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From the Departments of
Received for publication, May 28, 2002, and in revised form, September 27, 2002
Bile acid synthesis occurs mainly via two
pathways: the "classic" pathway, initiated by microsomal
cholesterol 7 The liver plays a pivotal role in the maintenance of cholesterol
homeostasis. Under normal physiologic conditions, cholesterol input
into the body equals cholesterol output (1, 2). Bile acid synthesis in
liver is the major pathway for cholesterol output. The
biotransformation of cholesterol to primary bile acids occurs via two
main pathways. In the "classic/neutral" pathway, metabolism of the
sterol nucleus occurs before side chain modifications and begins with
hydroxylation of cholesterol at the 7 In contrast to CYP7A1, which is found only in the liver, CYP27 has a
wide tissue distribution. The ability of peripheral cells to
27-hydroxylate cholesterol has been proposed to be important in
"reverse cholesterol transport" (4-6). According to this
hypothesis, CYP27 located in peripheral tissues generates oxysterols
that are more water-soluble than cholesterol. These metabolites can then be transported to the liver and converted to bile acids. It is
possible that CYP27 in peripheral tissues may both down-regulate cholesterol synthesis and enhance the efflux of cholesterol to the
liver for elimination. Thus, up-regulation of CYP27 could represent a
treatment of hyperlipidemia. However, overexpression of the gene
encoding CYP27 in primary rat and human hepatocytes or HepG2 cells led
to only an ~50% increase in bile acid synthesis (7). This led us to
hypothesize that increasing cholesterol delivery to and/or into the
mitochondria where CYP27 is located could potentially increase the rate
of bile acid synthesis via the acidic pathway. Precedence for this has
previously been demonstrated in other steroidogenic tissues. In the
adrenal gland, increased expression of the mitochondrial cholesterol
transport protein steroidogenic acute
regulatory
(StAR)1 protein was shown to
increase mitochondrial cholesterol delivery and steroidogenesis
(8).
This study shows that overexpression of the gene encoding StAR protein
in primary rat hepatocytes dramatically increases bile acid synthesis,
which suggests that cholesterol delivery to the inner mitochondrial
membrane is the rate-determining step for bile acid biosynthesis via
the alternative pathway rather than CYP27. Furthermore, it is shown
that increasing cholesterol transport to inner mitochondrial CYP27
bypasses the highly regulated CYP7A1 of the
"classic/neutral" pathway of bile acid biosynthesis. These findings provide an entirely new insight into how bile acid
biosynthesis is regulated.
Materials
Cell culture reagents and supplies were purchased from
Invitrogen. The RPA II kit was purchased from Ambion Inc.
(Austin, TX). [14C]Cholesterol and
25-[3H]hydroxycholesterol were purchased from PerkinElmer
Life Sciences. 25-, 7 Isolation and Culture of Primary Rat Hepatocytes
Hepatocytes were isolated from male Sprague-Dawley rats
(250-300 g) as previously described by us (10) using the
collagenase perfusion technique of Bissell and Guzelian (9). Cells were routinely harvested after 72 h of culture as previously described (10). Unless specified, cells were maintained under conditions in which
CYP7A1 activity is undetectable (i.e. cultured as previously described (10) in the absence of thyroid hormone).
Generation of Recombinant Adenoviruses and Their Use
The adenovirus constructs used in this study were obtained
through the Massey Cancer Center Shared Resource Facility of the Virginia Commonwealth University. The CMV-CYP27 recombinant adenovirus clone (Ad-CMV-CYP27) was constructed as previously described (7, 11).
Briefly, the CMV-StAR adenovirus construct (Ad-CMV-StAR) was obtained
using a pTG-CMV system as previously described (7, 11). A 1.6-kb human
adrenal cortex StAR cDNA (a generous gift from Dr. Jerome Strauss,
Department of Obstetrics and Gynecology, University of Pennsylvania,
Philadelphia, PA) was cloned into the
SalI/NotI restriction sites of
pZeroTG-CMV, a plasmid containing the CMV promoter, multiple cloning
sites, and a partial DNA sequence from Ad5dl324 (12).
The resulting pZeroTG-CMV/HStAR plasmid was cotransformed with
ClaI-linearized pTG-CMV (containing the entire
Ad5dl324 genome) into Escherichia coli.
Recombinant plasmids were transfected into human embryonic kidney 293 cells (American Type Culture Collection, Manassas, VA). Adenovirus DNA
from the resulting plaques was further screened by Southern blotting
for the presence of the insert.
