From the Division of Molecular Medicine, Department
of Medicine, Columbia University, New York, New York 10032 and the
§ Department of Biochemistry, Weill Medical College, Cornell
University, New York, New York 10021
Received for publication, August 23, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Liver X receptor/retinoid X receptor
(LXR/RXR) transcription factors have been found to induce a number of
genes involved in the regulation of cellular cholesterol efflux,
including the ATP-binding cassette transporter A1 (ABCA1), which
mediates the active efflux of cellular cholesterol and phospholipids to
extracellular acceptors, such as apolipoprotein A-I (apoA-I). In a
screen for macrophage LXR/RXR target genes, we identified
stearoyl-CoA desaturases 1 and 2 (Scd1 and Scd2), and subsequently tested the
hypothesis that SCD activity might modulate cellular cholesterol
efflux. In HEK 293 cells co-transfection of ABCA1 with either SCD1 or SCD2 inhibited ABCA1-mediated cholesterol efflux but not phospholipid efflux. In Chinese hamster ovary (CHO) cells with moderate stable overexpression of SCD1, cholesterol efflux to apoA-I was inhibited by
73%, whereas phospholipid efflux and ABCA1 protein levels were unchanged. In contrast, cholesterol efflux to HDL2, which
is not dependent on ABCA1, was increased 2-fold in CHO-SCD1 cells. The effect of SCD on cholesterol efflux to apoA-I was independent of
acyl-CoA:cholesterol acyltransferase (ACAT) activity. SCD activity led
to an increased content of plasma membrane monounsaturated fatty acids
(18:1) at the expense of saturated fatty acids (18:0). As shown by
confocal microscopy, SCD overexpression led to a decrease of Triton
X-100-resistant domains in the plasma membrane, indicating a decrease
in membrane-ordered regions. The data suggest that SCD changes membrane
organization and depletes a specific pool of membrane cholesterol
supporting ABCA1-mediated efflux, whereas increasing availability of
cholesterol for passive efflux by HDL2. ABCA1-mediated
cholesterol and phospholipid efflux may be uncoupled in pathological
states associated with high SCD activity, as in hyperinsulinemic obese
mice, or in animals treated with LXR activators.
Cellular cholesterol efflux is central to the anti-atherogenic
role of HDL1 and its
apolipoproteins (1). Cholesterol efflux can involve several different
pathways, including an active efflux to lipid-poor apolipoproteins
mediated by ABCA1, and passive pathways mediated by diffusion through
the aqueous medium or the binding of HDL to its receptor, scavenger
receptor BI (SR-BI) (2, 3). Recent studies have identified ATP-binding
cassette transporter A1 (ABCA1) as the mutant gene in patients with
Tangier disease (4-6). The accumulation of cholesterol esters
in the macrophages of Tangier disease patients indicates the key role
of ABCA1 in mediating cellular cholesterol efflux. ABCA1 facilitates
cholesterol and phospholipid efflux to extracellular lipid-poor
apolipoproteins in liver, macrophages, and other tissues, initiating
the formation of HDL (7, 8). Studies in Tangier disease patients and
mouse models of ABCA1 overexpression or ablation suggest that ABCA1 has
a protective role against atherosclerosis (9-13).
ABCA1 gene and protein expression are highly regulated on several
different levels. Cholesterol efflux to apoA-I is increased in cells
loaded with cholesterol (14). Cholesterol loading induces ABCA1 gene
transcription, as a result of oxysterol activation of LXR (LXR The ability of ABCA1 to mediate cholesterol efflux is also modulated by
trafficking and compartmentalization of cholesterol in cells.
Overexpression of SR-BI in cells inhibits cholesterol efflux by the
ABCA1 pathway, either by sequestering cholesterol in the membrane, or
by promoting reuptake of newly effluxed cholesterol (18). In
macrophages lipoprotein cholesterol deposited in late endosomes/lysosomes acts as a preferential source of cholesterol for
ABCA1-mediated efflux (19). In these studies LXR/RXR activation increased cholesterol efflux to apoA-I, but the magnitude of the change
was much less than the increase in ABCA1 protein levels. This finding
raised the possibility that there might be other LXR/RXR target genes
that could act to oppose ABCA1-mediated cholesterol efflux. In this
study, we identified the LXR/RXR target gene, stearoyl-CoA desaturase
(SCD), as an inhibitor of ABCA1-mediated cholesterol efflux.
Cell Culture--
All cells were grown at 37 °C in a
humidified 5% CO2 incubator. Tissue culture reagents were
from Invitrogen. All cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
Transient and stable transfections were performed with LipofectAMINE
2000 (Invitrogen). The CHO-SCD1 cell line was established by
transfecting CHO cells with pcDNA3.1/Hygro-mSCD1. Hygromycin-resistant clones were pooled for experiments.
