Deficiency in apolipoprotein E has a protective effect on 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
the efficient catabolism of
Apolipoprotein E (apoE) mediates 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
[1]. The importance of
Abbreviations apoE, apolipoprotein E; apoE) ⁄ ), apoE deficient; apoE3knock-in mice, 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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Apolipoprotein E (apoE) is a 34.2-kDa glycoprotein synthesized by the liver and other peripheral tissues. In humans, isoforms, there are three major natural 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) this protein in the maintenance of plasma lipid homeostasis and ath- eroprotection was first established with the generation the apoE-deficient mouse [8,9], which develops of
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hypercholesterolemia and spontaneous atherosclerosis [8,9]. Recently, using apoE-deficient
(apoE) ⁄ )) mice, C57BL ⁄ 6 mice and apoE3knock-in mice (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].
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
for a total period of 24 weeks. As 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 shown in Fig. 1A, hematoxylin and eosin staining of liver 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].
A
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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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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
expected from previously published (Fig. 1D), the liver
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 significantly elevated in C57BL ⁄ 6 relative to was 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 relative to apoE) ⁄ ) mice of (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).
**
As results, apoE) ⁄ ) mice were less sensitive than C57BL ⁄ 6 mice to the development of diet-induced obesity [10,18]. the experiment, Specifically, during the course of 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 To further confirm that deficiency in apoE prevented the accumulation of hepatic triglycerides in the liver of
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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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their
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).
consumed 3.8 ± 0.2
starting weight of 25.7 ± 0.2 g at week 0, P < 0.05). average body weight was At week 12, 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 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)1 at weeks 12 and 24, respectively (P > 0.05). Similarly, C57BL ⁄ 6 mice 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
at week 24 (126.7 ± 60.9 mgÆdL)1
versus
at week 24 versus
One mechanism that could affect the hepatic triglycer- ide content is the secretion of hepatic triglycerides 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) ⁄ ) 11.2 ± 0.9 mgÆdL)1Æmin)1 (minimum, mice 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. 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 levels of apoE) ⁄ ) mice were 1475 ± cholesterol 48 mgÆdL)1 (Fig. 2B), whereas their plasma triglyceride levels increased but remained within the physiological versus range 18.3 ± 1.9 mgÆdL)1 at 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 levels showed slightly elevated fasting cholesterol (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 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).
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Surprisingly, apoE) ⁄ ) mice, which do not develop NAFLD, had a higher plasma concentration of 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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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 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.
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Kinetics of post-prandial triglyceride clearance in apoE) ⁄ ) and C57BL ⁄ 6 mice
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in the next in these mice. Thus,
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) mice. **P < 0.005.
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 (
80.8 mgÆdL)1; SEM = 10.7). However,
significantly still
Another potential mechanism that could explain the reduced sensitivity of apoE) ⁄ ) mice to diet-induced NAFLD could be reduced clearance of plasma trigly- set of cerides experiments, we sought to determine the kinetics of post-prandial 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 levels reached (59.5 ± 10.7 mgÆdL)1; minimum, 20.7 mgÆdL)1; maxi- in mum, 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 rate. At the circulation at a significantly slower the plasma triglycerides of 240 min post-gavage, apoE) ⁄ ) mice were 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
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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- the rates were 11.9 ± 1.3 mgÆdL)1Æmin)1 for cally, and 14.5 ± 1.2 mgÆdL)1Æmin)1 apoE) ⁄ ) mice for C57BL ⁄ 6 mice (n = 5, P = 0.023). Then, by subtract- ing the rate of hepatic triglyceride secretion (deter- To address the potential role of LDLr in the apoE- mediated deposition of dietary triglycerides in the liver, (LDLr) ⁄ )) low density lipoprotein receptor-deficient 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.
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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 types were the microvesicular and macrovesicular 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
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 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 liver sections from control mice eosin staining of In order to identify the molecular basis for this phe- nomenon, we determined a number of parameters the delivery and deposition of which could affect
A
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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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for cannot account
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. rate of hepatic VLDL triglyceride
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 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 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.
