Supplemental Material can be found at:

http://jn.nutrition.org/content/suppl/2012/03/20/jn.111.15319
7.DC1.html
The Journal of Nutrition
Nutrition and Disease

High-Fat and Fructose Intake Induces Insulin

Resistance, Dyslipidemia, and Liver Steatosis
and Alters In Vivo Macrophage-to-Feces Reverse
Cholesterol Transport in Hamsters1–3
François Briand,* Quentin Thiéblemont, Elodie Muzotte, and Thierry Sulpice

Physiogenex SAS, Prologue Biotech, Rue Pierre et Marie Curie, Labège-Innopole, France

Abstract
Reverse cholesterol transport (RCT) promotes the egress of cholesterol from peripheral tissues to the liver for biliary and
fecal excretion. Although not demonstrated in vivo, RCT is thought to be impaired in patients with metabolic syndrome, in
which liver steatosis prevalence is relatively high. Golden Syrian hamsters were fed a nonpurified (CON) diet and normal

Downloaded from jn.nutrition.org by guest on June 7, 2015

drinking water or a high-fat (HF) diet containing 27% fat, 0.5% cholesterol, and 0.25% deoxycholate as well as 10%
fructose in drinking water for 4 wk. Compared to CON, the HF diet induced insulin resistance and dyslipidemia, with
significantly higher plasma non-HDL–cholesterol concentrations and cholesteryl ester transfer protein activity. The HF diet
induced severe liver steatosis, with significantly higher cholesterol and TG levels compared to CON. In vivo RCT was
assessed by i.p. injecting 3H-cholesterol labeled macrophages. Compared to CON, HF hamsters had significantly greater
3
H-tracer recoveries in plasma, but not HDL. After 72 h, 3H-tracer recovery in HF hamsters was 318% higher in liver and
75% lower in bile (P , 0.01), indicating that the HF diet impaired hepatic cholesterol fluxes. However, macrophage-derived
cholesterol fecal excretion was 45% higher in HF hamsters than in CON hamsters. This effect was not related to intestinal
cholesterol absorption, which was 89% higher in HF hamsters (P , 0.05), suggesting a possible upregulation of
transintestinal cholesterol excretion. Our data indicate a significant increase in macrophage-derived cholesterol fecal
excretion in a hamster model of metabolic syndrome, which may not compensate for the diet-induced dyslipidemia and
liver steatosis. J. Nutr. 142: 704–709, 2012.

Introduction
The prevalence of metabolic syndrome, type 2 diabetes, and
cardiovascular diseases is dramatically increasing worldwide
and more specifically in developing countries (1). The impact of
these metabolic diseases is expected to be even worse with a
world urbanization level projected to be ;60% in 2025 (2).
Increased consumption of energy-dense foods containing high
amounts of animal fats, SFA (3), and fructose (4) is thought to be
the major contributor to the metabolic disorders (obesity, insulin
resistance, nonalcoholic steatohepatitis, dyslipidemia, high
blood pressure, and atherosclerosis) seen in type 2 diabetes,
which increase cardiovascular risk and result in premature
mortality (5).
1
Supported by the Agence Nationale de la Recherche, Oséo, the Région Midi-
Pyrénées, France, and the European fund Fonds Européen de Développement
Régional.
2
Author disclosures: F. Briand, Q. Thiéblemont, E. Muzotte, and T. Sulpice are
employees of Physiogenex.
3
Supplemental Table 1 and Figure 1 are available from the “Online Supporting
Material” link in the online posting of the article and from the same link in the
online table of contents at http://jn.nutrition.org.
* To whom correspondence should be addressed: f.briand@physiogenex.com.
The RCT4 pathway would be one of the impaired biological
processes contributing to increase cardiovascular risk in meta-
bolic syndrome (6). The role of RCT is to return the excess
cholesterol accumulated in peripheral tissues to the liver for
excretion into the bile and ultimately into the feces (6). The key
player mediating RCT is HDL, which mediates the efflux of
cholesterol from peripheral tissues (e.g., macrophages in the
vessel walls) to the plasma for transport back to the liver (7).
Hence, an inverse relationship exists between HDL-C levels and
cardiovascular diseases (8). In type 2 diabetes, dyslipidemia is
characterized by increased TG and low HDL-C levels. These
changes are amplified by increased CETP activity, which
mediates the transfers of cholesteryl esters from HDL to apoB-
containing particles (VLDL and LDL) in exchange of TG (9).
Although it remains to be demonstrated in vivo, diabetic
dyslipidemia is thought to impair the rate of RCT, which would
4
Abbreviations used: CON, nonpurified diet; FPLC, fast protein liquid chroma-
tography; HDL-C, HDL-cholesterol; HF, high-fat diet; RCT, reverse cholesterol
transport; TICE, trans-intestinal cholesterol excretion.

ã 2012 American Society for Nutrition.

