Microbially-conjugated Bile Salts Found in Human Bile Activate the Bile Salt Receptors TGR5 and FXR

Background & Aims Bile salts of hepatic and microbial origin mediate inter-organ crosstalk in the gut-liver axis. Here, we assessed whether the newly discovered class of microbial bile salt conjugates (MBSCs), activate the main host bile salt receptors (TGR5 and FXR) and enter the human systemic and enterohepatic circulation. Approach & Results N -amidates of (chenodeoxy)cholic acid and leucine, tyrosine and phenylalanine were synthesized. Receptor activation was studied in cell-free and cell-based assays. MBSCs were quantified in mesenteric and portal blood and bile of patients undergoing pancreatic surgery. MBSCs were activating ligands of TGR5 as evidenced by recruitment of Gsα protein, activation of a cAMP-driven reporter, and diminution of LPS-induced cytokine release from macrophages. Intestine- and liver-enriched FXR isoforms were both activated by MBSCs, provided that a bile salt importer was present. Affinity of MBSCs for TGR5 and FXR was not superior to host-derived bile salt conjugates. Individual MBSCs were generally not detected ( i . e . <2.5 nmol/L) in human mesenteric or portal blood, but Leu- and Phe-variants were readily measurable in bile, where MBSCs comprised up to 213 ppm of biliary bile salts. Conclusions MBSCs activate the cell surface receptor TGR5 and the transcription factor FXR, and are substrates for intestinal (ASBT) and hepatic (NTCP) transporters. Their entry into the human circulation is, however, non-substantial. Given low systemic levels and surplus of other equipotent bile salt species, the studied MBSCs are unlikely to have an impact on enterohepatic TGR5/FXR signaling in humans. Origin and function of biliary MBSCs remain to be determined. ![Figure][1] Graphical Abstract Created with [BioRender.com][2] ### Competing Interest Statement The authors have declared no competing interest. * ASBT : apical sodium-bile salt transporter BRET : bioluminescence resonance energy transfer BSA : bovine albumin serum C. difficile : Clostridium difficile CA : cholic acid cAMP : cyclic adenosine monophosphate CDCA : chenodeoxycholic acid DCA : deoxycholic acid DMSO : dimethyl sulfoxide FXR : farnesoid X receptor GCA : glycocholic acid GCDCA : glycochenodeoxycholic acid GLCA : glycolithocholic acid IL-6 : interleukin 6 LCA : lithocholic acid LC-MS : liquid chromatography–mass spectrometry LeuCA : leucocholic acid LeuCDCA : leucochenodeoxycholic acid LPS : lipopolysaccharide MBSC : microbial bile salt conjugate MCA : muricholic acid NF- κ B : nuclear factor- κ B NTCP : Na+ taurocholate co-transporting protein NMR : nuclear magnetic resonance OCA : obeticholic acid PheCA : phenylalanocholic acid PheCDCA : phenylalanochenodeoxycholic acid TCA : taurocholic acid TCDCA : taurochenodeoxycholic acid TGR5 : Takeda G protein-coupled receptor 5 TLCA : taurolithocholic acid TNFα : tumor necrosis factor α TR-FRET : time-resolved fluorescence resonance energy transfer TyrCA : tyrosocholic acid TyrCDCA : tyrosochenodeoxycholic acid. [1]: pending:yes [2]: http://BioRender.com