Propagation of Ad-CMV-StAR and Ad-CMV-CYP27--
Large-scale
production of recombinant virus was accomplished by infecting confluent
monolayers of human embryonic kidney 293 cells (grown in 15-cm tissue
culture dishes) with stock adenoviruses at a multiplicity of infection
of 1 plaque-forming unit/cell. After 2 h of infection, unbound
virus was removed, and Dulbecco's modified Eagle's medium with 2%
fetal bovine serum was added. Infected monolayers were harvested by
scraping when >90% of the cells showed cytopathic changes and
centrifuged at 2700 × g for 10 min at 4 °C. The
pellet was suspended in Dulbecco's modified Eagle's medium with 2%
fetal bovine serum and subjected to five cycles of freeze/thaw lysis to
release the recombinant virus. Cell debris were removed by
centrifugation at 7700 × g for 10 min at 4 °C. To
purify the recombinant virus, the crude supernatant was carefully
layered over a two-step gradient containing 3 ml of CsCl
(d = 1.4 g/ml) in TD buffer (0.14 M
NaCl, 5 mM KCl, 19 mM Tris (pH 7.4), and 0.7 mM Na2HPO4), layered over 3 ml of
CsCl (d = 1.25 g/ml) in TD buffer, and centrifuged at
155,000 × g for 1 h at 20 °C. The viral band
was removed, layered over 8 ml of CsCl (d = 1.33 g/ml)
in TD buffer, and centrifuged at 155,000 × g for
18 h at 20 °C. The pure viral opalescent band was removed and
dialyzed overnight at 4 °C against 10 mM Tris-HCl (pH
7.4), 1 mM MgCl2, and 10% glycerol. The virus
was aliquoted and stored at Infection of Cells with Adenovirus encoding StAR Protein and
CMV-CYP27--
Primary rat hepatocyte cultures, prepared as previously
described (10), were plated on 150-mm tissue culture dishes
(~2.5 × 107 cells) in Williams' E medium
containing dexamethasone (0.1 µM). Unless otherwise
specified, cells were maintained in the absence of thyroid hormone,
a condition under which only the acidic pathway of bile acid
synthesis is functional (10). In selected studies, thyroid hormone
(L-thyroxine) was added as previously described (10)
at a concentration of 1.0 µM, a culture condition under which both bile acid biosynthesis pathways are fully functional. Twenty-four hours after plating, the culture medium was removed, and
2.5 ml of fresh medium was added. Cells were then infected with
unpurified adenovirus encoding either CMV-StAR or CMV-CYP27 at a
multiplicity of infection of 10 plaque-forming units/cell. All
experiments were compared with the Ad-CMV control virus and no-virus
cultures. The virus was allowed to dwell for at least 2 h in
minimal culture medium, with the plates being gently shaken every 15 min. After 2 h of infection, unbound virus was removed and
replaced with 20 ml of fresh medium. The cells were incubated at
37 °C in 5% CO2 for 48 h. Cells were then
harvested as previously described (10).
RNA Preparation and Quantification
RNA was isolated as previously described (13). CYP7A1 and CYP27
mRNAs were quantified using Northern blot assays (20 µg of total RNA).
Protein Levels
After infection, either cells were harvested by adding sample
buffer as indicated, or subcellular fractions were separated and
isolated by centrifugation as previously described (7). Proteins were
then solubilized by adding 2× SDS-PAGE sample buffer (5 mM
Tris buffer (pH 8.3), 29% (w/v) SDS, 10% mercaptoethanol, 10% (v/v)
glycerol, 38 mM glycine, and 0.2% (w/v) bromphenol blue), followed by heating in a boiling water bath for 5 min. Solubilized proteins (3 µg) were analyzed by 10% SDS-PAGE. Electrophoresis was
performed at 20 mA for 2 h in a Bio-Rad minigel system. StAR protein was identified by Western blot analysis. After electrophoresis, samples were transferred to nitrocellulose membranes. Membranes were
blocked with 3% nonfat dry milk in 10 mM HEPES buffer (pH 7.4) containing 25 mM EDTA, 0.5 M NaCl, and
0.05% NaN3; immunostained with a rabbit polyclonal
antibody (1:2000 dilution) against the human StAR protein (a generous
gift from Dr. Jerome Strauss) in HEPES buffer; washed with the same
buffer plus 0.05% Tween 20; and incubated with a goat anti-rabbit
secondary antibody (1:10,000; Sigma). Bands were visualized using
chemiluminescence reagent (PerkinElmer Life Sciences) and Kodak BioMax film.
CYP27 immunoblotting was performed as previously described (7). Rabbit
polyclonal antibody against rat CYP27 protein was a generous gift from
Dr. N. Avadhani (University of Pennsylvania).
Determination of Enzyme Specific Activities
Mitochondria and microsomes were prepared as previously
described (7, 10). The specific activities of CYP7A1 and CYP27 were
determined by HPLC assays as previously described (7, 10).
Quantification of Bile Acid Synthesis Rates
In Vitro Studies--
Bile acid synthesis rates were determined
by addition of 2.5 µCi of [14C]cholesterol to each
150-mm plate of confluent primary rat hepatocyte cultures
(~2.45 × 107 cells) 24 h after plating. The
medium and cells were harvested 48 h after viral infection.
