Interleukin-1β and interleukin-6 stimulate 2-methylaminoisobutyric acid uptake in HepG2 cells

Inr. .I. Biorhm.

Pergamon

Cell Bid Vol. 29, No. 4. pp. 661-674, 1997 (‘I 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain

PII: Sl357-2725(97)00002-2

1357-2725197

$17.00 + 0.00

Interleukin+ and Interleukin-6 Stimulate 24VIethylaminoisobutyric Acid Uptake in HepG2 Cells SYLVIE GOENNER,‘” CLAUDINE COSSON,’ ALAIN LEGRAND,’ NICOLE MOATTI’

AUDREY

BOUTRON,’

‘Laboratoire de Biochimie I, H6pital Biktre, 94275, Le Kremlin-Bicgtre, France and ‘FacultP des Sciences Pharmaceutiques et Biologiques de I’Universitt Paris-&d, 92290, Chatenay-Malabry, France The metabolic response to inflammation involves an increased uptake of amino acids in the liver. It has been suggested that cytokines, such as interleukin-I/? and interleukin-6, could be involved in this increased amino acid uptake. We investigated the role of these two inflammatory cytokines in regulating hepatic amino acid transport systems in the human hepatoma cell line, HepG2. Uptake of methylaminoisobutyric acid, the most specific known substrate of system A, and of glutamine, both transported by other sodiumdependent transport systems ASC and N, was assayed after incubation of the cells for various times with cytokines, using the cluster-tray method. Interleukin-lj and interleukin-6 (1000 U/ml) stimulated methylaminoisobutyric acid uptake by 36 & 6 and 41 f 4%, respectively (per cent &- SD, n 2 6). Under our experimental conditions, these cytokines had no effect on glutamine uptake. The stimulatory effect on methylaminoisobutyric acid uptake was not increased by combining the cytokines or by the presence of dexamethasone. The cytokine effect was abolished by cycloheximide, suggesting the involvement of de ~OUO protein synthesis in this activation of transport system A. These data demonstrate that, in our culture conditions, interleukin-lb and interleukin-6 indirectly exert a stimulatory effect on methylaminoisobutyric acid transport in HepG2 cells. 0 1997 Elsevier Science Ltd Keywords: Amino HepG2 cells Int. J. Biochem.

acid

transport

Glutamine

Interleukin-l/l

Interleukin-6

Cell Biol. (1997) 29, 667-674

INTRODUCTION

hepatocytes. The increased hepatic amino acid uptake is required for the synthesis of acute phase proteins and for accelerated gluconeogenesis (Wilmore et al., 1980), and involves alterations in hepatocyte plasma membrane transporter activities (Pacitti et al., 1993). The human hepatoma cell line HepG2 synthesizes and secretes major human plasma proteins, in particular a 1-acid glycoprotein (AGP) (Knowles et al., 1980). Baumann et al. (1987) reported that the regulation of acute phase protein synthesis by HepG2 cells is mediated by hepatocyte-stimulating factors and interleukin-1 (IL-l). Depraetere et al. (1991) reported that IL-6 with or without dexamethasone stimulates the production and secretion of

The liver is the major site of amino acid uptake in the body during inflammation (Augsten et al., 1991). Bereta et al. (1990) were the first to report that interleukin-6 (IL-6), the major inflammatory mediator, induced amino acid uptake in rat

*To

MeAIB

whom

all correspondence should be addressed. AGP, G(I -acid glycoprotein; AIB, s(aminoisobutyric acid; CHX, cycloheximide; Dex, dexamethasone; GLN, glutamine; IL, interleukin; IL-lfi, interleukin-l/j’; IL-6, interleukin-6; MeAIB, methylaminoisobutyric acid; PBS, phosphate-buffered saline solution; TNF, tumour necrosis factor. Received 6 June 1996; accepted 18 December 1996.

