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 Department of Medicine, Pharmacology Unit, University of Patras School of Health Sciences, Rio-Achaias, Greece 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 June 2011, accepted 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 lipoprotein 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 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 ⁄ mice were fed a western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5 kcalỈg)1) for 24 weeks and their sensitivity to NAFLD was assessed by histological and biochemical methods apoE-deficient mice were less sensitive than control C57BL ⁄ 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 development 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 atheroprotection was first established with the generation of the apoE-deficient mouse [8,9], which develops 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 FEBS Journal 278 (2011) 3119–3129 ª 2011 The Authors Journal compilation ª 2011 FEBS 3119 Apolipoprotein E and diet-induced NAFLD E A Karavia et al hypercholesterolemia and spontaneous atherosclerosis [8,9] Recently, using apoE-deficient (apoE) ⁄ )) mice, C57BL ⁄ 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 homeostasis, apoE plays a central role in the development of diet-induced obesity and related metabolic dysfunctions in mice [10,11] Additional studies in genetically predisposed obese mice further confirmed that deficiency in apoE protects mice from obesity, insulin resistance and other metabolic abnormalities [12,13] Nonalcoholic fatty liver disease (NAFLD) is a spectrum of metabolic abnormalities ranging from simple accumulation of triglycerides in the liver (hepatic steatosis) 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 models, NAFLD in humans is characterized by obesity, insulin resistance and associated metabolic perturbations [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 D Results apoE) ⁄ ) mice are less sensitive than control C57BL ⁄ 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 ⁄ 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 B C As apoE possesses a central role in the metabolism of plasma lipoproteins and the development of dietinduced obesity, in this study we sought to determine how apoE affects the development of diet-induced NAFLD in mice To address this question 10–12week-old male apoE) ⁄ ) and wild-type (WT) C57BL ⁄ mice were fed a standard western-type diet (17.3% protein, 48.5% carbohydrate, 21.2% fat, 0.2% cholesterol, 4.5 kcalỈg)1) for 24 weeks, and histological and biochemical analyses were performed We found that deficiency in apoE has a protective effect on dietinduced 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 triglycerides in the liver and NAFLD which, over long periods of time, may lead to nonalcoholic steatohepatitis 3120 Fig Histological analyses of liver sections from apolipoprotein E-deficient (apoE) ⁄ )) and C57BL ⁄ mice (A, B) Representative photographs of hematoxylin and eosin-stained hepatic sections from apoE) ⁄ ) (A) and C57BL ⁄ (B) mice at week 24 on a western-type diet (C, D) Representative photographs of reticulin-stained hepatic sections of apoE) ⁄ ) (C) and C57BL ⁄ (D) mice fed a western-type diet for 24 weeks All photographs were taken at an original magnification of ·20 FEBS Journal 278 (2011) 3119–3129 ª 2011 The Authors Journal compilation ª 2011 FEBS E A Karavia et al Apolipoprotein E and diet-induced NAFLD sections revealed that deficiency in apoE did not result in any significant distortion of liver microscopic morphology or accumulation of triglycerides in the liver of apoE) ⁄ ) mice fed a western-type diet for 24 weeks In contrast, control C57BL ⁄ 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 analysis 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 ⁄ relative to apoE) ⁄ ) mice (P = 0.0001) In agreement with these data, staining of hepatic sections with reticulin showed that, in C57BL ⁄ mice fed a western-type diet for 24 weeks, NAFLD had progressed much more extensively 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 visceral 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 samples were isolated from apoE) ⁄ ) and C57BL ⁄ mice and their triglyceride contents were determined biochemically, 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 mgỈ(g hepatic tissue))1, whereas C57BL ⁄ mice had a much higher hepatic triglyceride content [155.7 ± 10 mgỈ(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 ⁄ 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 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 200 ** ** 150 ** ** 100 50 0 12 