Thioglycollate-elicited mouse peritoneal macrophages were collected as
described (19). 22(R)-Hydroxycholesterol and the synthetic
LXR activator TO-901317 were purchased from Sigma.
9-cis-Retinoic acid was purchased from Biomol.
Subtractive Hybridization--
Selective subtractive
hybridization was performed as described (20). Poly(A)+
mRNA (2 µg) was made from treated or untreated
thioglycollate-elicited mouse peritoneal macrophages. Selective
subtractive hybridization was performed with a PCR-select cDNA
subtraction kit (Clontech) according to the
manufacturer's recommendations with modifications (20). The subtracted
cDNA libraries were generated by inserting the cDNA obtained
after hybridization and PCR amplification into a cloning vector
pCRII-TOPO (Invitrogen). Individual clones were sequenced.
Northern Blot Analysis--
Total RNA was extracted from
macrophage using RNAzol (Tel-Test, Inc.). For Northern analysis, total
RNA was separated on 1% agarose-formaldehyde gels (30 µg of RNA per
lane), and transferred to Zeta-probe GT membranes (Bio-Rad). The
membranes were hybridized with randomly primed 32P-labeled
probes of cDNA fragments overnight at 65 °C, and washed twice
each for about 15 min at 65 °C with 0.2% SDS in 0.2× SSC. The
membranes were then exposed to storage a phosphorimaging screen (Amersham Biosciences) to detect signals.
Quantitative Real Time PCR--
RNA was treated with DNase I
(DNA-freeTM; Ambion, Inc). First strand cDNA was
synthesized from 5 µg of DNase I-treated total RNA with oligo(dT)
primers using Superscriptase II (Invitrogen). cDNA from 6 individual samples was pooled for each condition for real time PCR
analysis. Real time PCR was performed in triplicate and analyzed by
using the MX4000 system (Stratagene).
Lipoproteins and Apolipoprotein--
High density lipoprotein 2 (HDL2, 1.063 < d < 1.125 g/ml) was
isolated from plasma by sequential ultracentrifugation using an L8
ultracentrifuge (Beckman). Apolipoprotein A-I (apoA-I) was purchased
from Biodesign International (Saco, ME).
[3H]Cholesterol Labeling of Cells--
Cells were
labeled with [3H]cholesterol by two different methods
(1). The first method was 5 mM
methyl- Cholesterol Efflux--
After labeling and equilibration, the
cells were incubated with 10 µg/ml purified human apoA-I or 15 µg/ml human HDL2 in DMEM, 0.2% BSA for the indicated
time. Then medium was collected and centrifuged at 6000 × g for 10 min to remove cell debris and cholesterol crystals.
The cells were lysed in 0.1 M sodium hydroxide, 0.1% SDS
and radioactivity was determined by liquid scintillation counting.
Phospholipid Efflux--
Cells were labeled for 24 h in
DMEM, 10% FBS supplemented with 1.0 µCi/ml [3H]choline
(PerkinElmer Life Sciences). After 2 h equilibration in DMEM,
0.2% BSA, the cells were washed and efflux was performed in DMEM,
0.2% BSA containing 10 µg/ml apoA-I. Both medium and cell lysates
were extracted with hexane:isopropyl alcohol (3:2) three times and the
organic phase was used to determine radioactivity. Efflux was expressed
as the percentage of radioactivity in the medium relative to the total
radioactivity in cells and medium.
Media Transfer--
As described before (21) the donor cells
were incubated with 10 mM methyl- Immunoblots--
Postnuclear lysates were fractionated in 7.5 or
4-15% SDS-polyacrylamide gel electrophoresis, and transferred to
0.2-µm nitrocellulose membranes (Bio-Rad). Polyclonal anti-ABCA1 and
anti-SR-BI antibodies were purchased from Novus (Littlton, CO).
Monoclonal anti-actin antibody was purchased from Sigma. Polyclonal
anti-SCD antibodies were developed in a rabbit using a peptide
corresponding to C-terminal 14 amino acids of SCD protein. This region
is highly conserved among human SCD and mouse SCD1 and SCD2.
Cellular Lipid Mass--
Confluent cells were lysed in 0.1 M sodium hydroxide and 0.1% SDS. The lysates were passed
through a 21-gauge needle five times, and analyzed for protein by the
Lowry method. Cellular lipids were extracted with hexane:isopropyl
alcohol (3:2) three times. The organic phases were combined and dried
under N2. The residue was dissolved in 0.5% Triton
X-100. Cholesterol and phosphatidylcholine were determined
enzymatically (Wako Chemicals USA, Richmond, VA).
Membrane Fatty Acid Composition--
Plasma membrane was
isolated by nitrocellulose-treated DEAE-Sephadex beads as described
(22). Lipids were extracted from the beads and methyl-esterified and
quantified by gas-liquid chromatography (23).
