Stimulation of lipolysis in cultured fat cells by tumor necrosis factor, interleukin-1, and the interferons is blocked by inhibition of prostaglandin synthesis.

0013-7227/92/1301-0010$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Vol. 130, No. 1 Printed in U.S.A.

Society

Stimulation of Lipolysis in Cultured Fat Cells by Tumor Necrosis Factor, Interleukin-1, and the Interferons Is Blocked by Inhibition of Prostaglandin Synthesis* KENNETH R. FEINGOLD, WILLIAM DOERRLER, WALTER FIERS, AND CARL GRUNFELDt

CHARLES

A. DINARELLO,

Department of Medicine (K.R.F., W.D., C.G.), University of California, and Metabolism Section, Medical Service, Department of Veterans Affairs Medical Center, San Francisco, California 94121; the Department Medicine, Tufts University School of Medicine, and New England Medical Center (C.A.D.), Boston, Massachusetts 02111; and the Laboratory of Molecular Biology, State University of Ghent (W.F.), Ghent, Belgium

ABSTRACT. Multiple cytokines induce a number of alterations in lipid metabolism which can produce hyperlipidemia. Recent studies have demonstrated that tumor necrosis factor (TNF) increases lipolysis, resulting in an increase in circulating FFA levels, which stimulates hepatic triglyceride production, thereby contributing to the hyperlipidemia induced by TNF. In the present investigation we have determined the effects of a variety of cytokines on lipolysis in cultured 3T3-F442A adipocytes. TNF increased lipolysis approximately 3-fold with a maximal effect at 100 rig/ml and a half-maximal increase at 5-10 rig/ml. This increase was first observed 8 h after incubation with TNF. Interleukin-1 (IL-l) and interferon-a (IFN), $3, and -y also stimulated lipolysis in cultured adipocytes. The half-maximal increase in lipolysis occurred at approximately 10 rig/ml IL-

of

1,5 rig/ml IFNa, 10 rig/ml IFN& and 8 rig/ml of 1FN-r. Maximal lipolysis was observed at approximately 100 rig/ml for each of these cytokines, with the exception of IFN& for which maximal stimulation was observed at 1000 rig/ml. Neither platelet-activating factor nor IL-6 stimulated lipolysis; therefore, it is unlikely that these compounds mediate the increase in lipolysis induced by cytokines. However, indomethacin, a well known inhibitor of prostaglandin synthesis, prevented the increase in lipolysis induced by TNF, IL-l, IFNa, IFN& or IFNr. Indomethacin did not affect basal lipolysis or the acute stimulation of lipolysis induced by epinephrine. These results demonstrate that multiple cytokines can increase lipolysis and that this increase is mediated by cytokine-induced stimulation of prostaglandin synthesis. (Endocrinology 130: lo-16,1992)

I

NFECTIONS are frequently accompanied by hypertriglyceridemia (1). This increase in serum triglyceride levels may be due to an inhibition of lipoprotein lipase activity, which could result in a decrease in lipoprotein clearance (2,3). Additionally, studies have shown that infection can increase hepatic very low density lipoprotein (VLDL) production by two mechanisms: 1) an increase in hepatic de nouo fatty acid synthesis (4), and 2) a stimulation of lipolysis, which leads to the increased delivery of FFA to the liver which are reesterified, thus increasing total hepatic triglyceride synthesis and secretion (5). It is now widely recognized that cytokines, such as tumor necrosis factor (TNF), mediate many of the met-

abolic changes that produce the hyperlipidemia associated with infection (6). Initial studies focused on adipose tissue and demonstrated that TNF inhibited adipose tissue lipoprotein lipase activity by decreasing the synthesis of this enzyme (7-9). Subsequently a number of other cytokines, including interleukin-1 (IL-l) and interferon-y (IFNr), have also been shown to decrease adipose tissue lipoprotein lipase activity (9-11). However, extensive studies by this and other laboratories have demonstrated that TNF increases serum triglyceride levels in rodents, primarily by stimulating hepatic triglyceride synthesis, rather than by inhibiting adipose tissue lipoprotein lipase activity (12-14). In intact animals, TNF administration did not decrease the clearance of triglyceride-rich lipoproteins (14, 15). Instead, TNF administration increases de novo hepatic fatty acid synthesis, total hepatic triglyceride synthesis, and VLDL secretion by the liver (12-14). In addition to TNF, other cytokines, including IL-l, IL-6, and IFNa, have been shown to stimulate hepatic lipogenesis (16, 17).

