The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes

321

Biochimica et Biophysics Acta 836 (1985) 321-334 Elsevier

BBA 52022

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes

Haya Herscovitz and Alisa Tietz * Department of Biochemistry,

The George S. Wise Faculty

of Life Sciences, Tel-Aviv University, 69978 Tel Aviv (Israel)

(Received February 20th, 1985)

Key words: Bile acid synthesis; Cholesterol; Taurochenodeoxycholic acid; Taurocholic acid; Mevalonate; HDL; (Chick embryo hepatocyte)

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes was studied. (1) Mevalonolactone was taken up by the cells and converted to cholesterol, cholesterol ester and tauroconjugates of bile acids. The addition of mevalonolactone had little effect on the conversion of endogenous cholesterol to taurocholic acid; however, taurochenodeoxycholic acid synthesis was stimulated. 25-30% of the cholesterol synthesized from mevalonolactone was converted to taurochenodeoxycholic, taurocholic and two so-far unidentified bile acids. All bile acids were secreted into the incubation medium. (2) When cholesterol was added as mixed liposomes with phosphatidylcholine, it was taken up by the cells and converted to bile acids. At low concentrations of liposomes, the greater part of the cholesterol which was taken up by the cells was converted to bile acids. At higher concentrations, considerable amounts of cholesterol and cholesterol ester accumulated inside the cells. (3) When mevalonolactone and cholesterol liposomes was added together, both substrates were used simultaneously for bile acids synthesis. (4) HDL cholesterol was the best substrate tested, yielding large amounts of two, so-far, unidentified bile acids (possibly allo-bile acids) and smaller amounts of taurocholic and taurochenodeoxycholic acid. Addition of HDL suppressed the conversion of endogenous cholesterol to taurocholic acid; taurochenodeoxycholic acid synthesis, however, was stimulated.

Introduction

The liver plays a central role in cholesterol synthesis and degradation. Cellular cholesterol may originate from three different sources: de novo synthesis, ingested cholesterol delivered to the liver as chylomicron remnants and lipoprotein cholesterol delivered as LDL or HDL [l]. This cholesterol may be used for storage, incorporation and secretion as VLDL, secretion as biliary cholesterol and degradation to bile acids which are secreted into the bile.

* To whom correspondence should be addressed. 0005-2760/85/%03.30

Several studies pointed to the existence of specific hepatic cholesterol precursor pools associated with formation and secretion of bile acids and biliary cholesterol [2]. Cronholm et al. [3] showed that the biliary bile acids were formed from a cholesterol pool, the turnover of which was faster than that of the cholesterol pool used for biliary excretion. Mitropoulos et al. [4] showed that in the rat newly synthesized cholesterol was the preferred substrate for bile acid synthesis. Similar results were reported by Bjorkhem and Lewenhaupt [5]. They also showed that only a very small part of the newly synthesized cholesterol was excreted as biliary cholesterol. Different results were reported by Turley and Dietschy [6]. Em-

0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

322

ploying 3H,0 they showed that the newly synthesized cholesterol contributed 16-18% of the total biliary cholesterol. Schwartz et al. [7-91 administered HDL and LDL containing labeled free or esterified cholesterol to man and showed that free cholesterol from HDL was more rapidly incorporated into biliary cholesterol and bile acids than LDL cholesterol; cholesterol ester was a very poor substrate. Similar results were reported by Portman et al. [lo] in squirrel monkeys. This paper presents an attempt to study the availability of different sources of cholesterol for bile acid synthesis in cultured hepatocytes. Freshly isolated hepatocytes were used by Botham et al. [ll-131 and Kempen et al. [14,15] to investigate bile acid synthesis. In these experiments the differences observed in bile acid synthesis and secretion were a reflection of the physiological state of the animal prior to its being killed for preparation of hepatocytes. Thus, it was shown that cholesterol feeding or the addition of cholestyramine to the diet markedly stimulated bile acid synthesis by the hepatocytes [13,14]; infusion of bile acid had the opposite effect. Davis et al. [16,17] studied bile acid synthesis by cultured rat hepatocytes. In the present study, chick embryo hepatocytes were incubated in a serum-free medium in the presence of mevalonolactone, mixed liposomes of cholesterol and phosphatidylcholine or HDL labeled with [‘4C]cholesterol. It will be shown that, under our experimental conditions, HDL-free cholesterol was the preferred substrate for bile acid synthesis. Experimental

(600 X g for 5 min). The isolated cells were washed three times with M-199. They were finally suspended in M-199 in the presence of 10%~new-born calf serum and plated (Nunc 9 cm diameter plates, 1.5 livers per plate or 3 cm diameter plates, 0.5 livers per plate). After 18 h incubation, the medium and unattached cells were removed. The adherent cells were washed and incubated in fresh M-199 medium. r4C- or 3H-labeled precursors were added as indicated in the text. To eliminate contamination, penicillin (10 000 units/ml) streptomycin (10 mg/ml) and mycostatin (1250 units/ml) were added to all media. Incubations were done at 37°C in a controlled CO,-enriched atmosphere. In more recent experiments, Hams F-10 (indicator low) medium was used. At the end of the incubation period, the medium was collected and kept separately for lipid and bile acid analysis. The cells were removed from the plates with EDTA-trypsin (0.25%) and were washed twice with phosphate-buffered saline. The washed cells were homogenized with a small volume of H,O. An aliquot was removed for protein determination [19] and the rest was kept for lipid analysis. Preparation of liposomes Cholesterol and egg yolk phosphatidylcholine were mixed at a molar ratio of 1 : 10. The organic solvent was removed, medium M-199 was added to yield a concentration of 3 mM phosphatidylcholine and the mixture was sonicated for 10 min at room temperature. Phosphatidylcholine was prepared from egg yolks [20].