704 Manuscript received October 7, 2011. Initial review completed November 2, 2011. Revision accepted January 2, 2012.
First published online February 22, 2012; doi:10.3945/jn.111.153197.
contribute to atherosclerosis and thus cardiovascular risk in
patients with type 2 diabetes.
RCT therefore represents an attractive target to reduce
cardiovascular risk and prevent premature death. Toward this
goal, several animal models (e.g., rabbit, mouse and hamster)
have been used over the last 30 y to investigate this biological
process and develop novel therapies to prevent cardiovascular
diseases (7,10,11). To our knowledge, however, the effects of
metabolic syndrome on the rate of RCT remain to be assessed in
vivo. Meanwhile, an animal model that reflects the human
situation (e.g., fat and fructose overconsumption) would be
helpful to evaluate novel therapies targeting metabolic syn-
drome. We recently demonstrated the relevance of the hamster
model for studying RCT (11–13), because this CETP-expressing
species has a similar lipoprotein metabolism to humans (11). In
the present study, Golden Syrian hamsters were fed a high fat/
fructose-enriched (HF) diet to induce the main features of
human metabolic syndrome. We then investigated the potent
effects of such a diet on macrophage-to-feces RCT in vivo.
Methods
Hamsters and diet. All animal protocols were approved by the local
ethical committee (Comité régional d’éthique de Midi-Pyrénées). Male
Golden Syrian hamsters (91–100 g, 6 wk old, Elevage Janvier) were
housed in plastic cages (4–6 hamsters/cage) containing wood shavings
and maintained in a room with a 12-h-light cycle with free access to food
and tap water. Hamsters were adapted to these conditions and fed a
commercial nonpurified diet containing 214 g/kg protein, 51 g/kg fat,
and 0.1 g/kg cholesterol (diet no. A03, Safe Diets) for 1 wk. This
nonpurified diet was defined as the control diet.
A total of 44 hamsters were used to perform the 3 in vivo experiments
described below (plasma and tissue collection, macrophage-to-feces RCT,
and intestinal cholesterol absorption). Hamsters were separated into 2
groups of 22 hamsters each and they consumed ad libitum over 4 wk either
the control diet (CON) with normal drinking water or a HF diet with 10%
fructose in the drinking water. Body weight was monitored weekly. Food and
water intake were measured over 24 h on the last week of diet using
individually caged hamsters (n = 8/group). The HF diet was prepared by
mixing 762.5 g of the CON diet with coconut oil (115 g), corn oil (115 g),
cholesterol (5 g), and deoxycholate (2.5 g) to obtain 1 kg of HF diet (pelleted
from Safe Diets). Essential nutrients were not adjusted in the HF diet and
were thus not equal in the 2 diets. The HF diet was previously described to
reliably induce insulin resistance, dyslipidemia, and liver steatosis (14).
Plasma and tissue collection. After 4 wk of treatment, 16 hamsters
(n = 8/group) were feed deprived overnight, blood glucose was
monitored using a glucometer (Roche Diagnostics), and a blood sample
(1 mL) was collected by retro-orbital bleeding under isoflurane anesthe-
sia. Plasma was immediately isolated by centrifugation and aliquots were
stored at 2808C before biochemical analysis. For each group, a 400-mL
pool of plasma was made from each individual plasma sample and was
stored at 2808C prior to FPLC analysis. Hamsters were then killed by
cervical dislocation, exsanguinated, and livers were harvested, weighed,
sliced into small parts, flash-frozen in liquid nitrogen, and stored at
2808C until biochemical analysis. Another part of liver sample was fixed
using frozen isopentane and kept at 2808C prior to the evaluation of
liver steatosis using the oil-red-O/hematoxylin staining method (as
described below). The medium part (jejunum) of the small intestine was
isolated using the 1:3:2 (duodenum:jejunum:ileum) length ratio methods
(15). Samples of both liver and jejunum were stored at 2808C in
RNAlater (Invitrogen) for hepatic and intestinal gene expression.
In vivo macrophage-to-feces RCT. Another set of 16 hamsters was fed
the nonpurified diet (n = 8) or the HF diet (n = 8) and underwent the in vivo
macrophage-to-feces RCT experiment after 4 wk of diet as described
below. 3H-cholesterol–labeled and oxidized LDL-loaded J774 cells (2.5 3
106 cells containing 10 3 106 dpm in 0.5 mL minimum essential medium)
were prepared as previously described (12) and i.p. injected into
individually caged hamsters. Hamsters had free access to food and water
for 72 h. Blood was collected by retro-orbital bleeding and under
isoflurane anesthesia at 24, 48, and 72 h to measure radioactivity released
