0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society
Vol. 85, No. 2 Printed in U.S.A.
Tumor Necrosis Factor-␣ Inhibits Leptin Production in Subcutaneous and Omental Adipocytes from Morbidly Obese Humans* RACHAEL L. FAWCETT, ANGELA S. WAECHTER, LLOYD B. WILLIAMS, PEILI ZHANG, RAYMOND LOUIE, ROSEMARIE JONES, MARGARET INMAN, JOHN HUSE, AND ROBERT V. CONSIDINE Division of Endocrinology and Metabolism, Departments of Medicine (L.B.W., R.L.F., A.S.W., Z.P., R.L., R.V.C.) and Surgery (R.M.J.), Indiana University School of Medicine, Indianapolis, Indiana 46202; and the Department of Surgery, St. Vincent’s Hospital Carmel (R.M.J., M.I., J.H.), Carmel, Indiana 46032 (0.1 mol/L)-stimulated leptin production in sc adipocytes. In omental adipocytes TNF␣ inhibited leptin release 21.0 ⫾ 9.6% and 40.8 ⫾ 6.3% at 10 and 100 ng/mL by 48 h (P ⬍ 0.05). Significant inhibition of leptin release from omental adipocytes was observed at 24 h with 100 ng/mL TNF␣ (P ⬍ 0.05). Anti-TNF␣ antibody completely blocked TNF␣ inhibition of leptin release. The ob messenger ribonucleic acid was significantly reduced (23.6 ⫾ 5.9%) after 48 h of TNF␣ (100 ng/mL) treatment (P ⬍ 0.025). TNF␣ had no effect on glucose uptake or lactate production in sc and omental adipocytes. The data suggest that the direct paracrine effect of adipose-derived TNF␣ is inhibition of leptin production. (J Clin Endocrinol Metab 85: 530 –535, 2000)
ABSTRACT This study was undertaken to examine the regulation of leptin production from human adipocytes by tumor necrosis factor-␣ (TNF␣). Adipocytes were isolated from adipose tissue obtained during bariatric surgical procedures (17 women and 3 men; body mass index, 52.5 ⫾ 2.4 kg/m2; age, 40 ⫾ 3 yr) and cultured in suspension. Leptin release from sc adipocytes was inhibited 17.7 ⫾ 5.2% (P ⬍ 0.01), 21.6 ⫾ 4.3% (P ⬍ 0.005), and 37.1 ⫾ 7.2% (P ⬍ 0.05) by 1, 10, and 100 ng/mL TNF␣, respectively, after 48 h in culture. At 100 ng/mL, significant inhibition of leptin release (25.8 ⫾ 9.7%; P ⬍ 0.05) was detected by 24 h. TNF␣ (10 ng/mL) had no effect on dexamethasone
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line 18 h after cytokine administration (16). A transient rise in leptin, occurring 12 h after TNF␣ infusion, was also observed in humans with solid tumors, but again serum leptin levels returned to baseline by 24 h after administration of the cytokine (19). It was concluded from these experiments that TNF␣ could acutely increase serum leptin in animals and humans, and it was suggested that leptin may play a role in the anorexia associated with infection (15). However, it was not established in these studies whether the cytokine acted directly on the adipose tissue. Observations of leptin production in obese transgenic TNF␣-deficient mice also suggest a stimulatory effect of TNF␣ on leptin production (20). Serum leptin is lower in transgenic mice lacking TNF␣ compared to wild-type obese mice of equivalent weight, suggesting that TNF␣ increases leptin production in wild-type mice and that in the absence of this stimulus, leptin production is lower in the knockout mice. However, serum insulin, which is a stimulus for leptin production (21), was also significantly lower in the TNF␣deficient mice (22). Therefore, as in the studies mentioned above, it is not clear whether TNF␣ acts directly on the adipose tissue to regulate leptin production. In vitro, TNF␣ has been reported to either stimulate (17) or inhibit (23) leptin release from murine adipocytes treated under identical conditions. In one study using 3T3-L1 adipocytes, a transient stimulation of leptin release was evident at 5 h of treatment, but by 24 h leptin release in the presence of TNF␣ was significantly less than that from untreated cells (20). A second report found inhibition of ob gene expression
