 |
INTRODUCTION |
The risk of developing coronary artery disease is directly related
to plasma levels of low density lipoprotein
(LDL)1 cholesterol and
inversely associated with the concentration of high density lipoprotein
cholesterol (HDL) (1, 2). Although numerous studies have revealed many
steps involved in LDL metabolism, significantly less is known about HDL
metabolism. Novel insights concerning the metabolism of HDL have come
from a recent series of experiments suggesting that the scavenger
receptor class B type I (SR-BI) is an HDL receptor (3-9). SR-BI, a
member of the CD36 superfamily, was originally cloned as a receptor for
modified LDL (10-12). Transfected cells expressing SR-BI show high
affinity binding with HDL and take up cholesteryl ester from the
particle by a selective uptake pathway markedly distinct from the LDL
receptor pathway. Further support for SR-BI being an HDL receptor comes from the observations that SR-BI is expressed in liver and
non-placental steroidogenic tissues, long hypothesized sites of HDL
cholesterol delivery (3-6).
The expression of SR-BI in tissues appears to be responsive to changes
in hormonal status and HDL cholesteryl ester supply (13-16). In the
adrenal glands of knockout mice deficient in apoA-I (13), hepatic
lipase (13), or lecithin-cholesterol acyltransferase (14), the
expression of SR-BI is increased markedly. Moreover, the expression of
SR-BI is up-regulated in the adrenal gland and down-regulated in the
liver of rats after high dose estrogen treatment (15). The
estrogen-induced increase of SR-BI is accompanied by enhanced uptake of
lipid from HDL. Similarly, the administration of human chorionic
gonadotropin also causes an increase in SR-BI expression in the
steroidogenic Lydig cells of rat testis (15). These observations
suggest that the SR-BI expression may be regulated in response to the
supply and demand for cholesterol by some tissues.
Some of the most convincing studies suggesting a role for SR-BI in HDL
metabolism come from the characterization of mice with genetically
manipulated SR-BI expression. Two studies have examined mice with
marked reductions in SR-BI expression caused by gene targeting in
embryonic stem cells (8, 9). Increased HDL cholesterol levels in
animals deficient in SR-BI suggest the importance of SR-BI in the
clearance of HDL cholesterol. The in vivo effect of
transient increases in SR-BI expression was studied in mice after
adenovirus-mediated SR-BI gene transfer (7). This was associated with a
marked decrease in plasma HDL cholesterol and an increase in biliary
cholesterol. The adenoviral study, although supporting the hypothesis
that increased SR-BI expression results in accelerated clearance of HDL
cholesterol, is complicated by the massive short term increase in the
expression of SR-BI and the hepatotoxicity that accompanies murine
adenoviral infections.
To investigate the effect of steady-state increases in SR-BI expression
on HDL metabolism we created several lines of SR-BI transgenic mice.
One of the transgenes was a mouse BAC clone containing the SR-BI gene
with the promoter region replaced by human liver-specific apoA-I
promoter. With this transgene two lines of SR-BI transgenic animals
were studied, one with a 10-fold increase in SR-BI transcripts compared
with control mice and a second line with a 2-fold increase. To
determine the effect of SR-BI on lipoprotein metabolism and plasma
cholesterol homeostasis we examined the impact of the increased levels
of SR-BI on HDL and non-HDL lipoproteins and associated apolipoproteins
with regard to plasma levels and clearance rates.
 |
MATERIALS AND METHODS |
Generation of SR-BI Construct and Transgenic Mice--
A mouse
BAC clone, BACM16, containing the full length of the SR-BI gene, was
obtained from Genome Systems (St. Louis). The multiple steps involved
in creating the apoA-I promoter SR-BI construct are outlined in Fig.
1a.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 1.
