Function Of MTORC1 Signaling Pathway In Lysine Regulated Skeletal Muscle Development In Growing Meat Rabbit

The purpose of this paper is to investigate the effects of lysine（Lys）on production performance,skeletal muscle development in growing meat rabbit and proliferation,autophagy and apoptosis of skeletal muscle satellite cells.（1）Animal trial was designed to investigate the effects of diet supplemented with different levels of Lys on growth,development,nutrient digestion and metabolism of rabbit fed low protein diet to identify possible potential signals related to the regulation of skeletal muscle development by Lys.A thousand healthy weaned rabbit with similar body weight（938.6 ± 6.54 g）were randomly divided into five groups（5 replicates per group,40 rabbit per replicate）.These groups consisted of normal protein group（NP group）,low protein group（LP group）,and LP group with an addition of 0.15%,0.3% or 0.45% Lys.The trial included 7 d of pre-feeding period and 28 d trial period.Compared with NP diet and LP diet,dietary addition of 0.3% Lys in LP fed rabbit group improved growth performance（P < 0.05）,full-bore weight and half-bore weight（P < 0.05）.The LP + 0.3% Lys treatment resulted in a decrease in the excretion of faecal nitrogen and urinary nitrogen and an increase in nitrogen utilisation rate（P< 0.05）.LP diet increased the m RNA levels of myostatin（MSTN）and e3 ubiquitin protein ligase 1（WWP1）and protein levels of phosphorylation p70 ribosomal protein S6 kinase 1（P-P70S6K1）,phosphorylation eukaryotic translation initiation factor 4E binding protein 1（P-4EBP1）and phosphorylation ribosomal S6 kinase 1（P-S6）（P < 0.05）,and decreased the m RNA levels of insulin like growth factor1（IGF1）（P < 0.05）.LP + 0.3% Lys treatmet attenuated the effects of LP diet on the gene expression of MSTN,WWP1,IGF1,P-P70S6K1,P-4EBP1 and P-S6（P < 0.05）.LP + 0.3% Lys treatment resulted in an increase in m RNA levels of myogenic differentiation antigen（Myo D）and protein expression of phosphorylation of mammalian rapamycin target protein（P-mTOR）relative to the NP and LP groups（P <0.05）.（2）Cell experiment was designed to investigate whether Lys directly stimulates the proliferation via mammalian rapamycin target protein complex 1（mTORC1）signaling pathway primary rabbit skeletal muscle satellite cells（SCs）.Cells were starved in Lys-free and fetal bovine serum medium for 12 h and then cultured in DMEM/F12 medium containing0.92 mmol/L Lys（Control）and 0.02 mmol/L Lys deficiency group（Lys Deficitency）with10% fetal bovine serum（FBS）for 48 h.Cells proliferation was observed.after 48 h of Lys deficiency,cells proliferation was extremely reduced.The proliferation inhibition was reversed after 48 hours by replacing the Lys Deficitency medium with a sufficient amount of Lys group（Lys Addition）.Potential mechanisms were also resolved by rapamycin（Lys Addition + 1μM Rap）and MHY1485（Lys Deficitency + 1μM MHY1485）groups.The results showed that the protein level related to mTORC1 signaling pathway,cells proliferation rate and protein synthesis rate in Lys deficiency group were significantly lower than control group（P < 0.05）.Compared with Lys deficiency group,Lys supplement group significantly activated mTORC1 signaling pathway,increased cells proliferation rate and protein synthesis rate（P < 0.05）.Compared with the control group,the light chain 3-II of microtubule-associated protein 1/light chain 3-I of microtubule-associated protein 1（LC3-II/LC3-I）protein ratio and apoptosis rate in Lys deficiency group were significantly increased,which is significantly attenuated when Lys supplement（P < 0.05）.Compared with the control group,the number of resting state/first gap（G0/G1）phase cells in Lys deficiency group was significantly higher,but the number cells in second gap/ mitosis（G2/M1）phase was significantly lower（P < 0.05）.Lysine supplementation resulted in a significant decrease in the number of cells in G0/G1 phase and a significant increase in the number of cells in G2/M1 phase.（P < 0.05）.Compared with Lys supplement group,protein phosphorylation level ralted to mTORC1 signaling pathway,cells proliferation rate and protein synthesis rate decreased significantly in rapamycin group（P < 0.05）.Compared with Lys deficiency group,MHY1485 group significantly increased protein phosphorylation level ralted to mTORC1 signaling pathway,cells proliferation rate and protein synthesis rate（P < 0.05）.Compared with Lys supplement group,the LC3-II/LC3-I protein ratio and apoptosis rate in rapamycin group increased significantly.Compared with Lys deficiency group,MHY1485 group significantly decreased LC3-II/LC3-I protein ratio and apoptosis rate（P < 0.05）.Compared with Lys supplement group,the cell number in G0/G1 phase increased significantly in rapamycin group,but cell number in G2/M1 phase decreased significantly in rapamycin group（P < 0.05）.Compared with Lys deficiency group,MHY1485 group significantly decreased cell number in G0/G1 phase and increased cell number in G2 / M1 phase（P < 0.05）.In conclusion,the addition of Lys in low protein diet could improve the production performance absorption efficiency of in growing meat rabbit and activate mTORC1 signaling pathway.Lys could promote muscle satellite cell proliferation and decrease cell autophagy and apoptosis via mTORC1 signaling pathway.