Conversion of [14C]cholesterol to
[14C]methanol/water-soluble products was determined by
scintillation counting after Folch extraction (14) with
chloroform/methanol (2:1, v/v) of cells and of the culture medium. The
rates of bile acid biosynthesis following recombinant adenovirus
infection were calculated as the ratio of
[14C]methanol/water-soluble counts to the sum of
chloroform/methanol/water-soluble counts.
Individual bile acids were identified as previously described (11).
Briefly, to identify the individual bile acids, the [14C]methanol/water phase was first base-hydrolyzed and
then separated by TLC in a solvent system of ethyl
acetate/cyclohexane/acetic acid (7.7:2.3:1, v/v/v).
14C-Labeled bile acids were visualized with a PhosphorImager.
Time points for conversion of [14C]cholesterol to
14C-labeled bile acids were carried out using 150-mm
tissue culture dishes Aliquots (100 µl) of the medium were collected
in duplicate in a microcentrifuge tube and kept frozen until
analysis. A mini-Folch extraction was carried out by adding the
following to the culture medium sample to help separate the phases: 50 µl of water, 250 µl of methanol, 537 µl of chloroform
(H2O/MeOH/CHCl3, 2:3:7), and 3 µl of 1 M Na2CO3. The tubes were vigorously
vortexed and centrifuged at 16,000 × g for 6 min. The
phases were collected separately and counted. Time points for the
7
In selected studies, the rate of 7 In Vivo Studies--
Conjugated bile acids in the bile collected
from biliary diverted rats were analyzed by reverse-phase HPLC as
previously described (13). In chronic biliary diverted rats, bile acid
synthesis is equivalent to biliary bile acid secretion.
Biliary Diverted Rat
Adult male Sprague-Dawley rats (250-300 g) were housed under
controlled lighting conditions on a natural light-dark cycle. Groups of
age- and weight-matched animals were used in all experiments. Under
brief methoxyflurane anesthesia, intravenous and biliary fistula
cannulas were placed as previously described (13, 15, 16). After
cannula placement, each rat was intravenously infused with 1-1.5 × 1011 virus particles of recombinant adenovirus
containing CMV-StAR or control virus. Following surgery, the rats were
housed in individual metabolic cages with free access to water and
chow. Diverted bile was collected in timed increments throughout the
course of the experiment. All animals received a continuous infusion of
glucose/electrolyte replacement solution at 1.07 ml/h. Throughout the
experiment, dietary intake, activity, and bile flow were monitored as
previously described (13). At the end of the experiments, animals were briefly anesthetized and decapitated, and blood was collected to
measure serum alanine aminotransferase and alkaline phosphatase levels as previously described (16). Animals were killed at 9-10
a.m.
Statistics
Data are reported as means ± S.E. Where indicated, data
were subjected to t test analysis and determined to be
significantly different if p is <0.05.
StAR Overexpression in Primary Rat Hepatocytes--
Infection of
primary rat hepatocytes with Ad-CMV-StAR produced high StAR mRNA
and protein levels with no evidence of cell toxicity. Fig.
1 shows the increase in StAR mRNA and
protein levels 48 h following infection (see "Experimental
Procedures"). Rat adrenal poly(A) RNA was used as a control. Two
mRNA species (1.6 and 4.0 kb) were observed (Fig. 1A),
representing parental and mature forms of StAR mRNA as previously
shown (18, 19). Western blot analysis of mitochondrial proteins showed
one major immunoreactive band with a molecular mass of 30 kDa,
consistent with the mature StAR protein (Fig. 1B), as
previously reported (8, 17). Primary rat hepatocyte subcellular
fractions were then isolated, and distribution of StAR protein was
examined by Western blot analysis. In hepatocytes overexpressing StAR,
StAR protein was found widely distributed in the cytosol, microsomes,
and mitochondria (data not shown). A comparison of StAR protein levels
in StAR-overexpressing primary hepatocytes and in rat testis and
adrenal gland is shown in Fig. 1C. The recombinant StAR
protein had a molecular mass similar to that in rat testis and adrenal
gland; however, the level of StAR protein in hepatocytes following StAR
overexpression was significantly higher than that expressed under
normal physiologic conditions in rat testis or adrenal gland.
Sterol 27-Hydroxylase (CYP27) Overexpression in Primary Rat
Hepatocytes--
CYP27 is responsible for the 27-hydroxylation of
cholesterol as the initial step in the acidic pathway of bile acid
synthesis. To compare the effects of StAR and CYP27 overexpression,
primary rat hepatocytes were infected with recombinant adenovirus
containing the CMV-driven gene encoding CYP27 (Ad-CMV-CYP27). The
infected cells produced very high CYP27 mRNA and protein levels
without inducing any evidence of cell toxicity. Northern blot analysis showed a 2.1-kb mRNA band representing mature CYP27 mRNA (Fig. 2A). Western blot analysis of
mitochondrial proteins showed one major immunoreactive band with a
molecular mass of 55 kDa (Fig. 2B). Overexpression of StAR,
either alone or in combination with CYP27, did not alter CYP27 protein
levels or catalytic activity (data not shown).
