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

Abstract

ApoE plays an important role in lipoprotein metabolism. This study investigated the effects of adenovirus-mediated human apoE overexpression (AdhApoE3) on sterol 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, whereas hepatic cholesterol content increased (P < 0.05). These effects were dependent on scavenger receptor class B type I (SR-BI) as confirmed using SR-BI-deficient mice. Kinetic studies demonstrated increased plasma HDL cholesteryl ester catabolic rates (P < 0.05) and higher hepatic selective uptake of HDL cholesteryl esters in AdhApoE3-injected wild-type mice (P < 0.01). However, biliary and fecal sterol output as well as in vivo macrophage-to-feces RCT studied with (3)H-cholesterol-loaded mouse macrophage foam cells remained unchanged upon human apoE overexpression. Similar results were obtained using hApoE3 overexpression in human CETP transgenic mice. However, blocking ABCA1-mediated cholesterol efflux from hepatocytes in AdhApoE3-injected mice using probucol increased biliary cholesterol secretion (P < 0.05), fecal neutral sterol excretion (P < 0.05), and in vivo RCT (P < 0.01), specifically within neutral sterols. These combined data demonstrate that systemic apoE overexpression increases i) SR-BI-mediated selective uptake into the liver and ii) ABCA1-mediated efflux of RCT-relevant cholesterol from hepatocytes back to the plasma compartment, thereby resulting in unchanged fecal mass sterol excretion and overall in vivo RCT.

Fig. 1.

Apolipoprotein E overexpression affects plasma cholesterol distribution in an SR-BI-dependent fashion. FPLC profiles 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 filtration 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.

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-expressing adenovirus, AdhApoE3 kinetic experiments were performed using autologous HDL double labeled with ¹²⁵I-tyramine-cellobiose and ³H-cholesteryl ether (CE) as described in Materials and Methods. A: FCRs calculated from the respective plasma disappearance curves. B: Uptake of ¹²⁵I-tyramine-cellobiose and ³H-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. * Significantly different from the respective AdNull-injected controls as assessed by Mann-Whitney U-test (at least P < 0.05).

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 ³H-cholesterol-loaded primary mouse macrophage foam cells as described in Materials and Methods. A: Time course of ³H-cholesterol recovery in plasma. B: ³H-cholesterol within liver 48 h after macrophage administration. C: ³H-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, AdNull-injected mice; black bars, AdhApoE3-injected mice. * Significantly different from the respective AdNull-injected controls as assessed by Mann-Whitney U-test (at least P < 0.05).

Fig. 4.

Apolipoprotein E overexpression increases cholesterol efflux from macrophage foam cells toward plasma. Thioglycollate-elicited peritoneal mouse macrophages were loaded with 50 μg/ml acetylated LDL and 1 μCi/ml [³H]cholesterol as described in Materials and Methods. Subsequently, 2% plasma was added to the cells. After 4 h and 8 h, radioactivity within the medium and radioactivity remaining within the cells was determined by liquid scintillation counting. Efflux is given as the percentage of counts recovered from the medium in relation to the total counts present on the plate (sum of medium and cells). Data are presented as means ± SEM. n = 8 mice for each condition. White bars, AdNull-injected mice; black bars, AdhApoE3-injected mice. * Significantly different from the respective AdNull-injected controls as assessed by Mann-Whitney U-test (at least P < 0.05).

Fig. 5.

Probucol treatment decreases plasma cholesterol levels. FPLC profiles 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 filtration 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.

Fig. 6.

Probucol treatment increases in vivo macrophage-to-feces reverse cholesterol transport in apolipoprotein 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 ³H-cholesterol-loaded primary mouse macrophage foam cells as described in Materials and Methods. A: Time course of ³H-cholesterol recovery in plasma. B: ³H-cholesterol within liver 48 h after macrophage administration. C: ³H-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. * Significantly different from the respective controls as assessed by Mann-Whitney U-test (at least P < 0.05).