Cold Triton Extractability and Confocal Microscopy--
Cold
Triton X-100 extractability of surface-bound DiI C18
incorporated into the plasma membrane of various cell lines was performed as described (24). Cells were transferred from growth medium
to Medium 1 (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 2 g/liter glucose, and 20 mM Hepes, pH 7.4). Lipid analogs
were transferred as monomers from fatty acid-free BSA carriers (25). Cells were double labeled with C6-NBD-SM (fluid preferring)
and DiI C18 (ordered preferring) at 37 °C for 20 s,
washed with ice-cold Medium 1, and extracted with 1% Triton X-100 on
ice for 30 min. Confocal microscopy was performed using an Axiovert
100M inverted microscope equipped with an LSM 510 laser scanning unit
and a 63X1.4 NA plan Apochromat objective (Carl Zeiss, Inc.). Cells labeled with DiI C18 were excited with a 1.0-milliwatt
helium/neon laser emitting at 543 nm, and a 560-nm long pass filter was
used for collecting emissions; C6-NBD-SM was excited with a
25-milliwatt argon laser emitting at 488 nm and a 505-530 band-pass
filter was used for emissions. To minimize cross-talk, the two channels were scanned alternately in a line-by-line fashion, having only one
laser line and one detector channel on at each time. For
quantification, cells were treated as described above, with the
exception of replacing 1% Triton X-100 with Medium 1 for control
cells, and imaged on a Leica DMIRB wide-field microscope (Leica
Mikroscopie und Systeme GmbH, Germany) equipped with a cooled CCD
camera (Frame Transfer MicroMAX camera with a 512 × 512 back-thinned EEV chip, number 512BFT; Princeton Instruments) driven by
Image-1/MetaMorph Imaging System software (Universal Imaging Corp.,
Wester Chester, PA). Images were acquired using a ×25 oil immersion
objective (0.75 NA) to include a large number of cells in one field and
to acquire fluorescence from the entire cell thickness. DiI
C18 was imaged using a standard rhodamine filter set.
Images were first background corrected (26), and then quantified by
manually outlining each cell and taking the average fluorescence power
associated with the cells. The ratio of average intensities obtained
from the control and extracted cells of each cell line was determined
as the percentage remaining cell associated.
Statistical Analysis--
Cholesterol and phospholipid efflux
were expressed as the percentage of the radioactivity in the medium
relative to the total radioactivity in cells and medium. Data shown are
the average of at least three independent experiments with triplicates
in each experiment unless specified otherwise. Data are expressed as
mean ± S.D. The tests for the significant differences between groups were performed by Student's t test.
Stearoyl-CoA Desaturase Is Induced by LXR/RXR in
Macrophages--
In an attempt to identify genes that modulate the
cholesterol efflux pathway, we carried out selective subtractive
hybridization on mouse peritoneal macrophages treated with or without
LXR/RXR activators, 22(R)-OH cholesterol (5 µM) and 9-cis-retinoic acid (10 µM). Among the genes induced by this treatment, we
identified stearoyl-CoA desaturase 2 (Scd2), and this was
confirmed by Northern blot analysis (Fig.
1A). Consistent with these
observations, the related hepatic Scd1 is induced in animals
treated with LXR ligand (27). Scd1 and Scd2,
expressed in an overlapping tissue distribution, catalyze the
conversion of stearoyl-CoA to oleoyl-CoA and thereby regulate the ratio
of monounsaturated to saturated fatty acids in cell membrane
phospholipids (28). Quantitative real time PCR showed that both
Scd1 and Scd2 are expressed in mouse peritoneal macrophages (11.7 and 35.4% of actin, respectively), and both were
induced by treatment of macrophages with the synthetic LXR activator
TO-901317 (1 µM) (Fig. 1B).
To evaluate SCD protein levels, we developed a specific SCD antibody
using a peptide recognizing a common region in SCD1 and SCD2. As shown
in 293 cells transfected with either SCD1 or SCD2, the antibody
provided specific recognition of SCD1 and SCD2 protein, with no signal
in control cells (Fig. 1C). In macrophages treated with
LXR/RXR activators, there was a 5-fold induction of SCD protein (the
combined signal from SCD1 and SCD2) (Fig. 1D). SCD activity is increased in the liver in ob/ob mice (29), and in transgenic mice
overexpressing sterol responsive element-binding protein (SREBP)-1.
Using our SCD antibody, we showed 2-3-fold increase in SCD protein in
liver of ob/ob mice compared with wild type controls (Fig.
1E), but no change in protein levels in mouse peritoneal macrophages (not shown).
