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Laboratoire de Physiologie de la Nutrition-INRA, Université Paris XI, F-91405 Orsay Cedex, France
3To whom correspondence should be addressed. E-mail: jacqueline.ferezou{at}ibaic.u-psud.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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-hydroxylase activity and mRNA level,
and stimulated sterol 27-hydroxylase and oxysterol 7-hydroxylase
activities. Hepatic levels of LDL receptor decreased by 
60% in both
strains, whereas HDL receptor scavenger class B type 1 (SR-BI) levels
were unaffected by dietary cholesterol. The greater resistance of LPN
hamsters to the hypercholesterolemic diet can be explained by a lower
capacity to store cholesterol in the liver and greater efficiency in
reducing the activity of 3-hydroxy-3-methyl glutaryl coenzyme A
reductase in response to cholesterol feeding [from 11263 to 261
pmol/(min · organ) in LPN hamsters and from 4530 to 694
pmol/(min · organ) in JAN hamsters]. These results highlight the
usefulness of this two-strain model, which offers some analogy with
the inverse association between the predisposition to cholelithiasis
and the risk of atherosclerosis in humans.
KEY WORDS: • dietary cholesterol • bile acid synthesis • hamsters • strain
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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) and cholelithiasis (Cohen et al. 1989
). Feeding cholesterol-enriched diets increases the
plasma cholesterol concentration in most animal species including
humans, but the degree of the responsiveness differs according to the
species and the individuals within a species (Chen et al. 1998
, Paigen, 1995
; Xu et al. 1998
) including humans (Mc Namara et al. 1987
). This heterogeneity could be explained by differences in
cholesterol absorption and hepatic catabolism (Jolley et al. 1999
, Sehayek et al. 1998
) because the liver is
the key organ in the elimination of cholesterol from the body, either
directly or after conversion into bile acids.
The Syrian golden hamster (Mesocricetus auratus) is now
commonly used in studies of cholesterol and bile acid metabolism
because it presents certain similarities with human metabolism
(Kris-Etherton and Dietschy 1997
). Indeed, this species
is sensitive to hypercholesterolemic diets (Cai and Carr 1999
, Horton et al. 1995
, Turley et al. 1997
) and can develop cholesterol gallstones induced by excess
dietary cholesterol (Cohen et al. 1989
, Trautwein et al. 1999
) or sucrose (Dam and Christensen 1952
, Khallou et al. 1991
). Hamsters basically
differ from rats, which are protected against cholelithiasis and
atherosclerosis, with their absence of a gallbladder, a circulating
pool of hydrophilic bile acids and a high capacity of bile acid
production as a response to hypercholesterolemic diets (Spady and Cuthbert 1992
).
Recent studies have shown that male hamsters bred in our laboratory
[Laboratoire de Physiologie et de la Nutrition
(LPN)4
strain] are highly susceptible to cholesterol gallstone induction by a
low fat/sucrose-rich diet (Boehler et al. 1999
), whereas
hamsters from a commercial strain, termed Janvier (JAN), do not develop
cholesterol gallstones under the same dietary condition
(Férézou et al. 2000
). Under basal
conditions (i.e., when fed a commercial rodent diet), LPN differ from
JAN hamsters by a lower cholesterolemia, a more active
cholesterogenesis and a lower capacity of the liver to store
cholesterol and convert it into bile acids. This relatively low
capacity to metabolize cholesterol for export from the liver likely
predisposes LPN hamsters to sucrose-induced cholelithiasis, whereas
JAN hamsters may be more sensitive to dietary cholesterol. To confirm
this hypothesis, males from the two strains were fed a commercial
rodent diet with or without 0.3% cholesterol added. This choice was
based on previous observations that unlike semipurified diets,
commercial diets induce a lipoprotein profile in hamsters more similar
to that of humans (Férézou et al. 2000
,
Nicolosi et al. 1998
and unpublished data). Five weeks
after feeding the control diet or the cholesterol-rich diet, plasma
and bile lipid variables, liver cholesterol content and activities of
hepatic and intestinal 3-hydroxy-3-methylglutaryl coenzyme A reductase
(HMG-CoAR), as well as the amounts of hepatic lipoprotein receptors
[LDL receptor and HDL scavenger receptor class B type 1 (SR-BI)] were
compared between the two strains. Special attention was paid to three
rate-limiting enzymes involved in the classical (cholesterol
7-hydroxylase, CYP7A) or the alternative (sterol 27-hydroxylase,
CYP27 and oxysterol 7-hydroxylase, CYP7B) pathway of bile
acid synthesis (Princen et al. 1997
). Moreover,
retrotranscription polymerase chain reaction (RT-PCR) experiments
were performed to determine whether differences in HMG-CoAR and
CYP7A activities were associated with differences in corresponding mRNA
levels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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Cholesterol monohydrate (Merck, Darmstadt, Germany) was recrystallized
from methanol before addition to the diet.
