Inhibitory Factors Of Casein Synthesis In Mammary Tissue Of Lactating Dairy Cows
Excess nitrogen waste excreted from dairy cows causes numerous harmful effects on the environment, such as air and water pollution. Nitrogen efficiency can be improved by feeding low protein diets and supplementing with select essential AA (EAA), as is practiced in the swine industry. However, AA metabolism within dairy animals must be further understood in order to achieve this goal. The objective of this study was to determine α-S1 casein synthesis responses to the cell signaling inhibitors rapamycin and AICAR and high and low concentrations of essential AA in mammary tissue. Three lactating Holstein cows from the Virginia Tech herd were slaughtered at a processing facility on campus. Mammary tissue from an uninfected rear quarter was collected and tissue slices (120±30 mg) were prepared and incubated 4 hours at 37°C in 5ml of treatment media enriched with [2H5] Phe. Experiment 1 examined the interaction of 3 concentrations of rapamycin and EAA in a 2 x 3 factorial design. Essential AA were included at either 5% or 100% of normal Dulbecco’s Modified Eagle Medium (DMEM) concentrations. Rapamycin was added to the medium at 0, 0.5, or 10 µM. Experiment 2 consisted of a 2 x 3 factorial design with the EAA at 5 and 100% of DMEM and AICAR at 0, 0.4, and 4.0 mM. Experiment 3 tested the interaction between six non-essential AA and protein synthesis rates. Following incubation, samples were homogenized and a 4.6 pH precipitate was isolated. [2H5] Phe enrichment of the 34-NLLRFFVAPFPE α-S1 casein peptide was determined in the precipitate via MALDI-TOF-TOF and α-S1 casein fractional synthesis rate (CFSR) was determined. Experiment 1 revealed no effect of rapamycin, EAA or their interaction on CFSR. AICAR tended to reduce CFSR. There was no effect of EAA on synthesis rate, nor any interaction between the two factors. These responses were consistent with the marginal changes in mTOR signaling changes caused by these drugs. Experiment 3 revealed no interaction between non-essential AA supply and CFSR. Further study is needed to determine other possible factors regulating CFSR beyond cell signaling and amino acid substrate supply.
Keywords: casein synthesis, rapamycin, AICAR