From the Departments of Biochemistry, and
Animal Science, University of Wisconsin-Madison, Madison,
Wisconsin 53706, ** Center for Molecular Medicine and
Therapeutics, University of British Columbia, Vancouver, British
Columbia V65Z 4H4, Canada, and
Xenon
Genetics, Burnaby, British Columbia V5G 4W8, Canada
Received for publication, December 5, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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The ATP-binding cassette transporter A1 (ABCA1)
participates in the efflux of cholesterol from cells. It remains
unclear whether ABCA1 functions to efflux cholesterol across the
basolateral or apical membrane of the intestine. We used a chicken
model of ABCA1 dysfunction, the Wisconsin hypoalpha mutant (WHAM)
chicken, to address this issue. After an oral gavage of radioactive
cholesterol, the percentage appearing in the bloodstream was reduced by
79% in the WHAM chicken along with a 97% reduction in the amount of tracer in high density lipoprotein. In contrast, the percentage of
radioactive cholesterol absorbed from the lumen into the intestine was
not affected by the ABCA1 mutation. Liver X receptor
(LXR) agonists have been inferred to decrease cholesterol absorption through activation of ABCA1 expression. However, the LXR agonist T0901317 decreased cholesterol absorption equally in both wild type and
WHAM chickens, indicating that the effect of LXR activation on
cholesterol absorption is independent of ABCA1. The ABCA1
mutation resulted in accumulation of radioactive cholesterol ester in
the intestine and the liver of the WHAM chicken (5.0- and 4.4-fold, respectively), whereas biliary lipid concentrations were unaltered by
the WHAM mutation. In summary, ABCA1 regulates the efflux of cholesterol from the basolateral but not apical membrane in the intestine and the liver.
HDL1 concentrations are
inversely correlated with risk of cardiovascular disease. It is
postulated that this is due to the role of HDL in reverse cholesterol
transport from the cells of the arterial wall (1, 2). Reverse
cholesterol transport involves the HDL-mediated transport of
cholesterol from extrahepatic tissues to the liver, allowing for cells
to rid themselves of excess cholesterol and maintain cholesterol
balance. The selective uptake of HDL cholesterol ester (CE) by the
liver is mediated by scavenger receptor-BI and subsequent conversion of
cholesterol into bile acids allows for excretion of cholesterol into
bile (2, 3). The incomplete intestinal reabsorption of these biliary
components, concomitant with the sloughing off of intestinal enterocytes, constitutes the major route of cholesterol elimination from the body via fecal excretion.
ATP binding cassette transporters comprise a large family of
membrane-spanning proteins that are responsible for transporting a
variety of substrates in prokaryotes and eukaryotes. Many of these
transporters are responsible for the translocation of lipophilic substrates such as phospholipids, bile acids, and sterols (4, 5).
Mutations in the ABCA1 transporter in humans are responsible for the HDL deficiency observed in Tangier disease and in the heterozygous form, familial hypoalphalipoproteinemia; both conditions lead to premature cardiovascular disease (6-12). A hallmark of Tangier
disease is CE accumulation in many tissues, particularly the
reticuloendothelial system (13), highlighting the importance of ABCA1
and HDL in maintaining cholesterol balance.
The HDL deficiency found in Tangier disease and familial
hypoalphalipoproteinemia is due to the hypercatabolism of lipid-poor apoAI. The amphipathic apoAI protein is synthesized and secreted in a
lipid-poor state by the liver and intestine and must acquire phospholipid and cholesterol during and after the secretory process to
form pre- Aside from the efflux of lipid to form HDL, ABCA1 has been implicated
in controlling cholesterol flux at the apical membrane of the liver and
the intestine, potentially influencing biliary lipid concentration and
dietary cholesterol absorption, respectively. Both LXR agonists and
agonists of the LXR heterodimeric partner, RXR, have been shown to
drastically decrease intestinal cholesterol absorption in mice while
simultaneously increasing intestinal ABCA1 expression due to the
transactivation of the ABCA1 promoter (16, 17). Although
this suggested a role for ABCA1 in intestinal cholesterol absorption,
the subsequent discovery of ABCG5 and ABCG8, two
other LXR-regulated ABC transporters involved in sterol trafficking
(18-24), has offered another potential target for the LXR effect on
cholesterol absorption. In Caco-2 cells, WIF-B hepatocytes, and gall
bladder epithelial cells, ABCA1 has been shown to be predominantly
localized to the basolateral surface and to promote basolateral
cholesterol efflux in an LXR-stimulated, apoAI-dependent pathway (25-28). Furthermore, it has been shown that the enhanced secretion of cholesterol into bile mediated by LXR stimulation is
independent of ABCA1 (29). However, the role of ABCA1 in intestinal
cholesterol absorption is still unresolved.
ABCA1 is primarily localized to the plasma membrane, and time lapse
fluorescence microscopy of an ABCA1-green fluorescent protein fusion
protein also revealed vesicular trafficking of ABCA1 occurring among
endosomes, lysosomes, and the plasma membrane (30, 31). Functional
ABCA1 increases the cell surface binding of apoAI (32-34). This
ABCA1-mediated apoAI binding is believed to promote the formation of
phospholipid-apoAI complexes, which then permit cholesterol,
predominantly of late endosomal and lysosomal origin, to be effluxed in
an ABCA1-independent pathway (2, 35, 36).
The Wisconsin hypoalpha mutant (WHAM) chicken is the only known
spontaneously occurring animal model of ABCA1 dysfunction. The WHAM
chicken ABCA1 gene has a G265A substitution that results in
a glutamic acid to lysine substitution at amino acid 89 of the ABCA1
protein (E89K) (37). Similar to Tangier disease, the WHAM chickens have
a 90% reduction in HDL and apoAI levels, resulting in a greater than
70% decrease in serum phospholipid and cholesterol levels (38). In
contrast to the autosomal location of ABCA1 in humans and
mice, ABCA1 in chickens is sex-linked on the Z chromosome, giving rise to hemizygous females (Z/ In vitro studies showed that the E89K mutation results in
intracellular accumulation of ABCA1, decreasing plasma membrane ABCA1
expression by 80-90% of wild type ABCA1 (determined by cell surface
biotinylation) and resulting in a complete loss of function assayed by
both annexin-V and apoAI binding (37). The structural consequence of
this mutation is not known. However, investigation into the membrane
topology of ABCA1 concludes that the 2261-amino acid protein adopts a
type-II transmembrane orientation, placing a loop of ~600 amino
acids, including position 89, into the exoplasmic space (40, 41).
