Correlation between insulin-induced estrogen receptor methylation and atherosclerosis

BACKGROUND Hyperinsulinemia and insulin resistance have been recently recognized as an important cause of atherosclerosis. Clinical studies have also found that expression of the estrogen receptor is closely related to the incidence of atherosclerosis. This study investigate the effects of insulin and estrogen receptor α (ER-α) in atherosclerosis. METHODS Double knockout ApoE/Lepr mice were given intraperitoneal injections of insulin, and their aortae were harvested for hematoxylin-eosin staining and immunohistochemical analysis. In addition, vascular smooth muscle cells (VSMCs) were treated with insulin or infected with a lentivirus encoding exogenous ER-α, and changes in gene expression were detected by real-time polymerase chain reaction and western blotting. The methylation levels of the ER-α gene were tested using bisulfite sequencing PCR, and flow cytometry and EdU assay were used to measure VSMCs proliferation. RESULTS Our results showed that insulin can induce the formation of atherosclerosis. Gene expression analysis revealed that insulin promotes the expression of DNA methyltransferases and inhibits ER-α expression, while 5-aza-2'-deoxycytidine can inhibit this effect of insulin. Bisulfite sequencing PCR analysis showed that methylation of the ER-α second exon region increased in VSMCs treated with insulin. The results also showed that ER-α can inhibit VSMCs proliferation. CONCLUSIONS Our data suggest that insulin promotes the expression of DNA methyltransferases, induces methylation of ER-α second exon region and decreases the expression of ER-α, thereby interfering with estrogen regulation of VSMCs proliferation, resulting in atherosclerosis.