procedures

Preparation of hepatocytes Hepatocytes were prepared from livers of 14day-old chick embryos according to the procedure of Belleman et al. [18]. The livers were cut into small pieces with scissors and incubated for 30 min at 37°C in Ca”and Mg2+-free Krebs Hanselit buffer at pH 7.6. This was followed by a second 30 min incubation in the presence of CaCl, collagenase (70 pg/ml) and hyaluronidase (1 mg/ml). Generally, 18-20 livers were incubated in 15 ml buffer. Individual cells were released by aspiration through a Pasteur pipette. 3 vol. of M-199 medium (Hanks salt base) were then added and the free cells were collected by centrifugation

Preparation of HDL labeled with [3HJ or (‘4CJcholesterol HDL was isolated from the serum of young roosters according to Have1 et al. [21]. VLDL was collected at a density of 1.006 g/ml and LDL at 1.063 g/ml. At this stage, HDL formed a loose precipitate. This precipitate was suspended in a salt solution of 1.21 g/ml density and floated by centrifugation. A mixture of penicillin, streptomycin and mycostatin was added to avoid contamination. All lipoproteins were dialyzed extensively against several changes of phosphatebuffered saline (pH 7.4). The dialyzed lipoproteins were finally passed through an 0.45 pm Millipore filter and kept at 4°C for several weeks. The

323

purified HDL contained apolipoproteins A-I, A-IV and C as determined by SDS-gel electrophoresis 1221. HDL was labeled with [4-14C]- or [1,2(n)3H]cholesterol (Amersh~, U.K.), according to the procedure of Spector and Hoak [23]. Approximately 30% of the cholesterol initially added onto the celite was incorporated into HDL. Extraction and isolation of lipids Cholesterol. Lipids were extracted from cells or

incubation media with chloroform-methanol according to Bligh and Dyer [24]. The pH was adjusted to 8.0 with NH,OH. After phase separation, cholesterol and cholesterol ester as well as all glycerides and fatty acids were concentrated in the chloroform layer; the conjugated bile acids were contained in the methanol water layer. Glycerides, cholesterol and cholesterol ester were separated by chromatography on thin layers of silica gel G (Merck, Darmstadt, F.R.G.) employing hexane/ diethyl ether/ acetic acid/ methanol (90 : 20 : 2 : 3, v/v) as solvent. The isolated lipids were located under ultraviolet light after spraying the plates with a 0.01% solution of 2,7-dichlorofluores&n in 50% ethanol. To determine the amount of radioacti~ty associated with the different lipids, the corresponding spots were scraped directly into scintillation vials and the radioactivity was determined in a Prias liquid scintillation spectrometer (Packard, U.S.A.) after addition of Hydroluma (Lumac Syst. Inc., The Netherlands). Corrections for quenching were made using an external standard. To separate cholesterol ester and squalene which migrated with a similar R, under our experimental conditions, the corresponding spot was collected and subjected to alkaline hydrolysis with 1 M methanolic KOH; cholesterol and squalene were extracted into hexane and separated by TLC as described above. In most experiments over 90% of the radioactivity was associated with cholesterol ester. All results are expressed as nmol products formed per mg protein assuming that 5 mol of [2-‘4C]mevalonolactone [25] and 12 mol of [l14C]acetate yielded 1 mol’ of cholesterol, 4 mol of mevalonolactone and 11 mol of acetate - 1 mol of bile acid. To determine the quantities of cholesterol or

cholestrol ester present in the lipid extract, cholestrol and cholesterol ester were separated by TLC as described above. The responding spots were collected, mixed with 1 M me~~olic KOH and kept for 1 h at 50°C. Cholesterol was extracted into hexane and its quantity was determined according to Zlatkis and Zak [26]. Conjugated bile acidr. As.will be mentioned in the text, bile acids were detected exclusively in the incubation medium and none were found in the cells. In the early experiments, conjugated bile acids were isolated by ion pairing on Lipidex-1000 columns according to Dijverman and Sjovall 12’71. The aqueous phase obtained after phase separation [24] was dried under reduced pressure and the residue was dissolved in a 0.03 M solution of sodium decylt~methyl~mo~um bromide and passed through a small column of Lipidex-1000. Radioactive precursors were washed off the column with H,O. The bile acids were then eluted with methanol, and separated by chromatography on thin layers of silica gel G employing butanol/ acetic acid/water (10: 1: 1, v/v). Bile acids were located under ultraviolet light after spraying with a 0.01% 2,7-dic~orofluoresc~n solution. The isolated spots were scraped off the plates, the silica gel was collected directly into scintillation vials and counted as described in the previous section. Recoveries were calculated by adding 3H-labeled glycocholic acid into the chloroform-methanol extract (method 1). Recently, we developed a method for separation and quantitative estimation of conjugated bile acids employing high-pressure liquid chromatography (HPLC). Bile acids were extracted from culture media according to De Mark et al. [28] using Sep-Pak C-18 cartridges (Waters, Milford, MA, U.S.A.). Lipoproteins were not retained by the column and were eluted with water. However, when [i4C]cholesterol was added to the medium, the bile acid-containing methanol eluate was extracted with chloroform [24] to remove all traces of [‘4C]cholesterol. The bile acids were then separated on a column of Lichrosorb RP-18 of 7 pm (Merck, F.R.G.) using methanol/ water/ acetic acid (340: 180 :0.58, v/v; the mixture was adjusted to pH 5.2 with 10 M NaOH). The bile acids were detected ~ectrophotomet~c~y at 207 nm and the amounts eluted were calculated from the

324

areas under the peaks using the Spectra-Physics model SP-4100 Computing Integrator. The relationship between area and pg bile acid was determined for each of the known bile acids. To determine the radioactivity associated with the separated bile acids the eluates were collected directly into scintillation vials using an LKB (LKB, Denmark) fraction collector. Fractions were collected at 30 s intervals and counted as described above (method 2).

