To understand molecular events in regulation of
hepatic neutral cholesteryl ester hydrolase (EC3.1.1.13; CEH),
catalytic activity, protein mass, and mRNA levels were measured in rats
with various perturbations of hepatic cholesterol
metabolism. Cholesterol feeding decreased activity (56 ± 2%), mass (44 ± 2%), and mRNA (14 ± 3%). The cholesterol precursor mevalonate also decreased activity (42 ± 6%), mass (76 ± 3%), and mRNA (23 ± 16%).
Inhibition of cholesterol biosynthesis by lovastatin increased activity
(65 ± 12%) and mRNA (31 ± 24%). Stimulation of cholesterol
efflux by chronic biliary diversion increased activity (138 ± 34%), mass (29 ± 7%), and mRNA (146 ± 28%).
Chenodeoxycholate feeding decreased activity (46 ± 6%) and mRNA
(26 ± 12%). These data suggest rational regulation of CEH in
response to changes in cholesterol flux through the liver. In primary
hepatocytes, steady-state mRNA markedly decreased during 72-h cultures
and addition of L-thyroxine and
dexamethasone synergistically maintained mRNA levels near control
values. Lovastatin increased mRNA levels by 103 ± 15%.
Taurocholate and phorbol 12-myristate 13-acetate suppressed mRNA (61 ± 4% and 49 ± 13%, respectively), suggesting that protein
kinase C mediated effects of bile acids on CEH mRNA levels. These data
suggest regulation of CEH by hormones and signal transduction in
addition to changes in cholesterol flux.
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INTRODUCTION |
THE LIVER PLAYS A CENTRAL role in both the maintenance
of whole body cholesterol homeostasis and the regulation of plasma lipoprotein concentrations. Whereas de novo cholesterol synthesis and
uptake of dietary cholesterol (as lipoproteins) represent the two input
pathways, conversion of hepatic cholesterol to bile acids and biliary
secretion of cholesterol are the only significant output pathways. In
response to changes in cholesterol influx or efflux, sterol balance
across the hepatocyte is maintained by altering the flux of cholesterol
through 1) endogenous cholesterol synthesis, 2) lipoprotein uptake,
synthesis, and secretion, 3) conversion of cholesterol to bile acids, and
4) reversible conversion of excess
cholesterol to cholesteryl esters. Whereas hepatic free cholesterol and
cholesteryl esters are maintained in dynamic equilibrium, a regulated
flux of cholesterol through these hepatic pools not only maintains free
cholesterol levels within the hepatocyte but also influences the
secretion of cholesteryl esters as components of plasma lipoproteins.
Neutral cholesteryl ester hydrolase (CEH) is the key enzyme
required for releasing the pool of metabolically active free
cholesterol from intracellular stores of cholesterol esters,
providing substrate for bile acid synthesis and for biliary secretion of cholesterol. A second free cholesterol pool is derived from the de novo synthesis of cholesterol, which is regulated at the level of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR). It has been suggested that
newly synthesized cholesterol is the preferred substrate for
cholesterol 7
-hydroxylase (C7
H), whereas free cholesterol
released by the hydrolysis of cholesteryl esters is the proximal source
of biliary cholesterol (31, 24). Treatments that affect cholesterol
flux through the liver (e.g., cholesterol, bile acid, or cholestyramine
feeding or lovastatin infusion) lead to compensatory changes in the
expression of C7
H and HMGCoAR (16, 27, 35). Although C7
H appears to be regulated largely by changes in the mRNA levels and the contribution of posttranscriptional mechanisms is minimal (30), regulation of HMGCoAR is known to occur at multiple levels (15). However, few data are available concerning the regulation of CEH expression by changes in the cholesterol flux. Indeed,
cholesterol-enriched diets decreased CEH activity in rats (16), and a
consistent increase in activity is observed in response to
cholestyramine feeding (13). CEH activity is also shown to be sensitive
to hormonal stimuli in adrenal cortex (33), testis (10), corpus luteum
(2), and aorta (17). Although hormonal regulation of hepatic enzymes
involved in glycogen and glucose metabolism is widely studied (4),
limited information is available on the modulation of hepatic CEH by
hormones. Gandarias et al. (11) correlated increases in hepatic
cholesterol content in response to estradiol treatment with decreases
in hepatic neutral CEH activity after a single injection of estradiol.
Thyroid hormone effectively lowers serum cholesterol levels (34), and
Day et al. (9) correlated this reduction in plasma cholesterol with
enhanced biliary secretion of cholesterol. These studies suggest a
shift in the balance between free cholesterol and cholesterol esters by
thyroxine, probably mediated by CEH. However, a direct effect(s) of
L-thyroxine on hepatic CEH has
not been reported.