Therefore, the WHAM chicken is a unique animal model for investigating
the role of ABCA1 in cholesterol and lipoprotein metabolism.
Here, we provide data suggesting that the WHAM chicken has impaired
basolateral, but not apical, cholesterol transport during dietary
cholesterol absorption, with concomitant CE accumulation in liver and
intestine. Furthermore, treatment of animals with the LXR agonist,
T0901317, decreased intestinal cholesterol absorption similarly in both
wild type and WHAM chickens, dispelling ABCA1 as the
LXR-regulated gene responsible for decreased cholesterol absorption in
the intestine.
Animals--
Chickens were obtained from a flock maintained by
the University of Wisconsin-Madison Poultry Science Department. The
chickens were fed ad libitum a standard University of
Wisconsin poultry diet (0.01% cholesterol and 4.3% fat). For the
experiments described, age-matched male chickens between 4 and 8 weeks
of age were used.
[3H]Cholesterol Plasma Appearance--
Animals
fasted for 5 h prior to the experiment were given 500 µl of
CAPTEX medium chain triglyceride oil 10 min prior to
[3H]cholesterol gavage, as well as 4 and 8 h later.
[1,2-3H]cholesterol (100 µCi/animal; American
Radiolabeled Chemicals) was dried under N2 and resuspended
in 50 µl of EtOH. 1 ml/animal of phosphate-buffered saline containing
4 mg/ml egg PC (type XVI-E; Sigma) was added and sonicated briefly.
Bovine serum albumin was added to 5 mg/ml, and the mixture was
centrifuged for 10 min at 10,000 × g in a SS-34 rotor.
1 ml of the liposome suspension was delivered by oral gavage and was
immediately followed by 1 ml of H2O. Blood was collected
from the wing vein, and a 300-µl aliquot of each plasma sample was
adjusted to d = 1.090 g/ml using a 1.30 g/ml NaBr
solution and underlaid beneath 12 ml of buffer of density 1.063 g/ml
and centrifuged at 100,000 × g for 42 h at
10 °C in a 70.1 Ti rotor. Folch extraction (42) was performed on
whole plasma or gradient fractions, and the organic extracts were
subsequently counted by liquid scintillation counting (LSC; Packard
Tri-Carb 2900TR). Density gradient radioactivity recovery was
comparable with total plasma radioactivity. Total animal plasma values
were estimated using the individual animal weights and the specific activity of the plasma or fraction (43). For HDL supplementation experiments, animals received either purified wild type HDL or saline
via intravenous injection prior to oral gavage.
HDL-cholesterol-supplemented WHAM chickens were maintained at plasma
cholesterol concentrations at least 13-fold higher than unsupplemented
WHAM animals for the time course of the experiment.
HDL Purification--
Plasma was collected from wild type male
chickens, adjusted to d = 1.063 g/ml, and
phenylmethylsulfonyl fluoride was added to 0.5 mM and
centrifuged at 100,000 × g for 28 h at 15 °C
in a 50.2 Ti rotor. The infranatant was adjusted to d = 1.21 g/ml and centrifuged at 100,000 × g for 36 h
at 15 °C. HDL was collected from the surface, dialyzed to
phosphate-buffered saline, and filter-sterilized with a 0.2-µm
filter. SDS-PAGE analysis confirmed the absence of apoB-containing
lipoproteins and enrichment of apoAI. The final protein/cholesterol
ratio of the purified HDL ranged between 1.5 and 1.95.
HDL-Cholesterol Clearance--
Purified wild type or WHAM HDL
was labeled with [4-14C]cholesterol (American
Radiolabeled Chemicals) by incubating 375 µg of HDL protein with 1 ml
of [14C]cholesterol-PC-liposomes (20 µg/ml PC)
overnight at 37 °C in the presence of 1 mM diethyl
p-nitrophenyl phosphate. Labeled HDL was recovered by
density gradient ultracentrifugation as described above. Fasted animals
were placed under isoflurane anesthesia, catheterized in the jugular
vein, and injected with [14C]cholesterol-HDL. Blood was
sampled from the wing vein, and radioactivity was quantitated by LSC.
HDL-cholesterol (HDL-C) was isolated from plasma using an equivalent
volume of an HDL-precipitating reagent (Sigma). The apparent fractional
clearance rates were determined gravimetrically by measuring the area
under the radioactivity disappearance curves.
Fecal Radiolableled Sterol Analysis--
PC-liposomes containing
1 µCi of [5,6-3H]- Plasma Lipid Analysis--
Plasma lipoproteins were fractionated
on a Superose 6HR 10/30 FPLC column (Amersham Biosciences). The
equivalent of 100 µl of plasma was injected onto the column. 500-µl
fractions were collected and assayed for total cholesterol and
triglyceride using the Infinity cholesterol reagent and Infinity
triglyceride reagent (Sigma). Plasma concentrations of unesterified and
esterified cholesterol were determined using the Free Cholesterol C and
Cholesterol CII kits (Wako, Richmond, VA).
Tissue Lipid Analysis--
For triglyceride analysis, tissues
were collected after a 4-h fast from 5-week old wild type and WHAM male
chickens on day 5 of treatment with T0901317 or vehicle alone. 100 mg
of whole tissue was homogenized in 2 ml of ice-cold phosphate-buffered saline with a Potter-Elvejhem homogenizer. The homogenate was centrifuged for 5 min at 3000 rpm at 4 °C, and the supernatant was
collected. Lipid from 45 µl of homogenate was extracted by the
Bligh-Dyer method (45), dried under N2, and measured
essentially as described in Briaud et al. (46). The dried
lipid was resuspended by the addition of 30 µl of Thesit (Fluka
Biochemika) and incubated at 37 °C for 10 min, with periodic
vortexing. After the addition of 50 µl of isopropyl alcohol,
triglyceride was determined with the GPO-Trinder colorimetric assay
(Sigma). Protein determination was by the Lowry method (47) using 5 µl of homogenate.
For cholesterol and cholesterol ester analysis, ~5 mg of tissue
homogenate protein was lipid-extracted, dried under N2, and resuspended in 10 µl of CHCl3. After the addition of 90 µl of 10% Triton-X100 in isopropyl alcohol, the sample was vortexed for 10 s, supplemented with 3 ml of Free Cholesterol C or
Cholesterol CII reagent (Wako), and assayed according to the
manufacturer's instructions except that samples were vortexed to
clarity prior to absorbance measurement.
