Ovariectomy Stimulates Hepatic Fat and Cholesterol Accumulation in High-fat Diet-fed Rats

Abstract

This study was designed to determine how estrogens withdrawal during a high-fat (HF) diet regimen affects liver triacylglycerol (TAG) and cholesterol accumulation. Female Sprague-Dawley rats were submitted to a HF (42% energy as fat) or a standard (SD) diet for 6 weeks before being either ovariectomized (Ovx) or sham operated (Sham). Thereafter, Ovx and Sham rats were kept on the same diet for another 6 weeks leading to euthanasia. Liver TAG content was increased (p<0.01) in Ovx rats but not by the HF diet alone. However, the combination of HF diet and Ovx resulted in a greater liver TAG accumulation (p<0.06) than that observed in Ovx-SD/SD. Measurement of molecular markers of liver lipid metabolism revealed an increase in transcripts of markers of lipid oxidation (CPT-1 and PGC1; p<0.05) in rats fed the HF diet. This increase was, however, substantially less if HF fed rats were Ovx. Liver total cholesterol levels were increased (p<0.01) only in the Ovx-HF/HF rats while plasma cholesterol levels were increased in Ovx-SD/SD and in SHAM-HF/HF and Ovx-HF/HF rats. Transcripts of molecular markers of cholesterol metabolism suggest that biliary acids synthesis (CYP7a-1) was reduced in Ovx-SD/SD and Sham-HF/HF rats and even more so in Ovx-HF/HF rats. It is concluded that the effects of a HF diet on liver TAG accumulation are especially observed in Ovx rats possibly through a reduction in hepatic lipid oxidation. The combination of Ovx and HF diet also acts synergistically to favor liver cholesterol accumulation.

Full text

283Original Basic
Ngo Sock ET et al. Liver Cholesterol in Ovariectomized Rat … Horm Metab Res 2013; 45: 283–290
received 14 . 06 . 2012
accepted 15 . 10 . 2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0032-1329964
Published online:
December 7, 2012
Horm Metab Res 2013;
45: 283–290
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0018-5043
Correspondence
Dr. J.-M. Lavoie
Département de Kinésiologie
Université de Montréal
C.P. 6128, Succ. centre-ville
Montréal
H3C 3J7 Québec
Canada
Tel.: + 1/514/343 7044
Fax: + 1/514/343 2181
jean-marc.lavoie@umontreal.ca
Key words
●
▶
Lipogenesis
●
▶
lipid oxidation
●
▶
bile acids synthesis
●
▶
cholesterol synthesis
Ovariectomy Stimulates Hepatic Fat and Cholesterol
Accumulation in High-fat Diet-fed Rats
Estrogens-defi cient state in Ovx animals has
been repeatedly shown to result in substantial
liver fat accumulation [ 7 – 9 ] . Using the Ovx
model, we reported that liver fat accumulation
was associated with changes in molecular mark-
ers that suggest an increase in the lipogenesis
pathway [ 10 ] , a reduction in lipid oxidation in
liver [ 11 ] , and a reduction in VLDL-TG production
[ 12 ] . On the other hand, nutritional manipula-
tions, especially a high-fat diet, have been repeat-
edly associated with the short-term development
of a state of hepatic steatosis [ 13 , 14 ] . We recently
conducted 2 time course studies that revealed a
rapid infi ltration of lipids (2 weeks) followed by a
substantial decrease in the following weeks
(2–16 weeks), which was not coupled to fat mass
gain [ 15 , 16 ] . On the opposite, Paquette et al. [ 7 ]
reported a progressive increase in liver lipid infi l-
tration after 3, 8, and 13 weeks in Ovx rats fed a
HF diet. It was hypothesized that the absence of a
normal estrogenic status synergistically favors
Introduction
▼
Postmenopausal women are particularly inclined
to the development of a state of hepatic steatosis
[ 1 ] . Following menopause, nonalcoholic liver stea-
tosis is more common in women than in men and
twice as common in postmenopausal compared
to premenopausal women [ 2 , 3 ] . The importance
of this phenomenon is enlightened by the fact that
nonalcoholic fatty liver disease (NAFLD) is becom-
ing a risk factor for diabetes and cardiovascular
diseases independently of usual risk factors [ 4 , 5 ] .
At the present time, there are no specifi c or eff ec-
tive pharmacological treatments for NAFLD, and
lifestyle modifi cations such as healthy nutrition
and regular exercise are regarded as fi rst line
treatments [ 6 ] . Menopause is, therefore, a period
in women’s life where understanding the interac-
tions between the progressive decrease in estro-
gens content and nutritional status, especially the
fat intake, might be critical to prevent the develop-
ment of hepatic steatosis.