Abbrer*intions:

667

668

Sylvie

Goenncr

C-reactive protein by this cell line. Amino acids needed for this synthesis may come from intracellular protein turnover as well as cellular uptake. Previously, we reported that the human hepatoma cell line HepG2 possesses major neutral amino acid transport systems (A, ASC, L) and the specific hepatic system N (Goenner ~?rtrl., 1992). Glutamine transport system N is similar in normal human liver and human HepG2 cells, but only partially mediates glutamine uptake (Bode et al., 1995). Our aim in this study was to investigate whether two inflammatory cytokines regulate hepatic amino acid transport systems in the human hepatoma cell line HepG2. We measured the effects of IL-lp and IL-6 on transport system A by MeAIB uptake and on transport systems ASC and N by glutamine uptake. MATERIALS

AND

METHODS

The radiolabelled amino acids used were a-[ 1-‘“Cl-methylaminoisobutyric acid (NEN Research Products, France) and L-[U-14C]glutamine (Dositek, France). Unlabelled amino acids, cycloheximide and dexamethasone were obtained from Sigma. Recombinant human IL-6, produced by Chinese hamster ovary cells and recombinant (SP. act.. 4 x lOh Ujmg) human IL-lfl, produced in E. coli (sp. act., 2 x 10’ Uimg), were purchased from Genzyme Co. (Cambridge, MA. U.S.A.). Cell c.ulture.s Human hepatoma cells (HepG2) were grown as monolayers in RPMI-1640 medium (Gibco), 11.1 mM D-glucose, suppH 7.40, with plemented with L-glutamine (2 mM), antibiotics (penicillin: 200 U/ml; streptomycin: 200 pg/ml) and 10% (v/v) foetal calf serum (Flow Laboratories) at 37°C under a humidified atmosphere of 5% CO, and 95% air. Stock cultures of HepG2 cells were maintained in 75-cm’ flasks (Falcon Plastics). Culture medium was changed every 2 days. For transport assays, confluent cells were detached from the flasks with 0.05% (p/v) trypsin plus 0.02% (p/v) EDTA in NaCl solution (Flow Laboratories). Transport assure Cells (1.2 x 10’) were seeded into the 2-cm’ wells of Costar 24-well cluster trays (Costar No. 3524) and allowed to grow for 4 days in 1 ml of

et trl.

culture medium, which was replaced 24 hr before experiments. The technique for amino acid transport measurements using the cluster trays was adapted from that described by Gazzola et al. (1981). Transport measurements were made in either sodium-containing or sodium-free phosphate-buffered saline solution (Dulbecco’s) supplemented with 6mM D-glucose (PBS-G). The sodium-free medium was prepared by replacing the sodium chloride and sodium hydrogen phosphate buffer with choline chloride and potassium hydrogen phosphate. respectively. To minimize /runs effects, cells in culture were washed twice with sodium or choline medium and depleted in 1 ml of the same buffer for 1 hr at 37‘C. Transport was initiated by adding simultaneously to the 24 wells. 0.3 ml of PBS-G containing 0.3 ,nCi of labelled glutamine or 0.5 FCi of labelled MeAIB plus any other test compounds. Uptake was measured for 1 min at 37°C in a shaking water bath and stopped by pouring off the medium and immediately (< 15 set) washing the wells three times with 2 ml of ice-cold 154 mM sodium or choline chloride. The cluster trays were then drained and the monolayers were solubilized by addition of 0.5 ml of 1 M NaOH and incubation with gentle shaking for at least 2 hours at room temperature. From each well, 400 ~1 of the extract was counted for radioactivity in 10 ml of liquid scintillation counter (Picofluor) and 50 1-11was used for protein determination by the method of Lowry et 01. (1951). Uptake of amino acid at an extracellular concentration of 0.1 mM by HepG2 cells was assayed after various incubation times with 100 or 1000 Ui’ml of IL-lb or IL-6 added to the culture medium, and with or without IO ’ M dexamethasone for 24 hr. To appreciate the dependence of amino acid transport cytokine stimulation on protein synthesis, HepG2 cells were incubated for I hr with culture medium containing cycloheximide (100 ,uM) prior to the addition of IL-l/I or IL-6. After another 24 hr of incubation, MeAIB and glutamine uptake were determined as described above. Amino acid transport values, expressed as nmol per mg of protein per min, are the mean + SD of at least six determinations in two The non-parametric separate experiments. Mann-Whitney U-test was used for statistical analysis of MeAIB and glutamine uptakes. The

