Tumor Necrosis

Factor-a, Interleukin-1, Triglyceride Synthesis

and Interferon Alpha Stimulate in HepG2 Cells

Carl Grunfeld, Charles A. Dinarello, and Kenneth R. Feingold Tumor necrosis factor-a (TNF-a), interleukin-1 (IL-l), and interferon alpha (IFN-a) stimulate hepatic lipogenesis in vivo. We now show that TNF-a, IL-l, and IFN-a stimulate lipogenesis as measured by the incorporation of 3H-glycerol into triglyceride in cultured HepGZ cells. Incubation of HepGZ cells for approximately 24 hours with TNF or IL-I was required to see stimulation of lipogenesis, with this effect increasing over the next four days. TNF stimulated lipogenesis by 2.4.fold after 72 hours of incubation, while a 3.5-fold stimulation was seen with IL-l. The half maximal concentration for TNF stimulation of hepatic lipogenesis was 4 ng/mL, while that for IL-I was 0.3 ng/mL. Cells treated with TNF or IL-1 also showed increased secretion of labeled triglyceride into the media. IFN-a stimulated the incorporation of ‘H-glycerol into triglyceride by 39% after 72 hour’s incubation. In contrast, IFN? had no effect on lipogenesis in HepG2 cells. These data suggest that cytokines can directly stimulate the synthesis of triglycerides in cultured Hep G2 liver cells in vitro. Copyright 0 1991 by W.B. Saunders Compeny

T

HE RESPONSE to infectious agents is mediated by cytokines, peptide hormone signals produced by lymphocytes, macrophages, and endothelial cells.‘-3 Infection is frequently accompanied by profound disturbances in metabolism, including hypertriglyceridemia,4 which is thought to be mediated by cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-l), and the interferons (IFN).‘.* During infection, both increased hepatic triglyceride synthesis’ and decreased storage of triglyceride in fat cells”’ can be seen. Recent data from our laboratory and others indicate that the dominant mechanism by which TNF increases serum triglycerides in vivo is by stimulation of hepatic lipogenesis, rather than inhibition of adipose tissue triglyceride storage. 7x’1-‘4First, the time course of the increase in serum triglycerides parallels the increase in de novo hepatic lipogenesis.’ Second, the increase in serum triglycerides precedes the decrease in lipoprotein lipase (LPL) activity in the epididymal fat pad; in addition, little or no decrease is seen in LPL activity from other adipose sites tissue or from muscle, while an increase in lipase activity is seen in postheparin p1asma.l” Third, TNF effectively increases serum triglyceride levels in streptozotocin-diabetic rats, despite the fact that adipose tissue LPL activity is markedly reduced secondary to the diabetes.14 More important, no further decrease in LPL activity is seen after TNF treatment, yet TNF still stimulates hepatic lipogenesis in diabetic animals.lg Fourth, TNF treatment does not decrease the clearance of triglyceride-rich lipoproteins from the circulation.“.” Fifth, the increase in hepatic lipogenesis is From the Department of Medicine, University of California, San Francisco, CA: the Metabolism Section, Medical Service, Department of Veterans Affairs Medical Center, San Francisco, CA; and the Department of Medicine, Tufts UniversitySchool of Medicine and New England Medical Center, Boston, MA. Supported by grants from the National Institutes of Health (No. DK40990 and AI15614) and the Veterans Administration. Dr Grunfeld is the recipient of a Clinical Investigator Awardfrom the VA. Address reprint requests to Carl Grunfeld, MD, PhD, Metabolism Section (11 IF), VA Medical Center, 4150 Clement St, San Francisco, CA 94121. Copyright 0 1991 by W.B. Saunders Company 0272-6386191/4009-0003$03.00l0

894

parallelled by an increase in the hepatic very-low-density lipoprotein VLDL production rate as measured by the Triton WR-1339 technique.“.” Finally, total hepatic lipogenesis can be assayed in vivo in TNF-treated animals by measuring the incorporation of “H-glycerol into triglyceride; using this technique, we have demonstrated that TNF can increase the synthesis of triglyceride in the liver and that labeled triglyceride appears at increased levels in the circulation.” In standard chow-fed rats, total lipogenesis most accurately reflects the contribution of the liver to serum triglyceride levels.” In mice, a single injection of TNF, IL-l, or IFN-(r produces a sustained increase in de novo hepatic lipogenesis.” In contrast, IL-2, IFN-B and IFN-7 have no effect on hepatic lipogenesis. TNF, IL-l, and the interferons have been shown to inhibit lipid storage when added to cultured mouse adipose cells.6~‘x~20 TNF and IL-1 induce some proteins of the acute-phase response in HepG2 cells and other cultured hepatoma lines.“-24 However, these cytokines have not been studied with regard to whether they have direct effects on lipogenesis in cultured liver cells. Therefore, we have now examined the effect of these cytokines on hepatic lipogensis in HepG2 cells to determine whether they can directly stimulate lipogenesis in an in vitro cell culture model.