EPTIN IS A signal to the central nervous system of energy stores in the adipose tissue (1– 4) and a regulatory signal for a variety of other physiological processes in addition to body weight maintenance (5). Tumor necrosis factor-␣ (TNF␣) is an inflammatory cytokine produced by macrophages (6) and in adipose tissue (7). Adipose tissuederived TNF␣ has been implicated in the development of insulin resistance in both muscle and adipose tissue (7–10). TNF␣ messenger ribonucleic acid (mRNA) is significantly elevated with increasing adiposity and is reduced after weight loss in both rodents (11) and humans (12, 13). TNF␣ alters adipose tissue lipoprotein lipase activity, fatty acid synthesis, and lipolysis (7–10, 14). As described below, the cytokine has also been reported to influence leptin production in animal models, primary cells, and cultured cell lines. The effect of TNF␣ on leptin production is complex. The ob mRNA expression and circulating leptin were increased within several hours (7– 8 h) after the administration of TNF␣ to hamsters (15) and mice (16 –18), but returned toward baseReceived August 12, 1999. Revision received October 12, 1999. Accepted October 27, 1999. Address all correspondence and requests for reprints to: Robert V. Considine, Ph.D., Indiana University School of Medicine, 541 North Clinical Drive, Clinical Building 455, Indianapolis, Indiana 46202-5111. E-mail:
[email protected]. * This work was supported in part by NIH Grant DK-51140, Grant M01-RR-00750 –28 to the General Clinical Research Center, and the Showalter Trust. Portions of this work were presented in preliminary form at the 59th Scientific Sessions of the American Diabetes Association, San Diego, California, June 1999.
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in differentiated 3T3-L1 cells when analyzed after 24 h of treatment (24). A recent study in rodent adipocytes anchored in a defined matrix of basement membrane protein found that a 48-h exposure to TNF␣ inhibited leptin release and that inhibition was maintained up to 96 h in culture (25). It was further observed that TNF␣ increased glucose uptake into the adipocytes by 96 h, but not the conversion of glucose to lactate. These investigators have previously demonstrated that glucose metabolism is an important stimulus for leptin production in rat adipocytes (26). It was therefore concluded that TNF␣ significantly inhibited leptin production in rat adipocytes, but that this inhibitory effect was independent of the effects of TNF␣ on glucose metabolism (25). To date there have been no reports on the effect of TNF␣ on leptin production in human adipocytes. Therefore, the current studies were undertaken to examine the direct effect of TNF␣ on leptin production in human sc and omental adipocytes. Leptin release was quantitated up to 48 h to model the chronic paracrine effect of adipose tissue-derived TNF␣. The interaction of TNF␣ with dexamethasone was examined to determine whether these two compounds regulated leptin production through similar mechanisms. The effect of TNF␣ on glucose uptake was also assessed. Our data suggest that the long term effect of TNF␣ on human adipocytes is inhibition of leptin synthesis and release. Materials and Methods Human sc and omental adipocytes were isolated by collagenase (Worthington Biochemical Corp., Freehold, NJ) digestion (27) of adipose tissue biopsies obtained during bariatric surgical procedures (17 women and 3 men; body mass index, 52.5 ⫾ 2.4 kg/m2; age, 40 ⫾ 3 yr). All subjects were morbidly obese, but none was diagnosed with or was receiving treatment for diabetes at the time of biopsy. All protocols were approved by the institutional review boards of Indiana UniversityPurdue University (Indianapolis, IN) and St. Vincent’s Hospital (Carmel, IN). All subjects gave informed consent for the collection of tissue samples. Adipocytes (2 mL packed cells) were suspension cultured in 10 mL DMEM-Ham’s F-12 plus 10% FBS in 125-mL sterile polycarbonate flasks (Corning, Inc., Corning, NY) at 37 C and 95% O2-5% CO2. The medium was replaced every 24 h with fresh medium. Control and treated adipocytes from each subject were cultured in parallel over each 48-h period. Culture medium was purchased from Sigma (St. Louis, MO), and FBS was obtained from Life Technologies, Inc. (Grand Island, NY). In initial experiments recombinant murine TNF␣ (Life Technologies, Inc.) was used, and in later