Apolipoprotein E predisposes to obesity and related metabolic dysfunctions in mice Iordanes Karagiannides 1,2 , Rami Abdou 1 , Aikaterini Tzortzopoulou 1 , Peter J. Voshol 3 and Kyriakos E. Kypreos 1,4 1 Whitaker Cardiovascular Institute, Boston University School of Medicine, MA, USA 2 Beth Israel Deaconess Medical Center, Gastrointestinal Neuropeptide Center, Division of Gastroenterology, Harvard Medical School, Boston, MA, USA 3 Department of Endocrinology, Leiden University Medical Center, The Netherlands 4 Department of Medicine, Pharmacology Unit, University of Patras Medical School, Rio, Greece Obesity and its related pathologies constitute a major cause of death, with rates increasing at an alarming pace [1]. By the beginning of the millennium, over- weight adults accounted for over 15% of the world’s population (body mass index > 30, World Health Organization) [2], with this number increasing to 50% within the USA and Europe [3]. Obesity develops as a result of disruption of the homeostasis between food intake and energy expenditure, and therefore factors affecting these processes are the focus of extensive Keywords ApoE3 knock-in mice; apolipoprotein E; glucose intolerance; insulin resistance; obesity Correspondence K. E. Kypreos, Department of Medicine, Pharmacology Unit, University of Patras Medical School, Panepistimioupolis, Rio, TK 26500, Greece Fax: +30 2610996103 Tel: +30 2610969120 E-mail: kkypreos@med.upatras.gr (Received 23 February 2008, revised 25 July 2008, accepted 30 July 2008) doi:10.1111/j.1742-4658.2008.06619.x Obesity is a central feature of the metabolic syndrome and is associated with increased risk for insulin resistance and type II diabetes. Here, we investigated the contribution of human apoliprotein E3 and mouse apoli- protein E to the development of diet-induced obesity in response to western-type diet. Our data show that apolipoprotein E contributes to the development of obesity and other related metabolic disorders, and that human apolipoprotein E3 is more potent than mouse apolipoprotein E in promoting obesity in response to western-type diet. Specifically, we found that apolipoprotein E3 knock-in mice fed western-type diet for 24 weeks became obese and developed hyperglycemia, hyperinsulinemia, hyperleptin- emia, glucose intolerance and insulin resistance that were more severe than in C57BL/6 mice. In contrast, apolipoprotein E-deficient mice fed western- type diet for the same period were resistant to diet-induced obesity, had normal plasma glucose, leptin and insulin levels, and exhibited normal responses to glucose tolerance and insulin resistance tests. Furthermore, low-density lipoprotein receptor-deficient mice were more sensitive to the development of diet-induced obesity and insulin resistance than apolipo- protein E-deficient mice, but were still more resistant than C57BL/6 mice, raising the possibility that low-density lipoprotein receptor mediates, at least in part, the effects of apolipoprotein E on obesity. Taken together, our findings suggest that, in addition to other previously identified mecha- nisms of obesity, apolipoprotein E and possibly the chylomicron pathway are also important contributors to the development of obesity and related metabolic dysfunctions in mice. Abbreviations ApoE, apolipoprotein E; ApoE )/), ApoE-deficient; ApoE3 knock-in mice, mice containing a targeted replacement of the mouse ApoE gene for the human ApoE3 gene; GTT, glucose tolerance test; IST, insulin sensitivity test; LDLr, low-density lipoprotein receptor; LDLr )/) , LDLr- deficient; LpL, lipoprotein lipase; VLDL, very low-density lipoprotein. 4796 FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS research for the development of effective antiobesity drugs, with only limited success being achieved thus far [4]. Aging, hormonal imbalance and genetic predis- position may also contribute to obesity [5–16]. How- ever, very few cases of human obesity are reported to be caused by genetic factors [17], leaving western-type diet and sedentary lifestyle, physical inactivity and imbalance in caloric load as the most common contrib- utors to the development of central obesity and the metabolic syndrome [2,18]. The risk of developing all other components of the metabolic syndrome increases with obesity, supporting the hypothesis that obesity is the central feature of the syndrome [19]. It is well established that abdominal obesity may result in insulin resistance and hyperinsu- linemia [19,20]. Epidemiological and population studies have established a direct correlation between obesity and the development of cardiovascular disease [21,22]. Despite the pivotal role of obesity and dyslipidemia in the development of the metabolic syndrome and heart disease, the functional interactions between adipose tissue and the lipid and lipoprotein transport system have not yet been investigated thoroughly. 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 [23], with ApoE3 being the most common [23–29]. ApoE is a major protein component of chylomicron remnants and very low-density lipopro- tein (VLDL) [23]. The importance of this protein in the maintenance of plasma lipid homeostasis and athero- protection was first established with the generation of the ApoE-deficient mouse [30,31], which develops hypercholesterolemia and spontaneous atherosclerosis [30,31]. Lipid-bound ApoE is the natural ligand of the low-density lipoprotein receptor (LDLr) [32–34], a cell surface receptor that is responsible for the catabolism of atherogenic lipoproteins [32,35–37]. In this study, we sought to determine the role of ApoE in the development of diet-induced obesity, glu- cose intolerance and insulin resistance in vivo. To address this question, female ApoE3 knock-in , wild-type C57BL/6, LDLr-deficient (LDLr )/) ) and ApoE-defi- cient (ApoE )/) ) mice were fed western-type diet for a period of 15 or 24 weeks, during which time their plasma lipid and glucose levels, body weight, body com- position, glucose tolerance and insulin sensitivity were monitored. We chose to study ApoE3 because it is the most common ApoE isoform in humans [24–29]. Our data establish that expression of ApoE predisposes mice to diet-induced obesity, hyperglycemia and insulin resis- tance, whereas deficiency in ApoE renders mice resistant to these conditions. Human ApoE3 appeared to be more potent than mouse ApoE in promoting obesity in response to western-type diet. Furthermore, LDLr )/) mice were more sensitive to the development of diet- induced obesity and insulin resistance than ApoE )/) mice, but still more resistant than wild-type C57BL/6 mice in response to western-type diet. Gavage adminis- tration of olive oil containing the nonhydrolyzable [ 3 H]cholesteryl-hexadecyl-ether to mice suggested that deficiency in LDLr and ApoE reduces the direct delivery of postprandial nonhydrolyzed lipids to the liver, one of the major tissues involved in glucose uptake from the circulation. A similar trend was also observed in the delivery of nonhydrolyzed dietary lipids to adipose tissue. Taken together, our data establish that ApoE is a key mediator of diet-induced obesity in response to western-type diet. Results ApoE promotes diet-induced weight gain in mice, whereas ApoE deficiency prevents it To test the effects of ApoE on weight gain in mice, groups of 4–6 weeks old female ApoE3 knock-in , ApoE )/) and wild-type C57BL/6 mice were placed on western- type or normal chow diet for a total period of 24 weeks. Mice in each group were weighed immedi- ately before the initiation of the experiment (week 0) and every 6 weeks thereafter up to week 24, using a Mettler precision microscale. It became apparent that as early as 6 weeks on high- fat diet, ApoE3-expressing mice gained weight and were significantly heavier than the wild-type C57BL/6 mice on the same diet (Fig. 1A). The weight of the ApoE3 knock–in mice was 31.37 ± 3.98 g (115 ± 34% higher than their initial weight of 17.03 ± 0.94 g, P < 0.05) (Fig. 1A). During the same period, C57BL/ 6 mice on a high-fat diet had an average body weight of 26.08 ± 1.89 g (66 ± 8% higher than their initial weight of 16.26 ± 0.61 g, P < 0.05) (Fig. 1A). There were no statistical differences between the weights of the ApoE3 knock-in and C57BL/6 control groups fed chow diet for 6 weeks (data not shown). At week 24 on high-fat diet, ApoE3-expressing mice showed a dramatic increase in body weight, with an average value of 50.23 ± 2.22 g (235 ± 32% higher than their starting weight at week 0, P < 0.05) (Fig. 1A). The body weight of the C57BL/6 mice was 43.10 ± 0.94 g (164 ± 9% higher than their starting weight at week 0, P < 0.05) (Fig. 1A). The control ApoE3 knock-in and C57BL/6 mouse groups on chow diet showed a much smaller increase in body weight, ranging between 22 and 24 g (29 ± 6% increase as compared to I. Karagiannides et al. ApoE and obesity FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS 4797 their starting weight at week 0, P < 0.05) (data not shown). In contrast to the ApoE3 knock-in and the C57BL/6 mice, ApoE )/) mice that were fed western-type diet showed only a modest increase in body weight dur- ing the course of the experiment (Fig. 1A). At week 6 of the experiment, the ApoE )/) mouse group had an aver- age body weight of 20.36 ± 1.37 g (32 ± 6% increase as compared to their starting weight of 16.26 ± 0.61 g at week 0, P < 0.05). At week 24, their body weight was 24.58 ± 1.07 g (41 ± 4% increase as compared to their starting weight at week 0, P < 0.05) (Fig. 1A). Similar increases in body weight were observed in the control ApoE )/) mice fed chow diet (data not shown). To compare the steady-state plasma ApoE levels between ApoE3 knock-in and C57BL/6 mice, at week 0 of the experiment plasma samples were isolated from three mice from each group and 5 lL of plasma was analyzed by western blotting using a polyclonal antibody that recognizes both human and mouse ApoE (Santa-Cruz Biotech, Santa Cruz, CA, USA; cat. no. sc-31821). This analysis showed that C57BL/6 mice had approximately four-fold higher steady-state plasma ApoE levels than ApoE3 knock-in mice, suggest- ing that the increased sensitivity of ApoE3 knock-in mice to obesity is not due to higher plasma ApoE levels in these mice compared to C57BL/6 mice (Fig. 1B). To determine whether body weight differences among groups fed western-type diet could be explained by differences in their average daily food consumption, at week 12 of the experiment we determined the average daily food consumption for each mouse group. It was found that ApoE3 knock-in mice consumed 0 6 12 18 24 0 250 500 750 1000 1250 1500 Cholesterol (mg·dL –1 ) A C B 0 6121824 0 25 50 75 Triglycerides (mg·dL –1 ) 0 6121824 0 400 300 200 100 ApoE3 knock in ApoE Ponseau S ApoE3 knock in ApoE3 knock in ApoE3 knock in C57BL/6 C57BL/6 apoE –/– ApoE3 knock in C57BL/6 apoE –/– C57BL/6 C57BL/6 ApoE –/– apoE –/– ApoE3 knock in C57BL/6 ApoE –/– % of initial body-weight Weeks Weeks Weeks * * * * * ** * 0 10 20 30 40 50 ** * * * % body TG content D E 0 25 50 75 100 125 150 175 week 0 week 24 ** ** ** ** ** Fasting plasma glucose (mg·dL –1 ) F 3213 2 1 Fig. 1. Percentage of initial body weight (A), plasma ApoE levels (B), plasma cholesterol levels (C) and plasma triglyceride levels (D) of C57BL/6, ApoE3 knock-in and ApoE )/) mice fed western-type diet for a period of 24 weeks. (E) Percentage body fat content of ApoE3 knock-in , ApoE )/) and C57BL/6 mice at week 24. (F) Fasting plasma glucose levels of ApoE3 knock-in , ApoE )/) and C57BL/6 mice at weeks 0 and 24. Each point on the graphs represents the mean value of the group, and