胰岛素抵抗中巨噬细胞M1/M2极化改变的新机制及药物干预

M1/M2 macrophage polarization plays an important role in insulin resistance of adipose tissue, which can be a check point for disease therapy. But the underlying mechanisms are not very clear, based on the previous researches, we first found that hnRNP A1-induced autophagy is involved in the M1/M2 polarization. Then, we will continue to identify effects of autophagy on saturated free fatty acid-induced macrophage polarization, and following in inflammatory cytokines secretion and insulin resistance of adipocytes; Furthermore, we will elucidate the functions of hnRNP A1 on mRNA stability and transcription of autophagy genes; and evaluate whether these functions of hnRNP A1 can be modified by phosphorylation and translocation in nuclear-cytosol. Use hnRNP A1-conditional knockout mice, we examine deficiency of hnRNP A1 on autophagy, phenotype and inflammatory cytokines in macrophages, and the insulin signaling transduction in adipose tissues, from that, we want to find the molecular mechanisms of hnRNP A1-induced autophagy, inflammation and following effects in adipocytes. We will setup the drug evaluation system targeting hnRNP A1 and detect the improvement in insulin resistance of candidate compounds. By this project, we will evaluate the contribution of macrophage "hnRNP A1-autophage-polarization-inflammation" axis to insulin resistance which maybe the base of drug treatment.

巨噬细胞M1/M2极化对脂肪组织胰岛素抵抗具有重要调控作用，成为干预胰岛素抵抗的重要切入点。胰岛素抵抗中巨噬细胞极化改变的详细机制不明，基于前期工作，首次发现核不均一核糖核蛋白（hnRNP A1，以下称A1）介导的细胞自噬参与其中。本项目拟进一步明确自噬参与调控饱和游离脂肪酸诱导巨噬细胞极化改变、影响炎症因子分泌和脂肪细胞胰岛素信号的分子机制。研究A1与自噬相关基因结合并调控其mRNA稳定性和转录活性的功能；观察A1的核-浆定位、磷酸化修饰对上述自噬基因调控的影响；构建巨噬细胞条件敲除A1的小鼠，比较条件敲除型和野生型巨噬细胞在自噬、极化表型、炎症因子分泌上的差异和脂肪细胞功能的改变，发现A1调控自噬和炎症、改变胰岛素信号转导的分子基础；建立药物评价体系，研究药物靶向A1对胰岛素抵抗的改善作用，确立巨噬细胞"A1-自噬-极化-炎症"轴对胰岛素抵抗病理机制的重要贡献，为新型药物治疗打下基础。

代谢综合症的重要致病因素是胰岛素抵抗和大量炎症因子的产生。在胰岛素抵抗的初期，脂肪组织中已有巨噬细胞浸润和炎症因子的释放，并且伴随整个发病过程；选择性去除体内CD11C+炎性巨噬细胞，可快速恢复脂肪组织的胰岛素敏感性。为此，本课题希望在疾病早期以控制炎症改善胰岛素抵抗为目标,发现胰岛素抵抗和代谢综合症的关键切入点及其干预手段。基于实验室前期对免疫细胞中RNA结合蛋白的工作基础，我们获得了巨噬细胞特异性缺失核不均一核糖核蛋白（heterogeneous nuclear ribonucleoprotein, hnRNP A1）的条件敲除鼠。高脂饮食 诱导下，敲除鼠的肥胖和胰岛素抵抗明显减轻。表现在：敲除鼠血清中甘油三酯和胆固醇水平明显下降；敲除鼠的能量消耗和呼吸代谢速率明显增加；GTT、ITT以及胰岛素相关信号通路的免疫印迹检测显示，巨噬细胞缺失hnRNP A1的脂肪和肝组织中胰岛素敏感性增强；敲除鼠脂肪组织中M1巨噬细胞的浸润显著减少，相关炎症因子表达下调。进一步通过RIP-chip分析，我们找到了hnRNP A1在巨噬细胞调控的靶基因—细胞因子信号转导抑制因子3(Suppressor of cytokine signaling 3，SOCS3)。实验结果显示，M1极化条件下，hnRNP A1蛋白与SOCS3的mRNA在胞浆中存在共定位，并且敲除hnRNP A1降低了SOCS3 mRNA的稳定性，对SOCS3的翻译效率无影响。我们随后证实hnRNP A1结合在SOCS3 mRNA的ARE结构，调控其稳定性，进而调控巨噬细胞M1极化。同时凝胶迁移实验和表面等离子体共振实验也证实了hnRNP A1与SOCS3 ARE domain的结合。我们还发现hnRNP A1实现稳定Socs3 mRNA的作用，是阻断由miR-221介导的SOCS3 mRNA向P小体聚集来实现的。因此，通过本项目我们首次发现并证实了hnRNP A1调控mRNA稳定性的新机制，并明确了其在巨噬细胞M1极化和周围脂肪组织胰岛素抵抗过程中的作用。这些发现丰富了巨噬细胞极化的调控网络，为实现通过干预炎症反应治疗胰岛素抵抗和代谢综合症提供了新的理论依据。