demonstrated to reverse insulin resistance. The aim of this study was to investigate whether NF-B regulates the release of adipocytokines in human adipose tissue and skeletal muscle. Human sc adipose tissue and skeletal muscle (obtained from normal pregnant women) were incubated in the absence (control) or presence of two NF-B inhibitors sulfasalazine (1.25, 2.5, and 5 mM) and BAY 11-7082 (25, 50, and 100 μM). After an 18-h incubation, the tissues were collected, and NF-B p65 DNA-binding activity and IB kinase (IKK-?) and insulin receptor-? protein expression were assessed by ELISA and Western blotting, respectively. The incubation medium was collected, and the release of TNF-, IL-6, IL-8, resistin, adiponectin, and leptin was quantified by ELISA. Treatment of adipose tissue and skeletal muscle with sulfasalazine and BAY 11-7082 significantly inhibited the release of IL-6, IL-8, and TNF-; NF-B p65 DNA-binding activity; and IKK-? protein expression (P < 0.05, by Newman-Keuls test). There was no effect of sulfasalazine and BAY 11-7082 on resistin, adiponectin, or leptin release. Both sulfasalazine and BAY 11-7082 increased the adipose tissue and skeletal muscle expression of insulin receptor-?. The data presented in this study demonstrate that the IKK-?/NF-B transcription pathway is a key regulator of IL-6, IL-8, and TNF- release from adipose tissue and skeletal muscle. Control of the IKK-?/NF-B pathway may therefore provide an alternative therapeutic strategy for regulating aberrant cytokine release and thereby alleviating insulin resistance in type 2 diabetes mellitus.
Introduction
INSULIN RESISTANCE IS defined as a failure of target tissues (including adipose tissue and skeletal muscle) to respond normally to insulin. Obesity is a major risk factor for insulin resistance and type 2 diabetes, and adipocytes secrete a number of cytokines, including TNF-, IL-6, IL-8, adiponectin, resistin, and leptin, all of which have been implicated in insulin resistance in type 2 DM (1, 2, 3, 4). TNF- and resistin appear to impair glucose tolerance, whereas leptin and adiponectin work to improve the metabolic status. We have recently demonstrated that in human gestational tissues, the nuclear factor-B (NF-B) signaling pathway is a regulator of TNF-, IL-6, and IL-8 release (5); therefore, the aim of this study was to determine whether NF-B also regulates cytokine release from adipose tissue and skeletal muscle, in particular TNF-, IL-6, IL-8, resistin, adiponectin, and leptin. Understanding how adipocytokines are regulated by drugs influencing insulin sensitivity will have major and important implications for the treatment of insulin resistance and obesity. The IB kinase (IKK-?)/NF-B pathway might impair insulin sensitivity at least in part via up- or down-regulation of adipocytokine synthesis. Pregnancy is a state of insulin resistance; therefore, the availability of adipose tissue and skeletal muscle from women undergoing elective cesarean section allows us the opportunity to study the regulation of cytokines in these tissues.
NF-B is a ubiquitous transcription factor that by regulating the expression of multiple inflammatory and immune genes plays a critical role in host defense and chronic inflammatory diseases (6). At least five genes belong to the NF-B family, with the most common NF-B dimer composed of the RelA (p65) and p50 subunits. NF-B dimers are usually sequestered in the cytoplasm bound to an inhibitory subunit, IB-. Upon stimulation, IB- is phosphorylated by the action of a specific IKK, ubiquitinated, and rapidly degraded resulting in the rapid translocation of NF-B to the nucleus, where it binds specific DNA elements (B motifs) in the promoter/enhancer region of target genes to initiate, enhance, or suppress the transcriptional process.
