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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

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diet-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.

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hypercholesterolemia 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.

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sections 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.

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starting 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

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triglyceride-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.

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In 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.

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intestinal 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

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possibility 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

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in 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

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the 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

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