experiments recombinant human TNF␣ (R&D Systems, Minneapolis, MN) was used. There was no difference in the extent of inhibition of leptin release by human or mouse recombinant TNF␣, and the data were pooled. Antihuman TNF␣ antibody was purchased from R&D Systems. Dexamethasone (D2915, water soluble) was obtained from Sigma. Leptin released into the medium during each 24-h period was quantitated with a commercially available RIA kit (Linco Research, Inc., St. Charles, MO). Leptin in the medium at 48 h represents the sum of that released during the first 24-h period plus that released during the second 24-h period. The within-assay variation (percent coefficient of variation) of the leptin assay is 8.3%, and the between-assay variation is 6.2% at 4.9 ng/mL. The limit of detection is 0.5 ng/mL. Samples (200 L) were measured in duplicate and ranged between 5–10 ng on the standard curve. The use of 200 L does not alter the sensitivity, reproducibility, or variability of the assay. The values obtained from the assay were divided by 2 to normalize for the use of twice the volume of sample called for in the assay. The culture medium containing 10% FBS contained no detectable leptin. RT-PCR was used to measure ob mRNA expression as previously described (28). All comparisons between samples were made on the
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linear portion of the amplification curve between 20 –35 cycles. No product was obtained in the absence of reverse transcriptase. The data are expressed as the ratio of ob complementary DNA (cDNA) to actin cDNA. There was no difference in the amount of actin cDNA among the samples studied. Glucose and lactate in the culture medium were measured on a glucose analyzer (model 2300, YSI, Inc., Yellow Springs, OH). Glucose uptake was determined by measuring medium glucose before and after each medium change and calculating the decrease. All data are expressed as the mean ⫾ sem. The percent inhibition of leptin release was calculated as the difference between 1 and the ratio of leptin release in the presence of TNF divided by that from untreated cultures, multiplied by 100. All statistical comparisons were made by Student’s paired t test (P ⬍ 0.05 was taken as statistically significant) where appropriate. A block-treatment ANOVA model was used to examine the effect on leptin release of coincubation with dexamethasone and TNF␣ (Fig. 2). Subjects were used as blocks to reduce possible error variability. After a significant ANOVA, Tukey’s test for multiple comparisons was used to determine which treatment pairs had significantly different rates of leptin release. A paired comparison (control and treatment) on adipocytes from each individual is denoted as n ⫽ 1.
Results
Subcutaneous adipocytes were suspension cultured in DMEM-Ham’s F-12 plus 10% FBS for 48 h. As shown in Fig. 1, leptin was released into the medium during each 24-h period, such that the cumulative concentration of leptin in the medium was significantly greater at 48 h than at 24 h (P ⬍ 0.05). At a concentration of 100 ng/mL, TNF␣ significantly inhibited leptin production at 24 and 48 h of culture (P ⬍ 0.05). Leptin production from sc adipocytes was inhibited by TNF␣ in a dose-dependent manner. At a concentration of 10 ng/mL, TNF␣ inhibited leptin production 21.6 ⫾ 4.3% (n ⫽ 16; P ⬍ 0.005) At the lowest concentration tested, 1 ng/mL TNF␣ significantly inhibited leptin release 17.7 ⫾ 5.2% by 48 h in culture (n ⫽ 11; P ⬍ 0.01). Neither 10 nor 1 ng/mL TNF␣ significantly attenuated leptin production within the first 24 h of treatment. Dexamethasone is a strong stimulus for leptin production. To determine whether TNF␣ could inhibit both basal and
FIG. 1. TNF␣ inhibits leptin release from sc adipocytes. Leptin release from untreated adipocytes was significantly greater at 48 h than at 24 h (P ⬍ 0.05). TNF␣ (100 ng/mL) significantly inhibited leptin production at 24 and 48 h of culture (P ⬍ 0.05). Values represent the mean ⫾ SEM for seven and eight independent observations at 24 and 48 h, respectively. *, P ⬍ 0.05, paired comparison between TNF␣treated and control cells.