Design of SR-BI construct and tissue
distribution of the expression of the transgene. Panel
a, a BAC clone, BACM16, containing the full length of the SR-BI
gene was fragmented at EcoRI sites in the first intron of
the gene and in the chloramphenicol gene in the vector sequence by RARE
cleavage. The fragments were separated by pulse field gel
electrophoresis. The SR-BI promoter-replacing vector, pBSAISRBI5'cm,
was constructed with a 1.7-kb EcoRI-KpnI fragment
of pCRAISRBI5', a 3.2-kb EcoRI- BssHII fragment
of the 5'-end of the SR-BI gene, and a 0.5-kb
NotI-EcoRI fragment of the chloramphenicol gene
in pBluescript SK(+). pBSAISRBI5'cm was digested with EcoRI
and ligated into the 65-kb EcoRI RARE fragment of BACM16.
The construct, pBACAISRBI, was screened by positive
selection on LB plates supplemented with ampicillin and
chloramphenicol. Panel b, 10 µg each of total RNA isolated
from various tissues of high SR-BI or wild type FVB was hybridized with
radiolabeled riboprobe complementary to the 5'-end of the SR-BI gene
and was digested with RNase A/T1 mix. The protected fragments were
separated on 5% polyacrylamide and 8 M urea gel and
detected by autoradiography. The expression of the endogenous gene and
the transgene was shown by 248- and 176-base protected bands,
respectively.
|
|
The human apoA-I promoter region from nucleotides
1613 to +72 of the
transcription start site was amplified from genomic DNA by PCR. The
forward primer contained a KpnI site at the 5'-end, and the
reverse primer contained SR-BI cDNA sequence +1 to +50. The PCR
product was cloned into the vector pCR21, using a TA Cloning kit
(Invitrogen, San Diego) to construct pCRAISRBI5'. The 4-kb EcoRI fragment of BACM16 containing the first exon of the
SR-BI gene was subcloned into pBluescript SK+ (Strategene, La Jolla, CA) to construct pBSSRBI5'. The SR-BI promoter-replacing vector pBSAISRBI5'cm was constructed by sequential insertion into pBluescript SK+ of a 1.7-kb EcoRI-KpnI fragment of
pCRAISRBI5', a 3.2-kb EcoRI-BssHII fragment of
pBSSRBI5' and a 0.5-kb NotI-EcoRI fragment of the chloramphenicol gene of the BAC vector, pBELOBAC11. BACM16 was fragmented at EcoRI sites in the first intron of the SR-BI
gene and in the chloramphenicol gene in the vector sequence by
recA-protected restriction enzyme (RARE) cleavage as described
previously with some modification (17). pBSAISRBI5'cm was digested with
EcoRI and ligated into the 65-kb EcoRI RARE
fragment of BACM16 separated by pulse field gel electrophoresis. The
BAC clone pBACAISRBI was screened by positive selection on LB plates
supplemented with ampicillin and chloramphenicol and then by PCR with
the forward primer within the apoA-I promoter sequence (apoA-I-620f)
and the reverse primer within the first exon of SR-BI (SR-BI r 176).
BAC DNAs were prepared for microinjection by modified alkaline lysis
methods followed by phenol-chloroform extraction, and then an overnight
dialysis against the injection buffer (10 mM Tris-HCl, pH
7.5, 0.1 mM EDTA, 100 mM NaCl) through
Millipore type VS membrane (0.025-µm pore size; Millipore, Bedford,
MA). The SR-BI transgenic mice in the inbred FVB background were
generated by standard microinjection methods (18). Mice were screened by PCR with a primer set, apoA-I-620f and SR-BI r 176.
Diets and Apolipoprotein and Lipoprotein Analyses--
Mice were
fed Purina mouse chow (no. 5001) until 6 weeks of age when blood
samples were collected from the tail vein after an overnight fast. The
animals were then fed an atherogenic diet high in fat and cholesterol
(1.25% cholesterol, 0.5% cholic acid, and 15% fat) (19). Blood
samples were collected again at 4 weeks after initiation of the
atherogenic diet.
Total cholesterol was determined by the standard enzyme method (Wako,
Osaka, Japan). HDL cholesterol was measured after selective precipitation of non-HDL lipoproteins by dextran sulfate and magnesium chloride.
