Ezetimibe normalizes metabolic defects in mice lacking ABCG5 and ABCG8

The ATP binding cassette transporters ABCG5 (G5) and ABCG8 (G8) limit the accumulation of neutral sterols by restricting sterol uptake from the intestine and promoting sterol excretion into bile. Humans and mice lacking G5 and G8 (G5G8-/-) accumulate plant sterols in the blood and tissues. However, despite impaired biliary cholesterol secretion, plasma and liver cholesterol levels are lower in G5G8-/- mice than in wild-type littermates. To determine whether the observed changes in hepatic sterol metabolism were a direct result of decreased biliary sterol secretion or a metabolic consequence of the accumulation of dietary noncholesterol sterols, we treated G5G8-/- mice with ezetimibe, a drug that reduces the absorption of both plant- and animal-derived sterols. Ezetimibe feeding for 1 month sharply decreased sterol absorption and plasma levels of sitosterol and campesterol but increased cholesterol in both the plasma (from 60.4 to 75.2 mg/dl) and the liver (from 1.1 to 1.87 mg/g) of the ezetimibe-treated G5G8-/- mice. Paradoxically, the increase in hepatic cholesterol was associated with an increase in mRNA levels of HMG-CoA reductase and synthase. Together, these results indicate that pharmacological blockade of sterol absorption can ameliorate the deleterious metabolic effects of plant sterols even in the absence of G5 and G8.     

ABCG5 and ABCG8 require MDR2 for secretion of cholesterol into bile

The major pathway for the removal of cholesterol from the body is via secretion into the bile. Three members of the ATP binding cassette (ABC) family, ABCG5 (G5), ABCG8 (G8), and ABCB4 (MDR2), are required for the efficient biliary export of sterols. Here, we examined the interdependence of these three ABC transporters for biliary sterol secretion. Biliary lipid levels in mice expressing no MDR2 (Mdr2-/- mice) were compared with those of Mdr2-/- mice expressing 14 copies of a human G5 (hG5) and hG8 transgene (Mdr2-/-;hG5G8Tg mice). Mdr2-/- mice had only trace amounts of biliary cholesterol and phospholipids. The Mdr2-/-;hG5G8Tg mice had biliary cholesterol levels as low as those of Mdr2-/- mice. Thus, MDR2 expression is required for G5G8-mediated biliary sterol secretion. To determine whether the reduction in fractional absorption of dietary sterols associated with G5G8 overexpression is secondary to the associated increase in biliary cholesterol, we compared the fractional absorption of sterols in Mdr2-/-;hG5G8Tg and hG5G8Tg animals. Inactivation of MDR2 markedly attenuated the reduction in fractional sterol absorption associated with G5G8 overexpression. These results are consistent with the notion that increased biliary cholesterol secretion contributes to the reduction in fractional sterol absorption associated with G5G8 overexpression.     

Ezetimibe stimulates faecal neutral sterol excretion depending on abcg8 function in mice

Ezetimibe stimulates faecal neutral sterol (FNS) excretion in mice, which cannot be explained by cholesterol absorption inhibition alone. We investigated whether these effects are mediated via the sterol exporter ATP binding cassette transporter G8 (abcg8). Ezetimibe increased FNS excretion 2.7-fold in WT mice and 1.5-fold in abcg8^−/− mice, without affecting biliary cholesterol secretion. Daily FNS excretion exceeded the sum of dietary cholesterol intake and biliary secretion by about 60%. Ezetimibe enhanced this ‘extra’ FNS excretion by 3.5-fold and 1.5-fold in wildtype (WT) and abcg8^−/− mice, respectively. Ezetimibe stimulates fecal sterol excretion of non-biliary and non-dietary origin, probably through stimulation of trans-intestinal cholesterol excretion. We show that this effect depends on intact abcg8 function.     

Stimulation of cholesterol excretion by the liver X receptor agonist requires ATP-binding cassette transporters G5 and G8

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.     

Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (-88%) and, to a lesser extent, LDL (-40%) and HDL (-35%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in diet-induced hyperlipidemic mice.     

Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.     

