Symposium review: Amino acid uptake by the mammary glands: Where does the control lie?

Milk protein yield responses to changes in the profile of essential amino acids absorbed by the gastrointestinal tract or circulating in blood plasma do not follow the classic limiting amino acid response, in part because of an ability of the mammary glands to modify their blood flow rate and net clearance of amino acids out of plasma. The hypothesis that mammary blood flow is locally regulated to maintain ATP balance accounts for observed changes in flow due to postruminal glucose, insulin, and essential amino acid (EAA) infusions. An additional hypothesis that net mammary uptakes of metabolites from blood are affected by perturbations in their respective arterial concentrations and the rate of mammary blood flow also appears to hold for the energy metabolites glucose, acetate, β-hydroxybutyrate, and fatty acids. However, net EAA uptakes by the mammary glands are poorly predicted by models considering arterial concentrations and blood flow rates only. Evidence points to intramammary protein synthesis and secretion as the determinant of net EAA uptake. The intracellular signaling network anchored by the mechanistic target of rapamycin complex 1 stands as an excellent candidate to explain nutritional effects on milk protein synthesis because it integrates information on physiological and nutritional state to affect protein synthesis and cell metabolism, growth, proliferation, and differentiation in many cell types. In mammary cells in vitro and in vivo, the mechanistic target of rapamycin complex 1, integrated stress response, and glycogen synthase kinase-3 networks that contribute to regulation of initiation of mRNA translation are responsive to acute changes in nutrient supply and EAA profile. However, after several days of postruminal infusion of balanced and imbalanced EAA profiles, these signaling networks do not appear to continue to account for changes in milk protein yields. Gene expression evidence suggests that regulation of components of the unfolded protein response that control biogenesis of the endoplasmic reticulum and differentiation of a secretory phenotype may contribute to effects of nutrition on milk protein yield. Connections between early signaling events and their long-term consequences should be sought.     

Casein synthesis is independently and additively related to individual essential amino acid supply

Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of α_S1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2⁴ factorial. Other AA were at the concentration in Dulbecco's modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4 h at 37°C in 5 mL of treatment medium containing ²H₅-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with ²H₅-Phe of the N[34]LLRFFVAPFPE α_S1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and α_S1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P < 0.1 and were removed from the model. Increasing concentrations of Ile, Leu, Met, and Thr simultaneously increased CFSR curvilinearly with a predicted maximum response of 4.32 ± 0.84%/h at 63% of DMEM concentrations. The maximum response to each of the 4 AA was at 71, 49, 60, and 32% of the concentration in DMEM, for Ile, Leu, Met, and Thr, respectively. These values correspond to 270, 120, 440, and 140% the plasma concentrations of Ile, Leu, Met, and Thr observed in lactating cows fed to meet National Research Council requirements, respectively. The CFSR estimated at those maxima were similar among AA (3.6 ± 0.6%/h). Individual AA effects on CFSR did not correlate with mammalian target of rapamycin (mTOR) signaling. Independent responses of CFSR to individual essential AA observed in this study contradict the single-limiting AA theory assumed in current requirement systems. The saturable responses in CFSR to these 4 AA also highlight the inadequacy of using a fixed postabsorptive AA efficiency approach for determining AA requirements for milk protein synthesis.     

Dietary energy source in dairy cows in early lactation: energy partitioning and milk composition

Metabolic problems related to negative energy balance suggest a role for the balance in supply of lipogenic and glucogenic nutrients. To test the effect of lipogenic and glucogenic nutrients on energy partitioning, energy balance and nitrogen balance of 16 lactating dairy cows were determined by indirect calorimetry in climate respiration chambers from wk 2 to 9 postpartum. Cows were fed a diet high in lipogenic nutrients or a diet high in glucogenic nutrients from wk 3 prepartum until wk 9 postpartum. Diets were isocaloric (net energy basis) and equal in intestinal digestible protein. There was no effect of diet on metabolizable energy intake and heat production. Cows fed the lipogenic diet partitioned more energy to milk than cows fed the glucogenic diet [1,175 ± 18 vs. 1,073 ± 12 kJ/(kg^0.75·d)] and had a higher milk fat yield (1.89 ± 0.02 vs. 1.67 ± 0.03 kg/d). The increase in milk fat production was caused by an increase in C16:0, C18:0, and C18:1 in milk fat. No difference was found in energy retained as body protein, but energy mobilized from body fat tended to be higher in cows fed the lipogenic diet than in cows fed the glucogenic diet [190 ± 23 vs. 113 ± 26 kJ/(kg^0.75·d)]. Overall, results demonstrate that energy partitioning between milk and body tissue can be altered by feeding isocaloric diets differing in lipogenic and glucogenic nutrient content.     

