Liver-specific induction of Abcg5 and Abcg8 stimulates reverse cholesterol transport in response to ezetimibe treatment

doi:10.1016/j.metabol.2014.06.014

Metabolism

Volume 63, Issue 10, October 2014, Pages 1334-1341

https://doi.org/10.1016/j.metabol.2014.06.014 Get rights and content

Abstract

Objective

Previous studies have shown ezetimibe treatment results in a 2–6-fold increase in reverse cholesterol transport (RCT). However, recent sterol balance studies question the role of biliary sterol secretion in RCT, and challenge the hypothesis that ezetimibe increases RCT through decreased absorption of biliary cholesterol in the intestine. We set out to determine whether ezetimibe may increase RCT by mechanisms that are independent of its well-established inhibition of intestinal cholesterol absorption.

Methods

C57BL/6J, Npc1l1-KO, and/or Abcg8-KO mice were fed a chow diet with or without ezetimibe and fecal [¹⁴C]-neutral and [¹⁴C]-acidic sterols were measured to examine macrophage-to-feces RCT. We measured the expression of RCT related genes in the liver and jejunum in these mice. To confirm our significant gene expression findings, we utilized primary human hepatocytes cultured with or without a glucuronated metabolite of ezetimibe.

Results

Our studies revealed that treatment with ezetimibe was associated with increased expression of hepatic Abcg5 and Abcg8. Ezetimibe did not directly affect expression in the liver, but this expression was due to the inhibition of intestinal cholesterol absorption. This conclusion was supported by the absence of an ABCG5/ABCG8 expression response to treatment with an ezetimibe metabolite in primary human hepatocytes. Finally, we found that the ezetimibe-dependent stimulation of RCT was attenuated in the absence of Abcg8.

Conclusions

Our study is the first to demonstrate ezetimibe treatment cooperatively stimulated macrophage-to-feces RCT by indirectly increasing liver Abcg5/Abcg8 expression in addition to its known suppression of intestinal cholesterol absorption.

Introduction

The process of reverse cholesterol transport (RCT) consists of the delivery of cholesterol from peripheral tissues to the liver via HDL particles, followed by secretion of cholesterol into bile, and subsequently its final elimination in the feces. RCT in cholesterol-loaded macrophages from atherosclerotic plaques plays a critical role in disease prevention by mediating the transfer of this cholesterol to the liver (or intestine) for disposal [1]. Excretion of cholesterol by the liver and intestine is mediated by the tissue-specific expression of the hemi-transporters Abcg5 and Abcg8 (Abcg5/g8) which ‘pump’ these sterols into the lumen of the biliary tree or intestine respectively [2]. Treatment with synthetic transcription factor liver-x-receptor alpha (Lxr-α) agonists induces liver and intestine Abcg5/g8 expression and stimulates macrophage-to-feces RCT [3], [4]. This stimulation is completely abolished in Abcg5/g8-knockouts [5]. Taken together, these studies suggest strongly that stimulation of Abcg5/g8 expression is an important determinant of macrophage-to-feces RCT.

We and others have reported previously that the absorption of cholesterol from the intestine is also an important determinant of macrophage-to-feces RCT [6], [7]. Treatment of mice with ezetimibe (EZ), a potent cholesterol absorption inhibitor, increased macrophage-to-feces RCT by 2–6-fold, probably due to the suppressed absorption of biliary cholesterol that reaches the intestine [6], [8]. Recent sterol balance studies suggested that EZ-dependent increase in fecal neutral sterol secretion is independent of biliary secretion and is largely attributable to direct secretion of cholesterol from enterocytes into the intestinal lumen [9]. In addition, Temel et al reported that compromised biliary cholesterol secretion in genetically manipulated mice or animals with surgically diverted bile flow displayed normal macrophage-to-feces RCT, questioning the role of biliary sterol secretion in macrophage-to-feces RCT [10]. Collectively, these reports challenged the hypothesis that EZ increases RCT through decreased absorption of biliary cholesterol that reaches the intestine. Therefore, we set out to determine whether EZ may increase RCT by mechanisms that are independent of its well-established inhibition of intestinal cholesterol absorption.

