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ATVB In Focus
Role of ABCA1 in Cellular Cholesterol Efflux and Reverse Cholesterol Transport
Series Editor:
Alan R. Tall
Previous Brief Reviews in this Series:
• Yancy PG, Bortnick AE, Kellner-Weibel G, de la Llera-Moya M, Phillips MC, Rothblat GH. Importance of different
pathways of cellular cholesterol efflux. 2003;23:712–719.
• Oram JF, HDL Apolipoproteins and ABCA1: partners in the removal of excess cellular cholesterol. 2003;23:720 –727.
• Joyce C, Freeman L, Brewer HB Jr, Sanatamarina-Fojo S. Study of ABCA1 function in transgenic mice. 2003;23:965–971.
• Aiello RJ, Brees D, Francone OL. ABCA1-deficient mice: insights into the role of monocyte lipid efflux in HDL
formation and inflammation. 2003;23:972–980.
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Liver X Receptor Agonists as Potential Therapeutic Agents
for Dyslipidemia and Atherosclerosis
Erik G. Lund, John G. Menke, Carl P. Sparrow
Abstract—The recent identification of liver X receptors (LXR) as regulators of the cholesterol and phospholipid export
pump ABCA1 has raised the possibility that LXR agonists could be developed as HDL-raising agents, possibly also
acting on the artery wall to stimulate cholesterol efflux from lipid-laden macrophages. Presently several pharmaceutical
companies are working to develop such compounds, which will require finding a path for separating these beneficial
effects from the detrimental stimulation of triglyceride synthesis also inherent to LXR agonists. Other challenges to the
drug development process include species differences, which makes prediction of in vivo effects of LXR agonists in
humans difficult. This review summarizes the present state of knowledge on LXR as a drug target and discusses possible
solutions for dissociating the favorable effects of LXR agonists from their unwanted effects. (Arterioscler Thromb Vasc
Biol. 2003;23:1169-1177.)
Key Words: atherosclerosis 䡲 liver X receptor 䡲 HDL 䡲 triglycerides
A
therosclerosis and its clinical sequelae, myocardial infarction and ischemic stroke, are a major cause of
premature death and disability in the industrialized world.1
Multiple studies have shown that statins, drugs that inhibit the
enzyme HMG-CoA reductase, improve clinical outcomes for
patients with atherosclerosis.2 There are also reports of
favorable clinical outcomes using fibrates, drugs that bind to
and activate the nuclear receptor PPAR␣.3 Neither statins nor
fibrates reduce the risk of a heart attack in dyslipidemic
patients by as much as 50%, however, so there is clearly a
need for additional drugs to combat atherosclerosis.
What biological effects might be desired in a new antiatherosclerosis drug? One possibility is HDL raising, which is
supported by the strong epidemiological evidence linking low
HDL levels to increased risk of disease.4 Although it is true
that HDL raising can be achieved using fibrates3 or certain
statins such as simvastatin,5 these effects are fairly modest
(usually ⬍20%). Niacin raises HDL more dramatically and
has shown clinical benefits in placebo-controlled trials; however, adverse effects associated with this drug makes compliance poor.6 A second desired effect of a new drug would be
to act on the cells of the artery wall to directly stimulate the
exit of cholesterol from existing atherosclerotic lesions; available evidence suggests that lipid-rich lesions are more prone to
rupture and thereby cause myocardial infarction.7 It may be
possible to induce these two desirable effects, HDL raising and
cholesterol efflux, through manipulation of a single drug target,
the nuclear receptor liver X receptor (LXR).
Nuclear receptors are ligand-activated transcription factors; the first members of this superfamily to be described
Received November 8, 2002; accepted January 6, 2003.
From the Departments of Atherosclerosis and Endocrinology and Metabolic Research (J.G.M), Merck Research Laboratories, Rahway, NJ.
Correspondence to Erik G. Lund, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065. E-mail [email protected]
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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DOI: 10.1161/01.ATV.0000056743.42348.59
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July 2003
TABLE 1.
