ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

Wijtske Annema; Arne Dikkers; Jan Freark de Boer; Thomas Gautier; Patrick C.N. Rensen; Daniel J. Rader; Uwe J.F. Tietge

doi:10.1194/jlr.m020743

ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

Annema, Wijtske;Dikkers, Arne;de Boer, Jan Freark;Gautier, Thomas;Rensen, Patrick C.N.;Rader, Daniel J.;Tietge, Uwe J.F. 2012-05-01 00:00:00 ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efﬂ ux to plasma 1, , † 1, § Wijtske Annema , * Arne Dikkers , * Jan Freark de Boer, * Thomas Gautier, †† 2, ,† Patrick C. N. Rensen , ** Daniel J. Rader, and Uwe J. F. Tietge * Department of Pediatrics,* Center for Liver, Digestive, and Metabolic Diseases, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands ; Top Institute Food and Nutrition , Wageningen, The Netherlands ; INSERM UMR866 Lipides, Nutrition, Cancer, Faculté de Médecine, Dijon, France ; Department of General Internal Medicine,** Endocrinology, and Metabolic Diseases, Leiden University Medical Center, Leiden , The Netherlands ; and Institute for Translational Medicine and †† Therapeutics, University of Pennsylvania School of Medicine , Philadelphia, PA Supplementary key words apolipoprotein E � reverse cholesterol Abstract ApoE plays an important role in lipoprotein me- transport � ATP-binding cassette transporter A1 � atherosclerosis � tabolism. This study investigated the effects of adenovirus- bile � cholesteryl ester transfer protein � feces � high density lipopro- mediated human apoE overexpression (AdhApoE3) on sterol tein � liver � macrophage � metabolism � mice � probucol metabolism and in vivo reverse cholesterol transport (RCT). In wild-type mice, AdhApoE3 resulted in decreased HDL cholesterol levels and a shift toward larger HDL in plasma, Apolipoprotein E (apoE) plays an important role in whereas hepatic cholesterol content increased ( P < 0.05). These effects were dependent on scavenger receptor class B lipo protein metabolism and atherosclerosis. ApoE is pro- type I (SR-BI) as conﬁ rmed using SR-BI-deﬁ cient mice. Ki- duced and secreted predominantly by the liver ( 1 ), but it netic studies demonstrated increased plasma HDL choles- is also expressed in a variety of other tissues, including teryl ester catabolic rates ( P < 0.05) and higher hepatic macrophages ( 2, 3 ). While loss of function of apoE in mice selective uptake of HDL cholesteryl esters in AdhApoE3- and in humans is associated with a proatherogenic lipo- injected wild-type mice ( P < 0.01). However, biliary and fecal protein proﬁ le and increased atherogenesis ( 4, 5 ), overex- sterol output as well as in vivo macrophage-to-feces RCT pression of apoE in various models has been shown to studied with H-cholesterol-loaded mouse macrophage foam protect against atherosclerotic lesion formation ( 6–11 ). cells remained unchanged upon human apoE overexpres- sion. Similar results were obtained using hApoE3 overex- Among other metabolic effects that are potentially anti- pression in human CETP transgenic mice. However, blocking atherogenic, apoE has been reported to promote choles- ABCA1-mediated cholesterol efﬂ ux from hepatocytes in terol efﬂ ux ( 12–14 ), and recent studies have suggested AdhApoE3-injected mice using probucol increased biliary cho- that lack of macrophage apoE might decrease overall re- lesterol secretion ( P < 0.05), fecal neutral sterol excretion ( P < verse cholesterol transport (RCT) ( 15 ). However, the pool 0.05), and in vivo RCT ( P < 0.01), speciﬁ cally within neutral of macrophage-derived apoE represents a small fraction of sterols. These combined data demonstrate that systemic total circulating apoE. apoE overexpression increases i ) SR-BI-mediated selective The classic RCT pathway is a multistep process that in- uptake into the liver and ii ) ABCA1-mediated efﬂ ux of RCT- relevant cholesterol from hepatocytes back to the plasma com- volves i ) HDL-mediated efﬂ ux of excess cholesterol from partment, thereby resulting in unchanged fecal mass sterol extrahepatic cells and most relevant for atherosclerosis excretion and overall in vivo RCT. —Annema, W., A. Dikkers, lipid-laden macrophages in the arterial wall, ii ) uptake of J. F. de Boer, T. Gautier, P. C. N. Rensen, D. J. Rader, and U. HDL cholesterol into the liver, and iii ) excretion of HDL J. F. Tietge. ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efﬂ ux to plasma. J. Lipid Res . 2012. 53: 929–940. Abbreviations: CE, cholesteryl ether; CETP, cholesteryl ester trans- fer protein; FCR, fractional catabolic rate; FPLC, fast protein liquid This work was supported by the Netherlands Organization for Scientiﬁ c Research chromatography; RCT, reverse cholesterol transport; SR-BI, scavenger VIDI grant 917-56-358 (U.J.F.T.), by the Top Institute (TI) Food and Nutrition receptor class B type I. (U.J.F.T.), and by grants HL022633 and HL059407 from the NHLBI 1 These authors contributed equally to this work. (D.J.R.). P.C.N.R. is an Established Investigator of the Netherlands Heart To whom correspondence should be addressed. Foundation (2009T038). e-mail: [email protected]. Manuscript received 21 September 2011 and in revised form 29 February 2012. The online version of this article (available at http://www.jlr.org) Published, JLR Papers in Press, March 1, 2012 contains supplementary data in the form of ﬁ ve tables, six ﬁ gures, and DOI 10.1194/jlr.M020743 supplemental Methods. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 53, 2012 929 This is an Open Access article under the CC BY license. liquid chromatography (FPLC) gel ﬁ ltration using a Superose 6 cholesterol into bile and ultimately feces either directly or column (GE Healthcare, Uppsala, Sweden) as described ( 21 ). after metabolic conversion into bile acids ( 16–18 ). Al- Individual fractions were assayed for cholesterol concentrations though the liver has a key function in the RCT pathway as described above. and the majority of circulating apoE is generated by hepa- To determine plasma HDL- and nonHDL-cholesterol levels, tocytes, the contribution of hepatic apoE to in vivo RCT apoB-containing lipoproteins were precipitated using polyethyl- has not been addressed. ene glycol in 10 mM HEPES (pH 8.0) as described ( 22 ). After Therefore, the aim of the current study was to investi- centrifugation of the samples at 2,000 g and 4°C for 30 min, the gate the effects of hepatic overexpression of human apoE HDL-containing supernatant was transferred to clean tubes. The nonHDL-containing pellet was dissolved in 0.5 M NaCO . Cho- on liver lipid metabolism, biliary sterol secretion, and in lesterol concentrations in the HDL and nonHDL fractions were vivo macrophage-to-feces RCT. Our data demonstrate that determined as described above. increasing plasma levels of liver-derived apoE enhances selective uptake of HDL cholesteryl esters into the liver Analysis of liver lipid composition and induces hepatic cholesterol accumulation in a scaven- Liver tissue was homogenized as described ( 23 ). Commercially ger receptor class B type I (SR-BI)-dependent manner. available kits were used to measure contents of total cholesterol, However, this does not affect fecal mass sterol excretion triglycerides (Roche Diagnostics), and free cholesterol (Diasys) and macrophage-speciﬁ c RCT due to an apoE-induced after extraction of lipids according to the general procedure of enhancement of ATP-binding cassette transporter A1 Bligh Dyer and redissolving lipids in water containing 2% Triton (ABCA1)-mediated efﬂ ux of RCT-relevant cholesterol X-100 ( 23 ). Phospholipid content of the liver was determined from hepatocytes back to the plasma compartment. These after lipid extraction essentially as described ( 23 ). ﬁ ndings suggest that systemic apoE overexpression pro- Analysis of gene expression by real-time quantitative PCR tects against atherosclerosis by mechanisms other than Total RNA from mouse livers was extracted with TriReagent modulation of RCT. (Sigma) and quantiﬁ ed using a Nanodrop ND-100 U-Vis spectro- photometer (NanoDrop Technologies, Wilmington, DE). cDNA synthesis was performed from 1 g of total RNA using reagents MATERIALS AND METHODS from Invitrogen (Carlsbad, CA). Real-time quantitative PCR was Animals carried out on an ABI-Prism 7700 (Applied Biosystems, Darm- stadt, Germany) sequence detector with the default settings ( 23 ). C57BL/6J mice were obtained from Charles River (Wilming- Primers and ﬂ uorogenic probes were designed with the Primer ton, MA). SR-BI knockout mice were obtained from The Jackson Express Software (Applied Biosystems) and synthesized by Euro- Laboratory (Bar Harbor, ME) and backcrossed to the C57BL/6J gentec (Seraing, Belgium). mRNA expression levels presented background for a total of eight generations. Probucol (Sigma, St. were calculated relative to the average of the housekeeping gene Louis, MO) was mixed into powdered chow (0.5% wt/wt). For cyclophilin and further normalized to the relative expression the RCT experiment, the diet was provided for 12 days before levels of the respective controls. and then throughout the 48-h period of the experiment. In all other experiments, the diet was provided for 14 days. Animals Bile collection and assessment of biliary excretion of bile were caged in animal rooms with alternating 12-h periods of light acids, phospholipids, and cholesterol (from 7:00 AM to 7:00 PM) and dark (from 7:00 PM to 7:00 AM), Bile was collected by cannulation of the gallbladder in mice with ad libitum access to water and mouse chow diet (Arie Blok, anesthetized by intraperitoneal injection of hypnorm (fentanyl/ Woerden, The Netherlands). Animal experiments were per- ﬂ uanisone, 1 mg/kg) and diazepam (10 mg/kg). During the bile formed in conformity with PHS policy and in accordance with collection, body temperature was maintained using a humidiﬁ ed the national laws. All protocols were approved by the responsible incubator. Bile collection was performed for 30 min, and secre- ethics committee of the University of Groningen and the Univer- tion rates were determined gravimetrically. Biliary bile salt, cho- sity of Pennsylvania. lesterol, and phospholipid concentrations were determined and Generation of recombinant adenoviruses the respective biliary excretion rates calculated as described pre- viously ( 23, 24 ). The empty control adenovirus AdNull ( 19 ) and the recombi- nant adenovirus encoding human apoE3 (AdhApoE3) ( 19 ) were Fecal sterol analysis ampliﬁ ed and puriﬁ ed as reported previously ( 20 ). For in vivo experiments, mice were injected with 1 × 10 particles/mouse of Mice were individually housed, and feces were collected over a period of 24 h and separated from the bedding. Fecal samples AdhApoE3 or AdNull. In vivo reverse cholesterol transport stud- ies were carried out between day 2 and day 4 after injection of were dried, weighed, and thoroughly ground. Aliquots thereof were used for determination of neutral sterol and bile acid con- recombinant adenoviruses, a time frame when high and stable expression from an adenovirus is achieved. All other experiments tent by gas-liquid chromatography as described ( 23, 24 ). described were performed on day 4 after injection of the recom- binant adenoviruses. HDL kinetics studies HDL kinetics studies were performed essentially as published Plasma lipid and lipoprotein analysis previously ( 21 ). Autologous HDL was prepared from pooled Mice were bled by heart puncture after a 4-h fast at the time of mouse plasma by sequential ultracentrifugation (density 1.063 < death. Aliquots of plasma were stored at 80°C until analysis. d < 1.21). After extensive dialysis against sterile PBS containing Commercially available reagents were used to measure plasma 0.01% EDTA, HDL was labeled with I-tyramine-cellobiose (TC) total cholesterol, triglycerides (Roche Diagnostics, Basel, Switzer- and cholesteryl hexadecyl ether (cholesteryl-1,2,- H; Perkin land), free cholesterol, and phospholipids (Diasys, Holzheim, Elmer Life Sciences) as previously described ( 25 ). For kinetic 125 3 Germany). Pooled plasma samples were subjected to fast protein studies, 0.4 µCi of I and 0.7 million dpm of the H tracer were 930 Journal of Lipid Research Volume 53, 2012 injected into the tail veins of fasted wild-type mice treated with 1640 medium (Invitrogen) supplemented with 1% FBS (Hy- AdNull or AdhApoE3. Blood samples were drawn by retroorbital Clone, Logan, UT) and penicillin (100 U/ml)/streptomycin bleeding at 5 min and at 1, 3, 6, 11, and 24 h after injection. (100 g/ml) (Invitrogen) and were allowed to adhere for 5 h at Plasma decay curves for both tracers were generated by dividing 37°C under 5% CO humidiﬁ ed air. Nonadherent cells were re- the plasma radioactivity at each time point by the radioactivity at moved by washing twice with PBS followed by loading of the mac- the initial 5-min time point after tracer injection. Fractional cata- rophages with 50 g/ml LDL and 1 Ci/ml H-cholesterol bolic rates (FCRs) were determined from the area under the (Perkin Elmer Life Sciences, Boston, MA) for 24 h. The cells plasma disappearance curves ﬁ tted to a bicompartmental model were washed again and equilibrated in RPMI 1640 medium sup- using the SAAM II program ( 26 ). The use of H-cholesteryl ether plemented with 2% BSA (Sigma) for 18 h. The cells were washed does not affect turnover rates in vivo compared with H-cholesteryl with PBS, and 2% mouse plasma was added. After 4 h and 8 h, ester-labeled HDL ( 21 ). Organ uptake of HDL apolipoproteins radioactivity within the medium was determined by liquid scintil- 125 3 ( I) and HDL-CEs ( H-cholesteryl ether) was determined by lation counting. The cell layer was washed twice with PBS, and 0.1 measuring the counts recovered in each organ expressed as a M NaOH was added. Plates were incubated 30 min at room tem- percentage of the injected dose, which was calculated by multi- perature, and the radioactivity remaining within the cells was as- plying the initial plasma