Authors E. T. Ngo Sock
1 , I. Côté
1 , J. S. Mentor
1 , D. Prud’homme
2 , R. Bergeron
1 , J.-M. Lavoie
1
Affi liations
1 Department of Kinesiology, Université de Montréal, Montréal, Québec, Canada
2 Faculty of Health Sciences, School of Human Kinetics, University of Ottawa, Ottawa, Ontario, Canada
Abstract
▼
This study was designed to determine how estro-
gens withdrawal during a high-fat (HF) diet regi-
men aff ects liver triacylglycerol (TAG) and
cholesterol accumulation. Female Sprague-Daw-
ley rats were submitted to a HF (42 % energy as
fat) or a standard (SD) diet for 6 weeks before
being either ovariectomized (Ovx) or sham oper-
ated (Sham). Thereafter, Ovx and Sham rats were
kept on the same diet for another 6 weeks lead-
ing to euthanasia. Liver TAG content was
increased (p < 0.01) in Ovx rats but not by the HF
diet alone. However, the combination of HF diet
and Ovx resulted in a greater liver TAG accumula-
tion (p < 0.06) than that observed in Ovx-SD/SD.
Measurement of molecular markers of liver lipid
metabolism revealed an increase in transcripts of
markers of lipid oxidation (CPT-1 and PGC1;
p < 0.05) in rats fed the HF diet. This increase was,
however, substantially less if HF fed rats were
Ovx. Liver total cholesterol levels were increased
(p < 0.01) only in the Ovx-HF/HF rats while
plasma cholesterol levels were increased in Ovx-
SD/SD and in SHAM-HF/HF and Ovx-HF/HF rats.
Transcripts of molecular markers of cholesterol
metabolism suggest that biliary acids synthesis
(CYP7a-1) was reduced in Ovx-SD/SD and Sham-
HF/HF rats and even more so in Ovx-HF/HF rats.
It is concluded that the eff ects of a HF diet on
liver TAG accumulation are especially observed
in Ovx rats possibly through a reduction in
hepatic lipid oxidation. The combination of Ovx
and HF diet also acts synergistically to favor liver
cholesterol accumulation.
Supporting Information for this article ( Fig. 1S ,
Table 1S , and Table 2 S ) is available online at http:/
www.thieme-connect.de/ejournals/toc/hmr
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284 Original Basic
Ngo Sock ET et al. Liver Cholesterol in Ovariectomized Rat … Horm Metab Res 2013; 45: 283–290
fat accumulation in liver when combined to HF feeding. This last
study, however, suff ers from the absence of a Sham group fed a
HF diet and a lack of documentation on possible mechanisms
substantiating this hypothesis. The present study was designed
to test the hypothesis that ovariectomizing rats in the middle of
a 13-week period of HF feeding will potentiate the eff ects of a HF
feeding, thus resulting in a higher liver fat accumulation. Fur-
thermore, we measured hepatic transcripts of molecular mark-
ers involved in lipogenesis, fat oxidation, and VLDL-TG
production in an attempt to determine which pathway is respon-
sible for the postulated higher fat accumulation in Ovx HF fed
rats [ 10 , 12 ] .
In addition to liver fat accumulation, liver cholesterol accumula-
tion with estrogens withdrawl is also a concern that has not
received a great deal of attention. Plasma cholesterol levels have
been reported to be increased in Ovx animals [ 17 ] . However, it is
not well established if estrogens withdrawal is associated with
an accumulation of cholesterol in liver and if a high-fat diet may
worsen this situation. We, therefore measured hepatic and
plasma levels of cholesterol along with transcripts of key mark-
ers of cholesterol metabolism including cholesterol
7α-hydroxylase (Cyp7a-1), the enzyme that catalyses the con-
version of cholesterol into 7α-hydroxycholesterol before its
transformation into bile acids, the fi rst step for cholesterol dis-
posal in mammals; 3-hydroxy-3-methylglutaryl-CoA reductase
(HMG-CoA-r), the main enzyme of cholesterol synthesis; and
ATP-binding cassette, subfamily G, member 5 and 8 (ABCG5,
ABCG8) that export cholesterol and other sterols from hepato-
cytes to the bile duct.
Materials and Methods
▼
Animal care and ethics statement
Female Sprague-Dawley strain rats (n = 32; Charles River, St Con-
stant, PQ, Canada), weighing 180–200 g (6 weeks of age) upon
their arrival were housed individually and had ad libitum access
to food and tap water. Their environment was controlled in
terms of light (12-h light-dark cycle starting at 06:00 AM),
humidity, and room temperature (20–23 °C). All experiments
described in this report were conducted according to the direc-
tives of the Canadian Council on Animal Care after institutional
approval (Comité de Déontologie de l’expérimentation sur les
animaux 11-082).
Diet protocol and surgery
Two days after their arrival in our laboratory, rats were randomly
divided into 2 groups: standard diet (SD) feeding (n = 16) and
high fat (HF) feeding (n = 16;
●
▶
Fig. 1S ). The HF diet was provided
in small pellets (42 % fat; 36 % CHO, 22 % protein; gross energy kJ,
ICN Pharmaceutical, NY, USA). The standard diet (12.5 % lipid,
63.2 % CHO, and 24.3 % protein; gross energy kJ) consisted of usual
pellet rat chow (Agribrands Purina Canada, Woodstock, Ontario,
Canada) [ 7 ] . Details of the diets are presented in
●
▶
Table 1S .
