Hepatic ATP-Binding Cassette Transporter A1 Is a Key
Molecule in High-Density Lipoprotein Cholesteryl Ester
Metabolism in Mice
Roshni R. Singaraja, Bjorn Stahmer, May Brundert, Martin Merkel, Joerg Heeren, Nagat Bissada,
Martin Kang, Jenelle M. Timmins, Rajasekhar Ramakrishnan, John S. Parks,
Michael R. Hayden, Franz Rinninger
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Objective—Mutations in ATP-binding cassette transporter A1 (ABCA1), the cellular lipid transport molecule mutated in
Tangier disease, result in the rapid turnover of high-density lipoprotein (HDL)–associated apolipoproteins that
presumably are cleared by the kidneys. However, the role of ABCA1 in the liver for HDL apolipoprotein and cholesteryl
ester (CE) catabolism in vivo is unknown.
Methods and Results—Murine HDL was radiolabeled with 125I in its apolipoprotein and with [3H]cholesteryl oleyl ether
in its CE moiety. HDL protein and lipid metabolism in plasma and HDL uptake by tissues were investigated in
ABCA1-overexpressing bacterial artificial chromosome (BAC)–transgenic (BAC⫹) mice and in mice harboring
deletions of total (ABCA1⫺/⫺) and liver-specific ABCA1 (ABCA1⫺L/⫺L). In BAC⫹ mice with elevated ABCA1
expression, fractional catabolic rates (FCRs) of both the protein and the lipid tracer were significantly decreased in
plasma and in the liver, yielding a diminished hepatic selective CE uptake from HDL. In contrast, ABCA1⫺/⫺ or
ABCA1⫺L/⫺L mice had significantly increased plasma and liver FCRs for both HDL tracers. An ABCA1 deficiency was
associated with increased selective HDL CE uptake by the liver under all experimental conditions.
Conclusions—Hepatic ABCA1 has a critical role for HDL catabolism in plasma and for HDL selective CE uptake by the
liver. (Arterioscler Thromb Vasc Biol. 2006;26:1821-1827.)
Key Words: ABCA1 䡲 HDL 䡲 selective uptake 䡲 cholesteryl ester
A
TP-binding cassette transporter A1 (ABCA1), a cellular
lipid transport molecule mutated in Tangier disease
(TD) patients, is essential for the maintenance of plasma
high-density lipoprotein (HDL) cholesterol levels.1 Studies
on the role of ABCA1 in the generation of HDL particles
indicate that the ABCA1 pathway is a major source for HDL
biogenesis.2
ABCA1 is widely expressed in mammalian tissues and is
most abundant in the liver, adrenal, and testis of mice.2,3 The
contribution of ABCA1 in specific tissues to HDL formation
has recently been a focus of investigation. Overexpression of
adenovirally delivered ABCA1 in the liver of wild-type (WT)
mice yields a significant increase in plasma HDL cholesterol
(HDL-C).4 In contrast, mice with ABCA1 selectively deleted
in the liver show an 80% decrease in plasma HDL-C.5 These
investigations suggest that the liver is the major site of
ABCA1-mediated lipidation of lipid-free apolipoprotein A-I
(apoA-I), the dominant protein component of HDL. Presumably, this process yields mature HDL particles.
Whether ABCA1 plays a role in HDL catabolism has not
been comprehensively investigated. ABCA1 may facilitate
the lipidation and conversion of small HDL particles into
large spherical HDL, thus slowing down the catabolism of
plasma HDL, prolonging its residence time in the circulation,
and contributing to the maintenance or raising of plasma
HDL.5 In patients with mutations in the ABCA1 gene,
intravenously injected apoA-I is rapidly cleared, suggesting a
role for ABCA1 in the catabolism of HDL-associated apolipoproteins.6,7 Such a rapid clearance of HDL apoA-I has also
been observed in the Wisconsin hypoalpha mutant chicken, a
naturally occurring model with mutations in the ABCA1
gene.8 Besides, a recent study showed that the catabolism of
HDL apoA-I was higher in liver-specific ABCA1 knockout
(ABCA1⫺L/⫺L) mice compared with controls.5 This apoA-I
hypercatabolism presumably is mediated by the kidneys,
whereas no major change in liver catabolism of apoA-I was
observed. However, in these studies, only the apolipoprotein
component of HDL was radiolabeled.5– 8 Therefore no information is available about the fate of cholesterol associated
with HDL. It is established that HDL protein and cholesteryl
ester (CE) moieties are metabolized at different rates in vivo.9
Original received December 8, 2005; final version accepted April 27, 2006.
From the University of British Columbia (R.R.S., N.B., M.K., M.R.H.), Vancouver, Canada; University Hospital Eppendorf (B.S., M.B., M.M., J.H.,
F.R.), Hamburg, Germany; Wake Forest University (J.M.T., J.S.P.), Winston-Salem, NC; and Columbia University (R.R.), New York, NY.
Correspondence to Franz Rinninger, University Hospital Eppendorf, Department of Medicine, Martinistrasse 52, 20246 Hamburg, Germany. E-mail
[email protected]
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1821
DOI: 10.1161/01.ATV.0000229219.13757.a2
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Arterioscler Thromb Vasc Biol.
August 2006
Plasma T-C and HDL-C in the Experimental Mouse Lines
T-C (mg/dL)
HDL-C (mg/dL)
WT
61.2⫾4.1
49.4⫾20.9
WT⫹LXR agonist
83.7⫾13.6
52.8⫾9.1
85.3⫾2.1
76.5⫾23.8
141.9⫾22.1
95.6⫾10.6
⫹
BAC
BAC⫹⫹LXR agonist
ABCA1⫺/⫺
10.7⫾5.2
1.2⫾0.5
BAC⫹ABCA1⫺/⫺
61.5⫾25.0
53.6⫾22.7
ABCA1⫺L/⫺L
11.4⫾3.0
6.5⫾2.4
ABCA1⫺/⫺
13.6⫾2.6
4.0⫾0.3
n.d.
4.4⫾0.4
ABCA1⫺/⫺⫹LXR agonist
n.d. indicates not determined.
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In addition, although catabolism by liver and kidneys were
addressed in some studies,5 the role of ABCA1 in HDL
metabolism of other organs has not been investigated.
In this study, the function of ABCA1 in plasma metabolism
of 2 major components of the HDL particle (ie, apolipoproteins and CE) was addressed. Besides, the role of ABCA1 in
HDL uptake by tissues and in particular by the liver was
investigated. It is shown that ABCA1 is essential for the
catabolism of HDL-associated apolipoproteins and lipids and
for the selective uptake of HDL-associated CE, in particular
by the liver.
