Stimulation of Cholesterol Excretion by the Liver X Receptor Agonist Requires ATP-binding Cassette Transporters G5 and G8*

Liqing YuDagger , Jennifer YorkDagger , Klaus von Bergmann§, Dieter Lutjohann§, Jonathan C. Cohen, and Helen H. HobbsDagger ||**

From the Dagger  McDermott Center for Human Growth and Development, the Departments of Molecular Genetics and Internal Medicine,  Center for Human Nutrition and || the Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, the § Department of Clinical Pharmacology, University of Bonn, Bonn 53105, Germany

Received for publication, February 6, 2003, and in revised form, February 21, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Liver X receptor (LXR) is a nuclear receptor that plays a crucial role in orchestrating the trafficking of sterols between tissues. Treatment of mice with a potent and specific LXR agonist, T0901317, is associated with increased biliary cholesterol secretion, decreased fractional cholesterol absorption, and increased fecal neutral sterol excretion. Here we show that expression of two target genes of LXRalpha , the ATP-binding cassette (ABC) transporters Abcg5 and Abcg8, is required for both the increase in sterol excretion and the decrease in fractional cholesterol absorption associated with LXR agonist treatment. Mice expressing no ABCG5 and ABCG8 (G5G8-/- mice) and their littermate controls were treated for 7 days with T0901317. In wild type animals, treatment with the LXR agonist resulted in a 3-fold increase in biliary cholesterol concentrations, a 25% reduction in fractional cholesterol absorption, and a 4-fold elevation in fecal neutral sterol excretion. In contrast, the LXR agonist did not significantly affect biliary cholesterol levels, fractional cholesterol absorption, or neutral fecal sterol excretion in the G5G8-/- mice. Thus Abcg5 and Abcg8 are required for LXR agonist-associated changes in dietary and biliary sterol trafficking. These results establish a central role for ABCG5 and ABCG8 in promoting cholesterol excretion in vivo.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cholesterol is an important structural component of animal cell membranes. The cholesterol required to maintain membrane integrity can be synthesized de novo from acetyl-CoA or can be obtained from cholesterol-containing foods in the diet. The typical Western diet includes ~400 mg of cholesterol per day, of which 40-50% is absorbed in the proximal small intestine. The major pathway by which cholesterol is eliminated from the body is by excretion into bile either as free cholesterol or after conversion to bile acids.

A variety of noncholesterol sterols are also present in the diet. The most plentiful of these are the two plant sterols, sitosterol and campesterol. The levels of these sterols in tissues are very low, because plant sterols are poorly absorbed from the intestine and are preferentially secreted into the bile by hepatocytes (1-3).

One mechanism by which excess cholesterol and other sterols are eliminated from the body involves the action of two ATP-binding cassette (ABC)1 half-transporters, ABCG5 and ABCG8 (4, 5). Mutations in either of these genes cause sitosterolemia, a rare autosomal recessive disorder of sterol trafficking (4, 5). Subjects with sitosterolemia have increased fractional absorption of dietary noncholesterol sterols and decreased biliary secretion of plant- and animal-derived sterols (6, 7). Consequently, these patients accumulate sitosterol, as well as other plant- and shellfish-derived neutral sterols, in the blood and tissues (8, 9). Subjects with sitosterolemia also are frequently hypercholesterolemic and develop tendon xanthomas and premature coronary artery disease (8, 10).

The pivotal role of ABCG5 and ABCG8 in enterohepatic sterol transport has been demonstrated directly by manipulating the expression of these genes in mice (11, 12). Transgenic mice containing ~14 copies of a human genomic DNA fragment, including both human ABCG5 and ABCG8 genes have a ~50% reduction in the fractional absorption of dietary cholesterol, dramatically elevated levels of biliary cholesterol, and a 4.5-fold increase in fecal neutral sterol excretion (11). Plant sterol levels are more than 50% lower in these mice than in their wild type littermates. Disruption of the mouse Abcg5 and Abcg8 genes has the opposite effect on dietary sterol trafficking. The G5G8-/- mice have 30-fold higher plasma levels of sitosterol than do their wild type littermates due to increased fractional absorption of dietary plant sterols and impaired biliary sterol excretion (12).