error bars indicate the SEM. The statistical significance of the observed differences among groups at each time-point is as indicated (*P < 0.05; **P < 0.005). In (B), plasma ApoE levels in ApoE3 knock-in and ApoE )/) mice were determined by western blot analysis using an antibody that recognizes equally mouse ApoE and human ApoE. Ponceau S staining of the nitrocellulose membrane was used to confirm equal loading and efficient transfer of proteins to the membrane. ApoE and obesity I. Karagiannides et al. 4798 FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS 3.04 ± 0.13 g per mouse per day, C57BL/6 mice con- sumed 3.31 ± 0.15 g per mouse per day, and ApoE )/) mice consumed 3.19 ± 0.17 g per mouse per day, and there was no statistical difference among groups (P > 0.05). Plasma lipid levels of the mice fed western-type diet for 24 weeks During the 24-week period of feeding mice with western-type diet, fasting plasma samples were taken every 6 weeks, and cholesterol, triglyceride and free fatty acid levels were measured as described in Experimental procedures. As shown in Fig. 1C, at week 24 both ApoE3 knock in and C57BL/6 mice on high-fat diet had slightly elevated fasting cholesterol levels (138 ± 10 mgÆdL )1 and 188 ± 14 mgÆdL )1 respectively) as compared to their starting cholesterol levels at week 0 (50 ± 3 mgÆdL )1 and 54 ± 6mgÆdL )1 , respectively), whereas their plasma triglyc- eride levels remained normal (24 ± 7 mgÆdL )1 and 24 ± 5 mgÆdL )1 , respectively) (Fig. 1D). FPLC anal- ysis of plasma from these mice showed that the small increases in the cholesterol levels of these mice at week 24 were due to a minor accumulation of chylomicron and VLDL remnants (Fig. 2A,B). In contrast, ApoE )/) mice showed a dramatic increase in their plasma cholesterol levels during the course of the experiment (Fig. 1C). At week 24 of the experiment, plasma cholesterol levels of the ApoE )/) mice were 1064 ± 198 mgÆdL )1 (Fig. 1C), whereas their plasma triglyceride levels remained normal (52 ± 28 mgÆdL )1 at week 24) (Fig. 1D). FPLC analysis showed that the hypercholesterolemia of these mice was due to increased accumulation of triglyceride-containing cholesterol-rich chylomicron remnants (Fig. 2A,B). No significant difference in the plasma free fatty acid levels among groups was observed during the course of the experiment. At week 24, plasma free fatty acid levels of the ApoE3 knock-in , C57BL/6 and ApoE )/) mice were 0.89 ± 0.08 mmol equiv., 0.81 ± 0.05 mmol equiv., and 0.99 ± 0.08 mmol equiv., respectively. Body composition analysis of the mice fed western-type diet for 24 weeks At the end of the 24-week period on western-type diet, at least six mice from each group (ApoE3 knock-in , ApoE )/) and C57BL/6 mice) were killed. The total weight, dry weight, water content, lipid content and lean body mass of the mice were determined as described in Experimental procedures. As shown in Fig. 1E, this analysis established that ApoE3 knock-in mice had a total body lipid content of 39 ± 4%. The wild-type C57BL/6 mice had a significantly lower total body lipid content of 32 ± 3% (P < 0.05). Thus, the increased body weight of the ApoE3 knock-in and C57BL/6 mice reflects excess accumulation of adipose tissue in these mice. In contrast, ApoE )/) mice remained lean during the course of the experiment, with a total body fat content of 11 ± 1% (Fig. 1E, P < 0.005). The complete body composition analysis of the mice fed western-type diet for 24 weeks is sum- marized in Table 1. Diet-induced obesity in ApoE3 knock-in and C57BL/6 mice is associated with elevated plasma glucose, insulin and leptin levels Epidemiological and animal studies have established that central obesity is associated with glucose intoler- ance and insulin resistance [20]. In addition, obesity is accompanied by increased levels of leptin [38], a hor- mone that reduces appetite and may function as the link between obesity and hypertension in individuals with the metabolic syndrome [39,40]. To determine whether the obesity observed in ApoE3 knock-in and C57BL/6 mice is associated with 0 100 200 300 400 Cholesterol (mg·dL –1 ) 0 2 4 6 810 FPLC fractions 12 14 16 18 20 22 24 0246810 FPLC fractions 12 14 16 18 20 22 24 ApoE3 knock-in C57BL/6 ApoE –/– ApoE3 knock-in C57BL/6 ApoE –/– 0 10 20 30 Week 24 Week 24 Triglycerides (mg·dL –1 ) A B Fig. 2. Representative FPLC cholesterol (A) and triglyceride (B) pro- files of C57BL/6, ApoE3 knock-in and ApoE )/) mice fed western-type diet for a period of 24 weeks. I. Karagiannides et al. ApoE and obesity FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS 4799 hyperglycemia, at weeks 0 and 24 of the experiment mice were fasted for 16 h and plasma glucose levels were then measured. Immediately prior to switching mice to western-type diet (week 0), the fasting plasma glucose levels of the ApoE3 knock-in , ApoE )/) and C57BL/6 mice were 84.2 ± 3.7 mgÆdL )1 , 60.7 ± 2.8 mgÆdL )1 , and 75.2 ± 3.5 mgÆdL )1 , respec- tively (Fig. 1F, P < 0.05). After 24 weeks on western-type diet, ApoE3 knock-in and control C57BL/6 mice developed hyperglycemia with fasting glucose levels of 146.5 ± 9.1 mgÆdL )1 (P < 0.05) and 135.4 ± 7.9 mgÆdL )1 (P < 0.05) respectively (Fig. 1F). In contrast, ApoE )/) mice, which remained lean during the course of the experiment, had only slightly elevated fasting glucose levels that were within the physiological range (94.3 ± 3.22 mgÆdL )1 , P < 0.005). To determine plasma insulin and leptin levels in these mice, serum samples were isolated at weeks 0 and 24 of the experiment and analyzed for insulin and leptin. At week 0, all three mouse groups had similar plasma insulin levels (0.17 ± 0.05 ngÆmL )1 for ApoE3 knock-in mice, 0.12 ± 0.05 ngÆmL )1 for ApoE )/) mice, and 0.16 ± 0.01 ngÆmL )1 for C57BL/6 mice). At week 24, ApoE3 knock-in and