Hydroxypropyl-ß-cyclodextrin was a gift from Roquette Frères
(Lestrem, France). Chemicals of the highest purity were purchased from
Sigma-Aldrich (St-Quentin Fallavier, France). Anion exchange AG1-X8
resin was purchased from Bio-Rad (Ivry/Seine, France).
L-3-[Glutaryl-3-14C] hydroxymethylglutaryl
coenzyme A, [5-3H]mevalonolactone,
[14C]cholesterol and
25-[26,27-3H2]hydroxycholesterol were
purchased from NEN (Les Ulis, France). 25-Hydroxycholesterol was kindly
provided by Roussel-Uclaf (Romainville, France); 7- and
7ß-hydroxycholesterol were prepared according to a previously
described procedure (Souidi et al. 1998
). A polyclonal
antibody raised against the bovine adrenal cortex LDL receptor was
kindly provided by Paul Roach (Adelaide, Australia). A rabbit
polyclonal antipeptide antibody, raised against amino acid residues
495–509 of murine SR-BI (Acton et al. 1996
), was
kindly prepared by André Mazur (Theix, France). Enhanced
chemiluminescence reagent was purchased from Amersham Pharmacia Biotech
(Les Ulis, France).
Diets.
The composition of the nonpurified rodent diet (CRF20 formula, produced
by UAR, Villemoisson/Orge, France) is detailed in Table 1
. This diet contained 11 mg of sterols/100 g, 20% of which consisted
of cholesterol and 80% phytosterols, as determined by GLC on
saponified lipid extracts (Férézou et al. 1993
). This powdered diet was used as the Control diet,
and the cholesterol-enriched (Chol-rich) diet was prepared by
mixing the powdered control diet with cholesterol (0.3 g/100 g)
dissolved in diethylether and dried under N2.
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All of the experiments started with 4-wk-old male golden hamsters
weighing 30–40 g, born in our breeding unit (LPN hamsters) or
purchased from CER Janvier (Villemoisson, France; JAN hamsters). They
were caged in pairs and received the Control diet during a 1-wk
adaptation period. They were then housed individually and assigned to
groups of 6–8 according to the diet fed (Control or Chol-rich) for the
next 5 wk. Food and water were consumed ad libitum. The room
temperature was maintained at 23 ± 1°C and lighting conditions
were controlled (lights on from 0700 to 2100 h). Body weight was
monitored throughout the experiments and food consumption measured
during the last week. The hamsters were killed in a postprandial state,
between 0900 and 1200 h, 30 min after food was withdrawn in all
experiments. The care and use of the experimental animals were in
accordance with the ethical standards of the French decree N°87–849
(19 October 1989).
Plasma, bile and organ sampling.
The hamsters were anesthetized using an intramuscular injection of Zoletil 50 (Reading Laboratory, Nice, France) at a dose of 250 mg/kg body and killed by intracardiac blood puncture using a 2-mL insulin syringe (Becton Dickinson Europe, Pont-de-Claix, France) containing 200 IU heparin. The abdomen was opened and the gallbladder was examined for the presence of gallstones; the bile was collected using a 0.5-mL syringe (Becton Dickinson Europe) and kept at -20°C. The liver was rapidly excised, weighed and apportioned for preparing cellular fractions or storing at -20 or -80°C for future use. The small intestine was excised, washed with physiologic saline, weighed and used in totality for preparing cellular fractions. Blood was centrifuged at 3500 x g at 4°C for 15 min to collect the plasma.
