Departments of 1Cell Biology, 2Molecular Pharmacology, and 3Medicine, Division of Endocrinology, 4Diabetes Research and Training Center, Albert Einstein College of Medicine, New York, New York 10461
Submitted 2 January 2004 ; accepted in final form 10 July 2004
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ABSTRACT |
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nuclear factor-B; inflammation
Both TNF- ligands as well as infectious pathogen cell wall constituents such as bacterial LPS and fungal zymosan mediate their inflammatory activities through NF-
B. These pyrogens activate NF-
B through the TLRs (34). We have previously shown that in the adipocyte, LPS-mediated activation of TLR4 causes upregulation of the fungal cell wall receptor TLR2 in an inflammatory feed-forward pathway (31). We observed that the time course and dose response of TLR2 induction by LPS were significantly different in the adipocyte compared with the preadipocyte, suggesting a change in NF-
B signaling during differentiation; however, there is no published data regarding the expression or regulation of NF-
B subunits during adipogenesis. With this intriguing new data indirectly suggesting regulation of this transcription factor during differentiation, and growing evidence implicating NF-
B as an important factor at the interface of metabolism and systemic inflammation, we felt further characterization of this important inflammatory transcription factor during adipocyte differentiation was necessary.
NF-B activation is at the core of many proinflammatory transcriptional programs (reviewed in Refs. 16, 26). NF-
B activation entails cytoplasmic deactivation/degradation of inhibitors of NF-
B (I
B-
, -
, and -
and the COOH terminus of p100) that then release the sequestered subunits of NF-
B transcription factors. These subunits, RelA (p65), p50, RelB (p68), and p52, then translocate to the nucleus where they bind their DNA response elements as hetero- and homodimers in various combinations. In a number of cell types, activated nuclear NF-
B induces transcription of serum amyloid A3 (SAA3), IL-6, and other secretory proteins of the innate immune response (49). A number of these acute-phase markers are expressed by the adipocyte (32). In the case of SAA3, we recently demonstrated that adipocytes (but not preadipocytes) specifically induce expression of SAA3 after inflammatory or hyperglycemic stimulation. Other inflammatory cytokines expressed by the adipocyte include TNF-
, IL-6, IL-1
, complement factors B and D, plasminogen activator inhibitor-1,
1-acid glycoprotein, lipocalin 24p3, migration inhibitory factor (MIF), IL-8, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-1
(7). Additional candidates that may exert pro- or anti-inflammatory activities are the adipokines leptin and adiponectin/Acrp30 (15, 50). In addition to the inflammatory signals secreted by adipocytes themselves, adipose tissue contains a significant population of interstitial macrophage cells and preadipocytes with macrophage-like activities (8, 9, 48). The heterotypic paracrine signaling events that may be occurring between these different cell populations have been completely unexplored to date. As a second aim of this investigation, we established an assay system that probes the effects of pre- and fully differentiated adipocytes on macrophages to begin characterizing these interactions. In this isolated system, adipocytes (but not preadipocytes) exert a strong proinflammatory effect on the macrophages. These effects are mediated by a secretory product of adipocytes that is unlikely to be IL-6 or TNF-
.
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METHODS |
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DMEM was purchased from Cellgro (Herndon, VA), and murine TNF- and IL-6 were purchased from Pharmingen (San Diego, CA). LPS (Escherichia coli) was purchased from Sigma (St. Louis, MO). Insulin was purchased from Sigma and used at 100 nM. All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).
Cell Culture
3T3-L1 murine fibroblasts (a generous gift of Dr. Charles Rubin, Dept. of Molecular Pharmacology, Albert Einstein College of Medicine) were propagated and differentiated according to the protocol described previously (12). In brief, the cells were propagated in FCS [DMEM containing 10% FCS (JRH Biosciences, Lenexa, KS) and penicillin-streptomycin (100 U/ml each)] and allowed to reach confluence (day 2). After 2 days (day 0), the medium was changed to IDX (containing FCS and 160 nM insulin, 250 µM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine). Two days later (day 2), the medium was switched to FCS containing 160 nM insulin. After another 2 days, the cells were switched back to FCS. Mouse macrophage J774 cells (a gift from Dr. Matthew Scharff, Albert Einstein College of Medicine) were cultured in FCS.