Abcg5 and Abcg8 are expressed predominantly in the liver and small intestine (4) and are coordinately up-regulated at the transcriptional level by dietary cholesterol. The response of Abcg5 and Abcg8 to cholesterol requires the liver X receptor alpha  (LXRalpha ) (13), a nuclear receptor that regulates the expression of many key genes in lipid metabolism, including ABCA1 (14, 15), the gene mutated in Tangier disease (16-19), murine (but not human) cholesterol 7alpha -hydroxylase (Cyp7A1) (20-22), the rate-limiting enzyme in bile acid synthesis, and sterol regulatory element-binding protein 1c (SREBP-1c) (23), an important transcription factor in the regulation of fatty acid biosynthesis (24). By regulating the expression of these genes, LXRalpha coordinates the synthesis and trafficking of cholesterol and fatty acids between tissues. Mice lacking LXRalpha accumulate large amounts of cholesterol in the liver when fed a high cholesterol diet (21), whereas wild type mice treated with an LXR agonist have decreased fractional absorption of dietary cholesterol (14) and increased biliary cholesterol excretion (25).

The mechanism by which LXRalpha prevents the accumulation of dietary cholesterol has not been fully defined. The decreased fractional absorption of dietary cholesterol associated with LXR agonist treatment was attributed initially to the action of ABCA1, because levels of ABCA1 mRNA increased dramatically in the small intestine of animals given the nonsteroidal synthetic LXR agonist: N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)phenyl]benzenesulfonamide (T0901317) (14). Subsequent characterization of mice expressing no ABCA1 (Abca1-/- mice) revealed no impairment in biliary cholesterol secretion or fecal neutral sterol excretion (25, 26). In the current study, we tested the hypothesis that ABCG5 and ABCG8 mediate the LXR agonist-associated increase in biliary and fecal excretion of cholesterol and reduction in cholesterol absorption.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The synthetic LXR agonist T0901317 was purchased from Cayman Chemical Company (Ann Arbor, MI). Sterols were obtained either from Steraloids Inc. (Newport, RI) or from Sigma-Aldrich (St. Louis, MO).

Animals and Diets-- Mice homozygous for a disrupted Abcg5 and Abcg8 allele (G5G8-/-) were generated as described previously (12). The mice used in these studies were offspring of G5G8+/- mice of mixed genetic background (129S6SvEv × C57BL/6J). The mice were housed in plastic cages in a temperature-controlled room (22 °C) with a daylight cycle from 6 am to 6 pm, and fed ad libitum a cereal-based rodent chow diet (Diet 7001, Harlan Teklad, Madison, WI) containing 0.02% cholesterol and 4% fat. All animal procedures were performed with approval of the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center.

T0901317 Treatment-- Diets containing 0.025% of T0901317 (T-diet) were made by mixing powdered chow diet (Diet 7001, Harlan Teklad) with pure T0901317. The T-diet was stored in aluminum foil-covered containers at 4 °C for no more than 3 days. Female mice were housed individually 1 week before initiation of the T-diet and then fed for 1 week with either the T-diet or the chow diet dispensed from a feeder jar. The dose and duration of T0901317 treatment were based on prior studies (14, 27).

Lipid Chemistries-- Sterol levels in plasma and liver were measured by gas chromatography (GC) as described (28, 29) with some modifications. Briefly, plasma and tissues were saponified in 3% potassium hydroxide/ethanol at 66 °C for 3 h after addition of 5alpha -cholestane as a quantitative recovery standard. Lipids were extracted using petroleum ether and dried under nitrogen. The residual lipids were re-dissolved in Tri-Sil reagent (product 48999, Pierce, Rockford, IL) for analysis by GC. Hepatic triglyceride levels were measured using Infinity triglycerides reagent (Sigma-Aldrich, St. Louis, MO). Lipoproteins were size-fractionated using fast protein liquid chromatography (FPLC), and the total sterol concentration in each fraction was measured using cholesterol/HP kits (catalog number 1127771, Roche Diagnostics Corp., Indianapolis, IN).

Biliary Lipid Composition-- Bile was collected from the gallbladder of anesthetized mice using a 30.5-gauge needle. The concentrations of cholesterol, phospholipids, and bile acids were measured as described previously (30).

