Departments of Nutrition, Biochemistry, and Pediatrics, Centre de Recherche, Hôpital Ste-Justine, Université de Montréal, Montreal, Quebec, Canada H3T 1C5
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ABSTRACT |
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Immortalized bile duct cells (BDC), derived from
transgenic mice harboring the SV40 thermosensitive immortalizing mutant
gene ts458, were utilized to investigate the
role of the biliary epithelium in lipid and sterol metabolism. This
cell model closely resembles the in vivo situation because it expresses
the specific phenotypic marker cytokeratin 19 (CK-19), exhibits the
formation of bile duct-like structures, and displays well-formed
microvilli projected from the apical side to central lumen. The BDC
were found to incorporate [14C]oleic acid (in
nmol/mg protein) into triglycerides (121 ± 6), phospholipids (PL;
59 ± 3), and cholesteryl ester (16 ± 1). The medium lipid
content represented 5.90 ± 0.16%
(P < 0.005) of the total
intracellular production, indicating a limited lipid export capacity.
Analysis of PL composition demonstrated the synthesis of all classes of
polar lipids, with phosphatidylcholine and phosphatidylethanolamine accounting for 60 ± 1 and 24 ± 1%, respectively, of the total. Differences in PL distribution were apparent between cells and media.
Substantial cholesterol synthesis was observed in BDC, as determined by
the incorporation of
[14C]acetate
suggesting the presence of hydroxymethylglutaryl-CoA (HMG-CoA)
reductase, the rate-limiting enzyme in the cholesterol biosynthetic
pathway. With the use of
[14C]acetate and
[14C]cholesterol as
precursors, both tauro- and glycoconjugates of bile acids were
synthesized, indicating the presence of cholesterol 7- and
26R-hydroxylases, the key enzymes involved in bile acid formation. The
transport of bile acids was not limited, as shown by their marked
accumulation in the medium (>6-fold of cell content). HMG-CoA
reductase (53.0 ± 6.7), cholesterol 7
-hydroxylase (15.5 ± 0.5), and acyl-CoA:cholesterol acyltransferase (ACAT; 201.7 ± 10.2) activities (in
pmol · min
1 · mg
protein
1) were present in
the microsomal fractions. Our data show that biliary epithelial cells
actively synthesize lipids and may directly contribute bile acids to
the biliary fluid in vivo. This BDC line thus represents an efficient
experimental tool to evaluate biliary epithelium sterol metabolism and
to study biliary physiology.
biliary epithelium; triglyceride; phospholipid; bile acid; sterol
metabolism; hydroxymethylglutaryl-CoA reductase; cholesterol
7-hydroxylase; acyl-CoA cholesterol acyltranferase
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INTRODUCTION |
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THE LIVER PLAYS A CENTRAL ROLE in the metabolism of plasma lipids and lipoproteins, serving as the major site of synthesis of several apoprotein components and various lipid species (13, 40, 41). It also secretes nascent lipoproteins (very-low-density lipoproteins and high-density lipoproteins) and degrades chylomicron remnants, as well as low- and high-density lipoproteins, after their uptake (9, 11). Furthermore, the liver also serves as a delivery system for the regulatory enzymes and conversion factors that promote and control the metabolic relationships and remodeling of plasma lipoproteins (26, 45). Finally, hepatic biliary cholesterol secretion and bile acid synthesis constitute the main routes of cholesterol elimination from the body (24, 33, 48).
Disturbances of plasma lipid and lipoprotein composition frequently occur in patients with chronic cholestatic liver disease (14, 20, 38, 40, 41). Despite the association between primary biliary cirrhosis and other cholestatic syndromes with major lipid and lipoprotein derangements, little is known about the contribution of the biliary epithelium to the metabolic disturbances. Although they make up only 3-5% of the overall population of liver cells, bile duct cells (BDC) provide a large surface area for exchange between blood and bile. They play a key physiological role in the formation of bile, producing as much as 40% of the daily volume (32, 47). BDC, in close proximity to the hepatic arterioles that serve as their vascular supply, also display absorptive and secretory capabilities (47). Furthermore, increasing evidence has been put forth indicating that BDC can modify the composition of bile by secreting water, protein, and bicarbonate and reabsorbing glucose, glutamate, and anions (8, 43, 46, 47, 49, 50). Although bile is a complex mixture of organic compounds, studies on biliary epithelium reported to date have been largely restricted to bile flow and ion permeability (8, 43, 46, 49, 50). It is noteworthy that bile flow is the result of plasma/bile transport of bile acids and other solutes, which create osmotic gradients that stimulate bile formation (4).
