Glucotoxicity Induces Insulin Promoter DNA Methylation in Beta Cells

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

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

<div><p>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 (<i>Ins1</i>) 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 <i>Ins1</i> 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 <i>Ins1</i> promoter; however, palmitate did not affect DNA methylation. Artificial methylation of <i>Ins1</i> 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 <i>Ins1</i> 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 <i>Ins1</i> 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 <i>Ins1</i> promoter in pancreatic beta cells. It provides new insights into the irreversible pathophysiology of diabetes.</p></div

Insulin mRNA levels and DNA methylation of the <i>Ins1</i> promoter in high-glucose conditions.

<p>(A–D) INS-1 cells were cultured for 14 days. (E and F) under normal-culture-glucose (11.2 mmol/l; white bar) or experimental-high-glucose (22.4 mmol/l; black bar) conditions. (G and H) INS-1 cells cultured in 11.2 mmol/l glucose conditions with palmitate for 14 days. Insulin mRNA levels (A, C, E, and G) were examined by real-time PCR analysis. DNA methylation of the <i>Ins1</i> promoter (B, D, F, and H) was examined by pyrosequencing analysis. (I) INS-1 cells were cultured for 14 days under the indicated conditions. Following this, GSIS was performed with low glucose (2.8 mmol/l; white bar) or high glucose (16.7 mmol/l; black bar) for 30 min. All results are mean ± SEM (<i>n</i> ≥ 4). Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p

Oxidative stress and endoplasmic reticulum (ER) stress did not induce DNA methylation of <i>Ins1</i> promoter.

<p>INS-1 cells were cultured for 14 days under the following conditions: (A and B) with H₂O₂ in 11.2 mmol/l glucose; (C and D) with N-acetyl-cysteine (NAC) in 22.4 mmol/l glucose; (E and F) with thapsigargin in 11.2 mmol/l glucose; and (G and H) with tauroursodeoxycholic acid (TUDCA) in 22.4 mmol/l glucose. Insulin mRNA levels (A, C, E, and G) were examined by real-time PCR. DNA methylation of the <i>Ins1</i> promoter (B, D, F, and H) was examined by pyrosequencing analysis. All results are means ± SEM (<i>n</i> ≥ 4). Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p

DNA methylation of <i>Ins1</i> promoter in pancreatic islets from male Zucker diabetic fatty (ZDF) rats.

<p>(A) DNA methylation of the <i>Ins1</i> promoter was examined by pyrosequencing analysis in the pancreatic islets isolated from 12-week-old ZDF rats. (B) The alpha/beta cell ratio was calculated in islets isolated from heterozygous and homozygous ZDF rats. (C) Isolated pancreases were immunostained for insulin (green), glucagon (red), and DAPI (blue) in heterozygous and homozygous ZDF rats. Scale bars indicate 100 μm. Results are mean ± SEM. A: <i>n</i> = 4 rats. B: <i>n</i> = 90 islets from 3 rats per group. Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p

Metformin improved insulin mRNA levels, intracellular triacylglycerol (TAG) content, and DNA methylation of <i>Ins1</i> promoter.

<p>(A) INS-1 cells were cultured in glucose and palmitate for 14 days. (B-D) INS-1 cells were cultured with metformin for 14 days. Intracellular TAG levels (A and D) were examined by ELISA, insulin mRNA levels (B) were examined by real-time PCR, and DNA methylation of the <i>Ins1</i> promoter (C) was examined by pyrosequencing. All results are means ± SEM (<i>n</i> ≥ 4). Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p

The effect of a high-glucose state on DNMT and TET in INS-1 cells.

<p>(A-D) INS-1 cells were cultured under normal-culture-glucose (11.2 mmol/l; white bar) or experimental-high-glucose (22.4 mmol/l; black bar) conditions for 14 days. The <i>Dnmt</i> (A) and <i>Tet</i> (C) mRNA levels were examined by real-time PCR. DNA methyltransferase (DNMT) (B) and ten-eleven-translocation methylcytosine dioxygenase (TET) (D) activities were examined by ELISA. All results are mean ± SEM (<i>n</i> ≥ 4). Asterisks indicate statistically significant difference (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p

The contribution of DNA methylation of the <i>Ins1</i> promoter.

<p>(A) INS-1 cells were cultured under normal-culture-glucose (11.2 mmol/l; white bar) or experimental-high-glucose (22.4 mmol/l; black bar) conditions for 14 days. DNA methylation of the <i>Ins1</i> promoter was examined by bisulfite sequencing analysis. (B) A diagram of the 496-bp rat <i>Ins1</i> promoter (position −304 to +191 bp relative to the transcription start site) in luciferase reporter plasmids. The positions of CpG sites are represented by lollipop markers. (C) Methylated (black bar) or mock-methylated (white bar) rat <i>Ins1</i> promoter-transfected INS-1 cells were incubated at 5.6 mmol/l glucose with/without cAMP-increasing agents, 1 μmol/l forskolin and 10 μmol/l IBMX (forskolin/IBMX), for 3 h. Luciferase activities are presented as relative expression compared with the mock-methylated vectors without forskolin/IBMX stimulation. The inset shows a magnified image of the methylated vector. (D and E) INS-1 cells were treated with 5-Aza-2′-deoxycytidine (DAC) for the last 3 days of the 14-day incubation under 22.4 mmol/l high glucose conditions, and the medium containing DAC was changed every 24 h. Insulin mRNA levels (D) were examined by real-time PCR. DNA methylation of the <i>Ins1</i> promoter (E) was examined by pyrosequencing analysis. All results are mean ± SEM (<i>n</i> ≥ 4). Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p