Fecal Sterol Excretion-- Mice were fed either the control or T-diet for 4 days prior to being moved to new cages containing fresh wood shavings. The diets were fed for an additional 3 days during which time the feces were collected. The feces were dried, weighed, and ground to a fine powder. An aliquot of 0.5 g of feces was used to determine fecal neutral and acidic sterol excretion (11, 28).

Fractional Absorption of Dietary Cholesterol-- Mice were fed either the control or T-diet for 4 days prior to administering an oil mixture containing deuterated cholesterol and sitostanol by gavage (12). Mice were then housed individually in new cages containing fresh wood shavings. The diets were continued, and the feces were collected for 3 days and processed for sterol absorption as described (12).

Quantitative Real-time PCR-- Total RNA was extracted from tissues using the RNA Stat-60 kit (Tel-Test Inc., Friendswood, TX), and real-time PCR was performed to assay the relative amounts of selected mRNAs as described (31, 32).

Statistical Analysis-- All data are reported as the mean ± S.E. The differences between the mean values were tested for statistical significance by the two-tailed Student's t test.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LXR Activation Increases Plasma Levels of Sitosterol and Campesterol in G5G8-/- Mice-- Plasma levels of sitosterol and campesterol were 30-fold higher in G5G8-/- than in G5G8+/+ female mice fed a chow diet. Addition of T0901317 (0.025%) to the diet for 7 days resulted in a fall of plasma plant sterol levels to barely detectable levels in wild type mice. Mean plasma sitosterol levels increased from 20.6 to 39.6 mg/dl in the LXR agonist-treated knockout animals (Fig. 1); an increase of similar magnitude occurred in the plasma level of campesterol, the other major dietary plant sterol.


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Fig. 1.   Plasma sterol levels in chow-fed mice with or without T0901317. 17-week-old female G5G8+/+ and G5G8-/- mice (n = 5 in each group) were fed a powdered chow diet (Diet 7001, Harlan Teklad, Madison, WI) with or without 0.025% of the synthetic LXR agonist T0901317 for 7 days. Mice were sacrificed during the daylight cycle after a 4-h fast, and venous blood was collected. Plasma was separated by centrifugation, and the levels of plasma sterols were determined by GC, as described under "Experimental Procedures." -T, chow diet; +T, chow diet plus T0901317 (0.025%); *, p < 0.05 between chow-fed and T0901317-fed mice of each genotype; **, p < 0.01 between chow-fed and T0901317-fed mice of each genotype.

Mean plasma levels of cholesterol were lower in chow-fed G5G8-/- mice than in wild type mice, as previously described (12) (Fig. 1). The levels of plasma cholesterol increased by 50% and by 90% in the wild type mice and G5G8-/- mice with T0901317 treatment. FPLC analysis was performed to determine the distribution of sterols in the plasma of these mice. No significant difference was seen in the distribution of sterols in the knockout and wild type mice. The increase in plasma sterol levels was due to increases of HDL sterols in both strains of mice (Fig. 2). Treatment with T019101317 was associated with widening of the HDL sterol peaks and a shoulder extending into the larger size fractions, as has been reported previously (26, 27).


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Fig. 2.   Sterol profiles of FPLC fractions from plasma of G5G8+/+ and G5G8-/- mice fed a chow diet with or without T0901317. Plasma isolated from the mice described in the legend to Fig. 1 was subjected to FPLC analysis. The sterol content of each fraction was assayed using a cholesterol/HP kit, as described under "Experimental Procedures." -T, chow diet only; +T, chow diet plus T0901317 (0.025%).

T0901317 Treatment Is Not Associated with Increased Biliary Cholesterol Levels in G5G8-/- Mice-- T0901317 treatment increased biliary cholesterol levels of wild type mice by almost 3-fold (from 7.13 to 20.12 µmol/ml), as was observed previously (26) (Fig. 3). In contrast to wild type mice, no significant increase in mean biliary cholesterol level was seen in the G5G8-/- mice after treatment with T0901317 (from 0.73 to 0.97 µmol/ml). These data are consistent with Abcg5 and Abcg8 being the target genes responsible for the increase in biliary cholesterol levels associated with LXR activation.


