Departments of 1 Medicine and 2 Environmental Health, Johns Hopkins University, Baltimore, Maryland 21205; and 3 Department of Medicine, Cornell University Medical College and Strang Cancer Prevention Center, New York, New York 10021
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ABSTRACT |
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Obesity is a
complex syndrome that involves defective signaling by a number of
different factors that regulate appetite and energy homeostasis.
Treatment with exogenous leptin reverses hyperphagia and obesity in
ob/ob
mice, which have a mutation that causes leptin deficiency, proving the
importance of this factor and its receptors in the obesity syndrome.
Cells with leptin receptors have been identified outside of the
appetite regulatory centers in the brain. Thus leptin has peripheral
targets. Because macrophages express signaling-competent leptin
receptors, these cells may be altered during chronic leptin deficiency.
Consistent with this concept, the present study identifies several
phenotypic abnormalities in macrophages from
ob/ob
mice, including decreased steady-state levels of uncoupling protein-2
mRNA, increased mitochondrial production of superoxide and hydrogen
peroxide, constitutive activation of CCAAT enhancer binding protein
(C/EBP)-, an oxidant-sensitive transcription factor, increased
expression of interleukin-6 and cyclooxygenase (COX)-2, two C/EBP-
target genes, and increased COX-2-dependent production of
PGE2. Given the importance of
macrophages in the general regulation of inflammation and immunity,
these alterations in macrophage function may contribute to
obesity-related pathophysiology.
obesity; uncoupling proteins; cytokines; cyclooxygenase-2; superoxide
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INTRODUCTION |
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OBESITY IS A MAJOR HEALTH problem in affluent societies. It is estimated that 30% of the current population in the United States are obese and that the incidence is increasing (19). Epidemiologic studies have demonstrated that obesity causes premature mortality in adults (38). This finding is consistent with evidence that obesity promotes a wide array of diseases, including cardiovascular disease, breast and colorectal cancer, osteoarthritis, diabetes, and cirrhosis (4, 23, 26, 29). Superficially, the treatment for obesity-related medical problems seems simple, i.e., weight reduction. However, once obesity has developed, efforts to return to normal body mass are typically unsuccessful and may actually increase mortality (1, 16, 29). Although much research has been devoted to understanding the mechanisms that drive hyperphagia and adiposity (reviewed in Ref. 34), very little work has focused on other end-organ consequences of obesity. However, efforts to delineate the mechanisms responsible for obesity-related organ damage are important because the latter increase morbidity and mortality in obese individuals.
Study of rodents with heritable obesity indicates that obesity is a complex syndrome that involves defective signaling by a number of different factors that regulate appetite and energy homeostasis (3, 10, 27, 34, 37). The hormone leptin and its receptors are important components of this system. Leptin is produced predominantly by adipocytes and acts on specific neuronal targets in the ventral median nucleus of the hypothalamus to inhibit appetite. Mice with homozygous loss-of-function mutations in the obese (ob) gene, which encodes leptin, or in the diabetes (db) gene, which encodes the leptin receptor, are hyperphagic and become obese (5). Both strains of mice are also insulin-resistant, hyperlipemic, and relatively infertile, and many of these problems are corrected by treatment with exogenous leptin (30). The fact that such a complex phenotype results from absolute or relative leptin deficiency suggests that leptin may have many targets. Consistent with this possibility is emerging evidence that many cells express either "short" (extracellular domains) or "long" (membrane spanning) forms of the leptin receptor (12, 39, 44). Short forms of the leptin receptor do not appear to transduce intracellular signals and probably provide a tissue reservoir of leptin. Long forms of the leptin receptor have homology to glycoprotein-130 (39) and activate many of the same intracellular signaling pathways that are initiated by interleukin-6 (IL-6), including induction of Janus kinases and signal-transducing activators of transcription (13).
Macrophages express transmembrane forms of the leptin receptor, and recombinant leptin has been shown to increase phagocytic activity and macrophage monocyte colony-stimulating factor release by peritoneal macrophages from normal mice (12, 25). Previously, we reported that phagocytic activity is decreased in macrophages harvested from leptin-deficient ob/ob mice and leptin-resistant db/db mice and demonstrated that recombinant leptin normalizes phagocytosis by cultured ob/ob macrophages but not db/db macrophages (which lack functional leptin receptors) (24). These results indicate that leptin interacts with its receptors to regulate important macrophage functions and suggest that chronic leptin deficiency may alter the phenotype of macrophages. If so, then macrophage dysfunction may be a primary abnormality in obesity syndromes that result from leptin insufficiency or leptin resistance. Furthermore, defective macrophages could play a role in many obesity-related diseases, since these cells are normally important regulators of inflammation and immunity.
