In contrast with apoE-deficient mice, LDLr-deficient mice fed a western-type diet for 24 weeks developed significant accumulation of hepatic triglycerides and NAFLD, suggesting that apoE-me
Trang 1diet-induced nonalcoholic fatty liver disease in mice
Eleni A Karavia1, Dionysios J Papachristou2, Ioanna Kotsikogianni2, Ioanna Giopanou2 and
Kyriakos E Kypreos1
1 Department of Medicine, Pharmacology Unit, University of Patras School of Health Sciences, Rio-Achaias, Greece
2 Department of Medicine, Anatomy, Histology and Embryology Unit, University of Patras School of Health Sciences, Rio-Achaias, Greece
Keywords
apoE-deficient mice; apolipoprotein E; low
density lipoprotein receptor; lipoproteins;
nonalcoholic fatty liver disease
Correspondence
K E Kypreos, Department of Medicine,
University of Patras School of Health
Sciences, Panepistimioupolis, Rio,
TK 26500, Greece
Fax: +302610994720
Tel: +302610969120
E-mail: kkypreos@med.upatras.gr
(Received 21 March 2011, revised 7 June
2011, accepted 6 July 2011)
doi:10.1111/j.1742-4658.2011.08238.x
Apolipoprotein E (apoE) mediates the efficient catabolism of the chylomicron remnants very low-density lipoprotein and low-density lipo-protein from the circulation, and the de novo biogenesis of high-density lipoprotein Lipid-bound apoE is the natural ligand for the low-density lipoprotein receptor (LDLr), LDLr-related protein 1 and other scavenger receptors Recently, we have established that deficiency in apoE renders mice resistant to diet-induced obesity In the light of these well-documented properties of apoE, we sought to investigate its role in the development of diet-induced nonalcoholic fatty liver disease (NAFLD) apoE-deficient, LDLr-deficient and control C57BL⁄ 6 mice were fed a western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5 kca-lÆg)1) for 24 weeks and their sensitivity to NAFLD was assessed by histo-logical and biochemical methods apoE-deficient mice were less sensitive than control C57BL⁄ 6 mice to diet-induced NAFLD In an attempt to identify the molecular basis for this phenomenon, biochemical and kinetic analyses revealed that apoE-deficient mice displayed a significantly delayed post-prandial triglyceride clearance from their plasma In contrast with apoE-deficient mice, LDLr-deficient mice fed a western-type diet for
24 weeks developed significant accumulation of hepatic triglycerides and NAFLD, suggesting that apoE-mediated hepatic triglyceride accumulation
in mice is independent of LDLr Our findings suggest a new role of apoE
as a key peripheral contributor to hepatic lipid homeostasis and the devel-opment of diet-induced NAFLD
Introduction
Apolipoprotein E (apoE) is a 34.2-kDa glycoprotein
synthesized by the liver and other peripheral tissues In
humans, there are three major natural isoforms,
apoE2, apoE3 and apoE4, with apoE3 being the most
common [1–7] apoE is a major protein component of
chylomicron remnants and very low-density lipoprotein (VLDL) [1] The importance of this protein in the maintenance of plasma lipid homeostasis and ath-eroprotection was first established with the generation
of the apoE-deficient mouse [8,9], which develops
Abbreviations
apoE, apolipoprotein E; apoE) ⁄ ), apoE deficient; apoE3knock-inmice, mice containing a targeted replacement of the mouse apoE gene for the human apoE3 gene; FFA, free fatty acid; HDL, high-density lipoprotein; IDL, intermediate density lipoprotein; LDL, low-density lipoprotein; LDLr, low-density lipoprotein receptor; LDLr) ⁄ ), LDLr deficient; NAFLD, nonalcoholic fatty liver disease; VLDL, very low-density lipoprotein;
WT, wild-type.
Trang 2hypercholesterolemia and spontaneous atherosclerosis
[8,9]
Recently, using apoE-deficient (apoE) ⁄ )) mice,
C57BL⁄ 6 mice and apoE3knock-inmice (mice containing
a targeted replacement of the mouse apoE gene for the
human apoE3 gene), we have shown that, in addition
to its role in the maintenance of plasma lipid
homeo-stasis, apoE plays a central role in the development of
diet-induced obesity and related metabolic
dysfunc-tions in mice [10,11] Additional studies in genetically
predisposed obese mice further confirmed that
defi-ciency in apoE protects mice from obesity, insulin
resistance and other metabolic abnormalities [12,13]
Nonalcoholic fatty liver disease (NAFLD) is a
spec-trum of metabolic abnormalities ranging from simple
accumulation of triglycerides in the liver (hepatic
stea-tosis) to hepatic steatosis with inflammation, fibrosis
and cirrhosis (steatohepatitis) [14,15] Although hepatic
steatosis is related to a number of clinical disorders
and has been studied in several different animal
mod-els, NAFLD in humans is characterized by obesity,
insulin resistance and associated metabolic
perturba-tions [14,15] For this reason, it has been proposed
that NAFLD should be included as a component of
metabolic syndrome [16] Aging, hormonal imbalance
and genetic predisposition may contribute to hepatic
triglyceride accumulation However, a western-type
diet and sedentary lifestyle, which result in excess body
fat, physical inactivity and imbalance in caloric load,
are the most common contributors to NAFLD [17]
As apoE possesses a central role in the metabolism
of plasma lipoproteins and the development of diet-induced obesity, in this study we sought to determine how apoE affects the development of diet-induced NAFLD in mice To address this question 10–12-week-old male apoE) ⁄ ) and wild-type (WT) C57BL⁄ 6 mice were fed a standard western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% choles-terol, 4.5 kcalÆg)1) for 24 weeks, and histological and biochemical analyses were performed We found that deficiency in apoE has a protective effect on diet-induced hepatic triglyceride accumulation, and the apoE-mediated development of diet-induced NAFLD
in mice is independent of the low-density lipoprotein receptor (LDLr) Our data establish that apoE plays a central role in the deposition of post-prandial triglyce-rides in the liver and NAFLD which, over long periods
of time, may lead to nonalcoholic steatohepatitis
Results
apoE) ⁄ )mice are less sensitive than control C57BL⁄ 6 mice to hepatic triglyceride
accumulation
To test the effects of apoE on hepatic triglyceride accumulation, groups of 10–12-week-old male apoE) ⁄ ) and WT C57BL⁄ 6 mice were placed on a western-type diet for a total period of 24 weeks As shown
in Fig 1A, hematoxylin and eosin staining of liver
Fig 1 Histological analyses of liver sections from apolipoprotein E-deficient (apoE) ⁄ )) and C57BL ⁄ 6 mice (A, B) Repre-sentative photographs of hematoxylin and eosin-stained hepatic sections from apoE) ⁄ ) (A) and C57BL ⁄ 6 (B) mice at week 24 on a western-type diet (C, D) Representative photographs of reticulin-stained hepatic sec-tions of apoE) ⁄ )(C) and C57BL⁄ 6 (D) mice fed a western-type diet for 24 weeks All photographs were taken at an original magnification of ·20.
