Tumor Necrosis Factor and Glucocorticoid Synergistically Increase ...

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The Journal of Clinical Endocrinology & Metabolism 91(4):1484 –1490 Copyright © 2006 by The Endocrine Society doi: 10.1210/jc.2005-1901

Tumor Necrosis Factor ␣ and Glucocorticoid Synergistically Increase Leptin Production in Human Adipose Tissue: Role for p38 Mitogen-Activated Protein Kinase Maria E. Trujillo, Mi-Jeong Lee, Sean Sullivan, Jianying Feng, Stephen H. Schneider, Andrew S. Greenberg, and Susan K. Fried Department of Nutritional Sciences (M.E.T., M.-J.L., S.S., S.K.F.), Rutgers University and Division of Endocrinology (S.H.S.), University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey 08901; Jean Mayer U.S. Department of Agriculture, Human Nutrition Research Center on Aging (A.S.G.), Tufts University, Boston, Massachusetts 02111; and Division of Endocrinology, Diabetes, and Nutrition (M.-J.L., J.F., S.K.F.), Department of Medicine, University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201 Context: TNF increases plasma leptin in humans in vivo, but previous studies showed it decreases leptin in vitro. Objective and Participants: The objective of this study was to determine the effect of TNF on leptin release from human adipose tissue (AT) from healthy subjects undergoing elective surgery or needle aspirations of AT at a university hospital. Design: Human omental and abdominal sc AT fragments from nonobese and obese subjects were placed in organ culture without or with TNF added in the presence or absence of insulin and/or dexamethasone (dex; a synthetic glucocorticoid) for up to 2 d. Results: In the absence of hormones, culture with TNF decreased

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NF␣ IS AN inflammatory cytokine that is overexpressed in adipose tissue (AT) in obesity (1, 2). The overexpression of TNF in AT is implicated in many of the metabolic perturbations associated with obesity and diabetes (2– 4). Both obesity and cachexia are associated with hyperleptinemia. Because several studies suggest that hyperleptinemia during cachexia due in part to increased systemic levels of TNF (5– 8), we hypothesized that the chronic overexpression of TNF within AT may contribute to hyperleptinemia in obesity. Previous in vitro studies show TNF has no effect or decreases leptin expression (9 –14). However, most of these studies examined the effects of TNF in the absence of hormones that are critical for the up-regulation of leptin expression and that are elevated systemically and/or locally in cachexia and obesity. To determine whether TNF could increase leptin expression in a relevant hormonal milieu, we tested the effects of TNF on leptin expression in human AT in the presence or absence of insulin and/or glucocorticoid. Because the effects of insulin and glucocorticoids on leptin First Published Online January 10, 2006 Abbreviations: AT, Adipose tissue; dex, dexamethasone; JNK, c-Jun NH2-terminal kinase; NS, not significant; Om, omental. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

leptin release. In contrast, when added in the presence of dex, TNF increased secreted leptin and leptin mRNA abundance in AT from nonobese and obese subjects. The TNF ⫹ dex-stimulated increase in leptin was associated with an increase in p38 MAPK activity and was totally blocked by p38 MAPK inhibitors. In contrast, inhibition of p38 MAPK only partially blocked the effect of TNF on IL-6 production. Culture of obese AT with either p38 or p44/42 MAPK inhibitors also blunted the spontaneous increase in media leptin that occurred from d 1–2 of culture in omental AT of obese subjects. Conclusion: Synergistic effects of increased local or systemic TNF in combination with glucocorticoids may contribute to increased leptin expression in response to stress, including infection and obesity. (J Clin Endocrinol Metab 91: 1484 –1490, 2006)

