title: 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.
abstract: 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.
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Kota
authors: K
authors: Ishikawa
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Shin
authors: S
authors: Tsunekawa
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Makoto
authors: M
authors: Ikeniwa
authors: Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Takako
authors: T
authors: Izumoto
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Atsushi
authors: A
authors: Iida
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Hidetada
authors: H
authors: Ogata
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Eita
authors: E
authors: Uenishi
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Yusuke
authors: Y
authors: Seino
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Nobuaki
authors: N
authors: Ozaki
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Yoshihisa
authors: Y
authors: Sugimura
authors: Department of Metabolic Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors: Yoji
authors: Y
authors: Hamada
authors: Diabetes Therapeutics and Research Center, The University of Tokushima, Tokushima, Japan.
authors: Akio
authors: A
authors: Kuroda
authors: Department of Epigenomics, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
authors: Keiko
authors: K
authors: Shinjo
authors: Department of Epigenomics, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
authors: Yutaka
authors: Y
authors: Kondo
authors: Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
authors: Yutaka
authors: Y
authors: Oiso
mesh_terms: D000818:Animals; D002460:Cell Line; D019175:DNA Methylation / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D007328:Insulin / Q000096:biosynthesis*; D050417:Insulin-Secreting Cells / Q000378:metabolism* / Q000473:pathology; D019308:Palmitic Acid / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D051381:Rats; D011924:Rats, Zucker; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D007328:Insulin; D019308:Palmitic Acid; D005947:Glucose