6 weeks after being submitted to the SD or the HF diet, each
group was randomly subdivided into 2 subgroups: sham-oper-
ated (Sham) or ovariectomized (Ovx), and maintained on their
respective diet for another 6 weeks (
●
▶
Fig. 1S ). Altogether, there
were 4 groups of 8 rats: Sham-SD/SD, Ovx-SD/SD, Sham-HF/HF,
and Ovx-HF/HF. Ovariectomy was conducted according to the
technique described by Robertson et al. [ 18 ] under isofl urane
Table 1 Anthropometric and plasma metabolic variables.
Sham-SD/SD Ovx-SD/SD Sham-HF/HF Ovx-HF/HF
Body weight (g) 332.7 ± 15.6 391.5 ± 7.8** 365.7 ± 7.1 416.3 ± 13.3**
Energy intake (kcal/day) (measured after the Ovx) 73.3 ± 1.3 81.2 ± 2.7* 65.7 ± 1.17 + 72.0 ± 2.58* +
Sum of 3 intra-abdominal fat pad weights (g) 30.8 ± 2.8 33.5 ± 2.6 35.8 ± 2.9 + 44.1 ± 3.3* + (* = 0.07)
Subcutaneous fat pad weight (g) 1.52 ± 0.18 2.39 ± 0.33* 2.42 ± 0.24 + 3.48 ± 0.44* +
Uterus weight (mg) 552 ± 58 180 ± 35*** 686 ± 70 127 ± 8***
Sum of 3 leg muscles weights (g) 2.1 ± 0.04 2.49 ± 0.07*** 2.2 ± 0.05 2.5 ± 0.06***
TAG (g/l) 1.26 ± 0.12 1.29 ± 0.19 1.85 ± 0.25 1.00 ± 0.14*
Glycerol (mg/l) 16.9 ± 1.68 19.2 ± 1.7 31.5 ± 4.2 + + 25.55 ± 2.62 + +
FFA (mmol/l) 0.51 ± 0.01 0.52 ± 0.01 0.54 ± 0.01 + + 0.56 ± 0.01 + +
Values are mean ± SE with n = 7–8 rats per group. TAG: triacylglycerol. FFAs: free fatty acids
* Signifi cantly diff erent from surgery counterparts p < 0.05; **p < 0.01; ***p < 0.001
+ Signifi cantly diff erent from diet counterparts p < 0.05;
+ + p < 0.01; + + + p < 0.001
Fig. 1 Changes in hepatic triacylglycerols (TAG) content in response to
high-fat (HF) diet and ovariectomy (Ovx). Biochemical a and histological
b analyses revealed that liver TAG content was increased following the
Ovx but not by the HF diet. However, the combination of Ovx and the
HF diet resulted in a larger (p < 0.06) liver TAG accumulation than that
observed in Ovx SD/SD. Values are mean ± SE with n = 7–8 rats per group.
** Signifi cantly diff erent from surgery counterparts p < 0.01;
+ signifi -
cantly diff erent from diet counterparts p < 0.05.
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285Original Basic
Ngo Sock ET et al. Liver Cholesterol in Ovariectomized Rat … Horm Metab Res 2013; 45: 283–290
anesthesia. Animals were injected with antibiotics (Tribrissen
24 %; 0.125 cc/kg, sc) for 3 days, beginning the day before sur-
gery. Details of the surgery have been previously described [ 19 ] .
Body weight and food intake were monitored twice a week in all
rats.
Sacrifi ce
Rats were euthanized between 09:00 and 12:00 AM. Food was
removed from the cage 2–3 h before sacrifi ce. Immediately after
complete anesthesia with isofl urane, the abdominal cavity was
opened following the median line of the abdomen and approxi-
mately 5 ml of blood was collected from the abdominal vena
cava into syringes pretreated with ethylenediaminetetraacetic
acid (EDTA; 15 %). Blood was centrifuged (3 000 rpm; 4 °C;
10 min; Beckman GPR Centrifuge) and the plasma kept for fur-
ther analyses. Several organs and tissues were removed in the
following order: liver, uterus, mesenteric, urogenital, retroperi-
toneal, and subcutaneous fat depots along with 4 skeletal mus-
cles of the right limb (soleus, plantaris, medial gastrocnemius,
and lateral gastrocnemius). All tissue samples were frozen in liq-
uid nitrogen immediately after being weighed (Mettler AE 100).
