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

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