Genetic background of cholesterol gallstone disease

AbstractCholesterol gallstone formation is a multifactorial process involving a multitude of metabolic pathways. The primary pathogenic factor is hypersecretion of free cholesterol into bile. For people living in the Western Hemisphere, this is almost a normal condition, certainly in the elderly, which explains the very high incidence of gallstone disease. It is probably because the multifactorial background genes responsible for the high incidence have not yet been identified, despite the fact that genetic factors clearly play a role. Analysis of the many pathways involved in biliary cholesterol secretion reveals many potential candidates and considering the progress in unraveling the regulatory mechanisms of the responsible genes, identification of the primary gallstone genes will be successful in the near future

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 1/1, Abcg8 1/2, and Abcg8 2/2 mice with hydrophilic and hydrophobic bile salts. In Abcg8 2/2 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 2/2 mice at a much lower infusion rate compared with Abc8 2/2 and Abcg8 1/2 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 2/2 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.—Kosters,A.,C.Kunne,N.Looije,S.B.Patel, R. P. J. Oude Elferink, and A. K. Groen. The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

Cardiomyopathy reverses with recovery of liver injury, cholestasis and cholanemia in mouse model of biliary fibrosis

BackgroundTriggers and exacerbants of cirrhotic cardiomyopathy (CC) are poorly understood, limiting treatment options in patients with chronic liver diseases. Liver transplantation alone reverses some features of CC, but the physiology behind this effect has never been studied.AimsWe aimed to determine whether reversal of liver injury and fibrosis in mouse affects cardiac parameters. The second aim was to determine whether cardiomyopathy can be induced by specifically increasing systemic bile acid (BA) levels.Methods6-8 week old male C57BL6J mice were fed either chow (n = 5) or 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) (n = 10) for 3 weeks. At the end of 3 weeks, half the mice in the DDC fed group were randomized to chow (the reversed [REV] group). Serial ECHOs and electrocardiographic analysis was conducted weekly for 6 weeks followed by liver tissue and serum studies. Hearts were analysed for key components of function and cell signalling. Cardiac physiological and molecular parameters were similarly analysed in Abcb11(-/-) mice (n = 5/grp) fed 0.5% cholic acid supplemented diet for 1 week.ResultsMice in the REV group showed normalization of biochemical markers of liver injury with resolution of electrocardiographic and ECHO aberrations. Catecholamine resistance seen in DDC group resolved in the REV group. Cardiac recovery was accompanied by normalization of cardiac troponin-T2 as well as resolution of cardiac stress response at RNA level. Cardiovascular physiological and molecular parameters correlated with degree of cholanemia. Cardiomyopathy was reproduced in cholanemic BA fed Abcb11(-/-) mice.ConclusionsCardiomyopathy resolves with resolution of liver injury, is associated with cholanaemia, and can be induced by BA feeding

Sexually Dimorphic Genome-Wide Binding of Retinoid X Receptor alpha (RXRα) Determines Male-Female Differences in the Expression of Hepatic Lipid Processing Genes in Mice

<div><p>Many hepatic functions including lipid metabolism, drug metabolism, and inflammatory responses are regulated in a sex-specific manner due to distinct patterns of hepatic gene expression between males and females. Regulation for the majority of these genes is under control of Nuclear Receptors (NRs). Retinoid X Receptor alpha (RXRα) is an obligate partner for multiple NRs and considered a master regulator of hepatic gene expression, yet the full extent of RXRα chromatin binding in male and female livers is unclear. ChIP-Seq analysis of RXRα and RNA Polymerase2 (Pol2) binding was performed livers of both genders and combined with microarray analysis. Mice were gavage-fed with the RXR ligand LG268 for 5 days (30 mg/kg/day) and RXRα-binding and RNA levels were determined by ChIP-qPCR and qPCR, respectively. ChIP-Seq revealed 47,845 (male) and 46,877 (female) RXRα binding sites (BS), associated with ∼12,700 unique genes in livers of both genders, with 91% shared between sexes. RXRα-binding showed significant enrichment for 2227 and 1498 unique genes in male and female livers, respectively. Correlating RXRα binding strength with Pol2-binding revealed 44 genes being male-dominant and 43 female-dominant, many previously unknown to be sexually-dimorphic. Surprisingly, genes fundamental to lipid metabolism, including Scd1, Fasn, Elovl6, and Pnpla3-implicated in Fatty Liver Disease pathogenesis, were predominant in females. RXRα activation using LG268 confirmed RXRα-binding was 2–3 fold increased in female livers at multiple newly identified RXRα BS including for Pnpla3 and Elovl6, with corresponding ∼10-fold and ∼2-fold increases in Pnpla3 and Elovl6 RNA respectively in LG268-treated female livers, supporting a role for RXRα regulation of sexually-dimorphic responses for these genes. RXRα appears to be one of the most widely distributed transcriptional regulators in mouse liver and is engaged in determining sexually-dimorphic expression of key lipid-processing genes, suggesting novel gender- and gene-specific responses to NR-based treatments for lipid-related liver diseases.</p></div

Correlation of gender differential RXRα, Pol2 and RNA expression levels.