[2~14C]Mevalonola~tone, [1-i4C]sodium acetate, [4-14C]cholesterol and (1,2(n)-3H]cholesterol were obtained from The Radiochemical Centre, Amersham, U.K. The [ l4 Clmevalonolactone was diluted with non-labeled mevalonolactone obtained from Sigma, U.S.A. The mixture was passed through a 0.45 pm Millipore filter. Since M-199 contained acetate, the [14C]acetate was not diluted before use. Collagenase from ~~ostridi~m histo~t~~um and ~ya~~~o~~d~~e from bovine testis (type I-S) were obtained from Sigma, St. Louis, MO, U.S.A. Media M-199 and F-10, new-born-calf serum, penicillin, streptomycin, mycostatin, trypsin-EDTA and glutamine were obtained from Biological Industries, Beth HaEmek, Israel. All solvents were Merck analytical grade and were used without further purification. Lipidex-1000 was obtained from Packard. U.S.A. Results Conuersion of me~~~o~oiaetone and acetate to cholesterol and Me acids Preliminary experiments showed that when chicken embryo hepatocytes were incubated with [i4C]mevalonolactone, the substrate was taken up by the cells and incorporated into cholesterol, cholesterol ester and conjugated bile acids. Whereas cholesterol and cholesterol ester were isolated from cells and medium, bile acids were found exclusively in the medium. As can be seen from Table I, the major products were taurochenodeoxycholic and taurocholic acids; small amounts of other conjugated bile acid which so far have not been identified were also detected by thin-layer chromatogrphy.

TABLE

I

BILE ACID SYNTHESIS FROM ACETATE AND CHOLESTEROL

MEVALONOI~ACTON~.

Hepatocytes were incubated for 5 days in medium M-199. Cholesterol was added as mixed liposomes with phosphatidylcholine (molar ratio 1 : 10). Bile acids were isolated and separated according to method 1. TCDC, taurochenodeoxycholic acid; TC. taurocholic acid. Substrate added

Bile acid synthesis (pmol/mg protein)

(PM)

TCDC

TC

total

16.8 62.5 236.0 81.0 260.0

4.9 94.5 35.0 42.0 98.0

26.1 157.0 211.0 123.0 558.0

Mevalonolactone, Mevalonolactone, Acetate, Acetate, Cholesterol.

20 100 600 600 30

As can be seen from Fig. 1 the amounts of cholesterol, cholesterol ester and bile acid formed increased when increasing concentrations of mevalonolactone were added to the incubation medium. At higher con~ntrations of mevalonolactone the increase in bile acid synthesis was greater

30 -

25-

20-

/

MVA

p!c+J

Fig. I. The effect of increasing concentratins of mevalonolactone (MVA) on cholesterol and bile acid synthesis. Hepatocytes were incubated for 5 days in M-199 in the presence of [2“C]mevaionolactone as indicated. Bile acids were isolated according to method 1. l , total cholesterol; 0, total cholesterol ester; A, conjugated bile acid.

325

than that of cholesterol. At 0.5 mM mevalonolactone in the incubation medium, 28.7% of the newly synthesized cholesterol was converted to bile acid (Table II). Under these conditions, considerable amounts of mevalonolactone were used for triacylglycerol biosynthesis, most likely after conversion to acetate by the Shunt pathway [29]. The results obtained show that under the conditions of our experiments, cholesterol synthesis was not maximal. Most likely, mevalonolactone uptake was rate limiting, thus its intracellular concentration was not sufficient to support maximal cholesterol synthesis. Table II also shows the conversion of exogenous [l- “Clacetate to cholesterol and bile acids. Since medium M-199 contains considerable amounts of acetate (50 mg/l) the dependence of bile acid synthesis on varying acetate concentrations was not studied. As shown in Table II approximately 30% of the newly synthesized cholesterol was released into the incubation medium. Cholesterol ester was secreted in much smaller quantities. Fig. 2 (a and b) shows the dependence of cholesterol, cholesterol ester and bile acid synthesis on the time of incubation. In the experiment

2

4 days

6

Fig. 2. The effect of increasingtime of incubation on cholesterol, cholesterol ester and bile acid synthesis. Cells were incubated in M-199 with 400 PM [2-14C]mevaIonoiactone, for (a) 24 h, (b) 6 days. l , total cholesterol; 0, total cholesteroi ester; A, bite acid.

shown, cholesterol synthesis increased at an almost linear rate for 6 days. Cholesterol ester and bile acid synthesis stopped after 4 days. Generally, the

TABLE II THE EFFECT OF INCREASING CONCENTRATIONS SYNTHESIS

OF MEVALONOLACTONE

ON CHOLESTEROL AND BILE ACID

Chick embryo hepatocytes were incubated for 5 days in M-199. Lipids were isolated from cells and medium as described in the Methods. Bile acids were isolated according to method 1. Since mevaionolactone is incorporated into fatty acids only after conversion to acetyl-CoA via the shunt pathway, the amounts of product formed could not be calculated and the results are expressed as nmole mevalonolactone incorporated. In the experiments in which mevalonolactone was added triacyIglycero1 was the major product. In the presence of acetate, both phospholipid and t~acyl~y~roI were labeled. n.d., not determined. Substrate added fPM1 MVA 50

loo 200 300 500 Acetate 600

Bile acid fnmoI/mg

(% “1

Chotesterol

Cholesterolester

@moI/mg)

