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(Received for publication, September 13, 1994; and in revised form, December 16, 1994) From the
Although dietary cholesterol raises plasma total and low density
lipoprotein (LDL) cholesterol concentrations, the response to a given
intake of cholesterol varies enormously among different species and
even among individuals of the same species. The mechanisms responsible
for differing sensitivity to dietary cholesterol were examined by
comparing the rat, which is able to adapt to large fluctuations in
sterol intake or loss with little change in plasma LDL levels, with the
hamster, where changes in sterol balance strongly influence plasma LDL
concentrations. When fed the same cholesterol-free diet, hepatic
7
Epidemiological studies in human populations show a strong
correlation between dietary cholesterol intake and coronary heart
disease(1) . Although some studies suggest that dietary
cholesterol may play a role in atherosclerosis independent of its
effects on plasma cholesterol(2, 3) , the link between
dietary cholesterol and atherosclerotic lesions is thought to be
primarily related to its effects on plasma low density lipoprotein
(LDL) ( Although
dietary cholesterol usually raises plasma total and LDL cholesterol
levels, the response to a given intake of cholesterol varies remarkably
among different species and even among individuals of the same
species(7) . This marked heterogeneity in sensitivity to
dietary cholesterol presumably represents polymorphisms at gene loci
involved in the absorption of dietary cholesterol, the hepatic
conversion of cholesterol to bile acids, the feedback inhibition of
endogenous cholesterol synthesis, or the regulation of the LDL receptor
pathway. Although heritable differences in several of these major
pathways have been correlated with responsiveness to dietary
cholesterol(8, 9, 10, 11) , the
actual gene(s) involved and the polymorphisms responsible for
variability are unknown. The two mechanisms most commonly implicated
in resistance to dietary cholesterol are 1) a diminished efficiency of
cholesterol absorption and 2) an enhanced capacity to convert
cholesterol to bile acids. Both mechanisms would reduce liver
cholesterol and potentially lead to up-regulation of the LDL receptor
pathway. Thus, rhesus monkeys and baboons that are sensitive to dietary
cholesterol appear to absorb cholesterol more efficiently than
resistant animals(11, 12) . On the other hand, studies
in squirrel monkeys (13) and in inbred strains of rabbits (9, 10) suggest that the ability of the liver to
convert cholesterol to bile acids may be more important in determining
responsiveness to dietary cholesterol. In humans, both mechanisms may
contribute to individual differences in responsiveness to a high
cholesterol
diet(14, 15, 16, 17, 18, 19, 20) . While heterogeneity of response to dietary cholesterol has been
observed among individuals of all outbred species examined, differences
between species are even more pronounced. In the present studies, the
mechanisms responsible for differing sensitivity to dietary cholesterol
were examined by comparing the rat, which is able to adapt to large
fluctuations in sterol input or loss with little change in plasma LDL
concentrations, with the hamster, where even modest changes in sterol
balance lead to alterations in plasma LDL
levels(21, 22) . The response to dietary cholesterol
in these two species, which are commonly used in studies of cholesterol
and bile acid metabolism, spans the range of individual responses
observed in humans.
Samples of rat and hamster liver were homogenized in guanidinium
thiocyanate, and the RNA was isolated by the method of Chomczynski and
Sacchi(29) . Total RNA (40 µg) was hybridized with the
Responsiveness to dietary cholesterol varies greatly among
different animal species. The rat is notoriously resistant to the
cholesterolemic effects of dietary cholesterol, whereas the hamster is
modestly responsive. Fig. 1shows the changes in the cholesterol
content of liver and plasma LDL in rats and hamsters fed a low-fat
semisynthetic diet supplemented with varying amounts of cholesterol. As
shown in the toppanel, liver cholesterol in animals
fed the cholesterol-free diet equaled 2.9 and 2.2 mg/g in the hamster
and rat, respectively. In the hamster, dietary cholesterol increased
the cholesterol content of the liver in a dose-dependent fashion with
levels reaching
Figure 1:
Liver and
plasma LDL cholesterol levels in rats and hamsters fed various amounts
of cholesterol. Animals were fed a low-fat semisynthetic diet
supplemented with various amounts (0-1%) of cholesterol for 6
weeks. Each value represents the mean ± 1 S.D. for data obtained
in six animals.