Section snippets

Animals and diets

C57BL/6J (stock # 000664, Jackson Laboratories, Bar Harbor, ME), Npc1l1-knockouts (Npc1l1-KO, courtesy of Yiannis A. Ioannou, Mount Sinai School of Medicine, New York, NY) and Abcg8-knockouts (Abcg8-KO, stock # 008763, Jackson Laboratories) mice were bred and housed in a controlled environment with 14/10 h light-dark cycle (5 am–7 pm light) at the Cleveland Clinic Biological Resources Unit. Mice (12–14 weeks old) were fed for 17 days with either rodent chow pellets (catalog # 2918, Harlan

Ezetimibe upregulates liver Abcg5/g8 expression

Treating C57BL/6J mice with 0.005% ezetimibe (EZ) resulted in a dramatic 86% decrease in cholesterol absorption and a 2–6-fold increase in macrophage-to-feces RCT [6], [7]. To determine whether EZ may increase RCT by mechanisms that are independent from its well-established cholesterol absorption blocking effect, we examined the effect of EZ on gene expression in the liver and intestines of C57BL/6J mice. As shown in Fig. 1A, EZ-treated animals displayed a 180% increase in liver mRNA Hmgcr

Discussion

Our studies demonstrate that decreased intestinal cholesterol absorption, whether due to EZ treatment or deletion of its molecular target Npc1l1, stimulates the expression of Abcg5/g8 in the liver, but not in the intestine (Figs. 1A and 2E). The expression of Abcg5/g8 in the mouse is regulated by three distinct transcriptional factors: Liver X receptor alpha (Lxr-α), Forkhead box O1 (Foxo1), and Hepatocyte nuclear factor 4 alpha (Hnf-4α) [18]. The best-studied regulator of Abcg5/g8 expression

Author contributions

ES and JBA contributed to study design, data collection and analysis, data interpretation and manuscript writing. SBP contributed the Abcg8-KO mice and manuscript writing.

Funding

This work was supported in part by the National Institutes of Health grant 1R01HL092991 and Merck/Schering-Plough grant MISP# 39658 (to ES) and by the Biomedical Laboratory Research and Development Program, Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development (SBP).

Disclosure

This research was partially funded by Merck/Schering-Plough, Whitehouse Station, NJ (grant MISP# 39658 to ES). However, they had no influence or input into the design, experimentation, analysis or interpretation of the data, or writing of the manuscript.

The following are the supplementary data related to this article.

. Primer sequences for qPCR analysis.

Acknowledgments

We appreciate Yiannis A. Ioannou, PhD for providing the Npc1l1-KO mice. The authors thank Charis Eng, MD, PhD and Winifred Wong, PhD for critical review of the manuscript. JBA is grateful to Dr. Jonathan Smith for helpful discussions.

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Basic ScienceLiver-specific induction of Abcg5 and Abcg8 stimulates reverse cholesterol transport in response to ezetimibe treatment

Abstract

Objective

Methods

Results

Conclusions

Introduction

Section snippets

Animals and diets

Ezetimibe upregulates liver Abcg5/g8 expression

Discussion

Author contributions

Funding

Disclosure

Acknowledgments

J Biol Chem

J Lipid Res

Eur J Pharmacol

FEBS Lett

Cell Metab

J Lipid Res

J Biol Chem

J Biol Chem

J Biol Chem

Macrophage reverse cholesterol transport: key to the regression of atherosclerosis?

Circulation

Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol

J Clin Invest

Pharmacological activation of liver X receptors promotes reverse cholesterol transport in vivo

Circulation

Basic Science
Liver-specific induction of Abcg5 and Abcg8 stimulates reverse cholesterol transport in response to ezetimibe treatment