Western Blot Analysis
Ten-centimeter plates of 3T3-L1 cells after treatment were washed twice with PBS and lysed in 1 ml of SDS-PAGE sample buffer (0.75% sodium dodecyl sulfate, 0.5 M Tris·HCl, pH 6.8, and 16 mM EDTA) plus 1 mM phenylmethylsulfonyl fluoride, and lysates were boiled for 5 min followed by brief sonication. Thirty micrograms of total protein were resolved by SDS-PAGE on 12% acrylamide gels and transferred to BA83 nitrocellulose (Schleicher and Schuell, Keene, NH). Blots were probed with antibodies to NF-B subunits from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit polyclonal antiserum against p52 (a kind gift of Dr. Ulrich Siebenlist, National Institutes of Health, Bethesda, MD), and rabbit polyclonal antibodies to the guanine nucleotide dissociation inhibitor (GDI; a generous gift from Dr. Perry Bickel, Washington Univ., St. Louis, MO). Western blots to nuclear proteins were performed on 5 µg of nuclear protein from the extracts obtained for the EMSA experiments.
Immunoblotting
After SDS-PAGE, proteins were transferred to BA83 nitrocellulose (Schleicher and Schuell). Nitrocellulose membranes were blocked in PBS or Tris-buffered saline with 0.1% Tween 20 and 5% nonfat dry milk. Primary and secondary antibodies were diluted in PBS or Tris-buffered saline with 0.1% Tween 20 and 1% bovine serum albumin. Bound antibodies were detected by enhanced chemiluminescence according to the manufacturer's instructions (NEN Life Science Products, Boston, MA).
Electrophoretic Gel Mobility Shift Analysis
3T3-L1 adipocytes or preadipocytes were incubated with the indicated agents for the indicated times and then washed with PBS. Nuclear proteins were extracted as follows. The cells were scraped into 10 mM HEPES, 0.5 mM MgCl2, 10 mM KCl, and 1 mM phenylmethylsulfonyl fluoride and vortexed for 10 s, and Nonidet P-40 was added to 0.1%. After vigorous vortexing, cells were pelleted in a microfuge at 1,000 rpm for 5 min, and cytosolic supernatant was removed. The extracted nuclei were washed and pelleted twice with the same hypotonic lysis buffer without detergent. The nuclear pellet was resuspended in 20 mM HEPES, 0.5 mM MgCl2, 400 mM NaCl, 0.1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation at 14,000 rpm for 10 min, the nuclear supernatant extract for each sample was quantitated for total protein by bicinchoninic acid assay (BCA; Pierce, Dallas, TX). The binding reaction was a 20-min incubation of 5 µg of nuclear protein with a 32P end-labeled, double-stranded oligonucleotide containing the NF-B binding site on the vascular cell adhesion molecule (VCAM)-1 promoter (5'-CCTTGAAGGGATTTCCCTCC-3') (37). Cold competition controls were performed by preincubating the nuclear proteins with unlabeled 50-fold molar excess of the NF-
B double-stranded oligonucleotide for 10 min before the addition of the 32P-labeled oligonucleotide. For SAA3 enhancer factor (SEF) binding studies, the oligonucleotide 5'-CACATTTCTGGAAATGCCTAGAT-3' was used and the corresponding mutant sequence 5'-CACATTTATCAAAATGCCTATAT-3' that lacks specific binding to SEF (43). Italicized nucleotides are the point mutations that differentiate the mutant from the wild-type sequence. The mixtures were resolved on native 5% polyacrylamide gels made and run with 0.5 x Tris-borate-EDTA buffer, which were dried and autoradiographed.
Quantitative Western and Gel Shift Data
Films from the Western blots of five independently differentiated sets of adipocytes were scanned, and the background-corrected signal from each band was quantitated by densitometry using an Alpha Innotech Multiimage Light Cabinet with Chemimager 4400 software. Signal for each sample lane (containing samples from day 0 to day 8) was normalized to levels found in the day 8 lane of that same blot. Similarly, to quantitate EMSA data, gel films were scanned and gel shift bands were quantitated by densitometry. Gel shift signal for every sample in each experiment was normalized to the amounts in unstimulated cells in that same experiment. The normalized relative levels for each experimental group are represented as means ± SE. For Fig. 1, these normalized averages were further converted to percent (expression levels in day 8 adipocytes = 100%), and results were graphed as percent change from preadipocyte to adipocyte.