IL-l/l

p-values inferior significant. Measurement

and

IL-6

stimulate

MeAIB

to 0.01 were considered to be

qf CI1-acid

glycoprotein

secretion

Before glutamine or MeAIB transport measurements, supernatant from each well was collected to examine AGP secretion. The AGP synthesized and secreted by HepG2 cells into the culture medium was quantified by immunoenzymatic assay (ELISA). Coating antibodies (anti-human AGP serum) and conjugated antibodies (anti-human AGP antibodies coupled to peroxidase) were obtained by immunization of rabbits with purified human AGP and were a generous gift from Dr C. Pous (Chatenay-Malabry, France). Purified preparations of plasma protein were used as standards and results are reported in pg of AGP secreted/mg protein/24 hr. RESULTS

To investigate cytokine effects on hepatic amino acid transport systems, HepG2 cells were exposed to two concentrations of IL-lfl or IL-6 (100 or 1000 U/ml) for various times, and the uptake of 0.1 mM MeAIB and 0.1 mM glutamine was then determined immediately (direct effect) or later (mediated effect) (Fig. 1). Figure l(a) and (d) report MeAIB and glutamine uptake by HepG2 cells immediately after an incubation of 1 hr with IL-l/I or IL-6. We measured also the amino acid uptake after contact times of 2, 5 and 10 min with cytokines (data not shown). Neither IL-lb nor IL-6, at 100 or 1000 U/ml, after the short incubation time tested, directly stimulated amino acid transport systems involved in MeAIB and glutamine uptake. Effects of IL-I/? and IL-6 were examined 24 hr after the start of the incubation with cytokines: either 24 hr incubation and uptake measured immediately after [Fig. 1(b) and (e)] or 1 hr incubation and uptake measured 24 hr later [Fig. l(c) and (f)]. After 24 hr of incubation with IL-l/J or IL-6, MeAIB uptake significantly increased (p < 0.005), whereas glutamine uptake was not modified whatever the incubation time conditions. Incubation for 1 hr with IL-l/I or IL-6 was sufficient to stimulate MeAIB uptake by HepG2 cells, but this effect was evident only 24 hr later. Thus, IL-l/? and IL-6 presumably act indirectly on MeAIB uptake. Uptake of MeAIB and glutamine were determined in control and cytokine-treated

uptake

in HepG2

cells

669

cells, after a contact of 24 hr with various concentrations of IL-lfl or IL-6 from 0.1 to 5000 U/ml (Fig. 2). The effect of the cytokines on MeAIB uptake was dose-dependent up to a concentration of 1000 U/ml for IL-lb, and at least up to 5000 U/ml for IL-6. Under the same conditions of cytokine exposure, no dose dependency of glutamine uptake by HepG2 cells was observed. Total MeAIB and glutamine influx observed immediately after a contact of 24 hr with IL-l/I or IL-6 are reported in Table 1. The MeAIB uptake occurred exclusively via Na+-dependent transport system A, as reported previously (Goenner et al., 1992). Thus, IL-l/I and IL-6 clearly stimulate transport system A activity. For glutamine uptake, it was necessary to distinguish the relative contribution of each amino acid transport system involved. Glutamine uptake was essentially Na+-dependent and mediated by transport system AX mainly and also by specific hepatic transport system N. Thus, we measured glutamine uptake in the absence of sodium, and in the absence of sodium and in the presence of lithium (Table 1) to distinguish between the Na+-independent component system L, the Na’-dependent component system ASC and the Na+-dependent, Li+-tolerant component system N. The cytokines tested had no effect on glutamine uptake, whatever amino acid transport system was involved. To assess the biological activity of the batches of IL-l/3 and IL-6 used in our experiments, we measured the secretion of AGP by HepG2 cells after 24 hr of incubation in the presence or absence of cytokines at 1000 U/ml (Table 2). The IL-I/3 and IL-6 significantly stimulated AGP secretion by these cells: stimulation of AGP secretion observed was similar to the stimulation of MeAIB uptake measured simultaneously. The effects of a combination of IL-l/3 and IL-6 and of dexamethasone addition were investigated (Table 2). The simultaneous presence of IL-lfl and IL-6 (both 1000 U/ml) in the culture medium of HepG2 cells for 24 hr stimulated MeAIB uptake, but only to a level similar to that in the presence of one cytokine. Cytokine combination had no effect on glutamine uptake. Dexamethasone, an effector of the inflammatory reaction, had no effect on amino acid uptake by control or by cytokinetreated HepG2 cells, or on AGP synthesis by these cells.