Thus, at any level of cholesterol intake, the
hamster accumulated much more cholesterol in the liver and plasma than
did the rat. A number of potential mechanisms may account for these
differences in response to dietary cholesterol. One possibility is that
the hamster simply absorbs dietary cholesterol more efficiently than
the rat. However, the hamster has an unusually short bowel, and
preliminary studies showed that the rat absorbed dietary cholesterol as
well as the hamster. ( The major form of regulation of 7
Figure 2:
Hepatic 7
Figure 3:
Measurement of hepatic 7
Figure 4:
Measurement of hepatic 7
Figure 5:
Hepatic 7
Figure 6:
Regulatory effects of cholic acid and
cholestyramine on hepatic 7
Figure 7:
Regulatory effects of cholic acid and
cholestyramine on hepatic 7
In response to changes in cholesterol influx or efflux, the
liver maintains cholesterol homeostasis by regulating the rates of de novo cholesterol synthesis and LDL cholesterol uptake. Fig. 8shows rates of hepatic cholesterol synthesis as a function
of hepatic cholesterol levels in rats and hamsters fed increasing
amounts of cholesterol. On the cholesterol-free diet, rates of hepatic
cholesterol synthesis at the mid-dark point of the light cycle equaled
2,245 nmol/h/g in the rat and 111 nmol/h/g in the hamster. Hepatic
cholesterol synthesis was suppressed 90-95% by dietary
cholesterol in both species; however, the absolute reduction in sterol
synthesis was much greater in the rat (2,245 to 135 nmol/h/g) than in
the hamster (111 to 8 nmol/h/g). Notably, hepatic cholesterol synthesis
was exquisitely sensitive to the cholesterol content of the liver in
both species. Thus, as the flux of cholesterol to the liver was
progressively increased, the cholesterol content of the liver began to
rise only after de novo sterol synthesis was completely
suppressed. As shown in Fig. 9, changes in HMG CoA reductase
mRNA could account for only a small fraction of the change in
cholesterol synthesis rates, suggesting that posttranscriptional
regulation of HMG CoA reductase accounts for much of the change in
cholesterol synthesis rates in both species.
Figure 8:
Hepatic cholesterol synthesis in the rat
and hamster as a function of the cholesterol content of the liver.
Animals were fed a low-fat semisynthetic diet supplemented with the
indicated amount of cholesterol for 6 weeks. Rates of hepatic
cholesterol synthesis were quantified in vivo using
[
Figure 9:
Hepatic HMG CoA reductase mRNA in the rat
and hamster as a function of the cholesterol content of the liver.
Animals were fed a low-fat semisynthetic diet supplemented with the
indicated amount of cholesterol for 6 weeks. Each value represents the
mean ± 1 S.D. for data obtained in six
animals.
The LDL receptor, like
HMG CoA reductase, is regulated by cellular cholesterol levels, and in
cultured cells, these two pathways are regulated in parallel in
response to changes in cholesterol availability. Fig. 10shows
rates of hepatic receptor-dependent LDL transport as a function of
liver cholesterol levels in rats and hamsters fed increasing amounts of
cholesterol. In the rat, receptor-dependent LDL uptake by the liver was
not down-regulated even at the highest level of cholesterol intake
(1%), despite the fact that hepatic cholesterol synthesis was
suppressed by nearly 95% in these animals. Similarly, in the hamster,
modest amounts of dietary cholesterol had little effect on hepatic LDL
receptor activity, even though hepatic sterol synthesis was largely
suppressed. Indeed, significant down-regulation of hepatic LDL receptor
activity was only observed at dietary cholesterol intakes that caused
massive accumulation of cholesterol in the liver. As shown in Fig. 11, sterol-dependent changes in receptor-dependent LDL
transport in the hamster were accompanied by similar changes in LDL
receptor mRNA levels.
Figure 10:
Hepatic receptor-dependent LDL transport
in the rat and hamster as a function of the cholesterol content of the
liver. Animals were fed a low-fat semisynthetic diet supplemented with
the indicated amount of cholesterol for 6 weeks. Rates of
receptor-dependent LDL uptake were quantified in vivo using
primed infusions of
Figure 11:
Hepatic LDL receptor mRNA in the rat and
hamster as a function of the cholesterol content of the liver. Animals
were fed a low-fat semisynthetic diet supplemented with the indicated
amount of cholesterol for 6 weeks. Each value represents the mean
± 1 S.D. for data obtained in six
animals.
Dietary cholesterol raises total and LDL cholesterol levels;
however, the response to a given level of dietary cholesterol varies
greatly among different species and even among individuals of the same
species(7) . In the present studies, the mechanisms responsible
for differing sensitivity to dietary cholesterol were investigated by
comparing the rat, which is able to adapt to large fluctuations in
sterol input or loss with little change in plasma LDL concentrations,
with the hamster, where changes in sterol balance usually lead to
alterations in plasma LDL levels(5) . These studies suggest
that differences in sensitivity to dietary cholesterol in the rat and
hamster are due, in large part, to differences in the regulation of
hepatic 7 When fed the
same cholesterol-free, low-fat diet, hepatic 7 Hepatic 7 Why
basal expression of 7 Not only was
the basal level of 7 In
the rat, 7 It was recently reported that
dietary cholesterol actually down-regulated 7 Understanding the
mechanisms responsible for resistance to dietary cholesterol may aid in
the development of strategies aimed at inducing resistance to, or
reversing, the cholesterolemic effect of Western diets. Cholesterol
absorption efficiency correlates with plasma cholesterol levels in some
human populations(15) , and agents that interfere with
cholesterol absorption have modest cholesterol-lowering
activity(57) . Hepatic 7
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5381-5387
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Hydroxylase Expression and Response to Dietary
Cholesterol in the Rat and Hamster (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-hydroxylase activity was 16-fold higher in the rat than in the
hamster. As a consequence, rates of hepatic cholesterol synthesis were
20-fold higher in the rat than in the hamster. In both species, hepatic
cholesterol synthesis was suppressed >90% in response to increasing
loads of dietary cholesterol. However, the quantitative importance of
this adaptive mechanism was much greater in the rat since the absolute
reduction in hepatic cholesterol synthesis in the rat (2,110 nmol/h/g)
was much larger than in the hamster (103 nmol/h/g). In the rat, the
high basal level of 7-hydroxylase expression was further induced
by substrate (cholesterol) allowing these animals to convert excess
dietary cholesterol to bile acids efficiently. In contrast, the low
basal level of enzyme expression in the hamster was not induced by
dietary cholesterol. Thus, the low basal rates of bile acid and
cholesterol synthesis coupled with a lack of 7-hydroxylase
induction by cholesterol render the hamster much more sensitive than
the rat to the cholesterolemic effects of dietary cholesterol.