Weeks 18 24 –1 ** 1250 ** ** 1000 750 500 250 175 C57BL/6 150 apoE–/– 125 100 75 50 25 HDL 150 125 100 75 50 25 12 Weeks 18 24 Triglycerides (mg·dL–1) F 150 100 50 12 Weeks FEBS Journal 278 (2011) 3119–3129 ª 2011 The Authors Journal compilation ª 2011 FEBS 9 Fraction number 200 CHYL/VLDL/IDL 175 C –1 ** 200 E 200 1500 Triglycerides (mg·dL ) Fig Biochemical parameters of apolipoprotein E-deficient (apoE) ⁄ )) and C57BL ⁄ 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, respectively (D) Average hepatic triglyceride content 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 corresponds to the top fraction [containing chylomicrons (CHYL) and very low-density lipoprotein (VLDL)] HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; Tg, triglyceride Cholesterol (mg·dL ) B D Hepatic Triglyceride content [mg Tg·(g tissue)–1] 250 apoE–/– C57BL/6 300 Cholesterol (mg·dL–1) % of initial body weight A 18 24 10 CHYL/VLDL/IDL 10 HDL 10 Fraction number 3121 Apolipoprotein E and diet-induced NAFLD E A Karavia et al 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 ⁄ mice showed a significant increase in their body weight during the course of the experiment At week 6, C57BL ⁄ 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 ± 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 relative 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 ⁄ mice corresponds 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 differences in hepatic triglyceride accumulation in apoE) ⁄ ) and C57BL ⁄ mice, fasting plasma samples were isolated every 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 mgỈdL)1 (Fig 2B), whereas their plasma triglyceride levels increased but remained within the physiological range (126.7 ± 60.9 mgỈdL)1 at week 24 versus 18.3 ± 1.9 mgỈdL)1 at week 0) (Fig 2C) Ultracentrifugation analysis of plasma samples showed that the hypercholesterolemia of these mice was caused by the increased accumulation of triglyceride-containing cholesterol-rich chylomicron remnants (Fig 2E,F) However, C57BL ⁄ mice on a high-fat diet for 24 weeks showed slightly elevated fasting cholesterol levels (224.6 ± 21 mgỈdL)1) relative to their starting cholesterol levels at week (91.9 ± 10 mgỈdL)1) (Fig 2B), whereas their plasma triglyceride levels remained normal (79.4 ± 7.4 mgỈdL)1 at week 24 versus 58.2 ± 1.1 mgỈdL)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 not develop NAFLD, had a higher plasma concentration of 3122 FFAs than C57BL ⁄ mice Steady-state FFA levels of apoE) ⁄ ) mice were 7.6 ± 1.2 mmol eq., whereas C57BL ⁄ 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 triglyceride 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 gỈmouse)1Ỉday)1 at weeks 12 and 24, respectively (P > 0.05) Similarly, C57BL ⁄ mice consumed 3.8 ± 0.2 and 3.4 ± 0.2 gỈmouse)1Ỉday)1 at weeks 12 and 24, respectively (P > 0.05) There was no statistically significant difference 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 gỈmouse)1Ỉday)1 for apoE) ⁄ ) and C57BL ⁄ 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 ⁄ mice One mechanism that could affect the hepatic triglyceride 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 ⁄ mice In accordance with previous studies [19–21], we found that the rate of hepatic triglyceride secretion decreased significantly in apoE) ⁄ ) relative to C57BL ⁄ mice Specifically, secretion rates were 2.1 ± 0.4 mgỈdL)1Ỉmin)1 (minimum, 1.7 mgỈdL)1Ỉmin)1; maximum, 3.5 mgỈdL)1Ỉmin)1; SEM = 0.4, n = 5) for apoE) ⁄ ) mice versus 11.2 ± 0.9 mgỈdL)1Ỉmin)1 (minimum, 9.8 mgỈdL)1Ỉmin)1; maximum, 13.7 mgỈdL)1Ỉmin)1; SEM = 0.9, n = 5) for C57BL ⁄ 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 FEBS Journal 278 (2011) 3119–3129 ª 2011 The Authors Journal compilation ª 2011 FEBS E A Karavia et al 15 10 C 57 BL / ap oE – 2000 Triglycerides (mg·dL–1) B mined above) from the total rate of plasma triglyceride supply, the rate of intestinal triglyceride secretion was determined as 9.8 ± 1.3 mgỈdL)1Ỉmin)1 for apoE) ⁄ ) mice and 2.0 ± 0.7 mgỈdL)1Ỉmin)1 for C57BL ⁄ mice (n = 5, P = 0.023) The data suggest that differences in intestinal triglyceride absorption or hepatic triglyceride secretion cannot account for the observed histological differences between apoE) ⁄ ) and C57BL ⁄ mice ** /– Rate of hepatic VLDL-triglyceride secretion (mg·dL–1·min–1) A Apolipoprotein E and diet-induced NAFLD apoE–/– Kinetics of post-prandial triglyceride clearance in apoE) ⁄ ) and C57BL ⁄ mice C57BL/6 1500 1000 SlopeapoE–/– =11.9 ± 1.3 (mg·dL–1·min–1) 500 SlopeC57BL/6 =14.5 ± 1.2 (mg·dL–1·min–1) 60 90 120 150 180 Time (min) C Plasma triglycerides (mg·dL–1) 200 C57BL/6 ** 150 ** apoE–/– 100 50 0 30 60 90 120 