production differ with depot (15), and some studies indicate that TNF expression is higher in omental (Om) than sc AT (16), we also compared the effects of TNF on leptin expression in these two AT depots. Although the signal transduction pathways that mediate the effect of TNF on leptin expression in human AT are not known, culture of differentiated human preadipocytes with TNF activates MAPKs (17, 18), and signaling through MAPKs mediates the stimulatory effect of insulin on leptin expression in 3T3-L1 adipocytes (19). Thus, we examined the role of MAPKs in the regulation of leptin expression in cultured AT from obese subjects and tested whether MAPKs mediate the effects of exogenous TNF on leptin production. Subjects and Methods Subjects AT samples were obtained from the Om and sc depots of morbidly obese subjects (body mass index, 50 ⫾ 2 kg/m2, n ⫽ 21, all female) who underwent gastric bypass surgery. AT was also obtained from the abdominal sc depots of lean volunteers (body mass index, 24 ⫾ 0.4 kg/m2, n ⫽ 4, two females, two males) via fat aspiration under local anesthesia (20). Subjects enrolled in this study were nondiabetic (by medical history) and not taking steroids, ␤-blockers, or statins. Those taking antihypertensives or antidepressants were not excluded. There was no indication of carryover effects from subjects taking antidepressants on the effects of dexamethasone (dex) in cultured human AT. All subjects gave informed consent, and protocols were approved by the Institutional

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Trujillo et al. • TNF Plus Glucocorticoids Increase Leptin

Review Boards of St. Peter’s University Hospital, the University of Medicine and Dentistry of New Jersey, the University of Maryland, and Rutgers University.

Tissue culture Samples of human AT were minced aseptically and placed in organ culture in serum-free M199 as described previously (21). Media were sampled and exchanged daily. At the end of culture, media were removed; tissue was quickly washed in saline and then frozen in liquid nitrogen. Media and tissue were stored at ⫺80 C until analysis.

Experiment 1 Om and sc ATs were cultured with 10 ng/ml (588 pm) TNF in the absence of hormones (basal), with 7 nm insulin (Humulin, Lilly, Indianapolis, IN), with 25 nm dex, or the combination of insulin and dex. Media from cultures was sampled and replenished daily with media containing fresh hormones and/or TNF for 2 d.

Experiment 2 Preliminary experiments had indicated that the most robust effects of TNF on leptin were obtained after 5 d of culture with dex, in which we had previously shown reduced endogenous cytokine production (22, 23). Thus, AT was cultured in the presence of dex for 5 d. On d 6, culture media were exchanged, and the effect of 2 d of culture with TNF in the presence of dex was tested as described in experiment 1.

Experiment 3 To test whether MAPKs regulate basal leptin in obese AT, Om and sc AT were cultured in the presence or absence of specific MAPK inhibitors for 2 d. To inhibit MAPKs, cultures with the following pharmacological inhibitors were employed: 25 ␮m SB203580 or SB202190 to inhibit p38, 100 ␮m PD098059 or 10 ␮m U0126 to inhibit p44/42, and 10 ␮m SP600125 to inhibit c-Jun NH2-terminal kinase (JNK).

Experiment 4 To determine whether the TNF-stimulated increase in secreted leptin is mediated by MAPKs, we cultured AT for 2 d with dex in the presence or absence of TNF and/or MAPK inhibitors.

Determination of fat cell size Fat cell number per gram of tissue was determined by Coulter counting as described by Hirsh and Gallian (24).

Measurement of secreted cytokines Leptin accumulation in culture media was measured via RIA (Linco, St. Charles, MO). Immunodetectable IL-6 was assayed in culture media via ELISA (R&D Systems, Minneapolis, MN).

Measurement of leptin gene expression Total RNA extraction and Northern analysis were carried out as previously described (25). For Western analysis, AT lysates were prepared as described by Wang et al. (25), and 10 –20 ␮g total protein was subjected to SDS-PAGE. After transfer to nitrocellulose, blots were probed with antibodies to total and phospho p44/42 (p44/42, Cell Signaling Technology, Beverly, MA) and visualized using chemiluminescence.

In vitro p38 MAPK activity assay p38 MAPK activity was analyzed using a p38 MAPK assay kit (Cell Signaling Technology). A DC Protein Assay Kit (BIO-RAD, Hercules, CA) was used to measure total protein in the homogenate. p38 activity was assayed in homogenates containing equivalent amounts of tissue protein.