C57BL/6 mice had elevated plasma insulin levels, with concentrations in the range of 4.73 ± 1.03 ngÆmL )1 (P < 0.05) and 1.28 ± 0.32 ngÆmL )1 (P < 0.05), respectively. In contrast, ApoE )/) mice fed western- type diet for 24 weeks had insulin levels of 0.23 ± 0.07 ngÆmL )1 , which were similar to the levels of ApoE )/) mice (0.317 ± 0.17 ngÆmL )1 ) on chow diet for the same period of time. Analysis of plasma leptin levels showed that at week 0, mice had similar leptin levels: 5.90 ± 0.40 ngÆmL )1 for ApoE3 knock-in mice, 4.51 ± 0.32 ngÆmL )1 for ApoE )/) mice, and 9.2 ± 0.30 ngÆmL )1 for C57BL/6 mice. At week 24, the plasma leptin levels of the ApoE )/) mice were reduced to 2.1 ± 0.4 ngÆmL )1 . In contrast, in ApoE3 knock-in mice fed western-type diet for 24 weeks, leptin levels increased dramatically to 41.14 ± 1.20 ngÆmL )1 (P < 0.005). A similar but lower increase was also observed in the plasma leptin levels of C57BL/6 mice fed western-type diet for 24 weeks (34.70 ± 1.50 ngÆmL )1 , P < 0.005). Diet-induced obesity in ApoE3 knock-in and C57BL/6 mice is associated with reduced glucose tolerance and insulin sensitivity To determine the role of ApoE in the development of obesity-associated insulin resistance and glucose intol- erance, we performed the standard glucose tolerance test (GTT) and insulin sensitivity test (IST). The GTT established that at week 0 all three mouse groups (ApoE3 knock-in , ApoE )/) and C57BL/6 mice) had simi- lar normal responses to intraperitoneal administration of glucose (Fig. 3A). However, at week 24 of the experiment, ApoE3 knock-in mice showed a significant deterioration in their ability to clear plasma glucose, as compared to C57BL/6 and ApoE )/) mice (Fig. 3B; P < 0.05). A similar but less severe effect was also observed in the C57BL/6 mice (Fig. 3A,B; P < 0.05). Remarkably, however, ApoE )/) mice (which are resis- tant to diet-induced obesity) fed western-type diet for 24 weeks cleared glucose from the circulation more efficiently than the two other groups, and there was no significant difference in their response to intraperito- neal glucose load between weeks 0 and 24 on western- type diet (compare Fig. 3A,B, P > 0.05). In a similar fashion, when an IST was performed at week 0, all three mouse groups exhibited a similar response to intraperitoneal administration of insulin (Fig. 3C). However, at week 24, ApoE3 knock-in mice fed western-type diet for 24 weeks displayed the poor- est response to insulin administration as compared to C57BL/6 and ApoE )/) mice (Fig. 3D; P < 0.05). C57BL/6 mice also exhibited reduced insulin sensitivity at week 24 that was less severe than in ApoE3 knock-in mice (Fig. 3D; P < 0.05). In contrast, ApoE )/) mice fed western-type diet for 24 weeks exhibited the highest sensitivity to insulin of all three mouse groups (P < 0.05). In addition, there was no significant differ- ence in their insulin sensitivity curves between weeks 0 and 24 of the experiment (compare Fig. 3C,D; P > 0.05). Table 1. Body composition of ApoE3 knock-in , C57BL/6 and ApoE )/) mice fed western-type diet for 24 weeks. Values are in grams expressed as mean ± SEM. Mouse strain Wet body weight Dry body weight Lean body mass Body fat Water ApoE3 knock-in 50.2 ± 2.2 33.2 ± 2.0 28.9 ± 3.3 21.3 ± 1.5 17.0 ± 3.9 C57BL/6 43.1 ± 0.9 34.8 ± 1.7 28.1 ± 1.9 15.0 ± 1.6 8.3 ± 2.6 ApoE )/) 24.6 ± 1.1 9.2 ± 0.7 22.0 ± 0.8 2.5 ± 0.3 14.4 ± 0.4 ApoE and obesity I. Karagiannides et al. 4800 FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS LDLr ) / ) mice are more sensitive to diet-induced obesity and hyperglycemia than ApoE ) / ) mice but less sensitive than C57BL/6 mice Low-density lipoprotein receptor is the major receptor involved in the clearance of ApoE-containing lipopro- teins from the circulation [41]. Therefore, one mecha- nistic explanation for the role of ApoE in the development of obesity would be that LDLr-mediated uptake of ApoE-containing chylomicron remnants promotes the direct deposition of dietary fat into the adipose tissue. If this was the case, deficiency in LDLr would prevent obesity and hyperglycemia. To address this possibility, groups of 8–10 female LDLr )/) or C57BL/6 or ApoE )/) mice were placed on western- type diet for a period of 15 weeks, and their body weight and composition, and plasma cholesterol, tri- glyceride and glucose levels were determined during the course of the experiment. At week 5 of the experi- ment, the average weight of the LDLr )/) mice was 22.28 ± 1.32 g (29.7 ± 4.1% higher than their initial weight of 17.13 ± 0.65 g, P < 0.05) (Fig. 4A). This increase was comparable to the 27.4 ± 3.8% increase observed in the ApoE )/) mice (from 16.83 ± 0.24 g to 21.43 ± 0.56 g, P < 0.05), but lower than the 55.4 ± 3.8% increase in the C57BL/6 mice (from 16.26 ± 0.28 g to 25.25 ± 0.50 g, P < 0.005) (Fig. 4A). At week 15 of the experiment, however, LDLr )/) mice showed an 84.5 ± 8.7% increase in body weight (with an average final weight of 31.63 ± 2.10 g, P < 0.05) (Fig. 4A). This increase was higher than the 51.4 ± 4.5% increase (P < 0.05) observed in the weight of the ApoE )/) mice (with an average final weight of 25.46 ± 0.55 g). However, it was still significantly lower than the 119.8 ± 7.6% increase observed in the weight of C57BL/6 mice (with an aver- age final weight of 37.28 ± 0.72 g) fed western-type diet for the same period of time (Fig. 4A; P < 0.05). Body composition analysis at the end of the experi- ment revealed that at week 15, LDLr )/) mice had a body fat content of 19.9 ± 1.2%, which was much higher than the body fat content of the ApoE )/) mice (13 ± 1.9%, P < 0.05) but still lower than the body fat content of the C57BL/6 mice (28.06 ± 3.92%, P < 0.05) fed western-type diet for the same period of time (Fig. 4D). The complete body composition analy- sis of