The liver median lobe was freeze-clamped and used for triacylg-
lycerol (TAG) and mRNA determinations. The mesenteric fat pad
consisted of adipose tissue surrounding the gastrointestinal
tract from the gastroesophageal sphincter to the end of the rec-
tum, with special care taken in distinguishing and removing
pancreatic cells. The urogenital fat pad included adipose tissue
surrounding the kidneys, ureters, and bladder as well as ovaries,
oviducts, and uterus. The retroperitoneal fat pad was taken as
that distinct deposit behind each kidney along the lumbar mus-
cles. For subcutaneous fat deposit measurement, a rectangular
piece of skin was taken on the right side of each animal, from the
median line of the abdomen to the spine and the right hip to the
fi rst rib, as described by Krotkiewski and Bjorntorp [ 20 ] . All rats
were visually inspected for presence or not of ovaries, and uterus
were excised and weighed to confi rm ovariectomy. All tissue
samples were stored along with plasma samples at − 78 °C until
analyses were performed.
Biochemical analyses
Plasma free fatty acid (FFA) and TAG concentrations were deter-
mined with an enzymatic colorimetric assay available from
Roche Diagnostics (Mannheim, Germany) and Sigma (Saint
Louis, Missouri, USA), respectively. Commercial kits from Sigma
(Sigma; St-Louis, Missouri, USA) were used to determine plasma
glycerol and TAG and liver TAG by colorimetric method. Liver
TAG concentrations were estimated from glycerol released after
KOH hydrolysis. The method described by Folch [ 21 ] was used to
extract hepatic total lipids content. Briefl y, 0.1 g of liver was
homogenized with chloroform-methanol mixture (2:1, v/v). The
chloroform layer was collected and evaporated overnight. After
adding 10 % Triton X-100 in isopropanol, the sample was assayed
for total cholesterol using commercial kits according to the man-
ufacturer’s instructions (Wako Diagnostics and Chemicals USA,
Richmond, VA, USA). Plasma total cholesterol was determined
using the same kits supplied by Wako.
RNA isolation and quantitative real-time (RT)
Polymerase Chain Reaction (PCR) RNA extraction and
cDNA preparation
Quick-frozen tissue samples of liver were powdered with cold
mortar and pestle, and ~100 mg was used for the isolation of
RNA. Total RNA was extracted by the guanidine thiocyanate
method and mRNA purifi ed using PureLink RNA Mini Kit (Invit-
rogen) according to the manufacturer’s instruction. Total RNA
was reverse transcribed in a fi nal volume of 20 μl using high
capacity cDNA reverse transcription kit with random primers
(Applied Biosystems, Foster City, CA, USA) as described by the
manufacturer. Reverse transcribed samples were stored at
–20 °C.
qPCR reactions – Taqman
® Gene Expression Assays –
endogenous controls
Gene expression level for endogenous controls was determined
using prevalidated Taqman Gene Expression Assays (Applied
Biosystems). qPCR reactions for 384-well plate formats were
performed using 2 μl of cDNA samples (5–25 ng), 5 μl of the Fast
Universal qPCR MasterMix (Applied Biosystems), 0.5 μl of the
TaqMan Gene Expression Assay (20 × ), and 2.5 μl of water in a
total volume of 10 μl. The following assays were used as endog-
enous controls: Hprt1 (Rn01527840) and Actb (Rn00667869).
qPCR reactions – Universal Probe Library (UPL) assays
Gene expression level for target genes was determined using
assays designed with the Universal Probe Library from Roche.
qPCR reactions for 384-well plate formats were performed using
2 μl of cDNA samples (5–25 ng), 5 μl of the Fast Universal qPCR
MasterMix (Applied Biosystems), 2 μM of each primer, and 1 μM
of a UPL probe in a total volume of 10 μl. The primer sets and
probe numbers used to generate amplicons are presented
in
●
▶
Table 2S .
Detection and analysis
The ABI PRISM ® 7900HT Sequence Detection System (Applied
Biosystems) was used to detect the amplifi cation level and was
programmed with an initial step of 3 min at 95 °C, followed by
40 cycles of: 5 s at 95 °C and 30 s at 60 °C. All reactions were run
in triplicate and the average values of Cts were used for quantifi -
cation. Hprt1 and Actb were used as endogenous control. The
relative quantifi cation of target genes was determined using the
ΔΔCT method. Briefl y, the Ct (threshold cycle) values of target
genes were normalized to an average Ct of both Hprt1 and Actb
(ΔCT = Ct target –Ct endo ) and compared with a calibrator:
ΔΔCT = ΔCt
sample – ΔCt
calibrator . Relative expression (RQ) was calcu-
lated using the formula is RQ = 2
–ΔΔ
CT
with the help of SDS2.2.2
and Data Assist 3.0 software (Applied Biosystems).
Statistical analysis
Values are expressed as mean ± S.E. Statistical analysis were per-
formed using a 2-way ANOVA for nonrepeated measures using
surgery and diet as main eff ects. Fisher’s post-hoc test was used
in the event of a signifi cant (p < 0.05) F ratio.
Results
▼
Body weight measured at the end of the experiment was higher
(p < 0.01) in Ovx compared to Sham rats in both dietary groups
(
●
▶
Table 1 ). Even though the HF diet seems to increase body
weight, the increase did not reach the signifi cant level (p = 0.07).