J Clin Endocrinol Metab, April 2006, 91(4):1484 –1490

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Data analysis All data are presented as means ⫾ sem. All data were log-transformed (unless noted), and statistical analysis was conducted using GraphPad Prism Software. The effects of TNF and/or hormones were evaluated via two-way ANOVA with repeated measures. Main effects or interactions with significant F values were assessed via post hoc Student’s t tests. Comparisons of the magnitude of the effects of Om vs. sc depots were assessed by paired Student’s t tests, and the effects in sc AT from obese vs. lean subjects were assessed via unpaired Student’s t tests, assuming equal variances. The effects of culture with MAPK inhibitors and time on basal leptin accumulation were assessed via two-way ANOVA. Evaluation of the effect of TNF on leptin mRNA levels was conducted using post hoc Student’s t tests. P ⬍ 0.05 was considered statistically significant.

Results Effect of TNF on leptin accumulation depends on hormonal milieu

Om. Culture of Om AT from obese subjects with TNF for 2 d in the basal condition (the absence of other hormones) decreased leptin accumulation in the medium (⫺49 ⫾ 6%, P ⬍ 0.005, vs. basal, n ⫽ 6, Fig. 1A). As expected, culture with insulin had no effect on leptin accumulation in Om, and dex increased leptin when added in the absence (⫹44 ⫾ 12%, P ⬍ 0.05, vs. basal) or presence of insulin (⫹151 ⫾ 29%, P ⬍ 0.0005, vs. insulin). Culture with TNF plus insulin, compared with insulin alone, decreased leptin accumulation in the medium (56 ⫾ 6%, P ⬍ 0.005). However, the combination of TNF plus dex increased leptin accumulation in the culture medium by ⫹75 ⫾ 25% vs. dex alone (TNF ⫻ dex interaction significant by ANOVA, P ⬍ 0.0001; P ⬍ 0.05 TNF ⫹ dex vs. dex alone, post hoc Student’s t test). Culture with 1 ng/ml TNF with dex was as effective as 10 ng/ml TNF (⫹50 ⫾ 19% vs. ⫹51 ⫾ 22%, n ⫽ 3, data not shown). TNF added in the presence of insulin plus dex did not further increase leptin accumulation. Subcutaneous. TNF effects on leptin were tested in sc AT from obese (n ⫽ 7, Fig. 1B) and lean (n ⫽ 4, Fig. 1C) subjects. Unlike results in Om AT, culture with TNF for 2 d had no effect on leptin accumulation in the medium under basal conditions. However, addition of TNF in the presence of dex synergistically increased leptin accumulation in the medium in cultures from both obese and lean subjects (P ⬍ 0.05). The addition of TNF in the presence of insulin alone or insulin plus dex had no further effect on leptin in cultures from obese or lean subjects. TNF did not affect leptin accumulation on d 1 of culture of sc AT from obese or lean under any conditions tested. The magnitude of the effect of TNF in the presence of dex was not significantly different in adipose samples from lean and obese subjects [not significant (NS)] or between paired cultures of Om (⫹81 ⫾ 29%) and sc (⫹137 ⫾ 20%) (NS, paired Student’s t test, n ⫽ 5). TNF-stimulated increase in leptin release is associated with increases in leptin mRNA

Because production of cytokines such as TNF and IL-6 is high during the first few days of culture (22, 26) and production of endogenous cytokines such as IL-6 is suppressed by culture with dex (22), we reasoned that preculture in the presence of dex might elicit a more robust TNF effect on leptin production. After 6 d preculture with dex, the addition of TNF increased leptin accumulation in the medium on d

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7– 8 of culture from 24 ⫾ 9 to 52 ⫾ 8 ng/106 cell/24 h in Om (⫹87 ⫾ 41%, n ⫽ 4) and from 22 ⫾ 5 to 39 ⫾ 8 ng/106 cell/24 h in sc cultures (⫹50 ⫾ 27%, n ⫽ 3). The magnitude of the TNF effect was similar to that previously observed on d 2. Nevertheless, the TNF-stimulated increases in secreted leptin were paralleled by an increase in leptin mRNA relative abundance. In Om tissue, leptin mRNA increased by 127 ⫾ 49% (P ⬍ 0.05, n ⫽ 4, Fig. 2), and in sc tissue, leptin mRNA tended to increase (P ⫽ 0.05, range from ⫹53 to ⫹139%, n ⫽ 3). Inhibition of p38 MAPK blocked the stimulatory effect of exogenous TNF on leptin accumulation