Effect of StAR on the Rate of Bile Acid
Synthesis--
Overexpression of StAR protein dramatically increased
the rates of bile acid synthesis in primary rat hepatocytes. Time
courses showing the increase in StAR protein levels, bile acid
synthesis, and [14C]cholesterol uptake in primary rat
hepatocytes after StAR overexpression are shown in Fig.
3. StAR protein was easily detected at
12 h following infection with recombinant adenovirus and steadily
increased up to 48 h (Fig. 3A). The effects of StAR
protein on the rates of bile acid synthesis in the cells were
determined via conversion of [14C]cholesterol to
[14C]methanol/water-extractable products (Fig.
3B). CYP27 overexpression only slightly increased bile acid
synthesis rates over that observed in controls (cells infected with
control recombinant adenovirus). In contrast, the rates of bile acid
synthesis increased dramatically upon expression of StAR protein (Fig.
3B). Furthermore, overexpression of CYP27 and StAR together
did not increase bile acid synthesis rates any more than StAR
overexpression alone (Fig. 4). These results show that an increase in StAR protein is capable of increasing bile acid synthesis more efficiently than an increase in CYP27 expression. The effects of StAR protein on cellular cholesterol uptake
are shown in Fig. 3C. To determine that this increase in bile acid synthesis was not the result of an increase in cholesterol uptake, the rates of cholesterol uptake were determined as
[14C]cholesterol "disappearance" (chloroform phase)
from the cell culture medium. As shown, neither StAR nor CYP27
overexpression affected cellular cholesterol uptake rates.
The rates of bile acid synthesis were further determined by quantifying
the bile acid levels in the culture medium and within cells at 48 h after infection, as shown in Fig. 4. Within the cells,
overexpression of StAR and co-overexpression of StAR plus CYP27
produced a >10-fold increase (p < 0.001) in the
amount of bile acids over that observed in control cells
(i.e. infected with control recombinant adenovirus), whereas
CYP27 overexpression alone only led to only a 1.4-fold increase (Fig.
4A). In the culture medium, an ~6-fold increase
(p < 0.001) in bile acids (i.e. less than
within the cell) was seen following StAR overexpression and StAR plus
CYP27 co-overexpression, with a 76 ± 58% increase following infection with recombinant adenovirus containing the CMV-driven gene
encoding CYP27 alone compared with the control virus (Fig. 4B). The differences in the rates of bile acid synthesis
following overexpression of the genes encoding StAR and StAR plus CYP27 were not significant. The increase in the rates of bile acid synthesis following addition of unlabeled cholesterol (5 µM) to
saturate and competitively slow [14C]cholesterol uptake
into cells was >2-fold (data not shown).
The steroid products in chloroform- and water/methanol-extractable
phases were further analyzed by TLC (Fig.
5). 14C-Labeled
steroid-extractable products in the chloroform phase were mainly
composed of cholesterol esters, cholesterol, 25- and 27-hydroxycholesterol, 3-oxo-7-hydroxycholesterol, and
7,27-dihydroxycholesterol (Fig. 5A). Cholesterol and
cholesterol esters were decreased in the culture medium of cells
overexpressing StAR or StAR plus CYP27. Conversely,
27-hydroxycholesterol and 7,27-dihydroxycholesterol levels increased
significantly in cells overexpressing StAR or StAR plus CYP27. Because
CYP27 is located in the inner mitochondrial membrane, it is assumed
that 27-hydroxycholesterol and 7,27-dihydroxycholesterol are products
of the alternative pathway of bile acid synthesis. The results from TLC
analysis of products in the methanol phase of the culture medium are
shown in Fig. 5B. A 5-fold increase was seen in soluble
steroids,
In selected studies, thyroid hormone was added to the culture medium as
described under "Experiment Procedures." Under these conditions.
CYP7A1 and the neutral (classic) pathway of bile acid synthesis are
fully active. Of note is that CYP7A1 mRNA levels under these
culture conditions are greater than those found in the up-regulated
cholestyramine-fed rat model (10). These studies were performed to
address the question, is StAR overexpression capable of increasing the
rate of bile acid synthesis over the basal rates found in the presence
of a fully functional neutral pathway? Using these culture conditions,
overexpression of StAR still led to a >2-fold increase
(p < 0.001) in the rates of bile acid synthesis (data
not shown).
