RNA m6A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 m6A methylation in insulin resistance in adipocytes

摘要:

目的 探讨RNA m⁶A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 m⁶A水平。高脂饮食诱导C57BL/6J小鼠构建胰岛素抵抗（insulin 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m⁶A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m⁶A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1 175个基因发生高m⁶A修饰，55个基因发生低m⁶A修饰，同时有182个基因呈现高m⁶A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m⁶A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m⁶A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。

Abstract:

Objective To explore the role of RNA m⁶A methylation in insulin resistance in adipocytes.Methods We collected redundant subcutaneous adipose tissue samples from patients with type 2 diabetes and patients without type 2 diabetes to measure the RNA m⁶A 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 m⁶A methylation using epitranscriptomic microarrays. The changes in insulin signaling-related 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 investigated.Results The overall m⁶A modification levels were significantly increased in the adipose tissue of patients with type 2 diabetes 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 m⁶A methylation levels in 1 175 genes and low m⁶A methylation levels in 55 genes；182 genes showed significantly high m⁶A 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 validated，and their m⁶A 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 m⁶A hypermethylation of AKT2，INSR，ACACA，and SREBF1 to downregulate their expression and block insulin signaling in adipocytes.