ATP binding cassette transporter G5 and G8 genotypes and plasma lipoprotein levels before and after treatment with atorvastatin

The mechanisms responsible for interindividual variation in response to statin therapy remain uncertain. It has been shown that hepatic cholesterol synthesis is associated with ATP binding cassette transporter G5 and G8 (ABCG5/8) activities. To test the hypothesis that genetic variation in ABCG5/8 might influence the plasma lipid response to statin therapy, we examined five nonsynonymous polymorphisms at the ABCG5/8 loci (Q604E, D19H, Y54C, T400K, and A632V) in 338 hypercholesterolemic patients treated with 10 mg atorvastatin. In carriers of the D19H variant, means of posttreatment values and adjusted percent reductions in LDL cholesterol (LDLC) were significantly lower (P = 0.028) and greater (P = 0.036) (112 mg/dl, 39.7%) than those of noncarriers (119 mg/dl, 36.2%), respectively, while no significant difference was observed in percent reductions in total cholesterol. Stepwise multiple regression analysis revealed significant and independent associations with absolute or percent reduction between D19H genotype and posttreatment LDL cholesterol levels. The other polymorphisms were not significantly associated with treatment effects. These results suggest that, in patients with hypercholesterolemia, the ABCG8 D19H variant is associated with greater LDLC-lowering response to atorvastatin therapy.     

ABCG5/G8 polymorphisms and markers of cholesterol metabolism: systematic review and meta-analysis

Genetic variation at the ABCG5/G8 locus has been associated with markers of cholesterol homeostasis. As data originate from small-scale studies, we performed a meta-analysis to study these associations in a large dataset. We first investigated associations between five common ABCG5/G8 polymorphisms (p.Q604E, p.D19H, p.Y54C, p.T400K, and p.A632V) and plasma sterol levels in 245 hypercholesterolaemic individuals. No significant associations were found. Subsequently, our data were pooled into a meta-analysis that comprised 3,364 subjects from 16 studies (weighted mean age, 46.7 ± 10.5 years; BMI, 23.9 ± 3.5 kg/m²). Presence of the minor 632V allele correlated with reduced LDL-C concentrations (n = 367) compared with homozygosity for the 632A variant [n = 614; −0.11 mmol/l (95% CI, range: −0.20 to −0.02 mmol/l); P = 0.01]. The remaining polymorphisms were not associated with plasma lipid levels. Carriers of the 19H allele exhibited lower campesterol/TC (n = 83; P < 0.001), sitosterol/TC (P < 0.00001), and cholestanol/TC (P < 0.00001), and increased lathosterol/TC ratios (P = 0.001) compared with homozygous 19D allele carriers (n = 591). The ABCG8 632V variant was associated with a clinically irrelevant LDL-C reduction, whereas the 19H allele correlated with decreased cholesterol absorption and increased synthesis without affecting the lipid profile. Hence, associations between frequently studied missense ABCG5/G8 polymorphisms and markers of cholesterol homeostasis are modest at best.     

Polymorphisms in the ABCG5 and ABCG8 genes associate with cholesterol absorption and insulin sensitivity

The roles of polymorphisms of the sitosterolemia genes ABCG5 and ABCG8 in the regulation of cholesterol metabolism and insulin sensitivity were studied in mildly hypercholesterolemic noncoronary subjects (n = 263, 144 men and 119 women) divided into tertiles by baseline serum cholestanol-to-cholesterol ratio (< or = 118.3 and > or = 147.7 10(2) x mmol/mol cholesterol), a surrogate marker of cholesterol absorption efficiency. The lowest cholestanol tertile was associated with high body mass index (BMI), plasma glucose, serum insulin and triglycerides, and cholesterol synthesis markers (cholestenol, desmosterol, lathosterol) and low HDL cholesterol and cholesterol absorption markers (campesterol, sitosterol) (P < 0.01 for all). The 19H allele of the ABCG8 gene accumulated in the lowest cholestanol tertile (P < 0.001) and was associated with low total and LDL cholesterol and absorption markers and with high synthesis markers (P < 0.05 for all). The 604E allele of the ABCG5 gene in men was associated with high BMI, plasma insulin, low serum sitosterol, and high serum cholestenol levels (P < 0.05 for all). In a subgroup of 71 men, the 604E allele was associated with insulin resistance measured with the hyperinsulinemic euglycemic clamp. In conclusion, low cholesterol absorption efficiency was associated with characteristics of the metabolic syndrome. Low serum cholesterol and cholesterol absorption were linked to the D19H polymorphism of the ABCG8 gene, and characteristics of the insulin resistance syndrome in men were linked with the Q604E polymorphism of the ABCG5 gene.     

