The deletion of the estrogen receptor α gene reduces susceptibility to estrogen-induced cholesterol cholelithiasis in female mice.

Compelling evidence has demonstrated that estrogen is a critical risk factor for gallstone formation and enhances cholesterol cholelithogenesis through the hepatic estrogen receptor α (ERα), but not ERß. To study the lithogenic mechanisms of estrogen through ERα, we investigated whether the deletion of Erα protects against gallstone formation in ovariectomized (OVX) female mice fed a lithogenic diet and treated with 17ß-estradiol (E2) at 0 or 6µg/day for 56days. Our results showed that the prevalence of gallstones was reduced from 100% in OVX ERα (+/+) mice to 30% in OVX ERα (-/-) mice in response to high doses of E2 and the lithogenic diet for 56days. Hepatic cholesterol secretion was significantly diminished in OVX ERα (-/-) mice compared to OVX ERα (+/+) mice even fed the lithogenic diet and treated with E2 for 56days. These alterations decreased bile lithogenicity by reducing cholesterol saturation index of gallbladder bile. Immunohistochemical studies revealed that ERα was expressed mainly in the gallbladder smooth muscle cells. High levels of E2 impaired gallbladder emptying function mostly through the ERα and cholecystokinin-1 receptor pathway, leading to gallbladder stasis in OVX ERα (+/+) mice. By contrast, gallbladder emptying function was greatly improved in OVX ERα (-/-) mice. This markedly retarded cholesterol crystallization and the growth and agglomeration of solid cholesterol crystals into microlithiasis and stones. In conclusion, the deletion of Erα reduces susceptibility to the formation of E2-induced gallstones by diminishing hepatic cholesterol secretion, desaturating gallbladder bile, and improving gallbladder contraction function in female mice.

The cholecystokinin-1 receptor antagonist devazepide increases cholesterol cholelithogenesis in mice.

BACKGROUND: A defect in gallbladder contraction function plays a key role in the pathogenesis of gallstones. The cholecystokinin-1 receptor (CCK-1R) antagonists have been extensively investigated for their therapeutic effects on gastrointestinal and metabolic diseases in animal studies and clinical trials. However, it is still unknown whether they have a potential effect on gallstone formation. DESIGN: To study whether the CCK-1R antagonists enhance cholelithogenesis, we investigated cholesterol crystallization, gallstone formation, hepatic lipid secretion, gallbladder emptying function and intestinal cholesterol absorption in male C57BL/6J mice treated by gavage with devazepide (4 mg/day/kg) or vehicle (as controls) twice per day and fed the lithogenic diet for 21 days. RESULTS: During 21 days of feeding, oral administration of devazepide significantly accelerated cholesterol crystallization and crystal growth to microlithiasis, with 40% of mice forming gallstones, whereas only agglomerated cholesterol monohydrate crystals were found in mice receiving vehicle. Compared to the vehicle group, fasting and postprandial residual gallbladder volumes in response to the high-fat meal were significantly larger in the devazepide group during cholelithogenesis, showing reduced gallbladder emptying and bile stasis. Moreover, devazepide significantly increased hepatic secretion of biliary cholesterol, but not phospholipids or bile salts. The percentage of intestinal cholesterol absorption was higher in devazepide-treated mice, increasing the bioavailability of chylomicron-derived cholesterol in the liver for biliary hypersecretion into bile. These abnormalities induced supersaturated bile and rapid cholesterol crystallization. CONCLUSIONS: The potent CCK-1R antagonist devazepide increases susceptibility to gallstone formation by impairing gallbladder emptying function, disrupting biliary cholesterol metabolism and enhancing intestinal cholesterol absorption in mice.

Estrogen induces two distinct cholesterol crystallization pathways by activating ERα and GPR30 in female mice.