SCD1 and SCD2 Specifically Inhibit ABCA1-mediated Cholesterol
Efflux--
We next carried out experiments to evaluate the effects of
increased SCD levels on cellular cholesterol efflux. Transient co-expression of either SCD1 or SCD2 with ABCA1 in 293 cells resulted in a significant inhibition of cholesterol efflux to apoA-I (Fig. 2A). In contrast, phospholipid
efflux to apoA-I was slightly increased in cells expressing ABCA1 and
either SCD1 or SCD2 (data not shown).
To further assess the role of SCD in modulating cellular cholesterol
efflux, we developed CHO cell lines with stable expression of SCD1
(CHO-SCD1), and carried out cholesterol efflux experiments using a pool
of clones. The CHO-SCD1 cells showed a moderate 4-fold increase in SCD
protein expression (Fig. 1F), comparable with the relative
SCD protein overexpression observed in macrophages treated with LXR/RXR
activators, and in liver in ob/ob mice, compared with wild type mice
(Fig. 1). Cholesterol efflux to apoA-I was decreased by 40% in
CHO-SCD1 cells compared with control CHO cells (Fig.
2B).
In these experiments, cells were labeled by 24 h incubation with
[3H]cholesterol:FBS. In contrast, when cells were labeled
by brief exposure to cyclodextrin, [3H]cholesterol (30),
there was a more pronounced 73% decrease in cholesterol efflux to
apoA-I in CHO-SCD1 cells compared with control CHO cells (Fig.
3A). Time course experiments
showed that the effect of SCD was observed throughout the 4-h efflux
period (Fig. 3B). In contrast to cholesterol efflux,
phospholipid efflux was slightly increased in CHO-SCD1 cells (Fig.
3C) and ABCA1 protein levels were identical in CHO and
CHO-SCD1 cells (Fig. 3D).
In contrast to these findings, cholesterol efflux to HDL2,
which is independent of ABCA1 (21), was increased 2-fold in CHO-SCD1 cells (Fig. 4A), and the
effect was observed over a wide range of HDL concentrations (Fig.
4B). Cholesterol efflux to HDL can occur by passive
diffusion (3) and can be facilitated by SR-BI (2). SR-BI protein levels
were identical in control and SCD overexpressing cells (Fig.
4A, inset). Moreover, incubation of CHO cells
with SR-BI neutralizing antibody showed only minor effects on
cholesterol efflux, reflecting the low level of SR-BI protein in this
cell type (2). Thus, SCD overexpression results in an increase in
passive cholesterol efflux to HDL2, which is independent of
ABCA1 and SR-BI.
Mechanisms Underlying Inhibition of ABCA1-mediated Lipid Efflux by
SCD--
Increased SCD activity is known to enhance cellular
ACAT activity and apoA-I promotes cholesterol efflux from an
ACAT-accessible pool (31, 32). Thus, we tested if an ACAT inhibitor
would reverse the effect of SCD. However, at concentrations shown to be
effective in inhibiting cellular cholesterol esterification (see
"Materials and Methods") the specific ACAT inhibitor, Dup128, did
not alleviate the inhibitory effect of SCD on apoA-I-mediated cholesterol efflux (Fig. 5).
We next considered that in cells expressing SCD, ABCA1 might generate
phospholipid·apoA-I complexes with a lower capacity to accept
membrane cholesterol. To evaluate this possibility we conducted a media
transfer experiment. Media containing complexes from control or SCD1
expressing CHO cells were transferred to a second set of cells (control
or SCD1 expressing). These experiments showed that inhibition of
cholesterol efflux was primarily a characteristic of cells expressing
SCD1, and not of the media obtained from SCD1 expressing cells (Fig.
6).
The effects of SCD were less pronounced in cells subjected to a
prolonged labeling procedure with FBS, [3H]cholesterol,
compared with the rapid cyclodextrin labeling procedure (cf.
Fig. 3 with Figs. 2 and 6). These results suggested that changes in
plasma membrane composition or organization might be responsible for
the effects of SCD. Cellular lipid measurements did not show any major
differences in total cholesterol, free cholesterol, cholesterol ester,
or phosphatidylcholine levels in control or SCD1 overexpressing cells
(Table I). However, in SCD1 expressing
cells there was a 71% increase in 18:1 to 18:0 ratio in plasma
membrane phospholipid fatty acids (Table
II). This -fold change in phospholipid
fatty acids is comparable with that occurring in ob/ob liver compared
with wild type liver (29).
Cholesterol-enriched membrane regions, known as liquid ordered domains
or "rafts" (33), are formed from sphingolipids and glycolipids
(34). Such regions are insoluble in Triton X-100. A new technique
involving confocal microscopy of cells containing a fluorescent
phospholipid marker (24) indicates that the major portion of plasma
membrane is in the liquid ordered state, as shown by resistance to cold
Triton X-100 extraction, whereas a minor part consists of Triton
X-100-soluble regions. We considered the possibility that SCD might
change the overall organization of the plasma membrane. The presence of
liquid-ordered domains was assessed by confocal microscopy, where we
observed the incorporation of the fluorescent phospholipid analog DiI
C18:0 into the plasma membrane of intact cells (Fig.