150 180 210 240 270 300 330 360 Time post-gavage (min) Fig Analysis of kinetic parameters associated with hepatic triglyceride 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 ⁄ (m) mice (C) Kinetics of post-prandial triglyceride clearance in apoE) ⁄ ) (h) and C57BL ⁄ ( ) mice **P < 0.005 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 ⁄ mice fed a western-type diet, following an oral fat load Groups of five apoE) ⁄ ) and C57BL ⁄ mice were fasted for 16 h, and then given an oral fat load of 300 lL of olive oil, as described in the Materials and methods section One hour post-gavage, mice were injected with Triton WR1339 and plasma triglyceride 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 ⁄ mice Specifically, the rates were 11.9 ± 1.3 mgỈdL)1Ỉmin)1 for apoE) ⁄ ) mice and 14.5 ± 1.2 mgỈdL)1Ỉmin)1 for C57BL ⁄ mice (n = 5, P = 0.023) Then, by subtracting the rate of hepatic triglyceride secretion (deter- Another potential mechanism that could explain the reduced sensitivity of apoE) ⁄ ) mice to diet-induced NAFLD could be reduced clearance of plasma triglycerides 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 mgỈdL)1, respectively, at 120 post-gavage (n = 5, P = 0.2195) (Fig 3C) However, there was a significant difference in post-prandial triglyceride clearance in apoE) ⁄ ) mice relative to C57BL ⁄ mice In particular, in C57BL6 mice, the rapid increase in plasma triglyceride levels at 120 after olive oil administration was followed by an immediate and steep decline At 240 post-gavage, the plasma triglycerides of C57BL ⁄ mice reached baseline levels (59.5 ± 10.7 mgỈdL)1; minimum, 20.7 mgỈdL)1; maximum, 80.8 mgỈdL)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 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 post-gavage, the plasma triglycerides of apoE) ⁄ ) mice were still significantly elevated (137.5 ± 21.9 mgỈdL)1; minimum, 106.5 mgỈdL)1; maximum, 184.0 mgỈdL)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 apoEmediated 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 triglyceride content by biochemical and histological analyses FEBS Journal 278 (2011) 3119–3129 ª 2011 The Authors Journal compilation ª 2011 FEBS 3123 Apolipoprotein E and diet-induced NAFLD E A Karavia et al In agreement with our previous studies, LDLr) ⁄ ) mice were more susceptible than apoE) ⁄ ) mice to dietinduced obesity, but more resistant than C57BL ⁄ mice [10] Surprisingly, however, we found that hepatic specimens from LDLr) ⁄ ) mice showed a higher triglyceride content than those of control C57BL ⁄ mice [233.0 ± 19 versus 155.7 ± 10 mgỈ(g hepatic tissue))1, respectively] Our biochemical results were in agreement with data from our histological analyses, which showed that LDLr) ⁄ ) mice developed NAFLD that had progressed even more than that of control C57BL ⁄ 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 protective effect against diet-induced NAFLD, which correlates mainly with the reduced clearance of postprandial triglycerides from the circulation Histological evaluation following hematoxylin and eosin staining of liver sections from control mice A B C D 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 microscopic 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 ⁄ mice, which showed heavy loading with fat The reticulin stain is a classical histopathological marker for the identification of hepatic architecture and structural damage within the liver parenchyma Therefore, 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 minimal 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 ⁄ mice (Fig 1D) corresponds to an extensive destruction of the hepatic architecture, primarily as a result of lipid accumulation within the hepatocytes and the development of NAFLD in these mice In order to identify the molecular basis for this phenomenon, we determined a number of parameters which could affect the delivery and deposition of 3124 Fig Histological analyses of liver sections from low-density lipoprotein receptordeficient (LDLr) ⁄ )) and C57BL ⁄ mice (A, B) Representative photographs of hematoxylin and eosin-stained hepatic sections from LDLr) ⁄ ) (A) and C57BL ⁄ (B) mice at week 24 on a western-type diet (C, D) Representative photographs of reticulin-stained hepatic sections of LDLr) ⁄ ) (C) and C57BL ⁄ (D) mice fed a western-type diet for 24 weeks All photographs were taken at an original magnification of ·20 FEBS Journal 278 (2011) 3119–3129 ª 2011 The Authors Journal compilation ª 2011 FEBS E A Karavia et al intestinal dietary triglycerides in the liver of the experimental mice In general, hepatic triglyceride content is a function of three parameters: (a) dietary triglyceride deposition in the liver; (b) endogenous triglyceride synthesis 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 ⁄ mice as it is well established that intracellular triglyceride turnover and synthesis, as well as the activities of diacylglycerol acyltransferase and microsomal triglyceride transfer protein, are comparable between apoE) ⁄ ) and WT C57BL ⁄ mice [22] Similarly, differences in the rate of hepatic VLDL triglyceride secretion between apoE) ⁄ ) and C57BL ⁄ mice could not explain the observed resistance of apoE) ⁄ ) mice to dietinduced NAFLD Consistent with previous data [19–21,23], we found that apoE) ⁄ ) mice displayed approximately five times slower hepatic VLDL triglyceride secretion compared with control C57BL ⁄ mice (2.1 ± 0.4 mgỈdL)1Ỉmin)1 for apoE) ⁄ ) mice versus 11.2 ± 0.9 mgỈdL)1Ỉmin)1 for C57BL ⁄ 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 triglycerides relative to C57BL ⁄ mice (2.0 ± 0.7 mgỈdL)1Ỉmin)1 in C57BL ⁄ mice versus 9.8 ± 1.3 mgỈdL)1Ỉmin)1 in apoE) ⁄ ) mice; P < 0.05) However, apoE) ⁄ ) mice displayed 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 baseline 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 Apolipoprotein E and diet-induced NAFLD interesting that, in our experiments, plasma cholesterol levels were inversely related to the hepatic accumulation of dietary triglycerides in mice Although, in our study, apoE) ⁄ ) mice appeared to be less sensitive to hepatic lipid deposition relative to control apoEexpressing C57BL ⁄ mice, previous work by Ma et al [25] has shown that artificially induced low-grade inflammatory stress triggered by subcutaneous injection of 10% casein increases the sensitivity of these mice to NAFLD development In the future, it would be interesting to compare how casein-induced inflammation affects the sensitivity of apoE) ⁄ ) and C57BL ⁄ 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 mgỈdL)1), although they were elevated compared with those of C57BL ⁄ 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 lipolysismediated release of FFAs is more efficient in apoE) ⁄ ) mice than in apoE-expressing C57BL ⁄ mice [27] In agreement with these studies, apoE) ⁄ ) mice showed elevated plasma FFA levels relative to C57BL ⁄ mice (apoE) ⁄ ) mice had steady-state FFA levels of 7.6 ± 1.2 mmol eq., whereas C57BL ⁄ 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 production and in steady-state plasma FFA levels, our apoE) ⁄ ) mice were resistant to diet-induced NAFLD and obesity Thus, our data not support the notion that elevated plasma FFAs are pivotal for the accumulation 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 deposition of post-prandial triglycerides in the liver, a process that, over long periods of time, may lead to NAFLD In vitro and in vivo studies have shown that lipoprotein-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 independent of LDLr, as LDLr) ⁄ ) mice fed a westerntype diet for 24 weeks developed significant NAFLD that was more severe than in C57BL ⁄ mice One FEBS Journal 278 (2011) 3119–3129 ª 2011 The Authors Journal compilation ª 2011 FEBS 3125 Apolipoprotein E and diet-induced NAFLD E A Karavia et al possibility is that the effects of apoE on hepatic lipid accumulation are mediated by LDLr-related protein or CD36, or, potentially, other apoE receptors However, 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 supported 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 triglycerides in the liver is associated with its lipoprotein-clearing function Our data extend our current knowledge on NAFLD development Although additional experiments will be needed in order to determine whether receptors mediate 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 dietinduced NAFLD in mice Materials and methods Animal studies The apoE) ⁄ ) [9], LDLr) ⁄ ) [35] and C57BL ⁄ mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA; http://www.jax.org); apoE) ⁄ ) mice were bred on the C57BL ⁄ 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, triglyceride and glucose levels, and body weights, of the individual animals Mice were fed a standard 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 western-type diet is composed of 17.3% protein, 48.5% carbohydrate, 21.2% fat and 0.2% cholesterol (0.15% added, 0.05% from fat source), and contains 4.5 kcalỈg)1 The contents of the main ingredients, expressed as gram per kilogram 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 carbonate, 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 histopathological analyses Carcasses were stored at )80 °C and 3126 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 