天然药物功能蛋白质学科组（赖仞）知识库

Adropin是由Enho基因所编码的一种肽类激素，相对分子质量为4.5KDa，广泛分布于大脑、肝脏、脐静脉、冠状动脉内皮细胞、肾脏和外周循环血液中，是维持能量动态平衡的内源性多肽，具有调节能量代谢，改善胰岛素抵抗，增加胰岛素敏感性的作用。本论文主要探索了adropin对HepG2肝癌细胞相关能量代谢的影响，并在此基础上探讨adropin可能作用的靶点和通路。 首先通过化学合成方法获得高纯度、具有活性的adropin多肽。检测发现，其能明显增强HepG2细胞对葡萄糖的吸收，并且存在剂量依赖关系。但是在小鼠体内adropin对血糖却没有明显影响。通过靶向代谢组学检测发现adropin能显著的降低HepG2细胞内ATP、GTP、FBP的含量，同时提高cAMP的含量，据文献报道以上改变能明显激活能量代谢调节的关键分子AMPK。 其次，通过Co-IP方法并结合质谱鉴定技术，最终找到可能与adropin结合的蛋白为血红蛋白β亚基（Hbb）。且通过表面等离子共振证明了adropin能与血红蛋白（Hb）直接结合，且存在浓度剂量依赖关系。实验中还发现在结合珠蛋白（Hp）存在情况下Hb与adropin结合更加紧密。因此推测adropin可能影响以Hp-CD163系统参与的体内游离血红细胞清除，通过体内实验证明在小鼠体内adropin的确能促进血红蛋白清除；在细胞水平，我们发现adropin与细胞内Hbb共定位，adropin处理细胞前Hbb定位于线粒体上，当adropin与Hbb结合后从线粒体解离并游离于胞质中，进而降低线粒体膜电位，最终导致ATP合成减少和活性氧产生减少，但不会导致细胞的凋亡。 最后通过相关信号通路研究，发现adropin主要是通过c-Src—ENO1信号通路促进细胞的葡萄糖酵解过程；通过cAMP/FBP—AMPK通路影响葡萄糖吸收。

Adropin is a peptide hormone encoded by the energy homeostasis-associated (Enho) gene. It has a relative molecular mass of 4.5KDa and is widely distributed in the brain, liver, umbilical vein, coronary endothelial cells, kidney and peripheral blood. This endogenous polypeptide has the effect of regulating energy metabolism, improving insulin resistance, and increasing insulin sensitivity. This paper mainly explores the effect of adropin on the energy metabolism of HepG2 hepatoma cells, and on this basis, explores the possible targets and correlated pathways of adropin.Firstly, a high purity, active adropin polypeptide was obtained by chemical synthesis. It significantly enhanced the glucose uptake of HepG2 cells in a dose-dependent manner. However, adropin had no significant effect on blood glucose level in mice. By targeted metabolomics analysis, adropin can significantly reduce the level of ATP, GTP and FBP in HepG2 cells, and increase the level of cAMP. According to the references, the above changes significantly activate the AMPK, which is key molecule of energy metabolism regulation.Secondly, through the Co-IP method combined with mass spectrometry identification technology, the protein that may bind to adropin was found to be hemoglobin β subunit (Hbb). By surface plasmon resonance, it was proved that adropin could directly bind to Hb, and there was a dose-dependent relationship. It was also found that Hb binds more closely to adropin in the presence of haptoglobin (Hp). Therefore, it is speculated that adropin may affect the free hemoglobin clearance in vivo by Hp-CD163 system, which proved in mice. At the cellular level, adropin colocalized with intracellular Hbb. The Hbb was localized on the mitochondria without adropin. When adropin bond to Hbb, it dissociated from the mitochondria and was released into the cytoplasm, thereby reducing the mitochondrial membrane potential, which ultimately led to a decrease in ATP synthesis and a decrease in reactive oxygen species, but didn't cause apoptosis.Finally, through the related signal pathway research, it was found that adropin promote the glucose glycolysis process through c-Src-ENO1 signaling pathway, and affect glucose absorption by cAMP/FBP-AMPK pathway.