The objective of this study was to evaluate local molecular adaptations proposed to regulate protein synthesis in the mammary glands. It was hypothesized that AA and energy-yielding substrates independently regulate AA metabolism and protein synthesis in mammary glands by a combination of systemic and local mechanisms. Six primiparous mid-lactation Holstein cows with ruminal cannulas were randomly assigned to 4 treatment sequences in a replicated incomplete 4 x 4 Latin square design experiment. Treatments were abomasal infusions of casein and starch in a 2 x 2 factorial arrangement. All animals received the same basal diet (17.6% crude protein and 6.61 MJ of net energy for lactation/kg of DM) throughout the study. Cows were restricted to 70% of ad libitum intake and abomasally infused for 36 h with water, casein (0.86 kg/d), starch (2 kg/d), or a combination (2 kg/d starch+0.86 kg/d casein) using peristaltic pumps. Milk yields and composition were assessed throughout the study. Arterial and venous plasma samples were collected every 20 min during the last 8h of infusion to assess mammary uptake. Mammary biopsy samples were collected at the end of each infusion and assessed for the phosphorylation state of selected intracellular signaling molecules that regulate protein synthesis. Animals infused with casein had increased arterial concentrations of AA, increased mammary extraction of AA from plasma, either no change or a trend for reduced mammary AA clearance rates, and no change in milk protein yield. Animals infused with starch had increased milk and milk protein yields, increased mammary plasma flow, reduced arterial concentrations of AA, and increased mammary clearance rates and net uptake of some AA. Infusions of starch increased plasma concentrations of glucose, insulin, and insulin-like growth factor-I. Starch infusions increased phosphorylation of ribosomal protein S6 and endothelial nitric oxide synthase, consistent with changes in milk protein yields and plasma flow, respectively. Phosphorylation of the mammalian target of rapamycin was increased in response to starch only when casein was also infused. Thus, cell signaling molecules involved in the regulation of protein synthesis differentially responded to these nutritional stimuli. The hypothesized independent effects of casein and starch on animal metabolism and cell signaling were not observed, presumably because of the lack of a milk protein response to infused casein.

Milk protein responses to protein nutrition are typically poor and, in part, may be due to the low efficiency (approximately 25 to 30%) of converting dietary N into milk. Posthepatic availability of amino acids (AA) is not limited, yet only approximately 30% is converted into milk. The poor capture of AA by the mammary gland may relate to the imbalanced and uncoordinated timing of nutrient delivery to the gland. The infusion of essential AA improves the efficiency of utilization (0.31); however, further catabolism of AA within the mammary gland suggests that AA transport is not a major limitation. These losses may serve ancillary or functional roles, but mammary oxidation of some AA occurs only when AA extraction exceeds the stoichiometric requirements for milk protein synthesis. Intracellular substrate supply may be more limiting than is the appartus for protein synthesis. Studies utilizing isotope labeling and conducted in vitro and in vivo now suggest that circulating peptides and proteins can serve as sources of perhaps all AA for casein synthesis, but the source of these remains elusive. Constitutive protein and casein turnover contribute significantly (42 to 72%) to mammary protein synthesis. All AA are extensively channeled through an intermediary protein pool or pools that have rapid turnover rates. The AA are then incorporated into casein, which appears to be fixed in association with protein turnover. The mammary gland is a major controller of its metabolism, and the mechanisms of AA extraction and conversion into milk protein are linked to secretion events. Blood flow may be a key point of regulation whereby mechanisms sense and respond to nutrient supply and balance to the gland via alterations in hemodynamics.

The aim of this study was to monitor amino acid (AA) exchange kinetics of the mammary gland in response to an imposed limitation on His supply for milk production. Lactating goats (n = 4, approximately 120 DIM) were fed a low protein ration that provided only 77% of metabolizable protein and 100% of energy requirements for milk production. The protein deficiency was alleviated by infusion into the abomasum of an AA mixture (67 g/d) including (+H; 4.4 g/d) or excluding (-H) His. Goats were assigned to treatments (6 to 7 d) according to a switchback design. On the last day of the first two periods, [U-13C]AA were continuously infused i.v. for 7 h and arterial and mammary vein blood was withdrawn to determine plasma AA concentration and enrichment. Flow probes monitored mammary blood flow. The secretion and enrichments of AA in milk casein were monitored each hour. A three-pool model of the gland was used to derive bi-directional rates of plasma AA exchange. Arterial plasma His concentration was lower during -H infusion (8 vs. 73 microM), but those of other AA changed little. Responses to low levels of plasma His were: 1) mammary blood flow increased by approximately 33%; 2) the gland's capacity to remove plasma His increased 43-fold, whereas the gland's capacity for other AA declined by two- to threefold; and 3) influx and efflux of His by the gland decreased. Thus, as the reduction in His efflux was insufficient to offset the reduced influx, milk protein yield decreased from 118 to 97 g/d.