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stimulated leptin release, sc adipocytes were coincubated with 0.1 mol/L dexamethasone and 10 ng/mL TNF␣. As illustrated in Fig. 2, leptin release from sc adipocytes treated with dexamethasone for 48 h was double that from untreated adipocytes. Dexamethasone-stimulated leptin release in the presence of TNF␣ was not different from that induced by dexamethasone alone. As a percentage of the control value, dexamethasone increased leptin production 193 ⫾ 29% in untreated adipocytes and by 206 ⫾ 31% in adipocytes treated with TNF␣. These observations suggest that TNF␣ and dexamethasone regulate leptin production through different mechanisms. TNF␣ also reduced leptin production in omental adipocytes. As observed for the sc adipocytes, leptin was released into the medium from omental adipocytes in a time-dependent manner (Fig. 3). At a concentration of 100 ng/mL, TNF␣ significantly inhibited leptin production within the first 24 h of culture (P ⬍ 0.05). By 48 h, TNF␣ inhibited leptin production by 40.8 ⫾ 6.3% (Fig. 3). At 10 ng/mL, TNF␣ had no effect on leptin production within the first 24 h of culture. By 48 h, 10 ng/mL TNF␣ significantly inhibited leptin release from omental adipocytes by 21.0 ⫾ 9.6% (6.2 ⫾ 1.6 vs. 5.2 ⫾ 1.9 ng/mL; n ⫽ 7; P ⬍ 0.05). There was no significant difference in TNF␣ inhibition of leptin release in omental vs. sc adipocytes. To demonstrate that the effect of TNF␣ was due to the cytokine and not to an unknown contaminant, sc adipocytes were treated with 10 ng/mL TNF␣, which had been previously incubated with 10 g/mL anti-TNF␣ antibody for 30 min before addition to the culture medium. Anti-TNF␣ antibody completely prevented the reduction in leptin release induced by TNF␣ (Fig. 4). TNF␣ inhibited leptin production by reducing ob gene expression. As shown in Fig. 5, ob mRNA at 48 h in sc adipocytes treated with 100 ng/mL TNF␣ was significantly less than that in untreated cells. The ob gene expression was also significantly reduced by the 48-h treatment with 10 ng/mL TNF␣ (53.9 ⫾ 9.3 vs. 38.4 ⫾ 8.4 arbitrary units for
FIG. 2. TNF␣ inhibits basal, but not dexamethasone-stimulated, leptin release from sc adipocytes. Cells were coincubated with 10 ng/mL TNF␣ and 0.1 mol/L dexamethasone for 48 h. Dexamethasone-stimulated leptin release is not significantly different in the presence or absence of TNF␣ as determined by a block-treatment ANOVA and Tukey’s test. Values represent the mean ⫾ SEM for seven independent experiments. *, P ⫽ 0.074 compared to untreated adipocytes. #, P ⫽ 0.012 compared to dexamethasone alone.
FIG. 3. TNF␣ inhibits leptin release from omental adipocytes. Leptin release from untreated adipocytes was significantly greater at 48 h than at 24 h (P ⬍ 0.05). TNF␣ (100 ng/mL) significantly inhibited leptin production at 24 and 48 h. Values represent the mean ⫾ SEM for five observations at each time point. *, P ⬍ 0.05, paired comparison to control cells.
FIG. 4. Inhibition of leptin release by TNF␣ is blocked by anti-TNF␣ antibody. TNF␣ (10 ng/mL) was incubated with 10 g/mL anti-TNF␣ antibody for 30 min before addition to the culture medium. Percent inhibition of leptin release in the presence TNF␣ and antibody is not different from zero. Values represent the mean ⫾ SEM for four observations. *, P ⬍ 0.05, paired comparison to percent inhibition of leptin release induced by TNF␣.