Received July 5, 1991. Address all correspondence and requests for reprints to: Kenneth R. Feingold, M.D., Metabolism Section (lllF), Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 94121. * This work was supported by grants from the Research Service of the Department of Veterans Affairs and the NIH (DK-40990 and AI15614). t Recipient of a Clinical Investigator Award from the Department of Veterans Affairs. 10

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After TNF administration, circulating FFA and glycerol levels are increased, suggesting that adipose tissue lipolysis is stimulated (18,19). Similar increases in serum FFA and glycerol levels also occur in man after TNF treatment (20). This increase in lipolysis is of importance, in that recent studies have demonstrated that the TNF-induced rise in circulating FFA significantly contributes to the increase in hepatic lipoprotein secretion and serum triglyceride levels (19). Inhibition of TNFstimulated lipolysis blunts the increase in serum triglyceride seen after TNF administration (19). Thus, an increase in lipolysis after cytokine treatment provides an important source of fatty acids for increasing hepatic triglyceride production and secretion. Because lipolysis is one of the least well studied metabolic effects of cytokines (9, 11, 21, 22), in the present manuscript we have examined the effects of TNF, IL-l,

IL-6, and the IFNs on lipolysis in cultured

adipocytes.

Additionally, we have explored the mechanisms by which cytokines stimulate lipolysis.

Materials

and Methods

Cytokines and other materials

Murine TNFa (SA, 2.9 x lo7 U/mg) and murine IFNr (SA, 5 x lo6 U/mg) were kindly provided by Genentech, Inc. (South San Francisco, CA). Recombinant human IL-l/3-(112-269) (SA, 5 x lo7 U/mg) was produced as described previously (23). Recombinant human IFNa (A/D) (SA, 7.9 x lo7 U/mg) was generously provided by Drs. M. Brunda and P. Sorter of Hoffman-LaRoche (Nutley, NJ). Human IFNa (A/D) hybrid has been shown to regulate mouse tissue in a manner similar to that of murine IFNa (24). Recombinant murine IFNP (SA, 1 x lo7 U/mg) was generously provided by Drs. A. Kitai and G. Kawana of Toray Industries, Inc. (Kanagawa, Japan). IL-6 (SA, 1.7 x 10’ U/mg) was purified from the medium of transformed yeast cells (25). Platelet-activating factor (PAF), enzymes, and substrates were purchased from Sigma (St. Louis, MO). Cell cultures

3T3-F442A mouse embryo preadipocytes were generously provided by Dr. Howard Green (Harvard Medical School). The cells were grown in six-well plates in Dulbecco-Vogt’s Modified Eagle’s Medium containing 10% fetal calf serum, as previously described (26). Differentiation of 3T3-F442A cells into adipocytes was induced by treating the confluent cells for 2 days with this medium supplemented with 0.5 mM 1-methyl-3-isobutyl-xanthine/0.25 mM dexamethasone-insulin (1 rg/mI), as previously described (27). Cells were then refed three times per week with standard medium without additives and used between 7-14 days after differentiation began. Cytokines were incubated with the differentiated adipocytes for the time periods indicated in the text and figure legends. In some long term experiments, the cells were incubated with the indicated cytokine for 16 h, the medium was removed and replaced with fresh medium containing the cytokine, and the

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11

incubation was continued for an additional l-2 h. The medium was then assayed for glycerol and/or FFA content. In all experiments simultaneous controls were employed. If the medium was replaced with fresh medium, this was also performed in the control as well as the cytokine-treated cultures. In these relatively short term experiments, none of these cytokines altered either the protein or DNA content of these cultures. Glycerol

assay

To 1 ml medium, 0.1 ml 30% HClO, was added, and the mixture was centrifuged at 2000 rpm for 10 min. The supernatant was adjusted to pH 9.5 with KOH, and after removal of KClO, by centrifugation at 2000 rpm for 10 min, a 0.2-ml aliquot was removed for enzymatic fluorometric determination of glycerol, as described previously (19). Fatty acid assay

