Lv Lixia, Chen Hewen, Sun Jiaying, Lu Di, Chen Chen, Liu Dongfang
Department of Endocrinology and Metabolism, The Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong District, Chongqing, 400010, China.
Endocrine. 2015 Aug;49(3):669-82. doi: 10.1007/s12020-015-0543-8. Epub 2015 Feb 10.
Protein N-arginine methyltransferase-1 (PRMT1), the major asymmetric arginine methyltransferase, plays important roles in various cellular processes. Previous reports have demonstrated that levels and activities of PRMT1 can vary in animals with type 2 diabetes mellitus. The aim of this study was to assess the expression and mechanism of action of PRMT1 during glucose toxicity-induced β cell dysfunction. Liposome-mediated gene transfection was used to transfect INS-1 cells with siPRMT1, which inhibits PRMT1 expression, and pALTER-FOXO1, which overexpresses forkhead box protein O1 (FOXO1). The cells were then cultured in media containing 5.6 or 25 mmol/L glucose with or without the small molecule PRMT1 inhibitor AMI-1 for 48 h. The protein levels of PRMT1, the arginine methylated protein α-metR, FOXO1, Phospho-FOXO1, pancreas duodenum homeobox-1 (PDX-1), and the intracellular localization of PDX-1 and FOXO1 were then measured by western blotting. FOXO1 methylation was detected by immunoprecipitated with anti-PRMT1 antibody and were immunoblotted with α-metR. The levels of insulin mRNA were measured by real-time fluorescence quantitative PCR. Glucose-stimulated insulin secretion (GSIS) and intracellular insulin content were measured using radioimmunoassays. Intracellular Ca(2+) ([Ca(2+)]i) was detected using Fura-2 AM. Intracellular cAMP levels were measured using ELISA. Chronic exposure to high glucose impaired insulin secretion, decreased insulin mRNA levels and insulin content, increased intracellular [Ca(2+)]i and cAMP levels, and abolishes their responses to glucose. Inhibiting PRMT1 expression improved insulin secretion, increased mRNA levels and insulin content by regulating the intracellular translocation of PDX-1 and FOXO1, decreasing the methylation of FOXO1, and reducing intracellular [Ca(2+)]i and cAMP concentrations. Transient overexpression of constitutively active FOXO1 in nuclear reversed the AMI-1-induced improvement of β cell function without changing arginine methylation. It is concluded therefore that PRMT1 regulates GSIS in INS-1 cells, through enhanced methylation-induced nuclear localization of FOXO1, which subsequently suppresses the nuclear localization of PDX-1. Our results suggest a novel mechanism that might contribute to the deficient insulin secretion observed under conditions of chronically hyperglycemia.
蛋白质N - 精氨酸甲基转移酶-1（PRMT1）是主要的不对称精氨酸甲基转移酶，在多种细胞过程中发挥重要作用。先前的报道表明，2型糖尿病动物体内PRMT1的水平和活性会有所不同。本研究的目的是评估PRMT1在葡萄糖毒性诱导的β细胞功能障碍过程中的表达及作用机制。采用脂质体介导的基因转染方法，用抑制PRMT1表达的siPRMT1和过表达叉头框蛋白O1（FOXO1）的pALTER - FOXO1转染INS - 1细胞。然后将细胞在含有5.6或25 mmol/L葡萄糖且有或无小分子PRMT1抑制剂AMI - 1的培养基中培养48小时。随后通过蛋白质印迹法检测PRMT1、精氨酸甲基化蛋白α - metR、FOXO1、磷酸化FOXO1、胰腺十二指肠同源盒-1（PDX - 1）的蛋白水平以及PDX - 1和FOXO1的细胞内定位。用抗PRMT1抗体进行免疫沉淀，并用α - metR进行免疫印迹检测FOXO1甲基化。通过实时荧光定量PCR检测胰岛素mRNA水平。使用放射免疫分析法测量葡萄糖刺激的胰岛素分泌（GSIS）和细胞内胰岛素含量。使用Fura - 2 AM检测细胞内Ca(2+)（[Ca(2+)]i）。使用酶联免疫吸附测定法测量细胞内cAMP水平。长期暴露于高葡萄糖会损害胰岛素分泌，降低胰岛素mRNA水平和胰岛素含量，增加细胞内[Ca(2+)]i和cAMP水平，并消除它们对葡萄糖的反应。抑制PRMT1表达可通过调节PDX - 1和FOXO1的细胞内转运、降低FOXO1的甲基化以及降低细胞内[Ca(2+)]i和cAMP浓度来改善胰岛素分泌，增加mRNA水平和胰岛素含量。在细胞核中瞬时过表达组成型活性FOXO1可逆转AMI - 1诱导的β细胞功能改善，且不改变精氨酸甲基化。因此得出结论，PRMT1通过增强甲基化诱导的FOXO1核定位来调节INS - 1细胞中的GSIS，进而抑制PDX - 1的核定位。我们的研究结果提示了一种新机制，可能与长期高血糖条件下观察到的胰岛素分泌不足有关。