Energy intake measured during the 6 weeks after surgery was
higher (p < 0.05) in the Ovx groups but lower (p < 0.05) in rats
submitted to the HF diet. A gain in intra-abdominal fat mass was
observed following the HF diet in both Sham and Ovx rats
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286 Original Basic
Ngo Sock ET et al. Liver Cholesterol in Ovariectomized Rat … Horm Metab Res 2013; 45: 283–290
(p < 0.05), whereas an eff ect of Ovx was observed (p = 0.07) only
in the Ovx-HF/HF group but not in the Ovx-SD/SD group of rats.
A gain in subcutaneous fat was also observed in Ovx and HF fed
rats (p < 0.05) and even more so in the Ovx HF/HF animals (syn-
ergic eff ects between HF diet and Ovx). Uterus weight was sig-
nifi cantly (p < 0.001) lower in all Ovx rats confi rming total
ovariectomy. The sum of 3 leg muscle weights was signifi cantly
(p < 0.001) higher in Ovx groups independently of the diet
(
●
▶
Table 1 ). Plasma TAG concentrations were signifi cantly
(p < 0.05) lower in the Ovx-HF/HF compared to Sham-HF/HF
group of rats. Plasma FFA and glycerol concentrations were sig-
nifi cantly (p < 0.01) higher with the HF diet in both Sham and
Ovx rats (
●
▶
Table 1 ) .
Liver TAG contents were signifi cantly higher (p < 0.01) in both
Ovx groups compared to respective Sham rats (
●
▶
Fig. 1 ). Even
though the diet × surgery interaction was signifi cant only at
p = 0.07 (F = 3.51) direct comparisons revealed that the combina-
tion of Ovx and the HF diet resulted in a higher liver TAG accu-
mulation (p < 0.06) than those observed in Ovx SD/SD [mean
diff erence (SD): − 13.1 (4.9) with a 95 % CI of − 26.7 to 0.41]. This
suggests a synergetic eff ect between the ovariectomy and the HF
diet. Similar results were observed with the histological sections
analysis of liver (
●
▶
Fig. 1b ). To explore potential mechanisms,
we measured gene expression of 2 of the main transcription fac-
tors involved in the lipogenic pathway. We found that SREBP-1c
mRNA levels were increased (p < 0.01) by the HF diet while
ChREBP mRNA levels were signifi cantly (p < 0.05) increased by
the Ovx and the HF diet in Sham rats (
●
▶
Fig. 2 ). There was no
synergistic eff ect on SREBP-1c and ChREBP mRNAs by the Ovx-
HF diet combination.
The HF diet in Sham rats resulted in increased (p < 0.05) expres-
sion of CPT-1 and PGC-1, 2 genes involved in mitochondrial oxi-
dative capacity (
●
▶
Fig. 3 ). Even though the diet × surgery
interactions for CPT-1 and PGC-1 were signifi cant only at the
level of p = 0.1, there was a strong tendency for a reduction of HF
diet-induced increase in these genes expression if rats were Ovx.
Direct comparisons between Sham-SD/SD and Sham-HF/HF
revealed a mean diff erence (SD) of − 3.04 (0.6) with a 95 % CI
of − 4.8 to − 1.28 for CPT-1 and − 1.1 (0.4) with a 95 % CI of − 2.1
to − 0.99 for PGC-1. By comparisons, Sham-SD/SD vs. Ovx-HF/HF
revealed a mean diff erence (SD) of − 1.7 (0.6) with a 95 % CI
of − 3.4 to 0.04 for CPT-1 and − 0.2 (0.4) with a 95 % CI of − 1.2 to
0.8, for PGC-1. These comparisons strongly suggest that indeed
withdrawal of estrogens decreased the HF-induced expression
of the lipid oxidation pathway.
To evaluate the possibility that VLDL production may be aff ected
by the HF/Ovx manipulations, we measured gene expression of
diacylglycerol acyltransferase-2 (DGAT-2) and microsomal trig-
lyceride transfer protein (MTP), 2 genes involved in VLDL syn-
thesis. We found a tendency for a decrease in MTP mRNA
(p < 0.08) in both groups of Ovx rats (
●
▶
Fig. 4 ). Similarly to the
molecular markers of the oxidative pathway, we found an
increase (p < 0.05) in DGAT-2 mRNA with the HF diet in Sham
rats that was signifi cantly (p < 0.01) decrease in Ovx-HF/HF ani-
mals (
●
▶
Fig. 4 ). This suggests a decrease in VLDL production in
Ovx rats that was particularly observed in Ovx rats fed a HF diet.
Fig. 2 Gene expressions of hepatic lipogenesis markers. Although
expressions of sterol regulatory element binding transcription factor 1
(SREBP-1c) mRNA was stimulated by the high fat (HF) diet and carbohy-
drate-responsive element-binding protein (ChREBP) mRNA expression
was stimulated by the Ovx and the HF diet, none of these responses were
potentialized by the Ovx-HF diet combination. Values are mean ± SE with
n = 7–8 rats per group.