Trang 3sections revealed that deficiency in apoE did not result
in any significant distortion of liver microscopic
mor-phology or accumulation of triglycerides in the liver of
apoE) ⁄ )mice fed a western-type diet for 24 weeks In
contrast, control C57BL⁄ 6 mice fed a western-type diet
for the same period exhibited remarkable steatosis,
characterized by excessive accumulation of lipids
within liver cells (Fig 1B) The observed steatosis was
diffuse and of the macrovesicular type Statistical
anal-ysis following histomorphometric evaluation of the
hematoxylin and eosin-stained sections revealed that
the number of lipid droplets within liver hepatocytes
was significantly elevated in C57BL⁄ 6 relative to
apoE) ⁄ ) mice (P = 0.0001) In agreement with these
data, staining of hepatic sections with reticulin showed
that, in C57BL⁄ 6 mice fed a western-type diet for
24 weeks, NAFLD had progressed much more
exten-sively and had resulted in significant disruption in the
normal architecture of the extracellular reticulin fibrils
of the liver (Fig 1D), relative to apoE) ⁄ ) mice
(Fig 1C) that displayed a normal hepatic histology
No significant differences in the size and shape of
vis-ceral adipocytes were detected between the two groups
of mice (data not shown)
To further confirm that deficiency in apoE prevented
the accumulation of hepatic triglycerides in the liver of
mice fed a western-type diet for 24 weeks, liver sam-ples were isolated from apoE) ⁄ ) and C57BL⁄ 6 mice and their triglyceride contents were determined bio-chemically, as described in the Materials and methods section This analysis showed that apoE) ⁄ )mice fed a western-type diet for 24 weeks had a triglyceride content of 98.6 ± 16.7 mgÆ(g hepatic tissue))1, whereas C57BL⁄ 6 mice had a much higher hepatic triglyceride content [155.7 ± 10 mgÆ(g hepatic tissue))1; P < 0.005], further confirming that apoE possesses a central role in the deposition of dietary triglycerides in the liver of mice and the development of diet-induced NAFLD (Fig 2D)
Body weight measurements and body composition analysis of mice fed a western-type diet for 24 weeks
As expected from previously published results, apoE) ⁄ ) mice were less sensitive than C57BL⁄ 6 mice
to the development of diet-induced obesity [10,18] Specifically, during the course of the experiment, apoE) ⁄ )mice showed only a modest increase in body weight (Fig 2A) At week 6 of the experiment, the apoE) ⁄ )mouse group had an average body weight of 26.7 ± 0.6 g (5.52 ± 1.45% increase relative to their
0 6 12 18 24 0
50 100 150 200 250
300 apoE–/–
C57BL/6
**
Weeks
25 50 75 100 125 150 175 200
C57BL/6 apoE–/–
**
–1 ]
10
10
0 6 12 18 24 0
250 500 750 1000 1250 1500
Weeks
–1 )
0 6 12 18 24 0
50 100 150 200
Weeks
0 1 2 3 4 5 6 7 8 9 10
Fraction number
CHYL/VLDL/IDL HDL
0 25 50 75 100 125 150 175
200 CHYL/VLDL/IDL HDL
Fraction number
Fig 2 Biochemical parameters of
apolipo-protein E-deficient (apoE) ⁄ )) and C57BL ⁄ 6
mice fed a western-type diet for a period of
24 weeks (A) Changes in average body
weight (B, C) Changes in average plasma
cholesterol and plasma triglycerides,
respec-tively (D) Average hepatic triglyceride
con-tent of mice fed a western-type diet for
24 weeks (**P < 0.005) (E, F) Cholesterol
and triglyceride contents, respectively, of
the different density fractions following the
separation of plasma lipoproteins by density
gradient ultracentrifugation Fraction 1
corre-sponds to the top fraction [containing
chylo-microns (CHYL) and very low-density
lipoprotein (VLDL)] HDL, high-density
lipoprotein; IDL, intermediate-density
lipoprotein; Tg, triglyceride.
Trang 4starting weight of 25.7 ± 0.2 g at week 0, P < 0.05).
