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Obesity: Original Article |
Endocrine Unit, Division of Endocrinology, Diabetes and Nutrition, Department of Internal Medicine (C.E.J.-A., E.S., A.P., C.A.M.); Hans Wilsdorf Laboratory, Division of Immunology and Allergy, Department of Internal Medicine (R.C., D.B., J.-M.D.); and Plastic Reconstructive Surgery Unit, Division of Reconstructive Surgery, Department of Surgery (B.C.-P.), University Hospital, CH-1211 Geneva 14, Switzerland; La Tour Hospital (P.Q.), CH-1217 Meyrin, Switzerland; and Division of Endocrinology, Diabetology and Metabolism, Department of Medicine (V.G.), University Hospital, CH-1011 Lausanne, Switzerland
Address all correspondence and requests for reprints to: Dr. Christoph A. Meier, Endocrine Unit, University Hospital Geneva, 24, rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: Christoph.Meier{at}medecine.unige.ch.
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Abstract |
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We demonstrate that IL-1Ra is mainly produced by adipocytes, rather than the stromal fraction of WAT, and that IL-1
and ß, as well as interferon-ß (IFN-ß), strongly up-regulate the expression and secretion of IL-1Ra in WAT. Moreover, human WAT expresses the receptors and proteins known to be required for the action of IL-1 (IL-1 receptor type I, IL-1 receptor accessory protein) and IFN-ß (IFN-/ß receptor subunits 1 and 2). Finally, human WAT actively secretes these regulatory cytokines, suggesting that they up-regulate IL-1Ra through a local autocrine/paracrine action, which is hypothesized to play a regulatory role in adipogenesis and metabolism.
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Introduction |
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, IL-6, and, more recently, IL-1 receptor antagonist (IL-1Ra) (1, 2, 3). These factors are speculated to have local and systemic metabolic actions, e.g. by modulating insulin sensitivity, but also to contribute to cardiovascular complications that are closely linked to obesity (2, 4, 5, 6).
We have recently shown that human and rodent white adipose tissue (WAT) is a major source of IL-1Ra, which is a member of the IL-1 family inhibiting the binding of IL-1 and IL-1ß to their receptor (3). The relevance of this observation in vivo is substantiated by the observation that the serum levels of IL-1Ra are markedly increased in obese patients and in patients with Cushing’s syndrome, correlating with body mass index (BMI) and the degree of insulin resistance (7, 8). The serum levels of IL-1Ra observed in obese patients are similar to those in patients with autoimmune inflammatory disorders or sepsis, suggesting that IL-1Ra may have immunomodulatory and/or metabolic activities in these patients. Indeed, it has not only been suggested that IL-1Ra may represent a link between obesity and an acquired resistance to leptin at the hypothalamic level (7, 9), but recent data demonstrate a metabolic role of IL-1Ra in the periphery for the control of fat mass and insulin sensitivity (6) (Somm, E., and C. A. Meier, unpublished data).
Because adipose tissue produces much more IL-1Ra than TNF- and IL-6 (3) and because IL-1 and IL-1Ra are involved in: 1) the regulation of lipid metabolism in vivo (6, 10) and in vitro (10, 11, 12, 13); 2) the control of adipocyte differentiation (14, 15); and 3) the regulation of glucose uptake (16), we now define the cellular source as well as the regulators of IL-1Ra in human adipose tissue.
We show that adipocytes and stromal cells secrete IL-1Ra and that IL-1, IL-1ß, and interferon (IFN)-ß are stimulators of IL-1Ra production. The observation that some of these factors are also produced by WAT indicates the presence of a novel paracrine signaling network in human adipose tissue.
