Prevention of cholesterol gallstone disease by FXR agonists in a mouse model

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1 24 Nature Publishing Group Prevention of cholesterol gallstone disease by FXR agonists in a mouse model Antonio Moschetta, Angie L Bookout & David J Mangelsdorf Cholesterol gallstone disease is characterized by several events, including cholesterol precipitation in bile, increased bile salt hydrophobicity and gallbladder inflammation. Here, we describe the same phenotype in mice lacking the bile acid receptor, FXR. Furthermore, in susceptible wild-type mice that recapitulate human cholesterol gallstone disease, treatment with a synthetic FXR agonist prevented sequelae of the disease. These effects were mediated by FXR-dependent increases in biliary bile salt and phospholipid concentrations, which restored cholesterol solubility and thereby prevented gallstone formation. Taken together, these results indicate that FXR is a promising therapeutic target for treating or preventing cholesterol gallstone disease. Cholesterol gallstone disease (CGD) has a high prevalence in the United States, where 2 million patients are treated for this disease annually 1. The disease is common in Native Americans and Hispanics, and in western Europe. The major events leading to the disease include supersaturation of bile with cholesterol, rapid precipitation of cholesterol crystals in the gallbladder, increased bile salt hydrophobicity and inflammation of the gallbladder 2,3.Ofthese events, precipitation of cholesterol crystals from supersaturated bile is a prerequisite for gallstone formation 4, and has been observed in 7% of patients with acute, formerly idiopathic, pancreatitis 5.In bile, cholesterol is solubilized in mixed micelles together with bile salts and phospholipids. Under supersaturated conditions, the sterol is solubilized by phospholipids into vesicles, called liquid crystals. As monohydrate crystals enucleate from these cholesterol-enriched vesicles, they aggregate, fuse and eventually precipitate into larger pathogenic crystals which lead to the disease. In mice, CGD can be mimicked by feeding them a lithogenic diet containing cholesterol and cholic acid. As in humans, susceptibility to gallstone formation varies greatly according to genetic background 6. For example, feeding a lithogenic diet to susceptible C57L mice results in cholesterol monohydrate crystals in bile after 5 d and cholesterol gallstones after 28 d, while resistant AKR mice do not form crystals until day 37 and gallstones until day 46 (ref. 7). The genetic background of susceptible and resistant strains of mice has been studied by quantitative trait locus analysis to define genomic regions associated with this disease. Several genes have been implicated in this process, including the farnesoid X receptor (FXR), a key regulator of bile acid homeostasis 8.In mouse strains susceptible to cholesterol gallstone formation, the expression of FXR and its cognate target genes is lower than in resistant strains. In addition, polymorphisms in the FXR gene are detected in susceptible strains 8. FXR (NR1H4) was first characterized as an orphan member of the nuclear receptor superfamily, but is now known to function as a bile acid receptor that regulates transcription of numerous genes involved in maintaining cholesterol and bile acid homeostasis 9 11.The primary bile acids, chenodeoxycholic and cholic acid, are the highest affinity endogenous ligands characterized for FXR in the enterohepatic system 9.To function as a bile acid receptor, FXR must form an obligate heterodimer with the retinoid X receptor (RXR) and bind to specific regulatory sequences within the target genes it regulates. In addition to FXR, a second subfamily of nuclear receptors, the liver X receptors (LXRs), are also known to play important roles in maintaining cholesterol homeostasis 12.The LXRs consist of two members, LXRα (NR1H3) and LXRβ (NR1H2), which also function as RXR heterodimers. The LXRs act as cholesterol sensors that activate transcription of genes involved in cholesterol transport and metabolism. The endogenous ligands for LXRs are oxysterols that accumulate in cells when cholesterol concentrations are elevated 13,14. FXR and LXRs work together to maintain proper bile acid and cholesterol metabolism in the enterohepatic system. For example, LXRs (positively) and FXR (negatively) regulate transcription of cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the classic bile acid biosynthesis pathway 15.Increased