GASTROENTEROLOGY 2000;119:1708 –1719
Sterol Carrier Protein 2 Gene Transfer Changes Lipid
Metabolism and Enterohepatic Sterol Circulation in Mice
SILVANA ZANLUNGO,* LUDWIG AMIGO,* HEGALY MENDOZA,* JUAN FRANCISCO MIQUEL,*
CARLOS VÍO,‡ JANE M. GLICK,§ ANNABELLE RODRÍGUEZ,储 KAREN KOZARSKY,¶
VERÓNICA QUIÑONES,* ATTILIO RIGOTTI,* and FLAVIO NERVI*
*Departamento de Gastroenterologı́a, Facultad de Medicina; ‡Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas,
Pontificia Universidad Católica, Santiago, Chile; §Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, Pennsylvania;
储Department of Endocrinology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and ¶Department of Cardiovascular
Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania
Background & Aims: Sterol carrier protein 2 (SCP-2) enhances sterol cycling and facilitates cholesterol translocation between intracellular organelles and plasma
membrane in cultured cells, including hepatocytes. We
examined the role of SCP-2 in hepatic cholesterol and
lipid trafficking through the sinusoidal and canalicular
secretory pathways of the liver in vivo. Methods: Recombinant adenovirus–mediated SCP-2 gene transfer was
used to obtain hepatic overexpression of SCP-2 in
C57BL/6 mice. Results: SCP-2 overexpression in the
mouse liver resulted in an 8-fold increase of SCP-2
protein levels and determined various effects on lipid
metabolism. It decreased high-density lipoprotein cholesterol and increased low-density lipoprotein (LDL) cholesterol concentrations. The expressions of hepatic LDL
receptor, apolipoprotein (apo) A-I, apoB, and apoE were
decreased. SCP-2 overexpression also increased hepatic
cholesterol concentration, associated with decreased
cholesterol neosynthesis. Increased biliary cholesterol
and bile acid secretion, bile acid pool size, and intestinal
cholesterol absorption were also observed. Conclusions:
These results indicate that modulation of SCP-2 expression in the liver determines important modifications on
lipoprotein metabolism, hepatic cholesterol synthesis
and storage, biliary lipid secretion, bile acid metabolism,
and intestinal cholesterol absorption.
he liver plays a central role in the maintenance of
cholesterol homeostasis in vivo.1– 4 Hepatocytes obtain cholesterol primarily through its biosynthesis from
acetate in the endoplasmic reticulum and peroxisomes,4,5
and its uptake from apolipoprotein (apo) B– containing
lipoproteins by low-density lipoprotein (LDL) receptor
family protein–mediated endocytosis1,2 or from highdensity lipoprotein (HDL) by selective cholesterol uptake
mediated by the scavenger receptor class B type I (SRBI).6,7 In addition, hepatocytes are relevant for cholesterol disposal by incorporating cholesterol and triglycerides into very-low-density lipoproteins, which, after the
T
delivery of endogenously synthesized lipids to peripheral
tissues, are converted into LDL.8 Liver cells also produce
a significant fraction of apoA-I, which is secreted into the
plasma complexed with phospholipids in HDL particles.8 Finally, the liver represents a unique organ involved in sterol elimination through the bile as cholesterol and newly synthesized bile acids.1,4,9 Given the
complex function of the liver in cholesterol metabolism,
sterol trafficking within the hepatocytes must be particularly intricate as well as exquisitely regulated.
Under steady-state conditions, the plasma membrane
and the endocytic apparatus contain approximately
80%–90% of the total cell cholesterol.10 This cholesterol
gradient between plasma membrane and intracellular
compartments implies the existence of a complex, efficient, and well-controlled system for sterol trafficking
among cell compartments through different intracellular
cholesterol transport mechanisms, including vesicles and
a variety of carrier proteins.10,11 Based on several in vitro
experiments, it has been postulated that sterol carrier
protein 2 (SCP-2) and sterol carrier protein X (SCP-X),
which originate from different transcripts of the same
gene,12 may have an important role in intracellular cholesterol trafficking and metabolism.13 Purified SCP-2
stimulated interorganellar cholesterol transfer14,15 as well
as the conversion of free cholesterol into cholesteryl
esters,16,17 steroid hormones,18 and 7␣-hydroxycholesterol.19,20 Similarly, SCP-X increased the transfer of sterols among intracellular membranes in vitro.21 In addiAbbreviations used in this paper: apo, apolipoprotein; ⑀-COP,
⑀-coatomer protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; PCR,
polymerase chain reaction; SCP-2, sterol carrier protein 2; SCP-X,
sterol carrier protein X; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SR-BI, scavenger receptor class B type I.
© 2000 by the American Gastroenterological Association
0016-5085/00/$10.00
doi:10.1053/gast.2000.20198
December 2000
tion, SCP-X had thiolitic activity on the side chain of
cholesterol, suggesting its relevance in bile acid biosynthesis.22
We have shown that SCP-2 appears to be critical for
rapid transport of newly synthesized cholesterol from the
endoplasmic reticulum to the plasma membrane in cultured human fibroblasts.23 More recently, Baum et al.24
reported that overexpression of SCP-2 in rat hepatoma
cells enhanced intracellular cholesterol cycling, increased
plasma membrane cholesterol content, and decreased
cholesterol esterification and HDL production. Furthermore, we found that SCP-2 expression was important to
control secretion of hepatic newly synthesized cholesterol
into bile in the rat, suggesting a role of SCP-2 function
in biliary cholesterol secretion.25 Consistent with this
latter proposal, Fuchs et al.26 showed that hepatic SCP-2
expression levels correlated with biliary cholesterol hypersecretion in mice with genetic predisposition to cholesterol gallstone disease.26 Similarly, hepatic SCP-2 levels were also elevated in patients with cholesterol
gallstones.27 These various in vivo studies strongly support the hypothesis that SCP-2 constitutively participates in hepatocellular cholesterol trafficking and suggest
that hepatic SCP-2 expression can influence overall cholesterol metabolism under physiologic and pathophysiologic conditions.
