Visceral adipocyte-derived extracellular vesicle miR-27a-5p elicits glucose intolerance by inhibiting pancreatic b-cell insulin secretion

Pancreatic ß-cell dysfunction caused by obesity can be associated with alterations in the levels of microRNAs (miRNAs). However, the role of miRNAs in such processes remains elusive. Here, we show that pancreatic islet miR-27a-5p, which is markedly increased in obese mice and impairs insulin secretion, is mainly delivered by visceral adipocyte-derived extracellular vesicles (EVs). Depleting miR-27a-5p significantly improves insulin secretion and glucose intolerance in db/db mice. Supporting the function of EVs' miR-27a-5p as a key pathogenic factor, intravenous injection of miR-27a-5p-containing EVs shows their distribution in mouse pancreatic islets. Tracing the injected AAV-miR-27a-5p (AAV-miR-27a) or AAV-FABP4-miR-27a-5p (AAV-FABP4-miR-27a) in visceral fat results in upregulating miR-27a-5p in EVs and serum, and elicits mouse pancreatic ß-cell dysfunction. Mechanistically, miR-27a-5p directly targets L-type Ca2+ channel subtype CaV1.2 (Cacna1c) and reduces insulin secretion in ß-cells. Overexpressing mouse CaV1.2 largely abolishes the insulin secretion injury induced by miR-27a-5p. These findings reveal a causative role of EVs' miR-27a-5p in visceral adipocyte-mediated pancreatic ß-cell dysfunction in obesity-associated type 2 diabetes mellitus. Overall design: Total RNA from islets of NCD and HFD mice was isolated using the RNeasy mini kit (Qiagen), following the protocol for total RNA isolation. RNA integrity and concentration were assessed using an RNA Nano 6000 Assay Kit for the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA), according to the manufacturer's instructions.

肥胖诱导的胰腺β细胞功能障碍，可与微小核糖核酸（microRNAs，miRNAs）的水平异常相关联，但此类进程中miRNAs的具体作用仍尚不明确。本研究证实，在肥胖小鼠中显著上调、可损伤胰岛素分泌的胰岛miR-27a-5p，主要由内脏脂肪细胞来源的细胞外囊泡（extracellular vesicles，EVs）递送。敲低miR-27a-5p可显著改善db/db小鼠的胰岛素分泌功能与葡萄糖耐受不良状态。为验证EVs携带的miR-27a-5p作为关键致病因子的功能，研究人员将携带miR-27a-5p的EVs经静脉注射入小鼠体内，观察到其可分布于小鼠胰岛中。对注射的AAV-miR-27a-5p（以下简称AAV-miR-27a）或AAV-FABP4-miR-27a-5p（以下简称AAV-FABP4-miR-27a）在内脏脂肪中的追踪结果显示，二者可上调囊泡与血清中的miR-27a-5p水平，并诱发小鼠胰腺β细胞功能障碍。机制层面，miR-27a-5p可直接靶向L型钙通道亚型CaV1.2（Cacna1c），并降低β细胞的胰岛素分泌功能。过表达小鼠CaV1.2可几乎完全逆转miR-27a-5p诱导的胰岛素分泌损伤。本研究结果揭示，EVs携带的miR-27a-5p在肥胖相关2型糖尿病中，介导了内脏脂肪细胞诱发胰腺β细胞功能障碍的致病作用。

实验整体设计：依照总RNA提取标准流程，使用RNeasy迷你试剂盒（Qiagen）提取正常饮食组（NCD）与高脂饮食组（HFD）小鼠胰岛的总RNA。按照制造商提供的说明书，使用适配Bioanalyzer 2100系统的RNA Nano 6000检测试剂盒（安捷伦科技公司，美国加利福尼亚州圣克拉拉市）评估RNA的完整性与浓度。