counts (5-min time point) with the esti- sessed by liquid scintillation counting. Wells incubated with RPMI mated plasma volume (3.5% of total body weight). Selective without added plasma were used as blanks to determine plasma- uptake into organs was determined by subtracting the percent- independent efﬂ ux, and these values were subtracted from the age of the injected dose of I-HDL recovered in each organ respective experimental values. Efﬂ ux is given as the percentage from the percentage of the injected dose of H-HDL-CE. of counts recovered from the medium in relation to the total counts present on the plate (sum of medium and cells). In vivo RCT studies Statistical analysis In vivo RCT studies were performed essentially as published previously ( 27 ). Wild-type C57BL/6J donor mice were injected with Statistical analyses were performed using the Statistical Pack- 1.0 ml of 4% Brewer thioglycollate medium (Becton Dickinson, age for Social Sciences version 16.0 (SPSS Inc., Chicago, IL). Le Point de Claix, France). Peritoneal macrophages were har- Data are presented as means ± SEM. The Mann-Whitney U-test vested 4 days after thioglycollate injection as described ( 28 ). Mac- was used to compare different groups. Statistical signiﬁ cance for rophages were plated in RPMI 1640 medium (Invitrogen) sup- all comparisons was assigned at P < 0.05. plemented with 1% FBS (HyClone, Logan, UT) and penicillin (100 U/ml)/streptomycin (100 g/ml) (Invitrogen) and were allowed to adhere for 5 h at 37°C under 5% CO humidiﬁ ed air. RESULTS Nonadherent cells were removed by washing twice with PBS fol- lowed by loading of the macrophages with 50 g/ml acetylated Hepatic apoE overexpression affects HDL size LDL and 3 Ci/ml H-cholesterol (Perkin Elmer Life Sciences, distribution but not plasma lipid levels Boston, MA) for 24 h. Thereafter, cells were washed again and To assess the effects of hepatic overexpression of equilibrated in RPMI 1640 medium supplemented with 2% BSA human apoE3 on plasma lipid levels, wild-type mice were (Sigma) for 18 h. Immediately before injection, cells were har- vested and resuspended in RPMI 1640 medium. For in vivo mac- injected with an empty control adenovirus AdNull or with rophage-to-feces RCT studies, 2 million H-cholesterol-loaded an adenovirus expressing human apoE3. Plasma levels of macrophage foam cells were injected intraperitoneally into indi- total cholesterol, free cholesterol, esteriﬁ ed cholesterol, vidually housed recipient mice. Plasma was collected 6 h and 24 phospholipids, and triglycerides remained essentially un- h after macrophage injection by retroorbital puncture and for changed in response to hepatic apoE overexpression the ﬁ nal blood draw (48 h) by heart puncture. At the end of the ( Table 1 ). However, FPLC analysis revealed a lower HDL experimental period, livers were harvested, snap-frozen in liquid cholesterol peak and a shift toward larger particles in the nitrogen, and stored at 80°C until further analysis. Feces were AdhApoE3-injected mice compared with controls ( Fig. 1A ). collected continuously for 48 h. In parallel, plasma levels of apoA-I ( P = 0.055; Supplemen- Counts in plasma were assessed directly by liquid scintillation counting (Packard 1600CA Tri-Card, Packard, Meriden, CT). tary Figure IA) and apoB100 ( P < 0.01; Supplementary Fig- Counts within liver were determined after solubilization of the ure IB) were lower in the mice overexpressing human tissue using Solvable (Packard) exactly as previously reported apoE, whereas plasma apoB48 was not altered (n.s.; Sup- ( 26 ). Counts recovered from the respective liver piece were back- plementary Figure IC). To explore the distribution of hu- calculated to total liver mass. Feces were separated from the bed- man apoE across the different lipoprotein classes, Western ding, dried, weighed, and thoroughly ground. Aliquots were blot analysis for apoA-I and human apoE was performed separated into neutral sterol and bile acid fractions as previously on the individual FPLC fractions. In the mice adminis- reported ( 27 ). Brieﬂ y, samples were heated for 2 h at 80°C in al- tered AdhApoE3, human apoE was present in the apoA-I- kaline methanol and then extracted three times with petroleum containing HDL fractions and in the nonHDL lipoprotein ether. In the top layer, radioactivity within the neutral sterol frac- tion was determined by liquid scintillation counting, whereas ra- fractions lacking apoA-I expression (Supplementary Fig- dioactivity incorporated into bile acids was assessed from the ure II). Because cholesteryl ester transfer protein (CETP) bottom layer. Counts recovered from the respective aliquots were plays an important role in human lipoprotein metabolism related to the total amount of feces produced over the whole ex- but is absent in wild-type mice ( 29 ), apoE overexpression perimental period. All obtained counts were expressed relative experiments were carried out in transgenic mice express- to the administered dose. ing human CETP under the control of its endogenous In vitro cholesterol efﬂ ux assay promoter (hCETP tg). Comparable to the results in wild- type mice, in hCETP tg mice no major changes in plasma Thioglycollate-elicited peritoneal mouse macrophages were lipids occurred in response to hepatic apoE overexpression harvested as described above. Macrophages were plated in RPMI ApoE overexpression does not impact in vivo RCT 931 TABLE 1. Plasma lipids, liver lipid composition, and biliary excretion of sterols in wild-type and SR-BI knockout mice in response to hepatic apolipoprotein E overexpression Wild-type SR-BI knockout AdNull AdhApoE3 AdNull AdhApoE3 Plasma Total cholesterol (mg/dl) 69.5 ± 1.9 65.5 ± 2.6 147.6 ± 9.5 140.5 ± 12.1 Free cholesterol (mg/dl) 29.0 ± 0.7 31.3 ± 0.9 86.4 ± 4.9 81.3 ± 7.8 Esteriﬁ ed cholesterol (mg/dl) 40.5 ± 1.6 34.3 ± 3.1 61.1 ± 6.4 59.2 ± 5.4 Phospholipids (mg/dl) 178.6 ± 12.7 148.6 ± 8.0 191.8 ± 10.8 214.7 ± 16.5 Triglycerides (mg/dl) 82.9 ± 9.2 80.0 ± 8.9 59.8 ± 6.9 79.9 ± 10.8 Liver Total cholesterol (nmol/mg liver) 6.3 ± 0.5 7.8 ± 0.4 8.5 ± 0.3 8.3 ± 0.4 Free cholesterol (nmol/mg liver) 5.4 ± 0.4 5.9 ± 0.3 6.7 ± 0.1 6.4 ± 0.1 Esteriﬁ ed cholesterol (nmol/mg liver) 0.8 ± 0.2 2.0 ± 0.1 1.7 ± 0.3 2.0 ± 0.3 Phospholipids (nmol/mg liver) 28.2 ± 1.5 25.8 ± 0.9 31.9 ± 0.7 29.5 ± 0.6 Triglycerides (nmol/mg liver) 12.8 ± 3.1 58.3 ± 5.0 22.2 ± 1.6 32.8 ± 4.5 Bile Bile ﬂ ow ( l/min/100 g bw) 10.3 ± 0.6 10.2 ± 0.2 ND ND Biliary bile acid secretion (nmol/min/100 g bw) 866 ± 86 762 ± 45 ND ND Biliary phospholipid secretion (nmol/min/100 g bw) 61.6 ± 5.0 82.6 ± 6.8 ND ND Biliary cholesterol secretion (nmol/min/100 g bw) 3.9 ± 0.2 4.0 ± 0.3 ND ND On day 4 after adenovirus injection, bile was collected continuously for 30 min, plasma samples were taken, and livers were harvested and snap- frozen in liquid nitrogen. Plasma lipids, liver lipids, and biliary output rates of bile acids, phospholipids, and cholesterol were determined as described in Materials and Methods. Values are means ± SEM; n = 5–8 mice for each condition. AdhApoE3, recombinant adenovirus expressing human apoE3; AdNull, empty control adenovirus; SR-BI, scavenger receptor class B type I; bw, body weight; ND, not determined. Signiﬁ cantly different from the respective AdNull-injected controls as assessed by Mann-Whitney U-test (at least P < 0.05). (Supplementary Table I), and the HDL cholesterol peak and triglyceride content were observed in response to was similarly decreased and was shifted toward larger HDL AdhApoE3 in hCETP tg mice (Supplementary Table II). particles (Supplementary Figure III). To test the hypothesis that the decrease in plasma HDL cholesterol and the concomitant increase in hepatic cho- lesterol content in response to apoE overexpression were Hepatic apoE overexpression increases hepatic cholesterol content by stimulating selective uptake into due to an enhanced selective uptake of cholesteryl esters the liver from HDL, HDL kinetic studies were carried out in wild- Next, we determined whether hepatic overexpression of type mice using autologous HDL. Hepatic apoE overex- human apoE would affect hepatic lipid composition. He- pression caused an increase in the HDL cholesteryl ester FCR (0.142 ± 0.009 vs. 0.196 ± 0.013 pools/h; P < 0.05; Fig. patic total cholesterol content was signiﬁ cantly increased by 24% in mice administered AdhApoE3 ( P < 0.05; Table 1 ), 2A ) without having signiﬁ cant effects on the HDL protein largely due to a higher hepatic esteriﬁ ed cholesterol FCR (0.084 ± 0.007 vs. 0.091 ± 0.013 pools/h; n.s.; Fig. 2A ). content (+150%; P < 0.01; Table 1 ). Whereas hepatic phos- Therefore, the apparent whole body selective uptake as calculated by the difference between the HDL cholesteryl pholipids were identical between AdNull-injected and AdhApoE3-injected mice ( Table 1 ), apoE overexpression ester and HDL protein FCRs was signiﬁ cantly higher in apoE- resulted in an elevated hepatic triglyceride content (+355%; overexpressing mice compared with controls (0.058 ± 0.011 P < 0.01; Table 1 ). Similar changes in hepatic cholesterol vs. 0.106 ± 0.010 pools/h; P < 0.05; Fig. 2A ). In agreement Fig. 1. Apolipoprotein E overexpression affects plasma cholesterol distribution in an SR-BI-dependent fashion. FPLC proﬁ les in response to apolipoprotein E overexpression in (A) wild-type mice and (B) SR-BI knockout (ko) mice. Pooled plasma samples collected on day 4 after injection with the control adenovirus AdNull or with the human apolipoprotein E3 expressing adenovirus AdhApoE3 were subjected to gel ﬁ ltra- tion chromatography analysis using a Superose 6 column as described in Materials and Methods. n = 6–8 mice for each condition. Open circles, AdNull-injected controls; closed squares, AdhApoE3-injected mice. 932 Journal of Lipid Research Volume 53, 2012 Fig. 2. Apolipoprotein E overexpression increases selective uptake of HDL cholesteryl esters into the liver. On day 4 after injection with the control adenovirus AdNull or with the human apolipoprotein E3-express- ing adenovirus, AdhApoE3 kinetic experiments were performed using autologous HDL double labeled with 125 3 I-tyramine-cellobiose and H-cholesteryl ether (CE) as described in Materials and Methods. A: FCRs calculated 125 3 from the respective plasma disappearance curves. B: Uptake of I-tyramine-cellobiose and H-CE by the liver. Data are presented as means ± SEM. n = 6 mice for each condition. White bars, AdNull-injected mice; black bars, AdhApoE3-injected mice. * Signiﬁ cantly different from the respective AdNull-injected controls as as- sessed by Mann-Whitney U-test (at least P < 0.05). with the above results, the uptake of HDL protein into the stimulating selective uptake of HDL cholesteryl esters into liver remained unchanged after hepatic apoE overexpres- the liver. sion (26.2 ± 3.7 vs. 24.6 ± 3.3%; n.s.; Fig. 2B ), whereas up- Increased hepatic cholesterol content in response to take of HDL cholesteryl ester into the liver tended to be hepatic apoE overexpression is dependent on SR-BI higher (40.4 ± 2.4 vs. 52.8 ± 4.2%; P = 0.07; Fig. 2B ). Over- SR-BI is the major receptor responsible for the selective all, this translated into an almost 2-fold increase in hepatic uptake of HDL cholesterol into the liver ( 21, 30 ). To con- selective uptake in the AdhApoE3-injected group (14.2 ± ﬁ rm a critical role of SR-BI mediating altered plasma 2.7 vs. 28.2 ± 1.6; P < 0.01; Fig. 2B ). Although selective up- lipoprotein distribution and hepatic cholesterol content take of HDL cholesteryl esters in the liver was enhanced, as a consequence of apoE overexpression, the effects of Sr-b1 mRNA expression was lower in wild-type mice ( P < AdNull or AdhApoE3 were investigated in SR-BI-deﬁ cient 0.01; Table 2 ) and in hCETP tg mice overexpressing apoE mice. In agreement with results in wild-type mice, injec- (Supplementary Table III). However, neither total nor tion of a human apoE expressing adenovirus did not alter membrane-associated hepatic SR-BI protein levels were plasma levels of total cholesterol, free cholesterol, esteri- changed in the two mouse models (Supplementary Figure ﬁ ed cholesterol, phospholipids, and triglycerides in SR-BI IV). Combined, these data demonstrate that hepatic apoE knockout mice ( Table 1 ). However, the marked altera- overexpression increases hepatic cholesterol content by tions observed in the lipoprotein distribution in response to apoE overexpression in wild-type mice were not present TABLE 2. Hepatic mRNA expression in wild-type mice in response in SR-BI knockouts, as reﬂ ected by virtually identical FPLC to hepatic apolipoprotein E overexpression proﬁ les in the AdNull-injected compared with the AdhA- poE3-injected group ( Fig. 1B ). In line with these results, Wild-type the hepatic content of total cholesterol ( Table 1 ), free AdNull AdhApoE3 cholesterol ( Table 1 ), and esteriﬁ ed cholesterol ( Table 1 ) Sr-b1 1.00 ± 0.03 0.70 ± 0.03 Abcb11 1.00 ± 0.06 0.60 ± 0.04 was not affected by apoE overexpression in SR-BI knock- Abcb4 1.00 ± 0.07 0.91 ± 0.05 out mice. Nevertheless, AdhApoE3 injection in the SR-BI Abcg5 1.00 ± 0.08 0.62 ± 0.04 knockouts caused a slight but signiﬁ cant decrease in he- Abcg8 1.00 ± 0.06 0.69 ± 0.06 Cyp7a1 1.00 ± 0.23 0.91 ± 0.13 patic phospholipid content ( 8%; P < 0.05; Table 1 ), Cyp27a1 1.00 ± 0.09 0.46 ± 0.04 whereas the hepatic triglyceride content tended to be Cyp8b1 1.00 ± 0.07 0.74 ± 0.02 a higher (+48%; P = 0.06; Table 1 ). These data indicate that Srebp2 1.00 ± 0.10 0.70 ± 0.02 the apoE-mediated changes in lipoprotein distribution Ldlr 1.00 ± 0.09 0.71 ± 0.04 Hmgcr 1.00 ± 0.16 0.74 ± 0.09 and hepatic cholesterol content are dependent on SR-BI. Abca1 1.00 ± 0.02 0.92 ± 0.06 Hepatic apoE overexpression does not affect biliary Livers of mice administered the respective adenoviruses were and fecal sterol excretion harvested on day 4 after adenovirus injection and snap-frozen in liquid nitrogen. mRNA expression levels were determined by