Total tissue [14C]cholesterol retention in the various
tissues was determined by solubilizing tissue in alcoholic KOH (6 parts 50% KOH, 94 parts EtOH) at 65 °C and analyzing an aliquot by LSC. An aliquot of the liver and jejunum was used to make a 10% (w/v) tissue homogenate in phosphate-buffered saline, Folch-extracted, and
analyzed by thin layer chromatography (Silica Gel 150; Whatman) using a
solvent system of hexane/ethyl ether/glacial acetic acid (90:20:1).
Delivery of LXR Agonist T0901317--
Animals were given 30 mpk/day of the nonsteroidal LXR agonist T0901317 (Tularik) (16, 48)
once a day for 5 days in 600 µl of a vehicle consisting of 1.5%
carboxymethylcellulose and 0.15% Tween 20 (Sigma). Untreated animals
received the vehicle only. Animals were given ad libitum
access to food and water during the experiment except when 4-h fasting
blood samples were taken at 0, 72, and 96 h post-treatment. While
collecting data for subsequent analyses, on rare occasions we found
that individual animals treated with T0901317 gave values indicative of
hypersensitivity to the compound. These individuals yielded values far
greater than three S.D. values from the mean for their respective
groups. These outliers were removed from the analyses. No
vehicle-treated animals were removed.
Real Time Quantitative PCR--
Animals used for mRNA
analysis were fasted for 4 h prior to tissue collection. Total RNA
was isolated using the RNA-Bee reagent (Tel-Test, Inc., Friendswood,
TX) and purified using the RNeasy kit with DNase digestion (Qiagen).
First strand cDNA synthesis was performed from 1 µg of total RNA
from each animal with random hexamer primers using the Superscript II
first strand cDNA synthesis kit (Invitrogen). Specific primers for
each gene were designed using Primer Express software (Applied
Biosystems). The real time PCR contained, in a final volume of 25 µl,
25 ng of reverse transcribed total RNA and 240 nM forward
and reverse primers and was performed using SYBR Green Core
Reagents (PerkinElmer Life Sciences). PCR was carried out in 96-well
plates using the GeneAmp 5700 Sequence Detection System (Applied
Biosystems). All reactions were done in duplicate. The relative amount
of all mRNAs was calculated using the comparative
C method (49). Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as the invariant control. Primer
sequences are available upon request.
Protein Isolation and Immunoblotting--
Chicken liver and
jejunum homogenates were prepared from frozen tissues, analyzed by
SDS-PAGE, transferred to polyvinylidene difluoride (Immobilon-P;
Millipore), and probed for ABCA1 and glyceraldehyde-3-phosphate
dehydrogenase (50). Liver nuclear extracts and membrane fractions were
prepared as described (51), except that animals were fasted for 4 h prior to tissue collection. Sterol response element-binding protein-1
(SREBP-1) was detected using the monoclonal 2A4 SREBP-1 antibody
(ATCC). SCD1 was detected using a polyclonal rabbit anti-human SCD1
antibody (Xenon Genetics). VLDL receptor/vitellogenin receptor was
detected using a polyclonal rabbit anti-human LDLR354
antibody (52). Detection of signal was performed with the ECL or ECF
Western blot detection kit (Amersham Biosciences). Band densities were
quantified using ImageQuant 5.0 software.
Statistical Methods--
Statistical significance was determined
using a two-tailed Student's t test assuming equal
variance. Multiple regression analysis was performed using PROC MIXED
in SAS (SAS 8.00).
ABCA1 Dysfunction Results in a Deficit of Dietary Cholesterol in
the Circulation--
To determine the role of ABCA1 in the absorption
of dietary cholesterol from the intestinal lumen, the appearance of
dietary [3H]cholesterol in the circulation was followed
over a 24-h time course after oral gavage with
[3H]cholesterol-liposomes. The WHAM chickens showed a
severely reduced level of plasma [3H]cholesterol relative
to wild type, reaching only 21% of wild type after 24 h (Fig.
1A; p < 0.001). This difference is mainly attributable to a lack of
[3H]cholesterol in HDL; WHAM HDL
[3H]cholesterol reached only 2.9% of wild type values by
24 h (Fig. 1B; p < 0.001). However,
the plasma appearance rate of non-HDL [3H]cholesterol in
the plasma was similar in wild type and WHAM chickens during the time
course (p = 0.50; multiple regression analysis),
suggesting that secretion of [3H]cholesterol on
portomicrons is not affected in the WHAM chicken (Fig. 1C).
Between 8 and 24 h after gavage, a significant difference in the
non-HDL [3H]cholesterol is observed (Fig. 1C;
p = 0.02), possibly due to a lack of
[3H]cholesterol in HDL to participate in the transfer of
CE to apoB-containing lipoproteins.
Avian species have a less developed lymphatic system than mammals.
Intestinal apoB-containing lipoproteins are secreted directly into the
portal vein in birds, whereas in mammals they are secreted into the
mesenteric lymph. As a result, these particles are immediately accessible to the liver in the chicken. Therefore, the uptake and
resecretion of dietary cholesterol by the liver may be a factor in the
level of dietary cholesterol appearing in the plasma.
We hypothesized that ABCA1 dysfunction in the WHAM chicken causes
decreased plasma dietary cholesterol in at least one of four ways: 1)
inefficient basolateral cholesterol efflux into the circulation due to
lack of an HDL acceptor; 2) impaired apical cholesterol transport into
the intestine; 3) rapid clearance of HDL-C from the plasma; or 4)
inefficient basolateral cholesterol efflux into the circulation as HDL
due to a defective transporter.
Exogenous HDL Supplementation Does Not Increase the Entry of
Dietary Cholesterol into the Bloodstream--
The deficit of dietary
[3H]cholesterol in the bloodstream could be a cause or a
consequence of the HDL deficiency. The latter possibility was directly
tested by increasing the HDL pool size in the WHAM chicken by 13-fold,
bringing the HDL level to >50% of wild type. The HDL supplementation
had no effect on the appearance of dietary
[3H]cholesterol in the bloodstream during the 8-h time
course of this experiment in whole plasma (Fig.
2) or the HDL fraction (data not shown).
This result shows that the reduced transport of dietary [3H]cholesterol is not a consequence of the HDL
deficiency of the WHAM chicken.
