应激影响猪GPR120受体功能及其內吞机制研究

G protein-coupled receptors (GPR) 120 is a kind of long chain fatty acid receptor. In recently years researches showed that GPR120 transduce a wide array of extracellular signals and regulate body physiology including glucose metabolism, lipid deposition, inflammation reaction. It has attracted considerable attention as a potential drug target. Yet in pigs the research on the function of GPR120 is less. In our previously studies, we found that after stress, the serum free fatty acid (FFA) of pigs increased significantly. According to G protein family characteristic, when the ligands existed for a long time, the GPR will desensitization and internalization to limit GPR signaling. Many proteins including GPR kinases (GRKs), β-arrestins et al play important roles in internalization. Whether the increased FFA content after stress influenced the GPR120 function, and whether this effect is exerted via internalization are unclear. Therefore in the present study, we will treat growing pigs and primary cultured hepatocyte with dexamethasone (Dex), analysis the GPR120 and its downstream signal changes to clarify the effect of stress on porcine GPR120 function. Meanwhile we will prepare a construct of GPR120 fused with enhanced fluorescent protein (GFP) at its C-terminus for direct imaging of the localization and internalization of GPR120 by confocal microscopy. In stable transfected cells, we add different type of fatty acids and Dex to explore the possible underlying mechanism of internalization. The results will help to further understand the porcine metabolic disorder resulted by stress, and provide new clues for stress prevention and lipid regulation in pigs. Considering the homology among FFA receptors, the findings could give new insight into the investigation of other members in this family.

G蛋白偶联受体（G protein-coupled receptor，GPR）120是一种长链脂肪酸受体，其参与调节机体糖脂代谢，介导炎症反应，近年来受人关注。但是GPR120的功能在猪上研究很少。前期实验中我们发现应激后动物血液游离脂肪酸（free fatty acid, FFA）显著升高，肝脏代谢发生变化。应激状态下升高的FFA是如何通过GPR120信号途径介导肝脏能量稳态调节，目前尚不清楚。内吞是GPR信号通路失敏的重要调控方式。应激状态下GPR120是否发生了內吞从而介导了肝脏代谢失衡？也未见报道。因此，本项目拟通过体内外的实验，深入研究猪应激状态下GPR120受体及其介导的下游信号通路的变化，并通过体外构建表达载体等实验，探索应激导致GPR120內吞的作用机制。研究结果对进一步认识应激导致的动物代谢紊乱，生长阻滞有重要的意义，为猪抗应激以及脂质代谢调控提供理论依据和新的靶点。

G蛋白偶联受体（G protein-coupled receptor，GPR）120是一种长链脂肪酸受体，其参与调节机体糖脂代谢，介导炎症反应，近年来受人关注。本课题组通过体内外的实验，深入研究应激导致GPR120內吞的机制及其介导的下游信号通路的变化，探索GPR120介导应激致机体代谢紊乱的机制。通过应激激素皮质酮和地塞米松建立模型，分析应激后GPR120在细胞中的定位以及表达变化，发现皮质酮处理显著降低了肝细胞中GPR120在细胞膜上的表达，而增加了其在细胞浆中的表达，应激状态下GPR120发生内吞进入胞浆中介导了下游信号发挥作用。通过添加GPR120的配体ALA分析GPR120在地塞米松模拟应激模型中介导的作用，发现地塞米松处理会造成小鼠肝脏损伤指标ALT显著升高，添加ALA后通过重新激活GPR120与下游的β-arrestin2结合，进而调节P-ERK1/2信号通路，缓解地塞米松导致的肝脏脂质代谢紊乱。在H2O2诱导的肝细胞氧化损伤中，添加GPR120配体DHA和TUG891可以通过激活线粒体自噬，改善线粒体功能障碍保护肝细胞免受氧化损伤。LPS刺激导致了肝脏Kupffer细胞发生焦亡，添加DHA和AA可以通过激活GPR120与NLRP3互作，从而抑制炎性小体复合物的组装，来发挥抗焦亡以及保护肝脏免受LPS损伤的作用。以断奶仔猪感染沙门氏菌为模型，发现沙门氏菌感染显著降低了仔猪肠道中GPR120的表达，导致了调节血糖的重要激素GLP-1分泌的减少，从而导致机体代谢紊乱。项目以GPR120为纽带，揭示了动物机体代谢和应激、炎症之间的关系，研究结果对进一步认识应激导致的动物代谢紊乱，生长阻滞有重要的意义，为猪抗应激以及脂质代谢调控提供理论依据和新的靶点。