中华鳖适应饥饿胁迫的糖代谢模式及调控机制

Animals often suffer from food deprivation under natural conditions and consequently face starvation due to the fluctuations of food resource. The adaptation capacity to starvation stress is important for the survival of the species. There are very big differences in enduring starvation among different animals. It is well known that reptile such as turtles can endure long periods of starvation, while the adaption mechanism is still open. .This project intends to study the adaptation mechanism for turtles in carbohydrate metabolism under starvation stress. we select the Chinese soft-shelled turtle as the experimental animal. Molecular and physiological methods are adopted in this study. The changes for the glucose metabolism pathways, the glucose sensing and the glucose control system of the turtle will be pursued during starvation and re-feedings peroids in this project. Glycogen contents in tissue, plasma glucose and lactate level will be determined; the activities of some key enzymes in glycolysis and gluconeogenesis will be analyzed; Plasma glucose regulation hormones such as insulin, glucagon and insulin-like growth factor-1(IGF-1) will be determined by radioimmunoassay. At the same time, the mRNA expression levels for enzymes and hormones will be also analyzed using fluorescence quantitative PCR methods. The expression Glucose sensoring (GLUT2 and IGFBP-1) will be determined by fluorescence quantitative PCR. .On the one hand, the desired results will reveal the adaptation strategy to starvation for Chinese soft-shelled turtle, and provide data for understanding the evolution significance in food habit. On the other hand, this project will clarify the carbohydrate mechanism, and it will be expected to give directions in improving the carbohydrate utilization efficeiency of Chinese soft-shelled turtle in the future.

动物常常在生活史的一定阶段面临饥饿胁迫，爬行动物有着很强的耐受饥饿的能力，但其适应机制尚不清楚。本项目拟以中华鳖为研究对象，综合运用动物生理学、生态学、营养学、生物化学及分子生物学等现代生物学研究技术与方法，从两个研究水平（生理和分子）、四个代谢层次（代谢产物、代谢酶、代谢激素及感知因子）系统地对中华鳖饥饿胁迫下的糖代谢机制展开研究。通过测定中华鳖体内糖原、血糖、糖酵解与糖异生关键酶的变化，阐明糖代谢模式的适应变化；通过测定糖代谢激素及感知与调控因子的变化，阐明糖代谢调控的适应机制；进一步揭示中华鳖适应饥饿胁迫的糖代谢机制；此外，确定对中华鳖有效的葡萄糖代谢调控的信号通路，提出改善中华鳖糖利用效率的措施。本项目预期研究结果将有助于提高对爬行动物适应生理学的认识，丰富中华鳖糖代谢的研究资料，为提高中华鳖的糖利用效率提供理论指导。

本研究对比探讨了中华鳖对内源糖及外源糖利用过程中糖代谢产物、糖代谢酶、糖代谢激素及感知因子的变化规律，阐述了中华鳖的糖利用模式及调控机制，阐释了中华鳖适应饥饿胁迫的糖利用机制。结论如下：①中华鳖在腹腔注射1000 mg/kg(BW)葡萄糖后出现持久高血糖现象，持续24 h，表现出与肉食性鱼类相似的对外源葡萄糖的较低耐受性。胰岛素分泌不足、糖异生作用没有受到显著抑制是中华鳖不能有效利用葡萄糖的主要原因。②本研究首次发现在中华鳖的胰腺中并没有刺激胰岛素分泌的葡萄糖感受器—葡萄糖转运蛋白2 (GLUT2)的表达，这与鸡的结果相同，其在物种进化上的意义有待进一步探明。但肝脏中GLUT2表达量随血糖水平升高而显著上升，峰值滞后于血糖峰值3h；血浆甘油三酯含量随血糖水平升高也显著上升，于血糖峰值1h后达峰值，肝脏糖原合成酶表达量和酶活都在血糖峰值6h后达到峰值，大部分葡萄糖由GLUT2转运到肝细胞中合成了脂肪和糖原，是外源葡萄糖被利用的主要途径。③胰岛素样生长因子结合蛋白-1—胰岛素样生长因子-1(IGFBP-1—IGF-1)不是有效调控葡萄糖代谢、降低血糖水平的信号通道，其在中华鳖饥饿再投喂后的补偿生长中发挥重要作用。④30℃恒温、220d饥饿期内，体重和肝体比分别下降32.5%和73.51%的中华鳖存活率仍高达100%。饥饿时中华鳖优先动用的是体脂肪和少量糖类，30d后开始动用体蛋白质。血糖在饥饿胁迫发生第7d显著下降，此后稳定。血糖内稳态的保持是中华鳖能够耐受220d饥饿的重要原因。⑤饥饿胁迫显著降低了糖酵解途径中的3个关键酶，但糖异生途径中的磷酸烯醇式丙酮酸羧激酶PEPCK却显著升高，胰岛素下降而胰高血糖素(GCG)上升。饥饿胁迫下糖酵解作用下降，糖异生作用的增强保证了非糖物质向葡萄糖的转化，维持了血糖稳定。⑥饥饿胁迫下中华鳖肝脏IGFBP-1被诱导表达，IGFBP-1表达的提高作为一个分子转换信号，抑制了IGF信号系统，IGF-1表达下降，从而将有限的能量从生长转移到保证动物生存的物质代谢中。长期饥饿胁迫下，IGFBP-1和GCG是维持血糖稳定的主要调节因子与激素。