Clinica Chimica Acta 428 (2014) 82–88

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Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Invited critical review

ABCG5/ABCG8 in cholesterol excretion and atherosclerosis

Xiao-Hua Yu a, Kun Qian c, Na Jiang c, Xi-Long Zheng d, Francisco S. Cayabyab e, Chao-Ke Tang a,b,⁎
a
Life Science Research Center, University of South China, Hengyang, Hunan 421001, China
b
Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang, Hunan 421001, China
c
The Second Affiliated Hospital, University of South China, Hengyang, Hunan 421001, China
d
Department of Biochemistry and Molecular Biology, The Libin Cardiovascular Institute of Alberta, The University of Calgary, Health Sciences Center, 3330 Hospital Dr NW, Calgary,
Alberta T2N 4N1, Canada
e
Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Cholesterol is essential for the growth and function of all mammalian cells, but abnormally increased blood cho-
Received 19 September 2013 lesterol is a major risk factor for atherosclerotic cardiovascular disease. ATP-binding cassette (ABC) transporters
Received in revised form 7 November 2013 G5 (ABCG5) and G8 (ABCG8) form an obligate heterodimer that limits intestinal absorption and facilitates biliary
Accepted 9 November 2013
secretion of cholesterol and phytosterols. Consistent with their function, ABCG5 and ABCG8 are located on the
Available online 16 November 2013
apical membrane of enterocytes and hepatocytes. Liver X receptor is the major positive regulator of ABCG5
Keywords:
and ABCG8 expression. Mutations in either of the two genes cause sitosterolemia, a condition in which cholester-
ABCG5 ol and plant sterols accumulate in the circulation leading to premature cardiovascular disease. Overexpression of
ABCG8 ABCG5 and ABCG8 in mice retards diet-induced atherosclerosis because of reduced circulating and hepatic cho-
Cholesterol lesterol. In the current review, we summarize recent developments and propose a future framework that
Sitosterolemia provides new perspectives on the regulation of cholesterol metabolism and treatment of atherosclerotic cardio-
Atherosclerosis vascular disease.
© 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
83
2. Structure and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3. Sterol efflux function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4. Genetic polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5. Expression and regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
6. Sitosterolemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
7. Role in atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Disclosure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Abbreviations: ABCG5, ATP-binding cassette transporter G5; ABCG8, ATP-binding cassette transporter G8; NPC1L1, Niemann–Pick C1-Like 1; NBD, nucleotide binding domain; ER, en-
doplasmic reticulum; apoA-I, apolipoprotein A-I; HDL, high-density lipoprotein; LDL-C, low-density lipoprotein-cholesterol; VLDL-C, very low-density lipoprotein-cholesterol; LXR, liver X
receptor; TH, thyroid hormone; HNF4α, hepatocyte nuclear factor 4α; GATA4, GATA-binding protein 4; SNP, single nucleotide polymorphisms; HNF4α, hepatocyte nuclear factor 4α;
GATA4, GATA-binding protein 4; ET, endotoxin; LDLR, low-density lipoprotein receptor; PUFAs, polyunsaturated fatty acids.
⁎ Corresponding author at: Institute of Cardiovascular Research, University of South China, Hengyang, Hunan 421001, China. Tel./fax: +86 734 8281853.
E-mail address: tangchaoke@qq.com (C.-K. Tang).

0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.cca.2013.11.010
 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 82–88
1. Introduction
Cholesterol is an essential structural component of mammalian cell
membranes and plays a crucial role in normal embryonic development,
cell differentiation, nerve conduction, membrane fluidity and steroid
synthesis. Therefore, the normal supply of cholesterol is important for
human health. Too much cholesterol, however, may lead to pathological
consequences. An uncontrolled buildup of cholesterol in cells is able to
disrupt membranes and facilitate apoptosis. Accumulation of excessive
cholesterol in the blood causes hyperlipidemia and atherosclerotic car-
diovascular disease, one of the major causes of death worldwide, and
might contribute to the early onset of Alzheimer's disease [1–3].
Cholesterol homeostasis in the body is maintained mainly by de
novo synthesis, intestinal absorption, and biliary and fecal excretion.
