Lipid and lipoprotein metabolism in Hep G2 cells

Biochimica Elsevier

et Biophysics

351

Acta, 961 (1988) 351-363

BBA 52886

Lipid and lipoprotein metabolism in Hep G2 cells S.R. Wang, M. Pessah, J. Infante, INSERM

U.55, HGpital Saint-Antoine,

(Revised

Key words:

D. Catala, C. Salvat and R. Infante Paris (France)

(Received 20 January 1988) manuscript received 2 May 1988)

Lipid; Apolipoprotein;

Insulin;

Glucose;

Fructose;

Mevalonate;

(Hep G2 cell)

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P < 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [ “C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mBNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 f 1.04 pg/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of ]3H]glycerol into the medium and cell triacylglycerols by 251 and 8!99%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [ 14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMGCoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.

Introduction The metabolism of lipids and lipoproteins is one of the prominent functions of the liver. The hepatocytes synthesize lipids in the form of tri-

Abbreviations: DME medium, Dulbecco’s modified Eagle’s medium; HDL, high-density lipoproteins; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.88); IDL, intermediate-density lipoprotein; LDL, lowdensity lipoproteins; VLDL, very-low-density lipoproteins; apo, apolipoprotein. Correspondence: R. Infante, INSERM (U.9) cherches d’Hepatologie, Hapital Saint-Antoine, Faubeurg Saint-Antoine, 75012 Paris, France. 0005-2760/88/%03.50

Unite de Re184 rue du

0 1988 Elsevier Science Publishers

acylglycerols, cholesterol and phospholipids; some of these are packaged with apolipoproteins and delivered to the blood for use in peripheral tissues. This complex process depends upon a sequential series of intracellular events. The synthesis and secretion of hepatic lipids and lipoproteins are exquisitely regulated by a considerable number of factors (hormone, nutrients and metabolites). Hepatocytes also take up the lipids carried by these lipoproteins, for example, LDL or HDL cholesterol for storage, secretion or degradation. Much information concerning this field has been gained from studies on cultured isolated hepatocytes derived from a variety of animals [l]. However, there are no relevant experiments with humans. Because of the difficulties encountered in

B.V. (Biomedical

Division)

352

obtaining human hepatocytes under physiological conditions and of interspecies differences in lipid metabolism, hepatocyte cell lines derived from human tumors could represent an alternative model for studies on human hepatic lipid metabolism. Recently, the human hepatoma cell line Hep G2 has been used to investigate a number of hepatocyte-specific functions [2-51. The aim of this article is to study the processes involved in lipid and lipoprotein synthesis and secretion by Hep G2 cells and to reexamine some factors which have been shown to stimulate or inhibit the lipid secretion or synthesis in cultured rat hepatocytes, such as glucose, fructose, fatty acids, mevalonic acid and insulin [6-81. This work confirms that the Hep G2 cell line may provide a useful system for the investigation of hepatic lipoprotein metabolism.

Materials and Methods Cell cultures. Hep G2 cells were kindly provided by Dr Barbara Knowles, (The Wistar Institute, Philadelphia) and grown in 25 cm2 tissue culture flasks (Falcon) with 5 ml of Dulbecco’s modified Eagle’s (DME) medium containing 5.5 mM glucose, 10% fetal bovine serum, 100 pg/ml streptomycin sulfate, 100 U/ml penicillin G and 25 pg/ml amphotericin B. Cells were trypsinized for subculture with ATV (1 g/l dextrose/8 g/l NaCl/0.4 g/l KCl/0.58 g/l NaHC0,/0.2 g/l EDTA (disodium salt dihydrate)/0.5 g/l trypsin) at a 1 : 4 split ratio every 5-6 days for continuous maintenance. In all experiments, confluent monolayers were preincubated with 5 ml of serum-free DME medium for 24 h, and then the cells were extensively washed with Dulbecco’s phosphatebuffered saline and incubated with 3 or 5 ml of serum and Phenol red-free DME medium containing the compounds indicated below. Lipid composition of cells. Cell monolayers were washed three times with 0.9% NaCl and harvested with ATV as described above. After centrifugation, cell pellets were washed once again and resuspended in 2 ml of 0.9% NaCl. Aliquots of cell suspensions were sampled for protein determination as described by Lowry et al. [9] using crystallized bovine serum albumin as standard. The cell