The effects of ABCG5/G8 polymorphisms on plasma HDL cholesterol concentrations depend on smoking habit in the Boston Puerto Rican Health Study

Low HDL-cholesterol (HDL-C) is associated with an increased risk for atherosclerosis, and concentrations are modulated by genetic factors and environmental factors such as smoking. Our objective was to assess whether the association of common single-nucleotide polymorphisms (SNPs) at ABCG5/G8 (i18429G>A, i7892T>C, Gln604GluC>G, 5U145A>C, Tyr54CysA>G, Asp19HisG>C, i14222A>G, and Thr400LysC>A) genes with HDL-C differs according to smoking habit. ABCG5/G8 SNPs were genotyped in 845 participants (243 men and 602 women). ABCG5/G8 (i7892T>C, 5U145A>C, Tyr54CysA>G, Thr400LysC>A) SNPs were significantly associated with HDL-C concentrations (P < 0.001–0.013) by which carriers of the minor alleles at the aforementioned polymorphisms and homozygotes for the Thr400 allele displayed lower HDL-C. A significant gene-smoking interaction was found, in which carriers of the minor alleles at ABCG5/G8 (Gln604GluC>G, Asp19HisG>C, i14222A>G) SNPs displayed lower concentrations of HDL-C only if they were smokers (P = 0.001–0.025). Also, for ABCG8_Thr400LysC>A SNP, smokers, but not nonsmokers, homozygous for the Thr400 allele displayed lower HDL-C (P = 0.004). Further analyses supported a significant haplotype global effect on lowering HDL-C (P = 0.002) among smokers. In conclusion, ABCG5/G8 genetic variants modulate HDL-C concentrations, leading to an HDL-C-lowering effect and thereby a potential increased risk for atherosclerosis only in smokers.     

Polymorphisms in ABCG5 and ABCG8 transporters and plasma cholesterol levels

ABCG5 and ABCG8 transporters play an important role in the absorption and excretion of sterols. Missence polymorphisms (Gln604Glu in the ABCG5 and Asp19His, Tyr54Cys, Thr400Lys, and Ala632Val in the ABCG8) in these genes have been described. In 131 males and 154 females whose dietary composition markedly changed and lipid parameters decreased over an 8-year follow-up study (total cholesterol decreased from 6.21+/-1.31 mmol/l in 1988 to 5.43+/-1.06 mmol/l in 1996), these polymorphisms were investigated using PCR. Plasma lipid levels and changes in plasma lipid levels were independent of the Gln604Glu polymorphism in ABCG5 and Asp19His and the Ala632Val polymorphisms in ABCG8. The Tyr54Cys polymorphism influenced the degree of reduction in total plasma cholesterol (delta -0.49 mmol/l in Tyr54 homozygotes vs. delta +0.12 mmol/l in Cys54 homozygotes, p<0.04) and LDL-cholesterol (delta -0.57 mmol/l in Tyr54 homozygotes vs. delta +0.04 mmol/l in Cys54 homozygotes, p<0.03) levels between 1988 and 1996 in females, but not in males. Male Thr400 homozygotes exhibited a greater decrease in total cholesterol (delta -0.90 mmol/l vs. delta -0.30 mmol/l, p<0.02) and LDL-cholesterol (delta -0.62 mmol/l vs. delta -0.19 mmol/l, p<0.04) than Lys400 carriers. No such association was observed in females. We conclude that Tyr54Cys and Thr400Lys variations in the ABCG8 gene may play a role in the genetic determination of plasma cholesterol levels and could possibly influence the gender-specific response of plasma cholesterol levels after dietary changes. These polymorphisms are of potential interest as genetic variants that may influence the lipid profile.     

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.     

Common sequence variations in ABCG8 are related to plant sterol metabolism in healthy volunteers

Polymorphisms in the ATP binding cassette (ABC) transporters ABCG5 and ABCG8 are related to plasma plant sterol concentrations. It is not known whether these polymorphisms are also associated with variations in serum plant sterol concentrations during interventions affecting plant sterol metabolism. We therefore decided to study changes in serum plant sterol concentrations with ABCG5/G8 polymorphisms after consumption of plant stanol esters, which decrease plasma plant sterol concentrations. Cholesterol-standardized serum campesterol and sitosterol concentrations were significantly associated with the ABCG8 T400K genotype, as were changes in serum plant sterol concentrations after consumption of plant stanols. The reduction of -57.1 +/- 38.3 10(2) x micromol/mmol cholesterol for sitosterol in TT subjects was significantly greater compared with the -36.0 +/- 18.7 reduction in subjects with the TK genotype (P = 0.021) and the -16.9 +/- 13.0 reduction in subjects with the KK genotype (P = 0.047). Changes in serum campesterol concentrations showed a comparable association. No association with serum LDL cholesterol was found. Genetic variation in ABCG8 not only explains cross-sectional differences in serum plant sterol concentrations but also determines a subject's responsiveness to changes in serum plant sterols during interventions known to affect plant sterol metabolism.     

