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There is mounting evidence that microbes residing 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 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.

Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here, we show that gut microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (Lepob/ob). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake but is instead associated with alterations in the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that gut microbial CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have potential as anti-obesity therapeutics.

Lipiddroplet-associated proteins play an important role in adipocyte triglyceride (TG) metabolism. Here, weshow that trans-10,cis-12 conjugated linoleic acid (CLA), but notcis-9,trans-11 CLA, increased lipolysis and altered human adipocyte lipid dropletmorphology. Before this change in morphology, there was a rapid trans-10,cis-12CLA-induced increase in the accumulation of perilipin A in the cytosol, followed by the disappearance ofperilipin A protein. In contrast, protein levels of adipose differentiation-related protein (ADRP) wereincreased in cultures treated with trans-10,cis-12 CLA. Immunostaining revealed that ADRP localized to the surface of small lipid droplets, displacing perilipin. Intriguingly,trans-10,cis-12 CLA increased ADRP protein expression to a much greater extent thanADRP mRNA without affecting stability, suggesting translational control of ADRP. To this end, we foundthat trans-10,cis-12 CLA increased activation of the mammalian target ofrapamycin/p70 S6 ribosomal protein kinase/S6 ribosomal protein (mTOR/p70S6K/S6)pathway.Collectively, these data demonstrate that the trans-10,cis-12CLA-mediated reduction of human adipocyte TG content is associated with the differential localization andexpression of lipid droplet-associated proteins. This process involves both the translational control ofADRP through the activation of mTOR/p70S6K/S6 signaling and transcriptional control of perilipinA.