Transport of cholesterol into mitochondria in rat limiting for bile acid biosynthesis via the alternative pathway

HEPATOLOGYVol. 34, No. 4, Pt. 2, 2001

AASLD ABSTRACTS

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TARGETED DELETION OF TIlE INTRONLESS STEROL 1 2 a HYDROXYLASE (CYP8B1) GENE IN MICE LEADS TO LOSS OF CHOLIC ACID PRODUCTION BUT UPREGULATED BILE ACID SYNTHESIS. Mats Gafv-

IMPAIRED EEEDBACK INHIBITION OF OXYSTEROL 7A-HYDROXYLASE IN MICE W I T H HOMOZYGOUS DISRUPTION OF TIlE STEROL CARRIER PROTEIN 2 (SCP2) GENE. Christian Muench, Division of Gas-

els, Div of Clin Chem Huddinge Univ Hsp, Stockholm Sweden; Julia LiHawkins, Mol Genetics, UTSW Med Center, Dallas, TX; Maria Olin, Div of Clin Chem Huddinge Univ Hsp, Stockholm Sweden; Erik Lund, Mol Genet UTSW Med Center, Dallas, TX; Ulla Andersson, Div of Clin Chem Huddinge Univ Hsp, Stockholm Sweden; Gertrud Schuster, Center for Biotechnol, Novum, K1, Stockholm Sweden; Ingemar Bjorkhem, Div of Clin Chem Huddinge Univ Hsp, Stockholm Sweden; David Russell, Mol Genet, UTSW"Med Center, Dallas, TX; Gosta Eggertsen, Div of Clin Chem, Huddinge Univ Hsp, Stockholm Sweden

troenterology, Medical University of Luebecki, Luebeck Germany; Nadine Katzberg, Division of Gastroenterology, Medical University of Luebeck, Luebeck Germany; Udo Seedorf, Institute for Arteriosclerosis Research, University of Muenster, Muenster Germany; Juergen Scheibner, Eduard F Stange, Michael Fuchs, Division of Gastroenterology, Medical University of Luebeck, Luebeck Germany

The study was performed to evaluate the biological significance of sterol 12ahydroxylase and cholic acid in the bile acid metabolism CYP8B1 (sterol 12a hydroxylase) is a branch-point enzyme that regulates the composition of bile acids in bile with conseqnences for intestinal absorption of cholesterol and degree of suppression of activity of the rate-limiting enzyme in bile acid synthesis, CYP7A1. To study the physiological role of CYP8B1 in vivo we used homologous recombination in embryonic stem cells to produce a strain of mice with a null mutation in the Cyp8bl gene The deletion led to an almost complete lack of cholic acid in bile, confirming that Cyp8bl is the only enzyme responsible for formation of cholic acid. The loss of cholic acid in bile was replaced by substantial amounts of chenodeoxycholate and muricholate. No physical or behavioural abnormalities were found. The bile acid pool size and fecal bAle acid excretion rate were increased by 37% and 53% in male knockout mice, respectively. Intestinal cholesterol absorption was decreased by 4-1% in CypSbl-/- mice, and a compensatory two-fold increase in hepatic sterol biosynthesis was detected. The upregulation of cholesterol synthesis was also reflected by an increase of HMG CoA reductase mRNA levels in the liver and lathosterol levels in circulation. Together these metabolic adaptations maintained plasma and hepatic cholesterol and triglycerides at normal levels in the mutant mice. Surprisingly, the deletion led to a significant upregulation of Cyp7al as judged from mRNA, Western blotting and enzyme activity levels, while the mRNA levels of short heterodimerizing partner (SHP) was decreased, indicating an attenuated FXR activity as a cause for reduced SHP expression. Cholic acid was found to suppress Cyp7al transcription to a higher extent than chenodeoxycholic acid in feeding experiments with both wild type and Cyp8bl deficient mice. The deletion did not cause significant changes in the mRNA levels of FXR, LRH-1 or LXRa. These results suggest that cholic acid is the major ligand for FXR in mouse liver. By use of DNA microarray technology we identified other genes not known to be directly involved in bile acid metabolism that were also regulated in cyp8bl -/-mice. We conclude that the loss of sterol 12a-hydroxylase have profound consequences for sterol metabolism and gene expression in the mouse