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Luciferase assays were performed essentially as described previously (14). 3T3-L1 preadipocytes were transfected with an NF-B-responsive luciferase reporter construct (a gift from Dr. Richard Pestell, described in Ref. 19) containing three tandem consensus binding sites from the major histocompatibility complex (MHC) II promoter. Control luciferase constructs with promoters shown to be activated by PPAR
, c-Fos, c-Myc, cyclin D, cyclin A, and junB were also graciously provided by the laboratory of Dr. Richard Pestell. The cells were cotransfected with a construct containing a hygromycin-resistance gene, and clones were selected by hygromycin. Resistant clones were picked and screened for both baseline luciferase activity and NF-
B responsiveness to TNF-
induction. The clones were checked for competence to hormonal differentiation and lipid accumulation both by appearance under the microscope as well as oil red O staining for lipids (as described in Ref. 14). After experimental treatments, cells were subjected to lysis in 200 µl of extraction buffer, 50 µl of which were used to measure luciferase activity, as described previously (40).
Enzyme-Linked Immunosorbant Assays for IL-6 and TNF-
Ten-centimeter plates of confluent preadipocytes, adipocytes, and J774 macrophage cells were given the indicated treatments, the cells were washed with PBS, and 3 ml of media were added for collection of secreted cytokines for 6 h. Fifty microliters of the resulting cell media supernatant were assayed for accumulation of IL-6 and TNF- using ELISAs purchased from R&D Systems (Minneapolis, MN).
Heterotypic Stimulation Experiments
Confluent preadipocytes and adipocytes were incubated in FCS medium for 24 h, and this conditioned medium was removed and assayed for cytokines. The conditioned medium was then diluted 1:5 in fresh FCS medium and used to stimulate J774 cells for 12 h. This medium was then assayed for its accumulated cytokine content as described in RESULTS. For immunodepletion experiments, the conditioned medium was incubated for 6 h at 4°C on protein G-Sepharose resin with anti-IL-6 antibody bound. Control mouse monoclonal antibodies generated by our laboratory were used in control immunodepletion.
Other Methods
Separation of proteins by SDS-PAGE, fluorography, and immunoblotting was performed as described previously (46). All statistical analyses were performed with numerical data represented throughout the figures as means ± SE. Student's t-test (P values 0.05) was used as a cutoff for statistical significance.
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RESULTS |
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To study changes in NF-B signal pathways associated with differentiation, 3T3-L1 cells were used to investigate expression of NF-
B subunits throughout the process of differentiation from fibroblastic preadipocyte to lipid-laden fat cell. Cells are differentiated after they reach confluence over a period of 8 days, after which triglyceride droplets are visible by microscopy. Protein extracts were made from cells at various stages of differentiation, and Western blots were processed for expression of NF-
B subunits as well as for GDI as an equal total protein loading control. Over the 8-day differentiation process, the most striking results are a significant induction of the NF-
B p65 (RelA) and p68 (RelB) subunits, as well as the regulatory subunit I
B
(Fig. 1A). This could be explained by the autoregulation of their promoters by increased NF-
B activity (6, 47). Even more strikingly, the p52 subunit increases from undetectable levels in the preadipocyte to a continuously and dramatically induced expression in the day 8 adipocyte. Interestingly, levels of the p52 precursor p100 remain relatively unchanged, implying that it is not necessarily expression of p52 that is changed but rather the proteolytic processing of the precursor to its smaller transcriptionally active form. p50 and its precursor p105 (NF-
B1) as well as Bcl-3 levels remain relatively unchanged. A number of the subunits have differential regulation specifically on day 2, with p65 and p68 displaying a downregulation and p105, p50, and Bcl-3 displaying an increase. This is, however, most likely due to the effects of the dexamethasone in the differentiation cocktail (1, 10, 29). c-Rel was not detectable on these Western blots at any stage of differentiation (data not shown). Replicates of this experiment with independently differentiated cells and subsequent assays measuring the expression of the DNA binding subunits of NF-
B (p65, p68, p50, p52) were performed and quantitated by densitometry, with amounts in each Western blot normalized to the signal present in the day 8 adipocyte samples (Fig. 1B). These results are presented graphically as percent change in expression after differentiation.
