Astrid Kosters

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

BackgroundMutations 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.MethodsIn 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.ResultsMice 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.ConclusionThese 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.

Probiotic Lactobacillus reuteri promotes TNF‐induced apoptosis in human myeloid leukemia‐derived cells by modulation of NF‐κB and MAPK signalling

The molecular mechanisms of pro‐apoptotic effects of human‐derived Lactobacillus reuteri ATCC PTA 6475 were investigated in this study. L. reuteri secretes factors that potentiate apoptosis in myeloid leukemia‐derived cells induced by tumour necrosis factor (TNF), as indicated by intracellular esterase activity, terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end‐labelling assays and poly (ADP‐ribose) polymerase cleavage. L. reuteri downregulated nuclear factor‐κB (NF‐κB)‐dependent gene products that mediate cell proliferation (Cox‐2, cyclin D1) and cell survival (Bcl‐2, Bcl‐xL). L. reuteri suppressed TNF‐induced NF‐κB activation, including NF‐κB‐dependent reporter gene expression in a dose‐and time‐dependent manner. L. reuteri stabilized degradation of IκBα and inhibited nuclear translocation of p65 (RelA). Although phosphorylation of IκBα was not affected, subsequent polyubiquitination necessary for regulated IκBα degradation was abrogated by L. reuteri. In addition, L. reuteri promoted apoptosis by enhancing mitogen‐activated protein kinase (MAPK) activities including c‐Jun N‐terminal kinase and p38 MAPK. In contrast, L. reuteri suppressed extracellular signal‐regulated kinases 1/2 in TNF‐activated myeloid cells. L. reuteri may regulate cell proliferation by promoting apoptosis of activated immune cells via inhibition of IκBα ubiquitination and enhancing pro‐apoptotic MAPK signalling. An improved understanding of L. reuteri‐mediated effects on apoptotic signalling pathways may facilitate development of future probiotics‐based regimens for prevention of colorectal cancer and inflammatory bowel disease.

Bile acid transporters in health and disease

In recent years the discovery of a number of major transporter proteins expressed in the liver and intestine specifically involved in bile acid transport has led to improved understanding of bile acid homeostasis and the enterohepatic circulation. Sodium (Na+)-dependent bile acid uptake from portal blood into the liver is mediated primarily by the Na+ taurocholate co-transporting polypeptide (NTCP), while secretion across the canalicular membrane into the bile is carried out by the bile salt export pump (BSEP). In the ileum, absorption of bile acids from the lumen into epithelial cells is mediated by the apical Na+ bile salt transporter (ASBT), whereas exit into portal blood across the basolateral membrane is mediated by the organic solute transporter α/β (OSTα/β) heterodimer. Regulation of transporter gene expression and function occurs at several different levels: in the nucleus, members of the nuclear receptor superfamily, regulated by bile acids and other ligands are primarily involved in controlling gene expression, while cell signalling events directly affect transporter function, and subcellular localization. Polymorphisms, dysfunction, and impaired adaptive responses of several of the bile acid transporters, e.g. BSEP and ASBT, results in liver and intestinal disease. Bile acid transporters are now understood to play central roles in driving bile flow, as well as adaptation to various pathological conditions, with complex regulation of activity and function in the nucleus, cytoplasm, and membrane.

The Role of Inflammation in Cholestasis: Clinical and Basic Aspects

Hepatobiliary transport systems are essential for the uptake and excretion of a variety of compounds including bile acids. Disruption and dysregulation of this excretory pathway result in cholestasis, leading to the intrahepatic accumulation of bile acids and other toxic compounds with progression of liver pathology. Cholestasis induced by inflammation is a common complication in patients with extrahepatic infections or inflammatory processes, generally referred to as sepsis-associated cholestasis. Microbial products, including endotoxin, induce signaling pathways within hepatocytes either directly, or through activation of proinflammatory cytokines, leading to rapid and profound reductions in bile flow. The expression and function of key hepatobiliary transporters are suppressed in response to inflammatory signaling. These proinflammatory signaling cascades lead to repressed expression and activity of a large number of nuclear transcriptional regulators, many of which are essential for maintenance of hepatobiliary transporter gene expression. Interestingly, recently discovered molecular crosstalk between bile acid activated nuclear receptors and proinflammatory nuclear mediators may provide new means of understanding adaptive processes within liver. Inflammation-induced cholestasis and the effects of retained molecules in cholestasis on inflammatory signals are interwoven in the liver, providing potential opportunities for research and therapeutics.

Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice

BACKGROUND/AIM Mutations in genes encoding the ATP-binding cassette (ABC)-transporters ABCG5 and ABCG8 underlie sitosterolemia, which is characterized by elevated plasma levels of phytosterols due to increased intestinal absorption and impaired biliary secretion of sterols. The aim of our study was to correlate the expression levels of Abcg5 and Abcg8 to biliary cholesterol secretion in various (genetically-modified) mouse models. METHODS Bile was collected from genetically-modified mice fed a chow diet, or from mice fed either a chow diet, or chow supplemented with either 1% diosgenin, 0.1% simvastatin, or a synthetic liver X receptor agonist, for determination of biliary lipids. Livers and small intestines were harvested and expression levels of Abcg5, Abcg8 and Abcb4 were determined by real-time polymerase chain reaction. RESULTS Intestinal expression of Abcg5 and Abcg8 did not show much variation between the various models. In contrast, a linear correlation between hepatic expression levels of Abcg5 and Abcg8 and biliary cholesterol secretion rates was found. This relation was independent of Abcb4-mediated phospholipid secretion. However, in diosgenin-fed mice showing cholesterol hypersecretion, hepatic Abcg5 and Abcg8 expression levels remained unchanged. CONCLUSIONS Our results strongly support a role for Abcg5 and Abcg8 in regulation of biliary cholesterol secretion, but also indicate the existence of a largely independent route of cholesterol secretion.

Curcumin improves sclerosing cholangitis in Mdr2 / mice by inhibition of cholangiocyte inflammatory response and portal myofibroblast proliferation

Background and aim Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. Curcumin, the yellow pigment of the spice turmeric, has pleiotropic actions and attenuates hepatic damage in animal models of chemically-induced liver injury. Whether curcumin has beneficial effects in cholangiopathies is unknown. Methods Potential anticholestatic, anti-inflammatory and antifibrotic mechanisms of curcumin were explored in vivo in Mdr2−/− mice as a murine model of chronic cholangiopathy; as well as in vitro in a cholangiocyte cell line (HuCCT1) and portal myofibroblasts (MFBs) isolated from Mdr2−/− mice. Results Liver damage, cholestasis and fibrosis were reduced in Mdr2−/− mice after curcumin feeding. Moreover, curcumin inhibited cholangiocyte proliferation and expression of activation marker vascular cell adhesion molecule-1 in Mdr2−/− mice. Curcumin—similar to PPARγ synthetic agonist troglitazone—directly inhibited TNF-α-induced inflammatory activation of cholangiocytes in vitro, whereas these beneficial effects of curcumin were largely blocked by a PPARγ synthetic antagonist. In addition, curcumin blocked proliferation and activation of portal MFBs by inhibiting ERK1/2 phosphorylation, thus contributing to reduced fibrogenesis. Conclusions These results show that curcumin may have multiple targets in liver including activation of PPARγ in cholangiocytes and inhibition of ERK1/2 signalling in MFBs, thereby modulating several central cellular events in a mouse model of cholangiopathy. Targeting these pathways may be a promising therapeutic approach to cholangiopathies.

Genetic background of cholesterol gallstone disease

Cholesterol 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+/+, 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.

Hypertrophic cardiomyopathy and dysregulation of cardiac energetics in a mouse model of biliary fibrosis

Cardiac dysfunction is a major cause of morbidity and mortality in patients with end‐stage liver disease; yet the mechanisms remain largely unknown. We hypothesized that the complex interrelated impairments in cardiac structure and function secondary to progression of liver diseases involve alterations in signaling pathways engaged in cardiac energy metabolism and hypertrophy, augmented by direct effects of high circulating levels of bile acids. Biliary fibrosis was induced in male C57BL/6J mice by feeding a 0.1% 3,5‐diethoxycarbonyl‐1,4‐dihydroxychollidine (DDC) supplemented diet. After 3 weeks, mice underwent live imaging (dual energy x‐ray absorptiometry [DEXA] scanning, two‐dimensional echocardiography [2DE], electrocardiography, cardiac magnetic resonance imaging), exercise treadmill testing, and histological and biochemical analyses of livers and hearts. Compared with chow‐fed mice, DDC‐fed mice fatigued earlier on the treadmill, with reduced VO2. Marked changes were identified electrophysiologically (bradycardia and prolonged QT interval) and functionally (hyperdynamic left ventricular [LV] contractility along with increased LV thickness). Hearts of DDC‐fed mice showed hypertrophic signaling (activation of v‐akt murine thymoma viral oncogene/protein kinase B [AKT], inhibition of glycogen synthase kinase‐3β [GSK3β], a 20‐fold up‐regulation of β myosin heavy chain RNA and elevated Gsα/Giα ratio. Genes regulating cardiac fatty acid oxidation pathways were suppressed, along with a threefold increase in myocardial glycogen content. Treatment of mouse cardiomyocytes (which express the membrane bile acid receptor TGR5) with potent natural TGR5 agonists, taurochenodeoxycholic acid and lithocholic acid, activated AKT and inhibited GSK3β, similar to the changes seen in DDC‐fed mouse hearts. This provides support for a novel mechanism whereby circulating natural bile acids can induce signaling pathways in heart associated with hypertrophy. Conclusion: Three weeks of DDC feeding‐induced biliary fibrosis leads to multiple functional, metabolic, electrophysiological, and hypertrophic adaptations in the mouse heart, recapitulating some of the features of human cirrhotic cardiomyopathy. Hepatology 2010;51:2097–2107

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 heterodimer. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;41:141–150.)