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Materials and Methods |
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IL-1Ra and IL-1ß concentrations in the supernatant of WAT cultures were determined by the Quantikine ELISA (R&D Systems Europe Ltd., Abingdon, UK) and Immunotech (Marseille, France) kits, respectively, whereas IFN-ß and IL-10 concentrations were measured with the kits from Biosource (Lucerna Chem AG, Luzern, Switzerland). Recombinant human IL-1 and -ß and TNF- were a kind gift of Biogene (Boston, MA), and IFN-ß was provided by Ares-Serono (Rehovot, Israel). IL-4 was a kind gift of Christophe Caux (Schering-Plough, Dardilly, France). Recombinant murine leptin and IL-10 were from Peprotech (Juro, Luzern, Switzerland), and IL-6 was from R&D Systems.
Culture and analysis of human adipose tissue
Authorizations from the Ethical Commission of the University Hospitals of Geneva and the Centre Hospitalier Universitaire Vaudois were obtained for this study, including the informed consent from the patients.
For the analysis of cytokine expression and production, sc and visceral adipose tissue samples were obtained from 28 morbidly obese patients with a median BMI of 45 kg/m2 (range, 37–65 kg/m2) without comorbidity, undergoing gastric banding or gastric bypass surgery. Subcutaneous adipose tissue samples were also obtained from 10 patients with a BMI of less than 30 kg/m2. Tissues were immediately frozen in liquid nitrogen before the preparation of tissue extracts and RNA.
For short-term culture experiments, human sc WAT was obtained from patients undergoing plastic surgery and prepared as described (3, 17). Briefly, tissue was placed in PBS [prewarmed at 37 C, containing penicillin (100 U/ml) and streptomycin (100 mg/ml)], and connective tissue and blood vessels were removed by dissection before mincing the tissue into 5- to 10-mg pieces. Minced tissue fragments were filtered through nylon mesh (350 µm) and washed with medium [M199, supplemented with 5% fetal bovine serum (FBS), penicillin, and streptomycin]. A total of 0.3 g of minced tissue were then placed into 1 ml M199 (with 5% FBS, penicillin, and streptomycin) for 18 h before stimulation with the appropriate compounds for 48 h. At the end of the stimulation period, supernatants were collected for cytokine determination, and the explants were recovered and placed directly into TRIzol reagent (Invitrogen, Basel, Switzerland) for immediate RNA preparation.
Preparation and culture of stromal fraction and adipocytes from human WAT
Minced human adipose tissue fragments were first digested with collagenase type I (200 U/ml; Worthington Biochemical Corp., Lakewood, NJ) (3 ml/g adipose tissue) in low-glucose (1000 mg/liter) DMEM containing penicillin and streptomycin, as well as the BSA fraction V (20 mg/ml) (Sigma, Buchs, Switzerland) at 37 C in a rocking bath. After 30 min of incubation, the digestion was stopped by the addition of FBS (0.3 ml/ml digestion medium). Adipocytes and the stromal fraction were then separated from undigested tissue by filtration through a 350-µm nylon mesh before the separating centrifugation (10 min at 200 x g). Adipocytes prepared from 0.6 g of adipose tissue were resuspended in 2 ml of low-glucose DMEM (10% FBS, glutamine 200 mM, and 1% penicillin and streptomycin). The pellet of the stromal fraction was resuspended in low-glucose DMEM containing 10% FBS, 1% glutamine, and 1% penicillin and streptomycin to promote cell adhesion. Twenty-four hours after plating, the medium was changed, and cells were grown for 4 d before stimulation with the appropriate compounds for another 48 h.
Preparation and quantification of DNA and RNA
Total DNA and RNA from 500 mg of human sc or visceral WAT, as well as from the cultured explants, were prepared using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. DNA was measured with the PicoGreen Kit (Molecular Probe, Juro, Luzern, Switzerland). Total RNA (5 µg) was reverse-transcribed using 800 U Moloney murine leukemia virus reverse transcriptase (Invitrogen) in the presence of 0.3 U/µl RNAsin (Promega Corp., Madison, WI), 7.5 µM of random primers [oligo(dN)6], 1.2 mM of deoxynucleotide triphosphate, and 12 µM of dithiothreitol.