untreated and TNF␣-treated adipocytes, respectively; n ⫽ 10; P ⬍ 0.05). The extent of inhibition of ob gene expression achieved with 100 and 10 ng/mL TNF␣ was not different when measured after 48 h of treatment (23.6 ⫾ 5.9% vs. 23.1 ⫾ 9.3% inhibition for 100 and 10 ng/mL, respectively). TNF␣ treatment had no effect on glucose metabolism in sc or omental adipocytes. As illustrated in Table 1, there was no difference in glucose uptake or lactate release over the 48-h incubation in adipocytes treated with 100 ng/mL TNF␣ compared to untreated cells. At a concentration of 10 ng/mL, TNF␣ also had no effect on glucose metabolism in either sc or omental adipocytes (data not shown). Discussion
The current studies were undertaken to examine the effect of TNF␣ on leptin production in human adipocytes. We
LEPTIN PRODUCTION IN HUMAN ADIPOCYTES
FIG. 5. TNF␣ inhibits ob gene expression in sc adipocytes. Relative ob mRNA expression was determined by RT-PCR in adipocytes treated with 100 ng/mL TNF␣ for 48 h. Top, Ethidium bromide staining of 350 bp ob cDNA and 650 bp -actin cDNA in control and TNF␣-treated adipocytes. Three paired experiments using adipocytes from three different subjects are shown (lanes 1, 3, and 5, untreated adipocytes; lanes 2, 4, and 6, TNF␣-treated adipocytes). Bottom, The ob gene expression normalized to -actin expression in control and TNF␣-treated adipocytes (n ⫽ 8). *, P ⬍ 0.025, paired comparison to control cells. TABLE 1. Lack of effect of TNF␣ on glucose uptake and lactate production in human adipocytes
Subcutaneous adipocytes Untreated (n ⫽ 8) TNF␣ Omental adipocytes Untreated (n ⫽ 4) TNF␣
Glucose uptake (mol)
Lactate production (mol)
19.2 ⫾ 2.4 22.3 ⫾ 4.4
18.5 ⫾ 3.0 19.8 ⫾ 3.8
20.2 ⫾ 2.9 22.1 ⫾ 3.6
11.7 ⫾ 1.1 11.5 ⫾ 1.0
Adipocytes were cultured in medium containing 7.7 mmol/L glucose with or without 100 ng/ml TNF␣ for 48 h. Values represent the mean ⫾ SEM of the indicted number of paired experiments.
observed that TNF␣ inhibited leptin release from sc and omental adipocytes from morbidly obese subjects in a timeand dose-dependent manner. At the highest concentration tested, TNF␣ inhibition of leptin release was significant within the first 24 h of treatment, and the cytokine was equally effective in both sc and omental adipocytes. Although TNF␣ reduced basal leptin production, it had no effect on dexamethasone-stimulated release, a finding that
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suggests that the two compounds regulate leptin production through different mechanisms. The attenuation of leptin release by TNF␣ appears to be mediated at least in part through a reduction in ob gene expression and does not involve changes in glucose metabolism. Previous in vitro experiments using mouse adipocytes have been inconclusive regarding the direct effect of TNF␣ on leptin synthesis. In the study by Finck et al. (17) TNF␣ increased leptin release during a 24-h incubation of epididymal adipocytes obtained from both endotoxin-sensitive (OuJ) and endotoxin-insensitive (HeJ) mice. In contrast, at similar cytokine concentrations and treatment time, Yamaguchi et al. (23) observed TNF␣-induced inhibition of leptin release in parametrial adipocytes from 12- to 14-day pregnant mice. Although it is possible that TNF␣ could have opposite effects on adipocytes from different fat pads, in our hands TNF␣ inhibited leptin release to the same extent in both sc and omental adipocytes. Our findings are in accord with experiments in differentiated 3T3-L1 cells (20, 24) and cultured rat adipocytes (25) that exposure to TNF␣ for 24 h or longer inhibits ob gene expression and leptin release. Also in agreement with the present findings, Yamaguchi et al. (23) observed that TNF␣ (100 ng/mL) inhibited leptin release in sc adipocytes from pregnant women. Taken together, it therefore appears that chronic exposure to TNF␣ inhibits leptin production. Early observations in rodents of an acute stimulation of leptin release by TNF␣ led to the suggestion that leptin could mediate, in whole or in part, the reduced food intake and weight loss associated with infection and cancer (15–18). Similar findings in humans, that both TNF␣ (19) and interleukin-1␣ (29) acutely increase leptin release, would support this hypothesis. However, in both humans and rodents the effect of cytokines to increase serum leptin is transient, with a return to precytokine administration levels within 24 h (16, 19, 29). In the case of interleukin-1␣, leptin levels returned to baseline despite continued administration of the cytokine (29). Although we did not study time points earlier than 24 h, the results of the present study would suggest that the acute increase in leptin release with cytokine administration is not due to a direct effect of the cytokine on adipocytes, but may be a secondary response. Furthermore, as leptin is believed to be a long term regulator of food intake and energy balance, it is difficult to reconcile the transient increase in leptin as the mechanism for cytokine-induced anorexia. Indeed, lipopolysaccharide administration to leptin-deficient ob/ob and leptin receptor-deficient db/db mice increased TNF␣ to the same extent in both animal models and induced anorexia in the absence of a functional leptin signal system (30). An alternative possibility for the acute transient increase in leptin with TNF␣ is that it may serve as a signal to up-regulate the immune system (17). Leptin receptors are present on many different hemopoietic cells (5), and leptin stimulates the proliferation of naive and memory T cells (31). There are several possible mechanisms through which TNF␣ could inhibit leptin synthesis. TNF␣ decreases lipoprotein lipase activity and stimulates lipolysis in adipocytes (7–10, 14). These actions of TNF␣ to limit lipid accumulation and/or increase lipid release from the adipocyte could reduce leptin production, although it is not clear at this