Plasma levels of mouse apoA-I and apoB were determined by enzyme-linked
immunosorbent assays as described previously (20). Mouse apoB levels
were determined by enzyme-linked immunosorbent assay using a rabbit
polyclonal antibody prepared from an immunized animal injected with
mouse LDL. Antiserum was purified by ligand immunoaffinity
chromatography to isolate those antibodies reactive against apoB.
Antibodies were purified on a HiTrap protein A affinity column and biotinylated.
Plasma samples of five to seven animals of each group were combined and
subjected to FPLC fractionation analysis with two tandem Superose 6 columns (Amersham Pharmacia Biotech) as described previously (21).
The size distribution of the d
1.21 g/ml lipoprotein
fractions were obtained by nondenaturing polyacrylamide gradient gel electrophoresis essentially as described by Nichols et al.
(22). Lipoprotein fractions were isolated by ultracentrifugation of plasma pooled from five to seven animals of each group.
Gene Expression Analyses--
Total RNA was isolated from
brain, lungs, heart, liver, spleen, kidney, small intestine, adrenal
glands, and ovary or testis of the animals using RNA Stat 60 (Teltest
Inc., Friendswood, TX).
RNase protection assay was run by RPAII kit (Ambion, Austin, TX).
Briefly, total RNA (10 µg) was hybridized with labeled probes overnight at 45 °C and then digested with RNase A/T1 mix. The protected fragments were separated on a 5% polyacrylamide and 8 M urea gel and were detected by autoradiography.
To identify the transgene expression from that of the endogenous gene
we designed a riboprobe on the 5'-end of the SR-BI gene expanding from
the 5'-untranslated region to the first exon; the probe on the 3'-end
of the gene recognized both the transgene and endogenous gene. Primer
sets were generated to amplify the probes for SR-BI and a probe for
mouse LDL receptor from wild type FVB mouse liver cDNA as a
template by PCR with T7 promoter sequence supplemented on the
5'-upstream of the reverse primers. The probes were radiolabeled with
[
-32P]UTP (Amersham Pharmacia Biotech) using a
MAXiscript kit (Ambion).
Immunoblot analysis was carried out with liver membrane proteins
solubilized with Triton X-100. The indicated amounts of reduced protein
samples were electrophoresed on pre-cast SDS-polyacrylamide gels
(4-20% acrylamide; Novex, San Diego) and transferred to
nitrocellulose. Mouse SR-BI was detected by rabbit anti-mouse SR-BI
antiserum directed to residues 405-509 for mouse SR-BI after blocking
with 3% dry milk in phosphate-buffered saline. The primary antibody was detected using a peroxidase-conjugated goat anti-rabbit IgG (Sigma)
followed by ECL Western blotting detection reagents (Amersham Pharmacia Biotech).
In Vivo Turnover Studies of Lipoproteins--
In vivo
turnover studies were carried out as described previously (7). LDL
(d = 1.019-1.063 g/ml) was isolated from human plasma
and HDL (d = 1.063-1.210 g/ml) from FVB mouse plasma
by standard sequential ultracentrifugation and then subjected to iodination with 125I using IODO-BEADS (Pierce). The labeled
lipoproteins (10-15 µg of protein, 50-100 cpm/ng) were injected
into the tail vein of each animal. The injected lipoprotein was less
than 0.5% of the mouse LDL or HDL pool size. Blood (50 µl) was
withdrawn from the retroorbital plexus into heparinized capillary tubes
at the indicated time points after injection. The apolipoprotein
retention was measured by the radioactivity in the trichloroacetic acid
precipitate of the plasma. The radioactivity of the precipitate at 2 min after injection was defined as 100%.
 |
RESULTS |
Production of SR-BI Transgenic Mice--
A 120-kilobase pair BAC
clone, BACM16, containing the entire SR-BI gene was injected into FVB
mouse embryos as was a second DNA preparation containing a
construct engineered from this clone, BACAISRBI, whose SR-BI promoter
was replaced with the human apoA-I promoter (Fig. 1a). Two
BACM16 transgenic founder mice and seven transgenic founder mice
containing BACAISRBI were created (Table I). No detectable effect on plasma HDL
levels was observed in the two founder mice with the BACM16, whereas
four of the seven founder mice of BACAISRBI transgenics had
significantly lower HDL cholesterol levels. Among the BACAISRBI
transgenic lines B2611 (referred to as low SR-BI) and B2614 (referred
to as high SR-BI) with moderate and profound decreases in HDL
cholesterol, respectively, were chosen for further analysis.