At week 12, their average body weight was
30.7 ± 1.1 g and, at week 24, it showed a further
slight increase to 31.6 ± 1.7 g (19.7 ± 7.3% increase
relative to their starting weight at week 0, P < 0.05)
(Fig 2A) In contrast, C57BL⁄ 6 mice showed a
signifi-cant increase in their body weight during the course of
the experiment At week 6, C57BL⁄ 6 mice had an
average body weight of 31.8 ± 1.7 g (23.5 ± 3.9%
increase relative to their starting weight of 25.8 ± 1 g
at week 0, P < 0.05) At week 12, their body weight
was 35.3 ± 0.6 g and, at week 24, it showed a further
increase to 42.8 ± 1.7 g (66.7 ± 5.6% increase
rela-tive to their starting weight at week 0, P < 0.05)
(Fig 2A) In agreement with our previous findings
[10], the increased body weight of C57BL⁄ 6 mice
cor-responds to an increased body fat mass (data not
shown)
Plasma lipid levels and average daily food
consumption of mice fed a western-type diet for
24 weeks
To determine how plasma lipid levels may reflect
differ-ences in hepatic triglyceride accumulation in apoE) ⁄ )
and C57BL⁄ 6 mice, fasting plasma samples were
iso-lated every 6 weeks and cholesterol, triglyceride and free
fatty acid (FFA) levels were measured as described
in the Materials and methods section As shown
in Fig 2B, apoE) ⁄ )mice showed a dramatic increase in
their plasma cholesterol levels during the course of the
experiment At week 24 of the experiment, the plasma
cholesterol levels of apoE) ⁄ ) mice were 1475 ±
48 mgÆdL)1(Fig 2B), whereas their plasma triglyceride
levels increased but remained within the physiological
range (126.7 ± 60.9 mgÆdL)1 at week 24 versus
18.3 ± 1.9 mgÆdL)1at week 0) (Fig 2C)
Ultracentrifu-gation analysis of plasma samples showed that the
hypercholesterolemia of these mice was caused by the
increased accumulation of triglyceride-containing
cho-lesterol-rich chylomicron remnants (Fig 2E,F)
How-ever, C57BL⁄ 6 mice on a high-fat diet for 24 weeks
showed slightly elevated fasting cholesterol levels
(224.6 ± 21 mgÆdL)1) relative to their starting
choles-terol levels at week 0 (91.9 ± 10 mgÆdL)1) (Fig 2B),
whereas their plasma triglyceride levels remained normal
(79.4 ± 7.4 mgÆdL)1 at week 24 versus 58.2 ±
1.1 mgÆdL)1 at week 0) (Fig 2C) Ultracentrifugation
analysis of plasma samples showed that the cholesterol
of these mice was mainly distributed in the high-density
lipoprotein (HDL) fractions (Fig 2E,F)
Surprisingly, apoE) ⁄ ) mice, which do not develop
NAFLD, had a higher plasma concentration of
FFAs than C57BL⁄ 6 mice Steady-state FFA levels
of apoE) ⁄ ) mice were 7.6 ± 1.2 mmol eq., whereas C57BL⁄ 6 mice showed a much lower steady-state plasma FFA concentration of 1.4 ± 0.1 mmol eq (P = 0.0001)
To determine whether differences in hepatic triglycer-ide accumulation could be explained by differences in the average daily food consumption between the two groups of mice, at weeks 12 and 24 of the experiment
we determined the average daily food consumption for each mouse group It was found that apoE) ⁄ ) mice consumed 3.3 ± 0.2 and 3.5 ± 0.6 gÆmouse)1Æday)1at weeks 12 and 24, respectively (P > 0.05) Similarly, C57BL⁄ 6 mice consumed 3.8 ± 0.2 and 3.4 ± 0.2 gÆmouse)1Æday)1 at weeks 12 and 24, respectively (P > 0.05) There was no statistically significant differ-ence between the two groups (P > 0.05) Although, in this study (n = 5), we were unable to determine a statistically significant difference in the average daily food consumption between the two mouse strains at week 12 of the experiment (3.3 ± 0.2 versus 3.8 ± 0.2 gÆmouse)1Æday)1 for apoE) ⁄ ) and C57BL⁄ 6 mice, respectively; P = 0.0833), a trend towards lower food consumption existed for the apoE) ⁄ ) mice A future larger trial may be useful to confirm the similar average food consumption observed in the present study
Rate of hepatic triglyceride secretion and intestinal triglyceride absorption in apoE) ⁄ )and C57BL⁄ 6 mice
One mechanism that could affect the hepatic triglycer-ide content is the secretion of hepatic triglycertriglycer-ides in the circulation To determine the contribution of VLDL triglyceride secretion in apoE-mediated hepatic lipid accumulation, we compared the rate of hepatic VLDL triglyceride secretion between apoE) ⁄ ) and C57BL⁄ 6 mice In accordance with previous studies [19–21], we found that the rate of hepatic triglyceride secretion decreased significantly in apoE) ⁄ )relative to C57BL⁄ 6 mice Specifically, secretion rates were 2.1 ± 0.4
