Effects Of Threonine On Intracellular Free Amino Acid Concentrations And Signal Pathway Of Milk Protein Synthesis In MAC-T

The mammalian target of rapamycin(mTOR)links cellular metabolism and growth to a wide range of environmental changes and is important for energy homeostasis,changes in cell physiology and key growth regulators in all higher eukaryotes.Essential amino acids(EAA)can affect milk protein synthesis through the mTOR pathway,and threonine(Thr)is an important limiting AA after lysine(Lys)and methionine(Met).The objective of this study was to observe the effects on free amino acid(AA)concentrations and signaling pathways related to milk protein synthesis in mammary epithelial cells(MAC-T)of dairy cows by gradient deficiency or supplementation of Thr in the extracellular environment.Experiment 1: With a Thr concentration of 100% in complete medium,gradient deficiency of Thr at 0%,25%,50%,75% or 100% to explore the effects on each indicator after 2 h of treatment.The experiment results were(1)To observe the effects of gradient deficiency of Thr in serum starvation state on the degree of phosphorylation of signaling proteins related to milk protein synthesis.The results showed that the phosphorylation ratio of Ribosomal protein S6 kinase polypeptide1(S6K1)began to decrease significantly when Thr deficiency reached 25%,by 32.1% and 25.2% in that order(P < 0.01).When Thr deficiency reached 50%,the phosphorylation percentage of eukaryotic translation initiation factor 4E-binding protein 1(4EBP1)started to decrease significantly,by 31.9%,23.5% and 28.6%,in that order(P < 0.01).Gradient deficiency of Thr caused a significant increase in the percentage of phosphorylation of both eukaryotic initiation factor 2α(e IF2α)(P < 0.01)and protein kinase B(Akt)(P < 0.01).(2)To observe the effects of gradient deficiency of Thr on intracellular free AA concentration under serum starvation.The results showed a significant linear decrease in intracellular free Thr concentration(P < 0.01)and a significant increase in intracellular Asparagine(Asn)(P = 0.05),Serine(Ser)(P = 0.04),Histidine(His)(P = 0.03),and Aspartic acid(Asp)(P = 0.04)concentrations were significantly increased.All AA concentration except for cysteine(Cys)showed a significant linear increase or an increasing linear trend.(3)To observe the effects of gradient deficiency of Thr in serum starvation state on transcriptome results.The results showed that gradient deficiency of Thr affected functional genes and signalling pathways associated with AA transport and milk protein synthesis.The differential genes were mainly enriched in the regulation of TORC1 signaling,cell proliferation and apoptosis,cell signaling,inflammation genesis,pro-synuclein-activated protein kinase signaling pathway and extracellular growth factor stimulation response.Experiment 2: With a Thr concentration of 100% in complete medium,gradient supplementation of Thr at 0%,25%,50%,75% or 100% in EAA-free medium,one more group treated with the complete medium was set up as a positive control group,to explore the effects on each indicator after 12 h of treatment.The experiment results were(1)To observe the effects of gradient supplementation of Thr under serum starvation on the degree of phosphorylation of signaling proteins related to milk protein synthesis.The results showed that gradient supplementation of Thr resulted in a sustained increase in the phosphorylation percentage of S6K1(P < 0.01)and a significant increase in the phosphorylation percentage of 4EBP1(P <0.01),reaching a maximum at 75% Thr supplementation,and the phosphorylation percentage of both S6K1 and 4EBP1 was significantly higher in the positive control group than in the remaining groups.The phosphorylation ratios of Akt were all increased,with the 25%(P <0.01)and 50%(P < 0.01)groups showing significant differences and the positive control groups all showing significantly lower phosphorylation ratios than the remaining groups.(2)To observe the effects of gradient supplementation of Thr on intracellular free AA concentration under serum starvation conditions.The results showed a significant increase in intracellular free Thr concentration(P < 0.01),along with significant changes in intracellular concentrations of glutamine(Gln)(P = 0.04),Asp(P = 0.04),glutamate(Glu)(P = 0.04)and Met(P = 0.04).(3)To observe the effects of gradient supplementation of Thr in serum starvation state on transcriptome results.The results showed that gradient supplementation of Thr affected functional genes and signalling pathways associated with AA transport and milk protein synthesis.The differential genes were mainly enriched in mammary gland development,inositol catabolism,DNA replication,cell cycle,apoptosis,and development of inflammatory response.Overall,gradient deficiency or supplementation of Thr can affect milk protein synthesis through the two main protein downsteams of the mTOR signaling pathway,S6K1 and 4EBP1.Both gradient deficiency and supplementation of Thr had significant effects on free Thr concentration in MAC-T cells,and the trends of changes in intracellular and extracellular concentrations were similar,with changes in the extracellular environment altering the bidirectional transport of AA inside and outside the cell.By transcriptomic analysis,gradient deficiency and supplementation of Thr both affected functional genes and signalling pathways associated with milk protein synthesis,and affected different functional genes and signalling pathways respectively.