Energy and nitrogen partitioning in dairy cows at low or high metabolizable protein levels is affected differently by postrumen glucogenic and lipogenic substrates

This study tested the effects of energy from glucogenic (glucose; GG) or lipogenic (palm olein; LG) substrates at low (LMP) and high (HMP) metabolizable protein levels on whole-body energy and N partitioning of dairy cattle. Six rumen-fistulated, second-lactation Holstein-Friesian dairy cows (97 ± 13 d in milk) were randomly assigned to a 6 × 6 Latin square design in which each experimental period consisted of 5 d of continuous abomasal infusion followed by 2 d of rest. A total mixed ration consisting of 42% corn silage, 31% grass silage, and 27% concentrate (dry matter basis) was formulated to meet 100 and 83% of net energy and metabolizable protein requirements, respectively, and was fed at 90% of ad libitum intake by individual cow. Abomasal infusion treatments were saline (LMP-C), isoenergetic infusions (digestible energy basis) of 1,319 g/d of glucose (LMP-GG), 676 g/d of palm olein (LMP-LG; major fatty acid constituents are palmitic, oleic, and linoleic acid), or 844 g/d of essential AA (HMP-C), or isoenergetic infusions of 1,319 g/d of glucose + 844 g/d of essential AA (HMP-GG) or 676 g/d of palm olein + 844 g/d of essential AA (HMP-LG). The experiment was conducted in climate respiration chambers to determine energy and N balance in conjunction with milk production and composition, nutrient digestibility, and plasma constituents. Infusion of GG and LG decreased dry matter intake, but total gross energy intake from the diet plus infusions was not affected by GG or LG. Furthermore, GG or LG did not affect total milk, protein, or lactose yields. Infusing GG or LG at the HMP level did not affect milk production differently than at the LMP level. Infusion of GG stimulated energy retention in body tissue, increased plasma glucose and insulin concentrations, decreased lipogenic metabolites in plasma, and decreased milk fat yield and milk energy output. Nitrogen intake decreased and milk N efficiency increased in response to GG, and N retention was not affected. Infusion of LG tended to increase metabolizable energy intake, increased milk fat yield and milk energy output, increased plasma triacylglycerides and long-chain fatty acid concentrations, and had no effect on energy retention. Infusion of LG decreased N intake but did not affect milk N efficiency or N retention. Compared with the LMP level, the HMP level increased dry matter intake, gross and metabolizable energy intake, and total milk, fat, protein, and lactose yields. Milk energy output increased at the HMP level, and protein level did not affect total energy retention. Heat production increased at the HMP level, but only when GG and LG were infused. The HMP level increased N intake, milk N output, and plasma urea concentration, tended to increase N retention, and decreased milk N efficiency. Regardless of protein level, GG promoted energy retention and improved milk N efficiency, but not through increased milk protein yield. Infusion of LG partitioned extra energy intake into milk and had no effect on milk N efficiency.     

