456

The Abcc2-Lith2 Locus Induces Hypersensitivity to The Biliary Lithogenic Effect of Oestrogens by Precipitating The Onset of Lithogenic Bile Eve[yrte Morin, Diane Mlgnauh, Guylaine Bouchard

Background and Aim: We recently established Abcc2 to be the molecular basis underlying Lith2, one of the major cholesterol gallstone susceptibility loci identified in inbred mice fed a lithogenic diet. Based on the direct role of ABCC2 in the hepatohiliary transport of different drug and hormone conjugates, many of which are well-recognised to constitute high risk factors for human cholelithiasis, we hypotbetised that the Lith2/Abcc2 locus impacts not only the fithogenic response to dietary intervention but also to drugs and hormones. We tested our hypothesis here, using estrogen as a model compound. Methods: AKi.L/th2 s congenic mice (Lith2) and AKR controls were injected i.p. with ethinyl estradiol (EE) at the dose of 1X or 3X (5 or 15mg/kg-day) for 3 days. Bile flow and biliary lipids were assessed from hepatic bile with standard procedures. Hepatic bile acids were assayed by LC-MS-MS. Results: Body weights were identical between groups, establishing that all mice received an equivalent amount of EE Without EE-treatment, biliary lipid secretion rates, biliary lipid coupling and CSI were undifferentiable between groups. After EE 1X, however, while the controls increased their bile salts (BS) secretion rate and modestly but significantly decreased their biliary secretion of phospholipids (PL) and cholesterol (Ch) (most likely due to the uncoupling effect of EE-conjugates), the Lith2 group exhibited a declining BS secretion rate, a drop 2 times more pronounced in PL secretion rates and a sharp rise in biliary Ch, culminating in a marked elevation of Ch/BS or PL ratios and CSI. Tripling the dose of EE had no additional effect in lath2 mice but led the controls to now develop an hepatobiliary and lithogenic profile undistinguishable from the congenic animals. Percent of hepatic dihydroxylated BS were elevated in all Lith2 mice treated with EE but only in the controls receiving EE 3X, thereby being in direct relationship with biliary lithogenicity. Conclusions: The Lith2 locus, and most likely Abcc2, is not solely involved in the high-lithogeinc risk following a lithogenic diet, but also induces an hypersensitivity to the hthogenic effect of estrogens. Dihydroxylated hepatic bile acids appears central to the development of lithogenic bile, possibly through activation of nuclear receptor/ABCG5-8 expression. We speculate that such gene-estrogen interaction plays a role in the lithogenic risk associated with gender, pregnancy and estrogen-based therapies.

457

Homozygosity for the Q136X Mutation within the ARH Gene Results in Delayed In Vivo LDL Catabolism Janine Geoschel, Uwe J. Tietge, Robert Shamburek, Herbert Locks, Loren A. Zech, Bryan H Brewer Jr., Helen Hobks, Hartmut H. Schmidt

The strong positive association between elevated levels of plasma LDL cholesterol and atherosclerotic disease has been well established. Most commonly, the underlying genetic dekct are mutations in the gene encoding the LDL receptor (LDLR) resulting in autosomal dominant familial hypercholesterolemia (FH). In addition, certain mutations within the LBLR binding region of the apoB protein are known to cause a similar clinical phenotype. Most recently, mutations in a novel gene encoding the putative adaptor protein ARH have been identified to result in the extremely rare disorder of autosomal recessive hypercholesterolemia (ARH). ARH contains a phosphotyrosine-binding (IV'l'B) domain known to interact with the NPXY motif present e.g. on the cytoplasmic tail of the LDLR. We have identified a 44 year 01d male patient with the clinical expression of homozygous familial hypercholesterolemia presenting severe coronary artery disease, tendon and skin xanthomas, arcus lipoides, and joint pain. The genetic trait is autosomal recessive. Interestingly, serum concentrations of cholesterol did well respond to diet and statins. We bad no evidence of an abnormal LDL- apoB particle, which was isolated from the patient, using the U937 proliferation assay as a functional test of the LDL binding capacity. The apolipoprotein B (apoB) 3500 and apoB 3531 defects were ruled out by PCR. In addition, we found no evidence for a defect within the low density lipoprotein receptor by skin fibrobiasts analysis, linkage analysis, SSCP and Southern blot screening across the entire LDL receptor gene. However, we succeeded in identifying a homozygous mutation for Q136X within the ARH gene by direct sequencing. The in vivo kinetics of radioiodinated LDL-apoB were evaluated in the proband and three normal controls. The LDL-apoB isolated from the patient showed a normal catabolism, confirming an intact LDL particle. In contrast the fractional catabolic rate (FCR, d ~) of autologous LDL in the subject and the normal controls revealed a remarkable delayed catabolism of the patients LDL (015 versus 0.33-0.43 dl). In addition, the elevation of LDL-cholesterol in the patient resulted from an increased production rate with 22.8 mg/kg/ day versus 12.7-15.7 mg/kg/day. These data reveal for the first time, that the homozygous QI36X mutation within ARH results in in vivo delayed LDL catabolism as seen in LDL receptor dehciency. Therefore, the functional consequences of mutations within ARH are similar to mutations within the LDL-receptor and apoB gene