into the plasma and HDL fraction after phosphotungstate/MgCl2 precip-
itation (for both, 20 mL counted in a liquid scintillation counter). After 72
h, hamsters were then killed by cervical dislocation and exsanguinated.
Bile was collected and the whole volume was measured and used for
radioactivity recovery. Liver was harvested and weighed from each
hamster. Two pieces of ;50 mg liver were isolated and each sample was
homogenized using an ultrasound probe in 500 mL distilled water. The first
liver homogenate (400 mL) was used to measure the hepatic radioactivity.
The other liver homogenate (400 mL) was used for 3H-cholesterol and 3H-
bile acid extraction, as described for feces below.
Feces were collected for 72 h and were stored at 48C before extraction
of cholesterol and bile acid. Fecal cholesterol and bile acid extraction was
performed as previously described (12) to measure radioactivity in both
extracts. The remaining volume of both extracts was evaporated and
resuspended in ethanol, then commercial kits were used to determine fecal
total cholesterol (cholesterol RTU, Biomerieux) and total bile acids (total
bile acids colorimetric assay kit, Bioquant) mass.
Results were expressed as a percentage of the radioactivity injected
and recovered in plasma, liver, bile, and feces. The plasma volume was
estimated as 3.5% of the body weight (12).
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Intestinal cholesterol absorption. Intestinal cholesterol absorption
was measured in a separate set of 12 hamsters fed the nonpurified diet
(n = 6) or HF diet (n = 6). After 4 wk of diet treatment, hamsters were
feed deprived overnight, weighed, and given by oral gavage 4 mCi of 3H-
cholesterol in 750 mL olive oil containing 2 g/L cholesterol. Immediately
after oral gavage, hamsters were i.p. injected with poloxamer-407 (1 g/
kg body weight) as a lipase inhibitor. Blood (200 mL) was then collected
by retro-orbital bleeding under isoflurane anesthesia at 3, 5, and 6 h to
measure 3H-tracer appearance in plasma and HDL fraction after the
phosphotungstate/MgCl2 precipitation method. Results were expressed
as the percentage of injected dose in plasma and HDL fraction. Values
were used to calculate the AUC for plasma and HDL. The plasma
volume was estimated as 3.5% of the body weight (12).
Biochemical analysis. Plasma insulin was assayed using a commercial
ELISA kit (Insulin ELISA kit, Abcys). Commercial kits were used to
measure plasma total cholesterol (cholesterol RTU, Biomerieux), TG
(Triglyceride PAP 150, Biomerieux), alanine aminotransferase (Enzyline
ALAT/GPT, Biomerieux), and aspartate aminotransferase (Enzyline
ASAT/GOT, Biomerieux). HDL-C was determined using the phospho-
tungstate/MgCl2 precipitation method. Non-HDL-C levels were then
determined by subtracting HDL-C values from total plasma cholesterol.
Plasma CETP activity was measured by fluorescence using a commercial
kit (RB-CETP kit, Roarbiomedical). FPLC lipoprotein profiles analysis
(total cholesterol) using pooled plasma was performed as previously
described (12). Commercial kits were used to measure hepatic choles-
terol (cholesterol RTU, Biomerieux), TG (triglyceride PAP 150,
Biomerieux), and fatty acid [NEFA-HR (2),Wako Chemicals] levels
from liver homogenate incubated with deoxycholate, as described (16).
Oil red O/hematoxylin staining procedure. After fixation of a 5-mm
frozen liver section in a 10% formalin solution for 1 h, section were
washed and stained with Oil red O for 5 min. Sections were then washed
and stained with Harris’ hematoxylin for 1 min.
Gene expression analysis. Tissue for mRNA analysis was homogenized
and RNA was isolated using Trizol reagent (Invitrogen) according to the
manufacturer’s instructions. Real-time qPCR analysis was performed using
an Applied Biosystems 7300 sequence detector, as previously described
(17). Primers sequences are detailed in Supplemental Table 1. Relative
quantities of mRNA were calculated from CT values by the comparative CT
method. A serial dilution of a standard was run on each plate for each
mRNA and used to calculate the relative levels of mRNA. Data were
normalized to cyclophylin mRNA and expressed as fold of the CON group.
High-fat feeding in hamsters 705
Western-blot analysis. Protein expression of hepatic LDLR and SR-BI
was evaluated from liver homogenates by using Western-blot analysis as
previously described (18), using antibodies reacting with hamster LDLR
(Biovision) and SR-BI (Novus Biological). Pan-cadherin (AbCam)
antibody was used for loading control. For densitometry analysis of
bands obtained via Western-blot analysis, we applied ImageJ software
based analysis (19). The AUC of the specific signal was corrected for the
AUC of the loading control.