To assess whether activation of MAPKs mediated the ability of TNF to stimulate leptin production in the presence of dex, the effects of p38, p44/42 (ERK), and JNK inhibitors were tested. In this group of subjects, culture of OM AT with dex ⫹ TNF increased leptin accumulation by 98 ⫾ 31% (P ⬍ 0.05, n ⫽ 4, Fig. 3). Culture with p38 inhibitors almost completely blocked the TNF ⫹ dex-stimulated increase in leptin accumulation on d 2 (dex ⫻ inhibitor effects significant by ANOVA, P ⫽ 0.03, Fig. 3). The effects of chemical inhibition of p38 MAPK on TNF ⫹ dex-stimulated leptin production were specific to p38 because culture with either p44/42 or JNK MAPK inhibitors did not affect TNF ⫹ dex-stimulated leptin accumulation (data not shown). We next determined whether the effect of TNF ⫹ dex was associated with higher in vitro p38 MAPK activity by measuring its in vitro kinase activity, as assessed by phosphorylation ATF2. Because the effects of TNF on leptin accumulation in both Om and sc cultures were blocked by coculture with p38 MAPK inhibitors, Om and sc cultures were combined to facilitate analysis. Culture with dex alone tended to decrease p38 activity 0.5- ⫾ 0.1-fold in cultured AT (P ⬍ 0.1, n ⫽ 3, 2 Om, 1 sc; Fig. 4A). In the absence of hormones, TNF

FIG. 1. The effects of TNF on leptin accumulation in the medium of cultured Om (n ⫽ 7) (A) and sc AT from obese (n ⫽ 7) (B) and nonobese (n ⫽ 4) (C) subjects. Human AT was cultured in the presence or absence of TNF (10 ng/ml) in combination with insulin (7 nM) and/or dex (25 nM) for 2 d. Medium was exchanged daily, and samples were assayed for leptin. Data from d 2 of culture are expressed as mean ⫾ SEM. Different letters represent hormonal treatments that were significantly different (P ⬍ 0.05). Interactions between TNF and dex were significant by ANOVA in cultured AT from Om (P ⬍ 0.0001) and sc depots of obese (P ⬍ 0.0001) and lean. *, TNF effect, P ⬍ 0.05.

FIG. 2. The effects of TNF on leptin accumulation and mRNA levels in AT from Om (n ⫽ 4) and sc (n ⫽ 3) depots from obese subjects. Human AT was precultured in the presence of 25 nM dex for 5 d. On d 6, the media were exchanged, and tissue was cultured with dex in the presence or absence of 10 ng/ml TNF for 2 additional days (8 d total). *, TNF effect, P ⫽ 0.05.



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kinase activity assays, as determined by Western blots (not shown). Both p38 and p44/42 inhibitors decreased basal leptin production

FIG. 3. The effects of p38 inhibitors SB203580 (25 ␮M) or SB202190 (25 ␮M) on TNF-stimulated leptin accumulation were tested in cultured Om AT from obese subjects (n ⫽ 4). AT was cultured in the presence of 25 nM dex in combination with 10 ng/ml TNF and/or p38 inhibitors for 2 d. Media were assayed for leptin accumulation. TNF effect within hormonal condition, *, P ⬍ 0.01; ⫹, effect of inhibitor, P ⬍ 0.01 vs. dex ⫹ TNF; ⵩, P ⫽ 0.06 vs. dex alone.

Because culture with exogenous TNF increases p38 activity and leptin production in obese AT, we hypothesized that activation of p38 MAPK by endogenous TNF contributed to leptin overexpression in obese AT. Furthermore, we hypothesized that high endogenous cytokine production during d 1 compared with d 2 of culture (22) (Trujillo, M. E., and S. K. Fried, unpublished observations) may cause the spontaneous increase in leptin accumulation in the medium that occurred in Om AT cultured under basal conditions (⫹153 ⫾ 58% d 1 vs. 2, P ⫽ 0.01, n ⫽ 7, Fig. 5A). As predicted, inhibition of p38 MAPK with SB203580 blunted the spontaneous increase in leptin (inhibitor effect P ⬍ 0.05 by ANOVA); however, SB202190 did not have a consistent effect. In contrast to Om AT, in sc, the accumulation of leptin in the medium did not increase from d 1–2 of culture. Nevertheless, culture with p38 inhibitors tended to decrease leptin accumulation in the medium on d 2 of culture [SB203580, ⫺44 ⫾ 9% vs. basal, P ⫽ 0.01 (paired Student’s t test, nonlog-transformed data), n ⫽ 4, Fig. 5B; SB202190 (NS)], suggesting that high basal p38

