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

• Published in: PloS one Vol. 10; no. 2; p. e0115350
• Main Authors: Ishikawa, Kota, Tsunekawa, Shin, Ikeniwa, Makoto, Izumoto, Takako, Iida, Atsushi, Ogata, Hidetada, Uenishi, Eita, Seino, Yusuke, Ozaki, Nobuaki, Sugimura, Yoshihisa, Hamada, Yoji, Kuroda, Akio, Shinjo, Keiko, Kondo, Yutaka, Oiso, Yutaka
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 06.02.2015; Public Library of Science (PLoS)
• Subjects: Accumulation; Analysis; Animals; Beta cells; Bisulfite; Cell culture; Cell Line; CpG islands; Cyclic AMP; Deactivation; Deoxyribonucleic acid; Diabetes mellitus; Dioxygenase; DNA; DNA methylation; DNA Methylation - drug effects; DNA methyltransferase; Endoplasmic reticulum; Epigenetic inheritance; Epigenetics; Gene expression; Gene sequencing; Genes; Genetic aspects; Glucose; Glucose - pharmacology; Insulin; Insulin - biosynthesis; Insulin-Secreting Cells - metabolism; Insulin-Secreting Cells - pathology; Kinases; Luciferase; Metformin; Methylation; Nutrition; Oxidative stress; Palmitic acid; Palmitic Acid - pharmacology; Pancreas; Pancreatic beta cells; Promoter Regions, Genetic; Rats; Rats, Zucker; RNA; Rodents; Sulfites; Transcription; Transcription (Genetics); Transcription, Genetic - drug effects; Translocation; Triglycerides; Type 2 diabetes; United States > US; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0115350

Recent studies have implicated epigenetics in the pathophysiology of diabetes. Furthermore, DNA methylation, which irreversibly deactivates gene transcription, of the insulin promoter, 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 cultured 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. DNA methylation of the rat insulin 1 gene (Ins1) promoter was investigated using bisulfite sequencing and pyrosequencing analysis. Experimental-high-glucose conditions significantly suppressed insulin mRNA and increased DNA methylation 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 DNA methylation. Artificial methylation of Ins1 promoter significantly suppressed promoter-driven luciferase activity, and a DNA methylation inhibitor significantly improved insulin mRNA suppression by experimental-high-glucose conditions. Experimental-high-glucose conditions significantly increased DNA methyltransferase activity and decreased ten-eleven-translocation methylcytosine dioxygenase activity. Oxidative stress and endoplasmic reticulum stress did not affect DNA methylation of the Ins1 promoter. High glucose but not palmitate increased ectopic triacylglycerol accumulation parallel to DNA methylation. Metformin upregulated insulin gene expression and suppressed DNA methylation and ectopic triacylglycerol accumulation. Finally, DNA methylation of the Ins1 promoter increased in isolated islets from Zucker diabetic fatty rats. This study helps to clarify the effect of an over-nutrition state on DNA methylation of the Ins1 promoter in pancreatic beta cells. It provides new insights into the irreversible pathophysiology of diabetes.