Txnip和GnT-IVa与京尼平苷调节葡萄糖转运体内吞的相关性研究

An impressive number of evidence suggests that dysfunction and apoptosis of pancreatic beta cells challenged by high glucose plays an essential role on the development of type 2 diabetes mellitus. Our previous works showed that geniposide could regulate the phosphorylation of AMPK (Adenosine 5’-monophosphate (AMP)-activated protein kinase) and degradation of Txnip (thioredoxin-interacting protein), and influence the uptake and metabolism of glucose in pancreatic beta cells and isolated rat islets in a very short time (in one hour). At the same time, geniposide also mediated the distribution of GLUT (glucose transporter ) in membrane and cytoplasm in pancreatic beta cells. But unfortunately, the mechanisms and relationship about those keep unknown. So, we hypothesize that geniposide might regulate the phosphorylation of AMPK to affect the degradation of Txnip, and then influence the interaction of Txnip and GLUT, which changed the distribution of GLUT in membrane and plasma, and finally influence the uptake of glucose and metabolism, and insulin secretion in pancreatic beta cells. To verify that, we firstly design to probe the linkage between AMPK activity and Txnip degradation regulated by geniposide. Secondly, we want to investigate the effects of geniposide on the glycosylation and endocytosis of GLUT1/2, and the role of GLcNAc-IVa glycosyltransferase (GnT-IVa) during that. Finally, using RNAi, Txnip-/- knockout and transgenic model mice, we also want to prove the role of Txnip on geniposide regulating the glycosylation and endocytosis of GLUT1/2 in vitro and in vivo, and the role of geniposide on blood sugar, insulin level and the dysfunction of pancreatic beta cells in db/db mice. The aims of this study are to explore the mechanisms of geniposide regulating glucose-stimulated insulin secretion in high glucose cultured pancreatic β cells and isolated rat islets, and to supply more targets and ideas for the prevention and treatment of type 2 diabetes mellitus.

葡萄糖转运体GLUT内吞过程对于维持机体糖稳态具有十分重要的作用。前期研究证实，京尼平苷可通过调节AMPK磷酸化和Txnip降解影响胰腺β细胞和原代胰岛对葡萄糖的吸收、代谢及胰岛素分泌，而且还能调节GLUT在胰腺β细胞膜和细胞浆中的分布。但是，相关作用机制还不清楚。本项目中，我们将首先在多种不同的细胞中分析AMPK与京尼平苷调节Txnip降解之间的关系，明确AMPK和Txnip在京尼平苷调节GLUT内吞过程中的作用；然后，在胰腺β细胞中详细考察京尼平苷对GLUT内吞的作用机制，重点考察糖基化转移酶GnT-IVa对GLUT的修饰，及其与京尼平苷调节GLUT内吞的相关性；最后，在Txnip-/-和db/db小鼠动物模型上考察京尼平苷对GLUT糖基化及其内吞的影响，以及对2型糖尿病的改善作用，为进一步明确京尼平苷调节胰腺β细胞胰岛素分泌提供科学依据，为2型糖尿病的防治提供新的思路和靶点。

葡萄糖转运体GLUT的糖基化修饰及内吞过程对于维持其正常生理功能、调节糖稳态、防止2型糖尿病的形成尤为重要。本项目在前期研究证实京尼平苷能够短时间调节胰腺β细胞葡萄糖吸收和胰岛素分泌的基础上，分析了京尼平苷对AMPK介导的Txnip降解在GLUT2内吞过程中的调节作用及相关机制，以及GnT-IVa与京尼平苷调节胰腺β细胞GLUT2内吞的相关性。研究结果表明京尼平苷通过调节AMPK磷酸化影响Txnip降解，进而调节Txnip与GLUT2的相互作用，间接影响GLUT2的内吞作用；与此同时，在胰腺β细胞中，京尼平苷可调节GnT-IVa表达直接影响GLUT2的糖基化及其在细胞膜上的驻留，并最终调节胰腺β细胞对葡萄糖的吸收、代谢和胰岛素分泌，并在体内拮抗高糖高脂诱导的β细胞凋亡。本项目的研究结果初步阐明了Txnip和GnT-IVa在京尼平苷调节细胞GLUT2内吞过程中的作用，以及京尼平苷快速调节胰腺β细胞GSIS的分子机制，为京尼平苷的临床应用提供了实验证据，为2型糖尿病的防治提供了新的思路和策略。