Basal NF-B Activity Increases During Adipocyte Differentiation
Basal NF-B transcriptional activation increases through differentiation.
3T3-L1 preadipocytes were stably transfected with an NF-
B-responsive luciferase reporter construct containing three tandem binding sites from the MHC class II promoter to measure NF-
B transcriptional activity and induction (19). Clones were selected based on TNF-
-responsive luciferase activity. These clones could be differentiated into lipid-laden adipocytes efficiently and were used to look at constitutive NF-
B activity during the process of differentiation. Cells were harvested on days 0, 2, 4, and 8, and cell extracts were normalized for total protein. Extracts were then assayed for their unstimulated constitutive NF-
B-mediated luciferase activity. Figure 2A shows that, during the 8-day differentiation protocol, constitutive NF-
B activity increased significantly and remained significantly higher in the mature adipocyte compared with preadipocytes. To show that luciferase induction was specifically dependent on the NF-
B promoter and not a reflection of a general increase in transcriptional activity during differentiation, a number of other luciferase constructs driven by various other promoters were transfected into 3T3-L1 cells and similarly assayed in day 0 and day 8 3T3-L1 cells (Fig. 2B). The luciferase activity driven by these promoter constructs generally decreased or remained constant, whereas a construct that served as a positive control (a PPAR
-responsive construct) doubled its activity during differentiation. Significantly, the NF-
B reporter induced luciferase activity even more significantly than PPAR
, underlining that this increase is a specific differentiation-induced effect. In addition to fold activity, absolute luciferase activities (counts/min; cpm) are indicated within the data bars for preadipocytes.
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NF-B-responsive luciferase activity is stimulated by LPS in preadipocytes but suppressed in the adipocyte.
After observing the surprising induction of NF-
B luciferase activity through differentiation in the basal state, we tested what the effects on stimulatable NF-
B activity were during differentiation. 3T3-L1 cells carrying NF-
B luciferase reporter constructs were grown in six-well plates and analyzed at either differentiated or the preadipocyte level. Preadipocytes and adipocytes (days 0 and 8) were stimulated with either 1 µg/ml LPS or 10 ng/ml TNF-
for 8 h, and equal amounts of total cellular protein were assayed for luciferase activity. As shown in Fig. 3A, preadipocytes displayed a solid induction of NF-
B upon exposure to either LPS or TNF-
compared with unstimulated cells, whereas adipocytes were sensitive only to TNF-
. Likewise, during a time-course experiment of LPS stimulation (1 µg/ml), preadipocytes produced a significant induction of NF-
B transcriptional activation upon LPS treatment, whereas adipocytes had constitutively higher levels of NF-
B and were insensitive to further induction by LPS (Fig. 3B). Dose-response experiments further corroborate these results, showing that although preadipocytes are exquisitely sensitive to even the lowest doses of LPS (20 ng/ml), adipocytes are resistant to even the highest doses (Fig. 3C).