SR-BI Transcript and Protein Assays--
To examine tissue
specificity of expression of the transgene and to differentiate it from
that of the endogenous SR-BI gene, we performed RNase protection
assays. The riboprobe at the 5'-end of the SR-BI cDNA used for
these studies distinguishes the two SR-BI transcripts in the transgenic
mice generating a 176-base pair band for the transgene and a 248-base
pair band for the endogenous SR-BI gene. The expression of the
transgene is prominent in the liver of transgenic mice as expected from
previous studies (23) (Fig. 1b). In agreement with previous
studies in mice, the endogenous SR-BI expression though detectable in
the liver, ovary, and lung, was highest in the adrenal gland (3). The
liver expression of the SR-BI transgene did not alter the pattern or
levels of the endogenous SR-BI gene. Analyses of SR-BI expression in
these mice indicate that the expression levels of SR-BI in the liver of
low and high SR-BI transgenic mouse lines are 2-fold and >10-fold higher than those of control FVB mice, respectively (Fig.
2a).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 2.
RNase protection assay (panel
a), Western blot analysis of the expression level of SR-BI
(panel b), and the dietary effect on gene expression
in the liver (panel c). Panel a, three
levels of total liver RNA were subjected to RNase protection assay with
radiolabeled SR-BI 3'-riboprobe. Total SR-BI expression was shown by a
310-base protected band. Panel b, liver membrane protein was
solubilized with Triton X-100; the indicated amounts of reduced protein
samples were electrophoresed on SDS-polyacrylamide gels (4-20%
acrylamide) and transferred to nitrocellulose. Mouse SR-BI was detected
by rabbit anti-mouse SR-BI antiserum after blocking with dry milk. The
primary antibody was detected using a peroxidase-conjugated goat
anti-rabbit IgG and then by ECL Western blotting detection reagents.
Panel c, 10 µg of total liver RNA of animals before or
after high fat, high cholesterol diet treatment were hybridized with
the SR-BI 5', mouse LDLr, or mouse -actin riboprobes and were
digested with RNase A/T1 mix. The protected fragments were separated on
5% polyacrylamide and 8 M urea gel and detected by
autoradiography.
|
|
To examine the SR-BI protein product in the liver of transgenic mice,
cell membrane proteins were collected by ultracentrifugation, separated
by SDS-polyacrylamide gel electrophoresis, and immunoblotted using an
antibody against mouse SR-BI. In both transgenic lines studied, the
SR-BI protein levels reflected the results of the RNase protection
studies (Fig. 2b) and were about 2- and 10-fold higher than
endogenous levels in the low and high SR-BI transgenics, respectively.
Plasma Lipids and Lipoprotein Analysis--
The increases in SR-BI
expression levels observed in the low and high SR-BI transgenic mice
livers correlated inversely with plasma cholesterol concentrations. The
total cholesterol levels of the transgenics were 5.9% of control FVB
in the high SR-BI Tg line and 58.5% of control FVB in the low SR-BI Tg
line (Fig. 3a). The HDL
cholesterol levels of the high and low SR-BI transgenics were 4.0 and
61.6% of control FVB mice, respectively (Fig. 3b). To
determine whether the decrease in HDL cholesterol is accompanied by a
parallel decrease in apoA-I concentration, murine apoA-I levels were
determined by enzyme-linked immunosorbent assay. The concentration of
apoA-I was 1.0 and 69.2% of non-transgenic controls in high SR-BI
transgenics and low SR-BI transgenics, respectively (Fig.