Methods
For Methods, please see the online supplement, available at
http://atvb.ahajournals.org.
Results
Supplemental Figures I, II, III, and IV are available online at
http://atvb.ahajournals.org
Plasma Cholesterol in Mice With Overexpression
of ABCA1
To investigate the role of ABCA1 in HDL metabolism,
ABCA1– bacterial artificial chromosome (BAC)-transgenic
(BAC⫹) mice were used that express ABCA1 in the liver and
in other tissues.10,11 BAC⫹ animals showed an increase in
plasma total cholesterol (T-C; P⫽0.002; n⫽6) and HDL-C
compared with WT littermate controls (Table ).
Because untreated BAC⫹ mice had quantitatively only a
mild increase in ABCA1 protein expression (supplemental
Figure I) and in plasma HDL-C (Table), these animals were
fed the liver X receptor (LXR) agonist T0901317 to increase
ABCA1 and HDL-C.11 Feeding this compound to BAC⫹ mice
further increased ABCA1 protein expression (supplemental
Figure I). The LXR agonist yielded an additional and significant increase in plasma T-C of 69% (P⬍0.0001; n⫽7) and in
HDL-C of 81% (P⬍0.0001; n⫽7) compared with WT controls (Table).
Because LXR agonists regulate many genes in lipid metabolism,12 ABCA1⫺/⫺ mice were fed T0901317 to determine
whether the increase in HDL-C observed in the presence of
the compound was mediated through ABCA1. Indeed, no
change in plasma HDL-C was observed in treated ABCA1⫺/⫺
mice (Table). This result indicates that ABCA1 is responsible
for HDL-C elevation in response to LXR agonist treatment.
HDL Catabolism in Plasma in Mice With
Overexpression of ABCA1
After injection of 125I-TC-/[3H]CEt-HDL, the catabolism of
this preparation was investigated in WT, BAC⫹, and BAC⫹
mice fed the LXR agonist.13 The plasma fractional catabolic
rates (FCRs) for both HDL tracers were decreased in BAC⫹
mice compared with WT (Figure 1A). This decrease was not
statistically significant, presumably because of the low levels
of ABCA1 transgene expression in BAC⫹ mice. However, a
significant decrease in plasma FCRs was observed in the
LXR agonist-treated BAC⫹ mice (125I: WT, 68.9⫾5.7 versus
LXR agonist–fed BAC⫹, 51.2⫾5.2 pools⫻103 ⫻ h⫺1, n⫽7,
P⫽0.0001; [3H]CEt: WT, 157.1⫾13.5 versus LXR agonist–
fed BAC⫹, 118.2⫾11.9 pools⫻103 ⫻ h⫺1, n⫽7, P⫽0.0001;
Figure 1A). If the difference in plasma FCRs is calculated
Figure 1. Plasma decay and uptake of 125ITC-/[3H]CEt-HDL by tissues in WT and
BAC⫹ mice. 125I-TC-/[3H]CEt-HDL was
injected into WT, BAC⫹, or BAC⫹ mice fed
the LXR agonist. Blood was harvested
periodically for 24 hours, and tissues were
finally collected. Plasma and tissue samples were analyzed for both HDL tracers.
Plasma (A), liver (B), adrenal (C), and kidney (D) FCRs for 125I-TC (125I) and [3H]CEt
(3H) and selective HDL CE uptake (3H-125I)
were calculated. Values are means⫾SD.
n⫽6 mice per group.
Singaraja et al
ABCA1 and HDL Cholesteryl Ester Metabolism in Mice
([3H]CEt-125I-TC), then selective CE uptake from plasma
HDL by all tissues of a mouse is obtained.9 This selective CE
removal decreased in BAC⫹ mice and was further reduced in
the LXR agonist–fed BAC⫹ animals compared with WT
(WT, 88.3⫾14.7 and LXR agonist–fed BAC⫹, 67.0⫾13.0;
n⫽7; P⫽0.007). Thus, increasing ABCA1 expression decreased the selective CE removal from HDL and delayed
plasma HDL turnover.
HDL Uptake by Tissues in Mice With
Overexpression of ABCA1
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Tissue uptake of 125I-TC-/[3H]CEt-HDL was explored 24
hours after injection in mice.9,13 Liver, adrenals, and kidneys
showed the major changes in HDL tracer internalization in
these experiments (Figure 1). Remarkably, these tissues have
high levels of ABCA1 expression.2
The liver is the principal site of HDL catabolism in rodents,
and this is confirmed here (Figure 1B).9 In this organ, the
uptake of both HDL-associated tracers decreased in BAC⫹
mice (125I: WT, 28.8⫾4.0 versus BAC⫹, 25.9⫾1.5 pools⫻103
⫻ h⫺1, n⫽5, P⫽NS; [3H]CEt: WT, 114.9⫾21.2 versus
BAC⫹, 100.5⫾11.4 pools⫻103 ⫻ h⫺1, n⫽6, P⫽NS). This
decrease in hepatic HDL tracer uptake was significantly
different in LXR agonist–treated BAC⫹ mice (125I: WT,
28.8⫾4.0 versus LXR agonist–fed BAC⫹, 20.9⫾1.9
pools⫻10 3 ⫻ h ⫺1 , n⫽6, P⫽0.00005; [ 3 H]CEt: WT,
114.9⫾21.2 versus LXR agonist–fed BAC⫹, 78.3⫾14.0
pools⫻103 ⫻ h⫺1, n⫽6, P⫽0.0009). Hepatic HDL selective
CE uptake (ie, the difference between [3H]CEt and 125I-TC)
decreased in BAC ⫹ mice (WT, 86.1⫾21.5; BAC ⫹ ,
74.5⫾11.5; P⫽NS) and was significantly lower in LXR
agonist–treated BAC⫹ animals (WT, 86.1⫾21.5; LXR agonist–fed BAC⫹, 57.4⫾14.2; P⫽0.01; Figure 1B). As expected, quantitatively, the highest rates of HDL tracer uptake
from all tissues investigated were observed in the liver.
In adrenals, a trend toward a decrease in FCR for [3H]CEt
in BAC⫹ mice (Figure 1C) was observed. However, neither
the adrenal FCRs for [3H]CEt or 125I-TC nor HDL CE
selective uptake were significantly different in BAC⫹ mice
compared with WT, even when BAC⫹ mice were fed the
LXR agonist.
