The changing metabolic landscape of bile acids - keys to metabolism and immune regulation

Abstract

Bile acids regulate nutrient absorption and mitochondrial function, they establish and maintain gut microbial community composition and mediate inflammation, and they serve as signalling molecules that regulate appetite and energy homeostasis. The observation that there are hundreds of bile acids, especially many amidated bile acids, necessitates a revision of many of the classical descriptions of bile acids and bile acid enzyme functions. For example, bile salt hydrolases also have transferase activity. There are now hundreds of known modifications to bile acids and thousands of bile acid-associated genes, especially when including the microbiome, distributed throughout the human body (for example, there are >2,400 bile salt hydrolases alone). The fact that so much of our genetic and small-molecule repertoire, in both amount and diversity, is dedicated to bile acid function highlights the centrality of bile acids as key regulators of metabolism and immune homeostasis, which is, in large part, communicated via the gut microbiome.

Figure 1.. Diversity of known bile acid chemical modifications.

The outer circle (blue) reports the producer organism for these modifications if known. The inner circle (pink) shows where these bile acid modifications have been observed: humans, animals, or microorganisms. Substructures (red) represent the modification introduced by the specified reaction. Modifications of the bile acid core: Hydroxylation^,–; Dehydroxylation^,–; Epimerization^,–; Oxidation^,– and reduction; Dehydrogenation; Dehydration^– and reduction . Modifications of the carboxyl: Thioesterification; Taurine amidation; Glycine amidation; Other amino acid Amidation^,,–; Polypeptides/proteins amidation; Amine/polyamine amidation^,,; Carboxylate reduction^,; Esterification/addition of sugar; Oxidative amidation. Modifications of the hydroxyl: Sulfation/desulfation^,; Esterification; Methylation; Ether formation or addition of sugar,,; Acetylation; Acylation^,,; (Poly)amine substitution. Ring-opening and bio-transformations: Ring-opening; Pseudomonas sp; Rhodococcus ruber; Streptomyces rubescens.

Figure 2.. Modifications of known bile acids.

a) Bile acid overlap in the structural and mass spectrometry databases for GNPS/MassIVE BILELIB19, Human Metabolome Database (HMDB) (only considering detected bile acids), and LipidMAPS was derived from deposited SMILES codes (links to databases are provided in the code availability section). For the purpose of this figure to visualize the data in the three repositories, all SMILES with the (SMARTS string-defined) substructure “C-,=C12-,=C-,=C-,=C-,=C-,=C-,=1-,=C-,=C-,=C3-,=C4-,=C-,=C-,=C-,=C-,=4(-,=C-,=C-,=C-,=3-,=2)-,=C” and a carboxylic acid ester (“C(=O)O”), (thio)ester (“C(=O)S”), or amide (“C(=O)N”) in their caboxy-tail were considered as bile acids. Substructure searches were conducted via RDKit (http://www.rdkit.org). Diastereomeric information was not considered for the comparisons across bile acids. b) Mass shifts were calculated by subtracting the mass of the unconjugated bile acid (e.g., C₂₄H_40+xO_2+x with x = ‘number of hydroxyl modifications’ on the respective bile acid for C24 bile acids; formula was adjusted for C22-C28 bile acids (as defined in LipidMAPS), respectively) from the mass of the (potentially) conjugated bile acid. Only mass differences > 40 m/z are shown.

Figure 3.. Distribution of bile acids and related transcripts or proteins.

Distribution of observed bile acids as documented with the mass spectrometry reanalysis of data user interface (ReDU) in a) humans and b) mice. Briefly, sample information and metadata available in the public repositories were filtered for ‘homo sapiens’ and ‘mus/rattus’. A search list adapted specifically for bile acids was used to subset a list of files for which bile acids (only those that are curated in the Global Natural Product Social molecular networking (GNPS) spectral library) were detected. Total count of such files across different body regions as defined by the UBERONBodyPartName ontology in ReDU was calculated, grouped based on modification of bile acid (unconjugated, glycine-conjugated, taurine-conjugated, other amino acid-conjugated, methylated and oxidized to form ketones) and described for humans and mice. For transcript locations, all matches discussed are to “bile” in the c) Human Protein Atlas and d) mouse gene expression databases. Numbers in parts c and d represent the number of bile acid-associated genes or gene products observed in respective body locations. Overlaid is the information about the bile acid receptor proteins, FXR, SXR (humans), PXR (mouse), and TGR5 and the N-acyltransferase enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT). All panels are made using Biorender.com. Data and codes used for the reanalysis of public data to create this figure are made available in the Data availability section. It should be noted that inferences of differential distribution should not be inferred between organisms from this figure but rather as a documentation of where transcripts, proteins, and bile acids have been detected. It is also an incomplete picture as many organs or biofluids have not been studied by transcriptomics, proteomics, or metabolomics, let alone under all conditions that might alter the detection, but it gives an overview of just how widely the bile acid metabolic and gene network is distributed throughout the body. BSH, bile salt hydrolase.