mgÆ-dL)1Æmin)1 (minimum, 1.7 mgÆdL)1Æmin)1; maximum, 3.5 mgÆdL)1Æmin)1; SEM = 0.4, n = 5) for apoE) ⁄ ) mice versus 11.2 ± 0.9 mgÆdL)1Æmin)1 (minimum, 9.8 mgÆdL)1Æmin)1; maximum, 13.7 mgÆdL)1Æmin)1; SEM = 0.9, n = 5) for C57BL⁄ 6 mice (P = 0.0001) (Fig 3A) Thus, on the basis of these results, it appears that hepatic triglyceride secretion cannot account for the differences in hepatic triglyceride deposition seen between apoE) ⁄ )and C57BL6 mice
One additional mechanism that could explain the increased sensitivity of apoE) ⁄ ) mice to diet-induced NAFLD could be increased intestinal secretion of
Trang 5triglyceride-rich lipoproteins in the plasma of these
mice To determine the rate of intestinal triglyceride
secretion, we calculated the total rate (intestinal and
hepatic) of plasma triglyceride input in apoE) ⁄ ) and
C57BL⁄ 6 mice fed a western-type diet, following an
oral fat load Groups of five apoE) ⁄ ) and C57BL⁄ 6
mice were fasted for 16 h, and then given an oral fat
load of 300 lL of olive oil, as described in the
Materi-als and methods section One hour post-gavage, mice
were injected with Triton WR1339 and plasma
triglyc-eride levels were determined as a function of time As
shown in Fig 3B, apoE) ⁄ ) mice showed a lower rate
of total triglyceride input than C57BL⁄ 6 mice
Specifi-cally, the rates were 11.9 ± 1.3 mgÆdL)1Æmin)1 for
apoE) ⁄ ) mice and 14.5 ± 1.2 mgÆdL)1Æmin)1 for
C57BL⁄ 6 mice (n = 5, P = 0.023) Then, by
subtract-ing the rate of hepatic triglyceride secretion
(deter-mined above) from the total rate of plasma triglyceride supply, the rate of intestinal triglyceride secretion was determined as 9.8 ± 1.3 mgÆdL)1Æmin)1 for apoE) ⁄ ) mice and 2.0 ± 0.7 mgÆdL)1Æmin)1 for C57BL⁄ 6 mice (n = 5, P = 0.023) The data suggest that differ-ences in intestinal triglyceride absorption or hepatic triglyceride secretion cannot account for the observed histological differences between apoE) ⁄ )and C57BL⁄ 6 mice
Kinetics of post-prandial triglyceride clearance in apoE) ⁄ )and C57BL⁄ 6 mice
Another potential mechanism that could explain the reduced sensitivity of apoE) ⁄ ) mice to diet-induced NAFLD could be reduced clearance of plasma trigly-cerides in these mice Thus, in the next set of experiments, we sought to determine the kinetics of post-prandial triglyceride clearance As shown in Fig 3C, following gavage administration of olive oil, the mouse groups reached similar maximum plasma concentrations of 142.7 ± 29.6 and 161.4 ± 21.5 mgÆdL)1, respectively, at 120 min post-gavage (n = 5,
P= 0.2195) (Fig 3C) However, there was a signifi-cant difference in post-prandial triglyceride clearance
in apoE) ⁄ )mice relative to C57BL⁄ 6 mice In particu-lar, in C57BL6 mice, the rapid increase in plasma triglyceride levels at 120 min after olive oil administra-tion was followed by an immediate and steep decline
At 240 min post-gavage, the plasma triglycerides of C57BL⁄ 6 mice reached baseline levels (59.5 ± 10.7 mgÆdL)1; minimum, 20.7 mgÆdL)1; maxi-mum, 80.8 mgÆdL)1; SEM = 10.7) However, in apoE) ⁄ )mice, a similar increase in plasma triglyceride levels at the 2-h time point persisted over the period of the next 4 h (360 min), suggesting that, in the absence
of apoE, post-prandial triglycerides are cleared from the circulation at a significantly slower rate At
240 min post-gavage, the plasma triglycerides of apoE) ⁄ ) mice were still significantly elevated (137.5 ± 21.9 mgÆdL)1; minimum, 106.5 mgÆdL)1; maximum, 184.0 mgÆdL)1; SEM = 21.9)
LDLr-deficient (LDLr) ⁄ )) mice fed a western-type diet for 24 weeks developed significant
accumulation of hepatic triglycerides and NAFLD
To address the potential role of LDLr in the apoE-mediated deposition of dietary triglycerides in the liver, low density lipoprotein receptor-deficient (LDLr) ⁄ )) mice were fed a western-type diet for 24 weeks and liver specimens were isolated and analyzed for triglyc-eride content by biochemical and histological analyses
apoE
–/–
C57BL/6
0
5
10
15
**
–1 ·min
60 90 120 150 180
0
500
1000
1500
2000
apoE –/–
C57BL/6
Time (min)
SlopeapoE–/–=11.9 ± 1.3 (mg·dL –1 ·min –1 ) SlopeC57BL/6=14.5 ± 1.2 (mg·dL –1 ·min –1 )
0 30 60 90 120 150 180 210 240 270 300 330 360
0
50
100
150
apoE–/–
Time post-gavage (min)
A
B
C
Fig 3 Analysis of kinetic parameters associated with hepatic
tri-glyceride content (A) Rate of hepatic very low-density lipoprotein
(VLDL) triglyceride secretion (B) Rate of total triglyceride supply in
plasma in apolipoprotein E-deficient (apoE) ⁄ )) (h) and C57BL ⁄ 6 (m)
mice (C) Kinetics of post-prandial triglyceride clearance in apoE) ⁄ )
(h) and C57BL⁄ 6 ( ) mice **P < 0.005.
