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.     

A missense mutation in the Abcg5 gene causes phytosterolemia in SHR,stroke-prone SHR,and WKY rats

Sitosterolemia is an autosomal recessive disorder caused by mutations in the ABCG5 or ABCG8 half-transporter genes. These mutations disrupt the mechanism that distinguishes between absorbed sterols and is most prominently characterized by hyperabsorption and impaired biliary elimination of dietary plant sterols. Sitosterolemia patients retain 15-20% of dietary plant sterols, whereas normal individuals absorb less than 1-5%. Normotensive Wistar Kyoto inbred (WKY inbred), spontaneously hypertensive rat (SHR), and stroke-prone spontaneously hypertensive rat (SHRSP) strains also display increased absorption and decreased elimination of dietary plant sterols. To determine if the genes responsible for sitosterolemia in humans are also responsible for phytosterolemia in rats, we sequenced the Abcg5 and Abcg8 genes in WKY inbred, SHR, and SHRSP rat strains. All three strains possessed a homozygous guanine-to-thymine transversion in exon 12 of the Abcg5 gene that results in the substitution of a conserved glycine residue for a cysteine amino acid in the extracellular loop between the fifth and sixth membrane-spanning domains of the ATP binding cassette half-transporter, sterolin-1. The identification of this naturally occurring mutation confirms that these rat strains are important animal models of sitosterolemia in which to study the mechanisms of sterol trafficking.     

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/Abcg8-independent pathways contribute to hepatobiliary cholesterol secretion in mice

The ATP-binding cassette (ABC) half-transporters ABCG5 and ABCG8 heterodimerize into a functional complex that mediates the secretion of plant sterols and cholesterol by hepatocytes into bile and their apical efflux from enterocytes. We addressed the putative rate-controlling role of Abcg5/Abcg8 in hepatobiliary cholesterol excretion in mice during (maximal) stimulation of this process. Despite similar bile salt (BS) excretion rates, basal total sterol and phospholipid (PL) output rates were reduced by 82% and 35%, respectively, in chow-fed Abcg5(-/-) mice compared with wild-type mice. When mice were infused with the hydrophilic BS tauroursodeoxycholate, similar relative increases in bile flow, BS output, PL output, and total sterol output were observed in wild-type, Abcg5(+/-), and Abcg5(-/-) mice. Maximal cholesterol and PL output rates in Abcg5(-/-) mice were only 15% and 69%, respectively, of wild-type values. An infusion of increasing amounts of the hydrophobic BS taurodeoxycholate increased cholesterol excretion by 3.0- and 2.4-fold in wild-type and Abcg5(-/-) mice but rapidly induced cholestasis in Abcg5(-/-) mice. Treatment with the liver X receptor (LXR) agonist T0901317 increased the maximal sterol excretion capacity in wild-type mice (fourfold), concomitant with the induction of Abcg5/Abcg8 expression, but not in Abcg5(-/-) mice. In a separate study, mice were fed chow containing 1% (wt/wt) cholesterol. As expected, hepatic expression of Abcg5 and Abcg8 was strongly induced (fivefold and fourfold) in wild-type but not LXR-alpha-deficient (Lxra(-/-)) mice. Surprisingly, hepatobiliary cholesterol excretion was increased to the same extent, i.e., 2.2-fold in wild-type mice and 2.0-fold in Lxra(-/-) mice, upon cholesterol feeding. Our data confirm that Abcg5, as part of the Abcg5/Abcg8 heterodimer, strongly controls hepatobiliary cholesterol secretion in mice. However, our data demonstrate that Abcg5/Abcg8 heterodimer-independent, inducible routes exist that can significantly contribute to total hepatobiliary cholesterol output.     