)cholesterol. Dietary cholesterol is not incorporated
directly into LDL but rather increases plasma LDL concentrations
indirectly through effects on sterol metabolism in the
liver(4, 5) . The liver is the key organ involved in
adaptation to a high cholesterol diet since it is not only the
recipient of dietary cholesterol but also the major site through which
cholesterol is eliminated from the body, either directly or after
conversion to bile acids. In response to changes in cholesterol intake,
the liver compensates by reducing the rate of de novo cholesterol synthesis and, in some cases, the rate of
receptor-dependent LDL cholesterol uptake. Since the liver is the
principal site of LDL catabolism, changes in hepatic LDL receptor
expression usually result in reciprocal changes in plasma LDL
concentrations. Finally, in some species, 7-hydroxylase, which
catalyzes the initial and rate-limiting step in the bile acid
biosynthetic pathway, is subject to induction by substrate
(cholesterol), thereby making it possible for these animals to convert
excess dietary cholesterol to bile acids(6) .
Animals and Diets
Sprague-Dawley rats
and Golden Syrian hamsters (Sasco, Inc., Omaha, NE) were housed in
colony cages and subjected to light cycling for at least 3 weeks prior
to introduction of the experimental diets. When used in specific
experiments, the rats and hamsters were in the 190-240- and
120-150-g-weight range, respectively. The control semisynthetic
diet used in these studies contained (wt/wt) 20% soy protein, 58% corn
starch, 2% corn oil, 0.3% DLmethionine, 8.5% salt mix, 1%
vitamin mix, 0.2% choline bitartrate, and 10% cellulose. The
experimental diets were prepared by supplementing the control
semisynthetic diet with cholesterol at the 0.05% (148 mg/1,000 kcal),
0.1% (296 mg/1,000 kcal), 0.25% (740 mg/1,000 kcal), 0.5% (1,480
mg/1,000 kcal), and 1% (2,960 mg/1,000 kcal) levels. The experimental
diets were fed ad libitum for 6 weeks, and all studies were
carried out during the mid-dark phase of the light cycle.Determination of Hepatic 7
Hepatic 7-Hydroxylase
Activityhydroxylase activity was measured
using a high pressure liquid chromatography-spectrophotometric assay
that quantifies the mass of 7-hydroxycholesterol formed from
endogenous microsomal cholesterol after enzymatic conversion to
7-hydroxy-4-cholesten-3-one using cholesterol
oxidase(23) .Determination of Hepatic Cholesterol Synthesis
Rates
Rates of hepatic cholesterol synthesis were measured in vivo using [
H]water. As previously
described(24) , the animals were administered 
100 mCi of
[H]water intravenously and then returned to
individual cages under a fume hood. 1 h after the injection of
[H]water, the animals were anesthetized and
exsanguinated through the abdominal aorta. 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 the nmoles of
[H]water incorporated into digitonin-precipitable
sterols per hour per gram of liver (nmol/h/g).Determination of Hepatic LDL Uptake Rates in
Vivo
Plasma was obtained from donor rats and hamsters
maintained on standard rodent diet (Wayne Lab Blox, Allied Mills,
Chicago, IL). LDL was isolated from plasma by preparative
ultracentrifugation in the density range of 1.020-1.055 g/ml and
labeled with 
I- or 
I-tyramine cellobiose (25) . Rates of hepatic LDL uptake were measured using primed
infusions of I-tyramine cellobiose-labeled LDL as
previously described(26) . Changes in hepatic
receptor-dependent LDL transport (defined as the rate of
receptor-dependent LDL uptake in an experimental animal relative to the
rate of receptor-dependent LDL uptake in control animals having the
same plasma LDL concentration) were determined as
described(26, 27) .Determination of mRNA Levels
Hepatic
7-hydroxylase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA)
reductase, LDL receptor, and glyceraldehyde-3-phosphate dehydrogenase
(used as an invariant control) mRNA levels were determined by nuclease
protection as previously described(21) . Probes were
synthesized using 0.5 µM [
P]dCTP
and 1-2 µM (7-hydroxylase, HMG CoA reductase,
and LDL receptor) or 300 µM (glyceraldehyde-3-phosphate
dehydrogenase) unlabeled dCTP(21, 26, 28) . P-labeled cDNA probes simultaneously at 48 °C
overnight. Unhybridized probe, present in excess relative to the amount
of specific mRNA, was then digested with 40 units of mung bean nuclease
(Life Technologies, Inc.). The mRNA-protected P-labeled
probes were separated on 7 M urea, 6% polyacrylamide gels
together with P-labeled MspI-digested pBR322 size
standards. The radioactivity in each band, as well as background
radioactivity, was quantified using an isotopic imaging system (Ambis,
Inc., San Diego, CA). The level of glyceraldehyde-3-phosphate
dehydrogenase mRNA did not vary among the various experimental groups
and was used to correct for any procedural losses.Determination of Liver and Plasma Cholesterol
Distribution
Liver cholesterol was quantified by capillary
gas-liquid chromatography. The cholesterol distribution in plasma was
determined by gel filtration chromatography using a Superose 6 column
(Sigma). 2-ml aliquots were collected and assayed for cholesterol using
an enzymatic kit (Boehringer Mannheim).
