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Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR.

Abstract Background The farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR) are three closely related nuclear hormone receptors in the NR1H and 1I subfamilies that share the property of being activated by bile salts. Bile salts vary significantly in structure across vertebrate species, suggesting that receptors binding these molecules may show adaptive evolutionary changes in response. We have previously shown that FXRs from the sea lamprey (Petromyzon marinus) and zebrafish (Danio rerio) are activated by planar bile alcohols found in these two species. In this report, we characterize FXR, PXR, and VDR from the green-spotted pufferfish (Tetraodon nigriviridis), an actinopterygian fish that unlike the zebrafish has a bile salt profile similar to humans. We utilize homology modelling, docking, and pharmacophore studies to understand the structural features of the Tetraodon receptors. Results Tetraodon FXR has a ligand selectivity profile very similar to human FXR, with strong activation by the synthetic ligand GW4064 and by the primary bile acid chenodeoxycholic acid. Homology modelling and docking studies suggest a ligand-binding pocket architecture more similar to human and rat FXRs than to lamprey or zebrafish FXRs. Tetraodon PXR was activated by a variety of bile acids and steroids, although not by the larger synthetic ligands that activate human PXR such as rifampicin. Homology modelling predicts a larger ligand-binding cavity than zebrafish PXR. We also demonstrate that VDRs from the pufferfish and Japanese medaka were activated by small secondary bile acids such as lithocholic acid, whereas the African clawed frog VDR was not. Conclusions Our studies provide further evidence of the relationship between both FXR, PXR, and VDR ligand selectivity and cross-species variation in bile salt profiles. Zebrafish and green-spotted pufferfish provide a clear contrast in having markedly different primary bile salt profiles (planar bile alcohols for zebrafish and sterically bent bile acids for the pufferfish) and receptor selectivity that matches these differences in endogenous ligands. Our observations to date present an integrated picture of the co-evolution of bile salt structure and changes in the binding pockets of three nuclear hormone receptors across the species studied.

Abstract Background Bile salts are the major end-metabolites of cholesterol and are also important in lipid and protein digestion and in influencing the intestinal microflora. We greatly extend prior surveys of bile salt diversity in both reptiles and mammals, including analysis of 8,000 year old human coprolites and coprolites from the extinct Shasta ground sloth (Nothrotherium shastense). Results While there is significant variation of bile salts across species, bile salt profiles are generally stable within families and often within orders of reptiles and mammals, and do not directly correlate with differences in diet. The variation of bile salts generally accords with current molecular phylogenies of reptiles and mammals, including more recent groupings of squamate reptiles. For mammals, the most unusual finding was that the Paenungulates (elephants, manatees, and the rock hyrax) have a very different bile salt profile from the Rufous sengi and South American aardvark, two other mammals classified with Paenungulates in the cohort Afrotheria in molecular phylogenies. Analyses of the approximately 8,000 year old human coprolites yielded a bile salt profile very similar to that found in modern human feces. Analysis of the Shasta ground sloth coprolites (approximately 12,000 years old) showed the predominant presence of glycine-conjugated bile acids, similar to analyses of bile and feces of living sloths, in addition to a complex mixture of plant sterols and stanols expected from an herbivorous diet. Conclusions The bile salt synthetic pathway has become longer and more complex throughout vertebrate evolution, with some bile salt modifications only found within single groups such as marsupials. Analysis of the evolution of bile salt structures in different species provides a potentially rich model system for the evolution of a complex biochemical pathway in vertebrates. Our results also demonstrate the stability of bile salts in coprolites preserved in arid climates, suggesting that bile salt analysis may have utility in selected paleontological research.

Analysis of the sub-caudal gland samples of Owl monkeys, showed a unique and complex chemical profile for each family. This is used to determine individual gender and age and prevents inbreeding.

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3[alpha]-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3[alpha]-hydroxy bile acid concentration ([micro]mol bile acid/ml cecal content) was 0.4 [+ or -] 0.2 mM (mean [+ or -] SD); the total 3-hydroxy bile acid concentration was 0.6 [+ or -] 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 [+ or -] 9%, mean [+ or -] SD); sulfated, nonamidated bile acids were 7 [+ or -] 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 [+ or -] 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 [+ or -] 16%), lithocholic (26 [+ or -] 10%), and ursodeoxycholic (6 [+ or -] 9), as well as their primary bile acid precursors cholic (6 [+ or -] 9%) and chenodeoxycholic acid (7 [+ or -] 8%). In addition, 3[beta]-hydroxy derivatives of some or all of these bile acids were present and averaged 27 [+ or -] 18% of total bile acids, indicating that 3[beta]-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations. bile acid deconjugation; bile acid dehydroxylation; intestinal bile acids; mass spectrometry of bile acids doi:10.1152/ajpgi.00027.2007

Bile acid epimers and side-chain homologues are present in the human colon. To test whether such bile acids possess secretory activity, cultured T84 colonic epithelial cells were used to quantify the secretory properties of synthetic epimers and homologues of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). In our study, chloride secretion was measured as changes in short-circuit current ([DELTA][I.sub.SC], in [microLA/[cm.sup.2]) with the use of voltage-clamped monolayers of T84 cells mounted in Ussing chambers. Bile acids were added at 0.5 mM, a concentration that did not alter transepithelial resistance. Data were expressed as peak [DELTA][I.sub.SC] (means [+ or -] SD). When added bilaterally, DCA stimulated a [DELTA][I.sub.SC] response of 15.7 [+ or -] 12.5 [micro]A/[cm.sup.2]. The 12[beta]-OH epimer of DCA was less potent ([DELTA][I.sub.SC] = 8.0 [+ or -] 1.7 [micro]A/[cm.sup.2]), whereas its 3[beta]-OH epimer had no effect. CDCA stimulated secretion ([DELTA][I.sub.SC] = 8.2 [+ or -] 5.5 [micro]A/[cm.sup.2]), whereas both its 7[beta]-OH and 3[beta]-OH epimers were inactive, as was lithocholic acid. HomoDCA (1 additional side-chain carbon) was active ([DELTA][I.sub.SC] = 7.8 [+ or -] 4.8 [micro]A/[cm.sup.2]), whereas norDCA (1 fewer carbon) and dinorDCA (2 fewer carbons) were not. Taurine conjugates of DCA and CDCA stimulated secretion (Alsc = 12.3 [+ or -] 7.5 and 8.8 [+ or -] 4.8 [micro]A/[cm.sup.2], respectively) from the basolateral side but not the apical side. Uptake of taurine conjugates from the basolateral but not the apical side was shown by mass spectrometry. These studies indicate marked structural specificity for bile acid-induced chloride secretion and show that modification of bile acid structure by colonic bacteria modulates the secretory properties of these endogenous secretagogues. bile acid homologues; conjugated bile acids; bile acid physiology