Increased hepatobiliary and fecal cholesterol excretion upon activation of the liver X receptor is independent of ABCA1

The ATP-binding cassette transporter ABCA1 is essential for high density lipoprotein (HDL) formation and considered rate-controlling for reverse cholesterol transport. Expression of the Abca1 gene is under control of the liver X receptor (LXR). We have evaluated effects of LXR activation by the synthetic agonist T0901317 on hepatic and intestinal cholesterol metabolism in C57BL/6J and DBA/1 wild-type mice and in ABCA1-deficient DBA/1 mice. In wild-type mice, T0901317 increased expression of Abca1 in liver and intestine, which was associated with a similar to60% rise in HDL. Biliary cholesterol excretion rose 2.7-fold upon treatment, and fecal neutral sterol output was increased by 150-300%. Plasma cholesterol levels also increased in treated Abca1(-/-) mice (+120%), but exclusively in very low density lipoprotein-sized fractions. Despite the absence of HDL, hepatobiliary cholesterol output was stimulated upon LXR activation in Abca1(-/-) mice, leading to a 250% increase in the biliary cholesterol/phospholipid ratio. Most importantly, fecal neutral sterol loss was induced to a similar extent (+300%) by the LXR agonist in DBA/1 wild-type and Abca1(-/-) mice. Expression of Abcg5 and Abcg8, recently implicated in biliary excretion of cholesterol and its intestinal absorption, was induced in T0901317-treated mice. Thus, activation of LXR in mice leads to enhanced hepatobiliary cholesterol secretion and fecal neutral sterol loss independent of (ABCA1-mediated) elevation of HDL and the presence of ABCA1 in liver and intestine

Localization of ABCG5 and ABCG8 proteins in human liver, gall bladder and intestine

BACKGROUND: The molecular mechanisms that regulate the entry of dietary sterols into the body and their removal via hepatobiliary secretion are now beginning to be defined. These processes are specifically disrupted in the rare autosomal recessive disease, Sitosterolemia (MIM 210250). Mutations in either, but not both, of two genes ABCG5 or ABCG8, comprising the STSL locus, are now known to cause this disease and their protein products are proposed to function as heterodimers. Under normal circumstances cholesterol, but not non-cholesterol sterols, is preferentially absorbed from the diet. Additionally, any small amounts of non-cholesterol sterols that are absorbed are rapidly taken up by the liver and preferentially excreted into bile. Based upon the defects in sitosterolemia, ABCG5 and ABCG8 serve specifically to exclude non-cholesterol sterol entry at the intestinal level and are involved in sterol excretion at the hepatobiliary level. METHODS: Here we report the biochemical and immuno-localization of ABCG5 and ABCG8 in human liver, gallbladder and intestine using cell fractionation and immunohistochemical analyses. RESULTS: We raised peptide antibodies against ABCG5 and ABCG8 proteins. Using human liver samples, cell fractionation studies showed both proteins are found in membrane fractions, but they did not co-localize with caveolin-rafts, ER, Golgi or mitochondrial markers. Although their distribution in the sub-fractions was similar, they were not completely contiguous. Immunohistochemical analyses showed that while both proteins were readily detectable in the liver, ABCG5 was found predominately lining canalicular membranes, whereas ABCG8 was found in association with bile duct epithelia. At the cellular level, ABCG5 appeared to be apically expressed, whereas ABCG8 had a more diffuse expression pattern. Both ABCG5 and ABCG8 appeared to localize apically as shown by co-localization with MRP2. The distribution patterns of ABCG5 and ABCG8 in the gallbladder were very similar to each other. In the small intestine both ABCG5 and ABCG8 appear to line the brush border. However, at the level of the enterocyte, the cellular distribution patterns of ABCG5 and ABCG8 differed, such that ABCG5 was more diffuse, but ABCG8 was principally apical. Using standard deglycosylation methods, ABCG5 and ABCG8 do not appear to be glycosylated, suggesting a difference between human and mouse proteins. CONCLUSION: We report the distribution patterns of ABCG5 and ABCG8 in human tissues. Cell fractionation studies showed that both proteins co-fractionated in general, but could also be found independent of each other. As predicted, they are expressed apically in both intestine and liver, although their intracellular expression patterns are not completely congruent. These studies support the concept of heterodimerization of ABCG5 and ABCG8, but also support the notion that these proteins may have an independent function

