Screening Of Amino Acids Participating In The Regulation Of M TOR Signaling By T1R1/T1R3 And The Mechanism In Myoblast

The mammalian target of rapamycin(m TOR) is a central regulator of mammalian cell growth, which can form two distinct structural and functional complexes: m TORC1 and m TORC2. m TORC1 can sense a series of signals, such as energy states, growth factors and hormone, nutrients, etc, to control cell growth by regulating protein synthesis and degradation. Among the various nutrients, amino acid(AA) acts as not only the necessary substrate of protein synthesis, but also an essential regulator of m TOR activity. Of amino acids, leucine, glutamine, arginine is regarded as the effective regulator of m TORC1. Amino acid transporters involve in amino acid-dependent m TOR activation.In recent years, the study found that a G protein coupled receptor T1R1/T1R3 can be used as a broad spectrum of AA receptor, by promoting intracellular Ca2+ and activating ERK1/2 to participate in the regulation of m TOR. However, previous studies mainly were the extracellular total AA, specific which AA to participate in the process is not clear, and whether the process has AA specificity is not clear too.This experiment use C2C12 myoblast as the research model. The first part is study the expression pattern of T1R1/T1R3 in C2C12 myoblast, verify the function of T1R1/T1R3 regulate protein synthesis, and selecte extracellular AA to participate in T1R1/T1R3 mediated regulation of m TOR signaling. The second part use the AAs had been filtered out as experimental object, further explore the regulation mechanism and its AA specificity of T1R1/T1R3 activate m TORC1.The main results of first part were as follows:1. Using the Q-PCR analysis the T1R1/T1R3 expression information in the process of 2% horse serum induced C2C12 cells differentiation for 0, 2, 4, 8d. T1R1/T1R3 expressed during the process of C2C12 myoblast differentiation.2. Under the premise of AA stimulus, treated cell with signaling pathway inhibitor or agonist, western blot test p-S6K1, p-m TOR protein level, select the best inhibitor concentration. Using Ca2+ chelatingagent BAPTA-AM and ERK1/2 active inhibitor U0126 can significantly suppress the activation of m TOR induced by AA(P<0.01), and with concentration dependence effect. Pretreatment cells with T1R1 specificity of AA enhancer IMP could significantly increase the activation of m TOR induced by AA(P<0.05). Explained that in C2C12 myoblast T1R1/T1R3 can also mediate AA by promoting increase intracellular Ca2+ and activate ERK1/2 to participate in regulating the m TOR signal.2. Treated cell with different kinds of AA(50 mmol/L), western blot test p-S6K1, p-m TOR protein level, SUn SET nonradioactive method to detect the protein synthesis rate, Q-PCR detection of protein degradation marker gene. To selecte out those AA participated in T1R1/T1R3 mediated regulation of m TOR signaling. 7 kinds of AA reported could increased intracellular Ca2+, only methionine(Met) could significantly activated m TORC1(P<0.01). And Met can significantly promote the rate of protein synthesis, and significantly inhibit the protein degradation pathway key protein Mu RF-1 and Atrogin-1 levels(P<0.01). Illustrated that Met may become the main AA to research AA receptor T1R1/ T1R3 mediated regulation m TOR signal.The main results of second part were as follows:1. On the premise of Met stimulus, using signaling pathway inhibitors and agonist treatment, validated the molecular mechanism of Met regulated the m TOR signaling in C2C12 myoblast. Using Ca2+ chelatingagent BAPTA-AM, PLC specific inhibitor U73122, ERK1/2 activity inhibitor U0126, both significantly suppressed the activation of m TOR induced by AA(P<0.01). Pretreatment cells with T1R1 specificity of AA enhancer IMP could significantly increase the activation of m TOR induced by AA(P<0.05). Showed that Met can activate m TORC1, and Met regulate m TOR depended on T1R1/T1R3-PLC-Ca2+-ERK1/2 signal transduction pathways.2. Test access key protein p-ERK1/2 levels, validation of the importance of ERK1/2 in Met regulating m TOR. Ca2+ chelatingagent BAPTA-AM treatment can significantly increase p-ERK1/2 level by Met stimulate(P<0.01), and with concentration dependence effect, in contrast to the change of p-S6K1 level. IMP treatment can inhibit p-ERK1/2 levels caused by Met. But inhibition of ERK1/2 activity itself would not have this phenomenon. Illustrated that cells might have the ability compensatory remedy ERK1/2 change, ERK1/2 is indispensable in T1R1/T1R3 mediated Met regulation m TOR.3. Treated cells with different kinds of AA, test signal pathways key protein levels changes, exploring if T1R1/T1R3 mediated AA regulation m TOR has AA specificity. Met, Leu, Gln, Gly and Ala stimulate cells, along with the increasing concentration of AA, Met and Leu caused access key protein level increased gradually, and Gln, Gly and Ala did not change significantly. Leu activate m TORC1 also depended on the intracellular Ca2+ and ERK1/2. Explained that T1R1/T1R3 mediated AA regulation m TOR signal had AA specificity. Met can through T1R1/T1R3-PLC-Ca2+-ERK1/2 signal transduction pathways involved in regulating the m TOR, other AA had no significant effect. To sum up, the conclusion of this study were:1. The extracellular AA mediated by T1R1/T1R3 to regulate m TOR signaling in C2C12 myoblast mainly was Met.2. Met activate m TORC1 relied on T1R1/T1R3-PLC-Ca2+-ERK1/2 signal transduction pathways. T1R1/T1R3-PLC-Ca2+-ERK1/2 signal transduction pathways regulate m TOR had AA specificity.