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Endocrinology Vol. 148, No. 11 5478-5486
Copyright © 2007 by The Endocrine Society

Activation of Nuclear Factor-{kappa}B by High Molecular Weight and Globular Adiponectin

Fred Haugen and Christian A. Drevon

Department of Nutrition, Institute of Basic Medical Sciences, Medical Faculty, University of Oslo, 0316 Oslo, Norway

Address all correspondence and requests for reprints to: Fred Haugen, Ph.D., Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1046 Blindern, 0316 Oslo, Norway. E-mail: fred.haugen{at}medisin.uio.no.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Adipose tissue secretes a wide range of hormones named adipokines, and these may play a role in obesity-related inflammation. Adiponectin is an exceptional adipokine because low plasma concentrations are associated with obesity, type 2 diabetes, and cardiovascular diseases. It has been observed that plasma adiponectin concentrations are elevated during inflammatory conditions like preeclampsia and arthritis. Nuclear factor-{kappa}B (NF-{kappa}B) is an essential transcription factor for expression of inflammation-related proteins. We have used U937 cells stably transfected to express luciferase under the control of NF-{kappa}B to examine if adiponectin may modulate NF-{kappa}B activity. Physiological concentrations of native adiponectin induced NF-{kappa}B activity. This effect was relatively strong compared with proinflammatory adipokines like leptin, resistin, and IL-6. The enhanced NF-{kappa}B activity was attributed to the high molecular weight adiponectin isoforms. NF-{kappa}B was not activated by mutated adiponectin that is unable to form high molecular weight complexes. Furthermore, the C-terminal fragment, globular adiponectin, markedly increased NF-{kappa}B reporter activity, cytokine release, and mRNA expression of inflammation marker genes, at higher levels than stimulation with TNF-{alpha} and lipopolysaccharide. NF-{kappa}B activation by globular adiponectin was not affected by antibody inhibition of toll-like receptor 4 or TNF receptors 1 and 2 but was attenuated by inhibitors of p38 MAPK, phosphatidylinositol 3-kinase, and protein kinase C. Analyses of the p65 subunit of NF-{kappa}B in different leukocyte cell lines showed activation of two monocytic cell lines (U937 and THP-1) by native and globular adiponectin. Our results indicate that adiponectin has proinflammatory properties in monocytic cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
ADIPOSE TISSUE IS a source of several adipokines, some of which are specifically secreted by adipocytes. Adipokines may act locally and at the systemic level, influencing a variety of biological processes like energy metabolism, neuroendocrine function, and immune response (1).

Adiponectin is an adipokine mainly secreted from adipocytes (2, 3, 4, 5), whereas other cell types may express low levels of adiponectin (6). Native adiponectin has a multimeric structure (5); polypeptides form homotrimers via noncovalent interactions of their collagenous domains (7). Moreover, trimeric adiponectin can link, via disulfide bonds, into hexamers and a group of larger complexes, referred to as high molecular weight (HMW) adiponectin (8, 9). Intracellular formation of HMW adiponectin involves posttranslational modifications, e.g. hydroxylation and glycosylation in the collagenous domain (10). Adipocytes secrete HMW adiponectin, which can be retained in the endoplasmic reticulum by chaperone molecules differentially regulated by changes in nutrient status (11, 12). Globular adiponectin, which only consists of the C-terminal globular domain of the adiponectin protein, may also be present in blood (13, 14, 15). Adiponectin may be cleaved into globular fragments by proteases (16).

Based on the observation that plasma concentrations of total adiponectin are reduced in vascular diseases, type 2 diabetes, and the metabolic syndrome (17, 18), adiponectin is likely to promote health benefits. Furthermore, HMW adiponectin has distinctive effects on metabolism (8). Impaired multimerization of adiponectin is associated with diabetes (19), and increased circulating levels of HMW adiponectin are associated with weight loss (20), high glucose tolerance (21), and improvement in insulin sensitivity (22).

It is often suggested that adiponectin is an antiinflammatory adipokine. However, this view has been challenged in recent reports of elevated adiponectin plasma levels in several diseases associated with inflammation: arthritis (23, 24), preeclampsia (25, 26, 27, 28, 29, 30, 31, 32, 33), and end-stage renal disease (34, 35, 36). Therefore, adiponectin may have proinflammatory as well as antiinflammatory properties, depending on the biological context.

Adipose tissue is composed of adipocytes and several other cell types like immune cells, linked to low-grade inflammation (37, 38, 39). Many transcription factors are important in orchestrating the inflammatory response. Nuclear factor-{kappa}B (NF-{kappa}B) is an essential transcription factor for expression of inflammatory and stress-related proteins (40). Here, we have studied the ability of adiponectin to modulate NF-{kappa}B activity in human monocytic cells to delineate potential paracrine communication between adipocytes and immune cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell culture and supplements
U937, THP-1, Raji, Ramos, Jurkat cells (American Type Culture Collection, Manassas, VA) were cultured in growth medium [RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L glutamine, penicillin (50 U/ml), and streptomycin (50 µg/ml) stabilized solution (all from Sigma-Aldrich, St. Louis, MO)] at 37 C in 5% CO2.

Generation of U937-3x-{kappa}B-LUC stably transfected reporter cells. U937 cells were cotransfected by electroporation with an NF-{kappa}B reporter plasmid construct containing a firefly luciferase gene under transcriptional control by a promoter containing three NF-{kappa}B elements (5'-GGGACTTTCC-3') derived from the Ig-{kappa}-light chain gene promoter region, together with the pMEP4 plasmid containing a hygromycin B resistance element, and stable transfectants of both plasmids were cloned as previously described (41). U937-3x-{kappa}B-LUC reporter cells were maintained in the presence of hygromycin B (75 µg/ml) (Invitrogen, Carlsbad, CA).

Cells were seeded in fresh growth medium on 24-well cluster plates (Corning Life Sciences, Schiphol-Rijk, The Netherlands) just before incubations (0.2 ml volume containing 0.6 x 106 cells in each well) with different substances: vehicle alone (PBS; Sigma-Aldrich); recombinant native and mutated (C39A) human adiponectin produced in HEK cells (BioVendor, Heidelberg, Germany); C-terminal globular domain of human adiponectin produced recombinantly in Escherichia coli (PeproTech, London, UK); recombinant human resistin (PeproTech); recombinant human TNF-{alpha} and IL-6 (R&D Systems, Minneapolis, MN); and E. coli lipopolysaccharide (LPS) (catalog no. L2880; Sigma-Aldrich).