The biosynthesis of cholesterol is a well-defined energy-consuming
and feedback-regulated process, which is involved in a variety of enzy-
matic reactions [4]. Niemann–Pick C1-Like 1 (NPC1L1) mediates intesti-
nal cholesterol absorption and biliary cholesterol re-absorption [5]. It is
also the target of ezetimibe, an inhibitor of dietary cholesterol uptake
which has been approved for the treatment of hypercholesterolemia.
In direct opposition of NPC1L1, the heterodimer of ATP-binding cassette
(ABC) transporters G5 (ABCG5) and G8 (ABCG8) has been shown to in-
hibit the absorption of cholesterol and plant sterols from the diet by me-
diating the efflux of these sterols from enterocytes back into gut lumen,
and by promoting efficient secretion of cholesterol and plant sterols
from hepatocytes into bile [6]. Thus, the heterodimer constitutes the
body's predominant defense against the accumulation of sterols derived
from the diet. This review focuses on the molecular characterization,
physiology, genetic polymorphisms, and regulation of ABCG5/ABCG8,
and explores their emerging pathogenic significance and therapeutic
potential in atherosclerosis.
2. Structure and characterization
The genes of ABCG5 and ABCG8 map to chromosome 2p21 in a head-
to-head orientation with only 140 bases separating their first exons.
Each is composed of 13 exons and 12 introns. The two genes encode
two different proteins, namely ABCG5 (sterolin-1) and ABCG8 (sterolin-
2). Each protein has an ATP-binding cassette near the N terminus, a trans-
membrane domain containing six helices, and a nucleotide binding do-
main (NBD) containing two conserved peptide motifs (Walkers A and
B) and a signature motif (Fig. 1) [7].
ABCG5 or ABCG8 serves only as a non-functional half-transporter
when expressed alone. Thus, they must form the heterodimer to obtain
sterol transport functionality. A variety of studies have shown that
ABCG5 and ABCG8 heterodimerize in the endoplasmic reticulum (ER),
transfer together through the Golgi apparatus, and eventually move to
apical subdomains in the plasma membrane [8,9]. Mice lacking leptin,
Fig. 1. Schematic of ABCG5 and ABCG8 protein structure. ABCG5 and ABCG8 belong to half-
ABC transporters, which consist of a transmembrane domain containing six helices and a
NBD region containing two conserved peptide motifs (Walkers A and B) and a signature
motif at the N-terminus.
83
an adipocyte-derived hormone, exhibit decreased abundance of ABCG5
and ABCG8 proteins in the liver, which may occur at the level of the
ABCG5/ABCG8 heterodimer assembly within the ER, whereas leptin ad-
ministration restores both proteins, suggesting that leptin is required for
the heterodimer formation [10]. As a lectin-like chaperone, calnexin or
its homolog calreticulin interacts with immature forms of ABCG5 and
ABCG8 to stimulate their productive folding by inhibiting their degrada-
tion [11]. Moreover, calnexin has been shown to enhance the ER retention
of ABCG5 and ABCG8; however, calreticulin predominantly promotes the
cell surface expression of mature ABCG5 and ABCG8 [11]. These findings
indicate a role for calnexin and calreticulin in the biosynthesis and quality
control of ABCG5/ABCG8.
Lack of expression of either half-transporter leads to the accumula-
tion of the other in the ER as a result of impaired translocation to the
plasma membrane, further indicating that ABCG5 and ABCG8 are
obligate heterodimers for proper trafficking to the cell surface [12].
N-terminal cytosolic domains of ABCG5 and ABCG8 are also involved
in ER retention of their homodimers [13]. Interestingly, N-linked glyco-
sylation of ABCG8 but not ABCG5 is essential for efficient trafficking of
the heterodimer [14]. The two NBDs of the heterodimer are not func-
tionally equivalent, because the integrity of the canonical NBD formed
by the Walker A and B motifs of ABCG5 and the signature motif of
ABCG8 is essential for their sterol efflux function [15]. In addition, sub-
stitutions of the D-loop aspartate and Q-loop glutamine in either NBD
do not interfere with sterol transport [16].
3. Sterol efflux function
ABCG5 and ABCG8 can promote cholesterol and plant sterol elimina-
tion from the body through hepatobiliary secretion (Fig. 2). Disruption
of both ABCG5 and ABCG8 in mice leads to extremely low biliary choles-
terol concentrations while biliary phospholipids and bile acid concentra-
tions are not altered, revealing transport selectivity of the heterodimer
[17]. Similar effects are also observed in mice lacking either ABCG5 or
ABCG8 [18,19]. Conversely, overexpression of ABCG5 and ABCG8 in
mice increases biliary cholesterol levels by more than five fold [20]. It is
worth noting that ABCG5/ABCG8-independent biliary cholesterol excre-
tion has been reported under some conditions [21,22], which needs fur-
ther exploration.