Disruption of the Scp2 gene impairs the thiolytic step in ~-oxidation of the steroid side chain in bile acid synthesis, but regulation of bile acid synthesis has not been studied so far. Twelve week old male C57BL/6 and C57BL/6Scp2('/-) mice (n=6-8) were fed a standard or a lithogenic diet (15% fat, 1.25% cholesterol, 0.5% cholic acid) for 3 months. Hepatic bile was collected for lh via an acute bile fistula and livers were harvested thereafter. Bile acid pool size and composition was analyzed by gas chromatography-massspectrometry and bile acid secretion rates were calculated. RT-PCR and Western Blotting was employed to analyze steady-state gene expression of key enzymes (Cyp7al, Cyp7bl,Cyp8b, Cyp39al) and transcription factors (Lxr, Fxr, Lrh, Shp) involved in bile acid synthesis. Under basal conditions bile acid pool size was 2.2-fold smaller in C57BL/6S~p~-(4-)mice reflected by a lower (p<0.01) bile acid secretion rate (83_+40 vs. 258_+110nmol/100g/h). The bile acid pool however was enriched with 23-norcholic acid (10.1 vs. 0.2p~g; p<0.05) whereas chenodeoxycholic acid was decreased (0.8 vs. 5.7tzg; p<0.005). Expression of regulatory enzymes and transcription factors was unaltered. When both groups of mice received the lithogenic diet, the bile acid pool became enriched with cholic acid (96%) and deoxycholic acid (2%) and bile acid secretion rates increased 9-11-fold (p<0.001). In C57BL/6 mice Cyp7bl mRNA and protein levels decreased by 65% (p<0.00l) and 80% (p<0.01), respectively. In contrast, no feedback inhibition occurred in C57BL/6scp2(-/-)mice. In both strains Cyp8b mRNA levels decreased by 90% (p<0.01) whereas Cyp39al mRNA expression remained constant. Similarly, Cyp7al mRNA levels decreased by 80% (p<0.01), which translated to a smaller 20% (p>0.05) inhibition of CYPTA1 protein levels. The lithogenic diet did not alter Lxr and Fxr mRNA expression. In contrast to C57BL/6sop2(-/-)mice, mRNA expression of Shp and Lrh increased 2.2-fold (p<0.001) and 1.5-fold (p>0.05) in C57BL/6 mice, respectively. In summary, not only the bile acid synthetic pathway, but also feedback inhibition of bile acid synthesis in response to the lithogenic diet is impaired in C57BL/6Sop2(4) mice.

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BILE ACID TRANSPORTER (NTCP, BSEP) PROTEIN LEVELS AND BILE ACID SYNTHETIC ENZYME ACTIVITIES 1N WILD TYPE AND FXR-NULL MICE FED CHOLIC ACID AND CHOW DIETS: DIFFERENTIAL REGULATION OF MRNA AND PROTEIN EXPRESSION. Jaya Bollineni, UMDNJ

TRANSPORT OF CHOLESTEROL INTO M1TOCIIONDRIA IS RATE LIMITING FOR BILE ACID BIOSYNTHESIS VIA THE ALTERNATIVE PATHWAY. William Pandak Jr., Dalila Marques, Shunlin Ren, Elizabeth Hail, Kay