MATERIALS

AND METHODS

Materials

[2-ZH]-glycerol(200 mCi/mmol/L) and carboxyl [“Cl-triolein (80 to 100 mCi/mmol/L) were purchased from New England Nuclear (Boston, MA). Ready-safe scintillation fluid was purchased from Beckman (Irvine. CA). Thin-layer chromatography (TLC) polygram sil G plates were purchased from Brinkmann Instruments (Westbury, NY). Human TNF-o with a specific activity of 5 X 10’ U/mg and human IFN-y with a specific activity of 2.5 x lo-’ Uimg were kindly provided by Genentech (South San Francisco, CA). Recombinant human IL-lf3 (112-269), with a specific activity of 5 X lo7 U/mg, was produced as described previously.” Recombinant human IFN-a AD. with the specific activity of 7.9 x 10’ U/mg, was generously provided by Drs M. Brunda and P. Sorter of Hoffmann LaRoche (Nutley. NJ). Fetal calf serum was purchased from Hyclone [Logan, UT). Oleic acid was purchased from Sigma (St Louis, MO). Bovine serum albumin (BSA) low in fatty acid content (CRG-7) was purchased from Armour Pharmaceutical (Kankakee. Metabolism, Vol40, No 9 (September), 1991: pp 894-898

CYTOKINE-STIMULATED

LIPOGENESIS IN HEPGZ CELLS

IL). Eagle’s minimal essential media (MEM) was purchased from the University of California Cell Culture Facility (San Francisco, CA). All other chemicals were of reagent grade.

a95

l.O-

0.8 --

l pl ,002 ** p( ,001

1.5

Cell Culture The human hepatoma cell line HepG2 was generously provided by Dr Barbara Knowles of the Wistar Institute (Philadelphia, PA). Cells were grown in MEM containing 10% fetal bovine serum and split at a ratio of 1:3 weekly. Measurement

0.6-.

0.5

of Lipogenesis

The effects of cytokines on lipogenesis in HepG2 cells were studied after the cells had reached confluence ( - 2 x 10hcells per 35-mm well). For studies in which HepG2 cells were preincubated with cytokine, the growth media was replaced with MEM containing 3% BSA (MEM-B)2”,27and incubated with cytokine or in parallel with control media for the total time indicated in the text and figure legends. The incorporation of ‘H-glycerol into lipid was measured by the same method regardless of whether cells were preincubated with cytokine or treated acutely. The media was changed to MEM containing 3% BSA, 1 mmol/L oleic acid, and 2.5 FCi of ‘Hglycerol per mL (MEM-BGO). OIeate was included during incorporation to optimize the synthesis of triacylglycerol.“0-29Oleate was bound to albumin as previously described.” There was no further effect on triglyceride synthesis or cytokine stimulation when oleate was included in the preincubation media. The incorporation of ‘H-glycerol into cellular triglyceride was linear for greater than 6 hours using this technique. In general, cells were incubated under these conditions for 6 hours, then glycerol incorporation was stopped by removing media and rinsing cells three times with phosphate-buffered saline containing .Ol% CaCl, and .Ol% MgCI, at 4°C. The labeled cells were rapidly solubilized in 1 mL of O.lN NaOH at room temperature. An internal standard of [carboxy-“C] triolein was added to both the media and the solubilized cells. Aliquots of the cell digest were taken for protein assay. The media and an aliquot of the cell digest underwent lipid extraction by the Folch technique.“’ Radioactive incorporation was determined by liquid scintillation counting in Ready-safe scintillation fluid correcting for recovery using the internal standard. In studies performed in parallel. the lipids in the Folch extraction were fractionated by TLC using petroleum ether:diethyl ether:glacial acetic acid, :80: 20:1, to determine the extent of incorporation of ‘H-glycerol into triglycerides and phospholipids. Under the conditions of these incubations, the cytokines tested had no effect on DNA content of cells. Basal incorporation varied from cell batch to cell batch, so parallel control incubations were performed for each experiment. The data presented represent a typical example of experiments performed at least twice. Statistics Values presented are mean + SEM. The Student’s t test was used to compare significance versus controls. RESULTS