real-time To explore whether higher SR-BI-mediated hepatic quantitative PCR as described in Materials and Methods. Values are cholesterol uptake after apoE overexpression in wild-type means ± SEM; n = 6 mice for each condition. Within each set of experiments, gene expression levels are related to the respective AdNull- mice would translate into changes in biliary sterol secretion, injected controls. AdhApoE3, recombinant adenovirus expressing a continuous bile cannulation experiment was performed human apoE3; AdNull, empty control adenovirus. in wild-type mice receiving AdNull or AdhApoE3. Neither Signiﬁ cantly different from the respective AdNull-injected controls as assessed by Mann-Whitney U-test (at least P < 0.05). bile ﬂ ow ( Table 1 ) nor biliary secretion rates of bile acids ApoE overexpression does not impact in vivo RCT 933 ( Table 1 ) were affected by hepatic overexpression of 0.3% injected tracer dose; n.s.), and 48 h (2.3 ± 0.2 vs. human apoE3. Although the biliary secretion rate of 2.4 ± 0.2% injected tracer dose; n.s.) after macrophage phospholipids was 1.3-fold higher ( P < 0.05; Table 1 ), the injection ( Fig. 3A ). Although apoE overexpression led to a secretion rate of cholesterol into bile remained un- 63% increase in macrophage-derived H-cholesterol within changed in wild-type mice ( Table 1 ). However, in hCETP the liver (7.0 ± 0.9 vs. 11.3 ± 1.2% injected tracer dose; tg mice, lower biliary output of cholesterol was noted in P < 0.05; Fig. 3B ), overall, in vivo RCT remained essentially the group injected with AdhApoE3, whereas there was no unchanged as reﬂ ected by no effect on the total excretion effect on the biliary secretion rates of bile acids and phos- of H-tracer into the feces (10.5 ± 1.0 vs. 10.7 ± 0.6% in- pholipids (Supplementary Figure V). jected tracer dose; n.s.; Fig. 3C ). In addition, no signiﬁ cant Hepatic mRNA expression of the hepatobiliary phos- changes were observed in the fecal loss of H-tracer within pholipid transporter Abcb4 (also known as multidrug neutral sterols (1.9 ± 0.2 vs. 2.2 ± 0.3% injected tracer dose; resistance protein 2, Mdr2 ) was not changed by apoE over- n.s.) or within the bile acid fraction (8.6 ± 0.9 vs. 8.5 ± expression in wild-type mice, whereas expression levels of 0.7% injected tracer dose; n.s.) in response to apoE over- the bile salt export pump Abcb11 (also known as Bsep ; P < expression ( Fig. 3C ). Moreover, in the CETP transgenic 0.01) and the cholesterol half-transporters Abcg5 and Abcg8 mouse model, RCT was not inﬂ uenced by AdhApoE3 were decreased ( P < 0.01 for both) ( Table 2 ). ApoE over- (Supplementary Figure VI). These observations indicate expression reduced expression of the key enzyme for the that hepatic apoE overexpression has no apparent effect alternative bile acid synthesis pathway in the liver, Cyp27a1 on macrophage RCT. ( P < 0.01) and the enzyme responsible for cholate synthe- sis, Cyp8b1 ( P < 0.05) but did not affect expression of the Probucol treatment decreases plasma lipid and lipoprotein levels rate-limiting enzyme for classic bile acid synthesis, Cyp7a1 ( Table 2 ). Hepatic gene expression of the sterol regula- It has been suggested recently that inhibition of hepatic tory binding protein 2 ( Srebp2 ) was suppressed by apoE ABCA1 activity by probucol reduces ABCA1-mediated cho- overexpression ( P < 0.05), whereas mRNA expression of its lesterol efﬂ ux from hepatocytes, thereby increasing cho- two target genes LDL receptor ( Ldlr ) and the rate-limiting lesterol excretion into the bile and feces ( 31 ). Cholesterol enzyme for cholesterol synthesis, HMG-CoA reductase efﬂ ux from lipid-laden macrophages in vitro was signiﬁ - ( Hmgcr ), was not signiﬁ cantly affected ( Table 2 ). There cantly higher toward plasma from ApoE-overexpressing was also no difference in Abca1 mRNA levels in the liver wild-type mice compared with controls after 4 h (6.5 ± 0.1 between AdhApoE3 injected wild-type mice and controls. vs. 7.5 ± 0.2%; P < 0.01) and 8 h (9.5 ± 0.1 vs. 10.8 ± 0.2%; In hCETP tg mice, virtually identical changes in liver gene P < 0.01) of incubation ( Fig. 4 ). We therefore hypothe- expression were found in response to apoE overexpres- sized that potentially increased ABCA1-mediated cho- sion (Supplementary Table III). lesterol efﬂ ux from hepatocytes might explain the Consistent with unaltered biliary secretion of choles- unchanged biliary and fecal sterol output in response to terol and bile acids in wild-type mice, the fecal mass output apoE over expression. To explore this hypothesis, wild-type of neutral sterols (3.11 ± 0.22 vs. 2.49 ± 0.34 mol/day; mice were fed a control chow diet or a chow diet supple- Supplementary Table IV) and bile acids (2.33 ± 0.19 vs. mented with 0.5% (wt/wt) probucol and were injected on 2.65 ± 0.11 mol/day; Supplementary Table V) was not day 10 of diet feeding with AdNull or AdhApoE3. Treat- inﬂ uenced by overexpression of apoE. Likewise, hCETP tg ment with probucol decreased plasma total cholesterol mice injected with AdhApoE3 also excreted similar amounts concentrations in AdNull-injected ( 51%; P < 0.01) and of neutral sterols (Supplementary Table IV) and bile acids AdhApoE3-injected mice ( 66%; P < 0.01; Table 3 ). This (Supplementary Table V) into the feces compared with was due to a signiﬁ cant lowering of plasma free cholesterol AdNull administered controls. Taken together, these re- ( 52% and 71% for AdNull and AdhApoE3, respec- sults indicate that apoE overexpression promotes hepatic tively; P < 0.01 for both) as well as esteriﬁ ed cholesterol cholesterol uptake without increasing biliary and fecal ste- ( 51% and 61% for AdNull and AdhApoE3, respec- rol excretion. tively; P < 0.01 for both) in the mice administered probu- col ( Table 3 ). As expected from the role of liver ABCA1 in Hepatic apoE overexpression does not affect HDL formation, HDL-cholesterol levels in plasma were macrophage-to-feces RCT markedly reduced by dietary probucol ( 41% and 42% Because apoE overexpression increased hepatic selec- for AdNull and AdhApoE3, respectively; P < 0.01 for both). However, probucol also resulted in decreased plasma non- tive uptake via SR-BI, an important step in the RCT path- way, but did not inﬂ uence mass biliary and fecal sterol HDL-cholesterol levels ( 66% and 79% for AdNull and excretion, we next investigated whether apoE overexpres- AdhApoE3, respectively; P < 0.01 for both). In line with sion might affect overall RCT. In vivo RCT was traced after these results, FPLC analysis demonstrated a clear reduc- tion in all lipoprotein classes in AdNull-injected mice fed intraperitoneal injection of primary mouse macrophages loaded with H-cholesterol in control and apoE-overex- the probucol diet compared with mice fed the control diet pressing wild-type mice. The appearance of tracer in ( Fig. 5A ). Similar changes in the plasma lipoprotein distri- plasma was not signiﬁ cantly different between controls bution were observed in human apoE-overexpressing mice in response to probucol ( Fig. 5B ). Finally, administration and mice injected with AdhApoE3 at 6 h (2.1 ± 0.6 vs. 3.0 ± 0.5% injected tracer dose; n.s.), 24 h (2.8 ± 0.4 vs. 2.8 ± of probucol resulted in a signiﬁ cant decrease in plasma 934 Journal of Lipid Research Volume 53, 2012 Fig. 3. Apolipoprotein E overexpression does not affect in vivo macrophage-to-feces reverse cholesterol transport in wild-type mice. On day 2 after injection with the control adenovirus AdNull or with the human apolipoprotein E3-expressing adenovirus AdhApoE3, mice received intraperitoneal injections with H-cho- lesterol-loaded primary mouse macrophage foam cells as described in Materials and Methods. A: Time 3 3 course of H-cholesterol recovery in plasma. B: H-cholesterol within liver 48 h after macrophage administra- tion. C: H-cholesterol appearance in feces collected continuously from 0 to 48 h after macrophage admin- istration and separated into bile acid and neutral sterol fractions as indicated. Data are expressed as percentage of the injected tracer dose and presented as means ± SEM. n = 8 mice for each condition. White bars, AdNull-injected mice; black bars, AdhApoE3-injected mice. * Signiﬁ cantly different from the respec- tive AdNull-injected controls as assessed by Mann-Whitney U-test (at least P < 0.05). phospholipids and triglycerides in mice injected with 66% for phospholipids and triglycerides, respectively; the control adenovirus AdNull ( 37% and 38% for P < 0.01 for both) (Table 3). phospholipids and triglycerides, respectively; P < 0.01 Probucol treatment does not change the hepatic and P < 0.05, respectively) or AdhApoE3 ( 57% and cholesterol content in response to hepatic apoE overexpression In mice that received the control adenovirus AdNull, the hepatic content of total cholesterol was not differ- ent between groups on control and probucol-containing diet ( Table 3 ). ApoE overexpression consistently increased total cholesterol levels in the liver; however, there was no additional effect of probucol ( Table 3 ). The amount of free cholesterol and esteriﬁ ed cholesterol in the liver was not changed in response to probucol in both the mice administered AdNull and AdhApoE3 ( Table 3 ). Probucol treatment resulted in a lower hepatic phos- pholipid content in the AdNull ( 9%; P < 0.05) but Fig. 4. Apolipoprotein E overexpression increases cholesterol not the AdhApoE3 group ( Table 3 ). There was no efﬂ ux from macrophage foam cells toward plasma. Thioglycollate- effect of probucol on the hepatic triglyceride content elicited peritoneal mouse macrophages were loaded with 50 g/ml ( Table 3 ). Thus, probucol does not change the hepatic acetylated LDL and 1 Ci/ml [ H]cholesterol as described in cholesterol mass content in response to hepatic apoE Materials and Methods. Subsequently, 2% plasma was added to the cells. After 4 h and 8 h, radioactivity within the medium and radio- overexpression. activity remaining within the cells was determined by liquid scintil- lation counting. Efﬂ ux is given as the percentage of counts Probucol treatment increases biliary and fecal sterol recovered from the medium in relation to the total counts present secretion in apoE-overexpressing mice on the plate (sum of medium and cells). Data are presented as Bile cannulation experiments revealed that dietary means ± SEM. n = 8 mice for each condition. White bars, AdNull- probucol had no effect on bile ﬂ ow or on the biliary se- injected mice; black bars, AdhApoE3-injected mice. * Signiﬁ cantly cretion of bile acids, phospholipids, and cholesterol in different from the respective AdNull-injected controls as assessed mice injected with the control adenovirus ( Table 3 ). by Mann-Whitney U-test (at least P < 0.05). ApoE overexpression does not impact in vivo RCT 935 TABLE 3. Plasma lipids, liver lipid composition, and biliary excretion of sterols in response to probucol treatment AdNull AdhApoE3 Control Probucol Control Probucol Plasma a a Total cholesterol (mg/dl) 72.3 ± 1.7 35.3 ± 1.3 63.7 ± 2.8 21.9 ± 1.3 a a Free cholesterol (mg/dl) 22.2 ± 0.4 10.7 ± 0.6 29.8 ± 2.0 8.5 ± 1.1 a a Esteriﬁ ed cholesterol (mg/dl) 50.1 ± 1.8 24.6 ± 1.1 34.0 ± 2.0 13.4 ± 0.6 a a Phospholipids (mg/dl) 154.1 ± 7.4 97.7 ± 5.8 137.8 ± 9.5 59.8 ± 1.3 a a Triglycerides (mg/dl) 58.2 ± 7.1 36.0 ± 3.5 95.3 ± 7.9 32.4 ± 7.0 Liver Total cholesterol (nmol/mg liver) 7.2 ± 0.2 7.5 ± 0.2 9.1 ± 0.4 9.2 ± 0.5 Free cholesterol (nmol/mg liver) 5.9 ± 0.1 6.0 ± 0.2 7.4 ± 0.5 6.9 ± 0.3 Esteriﬁ ed cholesterol (nmol/mg liver) 1.3 ± 0.1 1.5 ± 0.1 1.7 ± 0.2 2.3 ± 0.2 Phospholipids (nmol/mg liver) 29.2 ± 0.7 26.5 ± 0.6 26.7 ± 0.6 27.8 ± 0.6 Triglycerides (nmol/mg liver) 21.4 ± 1.7 22.3 ± 3.3 63.7 ± 7.1 59.4 ± 8.6 Bile Bile ﬂ ow ( l/min/100 g bw) 9.3 ± 0.6 8.2 ± 0.7 7.3 ± 1.1 10.6 ± 0.9 Biliary bile acid secretion (nmol/min/100 g bw) 469 ± 90 357 ± 73 427 ± 81 466 ± 51 Biliary phospholipid secretion (nmol/min/100 g bw) 41.6 ± 3.1 37.7 ± 5.6 43.0 ± 5.4 64.3 ± 5.1 Biliary cholesterol secretion (nmol/min/100 g bw) 3.5 ± 0.4 3.6 ± 0.5 2.9 ± 0.4 5.2 ± 0.7 Mice were fed a control chow diet or a chow diet containing 0.5% probucol for 2 weeks. On day 4 after adenovirus injection, bile was collected continuously for 30 min, plasma samples were taken, and livers were harvested and snap- frozen in liquid nitrogen. Plasma lipids, liver lipids, and biliary output rates of bile acids, phospholipids, and cholesterol were determined as described in Materials and Methods. Values are means ± SEM; n = 6 mice for each condition. AdhApoE3, recombinant adenovirus expressing human apoE3; AdNull, empty control adenovirus; bw, body weight. Signiﬁ cantly different from the respective controls as assessed by Mann-Whitney U-test (at least P < 0.05). However, in AdhApoE3-administered mice, bile flow of Srebp2 and its target genes ldlr and hmgcr in the liver was tended to increase in response to probucol ( P = 0.055; not different between mice fed a chow diet and mice fed a Table 3 ). Whereas the biliary output rate of bile acids re- probucol-enriched diet ( Table 4 ). Finally, as has been re- mained unchanged, biliary secretion rates of phospho- ported previously ( 31 ), no change in the hepatic mRNA lipids were 1.5-fold higher in apoE-overexpressing mice expression of Abca1 was detected in the probucol-treated in response to probucol ( P < 0.05), and the biliary secre- mice ( Table 4 ). tion rate of cholesterol increased signiﬁ cantly by 1.8-fold Analysis of fecal contents showed that treatment with ( P < 0.05) ( Table 3 ). probucol did not inﬂ uence the fecal excretion of neutral Hepatic mRNA expression of the bile acid transporter sterols (4.74 ± 0.28 vs. 5.07 ± 0.18 mol/day) and bile ac- Abcb11 , the phospholipid transporter Abcb4 , and the cho- ids (2.52 ± 0.22 vs. 3.02 ± 0.28 mol/day) in mice that re- lesterol half-transporters Abcg5 and Abcg8 was not modi- ceived the control adenovirus AdNull. In contrast and in ﬁ ed by probucol treatment in control mice or in mice that good agreement