Quercetin ameliorated insulin resistance via regulating METTL3-mediated N6-methyladenosine modification of PRKD2 mRNA in skeletal muscle and C2C12 myocyte cell line

Abstract Background: N6-Methyladenosine (m6A) is involved in many of pathological processes including insulin resistance (IR). Quercetin (Que), a bioactive compound with strong antioxidant activity, has potential therapeutic effects on IR-related metabolic diseases. The aim of this study is to investigate the roles of m6A and Que in hyperinsulinemia. Methods: Male C57Bl/6 mice received a high fat diet (HFD) for 8 weeks to establish an IR model. Biochemical analyzes, oral glucose tolerance test and homeostasis model assessment of IR (HOMA-IR) were conducted. C2C12 cells were transfected with methyltransferase-like 3 (METTL3) siRNA before treatment with palmitate (PA). 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG) was used to detect glucose uptake. Malondialdehyde (MDA) level, superoxide dismutase (SOD) activity, and glutathione (GSH) level were used to evaluate the levels of oxidative stress. SRAMP, a predicted site of m6A modification, was used to predict the relationship between METTL3 and erine-threonine kinase protein kinase D2 (PRKD2). Then, cells were treated with Que or/and transfected with pcDNA-METTL3 or PRKD2 siRNA. indirect calorimetry, Western blotting and qPCR were used to analyze protein and mRNA expression. Results: Que treatment reduced the body weight, blood glucose, plasma TG and serum insulin, ameliorated IR, and decreased oxidative stress in HFD-fed mice. Que at noncytotoxic doses promoted PA-induced glucose uptake and inhibited oxidative stress in C2C12 cells. Moreover, METTL3 and PRKD2 was downregulated in PA-induced C2C12 cells, and SRAMP revealed multiple m6A modification sites in the PRKD2 mRNA sequence. Downregulation of METTL3 enhanced PRKD2 expression by reducing m6A level and mRNA stability in PRKD2 mRNA transcript. C2C12 cells treatment with Que after inducing with PA inhibited m6A, METTL3 and phosphorylated insulin receptor substrate 1 (p-IRS1) levels, increased the protein expression of PRKD2, glucose transporter type 4 (GLUT4) and p-AKT and glucose uptake, and reduced oxidative stress. However, METTL3 overexpression or PRKD2 silence reversed the inhibitory effects of Que on the levels of MDA, GLUT4 and p-AKT, and the promotive effects on glucose uptake, SOD, GSH and p-IRS1 levels. Conclusion: Que promoted glucose uptake, repressed oxidative stress and improved IR through METTL3-mediated N6-methylation of PRKD2 mRNA.