Regulation of Protein Synthesis and Amino Acid Transport in Mammary Tissue of Lactating Dairy Cattle

REGULATION OF PROTEIN SYNTHESIS AND AMINO ACID TRANSPORT IN MAMMARY TISSUE OF LACTATING DAIRY CATTLE

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

HATCH

Reporting Frequency

Annual

Accession No.

0211936

Grant No.

(N/A)

Cumulative Award Amt.

(N/A)

Proposal No.

(N/A)

Multistate No.

(N/A)

Project Start Date

Oct 1, 2007

Project End Date

Sep 30, 2012

Grant Year

(N/A)

Program Code

[(N/A)]- (N/A)

Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061

Performing Department
Dairy Science

Non Technical Summary
The efficiency of conversion of dietary N to milk N is poor leading to excessive release of N to the environment. This leads to algal blooms in surface water and small particulate formation in air. The purpose of this project is to determine whether energy supply to the animal plays a role in determining N efficiency.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3410	1010	60%
302	3410	1030	30%
302	3410	1060	10%

Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1060 - Biology (whole systems); 1030 - Cellular biology; 1010 - Nutrition and metabolism;

Keywords

Goals / Objectives
The long-term goal is to identify and quantify cellular and biochemical mechanisms that regulate productive and catabolic use of amino acids and to quantify relationships among those mechanisms and animal inputs. Such work will lead to the development of N requirement equations that more accurately reflect animal needs and consequently improve the efficiency of N use and minimize N release to the environment. The specific objectives of this work are: 1. to assess the interactions between energetic substrate supply/hormonal signaling and amino acid supply on a) mammary AA transport, b) protein phosphorylation status of key intracellular signaling proteins that regulate protein synthesis, and c) rates of protein synthesis; 2. to development an intracellular signaling protein model and use it to improve existing models of mammary tissue and whole animal metabolism.

Project Methods
The 1st experiment will examine the effects of dietary MP and energy supply on milk production and composition of lactating dairy cows. Forty lactating dairy cattle will be selected from the Virginia Tech dairy herd and randomly assigned to 1 of 4 treatments. All cows will be fed a 17% crude protein (CP) diet containing 1.55 MCal NEL/kg diet DM (Diet HH, Positive Control) for the first 3 weeks of the study. During weeks 4 to 6 (Period 2), cows will be fed 1 of 4 dietary treatments: HH) the positive control diet from period 1, HL) a diet balanced for 37 kg of energy allowable milk and 29 kg of MP allowable milk, LH) a diet balanced for 33 kg of energy allowable milk and 39 kg of MP allowable milk, and LL) a diet balanced for 33 kg of energy allowable milk and 29 kg of MP allowable milk (the negative control). The response variables will be milk yield and composition. The 2nd experiment will utilize mammary arterio-venous difference techniques, stable isotope infusions, and mammary biopsies to evaluate the interactions of starch and absorbed amino acid supplies on mammary protein synthesis, signal transduction, and amino acid transport activity. Six primiparous Holstein cows will be selected for the study and surgically prepared with ruminal cannulae. Main factors will be abomasally infused starch and casein. Mammary A-V difference studies will be conducted over an 8 h period on day 14 of each period with a mammary biopsy at the end of the A-V. Blood will be analyzed for metabolite concentations, and biopsies will be analyzed for signallying protein phosphorylation status and rates of protein synthesis. Data collected on intracellular signaling will be utilized to construct an initial representation of intracellular signaling. The mammary model of Hanigan et al. will be evaluated for accuracy in predicting metabolite fluxes and concentrations in venous blood and milk protein output. After the initial evaluation, a simplified version of the signaling pathway model will be introduced, and the model will be reevaluated. If work with the mammary model indicates that energy supply or hormonal signaling are linked to milk protein output, a simplified representation of that subsystem will be developed and introduced into the whole animal model. The model would then be re-evaluated and any improvement in prediction accuracy quantified and assessed for statistical significance.