90 mg/g in animals fed 1% cholesterol. In
contrast, dietary cholesterol had little effect on liver cholesterol in
the rat with levels rising to only 7 mg/g in animals fed 1%
cholesterol. Changes in plasma LDL cholesterol levels paralleled the
changes in liver cholesterol. As shown in Fig. 1, bottompanel, plasma LDL cholesterol concentrations in the
hamster increased from 24 mg/dl to 88 mg/dl as the cholesterol content
of the diet was increased from 0 to 1%. In contrast, plasma LDL
cholesterol concentrations in the rat were only minimally affected by
dietary cholesterol.
)A second possibility is that the rat
is more efficient than the hamster at eliminating excess cholesterol
from the body. In both species, conversion of cholesterol to bile acids
is the major regulated pathway by which cholesterol is removed from the
body. Thus, differences in the regulation of this pathway may strongly
influence responsiveness to dietary cholesterol. The initial and
rate-limiting step in the conversion of cholesterol to bile acids is
catalyzed by hepatic 7-hydroxylase, and further studies were
undertaken to examine the regulation of this pathway by sterols in the
rat and hamster.-hydroxylase
is thought to be feedback repression of gene transcription by bile
acids returning to the liver in the enterohepatic circulation. In
addition, 7-hydroxylase is subject to induction by substrate
(cholesterol) in some species, including the rat. Fig. 2shows
7-hydroxylase activity in rats and hamsters as a function of the
cholesterol content of the liver in animals fed increasing amounts of
cholesterol. As indicated by the opensymbols, basal
7-hydroxylase activity in animals fed the same cholesterol-free,
low-fat diet were significantly higher in the rat than in the hamster.
Thus, at the mid-dark point of the light cycle, 7-hydroxylase
activity in the rat and hamster equaled 1,410 and 88 pmol/h/mg of
microsomal protein, respectively. Moreover, the high basal level of
7-hydroxylase activity in the rat was further increased when
cholesterol was added to the diet, whereas the low basal activity in
the hamster was not significantly altered by dietary cholesterol in
amounts that markedly raised the cholesterol content of the liver.
Aliquots of liver were taken from these same animals for the
determination of 7-hydroxylase mRNA levels by nuclease protection.
Autoradiograms illustrating the effect of dietary cholesterol on
7hydroxylase mRNA levels in the rat and hamster are shown in Fig. 3and Fig. 4, respectively, and the mean changes in
7hydroxylase mRNA levels are summarized in Fig. 5. As shown
in Fig. 5, dietary cholesterol increased 7-hydroxylase mRNA
levels in the rat at liver cholesterol levels that were only modestly
(2-3-fold) elevated. In the hamster, on the other hand, dietary
cholesterol, in amounts that increased liver cholesterol levels by up
to 10-fold, had no effect on 7-hydroxylase mRNA levels. Hepatic
7-hydroxylase mRNA levels did tend to increase (though not
significantly so) in hamsters fed cholesterol in amounts that raised
liver cholesterol levels by 15-30-fold. Higher levels of
cholesterol intake resulted in massive cholesterol accumulation in the
liver and a trend toward lower 7-hydroxylase mRNA levels. It was
not possible to compare the absolute amount of 7-hydroxylase mRNA
in the rat and hamster directly; however, based on the length,
nucleotide content, and specific activity of the P cDNA
probes that were used in these studies, 7-hydroxylase mRNA was
severalfold more abundant in rat than in hamster liver. Thus, in terms
of the regulation of 7-hydroxylase expression, the rat differed
from the hamster both with respect to basal levels of expression and
with respect to the ability to up-regulate expression in response to
increasing levels of substrate. In contrast, regulation of hepatic
7-hydroxylase by bile acids appeared to be similar in the two
species. As shown in Fig. 6, 7-hydroxylase activity was
suppressed by dietary cholate and derepressed by cholestyramine in both
species. As summarized in Fig. 7(with representative
autoradiograms in Fig. 3and Fig. 4), these changes in
7-hydroxylase activity were accompanied by similar changes in mRNA
levels.
-hydroxylase activity in the
rat and hamster as a function of the cholesterol content of the liver.
Animals were fed a low-fat semisynthetic diet supplemented with the
indicated amount of cholesterol for 6 weeks. Each value represents the
mean ± 1 S.D. for data obtained in six
animals.
-hydroxylase
mRNA levels in the rat. Hepatic RNA was isolated from rats fed a
low-fat semisynthetic diet supplemented with 0.25% cholic acid, 1%
cholestyramine, or 1% cholesterol. Total RNA (40 µg) was hybridized
with P-labeled single-stranded cDNA probes, and the
protected bands resistant to mung bean nuclease digestion were
separated by polyacrylamide gel electrophoresis and autoradiographed. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FABP, fatty acid binding protein; nt,
nucleotides.
-hydroxylase
mRNA levels in the hamster. Hepatic RNA was isolated from hamsters fed
a low-fat semisynthetic diet supplemented with 0.25% cholic acid, 1%
cholestyramine, or the indicated amount of cholesterol. Total RNA (40
µg) was hybridized with P-labeled single-stranded cDNA
probes, and the protected bands resistant to mung bean nuclease
digestion were separated by polyacrylamide gel electrophoresis and
autoradiographed. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; nt, nucleotides.
-hydroxylase mRNA in the rat
and hamster as a function of the cholesterol content of the liver.
Animals were fed a low-fat semisynthetic diet supplemented with the
indicated amount of cholesterol for 6 weeks. Each value represents the
mean ± 1 S.D. for data obtained in six
animals.
-hydroxylase activity in the rat and
hamster. Animals were fed a low-fat semisynthetic diet supplemented
with 0.25% cholic acid or 1% cholestyramine for 3 weeks. Each value
represents the mean ± 1 S.D. for data obtained in six
animals.
-hydroxylase mRNA in the rat and
hamster. Animals were fed a low-fat semisynthetic diet supplemented
with 0.25% cholic acid or 1% cholestyramine for 3 weeks. Each value
represents the mean ± 1 S.D. for data obtained in six
animals.
H]water. Each value represents the mean ±
1 S.D. for data obtained in six animals.
I-tyramine cellobiose-labeled LDL.
Each value represents the mean ± 1 S.D. for data obtained in six
animals.
-hydroxylase, the initial and rate-limiting enzyme in the
bile acid biosynthetic pathway. Thus, a low basal level of
7hydroxylase expression coupled with a lack of enzyme induction by
dietary cholesterol render the hamster more sensitive than the rat to
the cholesterolemic effects of dietary cholesterol.-hydroxylase
activity was 16-fold higher in the rat than in the hamster. Under
steady-state conditions, the rapid conversion of cholesterol to bile
acids in the rat must be balanced by a high rate of endogenous
cholesterol synthesis, and, indeed, basal rates of hepatic cholesterol
synthesis were 20-fold higher in the rat than in the hamster. The
significance of a high basal rate of hepatic cholesterol synthesis is
that a larger load of dietary cholesterol can be accommodated through
feedback repression of endogenous synthesis. Dietary cholesterol
suppressed hepatic sterol synthesis by >90% in both species;
however, the quantitative importance of this adaptive mechanism was far
greater in the rat since the absolute reduction in sterol synthesis in
this species (2,110 nmol/h/g) was much larger than in the hamster (103
nmol/h/g). Hepatic cholesterol synthesis was exquisitely sensitive to
cellular cholesterol levels in both species. Thus, as the flux of
cholesterol to the liver was progressively increased by raising the
cholesterol content of the diet, liver cholesterol levels began to rise
only after de novo sterol synthesis was completely suppressed (Fig. 8). Feedback repression of endogenous cholesterol
synthesis also appears to be an important adaptive mechanism for
dealing with dietary cholesterol in
humans(16, 17, 19) . In sterol balance
studies carried out by Nestel and Poyser (19) , the greater the
reduction in endogenous cholesterol synthesis, the less likely was
plasma cholesterol to rise in response to a high cholesterol diet. -hydroxylase activity in the rat is characterized by
marked diurnal fluctuations with maximal activity occurring in the
mid-dark phase of the light cycle(30, 31) . Diurnal
changes in 7-hydroxylase expression have not been systematically
examined in the hamster. However, we have found similar levels of
7-hydroxylase activity and mRNA at the mid-dark and mid-light
points of the light cycle, suggesting that diurnal fluctuations in
enzyme expression are much less pronounced in the hamster than in the
rat(28) . This is consistent with the finding that hamsters
consume approximately as much food during the two phases of the light
cycle(32) , whereas rats eat exclusively during the dark phase
of the light cycle. In any case, differences in integrated enzyme
activity over the entire 24-h light cycle would certainly be less than
the 16-fold difference in activity observed at the mid-dark point in
the light cycle. In addition, it is possible that alternative pathways
that circumvent 7-hydroxylase (33, 34) might
contribute to bile acid synthesis in the rat and/or hamster. Sterol
balance studies suggest that rats synthesize 3-5-fold more
bile acids over a 24-h period of time than hamsters, although direct
comparisons have not been
reported(35, 36, 37, 38) .-hydroxylase is higher in rats than in
hamsters is not known. In both species, 7hydroxylase expression is
up-regulated severalfold by interventions that interrupt the
enterohepatic cycling of bile
acids(6, 28, 39) . The conventional
interpretation of these data is that the enzyme is under feedback
control by bile acids fluxing through the liver in the enterohepatic
circulation. If this interpretation is true, one possible explanation
for the unusually high basal level of 7-hydroxylase expression in
the rat is that they malabsorb bile acids. Under physiological
conditions, bile acids are absorbed from the gut primarily by bile acid
transporters located in the brush border membranes of ileal enterocytes (40, 41) . It is possible that differences in the
expression of the ileal bile acid transporter could lead to differences
in the conservation of bile acids in the enterohepatic circulation and
differences in 7-hydroxylase expression. However, the
enterohepatic pool of bile acids is 3-fold larger in the rat (42, 43) than in the hamster, suggesting that the
differences in basal 7-hydroxylase expression are primary rather
than secondary to bile acid malabsorption in the rat.-hydroxylase expression very low in the
hamster, enzyme expression was not induced by dietary cholesterol in
this species. Induction of 7-hydroxylase expression and bile acid
synthesis by dietary cholesterol has been observed in several species,
including the rat(37, 44) , mouse(45) ,
dog(46) , and certain species of nonhuman
primates(13) , all of whom adapt to large amounts of dietary
cholesterol with little change in plasma cholesterol. For the most
part, dietary cholesterol does not lead to an increase in
7-hydroxylase expression or bile acid synthesis in
humans(16, 17, 18, 19, 20) .