Inflammation has been implicated as a major pathogenic mediator of type 2 diabetes mellitus and/or insulin resistance. Elevated levels of acute phase reactant and inflammatory mediators circulate in patients with type 2 diabetes mellitus (7), and inflammatory cytokines have been shown to attenuate insulin signaling in experimental models (3). Furthermore, the ability of salicylates, including sulfasalazine, aspirin, and sodium salicylate, to lower blood sugar has been recognized since the late 19th century (8). Recently, Juan et al. (9) demonstrated that high doses of salicylates reverse high blood sugar, high insulin, and high blood fat levels in obese rodents by inhibiting the NF-B and its upstream activator, IKK-?. In recent studies we have demonstrated that the antiinflammatory effects of sulfasalazine could also be attributed to its ability to inhibit NF-B DNA-binding activity (5). However, the effects of salicylates on cytokine release and insulin signaling from human adipose tissue and skeletal muscle are not known and may have important implications for the treatment of type 2 diabetes.
The hypothesis to be tested is that inhibition of NF-B DNA-binding activity suppresses the release of TNF-, IL-6, IL-8, resistin, adiponectin, and leptin from adipose tissue and skeletal muscle. Tissues were incubated in the presence of sulfasalazine (1.25, 2.5, and 5 mM) and BAY 11-7082 (an inhibitor of IB kinase (10); (E)-3-[4-methylphenylsulphonyl]2-propenenitrile); NF-B p65 DNA-binding activity in nuclear extracts was analyzed by a ELISA; IKK-?, and insulin receptor-? (IR-?) tissue protein expression were analyzed by Western blotting; and the release of TNF-, IL-6, and IL-8 into the incubation medium was quantified by ELISA.
Materials and Methods
Reagents
All chemicals were purchased from BDH Chemicals Australia (Melbourne, Victoria, Australia) unless stated otherwise. DMEM was obtained from Invitrogen Life Technologies, Inc. (Grand Island, NY). BSA (RIA grade), dithiothreitol, EDTA, leupeptin, lipopolysaccharides (from Escherichia coli 026:B6), ?-NADH (disodium salt), 3,3',5,5'-tetramethylbenzidine, pyruvic acid (dimer free), and sulfasalazine were supplied by Sigma-Aldrich Corp. (St. Louis, MO). BAY 11-7082 was purchased from Sapphire Bioscience (Crows Nest, Australia). Pefabloc SC [4-(2-amnoethyl)-benzenesulfonyl fluoride] was purchased from Roche (Mannheim, Germany). Streptavidin-horseradish peroxidase conjugate and the IL-6, IL-8, TNF-, and leptin kits were supplied by BioSource International (Camarillo, CA). The resistin and adiponectin ELISA kits were supplied by CytoLab (Rehovot, Israel) and R&D Systems (Minneapolis, MN), respectively. NF-B p65 ELISA was purchased from BD Clontech (Palo Alto, CA). Rabbit polyclonal IKK-? polyclonal antibody, rabbit polyclonal IR-? polyclonal antibody, and horseradish peroxidase-conjugated goat antirabbit immunoglobulin G were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Tissue collection and preparation
Subcutaneous adipose tissue (from the anterior abdominal wall) and skeletal muscle (pyramidalis) where obtained from 10 pregnant women who delivered healthy, singleton infants at term (37 wk gestation) after undergoing elective cesarean section (indications for cesarean section were breech presentation and/or previous cesarean section). The clinical characteristics of the subjects are collated in Table 1. Approval for this study was obtained from the Mercy Hospital for Women’s research and ethics committee, and informed consent was obtained from all participating subjects.
TABLE 1. Characteristics of the study group
Tissues were obtained within 10 min of delivery, and dissected fragments were placed in ice-cold PBS. Tissue were dissected into small fragments visibly free of connective tissue and blood. Tissue fragments were placed in DMEM at 37 C in a humidified atmosphere of carbogen gas (95% O2 and 5% CO2) for 1 h. Explants were blotted dry on sterile filter paper and transferred to 24-well tissue culture plates (100–150 mg wet weight/well). The explants were incubated in duplicate in 2 ml DMEM containing penicillin G (100 U/ml) and streptomycin (100 μg/ml). Tissues were incubated in the absence (control) or presence of sulfasalazine (1.25, 2.5, and 5 mM) or BAY 11-7082 (25, 50, and 100 μM). The concentrations used in this study were chosen according to our previous studies (5).