Because there is little published information on the regulation of
hepatic CEH, we have examined the regulation of CEH expression (activity, protein mass, and mRNA levels) in rats in response to
cholesterol feeding and drugs or metabolites that perturb cholesterol biosynthesis. Hepatic cholesterol flux was also altered by perturbing bile acid metabolism with chronic biliary diversion or bile acid feeding. To confirm and simplify interpretation of results from in vivo
studies, we have also examined the direct effects of hormones and
signal transduction pathways on CEH mRNA levels in primary rat
hepatocyte cultures. Our results indicate complex regulation of CEH at
multiple levels.
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EXPERIMENTAL PROCEDURES |
Materials.
Cholesterol, cholesteryl oleate, chenodeoxycholic acid, taurocholic
acid, tauroursodeoxycholic acid, mevalonolactone,
L-thyroxine, dexamethasone,
dextran sulfate, phorbol 12-myristate 13-acetate (PMA), 4
-PMA, and
N-lauroylsarcosine were
purchased from Sigma Chemical (St. Louis, MO). Lovastatin
was a generous gift from Merck Sharp & Dohme (Rahway, NJ). Guanidine
isothiocyanate and cesium chloride were purchased from Fisher
Scientific (Springfield, NJ). Williams' medium E and the
nick-translation kit were obtained from Gibco-BRL (Gaithersburg, MD).
GeneScreen membrane and radioisotopes ([
-32P]dCTP and
cholesteryl-[1-14C]oleate)
were purchased from NEN (Boston, MA). All other reagents were of the
highest quality available commercially.
Experimental design.
Male Sprague-Dawley rats (Charles River, Cambridge, MA) weighing
250-350 g were housed under controlled lighting conditions on a
12:12-h light-dark cycle (0600-1800 light phase). Groups of age-
and weight-matched animals were used for all studies. The treatments
were divided into two categories. 1)
To change intracellular cholesterol levels, rats were either fed 2%
cholesterol mixed with powdered lab chow or infused with mevalonate
(180 µmol/h for 48 h) or lovastatin (2 mg · kg
1 · h
1
for 24 h) intravenously. 2) To
increase cholesterol efflux and perturb bile acid metabolism, rats
either had biliary diversion as described below or were fed 1%
chenodeoxycholic acid mixed with powdered chow. Animals were pair fed
with modified diets as described above for 14 days. Rats were killed by
decapitation, and blood was collected for measurement of serum
aspartate aminotransferase, alanine aminotransferase, and alkaline
phosphatase levels as indicators of normal liver function. Livers were
harvested, and two 1-g pieces were removed. One piece was used to
prepare cytosol (12) and the other to isolate RNA.
Chronic biliary diverted rat model.
Under brief methoxyflurane anesthesia, biliary fistula and
intraduodenal cannulas were placed as described previously (19). After
surgery, rats were placed in individual metabolic cages with free
access to water and Purina laboratory chow. All animals received
continuous intraduodenal infusion of glucose-electrolyte replacement
solution. Dietary intake, activity, and bile flow of the rats were
carefully monitored. After 72 h of chronic biliary diversion, rats were
killed by decapitation and blood was collected to assess the normal
liver function as described above. Livers were harvested and processed
as described above for the preparation of RNA and cytosol.
Isolation and culture of primary rat hepatocytes.
Hepatocytes were isolated from male Sprague-Dawley rats (250- 300 g) using the collagenase perfusion technique of Bissell and Guzelian
(3). Before plating, cells were judged to be >90% viable using
trypan blue exclusion. Parenchymal cells (2.5 × 107) were plated onto 150-mm
petri dishes previously coated with rat tail collagen. Cells were
incubated in Williams' medium E (20 ml/plate) supplemented with
insulin (0.25 U/ml) and penicillin (100 U/ml) at 37°C in a 5%
CO2 atmosphere. The medium was
changed every 24 h and supplemented with various hormones where
indicated. Cells were routinely harvested after 48-72 h.
Measurement of CEH activity and protein.
CEH activity was measured in the cytosol by the radiometric assay
described previously (12). The specific activity is expressed as
nanomoles of oleate released per hour per milligram of protein. Relative CEH protein mass was determined by densitometric analysis of
the Western blots (23). Protein was estimated using the Pierce bicinchoninic acid protein assay kit (Rockford, IL).
Preparation of total RNA.