Progress 10/01/07 to 09/30/12

Outputs
OUTPUTS: Five in vivo and 3 in vitro experiments were conducted. The data resulting from those experiments was used to construct a model of cell signaling in mammary tissue which was linked to a model of protein synthesis. Experiment 1: The independent and additive effects of dietary energy and metabolizable protein supplies on blood amino acid concentrations and milk protein synthesis were assessed in vivo. Experiment 2: The independent and additive effects of abomasally infused starch and casein on blood amino acid concentrations, mammary amino acid uptake, mammary cell signaling, and milk protein synthesis were assessed. Experiment 3: The effects of infused methionine, lysine, and branched chain amino acids on blood amino acid concentrations and milk protein synthesis were assessed. Experiment 4: The effects of metabolizable protein and insulin on blood amino acid concentrations, mammary amino acid uptake, mammary cell signaling, and milk protein synthesis were assessed. Experiment 5: The independent and additive effects of ruminally protected lysine, methionine, and leucine on blood amino acid concentrations, mammary cell signaling, and milk protein synthesis were assessed. Experiment 6: The independent and additive effects of insulin and essential amino acid concentrations on cell signaling and milk protein synthesis was assessed in MAC-T cells and lactogenic mammary tissue slices. Experiment 7: The independent effects of individual essential amino acid concentrations on cell signaling and milk protein synthesis was assessed in MAC-T cells and lactogenic mammary tissue slices. Experiment 8: The independent and additive effects of acetate plus glucose and essential amino acid concentrations on cell signaling and milk protein synthesis was assessed in MAC-T cells and lactogenic mammary tissue slices. The experimental data was used to develop and parameterize a mammary cell signaling model which was linked to a protein synthesis model. All work was presented at the American Dairy Science Meetings and several invited talks have been given at regional and international meetings. Experiments 1-3 were published in the Journal of Dairy Science and Experiments 6 and 7 were published in the Journal of Nutrition. Manuscripts for the remaining work are in preparation. Three graduate students and 10 undergraduate students were mentored while conducting the work. PARTICIPANTS: Mark Hanigan was the project PD. He collaborated with Brian Bequette (Maryland) who provided help with mass spectrometry analyses; R. Michael Akers at Virginia Tech who provided help in training the students to conduct hormone assays and the tissue slice experiments, and Jeffery Escobar of Virginia Tech who helped with animal experiments and provided student training on cell signaling analyses. Agustin Rius, Ranga Appuhamy, and Ashley Bell worked on the project as graduate students and were responsible for experimental design, conduct of the experiment, all sample analyses, statistical analyses, and manuscript writing. TARGET AUDIENCES: The target audience for this work is the academic research community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The work has resulted in new basic knowledge regarding how metabolites and hormones interact to regulate rates of protein synthesis. Previously, and individual nutrient was considered to be the sole limiter of production. However, our findings have shown that all of these factors act independently and additively to set the rates of milk protein synthesis. The model that was developed will be used to develop new amino acid requirement equations which should have improved accuracy allowing lactating animals to be fed more efficiently.