Effect of StAR and/or CYP27 Gene Overexpression on Mitochondrial
Levels of 27-Hydroxycholesterol--
To demonstrate that StAR
overexpression leads to an increase in the product of CYP27,
mitochondrial 27-hydroxycholesterol levels were determined following
StAR and CYP27 overexpression (7). Mitochondrial sterol analysis
revealed an easily detectable retention peak for endogenous
27-hydroxycholesterol in the mitochondria of StAR-overexpressing
hepatocytes (Fig. 6). Of note is that
neither control cells nor cells overexpressing CYP27 had detectable
27-hydroxycholesterol levels in their mitochondria (previously
determined detection sensitivity of ~20 pmol). 27-Hydroxycholesterol
accumulated only following StAR overexpression. Following the
determination of endogenous mitochondrial 27-hydroxycholesterol levels,
mitochondria were assayed for CYP27 specific activity. Interestingly,
no detectable increase in CYP27 activity over controls was found
following StAR overexpression. These findings suggest that the ability
of StAR to increase mitochondrial cholesterol transport and its
subsequent conversion to 27-hydroxycholesterol occurred prior to
mitochondrial isolation for CYP27 activity analysis. Furthermore, these
results show, given the existing basal cellular CYP27 protein levels, that cholesterol delivery to the inner mitochondrial membrane is the
key rate-determining step in bile acid synthesis via the alternative
pathway. The above findings, coupled with the inability of
overexpression of the genes encoding CYP27 plus StAR to further increase bile acid synthesis above overexpression of the StAR gene
alone, suggest that there exists an abundance of CYP27 under normal
physiologic conditions.
Effect of StAR on 7 Effect of StAR Overexpression on Bile Acid Synthesis in the Biliary
Diverted Rat--
Infection of biliary diverted rats with recombinant
adenovirus encoding CMV-StAR markedly increased StAR mRNA and
protein levels (data not shown). Biliary diverted rats infected with
StAR 3 days earlier at the time of their biliary diversion increased their rates of bile acid synthesis by 2.5-fold (n = 6;
p < 0.001) over their basal synthesis rates at 20-24
h (Fig. 8). This represented a 1.8-fold
(n = 3; p < 0.03) increase over 3-day
biliary diverted controls (i.e. infected with control
recombinant adenovirus). Thus, overexpression of StAR was able to
dramatically increase bile acid synthesis rates over and above the
usual ~1.5-2-fold increase in basal rates observed in control
biliary diverted rats 3 days following the loss of negative bile acid
feedback. The bile acid concentration following both StAR and CYP7A1
overexpression was also similarly increased (data not shown).
StAR protein has previously been shown to mobilize cholesterol
from the outer to the inner mitochondrial membrane in steroidogenic cells (i.e. adrenal cortex and gonads) (8, 17). Cholesterol transport is a principal control point for regulation of
steroidogenesis by ACTH and other hormones acting through the adenylyl
cyclase and Ca2+ pathways (18). Observations by Sugawara
et al. (19) have subsequently provided evidence that not
only is mitochondrial cholesterol transport rate-determining for
steroidogenesis, but that StAR-induced mitochondrial cholesterol
transport is capable of enhancing mitochondrial cholesterol metabolism
by enzymes other than the steroidogenic cytochrome P450scc.
There is, however, an abundance of StAR protein within the adrenal
mitochondria. In contrast, StAR mRNA or protein has not been
detected in liver tissue (18, 20). Still, the existence of StAR or a
StAR-like protein in liver hepatocytes seems to be necessary for bile
acid synthesis to occur via the alternative pathway, as cholesterol must first be transported to the inner mitochondrial membrane before it
can undergo 27-hydroxylation. Furthermore, the inability to
dramatically increase bile acid synthesis in hepatocytes overexpressing CYP27 suggests that 27-hydroxylation of cholesterol is not
rate-limiting for the alternative pathway of bile acid synthesis.
The contribution of the "alternative" pathway to total bile acid
synthesis is unclear, as under most physiologic conditions, the
"classic" pathway appears to be the dominant pathway (3, 21). It is
currently believed that the "alternative" pathway of bile acid
synthesis may play at least three roles in cholesterol homeostasis (3).
1) The 27-hydroxylation of cholesterol, both in the periphery and the
liver, forms a regulatory oxysterol (i.e. 27-hydroxycholesterol) (3, 7). The liver is capable of hydroxylating these regulatory oxysterols, leading to their subsequent metabolism to
bile acids. 2) The "alternative" pathway may act as a "backup pathway" when the "classic" pathway is down-regulated. In CYP7A1 "knockout" animals, alternative pathways appear to be capable of
producing adequate amounts of bile acids for survival and growth (22).
3) The "alternative" pathway may serve to regulate the ratios of
bile acid species in bile, as this pathway is thought to generate
mostly chenodeoxycholic acid in humans (3). It has been shown that up
to 50% of bile acid biosynthesis may occur via an alternative pathway
in the rat (23, 24). Studies in humans have found a lower contribution
under normal physiologic circumstances (25). However, in human liver
cholestatic conditions, this contribution has been found to be much
higher, suggesting that the "alternative" pathway can be a major
pathway under certain pathophysiologic conditions (26).