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- time, may lead to cess that, over long periods of NAFLD.
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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 levels of apoE) ⁄ ) mice. It is in plasma cholesterol In vitro and in vivo studies have shown that lipopro- tein-bound apoE is ligand for LDLr the natural [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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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 in vivo mechanisms leading to NAFLD, making the use of 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.
Following a 16-h fasting period, plasma cholesterol, triglyc- eride and FFA levels were measured as described previously [36].
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. Plasma lipid determination
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. Fractionation of plasma lipoproteins by density gradient ultracentrifugation
Materials and methods
Pools (0.5 mL) of plasma from five apoE) ⁄ ) and five C57BL ⁄ 6 mice were fractionated by density gradient ultra- centrifugation over a 10-mL KBr density gradient, as described previously [37].
Animal studies
Body weight and body composition analyses were per- formed as described previously [10].
Body weight determination and body mass composition analysis
triglyceride and glucose
For hepatic triglyceride determination, a liver sample was collected, weighed and dissolved in 0.5 mL of 5 m KOH in 50% ethanol solution by overnight incubation at 65 (cid:2)C. 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.
Measurement of hepatic triglyceride content
At the end of the 24-week period, mice were sacrificed, and liver and visceral fat specimens were collected and stored at either )80 (cid:2)C or fixed in 10% formalin. Four-micrometer- thick sections were obtained from the formalin-fixed, paraffin-embedded tissue for further histological analyses. Conventional hematoxylin and eosin stain was performed
The apoE) ⁄ ) [9], LDLr) ⁄ ) [35] and C57BL ⁄ 6 mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA; http://www.jax.org); apoE) ⁄ ) mice were bred on the C57BL ⁄ 6 background for at least 10 generations. Male 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, 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, 0.05% from fat source), and contains 4.5 kcalÆg)1. The 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. liver and adipose tissue At the end of each experiment, specimens were collected and fixed in formalin for histo- pathological analyses. Carcasses were stored at )80 (cid:2)C and
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Histological analysis of liver samples
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[39]),
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 images camera (original magnification, ·10). The 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.
for 16 h. On the following mice were fasted overnight 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 Tri- ton-WR1339 at a dose of 500 mgÆ(kg body weight))1 using a 15% solution (w ⁄ v) in 0.9% NaCl (Triton-WR1339 has been shown to completely inhibit the catabolism of triglyc- eride-rich lipoproteins as described previously [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 accumulation, expressed as mgÆdL)1Æmin)1, was calculated 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.
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].
To measure the rate of hepatic VLDL triglyceride secre- tion, groups of four to six apoE) ⁄ ) and C57BL ⁄ 6 mice were injected with Triton-WR1339 at a dose of 500 mgÆ(kg body weight))1 using a 15% solution (w ⁄ v) in 0.9% NaCl, as described previously [26,36,37,40].
secretion of fat
Determination of daily food consumption
Subtraction of the rate of hepatic triglyceride secretion from the total plasma triglyceride supply yielded the rate triglyceride-rich chylomicrons intestinal of following an oral load, expressed as the mean ± SEM.
Determination of post-prandial triglyceride kinetics following the oral administration of olive oil
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.
Groups of five apoE) ⁄ ) and C57BL ⁄ 6 mice were tested. 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 isolated 1 min prior to the gavage determination was 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 mgÆdL)1 ± SEM.
Statistical analysis
Acknowledgements
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.
Rate of secretion of triglyceride-rich chylomicrons and VLDL
Briefly, to determine the total rate of triglyceride input in the plasma of mice, groups of five apoE) ⁄ ) and C57BL ⁄ 6 mice were tested. Prior to the experiment, the
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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 (awarded agreement PIRG02-GA-2009-256402 to the activities of D.J.P.). This work was part of the study of the research network ‘MetSNet’ for
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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.
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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