Because leptin deficiency affects multiple hormonal and metabolic responses, it is often difficult to determine which, if any, aspects of obesity-related pathophysiology result directly from the interruption of leptin-leptin receptor interactions. Several leptin-regulated genes have been identified in cells (e.g., adipocytes) that express transmembrane leptin receptors. For example, leptin is known to induce the expression of uncoupling protein (UCP)-2 in adipocytes (35). Macrophages also express UCP-2 (12, 20, 28), although it is not clear whether or not leptin regulates UCP-2 expression in these cells. If, however, leptin does directly or indirectly modulate macrophage UCP-2 activity, then leptin deficiency might alter energy homeostasis and oxidant production by these cells, since UCP are known to regulate mitochondrial respiration (11, 17, 28, 33).
The purpose of the present work was to determine whether the chronic leptin-deficient state alters the phenotype of macrophages and to evaluate the role of UCP-2, a leptin-regulated gene product, in this process. Our results demonstrate that UCP-2 expression is suppressed in macrophages from leptin-deficient mice and suggest that this is associated with increased mitochondrial oxidant generation, the activation of oxidant-sensitive transcription factors, and the induction of some genes that are transactivated by these proteins.
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MATERIALS AND METHODS |
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Animals. The ob/ob mice and their lean (?/ob) littermates were purchased from Jackson Laboratories. Animals were maintained in a temperature-controlled environment with a 12:12-h light-dark cycle and fed standard pellet chow diet. All animal experiments were done in accordance with National Institutes of Health and Johns Hopkins University guidelines for the humane use of laboratory animals.
Reagents. All chemicals were purchased from Sigma, with the following exceptions. Bicinchoninic acid (BCA) kits for protein assays came from Pierce. Sodium isothiocyanate, Taq polymerase, RT, and proteinase inhibitor solution were purchased from Boehringer Mannheim (Indianapolis, IN). Nylon membranes were purchased from DuPont (Boston, MA). Medium (DMEM), bovine serum, and plastic dishes for the cell culture experiments came from GIBCO. Enzyme immunoassay reagents for PGE2 assays were from Cayman (Ann Arbor, MI). The oligomer containing the CCAAT enhancer binding protein (C/EBP) binding site in the 422 promoter and the UCP-2 cDNA were gifts from M. D. Lane (Johns Hopkins University, Baltimore, MD). Dr. Gennaro Ciliberto (Research Institute, Rome, Italy) provided the cDNA for IL-6. The cyclooxygenase (COX)-2 cDNA was given to us by Dr. Raymond DuBois (Vanderbilt University, Nashville, TN)
Peritoneal macrophage experiments. Mice were injected with 3 cm3 of 3% thioglycollate intraperitoneally. Five days later, animals were euthanized by CO2 inhalation, and the peritoneal cavity was immediately lavaged with Hanks' balanced salt solution with EDTA (pH 7.2) to harvest the thioglycollate-elicited macrophages. Macrophages from ~10 mice/group were pooled and plated on plastic culture dishes (107 cells/10-cm dish) and cultured overnight in DMEM with 10% bovine serum (in studies to evaluate nuclear proteins, RNA, or mitochondrial oxidant generation) or DMEM without serum (to evaluate PGE2 production). Experiments were done the following morning. All experiments were repeated at least twice. Each experiment studied macrophages pooled from 10 ob/ob mice or from 10 control mice. Thus the final results were obtained from 3 different sets of macrophages pooled from a total of 30 ob/ob and 30 control mice.