In the kidneys, ABCA1 overexpression decreased the
uptake of both tracers (Figure 1D). The most dramatic fall in
renal 125I-TC organ-FCR was detected in the LXR agonist–
treated BAC⫹ mice. The renal uptake rates for 125I-TC were
substantially higher than those for [3H]CEt, and this observation is consistent with a role of the kidneys in HDL apolipoprotein catabolism.14
HDL uptake by brain, heart, lungs, spleen, stomach,
intestine, and carcass was also explored (data not shown).
Organ FCRs for both HDL tracers of these tissues were not
significantly changed in BAC⫹ and in BAC⫹ mice fed the
LXR agonist compared with WT littermates.
HDL Catabolism in Plasma in Mice With Total
Deficiency of ABCA1
Because ABCA1 overexpression decreased HDL catabolism,
it was hypothesized in analogy to TD patients6 that in mice,
the HDL decay is increased in the absence of ABCA1. To
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address this question, mice with an induced deficiency of
ABCA1 (ABCA1⫺/⫺) were used.15 ABCA1⫺/⫺ mice harboring
the human ABCA1 BAC (BAC⫹ABCA1⫺/⫺) were included in
this study because any ABCA1-mediated differences in HDL
catabolism between ABCA1⫺/⫺ and WT mice should be
reversed when ABCA1 is re-expressed.11 Plasma T-C and
HDL-C of these animals are presented in the Table.
The complete absence of ABCA1 resulted in a large
increase in plasma FCRs of both HDL tracers when compared
with WT ( 125 I: WT, 81.5⫾11.6 versus ABCA1 ⫺/⫺ ,
355.0⫾71.0 pools⫻103 ⫻ h⫺1, n⫽6, P⫽0.0002; [3H]CEt:
WT, 177.8⫾74.9 versus ABCA1⫺/⫺, 1273.9⫾203.5 pools⫻
103 ⫻ h⫺1, n⫽6, P⫽0.00001; Figure 2A). Selective CE
removal from HDL was increased in ABCA1⫺/⫺ mice (WT,
96.3⫾36.7 versus ABCA1⫺/⫺; 918.9⫾215.6; n⫽6;
P⬍0.0001; Figure 2A). When rescued by BAC⫹ABCA1⫺/⫺,
the plasma FCRs for both HDL tracers were significantly
decreased to near those of WT mice (125I: ABCA1⫺/⫺,
355.0⫾71.0 versus BAC⫹ABCA1⫺/⫺, 140.3⫾93.1 pools⫻103
⫻ h⫺1, n⫽6, P⬍0.0001; [3H]CEt: ABCA1⫺/⫺, 1273.9⫾203.5
versus BAC⫹ABCA1⫺/⫺, 411.5⫾293.8 pools⫻103 ⫻ h⫺1,
n⫽6, P⬍0.0001), as were the rates of selective CE uptake
from HDL (ABCA1⫺/⫺, 918.9⫾215.6 versus BAC⫹ABCA1⫺/⫺,
278.2⫾308.2; n⫽6; P⫽0.0009). Thus, plasma HDL catabolism and HDL-selective CE uptake by all tissues of the mice
are increased in the absence of ABCA1, and this effect is
reversed on ABCA1 expression.
HDL Uptake by Tissues in Mice With Total
Deficiency of ABCA1
HDL uptake by the liver of ABCA1⫺/⫺ mice revealed that the
hepatic FCRs for both HDL tracers increased significantly in
these animals compared with WT (125I: WT, 28.8⫾4.0 versus
ABCA1⫺/⫺, 178.7⫾42.7 pools⫻103 ⫻ h⫺1, n⫽6, P⬍0.0001;
[3H]CEt: WT, 114.9⫾21.2 versus ABCA1⫺/⫺, 957.5⫾167.1
pools⫻103 ⫻ h⫺1, n⫽6, P⬍0.0001; Figure 2B). This stimulation yielded a substantial rise in selective CE uptake in
ABCA1⫺/⫺ mice (WT, 86.1⫾21.5, n⫽6, versus ABCA1⫺/⫺,
778.8⫾172.5, n⫽6; P⬍0.0001). This increase in HDLselective CE uptake by the liver of ABCA1⫺/⫺ mice was
reduced in the BAC⫹ABCA1⫺/⫺ animals (ABCA1⫺/⫺,
778.8⫾172.5 versus BAC⫹ABCA1⫺/⫺, 260.5⫾198.2; n⫽6;
P⫽0.0003).
In adrenals from ABCA1⫺/⫺ mice, the FCRs for both HDL
tracers increased significantly (P⬍0.0001), and this was in
particular true for [3H]CEt (Figure 2C). In ABCA1⫺/⫺ adrenals, selective HDL CE uptake increased significantly
(P⬍0.0001) but was decreased to the WT range in this
steroidogenic gland from BAC⫹ABCA1⫺/⫺ mice.
In kidneys, an ABCA1 deficiency was associated with a
significant (P⬍0.0001) increase in the FCR for 125I-TC
(Figure 2D). However, this uptake was normalized close to
WT in BAC⫹ABCA1⫺/⫺ mice. The renal organ FCRs for
[3H]CEt were quantitatively similar in all experimental
groups. Thus, in the kidneys, the increase in HDL apolipoprotein uptake was a prominent change in ABCA1⫺/⫺
mice. However, comparing quantitatively HDL tracer uptake between liver and kidneys, the liver was the dominant
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Arterioscler Thromb Vasc Biol.
August 2006
Figure 2. Plasma decay and uptake of
125
I-TC-/[3H]CEt-HDL by tissues in WT,
BAC⫹, ABCA1⫺/⫺, and BAC⫹ABCA1⫺/⫺
mice. 125I-TC-/[3H]CEt-HDL was injected
in WT, BAC⫹, ABCA1⫺/⫺, and
BAC⫹ABCA1⫺/⫺ mice. Blood was harvested periodically for 24 hours, and tissues were finally collected. Plasma and
tissue samples were analyzed for both
HDL tracers. Plasma (A), liver (B), adrenal
(C), and kidney (D) FCRs for 125I-TC (125I)
and [3H]CEt (3H) and selective HDL CE
uptake (3H-125I) were calculated. Values
are means⫾SD. n⫽6 mice per group.
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organ site for HDL catabolism under any experimental
conditions (Figure 2).
In ABCA1⫺/⫺ mice, the ABCA1 deficiency had no impact
on HDL catabolism by brain, heart, lungs, spleen, stomach,
intestine, and carcass (data not shown).