To further examine whether SCP-2 indeed plays a
major physiologic role in whole-body cholesterol homeostasis in vivo, we determined whether adenovirusmediated transfer of SCP-2 into the mouse liver altered
lipoprotein and hepatic cholesterol metabolism and biliary lipid secretion. We show that transient hepatic
overexpression of SCP-2 in mice decreased HDL cholesterol levels, whereas it increased plasma LDL cholesterol
concentrations. These changes were associated with decreased hepatic apoA-I, apoB, apoE, and LDL receptor
expression. In addition, SCP-2 overexpression enhanced
enterohepatic circulation of cholesterol and bile acids,
bile acid pool size, and intestinal cholesterol absorption.
These results show that hepatic SCP-2 expression can
regulate lipoprotein metabolism, hepatic sterol synthesis
and content, biliary lipid secretion, and enterohepatic
circulation of cholesterol and bile acids.
Materials and Methods
Animals and Diet
Adult male C57BL/6 mice over 8 weeks of age were
used in all experiments. Mice had free access to commercial
rodent diet Prolab RMH 3000 (PMI Nutritional International
Inc., Brentwood, MO), except where otherwise specified. This
diet contained small amounts of cholesterol (0.02%, wt/wt).
STEROL CARRIER PROTEIN 2: HEPATIC LIPID METABOLISM
1709
The animals were housed at 25°C in a well-ventilated room
with controlled reverse light cycling (middark was set at 10 AM
and midlight at 10 PM). All experiments were carried out
during the dark phase of the diurnal cycle.
Recombinant Adenoviruses Preparation
and Administration
The recombinant adenovirus Ad.rSCP2 was generated
by homologous recombination in 293 cells, essentially as described previously.28,29 The adenoviral backbone used for the
construction of the vector containing rat SCP-2 complementary DNA (cDNA) under control of the cytomegalovirus enhancer/promoter was derived from a replication-deficient firstgeneration type 5 adenovirus with deletions of E1 and E3
genes. The control adenovirus, AdE1⌬, contained the same E1
and E3 deletions without the transgene expression cassette.
Large-scale production of recombinant adenoviruses was performed from infected 293 cells, as described previously.30
For administration of viruses, mice were anesthetized by
intraperitoneal injection of 45 mg/k body wt sodium pentobarbital (Abbott Laboratories, North Chicago, IL). A jugular
vein was exposed, and 1 ⫻ 1011 particles (in 0.1 mL of isotonic
saline buffer) of control or recombinant adenoviruses were
injected intravenously. An additional control group received
0.1 mL of saline buffer only. In preliminary experiments, we
observed that the effects of adenovirus-mediated transfer of
SCP-2 cDNA on cholesterol metabolism peaked 5– 8 days after
infection and began to decline within 10 days. Therefore, we
performed all subsequent experiments 7 days after adenoviral
infections.
Bile and Blood Sampling
After 12 hours of fasting, mice were anesthetized as
described above. The cyst duct was ligated, and a common bile
duct fistula was performed using a PE10 polyethylene catheter
(Clay-Adams, New York, NY). Hepatic bile specimens were
collected for 30 – 60 minutes in preweighed tubes, and constant body temperature was maintained under a heating lamp.
At the end of the experiments, blood was removed from the
inferior vena cava. Plasma was immediately separated by centrifugation at 10,000 rpm for 10 minutes at 4°C. In some
experiments, consecutive 30-minute bile samples were obtained under depletion of the bile acid pool or under intravenous infusion of sodium taurocholate (1 mmol/min).
cDNA Probes
cDNA probes for apoB, cholesterol-7␣-hydroxylase,
and oxysterol-7␣-hydroxylase were kindly provided by Dr.
Roger Davis (San Diego State University, San Diego, CA).
cDNA probes for apoA-I and apoE were obtained as follows.
First-strand cDNA was prepared by random priming and
reverse transcription from liver (for the apoA-I probe) and
small bowel (for the apoE probe) total RNA and used as
a template in polymerase chain reaction (PCR) reactions.
The PCR primer pair for the apo A-I probe was (5⬘) 5⬘AGTTGGTACCTCCTGGAAAACTGGGACA-3⬘ and (3⬘)
1710
ZANLUNGO ET AL.
5⬘-CGGAAAGCTTTCAGAGTCTCGCTGGCCTTGT-3⬘. The
primer pair for the apo E probe was: (5⬘) 5⬘-CCTGAACCGCTTCTGGGATTAC-3⬘ and (3⬘) 5⬘-CCTAGCTGGTCATGGATGTTGC-3⬘. PCR products were subcloned into pGEM-T
(Promega, Madison, WI), sequenced, and purified on agarose
gel before radiolabeling.
Quantitative Immunoblotting Analysis
For SCP-2 immunoblotting, liver homogenates were
prepared as previously described.25 Each sample (30 ␮g) was
subjected to 12% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blotting using a
rabbit polyclonal anti-rat SCP-2 serum. An antialbumin antibody was used for protein loading control. For lipoprotein
receptor expression analysis, total membrane extracts (postnuclear 100,000g membrane pellets) from mouse liver were
prepared.31 For SR-BI immunoblotting, membranes (40 –100
␮g of protein/sample) were size-fractionated on 8% SDSPAGE and immunoblotted with a rabbit polyclonal antipeptide antibody against murine SR-BI provided by Dr. Monty
Krieger (Massachusetts Institute of Technology, Cambridge,
MA). For LDL receptor Western blotting, membrane extracts
(100 ␮g of protein/sample) were separated on 8% SDS-PAGE
under nonreducing conditions and immunoblotted with a
rabbit polyclonal anti-bovine LDL receptor antibody provided
by Dr. Helen Hobbs and Dr. Joachim Herz (Southwestern
Medical Center, Dallas, TX). Anti–⑀-coatomer protein (anti–
⑀-COP) antibody obtained from Dr. Monty Krieger was used
for membrane protein loading control. Antibody binding to
protein samples was visualized using the enhanced chemiluminescence procedure. Densitometric analysis was performed
with a Macintosh Color One scanner (Cupertino, CA) and NIH
Image software.
