From the Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8887
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ABSTRACT |
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This study was undertaken to determine the effect
of transient overexpression of hepatic cholesterol 7-hydroxylase on
low density lipoprotein (LDL) cholesterol transport in mice lacking LDL
receptors (LDL receptor
/
). Primary overexpression
of hepatic 7
-hydroxylase in LDL receptor
/
mice
was accompanied by a dose-dependent decrease in the rate of
LDL cholesterol appearance in plasma (whole body LDL cholesterol transport) and a corresponding reduction in circulating LDL cholesterol levels. The increase in hepatic 7
-hydroxylase activity necessary to
achieve a 50% reduction in plasma LDL cholesterol concentrations was
~10-fold. In comparison, cholestyramine increased hepatic 7
-hydroxylase activity ~3-fold and reduced plasma LDL cholesterol concentrations by 17%. This study demonstrates that augmentation of
hepatic 7
-hydroxylase expression is an effective strategy for
lowering plasma LDL concentrations even in animals with a genetic
absence of LDL receptors.
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INTRODUCTION |
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Conversion of cholesterol to bile salts is the principal regulated
pathway whereby cholesterol is removed from the body. The initial and
rate-limiting enzyme in the major bile salt biosynthetic pathway is
hepatic cholesterol 7-hydroxylase (1). Hepatic cholesterol
7
-hydroxylase is regulated at the transcriptional level in response
to bile salts fluxing through the liver in the enterohepatic
circulation (2-5). Bile salt sequestrants such as cholestyramine bind
bile salts in the intestinal lumen, thereby preventing their
reabsorption and decreasing the return of bile salts to the liver. Loss
of bile salts from the enterohepatic circulation results in
derepression of hepatic 7
-hydroxylase expression and an increase in
the synthesis of bile salts from cholesterol (6-9). Interventions that
accelerate the conversion of cholesterol to bile salts reduce plasma
LDL1 concentrations and
prevent coronary events (10-12). The efficacy of these interventions
is postulated to result from a reduction in the availability of
unesterified cholesterol within hepatocytes. Depletion of unesterified
cholesterol within the hepatocyte triggers a compensatory increase in
de novo cholesterol synthesis and induction of the LDL
receptor pathway, the latter leading to enhanced clearance of LDL from
plasma (7, 13-15). Although induction of hepatic LDL receptor activity
has been emphasized as the major mechanism responsible for the
cholesterol-lowering effects of bile salt sequestrants, a recent study
suggests that these agents may also reduce the rate of LDL cholesterol
entry into plasma (16).
Humans (and animals) who genetically lack LDL receptors are characterized by massive elevations of circulating LDL levels, accelerated atherosclerosis, and premature coronary heart disease (17). In the absence of functional LDL receptors, the clearance of LDL from plasma is reduced, and the conversion of VLDL to LDL is increased. In Watanabe heritable hyperlipidemic (WHHL) rabbits, the absence of functional LDL receptors leads to a ~20-fold elevation of the plasma LDL concentration that is the result of a 60-75% reduction in the rate of LDL clearance from plasma coupled with a 2-5-fold increase in the rate of LDL entry into the plasma space (whole body LDL transport) (18, 19). In the mouse, targeted disruption of the LDL receptor gene leads to a 14-fold increase in the plasma concentration of LDL that is due to an 88% reduction in the rate of LDL clearance from plasma coupled with a 70% increase in whole body LDL transport (20).
In a recent study, we transiently overexpressed an exogenous
7-hydroxylase gene in hamsters using adenovirus-mediated gene transfer and determined the effects on hepatic sterol balance and LDL
transport (21). This demonstrated that primary overexpression of
hepatic 7
-hydroxylase markedly lowered plasma LDL concentrations in
animals fed control or Western-type diets. Notably, the reduction in
plasma LDL levels was due, in large part, to a decrease in the rate of
LDL cholesterol entry into the plasma space (whole body LDL transport).
This observation raised the possibility that enhancing hepatic
7
-hydroxylase activity might also be effective in lowering plasma
LDL concentrations in animals lacking LDL receptors. The present study
characterizes the response to hepatic 7
-hydroxylase overexpression
in mice with targeted disruption of the LDL receptor gene. The
results of this study indicate that enhancement of hepatic 7
-hydroxylase expression is an effective strategy for lowering plasma LDL concentrations not only in animals with diet-induced hypercholesterolemia, but also in animals that genetically lack LDL
receptors.
