RNA m^(6)A甲基化修饰在脂肪细胞胰岛素抵抗中的作用机制  

The role of RNA m^(6)A methylation in insulin resistance in adipocytes

作　　者：叶棣文 张炳杨 张丹彤 马万山[3] 逯素梅[3] Ye Diwen;Zhang Bingyang;Zhang Dantong;Ma Wanshan;Lu Sumei(School of Medical Laboratory,Weifang Medical University;Cheeloo College of Medicine,Shandong University;Department of Laboratory Medicine,The First Affiliated Hospital of Shandong First Medical University/ShandongProvincial Qianfoshan Hospital,Shandong Provincial Medicine and Health Key Laboratory of Clinical LaboratoryDiagnostics)

机构地区：[1]潍坊医学院医学检验学院,潍坊261000 [2]山东大学齐鲁医学院,济南250000 [3]山东第一医科大学第一附属医院/山东省千佛山医院检验医学,山东省医药卫生临床检验诊断学重点实验室,济南250000

出　　处：《重庆医科大学学报》2024年第2期132-140,共9页Journal of Chongqing Medical University

基　　金：山东省自然科学基金资助项目(编号:ZR2023MH031);山东第一医科大学青年科学基金培育资助计划项目(编号:202201-083)。

摘　　要：目的:探讨RNA m^(6)A甲基化修饰在脂肪细胞胰岛素抵抗中的作用及机制。方法:收集2型糖尿病患者术中赘余皮下脂肪组织,以非2型糖尿病患者同样组织为对照,检测组间RNA m^(6)A水平。高脂饮食诱导C57BL/6J小鼠构建胰岛素抵抗(in⁃sulin resistance,IR)模型(HFD组,n=5,60%高脂饲料喂养16周),对照组10%低脂饲料喂养16周(CD组,n=5)。模型构建成功后,取附睾周围脂肪组织行表观转录组学m^(6)A甲基化修饰芯片检测,并借助MeRIP-qPCR实验、RT-qPCR以及RNA结合蛋白免疫沉淀测定(RNA Binding Protein Immunoprecipitation Assay,RIP)实验验证胰岛素信号转导相关基因变化;进一步观察METTL3小分子抑制剂STM2457对高脂饮食诱导下小鼠胰岛素信号转导基因的影响。结果:2型糖尿病患者和小鼠IR模型脂肪组织中总体m^(6)A修饰水平均升高(患者200 ng RNA t=-8.375,P<0.001;患者100 ng RNA t=-3.722,P=0.006;患者50 ng RNA t=-4.937;P=0.001;小鼠100 ng RNA t=-3.590,P=0.023;小鼠50 ng RNA t=-2.760,P=0.025)。表观转录组学检测证实IR的脂肪组织中1175个基因发生高m^(6)A修饰,55个基因发生低m^(6)A修饰,同时有182个基因呈现高m^(6)A修饰且低表达,包括AKT2、INSR、PIK3R1、ACACA、SREBF1等5个胰岛素信号转导关键基因,其中AKT2、INSR、ACACA、SREBF1等4个基因被确证并证实其与METTL3存在直接结合,其m^(6)A修饰水平受METTL3正向调控。STM2457作用下,胰岛素敏感性提高,且AKT2、INSR、ACACA、SREBF1转录水平上调,提示IR表型改善明显。结论:高脂饮食通过METTL3诱导脂肪细胞胰岛素信号转导基因AKT2、INSR、ACACA、SREBF1发生m^(6)A高甲基化修饰,诱导其低表达,阻滞胰岛素信号转导,进而参与诱发IR。Objective:To explore the role of RNA m^(6)A methylation in insulin resistance in adipocytes.Methods:We collected redun⁃dant subcutaneous adipose tissue samples from patients with type 2 diabetes and patients without type 2 diabetes to measure the RNA m^(6)A modification level.A insulin resistance(IR)model was established by feeding C57BL/6J mice with a 60%high-fat diet for 16 weeks(n=5),while the control group was fed with a 10%low-fat diet for 16 weeks(n=5).After successful modeling,the adipose tissue around the epididymis was taken to detect m^(6)A methylation using epitranscriptomic microarrays.The changes in insulin signalingrelated genes were determined by MeRIP-qPCR assay,RT-qPCR,and RIP assay.The effects of the small-molecule inhibitor STM2457 targeting methyltransferase like 3(METTL3)on insulin signaling-related genes in mice feeding a high-fat diet were investi⁃gated.Results:The overall m^(6)A modification levels were signifi⁃cantly increased in the adipose tissue of patients with type 2 diabe⁃tes and IR mice(patients 200 ng RNA,t=-8.375,P<0.001;patients 100 ng RNA,t=-3.722,P=0.006;patients 50 ng RNA,t=-4.937,P=0.001;mice 100 ng RNA,t=-3.590,P=0.023;mice 50 ng RNA,t=-2.760,P=0.025).The epitranscriptomic assay detected high m^(6)A methylation levels in 1175 genes and low m^(6)A methylation levels in 55 genes;182 genes showed significantly high m^(6)A modification and low expression,including five key insulin signaling-related genes(AKT2,INSR,PIK3R1,ACACA,and SREBF1).Direct binding between AKT2,INSR,ACACA,and SREBF1 and METTL3 was vali⁃dated,and their m^(6)A modification levels were positively regulated by METTL3.STM2457 significantly increased insulin sensitivity,and significantly upregulated the transcriptional levels of AKT2,INSR,ACACA,and SREBF1,suggesting a significant improvement in IR phenotype.Conclusion:High-fat diet induces IR through METTL3,which mediates m^(6)A hypermethylation of AKT2,INSR,ACACA,and SREBF1 to downregulate their expression and block insulin signaling in adipocytes.

关 键 词：高脂饮食 胰岛素抵抗 RNA m^(6)A甲基化修饰 胰岛素信号转导通路 

分 类 号：R34[医药卫生—基础医学]