+ Signifi cantly diff erent from diet counterparts
p < 0.05; + + p < 0.01; * signifi cantly diff erent from surgery counterparts
p < 0.05. Diet × surgery interaction for ChREBP: F = 5.55, p < 0.02.
Fig. 3 Gene expressions of key molecular markers of hepatic lipid oxidation.
Gene expression of carnitine palmitoyl transferase family member (CPT-1),
peroxisome proliferative activated receptor gamma coactivator 1 (PGC-1)
and peroxisome proliferator activated receptor alpha (PPAR-α). CPT-1 and
PGC-1 were increased in HF fed animals and this increase was somewhat at-
tenuated if rats were Ovx. Values are mean ± SE with n = 7–8 rats per group.
+
Signifi cantly diff erent from diet counterparts p < 0.05;
+ + p < 0.01.
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287Original Basic
Ngo Sock ET et al. Liver Cholesterol in Ovariectomized Rat … Horm Metab Res 2013; 45: 283–290
To evaluate if cholesterol metabolism is aff ected by the Ovx/HF
manipulations, we measured and found that liver cholesterol
accumulation was signifi cantly higher (p < 0.05) in the Ovx-HF/
HF animals, while plasma cholesterol concentrations were
increased (p < 0.05) by both the Ovx and the HF diet and by the
combination of the Ovx and the HF diet (
●
▶
Fig. 5 ). Furthermore,
we measured key molecular markers of liver cholesterol metab-
olism. HMGCoA-r mRNA the key enzyme of cholesterol synthe-
sis was signifi cantly decreased (p < 0.01) by Ovx independently
of the diet (
●
▶
Fig. 6a ). The transporter ABCG5 mRNA was
increased (p < 0.05) by the HF diet in Sham and Ovx rats, while
ABCG8 mRNA was not aff ected by the diet or the surgery (
●
▶
Fig.
6b, c ). Finally, Cyp7a-1 mRNA, a key enzyme of biliary acids syn-
thesis, was decreased in Ovx (p < 0.05) and Sham-HF/HF fed rat
(p < 0.001) which decrease was more accentuated (p < 0.05) in
OVX-HF/HF rats (
●
▶
Fig. 6d ) .
Discussion
▼
The main fi nding of the present study is that the combination of
Ovx and the HF diet signifi cantly increased liver TAG and choles-
terol accumulation. The large increase in TAG indicates that liv-
ers of Ovx rats were unable to properly manage the increase in
nutritional fat intake so as to keep TAG accumulation to similar
level found in Ovx rats fed the SD diet. On the opposite, Sham
rats fed the HF diet for 13 weeks were able to cope with the HF
feeding so that liver TAG accumulation was comparable to the
level observed in Sham rats fed the SD diet. Similar fi ndings have
been previously reported after 8 and 16 weeks of HF feeding
(40 % kcal) in rats [ 15 , 16 ] . In these previous studies [ 15 , 16 ] , liver
TAG accumulation was increased during the fi rst few wk of HF
feeding but then decreased between the 2
nd
and 6
th
weeks and
remained low for another 6–8 weeks, suggesting an adaptation
to the HF diet. On the opposite when Ovx rats were submitted to
the same HF diet, liver kept on accumulating TAG over a 16-week
period [ 7 ] . Based on previous [ 15 , 16 ] and the present fi ndings, it
seems, therefore, that rats fed a HF (40 %, kcal) diet develop
mechanisms that allow them to limit liver TAG accumulation
over a certain time (6–16 weeks). Ovx rats, on the other hand,
seem unable to properly activate these adaptation mechanisms
over the same time, thus potentializing the HF diet eff ect on liver
TAG accumulation. Even though it might still be argued that the
present HF diet should have resulted in some accumulation of
liver TAG, as reported in some studies using diff erent nutritional
fat content (i. e., 60 % kcal from fat) [ 22 ] and durations [ 23 ] , it is
clear that a HF diet in estrogens withdrawal OVX rats largely
favors hepatic fat accumulation.
It might be argued that the higher liver fat accumulation found
in Ovx-HF/HF rats compared to Sham-HF/HF rats might be
explained by the higher energy intake in the former. This inter-
pretation is, however, largely tempered by the fact that energy
intake was lower in Ovx-HF/HF than in Ovx-SD/SD rats while
liver TAG accumulation was higher in the former. Alternatively,
Ovx-HF/HF rats show the highest intra-abdominal and subcuta-
neous fat pad weights. This is, at fi rst glance, a further indication
that Ovx animals were unable to manage increased nutritional
fat intake as well as Sham rats. It is well documented that fat
accumulation in adipose tissue is associated with a higher lipol-
ysis rate, thus providing more circulating free fatty acids to be
taken up by the liver [ 24 ] . This might have contributed to the
Fig. 4 Hepatic gene expressions of molecular markers of VLDL synthesis.