In toll-like receptor 4 (TLR4) inhibition experiments, we used functional grade antihuman TLR4 clone HTA125 and mouse IgG2a isotype control, both at 20 µg/ml (eBioscience, San Diego, CA). For TNF receptor (TNFR) inhibition, we used antihuman TNFR1 clone 16805.21, antihuman TNFR2 clone 22210, and mouse IgG1 as isotype control, all at 10 µg/ml (eBioscience). Cells were first preincubated for 30 min with antibodies alone and then for 6 h, together with globular adiponectin (5 µg/ml), TNF-{alpha} (0.2 ng/ml), or vehicle only.

Different pharmacological inhibitors (Calbiochem, La Jolla, CA) were dissolved in dimethyl sulfoxide, which was used as a vehicle for succeeding dilutions (final dimethyl sulfoxide concentration 0.05%) with growth medium (final concentrations in parenthesis): PD 98059 (10 µM), SB 203580 (10 µM), Jun N-terminal kinase (JNK) II inhibitor (10 µM), LY 294002 (10 µM), bisindolylmaleimide (1 µM), H-89 dihydrochloride (10 µM), Compound C (20 µM), and BAY 11-7082 (50 µM).

Luciferase activities were measured directly on living cells in cluster plates. We added 10 µl D-luciferin (Biosynth, Staad, Switzerland) solution (20 mg/ml in PBS) to the culture media; after 4-min incubation at 37 C, light emitted in the course of 1 min was imaged with the IVIS Imaging System 100 (Xenogen, Alameda, CA). Luciferase activity (photons/sec·cm2·steradian) in each well was quantified by image analysis using the Living Image Software (Xenogen).

Fold changes in NF-{kappa}B activities were calculated as the ratio of luciferase activity (photons/sec·cm2·steradian) in cells incubated with different test substances and control cells incubated with vehicle only.

Protein analyses
Adiponectin (10 µg; native and mutated recombinant; BioVendor) was analyzed using a Pharmacia SMART apparatus fitted with a Superdex 200 gel filtration column (10/300 GL), and a HMW Gel Filtration Calibration Kit (both from GE Healthcare Bio-Sciences, Piscataway, NJ). Samples loaded onto the column were eluted with PBS (100 µl/min; Sigma-Aldrich), and 24 fractions of 50 µl were collected.

The largest part (45 µl) of the gel fractions was tested for NF-{kappa}B inducing activity using U937-3x-{kappa}B-LUC cells as previously described, whereas an aliquot (2 µl) was analyzed by Western blotting. Proteins were separated together with the Dualcolor molecular weight standard (Bio-Rad Laboratories, Hercules, CA) by SDS-PAGE at 4 C overnight using 3–8% polyacrylamide gradient Tris-acetate buffered Criterion XT precast gels with or without the supplied Reducing Agent (Bio-Rad); protein was transferred to an activated polyvinylidene fluoride membrane (Immobilon-P, 0.45 µm; Millipore, Billerica, MA). Blots were incubated sequentially at room temperature in Tris-buffered saline (pH 7.5) + 0.05% Tween 20 (TTBS) (Sigma-Aldrich) with different additions: 1) 30-min blocking with 5% dry milk; 2) 1-h incubation with 1:10,000 diluted rabbit polyclonal antiserum directed against human adiponectin (catalog no. ALX-210-377; Alexis Biochemicals, San Diego, CA), followed by four TTBS washes; and 3) 1-h incubation with 1:50,000 dilution of goat antirabbit IgG (heavy and light chain)-horseradish peroxidase mouse/human absorbed antibodies (Southern Biotechnology, Birmingham, AL), followed by a final four TTBS washes. Adiponectin was visualized on Amersham Hyperfilm using the ECL+ chemoluminescence kit (GE Healthcare Bio-Sciences).

Concentration of cytokines and chemokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, granulocyte macrophage colony stimulating factor, interferon-{gamma}, and TNF-{alpha}) in conditioned media (50 µl) was determined in duplicates with a multiplexed immunoassay (13-plex High Sensitivity Human Cytokine LINCOplex kit; Millipore) on a Luminex instrument (Bio-Rad).

Nuclear extracts were prepared after washing cells with PBS (Sigma-Aldrich) added Complete proteinase inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), followed by a spin; pelleted cells were resuspended in hypotonic buffer [20 mM HEPES (pH 7.5), 5 mM NaF, 10 µM Na2MoO4, 0.1 mM EDTA] and allowed to swell on ice for 15 min. Nonidet P-40 (Sigma-Aldrich) was added to a final concentration of 0.5%, and the nuclei were pelleted by centrifugation. The nuclear pellet was resuspended in 50 µl Complete Lysis Buffer (Active Motif Europe, Rixensart, Belgium) and incubated for 30 min on ice, followed by a centrifugation (14,000 x g for 10 min at 4 C). The supernatants were collected, and protein concentrations were assayed using the bicinchoninic acid reagent kit (Pierce, Rockford, IL).

Immunoreactive p65 binding of to the NF-{kappa}B consensus DNA sequence (5'-GGGACTTTCC-3') was quantified in the nuclear extracts (3.5 µg total protein) with a Trans-AM NF-{kappa}B transcription factor assay kit, including a recombinant p65 standard curve, according to the manufacturer’s instructions (Active Motif Europe).

mRNA analyses
Cells were cultured as above (see Cell culture and supplements) harvested in TRIzol Reagent (Invitrogen) and stored at –70 C until RNA isolation, according to the manufacturer’s instructions. RNA quality and quantity were determined with a spectrophotometer (NanoDrop Technologies, Wilmington, DE).

PCR products were first amplified from U937 mRNA with the GeneAmp EZ rTth RNA PCR kit (Applied Biosystems, Langden, Germany) and primer pairs specific for human genes: I{kappa}B-{alpha} (official symbol: NFKBIA) forward (5'-AACCTGCAGCAGACTCCACT-3') and reverse (5'-GACACGTGTGGCCATTGTAG-3'); monocyte chemoattractant protein-1 (MCP-1) (CCL2) forward (5'-AGATGCAATCAATGCCC-3') and reverse (5'-GTTGTGGAGTGAGTGTTC-3'); and ribosomal protein L-27 (RPL27) forward (5'-CCTACAGCCATGCTCT-3') and reverse (5'-CATCCTTATTGACGACAGT-3'). Sizes and specificities of PCR products were confirmed on agarose gels and cloned into pCR2.1 using the TOPO TA cloning kit (Invitrogen), and the resulting plasmids were control sequenced. PCR positive controls were made by isolating plasmid inserts by restriction analysis and agarose gel extraction, and final quantification by spectrophotometry and semiquantitative agarose gel analysis.