Validation of explant cultures and viability
To determine the effects of sulfasalazine and BAY 11-7082 treatment on cell membrane integrity, the release of the intracellular enzyme lactate dehydrogenase (LDH) into incubation medium was determined as described previously (5). LDH release was investigated over the 18-h time course of tissue explants. Explants were incubated in either medium alone or medium containing sulfasalazine (1.25, 2.5, and 5 mM) or BAY 11-7082 (25, 50, and 100 μM). Neither in vitro incubation nor experimental treatment with sulfasalazine or BAY 11-7082 significantly affected LDH activity in the incubation medium (data not shown). These data indicate that the concentrations used in this study did not affect cell viability.
Experimental assays
After an 18-h incubation, the explant incubation medium was collected, and the release of IL-6, IL-8, TNF-, and leptin was determined by sandwich ELISA according to the manufacturer’s instructions (BioSource International, Camarillo, CA). The limits of detection of the IL-6, IL-8, TNF-, and leptin assays (defined as 2 SD from the zero standard) were 3, 2.8, 7.2, and 7.2 pg/ml, respectively. The release of resistin (CytoLab, Rehovot, Israel) and adiponectin (R&D Systems) in the explant incubation medium was determined by sandwich ELISA according to the manufacturer’s instructions. The intra- and interassay coefficients of variation for the resistin ELISA were 3.4% and 4.5%, respectively, and the minimum detectable limit of the assay was 16 pg/ml. The intra- and interassay coefficients of variation for the adiponectin ELISA were 4.2% and 4.7%, respectively, and the minimum detectable limit of the assay was 32 pg/ml. All data were corrected for total protein and expressed as picograms per milligram of protein. The protein content of tissue homogenates was determined using the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL) using BSA as a reference standard, as previously described (5).
Assessment of NF-B p65 DNA-binding activity by ELISA
After the 18-h incubation, tissues were collected, and protein was extracted as previously described (5). NF-B DNA binding was assessed using a commercially available NF-B p65 ELISA according to manufacturer’s instructions (BD Clontech) in which TNF--stimulated HeLa nuclear protein extract was used as a positive control for NF-B activation, and the specificity of NF-B binding was assessed using wild-type and mutated consensus oligonucleotides. A Benchmark Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA) was used to read the sample absorbance, and data are expressed as absorbance at 655 nm.
Assessment of IKK-? and IR-? protein expression by Western blotting
Assessment of IKK-? and IR-? protein expression in tissues was analyzed by Western blotting. Forty micrograms of tissue protein extracts were separated on a 10% polyacrylamide gel and transferred to nitrocellulose as previously described (11). Protein expression of IKK-? or IR-? was identified by comigration with a positive control and by comparison with the mobility of protein standard.
Statistical analysis
Statistical analyses were performed using a commercially available statistical software package (STSC, Statgraphics, Rockville, MD). The homogeneity of data was assessed by Bartlett’s test, and when significant, data were logarithmically transformed before additional analysis. Comparisons between groups were performed using Newman-Keuls multiple range tests. Statistical difference was indicated by P < 0.05. Data are expressed as the mean ± SEM and represent replication with independent tissue explants obtained from at least three women.
Results
Effects of sulfasalazine and BAY 11-7082 on NF-B-binding activity
The binding ability of NF-B p65 to DNA consensus sequences was measured using a commercially available kit. An NF-B wild-type consensus oligonucleotide was used to monitor the specificity of the assay. The wild-type oligonucleotide, by competing for NF-B binding to the probe immobilized on the plate, acted as an effective competitor for NF-B p65 binding (data not shown). The specificity of binding was also demonstrated using wells coated with mutated consensus oligonucleotide. In these experiments no binding was detected in the presence of the positive control (data not shown). Incubation of adipose tissue and skeletal muscle extracts with sulfasalazine at 2.5 and 5 mM significantly inhibited NF-B p65 DNA-binding activity (Fig. 1A; n = 3). Incubation of adipose tissue with BAY 11-7082 at all concentrations tested significantly inhibited NF-B p65 DNA-binding activity (Fig. 1B; n = 3), whereas in skeletal muscle, BAY 11-7082 at 50 and 100 μM significantly inhibited NF-B p65 DNA-binding activity (Fig. 1B; n = 3).