Hepatic Mttp deletion reverses gallstone susceptibility in L-Fabp knockout mice

Previous studies demonstrated that L-Fabp KO mice are more susceptible to lithogenic diet (LD)-induced gallstones because of altered hepatic cholesterol metabolism and increased canalicular cholesterol secretion. Other studies demonstrated that liver-specific deletion of microsomal triglyceride transfer protein (Mttp-LKO) reduced LD-induced gallstone formation by increasing biliary phospholipid secretion. Here we show that mice with combined deletion (i.e., DKO mice) are protected from LD-induced gallstone formation. Following 2 weeks of LD feeding, 73% of WT and 100% of L-Fabp KO mice developed gallstones versus 18% of Mttp-LKO and 23% of DKO mice. This phenotype was recapitulated in both WT and L-Fabp KO mice treated with an Mttp antisense oligonucleotide (M-ASO). Biliary cholesterol secretion was increased in LD-fed L-Fabp KO mice and decreased in DKO mice. However, phospholipid secretion was unchanged in LD-fed Mttp-LKO and DKO mice as well as in M-ASO-treated mice. Expression of the canalicular export pump ABCG5/G8 was reduced in LD-fed DKO mice and in M-ASO-treated L-Fabp KO mice. We conclude that liver-specific Mttp deletion not only eliminates apical lipoprotein secretion from hepatocytes but also attenuates canalicular cholesterol secretion, which in turn decreases LD-induced gallstone susceptibility.     

ABC转运体超家族对胆固醇吸收和血浆植物甾醇水平的调节作用

人们对控制胆固醇吸收和血浆植物甾醇水平的分子基础了解尚少.ABCG5和ABCG8的发现使得理解甾醇吸收的分子基础获得突破.ABCG5和ABCG8主要涉及植物甾醇代谢,而其他基因涉及胆固醇吸收.最近,一种新胆固醇吸收阻止剂（ezetimibe）的问世,给胆固醇吸收和血浆植物甾醇水平基因控制研究提供新的亮点.主要综述胆固醇吸收和血浆植物甾醇水平的基因控制,关注调节它们的共同点和不同点,讨论这一领域的最近发展和展望未来希望.     

Hepatic ABCG5 and ABCG8 overexpression increases hepatobiliary sterol transport but does not alter aortic atherosclerosis in transgenic mice

The individual roles of hepatic versus intestinal ABCG5 and ABCG8 in sterol transport have not yet been investigated. To determine the specific contribution of liver ABCG5/G8 to sterol transport and atherosclerosis, we generated transgenic mice that overexpress human ABCG5 and ABCG8 in the liver but not intestine (liver G5/G8-Tg) in three different genetic backgrounds: C57Bl/6, apoE-KO, and low density lipoprotein receptor (LDLr)-KO. Hepatic overexpression of ABCG5/G8 enhanced hepatobiliary secretion of cholesterol and plant sterols by 1.5-2-fold, increased the amount of intestinal cholesterol available for absorption and fecal excretion by up to 27%, and decreased the accumulation of plant sterols in plasma by approximately 25%. However, it did not alter fractional intestinal cholesterol absorption, fecal neutral sterol excretion, hepatic cholesterol concentrations, or hepatic cholesterol synthesis. Consequently, overexpression of ABCG5/G8 in only the liver had no effect on the plasma lipid profile, including cholesterol, HDL-C, and non-HDL-C, or on the development of proximal aortic atherosclerosis in C57Bl/6, apoE-KO, or LDLr-KO mice. Thus, liver ABCG5/G8 facilitate the secretion of liver sterols into bile and serve as an alternative mechanism, independent of intestinal ABCG5/G8, to protect against the accumulation of dietary plant sterols in plasma. However, in the absence of changes in fractional intestinal cholesterol absorption, increased secretion of sterols into bile induced by hepatic overexpression of ABCG5/G8 was not sufficient to alter hepatic cholesterol balance, enhance cholesterol removal from the body or to alter atherogenic risk in liver G5/G8-Tg mice. These findings demonstrate that overexpression of ABCG5/G8 in the liver profoundly alters hepatic but not intestinal sterol transport, identifying distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism.     