Intestinal Apical Cholesterol Transport--
Reduced appearance of
dietary cholesterol in the bloodstream could be due to a defect in
apical transport from the intestinal lumen into the intestine or
basolateral transport from the intestine to the bloodstream. The
ABCA1-dependent absorption of cholesterol from the
intestinal lumen was defined as the percent of radiolabeled [14C]cholesterol gavage that was not recovered in the
total feces. No significant difference in cholesterol absorption was
detected in the 48-h fecal samples between wild type (76 ± 3.8%;
n = 5) and WHAM (67 ± 4.9%; n
= 5) chickens (Fig.
3A).
To determine whether an increase in intestinal ABCA1 expression reveals
an ABCA1-dependent cholesterol absorption phenotype, wild
type and WHAM male chickens were treated with the LXR agonist T0901317
or vehicle for 72 h prior to determining cholesterol absorption.
No significant difference was observed between wild type (49 ± 4.3%) and WHAM (46 ± 6.9%) chickens treated with T0901317 (Fig.
3A). However, treatment with T0901317 increased jejunum ABCA1 mRNA 10-fold (Table I) and
protein 4.5-fold (Fig. 4) and decreased
cholesterol absorption equally in wild type and WHAM chickens (by 35.1 and 31.9%, respectively; p < 0.05, both). Similar results were obtained using the fecal dual isotope method (data not
shown). Therefore, the LXR agonist-mediated effect on apical cholesterol transport is ABCA1-independent.
The Effect of an LXR Agonist on Plasma Levels of Dietary
Cholesterol--
The effect of the T0901317 LXR agonist on the amount
of the dietary cholesterol in the plasma was determined in the same
vehicle-treated and drug-treated animals. Plasma was collected 24 and
48 h after the [14C]cholesterol gavage, analyzed for
radioactivity, and expressed as a percentage of the tracer absorbed
into the intestine rather than a percentage of the initial tracer.
Because all of the feces was collected, we are able to confidently
determine the fraction of the initial gavage that was not recovered in
the feces and was absorbed into the intestine. By correcting for the
variation in apical absorption between vehicle and drug-treated
animals, the results highlight the processes occurring after the tracer enters the intestine. Consistent with the results shown in Fig. 1, the
vehicle-treated WHAM chickens had a much smaller proportion of absorbed
dietary cholesterol appearing in the plasma than the vehicle-treated
wild type chickens (2.0 ± 0.30% versus 6.7 ± 0.46%, p < 0.0001) (Fig. 3B). The LXR
agonist treatment resulted in a small, but not significant elevation of
plasma [14C]cholesterol relative to vehicle treatment in
the wild type but not in the WHAM chicken.
ABCA1 Dysfunction Does Not Affect Biliary Lipid Levels--
If
ABCA1 does not serve a significant role in the apical membrane of the
intestine and if ABCA1 localization is conserved between intestine and
liver, then the transport of biliary lipids across the hepatocyte
apical (canalicular) membrane should also be unaffected. The
concentrations of biliary lipids were analyzed in gall bladder bile
from wild type males, wild type females, and WHAM males and are
summarized in Table II. We found no
significant difference in the concentration of bile acids or
cholesterol; there was a nonsignificant reduction in the average
biliary phospholipid concentration between wild type and WHAM males.
Since ABCA1 in chickens is a sex-linked gene carried on the
Z chromosome, wild type males and females are homozygous and hemizygous
for ABCA1, respectively (in avian species, the female is
heterogametic). Unlike the difference in apoAI concentrations (38),
there was no significant difference in biliary lipids between wild type males and females. Therefore, the apical transport of cholesterol in
both the intestine and liver is unaffected by ABCA1 gene
dosage or dysfunction.
Dietary Cholesterol Is Retained in the Intestine and Liver of WHAM
Chickens--
The hypothesis that the low level of dietary cholesterol
in the WHAM plasma is due to a defect in basolateral efflux rather than
in intestinal absorption predicts that WHAM tissues accumulate dietary
cholesterol. Taking into account the amount of
[14C]cholesterol not recovered in the feces, we estimate
that 37% more 14C-labeled total cholesterol was found in
WHAM liver than in wild type liver (9.7 ± 0.83% versus 6.1 ± 0.29%, p < 0.005) (Fig.
5A) and 20% more in WHAM
intestine than in wild type intestine (13.8 ± 1.13%
versus 11.1 ± 0.50%, p < 0.005)
(Fig. 5A). The majority of accumulation of
[14C]cholesterol in WHAM intestine was in the jejunum
(data not shown), consistent with this segment having the maximal ABCA1
expression in mice (16). The [14C]cholesterol ester
content of WHAM liver and jejunum was 4.4- and 5.0-fold higher,
respectively, compared with wild type levels (Fig. 5B)
(p < 0.005). The level of 14C-free
cholesterol did not differ significantly between wild type and WHAM
chickens in the liver or jejunum (Fig. 5B). The accumulation of dietary [14C]cholesterol ester in the liver and
intestine of the WHAM predicts that the tissue cholesterol ester mass
of liver and jejunum should also be elevated in the WHAM chicken.
Indeed, the WHAM accumulates 2.0- and 2.8-fold (p < 0.005) more cholesterol ester mass in the liver and jejunum,
respectively, versus wild type animals (Fig. 5C).
We do not know whether the accumulation of cholesterol in the intestine
of the WHAM chicken is localized to enterocytes, macrophages, or
another cell type.
In both wild type and WHAM chickens, the T0901317 LXR agonist decreased
the amount [14C]cholesterol retained in the liver and
intestine (Fig. 5A). Treatment with T0901317 decreased the
[14C]cholesterol ester and cholesterol ester mass in the
WHAM liver ~55% (p < 0.05) relative to the
vehicle-treated WHAM but did not have a significant effect on the
[14C]cholesterol ester content in WHAM jejunum or in wild
type liver and jejunum; the wild type tissues already have low
[14C]cholesterol ester in the vehicle-treated state. Both
wild type and WHAM animals showed comparable decreases in
14C-free cholesterol in liver and jejunum. Since the amount
of [14C]cholesterol absorbed from the lumen was accounted
for, this suggests that the net effect of LXR activation is to reduce
the amount of cholesterol in these tissues.