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MATERIALS AND METHODS |
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Animals and Diets-- All studies were performed in female mice with targeted disruption of the LDL receptor gene (22). All animals were housed in colony cages (five animals/cage) in a room with light cycling (12 h of light and 12 h of dark) and controlled temperature and humidity. All measurements were made at the mid-dark phase of the light cycle. The animals were maintained on a low fat (50 mg/g of diet), low cholesterol (0.23 mg/g of diet) cereal-based diet (Wayne Lab Blox, Allied Mills, Chicago, IL).
Recombinant Viruses--
The recombinant adenoviruses AdCMV7
(carrying a gene encoding rat 7
-hydroxylase), AdCMVluc (carrying a
gene encoding firefly luciferase), and AdCMV
gal (carrying the
Escherichia coli lacZ gene) have been described previously
(21, 23). Large-scale production of recombinant adenovirus was
performed as described (24) by infecting confluent monolayers of 293 cells grown in 15-cm tissue culture plates with primary stock at a
multiplicity of infection of 0.1-1.0. Infected monolayers were lysed
with Nonidet P-40 when >90% of the cells showed cytopathic changes,
and recombinant virus was purified by precipitation with polyethylene
glycol 8000, centrifugation on a discontinuous CsCl density gradient,
and desalting by chromatography on Sepharose CL-4B. Purified virus
eluting in the void volume was collected, snap-frozen in liquid
N2, and stored at
80 °C until used.
Hepatic 7-Hydroxylase Activity--
Hepatic 7
-hydroxylase
activity was determined using an HPLC/spectrophotometric assay that
quantifies the mass of 7
-hydroxycholesterol formed from endogenous
microsomal cholesterol after enzymatic conversion to
7
-hydroxycholesten-3-one using cholesterol oxidase (25).
Determination of Hepatic LDL Uptake Rates in Vivo--
Plasma
was obtained from LDL receptor/
donor mice. The LDL was
isolated from plasma by preparative ultracentrifugation in the density
range of 1.020-1.055 g/ml and labeled with 125I- or
131I-tyramine cellobiose as described previously (26).
Rates of hepatic LDL uptake were measured using primed infusions of
125I-tyramine cellobiose-labeled LDL. The infusions of
125I-tyramine cellobiose-labeled mouse LDL were continued
for 4 h, at which time each animal was administered a bolus of
131I-tyramine cellobiose-labeled LDL as a marker of plasma
volume and killed 10 min later by exsanguination through the inferior vena cava. Tissue samples along with aliquots of plasma were assayed for radioactivity in a
-counter (Packard Instrument Co.). The tissue
spaces achieved by the labeled LDL at 10 min (131I dpm/g of
tissue divided by 131I dpm/µl of plasma) and at 4 h
(125I dpm/g of tissue divided by 125I dpm/µl
of plasma) were then calculated and have the units of µl/g. The
increase in tissue space over the 4-h experimental period equals the
rate of radiolabeled LDL movement into each organ and is expressed as
µl of plasma cleared of its LDL content per h per g of tissue or per
whole organ. Clearance values were multiplied by the plasma LDL
concentration to obtain the absolute rates of LDL uptake.
VLDL ApoB Turnover-- VLDL (d < 1.006 g/ml) was isolated from fasted rabbits by preparative ultracentrifugation and radioiodinated using iodine monochloride (27). VLDL turnover studies were performed in mice as described by Ishibashi et al. (22).
Determination of Hepatic Cholesterol Synthesis Rates-- Rates of hepatic cholesterol synthesis were measured in vivo using [3H]water. As described previously (28), the animals were administered ~20 mCi of [3H]water intravenously and then returned to individual cages under a fume hood. One h after the injection of [3H]water, the animals were anesthetized and exsanguinated through the inferior vena cava. Aliquots of plasma were taken for the determination of body water specific activity, and samples of liver were taken for the isolation of digitonin-precipitable sterols. Rates of sterol synthesis are expressed as nmol of [3H]water incorporated into digitonin-precipitable sterols/h/g of liver.
Determination of mRNA Levels--
Hepatic 7-hydroxylase
and glyceraldehyde-3-phosphate dehydrogenase (used as an invariant
control) mRNA levels were determined by nuclease protection as
described previously (29). Probes were synthesized using 0.5 µM [32P]dCTP and 1 µM (mouse
7
-hydroxylase), 5 µM (rat 7
-hydroxylase), or 300 µM (mouse glyceraldehyde-3-phosphate dehydrogenase)
unlabeled dCTP.