458

Bile Acid (BA) Metabolism Is Impaired In Mice w i t h Defective Peroxisomal BA Synthesis Christian Muench, Sandra Teichmann, Eduard F. Stange, Udo Seedorf, Michael Fucks

Impairment of peroxisomal BA synthesis is characteristic for sterol carrier protein 2 knockout mice. To study how this may impact BA metabolism, lipid analysis of hepatic and gallbladder bile was performed with standard methods to calculate biliary lipid secretion rates in chow- fed mice (n = 6-9). Livers and sequential fractions of equal length from duodenum to ileum were harvested to isolate total RNA, homogenate and plasma membranes, respectively. Steady-state hepatic gene expression of basolateral (SlclOal, Slc21al) and canalionlar (Abcb4, Akbll, Abcc2) transporters with relevance for lipid secretion was measured and correlated with serum and biliary lipids. Size and composition of the BA pool was measured by GCMS. Measurement of fecal BAs employed a dual isotope ratio method. Gene expression of intestinal 8A transporters (Fabp6, SlclOa2) was also investigated. Serum BAs were higher in -/- mice and composed of nor-BAs and bile alcohols which were absent m controls. The BA pool

of 4- mice was reduced 3-fold and enriched more than 30-fold (p<0.01) with nor-cholate whereas chenodeoxycholic acid was reduced 7-fold (p<0.01). Despite unchanged Abet2 expression, bile flow was 1.5-fold higher (p<0.05) in 4- mice. BA output was reduced by 68% (p<0.01) which correlated with a significant suppression of Abcbll expression. Biliary excretion of cholesterol and phospholipids were unchanged in -/- mice and no sigrnficant change of Abcb4 expression was detected. In contrast to controls, SlclOal expression was induced by 40% (p<0.05) whereas no significant changed was observed for Slc21al. Fecal BA excretion was reduced to 50% (p<0.001) and bile alcohols were present in 4- mice only. In all mice, SlclOa2 and Fabp6 expression increased from proximal to distal with highest levels in the ileum. However, Sic10a2 and Fabp6 expression was significantly (p < 0.01 ) lower in -/-mice. Our findings suggest that impaired peroxisomal BA synthesis is associated with adaptive changes of BA metabolism. More hydrophilic BAs of a smaller BA pool may be poor substrates for the canalicular BA export pump and thus explain the lower Abcb11 expression with a reduction of the BA output. 1"his may further result in increased serum BA concentrations although we were unable to document inhibition of Slc10al or Slc21a1 expression. Decreased intestinal SlclOa2 and Fabp6 expression implies that conservation of the smaller but more hydrophitic BA pool was likely achieved by inducing BA absorption in more proximal parts of the intestine.