For quantitative RT-PCR, 2 µg total RNA was reversely transcribed into cDNA with MultiScribe (Applied Biosystems) in 50-µl reactions, according to the manufacturer’s instructions. Aliquots (2 µl) of the reverse transcriptase reaction, or water only (negative control), were PCR-amplified with the LightCycler-FastStart DNA Master SYBER Green kit (Roche Diagnostics) and the LightCycler instrument (Roche Diagnostics). The reaction was monitored real-time after every cycle (40 in total) of the temperature steps 95 C (10 sec), 55 C (3 sec), and 72 C (10 sec) [an additional 86 C (1 sec) step for I{kappa}B only]. PCR specificities were confirmed by melting curve analysis of amplified PCR positive controls and cDNA samples using the LightCycler Software (Roche Diagnostics). Target gene cDNA levels were determined using the second derivative maximum method in the LightCycler Software by relating to standard curves made of respective serial dilutions of PCR positive controls.

The gene encoding ribosomal protein L27 (RPL27) was used as an internal control and dividing target gene copy number by RPL27 cDNA copy number estimated relative target gene mRNA levels.

Presentation of data and statistical analysis
Data are presented as mean ± SEM. The statistical significance of differences between incubations was assessed with the two-tailed Student’s t test, and P < 0.05 was considered significant.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Native adiponectin reduces LPS-induced NF-{kappa}B activity and induces NF-{kappa}B activity
To test whether adiponectin could modulate cellular NF-{kappa}B activity, monocytic U937-3x-{kappa}B-LUC reporter cells were preincubated with or without native adiponectin for 18 h before an additional 4 h inflammatory stimulus with TNF-{alpha}, LPS, or vehicle alone. NF-{kappa}B reporter activity was increased 10.4 ± 0.8 and 22.3 ± 2.2-fold by TNF-{alpha} and LPS alone, respectively (both P ≤ 0.001) (Fig. 1Go). Cells preincubated with adiponectin displayed significantly reduced NF-{kappa}B reporter activities in response to LPS, but not TNF-{alpha} (Fig. 1Go). NF-{kappa}B reporter activity was significantly increased 3.5 ± 0.3-fold in the controls incubated with adiponectin alone (Fig. 1Go, white bars).


Figure 1
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  		FIG. 1. Effect of preincubation with adiponectin on NF-{kappa}B activity induced by TNF-{alpha} and LPS. U937 monocytic cells stably transfected with a NF-{kappa}B responsive luciferase reporter gene (U937-3x-{kappa}B-LUC reporter cells) were preincubated 18 h with (+) or without (–) native adiponectin (5 µg/ml) before 4-h incubation with either TNF-{alpha} (2 ng/ml) or LPS (1 µg/ml), or vehicle only (control). NF-{kappa}B activity (fold induction) is shown relative to control cells. Data represent means ± SEM (n = 3). **, P < 0.01

 
Confronted with the numerous studies reporting reduced concentrations of total plasma adiponectin in various conditions like vascular diseases, type 2 diabetes, and the metabolic syndrome (17, 18), we expected adiponectin to possess antiinflammatory properties. U937-3x-{kappa}B-LUC reporter cells preincubated with adiponectin had reduced LPS-induced NF-{kappa}B activity in our experiments. Thus, the first interpretation was that adiponectin inhibits NF-{kappa}B activation. In support of this notion, a previous in vitro study has shown that preincubation with adiponectin suppresses LPS-induced NF-{kappa}B activation in pig primary adipocytes and 3T3-L1 adipocytes (42). In another study adiponectin suppressed TNF-{alpha} induced NF-{kappa}B activity in human aortic endothelial cells (43). Alternatively, our observations raised the possibility that adiponectin induces NF-{kappa}B activity per se, although to a smaller extent than TNF-{alpha} and LPS.

Native adiponectin activates NF-{kappa}B in a time- and dose-responsive manner at physiological concentrations
Six hours after U937-3x-{kappa}B-LUC reporter cells were exposed to native adiponectin, a peak in NF-{kappa}B reporter activity was measured (2.7 ± 0.3-fold increase with 10 µg/ml; P = 0.014). The NF-{kappa}B reporter was increasingly activated at concentrations ranging from 1–10 µg/ml (Fig. 2AGo).


Figure 2
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  		FIG. 2. Time and dose response of different adipokines on NF-{kappa}B activity. U937-3x-{kappa}B-LUC reporter cells were incubated with adiponectin (A), leptin (B), resistin (C), and IL-6 (D) up to 30 h. NF-{kappa}B activity (fold induction) is shown relative to control cells incubated with vehicle only. Data points are means ± SEM (n = 3).

 
There have been reports of NF-{kappa}B activation by other adipokines (44, 45, 46), so we also tested if recombinant leptin, resistin, and IL-6 affected U937-3x-{kappa}B-LUC reporter activity (Fig. 2Go, B–D). Resistin, leptin (both 200 ng/ml), and IL-6 (40 ng/ml) increased mean NF-{kappa}B activity at 6 h by 1.8 ± 0.2 (P = 0.063), 1.5 ± 0.1 (P = 0.090), and 1.4 ± 0.0 (P = 0.003) fold, respectively. In the short term, there was no obvious dose-response relationship between the adipokines leptin and IL-6, and NF-{kappa}B reporter activity (Fig. 2Go, B and C). On the other hand, both adiponectin and resistin increased NF-{kappa}B reporter activity dose dependently (Fig. 2Go, A and C).

These data indicate that NF-{kappa}B responds to native adiponectin both rapidly within 4–12 h, and dose dependently within the physiological range. Different adipokines were compared, revealing that physiological relevant concentrations of both adiponectin and resistin activate NF-{kappa}B directly and dose dependently. Resistin is an adipokine with potent proinflammatory properties (46). Because adiponectin activated NF-{kappa}B to levels comparable with resistin, it may be a relatively strong activator of NF-{kappa}B. Leptin and IL-6 did also increase NF-{kappa}B activity, but only at supraphysiological concentrations and to lower levels than adiponectin.

Other investigators have reported that adiponectin stimulates the release of proinflammatory cytokines and prostaglandins from human placenta and adipose tissue (45). Recent studies suggest that the role of adiponectin in immunological and inflammatory diseases is somewhat different than in metabolic and cardiovascular diseases. Elevated plasma concentrations of adiponectin are observed in several diseases associated with inflammation: arthritis (23, 24), preeclampsia (25, 26, 27, 28, 29, 30, 31, 32, 33), and end-stage renal disease (34, 35, 36). Furthermore, adiponectin is expressed in bone (6) and enhanced in the synovial fluid of patients with arthritis (47, 48). Therefore, it has been suggested that an increased level of adiponectin is a compensatory mechanism to dampen inflammation. Our present data raise an alternative explanation: that adiponectin is a proinflammatory hormone.

