Mibefradil调节CaMKII-FoxO1通路在糖尿病糖代谢中的作用及其机制研究

Diabetes is clinical syndrome with a disorder of carbohydrate metabolism. Studies have been shown that the intracellular calcium disorders in diabetes.The disorders of hepatocytes can activate intracellular CaMKII,and increase hepatic glucose output. But it has not been reported that treat diabetes through the regulation of calciumion to improve glucose metabolism disorders. Our studies found that calcium channel antagonists Mibefradil significantly improve hyperinsulinemia in diabetes db/db mice, while reducing high blood sugar, but had no significant effect on blood glucose in normal mice, but the hypoglycemic mechanism is unknown. Further we found that mibefradil can inhibit FoxO1 nuclear translocation of diabetic hepatocyte in the preliminary experiments. Therefore, we suspect that the disorders of intracellular calcium increase liver glycogen output in diabetes , Mibefradil can lower blood sugar and to regulate activity of enzyme of glucose metabolism by CaMKII-FoxO1 pathway. For this purpose we will explore that mibefradil regulate glucose metabolism and its molecular mechanism by biochemistry, molecular biology and the related technologies of signaling pathways in db/db mice and and the liver FoxO1 knockout diabetic mice. The results can be further clarify the pathogenesis of diabetes, and it perhaps will provide new ideas of prevention of diabetes.

糖尿病是以糖代谢紊乱为特此的临床综合征。有研究表明糖尿病存在细胞内钙离子紊乱，肝细胞内钙离子紊乱可激活CaMKII，引起肝糖输出增加。但通过调节钙离子紊乱改善糖代谢异常治疗糖尿病的研究还未见报道。我们研究发现钙通道拮抗剂Mibefradil显著改善糖尿病db/db小鼠高胰岛素血症同时降低高血糖，而对正常小鼠血糖无明显影响。但其具体降糖机制不明。我们进一步预实验发现Mibefradil可以抑制糖尿病肝细胞FoxO1核转位。因此，我们猜想糖尿病肝糖输出增加与肝细胞内钙离子紊乱有关，Mibefradil可通过CaMKII-FoxO1通路调节糖代谢酶的活性而降低血糖。为此我们将运用db/db小鼠和肝脏FoxO1敲除糖尿病鼠以及生化、分子生物学和信号通路相关技术等手段，探讨Mibefradil调节糖尿病糖代谢的作用及其分子机制。其结果可进一步阐明糖尿病的发病机制，或许将为糖尿病的防治提供新思路。

本项目是在我们前期发现钙通道拮抗剂Mibefradil显著改善糖尿病db/db小鼠高胰岛素血症同时降低高血糖的基础上，旨在探讨探讨Mibefradil调节糖尿病糖代谢的作用及其分子机制。运用糖尿病模型小鼠及生化、分子生物学和信号通路相关技术等手段方法，观察Mibefradil对糖尿病模型鼠及胰岛素抵抗细胞模型的糖代谢通路的影响。以HepG2细胞作为研究对象，棕榈酸盐（palmitate，PA） 诱导 HepG2细胞成为胰岛素抵抗模型；采用链脲佐菌素（STZ）腹腔注射加高热量饲料喂养建立实验性 2 型糖尿病小鼠模型；经研究发现 Mibefradil能改善糖尿病模型小鼠血糖浓度，肝糖原贮存，提高外周组织对胰岛素的敏感性；Mibefradil调节糖尿病糖代谢的作用与两个糖异生限速酶—磷酸烯醇式丙酮酸羧基激酶(phosphoenolpyruvate carboxykinase ，PEPCK) 和葡萄糖-6-磷酸酶(Glucose-6-phosphate Dehydrogenase ，G6PC )下调有关。这一调节功能是通过调节钙离子稳态，钙调蛋白激酶(CaMKII)、AKT、FoxO1的磷酸化水平及 FoxO1的核定位实现的。