Progress in understanding the cellular and molecular basis of biliary epithelium metabolism and transport of sterols and lipoproteins has lagged behind studies on hepatocytes, largely because of technical problems in isolating pure BDC. However, the recent availability of immortalized BDC, originating from H-2Kb-ts458 transgenic mice (34), allows for more rigorous studies of the biology and function of the biliary epithelium. In the present study, we utilized this cell model to examine the synthesis of neutral lipid and phospholipid classes, as well as the production and conjugation of bile acids.
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METHODS |
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Cell culture. The immortalized BDC
line, originating from the transgenic mouse harboring the SV40
thermosensitive mutant gene ts458, was established as
previously described (22). Cells were allowed to grow on Matrigel in
75-cm2 ventilated flasks (Corning
Costar, Cambridge, MA). The culture medium consisted of a 50:50 mix of
Dulbecco's modified Eagle's medium with L-glutamine and
D-glucose-Ham's nutrient mixture F-12 with
L-glutamine (0.1 mmol/l), minimal essential medium
nonessential amino acid solution,
D-glucose (5.4 g/l), and HEPES
(10 mmol) buffer adjusted at pH 7.40. The medium was supplemented with
100 µg/l penicillin G and 100 µg/ml streptomycin sulfate (both from GIBCO, Grand Island, NY); 10 µg/ml epidermal growth factor, 5 ng/ml
each of insulin and transferrin, and 5 µg/ml selenium (all from
Collaborative Biomedical Products, Bedford, MA); and 32 ng/ml thyroxin,
10 ng/ml prostaglandin E1, 40 ng/ml hydrocortisone, and 10 µg/ml mouse recombinant interferon-
(all from Boehringer Mannheim). Cells were grown at
33°C and were used for experimental studies after confluence
(7-10 days). Their viability was assessed by trypan blue exclusion.
Immunofluorescence. Standard immunofluorescence microscopy techniques were used to detect cytokeratin-19 (CK-19), albumin, and macrophage F4/80 antigen. Briefly, cells were washed with PBS at pH 7.40, fixed in cold acetone for 10 min, and air dried. After nonspecific antibody binding was blocked, the slides were incubated with primary mouse anti-CK-19 (Amersham, Oakville, ON, Canada), the macrophage-specific antibody F4/80, or anti-albumin (Cedarlane Laboratories, Hornby, ON, Canada). This was followed by incubation with fluorescein isothiocyanate-labeled sheep anti-mouse immunoglobulin G2b (Biodesign, Kennebunkport, ME). Slides were washed with PBS, mounted, and photographed.
Electron microscopy. Cells with apparent ductlike structures were fixed in glutaraldehyde, embedded in Epon, and then sectioned using routine methods for electron microscopy (23).
Lipid synthesis. [14C]oleic acid (specific activity 53.9 mCi/mmol; Amersham, Montreal, PQ, Canada) complexed with albumin (25) was added to the medium (final specific activity 1,000,000 dpm/mol). Lipids were extracted from aliquots of cell homogenates and their respective incubation media with chloroform-methanol (2:1, vol/vol) (25). Tracer amounts of lipid standards were added to the samples before separation of individual lipid classes by unidimensional thin-layer chromatography (TLC) (silica gel, Eastman-Kodak, Rochester, NY) as described previously (25). The apolar solvent system was hexane-diethylether-glacial acetic acid (80:23:3, vol/vol/vol) and the polar solvent was chloroform-methanol-water-acetic acid (65:25:4:1, vol/vol/vol/vol). After scraping, the radioactivity of the separated fractions was measured in a liquid scintillation counter (Beckman Instruments, Mississauga, ON, Canada). Quench correction was done using computerized curves generated with external standards. Proteins were measured according to Lowry et al. (27) using BSA as standard. Results are expressed as disintegrations per minute per milligram protein.