Selective activation of NF-{kappa}B by the HMW isoforms of adiponectin
Because native adiponectin exists in blood as several multimers with different metabolic effects (5, 8), we hypothesized that the multimers may differ as activators of NF-{kappa}B. Multimers of native recombinant adiponectin with different apparent molecular weights were separated by gel filtration chromatography (Fig. 3AGo, upper panel), and we identified three peaks representing the different adiponectin multimer species: HMW [relative molecular weight (Mr) > 669,000], hexamers (Mr ~ 440,000), and trimers (440,000 > Mr > 232,000). The separate fractions collected during gel filtration of adiponectin were tested for activation of NF-{kappa}B. Only fraction numbers 5–7, corresponding to HMW adiponectin, increased cellular NF-{kappa}B reporter activity (Fig. 3AGo, lower panel). As a negative control, mutated recombinant adiponectin (C39A) was fractionated in a similar manner, yielding only one major peak representing trimeric adiponectin. Mutated adiponectin (C39A) did not alter NF-{kappa}B reporter activity, either before or after fractionation (data not shown).


Figure 3
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  		FIG. 3. Effect of different adiponectin isoforms on NF-{kappa}B activity. A, Native recombinant adiponectin was gel fractionated (top panel) and aligned with molecular weight (Mr) standards (x103). U937-3x-{kappa}B-LUC reporter cells were exposed to the indicated fractions. NF-{kappa}B activity is shown as the number of photons emitted from the cells (bottom panel). B, Fractions (numbered one to 24) of native adiponectin and mutated (C39A) adiponectin were eluted from a gel filtration column (Superdex 200), and proteins were separated by nonreducing and reducing SDS-PAGE. Immunoreactive adiponectin was detected by Western blotting and aligned with Mr standards (x103). Representative data of three similar experiments are shown.

 
To ensure that the fractions tested contained adiponectin, we analyzed the samples by SDS-PAGE and Western blotting using an adiponectin-specific antibody. Immunoreactive adiponectin was detected in fraction numbers 5–13 (Fig. 3BGo, upper panels), presented as multiple bands of different Mr. Under nonreducing conditions, fractions with HMW, hexameric, and trimeric adiponectin all presented an intense band with apparent Mr 50,000 (Fig. 3BGo, upper left panel), suggesting that the large complexes of adiponectin are unstable during SDS-PAGE. After chemical reduction and boiling (Fig. 3BGo, upper right panel), monomeric adiponectin was detected as an intense band with Mr 30,000 (8). We confirmed by Western blotting that most mutated adiponectin (C39A) had eluted in fractions 10–13 corresponding to trimeric adiponectin (Fig. 3BGo, lower panels).

Our data suggest that among the different adiponectin isoforms, only HMW adiponectin activates NF-{kappa}B. Theoretically, the adiponectin peptide (Mr ~ 30,000) at 5 µg/ml has molar concentration of approximately 170 nM. Native adiponectin consists of different isoforms, and about one third of its mass may be ascribed to HMW adiponectin. Furthermore, HMW adiponectin may consist of 18 or more adiponectin peptides (8). All this considered, the molar concentration of HMW adiponectin may be as low as 3 nM when cells are incubated with native adiponectin at 5 µg/ml.

In accordance with the present results, HMW adiponectin induced secretion of IL-6 in human monocytes (49). Furthermore, Tsao et al. (9, 50) have reported effects of adiponectin on NF-{kappa}B in murine muscle cells, and these were ascribed to both hexameric and HMW adiponectin. In our experiments using human cells, only the HMW isoform of native adiponectin induces NF-{kappa}B activity (Fig. 3Go). This difference may be due to species differences but may also suggest that different cell types respond differently to the adiponectin isoforms.

In the literature, papers reporting proinflammatory effects of adiponectin stand out as exceptions. In general, adiponectin is referred to as antiinflammatory. We tie these concepts together showing that adiponectin does inhibit inflammatory stimuli by LPS (Fig. 1Go) but that this is only after an initial activation of NF-{kappa}B by adiponectin (Fig. 2Go). This finding may be essential to understand the role of adiponectin in inflammation.

Strong activation of NF-{kappa}B by globular adiponectin involves p38 MAPK, phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC)
Based on structural homology between globular adiponectin and TNF-{alpha}, it may be predicted that globular adiponectin has proinflammatory properties (7). Globular adiponectin induced NF-{kappa}B reporter activity in a time- and dose-dependent manner, with activities peaking between 4 and 6 h in cells incubated with 5–10 µg/ml (Fig. 4AGo). At lower concentrations, globular adiponectin induced NF-{kappa}B reporter activity more slowly (Fig. 4Go, A and B). These results indicate that globular adiponectin at high concentrations is a direct activator of NF-{kappa}B in U937 cells.


Figure 4
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  		FIG. 4. Effect of globular adiponectin on NF-{kappa}B activity. A, U937-3x-{kappa}B-LUC reporter cells were incubated with: different concentrations of globular adiponectin (gAdn) up to 30 h [NF-{kappa}B activity (fold induction) is shown relative to cells incubated with vehicle only]. B, U937-3x-{kappa}B-LUC reporter cells were incubated with low concentrations of globular adiponectin (down to 32 ng/ml) [the heat map shows NF-{kappa}B activity as emitted light (photons/sec cm2 steradian)]. C, U937-3x-{kappa}B-LUC reporter cells were incubated with combinations of globular adiponectin (5 g/ml), TNF-{alpha} (20 ng/ml), and LPS (1 g/ml) for 6 h [NF-{kappa}B activity (fold induction) is shown relative to control]. D, U937-3x-{kappa}B-LUC reporter cells were first preincubated for 30 min in media with TLR4-specific inhibitory antibody, an irrelevant antibody, or no antibody added. Cells were then incubated 6 h after adding globular adiponectin (5 µg/ml), LPS (1 µg/ml), or vehicle only. NF-{kappa}B activity (fold induction) is shown relative to control. E, U937-3x-{kappa}B-LUC reporter cells were preincubated for 30 min with different inhibitors (in parentheses) of cellular signaling systems (bold) before 6-h incubation with (+) and without (–) globular adiponectin and NF-{kappa}B activity (photons/sec·cm2·steradian) measured. Data are presented as means ± SEM (n = 3). *, P < 0.05. **, P < 0.01. AMPK, AMP-activated protein kinase; MEK1, MAPK kinase 1.

 
TNF-{alpha} (0.2–20 ng/ml) and LPS (0.2–2 µg/ml) induced NF-{kappa}B activity in the same order of magnitude as globular adiponectin (1–10 µg/ml) (data not shown) (Fig. 4CGo). Cells incubated with matching exposures of LPS and globular adiponectin (both 1 µg/ml for 6 h) had 11.1 ± 1.26 and 6.7 ± 0.48-fold increases in NF-{kappa}B activity, respectively, but these were not significantly different (P = 0.110) (data not shown). This indicates that globular adiponectin is a potent activator of NF-{kappa}B.