Liver X receptor-mediated activation of reverse cholesterol transport from macrophages to feces in vivo requires ABCG5/G8

Liver X receptor (LXR) agonists increase both total fecal sterol excretion and macrophage-specific reverse cholesterol transport (RCT) in vivo. In this study, we assessed the effects of ABCG5/G8 deficiency as well as those of LXR agonist-induction of RCT from macrophages to feces in vivo. A [(3)H]cholesterol-labeled macrophage cell line was injected intraperitoneally into ABCG5/G8-deficient (G5/G8(-/-)), heterozygous (G5G8(+/-)), and wild-type G5/G8(+/+) mice. G5/G8(-/-)mice presented increased radiolabeled HDL-bound [(3)H]cholesterol 24 h after the label injection. However, the magnitude of macrophage-derived [(3)H]cholesterol in liver and feces did not differ between groups. A separate experiment was conducted in G5G8(+/+) and G5G8(-/-) mice treated with or without the LXR agonist T0901317. Treatment with T0901317 increased liver ABCG5/G8 expression, which was associated with a 2-fold increase in macrophage-derived [(3)H]cholesterol in feces of G5/G8(+/+) mice. However, T0901317 treatment had no effect on fecal [(3)H]cholesterol excretion in G5G8(-/-) mice. Additionally, LXR activation stimulated the fecal excretion of labeled cholesterol after an intravenous injection of HDL-[(3)H]cholesteryl oleate in G5/G8(+/+) mice, but failed to enhance fecal [(3)H]cholesterol in G5/G8(-/-) mice. Our data provide direct in vivo evidence of the crucial role of ABCG5 and ABCG8 in LXR-mediated induction of macrophage-specific RCT.     

ABCA1 and ABCG1 or ABCG4 act sequentially to remove cellular cholesterol and generate cholesterol-rich HDL

Recent developments in lipid metabolism have shown the importance of ATP binding cassette transporters (ABCs) in controlling cellular and total body lipid homeostasis. ABCA1 mediates the transport of cholesterol and phospholipids from cells to lipid-poor apolipoprotein A-I (apoA-I), whereas ABCG1 and ABCG4 mediate the transport of cholesterol from cells to lipidated lipoproteins. ABCA1, ABCG1, and ABCG4 are all expressed in cholesterol-loaded macrophages, and macrophages from ABCA1 and ABCG1 knockout mice accumulate cholesteryl esters. Here, we show that the lipidated particles generated by incubating cells overexpressing ABCA1 with apoA-I are efficient acceptors for cholesterol released from cells overexpressing either ABCG1 or ABCG4. The cholesterol released to the particles was derived from a cholesterol oxidase-accessible plasma membrane pool in both ABCG1 and ABCG4 cells, which is the same pool of cholesterol shown previously to be removed by high density lipoproteins. ABCA1 cells incubated with apoA-I generated two major populations of cholesterol- and phospholipid-rich lipoprotein particles that were converted by ABCG1 or ABCG4 cells to one major particle population that was highly enriched in cholesterol. These results suggest that ABCG1 and ABCG4 act in concert with ABCA1 to maximize the removal of excess cholesterol from cells and to generate cholesterol-rich lipoprotein particles.     

ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

ApoE plays an important role in lipoprotein metabolism. This study investigated the effects of adenovirus-mediated human apoE overexpression (AdhApoE3) on sterol metabolism and in vivo reverse cholesterol transport (RCT). In wild-type mice, AdhApoE3 resulted in decreased HDL cholesterol levels and a shift toward larger HDL in plasma, whereas hepatic cholesterol content increased (P < 0.05). These effects were dependent on scavenger receptor class B type I (SR-BI) as confirmed using SR-BI-deficient mice. Kinetic studies demonstrated increased plasma HDL cholesteryl ester catabolic rates (P < 0.05) and higher hepatic selective uptake of HDL cholesteryl esters in AdhApoE3-injected wild-type mice (P < 0.01). However, biliary and fecal sterol output as well as in vivo macrophage-to-feces RCT studied with (3)H-cholesterol-loaded mouse macrophage foam cells remained unchanged upon human apoE overexpression. Similar results were obtained using hApoE3 overexpression in human CETP transgenic mice. However, blocking ABCA1-mediated cholesterol efflux from hepatocytes in AdhApoE3-injected mice using probucol increased biliary cholesterol secretion (P < 0.05), fecal neutral sterol excretion (P < 0.05), and in vivo RCT (P < 0.01), specifically within neutral sterols. These combined data demonstrate that systemic apoE overexpression increases i) SR-BI-mediated selective uptake into the liver and ii) ABCA1-mediated efflux of RCT-relevant cholesterol from hepatocytes back to the plasma compartment, thereby resulting in unchanged fecal mass sterol excretion and overall in vivo RCT.     