Determination of cholesterol and bile acid synthesis. BDC cholesterol biogenesis was evaluated employing [14C]acetate as precursor (53.4 Ci/mmol) after a 20-h incubation period. Bile acid synthesis was assessed by measuring the incorporation of [14C]acetate or the conversion of [14C]cholesterol (2 µCi) into acidic products that were extracted into H2O-MeOH (21, 22). Tracer amounts (12 ng) of bile acid standards (taurolithocholic, taurochenodeoxycholic, taurocholic, glycolithocholic, glycochenodeoxycholic, glycocholic, lithocholic, chenodeoxycholic, and cholic acids) were added to the extract before individual bile acid classes were separated by unidimensional TLC using two consecutive migration solvent systems. The apolar solvent system was composed of isoctane:isopropyl ether-isopropanol:acetic acid (1:1:1:1, vol/vol/vol/vol), and the polar solvent was chloroform-methanol-water-acetic acid (65:25:4:1, vol/vol/vol/vol). The radioactive spots corresponding to the migration of bile acid standards were visualized by iodine vapor, scraped, and counted.
Preparation of BDC microsomes.
Cultured cells were removed and placed in ice-cold buffer (pH 7.4)
containing (in mmol/l) 250 sucrose, 20 Tris · HCl, 1 EDTA, 5 glutathione, and 20 dithiothreitol. Cells were rinsed,
homogenized, and centrifuged for 15 min at 12,000 g at 4°C. The supernatant fraction
was then centrifuged for 60 min at 100,000 g at 4°C as described previously
(21). The last step was repeated once. The purity of the microsomal fraction was assessed by the determination of glucose-6-phosphatase activity. The washed microsomal pellets were quick-frozen and stored at
80°C before use.
Enzyme activity assays. Microsomal
activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase,
cholesterol 7-hydroxylase, and acyl-CoA: cholesterol acyltransferase
(ACAT) was determined as previously described (21).
Statistics. Statistical evaluation of the results was performed by the Student's two-tailed t-test.
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RESULTS |
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Morphology and immunofluorescence
studies. Morphological properties characteristic of
bile duct epithelial cells were observed in our BDC line. The majority
of BDC consisted of homogeneous populations of small round cells,
readily distinguished from hepatocytes by their size and morphology.
The epithelial origin of the immortalized cell line was confirmed by
immunofluorescent microscopy technique showing the presence of CK-19
(Fig. 1, A
and B). Negative staining using
specific antibodies against F4/80 and albumin excluded contamination by
macrophages and hepatocytes, respectively. After the first 24-h period
in culture, BDC formed small islands on Matrigel, a basement membrane
gel complex. Thereafter, they developed branched, ductlike structures
(Fig. 1C). Electron microscopy
revealed well-formed microvilli and apical tight intercellular
junctions (Fig. 1D).
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Lipid synthesis and secretion. To
determine whether biliary epithelial cells had the ability to
synthesize and secrete newly formed lipids, BDC were incubated with
[14C]oleic acid. The
incorporation of this radioactive substrate into BDC was linear over
time for up to 20 h (results not shown). We observed substantial
incorporation into cellular triglycerides, phospholipids, and
cholesteryl esters (Fig. 2). In all
experiments, the amount of lipids was higher in cells than in the
media, suggestive of a limited secretory capacity of BDC. Table
1 depicts the composition of total lipids
analyzed by TLC. Triglycerides were the predominant lipids, followed by
phospholipids and cholesteryl esters. The same profile was observed for
both cells and media. Although the medium was slightly enriched in
triacylglycerol, diacylglycerol, and free cholesterol, it had a low
content of phospholipids plus monoacylglycerol and cholesterol ester.
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Phospholipid profile. The BDC were
also found to be capable of producing all major phospholipid classes.
The absolute amount of phospholipids synthesized was higher in cells
than in media, the latter accounting for <5% of cell phospholipids
(Table 2). Phosphatidylcholine was the
predominant form of 14C-labeled
lipids elaborated. Major differences were noted in phospholipid composition between the cellular compartment and the media (Fig. 3). The percentage of sphingomyelin,
phosphatidylserine, and phosphatidylinositol was higher in the medium,
whereas the proportion of phosphatidylcholine and
phosphatidylethanolamine was preponderant within the cells.