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Biochemical assays |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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Plasma lipids were measured by enzymatic procedures using commercial
kits with an automatic analyzer (Abbott VP, Rungis, France); total
cholesterol was measured by the CHOD-PAP method (Boehringer
Mannheim, Meylan, France), triglycerides and phospholipids by the Wako
method (Oxoid, Rungis, France). Lipids were extracted from homogenates
of liver samples (0.5 g) with isopropanol (Boehler et al. 1999
), directly assayed enzymatically for free and total
cholesterol (CHOD-PAP method, Boehringer Mannheim), and esterified
cholesterol concentration was calculated from the difference between
total and free cholesterol concentrations. Bile samples were diluted
(1/20) with physiologic saline and analyzed for cholesterol and
phospholipids as previously described (Boehler et al. 1999
). The total bile acid concentrations were assayed using
the 3-hydroxysteroid dehydrogenase method (Boehler et al. 1999
) and the cholesterol saturation index (CSI) was calculated
(Thomas and Hofmann, 1973
). Bile acid composition was
analyzed by HPLC and the hydrophobicity index was calculated
(Eckhardt et al. 1997
).
Plasma lipoproteins.
Lipoproteins were fractionated by ultracentrifugation of plasma samples
(0.4 mL) in a saline density gradient, using a SW41 rotor in a L8–70
apparatus (Beckman Instruments, Gagny, France) as described
(Férézou et al. 1997
). Twenty-two
fractions (0.5 mL) were collected and analyzed for cholesterol as
described above.
Liver cellular fractions.
Microsomal and mitochondrial fractions were prepared from fresh liver
samples according to procedures already described (Souidi et al. 1999
). Total liver membranes were prepared according to
Kovanen et al. (1979)
. Protein content of the fractions
was assayed by the Lowry method (Lowry et al. 1951
)
using bovine serum albumin as a standard. Total RNA was
extracted from frozen (-80°C) liver samples (100 mg) using a
commercial kit (Quickprep Total RNA Extraction, Amersham Pharmacia
Biotech) and stored at -80°C.
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Enzymatic assays |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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. Each assay
tube, containing 80–300 µg protein in phosphate Beg
buffer [final dithiothreitol concentration of 10 mmol/L] was
preincubated for 60 min at 37°C in the presence of 0.5 IU
phosphatase and incubated for 10 min at 37°C after the addition of
[14C]HMG-CoA (20 nmol, 100,000 dpm) and NADPH
(4.2 mmol/L). The reaction was stopped using 5 mol/L HCl to convert the
reaction product into [14C]mevalonolactone.
After the addition of[3H]mevalonolactone
(10,000 dpm) as an internal marker, each sample was passed through a
minicolumn of AG1-X8 anionic resin and the radioactivity of the eluate
was measured. All assays were performed in duplicate and four tubes
containing boiled microsomes were treated in parallel, as controls.
The radioisotopic assays for microsomal CYP7A (Souidi et al. 1998
), mitochondrial CYP27 (Souidi et al. 1999
)
and CYP7B (Souidi et al. 2000
) activities in the hamster
liver have been described in detail.
All of the enzymatic activities, calculated in pmol/(min · mg microsomal or mitochondrial protein), were expressed as pmol/(min · liver or intestine) to take into account the variations in the weight of organs due to strain or diet.
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Immunoassays |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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). Briefly, proteins of
total membrane fractions from liver were fractioned by 7.5%
SDS-glycerol polyacrylamide electrophoresis (Connelly and Kuksis 1982
) using a Mini Protean II apparatus (Bio-Rad) and
electrotransferred onto 0.45-µm nitrocellulose membranes
as previously described. After incubation with antibodies against the
LDL receptor or SR-BI, antibody binding was visualized using a
rabbit immunoglobulin G labeled with horseradish peroxydase and the
enhanced chemiluminescence method. The films (Hyperfilm, Amersham
Pharmacia Biotech) were scanned by a laser densitometer (LKB 2222
Ultroscan XL, Bromma, Sweden) interfaced with a microcomputer. Results
were expressed in arbitrary units/mg protein, using the peak area of
each band. |
Liver mRNA levels of HMG-CoAR, CYP7A |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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). Statistical analysis.