The expression of RNAs for human secreted IL-1Ra, IL-1, IL-1ß, IL-1 receptor type I (IL-1RI), IL-1 receptor type II (IL-1RII), IL-1 receptor-associated protein (IL-1RAcP), IFN- and -ß receptor subunits 1 and 2 (IFNAR1 and IFNAR2), and 28S was determined by quantitative real-time PCR using a LightCycler (Roche Diagnostics, Basel, Switzerland) with the DNA Master SYBR Green I or Fast Start DNA Master SYBR Green I (Roche Molecular Biochemicals, Rotkreuz, Switzerland) kits as appropriate. The primers used were as follows: for IL-1Ra, sense 5'-tccgcagtcacctaatcactc-3', antisense 5'-ttgacacaggacaggacaat-3'; IL-1, sense 5' gtaagctatggcccactcca-3', antisense 5'-aggtgctgacctaggcttga-3'; IL-1ß, sense 5'-gctgaggaagatgctggttc-3', antisense 5'-gtgatcgtacaggtgcatcg-3'; IL-1RI, sense 5'-aaactacccgttgcaggaga-3', antisense 5'-cacattcatcacgatgagcc-3'; IL-1RII, sense 5'-gcgcttgtacgtgttggtaa-3', antisense 5'-acagaggcccacaaccagta-3'; IL-1RacP, sense 5'-ctgcaaagtgatgcctcaga-3', antisense 5'-cggtcctgcctagtccaata-3'; IFNAR1, sense 5'-atcggtgctccaaaacagtc-3', antisense 5'-gtgctctggctttcacacaa-3'; and IFNAR2, sense 5'-cccttaaaatgcaccctcct-3', antisense 5'-tcaagactttggggaggcta-3'.
Statistics
Results are expressed as means ± 1 SEM. The nonparametric Mann-Whitney U test was used for comparing the effects of the various stimuli (SYSTAT 10.01; SPSS, Inc., Chicago, IL) and comparison among tissues.
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Results |
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We have previously shown that human adipose tissue expresses and secretes IL-1Ra, but it remained unclear whether the adipose and/or stromal fractions were the main source. As shown in Fig. 1
, the adipose and stromal fractions both secreted IL-1Ra, with adipocytes producing 1.9-fold more IL-1Ra when normalized for DNA content. However, when normalized for the initial tissue mass used for cell fractionation, adipocytes account for about 95% of the production of IL-1Ra (18-fold higher production compared with the stromal fraction), and the stromal fraction accounts for 5% of the production of IL-1Ra.
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To examine which factors might regulate the production of IL-1Ra in WAT, we tested several candidate agents, such as phorbol myristate acetate (PMA), lipopolysaccharide (LPS), dexamethasone, and certain cytokines (Fig. 2). PMA and LPS enhanced the expression and secretion of IL-Ra 2.4 ± 0.2-fold and 4.6 ± 1-fold, respectively, whereas dexamethasone was a strong inhibitor of basal IL-1Ra production. IL-1 and ß, as well as IFN-ß, the fibroblast-derived form of IFN, all induced IL-1Ra production, the latter more than 35-fold. However, IL-6 and TNF-, both of which were previously shown to be produced by WAT, did not regulate IL-1Ra. Similarly, leptin, which we have previously shown to stimulate IL-1Ra secretion in monocytes, had no effect on IL-1Ra production in WAT (18).
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IFN-ß is not only a known regulator of IL-1Ra in monocytes, but it also up-regulates IL-1Ra in human WAT (3). IFN-ß stimulated the secretion of IL-1Ra in a dose-dependent manner with a maximal effect at a concentration of 2000 U/ml (Fig. 3A), and this response could already be observed at 8 h (Fig. 3B). Moreover, we assessed whether the stimulatory effect of IFN-ß on IL-1Ra can be observed on the isolated adipose and stromal fractions of WAT, which was the case as shown in Fig. 3C.
