Metformin alleviates choline diet-induced TMAO elevation in C57BL/6J mice by influencing gut-microbiota composition and functionality

Abstract

Trimethylamine-N-oxide (TMAO), a gut-microbiota-dependent metabolite generated from its dietary precursors such as choline, has been identified as an independent risk factor for atherosclerosis. Metformin is the most widely used drug for the treatment of type 2 diabetes (T2D), which has therapeutic effects on hyperglycemia accelerated atherosclerosis. A growing body of evidence suggest that metformin plays a therapeutic role by regulating the structure and metabolic function of gut microbiota. However, whether metformin has an impact on gut-microbiota-mediated TMAO production from choline remains obscure. In this study, the oral administration of metformin significantly reduced choline diet-increased serum TMAO in choline diet-fed C57BL/6J mice. The diversity analysis based on 16S rRNA gene sequencing of C57BL/6J mice fecal samples indicated that metformin markedly changed the gut-microbiota composition. Metformin was positively correlated with the enrichment of different intestinal bacteria such as Bifidobacterium and Akkermansia and a lower cutC (a choline utilization gene) abundance. Furthermore, the ex vivo and in vitro inhibitory effects of metformin on choline metabolism of TMA-producing bacteria were confirmed under anaerobic condition. The results suggested that metformin suppresses serum TMAO level by remodeling gut microbiota involved in TMA generation from choline.

Fig. 1. The effects of metformin on serum TMA and TMAO levels in C57BL/6J mice under chow or choline diet.

a, b Female C57BL/6J mice were fed chow with or without 100 mg/kg metformin (Met-100) or 200 mg/kg metformin (Met-200) for 16 days, then fed 1% choline diet with or without metformin (veh (c), Met-100 (c), Met-200 (c)) for another 9 days. Mice administration as illustrated in schematic diagram (a). TMA and TMAO levels were detected at indicated time points by HPLC-MS/MS (b). veh/veh (c), n = 5; chow, n = 5; Met-100/Met-100 (c), n = 8; Met-200/Met-200 (c), n = 8. c 2% choline fed C57BL/6J mice were applied to evaluate the effects of metformin on TMA and TMAO levels. C57BL/6J mice were initially fed on chow diet with or without 200 mg/kg metformin for 6 weeks and sequentially converted to 2% choline diet for another 3 weeks. Serum TMA and TMAO levels were determined. n = 5 for each group. Values are presented as means ± SEM (*p < 0.05, ***p < 0.001).

Fig. 2. Metformin treatment altered composition of gut microbiota and inhibited TMA production from choline.

a Effects of metformin on enterotype of C57BL/6J mice fed with chow or choline diet. Samples were calculated using Jensen-Shannon distance and separated into two clusters by the PAM method. The left panel showed the clustering of two top principal components. b Linear regression analysis of the correlation between serum TMAO level and microbiota beta diversity based on OTU levels with bray curtis algorithm. c The abundance of significant different genera among four groups. Metformin, 200 mg/kg. Boxes show the median with interquartile ranges, whiskers show the minimum and maximum values. p-values are from Kruskal–Wallis H test (*p < 0.05, **p < 0.01, ***p < 0.001). d The abundances of cutC gene in fecal samples were measured by qPCR. veh/veh (c), n = 5; chow, n = 5; Met-100/Met-100 (c), n = 8; Met-200/Met-200 (c), n = 8. The error bars represented the mean ± SEM (*p < 0.05, ***p < 0.001). e Effects of metformin on TMA-producing bacteria under anaerobic condition. The production of d9-TMA from d9-choline (bar) and OD₆₀₀ (line) were measured after metformin treatment for 24 h.