However, certain individuals maintain normal plasma cholesterol levels
despite a high intake of cholesterol, and induction of bile acid
synthesis appears to account, at least in part, for the resistance to
dietary cholesterol observed in these individuals(14) .-hydroxylase expression is inhibited by bile acids
fluxing through the liver in the enterohepatic circulation and is
subject to induction by substrate (cholesterol). Presumably, the
7-hydroxylase promoter contains sequences that mediate regulation
by bile acids and cholesterol, i.e. sterol and bile acid
response elements(47, 48, 49) . It has been
suggested that expression of the enzyme may be titrated by these
positive and negative response elements that somehow sense the levels
of substrate and end products, respectively(6) . Hepatic
7hydroxylase expression was not induced by cholesterol in the
hamster, suggesting that either the sterol response element or the
proteins that interact with it may be nonfunctional in this species. If
the basal level of 7-hydroxylase expression is indeed titrated by
negative and positive response elements in the rat, absence of the
positive response mechanism could also explain the very low level of
basal expression in the hamster. We previously showed in the rat that
dietary cholesterol completely blocked the suppressive effects of bile
acids on 7-hydroxylase expression(21) . Indeed the highest
levels of expression were observed in animals fed cholesterol plus
cholic acid. Since cholic acid promotes cholesterol absorption (50) and greatly increased the cholesterol content of the
liver(21) , these findings suggested that the putative sterol
regulatory element contributed more to the regulation of gene
expression than the putative bile acid response element. Although
cholesterol regulates 7-hydroxylase expression in the rat
primarily at the level of the
hepatocyte(44, 48, 51) , it has been
suggested that large amounts of dietary cholesterol may also interfere
with bile acid absorption, thereby leading to derepression of hepatic
7-hydroxylase (52) . If true, events in the gut may also
contribute to induction of hepatic 7-hydroxylase expression by
dietary cholesterol in the rat.-hydroxylase activity
in African green monkeys(53) . However, the
cholesterol-enriched diets used in these studies also contained large
amounts of triglyceride. Dietary triglyceride has been shown to
suppress 7-hydroxylase activity in the rat(54) , and
diverting dietary triglyceride away from the liver by lymph
fistulization reportedly up-regulates enzyme activity(55) .
While the production of bile acids is important for the maintenance of
cholesterol homeostasis, bile acids serve an equally important role in
the gut where they emulsify lipids, thereby promoting fat digestion and
absorption. Thus, it would make mechanistic sense if one or more
products of lipolysis regulated the rate of bile acid synthesis. Oleic
acid has been shown to suppress bile acid secretion in the isolated
perfused rat liver, suggesting that fatty acids may directly inhibit
hepatic 7-hydroxylase(56) .-hydroxylase represents another
promising target. In recent studies, adenovirus-mediated transfer of a
gene encoding cholesterol 7-hydroxylase into hamsters increased
hepatic 7-hydroxylase activity by 10-15-fold (to levels seen
in the rat) and rendered these animals highly resistant to the
cholesterolemic effects of a high cholesterol diet(58) .
Currently available bile acid sequestrants increase hepatic
7-hydroxylase expression by only 2-3-fold and, as a
consequence, have only modest cholesterol lowering activity. More
effective strategies for increasing 7-hydroxylase expression
should prove proportionately more active in lowering plasma cholesterol
levels.