Vascular sources of amino acids for milk protein synthesis in goats at two stages of lactation

An arteriovenous technique, combined with a 30-h i.v. infusion of [5-(13)CH3]Met and [5,5,5-(2)H]Leu, was used to monitor mammary uptake of free amino acid (AA) and to estimate the proportion of casein synthesized from circulating peptides in goats in early and late lactation. At both stages, kinetics was performed on the last day of consecutive 5.5-d periods. The first period was an i.v. infusion of saline and the second an i.v. infusion of lysine (8.9 g/h) plus methionine (2 g/h). Net uptake of essential AA and protein yields were higher in early than in late lactation. Uptake of free Met, His, and Pro was less than, uptake of Tyr and Lys was equal to, and uptake of Arg, Leu, Val, and Ile was greater than milk protein synthesis. Peptide uptake, estimated from the difference in casein and plasma free AA enrichment, accounted for a larger fraction of casein-Met (17 vs. 8%) and casein-Leu (27 vs. 12%) in late than in early lactation. Small decreases in mammary blood flow, AA transport activity, and AA concentrations accounted for the lower uptake of AA in late compared with early lactation. Based on our studies of several AA, the utilization of circulating peptides for casein synthesis appears to be a general phenomenon.     

Invited review: Current representation and future trends of predicting amino acid utilization in the lactating dairy cow

In current dairy production systems, an average of 25% of dietary N is captured in milk, with the remainder being excreted in urine and feces. About 60% of total N losses occur postabsorption. Splanchnic tissues extract a fixed proportion of total inflow of each essential AA (EAA). Those EAA removed by splanchnic tissues and not incorporated into protein are subjected to catabolism, with the resulting N converted to urea. Splanchnic affinity varies among individual EAA, from several fold lower than mammary glands’ affinity for the branched-chain AA to similar or higher affinity for Phe, Met, His, and Arg. On average, 85% of absorbed EAA appear in peripheral circulation, indicating that first-pass removal is not the main source of loss. Essential AA in excess of the needs of the mammary glands return to general circulation. High splanchnic blood flow dictates that a large proportion of EAA that return to general circulation flow through splanchnic tissues. In association with this constant recycling, EAA are removed and catabolized by splanchnic tissues. This results in splanchnic catabolism equaling or surpassing the use of many EAA for milk protein synthesis. Recent studies have demonstrated that EAA, energy substrates, and hormones activate signaling pathways that in turn regulate local blood flow, tissue extraction of EAA, and rates of milk protein synthesis. These recent findings would allow manipulation of dairy diets to maximize mammary uptake of EAA and reduce catabolism by splanchnic tissues. Dairy cattle nutrient requirement systems consider EAA requirements in aggregate as metabolizable protein (MP) and assume a fixed efficiency of MP use for milk protein. Lysine and Met sufficiency is only considered after MP requirements have been met. By doing so, requirement systems limit the scope of diet manipulation to achieve improved gross N efficiency. Therefore, this review focuses on understanding the dynamics of EAA metabolism in mammary and splanchnic tissues that would lead to improved requirement prediction systems. Inclusion of variable individual EAA efficiencies derived from splanchnic and mammary responses to nutrient and hormonal signals should help reduce dietary protein levels. Supplementing reduced crude protein diets with individual EAA should increase gross N efficiency to more than 30%, reducing N excretion by the US dairy industry by 92,000 t annually.     

Mammary gland utilization of amino acids and energy metabolites differs when dairy cow rations are isoenergetically supplemented with protein and fat

