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Simple Summary  This study mainly employed metabolomics technology to determine changes of intracellular metabolite concentrations related to milk protein synthesis induced by heat stress (HS) in bovine mammary epithelial cells. HS was associated with significant differences in intracellular amino acid metabolism resulting in an increase in the intracellular amino acid concentrations. Moreover, HS promoted amino acid transportation and the activity of the mammalian target of rapamycin (mTOR) signaling pathway, which plays an important role as a central regulator of cell metabolism, growth, proliferation and survival. Greater expression of the alpha-S2-casein gene ( CSN1S2 ) was also observed during HS. Overall, our study indicated that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS.    Abstract  Heat stress (HS) is one of the most serious factors to negatively affect the lactation performance of dairy cows. Bovine mammary epithelial cells are important for lactation. It was demonstrated that HS decreases the lactation performance of dairy cows, partly through altering gene expression within bovine mammary epithelial tissue. However, the cellular metabolism mechanisms under HS remains largely unknown. The objective of this study was to determine whether HS induced changes in intracellular metabolites and gene transcription related to amino acid metabolism, amino acid transportation and the mTOR signaling pathway. Immortalized bovine mammary epithelial cell lines (MAC-T cells,  n  = 5 replicates/treatment) were incubated for 12 h at 37 °C (Control group) and 42 °C (HS group). Relative to the control group, HS led to a greater mRNA expression of heat shock protein genes  HSF1, HSPB8, HSPA5, HSP90AB1  and  HSPA1A . Compared with the control group, metabolomics using liquid chromatography tandem–mass spectrometry identified 417 differential metabolites with  p  < 0.05 and a variable importance in projection (VIP) score >1.0 in the HS group. HS resulted in significant changes to the intracellular amino acid metabolism of glutathione, phenylalanine, tyrosine, tryptophan, valine, leucine, isoleucine, arginine, proline, cysteine, methionine, alanine, aspartate and glutamate. HS led to a greater mRNA expression of the amino acid transporter genes  SLC43A1 ,  SLC38A9 ,  SLC36A1 , and  SLC3A2  but a lower mRNA expression of  SLC7A5  and  SLC38A2 . Additionally, HS influenced the expression of genes associated with the mTOR signaling pathway and significantly upregulated the mRNA expression of mTOR,  AKT ,  RHEB ,  eIF4E  and  eEF2K  but decreased the mRNA expression of  TSC1, TSC2  and  eEF2  relative to the control group. Compared with the control group, HS also led to greater mRNA expression of the  CSN1S2  gene. Overall, our study indicates that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS.

animals

Effect of Heat Stress on Bovine Mammary Cellular Metabolites and Gene Transcription Related to Amino Acid Metabolism, Amino Acid Transportation and Mammalian Target of Rapamycin (mTOR) Signaling