Genes Regulated by LXR in Mice and Humans
Gene Expression Response to LXR Activation in
Gene
ABCA1
SREBP-1c
CYP7A1
Function of Gene Product
Humans
Cellular cholesterol efflux and HDL formation
Mice
64,66,67
Induced54,64
21
Induced16,30
Induced
Master regulator of fatty acid synthesis
Induced
29,62,63
Rate determining enzyme of bile acid synthesis
No effect
Induced30,52
CETP
Transfer of neutral lipids between lipoprotein particles
Induced59
Gene not present68
PLTP
Phospholipid tranfer; lipoprotein assembly
Weakly induced (Menke, unpublished data)
Induced32
LXR␣
ABCG1
ABCG5/ABCG8
69,70
Sensor of cholesterol excess
No effect69,70
Induced
21,65
Unknown
Strongly induced
Efflux of plant sterols and cholesterol into bile and intestinal lumen
Not known
Induced24
Induced71
The authors’ laboratories have confirmed all of the effects listed in the table.
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were the steroid hormone receptors.8 The binding of a small
molecule ligand, such as estradiol for the estrogen receptor, to
a nuclear receptor modulates transcription of target genes
through the binding of the nuclear receptor/ligand complex to
appropriate response elements in the proximal promoter of
target genes. The nuclear receptors LXR␣ (NR1H3) and
LXR␤ (NR1H2) are important regulators of genes involved
in lipid metabolism.9 Originally cloned as orphan nuclear
receptors,10 it is now believed that both LXR isoforms are
intracellular sensors of cholesterol excess.11 Both LXR subtypes are different from the classical steroid receptors in that
they show a low level of constitutive activation in the absence
of ligand.12,13 LXR target genes include many of those known
to be important in the reverse cholesterol transport pathway,
from cellular cholesterol efflux through HDL metabolism to
biliary cholesterol excretion. A partial list of LXR-induced
genes, along with their presumed function, is given in Table
1. The apparent coordinate regulation of components of the
reverse cholesterol transport pathway makes LXR an attractive target for novel pharmaceutical agents.
From a drug development point of view, perhaps the most
interesting gene induced by LXR is ABCA1, which is a lipid
pump that functions to remove excess cholesterol from
cells.14 The critical role of ABCA1 in cellular cholesterol
homeostasis and HDL metabolism is supported by the fact
that genetic deficiency of ABCA1, which results in Tangier
disease, leads to cholesterol accumulation in several tissues
and vanishingly low HDL levels.14 The importance of
ABCA1 in atherosclerosis was supported by the observation
that patients heterozygous for ABCA1 deficiency have ⬇3fold increased risk for coronary artery disease.15 Therefore,
induction of the ABCA1 gene by LXR agonists might lead to
a decreased cholesterol burden in the artery wall as well as
increased HDL levels. The exciting possibilities for LXR
agonists as new therapeutics, however, are counterbalanced
by one significant liability: the induction of genes of fatty
acid synthesis and the accumulation of triglyceride. This
liability is, at least in part, mediated by LXR-induced transcription of SREBP-1c, a member of the sterol regulatory
element-binding protein family of transcription factors.16
SREBP-1c induces the transcription of many lipogenic genes,
including fatty acid synthase and acetyl-CoA carboxylase,
which are the 2 key enzymes of de novo fatty acid synthesis.17
Clearly the successful development of LXR agonists into
pharmaceuticals will require that a favorable balance be
found between the potentially beneficial effects of induction
of ABCA1 and the potentially detrimental effects of induction of SREBP-1c.
Can LXR Agonists Help When and Where
They Are Really Needed?
The great hope for LXR agonists as novel therapeutic agents
is that such molecules will be able to increase expression of
ABCA1 in the artery wall, leading to enhanced cholesterol
efflux and resolution of the lipid overload in atherosclerotic
lesions. For this hope to be realized, synthetic LXR agonists
must stimulate ABCA1 expression and cholesterol efflux in
artery wall cells that are already cholesterol-loaded. On first
principles, however, it is not clear that it would be feasible to
additionally activate LXR in foam cells. Although not universally accepted,18 it is generally believed that the physiological ligands of LXR are cholesterol-derived oxysterols,11
although the relative importance of different oxysterols in this
role is still a matter of debate. Oxysterols are present in
atherosclerotic plaques in such large amounts that even if
only 10% are present in an unesterified form,19 27hydroxycholesterol in particular seems to be present at
saturating concentrations with respect to LXR activation.20 It
is therefore conceivable that the LXRs may already be fully
activated in cholesterol-loaded cells such as those found in
atherosclerotic lesions in the artery wall and that no additional activation with an exogenous LXR agonist would be
feasible.