Statistical analysis. Values are presented as mean 6 SEM. Compar-

isons between groups were conducted using the Student’s t test (2-tailed).
A 2-tailed P-value of P , 0.05 was considered significant.

Results
HF diet induces insulin resistance, dyslipidemia, and liver
steatosis in Golden Syrian hamsters. After 4 wk of
treatment, the groups did not differ in body weight, food intake,
or water intake (Table 1). Blood glucose and plasma insulin
concentrations and HOMA-IR were 62, 237, and 547% greater,
respectively, in HF hamsters than in CON hamsters (P , 0.001).
Plasma TG and total cholesterol concentrations were 363 and
146% higher, respectively, in HF hamsters (P , 0.001).
Compared to CON hamsters, plasma HDL-C was 93% higher

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in HF hamsters (P , 0.001). However, total cholesterol in the
non-HDL–C fraction (VLDL+LDL) was 258% greater in HF
hamsters than in CON hamsters (P , 0.001), and this resulted in FIGURE 1 FPLC profiles for total cholesterol of pooled plasma
a 21% lower HDL-C:total cholesterol ratio in HF hamsters (P , samples collected from hamsters fed a CON or HF diet for 4 wk (A).
0.001). Plasma CETP activity was 55% higher in HF than in Protein expression of hepatic LDLR and SR-BI in CON and HF
CON hamsters (P , 0.001). FPLC analysis of pooled plasma hamsters was also evaluated by Western-blot analysis (B) with pan-
samples confirmed the dyslipidemic state of HF hamsters, with cadherin used as a loading control. CON, nonpurified diet; FPLC, fast
higher total cholesterol concentrations in fractions correspond- protein liquid chromatography; HF, high fat; LDLR, LDL-receptor; SR-
ing to VLDL, LDL, and HDL in HF hamsters than in CON BI, scavenger receptor class B type I.
hamsters (Fig. 1A).
Compared to CON hamsters, liver mass was 57% higher in
HF hamsters (P , 0.001) (Table 1), with concomitant liver
TABLE 1 Body weight, food intake, and biochemical variables steatosis, as shown by the oil-red-O analysis (Supplemental Fig.
in hamsters fed a CON or HF diet for 4 wk1 1). Compared to CON hamsters, hepatic total cholesterol, TG,
and fatty acid concentrations were 283, 115, and 239% higher
CON HF in HF hamsters, respectively (P , 0.001) (Table 1). Although
aspartate aminotransferase plasma concentrations did not differ
Body weight, g 120.3 6 2.0 121.7 6 2.3
in the 2 groups, alanine aminotransferase concentrations were
Food intake, g/d 6.5 6 0.7 6.1 6 0.3
47% higher in HF hamsters (P , 0.001) (Table 1).
Water intake, mL/d 7.9 6 0.9 7.3 6 0.8
Blood glucose, mmol/L 4.0 6 0.1 6.4 6 0.2*
HF diet impairs the expression of cholesterol metabolism
Plasma insulin, pmol/L 34.2 6 2.4 115 6 15.0*
regulators. To further evaluate the effects of the HF diet, we
HOMA-IR2 0.8 6 0.1 5.4 6 0.6*
measured the expression of genes regulating cholesterol metab-
Plasma TG, mmol/L 0.9 6 0.1 4.4 6 0.6*
olism and excretion in both liver and intestine (jejunum).
Plasma total cholesterol, mmol/L 3.2 6 0.1 7.9 6 0.3*
Compared to CON hamsters, expression of hepatic LDLR was
Plasma HDL-C, mmol/L 2.2 6 0.1 4.2 6 0.2*
48% lower (P , 0.001) in HF hamsters (Table 2). Also, hepatic
Plasma non HDL-C, mmol/L 1.0 6 0.1 3.7 6 0.4*
ACAT2 expression was 61% lower (P , 0.001). The expression
Plasma HDL-C:total cholesterol ratio 0.68 6 0.01 0.54 6 0.04*
of genes involved in biliary cholesterol excretion, namely ABCG5
Plasma CETP activity, mmol × L21 × h21 52 6 2 80 6 2*
and ABCG8, was 63 and 56% lower in HF hamsters (P , 0.001).
Plasma alanine aminotransferase, U/L 78.5 6 2.5 116 6 4.0*
In the intestine (Table 2), expression of both ABCG5 and
Plasma aspartate aminotransferase, U/L 62.0 6 2.4 59.0 6 4.6
ABCG8 did not differ between both groups. However, expression
Liver weight, g 3.7 6 0.1 5.8 6 0.3*
of NPC1L1, which mediates the intestinal absorption of choles-
Liver total cholesterol, mmol/mg tissue 22.7 6 1.1 87.0 6 1.3*
terol, was 56% lower in HF hamsters (P , 0.001).
Liver TG, mmol/mg tissue 14.5 6 1.2 31.1 6 1.3*
Whereas SR-BI expression did not differ between the groups,
Liver fatty acids, nmol/mg tissue 23.5 6 1.7 79.5 6 2.5*
LDLR expression was less in HF hamsters (65 6 11 arbitrary
1
Values are mean 6 SEM, n = 8. *Different from CON, P , 0.001. CON, nonpurified units) than in the CON group (100 6 11 arbitrary units) (P =
diet; HDL-C, HDL-cholesterol; HF, high fat. 0.013) (Fig. 1B).
2
HOMA-IR index was calculated from individual blood glucose (mmol/L) and plasma
insulin (mU/L) values as follows: HOMA-IR = [blood glucose (mmol/L) 3 plasma
insulin (mU/L)]/22.5. Conversion factor used for calculation: 1 mU/L insulin = In vivo macrophage-to-feces RCT is altered in hamsters
6 pmol/L. fed a HF diet. To investigate whether the HF diet affected the
706 Briand et al.
TABLE 2 Hepatic and intestinal mRNA expression in hamsters the bile (Fig. 2D) was 75% lower in HF compared to CON
fed a CON or HF for 4 wk1 hamsters (P , 0.01). However, 3H-tracer recovery in fecal
cholesterol (Fig. 2E) was 45% higher in HF hamsters (P , 0.01),
CON HF whereas 3H-tracer recovered in fecal bile acids did not differ
Liver mRNA expression, fold of CON between the groups. HF hamsters had a 55% higher fecal
LDLR 1.00 6 0.08 0.52 6 0.01* cholesterol excretion (P , 0.01) compared to CON hamsters
ACAT2 1.00 6 0.09 0.39 6 0.03* (Fig. 2F). Inversely, the mass of total bile acids excreted in feces
ABCG5 1.00 6 0.06 0.37 6 0.04* was 47% lower in HF hamsters (P , 0.01).
ABCG8 1.00 6 0.05 0.44 6 0.07* Finally, we investigated whether higher cholesterol fecal
Jejunum mRNA expression, fold of CON excretion after the HF diet was related to lower intestinal
ABCG5 1.00 6 0.13 1.22 6 0.07 cholesterol absorption. After 4 wk of the diet treatment, CON
ABCG8 1.00 6 0.06 1.01 6 0.15 and HF hamsters were given 3H-cholesterol labeled olive oil by
NPC1L1 1.00 6 0.08 0.44 6 0.04* oral gavage to measure 3H-tracer appearance in plasma and
HDL. Compared to CON hamsters, HF hamsters had 43, 105,
Values are mean 6 SEM, n = 8. *Different from CON, P , 0.001. CON, nonpurified and 114% higher 3H-tracer plasma appearance at 3, 5, and 6 h,
1

