基于AMPK/SIRT1信号网络探讨降糖消渴方调控骨骼肌线粒体能量代谢的机制

It has been shown that mitochondrial dysfunction in skeletal muscles is one of significant changes in the progress of insulin resistance and diabetes by multiple studies. AMPK/SIRT1 signal network, which is an important way in maintaining mitochondrial energy metabolism, plays a crucial role in oxidative metabolism and mitochondrial biosynthesis. We found that Jiang Tang Xiao Ke formula could regulate glucose and lipid metabolism, increase insulin sensitivity, and increase gene expression and activity of AMPK in diabetic rats in our previous studies. We speculated these functions may relate to its influence on mitochondrial, but the specific mechanism is unclear. So we design the present research to reveal the mechanisms of Jang Tang Xiao Ke formula on mitochondrial energy metabolism in skeletal muscles. Based on AMPK/SIRT1 signal network and PGC-1 α protein in the downstream, we will study the function of formula on mitochondrial energy metabolism in skeletal muscles in insulin resistant mice and C2C12 muscular cells, and AMPK low-expression cell lines. The study will be carried out in animal, cellar and molecular levels applying the technologies of Seahorse Bioscience XFe Analyzer, Western Blot, and real-time PCR, shRNA gene silencing, etc. In addition, the effective components of the formula on regulating energy metabolism will be further screened. The research will provide the experimental data and theoretical foundation for revealing the acting mechanisms of Jiang tang xiao ke formula and finding a new AMPK agonist.

研究表明骨骼肌线粒体功能紊乱是胰岛素抵抗和糖尿病进展的重要环节，AMPK/SIRT1信号网络为线粒体能量代谢的重要途径，在氧化代谢及线粒体生物合成方面至关重要。我们前期研究发现降糖消渴方能够调节T2DM大鼠糖脂代谢，增加胰岛素敏感性，上调AMPK的表达及活性，其作用与影响线粒体代谢有关，但具体机制不明。本项目拟以AMPK/SIRT1信号网络，及其下游重要调节蛋白PGC-1α为切入点，以线粒体能量代谢为主线，通过胰岛素抵抗小鼠与C2C12细胞模型及AMPK低表达细胞株，运用细胞能量代谢仪，Western Blot、实时荧光定量PCR、shRNA稳定基因沉默等技术，从整体、细胞及分子水平展开研究，从而揭示降糖消渴方调节骨骼肌线粒体能量代谢的机制及分子靶点，进一步验证并筛选出发挥调节能量代谢的有效组分，为揭示中药复方降糖消渴方的作用机制提供实验数据和理论依据，也为寻找新的AMPK激动剂奠定基础。

本项目以AMPK/SIRT1信号网络，及其下游重要调节蛋白PGC-1α为切入点，以线粒体能量代谢为主线，进行了降糖消渴方及其有效组分调节骨骼肌线粒体能量代谢的相关研究。研究主要分在体和离体实验两部分，应用高脂饮食诱导的胰岛素抵抗的肥胖小鼠为模型，结果揭示了降糖消渴方能够通调节骨骼肌AMPK信号通路中相关基因和蛋白的表达，实现改善IR小鼠胰岛素敏感性，调节其糖脂代谢的作用，该作用可能与调节小鼠骨骼肌线粒体的结构和功能有关。离体实验以C2C12骨骼肌细胞为研究对象展开，研究显示降糖消渴颗粒含药血清与其有效组分均能有效地改善高脂诱导的C2C12胰岛素抵抗细胞的葡萄糖消耗，并提高C2C12细胞的线粒体能量代谢，该作用与激活C2C12细胞的AMPK/SIRT1信号网络相关，降糖消渴颗粒的有效组分人参皂苷Rb1，丹酚酸B，马钱子苷可能是其发挥调节能量代谢的有效组分，本研究结果揭示了中药复方降糖消渴方的作用机制，亦为寻找新的AMPK激动剂奠定了基础。