Determination of Bile Salt Pool Size and Composition--
The
liver, gallbladder, and small bowel were removed en block from LDL
receptor/
mice that had been administered AdCMV7
or
control virus. The tissues and their contents were placed in a 400-ml
beaker with ~200 ml of ethanol (and trace amounts of
tauro[24-14C]cholic acid as an internal standard) and
refluxed for 4 h. After extraction with diethyl ether, bile acids
in the ethanolic extract were separated and quantified by HPLC.
Determination of Liver and Plasma Cholesterol Distribution-- Liver cholesterol was quantified by capillary gas-liquid chromatography. The cholesterol distribution in plasma was determined by FPLC using a Superose 6 column (Sigma). Two-ml fractions were collected and assayed for cholesterol using an enzymatic kit (Sigma).
Statistical Analysis-- The data are presented as means ± S.D. To test for differences among the dietary regimens, one-way analysis of variance was performed. Significant results were further analyzed using the Tukey multiple comparison procedure.
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RESULTS |
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To examine the role of hepatic 7-hydroxylase in controlling
plasma LDL concentrations in animals lacking LDL receptors, we first
determined the effect of agents known to suppress (cholate) or
up-regulate (cholestyramine) 7
-hydroxylase expression. LDL receptor
/
mice were fed a low cholesterol diet
supplemented with cholate (0.3%) or cholestyramine (3%) for 4 weeks.
The animals were then killed and used for the determination of hepatic
7
-hydroxylase expression and plasma cholesterol concentrations. As
shown in Fig. 1, orally administered
cholate suppressed hepatic 7
-hydroxylase activity by ~90%,
whereas cholestyramine increased hepatic 7
-hydroxylase activity
~3-fold. Parallel changes were observed in hepatic 7
-hydroxylase mRNA levels (data not shown). The effect of orally administered cholate and cholestyramine on the lipoprotein distribution of cholesterol in plasma was determined by FPLC and is shown in Fig. 2. In animals fed cholate, the amount of
cholesterol carried in the lower density lipoproteins (VLDL, IDL, and
LDL) was markedly increased, whereas high density lipoprotein
cholesterol concentrations were reduced. In animals fed cholestyramine,
LDL cholesterol concentrations were reduced by 15-20%, whereas VLDL
and high density lipoprotein cholesterol concentrations were unchanged.
Measurements of hepatic 7
-hydroxylase activity and plasma
cholesterol concentrations were also made in animals fed the cholate or
cholestyramine diets for 3 days. These studies showed that the effects
observed at 4 weeks were largely present at 3 days, although the
variability in serum cholesterol values was much greater at 3 days than
at 4 weeks (data not shown).
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We next investigated the effect of overexpressing an exogenous
7-hydroxylase gene on plasma lipoprotein concentrations in mice
lacking LDL receptors. LDL receptor
/
mice were
administered, by intravenous injection, recombinant adenovirus
expressing rat 7
-hydroxylase from the cytomegalovirus promoter
(AdCMV7
) or equivalent doses of control virus (recombinant adenoviruses expressing either the firefly luciferase gene or the
E. coli lacZ gene from the cytomegalovirus promoter).
Preliminary time course studies showed that plasma LDL concentrations
progressively declined for 2-3 days after the administration of
AdCMV7
, remained relatively constant for 4-5 days, and then
returned to preinjection values over the next 7-10 days; neither of
the control viruses altered plasma LDL cholesterol concentrations over
this time frame. Therefore, all subsequent studies were performed 3 days after the injection of recombinant adenovirus. Any animal that
lost weight during this 3-day period of time was not studied.
The relationship between hepatic 7-hydroxylase activity and plasma
LDL concentrations in mice lacking LDL receptors is illustrated in Fig.
3. LDL receptor
/
animals
were administered 1 or 2 × 109 pfu of AdCMV7
or
equivalent doses of AdCMVluc. Three days later, the animals were killed
and used for the determination of hepatic 7
-hydroxylase expression
and plasma lipoprotein concentrations. AdCMV7
increased hepatic
7
-hydroxylase activity and reduced plasma LDL cholesterol
concentrations in a dose-dependent manner. The increase in
hepatic 7
-hydroxylase activity necessary to achieve a 50% reduction
in plasma LDL concentrations was ~10-fold. By comparison, 3%
cholestyramine increased hepatic 7
-hydroxylase activity 3-fold and
reduced plasma LDL cholesterol concentrations by 17%.