We addressed this issue experimentally by studying the
regulation of ABCA1 mRNA and the stimulation of cholesterol
efflux in primary human monocyte-derived macrophages.21 We
confirmed previous observations22,23 that cholesterol loading
increased mRNA levels for ABCA1 (2-fold) and ABCG1
(5-fold; Table 1). The synthetic LXR agonist APD was more
effective, increasing these mRNA levels by 4- and 17-fold,
respectively.21 The combination of cholesterol loading plus APD
produced changes similar to those seen with APD alone. A
similar superiority of APD was seen in assays of cholesterol
efflux.21 This work strongly implies that synthetic LXR agonists
are more effective than cholesterol loading at inducing ABCA1
expression and cholesterol efflux in human macrophages. Sim-
Lund et al
LXR Agonists for Dyslipidemia and Atherosclerosis
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ilar conclusions for mouse macrophages were published by
Joseph et al.24 Joseph et al also showed that the synthetic LXR
agonist GW3965 decreased atherosclerosis in mice.24 This
promising data must be interpreted cautiously given the significant species differences in LXR-responsive genes between mice
and humans (see discussion below). Nevertheless, taken together, these results suggest that LXR agonists may be effective
in the treatment of human disease.
The observation that upregulation of ABCA1 expression by
synthetic LXR agonists is more effective than cholesterol loading is consistent with our recent findings implicating 27hydroxycholesterol as a major endogenous LXR ligand in
human monocyte-derived macrophages.25 We demonstrated that
cholesterol loading such cells leads to the production of micromolar levels of 27-hydroxycholesterol and its metabolite cholestenoic acid (another LXR agonist26) with little or no concomitant formation of other suggested natural LXR ligands.25
Furthermore, human fibroblasts deficient in CYP27, the enzyme
responsible for synthesis of 27-hydroxycholesterol, did not
upregulate LXR-regulated genes on cholesterol loading.25 Although 27-hydroxycholesterol is a bona fide LXR ligand,25,27,28
the maximal activation of LXR achieved by this ligand is less
than the maximal activation obtained by synthetic ligands even
at saturating conditions21,25,29 (and data not shown), thus mirroring the effects elicited by cholesterol loading.
Effects of LXR Agonists on Lipid Metabolism
in Small Animals
The data described above demonstrate that LXR action
induces ABCA1 expression and increases cholesterol efflux
in cultured cells. Several studies also showed that synthetic
LXR agonists can raise plasma HDL in experimental animals.
Schultz et al30 found a dose-dependent increase of HDL
cholesterol in C57Bl/6 mice using the LXR␣/␤ dual agonist
T0901317, a result later confirmed by Grefhorst at al31 and
Cao et al.32 We have also observed HDL elevation using
structurally unrelated LXR agonists (data not shown). Interestingly, the increase in HDL cholesterol chiefly occurred in
a population of large HDL particles of low abundance in
untreated animals.31,32 In contrast to the effects in C57Bl/6
mice, in mice deficient for either the LDL receptor or
apolipoprotein E, prolonged treatment with the synthetic
LXR agonist GW3965 did not raise HDL levels.24 The
difference could possibly be explained by the disturbances of
lipid metabolism induced by the gene deletions.
Encouragingly, recent studies have implicated LXR not
only in HDL formation but also directly in the protection of
the artery wall against atherosclerosis. Bone marrow transplantation from LXR double-knockout mice into mice deficient in either the LDL receptor or apolipoprotein E (apoE)
led to a significant increase in atherosclerosis without significant effects on plasma lipids.33 Similarly, deficiency of
LXR␣, LXR␤, or both led to increased aortic lipid deposition
in 18-month-old chow-fed mice, despite only minor changes
in plasma lipids.34 These results imply that the absence of
LXR signaling exacerbates atherosclerosis. The obverse of
this, that increased LXR signaling caused by administration
of an LXR agonist should decrease atherosclerosis, has
recently been demonstrated by Joseph et al,24 who showed
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that the synthetic LXR agonist GW3965 decreases the formation of atherosclerotic plaques in mice deficient for apoE
or the LDL receptor.24 In these studies, HDL cholesterol
levels were not altered. We have confirmed the antiatherosclerotic activity of LXR agonists in apoE-knockout mice
using structurally unrelated compounds. An interesting difference in our study was that serum triglyceride levels were
dramatically increased (unpublished data, 2001).
