Effects of p,p'-DDE on <i>H19</i> DMR methylation and insulin secretion of INS-1 cells

Yanqiu Zong; Huasen Xie; Xiaoqing LI; Lijun Wang; Lei Yang; Yang Song

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Effects of p,p'-DDE on H19 DMR methylation and insulin secretion of INS-1 cells

VernacularTitle:p,p'-DDE对胰岛素瘤细胞H19差异甲基化区甲基化及胰岛素分泌的影响
Author: Yanqiu ZONG ¹ ; Huasen XIE ¹ ; Xiaoqing LI ¹ ; Lijun WANG ¹ ; Lei YANG ¹ ; Yang SONG ¹
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1. Hangzhou Normal University, Hangzhou, Zhejiang 310016, China.
Publication Type:Experiment
Keywords: p,p'-dichlorodiphenyldichloroethylene; diabetes; islet function; H19; DNA methylation; insulin-like growth factor 2; insulin-like growth factor-1 receptor
From: Journal of Environmental and Occupational Medicine 2022;39(5):539-544
CountryChina
Language:Chinese
Abstract: Background Diabetes is a major threat to public health across the world. Studies have shown that exposure to p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) is closely related to the occurrence of type 2 diabetes mellitus. However, the relevant molecular mechanism is not clear. Objective To investigate the effects of p,p'-DDE on H19 differentially methylated region (DMR) methylation and insulin secretion of rat insulinoma cells (INS-1 cells). Methods INS-1 cells were cultured with different concentrations (0, 3.125, 6.25, 12.5, 25, 50, and 75 µmol·L⁻¹) of p,p'-DDE for 24 h, and the viability of INS-1 cells was detected by CCK-8 method. INS-1 cells were exposed to 0, 12.5, 25, and 50 µmol·L⁻¹ p,p'-DDE for 24 h in subsequent experiments. The methylation levels of 24 CpG sites in H19 DMR were analyzed by bisulfite genomic sequencing. The expression levels of insulin-like growth factor 2 (IGF2) mRNA were detected by real-time quantitative PCR. The expression levels of IGF2 and insulin-like growth factor-1 receptor (IGF1R) proteins were detected by Western blotting. The insulin secretion function of INS-1 cells was determined by glucose-stimulatedinsulin secretion test (5 and 25 mmol·L⁻¹ glucose, respectively). Results Compared with the control group, the viability of INS-1 cells increased significantly after treatment with 12.5 µmol·L⁻¹ p,p'-DDE; however, it was significantly inhibited after treatment with 50 or 75 µmol·L⁻¹ p,p'-DDE (P<0.01); therefore, 50 µmol·L⁻¹ was chosen as the maximum concentration of exposure for subsequent experiments. The 25 µmol·L⁻¹ p,p'-DDE treatment decreased the methylation levels of CpG18 and CpG22-CpG24 sites in H19 DMR, and the 50 µmol·L⁻¹ p,p'-DDE treatment decreased the methylation levels of CpG10-CpG24 sites (P<0.05 or P<0.05). Multiple concentrations (12.5, 25, and 50 µmol·L⁻¹) of p,p'-DDE down-regulated the mRNA and protein relative expression levels of IGF2 and the protein relative expression levels of IGF1R. The transcription level of IGF2 decreased to 67.8%, 68.6%, and 62.5% of the control group, the protein level of IGF2 decreased to 73.3%, 79.5%, and 80.9% of the control group, and the protein level of IGF1R decreased to 54.8%, 25.6%, and 12.9% of the control group, respectively (P<0.01). In the high glucose context, p,p'-DDE at selected concentrations inhibited the insulin secretion levels to 85.0%, 58.6%, and 49.5% of the control group, respectively (P<0.01). Conclusion p,p'-DDE could down-regulate methylation level of H19 DMR, interfere the IGF2/IGF1R signaling pathway, and inhibit insulin secretion of islet cells.