FIG. 4. The effects of culture with TNF in the presence or absence of dex on p38 kinase activity in cultured Om and sc AT from obese subjects. Human AT was cultured in the presence or absence of 10 ng/ml TNF in the presence or absence of 25 nM dex for 2 d. The p38 activity and phosphorylated p38 protein levels were assessed as described in Subjects and Methods. A representative Western blot is shown (A). TNF increased p38 activity 2.0- ⫾ 0.6-fold in the absence of hormones (P ⬍ 0.1, n ⫽ 3, 2 Om, 1 sc; B) and 5.0- ⫾ 2.0-fold in the presence of dex (P ⫽ 0.05, n ⫽ 5, 2 Om, 3 sc; C).

increased p38 MAPK activity 2.0- ⫾ 0.6-fold (P ⬍ 0.1, n ⫽ 3, 2 Om, 1 sc; Fig. 4B). Moreover, TNF increased p38 MAPK activity 5.0- ⫾ 2.0-fold in the presence of dex (P ⫽ 0.05, n ⫽ 5, 2 Om, 3 sc; Fig. 4C). The treatments did not affect the amount of p38 MAPK in the same homogenates used for

FIG. 5. The effects of p38 MAPK inhibitors on basal leptin accumulation in the medium of cultured Om (A) and sc (B) AT from obese subjects. Human AT was cultured in the presence or absence of p38 inhibitors for 2 d. Culture medium was sampled, then replenished daily. The increase in leptin production on d 1 and 2 of culture was significant in the controls (*, P ⬍ 0.05).

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MAPK also contributes to the up-regulation of leptin production in sc and Om AT in obesity. In Om, culture with p44/42 inhibitors also blunted the spontaneous increase in leptin accumulation in Om cultures from d 1–2 [⫹116 ⫾ 41%, n ⫽ 5, P ⬍ 0.05 (control), ⫹52 ⫾ 32%, PD98059, NS; ⫹29 ⫾ 17%, U0126, NS); Fig. 6A]. The effectiveness of the PD and U compounds were demonstrated by the decreased ratio of phospho to total p44/42 MAPK (⫺86 ⫾ 7%, PD98059, P ⫽ 0.06, ⫺97 ⫾ 1%, U0126, P ⫽ 0.01, n ⫽ 3, Fig. 6B). Culture with p44/42 inhibitors also tended to decrease leptin accumulation on d 2 in sc (⫺9 ⫾ 5%, PD98059, NS; ⫺18 ⫾ 6%, U0126, P ⫽ 0.1, n ⫽ 4). Culture with the JNK inhibitor SP600125 had no effect on basal leptin accumulation (n ⫽ 5, Om; n ⫽ 4, sc; data not shown). IL-6 is a known downstream target of p38 in 3T3-L1 adipocytes (27) so we also tested the ability of TNF to increase production of this cytokine in human AT. Culture of Om AT with p38 inhibitors dramatically decreased basal IL-6 accumulation in the medium on d 2 of culture (P ⬍ 0.05, SB203580; P ⬍ 0.01, SB202190, n ⫽ 4, Fig. 7A). As expected, culture with dex decreased IL-6. Moreover, in contrast to its effect on leptin, culture with p38 inhibitors blunted, but did not eliminate, the ability of TNF to increase IL-6 production in the presence or absence of dex (n ⫽ 3, Fig. 7B).

FIG. 7. The effects of culture with p38 inhibitors on TNF-stimulated IL-6 accumulation in the medium of Om AT from obese subjects cultured in the absence (A) or presence (B) of dex. Human AT was cultured in the presence or absence of 25 ␮M SB203580 or SB202190 for 2 d (n ⫽ 3). Medium was exchanged daily, and samples were assayed for IL-6. TNF significantly increased IL-6 accumulation in the absence and presence of SB203580 (n ⫽ 3) and SB202190 (n ⫽ 2). Culture with p38 inhibitors blunted, but did not eliminate, the ability of TNF to increase IL-6 production in the presence or absence of dex (n ⫽ 3). *, TNF effect (vs. control within hormonal condition).