(W medium)

nmoI,/mg

(W medium)

FAb

f~Wmg>

0.99

17.0

4.0

42.5

0.96 1.99 5.50 14.90

10.5 13.0 17.5 28.7

6.5 11.0 20.8 30.0

27.6 23.5 38.5 31.0

0.85 1.73 2.27 4.68 6.09

1.7 2.6 4.1 3.4

n.d. n.d. 12.01 18.01 31.21

0.27

15.6

59.8

0.09

27.2

6.1

l-64

3.3

a The percentage of newfy synthesized cholesterol converted to bile acid is caIcnIated according to: nmol bile acid

acid + nmol cholesterol and cholesterol ester of eelIs and medium. ’ FA, fatty acids, triacylglycerol and phospho~~ids.

(%medium)

2.7 4.8 7.0 53.3 X

lOO,/nmd bile

326

cultures were incubated strates for 3-5 days.

with “C-labeled

sub100

Effect of phenobarbital

Since phenobarbital is known to stimulate cytochrome P-450 synthesis [30], the effect of phenobarbital on cholesterol and bile acid synthesis from mevalonolactone was studied. In the presence of 0.5 mg/ml phenobarbital, bile acid synthesis doubled, while cholesterol synthesis was unchanged. To distinguish between cholesterol synthesis and its conversion to bile acid, cultures were first incubated with [~4C]mev~onolactone for 4 days and the cholesterol pool was labeled. The cells were then washed and incubated for 1 day in fresh medium in the presence of phenobarbital. As can be seen from Table III, in the presence of 1.0 mg/ml phenobarbital bile acid synthesis was increased 2.5fold.

150

00

I

Y

F 1 ii

c

-1 00

_J

100

Conversion of liposomai cholesterol to bile acids

Cholesterol

When hepatocytes were incubated with mixed liposomes of cholesterol and phosphatidylcholine, cholesterol was taken up by the cells and converted into bile acid. The dependence of cholesterol uptake, its esterification and conversion to bile acid on the concentration of cholesterol liposomes is shown in Fig. 3. Whereas cholesterol uptake increased as a function of its concentration in the medium, esterification and bile acid synthesis were maximal at approx. 150 PM liposomal cholesterol. At low exogenous cholesterol concentrations, the

TABLE

liposomes

200 PM

Fig. 3. The effect of increasing concentrations of cholesterol liposomes on cholesterol uptake and bile acid synthesis. Cells were incubated in M-199 for 5 days with different amounts of chol~terol/phosphatidyl~holine (1: 10, molar ratio) liposomes. 0. cellular cholesterol; cellular cholesterol ester; A. bile acid.

conversion of the absorbed cholesterol to bile acid is very efficient and relatively little accumulated inside the cells. As the concentration of cholesterol was increased, its conversion to bile acid became

III

THE EFFECT

OF PHENOBARBITAL

ON BILE ACID

SYNTHESIS

In the first experiment, cultures were incubated with 400 pm mevalonolactone for 5 days. Phenobarbital (PB) was added to one dish. In experiment 2, the cells were preincubated for 4 days with mevalonolactone. Fresh medium was then added and the secretion of bile acid, cholesterol and cholesterol was measured afater 1 day. The prelabeled cells contained, per mg protein, 11.4 nmol [‘4C]cholesterol and 2.85 nmol cholesterol ester. Expt.

Phenobarbital added

Bile acid (nmol)

(mg/ml) I

None 0.5

1.49 3.32

2

None 0.5 1.0

0.20 0.27 0.53

Cholesterol

Cholesterol

ester

(nmol)

(% released)

(nmol)

(% released)

13.13 13.96

21.2 12.8

2.53 4.66

17.1 4.0

14.2 12.7 14.9

5.5 1.3 2.3

321

less efficient. In these experiments, the major bile acid formed was taurochenodeoxycholic acid; it was accompanied by smaller amounts of taurocholic acid. In addition, cosiderable amounts of so-far unidentified conjugated bile acid were also formed (Table I). The dependence of bile acid formation on the time of incubation is shown in Fig. 4. Effect of mevinolin

Table IV shows the effect of mevinolin on liposomal cholesterol uptake and conversion to bile acid. At the concentration used, mevinolin completely inhibited cholesterol synthesis from acetate, while the incorporation of acetate into fatty acids, triacylglycerol and phospholipids was slightly augmented. Mevinolin did not effect cholesterol uptake but its conversion to bile acid was reduced by 30%. Simultaneous utilization of cholesterol and mevalonolactone When [ 3HIcholesterol-liposome and [ l4 C]meva-

lonolactone were added simultaneously, the conversion of mevalonolactone to cholesterol and the release of the newly synthesized cholesterol and cholesterol ester into the incubation medium was markedly reduced. However, the conversion of newly synthesized cholesterol to bile was augmented (Table V). [ 3H]Cholesterol uptake was not effected by the presence of mevalonolactone; however, its conversion to bile acid was stimulated. Mevalonolactone alone yielded 0.94 nmol bile acid and cholesterol liposomes 0.38 nmol. When added together, 0.62 nmol were formed from mevalono-

