Rafting for gallstones by slowing mass transit

Much has been learned about the predisposing factors that contribute to cholesterol gallstone disease and we now have a more granular understanding of the pathways that promote cholesterol monohydrate crystal formation and in turn lead to the formation of cholesterol gallstones. There are several sets of players involved, some of which have been invoked from studies using inbred lines of mice with differing susceptibility to map quantitative trait loci (QTLs) (Lyons & Wittenburg, 2006), while others have been inferred from gain- and loss-of-function approaches based on candidate genes (for example ABCG5/G8) identified through biochemical and molecular screens (Krawczyk et al. 2011). The emerging sense from these various approaches is that a range of genetic and acquired defects in intestinal, hepatic, canalicular and gallbladder signalling pathways can lead to the production of supersaturated bile. By comparison, there is a paucity of information regarding the influence of enterohepatic cholesterol cycling on intestinal motility and its possible contribution to gallstone susceptibility. In a recent issue of The Journal of Physiology, Xie and colleagues (2012) demonstrate that C57L inbred mice exhibit slowed intestinal transit of a non-absorbable marker when fed a lithogenic diet (LD). The authors demonstrate a predictable onset and reversal of this phenotype with dietary challenge and withdrawal, and show the effects mirror the time course for cholesterol supersaturation of bile. They show that the dietary component responsible for the altered intestinal motility is a combination of cholesterol and cholic acid rather than fat and they confirm this presumed role for cholesterol by showing that the effects of the LD are completely reversed by treatment with ezetimibe, which binds to the NPC1L1 transporter and blocks cholesterol absorption. To understand the mechanisms involved, they prepared small intestinal sarcolemmal membranes and showed that the abundance of the lipid raft protein Cav3 was reduced in the LD-fed group compared with chow-fed mice and that ezetimibe treatment at least partially reversed this reduction in expression. Taken together, the findings suggest that enrichment of sarcolemmal membranes with free cholesterol leads to disruption of lipid raft components, including decreased membrane Cav3 expression. How might these effects be linked to altered intestinal motility? Work in other model systems has demonstrated that shifts in membrane cholesterol content result in altered gallbladder muscle contractility whose effects can be reproduced in isolated culture and are reversible upon incubation with cholesterol-free liposomes (Yu et al. 1996). Further work revealed that the blunted response noted to CCK-8 administration following cholesterol enrichment of gallbladder muscle was associated with reduced binding to its cognate receptor, CCK-1R. This decreased interaction occurred through mechanisms that include sequestration of Cav3 and decreased CCK-1R turnover, reduced delivery to endosomal compartments and as a result, reduced recycling of the receptor to the cell surface which interrupts the normal interaction between CCK-8 and CCK-1R. The net result of this disrupted interaction between receptor and ligand is impaired muscle contraction (Cong et al. 2010). Other findings suggest that this paradigm for altered muscle function and defective CCK signaling in the setting of cholesterol enrichment may have relevance for gallstone disease. Earlier work showed that mice with targeted deletion of CCK-1R exhibited increased gallstone susceptibility, delayed small intestinal transit and increased biliary cholesterol secretion (Wang et al. 2004). Those findings reinforce the underlying plausibility of the current observations that alterations in signalling pathways that primarily influence smooth muscle function (in this case, gallbladder and small intestinal muscle) may in turn promote or augment biliary cholesterol supersaturation. Among the mechanisms invoked by those authors was an increase in cholesterol absorption in the CCK-1R−/− mice compared with wild-type controls, which the authors suggest led in turn to increased canalicular cholesterol secretion. The current findings from Xie and colleagues add to this body of literature and suggest an important and heretofore unanticipated role for cholesterol in regulating intestinal motility. Among the key questions that arise from this work are what is the source and carrier (chylomicron? high-density lipoprotein?) of the cholesterol that leads to sarcolemmal membrane enrichment? Certainly the overwhelming bulk of absorbed intestinal cholesterol is secreted in the form of chylomicron cholesterol ester, although Xie and colleagues suggest that since the sarcolemmal cholesterol enrichment is predominantly free rather than esterified cholesterol, that perhaps some of the cholesterol absorbed from the intestine is secreted as free cholesterol that enters the lamina propria and promotes enrichment of muscle plasma membranes. An alternative explanation, however, is that cholesterol ester delivered to intestinal smooth muscle cells undergoes endocytic degradation through neutral lipid esterases and the resultant free cholesterol is then trafficked to domains in the plasma membrane. Thus, it remains to be established just how (i.e. which carrier particle) and in what form (free versus ester) the excess lumenal cholesterol is transported to the muscle and whether similar alterations would be observed in the setting of systemic dyslipidaemia caused by low density lipoprotein receptor deficiency, for example. Thus, while much remains to be answered regarding the relative importance of the pathways influenced by defective CCK signalling in promoting cholesterol gallstone disease, there is a strong foundation to invoke alterations in lipid raft composition and function and particularly the role of Cav3 in the maintenance of CCK–CCK-1R interactions and with the regulation of intestinal motility.