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Although our results show altered NF-B sensitivity to LPS-induced signaling in the adipocyte, we had previously demonstrated a high level of sensitivity to LPS as judged by the transcriptional induction of TLR2 (31) as well as additional well-established NF-
B targets, such as IL-6 and SAA3 (32). To explain this apparent paradox, we sought possible additional adipocyte-specific intracellular inflammatory signals that could mediate the effects of LPS and/or TNF-
by modulating NF-
B activity in ways other than the classical nuclear I
B degradation/translocation paradigm. LPS and inflammatory cytokines induce the NF-
B target SAA3 only in adipocytes, not in preadipocytes. The ability of the mature adipocyte to induce SAA3 suggests the presence of a cofactor in addition to NF-
B that is present in the adipocyte but absent in preadipocytes or NIH-3T3 cells. The SAA3 promoter has a region called the distal regulatory element (DRE) that functions as a cytokine-inducible transcription enhancer (2, 3, 43). Within this DRE, there is an oligonucleotide that constitutes the binding site for a SEF and an overlapping NF-
B site. SEF is a transcription factor critically involved in SAA3 expression (43) (although the activity or presence of SEF has never been shown in adipocytes). Bing et al. (2) have demonstrated a functional synergy between SEF and NF-
B to achieve SAA expression and shown that this synergy may, in part, be due to the ability of SEF to recruit NF-
B through physical interactions that lead to enhancement or stabilization of NF-
B binding to the SAA3 promoter element. As in initial study, gel shift assays were performed on preadipocyte and adipocyte extracts using the SEF binding oligonucleotide, demonstrating that unstimulated and LPS-stimulated adipocytes had a strikingly increased amount of SEF binding activity compared with preadipocytes (Fig. 5A). LPS stimulation caused a modest increase in binding activity, so a time-course experiment was performed using both LPS and TNF-
as stimulants. To demonstrate specific binding, either wild-type oligos or oligos with a base pair mutation were coincubated with the SEF-binding radiolabeled probes. Notably, in this experiment, similar to the time course for p65/RelA translocation shown in Fig. 4, E and F, TNF-
treatment of mature adipocytes leads to a more rapid activation of SEF binding to DNA than treatment with LPS (Fig. 5B). Interestingly, although LPS-stimulated activity is delayed, it is significant and prolonged, whereas TNF-stimulated activity disappears after 5 h.
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In light of increased constitutive NF-B activity and acute-phase reactant secretion after adipocyte differentiation, we wanted to know whether this could be physiologically significant to inflammation in general. To simulate the paracrine interaction between adipocytes and adipose tissue-resident macrophages, cell culture medium was first conditioned by preadipocytes or differentiated adipocytes for 12 h. Subsequently, these conditioned media were diluted and used to treat J774 macrophages (1 ml of conditioned medium in 4 ml of fresh medium), and IL-6 as well as TNF-
secreted from the macrophages were assayed. When J774 cells were exposed to unstimulated preadipocyte medium, there was no increase in IL-6 or TNF-
production compared with untreated cells (Fig. 6C). In contrast, adipocyte-conditioned medium caused a significant increase in the secretion of both cytokines from macrophages. A comparison of the amounts of cytokines secreted from adipocytes with that from macrophages (Fig. 6, A and B) shows that the transfer of IL-6 and TNF-
secreted by preadipocytes and adipocytes does not contribute significantly to the levels of these cytokines found in the basal, unstimulated state of macrophages. This indicates that the macrophage-derived medium after exposure to conditioned medium containing IL-6 and TNF-
was almost completely derived from macrophages. Furthermore, this suggests that the factors stimulating the macrophages were adipocyte-specific inflammatory signals other than IL-6 and TNF-
. No detectable TNF-
could be found in the preadipocyte- or adipocyte-conditioned media. To further support the point that secreted IL-6 was not playing a role in J774 stimulation, the conditioned media from both preadipocytes and adipocytes were immunodepleted for IL-6 and used to stimulate J774 cells in a similar fashion. Immunodepletion with a resin coated in anti-IL-6 antibody compared with a control resin containing a nonrelevant antibody effectively depleted 90% of the IL-6 secreted from preadipocytes and adipocytes (Fig. 7A), yet there was no loss of J774-stimulating activity by these supernatants after removal of their IL-6 (Fig. 7B).
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DISCUSSION |
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No characterization of adipocyte NF-B expression has been reported thus far. Yet, the significant potential of the adipocyte as an inflammatory cell of systemic importance puts NF-
B regulation in adipocytes at center stage for the pathophysiology of diabetes. The patterns of expression through adipocyte differentiation, specifically of p65, p68, p52, and I
B
, suggest an increase in constitutive NF-
B activity. Luciferase reporter analysis of unstimulated preadipocytes and adipocytes corroborates these results, showing increased promoter activation by (at least) the p65/p50 NF-
B complex. Measurement of adipocyte-secreted IL-6 demonstrates that as a consequence of the increase in constitutive NF-
B activity through differentiation, the adipocyte constitutively produces inflammatory signals. The increased cytokine secretion by macrophages stimulated with adipocyte-conditioned media further amplifies this inflammatory activity. Thus we can infer that the adipocyte will contribute to systemic inflammation by secreting its own inflammatory cytokines, as well as inducing secretion of inflammatory signals via nearby resident tissue macrophages. This combined NF-
B-dependent inflammatory activity by adipose cellular constituents may be involved in the systemic inflammation and resulting metabolic dysregulation observed in obesity.
