Recent Advances in the Critical Role of the Sterol Efflux Transporters ABCG5/G8 in Health and Disease.

Cardiovascular disease is characterized by lipid accumulation, inflammatory response, cell death, and fibrosis in the arterial wall and is the leading cause of morbidity and mortality worldwide. Cholesterol gallstone disease is caused by complex genetic and environmental factors and is one of the most prevalent and costly digestive diseases in the USA and Europe. Although sitosterolemia is a rare inherited lipid storage disease, its genetic studies led to identification of the sterol efflux transporters ABCG5/G8 that are located on chromosome 2p21 in humans and chromosome 17 in mice. Human and animal studies have clearly demonstrated that ABCG5/G8 play a critical role in regulating hepatic secretion and intestinal absorption of cholesterol and plant sterols. Sitosterolemia is caused by a mutation in either the ABCG5 or the ABCG8 gene alone, but not in both simultaneously. Polymorphisms in the ABCG5/G8 genes are associated with abnormal plasma cholesterol metabolism and may play a key role in the genetic determination of plasma cholesterol concentrations. Moreover, ABCG5/G8 is a new gallstone gene, LITH9. Gallstone-associated variants in ABCG5/G8 are involved in the pathogenesis of cholesterol gallstones in European, Asian, and South American populations. In this chapter, we summarize the latest advances in the critical role of the sterol efflux transporters ABCG5/G8 in regulating hepatic secretion of biliary cholesterol, intestinal absorption of cholesterol and plant sterols, the classical reverse cholesterol transport, and the newly established transintestinal cholesterol excretion, as well as in the pathogenesis and pathophysiology of ABCG5/G8-related metabolic diseases such as sitosterolemia, cardiovascular disease, and cholesterol gallstone disease.

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.

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.

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.