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Fig. 4 shows the effect of
7-hydroxylase gene transfer on the lipoprotein distribution of
plasma cholesterol as determined by FPLC. LDL receptor
/
mice were administered 1 × 109 pfu of AdCMV7
or
the same dose of control virus (AdCMVluc). Three days later, the
animals were killed after a 6-h fast, and equal volumes of plasma were
pooled and injected onto a Superose 6 column. Primary overexpression of
hepatic 7
-hydroxylase markedly reduced the amount of cholesterol
carried in the lower density lipoproteins (92% reduction in VLDL and
53% reduction in IDL/LDL); high density lipoprotein cholesterol was
also reduced (19%), but to a lesser extent. Lipoproteins in fractions
corresponding to VLDL (tubes 1-6) and IDL/LDL (tubes 8-18) were
separated on precast 1% agarose gels and stained with Fat Red 7B (31).
Overexpression of hepatic 7
-hydroxylase markedly reduced the amount
of pre-
-migrating lipoproteins in FPLC fractions corresponding to
VLDL and of
- and pre-
-migrating lipoproteins in FPLC fractions
corresponding to IDL/LDL (data not shown).
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A change in the plasma concentration of LDL may be due to a change in
the rate of LDL entry into the plasma space or to a change in the rate
of LDL clearance by one or more tissues of the body. To determine the
mechanism responsible for the fall in plasma LDL concentrations
associated with 7-hydroxylase gene transfer, we performed LDL
transport studies using 125I-tyramine cellobiose-labeled
homologous LDL. LDL receptor
/
mice were administered
1 × 109 pfu of AdCMV7
or the same dose of control
virus (AdCMVluc). Three days later, LDL transport rates were determined
as described under "Materials and Methods." As shown in Table
I, rates of LDL clearance by the liver,
extrahepatic tissues, and whole body were not altered by primary
overexpression of hepatic 7
-hydroxylase. Multiplication of the
tissue clearance rates by the concentration of LDL cholesterol in
plasma yields the mass of LDL cholesterol transported by the liver,
extrahepatic tissues, and whole body, and these values are also shown
in Table I. Because 7
-hydroxylase gene transfer reduced plasma LDL
cholesterol concentrations but had no effect on LDL clearance rates,
the absolute rates of LDL cholesterol uptake fell in proportion to the
decrease in plasma LDL cholesterol concentrations. At steady state, the
rate of LDL cholesterol uptake by all tissues of the body must equal
the rate of LDL cholesterol entry into the plasma space (whole body LDL cholesterol transport). Overexpression of hepatic 7
-hydroxylase reduced the rate of whole body LDL cholesterol uptake by ~50% (from
27 to 14 µg/h/100 g of body weight), but had no effect on whole body
LDL cholesterol clearance, indicating that the fall in plasma LDL
cholesterol concentrations was due entirely to a decrease in the rate
of LDL cholesterol entering the plasma space.
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We also examined the effect of 7-hydroxylase gene transfer on rates
of VLDL apoB clearance from plasma. LDL receptor
/
mice
were administered 1 × 109 pfu of AdCMV7
or the
same dose of control virus (AdCMVluc). Three days later, each animal
was injected intravenously with 125I-labeled VLDL. Blood
was obtained from the retro-orbital sinus at 2 min and at 1, 2, 4, and
8 h. The plasma content of 125I-labeled apoB was
measured by isopropyl alcohol precipitation (32), and the radioactivity
present at each time point was expressed relative to the radioactivity
present 2 min after the injection of radiolabeled VLDL. As shown in
Fig. 5, the rate of disappearance of
125I-VLDL from plasma did not differ between the AdCMV7
-
and AdCMVluc-treated mice.
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To further characterize the compensatory response to primary
overexpression of hepatic 7-hydroxylase, we measured hepatic cholesterol levels and hepatic cholesterol synthesis rates in LDL
receptor
/
mice administered AdCMV7
or control virus.
LDL receptor
/
mice were administered 1 × 109 pfu of AdCMV7
or control virus (AdCMV
gal). Three
days later, the animals were administered 20 mCi of
[3H]water and killed 1 h later for the determination
of hepatic cholesterol levels and hepatic cholesterol synthesis rates.