Results are given as means ± SEM. Statistical analyses were performed by a two-way ANOVA, for strain effect, diet effect and the interaction of strain and diet. A Student’s t test was used as a post-hoc test to compare the diet effect between the two strains. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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JAN hamsters had higher final body weights (+10%) than LPN
hamsters, due to slightly greater body weight gain (calculated for the
last 5 wk of the experiment), (Table 2
). The cholesterol-rich diet did not affect the final body weight,
but it increased the liver weight in the two strains, more markedly in
JAN hamsters (+54%, P < 0.001) than in LPN hamsters
(+14%, NS). The small intestine weighed less in JAN hamsters than in
LPN hamsters and was not affected by the dietary cholesterol.
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LPN hamsters had lower concentrations of plasma lipids than JAN
hamsters (Table 3
). Compared with the control diet, the cholesterol-rich diet
increased plasma lipids in both strains, but less markedly in LPN than
in JAN hamsters. Cholesterol and phospholipids increased by 108 and
35% (P < 0.001), respectively, in LPN hamsters,
vs. 235 and 69% (P < 0.001), respectively, in JAN
hamsters. After cholesterol feeding, triglycerides increased only in
JAN hamsters (+51%, P < 0.01). As shown in
Figure 1
, the cholesterol distribution among the lipoproteins was similar in
both strains under basal conditions, i.e., LDL (fractions 4–12)
carried 35–40% and HDL (fractions 13–20) 55–60% of plasma
cholesterol. Feeding cholesterol increased the cholesterol in both LDL
(+168% in LPN, P < 0.0001; +300% in JAN,
P < 0.0001) and HDL (+79% in LPN, P
< 0.0001; +130% in JAN, P < 0.0001). These
effects were more marked in JAN hamsters than in LPN hamsters, as
shown by significant interactions for LDL (P = 0.0025) and HDL (P = 0.0019) The LDL cholesterol/HDL
cholesterol ratio varied with strain (higher in JAN hamsters than in
LPN hamsters, P = 0.05) and diet (increased by dietary
cholesterol, P = 0.013), without significant
interactions. After cholesterol feeding, the LDL peak in JAN hamsters
shifted toward a lower density.
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varied with strain
and the dietary conditions, due mainly to differences in the
concentration of esterified cholesterol. Due to cholesterol feeding,
these concentrations increased more dramatically in JAN hamsters than
in LPN hamsters.
A large proportion of JAN hamsters fed the commercial diet developed
pigmented gallstones (63%), an incidence that increased to 100% in
those fed cholesterol (Table 3)
. Although black gallstones were absent
in LPN hamsters fed the basal diet, they were induced in 6 of 8
hamsters in the group by cholesterol-feeding. In two cases, they
were associated with a sludge. No typical cholesterol gallstones were
found in any group. Lipid concentrations in the gallbladder bile were
higher in LPN than in JAN hamsters, whereas bile volumes were smaller
under the basal condition (P < 0.02). After
cholesterol feeding, bile volumes tended to increase in the LPN strain
(P = 0.06) without change in lipid concentrations,
whereas the cholesterol concentration increased (+130%,
P < 0.01) in JAN hamsters, leading to a 100% increase
in the CSI in this strain. Cholic (C) and chenodeoxycholic (CDC) acids
represented the major bile acids in all the groups (Table 4
). The glyco/tauroconjugation ratio was markedly higher in LPN hamsters
than in JAN hamsters. Cholesterol feeding increased the proportion of
CDC at the expense of C, leading to a greater hydrophobicity index of
the gallbladder bile. Although no significant interaction was found,
this diet effect tended to be more marked in LPN than in JAN hamsters.
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Under basal conditions, LPN hamsters had a higher HMG-CoAR
activity in both liver (+140%, P = 0.014) and
intestine (+260%, P < 0.0001) than JAN hamsters
(Table 5
). The cholesterol-rich diet decreased the liver HMG-CoAR
activity and mRNA level in the two strains, but more markedly in LPN
hamsters (to 2 and 8% of the baseline values, respectively) than in
JAN hamsters (to 15 and 24%, respectively). Intestinal HMG-CoAR
activity varied also with strain and the dietary conditions, but no
significant interaction was found. When expressed as pmol/(min · mg
protein) (results not shown), a 27% decrease (P = 0.028) in intestinal HMG-CoAR activity was induced by cholesterol
feeding in LPN hamsters. In both strains, the cholesterol challenge
reduced CYP7A activity and mRNA level and stimulated both the
CYP7B and CYP27 activities.