Trang 6In agreement with our previous studies, LDLr) ⁄ )mice
were more susceptible than apoE) ⁄ ) mice to
diet-induced obesity, but more resistant than C57BL⁄ 6
mice [10] Surprisingly, however, we found that hepatic
specimens from LDLr) ⁄ )mice showed a higher
triglyc-eride content than those of control C57BL⁄ 6 mice
[233.0 ± 19 versus 155.7 ± 10 mgÆ(g hepatic tissue))1,
respectively] Our biochemical results were in
agree-ment with data from our histological analyses, which
showed that LDLr) ⁄ ) mice developed NAFLD that
had progressed even more than that of control
C57BL⁄ 6 mice Liver steatosis was diffuse and both
the microvesicular and macrovesicular types were
observed (Fig 4A) A few lymphocytes were detected
within the liver parenchyma Reticulin stain revealed
that the liver architecture was disturbed, mainly
because of extensive steatosis (Fig 4C)
Discussion
In this study, we investigated the role of apoE in the
development of NAFLD in mice As consumption of
lipid-rich diets and sedentary lifestyle, resulting in excess
body fat, physical inactivity and imbalance in caloric
load, are the most common contributors to NAFLD in
humans [17], we focused our studies on diet-induced
NAFLD We found that deficiency in apoE has a
pro-tective effect against diet-induced NAFLD, which
correlates mainly with the reduced clearance of
post-prandial triglycerides from the circulation
Histological evaluation following hematoxylin and
eosin staining of liver sections from control mice
revealed increased levels of steatosis, as demonstrated
by the existence of a large number of lipid droplets within the vast majority of the examined hepatocytes Steatosis was diffuse and of the macrovesicular type,
in which a large fat vacuole within the hepatocyte pushed the nucleus towards the edge of the cell In contrast, however, hematoxylin and eosin-stained liver sections from apoE) ⁄ ) mice showed a normal micro-scopic appearance, the liver architecture was normal and there was no evidence of lipid accumulation within hepatocytes Our histological findings were in harmony with the results obtained by reticulin staining, which showed that, in the liver of apoE) ⁄ )mice, the reticulin network was not distorted, in contrast with the liver of C57BL⁄ 6 mice, which showed heavy loading with fat The reticulin stain is a classical histopathological mar-ker for the identification of hepatic architecture and structural damage within the liver parenchyma There-fore, the presence of more extensive reticulin network
in Fig 1C indicates that, in apoE) ⁄ )mice, the reticulin network is better preserved, further confirming that the structural damage in the liver of these animals is mini-mal following feeding with a high-fat diet In contrast, the destruction of the reticulin network (visualized as reduced reticulin stain) in the liver of C57BL⁄ 6 mice (Fig 1D) corresponds to an extensive destruction of the hepatic architecture, primarily as a result of lipid accumulation within the hepatocytes and the develop-ment of NAFLD in these mice
In order to identify the molecular basis for this phe-nomenon, we determined a number of parameters which could affect the delivery and deposition of
B A
D C
Fig 4 Histological analyses of liver sec-tions from low-density lipoprotein receptor-deficient (LDLr) ⁄ )) and C57BL ⁄ 6 mice (A, B) Representative photographs of hema-toxylin and eosin-stained hepatic sections from LDLr) ⁄ )(A) and C57BL⁄ 6 (B) mice at week 24 on a western-type diet (C, D) Rep-resentative photographs of reticulin-stained hepatic sections of LDLr) ⁄ )(C) and C57BL ⁄ 6 (D) mice fed a western-type diet for 24 weeks All photographs were taken
at an original magnification of ·20.
Trang 7intestinal dietary triglycerides in the liver of the
experi-mental mice In general, hepatic triglyceride content is
a function of three parameters: (a) dietary triglyceride
deposition in the liver; (b) endogenous triglyceride
syn-thesis and turnover; and (c) hepatic VLDL triglyceride
secretion in the circulation Endogenous triglyceride
clearance and turnover cannot account for the
observed differences between apoE) ⁄ ) and C57BL⁄ 6
mice as it is well established that intracellular
triglycer-ide turnover and synthesis, as well as the activities of
diacylglycerol acyltransferase and microsomal
triglycer-ide transfer protein, are comparable between apoE) ⁄ )
and WT C57BL⁄ 6 mice [22] Similarly, differences in
the rate of hepatic VLDL triglyceride secretion
between apoE) ⁄ )and C57BL⁄ 6 mice could not explain
the observed resistance of apoE) ⁄ ) mice to
diet-induced NAFLD Consistent with previous data
[19–21,23], we found that apoE) ⁄ ) mice displayed
approximately five times slower hepatic VLDL
triglyc-eride secretion compared with control C57BL⁄ 6 mice
(2.1 ± 0.4 mgÆdL)1Æmin)1 for apoE) ⁄ ) mice versus
11.2 ± 0.9 mgÆdL)1Æmin)1 for C57BL⁄ 6 mice) Thus,
we hypothesized that the resistance of apoE) ⁄ )mice to
diet-induced NAFLD must be caused by either a
decreased rate of intestinal absorption of dietary lipids
or reduced hepatic deposition of plasma triglycerides
Kinetic analysis showed that apoE) ⁄ ) mice exhibited
reduced rates of intestinal absorption of dietary
triglyce-rides relative to C57BL⁄ 6 mice (2.0 ± 0.7 mgÆdL)1Æmin)1
in C57BL⁄ 6 mice versus 9.8 ± 1.3 mgÆdL)1Æmin)1 in
apoE) ⁄ )mice; P < 0.05) However, apoE) ⁄ )mice
dis-played a significantly slower clearance of post-prandial
triglycerides from the circulation, consistent with a
slower rate of dietary lipid deposition in the liver and
other peripheral tissues
Previously, it has been suggested that 3–4-month-old
apoE) ⁄ ) mice on a chow diet have a slightly higher