Enthusiasm over the favorable effects of LXR agonists on
HDL metabolism and atherosclerosis may be tempered by the
triglyceride-raising effects of the same compounds. Schultz et
al30 found a 2- to 3-fold increase in plasma triglycerides in
C57Bl6/J mice treated with 5 to 50 mg/kg per day T0901317,
coupled with an up to 5- to 6-fold elevation of hepatic
triglyceride levels. These elevations were not seen in
LXR␣/␤ double knockout mice. The elevation of TG is at
least in part attributable to increased biosynthesis, because
Grefhorst et al31 found a 2.5-fold increase in hepatic
VLDL-TG secretion on treatment of C57Bl/6J mice with 10
mg/kg per day T0901317, an effect fully accounted for by the
formation of larger as opposed to more VLDL particles.
However, in that study, no elevation of plasma triglycerides
was found, indicating a compensatory increase in particle
clearance, a conclusion supported by the finding of massive
hypertriglyceridemia in mouse models of defective lipoprotein clearance on treatment with T0901317. We have also
measured varying degrees of hypertriglyceridemia and hepatic steatosis with different LXR agonists (not shown).
Speculation on Possible Effects of LXR
Agonists in Humans
Potential effects of LXR agonists in humans are suggested by
extrapolating from existing published evidence on the effects
of LXR antagonists in humans. It has recently been demonstrated that n-3 fatty acids, the major constituents of fish oil,
are LXR antagonists35,36 (Figure 1). Dietary n-3 fatty acids
have long been known to decrease serum triglyceride in
humans,37 an effect consistent with the triglyceride-raising
effects of synthetic LXR agonists described above. In general, changes in VLDL triglyceride and HDL cholesterol are
inversely correlated38,39 and, accordingly, most triglyceridelowering regimens (exercise, weight loss, niacin, and fibrates)
raise HDL. Interestingly, dietary n-3 fatty acids do not change
HDL significantly in humans; this failure of dietary n-3 fatty
acids to increase HDL may be attributable to their antagonism
of LXR, which could lead to decreased ABCA1 expression.
This model is consistent with our unpublished data showing
that feeding fish oil to mice decreases both serum triglyceride
and HDL.
Another clue to the possible effects of LXR agonists in
humans comes from the study of patients taking certain
CYP3A4-inducing anticonvulsants, including phenobarbital,
carbamazepine, and phenytoin. It was recently shown that
patients taking these drugs have highly elevated levels of
plasma 4␤-hydroxycholesterol. These authors propose that
this is a result of 4␤-hydroxylase activity of CYP3A4.40 In
contrast, valproic acid, another anticonvulsant, does not
induce CYP3A4 and did not raise 4␤-hydroxycholesterol.40
Interestingly, 4␤-hydroxycholesterol is a relatively potent
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Figure 1. Regulatory pathways controlling intracellular
cholesterol homeostasis. As a response to low intracellular cholesterol levels, SREBP-1c and SREBP-2 are
activated by proteolytic processing.17 The mature form
of SREBP-2 activates transcription of the LDL receptor
and cholesterol biosynthetic genes, including HMGCoA reductase.17 Also the transcription of SREBP-282
and SREBP-1c82,83 precursors is increased in a feedforward loop. The net result is an increase in cholesterol
synthesis and import. Under conditions of cholesterol
sufficiency or excess, SREBP-2 processing and cholesterol synthesis are inhibited.17 Instead, cholesterol loading leads to generation of oxysterols,25,84 activation of
LXR, and increased transcription of ABCA1,22 which
catalyzes efflux of excess cholesterol. LXR also stimulates the transcription of SREBP-1c precursor, but this
effect can be expected to be balanced by suppressed
SREBP-1c activation, and, therefore, the resulting
increase in fatty acid synthesis is modest. Synthetic
LXR agonists induce SREBP-1c transcription but do
not suppress activation, leading to an overshoot in fatty
acid synthesis. The activation of LXR can be antagonized by polyunsaturated fatty acids, which also suppress SREBP-1c processing.35,36,85,86 SREBP indicates
sterol regulatory element binding protein; HMGCR,
HMG-CoA reductase; LDLR, low-density lipoprotein
receptor; FAS, fatty acid synthase; LCE, long chain fatty
acyl-CoA elongase; TG, triglycerides; PL, phospholipids; and PUFA, polyunsaturated fatty acids.