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) indicated that the cholesterol-rich diet significantly decreased
the amount of LDL receptor in the liver (by 53% in LPN strain and by
66% in JAN strain). No differences were observed for the amounts of
SR-BI.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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). In these two strains,
dietary cholesterol excess produces hypercholesterolemia, increases
liver cholesterol and reduces cholesterogenesis and the expression of
the LDL receptor, as reported in various species (Dueland et al. 1993
, Férézou et al. 1997
,
Kovanen et al. 1981
, Roach et al. 1993
,
Stucchi et al. 1998
) including hamsters (Spady et al. 1986
) and humans (Duane 1994
, Jones et al. 1996
). The effect of cholesterol feeding on bile acid
synthesis is more controversial and is species dependent; hamsters, in
particular, differ from rats and mice in their more limited capacity to
increase bile acid synthesis (Cai and Carr 1999
,
Turley et al. 1997
).
The present results show that the degree of the responsiveness to
dietary cholesterol depends on the hamster strain. This can be related
to the important strain differences in major parameters controlling
cholesterol metabolism under basal conditions. Cholesterolemia is lower
in LPN hamsters than in JAN hamsters fed a control or
cholesterol-enriched diet, a strain difference already found in
hamsters fed the same commercial rodent diet or a sucrose-rich
semipurified diet (Férézou et al. 2000
). In
the two strains, the cholesterol-enriched diet increased the
cholesterol content in both the LDL and HDL subfractions, as already
reported in hamsters (Woollett et al. 1997
). This
contrasts with the alterations in the lipoprotein profile usually
observed in other species after long-term cholesterol feeding
(Férézou et al. 1997
, Sérougne et al. 1995
, Stucchi et al. 1998
). Compared with
LPN hamsters, hypercholesterolemia in JAN hamsters is associated with a
modest hypertriglyceridemia, especially after cholesterol feeding,
which probably produces less dense LDL in this strain. On the basis of
a relationship between the response to dietary cholesterol and the
efficiency of cholesterol absorption (Jolley et al. 1999
), the hyperresponsiveness of JAN hamsters could also be
explained in part by a more efficient dietary cholesterol absorption in
this strain, as shown in our previous study (Férézou et al. 2000
).
Cholesterol feeding also produces a large expansion of the cholesterol
stores in the liver, leading to hepatomegaly and fatty liver only in
JAN hamsters. Interestingly, the basal activity of the liver
acylcoenzyme A cholesterol acyltransferase was found to be twice as
high in JAN hamsters compared with LPN hamsters
(Férézou et al. 2000
), which could explain
the marked strain difference in liver cholesterol storage after
long-term cholesterol feeding (Table 3)
. Moreover, a strong
increase in the biliary cholesterol concentration occurs only in JAN
hamsters, leading to a significant increase (+100%,P
< 0.01) in the CSI, whereas this parameter is stable in LPN
hamsters. Both strains displayed a marked elevation in the CDC/C ratio,
leading to a significantly greater hydrophobic index of the bile. In
spite of these biliary modifications usually associated with the
cholesterol gallstone process (Trautwein et al. 1993
),
LPN or JAN hamsters did not develop typical cholesterol gallstones
after long-term cholesterol feeding. In contrast, the formation of
pigmented stones was clearly favored in both strains. The very high
incidence of spontaneous pigmented stone formation in JAN hamsters fed
the basal diet alone makes this strain a useful model for studying the
causes of this process. The susceptibility to cholesterol gallstone
formation by LPN hamsters consuming a sucrose-rich diet is possibly
insulin-related (Férézou et al. 2000
).
These observations confirm that the incidence of diet-induced
cholesterol or pigmented gallstones depends on the strain of hamster
(Cohen et al. 1989
, Hayes et al. 1992
,
Trautwein et al. 1993
).
Compared with LPN hamsters, the higher cholesterol accumulation in both
plasma and liver in JAN hamsters cannot be related to a strain
difference in the regulation of hepatic lipoprotein receptors by
dietary cholesterol. Indeed, the expression of the HDL receptor
SR-BI was unaffected by cholesterol feeding, which confirms other
recent results in hamsters (Woollett et al. 1997
), and
the expression of the LDL receptor decreased by the same proportion in
the two strains. Therefore, the liver production of lipoproteins could
be higher in JAN than in LPN hamsters, which in turn could contribute
to the strain difference in cholesterolemia.