suspensions were extracted with methanol/ chloroform (1 : 1, v/v) [lo]. The free cholesterol and cholesterol ester concentrations of cells were measured using a previously described enzymatic method [II]. Cellular phospholipid concentrations were determined as described by Ames [12]. Individual phospholipids were separated by silica gel thin-layer chromatography using chloroform/ methanol/isopropanol/0.25% KCl/ triethylamine (30 : 9 : 25 : 6 : 18, v/v) [13] as mobile phase and quantitated by phosphorus analysis. The triacylglycerol concentration of the cells was measured by an enzymatic method (Boehringer (Mannheim, F.R.G.). Separation of medium lipoproteins by densitygradient ultracentrifugation. After 24 h incubation of Hep G2 cells in a medium containing either [‘4C]palmitate (2 I*.Ci/flask; spec. act., 53.8 mCi/mmol) bound to 1% albumin or [35S]methionine (30 pCi/flask; spec. act., 1103 Ci/mmol), the medium was drawn off by suction and centrifuged at 1500 x g for 5 min to remove cells and debris. The same volume of 50% diluted human plasma was added as carrier. The density of samples was adjusted to d = 1.21 g/ml with solid KBr and 4.4 ml aliquots were pipetted into polyallomer centrifuge tubes (final volume 12.4 ml). A discontinuous density gradient was formed by carefully layering above the medium 3.0 ml of salt solution, d = 1.063 g/ml followed by 3.0 ml of salt solution, d = 1.019 g/ml. Finally, the tube was filled up with 2 ml of d = 1.006 g/ml solution [14]. In order to locate the lipoprotein fractions, one tube containing human plasma prestained with Sudan black B [15] was prepared under the same conditions. Ultracentrifugation was carried out in a SW-41 rotor at 40000 rpm at 20 o C for 24 h. The lipoprotein fractions were carefully transferred by aspiration into tubes and their density was measured with a digital precision densitometer. All samples were extensively dialyzed against four changes of 0.9% NaCl/O.Ol% EDTA (pH 7.4) for 48 h. Aliquots of the dialyzed samples were lyophilized and then solubilized in a buffer solution containing Tris base, SDS, glycerol and 2mercaptoethanol (Sigma), and heated at 80°C for 3 min. The apolipoproteins were separated by slab gel electrophoresis in an SDS-polyacrylamide gradient (3315%).

353

Dried gels were autoradiographed using X-ray films (Kodak X-omat). Standard proteins of known molecular weights were run on the same gel and the relative position of radioactive bands was compared to those of standard proteins stained with Coomassie blue. The 3sS-labeled apolipoprotein B (B-100) recovered in the fraction at a density of less than 1.21 was identified by immunoblotting analysis after SDS-PAGE separation. The gel was equilibrated for 30 mm in 20 mM Tris base/192 mM glycine/20% mercaptoethanol and then electcoblotted overnight onto a nitrocellulose filter at 20 volts using the same buffers as those used in the equilibration step. The post-transfer filter was then incubated in phosphate-buffers saline containing 0.05% Tween 20 and appropriate amounts of anti-human apo B antiserum, at 37 o C for 3 h. The filter was extensively washed with several changes of phosphate-buffered saline for 2-3 days. Autoradiography of the dried filter was performed as described above. The lipoprotein lipids were extracted with chloroform/methanol (2 : 1, v/v) and separated by thin-layer chromatography on silica gel plates developed in petroleum ether/diethyl ether/acetic acid (90 : 30 : 1, v/v). The lipid spots were scraped off into plastic vials containing a scintillating mixture (Lipoluma, J.T. Baker, The Netherlands), and the radioactivity was counted in a /3-spectrometer (Intertechnique, France). Preparation of Hep G.? RNA. Total RNA from Hep G2 cells was isolated by the gua~dine-HCl method [16]. In order to avoid ribonuclease activity, glassware was rinsed with a diethylpyrocarbonate solution and sterilized at 120 o C for 20 min. The aqueous solutions used in the preparation were added with 0.1% (v/v) diethylpyrocarbonate before sterilization. Cell-free translation system and immunoprecipitation. RNA was translated in a protein-synthesizing system from rabbit reticulocyte lysate [17]. Proteins were labeled by including 10 PCi of [35S]methionine in the translation medium. Nonspecific pr~ipitation of the translation medium 1181 and further immunoprecipitation of apolipoproteins with monospecific antisera were performed as previously described [19]. Effect of oieic acid on the incorporation of (2‘H~g~~ero~ into the medium and cell lipids. Con-

fluent cell monolayers were incubated with 3 ml of serum and Phenol red-free medium containing oleic acid (1 mM) bound to albumin (0.17 mM). The 2-I 3H]glycerol (5 pCi/flask, spec. act. 1 mCi/pmol) was added to the culture medium. Labeling glycerolipids with 2-[ 3H]glycerol could give underestimated values, since 3H in the 2-position is lost in the conversion of glycerol to dihydroxyacetone phosphate, a glycerohpid precursor. However, comparative experiments using [ 3H] glycerol labeled either in the 1,3- or in the 2-positions have shown very similar results, indicating that under our experimental conditions, more than 90% of the triacylglycerol derive from the rr~y~rophosphate pathway. After 12 h incubation, Hep G2 cells were harvested with ATV after removing the medium. The medium and cell lipids were extracted and processed as described. Effect of mevalonate on the incorporation of [‘4C]acetate into cell cholesterol. Confluent monolayers were incubated with 5 ml of serum and Phenol red-free DME medium (control) or the same medium containing 2.5-10 mM mevalonate, which was obtained by incubation at 56’ C for 15 min of mevalonolactone (Sigma) with 0.1 M NaOH. After 22 h incubation, [14C]acetate (5.5 pCi/flask, spec. act. 55 mCifmmo1) was added, and 3 h later, cells and medium were harvested. Hep G2 cells were extracted with chloroform/ methanol and the saponification of lipid extracts was carried out with ethanol/8 M KOH at 75 o C for 2 h [20]. The inco~oration of [‘4C]acetate into cholesterol was determined after thin-layer chromatography. The band of cholesterol was located by iodine vapor. The band was scraped off and the radioactivity was measured in a liquid scintillation spectrometer. In the study on the short-term effect of mevalonate, 2 PCi [14~]acetate/flask were added into the medium at the beginning of the incubation and the cells were harvested 60 min later. Effect of glucose and fructose on triacylglyceroi secretion. For the triacylglycerol secretion studies, different amounts of glucose and fructose were added into the serum and Phenol red-free medium. After 18 or 20 h incubation, the medium was aspirated and centrifuged at 1500 X g at 4’ C for 5 min. 1 ml of supernatant was lyophilized. The triacylglycerol concentration in the medium was