Evidence supporting mitochondrial cholesterol transport as the
rate-limiting step of bile acid synthesis via the "alternative" pathway would give rise to a new hypothesis regarding regulation of the
alternative pathway. It would also give strong evidence as to why CYP27
is localized in the mitochondria under highly regulated cholesterol
access. In the "alternative" pathway, the initial and presumed
rate-determining step is catalyzed by mitochondria CYP27. This study
shows that cholesterol transport into the inner mitochondrial membrane
is the rate-limiting step in the "alternative" pathway of bile acid
synthesis rather than CYP27. Furthermore, in an unregulated state
(i.e. increased expression of StAR protein with increased
mitochondrial cholesterol transport), the highly regulated "neutral
(classic)" pathway of bile acid synthesis can be bypassed,
demonstrating the absolute necessity of tight regulation of
mitochondrial cholesterol transport in the liver. The observation made
in primary rat hepatocytes cultured in the presence of thyroid hormone
is supportive of this statement. We have previously shown that upon
addition of thyroid hormone to our standard culture medium, CYP7A1 is
markedly up-regulated to levels greater than found in the up-regulated
cholestyramine-fed rat (13). StAR overexpression under these culture
conditions still led to a >2-fold increase in the rates of bile acid
synthesis. In in vivo studies, overexpression of the StAR
gene in the biliary diverted rat also led to a 1.8-fold increase in
bile acid synthesis over controls (Fig. 8), a model previously believed
to have maximal rates of bile acid synthesis.
Our results show that overexpression of the StAR gene or
co-overexpression of StAR and CYP27 led to the accumulation of
27-hydroxycholesterol in mitochondria, whereas overexpression of CYP27
alone did not. Meanwhile, overexpression of the gene encoding StAR
increased bile acid synthesis by 6-fold, whereas in direct comparison,
overexpression of the gene encoding CYP27 increased synthesis by
<2-fold. These findings are consistent with the increase in bile acid
synthesis seen in HepG2 cells following overexpression of the gene
encoding CYP27 (7).
StAR protein overexpression increases transport of cholesterol from the
outer to inner mitochondrial membrane, possibly leading to saturating
cholesterol concentrations within the inner mitochondrial membrane and
allowing maximal rates of bile acid synthesis. However, the
accumulation of 27-hydroxycholesterol and other bile acid intermediates
in the mitochondria suggests the possibility of another rate-limiting
step in bile acid synthesis, i.e. transport of
27-hydroxycholesterol and/or other bile acid intermediates across
mitochondrial membranes.
StAR (StARD1) is a member of a family of proteins, each containing an
~200-210 amino acid StAR-related lipid
transfer (START) domain (27). Recently, Soccio et
al. (28) have discovered several more members of a subfamily of START (i.e. "StAR") domain-containing proteins, StARD4,
StARD5, and StARD6. Both StARD4 and StARD5 are ubiquitously expressed, with the greatest abundance in the liver and kidney, whereas StARD6 is
exclusively expressed in the testis. Whether one of these liver START
domain-containing proteins could function as a liver mitochondrial cholesterol transporter is currently not clear. Of interest is that
most other previously identified START domain-containing proteins
contain an N-terminal domain that appears to be important in directing
their function (i.e. StARD1) (8, 17). However, StARD4, StARD5, and StARD6 are only 205-233-amino acid proteins consisting almost entirely of a START domain (28). Furthermore, StARD4-6 share only an ~20% identity with the
cholesterol-binding StARD1 and ~30% identity with each other,
allowing one to hypothesize that each may have a distinct function in
the maintenance of intracellular lipid homeostasis (28).
In summary, the results reported in this study show that the
"alternative" pathway of bile acid synthesis is primarily regulated by cholesterol transport into the mitochondria and suggest that the
hepatocyte must have StAR or homologs for transporting cholesterol into
the inner mitochondrial membrane. This study also suggests alternative
mechanisms for increasing the rates of bile acid synthesis and
cholesterol output from the body. Previously unsuspected, this study
also demonstrates that a sufficient increase in mitochondrial cholesterol transport within the hepatocyte is capable of bypassing the
usually dominant "classic" pathway of bile acid synthesis, obviating the rate-limiting function of the highly regulated CYP7A1.
We acknowledge the assistance of Dr. Jerome
Strauss, without whose help this study would not have been possible.
*
This work was supported by a grant from the Veterans Affairs
Medical Center and National Institutes of Health Grant PO1 DK38030.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. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Virginia Commonwealth
University, Medical College of Virginia Campus, P. O. Box 980711, Richmond, VA 23298-0711. Tel.: 804-828-3849; Fax: 804-828-7430; E-mail:
wmpandak@hsc.vcu.edu.