To distinguish the lithogenic effect of the classical estrogen receptor α (ERα) from that of the G protein-coupled receptor 30 (GPR30), a new estrogen receptor, on estrogen-induced gallstones, we investigated the entire spectrum of cholesterol crystallization pathways and sequences during the early stage of gallstone formation in gallbladder bile of ovariectomized female wild-type, GPR30((-/-)), ERα((-/-)), and GPR30((-/-))/ERα((-/-)) mice treated with 17ß-estradiol (E2) at 6 µg/day and fed a lithogenic diet for 12 days. E2 disrupted biliary cholesterol and bile salt metabolism through ERα and GPR30, leading to supersaturated bile and predisposing to the precipitation of cholesterol monohydrate crystals. In GPR30((-/-)) mice, arc-like and tubular crystals formed first, followed by classical parallelogram-shaped cholesterol monohydrate crystals. In ERα((-/-)) mice, precipitation of lamellar liquid crystals, typified by birefringent multilamellar vesicles, appeared earlier than cholesterol monohydrate crystals. Both crystallization pathways were accelerated in wild-type mice with the activation of GPR30 and ERα by E2. However, cholesterol crystallization was drastically retarded in GPR30((-/-))/ERα((-/-)) mice. We concluded that E2 activates GPR30 and ERα to produce liquid crystalline versus anhydrous crystalline metastable intermediates evolving to cholesterol monohydrate crystals from supersaturated bile. GPR30 produces a synergistic lithogenic action with ERα to enhance E2-induced gallstone formation.

Ezetimibe prevents the formation of oestrogen-induced cholesterol gallstones in mice.

BACKGROUND: Oestrogen is an important risk factor for cholesterol cholelithiasis not only in women of childbearing age taking oral contraceptives and postmenopausal women undergoing hormone replacement therapy, but also in male patients receiving oestrogen therapy for prostatic cancer. In women, hormonal changes occurring during pregnancy markedly increase the risk of developing gallstones. We investigated whether the potent cholesterol absorption inhibitor ezetimibe could prevent the formation of oestrogen-induced cholesterol gallstones in mice. DESIGN: Following ovariectomy, female AKR mice were implanted subcutaneously with pellets releasing 17ß-estradiol at 6 µg/day and fed a lithogenic diet supplemented with ezetimibe in doses of 0 or 8 mg/kg/day for 8 weeks. Cholesterol crystallization and gallstone prevalence, lipid concentrations and composition in bile, and biliary lipid output were analysed by physical-chemical methods. Intestinal cholesterol absorption efficiency was determined by faecal dual-isotope ratio methods. RESULTS: Ezetimibe inhibited intestinal cholesterol absorption, while significantly reducing hepatic secretion of biliary cholesterol. Consequently, bile was desaturated through the formation of numerous unsaturated micelles and gallstones were prevented by ezetimibe in mice exposed to high doses of oestrogen and fed the lithogenic diet. Ezetimibe did not influence mRNA levels of the classical oestrogen receptors α (ERα) and ERß, as well as a novel oestrogen receptor the G protein-coupled receptor 30 (GPR30) in the liver. CONCLUSIONS: Ezetimibe protects against the oestrogen-mediated lithogenic actions on gallstone formation in mice. Our finding may provide an efficacious novel strategy for the prevention of cholesterol gallstones in high-risk subjects, especially those exposed to high levels of oestrogen.

Sequence of events leading to primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease that is observed more frequently in middle-aged women. This disorder is considered an autoimmune disease, since liver injury is sustained by the presence of self-directed antimitochondrial antibodies targeting the bile duct cells. The prognosis may vary depending on an early diagnosis and response to therapy. However, nearly a third of patients can progress to liver cirrhosis, thus requiring a liver transplant. Traditional immunosuppressive therapies, commonly employed for other autoimmune diseases, have limited effects on PBC. In fact, dramatic functional changes that occur in the biliary epithelium in the course of inflammation play a major role in perpetuating the injury. In this minireview, after a background on the disease and possible predisposing factors, the sequential cooperation of cellular/molecular events leading to end-stage PBC is discussed in detail. The rise and maintenance of the autoimmune process, as well as the response of the biliary epithelia during inflammatory injury, are key factors in the progression of the disease. The so-called "ductular reaction (DR)", intended as a reactive expansion of cells with biliary phenotype, is a process frequently observed in PBC and partially understood. However, recent findings suggest a strict relationship between this pathological picture and the progression to liver fibrosis, cell senescence, and loss of biliary ducts. All these issues (onset of chronic inflammation, changes in secretive and proliferative biliary functions, DR, and its relationship with other pathological events) are discussed in this manuscript in an attempt to provide a snapshot, for clinicians and researchers, of the most relevant and sequential contributors to the progression of this human cholestatic disease. We believe that interpreting this disorder as a multistep process may help identify possible therapeutic targets to prevent evolution to severe disease.