354

determined by an enzymatic technique (Boehringer Mannheim, F.R.G.). The effect of insulin on t~iacy~~lyce~o~secwtion and on medial and ce~i~~~~protein synthesis. Confluent monolayers of Hep G2 cells were incubated with 3 ml/flask of serum and Phenol red-free DME medium containing 12 mM glucose, 1 mU or 7 pmol/ml insulin and 20 yCi/flask of ~35S]met~onine. Every 2 h, a dose of insulin (1 mU or 7 pmol/ml) was added. The same volume of medium without insulin was added to the control flasks. After 8 h incubation, the medium was removed, cells were harvested with ATV and lipids were extracted and quantitated as described above. Ahquots of the medium and cell suspension were precipitated with trichloroacetic acid and radioassayed as described by Mans and Novelli [Zl].

TABLE

I

LIPID LIVER

CONTENT

HEP

G2

CELLS

AND

HUMAN

Confluent Hep G2 monolayers were incubated in serum-free DME medium for 24 h, washed three times with Duibecco’s phosphate-buffered saline and finally incubated for 24 h in serum and Phenol red-free DME medium. Human liver samples were obtained from normal organ donors; lipids from harvested Hep G2 cells and liver samples were extracted and determined as described in Materials and Methods. Values are meansIt:S.E. from 12 cell flasks and three liver samples. The compositions (percent by weight) are shown in parentheses. Lipid content cell protein)

(~8 lipid/mg

Hep G2 celis

human

Triacyiglycerols

62.5 * 2.31 (24.5)

25.73 + 7.79 (17.7k4.9)

Phospholipids

175.5 t 9.02 (68.8)

103.99+11.33 (73.7 * 4.3)

Results Free cholesterol

Lipid composition of Hep ~3.2cells Cultured Hep G2 cells at confluence had a lipid composition somewhat different from that of adult liver tissue (Table I). Their total lipid content (255 pg/mg protein) was much higher than that of normal liver (142.5 pg/mg protein). Most of this difference was due to a higher triacylglycerol and phospholipid content and, to a lesser extent, to the free cholesterol concentration. The phospholipid composition of Hep G2 cells showed also differences compared with that of normal liver cells (Table II). The percentage of phosphatidylglycerol and phosphatidylethanola~ne was lower in the cell line, whereas that of phosphatidylcholine and sphingomyelin was significantly higher. The ratio phosphatidylcholine/ sphingomyelin, which is known to influence membrane fluidity [22], was much lower in Hep G2 cells than in normal liver (4.0 vs. 7.0). Culture conditions in a lipid-free medium might explain some of the differences in lipid composition. However, addition of 0.4 or 1.0 mM oleic acid to the medium still increased the cellular t~acylglycerol content by 135 and 300%, respectively, and that of cholesterol esters by 40 and 68%, a significant increase considering the relatively short time (18 h) of incubation and the moderate fatty acid molarity, similar to that found in human plasma (Table III).

OF

Cholesterol

esters

12.87+0&t

11.56

(5.0)

(8.0)

i 0.25

1.25kO.32 )

4.21 i 0.25 (1.7)

TABLE

liver

(1

II

PHOSPHOLIPID COMPOSITION NORMAL HUMAN LIVER

OF HEP G2 CELLS

AND

Cells and human liver samples were processed as in Table 1. Values are means f S.E. Significance (Student’s t-test) versus normal liver values are expressed as * P <: 0.025: * * P i 0.005. Composition

Phosphatidylglycerol Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserine Phosphatidylcholine Sphingomyelin

{percent

by weight)

HepG2(n=4)

human (n=3)

liver

6.45 + 0.62 23.8 + 2.29 7.08 f 0.24 4.05 f 0.80 46.88 + 1.43 * 11.53kO.86 **

9.0 20.58 36.0+ 5.2 8.33 i 0.33 4.67 f 1.20 37.67 + 2.60 5.33i0.88

Lip~Frotein secretion by Hep G2 cells Under the standard culture conditions, Hep G2 cells secreted into the medium newly synthesized triacylglycerols. These lipids are secreted as lipoprotein particles, which can be isolated by density gradient ultracentrifugation at different density cuts. As displayed in Table IV. most of the tri-

355 TABLE EFFECT

III OF OLEIC

ACID

ON LIPID

CONTENT

OF HEP G2 CELLS

Confluent cell cultures were incubated in serum-free DME medium for 24 h. After three washings with Dulbecco’s phosphate-buffered saline, each flask was added with 3 ml of serum and Phenol red-free DME medium containing either 0.4% (control 1) or 1% (control 2) defatted bovine serum albumin. Other flasks were supplemented with 0.4 mM oleic acid complexed to 0.4% bovine serum albumin or 1.0 mM oleic acid in 1% bovine serum albumin. After 18 h incubation, lipids were extracted and determined as described in different from control values at Materials and Methods. * Significantly different from control values at P < 0.05. * * Significantly P c 0.02 (Student’s r-test). Lipid content control

(ng lipid/mg

(1)

