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Department of Nutritional Sciences (M.E.T., S.S., I.H., S.K.F.), Rutgers University, New Brunswick, New Jersey 08901; Division of Endocrinology (S.H.S.), University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey 08901; Jean Mayer United States Department of Agriculture, Human Nutrition Research Center on Aging (A.S.G.), Tufts University, Boston, Massachusetts 02111; and Division of Gerontology (S.K.F.), Department of Medicine, University of Maryland School of Medicine, and Baltimore Veterans Affairs Medical Center, Baltimore Maryland 21201
Address all correspondence and requests for reprints to: Susan K. Fried, Ph.D., Division of Gerontology/GRECC, University of Maryland School of Medicine, Baltimore VA Medical Center, 10 North Greene Street, Baltimore, Maryland 21201. E-mail: sfried{at}grecc.umaryland.edu.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Limited studies have also examined the possibility that IL-6 could influence endocrine functions of adipose tissue, such as leptin production. Culture of 3T3L1 adipocytes or human adipose tissue with IL-6 inhibited or had no effect on leptin (7, 8). However, these studies were carried out in the absence of key endocrine regulators of leptin production in human adipose tissue such as glucocorticoids and/or insulin (9) and did not examine possible depot differences in response. Depot differences would be expected because omental adipose tissue of humans not only produces much higher levels of IL-6 (2) but also exhibit metabolic differences and sensitivity to hormonal influences on adipocyte metabolism (10).
Administration of IL-6 to healthy humans increased systemic lipolysis by approximately 60% after 90 min as assessed by measurement of free fatty acid (FFA) turnover (11) or the arteriovenous difference for FFA and glycerol across abdominal sc adipose tissue (12). However, whether IL-6 has a direct role in the regulation of lipolysis has been questioned because the protocols used incompletely controlled the levels of other potentially lipolytic hormones (13). Support for a direct stimulatory role for IL-6 on lipolysis comes from in vitro studies of newly differentiated human preadipocytes in culture (14), although no effect was observed in 3T3-F442A adipocytes (15, 16).
IL-6 may also directly influence human adipocyte metabolism by decreasing the activity of lipoprotein lipase (LPL), an enzyme that regulates uptake of circulating triglycerides into adipocytes, as suggested by studies in the 3T3-L1 adipocyte model system (15). A recent study demonstrated that IL-6 impairs insulin signaling in both 3T3-L1 and human adipocytes (17). However, no previous studies have tested IL-6 effects on lipid metabolism (lipolysis or LPL) in mature human adipocytes or adipose tissue.
The objectives of the current study were therefore to determine whether chronic exposure to IL-6 exerts a direct influence on leptin production, LPL, and lipolysis in human adipose tissues and to examine depot specific responses. We examined the effects of IL-6 in the presence and absence of insulin and/or glucocorticoid because these hormones normally circulate and are potent modulators of adipose function.
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Subjects and Methods |
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Adipose tissue samples were obtained from the omental (Om) and abdominal sc depots of severely obese subjects [body mass index (BMI) 57 ± 3 kg/m2, range 44–69 kg/m2, n = 12, 11 females, one male] who underwent gastric bypass surgery at St. Peter’s University Hospital (New Brunswick, NJ) or from overweight to obese subjects undergoing gynecological surgery at the University of Maryland (BMI 31 and 29 kg/m2, n = 2, both females). Results obtained from the one male subject and the females did not differ, so all data were combined. Abdominal sc tissue was also obtained using fat aspiration under local anesthesia (18) from nonobese subjects (BMI 24 ± 0.4 kg/m2, range 23–27 kg/m2, n = 5, 3 females, 2 males). The subjects enrolled in this study did not have a medical history of metabolic disease such as diabetes. Subjects taking ß-blockers (but not other antihypertensives) or glucosteroids were excluded. All samples were obtained with informed consent via protocols approved by the Human Subjects Institutional Review Committees of St. Peter’s University Hospital, the University of Medicine and Dentistry of New Jersey, Rutgers University, and the University of Maryland.
Tissue culture
Adipose tissue fragments from fat aspiration or minced surgical tissue was placed in organ culture as described previously in the presence or absence of hormones and/or cytokines (19). The hormonal treatments consisted of 7 nM insulin (Humulin, Eli Lilly, Indianapolis, IN), 25 nM dexamethasone (dex, a synthetic glucocorticoid), or the combination of insulin and dex. These hormonal concentrations were chosen because they have been shown to produce maximal responses and maintain leptin production in adipose tissue organ culture for up to 7 d at levels similar to those measured in fresh tissue (9).