We wanted to investigate the possibility that globular adiponectin may act via the same pathways as TNF-{alpha} or LPS. To reveal any additive effects, we incubated the NF-{kappa}B reporter cells with combinations of globular adiponectin, TNF-{alpha}, and LPS. The effect of globular adiponectin on NF-{kappa}B activity increased significantly by 31% together with TNF-{alpha} (P = 0.045); a corresponding increase was not observed with LPS (P = 0.108) (Fig. 4CGo). The additive effect of TNF-{alpha} and globular adiponectin suggests that they activate NF-{kappa}B via separate pathways. There were no additive effects of LPS and globular adiponectin, which implies that they may share some rate-limiting component of signaling transduction.

LPS and TNF-{alpha} act as extracellular agonists of TLR4 and TNFR1 and -2, respectively. We tested if globular adiponectin activates NF-{kappa}B via TLR4 or the TNFRs. An inhibitory antibody specific to TLR4 did not alter NF-{kappa}B activity induced by globular adiponectin, compared with irrelevant control antibody (P = 0.951) (Fig. 4DGo, black bars). On the contrary, LPS-induced NF-{kappa}B activity was 31% lower (P = 0.046) with the TLR4 inhibitory antibody compared with the control antibody (Fig. 4DGo, white bars). Similarly, inhibitory antibodies against TNFR1 and -2 did not inhibit NF-{kappa}B activity stemming from globular adiponectin, but only from TNF-{alpha} (data not shown). Together, these data suggest that globular adiponectin and TNF-{alpha} may activate NF-{kappa}B via separate pathways. Furthermore, globular adiponectin and LPS are likely to share some steps of signal transduction converging downstream of TLR4.

To elucidate further by which mechanism adiponectin activates NF-{kappa}B, we tested a panel of substances that inhibit key players in different signaling transduction pathways. BAY 11-7082, which inhibits phosphorylation of the intrinsic inhibitor NF-{kappa}B (I{kappa}B), was included as a positive control and reduced globular adiponectin-induced NF-{kappa}B reporter activity by 88% (Fig. 4EGo). Inhibitors of p38 MAPK (SB 203580), PI3K (LY 294002) and PKC (bisindolylmaleimide) reduced the effect of globular adiponectin significantly by 32% and 29% and 15%, respectively. Although the effect on PKC did not reach significance (P = 0.056), supplementary experiments combining all these three inhibitors suggested that they had an additive effect on NF-{kappa}B activity (data not shown). Inhibitors of p38 MAPK and PI3K also reduced NF-{kappa}B activity induced by native adiponectin by 47 and 19% (both P < 0.001), respectively (data not shown). Other inhibitors, targeting MAPK kinase 1, JNK, AMP-activated protein kinase, and protein kinase A (PKA), did not reduce NF-{kappa}B reporter activity induced by globular adiponectin (Fig. 4EGo). On the contrary, PKA inhibition increased the activity by 41% (P = 0.001). In summary, this suggests that p38 MAPK, PI3K, and PKC are partially involved when NF-{kappa}B is activated by globular adiponectin.

Globular adiponectin contributes to activities 1 order of magnitude higher than native adiponectin (Figs. 2AGo and 4AGo). At comparable concentrations both globular adiponectin and LPS induce similar levels of NF-{kappa}B activity (Fig. 4CGo) (data not shown), suggesting that globular adiponectin is a strong activator of NF-{kappa}B. Globular adiponectin was tested at concentrations 2 orders of magnitude higher than TNF-{alpha}, and these might not be comparable. It is not known if globular adiponectin forms HMW complexes that may alter the molar concentration.

Globular adiponectin has also induced TNF-{alpha} and IL-6 secretion in human macrophages (51), whereas globular adiponectin inhibits TLR-mediated NF-{kappa}B signaling in murine macrophages (52). It has recently been shown that proinflammatory actions of globular adiponectin on epithelial cells may involve NF-{kappa}B, ERK, and p38 MAPK (53, 54).

The presence of globular adiponectin has been demonstrated in human serum by immunoprecipitation (13), although it may account for less than 1% of the total adiponectin in blood (15). Data from direct measurements of globular adiponectin are very sparse. It has been observed that globular adiponectin is more potent than native adiponectin (13, 14). Cleavage of native adiponectin by proteases might represent a mechanism for making the globular fragment of adiponectin (16) but warrants further investigations.

Altered release of cytokines after incubation with adiponectin
Potentially, monocytic cells secrete several cytokines upon an inflammatory stimulus. In U937-conditioned media, the most highly concentrated cytokines were TNF-{alpha}, IL-6, IL-8, and IL-10. Incubating cells with native or globular adiponectin significantly increased TNF-{alpha} concentrations in the conditioned media 2.1 and 8.1-fold, respectively. Globular adiponectin increased IL-10 concentrations 7.2-fold at borderline significance (P = 0.078). Unexpectedly, native adiponectin reduced mean IL-6 concentrations by 67%, without reaching significance (P = 0.111) (Fig. 5AGo).


Figure 5
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  		FIG. 5. Effect of adiponectin on inflammation markers. A, U937 cells were incubated with native or globular adiponectin (both 5 µg/ml), or vehicle only as control for 6 h. Concentration of the indicated cytokines in U937-conditioned culture media was measured, and data are shown as means ± SEM (n = 4). B, U937 cells were incubated for up to 30 h with native or globular adiponectin (both 5–10 µg/ml), TNF-{alpha} (2–20 ng/ml), LPS (1–2 µg/ml), or vehicle only as control. MCP-1, I{kappa}B-{alpha}, and RPL27 mRNAs were quantified by RT-PCR, and mRNA expression levels presented are relative to the RPL27 control gene and to vehicle only incubated control cells. Data in panel B are presented on a log10 scale and represent means ± SEM (n = 4). *, P < 0.05. **, P < 0.01.

 
The genes encoding TNF-{alpha} and IL-8 are NF-{kappa}B target genes. Our results indicate that both native and globular adiponectin promote increased secretion of TNF-{alpha} and IL-8 from U937 cells, which is consistent with increased NF-{kappa}B activity. Other researchers have observed that HMW adiponectin may induce secretion of IL-6 in human monocytes (49), whereas we observed a tendency toward reduced IL-6 secretion with native adiponectin. IL-6 secretion is widely used as a bona fide marker of NF-{kappa}B activity, and this effect of adiponectin awaits a more through investigation.