the mice fed western-type diet for 15 weeks is summarized in Table 2. Plasma lipid and glucose analysis showed that during the 15-week period, LDLr )/) mice developed severe hypercholesterolemia (1338 ± 135 mgÆdL )1 ) that was accompanied by moderate hypertriglyceride- mia (242.6 ± 14.9 mgÆdL )1 ) (Fig. 4B,C). Plasma glucose levels were increased moderately (from 71.3 ± 6.7 mgÆdL )1 to 101 ± 4.9 mgÆdL )1 , P < 0.05) but were still lower than the levels of C57BL/6 mice (131 ± 5.3 mgÆdL )1 ) at week 15 of the experiment (Fig. 4E; P < 0.005). The GTT and IST revealed that the LDLr )/) mice fed western-type diet for 15 weeks had similar toler- ance to glucose and sensitivity to insulin as in their starting state (week 0) (Fig. 5; P > 0.05). In addition, there was no significant difference in the response to intraperitoneal administration of glucose or insulin 015 30 45 60 75 apoE3 knock-in C57BL/6 apoE –/– apoE3 knock-in C57BL/6 apoE –/– apoE3 knock-in C57BL/6 apoE –/– apoE3 knock-in C57BL/6 apoE –/– 90 105 120 0 100 200 300 400 0 100 200 300 400 0 50 100 150 200 0 50 100 150 200 Week 0 Week 24 Week 0 Week 24 Glucose levels (mg·dL –1 ) Time (min) 015 30 45 60 75 90 105 120 Time (min) 015 30 45 60 75 90 105 120 Time (min) 0 153045607590105120 Time (min) Glucose levels (mg·dL –1 ) * * * * A B C D Glucose levels (mg·dL –1 ) Glucose levels (mg·dL –1 ) * * * * Fig. 3. Glucose tolerance curves (A, B) and insulin sensitivity curves (C, D) of ApoE3 knock-in , C57BL/6 and ApoE )/) mice at weeks 0 and 24. Values indicate the average plasma glucose levels expressed as mean ± SEM. The statistical significance of the observed differences among groups at each time-point is as indicated (*P < 0.05). I. Karagiannides et al. ApoE and obesity FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS 4801 0 5 0 500 1000 1500 2000 Cholesterol (mg·dL –1 ) A C B 0 5 0 50 100 150 200 250 300 Triglycerides (mg·dL –1 ) 0 5 10 15 10 15 10 15 0 300 C57BL/6 LDLr –/– ApoE –/– C57BL/6 LDLr –/– ApoE –/– C57BL/6LDLr –/– ApoE –/– C57BL/6 LDLr –/– ApoE –/– C57BL/6 LDLr –/– ApoE –/– 200 100 * * Weeks Weeks Weeks % of initial body-weight * * * 0 25 50 75 100 125 150 175 Week 0 Week 15 Fasting plasma glucose (mg·dL –1 ) * * ** ns (P > 0.05) * * ** ** E 0 10 20 30 40 50 % body TG content * * * D Fig. 4. Percentage of initial body weight (A), plasma cholesterol levels (B) and plasma tri- glyceride levels (C) of C57BL/6, LDLr )/) and ApoE )/) mice fed western-type diet for a period of 15 weeks. (D) Percentage body fat content of ApoE3 knock-in , ApoE )/) and C57BL/6 mice at week 15. (E) Fasting plasma glucose levels of ApoE3 knock-in , ApoE )/) and C57BL/6 mice at weeks 0 and 15. Each point on the graphs represents the mean value of the group, and error bars indicate the SEM. The statistical significance of the observed differences among groups at each time-point is indicated (*P < 0.05; **P < 0.005). Table 2. Body composition of LDLr )/) , ApoE )/) and C57BL/6 mice fed western-type diet for 15 weeks. Values are in grams expressed as mean ± SEM. Mouse strain Wet body weight Dry body weight Lean body mass Body fat Water LDLr )/) 31.6 ± 2.1 14.5 ± 2.3 25.7 ± 2.5 5.3 ± 0.7 16.4 ± 1.0 ApoE )/) 25.5 ± 0.6 15.3 ± 1.5 19.9 ± 1.4 3.4 ± 0.6 8.0 ± 2.3 C57BL/6 37.3 ± 0.7 23.9 ± 1.2 26.9 ± 1.8 10.4 ± 1.3 13.4 ± 1.5 0 15304560759010512051 0 100 200 300 400 0 100 200 300 400 0 50 100 150 Glucose levels (mg·dL –1 ) Time (min) 0 153045607590105120 Time (min) 0 153045607590105120 Time (min) 015 30 45 60 75 90 105 120 Time (min) Week 0 Week 0 Week 15 Week 15 A B C D Glucose levels (mg·dL –1 ) * * 0 25 50 75 100 125 150 LDLr –/– C57BL/6 LDLr –/– C57BL/6 LDLr –/– C57BL/6 LDLr –/– C57BL/6 Glucose levels (mg·dL –1 ) * Glucose levels (mg·dL –1 ) * Fig. 5. Glucose tolerance curves (A, B) and insulin sensitivity curves (C, D) of LDLr )/) and C57BL/6 mice at weeks 0 and 15. Values indicate the average plasma glucose levels expressed as mean ± SEM. The statistical significance of the observed differences among groups at each time-point is as indicated (*P < 0.05). ApoE and obesity I. Karagiannides et al. 4802 FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS between LDLr )/) and C57BL/6 mice fed western-type diet for 15 weeks (Fig. 5). Interestingly, in our studies, feeding western-type diet to C57BL/6 for 15 weeks did not result in insulin resistance or glucose intolerance (Fig. 5B,D). Taken together, these data indicate that female LDLr )/) mice fed western-type diet for 15 weeks appear to be more sensitive than female ApoE )/) mice but still more resistant than female C57BL/6 mice in the development of diet-induced obesity and its related disorders. ApoE promotes diet-induced accumulation of excess triglycerides in the liver while ApoE or LDLr deficiency does not If ApoE and LDLr are involved in the direct delivery of dietary lipids to tissues, one would expect that feed- ing ApoE3 knock-in and C57BL/6 mice western-type diet would result in excess accumulation of triglycerides in the liver, whereas ApoE )/) and LDLr )/) mice would be resistant to excess hepatic triglyceride accumulation. To test this hypothesis, we isolated liver samples from mice fed western-type diet for 15 weeks and deter- mined their triglyceride content (Fig. 6A). Liver samples from ApoE )/) and LDLr )/) mice fed western-type diet for 15 weeks had similar triglycer- ide contents of 60.67 ± 4.12 mgÆg )1 and 58.40 ± 5.11 mgÆg )1 of hepatic tissue, respectively (Fig. 6A, P > 0.05). In contrast, ApoE3 knock-in and C57BL/6 mice had a much higher hepatic triglyceride content (215.00 ± 33.56 mgÆg )1 and 213.72 ± 11.89 mgÆg )1 respectively, P < 0.05), confirming that human ApoE3, murine ApoE, and the LDLr contribute to the accu- mulation of excess lipids in the liver in response to western-type diet (Fig. 6A). Effects of ApoE and LDLr