3d), suggesting that increased expression of SR-BI decreases not only HDL cholesterol levels but also HDL particle number.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Lipid and apolipoprotein analysis. Total
cholesterol (panel a), HDL cholesterol (panel b),
non-HDL cholesterol (panel c), mouse apoA-I (panel
d), and mouse apoB (panel e) levels in plasma of high
SR-BI, low SR-BI, and FVB animals before (solid bar) and
after (striped bar) high fat, high cholesterol diet
treatment. The bar graph represents the means ± S.D.
from 6-10 animals of each group. *** p < 0.0001; **
p < 0.01; * p < 0.03 compared
with FVB pre-diet;  p < 0.0001; p < 0.002 compared with FVB post-diet.
|
|
An unexpected result in the analysis of the SR-BI transgenics was that
the decrease in plasma cholesterol was not exclusively in the HDL
fraction but was also present in non-HDL fractions. The decrease in
non-HDL cholesterol, though not as great as the decrease in HDL
cholesterol, was significant in the high and low SR-BI transgenics, 90 and 45% lower, respectively, than that of the non-transgenic mice
(Fig. 3c). Plasma apoB levels were also decreased in mice
with elevated hepatic SR-BI expression (Fig. 3e). Compared
with non-transgenic controls, apoB was decreased by 35 and 26% in the
high and low transgenic mice, respectively. FPLC gel filtration
analysis using pooled plasma confirmed that there were
dose-dependent decreases in HDL and non-HDL cholesterol in
mice expressing SR-BI, compared with non-transgenic controls (Fig.
4a).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Panel a, FPLC profile of plasma
lipoprotein cholesterol. Plasma was pooled from six female animals of
each genotype and was subjected to FPLC analysis. Total cholesterol
concentration in each fraction was measured. Panels b and
c, nondenaturing gradient gel electrophoresis of plasma
lipoprotein fraction. Plasma was pooled from six animals of each
genotype, and the d 1.21 g/ml fraction was analyzed
on 4-30% (panel b) or 2-16% (panel c)
polyacrylamide gradient gels.
|
|
To examine the impact of SR-BI on the regulation of plasma cholesterol
levels of mice fed an atherogenic diet, we determined HDL cholesterol,
apoA-I, non-HDL cholesterol, and apoB levels in SR-BI transgenics and
non-transgenic littermates fed this diet for 4 weeks. Total plasma
cholesterol was increased significantly in all genotypes (Fig.
3a), and these changes were primarily the result of
increases in non-HDL cholesterol (Fig. 3c). Comparisons of
the transgenics and control animals demonstrate that the level of SR-BI
expression was roughly associated with a dose-dependent decrease in total cholesterol and non-HDL cholesterol. Plasma apoB
levels of high and low SR-BI transgenics were 36 and 19% lower,
respectively, than those of non-transgenic controls (Fig. 3e). As a consequence, the markedly decreased ratio of
non-HDL cholesterol to apoB (98.9 ± 54 in high SR-BI, 647 ± 85 in low SR-BI, and 778.3 ± 248 in FVB) could be used
as an indication of a decreased particle size in mice overexpressing
SR-BI. Taken together, these findings indicate that moderate increases
in SR-BI levels are associated with significant changes in lipoprotein particle size and suggest that SR-BI is also able to promote selective cholesteryl ester uptake from non-HDL as well as HDL particles.
SR-BI and LDLr Gene Expression Studies--
The effects of diet
and the SR-BI transgene on the expression of LDL receptor and
endogenous SR-BI gene were studied. RNase protection assays of total
liver RNA isolated from mice before and after high fat diet treatment
were performed and qualitatively compared (Fig. 2c).
Expression of the SR-BI transgene and the SR-BI endogenous gene was not
influenced by the diet. In contrast, LDLr was down-regulated after high
fat diet treatment in the control and low SR-BI transgenic mice,
whereas in the high SR-BI transgenics LDLr expression was not effected
by diet.