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time through what mechanism the adipocyte “senses” the amount of stored lipid and translates this into leptin release. TNF␣ also induces insulin resistance in adipocytes by reducing the tyrosine kinase activity of the insulin receptor (32, 33), and prolonged exposure of 3T3-L1 adipocytes to the cytokine (72–96 h) results in loss of insulin receptor substrate-1 message and protein (34). As ob gene expression is regulated by glucose metabolism (26) a TNF␣-mediated reduction in glucose uptake could down-regulate leptin production. This does not appear to be the case in our studies, as there was no effect of TNF␣ on basal glucose uptake or lactate production in either the sc or omental adipocytes during the 48-h incubation. These findings are in accord with those of Medina et al. (25), who did not observe a significant effect of TNF␣ on glucose uptake in rat adipocytes within the first 48 h of treatment. In contrast, these investigators found that a longer exposure to TNF␣ (96 h) actually resulted in a significant increase in glucose uptake and decreased leptin production. Medina et al. (25) therefore conclude, and we would concur, that TNF␣ significantly inhibits leptin production, but that this effect is not mediated through the effects of TNF␣ on glucose metabolism. One final mechanism through which TNF␣ may alter leptin production is through its effects on the expression of the transcription factor C/EBP␣. TNF␣ (⬃85 ng/mL) has been observed to reduce C/EBP␣ by 25% in 3T3-L1 cells exposed to the cytokine for 24 h (35). A C/EBP␣-binding site is located within the proximal 60 bp of the ob gene promoter and has been demonstrated to be necessary for ob gene transcription (36 –38). TNF␣ could directly attenuate ob gene expression by reducing the cellular content of C/EBP␣ protein. A TNF␣mediated reduction in C/EBP␣ would also explain the reduction in basal, but not dexamethasone-stimulated, leptin production, as dexamethasone increases ob gene promoter activity through cis-elements other than that for C/EBP␣ (39). In support of this possibility we observed a significant reduction in ob gene expression in TNF␣-treated cells. This finding is in agreement with the report of Medina et al. (25), who observed a significant reduction in ob mRNA in TNF␣treated rat adipocytes. Additional experiments will be necessary to completely elucidate the mechanism through which TNF␣ reduces ob gene expression and leptin production. The observation that TNF␣ inhibits leptin release from the adipose tissue could have important physiological implications in the regulation of adipose tissue deposition. It is possible that TNF␣-mediated attenuation of leptin release could contribute to the development of excess adipose tissue by masking the actual amount of adipose tissue that central mechanisms regulating body weight are sensing. In addition, other actions of leptin, such as its effects on glucose homeostasis, could also be attenuated (5). The positive correlation between TNF␣ mRNA and ob mRNA in human adipose tissue (40) as well as the positive correlation between serum leptin and serum TNF␣ receptor (measured as a surrogate for TNF␣) in humans (41, 42) suggest that the mechanisms regulating the production of these two cytokines by the adipose tissue are tightly linked. However, these correlations do not rule out the possibility of a dysregulation between TNF␣ and leptin production with increasing adiposity. In this regard it should be noted that our experiments
were performed on adipocytes obtained from extremely obese individuals, and it is possible that TNF␣ may have a different effect on leptin production in adipocytes from leaner individuals. In summary, TNF␣ inhibits leptin production in both sc and omental adipocytes from morbidly obese humans in a time- and dose-dependent manner. The attenuation in leptin secretion appears to be mediated through a reduction in ob gene expression and does not involve alterations in glucose metabolism. Additional experiments will be necessary to determine the exact mechanisms that regulate the production of both TNF␣ and leptin in the adipose tissue and the roles of both cytokines in the development of excess adipose tissue. Acknowledgments We thank the patients who donated adipose tissue for these studies. We also gratefully acknowledge the support of the nurses and staff of the Indiana University General Clinical Research Center and St. Vincent’s Hospital Surgical suite.