))
We thank Drs. David Russell, Diane Jelinek, Sohaib
Khan, Richard Tanaka, and Ray Wu for making the cDNAs available and
Jody Houston, Wendy Williams, and Matthew Zimmerman for excellent
technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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  K. T. Burke, P. S. Horn, P. Tso, J. E. Heubi, and L. A. Woollett Hepatic bile acid metabolism in the neonatal hamster: expansion of the bile acid pool parallels increased Cyp7a1 expression levels Am J Physiol Gastrointest Liver Physiol, July 1, 2009; 297(1): G144 - G151. [Abstract] [Full Text] [PDF]
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 I. Martinez, G. Wallace, C. Zhang, R. Legge, A. K. Benson, T. P. Carr, E. N. Moriyama, and J. Walter Diet-Induced Metabolic Improvements in a Hamster Model of Hypercholesterolemia Are Strongly Linked to Alterations of the Gut Microbiota Appl. Envir. Microbiol., June 15, 2009; 75(12): 4175 - 4184. [Abstract] [Full Text] [PDF]
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 H. B. Hartman, S. J. Gardell, C. J. Petucci, S. Wang, J. A. Krueger, and M. J. Evans Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR-/- and apoE-/- mice J. Lipid Res., June 1, 2009; 50(6): 1090 - 1100. [Abstract] [Full Text] [PDF]
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 Y.-K. Lee, D. R. Schmidt, C. L. Cummins, M. Choi, L. Peng, Y. Zhang, B. Goodwin, R. E. Hammer, D. J. Mangelsdorf, and S. A. Kliewer Liver Receptor Homolog-1 Regulates Bile Acid Homeostasis but Is Not Essential for Feedback Regulation of Bile Acid Synthesis Mol. Endocrinol., June 1, 2008; 22(6): 1345 - 1356. [Abstract] [Full Text] [PDF]
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 M. Torres-Gonzalez, S. Shrestha, M. Sharman, H. C. Freake, J. S. Volek, and M. L. Fernandez Carbohydrate Restriction Alters Hepatic Cholesterol Metabolism in Guinea Pigs Fed a Hypercholesterolemic Diet J. Nutr., October 1, 2007; 137(10): 2219 - 2223. [Abstract] [Full Text] [PDF]
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I. J. A. M. Jonkers, A. H. M. Smelt, H. M. G. Princen, F. Kuipers, J. A. Romijn, R. Boverhof, A. A. M. Masclee, and F. Stellaard Fish Oil Increases Bile Acid Synthesis in Male Patients with Hypertriglyceridemia J. Nutr., April 1, 2006; 136(4): 987 - 991. [Abstract] [Full Text] [PDF]
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 Z. Giricz, M. M. Lalu, C. Csonka, P. Bencsik, R. Schulz, and P. Ferdinandy Hyperlipidemia Attenuates the Infarct Size-Limiting Effect of Ischemic Preconditioning: Role of Matrix Metalloproteinase-2 Inhibition J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 154 - 161. [Abstract] [Full Text] [PDF]
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A. R. Tovar, I. Torre-Villalvazo, M. Ochoa, A. L. Elias, V. Ortiz, C. A. Aguilar-Salinas, and N. Torres Soy protein reduces hepatic lipotoxicity in hyperinsulinemic obese Zucker fa/fa rats J. Lipid Res., September 1, 2005; 46(9): 1823 - 1832. [Abstract] [Full Text] [PDF]
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 L. Yao and L. A. Woollett Adult sterol metabolism is not affected by a positive sterol balance in the neonatal Golden Syrian hamster Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2005; 288(3): R561 - R566. [Abstract] [Full Text] [PDF]
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K. E. Schmid and L. A. Woollett Differential effects of polyunsaturated fatty acids on sterol synthesis rates in adult and fetal tissues of the hamster: consequence of altered sterol balance Am J Physiol Gastrointest Liver Physiol, November 1, 2003; 285(5): G796 - G803. [Abstract] [Full Text] [PDF]
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 A. Onody, C. Csonka, Z. Giricz, and P. Ferdinandy Hyperlipidemia induced by a cholesterol-rich diet leads to enhanced peroxynitrite formation in rat hearts Cardiovasc Res, June 1, 2003; 58(3): 663 - 670. [Abstract] [Full Text] [PDF]
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B. Goodwin, M. A. Watson, H. Kim, J. Miao, J. K. Kemper, and S. A. Kliewer Differential Regulation of Rat and Human CYP7A1 by the Nuclear Oxysterol Receptor Liver X Receptor-{alpha} Mol. Endocrinol., March 1, 2003; 17(3): 386 - 394. [Abstract] [Full Text] [PDF]
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 K. Otte, H. Kranz, I. Kober, P. Thompson, M. Hoefer, B. Haubold, B. Remmel, H. Voss, C. Kaiser, M. Albers, et al. Identification of Farnesoid X Receptor {beta} as a Novel Mammalian Nuclear Receptor Sensing Lanosterol Mol. Cell. Biol., February 1, 2003; 23(3): 864 - 872. [Abstract] [Full Text]
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 J. G. Menke, K. L. Macnaul, N. S. Hayes, J. Baffic, Y.-S. Chao, A. Elbrecht, L. J. Kelly, M.-H. Lam, A. Schmidt, S. Sahoo, et al. A Novel Liver X Receptor Agonist Establishes Species Differences in the Regulation of Cholesterol 7{alpha}-Hydroxylase (CYP7a) Endocrinology, July 1, 2002; 143(7): 2548 - 2558. [Abstract] [Full Text] [PDF]
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R. A. Davis, J. H. Miyake, T. Y. Hui, and N. J. Spann Regulation of cholesterol-7{alpha}-hydroxylase: BAREly missing a SHP J. Lipid Res., April 1, 2002; 43(4): 533 - 543. [Abstract] [Full Text] [PDF]