7). In control cells, treatment with
Triton X-100 resulted in the appearance of a small number of holes in
the plasma membrane, indicating that in intact cells the major portion
of the membrane is resistant to Triton X-100 (Fig. 7A), as
reported (24). In CHO-SCD1 cells, there was a dramatic increase in the
formation of Triton X-100-soluble regions, resulting in a "Swiss
cheese" appearance (Fig. 7B). Thus, in SCD expressing
cells liquid domains are increased, whereas liquid-ordered domains are
decreased. Like the majority of plasma membrane proteins, ABCA1 has
been reported to be present in Triton-soluble regions (24). Using
antibodies against ABCA1-FLAG, we showed that the plasma membrane
signal was abolished by treatment with cold Triton X-100, consistent
with localization of ABCA1 in Triton-soluble regions (not shown)
(35).
Scavenger receptor BI inhibits ABCA1-mediated cholesterol efflux (18),
possibly as a result of direct binding of cholesterol to SR-BI or the
re-uptake of effluxed cholesterol via SR-BI. SR-BI has been localized
to Triton X-100-insoluble membrane regions (36). However, in CHO cells
or CHO-SCD1 cells overexpressing SR-BI, there was no significant change
in the formation of membrane liquid-ordered regions. SCD1 and SR-BI
overexpression resulted in a comparable inhibition of cholesterol
efflux, and in cells overexpressing both SCD1 and SR-BI the inhibition
of cholesterol efflux was additive, indicating that SCD and SR-BI
inhibit efflux by different mechanisms (Fig.
8).
In a screen for LXR target genes, we identified macrophage
Scd1 and Scd2, similar to the earlier findings
with hepatic Scd1 (37). To test the hypothesis that SCD
might decrease cholesterol efflux mediated by ABCA1, we overexpressed
SCD1 and SCD2 in 293 cells, and SCD1 in CHO cells, and showed a
selective defect in ABCA1-mediated cholesterol efflux to apoA-I. The
levels of overexpression of SCD1 protein were comparable with those
induced by LXR/RXR activation, or by leptin deficiency in ob/ob mice.
These findings suggest that SCD may be an important modulator of
ABCA1-mediated cholesterol efflux in animals treated with LXR
activators, or in different physiological states involving
up-regulation of SCD. Mechanistic studies reveal that SCD alters plasma
membrane phospholipid fatty acid composition and organization,
decreasing the availability of cholesterol for efflux to apoA-I.
In contrast, cholesterol efflux to HDL2 is increased by
SCD1 overexpression, indicating a clear difference in availability of
cholesterol efflux in the ABCA1 and passive diffusion pathways.
Cholesterol efflux to apoA-I and HDL are mediated by different
pathways. Plasma HDL promotes cholesterol efflux through passive aqueous diffusion. This process may be SR-BI-dependent or
-independent (3). This process is bidirectional and depends on a
cholesterol concentration gradient between cell and lipoproteins (38).
In this process, cholesterol must desorb from the lipid-water interface of plasma membrane before diffusing to and adsorbing into HDL, and the
desorption step is rate-limiting. Lund-Katz et al. (39) showed that cholesterol desorbs more rapidly from unsaturated phosphatidylcholine bilayers than from saturated phosphatidylcholine bilayer because of greater Van der Waals attraction in the latter system. This may explain why cholesterol efflux to HDL2 is
increased in CHO-SCD1 cells. Because SR-BI expression is unchanged by
SCD, it is most likely the cholesterol desorption step that is enhanced in SCD1 expressing cells. The effects of SCD could explain our previous
finding that LXR/RXR activation increased cholesterol efflux to
HDL2 in mouse peritoneal macrophages (19), i.e.
SCD expression was induced, resulting in increased efflux to
HDL2.
Our experiments add to the growing body of evidence that ABCA1-mediated
phospholipid efflux can be dissociated from cholesterol efflux (21, 40,
41), and provide the first clear evidence showing that these processes
may become uncoupled under physiological conditions. One interpretation
of the dissociation of cholesterol from phospholipid efflux is that
ABCA1 mediates lipid efflux in a two-step process, with initial
formation of a phospholipid·apoA-I complex by ABCA1, followed by
diffusion or insertion of cholesterol from a distinct
cholesterol-enriched membrane microdomain. Our results could indicate
decreased availability of cholesterol in membrane liquid-ordered
domains and could be taken to support this model. However, the kinetics
of phospholipid and cholesterol efflux are consistent with ABCA1
mediating coordinated efflux of cholesterol and phospholipid, possibly
as a result of the formation of cholesterol-enriched microdomains
contiguous with ABCA1 that are then solubilized by ABCA1-bound
apolipoprotein (42). Moreover, ABCA1 is localized to membrane Triton
X-100-soluble regions and mediates cholesterol efflux preferentially
from these regions (Ref. 35, and this study). Thus, we favor an
alternative interpretation that less cholesterol is available for
efflux in the expanded Triton X-100-soluble domains containing
ABCA1.