Discussion

FIG. 6. The effects of culture with p44/42 MAPK inhibitors on basal leptin accumulation (A), and phospho/total p44/42 MAPK levels (B) in Om AT from obese subjects. Human AT was cultured in the presence or absence of p44/42 inhibitors for 2 d. Culture medium was sampled then replenished daily. *, Time effect in control, P ⬍ 0.05. The inhibitors decreased phospho/total p44/42 levels in Om AT (⫺86 ⫾ 7%, PD98059, P ⫽ 0.06, ⫺97 ⫾ 1%, U0126, P ⫽ 0.01, n ⫽ 3).

When added in the presence of dex, TNF has a stimulatory effect on leptin release from both Om and sc fat of humans. The synergistic stimulatory effect of TNF ⫹ dex on leptin was dependent on activation of p38 MAPK, but not p44/42 or JNK, and appeared to be explained by the parallel increase in leptin mRNA. Our data suggest that the synergism of TNF and dex on leptin production involves specific cross-talk between glucocorticoid and p38 MAPK signaling pathways because the TNF effect on IL-6 was not dependent on the presence of dex. These data suggest that high systemic TNF may contribute to increased leptin production during stresses associated with hypercortisolemia such as infection and cachexia and that increased local levels of both TNF and cortisol contribute to overexpression of leptin in AT with obesity. In agreement with our finding that TNF added in the presence of dex increases leptin in vitro, in vivo studies have


shown that administration of TNF to rodents and humans increases serum leptin and leptin expression in AT (7, 8, 28). In contrast, our current results showing that TNF added under basal conditions (in the absence of dex) are in agreement with previous studies that showed inhibition (9 –14). It is apparent that the effects of TNF on leptin expression in vitro are dependent on the hormonal milieu. Our finding that TNF and dex synergistically increase leptin production in human AT fragments is in contrast to a study in isolated human fat cells by Fawcett et al. (13). Thus, we speculate that the stimulatory effect we observe in tissue fragments may require interaction of stromal cells and adipocytes. Inhibition of p38 activity almost completely blocked the stimulatory effect of TNF, in the presence of dex, on leptin in cultured human AT. Consistent with these results, we show that TNF increased p38 activity. Although TNF alone activated p38 to MAPK, this was not sufficient to increase leptin. On the other hand, dex alone increases leptin expression without affecting p38 MAPK activity (Fig. 4). Taken together, these data indicate that cross-talk between glucocorticoid and p38 MAPK signaling leads to an increase in leptin expression. The 2-fold increase in secreted leptin observed after longterm culture of AT in the presence of TNF plus dex appeared to be completely explained by a 2-fold increase in the level of leptin mRNA, suggesting an effect at the transcriptional level or an effect on leptin mRNA stability may provide a plausible mechanism for the p38-dependent synergistic increase in leptin production. Although the leptin promoter is known to include a glucocorticoid response element, the transcriptional regulation of the leptin gene by glucocorticoids is poorly understood (29). The combination of TNF and dex may amplify the effects of dex on leptin mRNA levels via a p38 MAPK-dependent alteration in the activity of a key transcription factor or coactivator that interacts with the glucocorticoid receptor or the glucocorticoid receptor itself. Alternatively, because leptin mRNA possesses several AU-rich RNA elements (30), activation of p38 by TNF may increase leptin mRNA by increasing its stability (31). Glucocorticoids antagonize many actions of proinflammatory cytokines such as TNF, yet they also work together to increase the expression of genes involved in innate immunity and cell survival (32, 33). Hyperleptinemia may also have a proinflammatory role (34). Thus, we speculate that the interactive effects of glucocorticoids and TNF on adipocyte gene expression contribute to the inflammatory state, including hyperleptinemia, as seen acutely in cachexia and chronically in obesity. The effect of exogenous TNF and dex on leptin was specific to p38 MAPK because culture with p44/42 or JNK MAPK inhibitors had no effect on the TNF-stimulated increase in leptin expression (data not shown) and is consistent with data from endotoxin-sensitive mice (35). In contrast to its suppressive effects on leptin, p38 inhibitors only partially blocked the TNF-stimulated increase in IL-6 production, suggesting the key role of p38 in mediating TNF effects on leptin. In contrast, TNF-stimulated increases in the NF␬B pathway partially mediate its effect on IL-6, as recently observed by Fain (36). Whether activation of p38 MAPK by other agonists would, like TNF, increase leptin is not known. The ability of TNF (with dex) to increase leptin was similar in AT of nonobese and obese and in Om and sc AT of obese,