References 1. Tataranni PA. 1998 From physiology to neuroendocrinology: a reappraisal of risk factors of body weight gain in humans. Diabetes Metab. 24:108 –115. 2. Friedman JM. 1998 Leptin, leptin receptors, and the control of body weight. Nutr Rev. 56:38 – 46. 3. Auwerx J, Staels B. 1998 Leptin. Lancet. 351:737–742. 4. Considine RV, Caro JF. 1997 Leptin and the regulation of body weight. Int J Biochem Cell Biol. 29:1255–1272. 5. Considine RV, Caro JF. 1999 Pleotropic cellular effects of leptin. Curr Opin Endocrinol Diabetes. 6:163–169. 6. Vassalli P. 1992 The pathophysiology of tumor necrosis factors. Annu Rev Immunol. 10:411– 452. 7. Peraldi P, Spiegelman B. 1998 TNF-␣ and insulin resistance: summary and future prospects. Mol Cell Biochem. 182:169 –175. 8. Skolnik E, Marcusohn J. 1996 Inhibition of insulin receptor signaling by TNF: potential role in obesity and Non-insulin dependent diabetes mellitus. Cytokine Growth Factor Rev. 7:161–173. 9. Kern P. 1997 Potential role of TNF-␣ and lipoprotein lipase as candidate genes for obesity. J Nutr. 127:1917S–1922S. 10. Argiles J, Lopez-Soriano J, Busquets S, Lopez-Soriano F. 1997 Journey from cachexia to obesity by TNF. FASEB J. 11:743–751. 11. Hofmann C, Lorenz K, Braithwaite SS, Colca JR, Palazuk BJ, Hotamisligil GS, Spiegelman BM. 1994 Altered gene expression for tumor necrosis factor-␣ and its receptors during drug and dietary modulation of insulin resistance. Endocrinology. 134:264 –270. 12. Kern PA, Saghizadeh M, Ong JM, Bosch RJ, Deem R, Simsolo RB. 1995 The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase. J Clin Invest. 95:2111–21119. 13. Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. 1995 Increased adipose tissue expression of tumor necrosis factor-␣ in human obesity and insulin resistance. J Clin Invest. 95:2409 –2415. 14. Grunfeld C, Feingold KR. 1996 Regulation of lipid metabolism by cytokines during host defense. Nutrition. 12(Suppl 1):S24 –S26. 15. Grunfeld C, Zhao C, Fuller J, Pollock A, Friedman J, Feingold K. 1996 Endotoxin and cytokines induce expression of leptin, the ob gene product in hamsters. J Clin Invest. 97:2152–2157. 16. Sarraf P, Frederich R, Turner E, et al. 1997 Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia. J Exp Med. 185:171–175. 17. Finck B, Kelley K, Dnatzer R, Johnson R. 1997 In vivo and in vitro evidence for the involovement of tumor necrosis factor-␣ in the induction of leptin by lipopoplysaccharide. Endocrinology. 139:2278 –2283. 18. Moshyyedi A, Josephs M, Abdalla E, Mackay S, Edwards C, Copeland E, Moldawer L. 1998 Increased leptin expressionon mice with bacterial peritonitis is partially regulated by tumor necrosis factor ␣. Infect Immun. 66:188 –1802. 19. Zumbach M, Boehme M, Wahl P, Stremmel W, Ziegler R, Nawroth P. 1997 Tumor necrosis factor increases serum leptin levels in humans. J Clin Endocrinol Metab. 82:4080 – 4082. 20. Kirchgessner T, Uysal K, Wiesbrock S, Marino M, Hotamsligil G. 1997 Tumor necrosis factor-␣ contributes to obesity-related hyperleptinemia by regulating leptin release from adipocytes. J Clin Invest. 100:2777–2782.