Adiponectin induces mRNA expression of inflammatory marker genes
Because reporter gene analysis is an indirect measure of gene expression, we examined if mRNA expression of endogenous inflammation-related genes was enhanced by adiponectin. Native and globular adiponectin, TNF-{alpha}, and LPS, all enhanced mRNA levels of both MCP-1 and I{kappa}B-{alpha} in U937 cells (Fig. 5BGo). MCP-1 mRNA levels peaked after 6–12 h (Fig. 5BGo, left panel), whereas the highest I{kappa}B-{alpha} mRNA levels were measured at 2 h (Fig. 5BGo, right panel). Globular adiponectin induced MCP-1 mRNA several times higher than TNF{alpha} and LPS (Fig. 5BGo, left panel), whereas globular adiponectin, TNF{alpha}, and LPS induced I{kappa}B-{alpha} mRNA to a similar degree (Fig. 5BGo, right panel).

Adiponectin activates both promoters that recruit NF-{kappa}B fast (e.g. I{kappa}B-{alpha}) as well as those that have a more delayed recruitment (e.g. MCP-1) (55). Our data also emphasize that globular adiponectin is an exceptionally strong activator of NF-{kappa}B, whereas native adiponectin has a more moderate effect.

Adiponectin induces nuclear translocation of p65 selectively in monocytic cell lines
We isolated nuclei from stimulated cells and assayed binding of the p65 subunit of NF-{kappa}B to its consensus promoter element in nuclear extracts as a measure of NF-{kappa}B activity. In accordance with the aforementioned NF-{kappa}B reporter analysis, native and globular adiponectin significantly increased levels of p65 in U937 cells by 1.5 ± 0.2 and 5.0 ± 0.3-fold, respectively (Fig. 6Go).


Figure 6
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  		FIG. 6. Effect of adiponectin on nuclear translocation of p65. U937, THP-1, Ramos, Raji, and Jurkat cells were incubated with 5 µg/ml native adiponectin, 1 µg/ml globular adiponectin, 20 ng/ml TNF-{alpha}, 1 µg/ml LPS, or vehicle only (control) for 6 h. DNA binding of NF-{kappa}B p65 to a consensus NF-{kappa}B promoter element was quantified in nuclear extracts and presented as ng p65 relative to µg total protein in the extracts. Data represent means ± SEM (n = 3). *, P < 0.05. **, P < 0.01.

 
We also tested if adiponectin could activate NF-{kappa}B in other leukocyte cell lines. THP-1 is another monocytic cell line that we incubated with native as well as globular adiponectin, which significantly increased levels of nuclear p65 5.5 ± 1.7 and 7.6 ± 3.2-fold, respectively (Fig. 6Go). This shows that adiponectin activates NF-{kappa}B in both the monocytic cell lines, U937 and THP-1. Native and globular adiponectin did not cause significant changes in the levels of nuclear p65 in B-cell lines (Ramos, Raji). In a T-cell line (Jurkat), globular adiponectin reduced nuclear p65 significantly by 30% (Fig. 6Go). TNF-{alpha} and LPS altered nuclear p65 levels significantly in all cell lines tested, except Raji that had chronically elevated levels. Earlier studies have shown that THP-1 cells incubated with globular adiponectin have enhanced secretion of TNF-{alpha} and IL-6 (51).

NF-{kappa}B plays a major role in the immune system. Different diseased tissues contain elevated numbers of inflammatory cells that act as a complex interface for cytokine signaling. For instance, immune cells present in adipose tissue may play an important role in diseases associated with increased adipose tissue mass. Immune cells are potentially a source of large quantities of cytokines. Knowledge of adiponectin’s ability to modulate NF-{kappa}B in immune cells as demonstrated here is probably relevant for understanding most inflammatory diseases.

Adipocytes may be prone to necrotic death in obese individuals, and macrophages residing in adipose tissue may scavenge dying adipocytes (39). Adiponectin can suppress the adherence of monocytic precursors of macrophages to endothelial cells (56), but it is not known if adiponectin affects recruitment of monocytes to the adipose tissue. Once monocytes extravasate through the endothelial cell layer of adipose tissue, adiponectin may activate NF-{kappa}B and modulate cytokine release.

We have undertaken a systematic and thorough investigation of adiponectin in relation to NF-{kappa}B activity. In summary, our data indicate that the HMW and globular isoforms of adiponectin activate NF-{kappa}B in monocytic cells. In adipose tissue, adiponectin may function as a paracrine signal from adipocytes to invading monocytic cells. Further studies are needed to understand fully the possible link between adiponectin and obesity related inflammation.


   Acknowledgments
 
We thank Harald Carlsen, Rune Blomhoff, and Kristin Hollung for establishing the U937-3x-{kappa}B-LUC reporter cells, Liv Austenaa for intrinsic nuclear factor-{kappa}B inhibitor-{alpha} and monocyte chemoattractant protein-1 primers and helpful scientific advice, Anne Randi Enget and Lie Jie for technical support, and Turid Veggan for technical advice.


   Footnotes
 
This work was supported by the Norwegian Cancer Society, Johan Throne Holst Foundation for Nutrition Research, Freia Medical Research Foundation, Joh. H. Andresen’s Medical Foundation, and Anders Jahre’s Foundation.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 16, 2007

Abbreviations: HMW, High molecular weight; I{kappa}B, inhibitor of nuclear factor-{kappa}B; JNK, Jun N-terminal kinase; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; Mr, relative molecular weight; NF-{kappa}B, nuclear factor-{kappa}B; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; RPL27, ribosomal protein L27; TLR4, toll-like receptor 4; TNFR, TNF receptor; TTBS, Tris-buffered saline (pH 7.5) + 0.05% Tween 20.

Received March 20, 2007.