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/ß receptor subunits IFNAR1 and IFNAR2 to be expressed in human adipose tissue, with higher levels of IFNAR2 being present in the sc and visceral WAT from obese patients (Fig. 3D
). However, although the expression of IFN-ß has been recently described in adipose tissue (19) where it correlated with the body mass, we were unable to reliably detect IFN-ß protein in supernatant of human WAT explants. This is possibly due to the limited sensitivity of the currently available assays, as well as the rapid degradation of the secreted IFN-ß by the producing cells (Chicheportiche, R., and J.-M. Dayer, personal communication). IL-1 induces IL-1Ra in a dose- and time-dependent manner
Similar to IFN-ß, IL-1 and IL-1ß also induced the secretion of IL-1Ra by WAT explants. Because we have shown previously that IL-1ß is produced by human WAT (3), we examined the dose-response curve as well as the time course of the regulatory effects of IL-1 and ß on IL-1Ra secretion by human WAT explants. A maximal effect was observed between 500 and 1000 pg/ml for both isoforms of IL-1, with a rapid effect within the first 8 h (Fig. 4, A and B). To exclude that IL-1 and IFN-ß regulated IL-1Ra through their mutual induction, we measured the production of IFN-ß after treatment of WAT explants with IL-1 for 48 h and vice versa with no measurable effect (data not shown), suggesting that the production of IL-1Ra is independently regulated by these two cytokines.
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). Indeed, human WAT expressed the functional type I receptor for IL-1 and the decoy IL-1RII, as well as the IL-1RAcP, which is important for signaling through the IL-1RI. IL-1Ra and IL-1RI were markedly up-regulated more than 9-fold and 3-fold, respectively, in WAT from obese patients. This increase was not accompanied by a concomitant higher expression of IL-1RII and IL-1RAcP. Furthermore, IL-1ß expression was selectively increased in the visceral fat from obese subjects.
Because IL-1 and the IL-1 receptor genes were up-regulated in obese subjects, we examined the regulation of these genes in human WAT ex vivo. WAT explants were treated for 48 h with PMA (25 nM) or LPS (5 µg/ml). As can be seen in Fig. 4D, treatment with neither compound up-regulated the expression of the IL-1RI or the IL-1RAcP, whereas IL-1ß was strongly induced by LPS, which is in keeping with an earlier observations (20). The decoy receptor IL-1RII is expressed at low levels in adipose tissue and its expression is undetectable in cultured explants either in the basal state or after stimulation by PMA or LPS (Fig. 4D).
IL-4 augments the secretion of IL-1Ra in WAT
As demonstrated above, IL-1 and IFN-ß are novel regulators of IL-1Ra in human WAT. To extend these observations, we assessed the effect of other putative coregulators on the stimulatory action of IL-1 and ß on IL-1Ra secretion by WAT. Given our findings described above with regard to IFN-ß as well as the known modulatory effects of IL-10 and IL-4 on IL-1Ra production by monocytes (21, 22), we tested the effect of these factors on human adipose tissue cultured for 48 h. Although we used a submaximal dose of IFN-ß (1000 U/ml), no additional effect of IL-1 or ß was seen on the stimulation by IFN-ß (Fig. 5). In contrast, the addition of IL-4 augmented the stimulatory effect of IL-1 1.9-fold, whereas neither IL-4 nor IL-10 was able to induce the production of IL-1Ra when used alone.
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Discussion |
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and IL-6. However, although these two proinflammatory cytokines have been speculated to contribute to insulin resistance and vascular complications, their serum levels are only marginally elevated in human obesity (5, 23, 24). In contrast, we have recently shown that IL-1Ra serum levels are highly elevated in human obesity to a degree that is similar to that present in systemic inflammation (7). Moreover, we have provided evidence that adipose tissue is the main source of this antiinflammatory cytokine in humans as well as rodents (3). We now demonstrate that IL-1 is produced by adipose tissue and regulates the secretion of IL-1Ra, with IFN-ß being another strong stimulator of IL-1Ra in WAT.