CYP7A1 activity catalyzes conversion of cholesterol into bile acids, which is increased in human cholesterol gallstone patients as well as in populations at risk for gallstones, like the Chilean Mapuche Indians 16. The process of nascent bile formation is mediated by an elaborate network of ATP-binding cassette (ABC) transporters on the hepatocyte canalicular membrane that regulate biliary secretion of bile salts, phospholipids and cholesterol. The sister of P-glycoprotein (SPGP) is an ATP-binding cassette transporter (ABCB11), also known as the bile salt export pump (BSEP), which represents the major canalicular bile salt export pump of mammalian liver 17.The human multidrug resistant (MDR) 3 P-glycoprotein, also known as ABCB4 (corresponding to the murine Mdr2 P-glycoprotein), functions as a flippase, translocating phosphatidylcholine molecules from the inner to the outer leaflet of the canalicular membrane 18.Other ABC transporters, namely ABCG5 and ABCG8, are directly involved in pumping cholesterol into bile 19. FXR and LXRs play important roles in transcriptional regulation of the genes encoding these proteins. The expression of Abcb11 (ref. 2), Howard Hughes Medical Institute and Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas , USA. Correspondence should be addressed to D.J.M. (davo.mango@utsouthwestern.edu). Published online 21 November 24; doi:1.138/nm VOLUME 1 NUMBER 12 DECEMBER 24 NATURE MEDICINE

2 24 Nature Publishing Group a c e Cholesterol saturation index Concentration (mm) b FXR +/+ FXR / ABCB4 (ref. 21) and Abcb4 (ref. 22) is controlled by FXR, whereas Abcg5 and Abcg8 are target genes of LXRs 23.In the present study, we show that after only one week on a lithogenic diet, FXR / mice exhibit all phenotypes that are pathognomonic for cholesterol gallstone disease. At the molecular level these changes are a consequence of decreased expression of the bile acid and phospholipid transporters, Abcb11 and Abcb4,relative to unchanged expression of the cholesterol transporters, Abcg5 and Abcg8.Furthermore, treatment of susceptible C57L mice with a synthetic FXR ligand prevented cholesterol precipitation and gallstone formation. In the treated mice, FXR-induced expression of Abcb11 and Abcb4 were coupled to increased biliary concentrations of bile salts and phospholipids, and a subsequent lowering of the cholesterol saturation index compared to untreated animals. We conclude that FXR function is critical for maintaining proper solubilization of sterols in bile, and that FXR agonists might be useful in preventing CGD. Bile salt f d Bile salt hydrophobicity index Phosopholipid Cholesterol FXR +/+ FXR / Figure 1 Biliary cholesterol crystallization, lipid profiles and gallbladder inflammation in lithogenic FXR / mice. (a) Gallbladder bile appears more turbid from FXR / than wild-type (FXR +/+ ) mice. Biliary bile salt, phospholipid and cholesterol concentrations (b); cholesterol saturation indices (c); and bile salt hydrophobicity indices (d) are shown from wild-type (black bars) and FXR / (gray bars) mice. (e,f) Histological examination of gallbladder mucosal epithelium by hematoxilin and eosin staining. Black arrowheads indicate gallbladder wall thickness, stromal granulocyte infiltration and damaged mucosae; white arrowheads indicate submucosal vasodilatation in FXR / (f), but not wild-type (e) mice (magnification, 4). Experiments were from male mice (n = 8) treated with a lithogenic diet for 1 week. P <.1. RESULTS Cholesterol crystals and biliary lipids in FXR / mice To study the susceptibility of FXR / mice to cholesterol gallstone formation, we gave age-matched male wild-type and FXR / mice a lithogenic diet for 1 week, and analyzed them for lipid composition and gene expression. Gallbladder bile from FXR / mice was notably more turbid compared to wild-type mice (Fig. 1a). Microscopic inspection of the bile showed the presence of cholesterol monohydrate crystals in all FXR / mice. In contrast, we detected aggregated vesicles in the bile of all but one of the wild-type mice. In conjunction with the presence of cholesterol crystals, biliary phospholipid and bile salt concentrations were significantly lower in FXR / compared to wild-type mice (Fig. 1b). In contrast, there was non significant change in the cholesterol concentration in bile of FXR / compared to wild-type mice (1.5 ± 1.2 versus 12.3 ± 1.1, respectively). As a consequence of the altered lipid levels, both cholesterol/phospholipid and bile salt/phospholipid ratios were significantly higher in