The response in whole-body and splanchnic tissue mass and isotope amino acid transfers in both plasma and blood has been studied in sheep offered 800 g lucerne (Medicago sutivu) pellets/d. Amino acid mass transfers were quantified over a 4 h period, by arteric+venous procedures, across the portal-drained viscera (PDV) and liver on day 5 of an intravenous infusion of either vehicle or the methylated products, choline (0.5 g/d) plus creatine (1.0 g/d). Isotopic movements were monitored over the same period during a 10 h infusion of a mixture of U-13C-labelled amino acids obtained from hydrolysis of labelled algal cells. Sixteen amino acids were monitored by gas chromatography-mass spectrometry, with thirteen of these analysed within a single chromatographic analysis. Except for methionine, which is discussed in a previous paper, no significant effects of choline plus creatine infusion were observed on any of the variables reported. Whole-body protein irreversible-loss rates ranged from 158 to 245 g/d for the essential amino acids, based on the relative enrichments (dilution of the U-I3C molecules by those unlabelled) of free amino acids in arterial plasma, and 206-519 g/d, when blood free amino acid relative enrichments were used for the calculations. Closer agreement was obtained between lysine, threonine, phenylalanine and the branched-chain amino acids. Plasma relative enrichments always exceeded those in blood (P < 0.001), possibly due to hydrolysis of peptides or degradation of protein within the erythrocyte or slow equilibration between plasma and the erythrocyte. Net absorbed amino acids across the PDV were carried predominantly in the plasma. Little evidence was obtained of any major and general involvement of the erythrocytes in the transport of free amino acids from the liver. Net isotope movements also supported these findings. Estimates of protein synthesis rates across the PDV tissues from [UJ3C] leucine kinetics showed good agreement with previous values obtained with single-labelled leucine. Variable rates were obtained between the essential amino acids, probably due to different intracellular dilutions. Isotope dilution across the liver was small and could be attributed predominantly to uni-directional transfer from extracellular sources into the hepatocytes and this probably dominates the turnover of the intracellular hepatic amino acid pools.Stable isotopes: Amino acids: Protein metabolism: Liver

We investigated the roles of insulin and amino acid (AA) in regulating milk production and the uptake of AA and blood flow (BF) by the mammary gland and hind-leg of goats (n = 4). During two periods, either saline or AA (65 g/d) was infused i.v. for 7.5 d, and, beginning on d 5, goats were subjected to a hyperinsulinemic-euglycemic clamp. The insulin clamp elevated plasma insulin levels threefold and insulin-like growth factor-1 by 27%, and euglycemia was maintained by the infusion of glucose. Arterial, mammary, and tarsal vein blood samples were obtained on d 4 and 8 of each period, and blood flow was monitored continuously by probes. Insulin and insulin plus AA infusions increased the yields of milk by 13 to 18% and protein by 10 to 21%, but AA infusion alone had no effect. The insulin clamp reduced milk fat content by 21 to 31% and yield by 8 to 19%, and reduced the yields of milk fatty acids >C16. The insulin clamp increased mammary blood flow by 42%, but insulin and AA infusions both increased hind-leg BF by 29 to 52% and by 25%, respectively. Net uptakes of most plasma AA by the udder were reduced by insulin, whereas AA infusion had no effect. For the leg, the uptake of His and Thr were decreased by insulin, whereas the infusion of AA stimulated the uptake of total essential AA. Insulin increased the uptake of glucose by the udder but not by the leg. This study suggests that the udder and leg tissues respond differently to infusions of insulin and AA; the udder was more responsive to insulin, while the leg was more responsive to AA concentralion (supply), at least in terms of AA uptake and net anabolism (protein gain or secretion).