A mouse model of sitosterolemia: absence of Abcg8/sterolin-2 results in failure to secrete biliary cholesterol

BACKGROUND: Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion. METHODS: In order to mimic the human disease, we created, by a targeted disruption, a mouse model of sitosterolemia resulting in Abcg8/sterolin-2 deficiency alone. Homozygous knockout mice are viable and exhibit sitosterolemia. RESULTS: Mice deficient in Abcg8 have significantly increased plasma and tissue plant sterol levels (sitosterol and campesterol) consistent with sitosterolemia. Interestingly, Abcg5/sterolin-1 was expressed in both liver and intestine in Abcg8/sterolin-2 deficient mice and continued to show an apical expression. Remarkably, Abcg8 deficient mice had an impaired ability to secrete cholesterol into bile, but still maintained the ability to secrete sitosterol. We also report an intermediate phenotype in the heterozygous Abcg8+/- mice that are not sitosterolemic, but have a decreased level of biliary sterol secretion relative to wild-type mice. CONCLUSION: These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol. Since biliary sitosterol secretion is preserved, although not elevated in the sitosterolemic mice, this observation suggests that mechanisms other than by Abcg8/sterolin-2 may be responsible for its secretion into bile

A common polymorphism in NR1H2 (LXRbeta) is associated with preeclampsia

<p>Abstract</p> <p>Background</p> <p>Preeclampsia is a frequent complication of pregnancy and a leading cause of perinatal mortality. Both genetic and environmental risk factors have been identified. Lipid metabolism, particularly cholesterol metabolism, is associated with this disease. Liver X receptors alpha (NR1H3, also known as LXRalpha) and beta (NR1H2, also known as LXRbeta) play a key role in lipid metabolism. They belong to the nuclear receptor superfamily and are activated by cholesterol derivatives. They have been implicated in preeclampsia because they modulate trophoblast invasion and regulate the expression of the endoglin (CD105) gene, a marker of preeclampsia. The aim of this study was to investigate associations between the <it>NR1H3 </it>and <it>NR1H2 </it>genes and preeclampsia.</p> <p>Methods</p> <p>We assessed associations between single nucleotide polymorphisms of <it>NR1H3 </it>(rs2279238 and rs7120118) and <it>NR1H2 </it>(rs35463555 and rs2695121) and the disease in 155 individuals with preeclampsia and 305 controls. Genotypes were determined by high-resolution melting analysis. We then used a logistic regression model to analyze the different alleles and genotypes for those polymorphisms as a function of case/control status.</p> <p>Results</p> <p>We found no association between <it>NR1H3 </it>SNPs and the disease, but the <it>NR1H2 </it>polymorphism rs2695121 was found to be strongly associated with preeclampsia (genotype C/C: adjusted odds ratio, 2.05; 95% CI, 1.04-4.05; <it>p </it>= 0.039 and genotype T/C: adjusted odds ratio, 1.85; 95% CI, 1.01-3.42; <it>p </it>= 0.049).</p> <p>Conclusions</p> <p>This study provides the first evidence of an association between the <it>NR1H2 </it>gene and preeclampsia, adding to our understanding of the links between cholesterol metabolism and this disease.</p

Cholesterol transport by the placenta:Placental liver X receptor activity as a modulator of fetal cholesterol metabolism?