HDL Catabolism in Plasma in Mice With
Liver-Specific Deficiency of ABCA1
To address the role of hepatic ABCA1 in HDL metabolism in more
detail, liver-specific ABCA1 knockout (ABCA1⫺L/⫺L) mice were
used.5 These animals show a substantial (P⫽0.0002) decrease in
plasma HDL-C compared with WT (Table).
ABCA1⫺L/⫺L mice had significantly increased plasma
FCRs for both HDL tracers (125I: WT, 73.9⫾14.0 versus
ABCA1⫺L/⫺L, 125.7⫾55.9 pools⫻103 ⫻ h⫺1, n⫽6, P⫽0.03;
[3H]CEt: WT, 125.6⫾33.2 versus ABCA1⫺L/⫺L, 339.7⫾145.4
pools⫻103 ⫻ h⫺1, n⫽6, P⫽0.003; Figure 3A). Besides,
selective CE removal from HDL by tissues increased in
ABCA1⫺L/⫺L rodents (WT, 51.7⫾36.0 versus ABCA1⫺L/⫺L,
214.0⫾155.9; n⫽6; P⫽0.02).
Figure 3. Plasma decay and uptake of
125
I-TC-/[3H]CEt-HDL by tissues in WT
and ABCA1⫺L/⫺L mice. 125I-TC-/[3H]CEtHDL was injected in WT and ABCA⫺L/⫺L
mice. Blood was harvested periodically
for 24 hours, and tissues were finally collected. Plasma and tissue samples were
analyzed for both HDL tracers. Plasma
(A), liver (B), adrenal (C), and kidney (D)
FCRs for 125I-TC (125I) and [3H]CEt (3H)
and selective HDL CE (3H-125I) uptake
were calculated. Values are means⫾SD.
n⫽6 mice per group.
Singaraja et al
ABCA1 and HDL Cholesteryl Ester Metabolism in Mice
HDL Uptake by Tissues in Mice With
Liver-Specific Deficiency of ABCA1
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The hepatic organ FCRs for both HDL tracers increased
significantly in ABCA1⫺L/⫺L mice compared with WT controls (125I: WT, 21.0⫾4.7 versus ABCA1⫺L/⫺L, 46.4⫾24.2
pools⫻103 ⫻ h⫺1, n⫽6, P⫽0.02; [3H]CEt: WT, 77.6⫾19.7
versus ABCA1⫺L/⫺L, 232.9⫾119.1 pools⫻103 ⫻ h⫺1, n⫽6,
P⫽0.006; Figure 3B). Selective CE uptake was also higher in
the liver of ABCA1⫺L/⫺L mice (WT, 56.6⫾20.2 versus
ABCA1⫺L/⫺L, 186.5⫾121.6; n⫽6; P⫽0.02).
In ABCA1⫺L/⫺L mice, the adrenal FCRs for 125I-TC-/
[3H]CEt-HDL were substantially higher compared with WT
(125I: WT, 0.11⫾0.04 versus ABCA1⫺L/⫺L, 0.42⫾0.22
pools⫻103 ⫻ h⫺1, n⫽6, P⫽0.003; [3H]CEt: WT, 0.74⫾0.26
versus ABCA1⫺L/⫺L, 7.2⫾3.7 pools⫻103 ⫻ h⫺1, n⫽6,
P⫽0.0003; Figure 3C). In addition, selective HDL CE uptake
increased in adrenals from ABCA1 ⫺L/⫺L mice (WT,
0.63⫾0.26 versus ABCA1 ⫺L/⫺L , 6.76⫾3.28; n⫽6;
P⫽0.0005).
In kidneys from ABCA1⫺L/⫺L animals, the FCRs for
HDL-associated 125I-TC increased substantially, whereas the
rate for [3H]CEt was quantitatively elevated to a smaller
extent (125I: WT, 5.27⫾1.21 versus ABCA1⫺L/⫺L, 15.74⫾7.87
pools⫻103 ⫻ h⫺1, n⫽6, P⫽0.005; [3H]CEt: WT, 0.64⫾0.16
versus ABCA1⫺L/⫺L, 1.39⫾0.31 pools⫻103 ⫻ h⫺1, n⫽6,
P⫽0.0002; Figure 3D).
The liver-specific ABCA1 deficiency had no substantial
effect on HDL tracer uptake by brain, heart, lungs, spleen,
stomach, intestine, or carcass (data not shown).
HDL Catabolism in Mice With
Adenovirus-Mediated ABCA1 Expression in
the Liver
To address the role of hepatic ABCA1 for HDL metabolism,
ABCA1 was delivered by adenovirus (Ad-ABCA1) to
ABCA1⫺/⫺ (supplemental Figure II) and to ABCA1⫺L/⫺L
(supplemental Figure III) mice.4 After Ad-ABCA1 injection,
plasma T-C and HDL-C of ABCA1⫺/⫺ and ABCA1⫺L/⫺L mice
significantly increased (ABCA1⫺/⫺: T-C⫽9.3⫾4.8, HDLC⫽3.9⫾1.3 and Ad-ABCA1–treated ABCA1⫺/⫺: T-C⫽39.8⫾
11.6, HDL-C⫽19.2⫾4.3, mg/dL, n⫽6; (ABCA1⫺L/⫺L:
T-C⫽10.3⫾4.8, HDL-C⫽6.5⫾2.4 and Ad-ABCA1–treated
ABCA1⫺L/⫺L: T-C⫽51.7⫾5.3, HDL-C⫽38.3⫾1.3 mg/dL,
n⫽6). In contrast, no change in plasma cholesterol was
observed in mice injected with the control adenovirus.
125
I-TC-/[3H]CEt-HDL metabolism was investigated in
ABCA1⫺/⫺ and ABCA1⫺L/⫺L mice injected with the control
virus or with Ad-ABCA1 (supplemental Figures II and III).
For comparison, WT mice were included in these studies. In
both groups of ABCA1-deficient mice, ABCA1 expression
decreased the plasma FCRs for 125I-TC and [3H]CEt significantly (P⬍0.001). Similarly, selective CE removal from HDL
([3H]CEt-125I-TC) by all tissues decreased significantly
(P⬍0.05) because of Ad-ABCA1 injection.
125
I-TC-/[3H]CEt-HDL uptake by the liver was investigated
in ABCA1⫺/⫺ and ABCA1⫺L/⫺L mice injected with AdABCA1 or with the control virus (supplemental Figures IIB
and IIIB). In both groups, ABCA1 expression decreased the
organ FCRs for 125I-TC and [3H]CEt significantly (P⬍0.05).