To test the effect of IL-6 on leptin and lipolysis, tissue was cultured with recombinant human IL-6 (Intergen, Purchase, NY) at a final concentration of 10 (0.48 nM) or 100 ng/ml (4.8 nM) in the presence or absence of insulin and/or dex for up to 2 d. After 24 h, media were exchanged with fresh media containing the same hormones and/or cytokines. Samples of media were obtained after 5, 24, and 48 h were stored at –80 C until analysis for lipolysis and leptin content.
Because our previous studies show that dex suppresses the expression of endogenous IL-6 in adipose tissue in organ culture (2), we hypothesized that the magnitude of the response to exogenous IL-6 would be higher after prior culture with dex. Furthermore, 6+ d of culture with the combination of insulin + dex ensures high expression of LPL activity (19) to allow us to test whether IL-6 could decrease LPL activity. Thus, in one set of experiments, tissue was first cultured for 6–8 d with dex or insulin + dex with media exchanges every 2 d. Then fresh media containing insulin + dex, with or without 100 ng/ml IL-6, were added, and culture was continued for an additional 24 or 48 h. Tissue from these experiments was frozen in liquid nitrogen and stored at –80 C for analysis of leptin mRNA and LPL activity, and media samples were saved for analysis of leptin content.
Determination of fat cell size
Fat cell number per gram of tissue was determined by Coulter counting as described by Hirsch and Gallian (20). The percent lipid in adipose tissue fragments was measured gravimetrically after extraction of total lipids (21). Average fat cell weight was then calculated by dividing lipid weight per gram of tissue by fat cell number per gram of tissue.
Measurement of lipolysis
Glycerol accumulation in culture medium was used to assess changes in the level of lipolysis. Glycerol accumulation was determined by fluorometric assay (22). Data are presented as micromoles of glycerol per 106 cells per 24 h of culture.
Measurement of leptin protein
Leptin accumulation in culture media was measured via RIA (Linco, St. Charles, MO). Data are presented as ng of leptin per 106 cells per 24 h of culture.
Measurement of LPL activity
LPL releasable by heparin from tissue fragments was collected, and activity was measured as described previously (23). One unit of LPL activity is defined as catalyzing the release of 1 µmol FFA in 1 h.
Measurement of leptin gene expression
Total RNA was extracted from frozen samples of cultured adipose tissue by a modified method described by Chomzczynski and Sacchi (24). Extracted RNA was separated by size by electrophoresis. Ethidium bromide-stained bands of 28S RNA were captured on film. The bands were then pixilated using a flat bed scanner and quantified with UN-SCAN-IT software for use as a loading control (Silk Scientific, Orem, UT). RNA separated by electrophoresis was then transferred onto GeneScreen (NEN Life Science Products, Boston, MA) via capillary action and then probed with a 32P-labled 1-kb cDNA fragment of human leptin (Redi Prime II, Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK). The blots were then washed and mRNA-bound probe was detected using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The absolute intensities of the bands corresponding to human leptin (3.5 kb) were assessed using ImageQuant software (version 5.0, Molecular Dynamics). Leptin mRNA abundance was then expressed as the ratio of leptin to 28S RNA. Samples from culture conditions that were to be compared were run on the same gel. Data from different blots were normalized to an arbitrary value for a pooled sample that was run on each gel.
Statistical analyses
Data are presented as means ± SEM. Preliminary analyses showed similar responses to IL-6 effects on leptin or lipolysis in adipose tissue from nonobese and obese subjects. There was no difference in the magnitude of the responses (percent increase due to IL-6) of nonobese (n = 4–5) and obese (n = 7) for leptin (P = 0.4, NS) or lipolysis (P = 0.6, NS) by independent t test. Furthermore, there was no significant correlation of BMI and magnitude of response and no lean-obese group effect by ANOVA (data not shown). We concluded that any lean-obese difference in response would be small in magnitude and require a large number of subjects to demonstrate. Thus, we focused on the question of whether IL-6 did affect human adipose tissue function and pooled all data for statistical analyses of IL-6 effects. The effects of IL-6, insulin, and dex on d 1 and 2 within each depot were determined by ANOVA with repeated measures (2 x 2 x 2) on log-transformed values. When main effects or interactions were significant, post hoc paired t tests were used to assess the significance of IL-6 effects within a hormonal condition. Time effects within a condition (d 1 vs. d 2) were also compared by paired t tests.
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Results |
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Under basal conditions (no hormones), culture of sc adipose tissue with 100 ng/ml IL-6 (4.8 nM) for 1 or 2 d tended to increase leptin accumulation in the medium (P < 0.1 d 1, P < 0.06 d 2, n = 11, Fig 1A
). In Om adipose tissue cultured under basal conditions, there was a spontaneous rise in leptin production from d 1 to d 2 (P < 0.005, Fig. 1B), and addition of 10 or 100 ng/ml IL-6 did not further increase leptin production.