FIG. 1. Effects of 1.25, 2.5, and 5 mM sulfasalazine (A; n = 5) and 25, 50, and 100 μM BAY 11-7082 (B; n = 3) on NF-B p65 DNA-binding activity in human adipose tissue () and skeletal muscle (). *, P < 0.05 vs. basal adipose tissue NF-B p65 DNA-binding activity; , P < 0.05 vs. basal skeletal muscle NF-B p65 DNA-binding activity.
Effects of sulfasalazine and BAY 141-7082 on IKK-?
A band corresponding to approximately 85 kDa was identified as IKK-? and was present in all samples of adipose tissue and skeletal muscle. Both sulfasalazine (Fig. 2A; n = 3) and BAY 11-7082 (Fig. 2B; n = 3) at the highest concentrations reduced IKK-? protein in adipose tissue and skeletal muscle.
FIG. 2. Effects of 1.25, 2.5, and 5 mM sulfasalazine (A) and 25, 50, and 100 μM BAY 11-7082 (B) on IKK-? protein expression in human adipose tissue () and skeletal muscle (). *, P < 0.05 vs. basal adipose tissue IKK-? protein expression; , P < 0.05 vs. basal skeletal muscle IKK-? protein expression. A representative Western blot is also shown, demonstrating the effects of sulfasalazine and BAY 11–7082 on IKK-? protein.
Effect of sulfasalazine on cytokine release
The release of TNF- from adipose tissue and skeletal muscle was detectable in only three of the six adipose tissue samples and three of the seven skeletal muscle samples tested. In these tissues, TNF- release (Fig. 3A) was significantly inhibited at all sulfasalazine concentrations tested. Treatment of adipose tissue with sulfasalazine completely suppressed TNF- release, whereas in skeletal muscle, complete suppression was observed at 5 mM sulfasalazine. IL-6 and IL-8 release was detected from all adipose and skeletal muscle tissue samples. Sulfasalazine concentrations greater than 2.5 mM significantly suppressed both IL-6 (Fig. 3B) and IL-8 (Fig. 3C) from adipose tissue and skeletal muscle. At the highest sulfasalazine concentration of 5 mM, there were 8- and 3-fold reductions in IL-6 release and 22- and 6-fold reductions in IL-8 release from adipose tissue and skeletal muscle, respectively. Treatment of adipose tissue and skeletal muscle with sulfasalazine had no effect on the release of adiponectin, resistin, and leptin (Table 2).
FIG. 3. Effects of 1.25, 2.5, and 5 mM sulfasalazine on TNF- (A), IL-6 (B), and IL-8 (C) release from human adipose tissue (; TNF-, n = 3; IL-6 and IL-8, n = 7) and skeletal muscle (; TNF-, n = 3; IL-6 and IL-8, n = 6). *, P < 0.05 vs. basal adipose tissue cytokine release; , P < 0.05 vs. basal skeletal muscle cytokine release.
TABLE 2. Effect of sulfasalazine on leptin, resistin, and adiponectin release from human adipose tissue and skeletal muscle
Effect of BAY 11-7082 on proinflammatory cytokine release
BAY 11-7082 at 100 μM significantly decreased the release of TNF- from adipose tissue (Fig. 4A), whereas the release of IL-6 (Fig. 4B) and IL-8 (Fig. 4C) was significantly inhibited at all concentrations of BAY 11-7082 tested. Treatment of skeletal muscle with BAY 11-7082 at concentrations greater than 50 μM significantly decreased the release of TNF- (Fig. 4A), IL-6 (Fig. 4B), and IL-8 (Fig. 4C). Treatment of adipose tissue and skeletal muscle with BAY 11-7082 had no effect on the release of adiponectin, resistin, and leptin (Table 3).
FIG. 4. Effects of 25, 50, and 100 μM BAY 11-7082 on TNF- (A), IL-6 (B), and IL-8 (C) release from human adipose tissue (; n = 3) and skeletal muscle (; n = 3). *, P < 0.05 vs. basal adipose tissue cytokine release; , P < 0.05 vs. basal skeletal muscle cytokine release.