Although TNF- and IL-6 were reported to be mainly produced by the stromal fraction of adipose tissue, we show that adipocytes are the main producers of IL-1Ra. Furthermore, IL-1 induces the production of IL-1Ra in a time- and dose-dependent manner, and this is compatible with a direct regulatory effect on the IL-1Ra gene, as is the case in monocytic cells (25). These findings are compatible with an autocrine/paracrine regulatory loop, because: 1) we have previously shown that IL-1ß is present in human WAT at quantities of around 6–8 pg/mg tissue (3), and 2) we now report that the various genes involved in IL-1 receptor signaling are expressed in human WAT. Moreover, the expression of not only IL-1Ra but also of IL-1ß and the IL-1RI are markedly increased in adipose tissue from obese subjects, suggesting that the augmented secretion of IL-1, as well as an increased sensitivity to this cytokine, might contribute to the overproduction of IL-1Ra in the obese state.
IFN-ß strongly up-regulates IL-1Ra expression in WAT ex vivo, and IFN- and -
showed no effect (data not shown). Recently published data from microarray experiments with WAT show that the expression of IFN-ß and IL-1Ra are positively correlated with body mass in rodents, suggesting the presence of a local autocrine/paracrine regulation of IL-1Ra not only by IL-1 but also by IFN-ß (19).
Although IL-1 and IFN-ß are positive regulators of IL-1Ra by themselves, we found that IL-4 can increase the stimulation of IL-1 further. Because IL-10 was described to be increased in the serum of obese patients (26) and to increase IL-1Ra production in monocytes, it was tempting to speculate that elevated amounts of IL-1Ra in the serum of obese patients might in part be due to the elevated IL-10 levels. However, our data obtained with human WAT ex vivo do not support this hypothesis.
Although WAT is clearly an important source of pro- and antiinflammatory cytokines, their true (patho)physiological role has only recently become more apparent, and it appears likely that IL-1Ra can negatively interfere with the anorexigenic activities of IL-1 at the hypothalamic and peripheral level. Specifically, IL-1 was shown to be involved in: 1) mediating some of the inhibitory effects of leptin on appetite as well as the stimulating effects on body temperature (9); and 2) inhibiting adipogenesis in vitro (11, 12). We and others have shown that this effect can be overcome by the administration of IL-1Ra (Ref. 9 and Juge-Aubry, C. E., and C. A. Meier, unpublished data) resulting in the promotion of adipogenesis and obesity. Finally, very recent data in IL-1Ra knockout mice have shown the impaired accumulation of fat in these animals, as well as a resistance to obesity after hypothalamic lesion (6), both of which are compatible with an obesity-promoting role of IL-1Ra. Moreover, IL-4 was shown to counteract the lipolytic activity of LPS on the perilymph node adipose tissue by an unknown mechanism (27), and we could postulate that this effect might be mediated through the locally induced production of IL-1Ra.
In summary, IL-1Ra is an antiinflammatory cytokine that is markedly up-regulated in human obesity, and the recent animal knockout studies support our concept of IL-1Ra being a promoter of obesity (6). We now show that IL-1Ra is mainly produced by human adipocytes, rather than the stromal fraction, and that it is up-regulated by IL-1ß, IFN-ß, and IL-4. Because human WAT expresses the receptors for IL-1 as well as for IFN-/ß, it will be of interest to elucidate whether these cytokines have additional regulatory effects on adipose tissue contributing to some of the metabolic and vascular complications related to obesity.
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Footnotes |
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C.E.J.-A. and E.S. contributed equally to this work.
Abbreviations: BMI, Body mass index; FBS, fetal bovine serum; IFN, interferon; IFNAR1 and IFNAR2, IFN- and -ß receptor subunit 1 and 2; IL-1Ra, IL-1 receptor antagonist; IL-1RAcP, IL-1 receptor-associated protein; IL-1RI and II, IL-1 receptor type I and II; LPS, lipopolysaccharide; PMA, phorbol myristate acetate; WAT, white adipose tissue.
Received July 14, 2003.
Accepted January 22, 2004.
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References |
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