Mammary gland utilization of AA and other metabolites in response to supplemental energy from protein (PT) and supplemental energy from fat (FT) was tested in a 2 × 2 factorial arrangement using a randomized complete block design. Fifty-six Holstein-Friesian dairy cows were adapted during a 28-d control period to a basal total mixed ration consisting of 34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter (DM) basis. Experimental rations were fed for 28 d immediately following the control period and consisted of (1) low protein, low fat (LP/LF), (2) high protein, low fat (HP/LF), (3) low protein, high fat (LP/HF), and (4) high protein, high fat (HP/HF). To obtain the high-protein (HP) and high-fat (HF) diets, intake of the basal ration was restricted and supplemented isoenergetically [net energy (MJ/d) basis] with 2.0 kg/d rumen-protected protein (soybean + rapeseed, 50:50 mixture on a DM basis) and 0.68 kg/d hydrogenated palm fatty acids on a DM basis. Arterial and venous blood samples were collected on d 28 of both periods. Isoenergetic supplements (MJ/d) of protein and fat independently and additively increased milk yield, PT increased protein yield, and FT increased fat yield. A PT × FT interaction affected arterial concentration of all essential AA (EAA) groups, where they increased in response to PT by a greater magnitude at the LF level (on average 35%) compared with the HF level (on average 14%). Mammary gland plasma flow was unaffected by PT or FT. Supplementation with PT tended to decrease mammary clearance of total EAA and decreased group 1 AA clearance by 19%. In response to PT, mammary uptake of total EAA and group 2 AA increased 12 and 14%, respectively, with significantly higher uptake of Arg, Ile, and Leu. Energy from fat had no effect on mammary clearance or uptake of any AA group. The mammary gland uptake:milk protein output ratio was not affected by FT, whereas PT increased this ratio for EAA and group 2 AA. Arterial plasma insulin concentration decreased in response to FT, in particular on the HP/HF diet, as indicated by a PT × FT interaction. Arterial concentrations of nonesterified fatty acids, triacylglycerol, and long-chain fatty acids increased in response to FT, and concentrations of β-hydroxybutyrate and acetate decreased in response to FT only at the HP level. Mammary clearance and uptake of triacylglycerol and long-chain fatty acids increased in response to FT. Energy from PT and FT increased lactose yield despite no change in arterial glucose concentration or mammary glucose uptake. Mammary-sequestered glucose with PT or FT was used in the same amount for lactose synthesis, and a positive net mammary glucose balance was found across all treatments. Results presented here illustrate metabolic flexibility of the mammary gland in its use of aminogenic versus lipogenic substrates for milk synthesis.     

发酵乳在发酵和低温储存过程中必需氨基酸变化研究

研究了混菌发酵乳与单菌发酵乳在发酵和4℃储存过程中必需氨基酸含量变化,并与作为底物的脱脂乳粉氨基酸组分进行对比。研究表明,在发酵和储存过程中总游离氨基酸含量增加;随储存时间延长,必需氨基酸含量增加,约占总游离氨基酸含量13%—17%;混菌发酵乳游离氨基酸和必需氨基酸含量高于单菌发酵乳。     

Regulation of protein synthesis in mammary glands of lactating dairy cows by starch and amino acids

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 × 4 Latin square design experiment. Treatments were abomasal infusions of casein and starch in a 2 × 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 8 h 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.     

Dietary energy source in dairy cows in early lactation: metabolites and metabolic hormones

Negative energy balance-related metabolic disorders suggest that the balance between available lipogenic and glucogenic nutrients is important. The objectives of this study were to compare the effects of a glucogenic or a lipogenic diet on liver triacylglycerides (TAG), metabolites, and metabolic hormones in dairy cows in early lactation and to relate metabolite concentrations to the determined energy retention in body mass (ER). Sixteen dairy cows were fed either a lipogenic or glucogenic diet from wk 3 prepartum to wk 9 postpartum (pp) and were housed in climate respiration chambers from wk 2 to 9 pp. Diets were isocaloric (net energy basis). Postpartum, cows fed a lipogenic diet tended to have higher nonesterified fatty acid concentration (NEFA; 0.46 ± 0.04 vs. 0.37 ± 0.04 mmol/L) and lower insulin concentration (4.0 ± 0.5 vs. 5.5 ± 0.6 μIU/mL). No difference was found in plasma glucose, β-hydroxybutyrate, insulin-like growth factor-I, and thyroid hormones. Liver TAG was equal between both diets in wk −2 and 2 pp. In wk 4 pp cows fed the glucogenic diet had numerically lower TAG levels, although there was no significant dietary effect. Negative relationships were detected between ER and milk fat and between ER and NEFA. A positive relationship was detected between ER and insulin concentration. Overall, results suggest that insulin plays a regulating role in altering energy partitioning between milk and body tissue. Feeding lactating dairy cows a glucogenic diet decreased mobilization of body fat compared with a lipogenic diet. The relative abundance of lipogenic nutrients, when feeding a more lipogenic diet, is related to more secretion of lipogenic nutrients in milk, lower plasma insulin, and higher plasma NEFA concentration.     