diet; HF, high fat.

rate of RCT in vivo, CON and HF hamsters were injected with
3
H-cholesterol labeled/cholesterol loaded macrophages after 4
wk of treatment (Fig. 2). Compared to CON hamsters, 3H-tracer
appearance was ;50% higher in HF hamsters at 24, 48, and
72 h (all P , 0.01) after 3H-cholesterol labeled macrophage
injection (Fig. 2A). However, 3H-tracer appearance in HDL did
respectively (Fig. 3A), indicating an increased cholesterol
absorption in HF hamsters. The calculated AUC was 89%
higher in HF hamsters (P , 0.05). Appearance of the 3H-tracer
in HDL was 32 and 25% higher in HF than in CON hamsters at
5 and 6 h, respectively (P , 0.05) (Fig. 3B). However, the
calculated AUC did not differ between the groups.
Discussion

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not differ between the groups at any time (Fig. 2B), suggesting
that most of the 3H-tracer was carried by non-HDL lipoproteins
(VLDL+LDL) in HF hamsters. After 72 h, the 3H-tracer recovery
in liver was 318% greater in HF hamsters than in CON hamsters
(P , 0.01) (data not shown). This difference was related to
greater hepatic 3H-cholesterol and 3H-bile acid recoveries,
which were 293 and 254% higher, resepectively (P , 0.01), in
HF hamsters (Fig. 2C). Inversely, the 3H-tracer recovered into
Macrophage-to-feces RCT has been extensively investigated over
the last 30 y. Despite the current epidemic of obesity and type 2
diabetes, little is known about the potent alterations of this
antiatherogenic process in the face of metabolic syndrome. The
aim of the present study was therefore to develop a nutritional
model to evaluate the effects of metabolic syndrome on macro-
phage-to-feces RCT in vivo. Although the goal was to mimic the

FIGURE 2 3H-tracer recovery in plasma (A), HDL (B), liver (C), bile (D), and feces (E) 72 h after injection of 3H-cholesterol labeled/cholesterol-
loaded macrophages and mass of total cholesterol and bile acids excreted in feces (F) in hamsters fed a CON or HF diet for 4 wk. Results are
mean 6 SEM, n = 8. *Different from CON, P , 0.01. CON, nonpurified diet; HF, high fat.