Some factors have also been identified to influence the effectiveness
of ABCG5/ABCG8 in secreting cholesterol from the liver. In canine gall-
bladder epithelial cells transfected with lentiviral constructs containing
mouse ABCG5 and ABCG8, cholesterol secretion is significantly in-
creased in the presence of bile salts, whereas other cholesterol acceptors
including apolipoprotein A-I (apoA-I), high-density lipoprotein (HDL)
or methyl-β-cyclodextrin fail to elicit this effect, suggesting that bile
salts serve as a cholesterol acceptor for the heterodimer [23]. ABCB4,
also called multidrug resistance protein 2 (MDR2), is a canalicular phos-
phatidylcholine flippase of hepatocytes. Deficiency of ABCB4 in mice
disrupts ABCG5/ABCG8-mediated secretion of cholesterol into the bile,
which indicates that ABCG5 and ABCG8 require ABCB4 for the efficient
biliary export of sterols [24].
Opposing the previously described function for NPC1L1 in the small
intestine, ABCG5 and ABCG8 transport cholesterol and plant sterols
from enterocytes into the gut lumen for fecal disposal (Fig. 2). Mice
overexpressing ABCG5 and ABCG8 exhibit a 50% decrease in the frac-
tional absorption of dietary cholesterol [20]. A recent study by Wang
et al. found that there is a negative correlation between the efficiency
of cholesterol absorption and the abundance of ABCG5 and ABCG8 in
the jejunum and ileum but not the duodenum in inbred mice, suggest-
ing that ABCG5 and ABCG8 in the jejunum and ileum are predominant
determinants of intestinal cholesterol absorption [25]. On the other
hand, ABCG5/ABCG8-deficient mice have a 2- to 3-fold increase in the
fractional absorption of dietary plant sterols as well as an approximately
30-fold elevation in plasma sitosterol levels, and display many charac-
teristics similar to those seen in sitosterolemic patients [17].
84 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 82–88

Fig. 2. Sterol efflux function of the ABCG5/ABCG8 heterodimer in vivo. The ABCG5/ABCG8 heterodimer locates to the brush border membrane of enterocytes and the canalicular membrane
of hepatocytes. ABCG5 and ABCG8 secrete sterols including phytosterols and cholesterol from hepatocytes into the bile. Biliary sterols account for more than two thirds of the total amount
of sterols in the intestinal lumen. Because the sterols are hydrophobic, they are transferred to the brush border membrane of enterocytes via bile salt micelles. Then, ABCG5 and ABCG8
mediate biliary and dietary sterol efflux from the brush border membrane back into the gut lumen for excretion. NPC1L1 is also present in the apical membrane of enterocytes and hepa-
tocytes, and can facilitate intestinal and biliary cholesterol absorption. Most cholesterol taken up into enterocytes by NPC1L1 is subsequently esterified, packaged into chylomicrons, and
then secreted into the lymph. The chylomicrons are finally delivered to hepatocytes through the circulatory system. This process is called the enterohepatic circulation of sterols. Ezetimibe
specially suppresses NPC1L1-dependent cholesterol absorption in the small intestine and liver.

Plant sterols are the non-saponifiable component of plant oils, and
have a chemical structure similar to cholesterol except for the addition
of an extra methyl or ethyl group. Dietary intake of plant sterols can de-
crease plasma levels of total cholesterol and low-density lipoprotein-
cholesterol (LDL-C) in humans because of their ability to displace cho-
lesterol from the mixed micelles in the upper small intestine, thereby
reducing fractional cholesterol absorption that is determined as the
ratio of [14C]cholesterol to [5,6-3H]sitostanol by a fecal dual isotope
method to indicate the efficiency of cholesterol absorption [26]. In addi-
tion to this, other mechanisms have been proposed. Plant sterol feeding
for 2 weeks results in a dose-dependent decrease of fractional choles-
terol absorption (2- to 7-fold reduction) in wild type mice and an 80%
reduction in mice lacking ABCG5, and stimulates intestinal cholesterol
excretion by 500% and 250% in wild type and ABCG5−/− mice, respec-
tively [27]. These observations indicate that the ABCG5/ABCG8 complex
is also involved in plant sterol-induced efflux of cholesterol.