Hepatic Immunohistochemistry
Liver slices (2– 4 mm thick) were fixed by immersion
in Bouin’s solution, dehydrated, embedded in Paraplast
(Monoject Scientific, St. Louis, MO), sectioned at 7-␮m thickness in a rotatory microtome, mounted on glass slides, and
stored until processing. Immunostaining was performed according to the peroxidase/antiperoxidase method with some
modifications previously described.32,33 After inhibition of endogenous pseudoperoxidase activity with 3% (vol/vol) hydrogen peroxide in absolute methanol, tissue sections were incubated with the anti–SCP-2 antibody (1:1000 –1:2000)
overnight at 22°C, followed by the secondary antibody (1:20),
the peroxidase/antiperoxidase complex (1:150), and color development with 3,3⬘-diaminobenzidine-hydrogen peroxide.
Sections were counterstained with hematoxylin, dehydrated,
cleared with xylene, and coverslipped. For high-resolution
morphologic analysis, a modification of a pre-embedding ultrastructural immunohistochemistry protocol was performed
as previously described.33 All tissue sections were visualized
and photographed with a Nikon Optiphot microscope with a
Nikon Microflex UFX IIA filter (Nikon, Tokyo, Japan).
GASTROENTEROLOGY Vol. 119, No. 6
Blot Hybridization of RNA
Total RNA was prepared from mouse liver using the
acid guanidinium thiocyanate–phenol– chloroform method.34
Aliquots of 15 ␮g of total RNA were size-fractionated on a 1%
(wt/vol) agarose-formaldehyde gel and transferred to nylon
membranes. Filters were hybridized with 32P-labeled probes
(1 ⫻ 106 cpm/mL) for 2 hours at 65°C using Rapid-hyb buffer
(Amersham Pharmacia Biotech, Piscataway, NJ). Membranes
were then washed with 0.1% (wt/vol) SDS/2⫻ standard saline
citrate for 10 minutes at room temperature, followed by another wash with the same solution for 10 minutes at 65°C, and
finally autoradiographed with Kodak film at ⫺80°C. Densitometric analysis was performed as described above. Results
were normalized to the signal of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) messenger RNA (mRNA). For cholesterol 7␣-hydroxylase expression analysis, poly(A)⫹-enriched
RNA was prepared from 200 ␮g of total liver RNA using the
PolyAT Tract kit (Promega, Madison, WI) and subjected to
blot hybridization, as described for the other cDNA probes.
Quantitation of Hepatic Cholesterol
Synthesis In Vivo
After a 2-hour fasting period, the rate of cholesterol
synthesis was measured at the middark phase of the diurnal
cycle (10 AM). Each mouse received 50 mCi [3H]water (Amersham Pharmacia Biotech, Piscataway, NJ) by intraperitoneal
injection as previously described.35 One hour after radiolabel
injection, animals were anesthetized and approximately 0.5
mL of blood was obtained for determination of water-specific
activity in plasma. After liver removal, tissue specimens were
saponified, and digitonin-precipitable sterols were isolated as
previously described.36 Results were expressed as micromoles
of [3H]water incorporated into digitonin-precipitable sterols
per hour per gram of liver weight.
Measurement of Bile Acid Pool Size and
Fecal Excretion
Bile acid pool size was quantified as the total mass of
bile acids extracted from the small intestine, liver, and gallbladder as previously described.35 Briefly, organs were minced
and extracted at 60°C for 4 hours in ethanol containing
[24-14C]taurocholic acid (New England Nuclear, Boston, MA)
as internal standard. After filtration, aliquots of the extracts
were dried in a vial and the recovery of radiolabeled taurocholic
acid was determined by scintillation counting. Recoveries were
always ⬎91%. Bile acid pool size was expressed as micromoles
of bile acids/100 g body wt. To determine daily fecal bile acid
excretion, stools were collected from each animal housed individually during a 24-hour period from the sixth to the
seventh day after intravenous administration of adenoviral
preparations or saline controls. Stools were extracted with
[14C]cholic acid (New England Nuclear, Boston, MA) added as
internal standard, and bile acids were quantified and corrected
for recovery.37
December 2000
STEROL CARRIER PROTEIN 2: HEPATIC LIPID METABOLISM
1711
Measurement of Intestinal
Cholesterol Absorption
Intestinal cholesterol absorption was measured by the
dual-isotope ratio method.38 Briefly, mice were individually
housed in wire cages, fasted for 4 hours, and at noon were
given an intragastric bolus of 100 ␮L of corn oil containing 1
mCi [14C]cholesterol (New England Nuclear) and 2 mCi
[3H]␤-sitostanol (American Radiolabeled Chemicals, St.
Louis, MO). Feces was collected for 24 hours, dried overnight
at 40°C, and extracted with chloroform-methanol (2:1 vol/
vol). After phase separation, a fraction of the chloroform phase
was transferred into scintillation vials and dried under a hood.
Radioactivity of each specimen was measured and corrected by
the channel ratio method using external standards. Intestinal
cholesterol absorption was calculated as the percent of cholesterol absorbed per day by using the formula: % Absorption ⫽
{1 - [ Fecal (14C/3H)]/[Administered (14C/3H)]} ⫻ 100.
Plasma Lipoprotein Separation and
Plasma, Hepatic, and Biliary Lipid Analyses
Plasma lipoprotein separation was performed by Superose 6 –fast protein liquid chromatography gel filtration of
fresh plasma specimens.39 For other determinations, liver, bile,
and plasma samples were frozen at 20°C until processing.