with the elevated biliary cholesterol se- overexpress apoE in the liver ( Table 4 ). No signiﬁ cant cretion, fecal excretion of neutral sterols was signiﬁ cantly changes in the hepatic gene expression level of the bile enhanced by probucol in mice overexpressing apoE (3.69 ± acid synthesizing enzymes Cyp7a1 , Cyp27a1 , and Cyp8b1 0.21 vs. 4.97 ± 0.37 mol/day; P < 0.05). Nonetheless, no were observed, except for higher relative mRNA levels effect of probucol on the fecal bile acid output was found of Cyp7a1 in apoE-overexpressing mice upon probucol in these mice (2.20 ± 0.14 vs. 2.66 ± 0.23 mol/day). Com- administration ( P < 0.05; Table 4 ). Furthermore, expression bined, these data demonstrate that biliary and fecal sterol Fig. 5. Probucol treatment decreases plasma cholesterol levels. FPLC proﬁ les in response to probucol treatment in (A) AdNull-injected and (B) AdhApoE3-injected mice. Mice were fed a control chow diet or a chow diet containing 0.5% probucol for 2 weeks. Pooled plasma samples collected on day 4 after injection with the control adenovirus AdNull or with the human apolipoprotein E3-expressing adenovirus AdhApoE3 were subjected to gel ﬁ ltration chromatography analysis using a Superose 6 column as described in Materials and Methods. n = 6 mice for each condition. Open circles, chow-fed controls; closed squares; probucol- treated mice. 936 Journal of Lipid Research Volume 53, 2012 TABLE 4. Hepatic mRNA expression in response to DISCUSSION probucol treatment This study demonstrates that hepatic overexpression of AdNull AdhApoE3 human apoE not only promotes SR-BI-mediated selective Control Probucol Control Probucol uptake of HDL cholesterol into the liver but also enhances a a Sr-b1 1.00 ± 0.05 0.88 ± 0.03 1.00 ± 0.03 1.08 ± 0.03 the resecretion of RCT-relevant cholesterol via hepatocyte Abcb11 1.00 ± 0.06 1.16 ± 0.04 1.00 ± 0.05 1.14 ± 0.08 ABCA1 back to the plasma compartment. As a result, bil- Abcb4 1.00 ± 0.07 1.05 ± 0.09 1.00 ± 0.06 1.12 ± 0.05 Abcg5 1.00 ± 0.10 0.91 ± 0.09 1.00 ± 0.06 1.20 ± 0.09 iary and fecal mass sterol excretion and RCT remain un- Abcg8 1.00 ± 0.07 0.93 ± 0.08 1.00 ± 0.06 1.13 ± 0.13 a changed in apoE-overexpressing mice with active ABCA1, Cyp7a1 1.00 ± 0.15 0.60 ± 0.09 1.00 ± 0.13 1.58 ± 0.12 whereas all of these parameters increase signiﬁ cantly when Cyp27a1 1.00 ± 0.05 0.90 ± 0.03 1.00 ± 0.07 1.11 ± 0.07 Cyp8b1 1.00 ± 0.08 0.96 ± 0.09 1.00 ± 0.06 1.04 ± 0.09 ABCA1 activity is blocked with probucol. Collectively, Srebp2 1.00 ± 0.04 0.96 ± 0.05 1.00 ± 0.05 0.81 ± 0.06 these results point to a metabolic shunt potentially con- Ldlr 1.00 ± 0.08 1.14 ± 0.08 1.00 ± 0.06 0.94 ± 0.06 necting the SR-BI and the ABCA1 pathway with a high rel- Hmgcr 1.00 ± 0.07 0.98 ± 0.09 1.00 ± 0.05 0.83 ± 0.09 Abca1 1.00 ± 0.04 1.07 ± 0.02 1.00 ± 0.04 1.04 ± 0.04 evance for the regulation of RCT. The RCT pathway represents an important atheropro- Mice were fed a control chow diet or a chow diet containing 0.5% tective functionality of HDL ( 16, 17 ). RCT comprises cho- probucol for 2 weeks. Livers of mice administered the respective adenoviruses were harvested on day 4 after adenovirus injection and lesterol efﬂ ux from macrophage foam cells within the snap-frozen in liquid nitrogen. mRNA expression levels were vessel wall, the transport of this cholesterol within HDL determined by real-time quantitative PCR as described in Materials and through the plasma compartment, the subsequent hepatic Methods. Values are means ± SEM; n = 6 mice for each condition. Within each set of experiments, gene expression levels are related to uptake via SR-BI or as a holoparticle, and excretion into the respective chow-fed controls. AdhApoE3, recombinant adenovirus the bile and feces ( 16, 17 ). In vitro, apoE expression by expressing human apoE3; AdNull, empty control adenovirus. macrophages has been shown to stimulate cholesterol ef- Signiﬁ cantly different from the respective controls as assessed by Mann-Whitney U-test (at least P < 0.05). ﬂ ux ( 12–14 ). In addition, in vivo macrophage-to-feces RCT was lower when apoE-deﬁ cient macrophages were injected into wild-type mice compared with wild-type macrophages secretion are increased upon probucol treatment in apoE- injected into wild-type mice ( 15 ). Combined with our pre- overexpressing mice. sent results, these data suggest that apoE expression by macrophages might determine the general availability of Probucol treatment increases macrophage-to-feces macrophage-derived cholesterol for the RCT pathway di- RCT in apoE-overexpressing mice rectly at the point of entry, whereas in subsequent steps of Because probucol enhanced biliary and fecal sterol se- RCT hepatic ABCA1 counteracts these effects. cretion in mice overexpressing human apoE, we investi- Experiments in SR-BI knockout mice as well as HDL ki- netic studies in the current report demonstrated that apoE gated whether this would also translate into an improvement overexpression speciﬁ cally increased selective uptake of in overall RCT from macrophages to feces. After intrap- eritoneal injection of H-cholesterol-loaded macro- HDL cholesterol into the liver. Importantly, the expression phages, counts within plasma were profoundly lower at the levels of hepatic SR-BI did not change in response to apoE 6 h (1.24 ± 0.16 vs. 0.48 ± 0.04% injected tracer dose; overexpression. Therefore, our present ﬁ ndings are com- plementary to previous work demonstrating impaired he- P < 0.01; Fig. 6A ), 24 h (1.62 ± 0.20 vs. 0.59 ± 0.04% injected tracer dose; P < 0.01; Fig. 6A ), and 48 h time point (1.48 ± patic selective HDL cholesterol uptake in mice lacking apoE 0.22 vs. 0.56 ± 0.06% injected tracer dose; P < 0.01; Fig. 6A ) ( 32 ), although the apoE knockout mouse model exhibits a in the probucol-treated apoE-overexpressing mice compared considerably altered plasma lipid proﬁ le with substantially increased levels of apoB-containing lipoprotein remnants with apoE-overexpressing controls. However, the amount of macrophage-derived tracer recovered within the liver was ( 32 ). In addition, selective uptake of HDL cholesterol into not affected by probucol in mice with hepatic apoE overex- HepG2 cells after treatment with a blocking antibody di- pression (7.6 ± 1.0 vs. 7.0 ± 0.7% injected tracer dose; n.s.; rected against apoE was reduced ( 33 ). It is possible that Fig. 6B ). Consistent with the higher biliary and fecal mass apoE stimulates SR-BI-mediated selective uptake due to excretion of sterols, probucol signiﬁ cantly enhanced the an improved interaction of the HDL particle with SR-BI total excretion of H-cholesterol originating from mac- ( 32 ), although the exact mechanisms will require further rophages into the feces of AdhApoE3-injected mice (6.6 ± investigation. 0.4 vs. 9.5 ± 0.5% injected tracer dose; P < 0.01; Fig. 6C ). Although hepatic human apoE overexpression in- Because tracer recovery in the fecal bile acid fraction creased the hepatic cholesterol content, biliary choles- remained unaltered (5.3 ± 0.4 vs. 5.7 ± 0.4% injected tracer terol excretion and the overall elimination of cholesterol dose; n.s.; Fig. 6C ), this was attributable to a 2.7-fold from the body did not increase in wild-type mice or higher excretion of H-cholesterol in the fecal neutral ste- hCETP tg mice. The current observation that increased rol fraction (1.4 ± 0.2% vs. 3.8 ± 0.5% injected tracer dose; SR-BI-mediated selective uptake does not necessarily P < 0.01; Fig. 6C ). These ﬁ ndings demonstrate that translate into enhanced biliary and fecal sterol excretion probucol results in an increased movement of choles- in the face of unaltered hepatic SR-BI expression is in terol from macrophages to the feces in mice with hepatic agreement with earlier observations made by us in mice apoE overexpression. ( 23 ) overexpressing group IIA secretory phospholipase A ApoE overexpression does not impact in vivo RCT 937 Fig. 6. Probucol treatment increases in vivo macrophage-to-feces reverse cholesterol transport in apolipo- protein E-overexpressing mice. Mice were fed a control chow diet or a chow diet containing 0.5% probucol for 12 days before and then throughout the 48-h period of the experiment. On day 2 after injection with the human apolipoprotein E- expressing adenovirus, AdhApoE3 mice received intraperitoneal injections with H-cholesterol-loaded primary mouse macrophage foam cells as described in Materials and Methods. A: 3 3 Time course of H-cholesterol recovery in plasma. B: H-cholesterol within liver 48 h after macrophage ad- ministration. C: H-cholesterol appearance in feces collected continuously from 0 to 48 h after macrophage administration and separated into bile acid and neutral sterol fractions as indicated. Data are expressed as percentage of the injected tracer dose and presented as means ± SEM. n = 8 mice for each condition. White bars, chow-fed controls; black bars, probucol-treated mice. * Signiﬁ cantly different from the respective con- trols as assessed by Mann-Whitney U-test (at least P < 0.05). or endothelial lipase (EL) ( 21, 34 ). Transgenic overex- ABCA1. Ideally, such studies should be carried out in po- pression of secretory phospholipase A or adenovirus- larized liver cells. To formally relate the cholesterol mass mediated overexpression of EL also resulted in an changes observed in our current study to each other, increased ﬂ ux of cholesterol into the liver, whereas bil- quantifying cholesterol ﬂ uxes would be required, which iary cholesterol secretion and mass fecal excretion of ste- has not been done in our present study and thus repre- rols remained unaffected ( 21, 23, 34 ). On the other sents a potential limitation in the interpretation of our hand, altering the hepatic expression level of SR-BI has current work. clear effects on biliary cholesterol secretion as well as Systemic apoE ( 7–11 ) and macrophage-derived apoE RCT with overexpression resulting in an increase and ( 36–39 ) have been shown to protect against atheroscle- knockdown in a decrease of both of these parameters rotic lesion formation due to effects not immediately involv- ( 24, 34, 35 ). How can this discrepancy be explained? Re- ing RCT. ApoE promotes the clearance of atherogenic cent data generated in probucol-fed mice demonstrated lipoproteins ( 6, 7, 10 ), and also a number of pleiotropic that a decrease in hepatic ABCA1 activity results in in- effects of apoE might be important. For example, apoE creased in vivo RCT ( 31 ). We therefore speculate that has antioxidative properties. ApoE prevented oxidative increased cholesterol uptake into the liver via SR-BI re- cell death in cultured neuronal cells and inhibited copper- sults in transport to a speciﬁ c intrahepatic compartment mediated oxidation of LDL, a key event in the initiation also accessible for ABCA1-mediated resecretion back into and progression of atherosclerotic lesions ( 40 ). Moreover, the plasma compartment to generate new HDL particles. apoE knockout mice have signiﬁ cantly increased in vivo Increasing hepatic SR-BI expression levels ( 24, 34 ) or de- oxidative stress, as determined by plasma, urinary, and vas- creasing the activity of ABCA1 might shift the balance cular isoprostane levels ( 41 ), whereas hepatic overexpres- toward biliary secretion (present report and Reference sion of apoE in LDL receptor knockout mice resulted in a 31 ). However, the nature of these intrahepatic choles- reduced oxidative stress burden and decreased atheroscle- terol pools is unknown. Future in vitro studies should rosis independent of plasma lipid and lipoprotein levels therefore focus on the intracellular trafﬁ cking of choles- ( 9 ). Anti-inﬂ ammatory activities of apoE also add to its terol and, using a pulse-chase set-up, on the incorpora- atheroprotective properties. In vitro studies have shown tion of HDL-derived cholesterol tracers into newly formed that apoE can suppress proliferation of cultured periph- HDL in models with altered expression of hepatocyte eral blood T lymphocytes ( 42 ) and shifts polarization of 938 Journal of Lipid Research Volume 53, 2012 11 . Kawashiri , M. , Y. Zhang , D. Usher , M. Reilly , E. Pure , and D. J. mouse macrophages to the anti-inﬂ ammatory M2 phenotype Rader . 2001 . Effects of coexpression of the LDL receptor and apoE ( 43 ). In vivo, apoE-deﬁ cient mice exhibit an exaggerated on cholesterol metabolism and atherosclerosis in LDL receptor- proinﬂ ammatory cytokine response after LPS injection deﬁ cient mice. J. 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Endogenous apolip- oprotein E modulates cholesterol efﬂ ux and cholesteryl ester knockout mice ( 46 ). Finally, apoE might be antiathero- hydrolysis mediated by high-density lipoprotein-3 and lipid-free genic by inhibiting platelet aggregation ( 47 ) as well as pro- apolipoproteins in mouse peritoneal macrophages. J Mol Med liferation of vascular smooth muscle cells ( 48 ). (Berl) . 78 : 217 – 227 . 15 . Zanotti , I. , M. Pedrelli , F. Poti , G. Stomeo , M. Gomaraschi , L. In summary, this study demonstrates that hepatic over- Calabresi , and F. Bernini . 2011 . Macrophage, but not systemic, expression of human apoE3 not only facilitates SR-BI- apolipoprotein E is necessary for macrophage reverse cholesterol mediated selective uptake of HDL cholesterol into the transport in vivo. Arterioscler. Thromb. Vasc. Biol. 31 : 74 – 80 . 16 . Lewis , G. F. , and D. J. Rader . 2005 . 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Invest. 113 : 609 – 618 . 940 Journal of Lipid Research Volume 53, 2012 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Lipid Research American Society for Biochemistry and Molecular Biology http://www.deepdyve.com/lp/american-society-for-biochemistry-and-molecular-biology/apoe-promotes-hepatic-selective-uptake-but-not-rct-due-to-increased-iyizO7n12S

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Publisher: American Society for Biochemistry and Molecular Biology
Copyright: Copyright © 2012 Elsevier Inc.