consideration the ‘3Rs’ (reduce, refine, replace) and minimized the number of animal experiments to the absolute minimum To date, there is no in vitro system to mimic satisfactorily 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 Plasma lipid determination Following a 16-h fasting period, plasma cholesterol, triglyceride and FFA levels were measured as described previously [36] Fractionation of plasma lipoproteins by density gradient ultracentrifugation Pools (0.5 mL) of plasma from five apoE) ⁄ ) and five C57BL ⁄ mice were fractionated by density gradient ultracentrifugation 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 performed 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 m KOH in 50% ethanol solution by overnight incubation at 65 °C The solution was adjusted to pH 7, and the final volume was recorded The total amount of triglycerides was determined 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 either )80 °C or fixed in 10% formalin Four-micrometerthick sections were obtained from the formalin-fixed, paraffin-embedded tissue for further histological analyses Conventional hematoxylin and eosin stain was performed FEBS Journal 278 (2011) 3119–3129 ª 2011 The Authors Journal compilation ª 2011 FEBS E A Karavia et al 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 camera (original magnification, ·10) The digital images were imported into Adobe Photoshop CS2 and a grid was added For each section, the number of lipid vacuoles intersected by the grid was determined and calculated independently 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 measurements, as described previously [38] Determination of post-prandial triglyceride kinetics following the oral administration of olive oil Groups of five apoE) ⁄ ) and C57BL ⁄ 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 isolated 30, 60, 120, 180 and 240 following olive oil administration A control sample for baseline triglyceride determination was isolated prior to the gavage administration of olive oil Triglyceride levels were quantified in plasma samples as described above, and then plotted on graphs as a function of time Values were expressed as mgỈdL)1 ± SEM Apolipoprotein E and diet-induced NAFLD 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 h (in our experimental set-up, dietary triglyceride absorption, measured as a post-gavage increase in plasma triglyceride levels, becomes apparent at approximately h following the oral administration of olive oil) The mice were then injected with Triton-WR1339 at a dose of 500 mgỈ(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 triglyceride-rich lipoproteins [39]), as described previously [26,36,37,40] Serum samples were isolated at 30, 60, 90, 120, 150 and 180 after injection with Triton-WR1339 As a control, serum samples were isolated approximately after injection with the detergent Plasma triglyceride levels at each time point were determined as described above, and linear graphs of triglyceride concentration versus time were generated The rate of plasma triglyceride accumulation, expressed as mgỈdL)1Ỉmin)1, was calculated from the slope of the linear graphs The slopes were reported as the mean ± SEM The total plasma triglyceride supply equals the sum of intestinal and hepatic triglyceride secretion To measure the rate of hepatic VLDL triglyceride secretion, groups of four to six apoE) ⁄ ) and C57BL ⁄ mice were injected with Triton-WR1339 at a dose of 500 mgỈ(kg body weight))1 using a 15% solution (w ⁄ v) in 0.9% NaCl, as 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 analyzed using ANOVA Data are reported as the mean ± SEM; n indicates the number of animals tested in the group Acknowledgements 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 in the plasma of mice, groups of five apoE) ⁄ ) and C57BL ⁄ mice were tested Prior to the experiment, the This work was supported by the European Community’s Seventh Framework Program [FP7 ⁄ 2007-2013] grant agreement PIRG02-GA-2007-219129, The University 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.) 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Havekes LM & Zannis VI (2005) Generation of a recombinant apolipoprotein E variant with improved biological functions: hydrophobic residues (LEU-261, TRP-264, PHE-265, LEU-268, VAL-269) of apoE can account for the apoE-induced hypertriglyceridemia J Biol Chem 280, 6276–6284 FEBS Journal 278 (2011) 3119–3129 ª 2011 The Authors Journal compilation ª 2011 FEBS 3129 ... 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... 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 homeostasis, apoE... 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