Total plasma and lipoprotein cholesterol concentrations were
measured using enzymatic kits (Sigma Chemical Co., St. Louis,
MO). Hepatic and biliary cholesterol, biliary phospholipids,
and bile acids were determined by routine methods.40 – 42 Bile
acid pool composition analysis was kindly performed by Dr.
Stephen Turley (Southwestern Medical Center, Dallas, TX).
The bile acid–independent bile flow fraction was calculated by
linear regression analysis of bile flow as a function of bile acid
output according to the equation y ⫽ a ⫹ bx, where a
represents the bile acid–independent bile flow at the interception of the y-axis.
Statistics
Data are presented as means ⫾ SE. The 2-tailed,
unpaired Student t test was used to compare the sets of data.
Statistically significant differences were considered at a P value
of ⬍0.05.
Results
To evaluate the relevance of SCP-2 in hepatic
lipid metabolism in vivo, we studied C57BL/6 mice that
transiently overexpressed SCP-2 in the liver by adenovirus-mediated gene transfer (Ad.rSCP2). Controls included noninfected saline-injected animals or mice infected with a control adenovirus that lack a cDNA
transgene (Ad.E1⌬). Hepatic SCP-2 expression was increased 8-fold in Ad.rSCP2–infected mice as evaluated
by immunoblotting of liver homogenates 7 days after
infection (Figure 1). As expected, hepatic SCP-X expression remained unchanged in Ad.rSCP2-infected mice.
Figure 1. Immunoblot analysis of SCP-2 expression in livers of mice
infected with SCP-2 recombinant adenovirus. Liver homogenates were
prepared and subjected to 12% SDS-PAGE and Western blotting with
anti–SCP-2 and antialbumin antibodies. The change in SCP-2 protein
expression of Ad.rSCP-2–infected mice compared with control
AdE1⌬–infected mice is shown after correction for albumin signal.
Immunostaining of liver tissue readily revealed SCP-2
protein overexpression in a large number of hepatocytes
from animals infected with Ad.rSCP2 (Figures 2A and C)
compared with tissue from noninfected mice (not shown)
or control Ad.E1⌬-infected mice (Figure 2B and D). In
Ad.rSCP2-infected mice, overexpression of SCP-2 protein was heterogeneous, ranging from faint to heavy
immunostaining. SCP-2–positive hepatocytes were
heavily stained with a subcellular pattern, suggesting a
granular distribution over the cytoplasm (Figure 2C).
When high-resolution immunohistochemistry was used
to examine thin liver sections, the subcellular distribution of SCP-2 was evidenced in round organelles highly
suggestive of peroxisomes (Figure 2E and F ), which are
a major subcellular compartment for SCP-2 localization
in liver parenchymal cells.43,44 No SCP-2 protein expression was found in liver endothelial cells or in Kupffer
cells.
Compared with those in control Ad.E1⌬-infected
mice, plasma cholesterol levels were unchanged in Ad.rSCP2 animals (Table 1). These results were consistent
with previous studies showing that recombinant adenoviral infection per se does not change total plasma cholesterol levels.45 However, plasma lipoprotein cholesterol
distribution was altered in Ad.rSCP2 mice compared
with that in Ad.E1⌬ mice (Figure 3); the plasma LDL
cholesterol concentration increased by 100%, and HDL
cholesterol levels decreased by 25%. Very-low-density
lipoprotein cholesterol was not changed. Together, these
results indicate that hepatic SCP-2 expression can regulate lipoprotein cholesterol metabolism in vivo.
Because the modifications observed in plasma lipoprotein cholesterol distribution in Ad.rSCP2-overexpressing
mice might have been caused by changes in hepatic
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GASTROENTEROLOGY Vol. 119, No. 6
Figure 2. Immunohistochemical localization
of SCP-2 in liver tissue of SCP-2 recombinant
adenovirus–infected mice. Low-resolution
immunohistochemistry was performed in
liver sections of (A and C ) Ad.rSCP2- and
(B and D) AdE1⌬-infected mice. (E and F )
High-resolution hepatic immunohistochemistry of Ad.rSCP2-infected mice. Bar ⫽ 50 ␮m
(A and B); bar ⫽ 25 ␮m (C and D); bar ⫽ 10
␮m (E ); bar ⫽ 5 ␮m (F ).
lipoprotein synthesis and/or receptor-mediated lipoprotein cholesterol clearance, we evaluated hepatic expression of key apolipoproteins and cell surface receptors
involved in lipoprotein metabolism in vivo. The expres-
sions of apoA-I, apoB, and apoE were analyzed by Northern blot and normalized to GADPH expression (Figure
4). Liver mRNA levels of apoA-I and apoE, 2 major
apolipoprotein constituents of HDL in rodents, and
Table 1. Effect of SCP-2 Recombinant Adenoviral Infection on Body and Liver Weight and Serum Lipid Concentrations in Mice
Total plasma triglyceride
(mg/dL)a
Group
Body wt
( g)
Liver wt
( g)
Total plasma cholesterol
(mg/dL)
Fed
Fasting
A. Saline (n ⫽ 17)
B. Ad.E1⌬ (n ⫽ 15)
C. Ad.rSCP2 (n ⫽ 12)
21 ⫾ 0.6
22 ⫾ 0.6
21 ⫾ 0.3
0.92 ⫾ 0.02
0.94 ⫾ 0.03
1.48 ⫾ 0.06b
88 ⫾ 2.7
87 ⫾ 3.9
98 ⫾ 5.1
60 ⫾ 3
104 ⫾ 6
84 ⫾ 5b
21 ⫾ 3
38 ⫾ 6
32 ⫾ 4
NOTE. Determinations were made 7 days after intravenous administration of saline or adenoviruses. Values represent means ⫾ SE. The number
of mice in each group is shown in parenthesis.
aPlasma triglyceride values were determined in 5– 6 mice in each group.
bSignificant difference (P ⬍ 0.001) compared with groups A and B.