Hypercatabolism of HDL-C Is Not Responsible for Intestinal
Accumulation of Dietary Cholesterol in the WHAM Chickens--
If ABCA1
is predominantly a basolateral cholesterol transporter in the
intestine, then there should be impaired free cholesterol efflux to HDL
and/or HDL precursors, resulting in cholesterol accumulation in the
intestine in the WHAM chicken. There is a possibility that there is
normal cholesterol efflux from the intestine and that HDL in the WHAM
chicken turns over rapidly and delivers its cholesterol to the
intestine. To test this possibility, autologous [14C]cholesterol-labeled HDLs were injected intravenously
into wild type and WHAM chickens. The apparent fractional clearance
rate was 2.3-fold higher in the WHAM than the wild type chickens (Fig. 6A). Similarly, the amount of
[14C]cholesterol that accumulated in the livers was
2.3-fold higher in WHAM than in wild type animals (Fig. 6B).
However, there was no increase in [14C]cholesterol
accumulation in WHAM intestine (Fig. 6B). Therefore, the
intestinal accumulation of dietary cholesterol in the WHAM chicken
cannot be due to intestinal reuptake of cholesterol from HDL. Because
dietary cholesterol is secreted into the portal circulation rather than
into the lymph, a liver-specific defect in efflux could lead to the low
level of dietary cholesterol observed in the WHAM plasma. The
accumulation of dietary cholesterol in both the intestine and liver
suggests that impaired efflux of cholesterol from both tissues in the
WHAM chicken contributes to the lack of dietary cholesterol in the
bloodstream (Fig. 1).
The Effect of an LXR Agonist on Plasma and Tissue Lipid
Mass--
LXR activation with T0901317 had a dramatic effect on plasma
cholesterol and triglyceride in wild type chickens but, surprisingly, had little effect in the WHAM chickens (Fig.
7A). The most pronounced increases were in the apoB-containing lipoproteins. The wild type chickens had a 28.8-fold (p < 0.001) and 27.7-fold
(p < 0.01) increase in VLDL cholesterol and
triglyceride, respectively, whereas the WHAM chickens showed only a
2-3-fold increase in both lipids (p < 0.01) (Fig. 7,
B and C). LXR activation elicited a small but
significant (p < 0.05) increase in HDL-C in wild type
but not WHAM animals. LXR activation resulted in a 5-fold increase in
triglyceride levels in the livers of the wild type chicken (p < 0.05) (Fig. 8) but
no significant effect in the WHAM liver or in the jejunum of either
chicken.
LXR Agonist-induced Triglyceride Synthesis Is Impaired in the
WHAM Chicken--
Many lipogenic genes are transcriptionally regulated
by LXRs (48) and/or SREBP-1c, which exists as an inactive
membrane-bound precursor, and a mature cytosolic form that enters the
nucleus and transactivates promoters that contain a sterol response
element (53). The cleavage of SREBP-1c to its mature form is inversely correlated with intracellular cholesterol levels. Therefore, we hypothesized that the decreased level of LXR agonist-induced
triglyceride synthesis in the WHAM chicken (Figs. 7 and 8) is caused by
a difference in the amount of mature SREBP-1c. The level of hepatic
SREBP-1c mRNA increased 6-fold in both wild type and WHAM chickens
in response to the LXR agonist (Table I). This is consistent with the
LXR agonist-induced increase in SREBP-1c previously observed in wild type mice (54). However, immunoblot analysis of SREBP-1c protein revealed that the amount of hepatic SREBP-1c protein in the WHAM chicken, for both the membrane-bound precursor and mature nuclear form,
was lower than that of wild type in the untreated and LXR agonist-treated states (Fig.
9A). The plasma triglyceride
levels correlated with the levels of SREBP-1c protein; WHAM plasma
triglycerides are decreased in the vehicle-treated condition relative
to wild type (0.2 mg/ml versus 0.5 mg/ml, p = 0.03), consistent with previous observations in the WHAM chicken
(38). The wild type and WHAM levels of membrane-bound and mature
SREBP-1c protein show comparable increases in response to the LXR
agonist, but the increase in mature SREBP-1c elicited by the LXR
agonist in the WHAM is still below the level of untreated wild type.
However, the levels of four SREBP-1c-regulated mRNAs were brought
to the same level in response to the LXR agonist in the wild type and
WHAM chickens (SCD1, fatty acid synthase, malic enzyme, and acetyl-CoA
carboxylase) (Table I). This suggests that in chickens, the
transcription of some lipogenic genes may be subject to direct LXR
transactivation as well as indirectly through increased expression of
SREBP-1c. This is consistent with T0901317 treatment of the SREBP-1c
knockout mouse, where the same lipogenic mRNAs showed an
SREBP-1c-independent response to LXR activation (55). It is possible
that the potent transcriptional activation effect of the LXR agonist
overrides any difference in mature SREBP-1c with respect to these
co-regulated genes. Interestingly, as observed with SREBP-1c, liver
SCD1 mRNA levels did not correlate with SCD1 protein (Fig.
9B and Table I). As a control protein, we probed for chicken
very low density lipoprotein receptor/vitellogenin receptor and
observed no difference between the wild type and WHAM chickens and no
difference between control and agonist-treated animals of either
genotype. This suggests impaired post-transcriptional regulation of
SREBP-1c and SCD1, as observed in the comparison of mRNA and
protein data, and possibly impaired SREBP-1c-regulated transcription,
as evidenced by the slightly decreased mRNA levels of some SREBP-1c
targets in vehicle-treated WHAM chickens. No significant difference was
observed for SREBP-2 or HMG-CoA-reductase due to LXR-activation or
between wild type and WHAM animals.
In this study, we have attempted to determine the polarity of
ABCA1-mediated cholesterol efflux from the intestine by measuring two
distinct steps in the absorption of dietary cholesterol: transport from
the intestinal lumen into the intestinal epithelial cells and transport
from the intestine to the bloodstream. Since macrophages apparently are
not a major source of ABCA1-mediated cholesterol and phospholipid for
HDL (56), it is likely that the liver and/or intestine supply most
of the cholesterol and phospholipid for HDL in an
ABCA1-dependent manner. This would predict that ABCA1 functions to transport lipids across the basolateral membrane in
hepatocytes and enterocytes. This prediction is borne out in recent
studies in WIF-B and Caco-2 cells (26-28).