Time:

hours

Fig. 4. The effect of increasing time of incubation on bile acid synthesis from cholesterol liposomes. Ceils were incubated in M-199 with (a) 30 pM cholesterol liposomes, (b) 150 pM cholesterol liposomes. l, cellular cholesterol; 0, cellular cholesterol ester; A, bile acid.

lactone and 0.77 nmol from cholesterol. Thus, the total amount formed from the two substrates together was 1.38 nmol, equal to the sum of bile acid formed separately from each substrate in different dishes. Table V shows the results obtained after 24 h of incubation. Similar effects were seen after 2.5 and 5 h of incubation. These data suggest that under our experimental conditions the capacity of the hepatocytes to make bile acid has not been saturated. To try and evaluate the contribution of the endogenous cholesterol pool, hepatocytes were incubated with [3H]cholesterol liposomes for 3 days. Fresh medium containing either [‘4C]meva-

TABLE IV EFFECT OF MEVINOLIN ON THE CONVERSION OF CHOLESTEROL TO BILE ACID Cells were incubated for 4 days in the presence of [14C]acetate or [‘4C]cholesterol liposomes as indicated Sterols indicated cholesterol and cholesterol ester ’ inside the cells b inside the cells + secreted into the medium. FA indicates triacylglycerol and phospholipids calculated as equivalents of fatty acids. n.d., not determined. Substrate (PM)

Mevalonolactone (PM) 100

1.87 a 2.06 a

FA (nmol/mg) _ _

100

7.78 b 0

1.34 b 2.07 b

Cholesterol (300 pM Acetate (600 @I)

Sterols

Bile acid

B of cell sterols

3.88 2.66

61.5 57.1

n.d. n.d.

328

TABLE

V

SIMULTANEOUS

UTILIZATION

OF [‘4]M~VALONOLACTONE

AND

[ ‘HICHOLESTEROL

LIPOSOMES

Hepatocyres were incubated in M-199 for 3 days. 400 pM mevalonola~tone and/or 150 pM cholesterol tiposomes were added. When mevalonolactone (MVA) and liposomes were added simultaneously. the results were calculated separately for [‘4C]mevalonolactone (line 2) and [ -‘H)cholesterol (line 4). Total bile acid formed, 0.62+0.77 nmol/mg. Substrate added

Products

(nmol/mg)

cholesterol

cholesterol

cell [‘4C]MVA [14H]MVA ( + liposomes)

[’ HjCholesterol [’ HJCholesterol

ester

bile acid

9; of total

medium

cell

medium

5.80

5.16

0.59

0.94

0.94

6.5 a

38.6 2.00 1.9

1.78 _ _

0.36 0.18 0.12

0.12 2.00 0.80

0.62 0.38 0.77

9.2 a 15.8 h 28.7 h

( + MVA) ’ % of total cholesterol synthesized: bile acidX lOO/(cholesterol h % of cholesterol and cholesterol inside cells.

+cholesterol

lonolactone or [ l4 Clcholesterol liposomes was then added and the metabolism of the endogenous [3H]cholesterol and of the newly acquired or synthesized [‘4C]cholesterol was studied. Addition of mevalonolactone did not alter the metabolism of the [3H]cholesterol. Addition of Iiposomal cholesterol, however, almost completely abolished the release of cholesterol and cholesterol ester from the cells and stimulated the conversion of endogenous cholesterol to bile acid. Cholesterol synthesis from [i4C]mevalonolactone by ‘preloaded’ cells was considerably lower than in cells TABLE

ester of cells and medium)+biIe

acid.

which had not been exposed to cholesterol. .Bile acid synthesis was slightly reduced (Table VI). [‘4C]Cholesterol uptake, however, was unchanged but its conversion to bile acid was markedly stimulated, confirming in vivo data showing the stimulation of bile acid synthesis by feeding cholesterol to rats. Bile acid s.vnth~sis from endoggno~s ~hoiest~rol and ~el?a~ono~actone In the previous experiment (Table VI). an attempt was made to label the endogenous

VI

METABOLISM

OF [‘4C]MEVALONOLACTONE

OR [‘4C]CHOLESTEROL

BY PRELOADED

HEPATOCYTES

Hepatocytes were incubated with 150 pM [ ‘H]cholesterol liposomes for 3 days. The medium was removed. cultures washed with fresh or 150 pM [‘4C]cholesterol liposomes. The metbolism of medium and incubated inthe presence of 400 PM (‘JC] mevalonolactone mevalonolactone (MVA) and cholesterol liposomes by hepatocytes of the same batch which had not been preincubated with liposomes can be seen in the previous table. Percent conversion to bile acids was calcualted as previously described. Time

(h)

Substrate added

[’ H]cholesterol

Endogenous cholesterol

’

cholesterol

Exogenous

(nmol/mg) ester a

bile acid

%

substrate

(nmol/mgf

cholesterol

chol-

ester01 20 70 20 70 20 70

None None [14C]MVA [‘JC]MVA 1“C]Cholesterol [‘4C]Cholesterol

3.78 3.74 2.66 3.69 1.50 1.20

0.30 055 0.37 0.60 0.32 1.06

0.23 0.31 0.20 0.36 0.26 0.60

5.2 6.5 6.2 7.x 12.4 21.0

5.42 J 11.43’ 1.76 h 3.35 h

0.95 2.09 0.10 0.85

ester

bile acid

CT’)

,’ ’ h h

0.28 0.72 1.69 5.00

4.1 5.1 475 54.3 --

“ Calculated ’ Calculated

for cells and medium. for cells only.