Dietary xenosterols lead to infertility and loss of abdominal adipose tissue in sterolin-deficient mice

The investigation of the human disease sitosterolemia (MIM 210250) has shed light not only on the pathways by which dietary sterols may traffic but also on how the mammalian body rids itself of cholesterol and defends against xenosterols. Two genes, ABCG5 and ABCG8, located at the sitosterolemia locus, each encodes a membrane-bound ABC half-transporter and constitutes a functional unit whose activity has now been shown to account for biliary and intestinal sterol excretion. Knockout mice deficient in Abcg5 or Abcg8 recapitulate many of the phenotypic features of sitosterolemia. During the course of our studies to characterize these knockout mice, we noted that these mice, raised on normal rodent chow, exhibited infertility as well as loss of abdominal fat. We show that, although sitosterolemia does not lead to any structural defects or to any overt endocrine defects, fertility could be restored if xenosterols are specifically blocked from entry and that the loss of fat is also reversed by a variety of maneuvers that limit xenosterol accumulation. These studies show that xenosterols may have a significant biological impact on normal mammalian physiology and that the Abcg5 or Abcg8 knockout mouse model may prove useful in investigating the role of xenosterols on mammalian physiology.     

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.     

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.     

Heritability of plasma noncholesterol sterols and relationship to DNA sequence polymorphism in ABCG5 and ABCG8

The plasma concentrations of cholesterol precursor sterols and plant sterols vary over a 5- to 10-fold range among normolipidemic individuals, and provide indices of the relative rates of cholesterol synthesis and fractional absorption. In the present study, we examined the relative contributions of genetic and environmental factors to variation in the plasma concentrations and sterol-cholesterol ratios of five noncholesterol sterols, including the 5alpha-saturated derivative of cholesterol (cholestanol), two precursors in the cholesterol biosynthesis pathway (desmosterol and lathosterol), and two phytosterols (campesterol and sitosterol). Plasma sterol concentrations were highly stable in 30 individuals measured over a 48 week period. Regression of offspring sterol levels on the parental values indicated that plasma levels of all five noncholesterol sterols were highly heritable. Analysis of monozygotic and dizygotic twin pairs also indicated strong heritability of all five sterols. Two common sequence variations (D19H and T400K) in ABCG8, an ABC half-transporter defective in sitosterolemia, were associated with lower concentrations of plant sterols in parents, and in their offspring.Taken together, these findings indicate that variation in the plasma concentrations of noncholesterol sterols is highly heritable, and that polymorphism in ABCG8 contributes to genetic variation in the plasma concentrations of plant sterols.     

Functional analysis of candidate ABC transporter proteins for sitosterol transport

Two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8, have recently been associated with the accumulation of dietary cholesterol in the sterol storage disease sitosterolemia. These two 'half-transporters' are assumed to dimerize to form the complete sitosterol transporter which reduces the absorption of sitosterol and related molecules in the intestine by pumping them back into the lumen. Although mutations altering ABCG5 and ABCG8 are found in affected patients, no functional demonstration of sitosterol transport has been achieved. In this study, we investigated whether other ABC transporters implicated in lipid movement and expressed in tissues with a role in sterol synthesis and absorption, might also be involved in sitosterol transport. Transport by the multidrug resistance P-glycoprotein (P-gp; Abcb1), the multidrug resistance-associated protein (Mrp1; Abcc1), the breast cancer resistance protein (Bcrp; Abcg2) and the bile salt export pump (Bsep; Abcb11) was assessed using several assays. Unexpectedly, none of the candidate proteins mediated significant sitosterol transport. This has implications for the pathology of sitosterolemia. In addition, the data suggest that otherwise broad-specific ABC transporters have acquired specificity to exclude sitosterol and related sterols like cholesterol presumably because the abundance of cholesterol in the membrane would interfere with their action; in consequence, specific transporters have evolved to handle these sterols.     