A number of studies have explored the role of ABCA1 in whole body
cholesterol homeostasis in mice by disruption of the ABCA1 gene. Investigations into cholesterol metabolism in two different ABCA1 knockout mouse strains have found that ABCA1 is
required to maintain plasma HDL-C and phospholipid levels (57, 58). These mice have also been used to explore lipid metabolism in the
intestine and liver. In SV129/C57BL/6 hybrid mice, loss of ABCA1 resulted in a small but significant decrease in
intestinal cholesterol absorption on a cholesterol-free diet, with an
associated increase in the fecal neutral sterol excretion (57, 59). In contrast, another ABCA1 In contrast to the ABCA1 Unlike the ABCA1 Intravenously injected autologous HDL labeled with
[14C]cholesterol was cleared about 2-fold more rapidly in
WHAM than in wild type chickens (Fig. 6A). This did not
result in an increase in [14C]cholesterol accumulation in
the intestine. Therefore, the accumulation of cholesterol in the
intestine is not a consequence of enhanced cholesterol clearance. Since
the amount of cholesterol absorbed from the gut lumen was unaltered,
and portomicron secretion is unaffected, the accumulation of dietary
cholesterol in the intestine is best explained by a decrease in the
basolateral efflux from this tissue. Although hypercatabolism of HDL
cholesterol does occur in the WHAM chicken, a defect in dietary
cholesterol efflux from the intestine would precede this effect, and
only the small fraction of HDL that is formed is subject to rapid
clearance. Therefore, it is likely that the hypercatabolism of
HDL-cholesterol is quantitatively less important in the case of dietary
cholesterol than in the direct injection of HDL-cholesterol into the bloodstream.
Decreased basolateral efflux could be caused by a primary transport
defect or by the paucity of an HDL acceptor. To test the latter
hypothesis, we increased HDL levels 13-fold in the WHAM chicken. This
had no effect on the plasma appearance of dietary cholesterol, thus
ruling out HDL deficiency as the cause of the transport defect (Fig.
2). Furthermore, if hypercatabolism of unstable HDL particles was the
reason for the reduced level of plasma dietary cholesterol, then we
should have been able to rescue this defect when we supplemented the
animal with stable HDL. This did not happen (Fig. 2), suggesting once
again that hypercatabolism of dietary cholesterol is secondary to a
defect in the secretion of that cholesterol into the bloodstream. The
inability of HDL supplementation to rescue plasma cholesterol levels
should apply to the intestine and the liver. Together with the
accumulation of hepatic cholesterol, this result provides a case for
defective cholesterol efflux from the liver.
Mutations in other ABC transporters have been shown to affect biliary
lipid concentrations in both humans and mice. The canalicular membrane
protein, P-glycoprotein (MDR3 in humans, MDR2 in mice), encoded by the
ABCB4 gene, promotes the translocation of PC into the bile,
and mice lacking this protein show a complete absence of biliary PC and
reduced levels of biliary cholesterol (63). Additionally, ABCB11 (BSEP)
mediates the transport of bile salts from the liver, and mutations in
ABCB4 or ABCB11 in humans are responsible for
progressive familial intrahepatic cholestasis (64, 65). Sitosterolemic
patients, with mutations in ABCG5 or ABCG8, have
impaired biliary secretion of neutral sterols (18-22, 66). Indeed,
recent data show that ABCG5 and ABCG8 are localized in the canalicular
membrane, consistent with a role in biliary cholesterol secretion (24).
The data suggest, however, that ABCA1 is not involved in biliary
secretion; neither the WHAM chicken (Table II) nor the
ABCA1 Treatment of mice with LXR or RXR agonists elicited a dramatic decrease
in cholesterol absorption, concomitant with an increase in liver and
intestine ABCA1 mRNA (16). However, the discovery of
ABCG5 and ABCG8 (18-24) subsequent to these
studies beckons a re-evaluation of the role of LXR agonists in
cholesterol absorption. We found that treatment of wild type and WHAM
chickens with the LXR agonist T0901317 caused a similar decrease in
cholesterol absorption in both animals, as well as increases in ABCA1
mRNA and protein in the liver and intestine of both animals (Figs. 3A and 4; Table I). This suggests that ABCA1 does not
function at the apical membrane and that another mechanism is
responsible for the LXR-induced decrease in cholesterol absorption.
ABCA1+/+ and ABCA1 As observed in mice and hamsters receiving RXR or LXR agonists (48), as
well as humans receiving rexinoid therapy (69), wild type chickens
treated with an LXR agonist developed severe hypertriglyceridemia
(Figs. 7 and 8). Using LXR LXR agonist-treatment of C57BL/6J mice increased the VLDL triglyceride
(TG) secretion rate without elevating plasma TG, indicating efficient
plasma TG clearance (70). In contrast, mouse models of defective
lipoprotein TG clearance show dramatic plasma TG accumulation in
response to the LXR agonist. Chickens lack apoE and may have a lower
capacity for VLDL TG clearance than mice.
The WHAM chicken faithfully displays all of the known phenotypes
associated with Tangier disease. The chicken and human ABCA1 proteins
are 85% identical. Dysfunctional ABCA1 results in a profound HDL
deficiency despite normal production of apoA1 (15, 38). This results in
the production of lipid-deficient HDL particles, which are rapidly
catabolized by the kidneys (15). Defective cholesterol efflux results
in cholesterol accumulation in the spleen, liver, and small intestine.
The similarities between the chicken and human ABCA1 proteins and the
parallels between the WHAM chickens and patients with Tangier disease
suggest that our observations regarding the polarity of ABCA1
function are likely to occur in humans.
The basolateral function of ABCA1 in the intestine and the liver is
consistent with these two organs playing a major role in HDL production
(Fig. 10). Early studies involving
mesenteric lymph collection (71) and liver perfusion (72) concluded
that HDL was produced in these two organs. Tissue-specific knockout experiments will be required to sort out the relative contributions of
liver and intestine to HDL production. Although an intact ABCA1 pathway
is essential for macrophages to rid themselves of excess cholesterol,
it is unlikely that macrophages contribute to the bulk of phospholipid
and cholesterol that comprises the HDL pool (56). Thus, the basolateral
function of ABCA1 also implies that the bulk of HDL cholesterol and
phospholipid originates and terminates in the intestine and the liver
(Fig. 10). One implication is that modulation of the ABCA1 pathway in
macrophages might be anti-atherogenic without necessarily altering HDL
levels or significantly affecting reverse cholesterol transport (56,
73).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-HDL. The subsequent reaction catalyzed by
lecithin-cholesterol acyltransferase allows for the transfer of an acyl
group from phosphatidylcholine (PC) to cholesterol, forming CE that
partitions to the core of the HDL particle and allows for a high
capacity cholesterol sink. Dysfunctional ABCA1 prevents the early
lipidation step in HDL biogenesis, thereby preventing the ability of
HDL to serve in reverse cholesterol transport (14, 15).