RNA isolation and analysis. Vehicle or lipopolysaccharide (LPS; from Escherichia coli serotype 0111:B4; 1 µg/ml) was added to macrophage cultures, and 90 min later cells were harvested. Total RNA was isolated from the fresh cell pellets according to the method of Chomczynski and Sacchi (7), as we have described (41). RNA was quantitated by measuring its absorbance at 260/280 nm, and its quality was assessed by electrophoresis on ethidium bromide-stained agarose gels under denaturing conditions. To evaluate potential differences in the gene expression of ob/ob and lean macrophages, total RNA (20 µg/lane) was separated by agarose gel electrophoresis under denaturing conditions and transferred to nylon membranes by capillary diffusion. Membranes were rinsed with 1% methylene blue and photographed to document lane-to-lane variations in RNA. Membranes were hybridized overnight at 42°C with 32P-labeled cDNAs for UCP-2, IL-6, COX-2, or 18S. After a washing under stringent conditions, membranes were exposed to X-ray film. IL-6 gene expression was also evaluated by semi-quantitative RT-PCR as described (31, 32). Briefly, total liver RNA (3 µg) was reverse transcribed and amplified with specific internal oligonucleotide primers in a thermocycler under semi-quantitative conditions. The expression of glyceraldehyde-3-phosphate dehydrogenase, a constitutively expressed gene, was evaluated in parallel assays. PCR products were separated by agarose gel electrophoresis. After transfer to nylon membranes by capillary blotting and hybridization with IL-6-specific probes, the products were visualized by enhanced chemiluminescence. All RNA experiments, using pooled macrophages from 10 mice per group per experiment, were repeated 3 times to assure reproducibility of results.
Nuclear protein isolation and gel mobility shift assays.
Macrophages were harvested and cultured as described above, except that
cultures were harvested at several different time points (0, 0.5, 1, or
1.5 h) after vehicle or LPS treatment. Nuclear proteins were isolated
according to the method of Lavery and Schibler (21), as we have
described (42). Briefly, cells were homogenized in homogenizing buffer
[in mM: 2 HEPES, 0.1 spermidine, 0.03 spermine, 1 EDTA, 0.5 EGTA, and
1.4 -mercaptoethanol with 0.35 M sucrose and 0.5% Nonidet P-40
(NP-40), pH 7.2]. After centrifugation, the pelleted nuclei were
resuspended in nuclear suspension buffer (20 mM
Tris · HCl, pH 7.9, 75 mM NaCl, 0.5 mM EDTA, and 50%
glycerol) and NUN solution (1.1 M urea, 0.33 M NaCl, 1.1% NP-40, and
25.5 mM HEPES, pH 7.6). Each of these buffer solutions also contains proteinase inhibitors (Boehringer Mannheim). After incubation at
4°C for 30 min, the suspension was centrifuged to isolate the nuclear proteins in the supernatant. Protein concentration was determined by BCA kits with BSA as the standard.
PGE2 production. Macrophages were cultured in the absence or presence of 10 µM sodium arachidonate, the substrate for COX-2, to evaluate PGE2 production. Cells were cultured overnight on 3-cm plastic dishes (106 cells/dish) in DMEM without serum. The morning of the experiment, either LPS (1 µg/ml) or vehicle was added and the cells were cultured for an additional 6 h. The medium was harvested and flash frozen. Cells were scraped from the plates and homogenized in lysis buffer so that total cellular protein could be determined by the BCA assay as described above. The amount of PGE2 in the conditioned medium was measured by ELISA as described previously (43). Results were normalized to the amount of protein in each culture. All assays were performed in triplicate to assure data reproducibility. The entire experiment was repeated using DMEM with 3% bovine serum and produced similar results, although the magnitude of the difference in PGE2 concentration between the ob/ob and lean macrophages after LPS was not as striking as when the cells were cultured in the absence of serum.
Macrophage oxidant and ATP production.
Macrophages were harvested from the culture dishes and resuspended in
fresh PBS as described above. Either lucigenin (5 µM) or luminol (10 µM) plus horseradish peroxidase (HRP) was added to the cell
suspensions (0.5-1 × 106 cells/ml), and lucigenin- or
luminol-derived chemiluminescence was measured in a six-chamber
Berthold LB9505 luminometer at 37°C for 40 min, as described (22).