hepatic triglyceride content relative to control mice
[22] Our results showed that the slightly higher
base-line hepatic triglyceride content of apoE) ⁄ )mice fed a
chow diet does not predispose these mice to increased
sensitivity to NAFLD In contrast, we found that
apoE deficiency renders these mice less sensitive to
hepatic triglyceride accumulation following feeding
with a high-fat diet A more recent study has suggested
that hypercholesterolemia sensitizes apoE) ⁄ ) mice to
carbon tetrachloride-mediated liver injury [24] Our
data show that the hypercholesterolemia of apoE) ⁄ )
mice is not a causative factor in diet-induced NAFLD
in these mice Rather, our results have established that
apoE deficiency has a protective effect against hepatic
triglyceride accumulation, despite the apparent increase
in plasma cholesterol levels of apoE) ⁄ ) mice It is
interesting that, in our experiments, plasma cholesterol levels were inversely related to the hepatic accumula-tion of dietary triglycerides in mice Although, in our study, apoE) ⁄ ) mice appeared to be less sensitive to hepatic lipid deposition relative to control apoE-expressing C57BL⁄ 6 mice, previous work by Ma et al [25] has shown that artificially induced low-grade inflammatory stress triggered by subcutaneous injec-tion of 10% casein increases the sensitivity of these mice to NAFLD development In the future, it would
be interesting to compare how casein-induced inflam-mation affects the sensitivity of apoE) ⁄ )and C57BL⁄ 6 mice to the development of NAFLD
Despite the enhanced intestinal absorption and reduced deposition of post-prandial triglycerides in the liver and other peripheral tissues, steady-state plasma triglyceride levels of apoE) ⁄ ) mice fed a western-type diet remained within normal values (< 150 mgÆdL)1), although they were elevated compared with those of C57BL⁄ 6 mice for the duration of the experiment It is well established that apoE is a potent inhibitor of plasma lipoprotein lipase [26–28], and that lipolysis-mediated release of FFAs is more efficient in apoE) ⁄ ) mice than in apoE-expressing C57BL⁄ 6 mice [27] In agreement with these studies, apoE) ⁄ ) mice showed elevated plasma FFA levels relative to C57BL⁄ 6 mice (apoE) ⁄ ) mice had steady-state FFA levels of 7.6 ± 1.2 mmol eq., whereas C57BL⁄ 6 mice had a much lower steady-state plasma FFA concentration of 1.4 ± 0.1 mmol eq.; P < 0.005) Despite this apparent increase in lipoprotein lipase-mediated FFA produc-tion and in steady-state plasma FFA levels, our apoE) ⁄ ) mice were resistant to diet-induced NAFLD and obesity Thus, our data do not support the notion that elevated plasma FFAs are pivotal for the accumu-lation of triglycerides in the liver of experimental mice [29,30], and that enhanced plasma lipoprotein lipase activity promotes the deposition of plasma triglycerides
in peripheral tissues, including hepatic and adipose tissues [31] In our experiments, it is apoE, and not plasma FFAs, that plays a central role in the deposi-tion of post-prandial triglycerides in the liver, a pro-cess that, over long periods of time, may lead to NAFLD
In vitroand in vivo studies have shown that lipopro-tein-bound apoE is the natural ligand for LDLr [26,32], which is the main receptor involved in the clearance of apoE-containing lipoproteins in vivo [33] Our data indicate that the apoE-mediated mechanism
of hepatic triglyceride accumulation in mice is indepen-dent of LDLr, as LDLr) ⁄ ) mice fed a western-type diet for 24 weeks developed significant NAFLD that was more severe than in C57BL⁄ 6 mice One
Trang 8possibility is that the effects of apoE on hepatic lipid
accumulation are mediated by LDLr-related protein 1
or CD36, or, potentially, other apoE receptors
How-ever, other alternative mechanisms should also be
investigated A recent epidemiological study has shown
that the e2 allele may be protective against NAFLD in
humans, whereas another epidemiological study
sup-ported a correlation of the e4 allele with increased
pathogenesis of fatty liver disease [34] As the human
apoE2 isoform of apoE is far less efficient than apoE3
and apoE4 in removing triglyceride-rich lipoproteins
from the circulation [28], it is possible that the ability
of apoE to promote the deposition of hepatic
triglyce-rides in the liver is associated with its
lipoprotein-clear-ing function
Our data extend our current knowledge on NAFLD
development Although additional experiments will be
needed in order to determine whether receptors
medi-ate the effects of apoE, our data clearly support a new
function of apoE as a key peripheral contributor to
hepatic lipid deposition and the development of
diet-induced NAFLD in mice
Materials and methods
Animal studies
purchased from Jackson Laboratories (Bar Harbor, ME,
mice, 10–12 weeks of age, were used in these studies All
animals were housed separately (one mouse per cage) and
allowed free access to food and water To ensure similar
average cholesterol, triglyceride and glucose levels and
starting body weights for all animal experiments, groups of
five mice (n = 5) were formed after determining the fasting
cholesterol, triglyceride and glucose levels, and body
weights, of the individual animals Mice were fed a
stan-dard western-type diet (Mucedola, Milan, Italy) for the
indicated period, and the body weights and fasting plasma
cholesterol and triglyceride levels were determined at the
indicated time points after diet initiation The standard
wes-tern-type diet is composed of 17.3% protein, 48.5%
carbo-hydrate, 21.2% fat and 0.2% cholesterol (0.15% added,
con-tents of the main ingredients, expressed as gram per