329

cholesterol pool by preincubation with [ 3H]cholesterol liposomesl assuming complete mixing of the newly acquired cholesterol with the endogenous pool. In the following experiments, bile acid formation was measured qu~titatively using HPLC. Hepatocytes were incubated in the presence of [ l4 Clmevalonolactone. Bile acids were isolated according to method 2 and separated by HPLC. Two distinct peaks corresponding to taurocholic acid (peak 3) and taurochenodeoxycholic acid (peak 4) were obtained. Both peaks contained 14C. However, as can be seen from Fig. 5, considerable amounts of radioactivity were eluted from the RP-18 column in front of taurocholic acid as a broad peak. Assuming that the specific radioactivity of the bile acid formed from [ “C]mevalonolactone is identical to that of the added mevalonolactone, we calculated the amounts of bile acid formed from mevalonolactone and from the endogenous (non-labeled) cholesterol pool. As can be seen from Fig. 6, [14C]taurocholic

acid and taurochenodeoxycholic acid were formed at a molar ratio of 1: 3. However, the endogenous cholesterol pool was metabolized mainly to taurocholic acid. Upon addition of mevalonolactone, taurochenodeoxycholic acid was also formed and, as the concentration mevalonolactone was increased, the amounts of taurochenodeoxycholic acid formed from this pool increased, while those of taurocholic acid remained almost constant or dropped slightly. Thus, while the total amounts of bile acid secreted in the presence of mevalonolactone increased, the contribution of endogenous cholesterol changed relatively little, suggesting that the newly synthesized cholesterol did not mix freely with the endogenous pool. Uptake and metabolism of HDL. cholesterol: conversion to bile acids HDL cholesterol is readily taken up by cultured hepatocytes. The greater part of the absorbed a

I

t

'0

8

Y

b

12

24

I

i

2

t

0

0

/\

I

4

2

4

MVA: my

Fig. 5. Separation by HPLC of “C-labeled bile acids synthesized from [t4C]mevalonoIactone or [i4C]cholesterol HDL. Hepatocytes were incubated for 3 days in medium F-10. Bile acids were separated by HPLC according to method 2. The results are expressed as nmol/mg cellular protein and were calculated, assuming that the specific radioactivity of the bile acids formed was identical to that of the precursor [i4C]mevalonolactone or [i4C]cholesterol HDL. Peak 3, taurocholic acid; peak 4, taurochenodeoxycholic acid; peaks 1, 2 not identified. 0, bile acids synthesized in the presence of 4 mM [‘4C]mevalonolactone. X , bile acid synthesized from 72 pM [t4CJcholesterol HDL.

Fig. 6. The effect of increasing inundations of mevalonolactone @#VA) on the synthesis of taurochenodeoxycholic acid and taurocholic acid from endogenous cholesterol and from mevdonolactone. Cells were incubated for 3 days in medium F-10. Bile acids were isolated according to method 2. (a) cholesterol and bile acid synthesis. A, cellular cholesterol; #, cellular cholesterol ester; 0, taurocholic acid and taurochenodeoxycholic acid from endogenous cholesterol; 0, taurocholic acid and taurochenodeoxycholic acid from endogenous cholesterol + [ I4Clmevalonolactone. (b) l, [I4 Cltaurocholic acid; 0, taurochohc acid from endogenous cholesterol; A, [14C]taurochenodeoxychoIic acid; A, taurochenodeoxycholic acid from endogenous cholesterol. The results are calculated as described in the legend to Table VII.

330

cholesterol was converted to bile acid and only relatively small amounts were retained by the cells as free cholesterol or cholesterol ester (Fig. 7). Bile acid synthesis from HDL cholesterol was directly related to the amount of HDL in the medium. The results obtained in the first experiment suggested that at HDL concentration above 50 PM cholesterol, the utilization of the endogenous cholesterol pool for bile acid synthesis was suppressed. Thus, in the second experiment, the HDL cholesterol concentrations were increased to 150 PM. The results of this experiment will be discussed in detail. Separation by HPLC of the bile acid synthesized from [t4C]cholesterol yielded, in addition to taurocholic acid and taurochenodeoxycholic acid (the identification of which was confirmed by TLC), at least two additional bile acids which were eluted from the RP-18 columns with a retention time shorter than taurocholic acid. Peak 1 almost coincided with the solvent front. In the experiments in which [ 14C]mevalonolactone or

60

r

HDL-

cholesterol:

p M_

Fig. 7: The effect of increasing concentrations of HDL on bile acid synthesis. The cells were incubated for 3 days in F-10. Bile acids were isolated according to method 2. 0, cellular j’4C]cholesterol; 0, cellular [‘4C]cholesterol ester; A. 14Clabeled bile acid (calculated as sum of radioactivity in peaks 1. 2. 3 and 4).