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Similarly, selective CE removal from plasma HDL ([3H]CEtI-TC) by the liver decreased significantly (P⬍0.05) because of Ad-ABCA1 injection.
In adrenals and kidneys of ABCA1⫺/⫺ and ABCA1⫺L/⫺L
mice, Ad-ABCA1 injection decreased the organ FCRs for
125
I-TC and for [3H]CEt close to those of WT (supplemental
Figures II and III).
125
Hepatic Scavenger Receptor Class B Type I in
Mice With Modified ABCA1 Expression
Hepatic HDL selective CE uptake is decreased in BAC⫹ and
increased in ABCA1⫺/⫺ and in ABCA1⫺L/⫺L mice. Scavenger
receptor class B type I (SR-BI) is an HDL receptor that
mediates selective lipid uptake.13,16 Therefore, SR-BI expression in liver lysates isolated from WT, BAC⫹, ABCA1⫺/⫺,
and ABCA1⫺L/⫺L mice was assessed by immunoblotting. In
BAC⫹ mice treated with the LXR agonist, hepatic SR-BI
expression decreased compared with WT (supplemental Figure I). In contrast, in ABCA1⫺/⫺ (supplemental Figure IVA)
and in ABCA1⫺L/⫺L mice (supplemental Figure IVB), no
increase in SR-BI expression was observed compared with
WT liver. Analogously in BAC⫹ABCA1⫺/⫺ mice, there was
no change in hepatic SR-BI (supplemental Figure IVA).
Discussion
This study reinforces that ABCA1 has a dominant effect on
plasma HDL-C levels. A deficiency of ABCA1 is associated
with low and a high expression of this protein with increased
HDL-C. The low HDL-C in liver-specific ABCA1-deficient
mice and the normalized HDL-C after the adenovirusmediated transfer of ABCA1 to the liver point to a major role
of hepatic ABCA1 as primary molecule that determines
HDL-C in vivo.
ABCA1-overexpressing BAC (BAC⫹) mice, ABCA1
knockout (ABCA1⫺/⫺), and liver-specific ABCA1 knockout
(ABCA1⫺L/⫺L) rodents were the models of this study.5,10,15
Besides, in ABCA1-deficient mice, this protein was expressed via transgene or adenovirus.4,11 BAC⫹ mice have an
increase in HDL-C, and this is attributable to a rise in
lipoprotein particle number.10 The composition of HDL in
BAC⫹ and WT mice is virtually the same.10 ABCA1⫺/⫺ mice
have essentially no HDL in plasma.15 ABCA1⫺L/⫺L mice have
⬇20% of normal HDL-C levels, and this decrease is attributable to a reduction in HDL particle number.5 The size and
composition of HDL from ABCA1⫺L/⫺L is very similar to the
one from WT animals.5 Based on these characteristics of the
endogenous HDL in the experimental animals, radiolabeled
murine WT-HDL was used as tracer. This preparation is an
appropriate tracer for the endogenous HDL pool, and this is
true at least in BAC⫹ and ABCA1⫺L/⫺L mice. This HDL
fraction was labeled in its protein and lipid moieties, and thus,
the metabolic fate of both HDL components could be independently explored.9
A key result of this study is that ABCA1 expression in
mice delayed the catabolism of both HDL tracers in plasma,
and this yielded a reduced selective CE uptake from plasma
HDL by tissues. Strikingly, ABCA1 expression decreased the
HDL tracer uptake by the liver, and this yielded a reduced
selective CE uptake. The decrease in HDL apolipoprotein
1826
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August 2006
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catabolism that was observed here is in line with another
study using ABCA1-transgenic mice.17 With respect to
whole-body HDL catabolism, quantitatively, the highest
HDL tracer uptake rates were detected in the liver, and this
was true for BAC⫹ and for WT mice. These results show that
the liver is the dominant site for HDL catabolism in mice with
physiological and induced ABCA1 overexpression.
These BAC⫹ mice with transgenic ABCA1 expression
display an increase in plasma HDL-C. This rise and the
decreased HDL catabolism by issues suggests that the
ABCA1-mediated delay in HDL catabolism is a mechanism
that contributes to the increased plasma HDL-C. However,
ABCA1 is a key molecule in the formation of HDL particles
as well.15 Therefore, it seems possible that ABCA1 has a dual
effect on both HDL catabolism and synthesis. Both mechanisms presumably contribute to the ABCA1-dependent increase in plasma HDL-C.
HDL catabolism in mice with total or liver-specific
ABCA1 deficiency was accelerated. The stimulated decay of
both HDL tracers yielded an increase in selective CE uptake
from plasma HDL by tissues. With respect to the increased
decay of 125I-labeled apolipoproteins, these experiments agree
with previous studies in ABCA1⫺L/⫺L mice5 and in TD
patients.6 The selective CE clearance from plasma HDL was
increased in ABCA1⫺/⫺ and ABCA1⫺L/⫺L mice. Similarly,
HDL holo-particle catabolism, as represented by 125I-TC, is
stimulated in both models. With respect to specific tissues,
liver, adrenal, and kidney uptake of HDL tracers were
significantly increased in both ABCA1-deficient animal models, and this resulted in upregulated rates of hepatic and
adrenal-selective CE uptake. With respect to whole-body
HDL catabolism, again, the HDL uptake rates were highest in
the liver. These results provide evidence that ABCA1 in the
liver plays an essential role in whole-body HDL metabolism
by delaying HDL turnover and by diminishing selective CE
uptake from HDL. For ABCA1⫺L/⫺L mice, it has been
suggested that the increased catabolism of HDL particles and
of HDL apolipoproteins occurs in the kidneys and in the
liver.5 This conclusion is confirmed in this study. Besides the
previously described increase in HDL protein uptake in the
liver of ABCA1⫺L/⫺L mice, it is demonstrated here that the
ABCA1-deficient murine liver displays a substantial increase
in selective HDL CE uptake as well.5
ABCA1-deficient mice have very low HDL-C in plasma.5,15 Previously, it was suggested that this finding is
attributable to a decreased HDL synthesis.15 However, in this
study, HDL catabolism is accelerated in ABCA1-deficient
mice. Therefore, the mechanism(s) underlying the low
plasma HDL presumably is both attributable to a decrease in
HDL synthesis and an increase in HDL catabolism.