FXR / than in wildtype mice (.9 ±.1 versus.4 ±.1 and 13.7 ± 1.6 versus 8. ± 1.2, respectively, P <.1). Differences in the relative amounts of bile salts versus phospholipids are known to play crucial roles in cholesterol precipitation and crystallization behavior, as predicted by the equilibrium cholesterol bile salt phospholipid ternary phase diagram 24.In the case of excess bile salts (resulting in a high bile salt/phospholipid ratio), crystals precipitate at fast rates, whereas with excess phospholipids (resulting in a low bile salt/phospholipid ratio) crystal precipitation proceeds at slower rates, and large amounts of cholesterol are solubilized in vesicles together with phospholipids. Accordingly, gallbladder bile from FXR / mice was supersaturated with cholesterol as shown by the significantly higher cholesterol saturation index (Fig. 1c). Although there was a trend toward increased gallbladder volume in FXR / versus wild-type mice (35 ± 5 µl versus 28 ± 4 µl, respectively), gallbladder bile from FXR / mice was less concentrated than that of wild-type (total lipid concentration n = 9.5 ± 1 g/dl versus 13.7 ± 1 g/dl, respectively, P <.1). Bile acid hydrophobicity and inflammation in FXR / mice A key contributing factor to increased rates of cholesterol precipitation and crystallization in bile is an increase in bile salt hydrophobicity 25.Consequently, a hallmark characteristic of cholesterol gallstone patients 26 and animal models 27 is the presence of an increased bile salt hydrophobicity index. We analyzed biliary bile salt composition in the FXR / and wild-type animals after 1 week on the lithogenic diet. Significant differences in the relative levels of hydrophobic deoxycholate (2.1 ± 2.2% FXR / versus 12.6 ± 1.9% wild-type, P <.1) and hydrophilic β-muricholate (3.2 ± 1.1% FXR / versus 13.6 ± 2.5% wild-type, P <.1) were observed between the two genotypes. As a result, bile salt hydrophobicity indices were higher in FXR / mice than in wild-type (Fig. 1d). Gallbladder mucosal inflammation is an important event in CGD. The toxic detergent effects of bile salts are related directly to the degree NATURE MEDICINE VOLUME 1 NUMBER 12 DECEMBER

3 24 Nature Publishing Group Table 1 Total cholesterol and triglyceride levels in plasma (mg/dl) and liver (mg/g tissue) from FXR / and wild-type mice after 1 week on a lithogenic diet. Cholesterol Triglycerides FXR +/+ FXR / FXR +/+ FXR / Plasma ± ± ± ± 21.2 Liver 2.9 ± ± ± ± 1.5 P <.1 versus wild-type mice of bile salt hydrophobicity 28,29.Thus, as a consequence of increased bile salt hydrophobicity, gallbladder epithelia of the FXR / mice under lithogenic conditions showed increased thickness, damage and inflammation (stromal granulocyte infiltration, vasodilatation) as compared to wild-type controls (Fig. 1e,f). Cholesterol and triglyceride levels in FXR / mice Bile salts are known to affect both cholesterol and triglyceride homeostasis, and treatment of cholesterol gallstones with chenodeoxycholic acid reduces hypertriglyceridemia in humans 3.In agreement with these effects, FXR / animals showed increases in both plasma and liver cholesterol and triglyceride levels after 1 week on the lithogenic diet (Table 1). Figure 2 Gene expression in lithogenic FXR / mice. Quantitative real-time PCR analysis was performed on mrna from livers of wild-type (black bars) and FXR / (gray bars) mice. Expression patterns of genes involved in canalicular bile secretion (a); bile salt synthesis and uptake (b); cholesterol, phospholipid, and fatty acid synthesis and metabolism (c) were characterized. Data (P <.1) were derived from the same experiment shown in Figure 1. Altered gene expression in FXR / mice A plausible explanation for the dramatic changes seen in bile lipid metabolism in the results shown above was the altered expression of FXR-dependent genes, such as the ABC transporters that normally regulate biliary bile salt and phospholipid secretion (i.e., Abcb11 and Abcb4,respectively). Indeed, expression of Abcb11 and Abcb4 was significantly lower in FXR / mice compared to wild-type on the lithogenic diet (Fig. 2a). In contrast, no differences were detected in the expression of the cholesterol transporters, Abcg5 and Abcg8.Although actual hepatic bile flow was not measured in the present study, the expression pattern of these ABC transporters correlates directly with gallbladder lipid concentration 19. Abcb11 and Abcb4 are Fxr target genes and therefore were upregulated when mice were fed a diet containing.5% cholic acid, an endogenous Fxr