Figure 4.. Proportion of bile acids in humans.

Data available in ReDU represented as normalized ion intensities across three biological matrices - a) urine b) blood, c) fecal. Each stacked bar represents a unique individual. Colors represent the seven bile acid groups - unconjugated, glycine conjugated, taurine conjugated, amino acid conjugated, oxidized, sulfated and tetrahydroxylated. The information that is visualized is from untargeted MS/MS datasets acquired on a high-resolution mass spectrometer, a Q-Exactive, and with associated metadata filtered from ReDU. Reprocessing these files by living data in Global Natural Product Social molecular networking (GNPS) yielded spectral matches to bile acids in the GNPS libraries. The precursor ion intensities of these bile acids were extracted from the classical molecular network and represented as sum normalized ion intensities of all female and male samples across three biological matrices.

Figure 5.. The known deconjugation and re-conjugation of bile acids by BAAT and BSH enzymes.

a) Representative reactions carried out by BSH and BAAT. Although other amidates, aside from taurine, can serve as substrates for the transamination reaction carried out by bile salt hydrolase (BSH), the taurine conjugates are the key molecules converted to the microbial bile acid amidates. Different BSH enzymes have different specificities of amine and bile acid for the transamination reaction. b) Representative structures of amino acid conjugated to cholic acid catalyzed by bacterial BSH belonging to the genera Lactobacillus, Enterocluster, Bifidobacteria and Bacteroides. BAAT, bile acid-CoA:amino acid N-acyltransferase.

Figure 6.. Bile acids and receptor interactions.

Interactions of known microbially modified bile acids (conjugated and unconjugated) with common host-ligand receptors and the phenotypic response, when characterized, are highlighted. The unconjugated bile acids isoallolithocholic acid (isoallo-LCA), 3-oxo cholic acid (3-oxo-CA), isodeoxycholic acid (isoDCA), deoxycholic acid (DCA), and lithocholic acid (LCA) bind to the nuclear receptors VDR, FXR and TGR5 and induce cytokines production and increases differentiation of regulatory T (T_reg) cells, reducing inflammation^,,,. DCA and LCA activate FXR, which downregulates interleukin-1 (IL-1), leading to vasodilation in endothelial cells. Cholic acid (CA) binds to FXR and increases production of IL-33, activating eosinophilia in stromal cell.Taurine-conjugated ursodeoxycholic acid (Tau-UDCA) is an agonist of the membrane receptor TGR5, leading to an increase in intracellular cAMP levels in microglia, which induces anti-inflammatory markers providing neuroprotection in conditions such as Alzheimer disease, Parkinson disease and amyotrophic lateral sclerosis^,. Glycine-conjugated deoxycholic acid (Gly-DCA) induces production of IL-22 on binding to TGR5, causing recovery of ovarian function in women with PCOS. In the case of microbially conjugated bile acids, the studies highlighting ligand interactions are limited given the novelty of such discoveries, but highlighted in this figure are some important observations from recent studies. Although only tested in mice ileum so far, tyrosine (Tyr), phenylalanine (Phe) and isoleucine (Ile) conjugated to cholic acid have binding affinity to FXR and regulate bile acid biosynthesis genes. Elevated amounts of these amino acid conjugates furnished by treating mice with a cocktail of BSH enzymes also led to inhibition of C. difficile spore germination and prevented its colonization. The mechanism for the inhibition of spore germination and the bile acid receptors involved are still unknown. Glutamate (Glu) conjugated to cholic and chenodeoxycholic acid are agonists of multiple nuclear receptors in vitro in human cells, however the phenotypic response has not yet been explored. The amino acids alanine (Ala), serine (Ser), tryptophan (Trp), Tyr, Phe, and Ile bind to FXR and TGR5, increasing WNT signaling and stem cell differentiation

Figure 7.. The bile acid encoder-decoder hypothesis.

Visualization of the encoder-decoder hypothesis. In this hypothesis, cholesterol is modified by host and microbial enzymes to encode the message that is then decoded by the receptors and transporters to enable communications at the organelle, cell (microbial or host cells) and tissue/organ levels. Figure 7 was created with BioRender.com.