LXR agonist in a cell-based transactivation assay.27 Numerous studies have been published describing effects on plasma
lipids by anticonvulsants, and although some of these studies
show an increase in HDL or triglyceride by the CYP3A4inducing drugs but not valproic acid,41 others do not.42
Nevertheless, it was recently speculated that the effects of
anticonvulsants on plasma lipids were attributable to LXR
activation.43 Although caution must be exercised in interpreting these studies, taken together with the evidence on n-3
fatty acids, it is reasonable to expect that LXR agonists will
increase both HDL and triglyceride in humans. Dissociating
this expected dual effect is the great challenge for the
development of LXR agonists as new therapeutics (see
discussion below).
Other possible effects of LXR agonists should also be
considered. Both isoforms of LXR are expressed in the brain,
and disruption of the LXR genes44 as well as treatment with
LXR agonist45 alters brain lipid homeostasis. In addition, a
recent study demonstrated increased secretion of A␤ from
neuronal cells treated with LXR agonists.46 In diabetic
rodents, the LXR agonist T0901317 was recently shown to
suppress genes involved in gluconeogenesis.47 Also recently,
LXR␤ was implicated in regulation of angiogenesis.48 It is at
present not clear what the physiological importance of the
above findings might be; however, it could be reasonably
expected that a brain-penetrant LXR agonist would have
effects on cholesterol homeostasis in that organ. An extended
list of genes regulated in liver and adipose tissue of mice fed
an LXR agonist was recently published.49
Understanding the Multiple Effects
of LXR Agonists
If we accept that LXR can detect cholesterol overload, it is
possible to rationalize why LXR would increase the expres-
sion of the cholesterol pump ABCA1. More difficult to
understand is why LXR activation should increase fatty acid
synthesis through induction of SREBP-1c. Whereas requirement of fatty acyl-CoAs for the synthesis of cholesterol esters
may provide a partial explanation,16,30 another part of the
answer may reside in the proposed mechanism of action of
ABCA1. According to one model, LXR-stimulated cholesterol efflux through the ABCA1 pathway requires prior
formation of acceptor vesicles containing phospholipids and
apo A-I.50,51 Nevertheless, the increase in fatty acid synthesis
seems to exceed the requirements for the formation of
phospholipid acceptor vesicles and cholesterol esters, as
shown in the resulting accumulation of triglycerides.
To understand the seemingly paradoxical hyperstimulation
of fatty acid synthesis by LXR, it may help to recall that the
underlying cause for nonpharmacologic LXR activation in a
physiological setting is cholesterol excess and to consider
what other effects on cellular lipid homeostasis are induced
by that state. A cell exposed to excess cholesterol responds
not only by activating LXR but also by suppressing SREBP
processing.17 The SREBP family of lipogenic transcription
factors, including the LXR-regulated gene SREBP-1c, is
synthesized as inactive precursors whose activation by proteolytic processing is blocked by intracellular cholesterol
excess.17 Thus, cholesterol loading seems to modulate the
expression of SREBP-1c–regulated genes by the following 2
independent and counteracting mechanisms: (1) generation of
LXR agonists, which would tend to raise SREBP-1c expression; and (2) suppression of SREBP-1c activation through
inhibition of proteolytic processing. Thus, activation of LXR
induced by cholesterol excess would not be expected to
increase lipogenesis dramatically. This effect of cholesterol
stands in sharp contrast to synthetic LXR agonists, which
induce the transcription of the SREBP-1c gene but do not
Lund et al
LXR Agonists for Dyslipidemia and Atherosclerosis
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Figure 2. Differential effects of cholesterol loading and a synthetic
LXR agonist on gene expression in human monocyte-derived macrophages. Treatment of macrophages with 1 ␮mol/L of the synthetic agonist APD21 for 24 hours boosts the expression of its
direct target ABCA1 as well as SCD through SREBP-1c. Cholesterol loading with Ac-LDL (72 hours, 300 ␮g/mL) also increases the
expression of ABCA1, although to a lesser degree, because
27-hydroxycholesterol, the major natural LXR ligand under these
conditions, is a partial agonist of LXR.25 Despite increased LXR
signaling, SCD expression is reduced. Combination of Ac-LDL and
APD leads to an intermediate response; however, the expression
of SCD is much suppressed compared with what is seen with APD
alone. This apparent attenuation of LXR-mediated SCD induction
by cholesterol loading can likely be explained by suppression of
SREBP processing.