ligand (see Supplementary Fig. 1 online). Likewise, Abcg5 and Abcg8 are LXR target genes that were upregulated when mice were fed a diet containing 1% cholesterol (Supplementary Fig. 1 online). As expected, the lithogenic diet (containing both.5% cholic acid and 1% cholesterol) was able to upregulate Abcg5 and Abcg8 in the FXR / mice similar to wild-type mice, whereas the expression of Abcb11 and Abcb4 was lower, thus explaining the differences in biliary bile salt and phospholipid concentrations. We also characterized the expression of genes involved in bile acid synthesis and transport (Fig. 2b), cholesterol synthesis and uptake, fatty acid synthesis, and phosphatidylcholine transport (Fig. 2c). As previously reported 31,decreased expression of the gene encoding small heterodimer partner (Shp) and increased expression of Cyp7a1 and sterol 12α-hydroxylase (Cyp8b1) were observed in FXR / mice, underscoring the importance of FXR-driven negative feedback regulation on bile acid synthesis. In keeping with decreased biliary phospholipid secretion resulting from downregulation of the phosphatidylcholine flippase Abcb4, expression of the gene encoding phosphatidylcholine transporter (Pctp) also was decreased in the FXR / mice, reflecting a reduced need for delivery of phosphatidylcholine from the Golgi apparatus to the inner leaflet of the hepatocyte canalicular membrane (Fig. 2c). In contrast, expression of genes encoding proteins involved in fatty acid and triglyceride synthesis, sterol regulatory element-binding protein-1c (Srebp1c) and fatty acid synthase (Fasn), were upregulated in FXR / compared to wild-type mice. These data are consistent with these genes being LXR targets and the well-known triglyceride-lowering effect induced by bile salts that is likely to result from FXR-driven negative feedback regulation on these genes 32.No differences were found in the expression of genes encoding proteins involved in cholesterol uptake and synthesis, including low-density lipoprotein receptor (Ldlr), scavenger receptor class B type 1 (Scarb1) and 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr). Taken together, these data further support the conclusion that the absence of FXR confers susceptibility to biliary cholesterol precipitation. FXR agonists prevent cholesterol gallstone formation The results above suggested the possibility that activation of the FXR pathway may prevent biliary cholesterol crystallization and gallstone formation in the susceptible C57L mouse model 33.Age-matched male a b c mrna (arbitrary units) mrna (arbitrary units) mrna (arbitrary units) Abcb4 Abcg5 Abcg8 Abcb11 Cyp27a1 Slc1a1 Cyp7a1 Cyp8b1 SHP Srebp1c Abca1 Hmgcr Ldlr Scarb1 Fan Lpl Pctp 1354 VOLUME 1 NUMBER 12 DECEMBER 24 NATURE MEDICINE

4 24 Nature Publishing Group a b c mrna (arbitrary units) Cholesterol saturation index C57L (vehicle) 5 C57L FXR / C57L (GW464) C57L BS PL Chol BS PL Chol Veh GW Veh GW C57L FXR / mrna (arbitrary units) d e f 5 FXR / (vehicle) FXR +/+ (GW464) FXR / susceptible C57L and FXR / mice were fed a lithogenic diet for 1 week in combination with a synthetic FXR ligand (GW464) 34,35 or vehicle. We then analyzed mice for lipid composition and gene expression. As expected, the vehicle-treated C57L and FXR / mice showed turbid gallbladder bile and large aggregates of cholesterol precipitates (Fig. 3a,b). In addition, two out of five vehicle-treated C57L mice had more advanced stages of the disease, with macroscopic cholesterol gallstones clearly visible in the gallbladder. In contrast, there were no sequelae of the disease observed in C57L mice treated with GW464. Instead, these mice showed a visible decrease in gallbladder bile turbidity (Fig. 3a) and an absence of cholesterol precipitates as judged by microscopic inspection under polarizing light conditions (Fig. 3b). Under the same lithogenic conditions, GW464 had no effect in the FXR / mice, showing that its effects were FXR-dependent. In line with these results, GW464-treated C57L mice had significantly higher bile salt and phospholipid concentrations in bile, whereas no differences in biliary lipid profiles were found in vehicle-treated C57L mice or both vehicle- and GW464-treated FXR / mice (Fig. 3c). Cholesterol concentrations in bile were not significantly affected by GW464 in any of the mice. Thus, GW464 treatment resulted in a significant decrease of the cholesterol saturation index for C57L but not FXR / mice (Fig. 3d), thereby explaining the higher biliary cholesterol solubility in these mice. Finally, gallbladder