December 2000
Figure 3. Plasma lipoprotein cholesterol distribution in mice with
adenovirus-mediated SCP-2 gene transfer. Plasma lipoproteins were
separated by fast protein liquid chromatography gel filtration, and
cholesterol was measured in lipoprotein fractions. Values are expressed as percentages of total plasma cholesterol for 5– 6 nonfasted mice in each experimental group. *P ⬍ 0.001.
apoB, the unique protein component of LDL, were decreased in Ad.rSCP2-infected mice by 50%, 80%, and
40%, respectively. Liver expression of LDL receptor and
HDL receptor SR-BI were evaluated by immunoblotting
in hepatic membrane extracts (Figure 5). Compared with
control mice, hepatic LDL receptor expression decreased
by 40%, whereas SR-BI levels remained unchanged.
These findings showed that SCP-2 can influence hepatic
apolipoprotein and LDL receptor expression.
As shown in Table 2, SCP-2 overexpression increased
hepatic-free cholesterol concentration 1.6-fold and decreased in vivo hepatic synthesis of cholesterol by 40%.
The increase in cholesterol content was consistent with
feedback inhibition of hepatic cholesterol synthesis and
LDL receptor expression (Figure 5) found in Ad.rSCP2infected mice compared with control virus-infected ani-
STEROL CARRIER PROTEIN 2: HEPATIC LIPID METABOLISM
1713
mals. Although no change was observed in absolute
hepatic cholesteryl ester concentration, the relative levels
of esterified cholesterol and total cholesterol were significantly reduced in Ad.rSCP-infected mice. These results
indicate that SCP-2 expression regulates intrahepatic
cholesterol metabolism.
Next, we studied the effects of hepatic SCP-2 overexpression on bile flow and biliary lipid concentrations
(Table 3). Total bile flow increased by 73% in Ad.rSCP2infected mice. This increased bile flow was most likely
caused by an approximately 3-fold increase in the bile
acid–independent bile flow in Ad.rSCP2 mice compared
with Ad.E1⌬-infected mice. Biliary lipid concentrations
remained within normal range, with the exception of
biliary cholesterol levels, which were slightly but significantly increased by 20% in Ad.rSCP2 mice. Biliary bile
acid and phospholipid outputs increased by approximately 50%– 60%, whereas biliary cholesterol output
doubled in Ad.rSCP2 mice compared with Ad.E1⌬ animals (Table 4). Cholesterol– bile acid and cholesterol–
phospholipid molar ratios were significantly increased in
hepatic bile from Ad.rSCP2 mice by 34% and 25%,
respectively, indicating that hepatic SCP-2 gene overexpression had a more important effect on biliary cholesterol secretion, which is independent of the observed
effect on biliary bile acid secretion.
Consistent with the increase in biliary bile acid output, the bile acid pool size was significantly expanded by
approximately 15% in Ad.rSCP2-infected mice, whereas
bile acid pool composition remained unchanged (Table
5). The daily fecal bile acid excretion rate, which is a
good indicator of hepatic bile synthesis, was also similar
in the different experimental groups, a finding that was
consistent with unchanged cholesterol 7␣-hydroxylase
expression, the most relevant rate-limiting enzyme in
Figure 4. Hepatic expression of apoA-I, apoB, and apoE in Ad.rSCP2-infected mice. Total hepatic RNA was prepared, electrophoresed, and
transferred to nylon membranes. Apolipoprotein gene expression was evaluated by RNA blot hybridization with 32P-labeled cDNA probes and
densitometric analysis. The fold change in mRNA expression in Ad.rSCP-2 mice compared with AdE1⌬-infected mice is shown after normalization
against GAPDH mRNA. AP ⬍ 0.05.
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GASTROENTEROLOGY Vol. 119, No. 6
Figure 5. Hepatic LDL receptor and SR-BI expression in SCP-2– overexpressing mice. Total membrane extracts were prepared, size fractionated
by SDS-PAGE, immunoblotted with anti–SR-BI and LDL receptor antibodies, and subjected to densitometric analysis. Anti-⑀COP antibody was used
for membrane protein loading control. The fold change in receptor expression of Ad.rSCP-2 mice compared with control Ad.E1⌬ mice is shown
after correction for ⑀COP signal. AP ⬍ 0.05.
bile acid synthesis (Figure 6, left panel). However, mRNA
levels for the oxysterol-7␣-hydroxylase were decreased by
50% in Ad.rSCP2 mice (Figure 6, right panel), suggesting
selective down-regulation of the alternative pathway for
hepatic bile acid synthesis. Given the measured basal
biliary bile acid outputs and assuming that the intrinsic
motility of the biliary tree remained unchanged, it can be
calculated that the bile acid pool recirculated 5.5 times/
day in the Ad.rSCP2 mice compared with 4 times/day in
control noninfected and Ad.E1⌬-infected mice, suggesting that the turnover of the bile acid pool was increased
in the Ad.rSCP2 mice. This increased enterohepatic circulation of bile acids could have had an effect on intestinal cholesterol absorption. In fact, dietary cholesterol
absorption was significantly increased from 63% in the
saline-injected and Ad.E1⌬-infected animals to 71% in
Ad.rSCP2 mice. Taken together, these studies indicate
that hepatic overexpression of SCP-2 results in increased
bile acid pool size and turnover and, subsequently, enhanced intestinal cholesterol absorption.
hepatic cholesterol and bile acid metabolism were found:
(1) unesterified cholesterol accumulation and cholesterol
synthesis inhibition; (2) increased bile flow and biliary
lipid secretion; and (3) increased bile acid pool size
associated with normal cholesterol 7␣-hydroxylase expression and fecal bile acid excretion. In addition, adenovirus-mediated SCP-2 gene transfer resulted in increased intestinal cholesterol absorption.
SCP-2 protein is mainly localized to peroxisomes.43,44
As a consequence, the mechanism by which peroxisomal
SCP-2 overexpression can regulate extraperoxisomal cholesterol trafficking in the liver remains to be elucidated.
