Proatherogenic gut-microbiome metabolite trimethylamine elicits a gut-liver circuit to regulate TMAO metabolism and atherosclerosis via mediation of CREBH

Abstract Introduction In humans, circulating metabolite Trimethylamine N-oxide (TMAO) is closely associated with higher risk of cardiovascular disease. Trimethylamine (TMA), a precursor of TMAO, is produced by gut microbiome using dietary components, i.e., choline and carnitine, as substrates. The gut-derived TMA is then transferred to the liver where it is further oxidized to TMAO by the flavin-containing monooxygenases (FMOs). The ER-resident transcription factor c-AMP responsive element binding protein H (CREBH/CREB3L3) is exclusively expressed in the liver and intestine. Perturbation of CREBH activity contributes to the development of hyperlipidemia and cardiovascular disease. Therefore, the Purpose of this study is to investigate the regulatory effect of a gut bacterium, Akkermansia muciniphila (A. muciniphila), on TMA and TMAO metabolism and the role of CREBH in this process. Methods Two groups of wild type (WT) and CREBH knockout (CREBH-KO) mice were inoculated with 200 μL of A. muciniphila (2×108 cfu/0.2 mL) in PBS or the vehicle (PBS) alone as control every other day through oral gavage for 2 weeks. Plasmas, liver and intestinal tissues were collected for metabolomics analysis, immunoblotting analysis and q-RT-PCR. Results Metabolomics analysis of the plasmas from the experimental mice revealed that increased colonization of A. muciniphila in the gut significantly reduced circulating TMA in the WT mice but not in CREBH-KO mice (P<0.05), suggesting that depletion of CREBH altered the microenvironment of gut microbiome which affected the metabolism of TMA by gut bacteria. In the livers, A. muciniphila treatment markedly reduced mRNA expression of FMO1 and FMO3 (P<0.05), which subsequently inhibited the enzymatic conversion of TMA to TMAO in hepatocytes. Immunoblotting analysis further revealed that LDL receptor was upregulated whereas ER stress markers, GRP94 and JNK1/2, were downregulated in the A. muciniphila treated KO mice, indicating an acceleration in lipoprotein (VLDL remnant) clearance from the circulation and the improvement of metabolic inflammation. In vitro, incubation of mouse hepatocytes AML12 with TMA (600 mM) for 12 hours stimulated expression of FMOs to facilitate the conversion of TMA to TMAO and induced lipotoxicity. Conclusion CREBH mediates the crosstalk between gut microbiome and liver metabolic system that regulates TMA and TMAO metabolism, which contributes to the induction of metabolic inflammation and atherogenesis. This novel finding may lend support to the therapeutic strategy of atherosclerosis. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation