Unexpected cholesterol gallstones
Authors: Albert K. Groen
Journal: Hepatology (2016)

Potential conflict of interest: Nothing to report. See Article on Page 853 Gallstone disease is one of the most common gastrointestinal diseases, with an estimated prevalence of 10%‐15% in the adult population.1 In Western countries, approximately 80% of gallstones consist of 80% pure crystalline cholesterol.1 Despite decades of research, the etiology of cholesterol gallstone disease remains enigmatic. Crystallization of cholesterol is the first step in gallstone formation and is caused by supersaturation of bile with cholesterol. Cholesterol solubilization in bile is a complex process in which the other main biliary components—bile salts and phospholipids—play a crucial role, making them key players in the regulation of cholesterol crystallization.3 Cholesterol gallstones develop almost exclusively in the gallbladder, hence conditions prevailing in this organ are essential as well.3 The striking differences in prevalence rates of gallstones between different ethnic populations suggests that genetic background has a prominent role in the pathogenesis of the disease. Native Americans have the highest incidence of gallstone disease; a high prevalence (>70%) was found in Pima Indians as well as other Native American populations.4 Yet the genes involved have not been found, suggesting a polygenic background of gallstone disease in combination with environmental factors. A large study in identical twins in Sweden revealed a relatively low concordance of gallstone disease of 25%,4 indicating an important contribution of environmental or epigenetic factors. One candidate gene came from a large genome‐wide association study encompassing German and Chilean gallstone patients.5 In that study, the analysis revealed a strong association between a single nucleotide polymorphism in the gene encoding the cholesterol transporter ABCG5/G8. In patients homozygous for this D19H variant, an odds ratio of 7.1 was found. The mutation was associated with higher biliary cholesterol levels, suggesting a gain of function mutation; however, this theory has not yet been proven in experiments in animal models expressing this variant. Because biliary cholesterol solubilization depends on the concentration of biliary bile salts and phospholipids, mutations in the genes encoding transporters for these compounds have been associated with gallstone disease as well. Bile salts are transported by the adenosine triphosphate‐binding cassette transporter ABCB11, whereas phospholipids are transported by ABCB4 (Fig. 1). The importance of ABCB11 and ABCB4 in bile formation is evident; in the absence of these proteins, transport of bile salts and phospholipids is almost abrogated, hence cholesterol can no longer be solubilized in the bile and therefore crystallizes. Disruption of the genes encoding ABCG5/G8 has much less of an effect. Under baseline conditions, approximately 30%‐40% of biliary cholesterol secretion is still found the absence of ABCG5/G8.6Figure 1: Simplified scheme of biliary lipid secretion in bile. The first step in bile formation is the transportation of bile salts across the canalicular membrane by ABCB11. These bile salts aggregate to simple micelles, which are able to pick up phosphatidylcholine. The formed mixed micelles pick up cholesterol from the heterodimer ABCG/G8. This pathway accounts for approximately 65% of biliary cholesterol secretion; the remaining 35% can be transported by an as‐yet unknown protein or can be solubilized directly from the membrane. When mixed micelles are supersaturated with cholesterol, vesicles containing phospholipids and cholesterol but no bile salts form.7 Abbreviations: ABC, adenosine triphosphate‐binding cassette transporter; ATP, adenosine triphosphate.In this issue of Hepatology, Wang et al.7 suggest that this ABCG5/G8‐independent cholesterol transport may be involved in gallstone disease. Abcg5/g8 and Abcg8 knockout mice were fed a gallstone‐promoting (lithogenic) diet, and surprisingly, these mice still developed cholesterol gallstones, albeit at a lower rate than wild‐type mice. Detailed studies of the lipid composition of gallbladder bile revealed a progression to supersaturation in phase diagrams. To reconcile these findings with the known information about the mechanism of biliary cholesterol secretion, Wang et al. suggest that an as‐yet unknown protein must be involved, though this is by no means certain. Before the discovery of the presence of ABCG5/G8 in the canalicular membrane, cholesterol was generally thought to be solubilized directly from the membrane through the action of bile salt/phospholipid mixed micelles.8 Indeed, several studies have demonstrated that mixed micelles readily solubilize cholesterol from model membranes.9 So why is a protein required to mediate the ABCG5/G8‐independent pathway? Wang et al. argue that the steroid diosgenin has been shown to hyperstimulate biliary cholesterol secretion independent of bile salt and phospholipid secretion. Because diosgenin did not increase gene or protein expression of ABCG5/G8, they suggest that the G5/G8‐independent pathway must have been involved7; this is, however, a misunderstanding. Although diosgenin did not influence expression of ABCG5/G8, the impact of the steroid on biliary cholesterol secretion was abrogated in the absence of the protein, indicating that diosgenin induced activity of the supply route to ABCG5/G8.10 Wang et al. also report experiments with the LXR agonist T01901317. This compound increased expression of ABCG5/G8 and biliary cholesterol secretion in wild‐type mice but did not influence biliary cholesterol secretion in G5/G8−/− mice. Consequently, T01901317 increased gallstone incidence in wild‐type mice but had no effect on Abcg5/g8‐deficient mice. Clearly, LXR activation does not induce the G5/G8‐independent pathway. Increasing the cholesterol content of the diet did induce the G5/G8‐independent pathway, which could indicate up‐regulation of an unknown protein. Wang et al. suggest that they have opened the hunt for the missing genes potentially involved in gallstone formation in humans; however, a note of caution should be added here. Novel proteins may not be necessary to explain G5/G8‐independent biliary cholesterol secretion. Direct solubilization of cholesterol from the canalicular membrane by mixed bile salt/phosphatidylcholine micelles can also account for the ABCG5/G8‐independent biliary cholesterol secretion.