4. Genetic polymorphisms
Recently, a variety of studies have focused on the genetic polymor-
phisms of ABCG5 and ABCG8. It is found that cholesterol absorption is
about 24% lower in individuals carrying the p.D19H allele of ABCG8
compared to wild type although there is no significant difference in
ABCG5 and ABCG8 expression levels, indicating an important role for
the ABCG8 19H allele in promoting cholesterol excretion [28]. The inter-
action between ABCG8 1199A and the bile acid biosynthetic enzyme
7α-hydroxylase (CYP7A1)-204A allele has been shown to affect lipid-
lowering response to atorvastatin in a Chinese population [29]. In hy-
percholesterolemic Japanese subjects, serum sitosterol levels and the
sitosterol/cholesterol ratio are higher in carriers of the ABCG8 M429V
variant than non-carriers [30]. A recent report in a heterozygous familial
hypercholesterolemia cohort indicates that the T400K polymorphism
in the ABCG8 gene has an increased risk of cardiovascular disease but
is not associated with cardiovascular endpoints [31]. Carriers of the
minor G allele at the ABCG5_Gln604GluCN G single nucleotide polymor-
phism (SNP) show significantly lower very low-density lipoprotein-
cholesterol (VLDL-C) and triglyceride concentrations than homozygous
C/C in patients with familial hypercholesterolemia [32]. Additionally, the
frequency of the CC homozygous genotype for the ABCG5 1950CNG poly-
morphism is significantly increased in hypercholesterolemic patients as
compared to healthy controls, whereas no significant difference for the
251AN G polymorphism of the ABCG8 gene is found, revealing a possible
link between ABCG5 1950CNG polymorphism and hypercholesterolemia
[33].
5. Expression and regulation
ABCG5 and ABCG8 are expressed almost exclusively on the brush
border membrane of enterocytes and the canalicular membrane of he-
patocytes [12]. More recently, Yoon et al. observed that ABCG8 forms
a diffuse intracellular pattern at the apical pole of human gallbladder ep-
ithelial cells, and that ABCG5 and ABCG8 protein levels are increased in
human gallbladder with cholesterol-related gallbladder diseases [34].
This suggests a role for the heterodimer in the pathogenesis of human
cholesterol-related gallbladder diseases.
The expression of ABCG5 and ABCG8 is primarily regulated at the
transcriptional level (Fig. 3). Liver X receptor α (LXRα), a ligand-
activated nuclear transcription factor involved in the control of lipid me-
tabolism, is regarded as the major regulator of ABCG5 and ABCG8 mRNA
expression. In support of this concept, both soy protein (SP) diet and
LXR agonist T0901317 markedly upregulate ABCG5 and ABCG8 mRNA
expression in the small intestine and liver of wild type mice, but not
 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 82–88
Fig. 3. Regulation of ABCG5 and ABCG8 expression. Both SP and T0901317 can induce ABCG5/ABCG8 expression by activating LXRα. Their expression is also upregulated by n−3 PUFAs in
a HNF4α-dependent manner. The interaction between HNF4α and GATA4 promotes ABCG5/ABCG8 gene transcription. Fish oil, açaí pulp, TH, CBE and HF/HG significantly increase ABCG5/
ABCG8 levels, whereas atorvastatin and ET decrease their levels. Additionally, binding of miR-200C to ABCG5 leads to a significant decrease in ABCG5 protein.
in LXRα knockout mice [35–37]. Moreover, two LXR response elements
in the ABCG5 and ABCG8 genes have been identified [38]. Thyroid hor-
mone (TH), an important regulator of cholesterol metabolism, has been
shown to raise hepatic gene expression of ABCG5 and ABCG8 in a LXR-
independent mechanism [39]. Hepatocyte nuclear factor 4α (HNF4α)
and GATA-binding protein 4 (GATA4) are also transcription factors,
and they can synergistically stimulate ABCG5 and ABCG8 gene tran-
scription in HepG2 cells [40]. Recently, Kim et al. generated fat-1 trans-
genic mice expressing the Caenorhabditis elegans fat-1 gene encoding an
n−3 fatty acid desaturase, an enzyme absent in mammals, which con-
verts n − 6 polyunsaturated fatty acids (PUFAs) to n − 3 PUFAs [41].