[ “C]cholesterol liposomes were added to the incubation medium, the amount of radioactivity isolated in this area was always less than that contained in taurocholic acid and taurochenodeoxycholic acid (Fig. 5). However, these peaks became very prominent when hepatocytes were incubated with [t4C]cholesterol HDL and, at high concentrations of HDL, contained lo-times more radioactivity than taurochenodeoxycholic acid (Table VII). The specific radioactivity of bile acid included in peak 2 was almost identical to that of the exogenous [‘4C]cholesterol added. Thus, it was synthesized from HDL cholesterol and not from the endogenous cholesterol pool. The amounts corresponding to peak 1 could not be measured, since nonspecific ultraviolet-absorbing materials were always eluted at the solvent front. The retention time of peak 2 coincided with that of tauroursodeoxycholic acid. The material contained in these peaks was recovered and separated by TLC under conditions employed by Batta et al. [31] for the separation and identification of tauroursodexocholic acid. Most of the radioactivity of peak 2 migrated in a zone similar to glycocholic acid. However. marker [ 3H]glycholic acid was eluted from the RP-18 columns with a different retention time. Peak 1 yielded some radioactivity in the tauroursodeoxycholic acid zone; however, most of the radioactivity showed a clear peak with slower mobility. As can be seen from Table VII, taurocholic acid was the major bile acid formed by the hepatocytes from the endogenous cholesterol pool; taurochenodeoxycholic acid was formed in much smaller amounts. Addition of HDL to the incubation medium markedly suppressed taurocholic acid formation and, in the presence of 144 nmol/ml HDL cholesterol, only 0.8 nmol taurocholic acid were formed from the endogenous cholesterol pool. In contrast. taurochenodeoxycholic acid synthesis from both the endogenous cholesterol and from HDL increased as a function of the concentrations of HDL cholesterol in the medium. However, the contribution of the endogenous cholesterol to the total amount of bile acid secreted decreased constantly. This observation suggests that HDL cholesterol became the preferred source for bile acid formation. However, if HDL cholesterol rapidly equilibrated with cellular cholesterol [32],

331 TABLE VII BILE ACID SYNTHESIS FROM HDL CHOLESTEROL Hepatccytes were incubated in medium F-10 for 3 days. Bile acids were separated by HPLC according to method 2. For the identification of peaks 1 to 4 (given in parentheses) see Fig. 5. a, nmol/mg calculated from: total radioactivity in the peak/specific radioactivity of HDL cholesterol, assuming that the specific radioactivity of the bile acid formed is equal to that of HDL cholesterol. b, nmol/mg calculated from the HPLC data (area under the peaks minus nmol/mg calculated from the radioactivity data (a). TC, taurocholic acid; TCDC, taurochenodeoxycholic acid. Total a+b

Bile acid formed (nmol/mg)

HDL

TCDC(4)

cholesterol

(I) =

(2) a

TC (3) b

(yg/mB

(nmol/mB

a

a

a

b

a

b

None 154 462 924

None 24 72 144

5.14 19.48 41.40

0.97 3.33 8.98

0.84 1.73 4.19

10.5 11.7 3.5 0.8

0.33 1.16 2.96

1.0 3.1 4.6 1.8

protein

the data obtained merely reflect the mixing of the two pools, and our assumption that the specific radioactivity of the bile acid produced from [‘4C]cholesterol is identical to that of the HDL cholesterol is inaccurate and corrections taking into consideration the endogenous cellular cholesterol are required. At low HDL concentrations, this factor would be quite considerable. Discussion

In the experiments presented in this paper, the biosynthesis of bile acids by cultured chick embryo hepatocytes was investigated. The availability of endogenous and newly synthesized as well as liposomal and HDL cholesterol as sources for bile acid synthesis were compared. A comparison of the results obtained with rat and chick embryo hepatocyte shows that in chick embryo preparations the rate of bile acid synthesis was much higher than in the rat hepatocyte. Furthermore, in our experiments, the bile acids were secreted into the medium; none were detected inside the cells. This is in contrast to the result of Davis et al. [16] who detected small amounts of bile acid inside cultured rat hepatocytes. The method of Davis et al. [16,17] included quantitative estimation of the isolated bile acids by gas chromatography. This method gave an accurate measure of bile acid secretion, and it showed the overall effect of the addition of mevalonolactone or lipoproteins to the incubation medium. However, it could not differentiate between the bile

11.5 23.4 33.8 66.2

acids which were synthesized from the endogenous cholesterol pool and those which were formed from newly synthesized cholesterol or lipoprotein cholesterol. We tried to overcome this difficulty by adding 14C- or 3H-labeled precursors to the medium and measuring simultaneously the amounts and radioactivity of the isolated bile acid. Assuming that the specific radioactivity of the precursor mevalonolactone or HDL-cholesterol and the bile acid produced from them was identical, we attempted to calculate the contribution of the endogenous cholesterol pool and of the external sources. Cultured rat hepatocytes secreted daily 0.35 nmol/mg protein of cholic acid and 0.30 nmol/mg muricholic acid (which is formed from taurochenodeoxycholic acid by rat hepataocytes and can be compared with that secreted by the chick embryo hepatocytes). Upon addition of 10 mM mevalonolactone, bile acid secretion was increased by 50% and muricholic acid became the major product. In chick embryo hepatocytes, 3.5 nmol/mg taurocholic acid and 0.3 nmol/mg taurochenodeoxycholic acid were formed daily from endogenous cholesterol. Approx. 3-times more [‘4C]taurochenodeoxycholic acid than [i4C]taurocholic acid were synthesized from [i4C]mevalonolactone. Addition of mevalonolactone also induced taurochenodeoxycholic acid synthesis from endogenous cholesterol. Whereas the synthesis of taurocholic acid did not change significantly, taurochenodeoxycholic acid synthesis increased as a function of mevalonolactone con-