ATP binding cassette G1-dependent cholesterol efflux during inflammation

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.     

Selective sterol accumulation in ABCG5/ABCG8-deficient mice

The ATP binding cassette (ABC) transporters ABCG5 and ABCG8 limit intestinal absorption and promote biliary secretion of neutral sterols. Mutations in either gene cause sitosterolemia, a rare recessive disease in which plasma and tissue levels of several neutral sterols are increased to varying degrees. To determine why patients with sitosterolemia preferentially accumulate noncholesterol sterols, levels of cholesterol and the major plant sterols were compared in plasma, liver, bile, and brain of wild-type and ABCG5/ABCG8-deficient (G5G8(-/-)) mice. The total sterol content of liver and plasma was similar in G5G8(-/-) mice and wild-type animals despite an approximately 30-fold increase in noncholesterol sterol levels in the knockout animals. The relative enrichment of each sterol in the plasma and liver of G5G8(-/-) mice (stigmasterol > sitosterol = cholestanol > bassicasterol > campesterol > cholesterol) reflected its relative enrichment in the bile of wild-type mice. These results indicate that 24-alkylated, Delta22, and 5alpha-reduced sterols are preferentially secreted into bile and that preferential biliary secretion of noncholesterol sterols by ABCG5 and ABCG8 prevents the accumulation of these sterols in normal animals. The mRNA levels for 13 enzymes in the cholesterol biosynthetic pathway were reduced in the livers of the G5G8(-/-) mice, despite a 50% reduction in hepatic cholesterol level. Thus, the accumulation of sterols other than cholesterol is sensed by the cholesterol regulatory machinery.     

ABCG1 regulates pulmonary surfactant metabolism in mice and men

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.     

ABCG5 and ABCG8 are obligate heterodimers for protein trafficking and biliary cholesterol excretion

ABCG5 (G5) and ABCG8 (G8) are ATP-binding cassette (ABC) transporters that limit intestinal absorption and promote biliary excretion of neutral sterols. Mutations in either ABCG5 or ABCG8 result in an identical clinical phenotype, suggesting that these two half-transporters function as heterodimers. Expression of both G5 and G8 is required for either protein to be transported to the plasma membrane of cultured cells. In this paper we used immunofluorescence microscopy to confirm, in vivo, that G5 is localized to the apical membranes of mouse enterocytes and hepatocytes. Other ABC half-transporters function as homodimers or as heterodimers with other subfamily members. To determine whether G5 or G8 complex with other ABCG half-transporters, we co-expressed G1, G2, and G4 with either G5 or G8 in cultured cells. G1, G2, and G4 co-immunoprecipitated with G5, and G4 co-immunoprecipitated with G8, but the putative dimers were retained in the endoplasmic reticulum (ER). Adenovirus-mediated expression of either G5 or G8 in the liver of G5G8 null mice resulted in ER retention of the expressed proteins and no increase in biliary cholesterol. In contrast, co-expression of G5 and G8 resulted in transit of the proteins out of the ER and a 10-fold increase in biliary cholesterol concentration. Finally, adenoviral expression of G2 in the presence or absence of G5 or G8 failed to promote sterol excretion into bile. These experiments indicate that G5 and G8 function as obligate heterodimers to promote sterol excretion into bile.     