Sylvie

670

Goenner

The HepG2 cells were treated with IL-l/I or IL-6 at 1000 U/ml in the absence or in the presence of the protein synthesis inhibitor cycloheximide (100 PM) and amino acid uptake was measured after a contact of 24 hr as

MeAlB

(b)

described in the Materials and Methods section (Fig. 3). Treatment by cycloheximide alone induced a decrease of MeAIB uptake by HepG2 cells from 0.458 k 0.020 to 0.359 + 0.015 nmol/ mg of protein/min (100 to 78%). Cycloheximide (4

I HI--

(4

rf ul.

MeAlB

GLN

GLN

(e)

24 H I -

**

24 H I

I,

MeAlB

(cl s-

1 H/24H

I50 .-

** -

r

GL.N

(f)

I H/24H

150

MeAlB

GLN

**

0 MeAlB

4 Control IZILIB = 100 U/ml ILlB = 1000 U/ml

q

GLN

MeAlB

n q

Control IL6 = 100 U/ml 0 IL6 = 1000 U/ml

or IL-6 [(d)-(f)] on MeAIB and Fig. 1. Effects of various incubation conditions with IL-l/I [(a)-(c)] glutamine uptake by HepG2 cells. (a) and (d) I hr incubation with IL and uptake measured immediately after (1 H/-). (b) and (e) 24 hr incubation with IL and uptake measured immediately after (24 Hi-). (c) and (f) I hr incubation with IL, then culture medium was renewed without IL and uptake measured 24 hr later (1 H/24 H). Data represent means f SD of at least six determinations and are expressed as percentages of control uptake without interleukin (100% of uptake); *p < 0.01 or **p < 0.005 in comparison to control.

IL-lp

and

IL-6

stimulate

MeAIB

uptake

in HepG2

DISCUSSION

(a) 160-

0 MeAlB . GLN

150l40-

90

0

I 0.1

IL-I

I I

I IO

I

I

I

100

1000

5000

B concentration

(U/ml)

(b) 160

r

0

T

0.1

IL-6

I

100

IO

concentration

1000

5000

(U/ml)

Fig. 2. Effects of (a) IL-Ip or (b) IL-6 at concentrations from 0. I to 5000 U/ml on MeAIB and glutamine uptake HepG2 cells. The 1 min amino acid transport rates from least six replicate wells are expressed as percentages control uptake.

by at of

did not modify the influx of glutamine (4.716 f 0.310 to 4.891 + 0.279 nmol/mg protein/min). When cycloheximide-treated HepG2 cells were exposed to IL-lp or IL-6, MeAIB uptake decreased from 138 to 75% and from 135 to 76%, respectively, to the same level as that observed without cytokine. Thus, for MeAIB uptake, the cytokine effect was abolished completely by cycloheximide treatment.

Table

1. Cytokine

stimulation

of amino MeAIB

Control + IL-1B (100 U/ml) + IL-lp (1000 U/ml) Control + IL-6 (100 U/ml) + IL-6 (1000 U/ml)

0.523 0.663 0.713 0.468 0.561 0.658

+ rf: & rt f +

acid transport

systems

We report the effects of IL-lb and IL-6 on MeAIB and glutamine uptake by the human hepatoma cell line HepG2. The HepG2 cells accumulate MeAIB and glutamine via major Na+-dependent transport systems A and AX. The IL-lb and IL-6 significantly stimulated MeAIB uptake, but had no effect on glutamine uptake by HepG2 cells in our culture conditions. This increase of MeAIB uptake was due to the activation of transport system A and depended on the concentration of the cytokine involved. This is in agreement with Bereta et uf. (1989, 1990) who demonstrated that IL-6 enhanced AIB accumulation in rat hepatocytes in a dose-dependent manner. Recently, in isolated human hepatocytes, Fischer et al. (1996) reported a stimulatory effect of IL-6 on alanine uptake similar to that which we observed for MeAIB uptake by HepG2 cells, and also they reported no effect on glutamine transport in isolated human hepatocytes as in HepG2 cells. The study of the different transport systems responsible for glutamine uptake in HepG2 cells showed no modification by cytokines whatever the transport system involved: system ASC or specific hepatic system N. The absence of cytokine effects on glutamine uptake probably is linked to a minor participation of system N and/or to an absence of response of system ASC. Transport system A, like transport system N, is regulated by amino acid starvation and hormones (Kilberg et al., 1985; Gebhardt and Kleemann, 1987; Collarini and Oxender, 1987). For transport system ASC, a carrier described as unresponsive to hormonal regulation in several tissues, Souba et al. (1991) reported that tumour necrosis factor (TNF) and IL-l stimulate system ASC activity on glutamine transport, but in cultured pulmonary endothelial cells. In cultured human fibroblasts,