Phenotypic heterogeneity of sitosterolemia

Sitosterolemia is a rare autosomal recessive disorder of lipoprotein metabolism characterized by xanthomas and increased plasma concentrations of plant sterols, such as sitosterol. Causative mutations occur in either the ABCG5 or ABCG8 gene, each of which encodes a sterol half-transporter expressed in the intestine. We report five Canadian subjects with nonsense mutations in these half-transporters: four related Caucasian subjects were homozygous for the ABCG8 S107X mutation, and one unrelated Japanese-Canadian subject was homozygous for a complex insertion/deletion (I/D) mutation in ABCG5 exon 3. A female subject with each mutation was symptomatic with coronary atherosclerosis: a 5-year-old ABCG8 S107X homozygote and a 75-year-old ABCG5 exon 3 I/D homozygote; these represent the extreme ends of the spectrum of vascular involvement in sitosterolemia. The largest reductions in plasma concentrations of sitosterol and LDL-cholesterol were seen with ezetimibe or bile acid sequestrant treatment, and less dramatic reductions were seen with statin drug treatment. These findings extend the range of clinical phenotypes in sitosterolemia caused by nonsense mutations in either ABCG5 or ABCG8.     

Genetic basis of sitosterolemia

The molecular mechanisms regulating the amount of dietary cholesterol retained by the body, as well as the body's ability to exclude other dietary sterols selectively, are poorly understood. An average Western diet will contain approximately 250-500 mg of dietary cholesterol and approximately 200-400 mg of non-cholesterol sterols, of which plant sterols are the major constituents. Approximately 50-60% of dietary cholesterol is absorbed and retained by the normal human body, but less than 1% of the non-cholesterol sterols are retained. There thus exists a subtle mechanism that allows the body to distinguish between cholesterol and non-cholesterol sterols. In sitosterolemia, a rare autosomal recessive disorder, affected individuals hyperabsorb and retain not only cholesterol but also all other sterols, including plant and shellfish sterols from the intestine. Consequently, patients with this disease have very high levels of plant sterols in the plasma, and develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. The STSL locus has been mapped to human chromosome 2p21. Mutations in two tandem ABC genes, ABCG5 and ABCG8, encoding sterolin-1 and -2, respectively, are now known to be mutant in sitosterolemia. The identification of these genes should now lead to a better understanding of the molecular mechanism(s) governing the highly selective absorption and retention of cholesterol by the body. Indeed, it is the very existence of this disease that has given credence to the hypothesis that there is a molecular pathway that regulates dietary cholesterol absorption and sterol excretion by the body.     

Dietary plant sterols accumulate in the brain

Dietary plant sterols and cholesterol have a comparable chemical structure. It is generally assumed that cholesterol and plant sterols do not cross the blood-brain barrier, but quantitative data are lacking. Here, we report that mice deficient for ATP-binding cassette transporter G5 (Abcg5) or Abcg8, with strongly elevated serum plant sterol levels, display dramatically increased (7- to 16-fold) plant sterol levels in the brain. Apolipoprotein E (ApoE)-deficient mice also displayed elevated serum plant sterol levels, which was however not associated with significant changes in brain plant sterol levels. Abcg5- and Abcg8-deficient mice were found to carry circulating plant sterols predominantly in high-density lipoprotein (HDL)-particles, whereas ApoE-deficient mice accommodated most of their serum plant sterols in very low-density lipoprotein (VLDL)-particles. This suggests an important role for HDL and/or ApoE in the transfer of plant sterols into the brain. Moreover, sitosterol upregulated apoE mRNA and protein levels in astrocytoma, but not in neuroblastoma cells, to a higher extend than cholesterol. In conclusion, dietary plant sterols pass the blood-brain barrier and accumulate in the brain, where they may exert brain cell type-specific effects.     

A reappraisal of the mechanism by which plant sterols promote neutral sterol loss in mice

Dietary plant sterols (PS) reduce serum total and LDL-cholesterol in hyperlipidemic animal models and in humans. This hypocholesterolemic effect is generally ascribed to inhibition of cholesterol absorption. However, whether this effect fully explains the reported strong induction of neutral sterol excretion upon plant sterol feeding is not known. Recent data demonstrate that the intestine directly mediates plasma cholesterol excretion into feces, i.e., without involvement of the hepato-biliary route.

Objective

Aim of this study was to determine whether stimulation of fecal neutral sterol loss during PS feeding is (partly) explained by increased intestinal cholesterol excretion and to assess the role of the cholesterol transporter Abcg5/Abcg8 herein.

