RESEARCH ARTICLE Long-Term Pancreatic Beta Cell Exposure to High Levels of Glucose but Not Palmitate Induces DNA Methylation within the Insulin Gene Promoter and Represses Transcriptional Activity

Recent studies have implicated epigenetics in the pathophysiology of diabetes. Further-more, DNA methylation, which irreversibly deactivates gene transcription, of the insulin pro-moter, particularly the cAMP response element, is increased in diabetes patients. However, the underlying mechanism remains unclear. We aimed to investigate insulin promoter DNA methylation in an over-nutrition state. INS-1 cells, the rat pancreatic beta cell line, were cul-tured under normal-culture-glucose (11.2 mmol/l) or experimental-high-glucose (22.4 mmol/l) conditions for 14 days, with or without 0.4 mmol/l palmitate. DNAmethylation of the rat insulin 1 gene (Ins1) promoter was investigated using bisulfite sequencing and pyrosequencing anal-ysis. Experimental-high-glucose conditions significantly suppressed insulin mRNA and in-creased DNAmethylation at all five CpG sites within the Ins1 promoter, including the cAMP response element, in a time-dependent and glucose concentration-dependent manner. DNA methylation under experimental-high-glucose conditions was unique to the Ins1 promoter; however, palmitate did not affect DNAmethylation. Artificial methylation of Ins1 promoter sig-nificantly suppressed promoter-driven luciferase activity, and a DNAmethylation inhibitor sig