Mice with a total ABCA1 deficiency yielded qualitatively
identical results as animals with a liver-specific lack of this
protein. This is true for both HDL plasma FCRs and organ
FCRs. Besides, adenovirus-mediated hepatic ABCA1 expression in ABCA1⫺/⫺ and in ABCA1⫺L/⫺L mice or transgeneinduced ABCA1 expression reversed the changes in HDL
metabolism induced by a deficiency of this protein. Also,
these results provide evidence that ABCA1 in the liver plays
a dominant role in HDL homeostasis in vivo.
The question has to be addressed whether the increase in
HDL catabolism in the absence of ABCA1 is caused by the
decreased HDL plasma pool in ABCA1⫺/⫺ and ABCA1⫺L/⫺L
mice. Patients with TD have an almost complete lack of HDL
in plasma, and radiolabeled HDL is rapidly cleared from the
circulation.6,7 However, this rapid HDL catabolism was
independent of HDL pool size because it did not change
despite the acute infusion of HDL to bring plasma HDL
concentrations back to near normal.7 In TD patients, only the
apolipoprotein moiety of HDL was labeled.7 Therefore, the
fate of HDL CE in the face of a diminished HDL pool was
unknown. However, another study injected HDL radiolabeled
in its unesterified and esterified cholesterol moieties into
apoA-I– deficient (apoA-I⫺/⫺) mice with low plasma HDL-C
levels and found that compared with WT, there was no
difference in the turnover of HDL lipids in apoA-I⫺/⫺ mice.18
These observations suggest that the HDL pool size does not
affect HDL turnover rates. In summary, it is unlikely that an
altered HDL pool size of ABCA1-deficient mice affected the
result of this study.
A mechanism by which ABCA1 presumably can alter
HDL catabolism in the liver is through the HDL receptor
SR-BI.16 Overexpression of SR-BI decreases plasma HDL-C,
accelerates HDL catabolism, and increases selective CE
uptake by the liver.19 In contrast, an induced deficiency of
SR-BI increases plasma HDL-C, delays HDL catabolism, and
decreases hepatic selective CE uptake.13 In BAC⫹ mice,
hepatic SR-BI expression was reduced, and this observation
is consistent with the diminished selective HDL CE uptake by
the liver in metabolic studies. However, in ABCA1⫺/⫺ and in
ABCA1⫺L/⫺L mice, the hepatic expression of SR-BI was
similar as in WT controls, although selective HDL CE uptake
by the liver was substantially upregulated in vivo. This lack
of regulation of SR-BI is consistent with a previous report.20
One possibility that might explain these discrepancies is an
SR-BI–independent mechanism for HDL lipid uptake; such a
pathway could mediate hepatic HDL CE uptake at least in
part.13 Besides, a novel HDL receptor has been defined that
may play a role in HDL degradation as well.21
In summary, ABCA1 has a substantial impact on HDL
metabolism in plasma and by tissues in mice. ABCA1
overexpression retards the catabolism of HDL in plasma and
decreases selective HDL CE uptake by the liver. In contrast,
an ABCA1 deficiency accelerates HDL catabolism in plasma
and increases HDL-selective CE uptake by the liver. All
experiments are in line with a key function of ABCA1 in the
liver for HDL metabolism in vivo. Besides, the liver also
quantitatively has a dominant function in HDL catabolism in
mice with a deficiency or a high level of ABCA1 expression.
Acknowledgments
For Acknowledgments, please see the online supplement.
Disclosures
None.
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Hepatic ATP-Binding Cassette Transporter A1 Is a Key Molecule in High-Density
Lipoprotein Cholesteryl Ester Metabolism in Mice
Roshni R. Singaraja, Bjorn Stahmer, May Brundert, Martin Merkel, Joerg Heeren, Nagat
Bissada, Martin Kang, Jenelle M. Timmins, Rajasekhar Ramakrishnan, John S. Parks, Michael
R. Hayden and Franz Rinninger
Arterioscler Thromb Vasc Biol. 2006;26:1821-1827; originally published online May 25, 2006;
doi: 10.1161/01.ATV.0000229219.13757.a2
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Copyright © 2006 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
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Online supplement
Hepatic ABCA1 is a key molecule in HDL cholesteryl ester metabolism in mice
Roshni R. Singaraja, Bjorn Stahmer, May Brundert, Martin Merkel, Joerg Heeren, Nagat
Bissada, Martin Kang, Jenelle M. Timmins, Rajasekhar Ramakrishnan, John S. Parks,
Michael R. Hayden and Franz Rinninger.
Methods
Mice
Mice transgenic for the human ABCA1 gene (BAC+) were described (1,2) and
were pure C57BL6/J. ABCA1 knockout (ABCA1-/-) (3) and liver-specific ABCA1
knockout (ABCA1-L/-L) (4) mice were obtained from Dr. Omar L. Francone (Pfizer
Global Research) and Dr. John S. Parks (Wake Forest University) respectively, and were
approximately 80% C57BL6/J. Littermate controls of the same sex and age were used in
all studies. In some cases, mice were fed orally the LXR agonist T-0901317 (Tularik®,
Sigma) or the solvent for 4 days at a dose of 10 mg/kg/day (2). All mice were maintained
on chow diet.
For adenovirus-mediated expression of ABCA1 in the liver, 5x108 pfu/mouse of
control adenovirus were injected through the tail vein in order to saturate the uptake of
viral particles by Kupffer cells (5,6). Three hours later, 5x108 pfu/mouse of adenovirus
harboring human ABCA1 (Ad-ABCA1) or the respective control virus were tail vein
injected. Three days later, HDL metabolism was investigated in these rodents. All animal
protocols were approved by the institutional animal care committees.
Isolation and radiolabeling of HDL
HDL (d = 1.063-1.21 g/ml) was isolated from WT mice (C57BL6/J) by
ultracentrifugation and labeled with 125I-Tyramine Cellobiose (125I-TC) in its protein and
1
[3H]Cholesteryl Oleyl Ether ([3H]CEt) (Amersham) in its lipid moiety (7,8). Both tracers
are intracellularly trapped after uptake and are not released from tissues (8).
HDL metabolism in mice
Plasma decay rates for 125I-TC-/[3H]CEt-HDL. Mice were fasted for 4 hours and
injected with 125I-TC-/[3H]CEt-HDL via the tail vein (9,10). Plasma decay data were
obtained by drawing periodic blood samples (30 µl per time point) at 0.16, 0.5, 2, 6, 20
and 24 hours after injection. Animals were fasted throughout the 24 hour study period but
had unlimited access to water. Aliquots of plasma were directly assayed for 125I
radioactivity and [3H] was analyzed after lipid extraction (11). Computer analysis was
used to fit a least-squares multiexponential curve to each set of plasma decay data and to
calculate plasma-fractional catabolic rates (plasma-FCR) (7,12).