involved

uptake

Total System

After

671

cells

0.024 0.045 0.044 0.022 0.027 0.026

and glutamine

Glutamine

A

Na+-independent System L

Total (100) (127) (136) (100) (120) (141)

in MeAIB

4.604 4.949 5.038 4.288 4.587 4.754

k k k + + +

0.221 0.264 0.348 0.153 0.190 0.377

(100) (107) (109) (100) (107) (110)

0.471 0.429 0.515 0.439 0.402 0.468

uptake

by HepG2

cells

uptake System

Na+-dependent ASC System

4.133 4.520 4.523 3.849 4.185 4.286

N

0.626 0.645 0.625 0.583 0.525 0.553

24 hr of treatment with IL-ID or IL-6, uptake of MeAIB and glutamine (0.1 mM) by HepG2 cells was measured for I min at 37°C in the presence of sodium, lithium or choline. Data are means + SD of at least six determinations and are expressed in nmol/mg proteimmin. Results expressed as percentages of control values are given in parentheses.

672

Sylvie Table

2. Modulation

Treatment No addition (Control) IL-lp 1000 U/ml IL-6 1000 U/ml IL-6 + IL-1p Dex I pM Dex + IL-IB Dex + IL-6

of xl-acid

glycoprotein

Goenner

secretion

100 (1.744 144 128

100 * (0.438 Ifr 138 k 130+9 I35 * 98 + 121 + I.11 &

18 0.309) 30 10 22

0 No addition aILID= IOOUlml q IL6 = 1000 U/ml

n q q

4 0.016) 5 I 4 5 3

uptake

by HepG2

cells

0.1 mM Glutamine (% of uptake) loo&h (4.294 + 107 + 108 + 10415 100 * 99 + Y4 +

0.275) x 3 6 4 6

IL-6 and/or dexamethasone (Dex). Results of amino acid transport of control values, and are means f SD of at least six determinations. as prnol of AGP secretedjmg protein/24 hr and as nmol of amino

Dudrick et al. (1992) demonstrated TNF-mediated inhibition of glutamine transport by system AK. However, in rat hepatocytes, TNFa did not exert a direct effect on MeAIB transport (Lim et al., 1995). The effects of cytokines seem to differ according to cell culture models and culture conditions. Indeed, Roh et al. (1986) showed that in vivo administration of IL-l stimulated alanine uptake in freshly isolated rat hepatocytes, but in vitro IL-l treatment of hepatocytes did not have the stimulatory effect. Similar results were reported by Warren et al. (1987) for isolated rat hepatocytes after in L>izw or in vitro administration of TNF. We investigated the effects of simultaneous IL-lp and IL-6 treatment on glutamine and MeAIB uptake by HepG2 cells. We observed no additive, synergic or inhibitory effects, although many authors have reported modulation of the effects on HepG2 cells of IL-6 by other cytokines, but only as concerns acute phase protein synthesis (Baumann et al., 1987; Smith and McDonald, 1992).

MeAlB

and glutamine

0.1 mM MeAIB (% of uptake)

HepG2 cells were treated for 24 hr with IL-l/I, AGP secretions are expressed as percentages 100% control rates are given in parentheses transported/mg protein/min

3 140 .? % 120 k

and MeAIB

AGP secretion (% of secretion) * * * -t ND 101 * ND ND

- I60 r ** **

et al.