Lipoprotein Particle Size Distribution--
The lipoprotein size
distribution of control and SR-BI transgenic mice is shown in Fig. 4,
b and c. In agreement with previous studies (24),
HDL isolated from wild type mice is shown as a monodisperse population
of particles with a peak diameter of 10.2 nm. HDL isolated from low
SR-BI mice also consisted of a unimodal distribution, but its peak
diameter is decreased markedly, suggesting a denser, less lipid-rich
particle (Fig. 4b). Surprisingly, the same phenomenon was
observed in apoB-containing lipoprotein fractions (Fig. 4c).
The peak diameter of the VLDL fraction in high SR-BI shows a striking
decrease, 35.5 nm compared with 37.3 nm in FVB mice. The peak size of
the LDL/IDL fraction also shows a remarkable and
dose-dependent reduction in SR-BI transgenics: 27.4 nm in high SR-BI, 29.4 nm in low SR-BI, and 30.8 nm in FVB mice. These observations support the hypothesis that SR-BI promotes the selective uptake of cholesteryl ester from apoB-containing lipoproteins leading
to the formation of smaller particles.
In Vivo Turnover Studies of Lipoproteins--
To evaluate the
effect of SR-BI overexpression on the metabolism of lipoproteins
in vivo, 125I-labeled human LDL and mouse HDL
were injected into mice of different genotypes. The disappearance of
radioactivity from plasma was followed for 24 h. The plasma
clearance rate of labeled HDL was increased significantly in the high
SR-BI and low SR-BI transgenics compared with control mice: 0.36, 0.19, and 0.12 pools of protein/h, respectively (Fig.
5a). This suggests that SR-BI
determines, at least in part, the rate of metabolism of HDL and that
the decrease in the plasma HDL pool size observed in mice expressing
SR-BI may be attributable to a dose-dependent increase in
the clearance of HDL.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Turnover studies of 125I-labeled
mouse HDL (panel a) and 125I-labeled human
LDL (panel b). Labeled lipoproteins were injected
into the tail vein of high SR-BI (closed circles), low SR-BI
(closed triangles), or FVB (open circles) mice.
After the indicated time, blood samples were collected from the
retroorbital sinus into heparinized capillary tubes. The radioactivity
in trichloroacetic acid precipitate of plasma was determined. The value
at 2 min after injection was defined as 100%. Each point represents
the mean remaining label from two to three animals of each group.
|
|
To examine whether SR-BI participates in the metabolism of
apoB-containing lipoproteins and thus contributes to the decrease in
plasma non-HDL cholesterol and apoB levels observed in SR-BI transgenic
mice, human LDL clearance rates were determined in SR-BI transgenic and
control mice. Labeled human LDL was used instead of autologous LDL
because human apoB binds less efficiently to the mouse LDLr. Thus
changes in the rate of LDL clearance would more likely be attributable
to SR-BI transgenes and not to the endogenous murine LDL receptor. Mice
with increased levels of SR-BI in the liver have an accelerated rate of
clearance of human LDL (Fig. 5b): 0.361 pools of apoB/h in
high SR-BI, 0.354 in low SR-BI, and 0.215 in FVB. These findings in the
SR-BI transgenics further suggest that SR-BI plays a part in non-HDL metabolism.
 |
DISCUSSION |
The transgenic mice created for these studies with increased
hepatic levels of SR-BI have enabled us to determine the effect of
steady-state overexpression of this gene on lipoprotein metabolism. The
inverse correlation between SR-BI expression and HDL turnover rates
suggests that the increased expression of SR-BI and its mediated
clearance of HDL result in lower plasma concentrations of this
lipoprotein. These findings are consistent with prior in
vitro as well as in vivo characterizations of SR-BI
(3-9) and add further support to the hypothesis that SR-BI is an
important participant in HDL metabolism.
HDL cholesterol uptake by the adrenal gland and liver is believed to
occur in part by the selective uptake pathway, a process whereby
cholesteryl esters are selectively removed from the particles. This
selective removal of cholesteryl esters would be predicted to result in
changes in HDL composition and particle size. The presence of the
smaller denser HDL particles in the SR-BI transgenics, demonstrated by
nondenaturing polyacrylamide gel electrophoresis, is thus consistent
with increased selective uptake of HDL cholesterol in the
SR-BI-overexpressing mice. The accelerated clearance of apoA-I in the
SR-BI transgenics may in part be a result of the changes in the HDL
particle size accelerating the metabolism of this apoA-I-containing
particle by the kidney (25), as has been noted previously in mice with
increased SR-BI expression caused by adenoviral gene transfer (7).
