Gut Microbial Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice
- Cleveland Clinic Lerner College of Medicine, United States
- Cleveland Clinic, United States
- University of Louisville, United States
- The University of Texas Southwestern Medical Center, United States
- National Institute of Health, United States
- University of Massachusetts Medical School, United States
- Beth Israel Deaconess Medical Center, United States
- Procter and Gamble, United States
- Cleveland Clinic, Lerner Research Institute, United States
Abstract
There is mounting evidence that microbes resident in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. We used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. We show the gut microbial choline metabolite trimethylamine (TMA) is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. The microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.
Data availability
Sequencing data have been deposited in GEO under accession code GSE157681.
Article and author information
Author details
Srinivasan Dasarathy
Jonathan Mark Brown
Funding
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All mice were maintained in an Association for the Assessment and Accreditation of Laboratory Animal Care, International-approved animal facility. All experimental protocols were approved by the institutional animal care and use committee (IACUC) at the Cleveland Clinic.
Human subjects: Patients with AH were classified as moderate (MELD < 20, n=112) and severe (MELD {greater than or equal to}20, n=152) according to the MELD score at admission as part of either of two independent clinical trials (ClincalTrials.gov identifier # NCT01809132 and NCT03224949) or the NOAC biorepository. These studies were approved by the Institutional Review Boards of all 4 participating institutions and all study participants consented prior to collection of data and blood samples. Written informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Institutional Review Boards at Johns Hopkins Medical Institutions.
Copyright
© 2022, Helsley et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Gut Microbial Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice
eLife 11:e76554.
https://doi.org/10.7554/eLife.76554
Further reading
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- Developmental Biology
- Medicine
From a forward mutagenetic screen to discover mutations associated with obesity, we identified mutations in the Spag7 gene linked to metabolic dysfunction in mice. Here, we show that SPAG7 KO mice are born smaller and develop obesity and glucose intolerance in adulthood. This obesity does not stem from hyperphagia, but a decrease in energy expenditure. The KO animals also display reduced exercise tolerance and muscle function due to impaired mitochondrial function. Furthermore, SPAG7-deficiency in developing embryos leads to intrauterine growth restriction, brought on by placental insufficiency, likely due to abnormal development of the placental junctional zone. This insufficiency leads to loss of SPAG7-deficient fetuses in utero and reduced birth weights of those that survive. We hypothesize that a ‘thrifty phenotype’ is ingrained in SPAG7 KO animals during development that leads to adult obesity. Collectively, these results indicate that SPAG7 is essential for embryonic development and energy homeostasis later in life.
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- Medicine
Background:
The development of obesity-associated comorbidities such as type 2 diabetes (T2D) and hepatic steatosis has been linked to selected microRNAs in individual studies; however, an unbiased genome-wide approach to map T2D induced changes in the miRNAs landscape in human liver samples, and a subsequent robust identification and validation of target genes are still missing.
Methods:
Liver biopsies from age- and gender-matched obese individuals with (n=20) or without (n=20) T2D were used for microRNA microarray analysis. The candidate microRNA and target genes were validated in 85 human liver samples, and subsequently mechanistically characterized in hepatic cells as well as by dietary interventions and hepatic overexpression in mice.
Results:
Here, we present the human hepatic microRNA transcriptome of type 2 diabetes in liver biopsies and use a novel seed prediction tool to robustly identify microRNA target genes, which were then validated in a unique cohort of 85 human livers. Subsequent mouse studies identified a distinct signature of T2D-associated miRNAs, partly conserved in both species. Of those, human-murine miR-182–5 p was the most associated with whole-body glucose homeostasis and hepatic lipid metabolism. Its target gene LRP6 was consistently lower expressed in livers of obese T2D humans and mice as well as under conditions of miR-182–5 p overexpression. Weight loss in obese mice decreased hepatic miR-182–5 p and restored Lrp6 expression and other miR-182–5 p target genes. Hepatic overexpression of miR-182–5 p in mice rapidly decreased LRP6 protein levels and increased liver triglycerides and fasting insulin under obesogenic conditions after only seven days.
Conclusions:
By mapping the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, validating conserved miRNAs in diet-induced mice, and establishing a novel miRNA prediction tool, we provide a robust and unique resource that will pave the way for future studies in the field. As proof of concept, we revealed that the repression of LRP6 by miR-182–5 p, which promotes lipogenesis and impairs glucose homeostasis, provides a novel mechanistic link between T2D and non-alcoholic fatty liver disease, and demonstrate in vivo that miR-182–5 p can serve as a future drug target for the treatment of obesity-driven hepatic steatosis.
Funding:
This work was supported by research funding from the Deutsche Forschungsgemeinschaft (KI 1887/2-1, KI 1887/2-2, KI 1887/3-1 and CRC-TR296), the European Research Council (ERC, CoG Yoyo LepReSens no. 101002247; PTP), the Helmholtz Association (Initiative and Networking Fund International Helmholtz Research School for Diabetes; MB) and the German Center for Diabetes Research (DZD Next Grant 82DZD09D1G).
