METHODS: High-fat diet (HFD)-fed mice were used as obesity-induced hepatic insulin resistance model, exosomes were isolated from BMMs which were extracted from HFD-fed mice by ultracentrifugation. Exosomes were analysed the spectral changes of microRNA expression using a microRNA array. The activation of the insulin signalling pathway and the level of glycogenesis were examined in hepatocytes after transfected with miR-143-5p mimics. Luciferase assay and western blot were used to assess the target of miR-143-5p.
RESULTS: BMMs from HFD-fed mice were polarized towards M1, and miR-143-5p was significantly upregulated in exosomes of BMMs from HFD-fed mice. Overexpression of miR-143-5p in Hep1-6 cells led to decreased phosphorylation of AKT and GSK and glycogen synthesis. Dual-luciferase reporter assay and western blot demonstrated that mitogen-activated protein kinase phosphatase-5 (Mkp5, also known as Dusp10) was the target gene of miR-143-5p. Moreover, the overexpression of MKP5 could rescue the insulin resistance induced by transfection miR-143-5p mimics in Hep1-6.
CONCLUSIONS: Bone marrow macrophage-derived exosomal miR-143-5p induces insulin resistance in hepatocytes through repressing MKP5.
方法:采用高脂饮食(HFD)喂养的小鼠作为肥胖诱导的肝脏胰岛素抵抗模型,从BMM中分离外泌体,BMM通过超速离心从HFD喂养的小鼠中提取。使用microRNA阵列分析外来体的microRNA表达的光谱变化。在用miR-143-5p模拟物转染后的肝细胞中检查胰岛素信号通路的激活和糖原水平。荧光素酶测定和蛋白质印迹用于评估miR-143-5p的靶标。
结果:HFD喂养小鼠的BMM向M1极化,miR-143-5p在HFD喂养小鼠的BMM外泌体中显著上调。miR-143-5p在Hep1-6细胞中的过表达导致AKT和GSK的磷酸化和糖原合成降低。双荧光素酶报告基因测定和蛋白质印迹表明,丝裂原活化蛋白激酶磷酸酶-5(Mkp5,也称为Dusp10)是miR-143-5p的靶基因。此外,MKP5的过表达可以挽救转染miR-143-5p模拟物在Hep1-6中诱导的胰岛素抵抗。
结论:骨髓巨噬细胞来源的外泌体miR-143-5p通过抑制MKP5诱导肝细胞胰岛素抵抗。