Methods and Results

Wild-type mice were fed a control diet or diets enriched with increasing amounts of PS (1%, 2%, 4% or 8%, wt/wt) for two weeks. In addition, Abcg5^-/- mice were fed either control or 8% PS diet. PS feeding resulted in a dose-dependent decrease of fractional cholesterol absorption (∼2–7-fold reduction) in wild-type mice and ∼80% reduction in Abcg5^-/- mice. Furthermore, PS feeding led to a strong, dose-independent induction of neutral sterol excretion (3.4-fold in wild-types and 2.7-fold in Abcg5^-/- mice) without changes in biliary cholesterol secretion. It was calculated that PS feeding stimulated intestinal cholesterol excretion by ∼500% in wild-type mice and by ∼250% in Abcg5^-/-.

Conclusions

Our data indicate that in mice the cholesterol-lowering effects of PS are to a large extent attributable to stimulation of intestinal, non-bile derived, cholesterol excretion. The Abcg5/Abcg8 heterodimer is involved in facilitating this PS-induced flux of cholesterol.     

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.     

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.     

Two Genes That Map to the STSL Locus Cause Sitosterolemia: Genomic Structure and Spectrum of Mutations Involving Sterolin-1 and Sterolin-2, Encoded by ABCG5 and ABCG8, Respectively

Sitosterolemia is a rare autosomal recessive disorder characterized by (a) intestinal hyperabsorption of all sterols, including cholesterol and plant and shellfish sterols, and (b) impaired ability to excrete sterols into bile. Patients with this disease have expanded body pools of cholesterol and very elevated plasma plant-sterol species and frequently develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. In previous studies, we have mapped the STSL locus to human chromosome 2p21. Recently, we reported that a novel member of the ABC-transporter family, named "sterolin-1" and encoded by ABCG5, is mutated in 9 unrelated families with sitosterolemia; in the remaining 25 families, no mutations in sterolin-1 could be identified. We identified another ABC transporter, located <400 bp upstream of sterolin-1, in the opposite orientation. Mutational analyses revealed that this highly homologous protein, termed "sterolin-2" and encoded by ABCG8, is mutated in the remaining pedigrees. Thus, two highly homologous genes, located in a head-to-head configuration on chromosome 2p21, are involved as causes of sitosterolemia. These studies indicate that both sterolin-1 and sterolin-2 are indispensable for the regulation of sterol absorption and excretion. Identification of sterolin-1 and sterolin-2 as critical players in the regulation of dietary-sterol absorption and excretion identifies a new pathway of sterol transport.     

Loss of ABCG1 results in chronic pulmonary inflammation

ABCG1, a member of the ATP-binding cassette transporter superfamily, is highly expressed in multiple cells of the lung. Loss of ABCG1 results in severe pulmonary lipidosis in mice, with massive deposition of cholesterol in both alveolar macrophages and type 2 cells and the accumulation of excessive surfactant phospholipids. These observations are consistent with ABCG1 controlling cellular sterol metabolism. Herein, we report on the progressive and chronic inflammatory process that accompanies the lipidosis in the lungs of Abcg1-/- mice. Compared with wild-type animals, the lungs of aged chow-fed mice deficient in ABCG1 show distinctive signs of inflammation that include macrophage accumulation, lymphocytic infiltration, hemorrhage, eosinophilic crystals, and elevated levels of numerous cytokines and cytokine receptors. Analysis of bronchoalveolar lavages obtained from Abcg1-/- mice revealed elevated numbers of foamy macrophages and leukocytes and the presence of multiple markers of inflammation including crystals of chitinase-3-like proteins. These data suggest that cholesterol and/or cholesterol metabolites that accumulate in Abcg1-/- lungs can trigger inflammatory signaling pathways. Consistent with this hypothesis, the expression of a number of cytokines was found to be significantly increased following increased cholesterol delivery to either primary peritoneal macrophages or Raw264.7 cells. Finally, cholesterol loading of primary mouse macrophages induced cytokine mRNAs to higher levels in Abcg1-/-, as compared with wild-type cells. These results demonstrate that ABCG1 plays critical roles in pulmonary homeostasis, balancing both lipid/cholesterol metabolism and inflammatory responses.     