Lucigenin-derived chemiluminescence measures intramitochondrial
superoxide anion (O2) generation, and luminol-derived chemiluminescence measures
extracellular, mitochondrion-derived hydrogen peroxide
(H2O2)
production. To confirm the importance of mitochondria as the source of
these oxidants, inhibitors of mitochondrial respiration, rotenone (10 µM; a complex I inhibitor) and myxothiazol (10 µM; a complex III inhibitor), were added to some assays to determine whether this reduces
either O
2 or
H2O2
generation. ATP production was evaluated according to the
manufacturer's protocols with commercially available
luciferin-luciferase ATP assay kits (Sigma), using similar approaches
to monitor chemiluminescence. Briefly, to each well, 50 µl complete
PBS, 50 µl H2O, and 100 µl
ATP-releasing reagent were added to lyse cells. Two minutes later, 100 µl of the cell lysate were added into a cuvette containing 100 µl
ATP assay mix. The luminescence was determined immediately on a
luminometer (Berthold, LB9505). ATP content was calculated using a
concurrently run standard curve and expressed as percentage of
untreated control cells.
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RESULTS |
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The yield and viability of macrophages harvested from the peritoneal
cavities of obese
(ob/ob)
mice and their lean (?/ob) littermates were similar. However, closer inspection of macrophages pooled from obese mice and those pooled from lean mice
revealed several phenotypic differences. As shown in Fig.
1, UCP-2 was expressed in cultured
peritoneal macrophages from lean mice, and a 90-min exposure of these
cultures to LPS decreased UCP-2 expression by ~40%. The
ob/ob
macrophages expressed only about one-half as much UCP-2 as the lean
macrophages basally. These low basal levels of UCP-2 transcripts were
not further reduced following LPS treatment.
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UCP-2 activity is thought to uncouple oxidative phosphorylation and
decrease O2 generation by normal
peritoneal macrophages (28). To determine whether differences in UCP-2 expression in lean and
ob/ob
macrophages were associated with differences in mitochondrial oxidant
generation, cell suspensions were incubated with either
lucigenin or luminol plus HRP to quantitate mitochondrion-derived
O
2 and
H2O2
generation, respectively. The
ob/ob
macrophages produced more O
2 (Fig.
2,
A and
C) and
H2O2
(Fig. 2B) than the lean macrophages basally. LPS exposure significantly increased oxidant production by
lean macrophages but had less of an effect in
ob/ob
cells, which were already producing high levels of
O
2 and
H2O2.
Thus there appeared to be an inverse correlation between oxidant
production and UCP-2 mRNA levels. Basal expression of UCP-2 was high in
lean macrophages, and these cells produced low levels of
O
2 and
H2O2
basally. In contrast, ob/ob
cells expressed much less UCP-2 and produced much more
O
2 and
H2O2
basally. In lean macrophages, LPS stimulation resulted in a significant
fall in UCP-2 mRNA and led to a marked increase in oxidant generation.
LPS had less of an effect on the already low levels of UCP-2 in
ob/ob
macrophage, and LPS treatment was also followed by a smaller increase
in the already high rates of oxidant production by these cells.
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Results displayed in Fig. 3 support the
concept that mitochondria are the major source of the oxidants that
were measured during the previous experiments. Addition of electron
transport chain inhibitors that block respiration at complex I
(rotenone) and complex III (myxothiazol) virtually eliminates
O2 generation in both
ob/ob
and lean macrophages (Fig. 3A).
Furthermore, treatment of
ob/ob
cells with carbonyl cyanide
p-(tri-fluoromethoxy)phenylhydrazone (FCCP), a known mitochondrial uncoupling agent, significantly reduces
their O
2 generation, despite continued low expression of endogenous UCP-2 (Fig.
3B). The latter finding suggests
that decreased UCP-2 may contribute to the dysregulation of oxidant
production by
ob/ob
macrophages.
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Mitochondrial uncouplers are also thought to influence ATP production.
Uncoupling of mitochondrial electron transport from ATP synthesis is
predicted to decrease cellular ATP stores (17). Commercially available
luciferase assays were used to evaluate ATP production in untreated
macrophage suspensions. As predicted, ob/ob
macrophages, which express less UCP-2 (Fig. 1) and which generate more
mitochondrion-derived oxidants (Fig. 2), also have greater ATP
concentrations than lean macrophages (Fig.
4).