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[14C]acetate
incorporation into cholesterol. The BDC cholesterol
biogenesis was assessed using
[14C]acetate. As can
be seen in Fig. 4, BDC incorporated
substantial amounts of
[14C]acetate into
cholesterol and cholesteryl ester. These findings confirm the data
(Table 1) obtained with
[14C]oleic acid and
suggest the presence, in BDC, of HMG-CoA reductase and ACAT, the two
key enzymes involved in cholesterol metabolism.
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Bile acid synthesis. BDC were
evaluated for their ability to synthesize bile acids in the presence of
[14C]acetate (Table
3). Several types of bile acids were
produced, including taurolithocholate, taurocholate, glycocholate,
lithocholate, and cholic acid (Fig. 5).
Similar results were obtained using [14C]cholesterol as
substrate (results not shown). The composition of the bile acids in the
cells was quite different from their distribution in the culture
medium. Intracellularly, a preponderance of taurolithocholate,
taurocholate, and lithocholate was found, whereas
glycocholate and glycochenocholate were dominant in the medium.
Overall, the total incorporation of
[14C]acetate into bile
acids in the medium consistently exceeded fivefold that in the cells,
indicating an active bile acid secretory capacity.
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Enzyme activity. We also determined
the activity of the three sterol enzymes that regulate hepatic
intracellular cholesterol homeostasis. The activity of HMG-CoA
reductase, cholesterol 7-hydroxylase, and ACAT was detected in
microsomes isolated from BDC (Fig. 6). The
mean activities measured (in
pmol · min
1 · mg
protein
1) were 53.0 ± 6.7 for HMG-CoA reductase, 15.5 ± 0.5 for cholesterol 7
-hydroxylase, and 201.7 ± 10.2 for ACAT.
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DISCUSSION |
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Until recently, study of the cell biology and function of biliary epithelial cells has been hampered by their inaccessibility. Attempts to isolate BDC have shed light on many properties of BDC (1, 2, 12, 18, 30, 35, 51, 52). However, none of these techniques yield pure populations of homogeneous BDC in sufficient quantity. With one exception (35), they do not recreate the tubular orientation of polarized BDC in vitro. Strategies have thus been designed to develop immortalized BDC clones that would mimic the in vivo situation as closely as possible. In this study, we confirm that immortalized BDC display well-differentiated features of mature biliary epithelium, with definite cellular polarization and formation of ductlike structures, well-developed apical microvilli, and tight junctions. The immortalized BDC also express cytokeratin-19, a phenotypic marker normally found in BDC. This cell line thus provides us with the opportunity to verify the emerging concept that intrahepatic biliary epithelial cells are actively involved in lipid and bile acid metabolism.
In the present study, we demonstrate the ability of BDC to incorporate fatty acids for lipid esterification. The mechanism of cellular uptake of these lipophilic compounds is not yet elucidated. Fatty acids may enter cells by passive diffusion or may be absorbed via an energy-independent, facilitated diffusion mechanism (6, 7, 36). Recent studies have also suggested that a saturable process mediated by specific binding sites on the cell surface is involved in the uptake of the albumin-ligand complex (36). The relative importance of these potential routes depends on the tissue and organ involved and the concentration and properties of the ligand (5, 7, 36). Additional work is needed to define the uptake and translocation of fatty acids to the endoplasmic reticulum for lipid esterification.
Triglycerides are the dominant class of lipids elaborated by BDC when using [14C]oleic acid as precursor. In the liver, after acylation in the rough endoplasmic reticulum, the majority of fatty acids are incorporated into triglycerides via the phosphatidic acid pathway (10). The conversion of phosphatidate to 1,2-diacylglycerol by phosphatidate phosphatase is the rate-limiting reaction of triglyceride biosynthesis. Our data suggest that BDC possess this important anabolic step. Furthermore, the high de novo-formed phospholipid content points to the active intracellular participation of the enzymes required for their synthesis.
In our experiments, the medium lipid content approaches only 7% of intracellular production, indicating a limited lipid export capacity of BDC. In this respect, BDC do not contribute a significant movement of locally manufactured lipids into bile, in contrast to hepatocytes. However, it is possible that lipid reabsorptive mechanisms are also operative in BDC.
