Pharmacologic suppression of hepatic ATP-binding cassette transporter 1 activity in mice reduces high-density lipoprotein cholesterol levels but promotes reverse cholesterol transport

Abstract

Background: The role of hepatic ATP-binding cassette transporter 1 (ABCA1) in maintaining plasma high density lipoprotein cholesterol (HDL-C) levels is well established, but its role in reverse cholesterol transport (RCT) is unclear. Probucol is a compound that reduces HDL-C levels but also reduces atherosclerosis in animal models and xanthomas in humans. The aim of the present study was to test the hypothesis that probucol inhibits hepatic ABCA1 activity, thereby reducing HDL-C levels but promoting RCT from macrophages.

Methods and results: Wild-type (WT) C57BL/6 mice and scavenger receptor class B type I (SR-BI) knockout mice were fed a chow diet containing 0.5% probucol or normal chow for 2 weeks. In WT mice, probucol, despite decreasing HDL-C by >80%, effectively maintained macrophage RCT. In SR-BI knockout mice, probucol also substantially reduced HDL-C but significantly increased macrophage RCT. Furthermore, probucol significantly enhanced the excretion of HDL-derived cholesterol into feces in both WT and SR-BI knockout mice. Probucol inhibited ABCA1-dependent cholesterol efflux from mouse primary hepatocytes, and this effect was shown to be responsible for the effect of probucol on increasing the fecal excretion of HDL-derived cholesterol in vivo.

Conclusions: We demonstrate that pharmacological inhibition of hepatic ABCA1 activity with probucol reduced HDL-C levels but promoted RCT through diversion of HDL-derived cholesterol from efflux back into plasma instead to excretion in the bile. These results explain the beneficial effects of probucol on atherosclerosis and xanthomas despite its HDL-lowering effects and suggest that inactivation of hepatic ABCA1 leads to increased RCT despite reducing plasma HDL-C levels.

Figure 1

Effects of probucol on reverse cholesterol transport (RCT) in mice. Mice (n=8 in each group) were fed the experimental diets for 2 weeks, and RCT studies were performed as described in Methods on C57BL6/SW129 (A through C) and scavenger receptor class B type I knockout (SR-BI KO; D through F) mice. A and D, ³H-cholesterol in plasma after macrophage injection. B and E, ³H-cholesterol in liver. C and F, ³H-cholesterol in feces. cpm Indicates counts per minute. Significant difference, *P<0.05 (vs control).

Figure 2

Effects of probucol on ³H-cholesteryl oleate high-density lipoprotein (³H-CEs-HDL) clearance in mice. Mice (n=6 in each group) were fed the experimental diets for 2 weeks, and HDL turnover studies were performed as described in Methods on C57BL6/SW129 (wild-type [WT]) and scavenger receptor class B type I knockout (SR-BI KO) mice. A, Change in ³H-CEs-HDL in plasma and plasma fractional catabolic rate (FCR). B, ³H-cholesterol in the liver. C, ³H-sterol in feces. D, ³H-free cholesterol in feces. E, ³H-bile acid in feces. cpm Indicates counts per minute. Significant difference, *P<0.05 (vs control).

Figure 3

Effects of probucol on plasma decay kinetics of ³H-cholesteryl hexadecyl ether (³H-CEt)/¹²⁵I-N-methyl tyramine cellobiose (¹²⁵I-NMTC) high-density lipoprotein (HDL) in mice. Mice (n=6 in each group) were fed the experimental diets for 2 weeks, and HDL turnover studies were performed as described in Methods on C57BL6/SW129 (A through C) and scavenger receptor class B type I knockout (D through F) mice. A and D, ³H-CEt and ¹²⁵I-NMTC levels in plasma after injection. B and E, The CE-selective plasma fractional catabolic rate (FCR). C and F, The CE-selective uptake in the liver. cpm Indicates counts per minute. Significant differences, *P<0.05 (vs control).

Figure 4

Effects of probucol on ATP-binding cassette transporter 1 (ABCA1) expression in mouse hepatocytes. A, The mRNA (left) and protein (right) expression of hepatic ABCA1 (n=3 in each group) B, The probucol effect on apolipoprotein AI–dependent free cholesterol (FC) efflux from wild-type (WT) or ABCA1-deficient (ABCA1 KO) primary hepatocytes (n=3 in each group).

Figure 5

Effects of probucol on ³H-cholesteryl oleate high-density lipoprotein (³H-CEs-HDL) clearance in ATP-binding cassette transporter 1 (ABCA1) knockout (KO) mice. Mice (n=6 in each group) were fed the experimental diets for 2 weeks, and HDL turnover studies were performed as described in Methods on DBA/J (wild-type [WT]) and ABCA1 KO mice. A, Change in ³H-CEs-HDL in plasma. B, Fractional catabolic rate (FCR) of ³H-CEs-HDL. C, ³H-cholesterol in the liver. D, ³H-sterol in feces. cpm Indicates counts per minute. Significant differences indicated with *P<0.05 (versus control).