As shown in Table II, rates of hepatic
cholesterol synthesis were up-regulated ~5-fold in animals
administered AdCMV7
, whereas control virus had no significant
effect. The concentration of esterified and unesterified cholesterol in
the liver was not significantly altered in animals administered
AdCMV7
. As also shown in Table II, cholestyramine increased the rate
of hepatic cholesterol synthesis ~3-fold, whereas cholate suppressed
hepatic cholesterol synthesis by 75%.
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To investigate the effect of primary overexpression of hepatic
7-hydroxylase on the enterohepatic pool of bile salts, we determined
the size and composition of the bile salt pool in LDL receptor
/
mice administered AdCMV7
. LDL
receptor
/
mice were administered 1 × 109 pfu of AdCMV7
. Three days later, the animals were
killed, and the amount and type of bile salts present in the liver,
gallbladder, and small intestine were determined. For comparative
purposes, bile salt pool size and composition were also determined in
animals fed cholestyramine (3%) or cholate (0.3%). As shown in Table
III, administration of AdCMV7
expanded
the size of the bile salt pool by 39%, but had little effect on the
composition. The size of the bile salt pool was also increased in mice
fed cholate, and in these animals, the pool was enriched with
cholate.
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The effect of primary overexpression of an exogenous 7-hydroxylase
gene on expression of the endogenous gene was next examined. LDL
receptor
/
mice were administered 1 × 109 pfu of AdCMV7
or control virus (AdCMVluc). Three
days later, the animals were killed, and samples of liver were taken
for the determination of 7
-hydroxylase mRNA levels using a
nuclease protection assay with probes specific for the exogenous (rat)
or endogenous (mouse) genes. Preliminary nuclease protection
experiments showed that the mouse probe did not yield a protected band
when hybridized with rat RNA, and the rat probe did not yield a
protected band when hybridized with mouse RNA (data not shown). As
illustrated in Fig. 6, expression of the
transgene had relatively little effect on expression of the endogenous
gene. The endogenous gene was suppressed by only ~30% even under
conditions in which the transgene was expressed at levels sufficient to
raise 7
-hydroxylase activity 10-fold. This observation is in
contrast to the hamster, in which overexpression of an exogenous
7
-hydroxylase gene reciprocally suppressed expression of the
endogenous gene (5).
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A final set of experiments was undertaken to determine if expansion or
depletion of the enterohepatic pool of bile salts had any effect on
7-hydroxylase activity or plasma LDL cholesterol levels in animals
overexpressing an exogenous 7
-hydroxylase gene. LDL
receptor
/
mice were maintained on a low cholesterol
control diet or the same diet supplemented with cholate (0.3%) or
cholestyramine (3%). After 4 weeks, the animals were administered
1 × 109 pfu of AdCMV7
and killed 3 days later for
the determination of hepatic 7
-hydroxylase expression and plasma LDL
cholesterol concentrations. As shown in Fig.
7, administration of AdCMV7
was
associated with an ~11-fold increase in hepatic 7
-hydroxylase activity. In animals administered AdCMV7
, hepatic 7
-hydroxylase activity was the same whether the animals were consuming the control diet or diets supplemented with cholate or cholestyramine. Nuclease protection assays demonstrated that mRNA encoding the endogenous (mouse) 7
-hydroxylase was completely suppressed (undetectable) in
animals ingesting the cholate diet. As also shown in Fig. 7, administration of AdCMV7
was associated with a ~50% reduction in
plasma LDL cholesterol levels. In animals administered AdCMV7
, plasma LDL cholesterol concentrations were similar whether the animals
were consuming the control diet or diets supplemented with cholate or
cholestyramine.