High-fat feeding in hamsters 707


FIGURE 3 3H-tracer appearance in plasma (A) and HDL (B) and
calculated AUC after an oral gavage of 3H-cholesterol labeled olive oil
in hamsters fed a CON or HF diet for 4 wk. Results are mean 6 SEM,
n = 6. *Different from CON, P , 0.05. CON, nonpurified diet; HF, high
fat.
human situation in which intakes of vitamins and other micro-
nutrients may be reduced due to overconsumption of energy-
dense foods (20), the lack of adjustment for essential nutrients for
the HF diet in this study is a limitation, as it may affect the
outcome studied. For instance, Zn and Cu deficiencies alter
lipoprotein metabolism (21,22). Despite this limitation, the main
features of metabolic syndrome were induced in hamsters fed the
HF diet: hyperglycemia, hyperinsulinemia, insulin resistance (as
measured by HOMA-IR), and dyslipidemia. Interestingly, this
hamster model also develops a severe liver steatosis, which
precedes the development of nonalcoholic fatty liver disease, a
common feature in patients with metabolic syndrome (23), which
likely contributes to increased cardiovascular risk (24).
Hamsters fed the HF diet had several alterations in macro-
phage-to-feces RCT. After 3H-cholesterol labeled macrophage
injection, we observed a higher 3H-tracer appearance in the
plasma, but not in the HDL fraction. This might be related to the
higher CETP activity (which transfers macrophage-derived cho-
lesterol from HDL to atherogenic VLDL and LDL lipoproteins)
and a lower hepatic uptake of the VLDL/LDL particles (through
lower LDLR expression). These deleterious effects of a HF/high
cholesterol diet increasing CETP activity and reducing LDLR-
dependent uptake have been previously described in hamsters
(12,25,26). Concomitant with the severe liver steatosis, the hepatic
3
H-tracer recovery was dramatically higher, whereas the 3H-tracer
recovery in bile was lower in HF hamsters. These data suggest an
accumulation of macrophage-derived cholesterol in the liver and a
708 Briand et al.
severe impairment of biliary excretion. In fact, we observed a
lower hepatic mRNA expression of ABCG5 and ABCG8, both
known to mediate biliary cholesterol excretion (27). Taken
together, these results highlight the harmful effects of liver steatosis
on macrophage-to-feces RCT, suggesting that liver steatosis might
contribute to increased cardiovascular risk through an impairment
of liver cholesterol metabolism and biliary excretion.
Despite the strong impairment of cholesterol fluxes at the liver
levels, a 45% increase in macrophage-derived cholesterol fecal
excretion was observed in hamsters fed the HF diet. Because the
mass of cholesterol excreted in feces was also higher, we therefore
hypothesized that higher fecal cholesterol excretion was related to
lower intestinal cholesterol absorption. Indeed, we observed a
lower NPC1L1 expression, the protein mediating intestinal
cholesterol absorption. Lower NPC1L1 expression promotes
macrophage-to-feces RCT by preventing the intestinal reabsorp-
tion of cholesterol deriving from bile (17). Surprisingly, intestinal
cholesterol absorption was found to be dramatically higher in HF
hamsters. This suggests that the higher fecal cholesterol excretion
may not be due to lower intestinal cholesterol absorption. Because
the contribution of both the “classical” biliary pathway and the
intestinal cholesterol absorption to promote fecal cholesterol
excretion seems unlikely, we therefore propose that other mech-
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anisms, such as increased intestinal cell shedding or upregulation
of the TICE pathway could play a role. As recently investigated,
TICE is a nonbiliary pathway mediating the direct excretion of
cholesterol from plasma to the intestinal lumen (28). Although the
molecular mechanism (29) and the contribution of TICE in the
overall RCT (30) remain to determined, some studies in mice have
suggested that TICE can be stimulated with pharmacologic
intervention, e.g., liver X receptor agonist (31) as well as dietary
manipulations. For instance, a Western-type diet, containing high
fat and high cholesterol levels, was found to upregulate TICE in
mice (32). In another study, Brown et al. (33) showed that mice
deficient in hepatic ACAT2 had an increased fecal cholesterol
excretion without a changed biliary cholesterol excretion. These
authors suggested that hepatic ACAT2 depletion might result in
the secretion of specific lipoprotein particles redirecting choles-
terol toward TICE (33). In the present study, we also observed a
reduction in hepatic ACAT2 mRNA expression in hamsters fed
the HF diet. It is thus possible that both the reduction in hepatic
ACAT2 and high levels of dietary fat may have contributed to