) and homozygous males (ZZ) (39). Plasma apoAI concentrations in normal chickens correlate with
ABCA1 gene dosage; females and heterozygous males have half the normal male apoAI levels (38). Cultured hepatocyte and intestinal loop experiments concluded that apoAI secretion is unaffected in the
WHAM chicken. Subsequent experiments showed that the WHAM chicken lacks
the ability to efficiently lipidate free apoAI, shown by the
drastically increased catabolism of 125I-labeled apoAI, in
contrast to a much slower rate of 125I-labeled HDL
catabolism (15, 38).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sitostanol (American Radiolabeled
Chemicals) and 1 µCi of [14C]cholesterol per animal
were delivered via oral gavage after a 4-h fast. Animals were given
ad libitum access to food and water after gavage. Total
feces were collected into Petri dishes after 24 and 48 h,
desiccated, ground, and weighed. Triplicate 30-mg aliquots of each
feces sample were solubilized and analyzed with a dual label program on
the LSC. Cholesterol absorption was determined by calculating the total
fecal recovery of [14C]cholesterol or by comparing the
initial and fecal 14C/3H isotopic ratio (44)
using the following equation: percentage of cholesterol absorption = (1
((14C/3H)feces/(14C/3H)initial))100.
However, the data used in this paper are derived from total fecal
[14C]cholesterol recovery, because the small amount of
sitostanol that is absorbed can lead to an overestimation of
cholesterol absorption (data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Plasma appearance of dietary
[3H]cholesterol in wild type and WHAM chickens.
Fasted animals were given an oral gavage of 100 µCi of
[3H]cholesterol-liposomes. Blood was drawn at the
indicated time points, and the specific activity of whole plasma
(A), HDL (B), and non-HDL (C) was
determined. Data represent mean and S.E. (n = 4).
Multiple regression analysis between wild type and WHAM is shown:
p < 0.0001 (a), p < 0.0001 (b), p = 0.50 (c).
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Fig. 2.
Plasma appearance of dietary
[3H]cholesterol following HDL supplementation in the WHAM
chicken. Fasted animals given an oral gavage of 100 µCi of
[3H]cholesterol-liposomes. WHAM chickens were injected
intravenously with saline or wild type HDL to raise the HDL pool size
13-fold. Wild-type chickens were injected with saline. Blood was drawn
at the indicated time points, and the specific activity of whole plasma
was determined. Data represent mean and S.E. (n = 3).
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Fig. 3.
Effects of an LXR agonist on intestinal
absorption and plasma appearance. Animals were treated for 5 days
with vehicle only, or 30 mpk T0901317 and were given an oral gavage of
1 µCi of [14C]cholesterol on day 3 of treatment. Feces
and blood were collected 24 and 48 h later and analyzed for
specific activity. Intestinal cholesterol absorption (A) was
calculated from the percentage fecal recovery of
[14C]cholesterol in pooled 24- and 48-h feces. Plasma
levels of [14C]cholesterol (B) were normalized
to individual animal absorption values determined in A. Data
represent mean and S.E. for wild type and WHAM, vehicle, and
T0901317-treated (n = 5 in each group); *, wild type
versus WHAM; #, untreated versus
T0901317-treated; */#, p < 0.05; **/##,
p < 0.01; ***/###, p < 0.001.
Real-time quantitative PCR of ABCA1, SREBP, and SREBP-regulated genes
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Fig. 4.
Effects of an LXR agonist on liver and
jejunum ABCA1 protein. Liver and jejunum from animals treated with
(n = 6) or without (n = 3) 30 mpk
T0901317 were homogenized, and ABCA1 protein levels were detected by
SDS-PAGE and immunoblot analysis. Equal portions of tissue from
vehicle-treated (n = 3) and T0901317 (n = 6) were pooled. 20 µg of liver (A) and 40 µg of
jejunum protein (B) were detected using a monoclonal ABCA1
antibody and normalized to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) levels. F, wild type female; ( ),
vehicle-treated; (+), T0901317-treated. Data are expressed as -fold
change relative to vehicle-treated wild type male.
Biliary lipid measurements
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Fig. 5.
Cholesterol accumulation in
tissues. Liver and intestine were collected from wild type and
WHAM animals used in Fig. 3 and analyzed for retention of
[14C]cholesterol. A, liver, duodenum, jejunum,
and ileum were solubilized in alcoholic NaOH and analyzed by LSC for
total [14C]cholesterol. Tissue radiolabel was normalized
to individual animal absorption determined in Fig. 3. B, an
aliquot of liver and jejunum was subjected to thin layer chromatography
to separate free (FC) and esterified (CE)
[14C]cholesterol and analyzed by LSC. The ratio of
[14C]FC to [14C]CE was used to determine
total tissue [14C]FC and [14C]CE.
C, cholesterol mass in liver and jejunum of vehicle- and
T0901317-treated animals. Data are expressed as mean and S.E. for
n = 5 animals of each group (wild type and WHAM,
vehicle-treated or T0901317-treated); *, genotype (wild type
versus WHAM); #, untreated versus
T0901317-treated; */#, p < 0.05; **/##,
p < 0.01; ***/###, p < 0.001.
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Fig. 6.
Plasma disappearance of injected HDL
cholesterol. Autologous HDL labeled with
[14C]cholesterol was injected into wild type and WHAM
chickens (n = 3), and total radioactivity in the plasma
(A) was determined at the indicated times and in the tissues
(B) after 24 h. Data represent mean and S.E.; *,
p < 0.05; **, p < 0.01; ***,
p < 0.001.
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Fig. 7.
Effect of an LXR agonist on lipoprotein
lipids. Plasma cholesterol and triglyceride values were monitored
in animals treated with (n = 6) or without
(n = 5) 30 mpk T0901317 for 5 days. Fasting
plasma was analyzed on day 0, day 3, and day 5 for total plasma
cholesterol and triglyceride (A), day 3 FPLC-fractionated
lipoprotein cholesterol (B), and day 3 FPLC-fractionated
lipoprotein triglyceride (C). Data are expressed as mean and
S.E.; *, genotype (wild type versus WHAM); #, untreated
versus T0901317-treated; */#, p < 0.05;
**/##, p < 0.01; ***/###, p < 0.001.
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Fig. 8.
Effect of an LXR agonist on tissue
lipids. Tissue triglyceride values were determined in animals
treated with (wild type, n = 7; WHAM,
n = 6) or without (n = 5) 30 mpk
T0901317 for 5 days. Samples were normalized to protein. Data are
expressed as mean and S.E.; *, genotype (wild type versus
WHAM); #, untreated versus T0901317-treated; */#,
p < 0.05; **/##, p < 0.01; ***/###,
p < 0.001.
