Butyrate alleviates high fat diet-induced obesity through activation of adiponectin-mediated pathway and stimulation of mitochondrial function in the skeletal muscle of mice

Chronic stress or excessive exposure to glucocorticoids (GC) contributes to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Glucocorticoid receptor (GR) mediates the action of GC, but its downstream signaling is not fully understood. Fat mass and obesity associated (FTO) protein and its demethylation substrate N6-methyladenosine (m⁶A) are both reported to participate in the regulation of lipid metabolism, yet it remains unknown whether they are involved in GC-induced hepatic lipid accumulation as new components of GR signaling. In this study, we use both in vivo and in vitro models of GC-induced hepatic lipid accumulation and demonstrate that the activation of lipogenic genes and accumulation of lipid in liver cells are mediated by GR-dependent FTO transactivation and m⁶A demethylation on mRNA of lipogenic genes. Targeted mutation of m⁶A methylation sites and FTO knockdown further validated the role of m⁶A on 3ʹUTR of sterol regulatory element-binding transcription factor 1 and stearoyl-CoA desaturase mRNAs. Finally, FTO knockdown significantly alleviated dexamethasone-induced fatty liver in mice. These results demonstrate a role of GR-mediated FTO transactivation and m⁶A demethylation in the pathogenesis of NAFLD and provide new insight into GR signaling in the regulation of fat metablism in the liver.

Transforming growth factor-beta1 (TGF-β1) secreted from activated Kupffer cells (KC) promotes the progression of nonalcoholic steatohepatitis (NASH) to liver fibrosis. N6-methyladenosine (m6A) RNA modification participates in various cell stress responses, yet it remains unknown whether it plays a role in TGF-β1 up-regulation in activated KC. Western blot, Dot blot and LC-MS/MS were used to determine the expression of m6A methyltransferase, METTL3 and METTL14, as well as global m6A modification. RNA-seq and m6A-seq were employed to screen differentially expressed genes and responsive m6A peaks. NF-κB-mediated METTL3/METTL14 transactivation were validated with ChIP-PCR and dual-luciferase reporter system, and the role of m6A in TGF-β1 up-regulation was further verified in METTL3/METTL14-deficient KC and myeloid lineage cell-specific METTL14 knockout mice. Serum lipopolysaccharide (LPS) concentration is increased in high-fat diet induced NASH rats. TGF-β1 up-regulation is closely associated with METTL3/METTL14 up-regulation and global m6A hypermethylation, in both NASH rat liver and LPS-activated KC. LPS-responsive m6A peaks are identified on 5'UTR of TGF-β1 mRNA. NF-κB directly transactivate METTL3 and METTL14 genes. LPS-stimulated TGF-β1 expression is abolished in METTL3/METTL14-deficient KC and myeloid lineage cell-specific METTL14 knockout mice. Mutation of m6A sites on 5'UTR of TGF-β1 mRNA blocks LPS-induced increase of luciferase reporter activity. NF-κB acts as transcription factor to transactivate METTL3/METTL14 genes upon LPS challenge, leading to global RNA m6A hypermethylation. Increased m6A on 5'UTR of TGF-β1 mRNA results in m6A-dependent translation of TGF-β1 mRNA in a cap-independent manner. We identify a novel role of m6A modification in TGF-β1 up-regulation, which helps to shed light on the molecular mechanism of NASH progression.