Metabolic dysfunction-associated gallstone disease: expecting more from critical care manifestations.

About 20% of adults worldwide have gallstones which are solid conglomerates in the biliary tree made of cholesterol monohydrate crystals, mucin, calcium bilirubinate, and protein aggregates. About 20% of gallstone patients will definitively develop gallstone disease, a condition which consists of gallstone-related symptoms and/or complications requiring medical therapy, endoscopic procedures, and/or cholecystectomy. Gallstones represent one of the most prevalent digestive disorders in Western countries and patients with gallstone disease are one of the largest categories admitted to European hospitals. About 80% of gallstones in Western countries are made of cholesterol due to disturbed cholesterol homeostasis which involves the liver, the gallbladder and the intestine on a genetic background. The incidence of cholesterol gallstones is dramatically increasing in parallel with the global epidemic of insulin resistance, type 2 diabetes, expansion of visceral adiposity, obesity, and metabolic syndrome. In this context, gallstones can be largely considered a metabolic dysfunction-associated gallstone disease, a condition prone to specific and systemic preventive measures. In this review we discuss the key pathogenic and clinical aspects of gallstones, as the main clinical consequences of metabolic dysfunction-associated disease.

Secondary bile acids and the biliary epithelia: The good and the bad.

The biliary tract has been considered for several decades a passive system just leading the hepatic bile to the intestine. Nowadays several researches demonstrated an important role of biliary epithelia (i.e. cholangiocytes) in bile formation. The study of biliary processes therefore maintains a continuous interest since the possible important implications regarding chronic cholestatic human diseases, such as primary biliary cholangitis or primary sclerosing cholangitis. Bile acids (BAs), produced by the liver, are the most represented organic molecules in bile. The physiologic importance of BAs was initially attributed to their behavior as natural detergents but several studies now demonstrate they are also important signaling molecules. In this minireview the effect of BAs on the biliary epithelia are reported focusing in particular on secondary (deriving by bacterial manipulation of primary molecules) ones. This class of BAs is demonstrated to have relevant biological effects, ranging from toxic to therapeutic ones. In this family ursodeoxycholic and lithocholic acid present the most interesting features. The molecular mechanisms linking ursodeoxycholic acid to its beneficial effects on the biliary tract are discussed in details as well as data on the processes leading to lithocholic damage. These findings suggest that expansion of research in the field of BAs/cholangiocytes interaction may increase our understanding of cholestatic diseases and should be helpful in designing more effective therapies for biliary disorders.

Animal models for cystic fibrosis liver disease (CFLD).

Liver disease is a severe complication in patients with Cystic Fibrosis (CF), a genetic disease caused by mutations in the gene encoding for cystic fibrosis transmembrane conductance regulator (CFTR) channel. The sequence of events leading to CFLD is still unclear and has limited the development of more specific treatments other than the bile acid UDCA. However, in the last twenty years, several gaps have been filled, which have mainly been possible due to the availability of different animal models that mimic CF. CF mice, although they lack a spontaneous liver manifestation, have been essential to better understand the multiple functions of CFTR expression on the apical membrane of cholangiocytes, from chloride channel to regulator of epithelial innate immunity. Additionally, we have learned that the gut microbiota might be a pathogenetic factor for the development of liver disease. The recent creation of novel CF animal models (i.e. pig and ferret) that better reproduce the human disease, will allow for comparative studies with species that spontaneously develop the liver disease and will hopefully lead to novel therapeutic treatments. In this review, we have compared and summarized the main features of the current available CF animal models and their applicability for the study of the liver phenotype.

Critical Care Aspects of Gallstone Disease.

Approximately twenty per cent of adults have gallstones making it one of the most prevalent gastrointestinal diseases in Western countries. About twenty per cent of gallstone patients requires medical, endoscopic, or surgical therapies such as cholecystectomy due to the onset of gallstone-related symptoms or gallstone-related complications. Thus, patients with symptomatic, uncomplicated or complicated gallstones, regardless of the type of stones, represent one of the largest patient categories admitted to European hospitals. This review deals with the important critical care aspects associated with a gallstone-related disease.

New insights into the role of Lith genes in the formation of cholesterol-supersaturated bile.