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Similar to our previous results (9), the combination of insulin and dex increased leptin in sc and Om of obese. Insulin alone was without effect on leptin in this sample. Addition of IL-6 in the presence of insulin, with or without dex, did not further increase leptin production in either Om or sc under these hormonal conditions. No significant effects of IL-6 on leptin were detected after 5 h of culture under any hormonal condition (data not shown).
IL-6 + dex increased leptin mRNA levels
To investigate the mechanism by which IL-6 + dex increased secreted leptin, leptin mRNA levels were measured. In these experiments, adipose tissue was first cultured with dex for 6 d (to suppress endogenous IL-6 expression), followed by culture with dex alone or IL-6 + dex for an additional 2 d. Similar to results during the first 2 d of culture, IL-6 added to tissue previously cultured with dex increased leptin by 90 ± 30% (P < 0.02, n = 5). IL-6 also increased leptin mRNA levels (leptin mRNA/28S RNA: 2.51 ± 0.43 vs. 1.90 ± 0.42 arbitrary units with dex alone, P < 0.02) with similar trends in both depots (n = 3 Om, n = 2 sc). With the caveat that Northern analysis is only semiquantitative, the small magnitude of the IL-6-induced increases in leptin mRNA (37 ± 11%) appeared insufficient to fully account for the increase (90%) in leptin production in the same subjects.
IL-6 increases lipolysis during culture in the absence of insulin
The possibility that IL-6 increases lipolysis in human adipose tissue was assessed by measuring the accumulation of glycerol in the culture medium. Culture with IL-6 under basal conditions significantly increased lipolysis in sc on d 2 of culture (79 ± 23%, P < 0.01, n = 11, Table 1). In Om, IL-6 also increased lipolysis on d 2 of culture (by 26 ± 9%, P < 0.01, n = 8, Table 1). The effects of 10 ng/ml IL-6 on lipolysis in sc and Om cultures were apparent in four or five subjects studied, but the difference did not reach statistical significance (n = 5 sc, n = 4 Om, data not shown). There was no significant depot difference in the magnitude of the response in paired samples of sc and Om cultures from six obese subjects (NS, data not shown).
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No significant effects of IL-6 on lipolysis were detected after 5 h of culture (data not shown)
Chronic insulin increases lipolysis, and IL-6 does not have an additive effect
Culture with insulin for 2 d increased lipolysis in sc but not Om cultures, compared with respective controls (P < 0.05). Addition of IL-6 in the presence of insulin did not further increase lipolysis in adipose tissue from either depot. Addition of insulin the presence of dex increased lipolysis in sc and Om (P < 0.05). Addition of IL-6 in the presence of insulin + dex did not further increase lipolysis in either depot.
IL-6 decreases LPL activity
To determine whether IL-6 affected LPL activity, adipose tissue from obese subjects was first cultured for 7–8 d with insulin and dex to induce a high level of LPL activity. Cultures were then provided with media containing insulin + dex, with or without IL-6 (100 ng/ml) for an additional 24 h. IL-6 decreased LPL activity by 56 ± 12% in Om (P < 0.05, Fig. 2) and 68 ± 8% sc (P < 0.02, paired samples from four severely obese subjects).
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Discussion |
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We predicted that the biological effects of IL-6 would differ in Om and sc adipose tissue because the former is known to express much higher levels of IL-6. This appeared to be the case with respect to the stimulatory effect of IL-6 on leptin production under basal conditions in culture. However, we found that both depots were similarly responsive to the synergistic effects of dex and IL-6 on leptin as well as to other biological effects of this cytokine. In addition, although high BMI is associated with higher adipose IL-6 expression (3), adipose tissue from the obese subjects in our sample was clearly responsive to IL-6. Thus, our data suggest that chronic exposure to high local levels of IL-6 in vivo does not appear to cause resistance to its effects. Furthermore, chronically high paracrine concentrations of IL-6 may contribute to altered adipocyte function in obesity as well as differences in adipocyte behavior between adipose tissue depots.
We show for the first time that IL-6 increases leptin production in both Om and sc adipose tissue, most markedly in combination with a glucocorticoid (dex). Both IL-6 and leptin are overexpressed in obese adipose tissue (3), and both IL-6 and leptin are increased in early cachexia (25). Thus, increased IL-6 may contribute to the hyperleptinemia associated with obesity or cachexia. In contrast to these findings in human adipose tissue, previous studies of rodent adipocyte models found that IL-6 decreases leptin (7). In addition, Bruun et al. (8) concluded IL-6 did not affect leptin production in human adipose tissue but tested its effects only in the presence of insulin. From a physiologic point of view, synergistic effects of glucocorticoid and IL-6 on leptin suggest a role in stress-induced increases in leptin production, either in obesity secondary to local increases in cortisol production (26) or in early infection when systemic cortisol production is high.