TABLE 3. Effect of BAY 11-7082 on leptin, resistin, and adiponectin release from human adipose tissue and skeletal muscle
Effect of sulfasalazine and BAY 141-7082 on IR-?
A band corresponding to approximately 95 kDa was identified as IR-? and was present in all samples of adipose tissue and skeletal muscle. Sulfasalazine at 5 mM (Fig. 5A) and BAY 11-7082 at 100 μM (Fig. 5B) increased IR-? protein in both adipose tissue and skeletal muscle.
FIG. 5. A representative Western blot demonstrating the effect of 5 mM sulfasalazine and 100 μM BAY 11-7082 on IR-? protein expression in human adipose tissue and skeletal muscle. Both sulfasalazine and BAY 11-7082 increased IR-? protein expression in adipose tissue and skeletal muscle. Similar results were obtained in three experiments.
Discussion
Current dogma suggests that there is a link between type 2 diabetes mellitus and inflammation (7, 12). Although early studies have demonstrated that antiinflammatory agents, including aspirin, improve glucose metabolism in patients with type 2 diabetes mellitus (8), it has only been recently that the mechanisms by which aspirin works is being uncovered. Salicylates inhibit the activity of IKK-?, an upstream activator of the NF-B pathway (13, 14). In this study adipose tissue and skeletal muscle obtained from normal pregnant women were used to determine the effects of two inhibitors of the IKK-?/NF-B pathway, sulfasalazine and BAY 11-7082. The data presented demonstrate that suppression of the IKK-?/NF-B signaling pathway results in inhibition of proinflammatory cytokine release from human adipose tissue and skeletal muscle. However, there was no effect of NF-B inhibition on adiponectin, resistin, and leptin release from either adipose tissue or skeletal muscle.
Sulfasalazine, at concentrations of at least 2.5 mM, suppressed NF-B p65 DNA-binding activity in human adipose tissue and skeletal muscle. This inhibition was associated with a significant and concomitant inhibition of IL-6, IL-8, and TNF- release. Although similar findings in both gestational and nongestational tissues have revealed that salicylate suppression of NF-B is associated with a reduction in cytokine expression and/or release (5, 14, 15, 16, 17), this is the first study to demonstrate that the NF-B signaling pathway coordinates cytokine release in adipose tissue and skeletal muscle and may therefore have important implications in the treatment of insulin resistance and type 2 diabetes mellitus. It has been demonstrated that sulfasalazine and other salicylates prevent nuclear translocation of NF-B p65 due to the inhibition of IKK-? or another upstream signal (13, 14). In this study we have demonstrated that sulfasalazine inhibits IKK-? protein expression in both adipose tissue and skeletal muscle.
There is now increasing evidence demonstrating salicylates as therapeutic agents for type 2 diabetes mellitus. Yuan et al. (9) demonstrated that treatment of insulin-resistant Zucker rats with aspirin and salicylate increased IR tyrosine phosphorylation in liver and skeletal muscle. Furthermore, treatment with aspirin reverses the TNF--induced reduction in IR-? and IRS-1 tyrosine phosphorylation in cultured 3T3-L1 adipocytes, and IRS-2 tyrosine phosphorylation in Fao hepatoma cells. Kim et al. (18) reported that high dose salicylate prevented fat-induced insulin resistance in skeletal muscle by blocking the activity of IKK-? and subsequent phosphorylation of IRS-1 by IKK-? and decreased activation of IRS-1-associated phosphatidylinositol 3-kinase. Similarly, in this study we have demonstrated that sulfasalazine and BAY 11-7082 increased IR-? protein expression. Taken together, these studies demonstrate the potential importance of IKK-? in mediating insulin resistance and the ability of high dose salicylate to inhibit IKK-? activity. The importance of IKK-? in the treatment of type 2 diabetes mellitus was also recently demonstrated by Hundal et al. (19), who reported improvements in glucose metabolism in type 2 diabetes mellitus subjects treated with high dose aspirin.