<p>A) Correlation of RXRα and Pol2 binding with gene expression levels. B) Scatterplot representation of gender differential RXRα binding with gender differential gene expression on the left-hand side, and scatterplot representation of gender differential Pol2 binding with gender differential gene expression on the right-hand side. C) Genes with a gender specific positive correlation between RXRα binding Pol2 binding and changes in RNA levels (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s004" target="_blank">Fig. S4A–B</a> and Table S6A–H in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>).</p

Representatives of gender differential RXRαregulated genes.

<p>A) Predominant RXRα and Pol2 binding for Cyp7b1 in male mouse liver. Left-hand panel: Top 2 tracks show Pol2 binding along the Cyp7b1 gene-span in male and female mouse liver. Bottom 2 tracks show RXRα peaks binding upstream of the TSS of Cyp7b1 TSS. Male enriched RXRα peaks are indicated by arrows P1, P2 and P3. Right-hand panel: Sexual dimorphic RNA levels of Cyp7b1 were confirmed by qPCR (n = 5–6) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s002" target="_blank">Fig. S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s003" target="_blank">S3A–G</a>). B) Predominant RXRα and Pol2 binding for Pnpla3 in female mouse liver. Left-hand panel: Top 2 tracks show Pol2 binding along the Pnpla3 gene-span in male and female mouse liver. Bottom 2 tracks show RXRα peaks binding upstream of the TSS of Pnpla3 TSS. Female enriched RXRα peaks are indicated by arrows P1, P2, P3 and P4. Right-hand panel: Sexual dimorphic RNA levels of Pnpla3 were confirmed by qPCR (n = 5–6) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s002" target="_blank">Fig. S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s003" target="_blank">S3A–G</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s007" target="_blank">S7</a> and S8). *p<0.05</p

Diosgenin-induced biliary cholesterol secretion in mice requires Abcg8

The plant sterol diosgenin has been shown to stimulate biliary cholesterol secretion in mice without affecting the expression of the adenosine triphosphate-binding cassette transporter heterodimer Abcg5/g8. The aim of this study was to investigate the mechanism of diosgenin-induced cholesterol hypersecretion and to identify the genes involved. Surprisingly, despite its lack of effect on Abcg5/g8 expression in wild-type mice, diosgenin did not stimulate biliary cholesterol secretion in mice deficient for Abcg8. Analysis of the kinetics of cholesterol secretion suggested that diosgenin probably activates a step before Abcg5/g8. To identify potential diosgenin targets, gene expression profiling was performed in mice fed a diosgenin-supplemented diet. Diosgenin feeding increased hepatic expression of genes involved in cholesterol synthesis as well as genes encoding for several cytochrome P450s. No significant change in expression of known cholesterol transporters was found. Comparison with published expression-profiling data for Srebp2-overexpressing mice, another mouse model in which biliary cholesterol secretion is elevated, revealed a number of genes with unknown function that were upregulated in both diosgenin-fed mice and mice overexpressing Srebp2. In conclusion, we found that although Abcg8 is essential for most diosgenin-induced biliary cholesterol hypersecretion, diosgenin probably does not interact directly with Abcg5/Abcg8, but rather increases cholesterol delivery to the heterodime

Sexual dimorphic RXRα-dependent gene regulation in mouse liver.

<p>A) Scatter plot of RXRα scores correlating with Pol2 scores. Genes identified having a RXRα and Pol2 score >6 for male and -<6 for females are represented by the black dots, with the grey dots representing correlation of all genes. Genes listed in order of RXRα score high to low (see also Table S5A and S5B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>). B) Ontology analysis and pathway analysis of genes with an RXRα and Pol2 score >6 for male and -<6 for females (see also Table S5C and S5D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>)</p

Genome wide mapping of RXRα and Pol2 binding sites in male and female mouse liver.

<p>A) Venn diagrams show number of RXRα binding sites in male and female livers with 37602 binding sites shared between genders, and 10243 unique RXRα male and 9275 unique RXRα female binding sites (see also Table S1A and S1B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>). B) Venn diagrams show number of Pol2 binding sites in male and female livers, with 19307 binding sites shared between genders, and 6037 unique male and 1332 unique female Pol2 binding sites. C) Venn diagrams representing number of genes associated with RXRα peaks shown in A), with 11660 genes shared between genders, and 1099 unique genes for male and 1021 genes for female. D) Western blot analysis shows increased nuclear RXRα protein levels in female mouse liver compared to male mouse liver. E) Number of RXRα peaks as stratified by peak height (see also Table S2A and S2B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s008" target="_blank">File S1</a>). F) Genomic positions of RXRα binding sites in male and female mouse liver. Data expressed as percentage of total peaks per gender. RXRα binding was significantly increased at TSS (+/−500 bp), TES, within exons and introns with a depletion in binding compared to the expected number of sites based on random distribution (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071538#pone.0071538.s001" target="_blank">Fig. S1A and S1B</a>).</p