Verapamil treatment of β-cells

In this study, we investigated the molecular and cellular mechanisms of verapamil treatment on β-cell function and survival using MIN6 cells. Verapamil's molecular processes were studied using transcriptomic data. MTT, cell count, and flow cytometry assessed cell proliferation, growth, and cycle. MIN6 cell function was assessed by glucose-stimulated insulin release and total insulin content. Seahorse and metabolic stress kits examined metabolic activity and oxygen consumption rate. The protective impact of verapamil on MIN6 cells was evaluated by challenging the verapamil treated cell with streptozotocin, T1D-cytomix, or T2D-cytomix cocktail. Our results demonstrate that verapamil treatment induced higher proliferation of MIN6 cells, altered expression of various proteins and genes, improved insulin secretion in hyperglycemic conditions, increased basal and maximal respiration levels, along with β-cell survival against streptozotocin, T1D-, or T2D-cytomix-induced toxicity, and rendered a protective effect. Min6 cells were cultured in DMEM media with 5.6 mM glucose supplemented with 15% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 50 μg/mL penicillin, 100 μg/mL streptomycin, and 70 mM β-mercaptoethanol at 37.0 °C, with 5% CO2. After 24 hours, cells were treated with 50 μM verapamil for another 24 hours. MIN6 cells at a passage of <25 were used in this study. RNA isolation was performed from the verapamil treated and untreated MIN6 cells (12 independent experiments) using the RNeasy kit (Qiagen, Hilden, Germany), following standard protocol. 40 ng of purified RNA was used for whole transcriptome sequencing. Transcriptome libraries were produced using Truseq stranded mRNA kit (Illumina Inc. USA), following the manufacturer’s protocol, and were validated and quantified using bioanalyzer (Agilent, California, United States) and qubit fluorometer (Thermofisher Scientific, Massachusetts, United States), respectively. Then paired-end sequencing on Novaseq 6000 system (Illumina Inc. USA) was carried on. The resulting BCL files were concerted to Fastq using the bcl2fastq v.2.20 tool and qualified ty control of Fastq files using FastQC (v0.11.9). Trimmomatic (v0.39) was used for adaptor and quality trimming, and for the removal of extremely short reads. HISAT2 (v2.1.0) was used for data alignment. To enumerate the number of reads that are associated with the genes, htseq-count tool in HTSeq (v 0.9.1) was used. Differential gene expression analysis was conducted using Bioconductor package edgeR, by adopting the default setting.

本研究以MIN6细胞为模型，探究维拉帕米（verapamil）处理对β细胞功能与存活的分子及细胞机制。本研究通过转录组数据分析维拉帕米的分子调控过程；采用MTT实验、细胞计数及流式细胞术检测细胞增殖、生长与细胞周期状态；通过葡萄糖刺激下的胰岛素分泌量与总胰岛素含量，评估MIN6细胞的功能；使用Seahorse细胞能量代谢分析仪及代谢应激检测试剂盒，检测细胞代谢活性与耗氧速率。通过链脲佐菌素（streptozotocin）、1型糖尿病细胞因子混合物（T1D-cytomix）或2型糖尿病细胞因子混合物（T2D-cytomix）处理维拉帕米预孵育的MIN6细胞，评估维拉帕米对细胞的保护作用。本研究结果显示，维拉帕米处理可促进MIN6细胞增殖，调控多种蛋白与基因的表达水平；在高糖环境下可改善胰岛素分泌功能，提升基础呼吸与最大呼吸速率；同时可提高β细胞对抗链脲佐菌素、T1D-及T2D-cytomix诱导的细胞毒性的存活能力，发挥保护作用。MIN6细胞培养于含5.6 mM葡萄糖的DMEM培养基中，该培养基添加15%热灭活胎牛血清（FBS）、2 mM L-谷氨酰胺、1 mM 丙酮酸钠、10 mM HEPES、50 μg/mL青霉素、100 μg/mL链霉素及70 mM β-巯基乙醇，培养条件为37 ℃、5% CO₂。细胞培养24小时后，用50 μM维拉帕米继续处理24小时；本研究均使用传代次数小于25代的MIN6细胞。按照标准实验流程，使用RNeasy试剂盒（Qiagen，德国希尔德）从维拉帕米处理组与未处理组的MIN6细胞中提取总RNA，共完成12次独立重复实验。取40 ng纯化后的总RNA进行全转录组测序。按照试剂盒说明书，使用Truseq链特异性mRNA建库试剂盒（Illumina公司，美国）构建转录组文库；分别使用生物分析仪（Agilent，美国加利福尼亚州）与Qubit荧光定量仪（Thermo Fisher Scientific，美国马萨诸塞州）对文库进行质检与定量。随后在NovaSeq 6000测序系统（Illumina公司，美国）上进行双端测序。使用bcl2fastq v2.20工具将生成的BCL文件转换为Fastq格式文件，并使用FastQC（v0.11.9）对Fastq文件进行质量控制。使用Trimmomatic（v0.39）进行接头序列切除、质量修剪及过短reads的过滤。使用HISAT2（v2.1.0）进行序列比对。使用HTSeq（v0.9.1）中的htseq-count工具统计与基因匹配的reads数目。采用Bioconductor的edgeR软件包，以默认参数进行差异基因表达分析。