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Fig. 3.   Biliary lipid levels in G5G8+/+ and G5G8-/- mice fed a chow diet with or without T0901317. Gallbladder bile was collected from the mice described in the legend to Fig. 1. The concentrations of biliary cholesterol, phospholipids, and bile acids were measured as described under "Experimental Procedures." -T, chow diet; +T, chow diet plus T0901317 (0.025%); *, p < 0.05 between chow-fed and T0901317-fed mice of each genotype; **, p < 0.01 between chow-fed and T0901317-fed wild type mice.

Biliary phospholipid levels were significantly lower in the knockout than in the wild type mice. A similar difference in biliary phospholipid levels was observed previously in female, but not male, G5G8-/- mice (12). Biliary phospholipids and bile acid levels fell after T0901317 treatment in the G5G8+/+ mice but not in the G5G8-/- mice (Fig. 3). Similar reductions in biliary lipid levels were seen previously in wild type mice treated with an LXR agonist (26).

Hepatic Cholesterol Levels in the G5G8-/- Mice Do Not Fall with T0901317 Treatment-- Levels of plant sterols were significantly higher in chow-fed G5G8-/- mice than in wild type animals. G5G8-/- mice have increased fractional absorption of dietary plant sterols and a decrease in the biliary excretion of sterols, which contribute to the higher hepatic levels of plant sterols in these mice (12). Treatment of the G5G8-/- mice with T0901317 resulted in a modest, but significant reduction in hepatic sitosterol and campesterol levels (Fig. 4). The hepatic cholesterol level fell by 30% in the wild type mice after treatment with T0901317, presumably in part due to the increase in biliary cholesterol secretion (Fig. 4). In contrast to the wild type mice, no change in hepatic cholesterol levels was seen in the knockout mice after LXR agonist treatment, presumably resulting from the lack of increase in biliary cholesterol secretion in these mice (Fig. 4).


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Fig. 4.   Hepatic lipid levels in G5G8+/+ and G5G8-/- mice fed a chow diet with or without T0901317. Livers were obtained from the mice described in the legend to Fig. 1. An aliquot of liver was weighed, saponified, and processed for the determination of tissue sterol contents by GC, as described under "Experimental Procedures." Hepatic triglyceride levels were measured enzymatically as described under "Experimental Procedures." -T, chow diet only; +T, chow diet plus T0901317 (0.025%); *, p < 0.05 between chow-fed and T0901317-fed mice of each genotype; **, p < 0.01 between chow-fed and T0901317-fed mice of each genotype.

Hepatic triglyceride levels were similar in the chow-fed G5G8+/+ and G5G8-/- mice. Treatment with T090137 was associated with an increase in hepatic triglycerides in both groups of mice (Fig. 4), as previously observed (27). These results are attributed to the activation of SREBP-1c, which stimulates the expression of multiple genes in the fatty acid biosynthetic pathway and is an LXR target gene (23).

LXR Agonist T0901317 Does Not Reduce Fractional Absorption of Dietary Cholesterol in G5G8-/- Mice-- The fractional absorption of dietary cholesterol was reduced from 72% to 52% in the wild type mice treated with T0901317, which is similar to that observed by Repa et al. (14). In contrast to wild type mice, the fractional absorption of cholesterol increased slightly (from 76% to 81%) in T0901317-treated G5G8-/- mice (Fig. 5).


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Fig. 5.   Fractional absorption of dietary cholesterol in G5G8+/+ and G5G8-/- mice treated with T0901317. Five-month-old female mice of the indicated genotype (n = 7-8) in each group) were housed individually. After treatment with either chow diet or chow plus T0901317 (0.025%) diet for 4 days, each mouse was gavaged with 50 µl of an oil mixture containing deuterated cholesterol and sitostanol. Feces were collected for 3 days while continuing the treatment, and the sterols were extracted. The fecal sterols were subjected to GC-MS as described previously (12). Deuterated sitostanol was used as a nonabsorbable fecal marker by which the fractional absorption of dietary cholesterol was calculated. *, p < 0.01 between chow-fed and T0901317-treated wild type mice.