A one-gram piece of liver was homogenized using a Dounce homogenizer in
a solution of 4 M guanidine isothiocyanate, 10 mM tris(hydroxymethyl)aminomethane · HCl, pH 7.4, and 7% 2-mercaptoethanol. N-lauroylsarcosine was added to a
final concentration of 2%, and the homogenate was passed through a
23-gauge needle. This suspension was passed through a 23-gauge needle
and underlaid with 5.7 M cesium chloride containing 10 mM EDTA (pH
7.4). The total RNA pellet obtained after centrifugation
at 100,000 g for 16 h was washed twice
with 100% ethanol, dissolved in diethyl pyrocarbonate (DEPC)-treated
water, and precipitated with 2 vol of 100% ethanol in the presence of
0.10 vol of 3 M sodium acetate, pH 5.0, and stored at
20°C
for overnight precipitation (5). Total RNA was pelleted by
centrifugation at 12,000 g for 30 min,
redissolved in DEPC-treated water, and quantified by measuring the
absorbance at 260 nm. For RNA preparation from cultured hepatocytes
after incubation, the media were aspirated and cells washed with 5 ml of phosphate-buffered saline (PBS). After the last traces of PBS were
removed, the cells were harvested in 7.5 ml of guanidine isothiocyanate
solution, and N-lauroylsarcosine was
added to a final concentration of 2%. The mixture was vortexed to lyse
the cells completely, passed through a 23-gauge needle, and processed as described above.
Determination of CEH mRNA levels.
The methods for determination of CEH mRNA levels have been described
previously (23). In brief, 10 µg of total RNA were electrophoresed on
a 1% agarose gel in the presence of formaldehyde, transferred to
GeneScreen membrane, and hybridized with
32P-labeled full-length cDNA probe
for hepatic CEH according to the manufacturer's instructions. The
blots were washed under high stringencies (0.2× SSC plus 0.1%
SDS at 65°C; 1× SSC is 0.15 M NaCl and 0.015 M sodium
citrate, pH 7.0). Positive hybridization was detected by exposure to
Kodak XAR-2 films for 18 h at
70°C (14). Radioactivity
associated with each band was quantified using the personal
densitometer from Molecular Dynamics. Rat cyclophilin was used as the
internal control (8).
Statistical analyses.
Data were analyzed by Student's
t-test.
P < 0.05 was considered significant.
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RESULTS |
Effects of treatments known to change intracellular cholesterol
levels.
Cellular cholesterol metabolism was perturbed by cholesterol feeding
and by intravenous infusion of mevalonate or lovastatin. As shown in
Fig. 1, cholesterol feeding significantly
decreased CEH activity (56 ± 2%,
P < 0.001), protein mass (44 ± 2%, P < 0.001), and mRNA levels (14 ± 3%, P < 0.005) compared with
the pair-fed controls. Intravenous infusion of mevalonate, which
increases cholesterol biosynthesis, led to a compensatory decrease in
CEH activity (42 ± 6%,
P < 0.005) and protein mass (74 ± 3%, P < 0.001). The mRNA
levels also showed a similar decreasing trend (22 ± 16%); however,
unlike with cholesterol feeding, the changes did not reach statistical
significance. Infusion of the HMGCoAR inhibitor lovastatin, on the
other hand, significantly increased CEH activity (65 ± 12%,
P < 0.001). Although CEH mRNA levels
also showed an increasing trend after lovastatin infusion, this
difference did not reach statistical significance (31 ± 24%) and no significant change was seen in CEH protein mass (Fig. 1).

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Fig. 1.
Effect of treatments known to change intracellular cholesterol on
cholesteryl ester hydrolase (CEH) activity, mRNA levels, and protein
mass. CEH activity, protein mass, and mRNA levels were measured in rats
receiving the following treatments: 2% cholesterol feeding (2% XOL;
n = 5), intravenous infusion of
mevalonate (iv MEV; n = 3), and
intravenous infusion of lovastatin (iv LOV;
n = 6). Data are expressed as
%control. Pair-fed and sham-operated rats were used as controls for
feeding and infusion studies, respectively. * Significant
(P < 0.05) change.
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Effects of increased cholesterol efflux and bile acid feeding.
Chronic biliary diversion, which drains hepatic cholesterol through
both biliary cholesterol and bile acid synthesis, increased CEH
activity (138 ± 34%, P < 0.025)
and mRNA (146 ± 28%, P < 0.05) relative to sham-operated controls (Fig.
2). The CEH protein mass also increased (29 ± 7%, P < 0.025; data not
shown). In contrast, chenodeoxycholate (fed as 1% of diet) decreased
CEH activity by 46 ± 6% (P < 0.005). Although CEH mRNA levels were also decreased by
chenodeoxycholate (26 ± 12%), this trend did not reach statistical significance. Cholic acid (1% of diet) and deoxycholic acid (fed at
0.25% to avoid toxicity) did not significantly change CEH activity and
mRNA levels (data not shown).

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Fig. 2.