Effect of TNF-(Y on Lipogenesti No significant effect of TNF on incorporation of ‘Hglycerol into lipid was seen after 6 hours of incubation, but by 24 hours of incubation, a trend toward increased lipogensis was seen (Fig 1). Although this increase was not significant in this experiment, the increase in lipogenesis at 24 hours did reach statistical significance in other experi-

1.0

o-0 0.0

a

TNF

24

0-v 48

IL-1

: 72

0.0

96

HOWS Fig 1. Time course of TNF and IL-1 stimulation of ‘H-glycerol incorporation into lipid. HepGZ cells were grown as described under Methods and allowed to come to confluence. Media was changed to MEM-B for 96 hours for all cells. TNF was added at a concentration of 100 ng/mL (0, left y-axis), while IL-1 was added at 10 ng/mL (0, right y-axis). Total exposure to cytokines equals the time indicated on the x-axis. Each time point represents the mean f SE of three plates. For the last 6 hours, media was replaced with MEWEGO and with TNF or IL-1 where indicated. The cells were rinsed and W-glycerol incorporation to lipid measured.

ments. In three similar experiments of 24-hour exposure, TNF stimulation averaged 1.33 t .07-fold over control (P < .02 in a paired t test for TNF ~2control). Longer incubation with TNF results in a progressive increase in the ability of TNF to stimulate incorporation of ‘H-glycerol into lipid in HepG2 cells (Fig 1). After 72 hours of preincubation, TNF-treated cells secreted significantly more labeled lipid into the media compared with control cells (control. 1.70 2 .43; 100 ng/mL TNF, 7.93 k 1.37 pmol 3H-glycerol incorporated/mgl protein/h, P < .Ol). In contrast, during brief incubations (6 hours) with TNF, there was no increase in the secretion of newly synthesized lipid (control, 3.77 k 1.5; 100 ng/mL TNF, 4.17 2 1.47, NS). The absolute amount of labeled lipid secreted by control and TNF-treated HepG2 cells is small compared with the amount of newly synthesized lipid in the cell. (Note that secreted lipid is reported in picomoles, while cellular lipid is reported in nanomoles incorporated.) Other laboratories have also found that cultured hepatocytes and HepG2 cells secrete only a small fraction of newly synthesized lipid.2b”X Further experiments were performed using a 72-hour preincubation with TNF-(w. The dose-response curve for TNF stimulation of ‘H-glycerol incorporation into lipid is shown in Fig 2. The maximal effect of TNF occurs at 1,000 ng/mL. In multiple similar experiments, incorporation of ‘H-glycerol into lipid was increased 2.4 k .O&fold by TNF. The half-maximal concentration for TNF stimulation is 4 ng/mL. Under these conditions, 85% of the ‘H-glycerol incorporated into lipid was in the triglyceride fraction in both control and TNF-treated cells, with the remainder in phospholipids. TNF maximally stimulates secretion of newly synthesized lipid into the media at a concentration of approximately 100 ng/mL (Fig 2). Half-maximal stimulation is seen at 3.3

896

GRUNFELD, DINARELLO, AND FEINGOLD

,,6__

l

p

(

.ool /

0.5 --

i-;

l

0.4--

TNF (ng/ml) Fig 2. Dose-response curve for TNF stimulation of cellular lipogenesis and lipid secretion. Cells were allowed to grow to confluence as described in Fig 1 and changed to MEM-B in the presence or absence of TNF at the concentration indicated on the x-axis. Total exposure to TNF was 72 hours. During the last 6 hours, media was changed to MEM-BGO in the presence or absence of TNF at the indicated concentrations. “H-glycerol incorporation into cellular lipid (0, left y-axis) or secreted lipid (0, right y-axis) was measured as described under Methods. Values are the mean k SE of five plates.

