Isoleucine and leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices

Abstract

Understanding the regulatory effects of individual amino acids (AA) on milk protein synthesis rates is important for improving protein and AA requirement models for lactation. The objective of this study was to examine the effects of individual essential AA (EAA) on cellular signaling and fractional protein synthesis rates (FSR) in bovinemammary cells. Omission of L-arginine, L-isoleucine, L-leucine, or all EAA reduced (P, 0.05) mammalian target of rapamycin (mTOR; Ser2448) and ribosomal protein S6 (rpS6; Ser235/236) phosphorylation inMAC-T cells. Phosphorylation ofmTOR and rpS6 kinase 1 (S6K1; Thr389) decreased (P,0.05) in the absence of L-isoleucine, L-leucine, or all EAA in lactogenicmammary tissue slices. Omission of L-tryptophan also reduced S6K1 phosphorylation (P = 0.01). Supplementation of L-leucine tomedia depleted of EAA increasedmTOR and rpS6 and decreased eukaryotic elongation factor 2 (Thr56) phosphorylation (P, 0.05) in MAC-T cells. Supplementation of L-isoleucine increased mTOR, S6K1, and rpS6 phosphorylation (P, 0.05). No single EAA considerably affected eukaryotic initiation factor 2-α (eIF2α; Ser51) phosphorylation, but phosphorylation was reduced in response to provision of all EAA (P, 0.04). FSR declined when L-isoleucine (P = 0.01), L-leucine (P=0.01), L-methionine (P = 0.02), or L-threonine (P = 0.07) was depleted in media and was positively correlated (R = 0.64, P < 0.01) with phosphorylation of mTOR and negatively correlated (R = 20.42, P = 0.01) with phosphorylation of eIF2α. Such regulation of protein synthesis will result in variable efficiency of transfer of absorbed EAA tomilk protein and is incompatible with the assumption that a single nutrient limits protein synthesis that is encoded in current diet formulation strategies. © 2012 American Society for Nutrition.