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

List of primary antibodies.

<p>List of primary antibodies.</p

Tomosyn is a SNAP25 binding protein in the posterior pituitary.

<p>(a) <i>In vitro</i> binding assay. Rat posterior pituitary lysates was incubated with GST-SNAP25 and glutathione (GSH) Sepharose. Eluted proteins bound to the beads were separated on SDS-PAGE followed by silver staining of proteins. The arrow indicates the band of interest. (b) Western blotting of pull-down samples. The arrowhead indicates the location of the immune reactive band reacting with anti-tomosyn antibody (Santa Cruz Biotechnology #sc-136105). Lane 1 = GST-SNAP25 with rat posterior pituitary lysates; lane 2 = GST-SNAP25 without rat posterior pituitary lysates (for both a, and b); Marker = molecular weight markers.</p

Tomosyn-1 negatively regulates secretion of AVP vesicles.

<p>(a–k) The effects of overexpression or siRNA treatments on tomosyn-1 expression. Dispersed SFEBq/gfCDM cultured cells were transfected with empty vector (vector) or tomosyn-1 vector (Tomosyn-1), scrambled siRNA (siScr) or Tomosyn-1 siRNA (siTomosyn-1 #1 or #2), or not treated (NT). (a, f, g) After 48 h, the expression levels of tomosyn were analysed by western blotting using anti-tomosyn antibody (Tomosyn, marked with arrowheads at the right). The levels of β-actin in the same samples were determined as a control for protein loading (bottom panels of a, f, g). (b–e) Representative immunostaining for tomosyn (green), copeptin (red), and DAPI (blue) at 48 h after overexpression of tomosyn-1 in dispersed SFEBq/gfCDM cultured cells. The merged image is shown in (e). (h–k) Representative immunostaining for tomosyn (green), copeptin (red), and DAPI (blue) at 48 h after knockdown of tomosyn-1 with siRNA in dispersed SFEBq/gfCDM cultured cells. The merged image is shown in (k). (l) AVP concentration in the media of artificial cerebrospinal fluid cultured cells (aCSF) (non-treated, NT; n = 13), KCl treatment (KCl; n = 13), empty vector with KCl (Vector + KCl; n = 12), and tomosyn-1 vector with KCl (Tomosyn-1 + KCl; n = 12). (m) AVP concentration from scrambled siRNA with KCl (siScr + KCl; n = 13), and siTomosyn-1 with KCl (siTomosyn-1 #1 + KCl group; n = 13, siTomosyn-1 #2 + KCl group; n = 12). Final KCl concentration was 100 mM. Values are expressed as the mean ± SEM. **P < 0.01.</p

Tomosyn is expressed in the rat hypothalamo-posterior pituitary axis.

<p>(a) Presence of tomosyn in the rat anterior and posterior pituitary as indicated by western blotting. Pituitary lysates were separated on SDS-PAGE followed by western blotting using anti-tomosyn, anti-SNAP25, anti-syntaxin 1A, anti-syntaxin 1B, anti-AVP, and anti-β-actin antibodies (as a loading control). (b) Subcellular fractionation of rat posterior pituitary samples. Fraction 1 (cytosolic proteins, F1) and Fraction 2 (membranes and membrane organelles, F2) were subjected to SDS-PAGE followed by western blotting using anti-tomosyn antibody. Five μg of proteins was loaded onto each lane. The subcellular fractionation was confirmed by western blotting with anti-Akt (cytosolic marker) and anti-N-cadherin (membrane fraction marker) antibodies. Tomosyn was present in both fractions. (c) Expression levels of tomosyn-1 mRNA in rat cortex and hypothalamus are similar. The amount of mRNA was determined using quantitative RT-PCR. The values are normalized to β-actin mRNA and are expressed as the mean ± SEM.</p

Arginine vasopressin (AVP)-secreting neurons from mouse embryonic stem cells express tomosyn.

<p>(a) Flow chart showing the method for culturing modified embryonic stem (ES) cells differentiating in serum-free medium (SFEBq)/growth factor-free chemically defined medium (gfCDM). DFNB = DMEM/F12 supplemented with 7 g/L glucose, N2 and B27. (b–e) Immunostaining with copeptin (red), NeuN (green), and DAPI (blue) in dispersed SFEBq/gfCDM cultured cells. A merged image is shown in the right panel. White scale bars indicate 20 μm. (f–k) Immunolocalization of proteins in dispersed SFEBq/gfCDM cultured cells, immunostaining with anti-tomosyn (green), anti-copeptin (red), or anti-SNAP25 (red) antibodies as analysed with confocal microscopy. Merged images are shown in the right panels. White scale bars indicate 25 μm. (l) AVP levels with or without 100 mM KCl stimulation in SFEBq/gfCDM cultured cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164544#sec010" target="_blank">Methods</a> section). AVP concentrations in the media of artificial cerebrospinal fluid cultured cells (aCSF) (non-treated, NT; n = 8), or 100 mM KCl treatment (KCl; n = 8) are shown. Values are expressed as the mean ± SEM. **P < 0.01 versus NT (non-treated artificial spinal fluid). (m) Tomosyn-1 mRNA expressions in dispersed SFEBq/gfCDM cultured cells. The amount of mRNA was determined using quantitative RT-PCR. The values are normalized to β-actin mRNA and are expressed as the mean ± SEM.</p

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

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

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