FFA were determined using a microfluorometric assay, described by Miles et al. (28). Briefly, to 5 ~1 medium, 1 ml of a Tris buffer [0.08 M Tris, 0.6 mM EDTA, 10 mM magnesium chloride, and 0.1% (vol/vol) Triton X-100, pH 81 containing NADH (47 nmol/ml), ATP (840 nmol/ml), phospholenolpyruvate (979 nmol/ml), myokinase (3.3 U/ml), pyruvate kinase (1.5 U/ml), lactic dehydrogenase (6 U/ml), acyl coenzyme-A synthetase (1.5 mu/ml) was added, vortexed, and allowed to incubate for 10 min at room temperature. After an initial reading, 80 nmol coenzyme-A were added to each sample, mixed, and incubated for 90 min at room temperature, after which a reading was obtained. The decrease in fluorescense due to NADH oxidation was determined by subtracting the final reading from the initial reading. Statistics

Statistical differences were determined by using two-tailed Student’s t test.

Results Because of the well described importance of TNF in mediating alterations in lipid metabolism, our initial studies focused on the effect of TNF on lipolysis in 3T3F442A adipocytes. As shown in Fig. 1, the addition of TNF to these adipocytes increased glycerol accumulation in the medium, indicating that TNF stimulates lipolysis. This increase in lipolysis was first observed 8 h after incubation with TNF, and by 16 h, the accumulation of

glycerol was approximately 2.5-fold greater in the TNFtreated cells than in the controls. In the next set of experiments, a protocol using a rinse after 16 h was employed to allow for assessment of the rate of lipolysis at time points after the induction of the increase in lipolysis by TNF or other cytokines. Figure 2 depicts the dose-response curve for TNF-induced stimulation of lipolysis in cultured adipocytes at 16 h. As little as 1 ng/ ml TNF induced a significant increase in glycerol accumulation, and 100 rig/ml caused maximal stimulation. The half-maximal

increase occurred at approximately


5-

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14

16

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FIG. 1. Time course of TNF-induced stimulation of lipolysis. 3T3F442A adipocytes were incubated with 100 rig/ml TNF. At the time points indicated, the medium was assayed for glycerol content, as described in Materials and Methods. The data are presented as the mean + SEM (n = 3 for each data point). *, P < 0.01; **, P c 0.001 (us. control). 600 l

c

.

.

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Endo. 1992 Voll30. No 1

than the expected 3:l ratio. We next examined the effects of other cytokines on lipolysis. After incubation for 2 h, neither TNF, IL-l, IFNa, IFNP, nor IFN+y increased lipolysis ‘(data not shown). In contrast, epinephrine, which is well recognized to rapidly stimulate lipolysis in cultured adipocytes (29), induced a marked increase in glycerol release into the medium (&&fold increase) during a 2-h incubation. With long term exposure (16 h), TNF, IL-l, IFNa, IFN& and IFNy all stimulated lipolysis in cultured adipocytes (Fig. 3). In multiple experiments, the average increase in lipolysis induced by TNF was 3.2-fold, that induced by IL-l was l&fold, that induced by IFNa was 2.2-fold, that induced by IFNP was 1.7-fold, and that induced by IFNr was 2.6-fold. Figure 4 shows the doseresponse curves of IL-lb, IFNa, IFNP, and IFNr, respectively. The half-maximal increase in lipolysis occurred at approximately 10 rig/ml IL-l, 5 rig/ml IFNcq 20 rig/ml IFNP, and 8 rig/ml IFNr. Maximal lipolysis was observed at approximately 100 rig/ml for each of these cytokines, with the exception of IFNP, for which maximum stimulation was observed at 1000 rig/ml. Thus, as seen with other aspects of lipid metabolism (g-11,16), multiple different cytokines are capable of stimulating lipolysis in adipocytes. Many of the actions of TNF and other cytokines are thought to be mediated by small molecular mediators, such as PAF (30, 31) or prostaglandins (32-39), or by

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FIG. 2. Dose response of TNF-induced lipolysis. 3T3-F442A adipocytes were incubated with the concentration of TNF indicated for 16 h. The medium was removed and replaced with fresh medium containing the indicated dose of TNF, and the incubation was continued for an additional 2 h. The medium was then assayed for glycerol content. In controls, the medium was also replaced with fresh medium at 16 h. The data are presented as the mean f SEM (n = 3 for each data point). *, P < 0.001 us. control.

