Abstract

Background Alcohol overuse disrupts liver function and alters microbial gut communities, with alcohol-related liver disease (ALD) accounting for half of all liver-related deaths worldwide. Bile acids (BAs) regulate liver and gut function, but their metabolism becomes disrupted in ALD. While it is known that gut microbes transform primary to secondary BAs, which are subsequently reabsorbed via the enterohepatic circulation, BA metabolism during ALD progression remains poorly understood.

Methods We investigated BA co-metabolism in a cross-sectional cohort of individuals with ALD (n=462) and healthy controls (n=148). We validated key findings in two independent ALD cohorts (n=34 and n=52). We integrated BA concentrations, measured by targeted mass spectrometry in feces and plasma, with liver proteomics, and gut microbiome profiles derived from metagenomic and metatranscriptomic sequencing of fecal samples.

Results Advanced fibrosis was associated with decreased hepatic BA synthesis and impaired BA transport. Despite this, disease progression corresponded with increased levels of primary and secondary BAs in plasma and feces. The abundance of microbial secondary BA dehydroxylation and epimerization pathways in the gut microbiome changed with disease severity. Genes encoding early steps in the multi-step dehydroxylation pathway increased, whereas those involved in later steps were depleted, indicating a community-level microbial imbalance. In ALD, we identified Eggerthella lenta as a key mediator of BA dehydroxylation, while Mediterraneibacter torques and Bacteroides thetaiotaomicron facilitated most of the BA epimerization as a detoxification mechanism.

Conclusion Fibrotic ALD is characterized by disrupted primary BA synthesis and transport, leading to BA accumulation in the gut and blood circulation. Altered microbial secondary BA metabolism reflects a compensatory mechanism that becomes impaired at advanced fibrosis stages. Our findings highlight the gut-liver axis as an important factor influencing ALD progression.

Impact and Implications

With the progression of alcohol-related liver disease (ALD), levels of bile acids (BA) in serum and feces increase, but BA production and transport are impaired in the liver.

Secondary microbial BA metabolism, particularly epimerization and dehydroxylation, increases in ALD. However, a key enzyme, baiN, is depleted, illustrating a microbial community-level metabolic dysbiosis.

The main contributing microbial species were, among others, Mediterraneibacter torques, Bacteroides thetaiotaomicron, and Eggerthella lenta, which could serve as potential targets for future microbial-targeted interventions.

Lay summary Alcohol-related liver disease (ALD) from long-term alcohol overuse affects how the liver and gut interact, especially in handling bile acids (BAs), which are molecules produced by the liver and transformed by gut bacteria. Our study found that in people with ALD, the liver produces fewer BAs, but BAs accumulate in the gut and blood because their transport is impaired. We also observed that bacterial transformations of these BA change as the disease progresses, most likely due to an imbalance in the gut microbiome. For the first time, we identify specific bacterial species that strongly influence BA levels in ALD.

Figure 0: Graphical AbstractCompeting Interest Statement

AK has served as a speaker for Novo Nordisk, Norgine and participated in advisory boards for Boehringer Ingelheim, GSK, and Novo Nordisk, all outside the submitted work. Research support: Astra, Siemens, Nordic Bioscience, GSK, Echosense. He is a board member and co-founder Evido. MT received speaker's fee from Echosens, Madrigal, Takeda, and Novo Nordisk, and advisory fee from Boehringer Ingelheim, Astra Zeneca, Novo Nordisk, and GSK, and a research grant from GSK. She is a co-founder of Evido. The interests are all outside of the submitted work. All other authors declare no competing interests.

Funding Statement

This work was supported by funding from the European Union's Horizon 2020 research and innovation program under grant agreement number 668031 (GALAXY). This reflects only the author's view, and the European Commission is not responsible for any use that may be made of the information it contains. The study was also supported by the Novo Nordisk Foundation through a Challenge Grant MicrobLiver (grant number NNF15OC0016692). The work was supported by the EMBL International PhD Programme (M.I.K.) and EMBL IT Services HPC resources.

Author Declarations

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Danish Data Protection Agency (13/8204, 16/3492) and the ethics committee for the Region of Southern Denmark (ethical ID S-20120071, S-20160021, S-20170087 and ID S-20160006G) approved these cohorts. The study followed the ethical principles of the Declaration of Helsinki in all methods involving participants.

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AbbreviationsALDAlcohol-related liver diseaseBALBile acid CoA ligasebaiBile acid-inducedBMIBody mass indexBSEPBile salt export pumpbshBile salt hydrolaseCACholic AcidCDCAChenodeoxycholic acidDCADeoxycholic acidGCAGlycocholic acidGTDBGenome taxonomy databaseGUDCAGlycoursodeoxycholic acidFXRFarnesoid X receptorHCHealthy controlHdhA7-alpha-hydroxysteroid dehydrogenaseKEGGKyoto encyclopedia of genes and genomesLCALithocholic acidMAGMetagenome-assembled genomeMELDModel of end-stage liver diseaseMetaGMetagenomics MetaT MetatranscriptomicsmOTUMetagenomic operational taxonomic unitsNTCPNA+ dependent taurocholate cotransporting polypeptide 28TCATaurocholic acidTCDCATaurochenodeoxycholic acidTUDCATauroursodeoxycholic acidUHPLC-MS/MSUltra-high performance liquid chromatography-tandem mass spectrometry