ISSN: 0022-2275
eISSN: 1539-7262
DOI: 10.1194/jlr.m020743
Publisher site: See Article on Publisher Site

Abstract

ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efﬂ ux to plasma 1, , † 1, § Wijtske Annema , * Arne Dikkers , * Jan Freark de Boer, * Thomas Gautier, †† 2, ,† Patrick C. N. Rensen , ** Daniel J. Rader, and Uwe J. F. Tietge * Department of Pediatrics,* Center for Liver, Digestive, and Metabolic Diseases, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands ; Top Institute Food and Nutrition , Wageningen, The Netherlands ; INSERM UMR866 Lipides, Nutrition, Cancer, Faculté de Médecine, Dijon, France ; Department of General Internal Medicine,** Endocrinology, and Metabolic Diseases, Leiden University Medical Center, Leiden , The Netherlands ; and Institute for Translational Medicine and †† Therapeutics, University of Pennsylvania School of Medicine , Philadelphia, PA Supplementary key words apolipoprotein E � reverse cholesterol Abstract ApoE plays an important role in lipoprotein me- transport � ATP-binding cassette transporter A1 � atherosclerosis � tabolism. This study investigated the effects of adenovirus- bile � cholesteryl ester transfer protein � feces � high density lipopro- mediated human apoE overexpression (AdhApoE3) on sterol tein � liver � macrophage � metabolism � mice � probucol metabolism and in vivo reverse cholesterol transport (RCT). In wild-type mice, AdhApoE3 resulted in decreased HDL cholesterol levels and a shift toward larger HDL in plasma, Apolipoprotein E (apoE) plays an important role in whereas hepatic cholesterol content increased ( P < 0.05). These effects were dependent on scavenger receptor class B lipo protein metabolism and atherosclerosis. ApoE is pro- type I (SR-BI) as conﬁ rmed using SR-BI-deﬁ cient mice. Ki- duced and secreted predominantly by the liver ( 1 ), but it netic studies demonstrated increased plasma HDL choles- is also expressed in a variety of other tissues, including teryl ester catabolic rates ( P < 0.05) and higher hepatic macrophages ( 2, 3 ). While loss of function of apoE in mice selective uptake of HDL cholesteryl esters in AdhApoE3- and in humans is associated with a proatherogenic lipo- injected wild-type mice ( P < 0.01). However, biliary and fecal protein proﬁ le and increased atherogenesis ( 4, 5 ), overex- sterol output as well as in vivo macrophage-to-feces RCT pression of apoE in various models has been shown to studied with H-cholesterol-loaded mouse macrophage foam protect against atherosclerotic lesion formation ( 6–11 ). cells remained unchanged upon human apoE overexpres- sion. Similar results were obtained using hApoE3 overex- Among other metabolic effects that are potentially anti- pression in human CETP transgenic mice. However, blocking atherogenic, apoE has been reported to promote choles- ABCA1-mediated cholesterol efﬂ ux from hepatocytes in terol efﬂ ux ( 12–14 ), and recent studies have suggested AdhApoE3-injected mice using probucol increased biliary cho- that lack of macrophage apoE might decrease overall re- lesterol secretion ( P < 0.05), fecal neutral sterol excretion ( P < verse cholesterol transport (RCT) ( 15 ). However, the pool 0.05), and in vivo RCT ( P < 0.01), speciﬁ cally within neutral of macrophage-derived apoE represents a small fraction of sterols. These combined data demonstrate that systemic total circulating apoE. apoE overexpression increases i ) SR-BI-mediated selective The classic RCT pathway is a multistep process that in- uptake into the liver and ii ) ABCA1-mediated efﬂ ux of RCT- relevant cholesterol from hepatocytes back to the plasma com- volves i ) HDL-mediated efﬂ ux of excess cholesterol from partment, thereby resulting in unchanged fecal mass sterol extrahepatic cells and most relevant for atherosclerosis excretion and overall in vivo RCT. —Annema, W., A. Dikkers, lipid-laden macrophages in the arterial wall, ii ) uptake of J. F. de Boer, T. Gautier, P. C. N. Rensen, D. J. Rader, and U. HDL cholesterol into the liver, and iii ) excretion of HDL J. F. Tietge. ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efﬂ ux to plasma. J. Lipid Res . 2012. 53: 929–940. Abbreviations: CE, cholesteryl ether; CETP, cholesteryl ester trans- fer protein; FCR, fractional catabolic rate; FPLC, fast protein liquid This work was supported by the Netherlands Organization for Scientiﬁ c Research chromatography; RCT, reverse cholesterol transport; SR-BI, scavenger VIDI grant 917-56-358 (U.J.F.T.), by the Top Institute (TI) Food and Nutrition receptor class B type I. (U.J.F.T.), and by grants HL022633 and HL059407 from the NHLBI 1 These authors contributed equally to this work. (D.J.R.). P.C.N.R. is an Established Investigator of the Netherlands Heart To whom correspondence should be addressed. Foundation (2009T038). e-mail: [email protected]. Manuscript received 21 September 2011 and in revised form 29 February 2012. The online version of this article (available at http://www.jlr.org) Published, JLR Papers in Press, March 1, 2012 contains supplementary data in the form of ﬁ ve tables, six ﬁ gures, and DOI 10.1194/jlr.M020743 supplemental Methods. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 53, 2012 929 This is an Open Access article under the CC BY license. liquid chromatography (FPLC) gel ﬁ ltration using a Superose 6 cholesterol into bile and ultimately feces either directly or column (GE Healthcare, Uppsala, Sweden) as described ( 21 ). after metabolic conversion into bile acids ( 16–18 ). Al- Individual fractions were assayed for cholesterol concentrations though the liver has a key function in the RCT pathway as described above. and the majority of circulating apoE is generated by hepa- To determine plasma HDL- and nonHDL-cholesterol levels, tocytes, the contribution of hepatic apoE to in vivo RCT apoB-containing lipoproteins were precipitated using polyethyl- has not been addressed. ene glycol in 10 mM HEPES (pH 8.0) as described ( 22 ). After Therefore, the aim of the current study was to investi- centrifugation of the samples at 2,000 g and 4°C for 30 min, the gate the effects of hepatic overexpression of human apoE HDL-containing supernatant was transferred to clean tubes. The nonHDL-containing pellet was dissolved in 0.5 M NaCO . Cho- on liver lipid metabolism, biliary sterol secretion, and in lesterol concentrations in the HDL and nonHDL fractions were vivo macrophage-to-feces RCT. Our data demonstrate that determined as described above. increasing plasma levels of liver-derived apoE enhances selective uptake of HDL cholesteryl esters into the liver Analysis of liver lipid composition and induces hepatic cholesterol accumulation in a scaven- Liver tissue was homogenized as described ( 23 ). Commercially ger receptor class B type I (SR-BI)-dependent manner. available kits were used to measure contents of total cholesterol, However, this does not affect fecal mass sterol excretion triglycerides (Roche Diagnostics), and free cholesterol (Diasys) and macrophage-speciﬁ c RCT due to an apoE-induced after extraction of lipids according to the general procedure of enhancement of ATP-binding cassette transporter A1 Bligh Dyer and redissolving lipids in water containing 2% Triton (ABCA1)-mediated efﬂ ux of RCT-relevant cholesterol X-100 ( 23 ). Phospholipid content of the liver was determined from hepatocytes back to the plasma compartment. These after lipid extraction essentially as described ( 23 ). ﬁ ndings suggest that systemic apoE overexpression pro- Analysis of gene expression by real-time quantitative PCR tects against atherosclerosis by mechanisms other than Total RNA from mouse livers was extracted with TriReagent modulation of RCT. (Sigma) and quantiﬁ ed using a Nanodrop ND-100 U-Vis spectro- photometer (NanoDrop Technologies, Wilmington, DE). cDNA synthesis was performed from 1 g of total RNA using reagents MATERIALS AND METHODS from Invitrogen (Carlsbad, CA). Real-time quantitative PCR was Animals carried out on an ABI-Prism 7700 (Applied Biosystems, Darm- stadt, Germany) sequence detector with the default settings ( 23 ). C57BL/6J mice were obtained from Charles River (Wilming- Primers and ﬂ uorogenic probes were designed with the Primer ton, MA). SR-BI knockout mice were obtained from The Jackson Express Software (Applied Biosystems) and synthesized by Euro- Laboratory (Bar Harbor, ME) and backcrossed to the C57BL/6J gentec (Seraing, Belgium). mRNA expression levels presented background for a total of eight generations. Probucol (Sigma, St. were calculated relative to the average of the housekeeping gene Louis, MO) was mixed into powdered chow (0.5% wt/wt). For cyclophilin and further normalized to the relative expression the RCT experiment, the diet was provided for 12 days before levels of the respective controls. and then throughout the 48-h period of the experiment. In all other experiments, the diet was provided for 14 days. Animals Bile collection and assessment of biliary excretion of bile were caged in animal rooms with alternating 12-h periods of light acids, phospholipids, and cholesterol (from 7:00 AM to 7:00 PM) and dark (from 7:00 PM to 7:00 AM), Bile was collected by cannulation of the gallbladder in mice with ad libitum access to water and mouse chow diet (Arie Blok, anesthetized by intraperitoneal injection of hypnorm (fentanyl/ Woerden, The Netherlands). Animal experiments were per- ﬂ uanisone, 1 mg/kg) and diazepam (10 mg/kg). During the bile formed in conformity with PHS policy and in accordance with collection, body temperature was maintained using a humidiﬁ ed the national laws. All protocols were approved by the responsible incubator. Bile collection was performed for 30 min, and secre- ethics committee of the University of Groningen and the Univer- tion rates were determined gravimetrically. Biliary bile salt, cho- sity of Pennsylvania. lesterol, and phospholipid concentrations were determined and Generation of recombinant adenoviruses the respective biliary excretion rates calculated as described pre- viously ( 23, 24 ). The empty control adenovirus AdNull ( 19 ) and the recombi- nant adenovirus encoding human apoE3 (AdhApoE3) ( 19 ) were Fecal sterol analysis ampliﬁ ed and puriﬁ ed as reported previously ( 20 ). For in vivo experiments, mice were injected with 1 × 10 particles/mouse of Mice were individually housed, and feces were collected over a period of 24 h and separated from the bedding. Fecal samples AdhApoE3 or AdNull. In vivo reverse cholesterol transport stud- ies were carried out between day 2 and day 4 after injection of were dried, weighed, and thoroughly ground. Aliquots thereof were used for determination of neutral sterol and bile acid con- recombinant adenoviruses, a time frame when high and stable expression from an adenovirus is achieved. All other experiments tent by gas-liquid chromatography as described ( 23, 24 ). described were performed on day 4 after injection of the recom- binant adenoviruses. HDL kinetics studies HDL kinetics studies were performed essentially as published Plasma lipid and lipoprotein analysis previously ( 21 ). Autologous HDL was prepared from pooled Mice were bled by heart puncture after a 4-h fast at the time of mouse plasma by sequential ultracentrifugation (density 1.063 < death. Aliquots of plasma were stored at 80°C until analysis. d < 1.21). After extensive dialysis against sterile PBS containing Commercially available reagents were used to measure plasma 0.01% EDTA, HDL was labeled with I-tyramine-cellobiose (TC) total cholesterol, triglycerides (Roche Diagnostics, Basel, Switzer- and cholesteryl hexadecyl ether (cholesteryl-1,2,- H; Perkin land), free cholesterol, and phospholipids (Diasys, Holzheim, Elmer Life Sciences) as previously described ( 25 ). For kinetic 125 3 Germany). Pooled plasma samples were subjected to fast protein studies, 0.4 µCi of I and 0.7 million dpm of the H tracer were 930 Journal of Lipid Research Volume 53, 2012 injected into the tail veins of fasted wild-type mice treated with 1640 medium (Invitrogen) supplemented with 1% FBS (Hy- AdNull or AdhApoE3. Blood samples were drawn by retroorbital Clone, Logan, UT) and penicillin (100 U/ml)/streptomycin bleeding at 5 min and at 1, 3, 6, 11, and 24 h after injection. (100 g/ml) (Invitrogen) and were allowed to adhere for 5 h at Plasma decay curves for both tracers were generated by dividing 37°C under 5% CO humidiﬁ ed air. Nonadherent cells were re- the plasma radioactivity at each time point by the radioactivity at moved by washing twice with PBS followed by loading of the mac- the initial 5-min time point after tracer injection. Fractional cata- rophages with 50 g/ml LDL and 1 Ci/ml H-cholesterol bolic rates (FCRs) were determined from the area under the (Perkin Elmer Life Sciences, Boston, MA) for 24 h. The cells plasma disappearance curves ﬁ tted to a bicompartmental model were washed again and equilibrated in RPMI 1640 medium sup- using the SAAM II program ( 26 ). The use of H-cholesteryl ether plemented with 2% BSA (Sigma) for 18 h. The cells were washed does not affect turnover rates in vivo compared with H-cholesteryl with PBS, and 2% mouse plasma was added. After 4 h and 8 h, ester-labeled HDL ( 21 ). Organ uptake of HDL apolipoproteins radioactivity within the medium was determined by liquid scintil- 125 3 ( I) and HDL-CEs ( H-cholesteryl ether) was determined by lation counting. The cell layer was washed twice with PBS, and 0.1 measuring the counts recovered in each organ expressed as a M NaOH was added. Plates were incubated 30 min at room tem- percentage of the injected dose, which was calculated by multi- perature, and the radioactivity remaining within the cells was as- plying the initial plasma counts (5-min time