suppress SREBP activation, thereby leading to an overshoot
in fatty acid synthesis. Confirmation of this model for the
excessive lipogenesis in animals treated with synthetic LXR
agonists will require immunoblot determination of the mature
and precursor forms of the hepatic SREBPs, as has been done
previously under other experimental conditions.17 A summary of these concepts is presented in Figures 1 and 2. Figure
1 is a representation of pathways pertinent to intracellular
cholesterol homeostasis, and Figure 2 is an example of
differential effects of an LXR agonist and cholesterol loading
on the expression of ABCA1 (a direct LXR target) and
stearoyl-CoA desaturase (SCD; an SREBP-1c target).
Potential Strategies for Dissociating
Antiatherosclerotic Effects From
Triglyceride Raising
Based on the evidence described above, it is reasonable to
believe that activation of LXR would decrease atherosclerosis
in humans. The challenge to drug discovery, however, is to
identify an LXR agonist that will not cause unacceptable
triglyceride accumulation. Below we shall discuss 3 potential
strategies for dissociating the HDL- versus triglycerideraising properties of LXR agonists.
1. LXR␤-Selective Agonists
Evidence from gene expression studies in LXR knockout
mice suggests that it may be possible to dissociate the
HDL-raising effects from the triglyceride-raising effects of
LXR agonists by identifying LXR␤-selective agonists, ie,
compounds that selectively bind and activate LXR␤ but not
LXR␣. The 2 LXR subtypes, LXR␣ and LXR␤, have very
similar sequences but probably do not play identical roles in
vivo, as judged by their pattern of expression and the
phenotypes of the knockout mice. LXR␣ is strongly expressed in a small number of tissues (liver, small intestine,
adipose, and macrophages), whereas LXR␤ is ubiquitously
expressed.10 LXR␣ knockout mice, but not LXR␤ knockout
mice, massively accumulate hepatic cholesterol on cholester-
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ol feeding.52,53 No observable phenotype has yet been ascribed to LXR␤ knockout mice.
LXR␣ knockout mice show reduced plasma triglyceride
levels as well as reduced hepatic mRNA levels for multiple
enzymes of fatty acid synthesis, including fatty acid synthase.52 LXR␤ knockout mice do not show these effects,
implying that LXR␣ is the subtype most responsible for
controlling SREBP-1c transcription in liver.53 Conversely,
peritoneal macrophages from LXR␤ knockout mice, but not
LXR␣ knockout mice, show altered basal expression of
ABCA1 mRNA,54 suggesting that LXR␤ is the subtype most
responsible for controlling ABCA1 transcription in macrophages. The possibility that the LXR subtypes control different genes can be rationalized by their tissue distribution,
combined with the idea that both LXR subtypes are activated
by oxysterols that signal cholesterol excess or high rates of
cholesterol synthesis. LXR␣ is expressed in tissues that
synthesize triglyceride. In these tissues, such as liver, a slight
excess of cholesterol could activate LXR␣, leading to enhanced triglyceride synthesis, which would drive lipoprotein
assembly and secretion of the excess cholesterol. Conversely,
in typical extrahepatic tissues, the appropriate response to
excess cholesterol would be activation of LXR␤ leading to
increased expression of ABCA1, allowing export of the
excess cholesterol. It is possible that these apparent gene
selectivities of LXR␣ and LXR␤ can be explained by
different ratios of LXR␣ versus LXR␤ expression in different
tissues. At present, the only bona fide gene selectivity of the
LXR subtypes that has been described is that the LXR
response element controlling the lipoprotein lipase gene
responds only to LXR␣ and not to LXR␤.55
Regardless of the true mechanistic origins of the apparent
gene selectivities, the observations and rationales described
above suggest the tantalizing possibility that agonists selective for LXR␤, with a low expression in liver, may offer a
solution to the triglyceride-raising problems of the LXR␣/␤
dual agonists that have been disclosed to date. Final proof that
LXR␤-selective agonists will be superior must await the
identification and description of such molecules. The search
for LXR␤-selective agonists may be an arduous one, however, because of the high degree of sequence identity between
the ligand-binding domains of LXR␣ and LXR␤ (Table 2).
As shown in Table 2, the ligand-binding domain of these two
receptors are more similar to each other than most other pairs
of related nuclear receptors.