Isoleucine,leucine, methionine,and threonine effects on mammalian target of rapamycin signaling in mammary tissue

Improved representation of postabsorptive N metabolism in lactating dairy cows requires a better understanding of protein synthesis regulation in the mammary glands. This study aimed to determine the quantitative effects of Ile, Leu, Met, and Thr on the phosphorylation state of signaling proteins that regulate protein synthesis. The experiment used a composite design with a central point, 2 axial points per AA, and a complete 2⁴ factorial. All of the other AA were provided at the concentrations in Dulbecco's modified Eagle's medium. The experiment was replicated with tissues from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4 h. Total and site-specific phosphorylated mammalian target of rapamycin (mTOR; Ser2448), eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 2α (Ser51) were determined by western immunoblotting. Tissue concentrations of the 4 AA studied responded linearly to media supply. Addition of Ile, Leu, Met, or Thr had no effect on eukaryotic initiation factor 2α phosphorylation. Isoleucine and Thr positively affected mTOR phosphorylation. However, the 2 AA had an antagonistic relationship. Similarly, Ile linearly increased ribosomal protein S6 phosphorylation, and Thr inhibited the Ile effect. In addition, eEF2 phosphorylation was linearly decreased by Ile and Leu. Threonine curvilinearly decreased eEF2 phosphorylation, Ile and Leu negatively interacted on eEF2, and Thr tended to inhibit Leu effects on eEF2. This work demonstrated saturable responses and interactions between AA on activation of the mTOR pathway. Incorporation of these concepts into milk protein response models will help to improve milk and milk protein yield predictions and increase postabsorptive N efficiency and reduce N excretion by dairy cows.     

Variations in mammary metabolism during the natural filling of the udder with milk over a 12-h period between two milkings

Two groups of four Holstein cows, one in their second and the other in their third or fourth lactation, were used to study temporal variations of mammary metabolism over a 12-h period between two milkings. Blood samples were collected every 30 min from an artery and a mammary vein during a 12-h interval between two milkings. Isoleucine, leucine, lysine, methionine, and phenylalanine mammary net fluxes varied or tended to change over time after milking with a similar pattern between whole blood and plasma. For these amino acids, whole blood and plasma net fluxes reached their maximum over the first 8 h after milking. Simultaneously, respiratory quotients decreased linearly and varied from 2.31 to 2.01 during the first 8 h of the period, suggesting active mammary lipogenesis. From 8 to 12 h after milking, mammary amino acid net fluxes decreased, while mammary oxygen uptake tended to increase with a concomitant decrease in the respiratory quotient reaching 1.84 to 1.40. These findings suggest that, beginning 8 h after milking, mammary uptake of amino acids starts to decrease and catabolic processes appear promoted; this phenomenon could help to explain the increase in milk production reported in the literature with increased milking frequency.     

Effect of level of metabolizable protein on splanchnic flux of amino acids in lactating dairy cows

The response of splanchnic tissue metabolism to different levels of metabolizable protein (MP) was measured in 6 catheterized multiparous lactating Holstein cows. Three diets, balanced to provide similar energy intakes and increasing amounts of MP (g/d)-1922 (low), 2264 (medium), and 2517 (high)-were fed during 21-d experimental periods according to a replicated Latin square. On d 18, 19, or 20, six hourly blood samples were collected simultaneously from the portal and hepatic veins plus an artery to determine net fluxes of nutrients across the portal-drained viscera and the liver. Yields of milk and protein increased, as did urinary N excretion with increasing MP. Portal absorption of essential amino acids (EAA) increased linearly with increasing MP supply, as did liver removal of His, Met, and Phe. In contrast, liver removal of the branched-chain AA (BCAA) and lysine was unaffected by diets. With increasing MP, the ratio of milk output to postliver supply of BCAA, Thr, and Lys decreased linearly, indicating oxidation of these AA in the peripheral tissues. Concomitant to a decreased catabolism of EAA in the liver (His, Met, Phe, and Thr) and/or in peripheral tissues (BCAA, Lys, and Thr), the efficiency of transfer of absorbed EAA into milk protein decreases markedly as protein supply increases. The efficiency of transfer of absorbed AA into milk also varies greatly between AA. These 2 important factors should be taken into account when building predictive schemes for milk protein output.