高支链氨基酸血症调控肝细胞Betatrophin表达影响甘油三酯代谢的机制研究

On the basis of our findings that high blood branched chain amino acids (BCAAs) may induce abnormal lipid metabolism (increase in triglycerides [TG]) and significantly increase the risk for cardiovascular diseases (Int J Endocrinol.2016. 2016:8173905.). In addition, we also found metabolic factor Betatrophin was positively related to TG, but had no relationship with LDL-C, which were similar to those of BCAAs; serum BCAAs level was also positively associated with serum Betatrophin. Further in vitro study showed BCAAs treatment could increase Betatrophin mRNA expression and TG secretion in HepG2 cells to different extents. Thus, we speculate that high blood BCAAs may regulate mTOR/SREBP signaling pathway to up-regulate Betatrophin expression in hepatocytes and then affect TG concentration, which increases the risk for cardiovascular diseases. In this study, LC-MS/MS, Real-time PCR, RNA silencing, RNA pull down and co-immunoprecipitation will be employed to investigate the role of mTOR/SREBP/Betatrophin in the pathogenesis of BCAAs related cardiovascular disease. Our findings may provide new theoretical evidence and scientific basis for the pathogenesis of amino acid metabolic disorder related lipid metabolism disorder and cardiovascular diseases.

基于前期研究，即高支链氨基酸（BCAAs）血症可引起脂代谢异常（甘油三脂TG升高），同时加剧心血管疾病风险（Int J Endocrinol. 2016:8173905.）。随后发现促代谢因子（Betatrophin）与脂代谢相关性结果和BCAAs相似，且血清BCAAs水平与血清Betatrophin水平成正相关。进一步体外研究表明，用BCAAs刺激HepG2后，Betatrophin mRNA和TG含量均有不同程度的上调。由此提出高BCAAs血症通过调控mTOR/SREBP信号通路上调肝细胞Betatrophin表达影响TG浓度，从而加剧心血管疾病风险的科学假说。本研究拟利用LC-MS/MS检测、RNA干扰、RNA pull down、免疫共沉淀等手段阐明mTOR/SREBP/Betatrophin在BCAAs致心血管疾病中的作用，将为氨基酸代谢紊乱导致心血管疾病的发病机制提供新的理论。

基于前期研究，本项目纳入了1302名受试者，通过队列研究纵向分析血清BCAAs水平与Betatrophin和甘油三酯（TG）的关系以及心血管疾病的相关性。构建了慢性代谢性高BCAAs血症大鼠模型，评估高BCAAs对机体代谢的影响及其与潜在机制。此外，在细胞水平研究BCAAs与通过betatrophin调控TG代谢机制。.通过人群研究发现，随着基线BCAAs的增加，心血管疾病发病率逐渐升高。有趣的是，TG呈现先降低后升高的趋势，其BCAAs转折点浓度在70.9 g/mL-79.8g/mL。此外，缬氨酸、亮氨酸、异亮氨酸及总BCAAs是心血管疾病发病的独立危险因素。.动物实验中，构建了高果糖诱导的高BCAAs血症这种慢性代谢紊乱引起的高BCAAs血症。高BCAAs大鼠血清TG明显升高，同时肝内TG蓄积增多，Betatrophin表达水平明显升高。心肌masson染色显示，高BCAAs血症大鼠心肌纤维化水平相对较高，并且BCAAs血症大鼠心肌凋亡水平亦升高。这一结果初步提示慢性高BCAAs可诱导产生心肌损伤，侧面揭示高BCAAs血症人群心血管疾病发病率升高的原因。.细胞水平中，重点研究BCAAs通过betatrophin调控TG代谢机制。与对照组相比，LO2在含5mmol/L BCAAs培养下Betatrophin mRNA及蛋白水平均显著升高。敲低LO2细胞中的Betatrophin表达后，TG浓度降低。进一步研究发现，BCAAs可能通过p38-MAPK依赖的mTOR/SREBP-1c信号通路调控 betatrophin 表达影响TG 代谢。.本项目证实BCAAs通过上调肝细胞Betatrophin表达影响TG代谢这一假说，阐明BCAAs升高致TG代谢紊乱的机制，为氨基酸代谢异常导致脂代谢紊乱，进而引发心血管疾病的发病机制提供新的理论依据和科学基础。