Measurements of HMG-CoAR activities in both the liver and intestine
indicated that basal cholesterogenesis activity was higher in LPN than
in JAN hamsters, which agrees with our previous report
(Férézou et al. 2000
). This observation
suggests that the two strains differ in their capacity to regulate
cholesterol synthesis in response to cholesterol feeding.
Interestingly, an earlier study showed that the greater the reduction
in cholesterol synthesis in humans, the less likely plasma cholesterol
was to increase in response to a high cholesterol diet (Nestel and Poyser 1976
). The present experiment shows that the
downregulation of HMG-CoAR is more efficient in LPN hamsters, which
could explain in part their greater resistance to dietary cholesterol.
The coordinate variations in both activities and mRNA levels of
HMG-CoAR in the liver suggest a transcriptional regulation of this
enzyme by cholesterol in hamsters (Spady et al. 1996
),
which contrasts with the reported regulation at the translation level
in rats (Chambers and Ness 1998
). The recent
characterization of the family of transcription factors, designated as
sterol regulatory element binding proteins (SREBP), helps to explain
the coordinate regulation of the mRNAs encoding for the multiple key
proteins involved in cell cholesterol homeostasis, including
HMG-CoAR and the LDL receptor (Horton and Shimomura, 1999
). In hamster liver, the levels of nuclear active forms of
SREBP are reduced by cholesterol feeding, explaining the reduction in
mRNAs levels for LDL receptor and HMG-CoAR (Shimomura et al. 1997
). We are currently investigating whether a difference in
the expression and/or activation of SREBP could explain why the basal
cholesterogenesis and the response to dietary cholesterol differ
between LPN and JAN hamsters.
Another mechanism that might explain the resistance to dietary
cholesterol is the increased conversion of cholesterol into bile acids.
It is now accepted that two pathways contribute to bile acid synthesis
(Princen et al. 1997
), i.e., the neutral (or classic)
pathway, initiated in the liver by CYP7A, and the acidic (or
alternative) pathway for which the key enzymes are CYP27 and CYP7B
(Schwartz et al. 1998
). The feedback control of CYP7A by
bile acids fluxing through the liver in the enterohepatic circulation
is well documented (Russell and Setchell 1992
), but the
effects of cholesterol feeding on CYP7A activity and gene transcription
vary according to the species, i.e., CYP7A activity is generally
increased in rats (Bjorkhem et al. 1991
) and mice
(Dueland et al. 1993
), whereas it is decreased in
rabbits (Xu et al. 1998
). These discrepancies are
explained in part by the different properties of the bile acids present
in the enterohepatic circulation; hydrophilic compounds are more poorly
absorbed in the gut (Schiff et al. 1972
), and only an
increased flux of hydrophobic bile acids through the hepatocytes
represses CYP7A (Vlahcevic et al. 1991
). These
differences account for the stimulation of CYP7A activity in
cholesterol-fed rats and mice, due to the reduced flux of
hydrophobic bile acids. Similarly, the downregulation of this activity
in rabbits is explained by the enlargement of the bile acid pool and
its enrichment with the hydrophobic deoxycholic acid (Xu et al. 1998
). In humans, increasing the proportion of chenodeoxycholic
acid in bile reduces CYP7A activity (Bertolotti et al. 1991
), and cholesterol feeding reduces the intestinal
absorption of cholic acid, without affecting that of chenodeoxycholic
acid, leading to an increase in the hydrophobicity index of circulating
bile acids (Duane 1994
). In the present study, CYP7A
activity decreased in hamsters after 5 wk of cholesterol consumption.
This effect tended to be stronger in LPN hamsters than in JAN hamsters
(Table 5)
, but the interaction (strain x diet) was not
significant, probably because of the limited number of hamsters in each
group. Nevertheless, the high sensitivity of the improved enzymatic
assay (Souidi et al. 1998
) probably allows this effect
to be detected, whereas no difference in CYP7A activity was reported in
cholesterol-fed hamsters, despite decreased CYP7A mRNA levels
(Horton et al. 1995
). Thus, the decreased CYP7A mRNA
levels in both the LPN and JAN strains confirm the reduction of the
CYP7A gene transcription induced by dietary cholesterol in hamsters.