epithelia of C57L mice treated with GW464 were substantially less damaged, and showed decreased thickness and inflammation compared to vehicle-treated mice (Fig. 3e,f). These data indicate that FXR agonists can prevent the entire sequelae of CDG. The protective effects of GW464 on susceptible C57L mice were explained by changes in gene expression (Fig. 4). Both Abcb11 and Abcb4 (genes encoding the bile salt and phospholipid transporters) were upregulated in C57L mice treated with GW464, but not in FXR / mice, whereas no significant differences in expression of Abcg5 and Abcg8 (genes encoding the sterol transporters) were detected. The effects of GW464 coincided with decreased expression of Cyp7a1 and Cyp8b1, as a result of increased expression of the repressor Shp, a known FXR target gene 15.Notably, we also observed a decrease in expression of Srebp1c in C57L mice on a lithogenic diet when treated with GW464 for 1 week, a result that was consistent with previous studies on FXR agonism in vivo 32.The decrease in Srebp1c expression was coupled with notably lowered plasma triglyceride levels in susceptible C57L mice on the lithogenic diet (138.3 ± 8.1 mg/dl vehicletreated versus ± 7.8 mg/dl GW464-treated mice). The GW464-induced changes in gene expression and lipid profiles were absent in the FXR / mice, underscoring the FXR-dependence of the pathway. Taken together, these data support the conclusion that FXR activation prevents cholesterol gallstone formation. DISCUSSION Here we show an important role for the bile acid receptor, FXR, in maintaining normal bile lipid homeostasis and as a potential therapeutic target for preventing or treating CGD. Using a well-established dietary model for lithogenesis in animals, we show that FXR / mice exhibit the rapid onset of phenotypes associated with human CGD, including supersaturation of bile with cholesterol, precipitation of cholesterol crystals in the gallbladder, increased bile salt hydrophobicity and gallbladder inflammation 2,3. Our data provide direct evidence that FXR deficiency confers susceptibility to the sequelae of events leading to cholesterol crystallization and gallstone disease. These results support the notion that mutations or polymorphisms in the FXR gene may underlie some forms of human CGD. Consequently, we believe this study may justify the use of FXR gene analysis in screening families with a history of CGD. The work presented here clarifies the molecular mechanisms leading to increased cholesterol precipitation under lithogenic conditions when the FXR signaling pathway is compromised, and demonstrates the potential utility of FXR agonists in treating CGD and restoring normal biliary lipid physiology. The observed effects on biliary lipid physiology resulting from either loss or gain of FXR function are explained by a simple model based on the expression profiles of several Figure 3 Effects of FXR agonist on cholesterol crystallization and gallstone formation in susceptible wild-type (C57L) and FXR / mice. (a) Turbidity of gallbladder bile in C57L and FXR / mice treated with vehicle or GW464. (b) Polarizing light microscopy of gallbladder bile from vehicle or GW464 treated C57L and FXR / mice (magnification, 2). (c) Bile salt, phospholipid, and cholesterol concentrations from bile of vehicle treated (black bars) and GW464 treated (gray bars) C57L and FXR / mice. (d) Cholesterol saturation indices calculated from the data samples in c. (e,f) Histological examination of gallbladder mucosal epithelium by hematoxilin and eosin staining in C57L mice (magnification, x4). Experiments were from male mice (n = 8) treated with a lithogenic diet for 1 week with GW464 versus vehicle. P <.1. NATURE MEDICINE VOLUME 1 NUMBER 12 DECEMBER

5 Figure 4 Liver gene expression in cholesterol gallstone susceptible C57L and FXR / mice. We performed quantitative real-time PCR analysis on mrna from livers of mice treated with vehicle (black bars) or GW464 (gray bars). We characterized expression patterns of Abcb11, Abcb4, Abcg5, Abcg8, Shp, Cyp7a1, Cyp8b1 and Srebp1c. Data (P <.1) were derived from the same experiment shown in Figure 3; mrna values were normalized to an internal standard relative to vehicle treated mice for each mouse strain Abcb Abcb4 24 Nature Publishing Group lipid transporters that are encoded by nuclear receptor target genes (Fig. 5). The main lipids in bile are cholesterol, bile salts and phospholipids. The proper balance of these components is determined by the coordinate expression of the lipid transporters