Published, JBC Papers in Press, October 3, 2002, DOI 10.1074/jbc.M205244200
The abbreviations used are:
StAR, steroidogenic acute regulatory;
HPLC, high-performance liquid chromatography;
CMV, cytomegalovirus;
Ad, adenovirus;
ACTH, adrenocorticotropin.
Transport of Cholesterol into Mitochondria Is Rate-limiting
for Bile Acid Synthesis via the Alternative Pathway in Primary Rat
Hepatocytes*
§,,,,,,
, and Medicine, ¶ Microbiology
and Immunology, and Biochemistry, Veterans Affairs Medical
Center, and Virginia Commonwealth University,
Richmond, Virginia 23298-0711
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase (CYP7A1), and an "alternative"
(acidic) pathway, initiated by sterol 27-hydroxylase (CYP27). CYP27 is
located in the inner mitochondrial membrane, where cholesterol content
is very low. We hypothesized that cholesterol transport into
mitochondria may be rate-limiting for bile acid synthesis via
the "alternative" pathway. Overexpression of the gene
encoding steroidogenic acute
regulatory (StAR) protein, a known mitochondrial
cholesterol transport protein, led to a 5-fold increase in bile acid
synthesis. An increase in StAR protein coincided with an increase in
bile acid synthesis. CYP27 overexpression increased bile acid synthesis
by <2-fold. The rates of bile acid synthesis following a combination
of StAR plus CYP27 overexpression were similar to those obtained with
StAR alone. TLC analysis of 14C-labeled bile acids
synthesized in cells overexpressing StAR showed a 5-fold increase in
muricholic acid; in chloroform-extractable products, a dramatic
increase was seen in bile acid biosynthesis intermediates (27- and
7,27-hydroxycholesterol). High-performance liquid chromatography
analysis showed that 27-hydroxycholesterol accumulated in the
mitochondria of StAR-overexpressing cells only. These findings suggest
that cholesterol delivery to the inner mitochondrial membrane is the
predominant rate-determining step for bile acid synthesis via
the alternative pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-position (3). This reaction is
catalyzed by cholesterol 7-hydroxylase (CYP7A1), the initial and
rate-determining step in this pathway of bile acid synthesis. In the
"alternative" pathway of bile acid synthesis, commonly called the
"acidic" pathway, side chain modifications precede modifications in
the sterol nucleus. The initial and presumed rate-determining step in
the acidic pathway is catalyzed by mitochondrial sterol 27-hydroxylase (CYP27).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-, and 7
-hydroxycholesterol were purchased
from Steraloids, Inc. (Newport, RI). Cyclodextrin was purchased from
Cyclodextrin Technologies Development Inc. (Gainsville, FL). Silica gel
TLC plates (LK6D) were from Whatman. Silica Gel 1B TLC sheets were purchased from VWR Scientific Products (Bridgeport, NJ).
HPLC-grade solvents were purchased from Fisher. All other reagents were
from Sigma unless otherwise indicated.
70 °C until used. The virus titer
(plaque-forming units) was determined by plaque assay, and virus
particles were determined by measuring the absorbance at 260 nm
by spectrophotometry.
-[14C]hydroxycholesterol conversion to bile acids
were taken from 60-mm tissue culture dishes plated for that purpose.
-hydroxycholesterol uptake and
subsequent metabolism to bile acids was determined. Twenty-four hours
after plating, isolated primary rat hepatocytes were infected with
recombinant adenovirus encoding the CMV-driven StAR gene, null virus
(control), or no-virus addition. Following infection, 7-[14C]hydroxycholesterol (1 × 105
dpm/60-mm plate) and unlabeled 7-hydroxycholesterol (5 µM) were added. Samples were collected in duplicate and
extracted, and methanol/water-soluble counts were determined. Bile acid
synthesis was measured as conversion of
7-[14C]hydroxycholesterol to
[14C]methanol/water-extractable counts.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
StAR mRNA and protein levels in primary
rat hepatocytes following StAR and CYP27 overexpression. Primary
rat hepatocytes (PRH) were infected with the indicated
recombinant adenoviruses as described under "Experiment
Procedures." Cells were harvested 48 h following infection, and
RNA or mitochondria were isolated. A, mRNA levels for
StAR and cyclophilin (as a control) were determined by Northern
analysis. B and C, StAR protein levels were
determined by Western analysis. Rat adrenal gland and testis RNA and
protein were used as positive controls compared with overexpressed StAR
levels.
View larger version (44K):
[in a new window]
Fig. 2.
CYP27 mRNA and protein levels in primary
rat hepatocytes following StAR and CYP27 overexpression. Primary
rat hepatocytes were infected with the indicated recombinant
adenoviruses as described under "Experimental Procedures." Cells
were harvested 48 h following infection, and RNA or mitochondria
were isolated. A, mRNA levels for CYP27 and cyclophilin
(as a control) were determined by Northern analysis. B,
CYP27 protein levels were determined by Western analysis.