The regulation of the neutral pathway of bile acid synthesis is now
better explained by the antagonistic roles of two orphan nuclear
receptors in the transcription of the CYP7A gene (Repa and Mangelsdorf 1999
). On the one hand, chenodeoxycholic acid (or
deoxycholic acid) has been identified as the preferred ligand of the
farnesoid X receptor (FXR) which, after binding with the hydrophobic
bile acid, becomes able to repress the transcription of CYP7A. On the
other hand, an isoform of the liver X receptor (LXR) has been
implicated in the induction of the transcription of CYP7A gene. This
nuclear receptor, which is activated by binding with oxysterols, is now
considered a key sensor of dietary cholesterol. However, the recent
demonstration that the activation by LXR was antagonized by FXR in
rat hepatocytes (Wang et al. 1999
) suggests that the
contribution of bile acids in the regulation of the gene encoding CYP7A
overrides that of oxysterols. This assumption is supported by comparing
the effects of long-term cholesterol feeding between the two
strains, i.e., the degree of the CYP7A inhibition (higher in LPN
hamsters, Table 5
) varies in parallel with the relative
elevation of the CDC/C ratio (higher in LPN hamsters, Table 4
).
Only few data are available concerning the effect of cholesterol
feeding on the key enzymes of the alternative pathway of bile acid
synthesis. In rats, guinea pigs and rabbits, a stimulation of CYP27
activity has been reported (Nguyen et al. 1999
). This
effect is shown also in hamsters in the present study (Table 5)
. Other
recent observations in rabbits have suggested that CYP27 is upregulated
by the pool of cholesterol stored in the liver (Xu et al. 1999
). This hypothesis is supported by comparing our two
strains of hamsters (Table 3)
. After cholesterol feeding, the CYP27
stimulation tended to be more marked in JAN hamsters than in LPN
hamsters, which store less cholesterol and also display a lower plasma
response to dietary cholesterol excess. The situation is different in
baboons because the degree of activation of the hepatic and
extrahepatic CYP27 activities modulates the responsiveness to dietary
cholesterol, even preventing the hypercholesterolemic effect of the
diet in hyporesponding baboons (Chen et al. 1998
). Our
results also show that cholesterol feeding upregulates CYP7B in the
same way as CYP27 in hamsters, whereas no modification of this
enzymatic activity was observed in cholesterol-fed mice
(Schwartz et al. 1998
). Moreover, in the latter report,
these two key enzymes in the alternative pathway were shown to be
insensitive to the enlarged pool of hydrophobic bile acids fluxing
through the liver, in contrast with CYP7A.
The present study shows that subtle differences in the regulation of cholesterol and/or bile acid synthesis may account for a different responsiveness to dietary cholesterol excess. The results show that the two pathways of bile acid synthesis are regulated independently in hamsters. It is noteworthy that our two-strain model offers an interesting analogy with the well-accepted notion of an inverse association between plasma cholesterol levels and cholesterol gallstones in humans, generally reported in epidemiologic studies. Although LPN hamsters are predisposed to sucrose-induced biliary cholesterol gallstones, they are more resistant to long-term cholesterol feeding than JAN hamsters, which are basically hypercholesterolemic with a low cholesterogenesis activity. Therefore, this JAN strain could represent a valuable experimental model with which to study the induction of aortic lesions by atherogenic diets.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: GI Division, Brigham and
Women’s Hospital, Boston MA 02215.
4 Abbreviations used: C, cholic acid; CDC,
chenodeoxycholic acid; CSI, cholesterol saturation index; CYP7A,
cholesterol 7-hydroxylase; CYP7B, oxysterol 7-hydroxylase; CYP27,
sterol 27-hydroxylase; FXR, farnesoid X receptor; HMG-CoAR,
3-hydroxy-3-methylglutaryl Coenzyme A reductase; JAN, Janvier; LPN,
Laboratoire de Physiologie de la Nutrition; LXR, liver X receptor;
RT-PCR, retrotranscription polymerase chain reaction; SR-BI,
scavenger receptor class B type 1; SREBP, sterol regulatory element
binding protein.
Manuscript received July 17, 2000. Initial review completed September 12, 2000. Revision accepted March 14, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSBiochemical assaysEnzymatic assaysImmunoassaysLiver mRNA levels of...RESULTSDISCUSSIONREFERENCES |
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