Accepted for publication August 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Kershaw EE, Flier JS 2004 Adipose tissue as an endocrine organ. J Clin Endocrinol Metab 89:2548–2556[Abstract/Free Full Text]
  2. Hu E, Liang P, Spiegelman BM 1996 AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271:10697–10703[Abstract/Free Full Text]
  3. Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K 1996 cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose most abundant gene transcript 1). Biochem Biophys Res Commun 221:286–289[CrossRef][Medline]
  4. Nakano Y, Tobe T, Choi-Miura NH, Mazda T, Tomita M 1996 Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J Biochem (Tokyo) 120:803–812[Abstract/Free Full Text]
  5. Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF 1995 A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 270:26746–26749[Abstract/Free Full Text]
  6. Berner HS, Lyngstadaas SP, Spahr A, Monjo M, Thommesen L, Drevon CA, Syversen U, Reseland JE 2004 Adiponectin and its receptors are expressed in bone-forming cells. Bone 35:842–849[Medline]
  7. Shapiro L, Scherer PE 1998 The crystal structure of a complement-1q family protein suggests an evolutionary link to tumor necrosis factor. Curr Biol 8:335–338[CrossRef][Medline]
  8. Pajvani UB, Du X, Combs TP, Berg AH, Rajala MW, Schulthess T, Engel J, Brownlee M, Scherer PE 2003 Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. Implications for metabolic regulation and bioactivity. J Biol Chem 278:9073–9085[Abstract/Free Full Text]
  9. Tsao TS, Tomas E, Murrey HE, Hug C, Lee DH, Ruderman NB, Heuser JE, Lodish HF 2003 Role of disulfide bonds in Acrp30/adiponectin structure and signaling specificity. Different oligomers activate different signal transduction pathways. J Biol Chem 278:50810–50817[Abstract/Free Full Text]
  10. Wang Y, Lam KS, Chan L, Chan KW, Lam JB, Lam MC, Hoo RC, Mak WW, Cooper GJ, Xu A 2006 Post-translational modifications of the four conserved lysine residues within the collagenous domain of adiponectin are required for the formation of its high molecular weight oligomeric complex. J Biol Chem 281:16391–16400[Abstract/Free Full Text]
  11. Qiang L, Wang H, Farmer SR 2007 Adiponectin secretion is regulated by SIRT1 and the endoplasmic reticulum oxidoreductase Ero1-L{alpha}. Mol Cell Biol 27:4698–4707[Abstract/Free Full Text]
  12. Wang ZV, Schraw TD, Kim JY, Khan T, Rajala MW, Follenzi A, Scherer PE 2007 Secretion of the adipocyte-specific secretory protein adiponectin critically depends on thiol-mediated protein retention. Mol Cell Biol 27:3716–3731[Abstract/Free Full Text]
  13. Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT, Bihain BE, Lodish HF 2001 Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA 98:2005–2010[Abstract/Free Full Text]
  14. Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T 2001 The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 7:941–946[CrossRef][Medline]
  15. Maeda N, Shimomura I, Kishida K, Nishizawa H, Matsuda M, Nagaretani H, Furuyama N, Kondo H, Takahashi M, Arita Y, Komuro R, Ouchi N, Kihara S, Tochino Y, Okutomi K, Horie M, Takeda S, Aoyama T, Funahashi T, Matsuzawa Y 2002 Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med 8:731–737[CrossRef][Medline]
  16. Waki H, Yamauchi T, Kamon J, Kita S, Ito Y, Hada Y, Uchida S, Tsuchida A, Takekawa S, Kadowaki T 2005 Generation of globular fragment of adiponectin by leukocyte elastase secreted by monocytic cell line THP-1. Endocrinology 146:790–796[Abstract/Free Full Text]
  17. Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K 2006 Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J Clin Invest 116:1784–1792[CrossRef][Medline]
  18. Ouchi N, Shibata R, Walsh K 2006 Cardioprotection by adiponectin. Trends Cardiovasc Med 16:141–146[CrossRef][Medline]
  19. Waki H, Yamauchi T, Kamon J, Ito Y, Uchida S, Kita S, Hara K, Hada Y, Vasseur F, Froguel P, Kimura S, Nagai R, Kadowaki T 2003 Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin. J Biol Chem 278:40352–40363[Abstract/Free Full Text]
  20. Bobbert T, Rochlitz H, Wegewitz U, Akpulat S, Mai K, Weickert MO, Mohlig M, Pfeiffer AF, Spranger J 2005 Changes of adiponectin oligomer composition by moderate weight reduction. Diabetes 54:2712–2719[Abstract/Free Full Text]
  21. Fisher FF, Trujillo ME, Hanif W, Barnett AH, McTernan PG, Scherer PE, Kumar S 2005 Serum high molecular weight complex of adiponectin correlates better with glucose tolerance than total serum adiponectin in Indo-Asian males. Diabetologia 48:1084–1087[CrossRef][Medline]
  22. Pajvani UB, Hawkins M, Combs TP, Rajala MW, Doebber T, Berger JP, Wagner JA, Wu M, Knopps A, Xiang AH, Utzschneider KM, Kahn SE, Olefsky JM, Buchanan TA, Scherer PE 2004 Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity. J Biol Chem 279:12152–12162[Abstract/Free Full Text]
  23. Otero M, Lago R, Gomez R, Lago F, Dieguez C, Gomez-Reino JJ, Gualillo O 2006 Changes in plasma levels of fat-derived hormones adiponectin, leptin, resistin and visfatin in patients with rheumatoid arthritis. Ann Rheum Dis 65:1198–1201[Abstract/Free Full Text]
  24. Senolt L, Pavelka K, Housa D, Haluzik M 2006 Increased adiponectin is negatively linked to the local inflammatory process in patients with rheumatoid arthritis. Cytokine 35:247–252[CrossRef][Medline]
  25. D’Anna R, Baviera G, Corrado F, Giordano D, De Vivo A, Nicocia G, Di Benedetto A 2006 Adiponectin and insulin resistance in early- and late-onset pre-eclampsia. BJOG 113:1264–1269[CrossRef][Medline]
  26. Haugen F, Ranheim T, Harsem NK, Lips E, Staff AC, Drevon CA 2006 Increased plasma levels of adipokines in preeclampsia: relationship to placenta and adipose tissue gene expression. Am J Physiol Endocrinol Metab 290:E326–E333
  27. Hendler I, Blackwell SC, Mehta SH, Whitty JE, Russell E, Sorokin Y, Cotton DB 2005 The levels of leptin, adiponectin, and resistin in normal weight, overweight, and obese pregnant women with and without preeclampsia. Am J Obstet Gynecol 193(3 Pt 2):979–983
  28. Kajantie E, Kaaja R, Ylikorkala O, Andersson S, Laivuori H 2005 Adiponectin concentrations in maternal serum: elevated in preeclampsia but unrelated to insulin sensitivity. J Soc Gynecol Investig 12:433–439[Medline]