Tissue analysis of 125I-TC-/[3H]CEt-HDL uptake. Tissue sites of uptake of HDLassociated 125I-TC and [3H]CEt were determined 24 hours after injection of 125I-TC/[3H]CEt-HDL when both tracers were predominantly cleared from plasma (9,10). Finally
the animals were anesthetized, the abdomen and chest were opened, and a catheter was
inserted into the heart. The inferior vena cava was cut, and the mice were perfused with
saline (50 ml per animal). After perfusion, liver, adrenals, kidneys, brain, heart, lungs,
spleen, stomach, intestine and carcass were harvested and homogenized. Homogenates
from each tissue and from carcass were directly assayed for 125I radioactivity and aliquots
were analyzed for [3H] after lipid extraction (11).
Total radioactivity recovered from all tissues and from carcass of each mouse was
calculated (9). The fraction of total tracer uptake attributed to a specific organ was
calculated as the radioactivity recovered in that organ divided by the total radioactivity
2
recovered from all tissues and carcass. Thus the % of recovered extravascular
radioactivity in tissues is determined 24 h after injection of labeled HDL.
To allow direct comparison of the specific activities of the various tissues in HDL
uptake and to directly compare the rates of uptake of the apolipoprotein component and
the CE moiety of HDL in tissues, the data are expressed as organ-fractional catabolic
rates (organ-FCR) (8,9). These rates refer to the whole organ.
The organ-FCR is calculated as follows:
(Organ-FCR in tissue X) = (Plasma-FCR) x (Fraction [%] of Total Body Tracer
Recovery in Tissue X)
These organ-FCR’s represent the fraction of the plasma pool of either HDL tracer cleared
by an organ per hour (8).
Selective HDL CE uptake is calculated as the difference in organ-FCR’s between
[3H]CEt and 125I-TC (8,9).
Immunoblots
Protein lysates were prepared from livers, separated by SDS-PAGE under nonreducing conditions, transferred to polyvinylidene fluoride (PVDF) membranes
(Millipore) and probed with anti-SR-BI (Novus Biochemicals), anti-ABCA1 (13) and
anti-GAPDH (Chemicon) antibodies. GAPDH was used as a loading control.
Densitometric scanning was performed using Quantity One software (BioRad).
Calculations and Statistics
All statistical analyses were performed using Students t-test. Values are means
±SD. ns = not significant at p=0.05.
3
Acknowledgements
FR is supported by Research Grant Ri 436/8-1 from Deutsche Forschungsgemeinschaft
(DFG), Bonn, Germany. MRH is supported by grants from the Canadian Institutes for
Health Research, and the Heart and Stroke Foundation of BC and the Yukon. He holds a
University Killam Professorship and a Canada Research Chair in Human Genetics. RRS
is supported by the Heart and Stroke Foundation of Canada and the Michael Smith
Foundation for Health Research. JSP is supported by NIH grant HL 49373 and JMT is
supported by a Cardiovascular Pathology Training grant (HL07115). Excellent technical
assistance by Mrs. B. Keller, Mrs. B. Schulz and Mrs. M. Thiel is appreciated. Dr.
Richard E. Morton and Mrs. Diane Greene donated CETP for radiolabeling.
4
References for methods/Online supplement
(1) Singaraja RR, Bocher V, James ER, Clee SM, Zhang LH, Leavitt BR, Tan B, BrooksWilson A, Kwok A, Bissada N, Yang Y, Liu G, Tafuri SR, Fievet C, Wellington CL,
Staels B, Hayden MR. Human ABCA1 BAC transgenic mice show increased hig
density lipoprotein cholesterol and apoA-I-dependent efflux stimulated by an internal
promoter containing liver X receptor response elements in intron 1. J Biol Chem.
2001;276:33969-33979.
(2) Coutinho JM, Singaraja RR, Kang M, Arenillas DJ, Bertram LN, Bissada N, Staels B,
Fruchart JC, Fievet C, Joseph-George AM, Wasserman WW, Hayden MR. Complete
functional rescue of the ABCA1-/- mouse by human BAC transgenesis. J Lipid Res.
2005;46:1113-1123.
(3) McNeish J, Aiello RJ, Guyot D, Turi T, Gabel C, Aldinger C, Hoppe KL, Roach ML,
Royer LJ, de Wet J, Broccardo C, Chimini G, Francone OL. High density lipoprotein
deficiency and foam cell accumulation in mice with targeted disruption of ATPbinding cassette transporter-1. Proc Natl Acad Sci U S A. 2000;97:4245-4250.
(4) Timmins JM, Lee JY, Boudyguina E, Kluckman KD, Brunham LR, Mulya A, Gebre
AK, Coutinho JM, Colvin PL, Smith TL, Hayden MR, Maeda N, Parks JS. Targeted
inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney
hypercatabolism of apoA-I. J Clin Invest. 2005;115:1333-1342.
(5) Wellington CL, Brunham LR, Zhou S, Singaraja RR, Visscher H, Gelfer A, Ross C,
James E, Liu G, Huber MT, Yang YZ, Parks RJ, Groen A, Fruchart-Najib J, Hayden
MR. Alterations of plasma lipids in mice via adenoviral-mediated hepatic
overexpression of human ABCA1. J Lipid Res. 2003;44:1470-1480.
(6) Tao N, Gao GP, Parr M, Johnston J, Baradet T, Wilson JM, Barsoum J, Fawell SE.
Sequestration of adenoviral vector by Kupffer cells leads to a nonlinear dose response
of transduction in liver. Mol Ther. 2001;3:28-35.
(7) Brundert M, Ewert A, Heeren J, Behrendt B, Ramakrishnan R, Greten H, Merkel M,
Rinninger F. Scavenger receptor class B type I mediates the selective uptake of highdensity lipoprotein-associated cholesteryl ester by the liver in mice. Arterioscler
Thromb Vasc Biol. 2005;25:143-148.
(8) Pittman RC, Taylor CA Jr. Methods for assessment of tissue sites of lipoprotein
degradation. Methods Enzym. 1986;129:612–628.
(9) Glass C, Pittman RC, Civen M, Steinberg D. Uptake of high-density lipoproteinassociated apoprotein A-I and cholesterol esters by 16 tissues of the rat in vivo and by
adrenal cells and hepatocytes in vitro. J Biol Chem. 1985;260:744–750.