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Cellular oxidants are known to influence the redox state of
oxidant-sensitive transcription factors, and this may influence the DNA
binding activity of these proteins (6, 18). Because the degree of
mitochondrial oxidants produced by
ob/ob
and lean macrophages differs, it is conceivable that these cells may
also exhibit differences in the DNA binding activities of
oxidant-sensitive DNA binding proteins, such as C/EBP- (18). To
evaluate this possibility, differences in the DNA binding activity of
this oxidant-sensitive transcription factor were evaluated by gel
mobility shift assays (Fig. 5). The DNA
binding activity of C/EBP-
is increased transiently by LPS treatment
in lean macrophages. However, C/EBP-
binding activity is apparent in
ob/ob
macrophages even before LPS exposure. Thus, compared with lean
macrophages,
ob/ob
macrophages, which produce more O
2 and
H2O2
basally (Fig. 2), also have more constitutive C/EBP-
binding
activity. LPS induces C/EBP-DNA binding activity transiently in lean
macrophages, and, as noted for UCP-2 and mitochondrial oxidant
production, LPS had less of an effect in
ob/ob
macrophages. However, C/EBP-DNA binding activity remained generally
greater in
ob/ob
macrophages than in lean macrophages after LPS treatment.
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Several genes that are induced during inflammation are
transcriptionally regulated by C/EBP- (2, 40). For example, the transcription of COX-2, which encodes an enzyme that metabolizes arachidonic acid to prostanoids, is activated by this DNA binding protein (8, 14, 36). Because
ob/ob
and lean macrophages exhibit differences in the DNA binding activities
of C/EBP-
, it is possible that these cells may have different levels
of COX-2, either basally or following treatment with LPS, a potent
inducer of COX-2. To evaluate this possibility, Northern blot analysis was used to compare steady-state levels of COX-2 mRNA in the two groups
of cells. As shown in Fig. 6, before LPS
treatment, COX-2 mRNAs could not be detected by Northern blot analysis
in either group. LPS treatment substantially induced COX-2 in both
groups, but COX-2 expression was about fivefold greater in
ob/ob
than in lean cells following LPS treatment.
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COX-2 catalyzes the synthesis of
PGE2 from arachidonate. Hence,
during conditions in which substrate (arachidonate) is not limiting,
PGE2 production is a good measure
of COX-2 activity (8, 14, 36). To determine whether COX-2 activity is
different in
ob/ob
and lean macrophages, PGE2
concentrations were evaluated in macrophage-conditioned medium
harvested from cells that were cultured in medium alone or
medium plus sodium arachidonate. Compared with macrophages
from lean mice, macrophages from
ob/ob
mice produce more PGE2 both
basally and when incubated with sodium arachidonate (Table
1). Thus COX-2 activity is generally
greater in
ob/ob macrophages than in macrophages from their lean littermates.
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PGE2 has many effects, including
the induction of IL-6 (15). Thus it is possible that
ob/ob
macrophages, which produce more PGE2, also produce more IL-6. To
evaluate this possibility, IL-6 expression in
ob/ob
and lean macrophages was evaluated by Northern blot analysis and RT-PCR
assay. As shown in Fig.
7A, IL-6
mRNA is not detected by Northern blot analysis in either
ob/ob
or lean macrophages before LPS treatment. LPS induces IL-6 in both
groups, but the degree of induction is much greater in
ob/ob
macrophages than in macrophages from lean controls. RT-PCR analysis is
a more sensitive technique than Northern blot analysis for
demonstrating low-abundance mRNAs. When this approach was used, basal
differences in IL-6 expression were identified between
ob/ob
and lean macrophages. IL-6 transcripts were barely detected by RT-PCR
assay of total RNA from lean macrophages but were easily identified in
ob/ob macrophages (Fig. 7B). Thus
ob/ob
macrophages produce more PGE2 and
more IL-6 than lean macrophages, both before and after LPS.
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DISCUSSION |
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Obesity is a complex syndrome that results from an imbalance in energy intake and utilization. Several different factors interact to regulate appetite by influencing the activity of neuronal melanocortin type 4 receptors in the ventral median nucleus of the hypothalamus (27). Many of these factors, including the appetite-suppressing hormone leptin, also trigger effects in peripheral tissues. Some of the latter appear to result from direct interactions between appetite-regulating factors and their receptors on cells that reside outside the central nervous system (5). It is likely that these peripheral effects contribute to defective energy utilization and some of the end-organ damage that occurs in obese individuals.
Results of the present study identify several phenotypic abnormalities in macrophages of chronically leptin-deficient ob/ob mice. These findings extend our initial observation that macrophage phagocytic function is abnormal in these genetically obese mice (25) and support the concept that macrophages are an important target cell in states of chronic leptin deficiency or resistance. Thus macrophage dysfunction may contribute to altered inflammatory or immunologic responses and, as such, play a role in the pathogenesis of obesity-related organ damage.