kilo-gram of diet, are as follows: casein, 195; dl-methionine, 3;
sucrose, 341.46; corn starch, 150; anhydrous milkfat, 210;
cholesterol, 1.5; cellulose, 50; mineral mix, 35; calcium
car-bonate, 4; vitamin mix, 10; ethoxyquin antioxidant, 0.04
At the end of each experiment, liver and adipose tissue
specimens were collected and fixed in formalin for
later subjected to body composition analysis as described below All animal studies were governed by the European Union guidelines on the ‘Protocol for the Protection and Welfare of Animals’ In our experiments, we took into con-sideration the ‘3Rs’ (reduce, refine, replace) and minimized the number of animal experiments to the absolute mini-mum To date, there is no in vitro system to mimic satisfac-torily the lipid and lipoprotein transport system and the
experimental animals mandatory All procedures used in our studies caused only minimal distress to the mice tested The work was authorized by the appropriate committee of the Laboratory Animal Center of The University of Patras Medical School
Plasma lipid determination
Following a 16-h fasting period, plasma cholesterol, triglyc-eride and FFA levels were measured as described previously [36]
Fractionation of plasma lipoproteins by density gradient ultracentrifugation
ultra-centrifugation over a 10-mL KBr density gradient, as described previously [37]
Body weight determination and body mass composition analysis
Body weight and body composition analyses were per-formed as described previously [10]
Measurement of hepatic triglyceride content
For hepatic triglyceride determination, a liver sample was collected, weighed and dissolved in 0.5 mL of 5 m KOH in
The solution was adjusted to pH 7, and the final volume was recorded The total amount of triglycerides was deter-mined in the resulting mixture as described above The results are expressed as milligrams of triglycerides per gram
of tissue ± SEM
Histological analysis of liver samples
At the end of the 24-week period, mice were sacrificed, and liver and visceral fat specimens were collected and stored at
Four-micrometer-thick sections were obtained from the formalin-fixed, paraffin-embedded tissue for further histological analyses Conventional hematoxylin and eosin stain was performed
Trang 9in order to evaluate the microscopic morphology of the
liver tissue samples In order to assess the tissue structural
integrity and architecture, the reticular fiber network was
outlined with the application of reticulin stain according to
the manufacturer’s instructions (Bioptica, Milan, Italy) All
sections were observed under an Olympus BX41 bright-field
microscope (Olympus Corp., Shinjuku-Ku, Tokyo, Japan)
Histomorphometry was performed using Adobe Photoshop
software More specifically, five representative sections of
the liver of each animal were used for histomorphometric
measurements Each section was photographed using a
Nikon Eclipse 80i microscope (Nikon Instruments Inc.,
Melville, NY, USA) with a Nikon DXM 1200C digital
were imported into Adobe Photoshop CS2 and a grid was
added For each section, the number of lipid vacuoles
inter-sected by the grid was determined and calculated
indepen-dently by one pathologist (D.J.P.) and one investigator
(K.E.K) in a blind fashion These data were then used to
assess the total number of fat vacuoles accumulated within
hepatocytes
Determination of daily food consumption
Food intake was assessed by determining the difference in
food weight during a 7-day period to ensure reliable
mea-surements, as described previously [38]
Determination of post-prandial triglyceride
kinetics following the oral administration of olive
oil
Prior to the experiment, mice were fasted overnight for
16 h On the following day, the animals were given an oral
load of 0.5 mL of olive oil, and plasma samples were
iso-lated 30, 60, 120, 180 and 240 min following olive oil
administration A control sample for baseline triglyceride
determination was isolated 1 min prior to the gavage
administration of olive oil Triglyceride levels were
quanti-fied in plasma samples as described above, and then plotted
on graphs as a function of time Values were expressed as
Rate of secretion of triglyceride-rich
chylomicrons and VLDL
To determine the rate of intestinal triglyceride secretion in
the plasma of our experimental mice, we measured the total
rate of plasma triglyceride input (intestinal and hepatic)
and subtracted the rate of hepatic triglyceride secretion
Briefly, to determine the total rate of triglyceride input
mice were fasted overnight for 16 h On the following day, animals were gavaged with 0.3 mL of olive oil and placed back in their cages for 1 h (in our experimental set-up, dietary triglyceride absorption, measured as a post-gavage increase in plasma triglyceride levels, becomes apparent at approximately 1 h following the oral adminis-tration of olive oil) The mice were then injected with
been shown to completely inhibit the catabolism of
[26,36,37,40] Serum samples were isolated at 30, 60, 90,
120, 150 and 180 min after injection with Triton-WR1339
As a control, serum samples were isolated approximately
1 min after injection with the detergent Plasma triglycer-ide levels at each time point were determined as described above, and linear graphs of triglyceride concentration ver-sus time were generated The rate of plasma triglyceride
from the slope of the linear graphs The slopes were reported as the mean ± SEM The total plasma triglycer-ide supply equals the sum of intestinal and hepatic triglyc-eride secretion
To measure the rate of hepatic VLDL triglyceride
injected with Triton-WR1339 at a dose of 500 mgÆ(kg body
described previously [26,36,37,40]