42.4& 3.5 147.7 f 25.1 10.2* 1.2 6.0+ 1.8

Triacylglycerols Phospholipids Free cholesterol Cholesterol esters

cell protein)

oleic acid (0.4 mM)

control

99.9* 8.9 ** 172.4 f 25.2 11.1* 0.5 8.4* 2.5

48.2* 4.3 170.7 f 26.9 10.3* 1.0 6.3f 2.1

acylglycerols are recovered at densities corresponding to very-lowand intermediary-density lipoproteins. Some 14C-labeled lipid was recovered in the 1.149-1.21 density fraction (HDL) and identified after TLC as unesterified cholesterol and phospholipid (data not shown). The amount of lipoproteins recovered from the medium was

TABLE

OF [‘4C]TRIACYLGLYCEROLS FRACTIONS

198.4 + 32.8 * 165.9* 19.1 10.9* 1.0 10.6* 1.7

insufficient to analyze the apolipoprotein composition of each density class. Thus, synthesis and secretion of apolipoproteins by Hep G2 cells were estimated after incubation in a medium containing [ 35S]methionine as protein precursor. Total lipo-

IN THE

Confluent cell monolayers were preincubated for 24 h in serum-free DME medium, washed three times with Dulbecco’s phosphate-buffered saline and then incubated for 24 h in serum and Phenol red-free DME medium containing 2 pCi [‘4C]palmitate (spec. act., 53.8 mCi/mmol) bound to 1% albumin. The medium was recovered after centrifugation and the lipoprotein fractions were separated by density-gradient ultracentrifugation. Lipids were extracted, isolated by TLC and their radioactivity was counted as described in Materials and Methods. Values are means f S.E. from three experiments. Density (g/ml)

I II III IV V VI VII VIII

oleic acid (1.0 mM)

IV

DISTRIBUTION LIPOPROTEIN

Fraction

(2)

< 1.006 1.006-1.020 1.020-1.036 1.036-1.063 1.063-1.103 1.103-1.148 1.149-1.21 21.21

Designation

VLDL IDL LDL _ HDL HDL _

[ “C]Triacylglycerol distribution % of total

cpm

16 47 14 4 11 1 1 0

8741+ 663 24711&1524 7732+ 519 2380f 554 6208f 431 6385 197 loll+ 59 356f 198

94 kDa67 kDa_ 43 kDa_ 30 kDa_ 20 kDa, 14 kDa, A

B

Fig. 1. Autoradiogram of 3-15% polyacrylamide gel of [ 35S]methionine-labeled apolipoproteins isolated at d < 1.21 g/ml from the culture medium of Hep G2 cells. Medium lipoproteins were isolated as described in Materials and Methods. The samples were analyzed by electrophoresis on 3-15% polyacrylamide gels containing sodium dodecyl sulfate. Standard proteins of known molecular weights were run on the same gel. Lane A: total 35S-labeled apolipoproteins isolated from medium lipoproteins at d -c 1.21 g/ml; lane B: autoradiogram of 35S-labeled apolipoprotein B-100 recovered in the fraction at d cl.21 g/ml and identified by immunoblotting analysis after SDS-PAGE separation.

356

A

B

C

.D

E

Fig. 2. Autoradiogram of [ 3sS]methionine-labeled proteins isolated by a single density-gradient ultracentrifugation from Hep G2 cell medium. Lane A: 35S-labeled apol i p o p roteins at d ~1.006;lane B: at 1.006 < d ~3.020;lane C: at 1.063 < d ~1.103;lane D: at 1.103 < d < 1.148 and lane E: at 1.148 < d < 1.21 g/ml.

proteins (d < 1.21 g/ml) were isolated by ultracentrifugation and delipidated. The apolipoproteins were separated by SDS-PAGE and located by autoradiography. On the basis of electrophoretic mobility compared to that of native human plasma apolipoproteins, we found that the cells produced apo B, apo A-I apo C and apo E in detectable amounts. Further immunoblotting analysis with anti-apo B antibodies revealed that the cell line secreted only the heaviest form of apo B (apo B-100) (Fig. 1). The distribution of 35S-labeled apolipoproteins among the different lipoproteins secreted into the medium was investigated by mixing the concentrated medium with human plasma as carrier before isolating the lipoproteins by density-gradient ultracentrifugation. As shown in Fig. 2, the VLDL and the IDL fractions (d < 1.006 g/ml and 1.006 < d < 1.020 g/ml) contain newly synthesized apo B-100, whereas the fractions heavier than 1.063 g/ml contain mainly apo A-I. In vitro translation of apo A-I and apo E from Hep G2 cells Total mRNA was isolated from Hep and translated in an mRNA-dependent synthesizing system derived from rabbit

cyte lysates using [35S]methionine as label. Preliminary experiments had shown that maximal protein synthesis was obtained after the addition

mRNAs G2 cells proteinreticulo-

Fig. 3. Autoradiogram

of PAGE slabs of translation of Hep G2 mRNA.

products

357

Fig. 4. Autoradiogram of [3SS]methionine-labeled apo A-I (arrow) synthesized in a cell-free system by Hep G2 mRNA. The protein was irmnunoprecipitated by an anti-human apo A-I antiserum.

