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Overexpression of ABCG5 and ABCG8 Promotes Biliary Cholesterol Secretion and Reduces Fractional Absorption of Dietary Cholestero

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Overexpression of ABCG5 and ABCG8 Promotes Biliary Cholesterol Secretion and Reduces Fractional Absorption of Dietary Cholestero Overexpression of ABCG5 See the related Commentary beginning on page 605. and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol Liqing Yu,1 Jia Li-Hawkins,1 Robert E. Hammer,2,3 Knut E. Berge,1 Jay D. Horton,1 Jonathan C. Cohen,4 and Helen H. Hobbs1 1Department of Molecular Genetics and McDermott Center for Human Growth and Development, 2Department of Biochemistry, 3Howard Hughes Medical Institute, and 4Center for Human Nutrition, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA Two ATP-binding cassette (ABC) transporters, ABCG5 and ABCG8, have been proposed to limit sterol absorption and to promote biliary sterol excretion in humans. To test this hypothesis, a P1 clone containing the human ABCG5 and ABCG8 genes was used to generate transgenic mice. The transgenes were expressed primarily in the liver and small intestine, mirroring the expression pattern of the endogenous genes. Transgene expression only modestly affected plasma and liver cholesterol levels but profoundly altered cholesterol transport. The fractional absorption of dietary cholesterol was reduced by about 50%, and biliary cholesterol levels were increased more than fivefold. Fecal neu- tral sterol excretion was increased three- to sixfold and hepatic cholesterol synthesis increased two- to fourfold in the transgenic mice. No significant changes in the pool size, composition, and fecal excretion of bile acids were observed in the transgenic mice. Transgene expression attenuated the increase in hepatic cholesterol content induced by consumption of a high cholesterol diet. These results demonstrate that increased expression of ABCG5 and ABCG8 selectively drives biliary neutral sterol secretion and reduces intestinal cholesterol absorption, leading to a selective increase in neu- tral sterol excretion and a compensatory increase in cholesterol synthesis. J. Clin. Invest. 110:671–680 (2002). doi:10.1172/JCI200216001. Introduction limited than that of cholesterol. Humans absorb less Multiple physiological mechanisms limit the entry of than 5% of dietary sitosterol (1), and the small amount dietary sterols into the bloodstream. Excess cholesterol of sitosterol that reaches the liver is preferentially is removed from the body either by direct secretion into secreted into the bile (2). Consequently, plasma levels the bile or after conversion to bile acids. Although the of sitosterol are very low (<1 mg/dl) in normal individ- mechanisms by which dietary sterols are absorbed by uals. A much higher proportion (45–55%) of dietary the intestine and secreted into the bile have been well cholesterol is absorbed by the proximal small intestine characterized physiologically, the machinery responsi- in humans, and the fractional excretion of sterols into ble for these two processes has not been molecularly the bile is lower for cholesterol than for sitosterol (3). defined. The most abundant sterols in the human diet Recently, a critical component of the transport are cholesterol, the principal animal-derived sterol, and machinery for dietary sterols was revealed by the find- sitosterol, the major plant sterol. These two sterols, ing that mutations in the genes encoding the ATP- although structurally very similar, are handled quite binding cassette (ABC) half-transporters ABCG5 and differently by the intestine and liver of normal mam- ABCG8 cause sitosterolemia, a rare autosomal reces- mals. The absorption of sitosterol is significantly more sive disorder of sterol metabolism (4, 5). Patients with sitosterolemia have increased fractional absorption Received for publication May 24, 2002, and accepted in revised form and decreased biliary secretion of all dietary neutral July 9, 2002. sterols (3, 6–8), which invariably leads to dramatically Address correspondence to: Helen H. Hobbs, Department of elevated plasma levels of sitosterol and other plant Molecular Genetics, University of Texas Southwestern Medical sterols. Most sitosterolemic individuals are also hyper- Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9046, USA. Phone: (214) 648-6724; Fax: (214) 648-7539; cholesterolemic (9). The disease phenotypically resem- E-mail: [email protected]. bles homozygous familial hypercholesterolemia in Conflict of interest: No conflict of interest has been declared. that both diseases are characterized by the develop- Nonstandard abbreviations used: ATP-binding cassette (ABC); ment of xanthomas in childhood accompanied by pre- 14C-taurocholic acid (TCA); HMG-CoA reductase (HMGCR); mature coronary atherosclerosis (6, 9, 10). ABCG5 and sterol regulatory element–binding