deficiency on the direct delivery of dietary lipids to adipose tissue As ApoE and LDLr play pivotal roles in the catabo- lism of chylomicron remnants, we attempted to evalu- ate the contribution of the direct delivery of nonhydrolyzed postprandial lipids to the development of obesity. To address this question, groups of ApoE3- knock-in , ApoE )/) , LDLr )/) and C57BL/6 mice that were maintained on western-type diet for 15 weeks were gavaged with 0.5 mL of olive oil containing 15 lCi of the nonhydrolyzable [ 3 H]cholesteryl-hexade- cyl-ether [cholesteryl-1,2- 3 H(N)]. Twenty-four hours later, mice were killed, and visceral fat and liver sam- ples were isolated, weighed, and homogenized. Then, the amount of 3 H radioactivity present in the homoge- nized tissues was determined using a liquid scintillation counter. ApoE3 knock-in mice showed a higher average accu- mulation of dietary [ 3 H]cholesteryl-hexadecyl-ether in adipose tissue (1718 ± 492 c.p.m. per gram of tissue) than C57BL/6 mice (1010 ± 202 c.p.m. per gram of tissue) (Fig. 6B), but this difference between the two groups did not reach statistical significance (P > 0.05). The hepatic accumulation of 3 H-label was similar between these two animal groups (3181 ± 585 c.p.m. per gram of tissue for the ApoE3 knock-in mice and 3281 ± 578 c.p.m. per gram of tissue for the C57BL/6 mice) (Fig. 6C). ApoE )/) and LDLr )/) mice showed lower average accumulation of 3 H-label in their fat (618 ± 41 c.p.m. per gram of tissue and 664 ± 65 c.p.m. per gram of tissue, respectively) and hepatic tissues (1624 ± 209 c.p.m. per gram of tissue 0 cpms per gram of hepatic tissue B A 0 cpms per gram of adipose tissue 0 50 100 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500 150 200 250 300 apoE3 knock-in apoE –/– LDLr –/– C57BL/6 * ** ** * * mg of TG per gram of hepatic tissue C Fig. 6. (A) Hepatic triglyceride content of ApoE3 knock-in , ApoE )/) , LDLr )/) and C57BL/6 mice fed western-type diet for 15 weeks. The statistical significance of the observed differences is indicated (*P < 0.05; **P < 0.005). (B, C) Total accumulation of tritiated label expressed as counts per minute per gram of adipose (B) or hepatic (C) tissue, 24 h after gavage administration of 0.5 mL of olive oil containing 15 lCi of [ 3 H]hexadecyl-cholesteryl-ether, per mouse. I. Karagiannides et al. ApoE and obesity FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS 4803 and 1759 ± 752 c.p.m. per gram of tissue, respec- tively) (Fig. 6B,C). No measurable radioactivity was found in the blood of the tested mice at the time of killing (not shown). Discussion ApoE is a major protein involved in the metabolism of dietary lipids and the removal of atherogenic lipo- proteins from the circulation. Following a lipid-rich meal, lipids are packaged into chylomicrons which, following partial lipolysis by lipoprotein lipase (LpL), are converted into chylomicron remnants and acquire ApoE [23]. Then, lipid-bound ApoE interacts with LDLr, which mediates the removal of ApoE-contain- ing lipoproteins from the circulation. In the present study, we show that ApoE3 knock-in mice are more sen- sitive to diet-induced obesity and related metabolic dysfunctions than wild-type C57BL/6 mice, whereas ApoE )/) mice are resistant to the development of these conditions. Furthermore, deficiency in LDLr results in reduced sensitivity to obesity in response to western-type diet, raising the possibility that the effects of ApoE may be mediated, at least in part, via its interactions with LDLr. Interestingly, there were no significant differences in plasma free fatty acid levels among mouse groups (ApoE3 knock-in versus C57BL/6 versus LDLr )/) versus ApoE )/) ), although previous studies suggested that increased plasma levels of free fatty acids are closely associated with obesity-induced insulin resistance [42,43]. Moreover, daily food consumption of the ApoE3 knock-in , C57BL/6 and ApoE )/) mice was similar among groups, suggesting that different responses to western-type diet cannot be attributed to differences in appetite. One possible explanation for the increased sensitivity of the ApoE3 knock-in mice to diet-induced obesity would be that higher plasma ApoE levels in these mice than in C57BL/6 mice are responsible for the enhanced deposition of dietary lipids in adipose tissue. To address this possibility, we compared the ApoE levels in plasma samples isolated from ApoE3 knock-in and C57BL/6 mice at week 0 of the experiment, using western blotting. This analysis showed that steady- state plasma ApoE levels in the ApoE3 knock-in mice used in our study are approximately four times lower than those in wild-type C57BL/6 mice. Thus, the increased sensitivity of ApoE3 knock-in mice to diet- induced obesity is not the result of elevated plasma ApoE levels in these mice as compared to C57BL/6 mice, and the difference in the ability of human ApoE3 and murine ApoE to promote obesity in response to high-fat diet may be due to intrinsic differ- ences between these two peptides. The data presented in Fig. 6B raise the possibility that chylomicron and VLDL remnants containing the human ApoE3 isoform are taken up more avidly by adipose tissue than the lipoproteins containing mouse ApoE. In a previous study, Sullivan et al. [44] reported that ApoE3 knock-in mice and C57BL/6 mice have similar plasma ApoE levels. Furthermore, using northern blot analysis, they also showed that ApoE mRNA levels were indistinguishable between ApoE3 knock-in mice and C57BL/6 mice, in all tissues tested except for the small intestine, where human ApoE3 mRNA expression was lower than mouse ApoE mRNA expression [44]. How- ever, the ApoE3 knock-in mice studied by these investiga- tors are different from the ApoE3 knock-in mice tested in our experiments, because our ApoE3 knock-in mice have