2. Sterol-Mimicking LXR Agonists
Another approach for dissociating the triglyceride-raising
from HDL-raising effects of LXR agonists is to emulate
cholesterol loading by developing compounds, perhaps steroid derivatives, that not only activate LXR but also suppresses SREBP processing. As discussed above, this suppression of SREBP activation would be expected to counteract
the upregulation of SREBP-1c synthesis by LXR and result in
an attenuated triglyceride response compared with other LXR
agonists. However, this is a problematic approach, taking into
the account the mechanism of action of statins. This class of
drugs acts by inhibiting HMG-CoA reductase, the ratelimiting enzyme of cholesterol biosynthesis. When the hepa-
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TABLE 2.
Sequence Similarities for Various Pairs of Nuclear Receptors
% Amino Acid Identity Between A and B in
Nuclear Receptor A
(Ligand Selective for A⬎B)
Nuclear Receptor B
(Ligand Selective for B⬎A)
Ligand That Binds
Both A and B
DNA-Binding
Domain, %
Ligand-Binding
Domain, %
LXR␣ (hypocholamide72)
LXR␤ (none described)
T090131730; APD21
75
77
PPAR␣ (fenofibrate73)
PPAR␦ (GW50151674)
F3methylAA29
86
74
PPAR␣ (fenofibrate )
PPAR␥ (rosiglitazone )
75
LY-465608
83
69
Estrogen receptor-␤ (DPN77)
17␤-Estradiol
96
60
Glucocorticoid receptor (prednisone)
Mineralocorticoid receptor (aldosterone)
Cortisol78
93
56
Glucocorticoid receptor (prednisone)
Progesterone receptor (progesterone)
Mifepristone (RU-486)79
90
53
60
31
73
Estrogen receptor-␣ (PPT76)
Retinoic acid receptor-␣
(all-trans-retinoic acid80)
73
Retinoid-X-receptor (RXR)-␣ (Targretin )
81
80
9-cis-retinoic acid
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The amino acid sequences for the human form of the nuclear receptors was obtained from SWISSPROT. For each nuclear receptor listed, the accession number,
DNA-binding domain amino acids, and ligand-binding domain amino acids, respectively, are as follows: ER␣: P03372, 185–250, 311–551; ER␤: Q92731, 149 –214,
215–530; LXR␣: Q13133, 98 –163, 215– 434; LXR␤: P55055, 87–154, 231– 461; PPAR␣: Q07869, 102–166, 281– 468; PPAR␥: P37231, 139 –203, 318 –505;
PPAR␦: Q03181, 74 –138, 254 – 441; GR: P04150, 421– 486, 528 –777; MR: P08235, 603– 668, 733–984; PR: P06401, 567– 632, 681–933; RAR: P10276, 88 –153,
200 – 419; RXR: P19793, 135–200, 225– 462.
tocyte experiences lower cholesterol levels because of the
inhibition of biosynthesis, it responds by an increase in
SREBP processing and, as a result thereof, LDL receptor
expression. This increase in LDL receptors leads to the
desired result—an efficient removal of LDL particles from
the circulation. Thus, an LXR ligand that also inhibits SREBP
processing would also be expected to counteract statin action.
This is clearly undesirable, especially because the intended
patient population to a large extent overlaps with the patients
who benefit from the statin class of drugs.
3. Tissue- or Gene-Selective Agonists
At least two more avenues toward suitable compounds can be
envisioned. First, LXR agonists with suitable pharmacokinetic properties, allowing a substantial distribution in the
extrahepatic tissues, may be identified. Such a compound
may be able to exert favorable effects on the artery wall at
doses sufficiently small to allow any increase in fatty acid
synthesis to be manageable. A second possibility is that
selective LXR modulators, analogous to the selective estrogen receptor modulators, can be found. The latter compounds
are characterized by mixed agonist profile; for example, the
estrogen receptor ligand tamoxifen functions as an estrogen
agonist in bone, but it is an antagonist in breast tissue.56 At
this point, however, it is unknown whether a similar behavior
can be obtained with LXR agonists. Presently, LXR␤selective agonists seem to offer the most promising path
toward LXR agonists having the desired properties with
respect to gene and tissue specificity.