The LDL turnover results, although not as dramatic as the HDL turnover
results, indicate faster clearance of labeled LDL in SR-BI transgenics
and suggest the involvement of SR-BI in LDL metabolism. The decreased
size of non-HDL lipoproteins coupled with their reduced concentrations
in mice overexpressing SR-BI suggests the possibility that the
selective uptake of cholesteryl esters via SR-BI is not restricted to
HDL but also includes non-HDL lipoproteins. Another possible
explanation for the reduced concentrations of non-HDL lipoproteins in
the SR-BI transgenics is that SR-BI overexpression may impact on the
production and secretion of VLDL in the liver or the uptake of
cholesterol-rich non-HDL particles. Although stimulated VLDL secretion
was observed in the recovery phase after adenovirus-mediated short term
overexpression of SR-BI (7), the persistent overexpression of SR-BI
obtained in the present study would be predicted to suppress this
secretion, which is regulated by pooled intracellular cholesterol.
The sensitive and quantitative RNase protection assays enabled
assessment of the effect of SR-BI activity and diet on the expression
of the endogenous murine SR-BI and LDLr. Although prior studies
demonstrated that adrenal SR-BI expression is affected by apoA-I (13),
hepatic lipase (13), and lecithin-cholesterol acyltransferase
deficiency (14), the expression of the endogenous hepatic SR-BI gene
was not affected by SR-BI transgene activity or diet in the present
study. LDLr expression was unchanged in the SR-BI transgenics when the
animals were fed mouse chow; however, the expression of LDLr was
diminished in the low SR-BI transgenics and nontransgenic control mice
when fed the high fat high cholesterol diet. This effect of diet on
LDLr expression was not detected in high SR-BI high expressors,
possibly suggesting that intracellular cholesterol pools and their
effect on LDLr expression may be dependent upon the route by which
cholesterol enters the cell. In summary, although it is not clear how
the mechanism of SR-BI overexpression impacts on LDLr expression, our
studies clearly indicate that the regulation of SR-BI and LDLr are in
many ways distinctly different.
An important but yet unanswered question concerning SR-BI is its effect
on an organism's susceptibility to diet-induced atherogenesis. The
predicted acceleration of reverse cholesterol transport, a process
whereby excess cholesterol accumulating in the peripheral tissues such
as the vasculature is mobilized, in the SR-BI overexpression transgenics would at first suggest that increasing the activity of
SR-BI should have an anti-atherogenic effect. An alternative perspective also consistent with the results of the present study would
suggest that increased expression of SR-BI should increase an
organism's susceptibility to atherosclerosis by decreasing both apoA-I
and HDL plasma concentration. This relates to the multiple in
vitro and in vivo studies that have attributed
antiatherogenic properties to the HDL particle itself, such as acting
as an antioxidant and preventing LDL modification (26, 27), effects
that would be diminished at reduced plasma concentrations of this
lipoprotein. The eventual analysis of atherosclerosis susceptibility in
the SR-BI transgenics, created in the inbred genetic background
described in this study, may offer an important vista into the role of
SR-BI as a potentially manipulable atherosclerosis susceptibility factor.
The present characterization of SR-BI-overexpressing mice has revealed
several sites where SR-BI may be involved in lipoprotein metabolism.
This includes its previously studied role in HDL metabolism as well as
a largely uncharacterized role in the metabolism of non-HDL
lipoproteins. This latter property might be viewed as having been
predicted by the fact that SR-BI was first identified by its ability to
bind native LDL as well as modified LDL (10). The major findings of the
present study that increased SR-BI expression in transgenic mice
dramatically lowers plasma levels and accelerates clearance of both HDL
and non-HDL cholesterol, highlights the potential major role of this
molecule in lipoprotein metabolism in vivo.