The Role And Mechanism Of RNA M~6A Methylation Modification In Insulin Signal Transduction In Adipocytes Induced By High-fat Diet

Objective To explore the role and mechanism of RNA m6A methylation modifications in insulin sensitivity of adipocytes,analyze the changes in m6A modification of insulin signal transduction gene induced by a high-fat diet,and uncover the key methyltransferases involved in insulin resistance(IR).Methods ①Reading and statistical literature reviews to find the scientific point of the research topic.Then,collect clinical intraoperative superfluous subcutaneous adipose tissue from patients with type Ⅱ diabetes mellitus(T2DM)and non-T2DM patients who need surgery due to benign lesions,and conduct dot-blot experimental to verify the overall level change of total RNA m6A modifications in adipose tissue of T2DM patients.②Male C57BL/6J mice(68 weeks)were used as experimental materials to prepare the insulin resistance(IR)model by feeding them with a basal diet and high-fat diet for 12-16 weeks.After validation by the intraperitoneal injection glucose tolerance test(IPGTT)and intraperitoneal injection insulin tolerance test(IPITT),gonadal adipose tissue was collected for epigenomic m6A methylation modification microarray assay.Further enrichment analysis around the insulin signaling pathway was conducted.The main differential genes were screened and validated by MeRIPPCR,and changes in methyltransferase transcripts were detected using RT-PCR.③3T3-L1 preadipocytes were induced in vitro to differentiate into mature adipocytes.Then mature adipocytes were collected for the RIP experiment to detect whether the main differential genes screened by the chip were directly combined with METTL3 protein.④METTL3 small molecule inhibitor STM2457(50 mg/kg)was applied to high-fat diet induced IR mice,daily for 14 consecutive days.Observe the effect of STM2457 on the change of m6A methylation in hyperlipidemic mice,and conduct IPGTT and IPITT experiments again to observe the difference of indicators between groups.The GLUT4 immunofluorescence staining of adipose tissue was further confirmed for the change in membrane expression,to verify the IR status.The expressions of differential gene transcripts in adipose tissue under STM2457 were checked.Results ①Elevated m6A methylation modifications in superfluous adipose tissue were found in T2DM patients,compared with controls;②There were 1175 genes hyper-methylated and 55 genes hypo-methylated in adipose tissue with IR,meanwhile,182 genes showed hypermethylated and low expressed significantly;Four genes,namely Akt2,Insr,Acaca and Srebf1,in the insulin signaling pathway were significantly enriched for hyper-methylated and low expressed.MeRIP-qPCR experiment confirmed the high m6A methylation modification of the above four genes,and the RT-qPCR experiment confirmed that the transcription level of methyltransferase writers(Mettl3,Mettl14,Wtap)in the adipose tissue around the genitals of mice fed with high-fat diet was increased;③RIP experiments confirmed that the RNAs of Akt2,Insr,Acaca and Srebf1,all bind to METTL3 in 3T3-L1 adipocytes;④Fluorescent staining indicated that inhibition of METTL3 activity in vivo by intraperitoneal injection of STM2457 antagonized the inhibition of GLUT4 membrane translocation induced by high-fat diet;METTL3 was inhibited and significantly decreased body weight,with improved insulin resistance status,evidenced by the remission of both IPITT and IPGTT experiments;And the transcriptional levels of Akt2,Insr,Acaca and Srebf1,were all elevated under STM2457 application.Conclusion High-fat diet induces the hypermethylation of m6A modification in Akt2,Insr,Acaca and Srebf1 genes in adipocytes,which is regulated by the methyltransferase METTL3.The m6A hypermethylation downregulates expression levels of Akt2,Insr,Acaca and Srebf1 genes,which in turn inhibits insulin signaling transduction and induces IR.It is suggested that METTL3 is expected to be a new target,and STM2457 to be a new drug for T2DM prevention and clinical treatment.