School of Medicine, Newark, NJ; I'Kyori Swaby, The Mount Sinai Medical Center, New York, NY; Christopher Sinal, Frank Gonzalez, National Institutes of Health, Bethesda, MD; Sarah Shefer, UMDNJ School of Medicine, Newark, NJ; Frederick Suchy, Meenakshisundaram Ananthanarayanan, The Mount Sinai Medical Center, New York, NY Recent studies from a number of laboratories have implicated the critical role of nuclear receptors in the regulation of bile acid synthesis and transport. Bile acids are ligands for Farnesoid-X-receptor (FXR)/13ileacid receptor (BAR,NR1H4). FXR/BAR has shown to be involved in the regulation of bile acid transporters, Ntcp and Bsep, and the enzymes involved in bile acid synthesis, Cyp7A (Cholesterol 7-hydroxylase), Cyp7B (Oxysterol 7-hydroxylase), Cyp8B (Stero112-hydroxylase)and Cyp27 (Stero127-hydroxylase). Targeted inactivation of FXR resulted in altered regulation in the mRNAs for the above genes in FXR-null mice and in their response to cholic acid (CA) feeding (Cell 102:731, 2000). The expected upregulation of Bsep and downregulation of Ntcp were impaired, while downregulation of Cyp7A, Cyp7B, Cyp8B and Cyp27 were also lost in FXR-null mice compared to wild-type controls (FXR+/+) fed 1% CA diet for 5 days. We analyzed the enzyme activities of bile acid synthetic enzymes in addition to Ntcp and Bsep protein levels in livers of wild type and FXR-null mice fed chow/CA to establish whether the changes in mRNA are reflected at the functional/protein level. Methods: Enzyme assaysfor Cyp7A, Cyp7B, Cyp8B and Cyp27 were those p r e v i o u s ~ s h e d in our laboratory. Polyclonal antibodies specific to rat Ntcp and Bsep, previously generated and characterized, were used in Western blot analysis to assess the transporter levels. Protein levels were quantitated by densitometry. Results: CypTA and Cyp8B activities in CA-fed ~a~ildtype mice were 27% and 30% controls, respectively, while their mRNAswere undetectable. CA-fed wild type mice had 30oB of chow-fed control Cyp7B mRNA while the enzyme activity" was 48% of controls. Cyp27 mRNA and protein levels between these two groups were similar. Cyp7A, Cyp8B and Cyp27 activities in CA-fed FXR-null mice were not significantly different from control animals and were in concordance with their mRNAlevels. In contrast, Cyp7B mRNA in CA-fed FXR-null mice were 20% while the enzyme activity was still at 52% of control mice. Bsep protein was present in significant quantities in FXR-nulImice in contrast to a 70% downregulation in its mRNA. CA feeding increased Bsep mRNAby 6-fold while the protein levels were increased by 1.7-fold. Ntcp protein levels upon CA feeding were decreased to 32% of controls, in agreement with a 40% decrease in its mRNA. Protein levels for the transporters were not significantly different in CA-fed FXRnull mice mirroring their mRNA amounts. Conclusion: There was significant discordance between the mRNA and enzyme activity/protein levels for bile acid synthetic enzyme activities and bile acid transporters in wild-type mice fed CA and in FXR-null mice fed chow diet. These data suggest that 1) regulation of these proteins is likely controlled by other factors in addition to FXR and 2) the half lives of the proteins are significantly longer than the corresponding mRNAs.

Redford, Gregorio Gil, Darrell Mallonee, Douglas Heuman, Phillip Hylemon, McGuire VAMC / Virginia Commonwealth University, Richmond, VA Bile acid biosynthesis (BAS) occurs predominantly via two pathways; the classic pathway, initiated by microsomal cholesterol 7a-hydroxylase (CYP7A1), and the alternative pathway, initiated by mitrocondrial sterol 27-hydroxylase (CYP27). CYP27 is located in the inner mitochondrial membrane where cholesterol content Asvery low. Hence, cholesterol transport to the inner mitochondrial membrane might be the rate-limiting step controlling BAS via the alternative pathway. Objective: To define the effects of increased mitrochondrial cholesterol transport on rates of BAS in primary rat hepatocytes (PRH) in culture. Methods: Rates of BAS were determined in PRH following over-expression of the StAR (steroidogenic acute regulator), protein), a mitochondrial cholesterol transporter, CYP27, or CYP27 plus STAR. Over-expression was mediated through infection of PRH with replication defective recombinant adenoviruses encoding either CMV-StAR or CMV-CYP27. PRH were maintained under culture conditions that limit CYP7Af expression to undetectahle levels (previously described). Results: StAR and CYP27 proteirdmRNA levels increased dramatically following infection with recombinant adenoviruses. StAR over-expression lead to >6-fold increase (n=9; p is less than or equal to 0.001) in rates of BAS, while BAS following over-expression of CYP27 increased only slightly (76_ + 58%; NS). The rates of BAS following the combination of StAR and CYP27 over-expression were similar to StAR alone. Analysis of [z4C]-bile acid biosynthetic intermediates extracted from whole cells and culture media showed dramatic increases in several intermediates including 27-hydroxycholesterol. Conclusion: Transport of cholesterol into the mitochondria is the rate-limiting step of BAS via the alternative pathway. A better understanding of cholesterol transport into mitochondria in the liver could lead to new therapeutic approaches for increasing cholesterol output from the liver.