T0901317 Treatment Fails to Increase Fecal Neutral Sterol Excretion in G5G8-/- Mice-- Decreased fractional cholesterol absorption and increased biliary cholesterol secretion both may contribute to the dramatic increase in fecal neutral sterol excretion that occurs with LXR agonist treatment (14, 26). The level of neutral sterols excreted into the feces over the 3-day collection period was ~30% lower in the chow-fed G5G8-/- mice than in the G5G8+/+ mice, and this level failed to increase with T0901317 treatment (Fig. 6). Thus, ABCG5 and ABCG8 are required for the stimulation of fecal neutral sterol excretion by the LXR agonist T0901317. Fecal bile acid excretion was similar in the G5G8+/+ and G5G8-/- mice and did not change significantly with T0901317 treatment (Fig. 6).


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Fig. 6.   Fecal neutral sterol excretion in G5G8+/+ and G5G8-/- mice fed a chow diet with or without T0901317. Feces were collected for the last 3 days of the experiment described in the legend to Fig. 1. The fecal neutral sterols and bile acids were measured as described under "Experimental Procedures." -T, chow diet only; +T, chow diet plus T0901317 (0.025%); *, p < 0.01 between chow-fed and T0901317-fed wild type mice.

LXR Agonist (T0901317) Treatment Increases mRNA Levels of Selected Target Genes in Liver and Small Intestine of G5G8+/+ and G5G8-/- Mice-- The mRNA levels of known LXR target genes, including Srebp-1c, Abca1, Abcg1, Abcg5, and Abcg8, were determined to confirm that T0901317 had the expected biological effects in the tissues of the treated mice. The expression levels of all selected LXR target genes were increased with T0901317 treatment in both strains of mice with the exceptions of Abcg5 and Abcg8 in G5G8-/- mice. The mRNA levels of Srebp-2, which is not a target gene of LXR, did not increase significantly with T0901317 treatment in either wild type or G5G8-/- mice (Fig. 7).


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Fig. 7.   Relative mRNA levels in livers and intestines of chow-fed mice with or without the addition of the LXR agonist T0901317. The livers and jejuna were harvested from the mice described in the legend to Fig. 1 and maintained at -80 °C after freezing in liquid nitrogen. Total RNA was extracted from the tissues using the RNA Stat-60 kit (Tel-Test Inc., Friendswood, TX). An equal amount of RNA from each sample was pooled in each group, and real-time PCR was performed using gene-specific primers as described previously (31, 32). Cyclophilin was used as an internal control for these studies. Values represent the mRNA levels relative to the mRNA level in the chow-fed wild type mice. -T, chow diet only; +T, chow diet plus T0901317 (0.025%).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The major finding of this study is that ABCG5 and ABCG8 are required in mice for the stimulation of biliary and fecal cholesterol excretion by the synthetic LXR agonist, T0901317. Disruption of Abcg5 and Abcg8 abolished the increase in biliary cholesterol levels, the reduction in fractional cholesterol absorption, and the increase in fecal neutral sterol excretion associated with LXR activation (14, 26). These data, taken together with the phenotypic characterization of mice expressing either no ABCG5 and ABCG8 or higher levels of both proteins (11, 12), suggest that LXR activation promotes the excretion of sterols by increasing Abcg5 and Abcg8 expression.

In wild type mice, treatment with the LXR agonist was associated with significantly lower plasma levels of both sitosterol and campesterol (Fig. 1). Similar reductions in plasma plant sterol levels were seen in transgenic mice containing 14 copies of the human ABCG5 and ABCG8 transgenes (11). In contrast to wild type mice, plasma levels of sitosterol and campesterol increased ~2-fold with T0901317 treatment in the G5G8-/- mice (Fig. 1). These data indicate that increased expression of Abcg5 and Abcg8 is both necessary and sufficient for the LXR agonist-associated reduction in plasma plant sterol levels observed in the wild type animals. In the absence of ABCG5 and ABCG8, plant sterols accumulate in the liver due to an inability to efficiently secrete sterols into the bile and an increased absorption of dietary plant sterols (12). The increased hepatic levels of plant sterols in the knockout animals likely result in an increased incorporation of these sterols into lipoproteins and secretion into plasma. Further studies are required to determine if LXR agonist treatment results in a greater increase in the incorporation of plant sterols into lipoproteins or an increased secretion of lipoproteins into the circulation in G5G8-/- mice. The LXR agonist also may promote the transport of sterols from peripheral tissues into the circulation of G5G8-/- mice.