Effect of increased cholesterol efflux and bile acid feeding on CEH
activity and mRNA levels. CEH activity and mRNA levels were measured in
rats either fed 1% chenodeoxycholic acid (1% CDCA;
n = 5) or with chronic biliary
diversion (CBD; n = 5 and 2, for
activity and mRNA, respectively). Data are expressed as %control.
Pair-fed or sham-operated rats were used as controls.
* Significant (P < 0.05)
change.
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Effects of L-thyroxine and
dexamethasone on CEH mRNA levels in primary hepatocytes.
Steady-state CEH mRNA levels were initially measured as a function of
culture age in a chemically defined medium without added serum. In the
absence of dexamethasone and
L-thyroxine, a large decrease
(98%) in CEH mRNA was observed compared with the levels in freshly
isolated hepatocytes (Fig. 3). Addition of
both hormones stabilized mRNA levels near those of fresh hepatocytes
(Fig. 3). In the presence of 0.1 µM dexamethasone,
L-thyroxine yielded a maximum response at concentrations between 1 and 10 µM, whereas 100 µM L-thyroxine
reduced mRNA levels to 29% of controls (Fig. 4). In the presence of 1 µM
L-thyroxine, a maximum response
was seen with 0.1 µM dexamethasone (Fig.
5). Although the specific activity of CEH
in freshly isolated hepatocytes is only 50% of that in liver cytosol,
activity of CEH could not be measured in cultured hepatocytes, even in
the presence of L-thyroxine and dexamethasone (data not shown).

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Fig. 3.
Effect of culture age on steady-state CEH mRNA levels. Primary
hepatocytes were plated in the presence or absence of 0.1 µM
dexamethasone (Dex) and 1 µM
L-thyroxine (T4). Total RNA was
isolated from cells at indicated times, and CEH mRNA levels were
determined by densitometric analysis of Northern blot as described in
EXPERIMENTAL PROCEDURES.
A: values are means ± SE;
n = 3. Control, mRNA level of
corresponding freshly isolated hepatocytes.
B: a representative blot probed for
CEH and the housekeeping gene cyclophilin (CYC).
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Fig. 4.
Effect of T4 concentration on CEH mRNA levels. Culture medium contained
0.1 µM dexamethasone with indicated T4 concentrations added at
time 0. Total RNA was isolated 72 h after
plating, and CEH mRNA levels were determined by densitometric analysis
of Northern blots. A: values are means ± SE; n = 5. Control, mRNA in
hepatocytes with dexamethasone alone.
B: representative blot probed for CEH
and CYC. Lane 1, dexamethasone alone;
lanes 2-7,
+0.001-100 µM T4.
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Fig. 5.
Effect of dexamethasone concentration on CEH mRNA levels. Culture
medium contained 1 µM T4 with indicated dexamethasone concentrations
added at time 0. Total RNA was isolated 72 h after
plating, and CEH mRNA levels were determined by densitometric analysis
of Northern blots. A: values are means ± SE; n = 6. Control, mRNA in
hepatocytes cultured with T4 alone. B:
representative blot probed for CEH and CYC.
Lane 1, no additions;
lane 2, T4 alone;
lanes 3-7,
+0.001-10 µM dexamethasone.
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Effects of modulators of signal transduction pathways on CEH mRNA
levels in hepatocytes.
The effects of dibutyryl-cAMP (DBcAMP), glucagon, and PMA on CEH mRNA
levels were determined under optimal culture conditions (in the
presence of insulin, 0.1 µM dexamethasone, and 1 µM
L-thyroxine). As shown in Table
1, although addition of glucagon or DBcAMP had no effect on CEH mRNA levels, PMA decreased CEH mRNA levels by 49 ± 13% (P < 0.01).
The response to PMA was time dependent with a maximum at 6 h (data not
shown). The effects of PMA were specific to 4
-PMA, because
incubation with the 4
-analog of PMA had no significant effect on CEH
mRNA (91% of control).
Effects of agents that perturb cholesterol metabolism on CEH mRNA
levels in hepatocytes.
Hepatic free cholesterol levels are coordinately regulated by
HMGCoAR, C7
H, acyl-CoA:cholesterol acyltransferase, and
CEH. To evaluate the molecular mechanisms underlying the compensatory role of CEH in cholesterol homeostasis in vivo, hepatocytes were treated with lovastatin, a competitive inhibitor of HMGCoAR,
or taurocholate, a repressor of C7
H and the primary bile acid in rat, before measurement of CEH mRNA levels. Lovastatin increased CEH
mRNA levels by 103 ± 15%
(P < 0.001). Suppression of CEH mRNA levels by taurocholate was both time (Fig.
6A)
and concentration dependent (Fig.
6B). The hydrophilic
bile acid tauroursodeoxycholate (50 µM), which does not
suppress bile acid synthesis (27), had no effect on CEH mRNA levels
(105% of controls with no additions).