332

centration and approached that of taurocholic acid (2.5 nmol/mg). Thus, whenever larger amounts of cholesterol were available, taurochenodeoxycholic acid became the favoured product. The results of Davis et al. [16,17] and our own results indicating a marked difference in taurocholic acid and taurochenodeoxycholic acid synthesis in response to the addition of mevalonolactone or HDL support the suggestion of Mitropolus et al. [4] that taurocholic acid and taurochenodeoxycholic acid were synthesized from different endogenous pools. It has been shown by Endo et al. [33] and by Alberts et al. [34] that compactin and its methylated derivative mevinolin inhibited /3-hydroxy, /?methylglutaryl-CoA reductase, thus inhibiting cholesterol synthesis from acetate. Kempen et al. [15] showed that compactin also inhibited the conversion of cholesterol to cholic and muricholic acid by isolated rat hepatocytes. A similar observation was made by Davis et al. [14] who showed a 30% reduction in cholic and muricholic acid secretion by cultured rat hepatocytes. We obtained a similar effect of mevinolin on the conversion of liposomal cholesterol to bile acid. Phenobarbital has been shown to induce the synthesis of several cytochrome-P-450-containing enzymes [30]. Shefer et al. [35,36] demonstrated that interperitoneal injection of phenobarbital to rats of the Wistar strain resulted in a marked stimulaion of the cholesterol 7a-hydroxylase activity. This stimulation was not seen in rats of the Sprague-Dawley strain. Rabbit liver microsomes showed a very low 7a-hydroxylase activity [37]; however, the 12a-hydroxylase of 7a-hydroxy-4-cholestene-3-one was stimulated by phenobarbital treatment. In chick embryo hepatocytes, phenobarbital stimulated the conversion of mevalonolactone and of newly synthesized cholesterol to bile acid. In contrast to the results with cultured rat hepatocytes [14,15], in our experiments, HDL cholesterol was a superior source for bile acid synthesis and the greater part of the cholesterol which was incorporated by the cells was converted to bile acid. Upon addition of HDL, a shift from taurocholic acid synthesis to taurochenodeoxycholic acid occurred; taurocholic acid synthesis from endogenous cholesterol was reduced from 3.5 nmol/mg to 0.3 nmol/mg and taurochenodeoxycholic acid synthesis increased from 0.3

nmol/mg ot 2.6 nmol/mg. Furthermore, large amounts of two bile acids, so far unidentified by us, were formed by the hepatocytes from HDL cholesterol. A comparison of the bile acid composition of biles of different animals compiled by Haslewood [38] shows that in germ-free domestic chicks taurocholic acid, taurochenodeoxycholic acid and tauroallocholic acid are the major components. In contrast, in germ-free rats, the bile acids contained 61% of tauro-/3-muricholic acid, traces of a-muricholic acid and 30% of taurocholic acid. Furthermore, the conversion of a-hydroxyl[14C]cholestanol to allocholic acid in hens with a bile fistula [39] and of cholesterol to allocholic acid by rat liver homogenates [40] have been shown. These observations suggest that the two unidentified bile acid peaks might be identical with allo-bile acids. Similar peaks were obtained when the conjugated bile acid of chick embryo bile were separated by HPLC. The very efficient conversion of HDL cholesterol to bile acid by chick embryo hepatocytes is in accordance with the observations of Schwartz et al. in man [7-91 and of Portman et al. [lo] in squirrel monkeys, which indicated that in vivo HDL cholesterol was the preferred source for biliary bile acid and cholesterol. The delivery of HDL cholesterol to the liver can occur in two different ways: (1) exchange of free cholesterol between plasma lipoproteins and cellular plasma membranes, (2) internalization of plasma lipoproteins and liberation of free cholesterol by proteolytic and hydrolytic activities of lysosomes [l]. The occurrence of the exchange process was studied by O’Malley et al. [32] in primary cultures of rabbit hepatocytes. Cells which had been incubated with HDL labeled with 3H in the protein moiety and with [‘4C]cholesterol showed an g-fold selectivity for the lipid. The uptake and degradation of HDL cholesterol ester has not been studied directly. In most experiments, the apolipoproteins were labeled with 125I or [‘251]tryaminecellobiose [41-441, whereas the cholesterol ester was labeled with cholesterol linoleyl ether which could not be degraded’ by lysosomal enzymes. In vivo experiments by Glass et al. [43] showed that, whereas the cholesterol ether was taken up by the liver, adrenals and ovary, the apolipoprotein A-I was found in the kidney. The

333

disparity between cholesterol ether and apolipoprotein uptake was also observed by Leitersdorf et al. [42] in cultured rat hepatocytes and adrenal cells. Binding and degradation studies of apolipoprotein E-free HDL by cultured hepatocytes of rats, rabbits and pigs [43,44] indicated that HDL was bound and degraded by a specific saturable process. However, relatively large amounts of protein were necessary. Protein degradation was inhibited by chloroquine [44], suggesting the involvement of lysosomes. In preliminary experiments, we compared the availability of HDL cholesterol and HDL cholesterol ester and showed that also HDL cholesterol ester provided cholesterol for bile acid synthesis. . The conversion of HDL cholesterol and HDL cholesterol ester to bile acid was not inhibited by chloroquine (100 PM). The mechanism of HDL degradation by chick embryo hepatocytes is now under investigation. Our results suggest that cultured chick embryo hepatocytes are a very good model for studying the control of cholesterol conversion to bile acid. There is however one important drawback in the cultured hepatocyte systems. Whereas in the intact organs bile acids are secreted into a limited space of the bile canaliculi and they do not mix with plasma proteins, in cultured hepatocytes bile acids as well as VLDL are secreted and diluted by the large volume of the medium. Thus, it seems unlikely that the secreted bile acids induce the secretion of phosphatidylcholine and cholesterol to form mixed micellesl Thus, it will be difficult to study the correlation between bile acid and cholesterol secretion, an observation which seems of great importance in the understanding of gallstone formation. Acknowledgements

This work was supported by a grant from the Israel Academy of Sciences. Mevinolin was kindly given to us by Dr. A.W. Alberts of the Merck, Sharp and Dome Research Laboratories, Rakway, NJ. References 1 Brow, M.S. and Goldstein, 743-747

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