ng/mL. Under these conditions, 70% of the ‘H-glycerol in the secreted lipid is in the phospholipid fraction, while the remainder was in triglyceride. Other laboratories have also found that cultured hepatocytes and hepatoma cells secrete lipoprotein particles that are relatively triglyceride-poor and phospholipid-rich.‘6’?X Effect of IL-1 on Lipogenesis The time course for IL-l stimulation of incorporation of ‘H-glycerol into lipid in HepG2 cells is identical to that seen with TNF (Fig 1). No significant effect of IL-1 on lipogenesis is seen after 6 hours of incubation, but a trend toward increased incorporation is seen after 24 hours of exposure. Longer incubations with IL-l produce a progressive increase in the ability of IL-1 to stimulate ‘H-glycerol incorporation into lipids. IL-l also stimulated the secretion by HepG2 cells of labeled lipid. After 6 hours of IL-l treatment, there was a trend toward increased secretion (control, 2.6 2 .82; 10 ng/mL IL-l, 4.5 2 .39 pmol incorporated/mg protein/h), but this did not quite reach statistical significance. IL-l did produce a statistically significant increase in the secretion of labeled lipid after a 72-hour preincubation (control, .55 2 .89; 10 ng/mL IL-l, 5.4 2 .96 pmol incorporated/mg protein/h, P < .Ol). In several similar experiments using 72 hours of preincubation, IL-1 (10 ng/mL) stimulated incorporation of “Hglycerol into lipid by 3.5 + .3-fold. Maximal effect is seen at 1 ng/mL (Fig 3). The half-maximal concentration for IL-1 stimulation of lipogenesis is 0.3 ng/mL (Fig 3).

0.0 ‘t 0

.Ol

.1 IL-l

10

1

(ng/ml)

Fig 3. Dose-response curve for IL-I stimulation of glycerol incorporation into lipid. Experimental design is identical to that in Fig 2, except that IL-1 was added at the concentrations indicated on the x-axis. Values are mean 2 SE of three plates.

1.39 k .Ol-fold over control. Concentrations up to 1,000 ngimL were tested and showed only slightly higher incorporation (1.55-fold). As seen with TNF, IFN-a was not able to increase secretion of labeled lipid after short-term incubations with IFN-cx (Table 1). However, with longer incubations, when IFN-(w had increased cellular lipogenesis, an increase was also seen in the secretion of labeled lipid into the media (Table 1). In contrast, there was no effect of IFN-?/ at concentrations up to 1,000 ng/mL on lipogenesis or secretion of labeled lipid when HepG2 cells were treated by either exposure for 6 hours or 72 hours with IFN-7 (Table 2). DISCUSSION

This report demonstrates that TNF-(w, IL-l, and IFN-a directly stimulate lipogenesis in HepG2 cells, a cultured hepatoma cell line. While earlier studies demonstrated that Table 1. IFN-a Stimulates Lipogenesis in HepG2 Cells CellularLipogenasis (nmollmg

protein/h)

Labeled Lipid Secreted (pmollmg

protein/h)

Acute Exposure (6 h) Control IFN-a (100 ng/mL) 72-hour incubation

.425 + ,041

5.9 t 0.7

,416 + ,013

3.7 2 1.0

Control

,368 + ,019

2.9 k .33

IFN-a (100 ng/mL)

,577 2 .023*

6.0 f .43**

NOTE. For the acute exposure, cells were incubated in MEM-BGO for 6 hours in the presence or absence of IFN-a. Forthe 72-hour incubation, cells were incubated in the presence or absence of IFN-a (100 kg/ml) for a total of 72 hours, with a media change after 48 hours; the initial media was MEM-B. This media was removed and replaced with MEM-BGO, in

Effect of IFNs on Lipogenesis Exposure of HepG2 cells to IFN-(U over the course of 6 hours produced no increase in the incorporation of 3Hglycerol into lipid (Table 1). However, after a 72-hour preincubation, IFN-a was able to stimulate the incorporation of ‘H-glycerol into lipid. In multiple experiments, IFN-a at 100 ng/mL showed an average incorporation of

the presence or absence of IFN-ufor the last 6 hours. At the end of the incorporation

period, the media was removed

and the cells were

rinsed. Lipid was extracted from media and cells, then prepared for counting as described under Methods. Values are the mean -t SE for three plates for acute exposure, preincubation. ‘P i

,005.

tP < ,001.

and five plates for the 72-hour

CYTOKINE-STIMULATED

897

LIPOGENESIS IN HEPGL CELLS

Table 2. IFN-y Does Not Stimulate Lipogenesir in HepG2 Cells Cellular Lipogenesis

(nmollmgprotein/h)

Labeled Lipid Secreted

(pmollmgprotein/h)

Acute ~exposure Con1rol

,271 2 .069

3.2 + .6

IFN-y (1,000 ng/mL)

.265 2 ,040

ND

Control

,285 + ,018

2.3 zz 1.4

IFN-y (1,000 ng/mL)

,226 -t ,016

3.1 * 1.1

72-hour incubation

NOTE, Experimental conditions are the same as Table 1, except that IFN-y was used at concentration of 1,000 ng/mL. Values are the mean 2 SEM for three plates. Abbreviation: ND, none detectable.