been bred for nine generations to the C57BL/6 back- ground. It is possible that back-crossing ApoE3 knock-in mice to C57BL/6 mice for nine generations resulted in the reduced plasma human ApoE3 levels that we observed. Human ApoE has three natural isoforms in humans: ApoE2, ApoE3 and ApoE4. In vitro receptor binding studies established that lipid-bound ApoE3 and ApoE4 have a similar affinity for LDLr, whereas lipid-bound ApoE2 has a much lower affinity [45,46]. In this study, we focused on ApoE3, mainly because it is the most common ApoE genetic polymorphism in humans [24– 29]. If the effects of ApoE3 on obesity are mediated by its lipid-lowering potential via LDLr, then we expect that both ApoE3 and ApoE4 will predispose to a simi- lar extent to diet-induced obesity and insulin resistance in mice, whereas ApoE2 may have a much lower potential to promote these conditions. Further studies are needed in order to address this point, and other mechanisms of ApoE-promoted diet-induced obesity should not be excluded. It is quite interesting that in all our experiments, plasma cholesterol levels correlated inversely with body weight gain and body fat accumulation (Figs 1 and 4). In the ApoE )/) mice, failure to clear chylomicron rem- nants due to deficiency in ApoE resulted in a steady increase in plasma cholester ol levels and rendered these mice resistant to diet-induced obesity. In contrast, in the ApoE3 knock-in mice, the efficient catabolism of chylomicron remnants resulted in only slightly elevated plasma cholesterol levels, but promoted obesity, insulin resistance and glucose intolerance. Similar to the ApoE3 knock-in mice, C57BL/6 mice, which express the mouse ApoE, developed only mild hypercholesterol- emia but became obese and insulin resistant following western-type diet for 24 weeks. ApoE and obesity I. Karagiannides et al. 4804 FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS Plasma triglyceride levels of the LDLr )/) mice were moderately elevated at week 0, and remained elevated during the 15 weeks on high-fat diet, whereas plasma triglyceride levels of the other animal groups remained normal for the duration of the experiment. This is not surprising, because in the absence of the LDLr, reduced clearance of ApoE-containing lipoproteins from the circulation results in elevated steady-state plasma ApoE levels. Since ApoE is a known inhibitor of LpL [47], and plasma triglyceride levels correlate with plasma ApoE levels [48], accumulation of ApoE in the blood of LDLr )/) mice results in reduced LpL- mediated lipolysis of plasma triglycerides and hyper- triglyceridemia. In our studies, LDLr )/) mice became more obese than ApoE )/) mice but less obese than C57BL/6 mice, raising the possibility that, in addition to LDLr, other ApoE-recognizing receptors may also promote the deposition of postprandial lipids in adi- pose tissue, thus contributing to diet-induced obesity and related metabolic dysfunctions. Indeed, a recent study showed that adipose-tissue-specific deletion of the LDLr-related protein makes mice less sensitive to obesity [49]. In the case of the LDLr )/) mice, LDLr- related protein and possibly other ApoE-recognizing ‘scavenger’ receptors may promote, to some extent, delivery of ApoE-containing chylomicron remnants to adipose tissue. However, in the case of the ApoE )/) mice, which lack the endogenous ApoE, all of these ApoE-mediated receptor processes are blocked, and ApoE )/) mice are more resistant to body fat gain than LDLr )/) mice. Even though the expression of LDLr in adipose tissue is much lower than in liver, our data support a functional role for this receptor in the ApoE-mediated mechanism of diet-induced obesity. In a previous study by Schreyer et al. [50], it was suggested that LDLr )/) mice fed a diabetogenic diet for 16 weeks were more susceptible to diet-induced obesity and hyperglycemia than C57BL/6 mice, whereas ApoE )/) mice appeared to be as susceptible to the development of these conditions as C57BL/6 mice. Furthermore, in that study, LDLr )/) mice developed severe hypertriglyceridemia during the course of the experiment. The diabetogenic diet used in that study contained a very high fat content of 35.5% (derived mainly from lard) (Bioserve, French- town, NJ, USA; cat. no. F1850). In all our experi- ments, mice were fed the standard western-type diet containing 21.1% fat (Harlan Teklad; cat. no. TD88137). It is possible that the high fat content of the diabetogenic diet predisposed ApoE )/) , LDLr )/) and C57BL6 mice to the development of diet-induced obesity, and resulted in saturation of the metabolic pathways that control body fat deposition and plasma lipid and glucose homeostasis. Under such conditions, ApoE or LDLr deficiency may not be sufficient to prevent obesity, as other mechanisms contributing to obesity may override the protective effect of ApoE or LDLr deletion that we observed in our experiments. Our data on LDLr )/) mice are in agreement with the data of MacDonald et al. [51], showing that female LDLr )/) mice do not become excessively obese, and do not develop hyperglycemia and glucose intolerance in response to western-type diet. In our experiments, we studied the role of ApoE in the development of obesity in response to dietary consumption of fat, one of the major causes of human obesity [2,18]. Our findings are supported by previous observations by Gao et al. [52] and Chiba et al. [53] showing that deficiency in ApoE renders genetically predisposed obese mice less sensitive to spontaneous development of obesity. Furthermore, in vitro studies suggested that ApoE promotes triglyc- eride uptake and deposition in in vitro differentiated adipocytes and in freshly isolated adipose tissue explants [54], whereas VLDL induces adipocyte differ- entiation in an ApoE-dependent manner [53]. In the present study, we report for the first time that human ApoE3 increases susceptibility to diet-induced obesity as compared to mouse ApoE. The functional role of ApoE-containing chylomicron and VLDL remnants in the development of diet-induced obesity is further supported by the observation that ApoE )/) mice remain lean when fed western-type diet for 15 or 24 weeks. One of the hallmarks of obesity-associated insulin resistance is the increase in the circulating levels of insulin [55]. Such a change is also evident in our stud- ies, further supporting the hypothesis that the benefi- cial effects of ApoE deletion on weight loss extend to increased insulin sensitivity. Furthermore, our data demonstrate a significant increase in plasma leptin levels in ApoE3 knock-in and, to a lesser extent, in C57BL/6 mice, an observation that is also consistent with their increased adiposity [56]. ApoE has long been known to be atheroprotective, mainly because of its ability to clear plasma lipids. However, our data show that if excess dietary lipids are present in the circulation, this atheroprotective property of ApoE may be counteracted by the enhanced deposition of lipids in adipose tissue. Over- all, our findings identify ApoE expression as a key peripheral contributor to the development of obesity and related metabolic dysfunctions in mice. I. Karagiannides et al. ApoE and obesity FEBS Journal 275 (2008) 4796–4809 ª 2008 The Authors Journal compilation ª 2008 FEBS 4805 [...]... least nine generations to ensure a similar genetic background Female mice, 4–6 weeks old, were used in these studies, and four to five mice were housed per cage To ensure similar average cholesterol triglyceride and glucose levels and starting body weights, groups of 8–10 mice were formed after determining the fasting cholesterol triglyceride and glucose levels and body weights of the individual mice. .. At the indicated time-points during the course of the experiments, mice in each group were brie y anesthetized using isofluorane, and their body weight was determined with a Mettler precision microscale (Mettler-Toledo, Columbus, OH, USA) At the end of each experiment, at least six mice from each group were killed Mouse carcasses were weighed to determine wet weight, and then they were dehydrated at... dextrose (for GTT, 1 g/kg) or insulin (for IST, 2 UÆkg)1 humulin; Eli-Lilly, Indianapolis, IN, USA) were injected intraperitoneally, serum samples were collected at 0, 15, 30, 60 and 120 min postinjection through the tail vein, and glucose levels were measured Determination of plasma insulin and leptin levels Insulin and leptin levels in the plasma of mice were determined spectrophotometrically by ELISA... mice Mice were fed the standard western-type diet (Harlan-Teklad, Madison, WI, USA; cat no TD88137) for the indicated period, and body weight and fasting plasma cholesterol and triglyceride levels were determined at the indicated time-points after diet initiation At the end of each experiment, mice were killed, and plasma and liver samples were collected and stored at 4 °C and )20 °C respectively Carcasses... The total amount of triglycerides was determined in the resulting mixture as described above Results are expressed as milligram of triglycerides per gram of tissue ± SEM Gavage administration of olive oil containing [3H]cholesteryl-hexadecyl-ether to mice Groups of ApoE3knock -in, ApoE)/), LDLr)/) and C57BL/6 mice containing 8–10 mice each were fed western-type diet for 15 weeks Prior to the experiment,... Carcasses were stored at )80 °C and later subjected to body composition analysis as described below All animal studies were governed by the EU guidelines of the Protocol for the Protection and Welfare of Animals In our experiments we took into consideration the ‘three Rs’ (reduce, refine, replace) and we kept the number of animal experiments to the absolute minimum To this date there is no in vitro system... mice were fasted overnight for 16 h On the next day, animals were weighed and then anesthetized by intraperitoneal injection of sodium pentobarbital Anesthetized mice were gavaged with 0.5 mL of olive oil containing 15 lCi of [3H]cholesteryl-hexadecylether [Cholesteryl-1,2-3H(N)] (Perkin Elmer, Waltham, MA, USA; cat no NET859-250UC), and placed back in their cages, where they were allowed to recover... system to mimic satisfactorily the lipid and lipoprotein transport system and the in vivo mechanisms leading to obesity and diabetes, making the use of experimental animals mandatory All procedures used in our studies cause only minimal distress to the mice tested The work was authorized by the appropriate committees of the Laboratory Animal Science Centers of Boston University and The University of... with the E2 /E2 phenotype Biochemistry 42, 9841–9853 46 Weisgraber KH, Innerarity TL & Mahley RW (1982) Abnormal lipoprotein receptor-binding activity of the human E apoprotein due to cysteine–arginine interchange at a single site J Biol Chem 257, 2518–2521 47 Rensen PC & van Berkel TJ (1996) Apolipoprotein E effectively inhibits lipoprotein lipase-mediated lipolysis of chylomicron-like triglyceride-rich... Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery J Clin Invest 92, 883–893 Kypreos KE, Van Dijk KW, van Der ZA, Havekes LM & Zannis VI (2001) Domains of apolipoprotein E contributing to triglyceride and cholesterol homeostasis in vivo Carboxyl-terminal region 203–299 promotes hepatic very low density lipoprotein-triglyceride secretion . diet for 24 weeks became obese and developed hyperglycemia, hyperinsulinemia, hyperleptin- emia, glucose intolerance and insulin resistance that were more. diet-induced obesity in response to western-type diet. Results ApoE promotes diet-induced weight gain in mice, whereas ApoE deficiency prevents it To test