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despite the expected desensitization due to higher cytokine levels in obese compared with nonobese and in Om compared with sc ATs (16, 22, 37). Thus, we conclude that the depot difference in TNF expression does not contribute to the lower leptin expression in Om compared with sc AT. We observed a spontaneous increase in leptin production during culture of Om but not sc AT. We speculate that this spontaneous increase in leptin production in cultured Om AT is secondary to heightened production of inflammatory adipokines in Om AT during culture (16, 22, 37). Interestingly, the spontaneous increase in Om AT leptin production was diminished by inhibition of either p38 or p44/42. We suspect that the production of higher cytokines in Om during culture led to higher basal MAPK activation, thereby stimulating a marked increase in leptin. Whether increased cytokine expression in Om AT results in depot differences in MAPK expression is unknown. Although culture with p44/42 MAPK inhibitors had no effect on the TNF ⫹ dex-stimulated increase in leptin production, they did blunt the spontaneous increase in leptin production in cultured Om AT from obese subjects. These data suggest that in Om AT, endogenous factors in addition to TNF increase p44/42 activity to elevate leptin production. Our data are in agreement with Fain et al. (36), who showed additive effects of p44/42 and p38 inhibitor on production of other adipokines such as IL-6, IL-8, and macrophage chemoattractant protein-1. These cytokines may act in a paracrine fashion to increase basal p44/42 and p38 MAPK activities and thereby contribute to increased basal leptin production in obesity through local effects in Om adipose tissue (22, 37). Our data show that culture with p38 inhibitors also decreased basal leptin production in sc AT, suggesting that high basal p38 activity in obesity may play an important role in up-regulating leptin expression in both Om and sc AT depots of obese subjects. p38 MAPK is basally phosphorylated in adipocytes from sc AT from obese, obese insulinresistant, and obese type 2 diabetics (38, 39), and this appears to be the case for the obese, nondiabetic subjects in our study. Previous studies of murine AT cultured with a p38 inhibitor show no effect on basal leptin production (35). We suspect that there was no effect of the inhibitor on leptin in this study because the AT was taken from lean mice with low levels of endogenous cytokines such as TNF that were capable of stimulating p38 activity. Therefore, it is possible that increased p38 activity, stimulated by endogenous TNF and other factors overexpressed in obese AT, may mediate the increases in leptin expression in obesity. In summary, our results are consistent with the hypothesis that in response to an inflammatory stress associated with hypercortisolemia, AT increases leptin expression resulting in hyperleptinemia. In early cachexia, hyperleptinemia has been attributed to increased systemic levels of inflammatory cytokines such as TNF in association with systemic elevations in glucocorticoid. In obesity, hyperleptinemia is associated with increases in insulin, local cortisol generated by adipocyte 11-␤-hydroxysteroid dehydrogenase (40), and cytokines such as TNF and IL-6 that also increase leptin synergistically with dex (41). These results emphasize the importance of the paracrine milieu within AT as a determinant of sensitivity to hormone effects.

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Acknowledgments Received August 24, 2005. Accepted December 30, 2005. Address all correspondence and requests for reprints to: Susan K. Fried, Ph.D., Division of Endocrinology, Diabetes, and Nutrition, University of Maryland School of Medicine, 660 West Redwood Street, Howard Hall 428, Baltimore, Maryland 21201. E-mail: sfried@ medicine.umaryland.edu. This work was supported by National Institutes of Health Grant DK59823 (to S.K.F.), by the American Diabetes Association (to S.K.F.), and by the Geriatric Education and Research Clinical Core, Baltimore Veterans Administration Medical Center. Present address for M.E.T.: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10468. Present address for M.-J.L.: Division of Endocrinology, Diabetes, and Nutrition, School of Medicine, University of Maryland, Baltimore, Maryland 21201. The authors have no conflicts to disclose.

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