However, a number of studies have suggested that at
least in hepatocytes, a significant proportion of SCP-2 is
also present in the cytosol.43,44 These observations have
been supported recently by studies in both mice that are
Table 2. Effect of SCP-2 Recombinant Adenoviral Infection
on Hepatic Cholesterol Synthesis and Content
in Mice
Discussion
These studies show that adenovirus-mediated
overexpression of SCP-2 in the mouse liver was associated
with marked changes in lipoprotein metabolism, hepatic
cholesterol synthesis and content, biliary lipid output,
and enterohepatic circulation of cholesterol and bile acids. Hepatic SCP-2 overexpression decreased HDL cholesterol concentration and increased LDL cholesterol levels, which were correlated with lowered expression of
hepatic apoA-I and LDL receptor. Multiple effects on
Group
A. Saline
B. Ad.E1⌬
C. Ad.rSCP2
Hepatic cholesterol
synthesis
(␮mol 䡠 h⫺1 䡠 100 g Cholesterol Cholesteryl esters
(mg/g liver)
body wt⫺1)
(mg/g liver)
1.63 ⫾ 0.09
3.14 ⫾ 0.19
1.91 ⫾ 0.33a
2.0 ⫾ 0.3
2.4 ⫾ 0.1
3.9 ⫾ 0.6b
0.32 ⫾ 0.3
0.39 ⫾ 0.1
0.52 ⫾ 0.2
NOTE. All values are expressed as means ⫾ SE. Experiments (4 – 6
animals in each group) were performed during the middark phase of
the diurnal cycle.
aSignificant difference (P ⬍ 0.001) compared with group B.
bSignificant difference (P ⬍ 0.001) compared with groups A and B.
December 2000
STEROL CARRIER PROTEIN 2: HEPATIC LIPID METABOLISM
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Table 3. Effect of SCP-2 Recombinant Adenoviral Infection on Bile Flow and Biliary Lipid Concentrations in Mice
Bile flow (␮L 䡠 min⫺1 䡠 100 g body wt⫺1)
Hepatic bile lipid concentration (mmol/L)
Group
Total
Bile acid independent
Bile acids
Phospholipids
Cholesterol
Gallbladder
cholesterol (mmol/L)
A. Saline
B. Ad.E1⌬
C. Ad.rSCP2
8.0 ⫾ 0.4
7.4 ⫾ 0.3
12.8 ⫾ 0.7a
6.0 ⫾ 0.8
3.8 ⫾ 0.8
10.7 ⫾ 0.7a
28.5 ⫾ 2.1
31.3 ⫾ 2.2
28.2 ⫾ 2.7
5.5 ⫾ 0.4
6.1 ⫾ 0.3
5.9 ⫾ 0.4
0.65 ⫾ 0.05
0.71 ⫾ 0.06
0.85 ⫾ 0.06b
2.1 ⫾ 0.27
2.1 ⫾ 0.41
2.0 ⫾ 0.19
NOTE. Values represent means ⫾ SE. Experiments were performed during the dark phase of the diurnal cycle. Measurements were performed
in 11 mice in each experimental group except for gallbladder cholesterol concentration (5– 6 mice in each group). Some animals were depleted
of bile salts or intravenously infused with 1 mmol sodium taurocholate. Linear regression analysis of bile flow as a function of bile acid secretion
rates were significantly correlated (r ⫽ 0.93, P ⬍ 0.001).
aP ⬍ 0.001, bP ⬍ 0.05; group C compared with groups A and B.
genetically susceptible to gallstones26 and cholesterol
gallstone patients,27 in whom hepatic SCP-2 mRNA
levels correlated with cytosolic SCP-2 protein concentration and hepatic cholesterol concentration. We hypothesize that the triggering event involved in the multiple
effects of hepatic SCP-2 overexpression on lipid metabolism in mice was the increased cholesterol cycling and
intracellular cholesterol redistribution previously reported in SCP-2–transfected cultured rat hepatoma
cells.24 These in vitro studies strongly suggest that the
velocity of the bidirectional flux of cholesterol between
intracellular and plasma membranes was a regulated
process dependent on SCP-2 expression levels. Consequently, SCP-2 transfection determined free cholesterol
accumulation in the plasma membrane and simultaneous
reduction in cholesterol esterification, HDL apolipoprotein expression, and HDL production. These changes in
cholesterol metabolism and transport in SCP-2–transfected rat hepatoma cells are consistent with our findings
in the SCP-2– overexpressing mouse model: a relatively
lower proportion of hepatic cholesteryl ester content,
decreased apoA-I and apoE expression, and reduced
plasma HDL cholesterol concentration. The effect of
SCP-2 overexpression in vivo on relative hepatic cholesteryl ester content might be explained by a shift in net
cholesterol transport from intracellular organelles to the
plasma membrane, as reported in SCP-2–transfected hepatic cells.24 Furthermore, this SCP-2– dependent control of intracellular cholesterol distribution may be a key
regulatory process determining the expression of choles-
terol-sensitive genes, such as apoA-I and apoE. In fact,
apoA-I and E gene expression have been correlated with
changes in cholesterol content in putative regulatory
sterol pools.46,47 Because apoA-I synthesis is the main
determinant of HDL production,8 it is likely that the
decreased plasma HDL cholesterol levels observed in
SCP-2– overexpressing mice were caused by decreased
hepatic HDL synthesis and secretion. Hepatic SCP-2
overexpression also had a major impact on hepatic cholesterol metabolism. The relative inhibition of hepatic
cholesterogenesis found in the Ad.rSCP2 mice was correlated with increased concentration of hepatic cholesterol and the expected feedback regulation in hepatic
LDL receptor expression. The mechanism by which
SCP-2 down-regulates apolipoprotein expression and
cholesterol synthesis is not known. It might be related to
changes in the cholesterol distribution within the cell,
presumably in the endoplasmic reticulum, where cholesterol sensing occurs. Although our measurements of LDL
receptor and SR-BI protein levels, as well as message
levels for apoproteins, can explain the physiologic basis
for changes in plasma lipoproteins in the SCP-2– overexpressing model, it is important to note that they are
indirect data and not direct measurements. Further studies are needed to establish the molecular basis and physiological significance of the observed changes.
