| Summary: | Vascular insulin resistance manifests in decreased production of nitric oxide subsequently leading to vasoconstriction and atherosclerosis. Inflammatory molecules such as IL-6 are known to induce insulin resistance in vascular endothelial cells. Epigenetic mechanisms including promoter DNA methylation have been demonstrated in development and progression of metabolic disorders and atherosclerosis. However, precise and underlying epigenetic mechanisms governing vascular insulin resistance are not known. Human endothelial cells treated with a) IL-6 and insulin together, b) pretreated with IL-6 and c) hyperinsulinemic conditions induced vascular insulin resistance leading to decreased Akt/eNOS activation and subsequently stabilized STAT3 phosphorylation. In 3D spheroid and matrigel assays, IL-6 abrogated insulin effects on angiogenesis. IL-6 induced insulin resistance was associated with down regulation of DNMT1 and DNMT3B, but not DNMT3A and resulted in decreased enzyme activity leading to global DNA hypomethylation. Protein levels of DNMT1 and DNMT3B were inversely correlated with S-phase of cell cycle. CpG microarray analysis revealed hypomethylation of promoters associated with 199 genes and promoter hypermethylation of 98 genes. Further, methylation status of promoters of genes associated with insulin signaling and angiogenesis such as RPS6KA2, PI3KR2, FoxD3, Exoc7, MAP3K8, ITPKB, EPHA6, IGF1R and FOXC2 were validated by bisulfite sequencing. Differentially methylated CpG sites of four out of five hypomethylated genes harboured putative binding site for HMGB1, a potent inflammatory mediator, suggesting HMGB1 might serve as epigenetic switch facilitating a pro-inflammatory milieu in response to IL-6 in endothelial cells. Our data indicates causal link between IL-6 induced DNMT1 changes and altered gene expression involved in insulin signaling and angiogenesis.
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