中国荷斯坦牛ABCG2基因编码区多态性检测及生物信息学分析

目的：研究中国荷斯坦牛ABCG2基因编码区（CDS）多态性,并进行生物信息学分析。方法：以中国荷斯坦牛为材料,利用PCR-SSCP技术对ABCG2基因CDS多态性进行检测,然后预测蛋白质序列的改变,并用生物信息学软件对蛋白质序列突变前后的结构及性质进行分析。结果：在外显子9中存在一个A→G碱基突变,导致氨基酸由酪氨酸突变为半胱氨酸,将此突变命名为Y367C;在外显子14中存在一个G→A突变,导致氨基酸由精氨酸突变为谷氨酰胺,将此突变命名为R578Q。2个突变一个位于功能区与跨膜区之间,一个位于跨膜区。生物信息学分析发现,蛋白质二级结构增加了1个卷曲（C）和2个转角（T）,同时减少了3个β折叠（E）,且ABCG2蛋白的组成和一些性质也发生了改变。结论：检测到的2个单核苷酸多态性引起了ABCG2蛋白性质和二级结构的改变;为进一步研究ABCG2蛋白对产乳性状的影响奠定了基础。     

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.     

Diosgenin regulates cholesterol metabolism in hypercholesterolemic rats by inhibiting NPC1L1 and enhancing ABCG5 and ABCG8

Hypercholesterolemia is a preventable risk factor for atherosclerosis and cardiovascular disease. However, the mechanisms of diosgenin (DG) that promote cholesterol homeostasis and alleviate hypercholesterolemia remain elusive. To investigate the effects and molecular mechanisms of the promotion of cholesterol metabolism by DG, a rat model of hypercholesterolemia was induced by providing a high-fat diet for 4 weeks. After 4 weeks, the rats were intragastrically administered high-dose DG (0.3 g/kg/d), low-dose DG (0.15 g/kg/d) or simvastatin (4 mg/kg/d) once a day for 8 weeks. The serum and hepatic cholesterol were tested, the mRNA and protein expression levels of Niemann-Pick C1-Like 1 (NPC1L1), liver X receptor-α (LXR-α) and the ATP-binding cassette G5/G8 (ABCG5/G8) transporters were measured. The results indicate that DG could reduce body weight, decrease the serum total cholesterol, triglyceride, low-density lipoprotein cholesterol, liver total cholesterol and free cholesterol levels compared to those in the controls. Simultaneously, liver tissue pathological morphology analyses revealed that DG could attenuate hepatic steatosis compared to that in the high-fat diet group. Further investigation demonstrated that DG significantly decreased the expression of NPC1L1 and LXR-α in the intestine and markedly increased the expression of ABCG5/G8 in the liver and intestine. Compared to the high-fat diet group, the rats in the DG-treated groups ameliorated hypercholesterolemia in a dose- and time-dependent manner. These data suggest that DG may not only inhibit intestinal cholesterol absorption by downregulating NPC1L1 but also enhance cholesterol excretion by increasing the expression of ABCG5/G8. DG could be a new candidate for the prevention of hypercholesterolemia.     

Structural Analysis of Cholesterol Binding and Sterol Selectivity by ABCG5/G8

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.     

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.     

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.     

High-level expression of ABCG5 and ABCG8 attenuates diet-induced hypercholesterolemia and atherosclerosis in Ldlr-/- mice

Transgenic mice expressing human ABCG5 (G5) and ABCG8 (G8) have decreased fractional absorption and increased biliary secretion of cholesterol, but their plasma cholesterol levels are unchanged (males) or modestly reduced (females). To determine whether increased expression of G5 and G8 can ameliorate hypercholesterolemia in mice lacking LDL receptors (LDLRs), we examined the effects of G5G8 transgene expression on cholesterol metabolism and atherosclerosis in Ldlr-/- mice. In chow-fed Ldlr-/- mice, the G5G8 transgene reduced fractional absorption of dietary cholesterol by 50% and increased biliary cholesterol levels by 60% but did not affect plasma cholesterol levels. On a Western diet (21% fat, 0.2% cholesterol), G5G8Tg; Ldlr-/- mice had a 30% reduction in the level of hepatic cholesterol and 45% lower plasma cholesterol levels than the Ldlr-/- mice. After 6 months on the Western diet, the atherosclerotic lesion area in the aortic root and arch was approximately 70% lower in the G5G8Tg;Ldlr-/- than in the Ldlr-/- mice and was correlated with the plasma cholesterol levels. These results demonstrate that increased expression of G5 and G8 attenuates diet-induced hypercholesterolemia in Ldlr-/- mice, resulting in a significant reduction in plasma levels of cholesterol and aortic atherosclerotic lesion area.     