Although increased hepatic SCP-2 expression stimulated secretion of the 3 major biliary lipids, it had a more
important effect on biliary cholesterol secretion. These
findings are consistent with the decreased transport of
Table 4. Effect of SCP-2 Gene Transfer on Biliary Lipid Secretion and Cholesterol Molar Ratio in Mice
Biliary lipid output (nmol 䡠 min⫺1 䡠 100 g body wt⫺1)
Cholesterol molar ratio
Group
Bile acids
Phospholipids
Cholesterol
Chol ⫻ 103/bile acids
Chol ⫻ 103/phospholipids
A. Saline
B. Ad.E1⌬
C. Ad.rSCP2
235 ⫾ 29
235 ⫾ 25
360 ⫾ 38a
45 ⫾ 5
46 ⫾ 4
74 ⫾ 4a
5.3 ⫾ 0.5
5.4 ⫾ 0.7
10.9 ⫾ 0.9a
24 ⫾ 2
23 ⫾ 1
31 ⫾ 2a
120 ⫾ 6
116 ⫾ 6
146 ⫾ 7a
NOTE. All values are means ⫾ SE. Each experimental group had 9 –11 animals.
aP ⬍ 0.01, group C compared with groups A and B.
1716
ZANLUNGO ET AL.
GASTROENTEROLOGY Vol. 119, No. 6
Table 5. Effect of SCP-2 Gene Transfer on Bile Acid Pool Size and Composition, Fecal Bile Acid Excretion, and Intestinal
Cholesterol Absorption in Mice
Cholesterol
group
A. Saline
B. Ad.E1⌬
C. Ad.rSCP2
Bile acid pool composition (%)
Bile acid pool size
(␮mol/100 g body
wt)
Muricholates
Cholate
81 ⫾ 3.2
82 ⫾ 2.8
94 ⫾ 4.3a
43 ⫾ 4
47 ⫾ 3
39 ⫾ 2
45 ⫾ 3
42 ⫾ 2
45 ⫾ 3
Other species
Fecal bile acid excretion
(␮mol 䡠 day⫺1 䡠 100 g
body wt⫺1)
Intestinal
absorption (%)
13 ⫾ 1
11 ⫾ 1
16 ⫾ 2
19.0 ⫾ 0.5
18.3 ⫾ 0.6
18.2 ⫾ 0.3
63 ⫾ 1
63 ⫾ 1
71 ⫾ 2a
NOTE. Values are means ⫾ SE. Bile acid pool size was determined in 11 mice in each group, bile acid pool composition in 4 – 6 animals/group,
and fecal bile acid outputs in 6 animals/group. Cholesterol absorption was measured in 4 – 8 mice/group.
aP ⬍ 0.02.
newly synthesized cholesterol from liver to bile observed
in SCP-2 antisense oligonucleotide–treated rats.25 They
are also consistent with the correlation between increased
SCP-2 expression and biliary cholesterol hypersecretion
in mice genetically predisposed to cholesterol gallstone
formation,26 as well as in patients with cholelithiasis.27
The major driving forces of biliary cholesterol secretion
are the rate of bile acid secretion, the hydrophobicity of
bile acid pool, and the availability of free cholesterol in
the metabolically active pool of the hepatocyte.1,9 In the
present study, we showed that 2 potential factors responsible for the enhanced biliary cholesterol output in Ad.rSCP2 mice were the increased biliary bile acid output
and the potentially augmented hepatic cholesterol availability as a consequence of increased intestinal cholesterol
absorption. Because the bile acid pool composition remained unchanged, increased biliary cholesterol secre-
tion was not attributable to a higher proportion of hydrophobic bile salt species.
We can postulate that the effect of SCP-2 overexpression on biliary cholesterol output might be explained by
vectorial enrichment of cholesterol in some specific canalicular plasma membrane domains, which are the immediate source of cholesterol to be recruited for bile
secretion. SCP-2 transfection in rat hepatoma cells determined cholesterol accumulation in the plasma membrane.24 Furthermore, SCP-2 can regulate cholesterol
distribution between different kinetic domains of the
plasma membrane.48 On the other hand, previous studies
have shown that cholesterol hypersecretion was correlated to a significant increase in the concentration of
cholesterol in the canalicular membrane.49 Cholesterolrich plasma membrane regions may exist in liver plasma
membranes and correspond to detergent-resistent do-
Figure 6. Hepatic expression of cholesterol 7␣-hydroxylase and oxysterol 7␣-hydroxylase in Ad.rSCP2-infected mice. Hepatic RNA was prepared,
electrophoresed, and transferred to nylon membranes. Hydroxylase gene expression was evaluated by RNA blot hybridization with 32P-labeled
cDNA probes and densitometric analysis. The fold change in mRNA expression of Ad.rSCP-2 mice compared with AdE1⌬-infected mice is shown
after normalization against GAPDH mRNA. AP ⬍ 0.05.