These fat-1 transgenic mice fed with a high-fat diet exhibit a significant
increase in hepatic mRNA levels of ABCG5 and ABCG8 by activating
HNF4α, suggesting that the endogenously synthesized n−3 PUFAs act
as a positive regulator for ABCG5/ABCG8 expression [41]. It has been
suggested that the expression of hepatic ABCG5 and ABCG8 genes in a
rat model of dietary-induced hypercholesterolemia is significantly in-
creased in the presence of açaí pulp, a fruit of the Euterpe oleracea
Martius tree [42]. Fish oil has been shown to strongly induce the
mRNA expression of biliary ABCG5 and ABCG8 in rats [43]. On the
other hand, atorvastatin, an inhibitor of 3-hydroxyl-3-methylglutaryl-
CoA reductase, has been shown to decrease the mRNA levels of both
ABCG5 and ABCG8 in the duodenum of hyperlipidemic men [44]. Endo-
toxin (ET), the major component of the outer membrane of Gram-
negative bacteria, is released during bacterial cell division or lysis [45].
It has been reported that ET markedly downregulates ABCG5 and
ABCG8 mRNA expression in the murine liver [46], but the molecular
mechanism involved in this response is not known. Taken together, it
is quite obvious that ABCG5 and ABCG8 are coordinately modulated
by multiple transcriptional processes. More work in this field may prove
to be key for future ABCG5/ABCG8-based therapeutic intervention.
ABCG5 and ABCG8 are also highly modulated posttranscriptionally
(Fig. 3). It has already been reported that protein levels of ABCG5 and
ABCG8 in bile canaliculi of mice are significantly increased by a high-
fat/high-sucrose (HF/HG) diet as compared to the standard diet [47].
Black chokeberry (Aronia melanocarpa) is a rich source of polyphenols.
The polyphenol-rich black chokeberry extract (CBE) significantly en-
hances the levels of ABCG5 and ABCG8 in Caco-2 cells [48]. MicroRNAs
(miRNAs) are short non-coding RNAs that bind to target mRNAs and
cause inhibition of translation or initiation of mRNA degradation. Over-
expression of miR-200c, a member of the miR-200 family, results in a
significant downregulation of ABCG5, indicating a novel regulatory
role of miR-200c in lipid metabolism [49]. Thus, miR-200c inhibitors
can potentially be used to treat atherosclerosis. Apart from evidence
85
showing N-linked glycosylation of ABCG5 and ABCG8 as reported by
Graf et al. [12], it is surprising that there are no reports to date regarding
other modes of potential posttranslational modifications of these ABC
transporters. For example, analyses of the intracellular domains of
mouse ABCG5 and ABCG8 revealed numerous consensus sites for ser-
ine, threonine and tyrosine phosphorylation. Future investigations in
assessing the functional consequences of protein kinase or phosphatase
modulation of ABC transporters should lead to the development of
other therapeutic options for regulating their function or expression.
Many other factors that modulate their expression at the posttranscrip-
tional level, such as protein–protein interactions with scaffolding mole-
cules, may be discovered in future research.
6. Sitosterolemia
Sitosterolemia, a rare autosomal recessive disorder, is characterized
by markedly elevated plasma levels of plant sterols and modest increases
in plasma cholesterol, which is attributable to the hyperabsorption of
these sterols from the small intestine and a reduced excretion into the
bile. Due to these abnormal circulating sterol levels, sitosterolemic pa-
tients develop atherosclerosis and premature cardiovascular disease
[50]. Other clinical manifestations include tendon xanthomas, hemolytic
anemia, hypersplenism, macrothrombocytopenia and bleeding. A recent
report indicates that intercalation of plant sterols into the plasma mem-
brane induces dysregulation of multiple platelet activation pathways,
resulting in macrothrombocytopenia and bleeding [51]. This disease is
caused by mutations in the genes encoding ABCG5 or ABCG8 [52]. More-
over, all missense mutations that have been investigated to date either in-
hibit formation of the ABCG5/ABCG8 heterodimers or impede their
efficient transfer from the ER to the plasma membrane [14].