Publications

Appuhamy, J. A. D. R. N..and M. D. Hanigan. 2010. Modeling the effects of insulin and amino acids on the phosphorylation of mTOR, Akt, and 4EBP1 in mammary cells. IN Modelling nutrient digestion and utilization in farm animals. D. Sauvant, J. Van Milgen, P. Faverdin, N. Friggens, eds. pp 225-232. Wageningen Academic Publishers, Wageningen.
Rius, A.G. , H.A. Weeks, J. Cyriac, R.M. Akers, B.J. Bequette, and M.D. Hanigan. 2012. 2012. Protein and energy intakes affected amino acid concentrations in plasma, muscle, and liver, and cell signaling in the liver of growing dairy calves. J Dairy Sci 95(4):1983-1991.
Appuhamy, J. A. D. R. N., N. Knoebel, J. Escobar, and M. D. Hanigan. 2012. Isoleucine and leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices. J. Nutr. 142:483-91.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: When fed to meet National Research Council (2001) protein recommendations, dairy cows consume an excess of many amino acids (AA) resulting in approximately 75% of dietary nitrogen (N) being lost to the environment as urine and feces. Reductions in environmental N release could be attained through an improvement of N efficiency in dairy cows. The objective of this study was to determine if the typical reduction in milk yield associated with feeding a low protein diet to lactating dairy cows could be avoided by dietary supplemention with one or more ruminally protected (RP) AA. Fourteen multiparous and 10 primiparous Holstein cows and 24 multiparous Holstein x Jersey crossbred cows were used in a Youden square design consisting of 8 treatments and 3 periods. The 8 dietary treatments were 1) a standard diet containing 17% crude protein (CP) (+Con), 2) a 14% CP diet (-Con), 3) -Con plus RP methionine (+Met, 16g/d), 4) -Con plus RP lysine (+Lys, 47g/d), 5) -Con plus RP leucine (+Leu, 181g/d), 6) -Con plus RP methionine and lysine (+Met+Lys), 7) -Con plus RP methionine and leucine (+Met+Leu), and 8) -Con plus RP methionine, lysine, and leucine (+Met+Lys+Leu). PARTICIPANTS: The work was conducted in collaboration with Micheal de Veth of Balchem Corp. and Ondrej Becvar of the Virginia-Maryland Regional School of Veterinary Medicine. A. L. Bell, a graduate student, completed the work. TARGET AUDIENCES: The target audience for this information is academic and industry research personnel. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Cows given the -Con and +Met+Lys+Leu diets had significantly lower milk production and milk protein yield than the +Con cows. The yield of milk and milk protein for all other AA treatments were not different from either the -Con or +Con treatments. Dry matter intake decreased for cows given the +Met+Leu diet, but all other treatments were not different from the +Con treatment. Milk urea N was significantly decreased for all diets compared to the +Con indicating N efficiency was improved for the low protein diets. Plasma isoleucine and valine were significantly reduced when CP was reduced. Leucine concentrations increased for the +Met+Leu treatment compared to -Con. All other AA were unchanged. In conclusion, supplementation of the individual AA or combinations of 2 AA, but not a combination of all 3 prevented a reduction in milk yield when dietary protein levels were reduced to 14% of dietary dry matter.