December 2000
mains, such as caveolae and lipid rafts that seem to
participate in cellular cholesterol efflux in nonhepatic
cells.50 In fact, caveolin-1 and -2, 2 major protein markers of caveolae, are highly expressed in hepatocytes and
copurify with Triton X-100 –resistent domains prepared
from hepatocellular membranes.51,52 Another consequence of an SCP-2– dependent cholesterol enrichment
in some specific plasma membrane domains may have
important functional implications for the activities of a
variety of plasma membrane enzymes and transporters.53
One of these potential implications might be related to
the increase in the bile acid–independent bile flow found
in Ad.rSCP2 mice. Increased cholesterol concentration in
the canalicular membrane could have stimulated the
activities of apical transporters (i.e., multidrug resistance–associated protein) that regulate secretion of key
bile constituents (i.e., glutathione) and control bile
flow.54
Previous studies show that cholesterol from HDL is
preferentially secreted into bile compared with cholesterol from other lipoproteins.55 However, the results of
the present study suggest that SR-BI may not be involved in the enhanced secretion of biliary cholesterol in
the SCP-2– overexpressing mice because hepatic SR-BI
protein levels do not change in these animals. Therefore,
in this model the decreased levels of plasma HDL cholesterol seem to be a consequence of decreased hepatic
HDL synthesis, rather than increased clearance of HDL
by the liver. More likely, the origin of the increase of
biliary cholesterol output was related to the enhanced
intestinal cholesterol absorption in the Ad.rSCP2 mice.
ApoE-rich chylomicron remnants and its sinusoidal
membrane receptor56 mediate this pathway of dietary
cholesterol delivery to the liver and bile.
A major finding in the SCP-2– overexpressing mice
was a greater than normal bile acid pool size, which was
associated with an increased enterohepatic circulation of
bile salts and correlated with enhanced absorption of
dietary cholesterol in the small intestine. Interestingly,
fecal bile acid excretion remained unchanged, suggesting
a normal rate of bile acid synthesis. This latter finding
was consistent with a normal hepatic expression of the
cholesterol 7␣-hydroxylase gene. The increase in bile
acid pool size and enhanced biliary secretion of bile acids
associated with a normal rate of synthesis may be related
to an increase in the turnover of the bile acid pool.
Another plausible explanation for these results is that the
bile acid pool size increased because of a transitory increase in bile acid synthesis that might have taken place
before we decided to measure bile acid kinetics. A potential weakness of this study was a lack of a temporal
STEROL CARRIER PROTEIN 2: HEPATIC LIPID METABOLISM
1717
vision of the events that took place between the moment
of the infection and the time chosen for performing the
protocols (7 days).
Unexpectedly, the expression of oxysterol-7␣-hydroxylase, an early enzyme of the alternative bile acid synthesis pathway that is coordinately regulated with sterol
27-hydroxylase but not with cholesterol 7-hydroxylase in
the mouse liver,57 was markedly reduced in Ad.rSCP2
mice. The functional significance of this finding is not
apparent, but it suggests that expression of this gene
might be under coordinate control together with other
genes sensitive to SCP-2– dependent intracellular cholesterol channeling, such as those genes encoding apoA-I,
apoB, and apoE.
In contrast to the present study, Seedorf et al.58 and
Kannereberg et al.59 have not reported a major abnormal
phenotype for cholesterol or lipoprotein metabolism in
mice with disruption of the SCP-2/SCP-X gene. These
investigators found only a failure in the oxidation of
2-methyl-branched fatty acids and the side chain of
cholesterol. The peroxisomal 58-kilodalton SPC-X or
SCP-2/thiolase22 normally performs these functions. We
speculate that this apparent discrepancy between the
SCP-2/SCP-X knockout mouse model and the present
study could be caused by compensatory mechanisms
activated in SCP-2/SCP-X knockout mice. These alternative mechanisms are normally able to transport cholesterol among intracellular organelles and might be
up-regulated as a consequence of the SCP-2 deficiency in
the gene-manipulated mouse model. We have previously
reported that the transfer of newly synthesized cholesterol from the endoplasmic reticulum to the plasma
membrane in normal human fibroblasts23 and from the
liver into the bile in the rat25 was a rapid SCP-2–
dependent transport process. However, when similar
studies were performed in fibroblasts obtained from patients with Zellweger disease that lack peroxisomes and
do not express SCP-2, cholesterol trafficking to the
plasma membrane was delayed, but not completely abolished. In fact, it was switched to a vesicle-mediated
intracellular cholesterol transport pathway that was sufficient to maintain normal cell viability.23
In summary, these studies support the concept that
hepatic SCP-2 lipid–mediated trafficking represents a
major physiologic regulatory mechanism for hepatic cholesterol metabolism, lipoprotein metabolism, biliary cholesterol secretion, and enterohepatic circulation of cholesterol and bile salts. The more striking new
contribution of these series of experiments is the finding
that hepatic SCP-2 overexpression determines a cascade
of sterol-regulatory mechanisms, which led to increased
1718
ZANLUNGO ET AL.
hepatic cholesterol concentration, a decrease in the rate of
tissue cholesterogenesis, and enhanced cholesterol absorption. It is possible that SCP-2 expression levels in the
liver play an important role in pathophysiologic processes related to abnormal hepatic cholesterol metabolism such as atherosclerosis and cholesterol gallstone
disease. Further studies would be required to elucidate
the complex interaction among different hepatocellular
sterol carrier proteins, the enterohepatic circulation of
sterol molecules, and whole body cholesterol homeostasis.
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Received May 5, 2000. Accepted July 26, 2000.
Address requests for reprints to: Flavio Nervi, M.D., Pontificia Universidad Católica de Chile, Departamento de Gastroenterologı́a, Marcoleta 387, Casilla 114-D, Santiago, Chile. e-mail: fnervi@med.puc.cl;
fax: (56) 2-639-7780.
Supported by a donation of Elliot Marcus, Fondo Nacional de Desarrollo Cientı́fico y Tecnológico (FONDECYT, grants 1971092 and
1000739 to F.N., 8990006 to A.R. and J.F.M., 7980016 to K.K. and
A.R., and 1000567 to S.Z.) and the National Institutes of Health
(grants HL55756 to J.M.G. and K08HL03067 to A.R.).
The authors thank Dr. J. Chianale and V. Vollrath for providing the
apoE cDNA probe; Drs. Helen Hobbs, Joachim Herz, and Monty Krieger
for providing antibodies; Dr. Roger Davis for supplying cDNA probes;
and Dr. Stephen Turley for measuring bile acid pool composition.