One possible limitation of the studies presented is that we use a tissue culture model to study the differentiation process. The 3T3-L1 cell line has been widely used for almost three decades (17, 18) and has proven to be a valuable tool for the elucidation of many processes relevant to primary adipocytes in vivo. We employed the classical differentiation cocktail containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine to induce adipogenesis in these cells in 25 mM glucose, conditions widely used in the literature. Compared with in vivo conditions this is comparable to hyperglycemic conditions. The adipocytes obtained by this differentiation protocol are therefore the product of these in vitro conditions, but offer nevertheless valuable insights into adipogenesis-related functional changes at the level of signal transduction and transcription.
In addition to the increase in constitutive NF-B activity during adipocyte differentiation, there is a concomitant repression of LPS-stimulatable NF-
B activity. Gel shift and luciferase activity assays show that adipocytes are nearly completely resistant to LPS stimulation via the classical NF-
B pathway. This resistance correlates with a decrease in LPS-mediated I
B degradation. Western analyses on nuclear NF-
B show that the adipocyte has constitutively increased nuclear p65/RelA, p68/RelB, p52, and p50, and that LPS stimulation causes little additional translocation.
Although LPS-induced NF-B activation is repressed after differentiation, adipocytes can induce NF-
B activity as judged by the ability of TNF-
to induce DNA binding and luciferase activity. Also, adipocytes are exquisitely sensitive to other modes of LPS-induced inflammatory signaling, as demonstrated by induction of IL-6, TLR2, and SAA3 (31, 32). Thus the significance of the changes in NF-
B signaling go beyond the previously observed phenomenon of endotoxin tolerance (53). The downstream signaling cascade initiated by the LPS receptor TLR4 may therefore differ and be unique in adipocytes.
We used an in vitro assay designed to mimic in vivo paracrine signaling between adipocytes and interstitial macrophages to demonstrate the constitutive inflammatory potential of the adipocyte. The results demonstrate that 3T3-L1 adipocytes, in addition to secreting significant amounts of IL-6, secrete additional factor(s) that have a significant impact on macrophages as judged by a massive induction of IL-6 and TNF- production at the level of the target cell. This is the first time that any direct evidence for a heterotypic signaling event between the adipocyte and a macrophage has been presented. The in vitro approach of using adipocyte-conditioned medium and measuring its effects on macrophages has the advantage that it represents a very clean and well-defined system; however, it has the drawback that we cannot account for effects mediated in vivo through direct cell-to-cell contact. We have recently started to characterize the effects of adipocyte-secreted products on surrounding breast cancer cells during tumor progression (23) and found that many adipocyte-derived factors have a profound impact on tumor cells, promoting tumor growth by stimulating breast cancer cell growth through activation of NF-
B and cyclin D1. These observations are equally borne out in both cell culture systems as well as in vivo. There is therefore ample evidence that the adipocyte can exert a very significant role on the transcriptional programs of neighboring cells. In vivo, adipocytes in different fat pads may significantly differ with respect to their inflammatory potential (11). The cross talk between adipocytes and macrophages may be particularly relevant in the context of obesity when the local inflammatory level is upregulated. The purification of the unknown soluble factor released by adipocytes that exerts this dramatic proinflammatory activity on macrophages is currently the focus of our efforts. Systemic elevation of inflammatory markers, in particular IL-6, is strongly associated with increased risks for cardiovascular problems (reviewed recently in Ref. 4). Adipose tissue has been shown to be a major contributor to systemic IL-6 levels (35). Its contribution may be particularly important in the subclinical inflammatory state associated with syndrome X. The elevated production of IL-6 under these circumstances may not only be the result of an upregulation of IL-6 in adipocytes, but the local macrophages may contribute significantly to this phenomenon.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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