NAFLD is defined as fat accumulation in the liver without excessive alcohol consumption [1], Over the past four decades, NAFLD has become the most common chronic liver disorder with an estimated global prevalence of 24% in 2016 [2]. With its adverse outcomes related to obesity, insulin resistance, and dyslipidemia, NAFLD consists of different subtypes, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) which together place a huge burden on public health and the health system. Given these conditions, growing interest has been sparked in understanding the molecular mechanisms leading to NAFLD to develop therapeutic approaches for the treatment of NAFLD. Nowadays, despite numerous exciting and significant discoveries having been made, very few pharmacological treatments have yet been approved for NAFLD treatment [1].

Autophagy is an evolutionarily conserved pathway in response to metabolic stress by digesting cytosolic macromolecules and damaged organelle and recycling materials through a lysosome-mediated system [3]. Recently research has demonstrated that dysregulation of hepatic autophagy has been found to contribute to NAFLD, while therapeutic targeting of hepatic autophagy may serve as a feasible way to treat NAFLD [[4], [5], [6]]. As a tightly regulated process, autophagy can be divided into six individual steps: initiation, nucleation, membrane expansion, closure, fusion, and cargo degradation, each involving spatiotemporal coordinated recruitment of specialized autophagy-related (ATG) proteins [4,7,8]. During membrane expansion, ATG12 is required for LC3 lipidation and subsequently autophagosome formation [8]. Notably, ATG12-deficiency-induced dysfunction of autophagy has been shown to promote lipid deposition in hepatocytes [9].

Folic acid, a water-soluble B9 vitamin that cannot be synthesized by mammals and must be obtained via dietary sources [10,11], is essential for cellular methylation reactions that regulate epigenetic processes. Several recent studies have indicated that serum folic acid levels are inversely associated with the risk of NAFLD [[12], [13], [14], [15], [16]]. While patients with severe NAFLD exhibited significantly decreased circulating folic acid levels compared with individuals with normal liver morphology [12]. Studies have suggested that dietary folate deficiency may also contribute to the development of hepatic steatosis in rodents [14,17]. Thus, dietary supplementation with folic acid has been thought of as a potential tactic to reduce and/or prevent NAFLD rates [13]. Dietary folic acid supply during high-fat diet feeding has been successful in preventing hepatic steatosis in mice [[18], [19], [20], [21]]. Specifically speaking, folic acid supplementation could attenuate high-fat diet-induced steatohepatitis via deacetylase SIRT1-dependent restoration of PPARα [19]. Activation of AMPK by folic acid supplementation might be responsible for the improvement of cholesterol and glucose metabolism by restoration of AMPK activation, leading to reduced hepatic cholesterol synthesis during high-fat diet feeding [18]. Collectively, all these studies have highlighted the importance of folic acid in regulating liver fat metabolism. However, the specific mechanisms underlying the protective role of folic acid on NAFLD remain largely unclear.

As the most prevalent modification of mRNA in eukaryotes, m6A is catalyzed by a large heteromultimeric methyltransferase complex and can be reversibly removed by demethylases fat mass and obesity-associated protein (FTO) and alkB homologue 5 (ALKBH5) [22,23]. Recent studies indicated that m6A methylation plays a key role in various physiologic processes and diseases [22,23]. Notably, m6A has been proposed to regulate hepatic fat metabolism through multiple pathways [22]. Folic acid has been reported to alter the abundance of m6A in the hippocampus in mice, which raises the question whether the anti‐NAFLD effect of folic acid is related to RNA methylation [24].

In this study, we revealed that folic acid alleviated HFD-induced NAFLD and metabolic disorders in mice. Further analysis confirmed that folic acid increased DNA methylation levels in the promoter of ALKBH5, leading to decreased expression of ALKBH5. The inhibition of ALKBH5 caused by folic acid supplementation subsequently enhanced the expression of ATG12 in a demethylase activity-dependent manner. Down-regulation of ATG12 induced by ALKBH5 overexpression could impair autophagy and the inhibitory effect of folic acid on lipid accumulation in hepatocytes. Together, dietary folic acid supplementation may represent a therapeutic candidate for the treatment of NAFLD.