point) with the esti- sessed by liquid scintillation counting. Wells incubated with RPMI mated plasma volume (3.5% of total body weight). Selective without added plasma were used as blanks to determine plasma- uptake into organs was determined by subtracting the percent- independent efﬂ ux, and these values were subtracted from the age of the injected dose of I-HDL recovered in each organ respective experimental values. Efﬂ ux is given as the percentage from the percentage of the injected dose of H-HDL-CE. of counts recovered from the medium in relation to the total counts present on the plate (sum of medium and cells). In vivo RCT studies Statistical analysis In vivo RCT studies were performed essentially as published previously ( 27 ). Wild-type C57BL/6J donor mice were injected with Statistical analyses were performed using the Statistical Pack- 1.0 ml of 4% Brewer thioglycollate medium (Becton Dickinson, age for Social Sciences version 16.0 (SPSS Inc., Chicago, IL). Le Point de Claix, France). Peritoneal macrophages were har- Data are presented as means ± SEM. The Mann-Whitney U-test vested 4 days after thioglycollate injection as described ( 28 ). Mac- was used to compare different groups. Statistical signiﬁ cance for rophages were plated in RPMI 1640 medium (Invitrogen) sup- all comparisons was assigned at P < 0.05. plemented with 1% FBS (HyClone, Logan, UT) and penicillin (100 U/ml)/streptomycin (100 g/ml) (Invitrogen) and were allowed to adhere for 5 h at 37°C under 5% CO humidiﬁ ed air. RESULTS Nonadherent cells were removed by washing twice with PBS fol- lowed by loading of the macrophages with 50 g/ml acetylated Hepatic apoE overexpression affects HDL size LDL and 3 Ci/ml H-cholesterol (Perkin Elmer Life Sciences, distribution but not plasma lipid levels Boston, MA) for 24 h. Thereafter, cells were washed again and To assess the effects of hepatic overexpression of equilibrated in RPMI 1640 medium supplemented with 2% BSA human apoE3 on plasma lipid levels, wild-type mice were (Sigma) for 18 h. Immediately before injection, cells were har- vested and resuspended in RPMI 1640 medium. For in vivo mac- injected with an empty control adenovirus AdNull or with rophage-to-feces RCT studies, 2 million H-cholesterol-loaded an adenovirus expressing human apoE3. Plasma levels of macrophage foam cells were injected intraperitoneally into indi- total cholesterol, free cholesterol, esteriﬁ ed cholesterol, vidually housed recipient mice. Plasma was collected 6 h and 24 phospholipids, and triglycerides remained essentially un- h after macrophage injection by retroorbital puncture and for changed in response to hepatic apoE overexpression the ﬁ nal blood draw (48 h) by heart puncture. At the end of the ( Table 1 ). However, FPLC analysis revealed a lower HDL experimental period, livers were harvested, snap-frozen in liquid cholesterol peak and a shift toward larger particles in the nitrogen, and stored at 80°C until further analysis. Feces were AdhApoE3-injected mice compared with controls ( Fig. 1A ). collected continuously for 48 h. In parallel, plasma levels of apoA-I ( P = 0.055; Supplemen- Counts in plasma were assessed directly by liquid scintillation counting (Packard 1600CA Tri-Card, Packard, Meriden, CT). tary Figure IA) and apoB100 ( P < 0.01; Supplementary Fig- Counts within liver were determined after solubilization of the ure IB) were lower in the mice overexpressing human tissue using Solvable (Packard) exactly as previously reported apoE, whereas plasma apoB48 was not altered (n.s.; Sup- ( 26 ). Counts recovered from the respective liver piece were back- plementary Figure IC). To explore the distribution of hu- calculated to total liver mass. Feces were separated from the bed- man apoE across the different lipoprotein classes, Western ding, dried, weighed, and thoroughly ground. Aliquots were blot analysis for apoA-I and human apoE was performed separated into neutral sterol and bile acid fractions as previously on the individual FPLC fractions. In the mice adminis- reported ( 27 ). Brieﬂ y, samples were heated for 2 h at 80°C in al- tered AdhApoE3, human apoE was present in the apoA-I- kaline methanol and then extracted three times with petroleum containing HDL fractions and in the nonHDL lipoprotein ether. In the top layer, radioactivity within the neutral sterol frac- tion was determined by liquid scintillation counting, whereas ra- fractions lacking apoA-I expression (Supplementary Fig- dioactivity incorporated into bile acids was assessed from the ure II). Because cholesteryl ester transfer protein (CETP) bottom layer. Counts recovered from the respective aliquots were plays an important role in human lipoprotein metabolism related to the total amount of feces produced over the whole ex- but is absent in wild-type mice ( 29 ), apoE overexpression perimental period. All obtained counts were expressed relative experiments were carried out in transgenic mice express- to the administered dose. ing human CETP under the control of its endogenous In vitro cholesterol efﬂ ux assay promoter (hCETP tg). Comparable to the results in wild- type mice, in hCETP tg mice no major changes in plasma Thioglycollate-elicited peritoneal mouse macrophages were lipids occurred in response to hepatic apoE overexpression harvested as described above. Macrophages were plated in RPMI ApoE overexpression does not impact in vivo RCT 931 TABLE 1. Plasma lipids, liver lipid composition, and biliary excretion of sterols in wild-type and SR-BI knockout mice in response to hepatic apolipoprotein E overexpression Wild-type SR-BI knockout AdNull AdhApoE3 AdNull AdhApoE3 Plasma Total cholesterol (mg/dl) 69.5 ± 1.9 65.5 ± 2.6 147.6 ± 9.5 140.5 ± 12.1 Free cholesterol (mg/dl) 29.0 ± 0.7 31.3 ± 0.9 86.4 ± 4.9 81.3 ± 7.8 Esteriﬁ ed cholesterol (mg/dl) 40.5 ± 1.6 34.3 ± 3.1 61.1 ± 6.4 59.2 ± 5.4 Phospholipids (mg/dl) 178.6 ± 12.7 148.6 ± 8.0 191.8 ± 10.8 214.7 ± 16.5 Triglycerides (mg/dl) 82.9 ± 9.2 80.0 ± 8.9 59.8 ± 6.9 79.9 ± 10.8 Liver Total cholesterol (nmol/mg liver) 6.3 ± 0.5 7.8 ± 0.4 8.5 ± 0.3 8.3 ± 0.4 Free cholesterol (nmol/mg liver) 5.4 ± 0.4 5.9 ± 0.3 6.7 ± 0.1 6.4 ± 0.1 Esteriﬁ ed cholesterol (nmol/mg liver) 0.8 ± 0.2 2.0 ± 0.1 1.7 ± 0.3 2.0 ± 0.3 Phospholipids (nmol/mg liver) 28.2 ± 1.5 25.8 ± 0.9 31.9 ± 0.7 29.5 ± 0.6 Triglycerides (nmol/mg liver) 12.8 ± 3.1 58.3 ± 5.0 22.2 ± 1.6 32.8 ± 4.5 Bile Bile ﬂ ow ( l/min/100 g bw) 10.3 ± 0.6 10.2 ± 0.2 ND ND Biliary bile acid secretion (nmol/min/100 g bw) 866 ± 86 762 ± 45 ND ND Biliary phospholipid secretion (nmol/min/100 g bw) 61.6 ± 5.0 82.6 ± 6.8 ND ND Biliary cholesterol secretion (nmol/min/100 g bw) 3.9 ± 0.2 4.0 ± 0.3 ND ND On day 4 after adenovirus injection, bile was collected continuously for 30 min, plasma samples were taken, and livers were harvested and snap- frozen in liquid nitrogen. Plasma lipids, liver lipids, and biliary output rates of bile acids, phospholipids, and cholesterol were determined as described in Materials and Methods. Values are means ± SEM; n = 5–8 mice for each condition. AdhApoE3, recombinant adenovirus expressing human apoE3; AdNull, empty control adenovirus; SR-BI, scavenger receptor class B type I; bw, body weight; ND, not determined. Signiﬁ cantly different from the respective AdNull-injected controls as assessed by Mann-Whitney U-test (at least P < 0.05). (Supplementary Table I), and the HDL cholesterol peak and triglyceride content were observed in response to was similarly decreased and was shifted toward larger HDL AdhApoE3 in hCETP tg mice (Supplementary Table II). particles (Supplementary Figure III). To test the hypothesis that the decrease in plasma HDL cholesterol and the concomitant increase in hepatic cho- lesterol content in response to apoE overexpression were Hepatic apoE overexpression increases hepatic cholesterol content by stimulating selective uptake into due to an enhanced selective uptake of cholesteryl esters the liver from HDL, HDL kinetic studies were carried out in wild- Next, we determined whether hepatic overexpression of type mice using autologous HDL. Hepatic apoE overex- human apoE would affect hepatic lipid composition. He- pression caused an increase in the HDL cholesteryl ester FCR (0.142 ± 0.009 vs. 0.196 ± 0.013 pools/h; P < 0.05; Fig. patic total cholesterol content was signiﬁ cantly increased by 24% in mice administered AdhApoE3 ( P < 0.05; Table 1 ), 2A ) without having signiﬁ cant effects on the HDL protein largely due to a higher hepatic esteriﬁ ed cholesterol FCR (0.084 ± 0.007 vs. 0.091 ± 0.013 pools/h; n.s.; Fig. 2A ). content (+150%; P < 0.01; Table 1 ). Whereas hepatic phos- Therefore, the apparent whole body selective uptake as calculated by the difference between the HDL cholesteryl pholipids were identical between AdNull-injected and AdhApoE3-injected mice ( Table 1 ), apoE overexpression ester and HDL protein FCRs was signiﬁ cantly higher in apoE- resulted in an elevated hepatic triglyceride content (+355%; overexpressing mice compared with controls (0.058 ± 0.011 P < 0.01; Table 1 ). Similar changes in hepatic cholesterol vs. 0.106 ± 0.010 pools/h; P < 0.05; Fig. 2A ). In agreement Fig. 1. Apolipoprotein E overexpression affects plasma cholesterol distribution in an SR-BI-dependent fashion. FPLC proﬁ les in response to apolipoprotein E overexpression in (A) wild-type mice and (B) SR-BI knockout (ko) mice. Pooled plasma samples collected on day 4 after injection with the control adenovirus AdNull or with the human apolipoprotein E3 expressing adenovirus AdhApoE3 were subjected to gel ﬁ ltra- tion chromatography analysis using a Superose 6 column as described in Materials and Methods. n = 6–8 mice for each condition. Open circles, AdNull-injected controls; closed squares, AdhApoE3-injected mice. 932 Journal of Lipid Research Volume 53, 2012 Fig. 2. Apolipoprotein E overexpression increases selective uptake of HDL cholesteryl esters into the liver. On day 4 after injection with the control adenovirus AdNull or with the human apolipoprotein E3-express- ing adenovirus, AdhApoE3 kinetic experiments were performed using autologous HDL double labeled with 125 3 I-tyramine-cellobiose and H-cholesteryl ether (CE) as described in Materials and Methods. A: FCRs calculated 125 3 from the respective plasma disappearance curves. B: Uptake of I-tyramine-cellobiose and H-CE by the liver. Data are presented as means ± SEM. n = 6 mice for each condition. White bars, AdNull-injected mice; black bars, AdhApoE3-injected mice. * Signiﬁ cantly different from the respective AdNull-injected controls as as- sessed by Mann-Whitney U-test (at least P < 0.05). with the above results, the uptake of HDL protein into the stimulating selective uptake of HDL cholesteryl esters into liver remained unchanged after hepatic apoE overexpres- the liver. sion (26.2 ± 3.7 vs. 24.6 ± 3.3%; n.s.; Fig. 2B ), whereas up- Increased hepatic cholesterol content in response to take of HDL cholesteryl ester into the liver tended to be hepatic apoE overexpression is dependent on SR-BI higher (40.4 ± 2.4 vs. 52.8 ± 4.2%; P = 0.07; Fig. 2B ). Over- SR-BI is the major receptor responsible for the selective all, this translated into an almost 2-fold increase in hepatic uptake of HDL cholesterol into the liver ( 21, 30 ). To con- selective uptake in the AdhApoE3-injected group (14.2 ± ﬁ rm a critical role of SR-BI mediating altered plasma 2.7 vs. 28.2 ± 1.6; P < 0.01; Fig. 2B ). Although selective up- lipoprotein distribution and hepatic cholesterol content take of HDL cholesteryl esters in the liver was enhanced, as a consequence of apoE overexpression, the effects of Sr-b1 mRNA expression was lower in wild-type mice ( P < AdNull or AdhApoE3 were investigated in SR-BI-deﬁ cient 0.01; Table 2 ) and in hCETP tg mice overexpressing apoE mice. In agreement with results in wild-type mice, injec- (Supplementary Table III). However, neither total nor tion of a human apoE expressing adenovirus did not alter membrane-associated hepatic SR-BI protein levels were plasma levels of total cholesterol, free cholesterol, esteri- changed in the two mouse models (Supplementary Figure ﬁ ed cholesterol, phospholipids, and triglycerides in SR-BI IV). Combined, these data demonstrate that hepatic apoE knockout mice ( Table 1 ). However, the marked altera- overexpression increases hepatic cholesterol content by tions observed in the lipoprotein distribution in response to apoE overexpression in wild-type mice were not present TABLE 2. Hepatic mRNA expression in wild-type mice in response in SR-BI knockouts, as reﬂ ected by virtually identical FPLC to hepatic apolipoprotein E overexpression proﬁ les in the AdNull-injected compared with the AdhA- poE3-injected group ( Fig. 1B ). In line with these results, Wild-type the hepatic content of total cholesterol ( Table 1 ), free AdNull AdhApoE3 cholesterol ( Table 1 ), and esteriﬁ ed cholesterol ( Table 1 ) Sr-b1 1.00 ± 0.03 0.70 ± 0.03 Abcb11 1.00 ± 0.06 0.60 ± 0.04 was not affected by apoE overexpression in SR-BI knock- Abcb4 1.00 ± 0.07 0.91 ± 0.05 out mice. Nevertheless, AdhApoE3 injection in the SR-BI Abcg5 1.00 ± 0.08 0.62 ± 0.04 knockouts caused a slight but signiﬁ cant decrease in he- Abcg8 1.00 ± 0.06 0.69 ± 0.06 Cyp7a1 1.00 ± 0.23 0.91 ± 0.13 patic phospholipid content ( 8%; P < 0.05; Table 1 ), Cyp27a1 1.00 ± 0.09 0.46 ± 0.04 whereas the hepatic triglyceride content tended to be Cyp8b1 1.00 ± 0.07 0.74 ± 0.02 a higher (+48%; P = 0.06; Table 1 ). These data indicate that Srebp2 1.00 ± 0.10 0.70 ± 0.02 the apoE-mediated changes in lipoprotein distribution Ldlr 1.00 ± 0.09 0.71 ± 0.04 Hmgcr 1.00 ± 0.16 0.74 ± 0.09 and hepatic cholesterol content are dependent on SR-BI. Abca1 1.00 ± 0.02 0.92 ± 0.06 Hepatic apoE overexpression