The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8(+/+), Abcg8(+/-), and Abcg8(-/-) mice with hydrophilic and hydrophobic bile salts. In Abcg8(-/-) mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8(-/-) mice at a much lower infusion rate compared with Abc8(-/-) and Abcg8(+/-) mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8(-/-) mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.     

ATP-binding cassette G5/G8 deficiency causes hypertriglyceridemia by affecting multiple metabolic pathways

Mutations in ABCG5 or ABCG8 transporters are responsible for sitosterolemia, an autosomal recessive disease characterized by the accumulation of plant sterols. The aim of this study was to investigate the effects of ABCG5 and ABCG8 deficiency on TG metabolism in mice. Experiments were carried out in wild-type (G5/G8+/+) mice, mice heterozygous for ABCG5 and ABCG8 deficiency (G5/G8+/-) and ABCG5/G8-deficient (G5/G8-/-) mice fed a chow diet. Plasma TG were 2.6 and 4.3-fold higher in fasted G5/G8+/- and G5/G8-/- mice, respectively, than in G5/G8+/+ mice. Postprandial TG were 5-fold higher in G5/G8-/- mice. TG metabolism studies indicate that: first, the fractional catabolic rate was significantly lower in G5/G8+/- (1.3-fold) and G5/G8-/- mice (1.5-fold) compared to G5/G8+/+ and postheparin plasma lipoprotein lipase activities were significantly lower in G5/G8+/- (1.8-fold) and G5/G8-/- mice (5.4-fold) than in G5/G8+/+. Second, liver TG secretion was 1.3-fold higher in G5/G8+/- and G5/G8-/- than in G5/G8+/+ mice and this was associated with an increase in liver LXR, FAS, ACAC and CD36 gene expression. Third, TG intestinal secretion, determined after an oral fat gavage of glycerol tri[9,10(n)-(3)H] oleate, was 5.8-fold higher in G5/G8-/- than in G5/G8+/+ mice. Also, the HOMA index was 2.6-fold higher in G5/G8-/- than in G5/G8+/+ mice, reflecting a degree of insulin resistance. In conclusion, ABCG5/G8 deficiency in mice fed a chow diet markedly raises TG levels by impairing TG catabolism and by increasing liver and intestinal TG secretion.     

SHRSP/Izm and WKY/NCrlCrlj rats having a missense mutation in Abcg5 deposited plant sterols in the body, but did not change their biliary secretion and lymphatic absorption-comparison with Jcl:Wistar and WKY/Izm rats

We had previously found plant sterols deposited in the bodies of stroke-prone spontaneously hypertensive rats (SHRSP)/Sea and Wistar Kyoto (WKY)/NCrlCrlj rats that had a missense mutation in the Abcg5 cDNA sequence that coded for ATP-binding cassette transporter (ABC) G5. We used SHRSP/Izm, WKY/NCrlCrlj, and WKY/Izm rats in the present study to determine the mechanisms for plant sterol deposition in the body. Jcl:Wistar rats were used as a control strain. A diet containing 0.5% plant sterols fed to the rats resulted in plant sterol deposition in the body of SHRSP/Izm, but not in WKY/Izm or Jcl:Wistar rats. Only a single non-synonymous nucleotide change, G1747T, resulting in a conservative cysteine substitution for glycine at amino acid 583 (Gly583Cys) in Abcg5 cDNA was identified in the SHRSP/Izm and WKY/NCrlCrlj rats. However, this mutation was not found in the WKY/Izm or Jcl:Wistar rats. No significant difference in the biliary secretion or lymphatic absorption of plant sterols was apparent between the rat strains with or without the missense mutation in Abcg5 cDNA. Our observations suggest that plant sterol deposition in rat strains with the missense mutation in Abcg5 cDNA can occur, despite there being no significant change in the biliary secretion or lymphatic absorption of plant sterols.