that is crucial to maintaining cholesterol in a solubilized state. In the basal state (i.e., on a normal chow diet), biliary lipid secretion in FXR / mice is not impaired 36,and bile salt and phospholipid concentrations in bile are normal. Thus, the proper balance of cholesterol, bile salts and phospholipids in bile is maintained 36.The situation is quite different under lithogenic conditions where the presence of cholic acid and high cholesterol increase the lipid load and bile hydrophobicity and stimulate both the FXR and LXR pathways. Under these conditions in susceptible C57L and FXR / mice (which lack a fully functioning FXR response), the biliary lipid balance becomes perturbed by decreased expression of the phospholipid and bile salt transporters (Supplementary Fig. 1 online), resulting in substantially lowered biliary salt and phospholipid concentrations (Fig. 5a). The difference in the relative amounts of these two lipids compared to unchanged biliary cholesterol (resulting from a functioning LXR response) provides the driving force that influences the saturation, precipitation and crystallization behavior of cholesterol, as predicted by the equilibrium cholesterol bile salt phospholipid ternary phase diagram 24. The higher bile salt hydrophobicity index that we observe in FXR / mice correlates predictably with the rapid onset of cholesterol crystallization in bile 25 and the severe gallbladder mucosal damage 29 seen in these animals. In the susceptible C57L strain of mice that have a functional (albeit reduced) FXR response, the constellation of CGD phenotypes was prevented by pharmacologic treatment with a synthetic FXR agonist. The ability of the FXR agonist to prevent CGD in these mice is explained by the increased transport of biliary phospholipids and bile salts (Fig. 5b), which promotes cholesterol solubilization and thereby decreases the saturation index. Precipitation of cholesterol crystals from supersaturated bile is a prerequisite for gallstone formation 4, and cholesterol crystals in bile are found in 7% of patients with acute idiopathic pancreatitis 5.Both diseases are severe and have a high prevalence rate in certain indigenous populations and those of western European descent 1.The only treatment for symptomatic CGD is the surgical removal of the gallbladder, cholecystectomy. Although immediately successful in pain management, surgical treatment is not always completely effective because cholecystectomized patients are frequently readmitted for symptoms and microlithiasis biliary pancreatitis 37.At present, the approved medical therapy for these patients is the oral administration of ursodeoxycholate, a hydrophilic bile salt that delays the cholesterol crystallization phenomena 25.Although associated with reduced risk of biliary pain and acute cholecystitis 38,ursodeoxycholate therapy is not completely successful in preventing or treating CGD. The finding that FXR may have a key role in gallstone formation suggests a new therapeutic target for this disease. The modulation of nuclear hormone receptors and their downstream targets has been validated repeatedly as a successful therapeutic strategy for the management of numerous diseases 39.In the case of gallstone disease, pharmacological activation of FXR is expected to increase expression of ABCB11 and ABCB4, providing selective Abcg5 Cyp7a1 Shp Vehicle GW464 Vehicle GW464 C57L FXR / increased secretion of bile salts and phospholipids, thereby maintaining dissolution and solubilization of cholesterol in bile. Given the identical physical-chemical behavior of biliary lipids in mice 7 and humans 4,41, and the direct regulation of both mouse and human ABCB11 (refs. 2,42) and ABCB4 (refs. 21,22) gene promoters by FXR, there is strong evidence to believe that the protective effects of FXR agonists can be translated from the mouse to the human cholesterol gallstone model. The current studies present direct evidence that FXR agonists may be used to prevent or treat patients at risk for cholesterol gallstone disease and acute microlithiasis pancreatitis. METHODS Animals and diets. FXR / mice were previously generated and characterized 31. FXR / and wild-type mice were on the same mixed background (C57BL/6N 129/SVJ FVB, backcross C57BL/6N). Gallstone-susceptible C57L/J mice were purchased from Jackson Labs. We housed the mice in a temperaturecontrolled room (22 23 C) with a 12 h light 12 h dark cycle and maintained them on a low cholesterol (.2%) chow diet (Purina 51, Harlan Teklad 752) until 9 weeks of age, followed by a lithogenic diet (Harlan Teklad 3451), containing 15% butter fat, 1% cholesterol,.5% cholic acid, 2% corn oil, 5% sucrose, 2% casein and essential minerals and vitamins, for 1 week. Given the cholate-induced liver toxicity of FXR / mice 31, in the present study we limited Abcg8 Cyp8b1 Srebp1c Vehicle GW464 Vehicle GW464 C57L FXR / Relative mrna levels 1356 VOLUME 1 NUMBER 12 DECEMBER 24 NATURE MEDICINE

