Microbially conjugated bile salts found in human bile activate the bile salt receptors TGR5 and FXR

Abstract

Background: Bile salts of hepatic and microbial origin mediate interorgan cross talk 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 (Takeda G protein-coupled receptor 5 [TGR5] and farnesoid X receptor [FXR]) and enter the human systemic and enterohepatic circulation.

Methods: 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.

Results: MBSCs were activating ligands of TGR5 as evidenced by recruitment of Gsα protein, activation of a cAMP-driven reporter, and diminution of lipopolysaccharide-induced cytokine release from macrophages. Intestine-enriched and liver-enriched FXR isoforms were both activated by MBSCs, provided that a bile salt importer was present. The affinity of MBSCs for TGR5 and FXR was not superior to host-derived bile salt conjugates. Individual MBSCs were generally not detected (ie, < 2.5 nmol/L) in human mesenteric or portal blood, but Leu-variant and Phe-variant 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 (apical sodium-dependent bile acid transporter) and hepatic (Na+ taurocholate co-transporting protein) transporters. Their entry into the human circulation is, however, nonsubstantial. Given low systemic levels and a surplus of other equipotent bile salt species, the studied MBSCs are unlikely to have an impact on enterohepatic TGR5/FXR signaling in humans. The origin and function of biliary MBSCs remain to be determined.

Graphical abstract

FIGURE 1

TGR5-activating potential of microbial bile salt conjugates. (A) Recruitment of a mini-G_s protein to ligand-activated TGR5 was studied in 293 cells expressing tagged protein versions, allowing their interaction to be monitored by bioluminescence resonance energy transfer. TGR5 activation was evaluated for both CDCA-based (left panel) and CA-based MBSCs (right panel). Experiments were replicated four times, with three technical replicates per condition in each experiment. (B) Signaling downstream of TGR5 was assessed in 293T cells carrying a cAMP-driven luciferase reporter in the absence (−TGR5) or presence of TGR5 overexpression (+TGR5). TGR5 activation was evaluated for both CDCA-based (left panel) and CA-based MBSCs (right panel) at 100 µM. Experiments were repeated 3 times, with four technical replicates per condition in each replication. (C) Inhibition of NF-κB signaling was tested in RAW264.7 cells treated with the indicated test compounds for 1 hour prior to exposure to solvent (−LPS) or 3 ng/mL LPS (+LPS). Cytokine levels in the medium were normalized to cellular protein content. Experiments were replicated twice, with 4 technical replicates per condition in each test. Data are presented as the average±SD of independent replications and were statistically evaluated using a linear mixed model. Treatment effects were compared versus the control situation, applying Bonferroni correction for multiple testing. Significance is depicted as *p < 0.05, **p < 0.01 and ***p < 0.001. Abbreviations: CA, cholic acid; CDCA, chenodeoxycholic acid; G(CD)CA, glyco(chenodeoxy)cholic acid; LPS, lipopolysaccharide; MBSCs, microbial bile salt conjugates; ns, not significant; T(CD)CA, tauro(chenodeoxy)cholic acid; TLCA, taurolitocholic acid.

FIGURE 2

FXR-activating potential of microbial bile salt conjugates. (A) Ligand-induced recruitment of SRC1 coactivator peptide to the ligand-binding domain of FXR was studied using a TR-FRET–based, cell-free assay. Test compounds were evaluated at concentrations ranging from 10 nM to 100 µM, with four technical replicates per condition. (B) The affinity of D-TyrCDCA and L-TyrCDCA to activate FXRα2 was studied in 293T cells expressing ASBT and an FXR-responsive element-driven luciferase reporter. Transiently transfected cells were exposed to various doses of L-TyrCDCA for 16–18 hours, with four technical replicates per condition. (C-F) Activation of FXRα2 and FXRα4 isoforms by MBSCs was investigated using a cell-based reporter assay. Hereto, 293T cells were transiently transfected with the indicated FXRα isoform, in the absence or presence of a bile salt importer (ASBT, NTCP), and incubated overnight with 50 µM CDCA-based MBSCs (C, E) or their CA-based equivalents (D, F). All conditions were tested in quadruplicate, with 3 independent replications. A linear mixed model was used to analyze data from replicated experiments. Treatment effects were compared versus the control situation, applying Bonferroni correction for multiple testing. Significance is depicted as *p < 0.05, **p < 0.01 and ***p < 0.001. Abbreviations: ASBT, apical sodium-dependent bile acid transporter; GCDCA, glycochenodeoxycholic acid; MBSC, microbial bile salt conjugate; NTCP, Na⁺ taurocholate co-transporting polypeptide; ns, not significant; OCA, obeticholic acid; SRC1, steroid receptor coactivator 1; TR-FRET, time-resolved fluorescence resonance energy transfer.