does not affect biliary Livers of mice administered the respective adenoviruses were and fecal sterol excretion harvested on day 4 after adenovirus injection and snap-frozen in liquid nitrogen. mRNA expression levels were determined by real-time To explore whether higher SR-BI-mediated hepatic quantitative PCR as described in Materials and Methods. Values are cholesterol uptake after apoE overexpression in wild-type means ± SEM; n = 6 mice for each condition. Within each set of experiments, gene expression levels are related to the respective AdNull- mice would translate into changes in biliary sterol secretion, injected controls. AdhApoE3, recombinant adenovirus expressing a continuous bile cannulation experiment was performed human apoE3; AdNull, empty control adenovirus. in wild-type mice receiving AdNull or AdhApoE3. Neither Signiﬁ cantly different from the respective AdNull-injected controls as assessed by Mann-Whitney U-test (at least P < 0.05). bile ﬂ ow ( Table 1 ) nor biliary secretion rates of bile acids ApoE overexpression does not impact in vivo RCT 933 ( Table 1 ) were affected by hepatic overexpression of 0.3% injected tracer dose; n.s.), and 48 h (2.3 ± 0.2 vs. human apoE3. Although the biliary secretion rate of 2.4 ± 0.2% injected tracer dose; n.s.) after macrophage phospholipids was 1.3-fold higher ( P < 0.05; Table 1 ), the injection ( Fig. 3A ). Although apoE overexpression led to a secretion rate of cholesterol into bile remained un- 63% increase in macrophage-derived H-cholesterol within changed in wild-type mice ( Table 1 ). However, in hCETP the liver (7.0 ± 0.9 vs. 11.3 ± 1.2% injected tracer dose; tg mice, lower biliary output of cholesterol was noted in P < 0.05; Fig. 3B ), overall, in vivo RCT remained essentially the group injected with AdhApoE3, whereas there was no unchanged as reﬂ ected by no effect on the total excretion effect on the biliary secretion rates of bile acids and phos- of H-tracer into the feces (10.5 ± 1.0 vs. 10.7 ± 0.6% in- pholipids (Supplementary Figure V). jected tracer dose; n.s.; Fig. 3C ). In addition, no signiﬁ cant Hepatic mRNA expression of the hepatobiliary phos- changes were observed in the fecal loss of H-tracer within pholipid transporter Abcb4 (also known as multidrug neutral sterols (1.9 ± 0.2 vs. 2.2 ± 0.3% injected tracer dose; resistance protein 2, Mdr2 ) was not changed by apoE over- n.s.) or within the bile acid fraction (8.6 ± 0.9 vs. 8.5 ± expression in wild-type mice, whereas expression levels of 0.7% injected tracer dose; n.s.) in response to apoE over- the bile salt export pump Abcb11 (also known as Bsep ; P < expression ( Fig. 3C ). Moreover, in the CETP transgenic 0.01) and the cholesterol half-transporters Abcg5 and Abcg8 mouse model, RCT was not inﬂ uenced by AdhApoE3 were decreased ( P < 0.01 for both) ( Table 2 ). ApoE over- (Supplementary Figure VI). These observations indicate expression reduced expression of the key enzyme for the that hepatic apoE overexpression has no apparent effect alternative bile acid synthesis pathway in the liver, Cyp27a1 on macrophage RCT. ( P < 0.01) and the enzyme responsible for cholate synthe- sis, Cyp8b1 ( P < 0.05) but did not affect expression of the Probucol treatment decreases plasma lipid and lipoprotein levels rate-limiting enzyme for classic bile acid synthesis, Cyp7a1 ( Table 2 ). Hepatic gene expression of the sterol regula- It has been suggested recently that inhibition of hepatic tory binding protein 2 ( Srebp2 ) was suppressed by apoE ABCA1 activity by probucol reduces ABCA1-mediated cho- overexpression ( P < 0.05), whereas mRNA expression of its lesterol efﬂ ux from hepatocytes, thereby increasing cho- two target genes LDL receptor ( Ldlr ) and the rate-limiting lesterol excretion into the bile and feces ( 31 ). Cholesterol enzyme for cholesterol synthesis, HMG-CoA reductase efﬂ ux from lipid-laden macrophages in vitro was signiﬁ - ( Hmgcr ), was not signiﬁ cantly affected ( Table 2 ). There cantly higher toward plasma from ApoE-overexpressing was also no difference in Abca1 mRNA levels in the liver wild-type mice compared with controls after 4 h (6.5 ± 0.1 between AdhApoE3 injected wild-type mice and controls. vs. 7.5 ± 0.2%; P < 0.01) and 8 h (9.5 ± 0.1 vs. 10.8 ± 0.2%; In hCETP tg mice, virtually identical changes in liver gene P < 0.01) of incubation ( Fig. 4 ). We therefore hypothe- expression were found in response to apoE overexpres- sized that potentially increased ABCA1-mediated cho- sion (Supplementary Table III). lesterol efﬂ ux from hepatocytes might explain the Consistent with unaltered biliary secretion of choles- unchanged biliary and fecal sterol output in response to terol and bile acids in wild-type mice, the fecal mass output apoE over expression. To explore this hypothesis, wild-type of neutral sterols (3.11 ± 0.22 vs. 2.49 ± 0.34 mol/day; mice were fed a control chow diet or a chow diet supple- Supplementary Table IV) and bile acids (2.33 ± 0.19 vs. mented with 0.5% (wt/wt) probucol and were injected on 2.65 ± 0.11 mol/day; Supplementary Table V) was not day 10 of diet feeding with AdNull or AdhApoE3. Treat- inﬂ uenced by overexpression of apoE. Likewise, hCETP tg ment with probucol decreased plasma total cholesterol mice injected with AdhApoE3 also excreted similar amounts concentrations in AdNull-injected ( 51%; P < 0.01) and of neutral sterols (Supplementary Table IV) and bile acids AdhApoE3-injected mice ( 66%; P < 0.01; Table 3 ). This (Supplementary Table V) into the feces compared with was due to a signiﬁ cant lowering of plasma free cholesterol AdNull administered controls. Taken together, these re- ( 52% and 71% for AdNull and AdhApoE3, respec- sults indicate that apoE overexpression promotes hepatic tively; P < 0.01 for both) as well as esteriﬁ ed cholesterol cholesterol uptake without increasing biliary and fecal ste- ( 51% and 61% for AdNull and AdhApoE3, respec- rol excretion. tively; P < 0.01 for both) in the mice administered probu- col ( Table 3 ). As expected from the role of liver ABCA1 in Hepatic apoE overexpression does not affect HDL formation, HDL-cholesterol levels in plasma were macrophage-to-feces RCT markedly reduced by dietary probucol ( 41% and 42% Because apoE overexpression increased hepatic selec- for AdNull and AdhApoE3, respectively; P < 0.01 for both). However, probucol also resulted in decreased plasma non- tive uptake via SR-BI, an important step in the RCT path- way, but did not inﬂ uence mass biliary and fecal sterol HDL-cholesterol levels ( 66% and 79% for AdNull and excretion, we next investigated whether apoE overexpres- AdhApoE3, respectively; P < 0.01 for both). In line with sion might affect overall RCT. In vivo RCT was traced after these results, FPLC analysis demonstrated a clear reduc- tion in all lipoprotein classes in AdNull-injected mice fed intraperitoneal injection of primary mouse macrophages loaded with H-cholesterol in control and apoE-overex- the probucol diet compared with mice fed the control diet pressing wild-type mice. The appearance of tracer in ( Fig. 5A ). Similar changes in the plasma lipoprotein distri- plasma was not signiﬁ cantly different between controls bution were observed in human apoE-overexpressing mice in response to probucol ( Fig. 5B ). Finally, administration and mice injected with AdhApoE3 at 6 h (2.1 ± 0.6 vs. 3.0 ± 0.5% injected tracer dose; n.s.), 24 h (2.8 ± 0.4 vs. 2.8 ± of probucol resulted in a signiﬁ cant decrease in plasma 934 Journal of Lipid Research Volume 53, 2012 Fig. 3. Apolipoprotein E overexpression does not affect in vivo macrophage-to-feces reverse cholesterol transport in wild-type mice. On day 2 after injection with the control adenovirus AdNull or with the human apolipoprotein E3-expressing adenovirus AdhApoE3, mice received intraperitoneal injections with H-cho- lesterol-loaded primary mouse macrophage foam cells as described in Materials and Methods. A: Time 3 3 course of H-cholesterol recovery in plasma. B: H-cholesterol within liver 48 h after macrophage administra- tion. C: H-cholesterol appearance in feces collected continuously from 0 to 48 h after macrophage admin- istration and separated into bile acid and neutral sterol fractions as indicated. Data are expressed as percentage of the injected tracer dose and presented as means ± SEM. n = 8 mice for each condition. White bars, AdNull-injected mice; black bars, AdhApoE3-injected mice. * Signiﬁ cantly different from the respec- tive AdNull-injected controls as assessed by Mann-Whitney U-test (at least P < 0.05). phospholipids and triglycerides in mice injected with 66% for phospholipids and triglycerides, respectively; the control adenovirus AdNull ( 37% and 38% for P < 0.01 for both) (Table 3). phospholipids and triglycerides, respectively; P < 0.01 Probucol treatment does not change the hepatic and P < 0.05, respectively) or AdhApoE3 ( 57% and cholesterol content in response to hepatic apoE overexpression In mice that received the control adenovirus AdNull, the hepatic content of total cholesterol was not differ- ent between groups on control and probucol-containing diet ( Table 3 ). ApoE overexpression consistently increased total cholesterol levels in the liver; however, there was no additional effect of probucol ( Table 3 ). The amount of free cholesterol and esteriﬁ ed cholesterol in the liver was not changed in response to probucol in both the mice administered AdNull and AdhApoE3 ( Table 3 ). Probucol treatment resulted in a lower hepatic phos- pholipid content in the AdNull ( 9%; P < 0.05) but Fig. 4. Apolipoprotein E overexpression increases cholesterol not the AdhApoE3 group ( Table 3 ). There was no efﬂ ux from macrophage foam cells toward plasma. Thioglycollate- effect of probucol on the hepatic triglyceride content elicited peritoneal mouse macrophages were loaded with 50 g/ml ( Table 3 ). Thus, probucol does not change the hepatic acetylated LDL and 1 Ci/ml [ H]cholesterol as described in cholesterol mass content in response to hepatic apoE Materials and Methods. Subsequently, 2% plasma was added to the cells. After 4 h and 8 h, radioactivity within the medium and radio- overexpression. activity remaining within the cells was determined by liquid scintil- lation counting. Efﬂ ux is given as the percentage of counts Probucol treatment increases biliary and fecal sterol recovered from the medium in relation to the total counts present secretion in apoE-overexpressing mice on the plate (sum of medium and cells). Data are presented as Bile cannulation experiments revealed that dietary means ± SEM. n = 8 mice for each condition. White bars, AdNull- probucol had no effect on bile ﬂ ow or on the biliary se- injected mice; black bars, AdhApoE3-injected mice. * Signiﬁ cantly cretion of bile acids, phospholipids, and cholesterol in different from the respective AdNull-injected controls as assessed mice injected with the control adenovirus ( Table 3 ). by Mann-Whitney U-test (at least P < 0.05). ApoE overexpression does not impact in vivo RCT 935 TABLE 3. Plasma lipids, liver lipid composition, and biliary excretion of sterols in response to probucol treatment AdNull AdhApoE3 Control Probucol Control Probucol Plasma a a Total cholesterol (mg/dl) 72.3 ± 1.7 35.3 ± 1.3 63.7 ± 2.8 21.9 ± 1.3 a a Free cholesterol (mg/dl) 22.2 ± 0.4 10.7 ± 0.6 29.8 ± 2.0 8.5 ± 1.1 a a Esteriﬁ ed cholesterol (mg/dl) 50.1 ± 1.8 24.6 ± 1.1 34.0 ± 2.0 13.4 ± 0.6 a a Phospholipids (mg/dl) 154.1 ± 7.4 97.7 ± 5.8 137.8 ± 9.5 59.8 ± 1.3 a a Triglycerides (mg/dl) 58.2 ± 7.1 36.0 ± 3.5 95.3 ± 7.9 32.4 ± 7.0 Liver Total cholesterol (nmol/mg liver) 7.2 ± 0.2 7.5 ± 0.2 9.1 ± 0.4 9.2 ± 0.5 Free cholesterol (nmol/mg liver) 5.9 ± 0.1 6.0 ± 0.2 7.4 ± 0.5 6.9 ± 0.3 Esteriﬁ ed cholesterol (nmol/mg liver) 1.3 ± 0.1 1.5 ± 0.1 1.7 ± 0.2 2.3 ± 0.2 Phospholipids (nmol/mg liver) 29.2 ± 0.7 26.5 ± 0.6 26.7 ± 0.6 27.8 ± 0.6 Triglycerides (nmol/mg liver) 21.4 ± 1.7 22.3 ± 3.3 63.7 ± 7.1 59.4 ± 8.6 Bile Bile ﬂ ow ( l/min/100 g bw) 9.3 ± 0.6 8.2 ± 0.7 7.3 ± 1.1 10.6 ± 0.9 Biliary bile acid secretion (nmol/min/100 g bw) 469 ± 90 357 ± 73 427 ± 81 466 ± 51 Biliary phospholipid secretion (nmol/min/100 g bw) 41.6 ± 3.1 37.7 ± 5.6 43.0 ± 5.4 64.3 ± 5.1 Biliary cholesterol secretion (nmol/min/100 g bw) 3.5 ± 0.4 3.6 ± 0.5 2.9 ± 0.4 5.2 ± 0.7 Mice were fed a control chow diet or a chow diet containing 0.5% probucol for 2 weeks. On day 4 after adenovirus injection, bile was collected continuously for 30 min, plasma samples were taken, and livers were harvested and snap- frozen in liquid nitrogen. Plasma lipids, liver lipids, and biliary output rates of bile acids, phospholipids, and cholesterol were determined as described in Materials and Methods. Values are means ± SEM; n = 6 mice for each condition. AdhApoE3, recombinant adenovirus expressing human apoE3; AdNull, empty control adenovirus; bw, body weight. Signiﬁ cantly different from the respective controls as assessed by Mann-Whitney U-test (at least P < 0.05). However, in AdhApoE3-administered mice, bile flow of Srebp2 and its target genes ldlr and hmgcr in the liver was tended to increase in response to probucol ( P = 0.055; not different between mice fed a chow diet and mice fed a Table 3 ). Whereas the biliary output rate of bile acids re- probucol-enriched diet ( Table 4 ). Finally, as has been re- mained unchanged, biliary secretion rates of phospho- ported previously ( 31 ), no change in the hepatic mRNA lipids were 1.5-fold higher in apoE-overexpressing mice expression of Abca1 was detected in the probucol-treated in response to probucol ( P < 0.05), and the biliary secre- mice ( Table 4 ). tion rate of cholesterol increased signiﬁ cantly by 1.8-fold Analysis of fecal contents showed that treatment