  29. Lu D, Yang X, Wu Y, Wang H, Huang H, Dong M 2006 Serum adiponectin, leptin and soluble leptin receptor in pre-eclampsia. Int J Gynaecol Obstet 95:121–126[CrossRef][Medline]
  30. Naruse K, Yamasaki M, Umekage H, Sado T, Sakamoto Y, Morikawa H 2005 Peripheral blood concentrations of adiponectin, an adipocyte-specific plasma protein, in normal pregnancy and preeclampsia. J Reprod Immunol 65:65–75[CrossRef][Medline]
  31. O’Sullivan AJ, Kriketos AD, Martin A, Brown MA 2006 Serum adiponectin levels in normal and hypertensive pregnancy. Hypertens Pregnancy 25:193–203[CrossRef][Medline]
  32. Ramsay JE, Jamieson N, Greer IA, Sattar N 2003 Paradoxical elevation in adiponectin concentrations in women with preeclampsia. Hypertension 42:891–894[Abstract/Free Full Text]
  33. Takemura Y, Osuga Y, Koga K, Tajima T, Hirota Y, Hirata T, Morimoto C, Harada M, Yano T, Taketani Y 2007 Selective increase in high molecular weight adiponectin concentration in serum of women with preeclampsia. J Reprod Immunol 73:60–65[CrossRef][Medline]
  34. Zoccali C, Mallamaci F, Tripepi G, Benedetto FA, Cutrupi S, Parlongo S, Malatino LS, Bonanno G, Seminara G, Rapisarda F, Fatuzzo P, Buemi M, Nicocia G, Tanaka S, Ouchi N, Kihara S, Funahashi T, Matsuzawa Y 2002 Adiponectin, metabolic risk factors, and cardiovascular events among patients with end-stage renal disease. J Am Soc Nephrol 13:134–141[Abstract/Free Full Text]
  35. Huang JW, Yen CJ, Chiang HW, Hung KY, Tsai TJ, Wu KD 2004 Adiponectin in peritoneal dialysis patients: a comparison with hemodialysis patients and subjects with normal renal function. Am J Kidney Dis 43:1047–1055[Medline]
  36. Shoji T, Shinohara K, Hatsuda S, Kimoto E, Fukumoto S, Emoto M, Tahara H, Koyama H, Ishimura E, Miki T, Tabata T, Nishizawa Y 2005 Altered relationship between body fat and plasma adiponectin in end-stage renal disease. Metabolism 54:330–334[CrossRef][Medline]
  37. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante Jr AW 2003 Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 112:1796–1808[CrossRef][Medline]
  38. Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ, Sole J, Nichols A, Ross JS, Tartaglia LA, Chen H 2003 Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 112:1821–1830[CrossRef][Medline]
  39. Cinti S, Mitchell G, Barbatelli G, Murano I, Ceresi E, Faloia E, Wang S, Fortier M, Greenberg AS, Obin MS 2005 Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans. J Lipid Res 46:2347–2355[Abstract/Free Full Text]
  40. Pahl HL 1999 Activators and target genes of Rel/NF-{kappa}B transcription factors. Oncogene 18:6853–6866[CrossRef][Medline]
  41. Carlsen H, Moskaug JO, Fromm SH, Blomhoff R 2002 In vivo imaging of NF-kappa B activity. J Immunol 168:1441–1446[Abstract/Free Full Text]
  42. Ajuwon KM, Spurlock ME 2005 Adiponectin inhibits LPS-induced NF-{kappa}B activation and IL-6 production and increases PPAR{gamma}2 expression in adipocytes. Am J Physiol Regul Integr Comp Physiol 288:R1220–R1225
  43. Ouchi N, Kihara S, Arita Y, Okamoto Y, Maeda K, Kuriyama H, Hotta K, Nishida M, Takahashi M, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Funahashi T, Matsuzawa Y 2000 Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-{kappa}B signaling through a cAMP-dependent pathway. Circulation 102:1296–1301[Abstract/Free Full Text]
  44. Bouloumie A, Marumo T, Lafontan M, Busse R 1999 Leptin induces oxidative stress in human endothelial cells. FASEB J 13:1231–1238[Abstract/Free Full Text]
  45. Lappas M, Permezel M, Rice GE 2005 Leptin and adiponectin stimulate the release of proinflammatory cytokines and prostaglandins from human placenta and maternal adipose tissue via nuclear factor-{kappa}B, peroxisomal proliferator-activated receptor-{gamma} and extracellularly regulated kinase 1/2. Endocrinology 146:3334–3342[Abstract/Free Full Text]
  46. Bokarewa M, Nagaev I, Dahlberg L, Smith U, Tarkowski A 2005 Resistin, an adipokine with potent proinflammatory properties. J Immunol 174:5789–5795[Abstract/Free Full Text]
  47. Schaffler A, Ehling A, Neumann E, Herfarth H, Tarner I, Scholmerich J, Muller-Ladner U, Gay S 2003 Adipocytokines in synovial fluid. JAMA 290:1709–1710[Free Full Text]
  48. Ehling A, Schaffler A, Herfarth H, Tarner IH, Anders S, Distler O, Paul G, Distler J, Gay S, Scholmerich J, Neumann E, Muller-Ladner U 2006 The potential of adiponectin in driving arthritis. J Immunol 176:4468–4478[Abstract/Free Full Text]
  49. Neumeier M, Weigert J, Schaffler A, Wehrwein G, Muller-Ladner U, Scholmerich J, Wrede C, Buechler C 2006 Different effects of adiponectin isoforms in human monocytic cells. J Leukoc Biol 79:803–808[Abstract/Free Full Text]
  50. Tsao TS, Murrey HE, Hug C, Lee DH, Lodish HF 2002 Oligomerization state-dependent activation of NF-{kappa}B signaling pathway by adipocyte complement-related protein of 30 kDa (Acrp30). J Biol Chem 277:29359–29362[Abstract/Free Full Text]
  51. Tsatsanis C, Zacharioudaki V, Androulidaki A, Dermitzaki E, Charalampopoulos I, Minas V, Gravanis A, Margioris AN 2005 Adiponectin induces TNF-{alpha} and IL-6 in macrophages and promotes tolerance to itself and other pro-inflammatory stimuli. Biochem Biophys Res Commun 335:1254–1263[Medline]
  52. Yamaguchi N, Argueta JG, Masuhiro Y, Kagishita M, Nonaka K, Saito T, Hanazawa S, Yamashita Y 2005 Adiponectin inhibits Toll-like receptor family-induced signaling. FEBS Lett 579:6821–6826[CrossRef][Medline]
  53. Hattori Y, Hattori S, Kasai K 2006 Globular adiponectin activates nuclear factor-kappaB in vascular endothelial cells, which in turn induces expression of proinflammatory and adhesion molecule genes. Diabetes Care 29:139–141[Free Full Text]
  54. Ogunwobi OO, Beales IL 2006 Adiponectin stimulates proliferation and cytokine secretion in colonic epithelial cells. Regul Pept 134:105–113[CrossRef][Medline]
  55. Saccani S, Pantano S, Natoli G 2001 Two waves of nuclear factor kappaB recruitment to target promoters. J Exp Med 193:1351–1359[Abstract/Free Full Text]
  56. Ekmekci H, Ekmekci OB 2006 The role of adiponectin in atherosclerosis and thrombosis. Clin Appl Thromb Hemost 12:163–168[Abstract/Free Full Text]



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