Since increased amounts of cholesterol and phytosterols in the blood
are deposited on the arterial walls, treatment to reduce plasma sterol
levels may be crucial to prevent life-threatening cardiovascular events
in patients with sitosterolemia. So far, low sterol diets, partial ileal by-
pass surgery that is defined as shortening of the ileum to decrease the
total small intestinal length, and drugs such as statins and sitostanol
have been used to cure sitosterolemia; however, these therapies are
only partially effective [53]. Thus, alternative strategies need to be pur-
sued. Recently, Tang et al. found that genetic inactivation of NPC1L1, a
cholesterol transporter with the opposite effect of ABCG5/ABCG8, mark-
edly protects against sitosterolemia in mice lacking ABCG5/ABCG8 [54].
Ezetimibe has been demonstrated to bind directly to NPC1L1 and acts by
blocking sterol-induced internalization of NPC1L1, subsequently sup-
pressing the absorption of sitosterol and cholesterol. Based on this,
86 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 82–88
ezetimibe should have the potential to be a revolutionary treatment
agent for sitosterolemia. Indeed, in a multicenter, double-blind, ran-
domized, placebo-controlled study, plasma sitosterol concentrations
were reduced by 21% in sitosterolemia patients treated with ezetimibe
10 mg/day for 8 weeks as compared to an insignificant 4% rise in
those on the placebo, with no serious treatment-related adverse events
or discontinuations due to adverse events [55]. Moreover, in patients
with homozygous sitosterolemia, long-term treatment with ezetimibe
10 mg/day for 2 years was also effective in decreasing plasma sitosterol
and LDL-C concentrations with an overall favorable safety and tolerabil-
ity profile [56].
7. Role in atherosclerosis
Because the ABCG5/ABCG8 heterodimer plays a key role in choles-
terol removal from the body, it seems logical to presume that overex-
pression of ABCG5 and ABCG8 should inhibit atherosclerosis while
knockout of ABCG5 and ABCG8 should promote atherosclerosis. In
agreement, high-level expression of ABCG5 and ABCG8 in both the in-
testine and liver of low-density lipoprotein receptor (LDLR)-deficient
mice leads to a significant reduction in plasma cholesterol levels and
aortic atherosclerotic lesion area [57]. Treatment of female Fischer rats
with a hypercholesterolemic diet supplemented with 2% açaí pulp
causes a significant decrease in serum total cholesterol, LDL-C, and ath-
erogenic index, as well as a significant increase in HDL-cholesterol and
cholesterol excretion in feces through upregulation of hepatic ABCG5
and ABCG8 expression [42]. Evodiamine, a main bioactive component
in the fruit of Evodia rutaecarpa, has been reported to reduce the size
of atherosclerotic lesions and alleviate the hyperlipidemia in apolipo-
protein E (apoE)-deficient mice via promotion of ABCG5/ABCG8-
dependent hepatic cholesterol clearance [58]. Li et al. have demonstrat-
ed that the levels of ABCG5 and ABCG8 in the liver are significantly
higher in the atherosclerosis-resistant than in the atherosclerosis-
susceptible Japanese quail (Coturnix japonica) strains [59]. In a large
cohort study of 2012 patients with heterozygous familial hypercholes-
terolemia, the ABCG8 rs11887534 variant shows an association with
higher risk of coronary heart disease [31]. Mice treated with low-dose
lipopolysaccharide have reduced bile and fecal counts as well as
decreased expression of hepatic ABCG5 and ABCG8, but there is
no effect on plasma or liver counts, suggesting that attenuation of
reverse cholesterol transport, particularly cholesterol efflux from
liver to bile and feces, may contribute to atherosclerosis in chronic
inflammatory states such as obesity, metabolic syndrome, and type
2 diabetes [60].
It is generally believed that many sitosterolemic patients suffer from
premature atherosclerotic cardiovascular disease. However, whether
the atherosclerotic cardiovascular disease can be attributed to an in-
creased concentration of plasma plant sterols remains uncertain. Coro-
nary heart disease still develops in some subjects with sitosterolemia,
despite normal plasma cholesterol levels, suggesting that high circulat-
ing levels of plant sterols may be atherogenic [61]. In contrast, rapeseed
oil fortified with phytosterols contributes to protection against athero-
genesis by improving plasma oxidative stress, lipid profiles as well as in-
flammation [62]. Horenstein et al. have demonstrated that although the
G574R variant of ABCG8 is associated with moderately elevated plant
sterol levels, carriers of the G574R allele have modestly thinner carotid
wall thickness than noncarriers [63]. More recently, Wilund et al. re-
ported that there is no statistically significant difference in aortic ath-
erosclerotic lesion area between ABCG5/ABCG8-deficient mice and
littermate controls, and that plasma levels of cholesterol, but not sitos-
terol or campesterol, are significantly higher in humans with coronary
atherosclerosis, indicating no association between plasma plant sterol
levels and atherosclerosis in mice and humans [64]. In this case, perhaps
increased plasma cholesterol levels are responsible for the progression
of premature atherosclerotic cardiovascular disease in patients with
sitosterolemia [64]. Overall, more studies are needed to clarify why
sitosterolemic patients are prone to premature atherosclerotic cardio-
vascular disease.