Cholesterol is an important sterol in mammals. Defects in cholesterol synthesis or intracellular routing have devastating consequences already in utero: the Smith-Lemli-Opitz syndrome, desmosterolosis and Niemann-Pick C I disease provide examples of severe human inherited diseases caused by mutations in cholesterol metabolism genes. On the other hand, elevated plasma cholesterol concentrations are associated with the development of atherosclerosis which represents a major health risk in Western societies. Moreover, several studies indicate that development of atherosclerosis may already start during fetal life. Hence, a carefully balanced regulation of cholesterol metabolism appears of critical importance for both the development of the fetus and health of the adult. In the adult, the liver X receptor is a key regulator of cholesterol metabolism. Its target genes regulate cellular cholesterol efflux and thereby modulate whole-body cholesterol fluxes. LXR and several of its target genes have recently been demonstrated to be expressed in the placenta, which would provide a means to control delivery of maternal cholesterol to the fetus. Here we discuss the potential role of the placenta in the regulation of fetal cholesterol homeostasis and strategies to influence maternal-fetal cholesterol transfer. (C) 2006 Elsevier Ltd. All rights reserved

Increased fecal neutral sterol loss upon liver X receptor activation is independent of biliary sterol secretion in mice

Background & Aims: Reverse cholesterol transport (RCT) is defined as high-density lipoprotein (HDL)-mediated flux of excess cholesterol from peripheral cells to liver, followed by secretion into bile and disposal via the feces. Various steps of this pathway are controlled by the liver X receptor (LXR). We addressed the role of the intestine in LXR-dependent stimulation of fecal cholesterol excretion. Methods: To segregate biliary from intestine-derived cholesterol, wild-type and Mdr2 P-glycoprotein-deficient mice (Mdr2(-/-)), which are unable to secrete cholesterol into bile, were treated with the LXR agonist GW3965. Results: Treatment with GW3965 increased biliary cholesterol secretion by 74% in wild-type mice but had no effect in Mdr2(-/-) mice. LXR activation increased fecal neutral sterol excretion 2.1-fold in wild-type mice. Surprisingly, an identical increase was observed in Mdr2(-/-) mice. Fractional cholesterol absorption was reduced on LXR activation in both strains but was more pronounced in Mdr2(-/-) mice, coinciding with reduced Npc111 expression. Intestinal gene expression of ATPbinding cassette transporters (Abc) Abca1, Abcg1, Abcg5, and Abcg8 was strongly induced upon LXR activation in both strains, whereas expression of HMGCoA reductase, controlling cholesterol synthesis, remained unaffected. Additionally, LXR activation stimulated the excretion of plasma-derived [H-3]cholesterol into the fecal neutral sterol fraction in Mdr2(-/-) mice. Conclusions: Increased fecal cholesterol loss upon LXR activation is independent of biliary cholesterol secretion in mice. An important part of excess cholesterol is excreted directly via the intestine, supporting the existence of an alternative, quantitatively important route for cholesterol disposal

The origin of fetal sterols in second-trimester amniotic fluid: endogenous synthesis or maternal-fetal transport?

OBJECTIVE: Cholesterol is crucial for fetal development. To gain more insight into the origin of the fetal cholesterol pool in early human pregnancy, we determined cholesterol and its precursors in the amniotic fluid of uncomplicated, singleton human pregnancies. STUDY DESIGN: Total sterols were characterized by gas chromatography-mass spectrometry in the second-trimester amniotic fluid of 126 healthy fetuses from week 15 until week 22. RESULTS: The markers of cholesterol biosynthesis, lanosterol, dihydrolanosterol, and lathosterol, were present in low levels until the 19th week of gestation, after which their levels increased strongly. beta-sitosterol, a marker for maternal-fetal cholesterol transport, was detectable in the amniotic fluid. The total cholesterol levels increased slightly between weeks 15 and 22. CONCLUSION: Our results support the hypothesis that during early life the fetus depends on maternal cholesterol supply because endogenous synthesis is relatively low. Therefore, maternal cholesterol can play a crucial role in fetal development