Increased expression of LXR alpha, ABCG5, ABCG8, and SR-BI in the liver from normolipidemic, nonobese Chinese gallstone patients

Cholesterol supersaturation of bile is one prerequisite for gallstone formation. In the present study of Chinese patients with gallstones, we investigated whether this phenomenon was correlated with the hepatic expression of genes participating in the metabolism of cholesterol and bile acids. Twenty-two nonobese, normolipidemic patients (female-male, 11:11) with gallstones were investigated with 13 age- and body mass index-matched gallstone-free controls (female-male, 10:3). The bile from the gallstone patients had higher cholesterol saturation than that from the controls. The mRNA levels of ABCG5, ABCG8, and liver X receptor alpha (LXRalpha) in the gallstone patients were increased by 51, 59, and 102%, respectively, and significantly correlated with the molar percentage of biliary cholesterol and cholesterol saturation index (CSI). The mRNA and protein levels of the hepatic scavenger receptor class B type I (SR-BI) were increased, and a significant correlation was found between the protein levels and the CSI. No differences were recorded between the two groups concerning the hepatic synthesis of cholesterol, bile acids, and esterification of cholesterol. Our results suggest that the upregulation of ABCG5/ABCG8 in gallstone patients, possibly mediated by increased LXRalpha, may contribute to the cholesterol supersaturation of bile. Our data are consistent with the possibility that increased amounts of biliary cholesterol may originate from plasma HDL cholesterol by enhanced transfer via SR-BI.     

Inhibition of cholesterol absorption by SCH 58053 in the mouse is not mediated via changes in the expression of mRNA for ABCA1, ABCG5, or ABCG8 in the enterocyte

Intestinal cholesterol absorption is a major determinant of plasma low density lipoprotein-cholesterol (LDL-C) concentrations. Ezetimibe (SCH 58235) and its analogs SCH 48461 and SCH 58053 are novel potent inhibitors of cholesterol absorption whose mechanism of action is unknown. These studies investigated the effect of SCH 58053 on cholesterol metabolism in female 129/Sv mice. In mice fed a low cholesterol rodent diet containing SCH 58053, cholesterol absorption was reduced by 46% and fecal neutral sterol excretion was increased 67%, but biliary lipid composition and bile acid synthesis, pool size, and pool composition were unchanged. When the dietary cholesterol content was increased either 10- or 50-fold, those animals given SCH 58053 manifested lower hepatic and biliary cholesterol concentrations than did their untreated controls. Cholesterol feeding increased the relative mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1), ABC transporter G5 (ABCG5), and ABC transporter G8 (ABCG8) in the jejunum, and of ABCG5 and ABCG8 in the liver, but the magnitude of this increase was generally less if the mice were given SCH 58053. We conclude that the inhibition of cholesterol absorption effected by this new class of agents is not mediated via changes in either the size or composition of the intestinal bile acid pool, or the level of mRNA expression of proteins that facilitate cholesterol efflux from the enterocyte, but rather may involve disruption of the uptake of luminal sterol across the microvillus membrane.     

Functional asymmetry of nucleotide-binding domains in ABCG5 and ABCG8

The ATP-binding cassette half-transporters ABCG5 (G5) and ABCG8 (G8) promote secretion of neutral sterols into bile, a major pathway for elimination of sterols. Mutations in either ABCG5 or ABCG8 cause sitosterolemia, a recessive disorder characterized by impaired biliary and intestinal sterol secretion, sterol accumulation, and premature atherosclerosis. The mechanism by which the G5G8 heterodimer couples ATP hydrolysis to sterol transport is not known. Here we examined the roles of the Walker A, Walker B, and signature motifs in the nucleotide-binding domains (NBD) of G5 and G8 using recombinant adenoviruses to reconstitute biliary sterol transport in G5G8-deficient mice. Mutant forms of each half-transporter were co-expressed with their wild-type partners. Mutations at crucial residues in the Walker A and Walker B domains of G5 prevented biliary sterol secretion, whereas mutations of the corresponding residues in G8 did not. The opposite result was obtained when mutations were introduced into the signature motif; mutations in the signature domain of G8 prevented sterol transport, but substitution of the corresponding residues in G5 did not. Taken together, these findings indicate that the NBDs of G5 and G8 are not functionally equivalent. The integrity of the canonical NBD formed by the Walker A and Walker B motifs of G5 and the signature motif of G8 is essential for G5G8-mediated sterol transport. In contrast, mutations in key residues of the NBD formed by the Walker A and B motifs of G8 and the signature sequence of G5 did not affect sterol secretion.