stimulate TICE in HF hamsters, which would have led to the
;50% increase in fecal cholesterol excretion. Although further
investigations are needed to measure the contribution of TICE in
hamster RCT, the present study suggests that HF-fed hamsters
could be a potential model to investigate this alternative pathway.
From a physiologic point of view, the upregulation of TICE
could actually represent an alternative mechanism against the
cholesterol overflow when consuming the HF diet. However, the
45% increase in macrophage-derived cholesterol fecal excretion
does not appear to compensate for metabolic disorders (e.g.,
hypercholesterolemia and liver steatosis) induced by dietary fat and
fructose overloading. In the present study, the most impaired steps
of RCT were localized in the liver. Upon liver steatosis, macro-
phage-derived cholesterol would accumulate in the liver, with a
concomitant reduction in biliary excretion, thereby contributing to
nonalcoholic fatty liver disease and cardiovascular risk (23).
Regarding novel treatments of diabetic dyslipidemia, it would be
important to evaluate whether a given therapy targeting RCT also
improves liver steatosis to promote cholesterol fluxes toward biliary
excretion. For instance, raising HDL levels with CETP inhibition
has emerged as a promising therapy in humans (34,35). CETP
inhibition has shown a potential to promote RCT in normolipi-
demic hamsters (36,37), but this remains to be investigated in
dyslipidemic hamsters with liver steatosis. Because the hamster has
a similar lipoprotein metabolism to humans, our data also suggest
that this animal model could be useful for evaluating the effects of
therapies targeting RCT in patients with metabolic syndrome.
Acknowledgments
The authors thank Dominique Lopes and Kenny Villevalois for
animal care, Noémie Burr, Isabelle Urbain, Emmanuel Brousseau,
and Hélène Guyraud for technical assistance, and Dr. Talal Al
Saati (INSERM US006, Toulouse) for histopathology expertise.
F.B. and T.S. designed research; F.B., Q.T., and E.M. conducted
research; F.B. analyzed data and wrote the paper; and T.S. had
primary responsibility for final content. All authors read and
approved the final manuscript.
Literature Cited
1. Astrup A, Dyerberg J, Selleck M, Stender S. Nutrition transition and its
relationship to the development of obesity and related chronic diseases.
Obes Rev. 2008;9 Suppl 1:48–52.
2. Misra A, Khurana L. Obesity and the metabolic syndrome in developing
countries. J Clin Endocrinol Metab. 2008; 93(11, Suppl 1):S9–30.
3. Misra A, Singhal N, Khurana L. Obesity, the metabolic syndrome, and
type 2 diabetes in developing countries: role of dietary fats and oils.
J Am Coll Nutr. 2010; 29 Suppl 3:S289–301.
4. Tappy L, Lê KA. Metabolic effects of fructose and the worldwide increase
in obesity. Physiol Rev. 2010;90:23–46.
5. Zoungas S, Patel A. Cardiovascular outcomes in type 2 diabetes: the
impact of preventative therapies. Ann N Y Acad Sci. 2010;1212:29–40.
6. Cuchel M, Rader DJ. Macrophage reverse cholesterol transport: key to
the regression of atherosclerosis? Circulation. 2006;113:2548–55.
7. Miller NE, La Ville A, Crook D. Direct evidence that reverse cholesterol
transport is mediated by high-density lipoprotein in rabbit. Nature.
1985;314:109–11.
8. Miller GJ, Miller NE. Plasma-high-density-lipoprotein concentration
and development of ischaemic heart-disease. Lancet. 1975;1:16–9.
9. Chan DC, Barrett PH, Watts GF. Recent studies of lipoprotein kinetics
in the metabolic syndrome and related disorders. Curr Opin Lipidol.
2006;17:28–36.
10. Escolà-Gil JC, Rotllan N, Julve J, Blanco-Vaca F. In vivo macrophage-
specific RCT and antioxidant and antiinflammatory HDL activity
measurements: new tools for predicting HDL atheroprotection. Ather-
osclerosis. 2009;206:321–7.
11. Briand F. The use of dyslipidemic hamsters to evaluate drug-induced
alterations in reverse cholesterol transport. Curr Opin Investig Drugs.
2010;11:289–97.
12. Briand F, Tréguier M, André A, Grillot D, Issandou M, Ouguerram K,
Sulpice T. Liver X receptor activation promotes macrophage-to-feces
reverse cholesterol transport in a dyslipidemic hamster model. J Lipid
Res. 2010;51:763–70.
13. Tréguier M, Briand F, Boubacar A, André A, Magot T, Nguyen P, Krempf
M, Sulpice T, Ouguerram K. Diet-induced dyslipidemia impairs reverse
cholesterol transport in hamsters. Eur J Clin Invest. 2011;41:921–8.
14. Wang PR, Guo Q, Ippolito M, Wu M, Milot D, Ventre J, Doebber T,
Wright SD, Chao YS. High fat fed hamster, a unique animal model for
treatment of diabetic dyslipidemia with peroxisome proliferator activated
receptor alpha selective agonists. Eur J Pharmacol. 2001;427:285–93.
15. Duan LP, Wang HH, Ohashi A, Wang DQ. Role of intestinal sterol
transporters Abcg5, Abcg8, and Npc1l1 in cholesterol absorption in