Because SCD activity is highly regulated, our findings provide the
first evidence to suggest that ABCA1-mediated phospholipid and
cholesterol efflux may sometimes be uncoupled in vivo. Under these circumstances formation of phospholipid-rich nascent HDL in
hepatocytes could induce cholesterol efflux from other cells or tissues
in a paracrine or endocrine fashion. Markedly increased SCD activity
occurs in association with increased SREBP1 activity in the liver, as
seen in SREBP1 transgenic mice (43), with fasting-refeeding (44) or in
association with insulin resistance and hyperinsulinemia (29). Our data
suggest that nascent HDL particles formed at the surface of hepatocytes
by ABCA1 and apoA-I in insulin-resistant states (e.g. in
ob/ob mice or in human metabolic syndrome) might be phospholipid-rich
and cholesterol-poor. Such HDL particles would have an increased
capacity to absorb cholesterol in the bloodstream, or in peripheral
tissues from macrophage foam cells. Conversely, the decreased
efflux of hepatic cholesterol via ABCA1 could lead to cholesterol
accumulation in hepatocytes, possibly leading to increased cholesterol
esterification or excretion into bile. Our observation that moderate
changes in SCD substantially change membrane domain organization also
raises the possibility that many different functions attributed to
membrane liquid-ordered regions or rafts, such as nitric-oxide synthase
activity (45), could be altered by changes in SCD expression.
In cells overexpressing SCD, ABCA1 protein is maintained and
phospholipid efflux is slightly increased. This is different to the
effects of the addition of exogenous fatty acids to cells (16). Thus,
Wang and Oram (16) found that unsaturated fatty acids inhibited
ABCA1-mediated phospholipid and cholesterol efflux by accelerating
ABCA1 protein degradation. Our results also contrast with the
inhibition of LXR-mediated gene transcription by exogenous polyunsaturated fatty acids (46). SCD overexpression led to increased
incorporation of monounsaturated fatty acids into cellular phospholipids, whereas polyunsaturated fatty acids were unchanged (Table II). Endogenous and exogenous fatty acids function differently because of different cellular compartmentalization (31). SCD promotes
formation of fatty acyl-CoAs that are readily incorporated into
membrane phospholipids, whereas this may not be the case for
exogenously added unsaturated fatty acids.
LXR activators have emerged as therapeutic targets in the treatment of
atherosclerosis (47). Our results indicate that the simultaneous
up-regulation of ABCA1 and SCD by these treatments will have opposing
effects on apoA-I-mediated cholesterol efflux. This may explain why
such agents appear to have relatively modest effects on cholesterol
efflux, despite marked up-regulation of ABCA1 (19). Because part of the
increased expression of SCD likely results from an LXR-mediated
increase in SREBP1c that in turn acts on the SCD promoter, LXR agonists
that are selective for the ABCA1 promoter but not the SREBP1c promoter,
would be predicted to have more favorable effects on macrophage
cholesterol efflux via the ABCA1 apolipoprotein pathway, in addition to
the benefit of not causing hypertriglyceridemia or fatty liver (29). Recently, mice deficient in SCD1 were shown to be resistant to dietary
and genetic obesity (48, 49), suggesting that SCD inhibitors could be
useful in the treatment of obesity. As noted above, inhibition of SCD
activity in the liver is likely to have complex effects on cholesterol
homeostasis and centripetal cholesterol transport. However, our results
suggest that inhibition of SCD in the macrophage is likely to result in
increased cholesterol efflux via the ABCA1 pathway and thus could be
anti-atherogenic.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
LXR
) that forms heterodimers with retinoid X receptor (RXR) on a
direct repeat 4 element in the promoter of the ABCA1 gene (15).
Emerging evidence suggests that ABCA1 is also regulated on a
post-transcriptional level. Wang et al. (16) showed that polyunsaturated fatty acids reduced cellular ABCA1 protein content by
enhancing ABCA1 degradation. In contrast, apoA-I and apoA-II stabilized
ABCA1 protein without changing the mRNA, as a result of decreased
degradation by thiol proteases (17).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin labeling. Cells were labeled with 1 µCi/ml
[1,2-3H]cholesterol (PerkinElmer Life Science) in DMEM
containing 5 mM methyl-
-cyclodextrin:cholesterol at a
molar ratio of 8:1 for 15 min at 37 °C. After washing, cells were
equilibrated in DMEM, 0.2% BSA for 30 min and then used for
efflux experiments (2). The second method was with 10%
FBS/DMEM. Cells were labeled with 1 µCi/ml
[3H]cholesterol in DMEM supplemented with 10% FBS for
24 h. The cells were then equilibrated in DMEM, 0.2% BSA for
2 h and then used for cholesterol efflux experiments. In
experiments with the acyl-CoA:cholesterol acyltransferase (ACAT)
inhibitor, Dup128, the compound was present for 24 h before efflux
at a concentration of 100 nM and during the efflux period.