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DISCUSSION |
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Clinical interventions that accelerate the conversion of
cholesterol to bile salts lower plasma LDL concentrations and reduce cardiovascular mortality (12, 33). The proposed mechanism is depletion
of unesterified cholesterol within hepatocytes, leading to compensatory
increases in 3-hydroxy-3-methylglutaryl-CoA reductase and LDL receptor
activities (7, 13-15). Up-regulation of hepatic LDL receptor activity,
in turn, results in enhanced clearance of LDL from plasma. We
previously showed that primary overexpression of hepatic
7-hydroxylase, the rate-limiting enzyme in the bile salt
biosynthetic pathway, markedly lowered plasma LDL concentrations in
hamsters fed control or Western-type diets (21). Moreover, the
reduction in plasma LDL concentrations was largely the result of a
decrease in the rate of LDL entry into the plasma space (whole body LDL
transport), suggesting that enhancing hepatic 7
-hydroxylase activity
might also be effective in lowering plasma LDL concentrations in
animals lacking LDL receptors. The present study demonstrates that
direct augmentation of hepatic 7
-hydroxylase by gene transfer markedly lowers plasma LDL concentrations even in the absence of LDL
receptors. These experimental results are consistent with recent
studies showing that interruption of the enterohepatic circulation by
ileal bypass or complete biliary diversion significantly lowers plasma
LDL cholesterol concentrations in WHHL rabbits lacking functional LDL
receptors (34, 35). Together, these observations indicate that major
reductions in LDL concentrations can be achieved through mechanisms
independent of LDL receptor induction.
The concentration of LDL in plasma is determined by the rate at which
LDL enters the plasma space relative to the rate at which LDL is
cleared from plasma by receptor-dependent and
receptor-independent pathways. In this study, primary overexpression of
hepatic 7-hydroxylase in LDL receptor
/
mice markedly
reduced the rate of LDL cholesterol entry into plasma. In contrast,
overexpression of hepatic 7
-hydroxylase had no effect on the rate of
LDL clearance in individual tissues or the whole body. These
observations are consistent with a recent study indicating that most of
the LDL cholesterol-lowering effect of bile salt sequestrants can be
attributed to a reduction in the rate of LDL cholesterol entry into
plasma (16). Enhanced conversion of cholesterol to bile salts
presumably decreases the content of unesterified cholesterol within
hepatocytes, resulting in a compensatory increase in de novo
cholesterol synthesis as discussed below. How depletion of hepatic
cholesterol leads to a decrease in LDL cholesterol entry into plasma is
less clear. A reduction in the rate of LDL cholesterol appearance in
plasma could be the result of decreased hepatic secretion of
apoB100-containing VLDL, a decrease in the proportion of
apoB100-containing VLDL that is converted to LDL, or a decrease in the
direct secretion of LDL from the liver. Overexpression of hepatic
7
-hydroxylase was associated with a marked reduction in plasma VLDL
concentrations, but no change in the rate of VLDL disappearance from
plasma, suggesting that decreased hepatic secretion of
apoB100-containing VLDL contributed to the decrease in LDL cholesterol
appearance in plasma. Whether or not the liver directly releases LDL
into the circulation is disputed (16, 36-39). Lipoprotein turnover
studies of apoB100-containing particles indicate that more LDL enters
the plasma space then can be accounted for by the metabolism of VLDL
(16, 38, 39). Whether this difference is due to the direct release of
LDL from the liver or to the release of a rapidly metabolized VLDL
precursor has not been resolved. In any event, recent reports suggest
that interruption of the enterohepatic circulation (by ileal bypass or
bile salt sequestrants) decreases the rate of LDL entry into plasma by
reducing the direct secretion of LDL by the liver (16, 38, 39).
The effect of 7-hydroxylase gene transfer was compared with the bile
salt sequestrant cholestyramine. Cholestyramine increased 7
-hydroxylase activity ~3-fold and modestly lowered plasma LDL concentrations (by ~17%), consistent with previous studies in which
bile salt sequestrants were used in animals or humans lacking LDL
receptors. This study suggests that the modest hypocholesterolemic effect of currently available bile salt sequestrants is related to the
limited capacity of these agents to interfere with bile salt absorption
and thereby increase hepatic 7
-hydroxylase activity and bile salt
synthesis. More impressive malabsorption of bile salts can be achieved
with ileal bypass or complete biliary diversion, and when these
procedures are performed in WHHL rabbits, plasma cholesterol
concentrations fall by ~40% (34, 35). However, marked depletion of
the enterohepatic bile salt pool is associated with a number of
undesirable side effects that make these procedures less than ideal as
therapy for clinical hypercholesterolemia (12, 40).
Augmentation of hepatic 7-hydroxylase activity did not increase the
rate of hepatic LDL clearance even in animals in which hepatic
7
-hydroxylase activity was increased >10-fold and plasma LDL
concentrations had fallen by >50%. This suggests that, in the absence
of the LDL receptor pathway, other receptors capable of binding LDL are
not up-regulated or derepressed in response to depletion of hepatic
cholesterol. Hepatic LDL uptake in WHHL rabbits and LDL
receptor
/
mice appears to occur entirely via
receptor-independent mechanisms (19, 20). Apparently, the role of the
LDL receptor is not subsumed by other lipoprotein transporters (LDL
receptor-related protein, very low density lipoprotein receptor, and
scavenger receptor type BI), at least under the conditions studied.