mice: gender and age effects. Am J Physiol Gastrointest Liver Physiol.
2006;290:G269–76.
16. Miao B, Zondlo S, Gibbs S, Cromley D, Hosagrahara VP, Kirchgessner
TG, Billheimer J, Mukherjee R. Raising HDL cholesterol without
inducing hepatic steatosis and hypertriglyceridemia by a selective LXR
modulator. J Lipid Res. 2004;45:1410–7.
17. Briand F, Naik SU, Fuki I, Millar JS, Macphee C, Walker M, Billheimer
J, Rothblat G, Rader DJ. Both the peroxisome proliferator-activated
receptor delta agonist, GW0742, and ezetimibe promote reverse
cholesterol transport in mice by reducing intestinal reabsorption of
HDL-derived cholesterol. Clin Transl Sci. 2009;2:127–33.
18. Sulpice T, Prunet-Marcassus B, Molveaux C, Cani PD, Vitte PA, Graber
P, Dreano M, Burcelin R. An adiponectin-like molecule with antidia-
betic properties. Endocrinology. 2009;150:4493–501.
19. Abramoff MD, Magalhaes PJ, Ram SJ. Image processing with ImageJ.
Biophotonics International. 2004;11:36–42.
20. Cordain L, Eaton SB, Sebastian A, Mann N, Lindeberg S, Watkins BA,
O’Keefe JH, Brand-Miller J. Origins and evolution of the Western diet:
health implications for the 21st century. Am J Clin Nutr. 2005;81:341–54.
21. Wu JY, Reaves SK, Wang YR, Wu Y, Lei PP, Lei KY. Zinc deficiency
decreases plasma level and hepatic mRNA abundance of apolipoprotein
A-I in rats and hamsters. Am J Physiol. 1998;275:C1516–25.
22. Lei KY. Dietary copper: cholesterol and lipoprotein metabolism. Annu
Rev Nutr. 1991;11:265–83.
23. Tsochatzis EA, Papatheodoridis GV. Is there any progress in the treatment
of non-alcoholic fatty liver disease? World J Gastrointest Pharmacol Ther.
2011;2:1–5.
24. Byrne CD, Olufadi R, Bruce KD, Cagampang FR, Ahmed MH.
Metabolic disturbances in non-alcoholic fatty liver disease. Clin Sci
(Lond). 2009;116:539–64.
25. Izem L, Morton RE. Molecular cloning of hamster lipid transfer
inhibitor protein (apolipoprotein F) and regulation of its expression by
hyperlipidemia. J Lipid Res. 2009;50:676–84.
Downloaded from jn.nutrition.org by guest on June 7, 2015
26. Woollett LA, Spady DK, Dietschy JM. Mechanisms by which saturated
triacylglycerols elevate the plasma low density lipoprotein-cholesterol
concentration in hamsters. Differential effects of fatty acid chain length.
J Clin Invest. 1989;84:119–28.
27. Graf GA, Yu L, Li WP, Gerard R, Tuma PL, Cohen JC, Hobbs HH.
ABCG5 and ABCG8 are obligate heterodimers for protein trafficking
and biliary cholesterol excretion. J Biol Chem. 2003;278:48275–82.
28. Vrins CL. From blood to gut: direct secretion of cholesterol via trans-
intestinal cholesterol efflux. World J Gastroenterol. 2010;16:5953–7.
29. Brufau G, Groen AK, Kuipers F. Reverse cholesterol transport revisited:
contribution of biliary versus intestinal cholesterol excretion. Arterios-
cler Thromb Vasc Biol. 2011;31:1726–33.
30. Nijstad N, Gautier T, Briand F, Rader DJ, Tietge UJ. Biliary sterol
secretion is required for functional in vivo reverse cholesterol transport
in mice. Gastroenterology. 2011;140:1043–51.
31. van der Veen JN, van Dijk TH, Vrins CL, van Meer H, Havinga R,
Bijsterveld K, Tietge UJ, Groen AK, Kuipers F. Activation of the liver X
receptor stimulates trans-intestinal excretion of plasma cholesterol. J
Biol Chem. 2009;284:19211–9.
32. van der Velde AE, Vrins CL, van den Oever K, Seemann I, Oude Elferink
RP, van Eck M, Kuipers F, Groen AK. Regulation of direct
transintestinal cholesterol excretion in mice. Am J Physiol Gastrointest
Liver Physiol. 2008;295:G203–8.
33. Brown JM, Bell TA III, Alger HM, Sawyer JK, Smith TL, Kelley K, Shah
R, Wilson MD, Davis MA, Lee RG, et al. Targeted depletion of hepatic
ACAT2-driven cholesterol esterification reveals a non-biliary route for
fecal neutral sterol loss. J Biol Chem. 2008;283:10522–34.
34. Stein EA, Stroes ES, Steiner G, Buckley BM, Capponi AM, Burgess T,
Niesor EJ, Kallend D, Kastelein JJ. Safety and tolerability of dalcetrapib.
Am J Cardiol. 2009;104:82–91.
35. Cannon CP, Shah S, Dansky HM, Davidson M, Brinton EA, Gotto AM,
Stepanavage M, Liu SX, Gibbons P, Ashraf TB, et al.. Safety of
anacetrapib in patients with or at high risk for coronary heart disease. N
Engl J Med. 2010;363:2406–15.
36. Tchoua U, D’Souza W, Mukhamedova N, Blum D, Niesor E, Mizrahi J,
Maugeais C, Sviridov D. The effect of cholesteryl ester transfer protein
overexpression and inhibition on reverse cholesterol transport. Car-
diovasc Res. 2008;77:732–9.
37. Niesor EJ, Magg C, Ogawa N, Okamoto H, von der Mark E, Matile H,
Schmid G, Clerc RG, Chaput E, Blum-Kaelin D, et al. Modulating
cholesteryl ester transfer protein activity maintains efficient pre-b-HDL
formation and increases reverse cholesterol transport. J Lipid Res.
2010;51:3443–54.
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