459

Role of Nuclear Bile Salt Receptors Fxr and Pxr in Mediating Adaptive Hepatobil iary Transporter Response to Cl'mtic Acid (CA) in Mouse Liver Gemot ZoUner, Peter Fickert, Dagmar Silbert, Andrea Puchsbichler, Martin Wagner, Grace Guo, Frank J. Gonzalez, Kurt 7~ttoukal, Helmut Denk, Michael Trauner

Background & Aims: Cholestasis is associated with adaptive transporter expression aimed at providing alternative excretory routes and limiting hepatocellufar overload with toxic bile salts. However, the role and relative contributions of the nuclear bile salt receptors FXR and PXR in mediating this adaptive response remains unclear. Methods: To answer these ques- tions, we compared RNA and protein expression of the major hepatocellular bile salt uptake and export systems (basofateral Ntcp, canalicular Bsep and basolateral Mrp3) in wild type (WT) and knockout mice for FXR (FXR/), PXR (PXR /) and in FXR/PXR (double nulls) fed with a diet containing 1% chofic acid (CA), a major bile salt retained during cholestasis. Results: Serum bile acid levels increased after CA feeding (1%wAy) in WT and PXR -/- and to an even greater extent in FXR /- and double nulls (p<0.01). CA reduced Ntcp in WT (p<0.05). Baseline Ntcp was reduced m FXR / and further decreased in response to CA (p<0.05). Similar findings were obtained in PXR ~- and double nulls. CA induced Bsep in WT and PXR 4- (p<O.O1). in contrast, baseline Bsep was reduced (p<0.01) and no longer inducible by CA in FXR ~. Mrp3 was induced by CA in FXR -/-, PXR / and double nulls to an even greater extent than in WT (p<0.05). Conclusion: Induction of Bsep and Mrp3 as the major components of the adaptive transporter defense against bile acids are independent of PXR, while Bsep induction strictly depends on FXR.

460

Overactivity of ABCC2 Is a Major Determinant of Murine Cholesterol Gallstone (ChGS) Risk as Evidenced by Positional Cloning, Sequencing, and Gene- Targeted Deletion Guylaine Boucbard, Stepbanie Casavant, Rrm Korstanje, Carol Soroka, James L. Boyer, Martin C. Carey, Beverly Paigen

Background and Aim: Despite the recognized genetic background of human ChGS disease, the predisposing molecular origins remain elusive. To uncover the genetic bases of ChGS and to better understand the biochemical pathways involved, we have exploited the genetic infrastructure of the inbred mouse. Phenomic studies in a strain survey first identified mice with low and high prevalence rates of ChGS, whereas quantitative trait locus (QTL) analysis in crosses derived from ChGS-snsceptible and -resistant strains revealed the location of the gallstone-predisposing alleles. Our aim now was to identify the gene underlying one of these QTLs, Lith2, shown earlier by us to strongly tntluence ChGS-susceptihifity in the C57L/J (C57L) compared to the resistant AKR/J (AKR). Methods and Results: The Lith2 susceptibility locus was refined to 300 kB using an overlapping congenic strategy. This locus carries only Abet2 as a candidate gene. Complete sequencing of Abet2 identified a single 1838G-->A (R613Q) missense mutation in the susceptible C57L compared to the AKR Presence of this mutation is linked in the AKi-Lith2 s congenic(constructed by introgressing the Lith2 locus into the AKR background), to heightened biliary secretion rates of bilirubin conjugates and glutathione, despite unchanged ABCC2 hepatic expression and subcellular localization. To ascertain whether this gain of function in ABCC2 is responsible for ChGS risk, we tested whether a reduction in ABCC2 function would have a salutary effect. Abcc2 +/- mice, with half-normal ABCC2 function (as evidenced by a 50% hepatic expression of ABCC2 and half biliary glutathione secretion levels compared to wild-type mice)were constructed by a gene- targeted deletion strategy. Upon feeding the lithogenic diet, they exhibit a significantly reduced risk (7% vs. 46%) for ChGS prevalence compared to Abcc2 +/+ mice. Prevention of ChGS is linked to decrease biliary cholesterol/phospholipid ratio and marked elevations in biliary lipid concentrations. Conclusions: A gain of function in Abcc2 strongly predisposes to murine ChGS formation. We suggest that the physiological mechanism includes unfavorable coupling of biliary lipids and bile dilution, both of which result in compromised micellar solubility of cholesterol. This discovery and others along these lines, promises to have appreciable impact on prevention and development of new treatments for this very common and costly digestive disease.

A-59 AGA Abstracts