ABCA1 Is Essential for Efficient Basolateral Cholesterol Efflux during the Absorption of Dietary Cholesterol in Chickens*

Jacob D. MulliganDagger §, Matthew T. FlowersDagger ¶¶, Angie TebonDagger , J. James Bitgood||, Cheryl Wellington**, Michael R. Hayden**Dagger Dagger , and Alan D. AttieDagger §§

From the Departments of Dagger  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 Dagger Dagger  Xenon Genetics, Burnaby, British Columbia V5G 4W8, Canada

Received for publication, December 5, 2002, and in revised form, January 22, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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).

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/-) 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).

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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]-beta -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).

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).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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).

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.


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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).

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).


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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.

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.


                              
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Table I
Real-time quantitative PCR of ABCA1, SREBP, and SREBP-regulated genes
Total RNA was isolated from liver and jejunum of vehicle-treated (n = 3) and T0901317-treated (+LXR, n = 7) wild type (WT) and WHAM male chickens and cDNA from individual animals subjected to real time PCR quantification on the listed genes as described. Glyceraldehyde-3-phosphate dehydrogenase was used as the invariant control. Values are expressed relative to vehicle-treated wild type animals. *, genotype (wild type versus WHAM); #, vehicle-treated versus T0901317-treated; */#, p < 0.05; **/##, p < 0.01; ***/###, p < 0.001. FAS, fatty acid synthetase; ACC, acetyl-CoA carboxylase.


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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.

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.


                              
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Table II
Biliary lipid measurements
Bile from 6-week-old wild type males (n = 6), wild type females (n = 6), and WHAM males (n = 7) was collected and analyzed for biliary concentrations of bile acids, cholesterol, and phospholipid. Data represent mean and S.E.

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.


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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.

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).


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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.

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.


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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.

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.


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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

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-/- 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).

In contrast to the ABCA1-/- 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.

Unlike the ABCA1-/- 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.

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-/- mouse strains show any abnormality in biliary levels of cholesterol or phospholipid (59).

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-/- 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).

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 LXRalpha and LXRbeta 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).

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).


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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.

    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.

    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

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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