6 a Figure 5 Model of increased susceptibility to cholesterol gallstone formation and its prevention by FXR agonists in lithogenic animals. Biliary secretion of bile salts (BS), phosphatidylcholine (PC) and cholesterol (Chol) is controlled at the outer leaflet of the hepatocyte canalicular membrane by Bsep, Mdr2 and Abcg5/Abcg8, respectively. In the presence of the lithogenic diet when the FXR pathway is compromised, the biliary lipid profile is unbalanced and bile becomes supersaturated with cholesterol (a). In contrast, stimulation of FXR activity by a synthetic agonist, rebalances the physical chemical interactions of lipids in bile, thereby preventing cholesterol gallstone formation (b). 24 Nature Publishing Group b the use of the lithogenic diet to 1 week, which did not significantly alter liver function in the FXR / compared to wild-type mice as measured by serum liver enzymes and histological examination (data not shown). This diet has been shown previously to induce precipitation of cholesterol monohydrate crystals in susceptible strains of inbred mice beginning at day 5 (day 7 in 1% of mice) and cholesterol gallstone formation by day 28 (refs. 7,33). Animals were fed ad libitum with free access to water. To investigate the role of FXR loss and gain of function in cholesterol precipitation in bile, the study was divided in two parts. For the first part of the study, we administered the lithogenic diet to wild-type and FXR / mice for 1 week. For the second part, susceptible C57L and FXR / mice fed lithogenic diet were treated daily, 2 h after beginning the light cycle, by oral gavage with 1 mg/kg mouse body weight of the selective, synthetic FXR agonist GW464 (refs. 34,35) (T. Willson, GlaxoSmithKline) or vehicle (PEG 4: Tween 8, 4:1, vol/vol) for 1 week. After 12 h fasting, animals were killed, cholecystectomies were performed, and gallbladder bile (obtained by a fundal incision) was examined immediately using polarizing light microscopy for the presence of cholesterol monohydrate crystals. Bile was stored at 2 C, and livers, gallbladders, small and large intestines, and tail tips were frozen in liquid nitrogen and stored at 8 C until further analysis could be done. All experiments were approved by the Institutional Animal Care and Research Advisory Committee of the University of Texas Southwestern Medical Center. Lipid measurements. We extracted biliary (1 µl) and hepatic (.2 g of liver) lipids according to Folch 43. Phospholipids were measured as inorganic phosphorus 44 and bile salts were quantified enzymatically using the 3α-hydroxysteroid dehydrogenase (Sigma Chemical Co.) method 45. We determined individual bile salt compositions by high-performance liquid chromatography 46, using the following bile salts as standards: tauro β-muricholate, tauroursodeoxycholate, taurohyodeoxycholate, glycohyodeoxycholate, taurocholate, glycocholate, taurochenodeoxycholate, taurodeoxycholate, glycochenodeoxycholate, glycodeoxycholate, taurolithocholate. Total bile salt hydrophobicity index was calculated as described 47, using a computer program that is available upon request. We measured cholesterol by enzymatic assay 48 using a colorimetric reagent from Roche Diagnostics Corp. The cholesterol saturation index of bile was calculated according to the critical tables for taurocholate-rich bile 49.We measured plasma and liver triglycerides using a reagent from Thermo Trace Ltd. and glycerol standards from Sigma Chemical Co. RNA extraction and gene expression analysis. We performed RNA extraction from liver using the RNA STAT-6 reagent (TEL-TEST B, Inc.). RNA was treated with RNAse-free DNAse (Roche), and reverse transcribed (Superscript II, Invitrogen) using random hexamers (Roche) to a final concentration of 2 ng/µl. We designed gene-specific primers using Primer Express Software (PE Biosystem; Supplementary Table 1 online ). Real-time quantitative PCR was performed as previously described 5, using SYBR Green I chemistry (SYBR Green PCR Master Mix, ABI) on the ABI Prism 79HT Sequence Detection System. We ran each sample in triplicate with 25 ng template and 15 nm for each primer. We calculated relative fold changes using the comparative cycle times method with cyclophilin as the reference gene and the wild-type mice from each strain as the calibrators. All real-time quantitative PCR data were generated using RNA isolated from tissues of individual animals. Statistics. Values are expressed as means ± s.e.m. Differences between groups were tested for statistical significance by analysis of variance. When a significant difference was detected, results were compared for contrasts using Fisher least significant difference test as post hoc test. Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS The authors thank F. Gonzalez for supplying the FXR / mice used in these studies and T. Willson (GlaxoSmithKline) for supplying the synthetic FXR agonist GW464; D. Russell, S. Kliewer, J. Repa, G. Palasciano, P. Portincasa, G. van Berge Henegouwen, K. van Erpecum and A. Hofmann for criticisms and discussions; C. Cummins, D. Jung, A. Liverman and X. Zhi from the Mango lab for contributing to the in vivo studies and S. Clark for expertise in the HPLC measurements of bile salts. D.J.M. is an investigator and A.M. is a research associate at the Howard Hughes Medical Institute (HHMI). This work was funded by HHMI, the Robert Welch Foundation (I-1275), and the National Institutes of Health (Atlas grant U19DK62434). COMPETING INTERESTS STATEMENT The authors declare competing financial interests; see the Nature Medicine website for details. Received 13 July; accepted 1 October 24 Published online at 1. Sandler, R.S. et al. The burden of selected digestive diseases in the United States. Gastroenterology 122, (22). 2. Hay, D.W. & Carey, M.C. Pathophysiology and pathogenesis of cholesterol gallstone NATURE MEDICINE VOLUME 1 NUMBER 12 DECEMBER

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9 Supplementary Figure 1 online. Gene expression in wild-type, Fxr / and C57L mice. Quantitative real-time PCR analysis was performed on mrna from livers of wild-type, Fxr /, and C57L mice fed a standard chow (black bars) or lithogenic diet (gray bars) for one week. Expression patterns of genes involved in the FXR (Bsep, Mdr2, Shp) and LXR (Abcg5, Abcg8) pathways were characterized. The lithogenic diet (which includes cholic acid) was able to upregulate all the FXR-target genes (i.e., Bsep, Mdr2, Shp) in wild-type but not in Fxr / mice. Importantly, the ability of these genes to be upregulated on the lithogenic diet in the C57L mice was significantly lower as compared to the wild-type mice. Also, expression of Fxr itself was upregulated on the lithogenic diet in the wild-type but not in the C57L mice. These findings support the hypothesis that CGD susceptible strains of mice may have a polymorphism in the Fxr promoter that affects FXR expression 8. In contrast to the differential expression of FXR targets, the lithogenic diet (which also contains cholesterol) was able to upregulate the LXR target genes (i.e., Abcg5 and Abcg8) identically in all three mouse models, independently of strain. P<.1 vs. chow diet in the same mouse model, #P<.1 vs. wild type mice in the same dietary regimen.

10 Supplementary Table 1 online. Sequences of the primers used in the real time quantitative PCR measurement of gene expression. Gene Forward primer Reverse primer Abca1 5'-TCCTCATCCTCGTCATTCAAA-3' 5'-GGACTTGGTAGGACGGAACCT-3' Abcb4 (Mdr2) 5'-CTTGAGGCAGCGAGAAATG-3' 5'-GGTTGCTGATGCTGCCTAGT-3' Abcg5 5'-TCAATGAGTTTTACGGCCTGAA-3' 5'-GCACATCGGGTGATTTAGCA-3' Abcg8 5'-TGCCCACCTTCCACATGTC-3' 5'-ATGAAGCCGGCAGTAAGGTAGA-3' Bsep 5'-AAGCTACATCTGCCTTAGACACAGAA-3' 5'-CAATACAGGTCCGACCCTCTCT-3' Cyclophilin 5'-GGAGATGGCACAGGAGGAA-3' 5'-GCCCGTAGTGCTTCAGCTT-3' Cyp7a1 5'-AGCAACTAAACAACCTGCCAGTACTA-3' 5'-GTCCGGATATTCAAGGATGCA-3' Cyp8b1 5'-TAGCCCTCTTTCCTCCACTCATA-3' 5'-GAACCGATCGAACCTAAATTCCT-3' Cyp27a1 5'-GCCTCACCTATGGGATCTTCA-3' 5'-TCAAAGCCTGACGCAGATG-3' Fas 5'-GCTGCGGAAACTTCAGGAAAT-3' 5'-AGAGACGTGTCACTCCTGGACTT-3' Fxr 5'-TCCGGACATTCAACCATCAC-3' 5'-TCACTGCACATCCCAGATCTC-3' Hmgcr 5'-CTTGTGGAATGCCTTGTGATTG-3' 5'-AGCCGAAGCAGCACATGAT-3' Ldlr 5'-AGGCTGTGGGCTCCATAGG-3' 5'-TGCGGTCCAGGGTCATCT-3' Lpl 5'-GGCCAGATTCATCAACTGGAT-3' 5'-GCTCCAAGGCTGTACCCTAAG-3' Lxrα 5'-AGGAGTGTCGACTTCGCAAA-3' 5'-CTCTTCTTGCCGCTTCAGTTT-3' Lxrβ 5'-AAGCAGGTGCCAGGGTTCT-3' 5'-TGCATTCTGTCTCGTGGTTGT-3' Ntcp 5'-GAAGTCCAAAAGGCCACACTATGT-3' 5'-ACAGCCACAGAGAGGGAGAAAG-3' Pctp 5'-TGGCATACTGGGAAGTGAAGTAC-3' 5'-GGCGGGTGTAGACGTAATCTC-3' Srb1 5'-TCCCCATGAACTGTTCTGTGAA-3' 5'-TGCCCGATGCCCTTGA-3' Srebp1c 5'-GGAGCCATGGATTGCACATT-3' 5'-GGCCCGGGAAGTCACTGT-3'