Leptin receptors have been identified in several peripheral tissues and localized to particular cell types, including adipocytes, macrophages, and pancreatic islet cells (12, 35, 44). Treatment of some of these cells with leptin results in the induction of genes that regulate energy homeostasis. For example, leptin increases UCP-2 expression in pancreatic cells and adipocytes (35, 44). Macrophages also express UCP-2 (20, 28). The present study identifies decreased steady-state UCP-2 mRNA levels in cultured peritoneal macrophages from chronically leptin-deficient animals. Although not conclusive, such evidence that UCP-2 expression is reduced in macrophages from ob/ob mice suggests that leptin may also regulate UCP-2 expression in these cells. However, preliminary studies in our laboratory indicate that acute exposure to leptin does not normalize many of the phenotypic abnormalities that were noted in ob/ob macrophages, including oxidant production and UCP-2 expression (data not shown). These observations suggest either that such macrophage dysfunction is an indirect consequence of the leptin-deficient state or that different doses or durations of leptin will be required to demonstrate an effect in vitro.
However, regardless of the exact molecular basis for decreased UCP-2
expression in
ob/ob
macrophages, suppression of UCP-2 has potentially important
physiological consequences because UCP can regulate both cellular ATP
stores and oxidant production (11). Decreased UCP activity is predicted
to increase mitochondrial ATP synthesis by maximizing the efficiency
with which substrate oxidation results in ADP phosphorylation. In some
circumstances, low UCP activity may also increase oxidant production,
since a slower rate of electron transport favors the escape of
electrons from electron carriers and this promotes
O2 generation (11, 28, 35). Consistent
with these predictions, we observed higher concentrations of ATP and
greater mitochondrion-derived O
2 and
H2O2
in
ob/ob
macrophages, in which UCP-2 expression is relatively suppressed.
Our data also demonstrate that, when normal macrophages are exposed to
bacterial products such as LPS, UCP-2 expression falls, mitochondrial
oxidant generation increases, and production of cytokines and
prostanoids increases. The downregulation of macrophage UCP-2 by LPS
appears to be a cell-specific response, since we have shown that UCP-2
transcripts are induced in hepatocytes following LPS exposure
(7a). Taken together, these results demonstrate that the basal phenotype of
ob/ob
macrophages resembles the phenotype of normal macrophages that have
been activated by LPS, an inflammatory stimulus. This concept is
supported by observations that
ob/ob macrophages exhibit higher constitutive DNA binding activity of the
LPS-regulated transcription factor C/EBP- and increased expression and/or activity of some of its target genes, such as those for COX-2 and IL-6. Thus macrophages from chronically leptin-deficient animals appear to be constitutively activated, such that they overproduce a variety of inflammatory mediators, including oxidants, cytokines, and prostanoids.
Although macrophages are critical cells in the immune response and in host defense mechanisms, they have also been implicated as mediators of a number of pathologies. Thus it is possible that these abnormalities in macrophage phenotype may contribute to some of the end-organ complications of obesity that have been observed in ob/ob mice. Circulating leptin levels are generally increased in human obesity and in several other genetically obese strains of mice and rats (e.g., with the diabetes, fatty, agouti, or tubby mutations) (34). Presumably, in the latter situations, obesity results, at least in part, from resistance to leptin-initiated signals in the hypothalamus, because hyperphagia occurs despite increased levels of a potent anorectic factor. Additional work will be required to determine whether macrophage dysfunction is a general feature of the obese phenotype and whether peripheral leptin resistance or other, as yet unrecognized, hormonal disturbances are responsible for the deficits. This effort appears well justified, however, since macrophages regulate inflammation and immunity and thus may participate in the pathogenesis of several diseases that contribute to obesity-related morbidity and mortality.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants R01-AA-10157 (to A. M. Diehl), K0200347 (to A. M. Diehl), R01-ES-03760 (to M. A. Trush), and ES-03818 (to M. A. Trush) and by a training grant from the First Chinese University, Hong Kong (to F.-Y. J. Lee).
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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. §1734 solely to indicate this fact.
Address for reprint requests: A. M. Diehl, Johns Hopkins University School of Medicine, 912 Ross Bldg., 720 Rutland St., Baltimore, MD 21205.
Received 19 June 1998; accepted in final form 11 November 1998.
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