Our results provide evidence for the capacity of BDC to synthesize and
secrete bile acids that, qualitatively, are similar to those produced
by hepatocytes. They differ, however, in their relative proportions.
Indeed, in our model, newly synthesized bile acids mostly consist of
lithocholic acid and its tauro- and glycoconjugates (83%). Cholic and
chenodeoxycholic acid derivatives account for ~11 and 6%,
respectively. Javitt (17) have clearly established that there are two
main pathways for bile acid synthesis. The first, which utilizes the
classical 7-hydroxylation pathway, yields cholic and
chenodeoxycholic acids, which account for 95% of the bile acids and
salts synthesized by Hep G2 liver cells in culture (15). It must be
stressed at this point that 7
-hydroxylase activity has been measured
in our cells (Fig. 6). The fact that its specific activity is lower
than that observed in liver microsomes in other models (19) supports
our [14C]acetate
incorporation results. We may thus surmise that this pathway is a minor
one in BDC. The C-26 hydroxylation of cholesterol is the
preferential pathway for the biosynthesis of lithocholic acid (15). It
can also be derived from the 7
-dehydroxylation of chenodeoxycholic
acid by the intestinal bacterial flora (16). Because our culture model
is devoid of such bacterial contamination and because the enzyme
responsible for C-26 hydroxylation of cholesterol is present in a
number of epithelial cells (37), it can legitimately be invoked as a
major pathway in our model.
Under physiological conditions, the coordination of the microsomal
enzymes HMG-CoA reductase, cholesterol 7-hydroxylase, and ACAT is
closely associated with the maintenance of liver cholesterol homeostasis (3). HMG-CoA reductase has been demonstrated to be the
rate-limiting enzyme in cholesterol biosynthesis (39). Cholesterol
7
-hydroxylase, a specific microsomal cytochrome
P-450 isoenzyme, is the initial and
rate-determining enzyme in the bile acid biosynthesis pathway (31, 42).
Based on the failure to observe detectable activity of the cytochrome
P-450 system (28, 29), it was assumed
that the biliary epithelium does not possess the de novo
sterol-synthesizing enzymes, which have not been tested in these
studies. However, our data unequivocally show that labeled bile acids
are formed from a radioactive precursor and that BDC microsomes contain
HMG-CoA reductase and cholesterol 7
-hydroxylase activities.
Furthermore, our results demonstrated the presence of microsomal ACAT,
which is not part of the cytochrome
P-450 system. It is therefore tempting
to speculate that the biliary epithelium, having the capacity to
elaborate and conjugate bile acids, may modify the composition of bile
acids secreted. It is important to emphasize that our BDC model is
devoid of any hepatocytes and macrophages, potential sources of
contamination. These BDC have furthermore been clearly characterized
with regard to their enzyme profiles and do not contain albumin, an
essential characteristic of hepatocytes.
ACAT is the enzyme responsible for the acylation of cholesterol to cholesterol esters, a transformation that strongly influences hepatic excretion of cholesterol (44). Activity of this enzyme is detected in BDC, suggesting a potential role in the biliary epithelium. Activation of ACAT would effectively result in an increment of cholesteryl ester, with deficient conversion to bile acids.
In conclusion, evidence has been presented that BDC synthesize various lipid classes albeit with a limited capacity of exporting them. The biogenesis and secretion of sterols, including bile acids, suggest an active role in the modification of biliary content by the biliary epithelium. This BDC line represents an experimental model to study biliary epithelial cell biology under normal and pathophysiological conditions.
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ACKNOWLEDGEMENTS |
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The authors thank Danielle St-Cyr for expert secretarial assistance.
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FOOTNOTES |
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This work has been supported by an operating grant from Natural Sciences and Engineering Research Council of Canada, Merck Frosst Canada, and Research Scholarship Awards (E. Seidman, E. Levy) from the Fonds de la Recherche en Santé du Québec.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: E. Levy, Gastroenterology-Nutrition Unit, Hôpital Ste-Justine, 3175 Côte Ste-Catherine, Montreal, Quebec, Canada H3T 1C5.
Received 25 June 1998; accepted in final form 21 October 1998.
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