METTL14‐Induced M ⁶ A Methylation Increases G6pc Biosynthesis, Hepatic Glucose Production and Metabolic Disorders in Obesity

METTL14 dimerizes with METTL3 to install N6-methyladenosine (m⁶A) on mRNA (m⁶A writers). Subsequently, m⁶A readers bind to m⁶A-marked RNA to influence its metabolism. RNA m⁶A emerges to critically regulate multiple intracellular processes; however, there is a gap in our understanding of m⁶A in liver metabolism. Glucose-6-phosphatase catalytic subunit (G6pc) mediates hepatic glucose production (HGP) and serves as the gatekeeper for glycogenolysis and gluconeogenesis; however, G6pc regulation is not fully understood. Here, METTL14 is identified as a posttranscriptional regulator of G6pc. Liver METTL14, METTL3, and m⁶A-methylated G6pc mRNA are upregulated in mice with diet-induced obesity. Deletion of Mettl14 decreases, whereas overexpression of METTL14 increases, G6pc mRNA m⁶A in hepatocytes in vitro and in vivo. Five m⁶A sites are identified, and disruption of them (G6pc^Δ ^5A) blocks METTL14-induced m⁶A methylation of G6pc^Δ ^5A mRNA. METTL14 increases both stability and translation of G6pc but not G6pc^Δ ^5A mRNA. YTHDF1 and YTHDF3 but not YTHDF2 (m⁶A readers) bind to m⁶A-marked G6pc mRNA to increase its synthesis. Deletion of hepatic Mettl14 decreases gluconeogenesis in primary hepatocytes, liver slices, and mice. Hepatocyte-specific restoration of G6pc reverses defective HGP in Mettl14 knockout mice. These results unveil a METTL14/G6pc mRNA m⁶A/G6pc biosynthesis/HGP axis governing glucose metabolism in health and metabolic disease.