Essential Amino Acid Regulation of Cell Signaling and Casein Synthesis in Mammary Tissue

Abstract

Specific AA have been demonstrated to activate signaling pathways that regulate<br />translation initiation and to stimulate protein synthesis in mammary tissue. The<br />objectives of this research were to determine the response to Ile, Leu, Met, and Thr in<br />cellular signaling and "-S1 casein fractional synthesis rates (CFSR). An experiment was<br />developed as a composite design. The experiment was replicated in tissue corresponding<br />to 5 cows. Mammary tissue slices (0.12 ± 0.02 g) from lactating dairy cows were<br />incubated 4 h in treatment media enriched with 2H5 Phe. Following incubation, slices<br />were homogenized in lysis buffer and caseins were precipitated by acidification to pH<br />4.6. An aliquot of the pellet was trypsinized and 2H5 Phe enrichment in the 34-<br />NLLRFFVAPFPE-45 peptide of "-S1 casein was measured by MALDI TOF-MS and<br />used to determine CFSR (%/h). Western immunoblotting was performed to identify total<br />and site-specific phosphorylated mammalian target of rapamycin (mTOR, Ser2448),<br />eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein (rp) S6 (Ser235/236),<br />and eukaryotic initiation factor (eIF) 2" (Ser51). Addition of Ile, Leu, Met, or Thr had<br />no effect on eIF2" phosphorylation. Isoleucine positively affected mTOR, and rpS6, and<br />negatively affected eEF2 phosphorylation. Leu had a similar effect on eEF2, but not on<br />mTOR or rpS6, and these two AA inhibited each other. Thr negatively interacted with<br />Ile on mTOR and rpS6, and with Leu on eEF2. Increasing concentrations of Ile, Leu,<br />Met, and Thr caused curvilinear increases in CFSR. The maximum response to Ile, Leu,<br />iii<br />Met, and Thr was at 71, 49, 60, and 65% of DMEM concentrations, respectively. All<br />maximums were above plasma AA concentrations observed in lactating cows fed to meet<br />NRC requirements. The CFSR estimated at those maximums were similar between AA<br />(3.6 ± 0.6 %/h). Individual AA effects on CFSR did not correlate with mTOR signaling.<br />Independent CFSR responses to individual essential AA observed in this study contradict<br />the single-limiting AA theory assumed in current requirement systems. The saturable<br />responses of CFSR to these 4 AA also demonstrate the deficiencies of a fixed postabsorptive<br />AA efficiency approach for determining AA requirements for milk protein<br />synthesis.