10 rig/ml TNF. To further demonstrate the effect of TNF on lipolysis, both glycerol and FFA were assayed in the medium. TNF stimulated the release of both glycerol and FFA into the medium (glycerol: control, 41.3 + 3.4; TNF, 179 f 14 nmol/well; P < 0.001; FFA: control, 41.0 + 6.0; TNF, 128 f 8.9 nmol/well; P < 0.001). Glycerol was a better marker of lipolytic rates, because unlike fatty acids, glycerol is not oxidized or reesterified by adipocytes. Oxidation and/or reesterification of fatty acids accounted for the ratio of fatty acid to glycerol being less

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FIG. 3. Effects of cytokines on lipolysis. 3T3-F422A adipocytes were incubated with 100 rig/ml of the indicated cytokine for 16 h. The medium was removed and replaced with fresh medium containing the cytokine, and the incubation was continued for 2 h. The medium was then assayed for glycerol content. The data are presented as the mean + SEM. The results represent the mean of 11 separate experiments for TNF, 4 experiments for IL-l, 7 experiments for IFNcx, 8 experiments for IFNP, and 7 experiments for IFNr. Each separate experiment was carried out with 3 cultures treated with cytokine and 3 controls. *, P < 0.001 us. control.


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FIG. 4. Dose response of cytokine-induced lipolysis. A, IL-l; B, IFNol; C, IFN@; D, IFNr. The experimental protocol employed was identical to that described in Fig. 2. The data are presented as the mean f SEM (n = 3 for each data point). *, P < 0.001; **, P < 0.01; ***, P c 0.05 (us. control).

other cytokines, such as IL-6 (40, 41). However, neither PAF nor IL-6 stimulated lipolysis (data not shown), and therefore, it is unlikely that these compounds mediate the increase in lipolysis induced by cytokines. Prostaglandins are well known to stimulate lipolysis in 3T3 adipocytes (29). Figure 5 demonstrates that indomethacin, a well known inhibitor of prostaglandin synthesis, prevents the TNF-induced increase in lipolysis. Moreover, indomethacin also inhibits the increase in lipolysis induced by IL-l, IFNa, IFN& and IFNr (Fig. 5). In contrast, the acute stimulation of lipolysis by epinephrine is not inhibited by indomethacin (control, 128 + 5.4; epinephrine, 1130 + 12.0; control with indomethacin, 143 +- 2.2; epinephrine and indomethacin, 1291 f 34 nmol glycerol/ml; n = 3). This observation as well as the absence of an effect of indomethacin on basal lipolysis (i.e. in the absence of cytokines) indicate that the inhibition of cytokine-induced lipolysis by indomethacin is not a nonspecific toxic effect. These results suggest that the cytokine-induced stimulation of lipolysis is mediated by prostaglandin production.

Discussion Several previous studies have examined the effects of highly purified recombinant cytokines on lipolysis in cultured adipocytes. Studies by this laboratory, measuring the release of 3H-labeled fatty acids that were prelabeled by incubating adipocytes with [3H]acetate, demonstrated that TNF and IFNr both increased the release of labeled fatty acids (9). This technique does not measure lipolysis directly and produced great variability in results, so that TNFa significantly increased fatty acid release after 8 h of incubation, but release was not significantly elevated after a 16-h incubation (9). Price and co-workers (11, 21) demonstrated an increase in lipolysis, measured as glycerol release, in 3T3-Ll adipocytes after 17 h of exposure to 12.5 rig/ml TNF or 16.6 rig/ml IL-l. Lastly, Kawakami et al. (22) studied the time course and dose response of TNF for lipolysis in 3T3-Ll adipocytes, demonstrating that an increase in lipolysis was not apparent until after 12 h of incubation with TNF, with a lowest effective dose of 42.5 rig/ml.