Fig. 5. Autoradiogram of [ 35S]methiorrine labeled apo E (arrow) synthesized in a cell-free system by Hep G2 mRNA. The protein was immunoprecipitated by an anti-human apo E antiserum.

to the assay of 12 pg Hep G2 RNA. Under optimal conditions, total mRNA synthesized numerous proteins of molecular weights ranging between 10 000 and 120 000, as estimated by electrophoretic mobility on SDS-PAGE (Fig. 3). After immunoprecipitation of the labeled polypeptides with apo A-I antiserum, autoradiography of PAGE slabs revealed a single protein band with an apparent molecular weight of 29000, slightly higher than that of mature apolipoprotein A-I from human plasma (Fig. 4). Moreover, immunoprecipita-

tion of translation products with apo E antiserum isolated a protein giving a single band on SDSPAGE with an estimated M, of 36000, slightly higher than that of native plasma apolipoprotein E (Fig. 5).

TABLE EFFECT

Secretion of triacylglycerols by Hep G2 cells. Effect of carbohydrate availability

During a 20 h incubation in a serum-free DME medium containing 5.5 mM glucose, the triacylglycerol secretion was 7.38 pg triacylglyc-

V OF GLUCOSE

CONCENTRATION

ON TRIACYLGLYCEROL

SECRETION

AND

Confluent monolayers were incubated as described in Table I, except that the medium used supplemented with 5.5, 12 or 25 mM glucose. Values are means f S.E. from three experiments. Lipid content Glucose Medium

triacylglycerols

Cell triacylglycerols Phospholipids

concentration

(pg lipid/mg

cell protein

LIPID

per 20 h)

12

(mM): 5.5

CELLULAR

25

1.04

14.03 f 2.78

24.39+

52.18 + 4.49

56.60 f 3.64

70.86 f 4.09

177.75 + 15.90

153.55 + 8.62

177.8Ozt 8.18

7.38&

CONTENT

in the last 24 h incubation

5.19

was

358 TABLE

VI

EFFECT OF GLUCOSE AND FRUCTOSE ON TRIACYLGLYCEROL SECRETION AND CONTENT OF HEP G2 CELLS Experiments were conducted as described in Table V. In the final 20 h incubation, the medium was supplemented with 12 mM glucose or 5 mM glucose+7 mM fructose. Values are means i SE. from three experiments. Lipid content (pg lipid/mg protein per 20 h)

Medium triacylglycerols Cell triacylglycerols

cell

12 mM glucose

7 mM fructose+ 5 mM glucose

14.14k1.22 53.71 + 1.34

14.14+0.41 57.88 f 2.36

erol/mg protein per 20 h (Table V). The rate of triacylglycerol secretion increased to 14.3 or 24.39 pg triacylglycerol/mg cell protein per 20 h when the glucose concentration in the medium was adjusted to 12 or 25 mM. The differences in triacylglycerol synthesis rates were also inferred from an increase in the cellular triacylglycerol concentration from 52.18 to 70.86 pg triacylglycerol/mg cell protein after 20 h of incubation with 5.5 and 2.5 mM glucose respectively. However, cellular phospholipid levels were not affected by glucose availability under our experimental conditions. Partial replacement of glucose by fructose (7 mM) induced the same increase in cell and medium triacylglycerols as glucose alone (Table VI). Effect of oleic acid on glycerolipid synthesis [ 3H]Glycerol is taken up by Hep G2 cells and incorporated into the cell and medium glyceroliTABLE EFFECT

pids at a linear rate (data not shown), indicating that these cells preserve the enzymes of exogenous glycerol activation and glycerolipid synthesis. Moreover, this cell line regulates the triacylglycerol synthesis rate as a function of fatty acid availability. Indeed, addition to the incubation medium of oleic acid (1 mM) was followed by an 8.0- and 2.5-fold increase, respectively, of [‘HIglycerol incorporation into cell and medium triacylglycerols. In contrast, ‘H incorporation into the cell or medium phospholipids was not affected by fatty acid influx (Table VII), in accordance with previous observations with isolated rat hepatocytes in suspension [23].

Ejject of insulin on triacylglycerol and protein synthesis and secretion Hep G2 cells have been shown to express functional insulin receptors [24]. Thus, we have investigated the effects of insulin on triacylglycerol synthesis and secretion in cell cultures in the presence of physiological concentrations of insulin and in its absence (Table VIII), in a medium containing 12 mM glucose. Since preliminary experiments had shown a rapid degradation of the hormone, appropriate concentrations were maintained by adding a priming dose of insulin followed by a supplement at 2 h intervals for a total 8 h of incubation. Under these conditions. insulin reduced by 50% the secretion of newly synthesized triacylglycerols and increased their intracellular concentration to such an extent that the total {medium + cell) triacylglycerol level was identical in the presence or absence of insulin. This indi-

VII OF OLEIC

ACID

ON THE INCORPORATION

The same conditions as those in Table red-free DME medium (control) or in added with S pCi [ ‘Hjglycerol (spec. separated by TLC on silica gel plates. at P < 0.02. * * Significantly different

OF [‘H]GLYCEROL

INTO CELLULAR

AND

MEDIUM

VI were used except that the final cell incubation was performed either in serum and Phenol the same medium supplemented with 1 mM oleic acid complexed to albumin. Both media were 12 h later. Lipids were extracted and act., 1 Ci/mmol) and media and ceils were recovered * Significantly different from control values and their radioactivity was counted as described. from control values at P < 0.01 (Student’s t-test). TG. triacylglycerol, PL, phospholipid. -.