Plasma cholesterol levels also increased significantly with T0901317 treatment in G5G8+/+ and G5G8-/- mice (Fig. 1). The increase in plasma cholesterol was limited to the HDL fraction (Fig. 2) and was associated with an increase in the size of HDL particles, as reported previously (27, 33), which may contribute to LXR agonist-induced expression of Abca1 in the intestine, liver (Fig. 7), and macrophages (14). ABCA1 participates in the efflux of excess cholesterol from peripheral cells to HDL (16-18, 34) and promotes formation of pre-beta -HDL particles by hepatocytes (35) and possibly enterocytes (36). The fall in hepatic cholesterol levels (Fig. 4) in the G5G8+/+ mice with LXR activation may also in part be due to an ABCA1-mediated increase in the efflux of cholesterol from the liver into the circulation (14, 26) in addition to the increase in biliary cholesterol secretion.

The most dramatic difference between G5G8+/+ and G5G8-/- mice in response to T0901317 treatment was in biliary cholesterol levels. Mean biliary cholesterol levels increased 3-fold in wild type mice but did not change significantly in knockout mice. Biliary phospholipid and bile acid levels were lower in the G5G8-/- mice than in their wild type littermates, which is comparable to the reductions observed previously in female mice (12). Both the biliary phospholipid and bile acid levels fell in wild type mice treated with T0901317 (Fig. 3), as was seen previously in mice treated with LXR agonists (26). No reduction in the levels of bile acids or phospholipids in the bile occurred in the T0901317-treated G5G8-/- mice. No change in fecal bile acid excretion was seen in either the knockout or the wild type animals treated with the LXR agonist (Fig. 6). Therefore, the increased excretion of biliary cholesterol associated with LXR agonist treatment was not quantitatively coupled to biliary bile acid or phospholipid excretion.

The results of these studies demonstrate that ABCG5 and ABCG8 mediate the effects of LXR agonists on the increase in fecal loss of cholesterol. These studies do not distinguish the relative contributions of the liver and the intestine to the increased fecal neutral sterol excretion. The stimulation of fecal cholesterol loss by the LXR agonist may result from an increase in biliary cholesterol secretion by hepatocytes and/or the decreased fractional absorption of dietary cholesterol by enterocytes. Tissue-specific disruptions of Abcg5 and Abcg8 will be required to assess the function of these transporters in the liver and small intestine.

Reverse cholesterol transport involves the efflux of cholesterol from peripheral tissues to the liver, the secretion of cholesterol into bile, and the excretion of sterols in feces. The molecular machinery that affects reverse cholesterol transport has not been fully characterized. The data in this report are consistent with the notion that two ABC half-transporters, ABCG5 and ABCG8, mediate the final step in this pathway.

    ACKNOWLEDGEMENTS

We thank Robert Guzman, Yinyan Ma, Anja Kerksiek, and Silvia Winnen for excellent technical assistance. We also thank Scott Clark, Anh Nguyen, Scott M. Grundy, and Gloria Vega for measuring lipids in bile, plasma, and tissues. We thank David W. Russell for helpful discussion.

    FOOTNOTES

* 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 the Howard Hughes Medical Institute, by National Institutes of Health Grants HL20948 and HL72304, by the Perot Family Foundation, by the W. M. Keck Foundation, by the Donald W. Reynolds Clinical Cardiovascular Research Center at Dallas, and by Bundesministerium für Bildung, Forschung, Wissenschaft und Technologie Grant 01EC9402. To whom correspondence should be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9046. Tel.: 214-648-6724; Fax: 214-648-7539; E-mail: helen.hobbs@utsouthwestern.edu.

Published, JBC Papers in Press, February 22, 2003, DOI 10.1074/jbc.M301311200

    ABBREVIATIONS

The abbreviations used are: ABC, ATP-binding cassette; G5G8+/+, wild type for Abcg5 and Abcg8 allele; G5G8-/-, homozygous for an allele with inactivated Abcg5 and Abcg8; LXR, liver X receptor; HDL, high density lipoprotein; LDL, low density lipoprotein; SREBP, sterol regulatory element-binding protein; GC, gas chromatography; FPLC, fast protein liquid chromatography.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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