14

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TNT &!4J

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FIG. 5. Effect of indomethacin (INDO) on cytokine-induced lipolysis. 3T3-F422A adipocytes were incubated with 100 rig/ml of the indicated cytokine with or without 100 pM indomethacin for 16 h. The medium was removed and replaced with fresh medium containing the indicated cytokines with or without indomethacin. The medium was then assayed for glycerol content. The data are presented as the mean & SEM (n = 3 for each data point). *, P < 0.01 US.control, indomethacin alone, and the respective cytokine plus indomethacin. **, P < 0.01 us. control, indomethacin alone, and IFNy alone.

The present investigation provides new information that confirms and extends these previous studies. The present study demonstrates that a variety of different cytokines, including TNF, IL-l, IFNcu, IFNP, and IFNr, which interact with different cellular receptors, can increase lipolysis in cultured adipocytes. The time course of this activation of lipolysis is similar to that described for the inhibition of lipoprotein lipase activity by several of these cytokines (7-11). Similarly, the doses of cytokine required to stimulate lipolysis are in the same range as those that inhibit lipoprotein lipase activity (7-11). Moreover, these findings indicate that cytokines can stimulate lipolysis by direct effects on adipose tissue. In contrast to the effects of TNF, IL-l, and the IFNs, there was no effect of IL-6 and PAF on lipolysis. This observation that multiple cytokines can effect lipolysis is similar to previous observations by this laboratory and others demonstrating that multiple cytokines can also stimulate hepatic lipogenesis and decrease adipose tissue lipoprotein lipase activity (9-11, 16). Thus, multiple cytokines induce a number of alterations in lipid metabolism that can contribute to increasing serum lipid levels. Another major finding of the present study is that the inhibition of prostaglandin synthesis can prevent the cytokine-induced increase in lipolysis. It is well recognized that some of the actions of cytokines are mediated by the production of prostaglandins and that prostaglandin synthesis inhibitors can prevent these effects (3239). For example, IL-l and TNF induce the classic febrile response by increasing the synthesis of PGE2 in the

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Endo. 1992 Vol 130. No 1

hypothalamic vasculature; this fever is blocked by inhibitors of prostaglandin synthesis (32, 42). Furthermore, in rodents, the shock syndrome induced by TNF or IL-l can be prevented by pretreatment with prostaglandin synthesis inhibitors (36-38). Lastly, many of the in uitro effects of cytokines on endothelial cells, fibroblasts, and other cells have been shown to be mediated by cytokine stimulation of the cyclooxygenase pathway (33, 35, 42). In the present study we demonstrate that indomethacin inhibits the increase in lipolysis induced by TNF, IL-l, and the IFNs. That this inhibition is a specific effect is shown by the fact that indomethacin did not affect either the basal rate of lipolysis or the increase in lipolysis induced by epinephrine. Additionally, studies by others have demonstrated that prostaglandins themselves directly stimulate lipolysis in cultured 3T3 adipocytes (29), indicating that a cytokine-induced increase in prostaglandin synthesis would be expected to increase lipolysis. In addition to the direct effects of cytokines on adipose tissue, it is likely that in the intact animal, cytokineinduced stimulation of the secretion of other hormones will also influence adipose tissue metabolism. Both TNF and IL-l have been shown to increase serum catecholamine and glucagon levels (6), which would stimulate lipolysis. Additionally, circulating cortisol levels are increased after TNF or IL-l treatment (6), which could also contribute to increased lipolysis (43). Finally, TNF has been shown to potentiate the effects of glucagon on hepatic amino acid metabolism (44). Thus, both the direct actions of cytokines on adipose tissue and the indirect effects of cytokines mediated via changes in hormonal levels could contribute to the increase in lipolysis that accompanies infection. However, adrenergic blockade does not inhibit the mobilization of fatty acids induced by TNF in uiuo (17). This increase in lipolysis could have either beneficial or detrimental effects on the host. The increase in lipolysis by depleting adipose stores could contribute to the potentially harmful cachexia that is observed during infection and inflammation. However, the increase in FFA flux from adipose tissue to other tissues could provide a source of energy, which may be beneficial, especially during infection-induced anorexia. Additionally, the increased delivery of FFA to the liver could contribute to the hyperlipidemia that accompanies infection and inflammation. Studies have suggested that this hyperlipidemia may be a beneficial response for the host. Specifically, experiments have demonstrated that lipoproteins bind and thereby protect the animal from the toxic effects of endotoxin (45-50). Furthermore, endotoxin is bound to VLDL in the circulation of normal individuals (50), suggesting that this detoxifying mechanism may be operating in the normal course of activity. Additionally, studies have shown that lipoproteins can