5
(10) Rinninger F, Pittman RC. Regulation of the selective uptake of high density
lipoprotein-associated cholesteryl esters. J Lipid Res. 1987;28:1313–1325.
(11) Dole VP. A relation between non-esterified fatty acids in plasma and the metabolism
of glucose. J Clin Invest. 1956;35:150-154.
(12) Le NA, Ramakrishnan R, Dell RB, Ginsberg HN, Brown WV. Kinetic analysis using
specific radioactivity data. Methods Enzym. 1986;129:384–395.
(13) Wellington CL, Walker EK, Suarez A, Kwok A, Bissada N, Singaraja R, Yang YZ,
Zhang LH, James E, Wilson JE, Francone O, McManus BM, Hayden MR. ABCA1
mRNA and protein distribution patterns predict multiple different roles and levels of
regulation. Lab Invest. 2002;82:273-283.
6
Supplemental Figure legends (online)
Figure I. ABCA1 and SR-BI expression in the liver of WT, BAC+ and BAC+ mice
fed the LXR agonist
Protein lysates (40 µg per lane) from the respective murine livers were immunoblotted
for ABCA1, SR-BI and GAPDH. Three independent similar blots yielded qualitatively
identical results.
Figure II. Plasma decay and uptake of 125I-TC-/[3H]CEt-HDL by liver, adrenals and
kidneys in WT, ABCA1-/- and ABCA1-/- mice injected with Ad-ABCA1
WT and ABCA1-/- mice were injected with a control virus or Ad-ABCA1 as described in
Methods. 72 hours later, 125I-TC-/[3H]CEt-HDL was injected in these animals. Then
blood was harvested periodically for 24 hours and tissues were finally collected. Plasma
and tissue samples were analyzed for both HDL tracers. Plasma (A), liver (B), adrenal
(C), and kidney (D) FCR’s for 125I-TC (125I) and [3H]CEt (3H) and selective HDL CE
uptake (3H – 125I) were calculated. Values are means ± SD. n=6 mice per group.
Figure III. Plasma decay and uptake of 125I-TC-/[3H]CEt-HDL by liver, adrenals
and kidneys in WT, ABCA1-L/-L and ABCA1-L/-L mice injected with Ad-ABCA1
WT and ABCA1-L/-L mice were injected with a control virus or Ad-ABCA1 as outlined in
Methods. 72 hours later, 125I-TC-/[3H]CEt-HDL was administered intravenously to these
animals. Thereafter blood was harvested periodically for 24 hours and tissues collected.
Plasma and tissue samples were analyzed for both HDL tracers. Plasma (A), liver (B),
adrenal (C) and kidney (D) FCR’s for 125I-TC (125I) and [3H]CEt (3H) and selective HDL
CE (3H – 125I) uptake were calculated. Values are means ± SD. n=6 mice per group.
7
Figure IV. ABCA1 and SR-BI expression in the liver of WT, ABCA1-/-,
BAC+ABCA1-/- and ABCA1-L/-L mice
Protein lysates (40 µg per lane) from the respective murine liver were immunoblotted for
ABCA1, SR-BI and GAPDH. In each case, at least three independent similar blots
yielded qualitatively identical results.
8
Ratio ABCA1 : GAPDH
Figure I
ABCA1
0,78
0,74
0,70
0,66
0,62
SR-BI
WT
BAC+
BAC+
+LXR agonist
WT
BAC+
BAC+
+LXR agonist
GAPDH
WT
+
BAC
+
BAC
+LXR agonist
Ratio SR-BI : GAPDH
1,2
1,0
0,8
0,6
0,4
0,2
Figure II
A Plasma-FCR
Liver-FCR
B
ns
800
600
*** ***
ns
ABCA1-/-
** ***
ABCA1-/+ Ad-ABCA1
ns
500
*
400
*
300
200
WT
500
400
*
ns
300
ABCA1-/-
ns
*
0
*** ***
200
0
I
3
3
H
H-
125
125
I
C Adrenal-FCR
*
*** ***
5.0
ns
4.0
*
*
3.5
ns
2.5
*** ***
3
H
3
H- 125I
ns
4.5
3.0
I
D Kidney-FCR
2.0
1.5
WT
50
ABCA1-/-
40
ABCA1-/+ Ad-ABCA1
Pool x 103 x h-1
125
WT
*** ***
ABCA1-/-
30
20
ns
10
** ***
ABCA1-/+ Ad-ABCA1
0
-10
1.0
125
I
3
H
-20
0.5
-30
0
125
I
3
H
3
H- 125I
-40
*** ***
* p<0.05
ABCA1-/+ Ad-ABCA1
100
100
Pool x 103 x h-1
*** **
WT
Pool x 103 x h-1
Pool x 103 x h-1
700
ns
600
** p<0.001
*** p<0.0001
ns
3
H- 125I
Figure III
A Plasma-FCR
B
Liver-FCR
ns
WT
Pool x 103 x h-1
600
ABCA1-L/-L
ns
500
400
*
ns
600
* **
ns
*
*
ABCA1-L/-L
+ Ad-ABCA1
*
300
200
Pool x 103 x h-1
700
ns
400
0
I
3
H
3
H-
125
* *
200
125
I
C Adrenal-FCR
D
* **
14
3
H
H- 125I
ns
ns
*** **
30
WT
ABCA1-L/-L
12
ABCA1-L/-L
+ Ad-ABCA1
10
8
6
ns
Pool x 103 x h-1
Pool x 103 x h-1
16
3
I
Kidney-FCR
ns
*
*
WT
20
ABCA1-L/-L
ns
10
*
ABCA1-L/-L
+ Ad-ABCA1
*
0
-10
125
I
3
H
* **
-20
0
I
3
H
3
H- 125I
-30
* **
125
ns
* p<0.05
ABCA1-L/-L
+ Ad-ABCA1
0
125
2
* *
ns
300
ABCA1-L/-L
100
100
4
WT
* **
500
** p<0.001
*** p<0.0001
3
H- 125I
Figure IV
A
Ratio SR-BI : GAPDH
1,2
ABCA1
SR-BI
GAPDH
1,0
0,8
0,6
0,4
0,2
WT
ABCA1-/- BAC+
ABCA1-/-
WT
ABCA1-/-
WT
ABCA1-L/-L
B
ABCA1
Ratio SR-BI : GAPDH
1,2
SR-BI
GAPDH
1,0
0,8
0,6
0,4
0,2
WT
ABCA1-L/-L
BAC+
ABCA1-/-