CHX IOOpM CHX+IL-Ip CHX + H-6

GLN

Fig. 3. Effect of 100 PM of cycloheximide (CHX) on MeAIB and glutamine uptake by HepG2 cells treated for 24 hr with 1000 U/ml of IL-lb or IL-6. Data are expressed as percentages of control uptake and are means + SD of at least six determinations; *p < 0.01 or **I, < 0.005 in comparison to control.

and The acid

Synergistic action between dexamethasone and cytokines have been observed on acute phase protein synthesis by rat and human liver cell cultures (Moshage et al., 1988; Depraetere et al., 1991; Smith and McDonald, 1992). We found that dexamethasone alone had no effect on amino acid uptake by HepG2 cells, although Lui et al. (1993) reported that glucocorticoid receptors were functional in this cell line. Watkins et al. (1994) studied in vice effects of IL-6 and dexamethasone on amino acid transport by hepatic plasma membrane vesicles isolated from normal rats: administration of IL-6 increased system A and system N activities, and this stimulatory effect was enhanced by dexamethasone. Recently, Fischer et al. (1996) showed that dexamethasone alone did not alter alanine or glutamine transport rates in isolated human hepatocytes, but pretreatment of the cells for 24 hr augmented the effects of both cytokines on carrier-mediated amino acid uptake. The absence of response of HepG2 cells probably is due to the short period of exposure to dexamethasone we used. The absence of an IL- 1/I and IL-6 direct effect on HepG2 cells suggests the involvement of a mediated effect. This is in agreement with the study of Pacitti et al. (1993) on hepatic plasma membrane vesicles prepared from normal rats. They reported that the incubation of this cell material with TNF, IL- 1 or IL-6 in vitro for 1 hr did not modify MeAIB and glutamine Na ‘dependent transport. To confirm a mediated effect, the ability of HepG2 cells to up-regulate MeAIB uptake was measured in the presence of cycloheximide. Cycloheximide alone decreased basal MeAIB uptake. In the presence of cycloheximide plus cytokines, the cytokinestimulated increase in MeAIB uptake was completely abolished, suggesting that de nom protein synthesis was required. Cycloheximide

IL-I/I

and IL-6

stimulate

MeAIB

alone and cycloheximide plus cytokines had no effect on glutamine uptake. The difference between MeAIB and glutamine uptake suggests that cycloheximide may prevent the synthesis of carrier protein with a short half-life. Cycloheximide decreased basal transport system A activity and this indicates a rapid turnover for system A carrier proteins in HepG2 cells, as previously reported for transport system A of rat isolated hepatocytes (Cheeseman, 199 1). This might not be the case for carrier proteins involved in glutamine uptake. We demonstrate that cytokines in citro regulate transport system A in HepG2 cells. The absence of response of transport systems involved in glutamine uptake as described in isolated human hepatocytes (Fischer et al., 1996) may be due to an unsuitable cytokine environment and/or to specific individual responses of amino acid transporters. The genes for several hepatic amino acid carriers have been isolated (e.g. system Y + and system AK) and probes developed from these genes will be useful for investigating the molecular regulation of HepG2 amino acid transport systems. AcknoMledgemc~nts~We

technical generous antibodies.

assistance gift of

are with AGP polyclonal

grateful to C. Pous for determinations and for the rabbit anti-human AGP

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Smith J. W. and McDonald T. L. (1992) Production of serum amyloid A and C-reactive protein by HepG2 cells stimulated with combinations of cytokines or monocyte conditioned media: the effects of prednisolone. C/in. E-Y/J. Immunol. 90, 293-299. Souba W. W., Salloum R. M., Bode B. P. and Herskowitr K. (1991) Cytokine modulation of glutamine transport by pulmonary artery endothelial cells. Surgery 110, 295-302. Warren R. S., Donner D. B., Fletcher Starnes H. and Brennan M. F. (1987) Modulation of endogenous

et N/.

hormone action by recombinant human tumor necrosis factor. Proc. Nail Acad. Sci. C’.S.A. 84, 8619-8622. Watkins K. T., Dudrick P. 55.. Copeland E. M. and Souba W. W. (1994) Interleukin-6 and dexamethasone work coordinately to augment hepatic amino acid transport. J. Trauma 36, 523-528. Wilmore D. W.. Goodwin C. W., Aulick L. H., Powanda M. C.. Mason A. D. and Pruitt B. A. (1980) Effect of injury and infection on visceral metabolism and circulation. Ann. Surg. 192, 491- 499.