Overexpression of hepatic 7-hydroxylase for 3 days increased the
size of the bile salt pool by ~40%. It may take longer than 3 days
for the size of the bile salt pool to plateau at a new steady-state
level where the rate of bile salt excretion in the feces equals the
rate of bile salt synthesis in the liver. However, the transient nature
of 7
-hydroxylase overexpression precluded extended time course
studies or studies of fecal bile salt excretion. If prolonged
overexpression of 7
-hydroxylase can be achieved, either in
transgenic animals or with improved somatic cell gene transfer
techniques, it will be important to compare changes in hepatic
7
-hydroxylase activity with changes in the dynamics of the
enterohepatic circulation and fecal bile salt excretion. It is possible
that massive and persistent overexpression of hepatic 7
-hydroxylase
could increase bile salt synthesis enough to cause diarrhea. However,
in this study, mice consuming ~70 µmol of cholate/day, which
exceeds by more than 10-fold the amount of bile salts normally excreted
in the feces (41), did not have diarrhea. Moreover, the size of the
bile salt pool in the cholate-fed animals increased by only ~70%.
The quantity of bile salts in the enterohepatic circulation is
regulated by a number of factors including the activity of the ileal
bile salt transporter (42). Recent studies suggest that expansion of
the enterohepatic bile salt pool results in down-regulation of the
ileal bile salt transporter in mice (43) and other rodents (44).
Regulation of the ileal bile salt transporter by luminal bile salts
would tend to prevent marked expansion of the enterohepatic pool of
bile salts in response to overexpression of hepatic 7
-hydroxylase or
bile salt feeding (43, 44).
Replacement of LDL receptors by adenovirus-mediated gene transfer has
been shown to normalize plasma LDL concentrations in LDL
receptor/
mice and WHHL rabbits (23, 45, 46). The
reduction in plasma LDL concentrations is transient, however, due to an
immune response mounted against the adenovirus vector. In addition,
animals completely void of LDL receptor protein expression (such as LDL
receptor
/
mice) generate both humoral and cellular
immune responses specific for the therapeutic transgene product (47).
This problem may limit the efficacy of replacement gene therapy for
genetic deficiency states where expression of the mutated gene product
is completely absent. One strategy for circumventing the destructive
immune response that is generated against the replacement gene product is to overexpress alternative proteins that result in similar metabolic
consequences. This strategy was recently demonstrated using transfer of
the VLDL receptor gene to lower LDL concentrations in LDL
receptor
/
mice (47). The present study indicates that
direct augmentation of hepatic 7
-hydroxylase expression can lower
plasma LDL cholesterol concentrations in animals with a genetic absence
of LDL receptors. The magnitude of the hypocholesterolemic effect
despite the marked compensatory increase in hepatic cholesterol
synthesis suggests that the combination of 7
-hydroxylase gene
transfer and an inhibitor of hepatic cholesterol synthesis might have
considerable efficacy in lowering plasma LDL cholesterol concentrations
in individuals lacking LDL receptors. Such a strategy might prove
useful in patients with familial hypercholesterolemia who are
completely devoid of residual LDL receptor expression if stable gene
transfer becomes practical in humans.
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ACKNOWLEDGEMENTS |
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We thank Brent Badger, Shari Herrick, and
Anna Lorenc for excellent technical assistance and David Russell for
providing a partial cDNA encoding mouse cholesterol
7-hydroxylase.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL-38049, HL-47551, and HL-1766; Grant-in-aid 92008850 from the American Heart Association; and the Specialized Center for Research in Ischemic Heart Disease.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: Dept. of Internal
Medicine, University of Texas Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, TX 75235-8887. Tel.: 214-648-4545; Fax:
214-648-9761; E-mail: spady{at}utsw.swmed.edu.
1 The abbreviations used are: LDL, low density lipoprotein; VLDL, very low density lipoprotein; IDL, intermediate density lipoproteins; WHHL, Watanabe heritable hyperlipidemic; HPLC, high pressure liquid chromatography; FPLC, fast protein liquid chromatography; pfu, plaque forming units.
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REFERENCES |
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