IL-6 did not further increase leptin production in the presence of maximally effective, superphysiologic concentrations of insulin and dex, although effects at subthreshold levels of these hormones cannot be ruled out from the current results. It is possible that this is because leptin expression was already maximal or that IL-6 and insulin + dex may modulate common signaling events to increase leptin.
Our experiments did not address the mechanism of the interaction of IL-6 and dex on leptin production. However, it appears that an increase in leptin mRNA is at least partly responsible for the observed increase in leptin release when IL-6 and dex are added together.
Our data extend the evidence available in the literature that demonstrate lipolytic effects of IL-6 in vivo or in newly differentiated cultured human adipocytes (11, 12, 14). In our study, the stimulatory effect of IL-6 on lipolysis required 24–48 h of culture. Thus, the influence of chronically high levels of IL-6 within adipose tissue may contribute to alterations in gene or protein expression that influence basal lipolysis. However, the moderate effect of IL-6 we show on basal lipolysis would not appear to account for the increase in lipolysis that occurs within hours when IL-6 is administered systemically to humans in vivo. It should be noted that we assessed lipolysis by the accumulation of glycerol in the culture medium under conditions that are not optimal for assessing short-term influences on lipolysis (e.g. no albumin in the medium; adipose tissue fragments instead of isolated adipocytes). Future studies should assess the influence of prior culture with IL-6 on lipolytic capacity in isolated adipocytes measured under optimized conditions to determine whether shorter-term effects of IL-6 on lipolysis on basal and stimulated lipolysis can be observed.
The lipolytic effect of IL-6 was not evident in the presence of insulin. We found, in agreement with previous work (27, 28, 29), that long-term culture with insulin itself increases lipolysis. It is possible that the effects of chronic culture with insulin on lipolysis may be mediated by increased expression of adipose-derived IL-6. Insulin increases IL-6 production by adipocytes and adipose tissue (30) (our unpublished results). Alternatively, insulin and IL-6 may act though common signaling pathways to increase rates of lipolysis.
The majority of IL-6 released from human adipose tissue is from the stromal-vascular fraction of adipose tissue, suggesting IL-6 may act on adipocytes in an autocrine and/or paracrine fashion (2). Recent studies (31, 32) indicate that resident macrophages might be an important source of cytokines in obese adipose tissue. The concentrations of IL-6 in the interstitial fluid of human adipose tissue was estimated by one study (33) to exceed plasma levels by more than 100-fold and by another (34) to range from 1 to 20 ng/ml with levels proportional to fat cell size. Thus, the concentrations of IL-6 tested in this work are likely to be relevant to pathophysiological concentration of IL-6 in the vicinity of adipocytes, but concentrations of IL-6 in plasma are unlikely to exert the effects on adipocyte metabolism observed here. Thus, current data contribute to accumulating evidence that IL-6 is a potent paracrine modulator of human adipose tissue function and gene expression (17). Further studies to establish the dose-response relationships for IL-6 effects in the absence and presence of subthreshold concentrations of modulating hormones are needed.
In summary, we add to a growing body of evidence that IL-6 is a paracrine regulator of human adipose metabolism. Relatively high concentrations of IL-6, which are likely to be present locally within adipose tissue in the obese (33, 34) or other stressed states, have multiple actions on human adipose tissue metabolism and endocrine function. In obesity, IL-6 within adipose tissue is elevated in association with insulin resistance (3, 17, 35). Thus, chronically elevated IL-6 expression in adipose tissue may contribute to the increased basal lipolysis and high rates of leptin production observed in adipose tissue of the insulin-resistant obese. Furthermore, IL-6, like TNF, may restrain lipid deposition within the adipocyte by down-regulating LPL activity, acting as an adipostat to control fat cell size. In cachexia, IL-6 and cortisol may promote the dyslipidemia and hyperleptinemia that contribute to wasting.
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Footnotes |
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Abbreviations: BMI, Body mass index; dex, dexamethasone; FFA, free fatty acid; LPL, lipoprotein lipase; Om, omental.
Received March 31, 2004.
Accepted August 23, 2004.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, overexpressed in human fat cells from insulin-resistant subjects. J Biol Chem 278:45777–45784 in 3T3–L1 adipocytes. Diabetologia 44:55–62[CrossRef][Medline]
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