| Objective Diabetes is a global health and economic problem.Type 2 diabetes(T2D)is the most common type of diabetes.Long-term hyperglycemia increases the risk of vascular complications in T2D patients.Studies have found that changes in nucleic acid methylation are related to diabetes,but the details remain to be further investigated.Methods Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to determine the content of N6-methyladenine(m6A),5-methylcytosine(5mC)and5-hydroxymethylcytosine(5hmC)in T2D patients and controls.qPCR was performed to detect the mRNA expression of target genes.HepG2 cells with high-glucose treatment and FTO knockout or overexpression were utilized to confirm the initial observations in patients.A highly sensitive 5hmC-Seal assay was used to profile genome-wide 5hmC in serum cell-free DNA(cfDNA).Differential 5hmC markers between T2D patients with or without vascular complications were analyzed with a case-control design.Results The m6A content was decreased(p=2.81×10-29),and mRNA expression levels of FTO,METTL3,METTL14 and WTAP were increased in T2D patients(all p<0.05).Interestingly,the m6A content was negatively associated with mRNA expression levels of METTL3,METTL14 and FTO(r=-0.237,p=0.001;r=-0.219,p=0.002;r=-0.247,p=0.019,respectively).Moreover,FTO was positively correlated with serum glucose(r=0.205,p=0.003).In HepG2 cells,high glucose upregulated FTO protein,whereas it had no significant effect on METTL3 or METTL14.Furthermore,mRNA expression levels of FOXO1,G6PC,and DGAT2were significantly increased(all p<0.05)and positively correlated with FTO(r=0.282,p=1.6×10-4;r=0.192,p=0.011;r=0.220,p=0.004,respectively)and serum glucose(r=0.160,p=0.035;r=0.279,p=1.9×10-4;r=0.204,p=0.008,respectively)in patients.Additionally,the global 5mC content was decreased(p=7.29×10-7)while 5hmC and TET2 mRNA expression were increased(p=0.038;p=0.034)in T2D patients.And the patients with macrovascular complications had lower5mC and higher 5hmC than those without macrovascular complications and controls(all p<0.05).Moreover,significant difference existed in the mean counts of cfDNA-5hmC markers(all p<0.05).These changes were associated with T2D-related vascular complications,involving genes and pathways relevant to vascular biology and diabetes,including insulin resistance and inflammation.Conclusions Our data revealed that,in T2D patients,high glucose enhanced FTO mRNA expression resulted in a decrease in m6A.The lower m6A content might be responsible for the upregulation of methyltransferases.FTO induced mRNA expression of FOXO1,G6PC,and DGAT2 and was closely associated with glucose metabolism.Moreover,TET2 promoted gDNA 5hmC increasing and 5mC decreasing in T2D patients,which also associated with macrovascular complication.The 5hmC markers in cfDNA could distinguish patients with or without vascular complications and patients with single or multiple complications,which showed better diagnostic performance than traditional clinical indicators. |
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