To test the efficiency of Dup128 at 100 nM, we labeled CHO
and CHO-SCD1 cells with 1 µCi/ml [3H]cholesterol in
DMEM supplemented with 10% FBS for 24 h in the presence or
absence of 100 nM Dup128. The cells were then equilibrated in DMEM, 0.2% BSA overnight. Cellular lipids were extracted with hexane:isopropyl alcohol (3:2) three times and the organic phase was
collected, evaporated under N2, and fractionated by TLC
using hexane:ethyl ether:methanol:acetic acid (85:15:1:1). Spots
corresponding to free cholesterol and cholesterol ester were collected
and counted by liquid scintillation. The radiolabeled cholesterol
ester:free cholesterol ratio in mock treated cells was 0.04 (CHO) and
0.12 (CHO-SCD1). In Dup128-treated cells, the cholesterol ester:free cholesterol ratio was decreased to 0.006 (CHO) and 0.009 (CHO-SCD1).
-cyclodextrin for 30 min, medium was changed to DMEM, 0.2% BSA containing 10 µg/ml
apoA-I. 4 h later this conditioned medium was transferred to
[3H]cholesterol-labeled recipient cells for the efflux
assay. The efflux was performed in recipient cells for 4 h.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SCD mRNA and protein expression.
A, peritoneal macrophages were isolated from C57BL/6
mice and plated in 6-well plates. Cells were treated with the indicated
agents for 24 h. Total RNA was extracted and 30 µg was loaded in
each lane. 22(R)HC,
22(R)-hydroxycholesterol (5 µM);
9-cis-RA, 9-cis-retinoic acid (10 µM). B, macrophages treated with the
synthetic LXR ligand TO-901317 (1 µM). cDNA from 6 individually treated wells was pooled for quantitative real time PCR.
The values are the mean ± S.D. (from triplicates).
C, total postnuclear lysates from HEK-293 cells
transfected with the indicated plasmids were used to test SCD antibody.
The membrane was also blotted with anti-FLAG M2 antibody (middle
panel). D-F, SCD protein levels as detected by Western
blot using a polyclonal antibody recognizing the C terminus of SCD.
D, SCD expression in mouse peritoneal macrophages with
or without LXR activation. Mouse peritoneal macrophages were treated
with 5 µM 22(R)-hydroxycholesterol and 10 µM 9-cis-retinoic acid for 24 h.
Postnuclear lysates were made and 75 µg of protein was loaded to each
lane. E, SCD expression in wild type and ob/ob mouse
liver postnuclear lysates. F, SCD expression in CHO and
CHO-SCD1 cells.
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Fig. 2.
SCD 1 and SCD2 inhibit cholesterol efflux to
apoA-I. A, HEK-293 cells were plated in 24-well
plates and transiently transfected with the indicated plasmid
constructs. Cells were labeled with [3H]cholesterol, 10%
FBS for 24 h and equilibrated in DMEM, 0.2% BSA. Efflux was
performed to 10 µg/ml apoA-I in DMEM, 0.2% for 20 h. *,
p < 0.05 compared with cells transfected with ABCA1
alone. B, CHO and CHO-SCD1 were labeled with
[3H]cholesterol, 10% FBS for 24 h and equilibrated
in DMEM, 0.2% BSA. Efflux was performed to 10 µg/ml apoA-I in DMEM,
0.2% for 4 h. *, p < 0.05 compared with control
CHO cells. Cholesterol efflux is [3H]cholesterol
radioactivity in the medium divided by total radioactivity (cells plus
medium) × 100. The values are the mean ± S.D.
View larger version (22K):
[in a new window]
Fig. 3.
SCD1 inhibits cholesterol efflux to apoA-I
without affecting phospholipid efflux or ABCA1 protein level.
Cells used for cholesterol efflux measurements (A and
B) were labeled with
[3H]cholesterol/cyclodextrin·cholesterol (8:1)
complexes for 15 min. After equilibration in DMEM, 0.2% BSA, efflux
was performed using 10 µg/ml apoA-I in DMEM, 0.2% BSA. Cholesterol
efflux is [3H]cholesterol radioactivity in the medium
divided by total radioactivity (cells plus medium) × 100. A, cholesterol efflux after 4 h incubation with
apoA-I; B, the time course of cholesterol efflux;
C, phospholipid efflux (4 h). For both cholesterol and
phospholipid efflux, the values are the mean ± S.D. *,
p < 0.05 compared with CHO cells. D,
immunoblot of ABCA1 following SDS-polyacrylamide gel electrophoresis of
cell lysates (n = 4). X-ray film was scanned by
densitometry, and relative intensities of bands were determined by
ImageQuant.