View larger version (26K):
[in a new window]
Fig. 3.
StAR overexpression increases cholesterol
uptake and rates of bile acid synthesis. Primary rat hepatocytes
were infected with the StAR recombinant adenovirus as described under
"Experimental Procedures." Cells and the tissue culture medium were
collected at the indicated time points following infection.
A, StAR protein in the mitochondria of infected cells at the
indicated times was analyzed by Western analysis. B, bile
acid synthesis rates were quantified as conversion of
[14C]cholesterol to methanol/water-extractable products
as described under "Experimental Procedures." C,
[14C]cholesterol was quantified as a function of change
in [14C]cholesterol counts in the medium.
View larger version (11K):
[in a new window]
Fig. 4.
Effect of StAR and/or CYP27 overexpression on
the rates of bile acid synthesis in primary rat hepatocytes.
Primary rat hepatocytes were infected with the indicated
recombinant adenoviruses and incubated with
[14C]cholesterol as described under "Experimental
Procedures." Forty-eight hours after infection, both cells
(A) and the culture medium (B) were harvested,
and bile acid synthesis levels were quantified as described under
"Experimental Procedures." Bile acid synthesis levels are expressed
as a percentage of control (null) virus and represent means ± S.E. of three to nine experiments.
-muricholic acid, and chenodeoxycholic acid (Fig.
5B) in the culture medium following StAR or StAR plus CYP27
overexpression.
View larger version (90K):
[in a new window]
Fig. 5.
StAR overexpression increases bile acids and
their intermediates. Primary rat hepatocytes were infected with
the indicated recombinant adenoviruses and incubated with
[14C]cholesterol as described under "Experimental
Procedures." Forty-eight hours after infection, both cells
(A) and the culture medium (B) were harvested,
extracted with methanol/water, and analyzed by TLC as described under
"Experimental Procedures." The migration of authentic standards is
indicated on the right.
View larger version (25K):
[in a new window]
Fig. 6.
27-Hydroxycholesterol accumulates in the
mitochondria of StAR-overexpressing cells, but not upon CYP27
overexpression. Primary rat hepatocytes were infected with the
indicated adenoviruses. Forty-eight hours after infection, mitochondria
were isolated, and endogenous 27-hydroxycholesterol levels were
determined as described under "Experimental Procedures." The graphs
represent the HPLC tracings showing 27-hydroxycholesterol peaks as
determined by an authentic standard.
-Hydroxycholesterol
Metabolism--
Overexpression of StAR did not alter the neutral
pathway of bile acid synthesis. 7-Hydroxycholesterol is the product
of the initial and rate-determining step in the neutral (classic)
pathway of bile acid synthesis. However, to be metabolized to bile
acids, 7-hydroxycholesterol must first be 27-hydroxylated by CYP27
in the mitochondria. To assess whether StAR protein might also induce the uptake and metabolism of 7-hydroxycholesterol to mitochondrial CYP27, 7-[14C]hydroxycholesterol was added to cells
(see "Experiment Procedures"). Shown in Fig.
7 is the time course for
7-[14C]hydroxycholesterol in cells overexpressing the
gene encoding StAR. The time course for
7-[14C]hydroxycholesterol utilization in primary rat
hepatocytes showed no effect on the metabolism of
7-hydroxycholesterol. These results further suggest that StAR
protein up-regulates bile acid synthesis via the alternative (acidic)
pathway via the delivery of cholesterol (and not bile acid
intermediates) to the inner mitochondrial membrane.
View larger version (11K):
[in a new window]
Fig. 7.
StAR overexpression does not
alter the rate of uptake or conversion of
7 -[14C]hydroxycholesterol to
bile acids. Primary rat hepatocytes were infected with the
indicated recombinant adenoviruses (NA, no virus) as
described under "Experimental Procedures." The cell culture medium
was collected at the indicated time points following infection.
A, bile acid synthesis rates were quantified as conversion
of 7-[14C]hydroxycholesterol to
methanol/water-extractable products as described under "Experimental
Procedures." B, 7-[14C]hydroxycholesterol
uptake was quantified as a function of change in
7-[14C]hydroxycholesterol extractable counts in the
medium.
View larger version (15K):
[in a new window]
Fig. 8.
Effects of StAR overexpression on the rate of
bile acid synthesis in chronic biliary diverted rats. Chronic
biliary diverted rats were infected with the indicated recombinant
adenoviruses (1.5 × 1011 virus particles) as
described under "Experimental Procedures." The 20-24-h time period
represents the time post-biliary diversion in which the pre-diversion
bile acid pool had drained and bile synthesis (i.e.
secretion) was at a basal level. The 70-h time period represents the
time of maximal up-regulation of bile acid synthesis that occurred
following pool drainage with loss of negative bile acid feedback. Data
are expressed as means ± S.E. (n = 6 for StAR and
n = 3 for controls).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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