LAUSR

Hyperthermia alters utilization of AA in protein synthesis and cell-signaling activity in bovine mammary cells. Essential AA and insulin regulate translation of proteins by controlling the activity of mammalian target of rapamycin (mTOR) signaling pathway. The objectives of this study were to evaluate (1) the effects of incubation temperature on the mTOR signaling pathway and transcription of AA transporters in a bovine mammary alveolar cell line (MAC-T) and (2) the combined effects of incubation temperature and insulin on the mTOR signaling pathway in this cell line. Cells were cultured in medium with 10% fetal bovine serum at 37°C and 5% CO2. In experiment 1, cells were subjected to 37°C (control) or 41.5°C (high incubation temperature; HT) for 12 h. In experiment 2, cells were assigned to 1 of 4 treatments as a 2 × 2 factorial arrangement, including 2 cell culture temperatures (control and HT) and absence or presence of 1.0 μg/mL of insulin. Proteins were harvested and separated by gel electrophoresis. In experiment 1, gene expression of AA transporters (SLC1A1, SLC1A5, SLC3A2, SLC7A1, SLC7A5, and SLC36A1) were evaluated, and changes of ≥2 fold were deemed significantly different. In experiments 1 and 2, immunoblotting was used to identify total and site-specific phosphorylated forms of protein kinase B (Akt1; Ser473), p70 S6 kinase (S6K1; Thr389), ribosomal protein S6 (rpS6; Ser235/236), and eukaryotic elongation factor 2 (eEF2; Thr56). Phosphorylated and total forms of Akt1, S6K1, rpS6, and eEF2 were quantified and expressed as the ratio of phosphorylated to total protein. In experiment 1, HT resulted in a ≥2-fold increase expression of SLC1A1 and SLC3A2. High incubation temperature reduced the phosphorylated to total ratio of Akt1 and rpS6 and increased the phosphorylated to total ratio of eEF2. In experiment 2, we found no temperature by insulin interactions on phosphorylation state of the protein factors of interest. High incubation temperature reduced the phosphorylated to total ratio of Akt1. The addition of insulin increased the phosphorylated to total ratio of Akt1, S6K1, and rpS6. In summary, HT reduced the activity of the mTOR signaling pathway and increased the expression of AA transporters. High incubation temperature possibly reduced protein translation by reducing the mTOR signaling pathway activity in an effort to adapt to thermal stress. These results may help explain the direct effect of elevated temperature on AA metabolism and protein translation in heat-stressed animals.