Publications

Storm, A. C., M. D. Hanigan, and N. B. Kristensen. 2011. Effects of ruminal ammonia and butyrate concentrations on reticuloruminal epithelial blood flow and volatile fatty acid absorption kinetics under washed reticulorumen conditions in lactating dairy cows. J. Dairy Sci. 94:3980-3994.
Sparks, J. A., J. Arogo Ogejo, J. Cyriac, M. D. Hanigan, K. F. Knowlton, S. W. Gay, and L. C. Marr. 2011. The effects of dietary protein content and manure handling technique on ammonia emissions during short-term storage of dairy cow manure. Transactions of the ASABE 54(2):675-683.
Appuhamy, J. A. D. R. N., A. L. Bell, W. A. D. Nayananjalie, J. Escobar, and M. D. Hanigan. 2011. Essential amino acids regulate both initiation and elongation of mRNA translation independent of insulin in MAC-T cells and bovine mammary tissue slices. J. Nutr. 141(6):1209-1215
Appuhamy, J. A. D. R. N., J. R. Knapp, O. Becvar, J. Escobar, and M. D. Hanigan. 2011. Effects of jugular infused lysine, methionine, and branched-chain amino acids on milk protein synthesis in high producing dairy cows. J. Dairy Sci. 94:1952-1960
Olson, K. M., B. G. Cassell, M. D. Hanigan, and R. E. Pearson. 2011. Interaction of energy balance, feed efficiency, early lactation health events, and fertility in first lactation Holstein, Jersey, and reciprocal F1 crossbred cows. J. Dairy Sci. 94:507-511.
Olson, K. M., B. G. Cassell, and M. D. Hanigan. 2010. Energy balance in first lactation Holstein, Jersey, and reciprocal F1 crossbred cows in a planned crossbreeding experiment. J. Dairy Sci. 93: 4374-4385.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Three experiments were completed to test the effects of amino acids and insulin on signaling pathways regulating protein synthesis. The first experiment used mammary tissue slices from lactating cows that were incubated in media deprived of individual essential amino acids. Rates of protein synthesis and phosphorylation of mTOR (Ser2448), rpS6 (Ser235/236), and eEF2 (Thr56)were measured. The second experiment was an in vivo experiment examining the effects of infused insulin and varying dietary protein on total and phosphorylated forms of Akt, mTOR, rpS6, 4EBP1, and eEF2 in mammary tissue. Cows were fed two levels of dietary crude proteins (17.5 or 14.0 percent of dietary DM) and infused with two levels of insulin (0 or 1 ug/kg BW) under euglycemic conditions in a 2 x 2 factorial design. At the end of each treatment period mammary tissue biopsies were harvested and assessed for total and phosphorylated forms of Akt (Ser473), mTOR (Ser2448), rpS6 (Ser235/236), 4EBP1 (Thr37/46), and eEF2 (Thr56). The third experiment explored the effect of deprivation of individual essential amino acids on the phosphorylation of eIF2-alpha at Ser 51 in MAC-T cells. Before the application of the treatments, cells were starved for 2 h in DMEM/F12, with essential amino acid concentrations of 5 percent of normal DMEM/F12 EAA concentrations. The experimental design was a randomized block design with three replications per treatment. Treatments were the starvation media, complete DMEM/F12 media, and the starvation media supplemented with individual EAA at 100 percent of normal DMEM/F12 concentrations. Cells were incubated in treatment media for 1 h. Cell lysates were assessed for the quantity of total and phosphorylated forms of eIF2-alpha (Ser 51). PARTICIPANTS: M. D. Hanigan (PD) J. Escobar (PI) O. Becvar (PI) R. M. Akers (PI) M. L. McGilliard (PI) K. F. Knowlton (PI) B. G. Cassell (Collaborator) D. Kirovski (Collaborator) J. R. Knapp (Collaborator) J. P. McNamara (Collaborator) J. G. Fadel (Collaborator) S. S. Donkin (Collaborator) B. J. Bequette (Collaborator) A. N. Hristov (Collaborator) J. A. D. R. N. Appuhamy (PhD student) A. G. Ruis (PhD student) W. A. D. Nayananjalie (PhD student) K. M. Olson (PhD student) J. Cyriac (PhD student) TARGET AUDIENCES: The target audience for this information is academic and industry research personnel. An invited presentation was made at the Joint Animal Meetings in Denver, CO in July. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Experiment 1: The -EAA treatment caused the phosphorylation of mTOR and rpS6 to decrease by 46 and 76 percent, respectively, phosphorylation of eEF2 to increase by 136 percent, and protein synthesis to decrease by 46 percent. Phosphorylation of mTOR was positively correlated (r=0.80) with Phe enrichment. Deprivation of Leu resulted in the greatest reduction of phosphorylation for mTOR (46 percent) and rpS6 (51 percent) whereas Met deprivation was associated with the greatest phosphorylation increase for eEF2 (64 percent). The greatest reductions in protein synthesis were associated with Ile (58 percent), Leu (47 percent), and Met (45 percent) deprivations. Essential amino acids, in particular Leu and Met had substantial regulatory effects on protein synthesis efficiency in bovine mammary tissues, and a significant proportion of the signaling appeared to be mediated by mTOR. Experiment 2: Increasing dietary protein level significantly increased PhS of Akt. Neither dietary protein nor insulin had significant effects on phosphorylation or total mTOR expression. However, increased dietary protein and insulin infusion significantly increased both the phosphorylation and total 4EBP1 expression. Total rpS6 expression but not phosphorylation was increased when insulin was infused. Increased dietary protein was associated with significantly greater total eEF2 expression. Increased rpS6 expression and increased phosphorylation of 4EBP1 should stimulate protein synthesis, but the lack of change in mTOR phosphorylation would not be stimulatory. Experiment 3: Supplementation of Phe, His, and Val affected eIF2-alpha phosphorylation ratios. Supplementation of Trp and Arg tended to reduce the phosphorylation ratio. These results indicate that protein synthesis would be better represented in our requirement models as a function of energy supply, hormonal signals, and individual amino acid availability at the mammary glands. Operation of these signaling pathways results in variably efficiencies of conversion of post absorptive N to milk N. Such a system is not consistent with the current single-limiting nutrient paradigm encoded in requirement models. Use of the single-limiting nutrient approach will prevent selection of dietary inputs that result in greater animal efficiency due to over predictions of production losses associated with a single nutrient deficiency.