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.     

Expression and regulation of the sterol half-transporter genes ABCG5 and ABCG8 in rats

The ABCG5 and ABCG8 genes encode half-transporter proteins that heterodimerize to form a transporter of plant sterols and cholesterol. The purpose of this study was to examine the expression and regulation of ABCG5 and ABCG8 at the mRNA level in Sprague-Dawley rats. Both ABCG5 and ABCG8 mRNA were expressed primarily in rat small intestine and liver, and gender-specific differences in expression were observed. The effects of treatment with a battery of microsomal enzyme inducers on ABCG5 and ABCG8 mRNA were examined; most treatments had no effect, but of three PXR ligands, PCN was an effective inducer, spironolactone was repressive, and dexamethasone was ineffective. The effects of a 1% cholesterol diet on the regulation of rat ABCG5 and ABCG8 were also examined, and compared with those in C57BL/6 mice. Cholesterol caused a suppression of ABCG5 and ABCG8 mRNA in rat liver, but the same treatment increased the expression of these genes in mouse liver. ABCG5 and ABCG8 mRNA was also induced by cholesterol in rat ileum, but not mouse ileum. These results suggest variation between rats and mice in regulatory mechanisms controlling ABCG5 and ABCG8 expression, and may explain some differences in lipid metabolism observed between these two species.     

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.     

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.     

Expression of ABCG5 and ABCG8 is required for regulation of biliary cholesterol secretion

The major pathway for elimination of cholesterol in mammals is via secretion into bile. Biliary cholesterol secretion is mediated by the ATP-binding cassette (ABC) transporters ABCG5 (G5) and ABCG8 (G8) and is stimulated by cholesterol and by the non-cholesterol steroids cholate and diosgenin. To define the relationship between G5G8 expression and biliary cholesterol secretion, we measured G5 and G8 mRNA levels and biliary cholesterol concentrations in genetically manipulated mice expressing 0, 1, 2, 5, 10, or 16 copies of the two genes. Biliary cholesterol levels varied directly with G5G8 copy number and hepatic mRNA levels over a >16-fold range. Thus neither delivery of cholesterol to the transporter nor levels of cholesterol acceptors in bile were limiting under these conditions. In wild-type mice, cholate and diosgenin both increased biliary cholesterol concentrations 2-3-fold. The increase in biliary cholesterol content was dependent on expression of G5 and G8; neither steroid increased biliary cholesterol levels in G5G8-/- mice. Cholate treatment was associated with a farnesoid X receptor (FXR)-dependent increase in hepatic mRNA and protein levels of G5 and G8. In contrast to cholate, diosgenin treatment did not affect G5G8 expression. Diosgenin increased the expression of several pregnane X receptor (PXR) target genes and the choleretic effect of diosgenin was reduced by approximately 70% in PXR knock-out mice. Thus G5 and G8 are required to modulate biliary cholesterol secretion in response to cholate and diosgenin, but the choleretic effects of these two steroids are mediated by different mechanisms requiring FXR and PXR, respectively.     

ABCG5 and ABCG8 are expressed in gallbladder epithelial cells

Gallbladder epithelial cells (GBEC) are exposed to high biliary cholesterol concentrations on their apical (AP) surface. The mechanisms of cholesterol absorption and efflux by these cells are not known. We hypothesized that ABCG5 and ABCG8 are expressed in GBEC and mediate AP cholesterol efflux. Human gallbladder cDNA expressed message for ABCG5 and ABCG8. Cultured murine GBEC also expressed abcg5 and abcg8 mRNA and protein, as did cultured canine GBEC. Interestingly, treatment with model bile containing supersaturating concentrations of cholesterol, or treatment with LXRalpha/RXR ligands, did not lead to differences in expression of ABCG5 or ABCG8 in the murine or the canine cells. The subcellular localization of ABCG5 and ABCG8 did show alterations, with predominantly intracellular localization at baseline and predominantly AP localization following treatment with model bile or LXRalpha ligand. GBEC therefore express ABCG5 and ABCG8; these sterol transporters may play a role in mediating AP cholesterol efflux in the gallbladder epithelium.