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 bovine mammary 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 in MAC-T cells. Phosphorylation of mTOR and rpS6 kinase 1 (S6K1; Thr389) decreased (P < 0.05) in the absence of L-isoleucine, L-leucine, or all EAA in lactogenic mammary tissue slices. Omission of L-tryptophan also reduced S6K1 phosphorylation (P = 0.01). Supplementation of L-leucine to media depleted of EAA increased mTOR 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 = -0.42, P = 0.01) with phosphorylation of eIF2α. Such regulation of protein synthesis will result in variable efficiency of transfer of absorbed EAA to milk protein and is incompatible with the assumption that a single nutrient limits protein synthesis that is encoded in current diet formulation strategies.

Current nutrient requirement models assume fixed efficiencies of absorbed amino acid (AA) conversion to milk protein. Regulation of mammary protein synthesis (PS) potentially violates this assumption by changing the relationship between AA supply and milk protein output. The objective of this study was to investigate the effects of essential AA (EAA) and insulin on cellular signaling and PS rates in bovine mammary cells. MAC-T cells were subjected to 0 or 100% of normal EAA concentrations in DMEM/F12 and 0 or 100 μg insulin/L in a 2 × 2 factorial arrangement of treatments. Lactogenic bovine mammary tissue slices (MTS) were subjected to the same treatments, except low-EAA was 5% of normal DMEM/F12 concentrations. In MAC-T cells, EAA increased phosphorylation of mammalian target of rapamycin (mTOR; Ser2448), ribosomal protein S6 kinase 1 (S6K1; Thr389), eIF4E binding protein 1 (4EBP1; Thr37/46), and insulin receptor substrate 1 (IRS1; Ser1101), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2; Thr56) and eukaryotic initiation factor (eIF) 2-α (Ser51). In the presence of insulin, phosphorylation of Akt (Ser473), mTOR, S6K1, 4EBP1, and IRS1 increased in MAC-T cells. In MTS, EAA had similar effects on phosphorylation of signaling proteins and increased mammary PS rates. Insulin did not affect MTS signaling, perhaps due to inadequate levels. Effects of EAA and insulin were independent and additive for mTOR signaling in MAC-T cells. EAA did not inhibit insulin stimulation of Akt phosphorylation. PS rates were strongly associated with phosphorylation of 4EBP1 and eEF2 in MTS. EAA availability affected translation initiation and elongation control points to more strongly regulate PS than insulin.

Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5 mmol/L EAA concentrations with 0 or 5 mmol/L acetate or 0 or 17.5 mmol/L glucose in 2 separate 2 × 2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18 mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h(-1), whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h(-1). Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.

The objectives of this study were to assess the effects of early grain feeding on acetate and glucose turnover rates and acetate and glucose preference for palmitate synthesis by subcutaneous fat (SCF), intramuscular fat (IMF), and visceral fat (VF) in finishing steers. Sixteen Angus × Simmental steers were used in the study; 8 were early weaned (EW) and fed a high-grain diet immediately after weaning for 100 or 148 d, and 8 remained with their dams on pasture until weaning at 202 ± 5 or 253 ± 5 d of age. Normal weaned (NW) and EW animals were combined and grazed to 374 ± 5 or 393 ± 5 d of age, when they were placed on a corn silage-based finishing ration until they achieved a SCF thickness of 1.0 to 1.2 cm (494 ± 17 d of age for EW steers and 502 ± 12 d of age for NW steers). Immediately before harvest, steers were continuously infused for 12 h with [2H3] acetate (1.63 mmol/min; n = 8) or [U-13C6] glucose (0.07 mmol/min; n = 8). Blood samples were collected before initiation of infusions and at the end of the infusion from 8 animals or at 1-h intervals for the first 11 h and at 15-min intervals for the last hour of infusion for the other 8 animals. Adipose tissue samples from SCF, IMF, and VF depots were collected at harvest, and lipids were extracted. Plasma enrichments of acetate and glucose and palmitate enrichment in each depot were used to calculate plasma turnover rates and fractional synthesis rates (FSR; % per h) of palmitate from each isotope. Early weaned steers had greater marbling scores compared to NW steers ( P< 0.05). Plasma turnover rates and FSR for EW and NW steers were similar except for SCF, where a greater FSR from acetate was observed for EW steers. It is possible the greater FSR for SCF was due to harvesting the animals at a slightly more advanced stage of conditioning as evidenced by the trend for greater 12th rib fat (P = 0.07). Plasma acetate turnover and palmitate FSR from acetate were much greater (P < 0.05) than the corresponding rates from glucose, supporting the primary role of acetate as an energy source and the primary substrate for lipid synthesis across fat depots. However, FSR from acetate and glucose were not different among depots, suggesting that any potential effects of dietary starch on differential deposition of energy in SCF and IMF are not substrate driven.

No abstract