BAY 11-7082 is a selective inhibitor of the actions of IKK-?, thus preventing the translocation of free NF-B to the nucleus (10), and was used in this study to confirm the results obtained using sulfasalazine. BAY 11-7082 treatment inhibited both IKK-? protein expression and NF-B p65 DNA-binding activity from adipose tissue and skeletal muscle, which was associated with a concomitant decrease in the release of TNF-, IL-6, and IL-8.
There is an apparent conflict between the doses of sulfasalazine and BAY 11-7082 required to inhibit TNF- and those that inhibit IKK-? and NF-B p65. However, this is most likely due to these inhibitors acting on more than one of the NF-B dimers and/or IKK isoforms. In mammals, the NF-B dimers arise from five polypeptides: p50, p52, p65, c-Rel, and RelB, with the p50/p65 heterodimer and the p50 homodimer being the most abundant. Some, but not all, of the other possible dimers have been shown to exist in cells. The homodimers of p50 and p52 and the p50/p52 heterodimer function as transcriptional repressors because they do not contain a distinct activation domain. The remaining combinations of dimeric NF-B proteins, containing at least one monomer of p65, c-Rel, or RelB, act as activators. Furthermore, IKK is an unusual kinase, in that in most cells IKK contains (at least) three distinct subunits: IKK-, IKK-?, and IKK-.
IL-6, IL-8, and/or TNF- protein and mRNA transcripts have been identified in both adipose tissue and skeletal muscle (7, 20, 21). In this study we have found the IKK-?/NF-B signaling pathway to be important for the formation of proinflammatory cytokines in these tissues. This has important implications because there is much evidence to indicate a role for cytokines, in particular TNF-, in insulin resistance in type 2 diabetes mellitus (reviewed in Ref. 3). TNF- down-regulates insulin-stimulated glucose uptake via effects on IR autophosphorylation and IRS-1. In adipose tissue and skeletal muscle, TNF- decreases both IR phosphorylation and tyrosine phosphorylation of IRS-1 (22, 23). In adipocytes, TNF- down-regulates the insulin-sensitive glucose transporter GLUT-4, resulting in decreased glucose uptake (24), whereas in skeletal muscle, glucose uptake is increased through the up-regulation of GLUT-1 (23). There is a direct correlation between insulin resistance and circulating IL-6 levels (25), and TNF- increases the release of IL-8 from adipose tissue (21).
Activation of the IKK-?/NF-B signaling pathway can initiate both extracellular and intracellular regulatory events that result in autoregulation of the inflammatory cascade (reviewed in Ref. 7). IKK-? activation may initiate NF-B-mediated transcription, which in certain cells would enhance the production of TNF-, which is capable of activating NF-B (26). This positive feedback loop could amplify and perpetuate a vicious cycle of low level inflammatory signaling, leading to insulin resistance.
In this study the IKK-?/NF-B pathway regulated TNF-, IL-6, and IL-8 release from adipose tissue and skeletal muscle; therefore, we hypothesized that this pathway may also regulate other adipocytokines that are known to influence insulin sensitivity and glucose metabolism, namely, adiponectin, resistin, and leptin. However, in this study we demonstrated that the IKK-?/NF-B pathway does not regulate adiponectin, resistin, or leptin synthesis from adipose tissue and skeletal muscle.
In this study we have established that in adipose tissue and skeletal muscle, the IKK-?/NF-B signaling pathway is a key regulator of the proinflammatory cytokines, IL-6, IL-8, and TNF-, as well as IR-?. By identifying the pathway by which obesity, fatty diet, and sedentary lifestyle cause insulin resistance, and because the IKK-?/NF-B pathway has already been shown to be a "drugable" target, efforts to identify better new drug treatments for type 2 diabetes mellitus can now be pursued.
Acknowledgments
We gratefully acknowledge the assistance of the clinical research midwives, Angie Denning, Val Bryant, Sarah Mitchell, Melissa Ryan, and Ellen Smith, and the obstetrics and midwifery staff of the Mercy Hospital for Women for their cooperation.
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