[ ‘H]GIyceroI

incorporation

(cpm/mg

TG 14500&1377 36602$2460

Ceil protein)

--

cell

medium

Control + 1 mM oleic acid

LIPID

*

PL

TG

PL

2636+95 2614+51

19355+ 849 174159+13448 **

2165411243 190.5012020

359 TABLE

VIII

EFFECT OF INSULIN ON TRIACYLGLYCEROL TION AND CELLULAR TRIACYLGLYCEROL TENT

SECRECON40

The final incubation medium was serum and Phenol red-free DME medium containing 12 mM glucose and supplemented with insulin solution, as described in Materials and Methods. After 8 h incubation, cell and medium lipids were extracted and assayed as described. Values are means i S.E. from six flasks. * Sig~ficantly different from control values at P < 0.005 (Student’s r-test). Lipid content

(ng lipid/mg

medium triacylglycerols

cell triacylglycerols

medium + cell triacylglycerols

Insulin-free

6.45 kO.53

46.05 k2.10

52.50 f 1.76

+ Insulin (1 mU or 7 pmol/ml)

3.45 f 0.21

49.06rt 2.12

52.51 f 2.28

DME medium

cell protein

32

per 8 h)

L

CONTROL

5.0mM

lO.OmM

Fig. 6. Effect of mevalonate on the incorporation of [14C]acetate into cholesterol. After 24 h of preincubation with serum-free medium, confluent monolayers were washed three times with Dulbecco’s phosphate-buffered saline and then incubated with 5 ml of serum and Phenol red-free DME medium (control) or containing mevalonate at concentrations ranging from 2.5 to 10 mM. After 22 h, [14C]acetate (5.5 pCi/flask; spec. act., 55 mCi/mmol) was added. 3 h later, cells and medium were harvested. Cellular lipids were extracted with chloroform/ methanol. After saponification of lipid extracts, the incorporation of [14C]acetate into cholesterol was determined after TLC separation. Values are means f SE. (n = 3).

cates a specific effect on lipid export. Incorporation of [35S]methionine into the cell and medium proteins showed differences which paralleled those described for triacylglycerols; lower amounts of newly synthesized proteins were secreted into the medium of cultures supplemented with insulin and correspondingly higher amounts of radioactive proteins were found in these cells. The total (medium + cell) incorporation of [ 35S]methionine was not modified by the hormone, suggesting that while overall protein synthesis is not affected, protein secretion is specifically inhibited (Table IX). TABLE

2.5 mM

Cholesterol synthesis by Hep G? cells. Effect of mevulon~te Hep G2 cells efficiently incorporate radioactive acetate and mevalonate into cell and medium

IX

EFFECT OF INSULIN ON INCORPORATION TEIN SECRETED INTO THE MEDIUM

OF [35S]METHIONINE

INTO

TOTAL

CELL

PROTEIN

AND

INTO

PRO-

The same culture conditions as those in Table VIII were used, except for the addition of 20 PCi [ 35S]methionine per flask followed by an 8 h incubation period. Cell and medium aliquots were precipitated with 10% trichloroacetic acid (TCA) followed by washing of the precipitate with trichloroacetic acid, solubilization of proteins and radioactivity count, as described in Materials and Methods. Values are means f S.E. from six flasks. * Significantly different from control values at P < 0.05. * * Significantly different from control values at P < 0.02 (Student’s t-test). DME medium

Incorporation

([ 35S]methionine

medium Insulin-free + Insulin 1 mU or 7 pmol/ml

166411+

119090*

TCA precipitate)

(cpm/mg

cell 14375

6805 *

428711 f

cell protein

per 8 h)

medium + cells 9996

484751~12004

595122+19317

**

60384O+18111

360

sterols. Administration of mevalonic acid to animals has been shown to inhibit the activity of HMG-CoA reductase [25]. This inhibitory effect, however, has not yet been demonstrated in normal human liver cells, although it has been reported in Hep G2 cells [26,27]. Prolonged exposure (22 h) to 2.5 or 10.0 mM mevalonate inhibited [i4C]acetate incorporation into cholesterol 3- to 6.2-fold, respectively, compared with control values (Fig. 6). On the other hand short exposure of cells to mevalonate, as described in Materials and Methods, did not influence [i4C]acetate incorporation (data not shown), suggesting that cholesterol synthesis inhibition after long-term incubation with exogenous mevalonate is not an artifact due to dilution of [i4C]mevalonic acid with unlabelled mevalonic acid from the medium. Discussion The human hepatoblastoma cell line Hep G2 maintains specialized functions of normal hepatocytes, including apolipoprotein synthesis, lipoprotein secretion and lipoprotein catabolism [28]. Moreover, recent reports [29,30] indicate that apolipoprotein synthesis by Hep G2 cultures may be modulated by the availability of lipid precursors or by several hormones. Little attention has been paid, however, to the regulation of lipid metabolism and cell lipid composition of Hep G2. Our results show that the lipid content and the lipid/protein ratio are higher in Hep G2 cells than in normal human liver biopsies (255 vs. 142 pg lipid/mg cell protein). This difference is largely due to triacylglycerol accumulation in the cytoplasm and to higher phospholipid levels. Our data are different from those reported by Araki et al. [31] on hepatocarcinoma lipid composition. Human and rat adult liver membranes are more fluid and have a higher phospholipid/cholesterol ratio than fetal liver membranes [22]. Other factors, such as fatty acid composition and saturation index or glycolipid content have been shown to determine the membrane microviscosity. With regard to the individual phospholipids of Hep G2 cells, the significantly lower phosphatidylcholine/ sphingomyelin molar ratio as compared with the normal human liver suggests a lower membrane fluidity. Alterations of these parameters may re-