with ( P < 0.05) ( Table 3 ). probucol did not inﬂ uence the fecal excretion of neutral Hepatic mRNA expression of the bile acid transporter sterols (4.74 ± 0.28 vs. 5.07 ± 0.18 mol/day) and bile ac- Abcb11 , the phospholipid transporter Abcb4 , and the cho- ids (2.52 ± 0.22 vs. 3.02 ± 0.28 mol/day) in mice that re- lesterol half-transporters Abcg5 and Abcg8 was not modi- ceived the control adenovirus AdNull. In contrast and in ﬁ ed by probucol treatment in control mice or in mice that good agreement with the elevated biliary cholesterol se- overexpress apoE in the liver ( Table 4 ). No signiﬁ cant cretion, fecal excretion of neutral sterols was signiﬁ cantly changes in the hepatic gene expression level of the bile enhanced by probucol in mice overexpressing apoE (3.69 ± acid synthesizing enzymes Cyp7a1 , Cyp27a1 , and Cyp8b1 0.21 vs. 4.97 ± 0.37 mol/day; P < 0.05). Nonetheless, no were observed, except for higher relative mRNA levels effect of probucol on the fecal bile acid output was found of Cyp7a1 in apoE-overexpressing mice upon probucol in these mice (2.20 ± 0.14 vs. 2.66 ± 0.23 mol/day). Com- administration ( P < 0.05; Table 4 ). Furthermore, expression bined, these data demonstrate that biliary and fecal sterol Fig. 5. Probucol treatment decreases plasma cholesterol levels. FPLC proﬁ les in response to probucol treatment in (A) AdNull-injected and (B) AdhApoE3-injected mice. Mice were fed a control chow diet or a chow diet containing 0.5% probucol for 2 weeks. Pooled plasma samples collected on day 4 after injection with the control adenovirus AdNull or with the human apolipoprotein E3-expressing adenovirus AdhApoE3 were subjected to gel ﬁ ltration chromatography analysis using a Superose 6 column as described in Materials and Methods. n = 6 mice for each condition. Open circles, chow-fed controls; closed squares; probucol- treated mice. 936 Journal of Lipid Research Volume 53, 2012 TABLE 4. Hepatic mRNA expression in response to DISCUSSION probucol treatment This study demonstrates that hepatic overexpression of AdNull AdhApoE3 human apoE not only promotes SR-BI-mediated selective Control Probucol Control Probucol uptake of HDL cholesterol into the liver but also enhances a a Sr-b1 1.00 ± 0.05 0.88 ± 0.03 1.00 ± 0.03 1.08 ± 0.03 the resecretion of RCT-relevant cholesterol via hepatocyte Abcb11 1.00 ± 0.06 1.16 ± 0.04 1.00 ± 0.05 1.14 ± 0.08 ABCA1 back to the plasma compartment. As a result, bil- Abcb4 1.00 ± 0.07 1.05 ± 0.09 1.00 ± 0.06 1.12 ± 0.05 Abcg5 1.00 ± 0.10 0.91 ± 0.09 1.00 ± 0.06 1.20 ± 0.09 iary and fecal mass sterol excretion and RCT remain un- Abcg8 1.00 ± 0.07 0.93 ± 0.08 1.00 ± 0.06 1.13 ± 0.13 a changed in apoE-overexpressing mice with active ABCA1, Cyp7a1 1.00 ± 0.15 0.60 ± 0.09 1.00 ± 0.13 1.58 ± 0.12 whereas all of these parameters increase signiﬁ cantly when Cyp27a1 1.00 ± 0.05 0.90 ± 0.03 1.00 ± 0.07 1.11 ± 0.07 Cyp8b1 1.00 ± 0.08 0.96 ± 0.09 1.00 ± 0.06 1.04 ± 0.09 ABCA1 activity is blocked with probucol. Collectively, Srebp2 1.00 ± 0.04 0.96 ± 0.05 1.00 ± 0.05 0.81 ± 0.06 these results point to a metabolic shunt potentially con- Ldlr 1.00 ± 0.08 1.14 ± 0.08 1.00 ± 0.06 0.94 ± 0.06 necting the SR-BI and the ABCA1 pathway with a high rel- Hmgcr 1.00 ± 0.07 0.98 ± 0.09 1.00 ± 0.05 0.83 ± 0.09 Abca1 1.00 ± 0.04 1.07 ± 0.02 1.00 ± 0.04 1.04 ± 0.04 evance for the regulation of RCT. The RCT pathway represents an important atheropro- Mice were fed a control chow diet or a chow diet containing 0.5% tective functionality of HDL ( 16, 17 ). RCT comprises cho- probucol for 2 weeks. Livers of mice administered the respective adenoviruses were harvested on day 4 after adenovirus injection and lesterol efﬂ ux from macrophage foam cells within the snap-frozen in liquid nitrogen. mRNA expression levels were vessel wall, the transport of this cholesterol within HDL determined by real-time quantitative PCR as described in Materials and through the plasma compartment, the subsequent hepatic Methods. Values are means ± SEM; n = 6 mice for each condition. Within each set of experiments, gene expression levels are related to uptake via SR-BI or as a holoparticle, and excretion into the respective chow-fed controls. AdhApoE3, recombinant adenovirus the bile and feces ( 16, 17 ). In vitro, apoE expression by expressing human apoE3; AdNull, empty control adenovirus. macrophages has been shown to stimulate cholesterol ef- Signiﬁ cantly different from the respective controls as assessed by Mann-Whitney U-test (at least P < 0.05). ﬂ ux ( 12–14 ). In addition, in vivo macrophage-to-feces RCT was lower when apoE-deﬁ cient macrophages were injected into wild-type mice compared with wild-type macrophages secretion are increased upon probucol treatment in apoE- injected into wild-type mice ( 15 ). Combined with our pre- overexpressing mice. sent results, these data suggest that apoE expression by macrophages might determine the general availability of Probucol treatment increases macrophage-to-feces macrophage-derived cholesterol for the RCT pathway di- RCT in apoE-overexpressing mice rectly at the point of entry, whereas in subsequent steps of Because probucol enhanced biliary and fecal sterol se- RCT hepatic ABCA1 counteracts these effects. cretion in mice overexpressing human apoE, we investi- Experiments in SR-BI knockout mice as well as HDL ki- netic studies in the current report demonstrated that apoE gated whether this would also translate into an improvement overexpression speciﬁ cally increased selective uptake of in overall RCT from macrophages to feces. After intrap- eritoneal injection of H-cholesterol-loaded macro- HDL cholesterol into the liver. Importantly, the expression phages, counts within plasma were profoundly lower at the levels of hepatic SR-BI did not change in response to apoE 6 h (1.24 ± 0.16 vs. 0.48 ± 0.04% injected tracer dose; overexpression. Therefore, our present ﬁ ndings are com- plementary to previous work demonstrating impaired he- P < 0.01; Fig. 6A ), 24 h (1.62 ± 0.20 vs. 0.59 ± 0.04% injected tracer dose; P < 0.01; Fig. 6A ), and 48 h time point (1.48 ± patic selective HDL cholesterol uptake in mice lacking apoE 0.22 vs. 0.56 ± 0.06% injected tracer dose; P < 0.01; Fig. 6A ) ( 32 ), although the apoE knockout mouse model exhibits a in the probucol-treated apoE-overexpressing mice compared considerably altered plasma lipid proﬁ le with substantially increased levels of apoB-containing lipoprotein remnants with apoE-overexpressing controls. However, the amount of macrophage-derived tracer recovered within the liver was ( 32 ). In addition, selective uptake of HDL cholesterol into not affected by probucol in mice with hepatic apoE overex- HepG2 cells after treatment with a blocking antibody di- pression (7.6 ± 1.0 vs. 7.0 ± 0.7% injected tracer dose; n.s.; rected against apoE was reduced ( 33 ). It is possible that Fig. 6B ). Consistent with the higher biliary and fecal mass apoE stimulates SR-BI-mediated selective uptake due to excretion of sterols, probucol signiﬁ cantly enhanced the an improved interaction of the HDL particle with SR-BI total excretion of H-cholesterol originating from mac- ( 32 ), although the exact mechanisms will require further rophages into the feces of AdhApoE3-injected mice (6.6 ± investigation. 0.4 vs. 9.5 ± 0.5% injected tracer dose; P < 0.01; Fig. 6C ). Although hepatic human apoE overexpression in- Because tracer recovery in the fecal bile acid fraction creased the hepatic cholesterol content, biliary choles- remained unaltered (5.3 ± 0.4 vs. 5.7 ± 0.4% injected tracer terol excretion and the overall elimination of cholesterol dose; n.s.; Fig. 6C ), this was attributable to a 2.7-fold from the body did not increase in wild-type mice or higher excretion of H-cholesterol in the fecal neutral ste- hCETP tg mice. The current observation that increased rol fraction (1.4 ± 0.2% vs. 3.8 ± 0.5% injected tracer dose; SR-BI-mediated selective uptake does not necessarily P < 0.01; Fig. 6C ). These ﬁ ndings demonstrate that translate into enhanced biliary and fecal sterol excretion probucol results in an increased movement of choles- in the face of unaltered hepatic SR-BI expression is in terol from macrophages to the feces in mice with hepatic agreement with earlier observations made by us in mice apoE overexpression. ( 23 ) overexpressing group IIA secretory phospholipase A ApoE overexpression does not impact in vivo RCT 937 Fig. 6. Probucol treatment increases in vivo macrophage-to-feces reverse cholesterol transport in apolipo- protein E-overexpressing mice. Mice were fed a control chow diet or a chow diet containing 0.5% probucol for 12 days before and then throughout the 48-h period of the experiment. On day 2 after injection with the human apolipoprotein E- expressing adenovirus, AdhApoE3 mice received intraperitoneal injections with H-cholesterol-loaded primary mouse macrophage foam cells as described in Materials and Methods. A: 3 3 Time course of H-cholesterol recovery in plasma. B: H-cholesterol within liver 48 h after macrophage ad- ministration. C: H-cholesterol appearance in feces collected continuously from 0 to 48 h after macrophage administration and separated into bile acid and neutral sterol fractions as indicated. Data are expressed as percentage of the injected tracer dose and presented as means ± SEM. n = 8 mice for each condition. White bars, chow-fed controls; black bars, probucol-treated mice. * Signiﬁ cantly different from the respective con- trols as assessed by Mann-Whitney U-test (at least P < 0.05). or endothelial lipase (EL) ( 21, 34 ). Transgenic overex- ABCA1. Ideally, such studies should be carried out in po- pression of secretory phospholipase A or adenovirus- larized liver cells. To formally relate the cholesterol mass mediated overexpression of EL also resulted in an changes observed in our current study to each other, increased ﬂ ux of cholesterol into the liver, whereas bil- quantifying cholesterol ﬂ uxes would be required, which iary cholesterol secretion and mass fecal excretion of ste- has not been done in our present study and thus repre- rols remained unaffected ( 21, 23, 34 ). On the other sents a potential limitation in the interpretation of our hand, altering the hepatic expression level of SR-BI has current work. clear effects on biliary cholesterol secretion as well as Systemic apoE ( 7–11 ) and macrophage-derived apoE RCT with overexpression resulting in an increase and ( 36–39 ) have been shown to protect against atheroscle- knockdown in a decrease of both of these parameters rotic lesion formation due to effects not immediately involv- ( 24, 34, 35 ). How can this discrepancy be explained? Re- ing RCT. ApoE promotes the clearance of atherogenic cent data generated in probucol-fed mice demonstrated lipoproteins ( 6, 7, 10 ), and also a number of pleiotropic that a decrease in hepatic ABCA1 activity results in in- effects of apoE might be important. For example, apoE creased in vivo RCT ( 31 ). We therefore speculate that has antioxidative properties. ApoE prevented oxidative increased cholesterol uptake into the liver via SR-BI re- cell death in cultured neuronal cells and inhibited copper- sults in transport to a speciﬁ c intrahepatic compartment mediated oxidation of LDL, a key event in the initiation also accessible for ABCA1-mediated resecretion back into and progression of atherosclerotic lesions ( 40 ). Moreover, the plasma compartment to generate new HDL particles. apoE knockout mice have signiﬁ cantly increased in vivo Increasing hepatic SR-BI expression levels ( 24, 34 ) or de- oxidative stress, as determined by plasma, urinary, and vas- creasing the activity of ABCA1 might shift the balance cular isoprostane levels ( 41 ), whereas hepatic overexpres- toward biliary secretion (present report and Reference sion of apoE in LDL receptor knockout mice resulted in a 31 ). However, the nature of these intrahepatic choles- reduced oxidative stress burden and decreased atheroscle- terol pools is unknown. Future in vitro studies should rosis independent of plasma lipid and lipoprotein levels therefore focus on the intracellular trafﬁ cking of choles- ( 9 ). Anti-inﬂ ammatory activities of apoE also add to its terol and, using a pulse-chase set-up, on the incorpora- atheroprotective properties. In vitro studies have shown tion of HDL-derived cholesterol tracers into newly formed that apoE can suppress proliferation of cultured periph- HDL in models with altered expression of hepatocyte eral blood T lymphocytes ( 42 ) and shifts polarization of 938 Journal of Lipid Research Volume 53, 2012 11 . Kawashiri , M. , Y. Zhang , D. Usher , M. Reilly , E. Pure , and D. J. mouse macrophages to the anti-inﬂ ammatory M2 phenotype Rader . 2001 . Effects of coexpression of the LDL receptor and apoE ( 43 ). In vivo, apoE-deﬁ cient mice exhibit an exaggerated on cholesterol metabolism and atherosclerosis in LDL receptor- proinﬂ ammatory cytokine response after LPS injection deﬁ cient mice. J. 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Invest. 113 : 609 – 618 . 940 Journal of Lipid Research Volume 53, 2012

Journal

Journal of Lipid Research – American Society for Biochemistry and Molecular Biology

Published: May 1, 2012

Keywords: apolipoprotein E; reverse cholesterol transport; ATP-binding cassette transporter A1; atherosclerosis; bile; cholesteryl ester transfer protein; feces; high density lipoprotein; liver; macrophage; metabolism; mice; probucol

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ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

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ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

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