stimulate hepatic lipogenesis in viva,‘,” there have been no previous data on the effect of these cytokines on lipid synthesis in cultured liver cells. Both TNF-a and IL-1 have been previously shown to mimic some, but not all, of the components of the acute-phase response using cultured hepatoma cell lines.“-23 The use of hepatoma cell lines, including HepG2 cells, have limitations in that, in most laboratories, these cells do not secrete enough lipoprotein to measure lipoprotein mass in most experiments and the sec:reted lipid is triglyceride-poor, properties that are also shared to some extent by primary hepatocyte cultures.‘6~‘XNonetheless, HepG2 cells have been used extensively as a model for studies of hepatic lipid metabolism.” Therefore, many studies of cultured hepatocytes and hepatoma cells use incorporation of label to measure lipogenesis and lipoprotein production, rather than measuring absolute mass.“.‘” Those studies and ours could be confounded by changes in intracellular carbohydrate metabolism, but this is unlikely, as it has been difficult to show any other effects of TNF or IL-l on hepatocyte metabolism in vitro in the absence of counter regulatory hormones such as glucagon.‘2-‘5 There are several interesting similarities between the properties of these cytokines in vivo and in vitro. TNF-a, IL-l, and IFN-a have all been shown to stimulate de novo hepatic fatty acid synthesis in intact mice.* In contrast, IFN-y has no effect on hepatic fatty acid synthesis in viva.* In this report, we show identical specificity for stimulation of total lipogenesis in vitro as measured by the incorporation of ‘H-glycerol into lipid in HepG2 cells: TNF-a, IL-l, and IFN-cx were active, whereas IFN-1/ was not. The effect of TNF on total lipogenesis has been studied in vivo by measuring the incorporation of ‘H-glycerol into lipid; using this technique, an increase was seen both in hepatic lipogenesis and in the appearance of labeled lipid in the circulation.1° We also report here that TNF, IL-l, and these cytokines

IFN-a produce an increase in the secretion of labeled lipid into the media by treated HepG2 cells, as well as increasing cellular lipid synthesis. Another parallel between the effects of these cytokines on HepG2 cells and that found in vivo is the relative sensitivity to TNF-a and IL-l. The half-maximal concentration for stimulating de novo lipogenesis in vivo in mice is 10 rig/g for TNF-a and 1 ngig for IL-l. In the present study, we find that the half-maximal concentration for stimulation of total lipogenesis in HepG2 cells is 4 ng/mL for TNF and 0.3 ng/mL for IL-l. a similar ratio with the response to IL-l again being lo-fold more sensitive than to TNF. It is also of interest that there is also a good correlation between the half-maximal concentration for TNF action (4 ng/mL) and the Kd’s for the binding of TNF to its receptor (.16-Sl nmol/L or 2.7 to 8.7 ng/mL) that have been reported by various laboratories.h.2b-‘R One difference between our in vitro studies with HepG2 cells and data from intact animals is the time course in which these cytokines stimulate lipogenesis. All three cytokines are capable of increasing lipogenesis in vivo within 30 minutes of injection,” whereas hours of incubation are required in vitro. This difference is similar to that found with two other metabolic effects of cytokines; serum zinc levels and hepatic amino acid transport are rapidly modulated when TNF is given to rats in vivo, but TNF has only been reported to increase zinc and amino acid transport after 16 to 20 hours of exposure in hepatocytes in vitro and requires treatment with glucagon.‘4.‘5 Whether these differences result from the metabolic and hormonal conditions under which tissue culture cells are grown or maintained is not yet known. Differences in diets and metabolic conditions in vivo are known to change the mechanism by which TNF stimulates total lipogenesis.” In addition, classic regulators of hepatic DNA replication also require longer times to be effective using in vitro systems.” The delayed effects of cytokines on lipid metabolism may mean that HepG2 cells provide a better model for the late effects in vivo.40 In summary, we have shown that three cytokines that affect hepatic lipid synthesis in vivo-TNF, IL-l, and IFN-a-also stimulate lipogenesis in cultured HepG2 cells in vitro. The specificity and the sensitivity of these cytokines parallels that seen in vivo. These data suggest that cytokines can directly stimulate lipid metabolism in HepG2 cells, an in vitro model for hepatic action, without requiring the intermediate action of other cells or organs. ACKNOWLEDGMENT The authors wish to thank M. Donahue for technical assistance, M. Pang for graphics, and P. Herranz for editorial assistance.

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