sult from incomplete maturation (fetal liver), rapid cell division or specific alteration of lipid metabolism due to cell malignancy. In the case of the Hep G2 line, the composition of the culture medium (i.e., fatty acid composition) is probably another factor which can potentially influence lipid metabolism and lipid membrane composition. Hep G2 cells synthesize and secrete lipids as lipoproteins of different densities. After 24 h incubation, most of the radioactive lipids were found in the triacylglycerol fraction of lipoproteins floating at very-low and intermediary densities. The major apolipoprotein in these fractions was apo B (B100). Unesterified cholesterol, phospholipids and apo A-I were the major components of the fractions of density greater than 1.063 g/ml. In vitro translation of mRNA extracted from the Hep G2 cells followed by immunoprecipitation with monospecific antisera allow recovery of apolipoproteins A-I and E. Anti-apo A-I antiserum precipitated a single protein with an apparent molecular weight higher than that of the mature plasma apo A-I, suggesting that the in vitro translated product is the pro-apolipoprotein. This immature form contains, in addition to the main apolipoprotein, a 6-amino-acid residue, which presumably is rapidly cleaved from the mature apo A-I in the circulation. Pro-apo A-I has been identified by Gordon et al. [32] and Zannis et al. [33] in the culture medium of Hep G2 cells and has also been found in the plasma of Tangier’s disease patients. In vitro translation of normal human liver mRNA also produces the pro-apo A-I 1341. Hep G2 mRNA synthesized in vitro apo E with a higher molecular weight than the native apo E from human plasma. Zannis et al. [35] have shown that Hep G2 cells secrete apo E with a relatively high content of sialic acid residues compared to the plasma apo E. Lipogenesis, lipid synthesis and lipoprotein secretion by the liver are modulated by substrate availability and by several hormones, such as insulin. In agreement with in vivo observations. variations in medium composition in cultured rat hepatocytes are followed by changes in lipid synthesis and secretion. The capacity of Hep G2 cells to regulate lipid synthesis and lipoprotein production was assayed

361

under different substrate and insulin concentrations in the medium. At a glucose concentration similar to that found in peripheral blood in vivo, the Hep G2 cells secreted 7 pg t~acyl~ycerol/mg cell protein/20 h. Increasing the glucose concentration to the range found in the portal blood after a meal (12 or 25 mM) raised proportionally the triacylglycerol secretion rate to 1 pg triacyl~y~rol/mg cell protein per h. Triacylglycerols recovered in the medium after 20 h incubation may reflect net secretion, provided no extracellular hydrolysis occurs during this period. Indeed, no lipase activity was detected in the medium after different incubation times of Hep G2 cells in agreement with previously published data on rat hepatocytes 1361.However, some uptake of triacylglycerol-rich lipoprotein particles by the cells cannot be excluded; in this case, the secretion rates should be underestimated. Glucose enhanced the cell triacylglycerol synthesis rate by a process which is not insulin-dependent. Experiments with perfused rat liver and cultured rat hepatocytes [37,38] have shown a poor uptake of glucose from the medium below a concentration of 12 mM; insulin does not significantly influence glucose utilisation 1393. Rat and human liver cells have a low glucokinase activity and, in addition, a high proportion of phosphorylated glucose is dephosphorylated back to glucose by glucose-6-phosphatase (futile cycle). This could explain why the substantial rates of glucose utilisation can only be observed when hepatocytes are exposed to very high carbohydrate levels. In vivo animal studies and perfused liver experiments have shown that fructose is a better substrate than glucose for hepatic lipogenesis. In contrast, our results with Hep G2 cells indicate a similar utilisation of fructose and glucose for triacylglycerol synthesis. Fructose metabolism is mainly controlled by ketohexokinase and fructosel-phosphate aldolase or aldolase B. The latter is virtually absent in fetal liver and expresses full activity only in adult liver. It is interesting to note that in spite of the fetal characteristics of human hepatoma cell lines, Hep G2 cells seem to express aldolase B activity, like mature hepatocytes. Under the standard culture conditions using fatty acid-poor media, Hep G2 cells synthesized lipids from endogenous fatty acids. However, when

the medium was supplemented with 1 mM oleic acid, [3H]glycerol incorporation into the medium and cell triacylglycerols increased to 2- and 9-fold, respectively, of basal values. This stimulatory effect has also been reported in rat hepatocyte monolayers [40], and confirms the capacity of cultured hepatocytes to adapt lipid synthesis to substrate availability, like the perfused livers and the intact animal. However, the effects of fatty acids on the secretion rate of apolipoproteins by the perfused rat liver have not been found in cultured hepatocytes [41]. Unlike rat hepatocytes, Hep G2 cells do not need insulin to facilitate adhesion to the support and rn~t~n viability. Yet it has been recently shown 124) that this cell line expresses membrane high-affinity receptors, which bound and intemalize [‘251]insulin. Under our culture conditions, insulin decreased the secretion by 50% and concomitantly increased the cell content of triacylglycerols. Indeed, insulin in the presence of 12 mM glucose didnot modify triacylglycerol synthesis but depressed only triacylglycerol export. Inhibition of triacylglycerol secretion by insulin has been demonstrated in perfused rat livers [42] and rat hepatocytes f43], although contradictory results have been published using these models [44,45]. Also human and animal studies have shown that insulin injection can either increase or decrease serum triacylglycerol levels. Interpretation of in vivo experiments is difficult, since, besides a direct effect of insulin on triacylglycerol synthesis or/and secretion, other factors, such as decreased free fatty influx and fatty acid synthesis inhibition by glucagon can ultimately modify the VLDL triacylglycerol secretion. In a careful study on the effects of glucose and insulin in cultured rat hepatocytes, Durrington et al. [7] have recently shown that, in the absence of insulin, the mass of VLDL triacylglycerol accumulated in the medium was directly related to the glucose concentration in the range of 2.5-25 mM, without significant variations of cell triacylglycerol concentration. Addition of insulin (50-500 $J/ml) to the medium significantly decreased the VLDL triacylglycerol secretion and increased the cell triacylglycerol content at any glucose concentration in the medium (O-25 mM). Changes in [3H]glycerol in-