Considering ABCG5/ABCG8 as cholesterol's way out of the body, ac-
tivation of the heterodimer may provide benefits for regulation of lipid
metabolism and treatment of atherosclerosis. Indeed, curcuma oil, a li-
pophilic component from Curcuma longa L., significantly decreases plas-
ma total cholesterol and LDL-C, but elevates HDL-C, in male golden
Syrian hamsters fed with a high-cholesterol and fat-rich diet, primarily
by upregulation of ABCG5/ABCG8 expression in the liver and jejunum
[65]. CM108, a flavone derivative, also improves lipid profiles in hyper-
lipidemic rats via enhancing hepatic ABCG5/ABCG8 levels [66]. Similar-
ly, the hypolipidemic effects of n-butanol extract (a LXRα agonist) of
Panax notoginseng root are associated with activation of ABCG5/ABCG8
[67]. It is worth noting that overexpression of human ABCG5 and
ABCG8 in the liver, but not intestine, enhances hepatobiliary secretion
of cholesterol and plant sterols by 1.5- to 2-fold, but does not affect
the development of proximal aortic atherosclerosis in apoE or LDLR
knockout mice [68]. This suggests that therapies which simply raise
biliary sterol output are not always effective, because these sterols can
be re-absorbed by intestinal NPC1L1. Thus, increased biliary cholesterol
secretion must be coupled with decreased intestinal cholesterol absorp-
tion to enhance net sterol loss from the body and inhibit atherosclerosis.
To test this hypothesis, the same authors found in a subsequent study
that treatment of LDLR knockout mice overexpressing hepatic ABCG5
and ABCG8 with ezetimibe markedly increases hepatobiliary cholester-
ol secretion rates and reduces intestinal cholesterol absorption, thereby
producing profound protection against atherosclerosis [69]. Therefore,
dual therapies targeting both ABCG5/ABCG8 and NPC1L1 may be a via-
ble approach to prevent atherosclerosis in the future.
8. Conclusions
The discovery of ABCG5 and ABCG8 is an exciting result in cardiovas-
cular research. In contrast to NPC1L1, the ABCG5/ABCG8 heterodimer is
a major route to facilitate excess cholesterol clearance from the body.
Defects in ABCG5 and/or ABCG8 are linked to sitosterolemia. Converse-
ly, upregulation of ABCG5 and ABCG8 levels can promote reverse
cholesterol transport and reduce atherosclerosis. Since cholesterol ex-
cretion is a complex and multistep process that is regulated by multiple
genes at the hepatocyte and enterocyte levels, we should also pay atten-
tion to the effect of other proteins such as NPC1L1 and ABCB4. Besides,
there are still many problems left to be solved. How is the heterodimer
transported from the endoplasmic reticulum to the plasma membrane?
Can these two transporters directly bind and translocate their sub-
strates? Are there efficient avenues to enhance their function? Can
a dual therapy of a potential ABCG5/ABCG8 activator plus ezetimibe
lead to improved protection against atherosclerosis in humans?
Understanding these questions will provide insightful knowledge
regarding the underlying mechanisms of cholesterol output from
the body and accelerate the future design of novel therapeutic
reagents.
Disclosure
The authors have declared no conflict of interest.
Acknowledgments
The authors gratefully acknowledge the financial support from
the National Natural Sciences Foundation of China (81070220 and
81170278), Aid Program for Science and Technology Innovative Re-
search Team in Higher Educational Institutions of Hunan Province,
China (2008-244) and the construct program of the key discipline in
Hunan province.
 X.-H. Yu et al. / Clinica Chimica Acta 428 (2014) 82–88
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