Cholesterol gallstone formation represents a failure of biliary cholesterol homeostasis in which the physical-chemical balance of cholesterol solubility in bile is disturbed. Lithogenic bile is mainly caused by persistent hepatic hypersecretion of biliary cholesterol and sustained cholesterol-supersaturated bile is an essential prerequisite for the precipitation of solid cholesterol monohydrate crystals and the formation of cholesterol gallstones. The metabolic determinants of the supply of hepatic cholesterol molecules that are recruited for biliary secretion are dependent upon the input-output balance of cholesterol and its catabolism in the liver. The sources of cholesterol for hepatic secretion into bile have been extensively investigated; however, to what extent each cholesterol source contributes to hepatic secretion is still unclear both under normal physiological conditions and in the lithogenic state. Although it has been long known that biliary lithogenicity is initiated by hepatic cholesterol hypersecretion, the genetic mechanisms that cause supersaturated bile have not been defined yet. Identification of the Lith genes that determine hepatic cholesterol hypersecretion should provide novel insights into the primary genetic and pathophysiological defects for gallstone formation. In this review article, we focus mainly on the pathogenesis of the formation of supersaturated bile and gallstones from the viewpoint of genetics and pathophysiology. A better understanding of the molecular genetics and pathophysiology of the formation of cholesterol-supersaturated bile will undoubtedly facilitate the development of novel, effective, and noninvasive therapies for patients with gallstones, which would reduce the morbidity, mortality, and costs of health care associated with gallstones, a very prevalent liver disease worldwide.

The Enriched Proanthocyanidin Extract of Ligaria cuneifolia Shows a Marked Hypocholesterolemic Effect in Rats Fed with Cholesterol-Enriched Diet.

BACKGROUND: Ligaria cuneifolia (Lc) (R. et P.) Tiegh. (Loranthaceae) (Argentine mistletoe) is usually used in local folk medicine. OBJECTIVE: We studied the effect of treatment with the Lc proanthocyanidin-enriched fraction (PLc) in rats fed with Cho-enriched diet on plasma lipids levels, the hemorheological parameters, and biliary secretion. METHOD: Adult male Wistar rats were fed ad libitum with a Cho-enriched diet (Cho (97% purity) 8 g/kg of diet and corn oil 280 g/kg of diet) during 28 days. Then, were separated in six experimental groups (n=5 each one), which were injected ip every 24 h with: 1) saline solution (control group, C) and 2) PLc, 3 mg/100 g body weight (treated group, C+PLc), during 3, 7 and 10 days. Group C presented an increase in plasma levels of Cho and Triglycerides (TG), and also, accumulation of hepatic lipid droplets. Also, cell shape and their corresponding morphological index (MI) were altered too. RESULTS: The treatment with PLc at 3, 7 and 10 days produces a diminution in the plasma Cho, LDL-Cho and serum TG levels, accompanied by a diminution of the lipid accumulation in the liver. The rates of bile acid output in bile can explain the diminution of plasma Cho, evidencing that some of the enzymes involved in the cholesterol conversion into bile acids could be up regulated by the treatment with PLc, leading to the observed increase bile flow. PLc treatment leads to a diminution of plasma levels of Cho and TG. CONCLUSION: Essentially, the treatment with PLc, despite the duration produces a modification in hemorheological parameters approaching the values of the experimental group with standard diet. Plasma levels of Cho, LDL-Cho and TG represent selected markers to evaluate the effect of enriched extract from Ligaria cuneifolia. Further work is necessary to better evaluate the mechanisms by which PLc induces modifications in the lipids metabolism.

Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells.

The HepaRG cell line is a well-differentiated human hepatoma cell line proposed as a surrogate for human hepatocytes, especially for hepatic detoxification studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, which represents a key hallmark of hepato-biliary drug transport, remains however incompletely documented in HepaRG cells. The present study was therefore designed to analyze transporter location in HepaRG cells, which exhibit mRNA expressions of most of hepatic transporters. HepaRG cells were demonstrated, through immunofluorescence staining, to express several drug transporters at their sinusoidal pole, especially the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of HepaRG cells. Moreover, saturable uptake of reference substrates for the sinusoidal transporters sodium-taurocholate cotransporting polypeptide, OATPs and OCT1 and canalicular secretion of reference substrates for the efflux transporters bile salt export pump and MRP2 were observed. This polarized and functional expression of various sinusoidal and canalicular transporters in HepaRG cells highlights the interest of using these hepatoma cells in xenobiotic transport studies.