View larger version (12K):
[in a new window]
Fig. 4.
SCD1 increases cholesterol efflux to
HDL2. Cells were labeled with
[3H]cholesterol/cyclodextrin·cholesterol (8:1)
complexes for 15 min. Efflux was performed to 15 µg/ml (A)
or the indicated amounts (B) of HDL2 in DMEM,
0.2% BSA for 4 h after equilibration in DMEM, 0.2% BSA.
Cholesterol efflux is expressed as the percentage of total
[3H]cholesterol radioactivity (cells plus medium)
recovered in medium. The values are the mean ± S.D. *,
p < 0.05 compared with CHO cells. The inset
shows a Western blot of SR-BI.
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Fig. 5.
ACAT inhibitor does not reverse the effect of
SCD1. Cells were labeled with
[3H]cholesterol/cyclodextrin·cholesterol (8:1)
complexes for 15 min. Dup128 (100 nM) or dimethyl sulfoxide
were added to cells 24 h before efflux. Efflux was performed to 10 µg/ml apoA-I in DMEM, 0.2% BSA for 4 h after equilibration in
DMEM, 0.2% BSA. Open bar, CHO cells; closed bar,
CHO-SCD1 cells. *, p < 0.05 compared with CHO cells in
same treatment.
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[in a new window]
Fig. 6.
Inhibitory effect of SCD1 on cholesterol
efflux is a feature of cells expressing SCD. Media transfer
experiments were performed as described under "Materials and
Methods." The donor cells were depleted of cholesterol by incubating
with 10 mM methyl- -cyclodextrin for 30 min. Then the
medium was changed to DMEM, 0.2% BSA containing 10 µg/ml apoA-I and
was incubated for 4 h. This conditioned medium was transferred to
[3H]cholesterol-labeled recipient cells for the efflux
assay. The recipient cells were labeled with
[3H]cholesterol, 10% FBS for 24 h and equilibrated
in DMEM, 0.2% BSA. Efflux was performed for 4 h in conditioned
medium, or to 10 µg/ml apoA-I in DMEM, 0.2% BSA as a control
(first two bars). Open bar, CHO cell;
closed bar, CHO-SCD1 cells. *, p < 0.05 compared with CHO cells in same treatment.
Lipid composition of CHO cells and CHO-SCD cells
Fatty acid composition of plasma membrane in CHO and CHO-SCD cells
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Fig. 7.
Cold Triton extractability of DiI
C18 is increased in SCD1 expressing cells. Cold Triton
extraction experiments were performed as described under "Materials
and Methods" in CHO (A), CHO-SCD1 (B),
CHO-SR-BI (C), and CHO-SCD1/SR-BI (D) cells.
Briefly, cells were first labeled with DiI C18 at 37 °C
for 20 s, washed, and extracted with 1% Triton X-100 on ice for
30 min. Confocal microscopy was done as described under "Materials
and Methods." Bar, 10 µm. E, percentage
of DiI C18 fluorescence remaining with cells after Triton
X-100 extraction. The fluorescent intensities of cells (before or after
extraction) were quantified by manually outlining each cell and taking
the average fluorescence power associated with the cells. The data are
presented as a ratio of fluorescence between extracted and control
cells.
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[in a new window]
Fig. 8.
SCD1 and SR-BI additively inhibit cholesterol
efflux to apoA-I. Cells were labeled with
[3H]cholesterol, 10% FBS for 24 h and equilibrated
in DMEM, 0.2% BSA. Efflux was performed to 10 µg/ml apoA-I in DMEM,
0.2% for 4 h. *, p < 0.01 compared with control
CHO cells. #, p < 0.05 compared with CHO-SCD1/SR-BI
cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. Toru Seo for help in plasma membrane separation.
![]() |
FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL 22682 (to A. R. T.) and DK27083 (to F. R. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: Pharmacia, 800 N. Lindbergh Blvd., T207W, St. Louis, MO 63141.
To whom correspondence should be addressed. Tel.:
212-305-9418; Fax: 212-305-5052; E-mail: art1@columbia.edu.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M208687200
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ABBREVIATIONS |
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The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP binding cassette transporter A1; apo, apolipoprotein; SCD, stearoyl-CoA desaturase; ACAT, acyl-CoA:cholesterol acyltransferase; SR-BI, scavenger receptor BI; LXR, liver X receptor; RXR, retinoid X receptor; SREBP, sterol regulatory element-binding protein; C6-NBD-SM, N-((6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosylphosphocholine; DiI, dialkylindocarbocyanine; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSA, bovine serum albumin.
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