A tendency for a decrease (p < 0.08) in microsomal triglyceride transfer
protein (MTP) mRNA in both groups of Ovx rats is observed while the
increase in diacylglycerol acyltransferase-2 (DGAT-2) mRNA observed with
the HF diet was attenuated if the rats were Ovx levels. Values are mean ±
SE with n = 7–8 rats per group. ** Signifi cantly diff erent from surgery
counterparts p < 0.01; + signifi cantly diff erent from diet counterparts
p < 0.05. Diet × surgery interaction for DGAT-2: F = 4.16, p < 0.05.
Fig. 5 Hepatic and plasma total cholesterol in response to ovariectomy
(Ovx) and high fat (HF) diet. Liver total cholesterol content was signifi -
cantly increased only in the Ovx-HF/HF rats while plasma total cholesterol
levels were increased in Ovx and HF fed animals and even more so in
Ovx-HF/HF rats. Values are mean ± SE with n = 7–8 rats per group.
+ + Sig-
nifi cantly diff erent from diet counterparts p < 0.01; * signifi cantly diff erent
from surgery counterparts p < 0.05; **p < 0.01. Diet × surgery interaction
for liver cholesterol: F = 5.27, p < 0.03.
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288 Original Basic
Ngo Sock ET et al. Liver Cholesterol in Ovariectomized Rat … Horm Metab Res 2013; 45: 283–290
higher liver fat accumulation in Ovx-HF/HF rats. Although this
possibility cannot be ruled out, plasma free fatty acids and glyc-
erol concentrations measured in Ovx and Sham rats fed the HF
diet do not support this interpretation. On the whole, it appears
that although an increased arrival of lipids constitutes a plausi-
ble explanation to the higher liver fat accumulation in Ovx rats
fed the HF diet compared to the Sham rats fed the HF diet, it can
hardly constitute the sole explanation.
A possible explanation for the higher liver TAG accumulation in
Ovx-HF/HF animals is an increase in the lipogenesis pathway.
Lipogenesis may be stimulated through the activation of the
SREBP-1c and/or the ChREBP transcription factors [ 25 ] . Although
gene expression of both of these transcription factors was
increased by the HF feeding, the response was similar in both
Sham and Ovx rats fed the HF diet. These results, therefore, do
not provide any evidence that an increased lipogenesis is respon-
sible for the synergetic eff ect of Ovx and the HF diet on the
development of hepatic steatosis in Ovx-HF/HF rats. On the
other hand, we found a strong tendency for key markers of lipid
oxidation in liver, PGC1 transcription factor and CPT-1 enzyme,
to have their mRNA expressed at higher level in Sham than in
Ovx rats fed the HF diet, both of these groups having higher
expression than the SD fed rats. This suggests that Sham rats fed
the HF diet were able to cope with the increased dietary lipids by
activating the oxidative pathway in liver. Indeed, increased
hepatic fat oxidation has been reported to be triggered by diff er-
ent mechanisms including increased liver TAG accumulation
and an induction of CPT1 [ 26 ] . In contrast, Ovx rats fed the HF
diet in the present study might have been unable to activate the
hepatic oxidative pathway to the same level as the Sham-HF/HF
rats thus resulting in higher liver fat accumulation. This inter-
pretation is supported by previous physiological and molecular
fi ndings indicating that indeed fat oxidation is reduced by estro-
gens withdrawal [ 10 , 11 , 27 ] . On the whole, the present molecu-
lar results suggest that a lack of proper activation of the
mechanisms involved in lipid oxidation in Ovx animals fed a HF
diet is one of the factors explaining the high level of liver fat
accumulation in these animals.
Besides a lack of increase in liver lipid oxidation, a decrease in
lipids exportation via VLDL-TAG production might also contrib-
ute to the high liver fat accumulation in Ovx-HF/HF fed rats. A
decline in VLDL-TAG production has been reported in estrogens-
defi cient animals [ 12 , 28 ] . DGAT-2 mRNA, the enzyme that cata-
lyzes the last step of the synthesis of TAGs that are going to be
incorporated into VLDL [ 29 ] , was stimulated by the HF diet but
reduced if the HF fed rats were Ovx. This suggests that one of the
adaptations to the HF diet in Sham rats is a programming for
enhanced lipid exportation from the liver, which by being atten-
uated in Ovx rats might contribute to the higher liver TAG accu-
mulation in the latter. In this regard, it is interesting to observe
that plasma TAG concentrations were lower and liver TAG con-
tents were greater in Ovx-HF/HF as compared to Sham-HF/HF
rats. This mirror image between liver and plasma TAG in rats has
been observed in other studies and has been associated with a
Fig. 6 Hepatic gene expressions of markers of biliary acids (CYP7A1), and cholesterol synthesis (HMGCoA-r) and cholesterol secretion (ABCG5/ABCG8).