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 J. H. Miyake, X. T. Duong-Polk, J. M. Taylor, E. Z. Du, L. W. Castellani, A. J. Lusis, and R. A. Davis Transgenic Expression of Cholesterol-7-{alpha}-Hydroxylase Prevents Atherosclerosis in C57BL/6J Mice Arterioscler. Thromb. Vasc. Biol., January 1, 2002; 22(1): 121 - 126. [Abstract] [Full Text] [PDF]
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M. Schwarz, D. W. Russell, J. M. Dietschy, and S. D. Turley Alternate pathways of bile acid synthesis in the cholesterol 7{alpha}-hydroxylase knockout mouse are not upregulated by either cholesterol or cholestyramine feeding J. Lipid Res., October 1, 2001; 42(10): 1594 - 1603. [Abstract] [Full Text] [PDF]
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W. M. Pandak, C. Schwarz, P. B. Hylemon, D. Mallonee, K. Valerie, D. M. Heuman, R. A. Fisher, K. Redford, and Z. R. Vlahcevic Effects of CYP7A1 overexpression on cholesterol and bile acid homeostasis Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G878 - G889. [Abstract] [Full Text] [PDF]
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M. Souidi, M. Combettes-Souverain, F. Milliat, E. R. Eckhardt, O. Audas, S. Dubrac, M. Parquet, J. Férézou, and C. Lutton Hamsters Predisposed to Sucrose-Induced Cholesterol Gallstones (LPN Strain) Are More Resistant to Excess Dietary Cholesterol than Hamsters That Are Not Sensitive to Cholelithiasis Induction J. Nutr., June 1, 2001; 131(6): 1803 - 1811. [Abstract] [Full Text]
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M. L. Fernandez Guinea Pigs as Models for Cholesterol and Lipoprotein Metabolism J. Nutr., January 1, 2001; 131(1): 10 - 20. [Abstract] [Full Text]
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J. Férézou, M. Combettes-Souverain, M. Souidi, J. L. Smith, N. Boehler, F. Milliat, E. Eckhardt, G. Blanchard, M. Riottot, C. Sérougne, et al. Cholesterol, bile acid, and lipoprotein metabolism in two strains of hamster, one resistant, the other sensitive (LPN) to sucrose-induced cholelithiasis J. Lipid Res., December 1, 2000; 41(12): 2042 - 2054. [Abstract] [Full Text]
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 J. J. Repa, G. Liang, J. Ou, Y. Bashmakov, J.-M. A. Lobaccaro, I. Shimomura, B. Shan, M. S. Brown, J. L. Goldstein, and D. J. Mangelsdorf Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta Genes & Dev., November 15, 2000; 14(22): 2819 - 2830. [Abstract] [Full Text]
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G. Xu, B. L. Shneider, S. Shefer, L. B. Nguyen, A. K. Batta, G. S. Tint, M. Arrese, S. Thevananther, L. Ma, S. Stengelin, et al. Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits J. Lipid Res., February 1, 2000; 41(2): 298 - 304. [Abstract] [Full Text]
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 G. J. Schroepfer Jr. Oxysterols: Modulators of Cholesterol Metabolism and Other Processes Physiol Rev, January 1, 2000; 80(1): 361 - 554. [Abstract] [Full Text] [PDF]
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M. L. Fernandez, T. A. Wilson, K. Conde, M. Vergara-Jimenez, and R. J. Nicolosi Hamsters and Guinea Pigs Differ in Their Plasma Lipoprotein Cholesterol Distribution when Fed Diets Varying in Animal Protein, Soluble Fiber, or Cholesterol Content J. Nutr., July 1, 1999; 129(7): 1323 - 1332. [Abstract] [Full Text]
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G. Xu, G. Salen, S. Shefer, G. S. Tint, L. B. Nguyen, T. T. Parker, T. S. Chen, J. Roberts, X. Kong, and D. Greenblatt Regulation of classic and alternative bile acid synthesis in hypercholesterolemic rabbits: effects of cholesterol feeding and bile acid depletion J. Lipid Res., August 1, 1998; 39(8): 1608 - 1615. [Abstract] [Full Text]
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S. M. Post, E. C. M. de Wit, and H. M. G. Princen. Cafestol, the Cholesterol-Raising Factor in Boiled Coffee, Suppresses Bile Acid Synthesis by Downregulation of Cholesterol 7{alpha}-Hydroxylase and Sterol 27-Hydroxylase in Rat Hepatocytes Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 3064 - 3070. [Abstract] [Full Text]
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 D. K. Spady, J. A. Cuthbert, M. N. Willard, and R. S. Meidell Feedback Regulation of Hepatic 7alpha -Hydroxylase Expression by Bile Salts in the Hamster J. Biol. Chem., August 2, 1996; 271(31): 18623 - 18631. [Abstract] [Full Text] [PDF]
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K. Alam, R. S. Meidell, and D. K. Spady Effect of Up-regulating Individual Steps in the Reverse Cholesterol Transport Pathway on Reverse Cholesterol Transport in Normolipidemic Mice J. Biol. Chem., May 4, 2001; 276(19): 15641 - 15649. [Abstract] [Full Text] [PDF]
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S. Gupta, R. T. Stravitz, P. Dent, and P. B. Hylemon Down-regulation of Cholesterol 7alpha -Hydroxylase (CYP7A1) Gene Expression by Bile Acids in Primary Rat Hepatocytes Is Mediated by the c-Jun N-terminal Kinase Pathway J. Biol. Chem., May 4, 2001; 276(19): 15816 - 15822. [Abstract] [Full Text] [PDF]
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