LEPTIN PRODUCTION IN HUMAN ADIPOCYTES 21. Saladin R, DeVos Piet, Guerre-Millo M, Leturque A, Girard J, Staels B, Auwerx J. 1995 Transient increase in obese gene expression after food intake or insulin administration. Nature. 377:527–529. 22. Uysal KT, Wiesbrock SM, Marino MW, Hotamisligil GS. 1997 Protection from obesity-induced insulin resistance in mice lack TNF-␣ function. Nature. 389:610 – 614. 23. Yamaguchi M, Murakami T, Tomimatsu T, et al. 1998 Autocrine inhibition of leptin production by tumor necrosis factor-␣ (TNF-␣) through TNF-␣ type-I receptor in vitro. Biochem Biophy Res Commun. 244:30 –34. 24. Granowitz E. 1997 Transforming growth factor-␣ enhances and pro-inflammatory cytokines inhibit ob gene expression in 3T3–L1 adipocytes. Biochem Biophys Res Commun. 240:382–385. 25. Medina EA, Stanhope KL, Mizuno TM, et al. 1999 Effects of tumor necrosis factor ␣ on leptin secretion and gene expression: relationship to changes of glucose metabolism in isolated rat adipocytes. Int J Obesity. 23:896 –903. 26. Mueller WM, Gregoire FM, Stanhope KL, et al. 1998 Evidence that glucose metabolism regulates leptin secretion from cultured rat adipocytes. Endocrinology. 139:551–558. 27. Considine RV, Nyce MR, Morales LM, et al. 1996 Paracrine stimulation of preadipocyte-enriched proliferation by mature adipocytes. Am J Physiol 270:E895–E899. 28. Considine RV, Sinha MK, Heiman ML, et al. 1996 Serum immunoreactiveleptin concentrations in normal-weight and obese humans. N Engl J Med. 334:292–295. 29. Janik J, Curti B, Considine RV, et al. 1997 Interleukin 1␣ increases serum leptin concentrations in humans. J Clin Endocrinol Metab. 82:3084 –3086. 30. Faggioni R, Fuller J, Moser A, Feingold K, Grunfeld C. 1997 LPS-induced anorexia in leptin deficient (ob/ob) and leptin receptor-deficient (db/db) mice. Am J Physiol 273:R181–R186. 31. Lord GM, Matarese G, Howard JK, Baker RJ, Bloom SR, Lechler RI. 1998 Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature. 394:897–901.
535
32. Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM. 1994 TNF-␣ inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA. 91:4854 – 4858. 33. Kanety H, Feinstein R, Papa M, Hemi R, Karasik A. 1995 Tumor necrosis factor ␣-induced phosphorylation of insulin receptor substrate-1 (IRS-1). J Biol Chem. 270:23780 –23784. 34. Stephens J, Lee J, Pilch P. 1997 Tumor necrosis factor-␣-induced insulin resistance in 3T3–L1 adipoctes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal transduction. J Biol Chem. 272:971–976. 35. Stephens J, Pekala P. 1991 Transcriptional repression of the GLUT4 and C/EBP genes in 3T3–L1 adipocytes by tumor necrosis factor-␣. J Biol Chem. 266:21839 –21845. 36. Miller SG, De Vos P, Guerre-Millo M, et al. 1996 The adipocyte specific transcription factor C/EBP ␣ modulates human ob gene expression. Proc Natl Acad Sci USA. 93:5507–5511. 37. Gong DW, Bi S, Pratley RE, Weintraub BD. 1996 Genomic structure and promoter analysis of the human obese gene. J Biol Chem. 271:3971–3974. 38. Mason MM, He Y, Chen H, Quon MJ, Reitman M. 1998 Regulation of leptin promoter function by Sp1, C/EBP, and a novel factor. Endocrinology. 139:1013–1022. 39. De Vos P, Lefebvre A-M, Shrivo I, Fruchart J-C, Auwerx J. 1998 Glucocorticoids induce the expression of the leptin gene through a non-classical mechanism of transcriptional activation. Eur J Biochem. 252:619 – 626. 40. Ranganathan S, Maffei M, Kern PA. 1998 Adipose tissue ob mRNA expression in humans: discordance with plasma leptin and relationship with adipose TNF ␣ expression. J Lipid Res. 39:724 –730. 41. Mantozoros C, Moschos S, Avramopoulos I, et al. 1997 Leptin concentrations on relation to body mass index and the tumor necrosis factor-␣ system in humans. J Clin Endocrinol Metab. 82:3408 –3413. 42. Corica F, Allegra A, Corsonello A, et al. 1999 Relationship between plasma leptin levels and the tumor necrosis factor-␣ system in obese subjects. Int J Obesity. 23:355–360.