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bind a variety of viruses with a resultant reduction in the toxic effects of these viruses (51-53). In summary, the present study demonstrates that a variety of different cytokines can directly stimulate lipolysis in adipose tissue and that this increase is mediated by cytokine-induced stimulation of prostaglandin synthesis. The ability of multiple different immune regulators, the cytokines, to alter lipid metabolism in a variety of different tissues emphasizes the importance of these changes in host intermediary metabolism. Acknowledgments We thank

Marvin

D. Siperstein

for his continued

interest

in

our work, and P. Herranz for excellent editorial assistance. References 1.

2. 3. 4. 5. 6. 7. 8.

9.

10. 11. 12. 13. 14.

15.

16.

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Stimulation of RNA synthesis in L-929 cells by rabbit tumor necrosis factor.

Synergistic stimulation of amnion cell prostaglandin E2 synthesis by interleukin-1, tumor necrosis factor and products from activated human granulocytes.

Cytokines stimulate lipolysis and decrease lipoprotein lipase activity in cultured fat cells by a prostaglandin independent mechanism.

Interleukin 1- and tumor necrosis factor-stimulation of prostaglandin E2 synthesis in MDCK cells, and potentiation of this effect by cycloheximide.

Transcription of the tumor necrosis factor alpha gene is rapidly induced by anti-immunoglobulin and blocked by cyclosporin A and FK506 in human B cells.

Inhibition by perilla juice of tumor necrosis factor production.

Tumor necrosis factor, tumor necrosis factor inhibition, and cancer risk.

Serotonin inhibition of tumor necrosis factor-alpha synthesis by human monocytes.

Inhibition of HIV-1 productive infection in hepatoblastoma HepG2 cells by recombinant tumor necrosis factor-alpha.

Insulin action in isolated fat cells. II. Effects of divalent cations on stimulation by insulin of protein synthesis, on inhibition of lipolysis by insulin, and on the binding of 125I-labelled insulin to isolated fat cells.

Inhibition of tumor necrosis factor-α enhances apoptosis induced by nuclear factor-κB inhibition in leukemia cells.

Inhibition of prostaglandin synthesis by lysolecithin.

[Modulation of hCG synthesis by alpha tumor necrosis factor in JAR choriocarcinoma cells].

Inhibition of human colony-forming-unit erythroid by tumor necrosis factor requires accessory cells.

Inhibition in fat-storing cell multiplication by a factor produced by normal cultured murine hepatocytes.

Induction of interleukin-2 receptor by tumor necrosis factor alpha on cultured ovarian tumor-associated lymphocytes.

Synthesis of factor VIII antigen by cultured human endothelial cells.

Prostaglandin synthesis by rheumatoid synovium and its stimulation by colchicine.

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α.

Oncolytic Herpes Simplex Virus Glioblastoma Therapy is Potentiated by Tumor Necrosis Factor-α Inhibition.

Prostate tumor-induced angiogenesis is blocked by exosomes derived from menstrual stem cells through the inhibition of reactive oxygen species.

Prostaglandin production by rheumatoid synovial cells: stimulation by a factor from human mononuclear Cells.

Inhibition of microRNA-25 by tumor necrosis factor α is critical in the modulation of vascular smooth muscle cell proliferation.

The current use of interferons, interleukin-2 and tumor necrosis factor in renal cell cancer.