RNA结合蛋白QKI调控团头鲂糖异生作用的分子机制研究

Carbohydrates are the cheapest energy source for fish, and have the protein-sparing potential. However, unlike the case of mammals, fish appears to have a limited capability to use dietary carbohydrate for energy purposes. Previous studies suggested that this might be ascribed to the impaired postprandial down-regulation of gluconeogenesis. However, the potential mechanisms are still poorly understood. Through transcriptome analysis, we obtained two functional genes, namely the RNA-binding protein QKI (Quaking) and SIRT1 (silent information regulation 2 homolog 1), which are closely involved in the gluconeogenesis pathway in fish. However, the activation processes of QKI mediated by SIRT1 are barely understood, as also holds true for their roles in the regulation of gluconeogenesis. Bearing this in mind, using Megalobrama amblycephala as the target animal, the present study aims to 1) ascertain the functions of QKI in the regulation of gluconeogenesis in hepatocytes in vitro through RNA interference; 2) illustrate the activation mechanisms of QKI through the deacetylation process mediated by SIRT1; 3) identify the target molecules of QKI through RNA-immunoprecipitation (RIP); 4) induce an altered gluconeogenesis of hepatocytes through glucose manipulations, and investigate the post-transcription regulation of QKI in gluconeogenesis. The results obtained here will partly illustrate the potential mechanisms by which QKI regulates the gluconeogenesis pathway in fish. It will also facilitate our understanding of the carbohydrate utilization by fish, as would benefit the development and application of high-carbohydrate feed in the aquaculture industry.

糖类较为廉价，且具有节约蛋白质的作用。但受生理条件限制，鱼类对糖类的利用率相对较低。研究表明，鱼类糖代谢障碍可能是由糖异生作用调控的缺失或受损所致，但潜在机制尚不明确。项目组前期通过转录组分析，确定RNA结合蛋白QKI和SIRT1与鱼体糖异生作用密切相关，但二者间的相互作用及QKI介导的转录后调控在鱼体糖异生调节中的作用尚不清楚。本项目以体外培养团头鲂肝细胞为研究对象，利用RNA干扰及质粒转染技术，明确QKI在鱼体糖异生调节中的作用；在细胞水平上干扰SIRT1活性，进而探讨其介导的去乙酰化修饰对QKI转录活性的调控作用，并利用RNA免疫沉淀技术确定QKI靶分子；在此基础上，添加不同浓度葡萄糖，诱导肝细胞糖异生强度发生变化。通过QKI介导的基因转录后调控的变化，初步阐明其对鱼体糖异生作用的调控机理，以期部分揭示鱼类糖代谢障碍的分子机制，并为高糖饲料的研发和应用提供参考。

糖类来源广泛、价格低廉，是最经济的能量物质。在饲料中添加一定糖类，可以节约蛋白质和脂肪用量，进而降低饲料生产成本。但受生理条件限制，鱼类对糖类的利用率相对较低。研究表明，鱼类糖代谢障碍可能是由糖异生作用调控的缺失或受损所致，但潜在机制尚不明确。研究表明，RNA结合蛋白可能在机体糖异生作用中发挥重要作用，但相关机制尚不明确。基于此，本项目进行了如下研究：1）克隆出了团头鲂QKIa全长序列，明确了其功能位点、保守性及组织分布规律；2）通过激素负荷改变鱼体糖异生强度，探讨了QKIa及糖异生相关基因表达变化，初步阐明了QKIa与糖异生途径间的相关性；3）通过葡萄糖负荷和高糖胁迫抑制鱼体糖异生途径，探讨了QKIa表达变化，进一步验证了其在糖异生中的作用；4）结合体内、外试验，初步明确了SIRT1对QKIa活性的调控作用，阐明QKIa活性调控的上游机制；5）初步筛选出了QKIa下游分子，确定了QKI调控鱼体糖异生作用的分子途径；6）探讨了不同糖异生强度下，肝细胞中QKIa及其下游基因表达变化，确定了QKIa调控鱼体糖异生作用的分子机制。在完成项目既定目标之外，还进行了部分拓展工作，初步明确了白藜芦醇和烟酰胺通过SIRT1调控QKIa进而改善高糖胁迫团头鲂血糖稳态的分子机制，筛选出了鱼类糖代谢调控物。上述研究初步阐明了QKIa调控鱼体糖异生作用的分子途径，部分揭示了高糖胁迫导致鱼类糖代谢障碍的分子机制，为鱼类营养代谢障碍研究提供了新的思路和借鉴，并为高糖饲料在水产养殖中的应用提供了指导。