桑叶1-脱氧野尻霉素（DNJ）对Ⅱ型糖尿病小鼠葡萄糖激酶的调控机理

Glucokinase (GK) mainly distributed in liver and pancreatic β cells, as one of the targets of a drug for treating diabetes. Many drugs, plant active substances treat the disorders of glucose metabolism through increase the activity and expression of glucokinase (GK). Mulberry leaves as a traditional medicine have been used in China to treat diabetes for many years. Deoxynojirimycin (DNJ), especially exists in mulberry, is a potent inhibitor of intestinal a-glycosidases. Our previous reported that DNJ could significantly increase the activity and the protein expression of GK in type I diabetes mice, these results indicated that DNJ may play a hypoglycemic effect by targeting GK. In this study, purified GK enzymes will be used as materials to confirm whether DNJ is an activator on GK, the effect of DNJ on the expressions of GK and its interaction proteins GKRP, PFK2/FBPase2 will be analyzed in liver and (or) in pancreatic β cell from high fat and low STZ induced type 2 diabetic mice, and the glucose metabolic regulation signal substance such as insulin, F1P, F6P, ATP/ADP and Ca2+ et al will also be observed. This study is expected to explore the underlying mechanisms of DNJ on the up-regulated expression of GK in liver, and the mechanism of DNJ on GK-glucose-insulin interaction in pancreas in diabetic mice. These results may provide a theoretical basis for research natural drug for treat type II diabetes mellitus.

葡萄糖激酶（GK）主要分布在肝细胞和胰岛β 细胞中，为糖尿病治疗药物的作用靶点之一。许多药物、天然活性物通过改善GK活性、表达来治疗糖代谢紊乱。桑叶为传统治疗糖尿病的中药，其特有的活性物质DNJ是一种α-葡糖苷酶抑制剂。我们前期研究发现DNJ能够显著提高Ⅰ型糖尿病小鼠肝脏GK活性和基因表达，初步证实DNJ可通过GK靶点发挥降糖作用。项目拟以GK纯化酶为材料，验证DNJ是否为GK激活剂，通过研究DNJ对高脂-STZ诱导的Ⅱ型糖尿病小鼠GK及其互作调控蛋白GKRP、PFK2/FBPase2的基因表达影响，分析葡萄糖代谢调控信号物质（胰岛素、F1P、F6P、ATP/ADP和Ca2+等）的水平变化，探索DNJ上调糖尿病小鼠肝脏GK 表达的作用机制及胰腺GK-葡萄糖-胰岛素互作调控机理。本项目的研究结果对阐明DNJ的降糖靶点及作用机制有重要意义，为研发天然的治疗Ⅱ型糖尿病药物奠定基础。

葡萄糖激酶（GK）是葡萄糖酵解代谢启动的第一个调节酶，GK 在体内的活性、表达受多种因素影响。除胰岛素对GK基因表达的调控、底物及产物对其反馈作用外，GK 还受到葡萄糖激酶调节蛋白（GKR）等互作调控蛋白的调节。本项目以此为切入点，主要研究内容为：（1）利用DNJ（ 纯度＞95％）与肝脏、胰腺GK纯化酶、10%肝脏和胰腺匀浆共孵育后，分析DNJ体外对GK活性的影响，确定DNJ是否为GK直接激活剂；（2）利用高脂饲料长期诱导、高脂联合低剂量STZ诱导建立的Ⅱ型模型小鼠，分析DNJ对Ⅱ型糖尿病小鼠肝脏内GK活性的影响，分析DNJ对肝脏GK及其互作调控基因GKRP、PFK2/FBPase2 等表达水平的影响；（3）排除体液中激素（胰岛素、等）对肝脏GK活性和表达的调控干扰，分析离体培养下DNJ对肝细胞GK及互作调控基因表达的影响；以及离体培养下DNJ对胰岛β细胞（INS-1）的影响；结果发现：DNJ可防治糖尿病早期阶段的胰岛素抵抗以及后期胰岛β恶化损伤；经DNJ治疗后，肝细胞的脂滴数量明显减少，肝脏病变得到明显改善，发挥这一保护作用可能是DNJ促进了肝细胞增殖；体外酶活力测试发现，DNJ是HK的激活剂，不直接激活GK酶；West blot结果证实：DNJ可显著下调Ⅱ型糖尿病小鼠外周器官（肝脏、肌肉）中胰岛素受体、葡萄糖异生关键酶（丙酮酸羧化酶（PCB）、1、6二磷酸果糖酶（FBPase）、磷酸烯醇式丙酮酸羧化激酶（PEPCK））的蛋白表达，同时上调胰腺或肝脏中胰岛素、糖酵解关键酶（GK、GKRP、 PFK、PK等）的蛋白表达；体外培养肝细胞LO2，结果发现DNJ可显著上调GK等蛋白表达，抑制葡萄糖异生酶的蛋白水平；综合结果表明：GK活性提高主要来源于蛋白表达水平的上调，但DNJ对HK具有明显的直接激活作用，共同促进己糖代谢，从而调控血糖。同时研究还发现：桑叶DNJ对高脂诱导的脂肪肝小鼠血脂、肝脂具有一定的调控作用，主要是通过加速血脂运输、肝脏葡萄糖、TG代谢发挥这一作用。