Profiling of RNA N6-Methyladenosine methylation reveals the critical role of m6A in betaine alleviating hepatic steatosis

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major predisposing factor of metabolic dysfunction-associated steatohepatitis, hepatic fibrosis and hepatocellular carcinoma. Abnormal RNA m6A modification leads to the disturbance of lipid metabolism, insulin resistance, and chronic inflammation, all of which are critical for the onset and progression of MASLD. It has been shown that betaine supplementation has an ameliorative effect on symptoms associated with MASLD. The purpose of this study is to explore the role of RNA m6A modification in the alleviation of MASLD by betaine, and to find out the possible targets of betaine. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were respectively employed to contrast m6A methylation profiles of livers from high-fat diet mice with betaine supplementation vs. those without betaine supplementation (HB vs. HFD). Functional enrichment analysis showed that up-methylated genes are mainly related to mTOR signaling pathway, Rap1 signaling pathway and MAPK signaling pathway. In addition, differentially expressed mRNAs were enriched in pathways such as promoting lipid catabolism and reducing lipid accumulation. By combining analyses of MeRIP-seq and RNA-seq, we identified 27 genes exhibiting significant differences in m6A abundance and mRNA levels. Notably, a new candidate gene, Trub2, was screened out and identified. Inhibition of Trub2 increased intracellular TG levels in AML12 cells, and this promotion was reduced by betaine, suggesting that betaine reduces intracellular TG levels by increasing Trub2 expression. Our findings in this study provide a new target and a new approach for the treatment of MASLD.