Species Differences in the Genes Controlled
by LXR: A Challenge to Drug Discovery
The development of new drugs requires the use of appropriate
animal models. A suitable animal model should mimic the
biochemistry and physiology of humans. One common obstacle to using small animals to develop novel therapeutics is
that drugs that interact with signaling receptors often have
dramatically different binding affinities for receptors from
different species; well-studied examples include ␤-adrenergic
receptors57 and PPAR␣.58 Significant species differences for
LXR ligands have not been reported to date; however, it has
become clear that there exist important species differences
with respect to which genes are controlled by LXR. Some of
these differences are summarized in Table 1, which compares
the effects of LXR activation on gene expression in mice and
humans. These two species were chosen because humans are
of greatest interest for drug discovery, whereas mice have
been studied most extensively with respect to LXR function.
The two most dramatic differences between the species are
CETP and CYP7A1 (see Table 1). Luo and Tall59 have shown
that the human CETP gene contains a functional LXR
response element; we have extended these results by demonstrating that synthetic LXR agonists increase CETP expression in primary human hepatocytes (unpublished data, 2001).
The finding that CETP is an LXR-responsive gene thus explains
the original observation in rabbits that CETP expression is
increased by cholesterol feeding.60 This potentially important
effect of LXR agonists in humans is not recapitulated in mice
because the mouse genome does not contain CETP.
Another important difference in LXR-mediated gene regulation between humans and mice concerns CYP7A1. The
mouse CYP7A1 gene figured prominently in the early seminal work on LXR. Lehmann et al61 showed that the mouse
CYP7A1 promoter contained an LXR response element,
which suggested that LXR might mediate the induction of
CYP7A1 expression by cholesterol feeding in mice. Proof
that LXR mediates this induction came from the work of Peet
et al,52 who showed that LXR␣ knockout mice fail to induce
CYP7A1 expression on cholesterol feeding. In contrast,
Chiang et al62 demonstrated that the human CYP7A1 promoter does not contain a functional LXR response element.
We have recently extended these findings by demonstrating
that a synthetic LXR agonist induced CYP7A1 expression in
rats in vivo and also in primary rat hepatocytes whereas the
same molecule had no effect on CYP7A1 expression in
primary human hepatocytes.29 The species difference was not
caused by a difference in affinity for rat versus human LXR
receptors, as judged by transactivation assays and the fact that
Lund et al
LXR Agonists for Dyslipidemia and Atherosclerosis
the synthetic agonist induced ABCA1 in the primary human
hepatocytes.29 In a similar vein, Chen et al63 demonstrated
that mice transgenic for human CYP7A1 on a mouse Cyp7A1
knockout background did not increase expression of the
transgene in response to cholesterol feeding.
Mice and humans show differences in LXR-mediated gene
regulation other than CETP and CYP7A1 (see Table 1). For
example, ABCG1 is very strongly induced in human cells by
LXR agonists.64 In our experience, ABCG1 is often induced
⬎20-fold by LXR agonists in human cells (data not shown).
In mouse cells, however, and in mouse tissues in vivo,
ABCG1 is usually only induced 2- to 3-fold by LXR
agonists21,24,65 (also data not shown). The differences between
mice and humans may complicate predictions of outcomes in
humans based on the effects of LXR agonists in mice.
Summary
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The last few years have seen an explosion of new information
on the orphan nuclear receptors, including LXR, and in our
understanding of the critical roles played by LXR-responsive
genes such as ABCA1 and SREBP-1c. It has very recently
become clear that LXR activation and ABCA1 expression
can mitigate atherosclerosis in animal models. Taken together, the evidence makes a compelling case for attempting
to develop LXR agonists as novel therapeutics for the
treatment of atherosclerosis and its clinical consequences.
The great challenges ahead include identifying means to
circumvent the LXR-mediated overproduction of triglyceride
and identifying appropriate animal models for the testing of
novel LXR agonists.
Acknowledgments
The authors gratefully acknowledge the advice received from Drs
Susan Rohrer, Anne Vosatka, Gerald Waters, and Hilary Wilkinson.
We also thank Dr Samuel Wright for stimulating discussions and
consistent support.
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Liver X Receptor Agonists as Potential Therapeutic Agents for Dyslipidemia and
Atherosclerosis
Erik G. Lund, John G. Menke and Carl P. Sparrow
Arterioscler Thromb Vasc Biol. 2003;23:1169-1177; originally published online January 16,
2003;
doi: 10.1161/01.ATV.0000056743.42348.59
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