Subtraction of the rate of hepatic triglyceride secretion from the total plasma triglyceride supply yielded the rate
of intestinal secretion of triglyceride-rich chylomicrons following an oral fat load, expressed as the mean ± SEM
Statistical analysis
Comparison of the data from the two groups of mice was performed using Student’s t-test When more than a two-group comparison was required, the results were ana-lyzed using ANOVA Data are reported as the mean ± SEM; n indicates the number of animals tested in the group
Acknowledgements
This work was supported by the European Commu-nity’s Seventh Framework Program [FP7⁄ 2007-2013] grant agreement PIRG02-GA-2007-219129, The Uni-versity of Patras Karatheodoris research grant (both awarded to K.E.K.) and the European Community’s Seventh Framework Program [FP7⁄ 2007-2013] grant agreement PIRG02-GA-2009-256402 (awarded to D.J.P.) This work was part of the activities of the research network ‘MetSNet’ for the study of
Trang 10the molecular mechanisms of metabolic syndrome at
the University of Patras We would like to thank
mathematician Mr Eleftherios Kypreos for his advice
on the statistical analysis of our results
References
1 Zannis VI, Kypreos KE, Chroni A, Kardassis D &
Zanni EE (2004) Lipoproteins and atherogenesis
In Molecular Mechanisms of Atherosclerosis (Loscalzo J
ed), pp 111–174 Taylor & Francis, New York, NY
2 Zannis VI & Breslow JL (1981) Human very low
den-sity lipoprotein apolipoprotein E isoprotein
polymor-phism is explained by genetic variation and
posttranslational modification Biochemistry 20,
1033–1041
3 Zannis VI, Just PW & Breslow JL (1981) Human
apoli-poprotein E isoprotein subclasses are genetically
deter-mined Am J Hum Genet 33, 11–24
4 Breslow JL, McPherson J, Nussbaum AL, Williams
HW, Lofquist-Kahl F, Karathanasis SK & Zannis VI
(1982) Identification and DNA sequence of a human
apolipoprotein E cDNA clone J Biol Chem 257,
14639–14641
5 Breslow JL, Zannis VI, SanGiacomo TR, Third JL,
Tracy T & Glueck CJ (1982) Studies of familial
type III hyperlipoproteinemia using as a genetic
1224–1235
6 Zannis VI, Breslow JL, Utermann G, Mahley RW,
Weisgraber KH, Havel RJ, Goldstein JL, Brown MS,
Schonfeld G, Hazzard WR et al (1982) Proposed
nomenclature of apoE isoproteins, apoE genotypes, and
phenotypes J Lipid Res 23, 911–914
7 Zannis VI & Breslow JL (1982) Apolipoprotein E Mol
Cell Biochem 42, 3–20
8 Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh
A, Verstuyft JG, Rubin EM & Breslow JL (1992)
Severe hypercholesterolemia and atherosclerosis in
apo-lipoprotein E-deficient mice created by homologous
recombination in ES cells Cell 71, 343–353
9 Zhang SH, Reddick RL, Piedrahita JA & Maeda N
(1992) Spontaneous hypercholesterolemia and arterial
lesions in mice lacking apolipoprotein E Science 258,
468–471
10 Karagiannides I, Abdou R, Tzortzopoulou A, Voshol
PJ & Kypreos KE (2008) Apolipoprotein E predisposes
to obesity and related metabolic dysfunctions in mice
FEBS J 275, 4796–4809
11 Kypreos KE, Karagiannides I, Fotiadou EH, Karavia
EA, Brinkmeier MS, Giakoumi SM & Tsompanidi EM
(2009) Mechanisms of obesity and related pathologies:
role of apolipoprotein E in the development of obesity
FEBS J 276, 5720–5728
12 Gao J, Katagiri H, Ishigaki Y, Yamada T, Ogihara T, Imai J, Uno K, Hasegawa Y, Kanzaki M, Yamamoto
TT et al (2007) Involvement of apolipoprotein E in excess fat accumulation and insulin resistance Diabetes
56, 24–33
13 Hofmann SM, Zhou L, Perez-Tilve D, Greer T, Grant
E, Wancata L, Thomas A, Pfluger PT, Basford JE, Gil-ham D et al (2007) Adipocyte LDL receptor-related protein-1 expression modulates postprandial lipid trans-port and glucose homeostasis in mice J Clin Invest 117, 3271–3282
14 Preiss D & Sattar N (2008) Non-alcoholic fatty liver disease: an overview of prevalence, diagnosis, pathogen-esis and treatment considerations Clin Sci (London)
115, 141–150
15 Angulo P (2002) Nonalcoholic fatty liver disease
N Engl J Med 346, 1221–1231
16 Marchesini G, Brizi M, Bianchi G, Tomassetti S, Bugia-nesi E, Lenzi M, McCullough AJ, Natale S, Forlani G
& Melchionda N (2001) Nonalcoholic fatty liver dis-ease: a feature of the metabolic syndrome Diabetes 50, 1844–1850
17 Browning JD & Horton JD (2004) Molecular mediators
of hepatic steatosis and liver injury J Clin Invest 114, 147–152
18 Hofmann SM, Perez-Tilve D, Greer TM, Coburn BA, Grant E, Basford JE, Tschop MH & Hui DY (2008) Defective lipid delivery modulates glucose tolerance and metabolic response to diet in apolipoprotein E-deficient mice Diabetes 57, 5–12
19 Kuipers F, Jong MC, Lin Y, Eck M, Havinga R, Bloks
V, Verkade HJ, Hofker MH, Moshage H, Berkel TJ
lipo-protein-triglycerides by apolipoprotein E-deficient mouse hepatocytes J Clin Invest 100, 2915–2922
20 Huang Y, Liu XQ, Rall SC Jr, Taylor JM, von Eckardstein A, Assmann G & Mahley RW (1998) Over-expression and accumulation of apolipoprotein E as a cause of hypertriglyceridemia J Biol Chem 273, 26388– 26393
21 Tsukamoto K, Maugeais C, Glick JM & Rader DJ (2000) Markedly increased secretion of VLDL triglyce-rides induced by gene transfer of apolipoprotein E iso-forms in apoE-deficient mice J Lipid Res 41, 253–259
22 Mensenkamp AR, Van Luyn MJ, Havinga R, Teusink
B, Waterman IJ, Mann CJ, Elzinga BM, Verkade HJ, Zammit VA, Havekes LM et al (2004) The transport
of triglycerides through the secretory pathway of he-patocytes is impaired in apolipoprotein E deficient mice
J Hepatol 40, 599–606
23 Mensenkamp AR, Jong MC, van Goor H, Van Luyn
MJ, Bloks V, Havinga R, Voshol PJ, Hofker MH, Van Dijk KW, Havekes LM et al (1999) Apolipoprotein E participates in the regulation of very low density