猪T1R1/T1R3介导氨基酸通过Gαi2促进骨骼肌mTORC1活化的作用与机制

Mammalian target of rapamycin complex 1(mTORC1) is a crucial element in controlling protein synthesis in skeletal muscle. Among amino acids, only leucine and arginine are commonly considered to active mTORC1.In our previous study, we showed that withdrawal of other amino acids,including glycine, proline, serine and alanine, reduced mTORC1 activation.However, the underlying mechanism until recently remains largely unclear.The aim of this proposed study is to explore this mechanism,and to provide theoretical base for optimalizing nutritional manipulation tactics in regulation of skeletal muscle growth.In this proposed study, we suppose that specific amino acids trigger the translocation of mTORC1 to lysosome via T1R1/T1R3 in a Gαi2/c-Src-dependent manner in porcine skeletal muscle,which promotes leucine or insulin-induced activation of mTORC1. By evaluating the expression and colocalization of T1R1, T1R3 and Gαi2, as well as the effect of amino acids on Gαi2 signaling, interaction between T1R1/T1R3 and Gαi2 will be identified. Then porcine T1R1/T1R3-responded amino acids will be identified by evaluating effect of specific amino acid on Gαi2 signaling. We will also evaluate effect of porcine T1R1/T1R3-responded amino acids on mTORC1 translocation, as well as interaction of T1R1/T1R3-responded amino acids-induced and leucine-induced or insulin-induced mTORC1 activation. At last, the of Gαi2 and c-Src in T1R1/T1R3-induced mTORC1 translocation will be study.This proposed study provide an opportunity to understand the role of T1R1/T1R3 in regulation of mTORC1 translocation. On the basis of this proposed study, the function of porcine T1R1/T1R3 will be realized.

哺乳动物雷帕霉素靶标复合物1（mTORC1）是控制骨骼肌蛋白质合成的关键元件。一般认为在氨基酸中，仅亮氨酸和精氨酸能激活mTORC1。本课题组前期研究发现，一些其它特定氨基酸的缺乏也会影响mTORC1活性，然而其中的机制尚不明确。本项目旨在解析其中机制，完善氨基酸调控mTORC1的机理，并为建立促进猪骨骼肌生长的营养调控技术提供理论依据。项目假设为：猪骨骼肌中，特定氨基酸在T1R1/T1R3的介导下，通过Gαi2/c-Src诱导了mTORC1向溶酶体的移位，促进了亮氨酸及胰岛素对mTORC1的活化。拟通过鉴定T1R1/T1R3与 Gαi2的关系；筛选猪T1R1/T1R3的效应氨基酸；研究T1R1/T1R3介导效应氨基酸对mTORC1移位的影响，及其对亮氨酸和胰岛素诱导mTORC1活化的影响；研究Gαi2及c-Src在T1R1/T1R3诱导mTORC1移位中的作用和机制，来证实该假设。

T1R1/T1R3是动物细胞膜表面的氨基酸受体，但是猪T1R1/T1R3的效应氨基酸尚不明确，同时肌肉细胞中T1R1/T1R3感应氨基酸后对蛋白质合成关键信号mTORC1的影响也不清楚。本项目首先筛选了猪T1R1/T1R3的效应氨基，并且研究了T1R1/T1R3在介导氨基酸激活mTORC1中的作用和机制。研究结果表明，丝氨酸是猪T1R1/T1R3的最有效的效应氨基酸，能激活下游的Ca2+-ERK信号。在成熟的骨骼肌细胞中，T1R1、T1R3和Gai2共表达。在鼠源肌肉细胞中，蛋氨酸能通过Ca2+-ERK信号激活mTORC1，并且T1R1/T1R3信号的增强剂IMP能增强蛋氨酸激活mTORC1的效应。当敲低敲低TIR1或用Ca2+或ERK抑制剂处理时，能显著抑制蛋氨酸对mTORC1的激活。对于猪而言，丝氨酸激活T1R1/T1R3后，也通过Ca2+-ERK信号激活了mTORC1。G蛋白组成型激活突变试验表明，仅有Gαi2能激活Ca2+-ERK信号，并激活mTORC1。以上结果表明，在骨骼肌细胞中，氨基酸通过T1R1/T1R3-Ca2+-ERK1/2 信号通路来激活mTORC1，并且由GPCR 受体信号中的Gαi2信号介导。该研究首次明确了猪T1R1/T1R3主要感应的氨基酸类型与人源T1R1/T1R3主要感应谷氨酸、鼠源T1R1/T1R3 能广谱性感应氨基酸存在明显不同，提示氨基酸的感应及下游信号存在典型种属特异性。此外，该研究明确了T1R1/T1R3 感应氨基酸促进mTORC1的作用和机制，突破了以往认为氨基酸必须转运进肌细胞内才能激活mTORC1 的传统观点，将氨基酸调控mTORC1的起始信号发生位点提前到细胞膜，是功能性氨基酸研究的重要发展。