362

corporation paralleled the medium and cell triacyiglycerol mass. Our own results indicate that in the Hep G2 cell line, insulin does not modify the net triacylglycerol synthesis but like in rat hepatocytes, depresses their secretion. Cholesterol synthesis in Hep G2 cells can be inhibited at different levels: competitive inhibitors of the key enzyme, HMG-CoA reductase, like compactin, are active on this cell line [46]. The same authors (271 have found that other drugs (U 18666 A, triparanol and but~obate) which block, respectively, the conversion of squalene, desmosterol or lanosterol into cholesterol in different cell types are also operative in Hep G2 cells. Finally, mevalonate, the conversion product of HMG-CoA reductase, can inhibit further cholesterogenesis in cells already having a partial blockade of cholesterol synthesis by preincubation with LDL or different drugs, suggesting a different mechanism of action. Our results in Fig. 6 show that long-term exposure of cells to exogenous mevalonate can almost completely suppress cholesterol synthesis from [‘4C]acetate. Since short-term exposure does not produce the same effect, this suggests that inhibition of [14C]acetate incorporation is not an artifact due to isotope dilution by non-radioactive mevalonate. The mecha~sm responsible for the inhibition of cholesterogenesis from acetate is unsettled. Mevalonolactone can inhibit HMG-CoA reductase in vivo and in cultured cells [25,47]. It has been suggested that an increased flux of mevalonate can produce sterols in sufficient amounts to inhibit cholesterogenesis by a feed-back mechanism. Consistent with this hypothesis are published data showing that mevalonate is inactive on enucleated cells [48] and that in intact cells, it represses the transcription of the HMG-CoA reductase gene 1491, like cholesterol ad~nistration, and consequently the reductase mRNA levels [50]. However, the fact that mevalonate increases further the reductase inhibition produced by cholesterol or oxygenated sterols [47] suggests a more complex mechanism involving sterol or maybe non-sterol derivatives of mevalonate [Sl]. Recently, Edwards et al. [52] have shown that mevalonolactone inhibits the synthesis and increases the degradation of HMG-CoA reductase in cultured rat hepato-

cytes. Whatever the mechanism, in our experiments, mevalonate had no effect on the reductase activity after short-term exposure of Hep G2 cells, and it exerted its inhibitory effect after long-term incubation. Since the fast-dividing Hep G2 cells express some traits of fetal hepatocytes, our findings are in agreement with published observations showing a lesser effect of mevalonate in other hepatoma cell lines [53] and in fetal rat hepatocytes [54] compared to mature cells or intact livers. This difference could be partly explained by a defect in the expression of some enzymes involved in the conversion of mevalonate to cholesterol in Hep G2 cells (Wang, S.R., unpublished data). In conclusion, the Hep G2 cell line expresses many features typical of the regulation of lipid metabolism in the liver and is certainly a very reliable and useful model in spite of some differences compared with the normal non-replicating cultures of human hepatocytes, which have other disadvantages, such as difficulty of obtaining liver fragments in a good metabolic condition, wide individual differences and shorter lifetimes, which hinder long-term regulation studies. Acknowledgements We thank Dr Isabel Beucler for providing human apolipoprotein B antiserum, Mrs. Michele Groussard, who prepared the figures, Ms. MariePierre Belot for excellent secretarial assistance and Mr Yves Issoulie for photographic work. References Forte, T.M. (1984) Annu. Rev. Physiol. 46, 403-415. Rash, J.M.. Rothblat, G.H. and Sparks, C.E. (1981) Biochim. Biophys. Acta 666. 294-298. Leichtner, A.M., Krieger, M. and Schwartz, A.L. (1984) Hepatology 4, 887-901. Dashti, N.. Wol~auer, G.. Koren, E.. Knowles, B. and Alaupovic, P. (1984) Biochim. Biophys. Acta 794. 373-384. Dashti, N., Wolfbauer, G. and Alaupovic, P. (1985) Biochim. Biophys. Acta 833, 100-110. Bell-Quint, J. and Forte, T. (1981) Biochim. Biophys. Acta 663, 83-98. Durrington, P.N., Newton, R.S., Weinstein, D.B. and Steinberg, D. (1982) J. Chn. Invest. 70, 63-73. Davis. R.A.. Hyde, P.M., Kuan. Jui-Chang, W., MaloneMcNeal, M. and Archambault-Schexnayder, J. (1983) J. Biol. Chem. 258. 3661-3667.

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