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Fig. 9.
The effect of the WHAM mutation on SREBP-1c
and SCD1 protein. Livers from animals treated for 5 days with
(n = 6) or without (n = 3)
30 mpk T0901317 were pooled and used to prepare membrane and nuclear
protein fractions as described elsewhere (47). 80 µg of membrane or
nuclear protein were subjected to SDS-PAGE and immunoblot analysis to
detect precursor and mature SREBP-1c (A) and SCD1 and the
control protein, VLDL receptor/vitellogenin receptor
(VLDLR/VTGR) (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mouse, created in a
DBA background, showed an increase in cholesterol absorption on both a
chow and Western diet, and the fecal excretion of neutral sterols was
unaffected (58, 60, 61). Despite heavy Oil Red O-positive lipid
staining in the liver, the SV129 ABCA1
/
mouse had no accumulation of cholesterol ester in the liver or intestine (57, 59). Interestingly, the DBA
ABCA1
/
mouse had either no accumulation or a
significant decrease in cholesterol ester levels in the liver and
intestine, depending on the study (61, 62).
/
mouse models, the
WHAM chicken accumulates cholesterol ester in the liver and intestine
(Fig. 5B). This phenomenon might be explained by the unique
avian physiology. Because dietary cholesterol is secreted into the
portal circulation rather than into the lymph, these particles are
immediately accessible to the liver in the chicken. This might expose
the avian liver to more cholesterol than the mammalian liver and could
result in the more dramatic hepatic cholesterol ester accumulation of the WHAM chicken.
/
mouse models, the
absorption of cholesterol from the intestinal lumen into the intestine
was unaltered in the WHAM chicken (Fig. 3). However, absorption of
cholesterol from the lumen into the bloodstream was severely reduced.
By 24 h after the oral gavage of radiolabeled cholesterol, the
plasma from the wild type chicken contained about 5-fold more tracer than did WHAM plasma. Two conclusions can be drawn from this
observation: 1) the WHAM mutation results in a decrease in the
secretion of cholesterol to the bloodstream, or 2) the WHAM mutation
results in rapid clearance of cholesterol from the bloodstream.
/
mouse strains show any abnormality in
biliary levels of cholesterol or phospholipid (59).
/
mice treated with T0901317 show similar increases in hepatobiliary and
fecal cholesterol excretion (29). Since biliary cholesterol excretion
has been shown to regulate the percentage of dietary cholesterol
absorption (67), it is possible that the decreased absorption elicited
by the LXR agonist is due to increased ABCG5/G8-mediated biliary
neutral sterol excretion as well as efflux of sterol from the
enterocyte into the lumen (21, 24, 29, 68).
and LXR
knockout mice, Repa et al. showed that this LXR-induced lipogenesis
is due to the transcriptional activation of SREBP-1c (54).
However, we unexpectedly observed a differential lipogenic response to the LXR agonist in wild type and WHAM chickens in both the plasma (Fig.
7) and the liver (Fig. 8). Consistent with this data, we found that the
WHAM chicken had a decreased amount of both membrane-bound SREBP-1c,
mature SREBP-1c and SCD1 protein, relative to wild type in both the
basal vehicle-treated state and the LXR agonist induced state (Fig. 9).
The mechanism for the effect of ABCA1 dysfunction on triglyceride
synthesis is unknown but may be caused by instability of SREBP-1c and
lipogenic enzymes in the endoplasmic reticulum membrane, possibly due
to the intracellular accumulation of the E89K ABCA1 variant (37).
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Fig. 10.
Polarity of cholesterol efflux from the
liver and intestine. ABCA1 is a basolateral
cholesterol/phospholipid transporter in both the liver and the
intestine (phospholipid is not shown for simplicity and may well be the
primary substrate). This results in transport to the lymph and
bloodstream from the enterocyte and across the sinusoidal membrane into
the bloodstream from hepatocytes. This localization predicts a major
contribution to HDL lipids and the profound HDL deficiency associated
with ABCA1 mutations. It also predicts a defect in the transport of
dietary cholesterol to the bloodstream, as reported here. ABCG5/G8 is
an apical cholesterol transporter and thus opposes cholesterol
absorption from the lumen of the intestine. In the liver, ABCG5/G8
transports cholesterol across the canalicular membrane into bile. This
orientation predicts the increased absorption of dietary cholesterol
and defective secretion of biliary cholesterol seen in patients with
mutations in ABCG5 or ABCG8. ABCA1 is
important for macrophages to efflux cholesterol and phospholipid, but
it is unlikely that this tissue source is quantitatively important for
HDL production.
In summary, we provide evidence that ABCA1 is involved in the
absorption of dietary cholesterol by mediating basolateral, but not
apical, efflux of cholesterol to apoAI/HDL from liver and intestine.
ABCA1 dysfunction in chickens results in increased levels of
intracellular cholesterol ester accumulation in the liver and
intestine, suggesting that these tissues are a major source of HDL
lipid. Additionally, the reduction of mature SREBP-1c and SCD1 is
indicative of a more global disruption of lipid homeostasis in the WHAM
chicken and perhaps also in humans with dysfunctional ABCA1.
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ACKNOWLEDGEMENTS |
---|
We thank Stephen Turley for biliary lipid analysis and Mark Cook and Bernard Wentworth for advice in avian physiology. We thank Louis Armentano, Peter Crump, and Jonathan Stoehr for help with the statistical and kinetic analyses of our data.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health (NIH) Grant HL56593 and Xenon Genetics, Inc.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.
§ Supported by NIH Biology of Aging Training Grant T32-AG00213.
¶ These two authors contributed equally to this work.
§§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706-1544. Tel.: 608-262-1372; Fax: 608-263-9609.
¶¶ Supported by National Institutes of Health Molecular Biosciences Training Grant T32-GM07215-25.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212377200
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ABBREVIATIONS |
---|
The abbreviations used are: HDL, high density lipoprotein; CE, cholesterol ester; PC, phosphatidylcholine; LXR, liver X receptor; RXR, retinoid X receptor; WHAM, Wisconsin hypoalpha mutant chicken; SREBP, sterol responsive element-binding protein; SCD, stearoyl-CoA desaturase; TG, triglyceride; LSC, liquid scintillation counting; HDL-C, HDL-cholesterol; FPLC, fast protein liquid chromatography; VLDL, very low density lipoprotein; mpk, mg/kg of body weight.
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