Publications

Appuhamy, J. A. D. R. N.*.and M. D. Hanigan. 2010. Modeling the effects of insulin and amino acids on the phosphorylation of mTOR, Akt, and 4EBP1 in mammary cells. IN Nutrient digestion and utilization in farm animals: modeling approaches. D. Sauvant, ed. Wageningen Academic Publishers, Wageningen.
Olson, K. M.*, B. G. Cassell, and M. D. Hanigan. 2010. Energy balance in first lactation Holstein, Jersey, and reciprocal F1 crossbred cows in a planned crossbreeding experiment. J. Dairy Sci. 93: 4374-4385
Rius, A. G.*, J. A. D. R. N. Appuhamy*, J. Cyriac*, D. Kirovski, J. Escobar, M. L. McGilliard, O. Becvar, B. J. Bequette, R. M. Akers, and M. D. Hanigan. 2010. Regulation of protein synthesis in mammary glands of lactating dairy cows by starch and amino acids. J. Diary Sci., 93: 3114-3127.
Rius, A.G.*, M.L. McGilliard, C. A. Umberger, and M. D. Hanigan. 2010. Interactions of energy and metabolizable protein in determining nitrogen efficiency in the lactating dairy cow. J. Dairy Sci. 2010, 93:2034-2043
Knowlton, K. F., M. L. McGilliard, Z. Zhao, K. G. Hall, W. Mims* and M. D. Hanigan. 2010. Effective nitrogen preservation during urine collection from Holstein heifers fed diets with high or low protein content. J. Dairy Sci. 93:323-329.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Task 2. The laboratory analyses of samples and statistical analyses of data derived from an infusion study designed to evaluate the interactions of starch and protein supply on mammary function, was completed and a publication has been submitted. Task 3: A simple model of the Akt and mTOR cell signaling pathways in mammary tissue has been developed and parameterized using cell culture data. PARTICIPANTS: M. D. Hanigan was the PD for the work and devoted 25 percent of this time to the project. He was responsible for project organization, overseeing 2 Ph.D. students and 1 technician working on the project. He also helped with sample collection and ongoing sample analyses. Drs. J. Escobar (Virginia Tech), R. M. Akers (Virginia Tech), O. Becvar (Virginia Tech), B. J. Bequette (Univ. of Maryland), and D. Kirovsky (Univ. of Belgrade) collaborated on the work. The project has provided a training opportunity for Dr. Kirovsky and the 2 Ph.D. students working on the project. They have gained experience in cannulation, mammary biopsy, Western blotting, and modelling techniques. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The work resulted in an incremental improvement in our fundamental knowledge of the regulation of milk protein synthesis in the dairy cow. A publication summarizing the work has been submitted to the Journal of Dairy Science.

Publications

Rius, A. G., J. Escobar, O. Becvar, D. Kirovski, and M. D. Hanigan 2009. Mammary cell signaling responses to abomasal starch and casein infusions in lactating dairy cows. J. Dairy Sci. 92 (e-Suppl1): 469.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Task 1. A production study designed to evaluate the dietary interactions of energy and protein supply in milk yield and nitrogen efficiency was completed. The data have been summarized and resulting information disseminated at the Federation of Animal Science Societies meeting and to dairy nutritionists and feed industry representatives at the VA State Feed Association meeting. Task 2. The animal work of an infusion study designed to evaluate the interactions of starch and protein supply on mammary function, was completed and sample analyses is underway. PARTICIPANTS: M. D. Hanigan was the PD for the work and devoted 25% of this time to the project. He was responsible for project organization, overseeing 2 Ph.D. students and 1 technician working on the project. He also helped with sample collection and ongoing sample analyses. Drs. J. Escobar (Virginia Tech), R. M. Akers (Virginia Tech), B. J. Bequette (Univ. of Maryland), and D. Kirovsky (Univ. of Belgrade) collaborated on the work. The project has provided a training opportunity for Dr. Kirovsky and the 2 Ph.D. students working on the project. They have gained experience in cannulation, mammary biopsy, and Western blotting techniques. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Task1. The work resulted in an incremental change in applied knowledge relative to protein requirements for lactation. A publication summarizing the work has been submitted to the Journal of Dairy Science.

Publications

Rius, A. G., J. A. D. R. N. Appuhamy, D. Kirovski, J. Cyriac, and M. D. Hanigan. 2008. Effect of starch and casein infusions in the abomasum of lactating dairy cows. J. Dairy Sci. 91 (E-Suppl. 1): 123.