CYP7a-1 transcripts was reduced by the Ovx and the HF diet and even more so in Ovx-HF/HF rats while HMGCoA-r was reduced in Ovx animals only. Liver
ABCG5 mRNA levels were increased by the HF diet, but no signifi cant change was observed with ABCG8 mRNA. Values are mean ± SE with n = 7–8 rats per
group. *Signifi cantly diff erent from surgery counterparts p < 0.05; **p < 0.01;
+ signifi cantly diff erent from diet counterparts p < 0.05;
+ + p < 0.01.
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289Original Basic
Ngo Sock ET et al. Liver Cholesterol in Ovariectomized Rat … Horm Metab Res 2013; 45: 283–290
decrease in VLDL-TAG production in Ovx rats [ 12 ] . This interpre-
tation must, however, be tempered by the fi nding that no diff er-
ence in MTP transcripts was found among all groups of rats.
Nevertheless, the present results suggest that in addition to a
decrease in liver lipid oxidation a decrease in VLDL-TAG produc-
tion may also constitute an explanation to the higher liver TAG
accumulation in Ovx-HF/HF rats.
Besides an increase in fat accumulation, the present study is the
fi rst, to our knowledge, to report an accumulation of cholesterol
in liver of Ovx rats submitted to a high-fat diet. It is well docu-
mented that increasing fat intake increases the risk of develop-
ing hypercholesterolemia [ 29 ] . An increase in plasma cholesterol
levels was also reported in Ovx rats [ 17 ] . The results of the
present study confi rm these fi ndings. Hepatic cholesterol levels,
on the other hand were not signifi cantly changed by the Ovx and
the HF diet alone. Liver total cholesterol level was reported not
to be aff ected by estrogens withdrawal in previous studies
[ 17 , 30 ] . Interestingly, however, large cholesterol accumulation
was found in liver of Ovx rats fed the HF diet. This synergistic
eff ect indicates a state of vulnerability to cholesterol accumula-
tion in Ovx animals when fed a HF diet suggesting an interaction
between liver fat and cholesterol accumulation in liver. Choles-
terol homeostasis depends on cholesterol synthesis, uptake, and
clearance. The maintenance of close to normal hepatic choles-
terol level in Ovx rats is interesting especially in view of the
decrease in HMGCoA-r and CYP7A1 transcripts, suggesting a
reduction in both cholesterol synthesis and clearance. Reduced
HMGCoA mRNA secondary to Ovx has been reported in other
studies [ 17 , 31 ] . Maintenance of hepatic cholesterol levels
despite higher total plasma cholesterol and reduced de novo
cholesterol synthesis in Ovx animals might suggest a decreased
cholesterol uptake by hepatic LDL receptors [ 30 ] . On the other
hand, conversion of cholesterol to bile acids is the major path-
way of cholesterol elimination and accounts for about 50 % of
daily cholesterol excretion [ 32 ] . The decrease in gene expression
of CYP7A1 that we found with the HF diet as well as with the
Ovx suggests that the elimination of cholesterol via bile acids
formation was reduced and might have contributed to the
hypercholesterolemic state associated with both of these condi-
tions [ 30 , 33 ] . In this regard, it is interesting to note that the
opposite, that is estrogens treatment, has been reported to result
in an increase in biliary cholesterol hypersecretion in mice [ 34 ] .
In the same vein, gene expression of ABCG5 and ABCG8 trans-
porters known to export cholesterol from the liver to the bile
ducts was unchanged by Ovx as observed in the SD diet condi-
tion. This is in line with prior observations made in aromatase
knockout mice showing that ABCG5 and ABCG8 do not appear to
be regulated by estrogens [ 35 ] . On the other hand, liver ABCG5
mRNA was increased by the HF diet. High-fat and high-sucrose
diets, known to cause an accumulation of lipids in liver have
been reported to increase the protein levels of ABCG5 and ABCG8
[ 36 ] . The impact of the present increase in ABCG5 with the HF
diet is, however, limited by the fact that ABCG8 gene expression
was not changed and based on results obtained in vitro and in
vivo, ABCG5 and ABCG8 are half-transporters proposed to func-
tion as obligate heterodimers [ 37 ] . On the whole, the present
results regarding the hepatic cholesterol metabolism indicate
that associated with a reduction in cholesterol and biliary acids
synthesis liver cholesterol level remained in normal range in
Ovx rats. Further studies are needed to better understand how
hepatic cholesterol metabolism is aff ected by estrogens with-
drawal.
In summary, results of the present study indicate that Ovx and
the feeding of a HF diet over a 13-week period act synergistically
in rats and result in an important liver fat and cholesterol accu-
mulation. On a clinical point of view, the present results suggest
that diets rich in fat are particularly deleterious after menopause
and that post-menopausal women must be encouraged to
reduce their daily fat intake.
Acknowledgements
▼
This work was supported by grants from the Natural Sciences
and Engineering Research Council of Canada (7594) and from
the Canadian Institutes of Health Research (JML; T 0602 145.02).
Confl ict of Interest
▼
The authors do not have any confl ict of interest.
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