protein (SREBP); LDL receptor (LDLR); ATP-binding cassette transporter A1 (ABCA1); ABCG8 are expressed predominantly in hepatocytes acyl-CoA:cholesterol acyltransferase-2 (ACAT-2). and enterocytes in the proximal small intestine of The Journal of Clinical Investigation | September 2002 | Volume 110 | Number 5 671 humans and mice (11). The expression levels of both genomic DNA was extracted from the tails of the off- genes are coordinately increased in these cells in mice spring. We estimated the copy number of the transgene consuming a high-cholesterol diet (4). LXRα, a nuclear by performing Southern blot analysis and comparing receptor that plays a key role in regulating genes band intensities to those of an equal amount of human involved in cholesterol trafficking (12), is required for genomic DNA. Three lines of human ABCG5 and the increased expression of mouse Abcg5 and Abcg8 in ABCG8 transgenic mice containing different copy num- response to dietary cholesterol (11). These findings bers of the transgene were established. implicate both proteins as key components of the Mice were housed in plastic cages in a temperature- pathways responsible for trafficking of dietary sterols. controlled room (22°C) with 12-hour-light/12-hour- The relationship between ABCG5/ABCG8 activity dark cycling (daylight cycle from 6:00 am to 6:00 pm). and sterol transport rates has not been defined. It is Animals were fed ad libitum a cereal-based rodent diet also not known whether increased expression of ABCG5 (Diet 7001; Harlan Teklad, Madison, Wisconsin, USA) and ABCG8 is sufficient to promote cholesterol secre- containing 0.2 mg of cholesterol and 40 mg of total tion, or whether other steps in the transport process are lipid per gram of diet. For cholesterol feeding studies, rate-limiting. Furthermore, the primary transport sub- cholesterol was added to a powdered form of the same strate of ABCG5/ABCG8 has not been identified, and cereal-based diet. All animal procedures were per- the effects of increased expression of these proteins on formed with the approval of the Institutional Animal other major biliary lipids (phospholipids and bile Care and Research Advisory Committee at the Univer- acids) are not known. To address these issues, we devel- sity of Texas Southwestern Medical Center. oped transgenic mice that contain a large fragment of Northern blot analysis. Total RNA was extracted from genomic DNA that includes the human ABCG5 and tissues using the RNA Stat-60 kit (Tel-Test Inc., ABCG8 genes under the control of their own regulato- Friendswood, Texas, USA). Northern blot analysis was ry sequences. We chose this approach since mutations performed exactly as previously described (14) using in either gene cause an identical phenotype in humans, [α-32P]-labeled cDNA probes (Megaprime DNA Label- presumably because ABCG5 and ABCG8 function as a ing System; Amersham Biosciences Corp., Piscataway, heterodimer (4). In this study, we show that increased New Jersey, USA) for ABCG5, ABCG8, mouse Abcg5, expression of ABCG5 and ABCG8 in liver and small mouse Abcg8, and cyclophilin (4) in Rapid-hyb buffer intestine dramatically increases biliary cholesterol (Amersham Biosciences Corp.). secretion, reduces cholesterol absorption, and increas- Plasma and hepatic lipid analysis. Plasma and hepatic lev- es hepatic cholesterol synthesis. els of cholesterol and triglyceride were measured by the methods described previously (15) using the Choles- Methods terol kit (catalog no. 1127771; Roche Molecular Bio- Generation of transgenic mice expressing human ABCG5 and chemicals, Mannheim, Germany) and Infinity Triglyc- ABCG8. A P1 clone (35B6) with a 90-kb insert contain- erides Reagent (catalog no. 343-500P; Sigma, St. Louis, ing both human ABCG5 and ABCG8 genes was pur- Missouri, USA). Plasma lipoprotein size-fractionation chased from Incyte Genomics Inc. (Palo Alto, Califor- was performed using fast protein liquid chromatogra- nia, USA). The clone was linearized with NotI (Promega phy as described (16). Plasma noncholesterol sterols Corp., Madison, Wisconsin, USA), size-fractionated on were measured using gas chromatography (8). a 1% SeaPlaque agarose gel (BioWhittaker Molecular Biliary lipid composition. Bile was removed from the gall- Applications, Rockland, Maine, USA) by pulse-field gel bladders of anesthetized mice using a 30.5-gauge needle. electrophoresis (Bio-Rad CHEF-DR III System; BioRad The concentrations of cholesterol, phospholipids, and Laboratories Inc., Hercules, California, USA), and puri- bile acids were measured as described previously (17). fied using GELase Agarose Gel-Digesting Preparation Quantitative real-time PCR. Real-time PCR was per- Kit (EPICENTRE, Madison, Wisconsin, USA). The puri- formed on pooled total RNA samples from five mice in fied DNA fragments were dissolved in microinjection each group as described previously (18).
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