Source: BAYLOR COLLEGE OF MEDICINE submitted to NRP
LEUCINE SUPPLEMENTATION TO PROMOTE LEAN GROWTH IN EARLY LIFE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0232061
Grant No.
2013-67015-20438
Cumulative Award Amt.
$400,000.00
Proposal No.
2012-04044
Multistate No.
(N/A)
Project Start Date
Jan 15, 2013
Project End Date
Jan 14, 2019
Grant Year
2013
Program Code
[A1241]- Dual use of animals for dual benefit
Recipient Organization
BAYLOR COLLEGE OF MEDICINE
(N/A)
HOUSTON,TX 77030
Performing Department
Department of Pediatrics
Non Technical Summary
Piglets are commonly weaned early from the sow to increase the sow productivity, however, this can lead to reduced food intake, restricted growth, and increased time required to attain market weight. We hypothesize that supplementing the diet of early weaned pigs with the amino acid, leucine, will increase lean growth by reducing the degradation of protein in muscle, even when food intake is reduced. Our objective is to determine the effects of leucine supplementation of an energy and protein restricted diet on the degradation of muscle protein, the intracellular signaling components that regulate protein degradation, and the oxidation of amino acids in young pigs. We expect that leucine supplementation of the diet will decrease the degradation of protein in muscle, reduce the oxidative loss of amino acids from the body, and improve the growth of lean tissue, even when dietary protein and energy intakes are reduced. The results are expected to provide important new information that will lead to the development of better diets to enhance the lean growth of pigs in nursery rooms.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023510101060%
3053510102020%
3023510103020%
Knowledge Area
302 - Nutrient Utilization in Animals; 305 - Animal Physiological Processes;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1030 - Cellular biology; 1010 - Nutrition and metabolism; 1020 - Physiology;
Goals / Objectives
The practice of early weaning of pigs is used to increase sow productivity and reduce investment cost. However, it is usually accompanied by reduced food intake of the piglets and the resulting growth retardation and mortality represents a major source of production loss in the swine industry. Our long-term goal is to develop strategies to optimize the nutritional management of piglets in order to improve lean growth. In the proposed experiments, we wish to specifically test a strategy that will promote better growth of lean mass during this time when full and normal feeding does not occur. Our specific objective is to determine the effects of leucine supplementation of a protein and energy restricted diet on rates of protein degradation in the hindlimb, intracellular signaling of the protein degradation pathways in skeletal muscle, and the oxidative loss of amino acids from the body. We expect to complete all animal care, feeding, and tracer infusions by the end of the first year, to complete all intracellular degradation signaling work by the end of the second year, to complete all measurement and calculation of whole body amino acid flux, whole body amino acid oxidation, and hindlimb protein degradation and synthesis by the end of the third year, and to complete submission of publications to journals by the end of the fourth year. We expect that leucine supplementation of the diet will decrease the degradation of protein in muscle, reduce the oxidative loss of amino acids from the body, and improve the growth of lean tissue, even if dietary protein and energy intakes are reduced. The work proposed is expected to demonstrate that leucine supplementation of the diet, even when intake is compromised, will suppress the degradation of protein in muscle and blunt the oxidative loss of amino acids from the body, thereby improving the efficiency with which the diet is used for lean growth.
Project Methods
Early weaned pigs will be fed for 21 d whey-based diets that are either 1) marginally restricted in protein and energy, 2) marginally restricted but supplemented with the amino acid, leucine, 3) marginally restricted but supplemented with the branched-chain amino acids, leucine, isoleucine and valine, or 4) complete in protein and energy. Muscle protein degradation will be assessed across the hindlimb in the multi-catheterized piglet model using amino acid kinetic measurements of the stable isotopes of phenylalanine and tyrosine. Whole body amino acid oxidation and flux also will be determined. The abundance and activation of components of signaling pathways that regulate protein degradation in muscle will be measured to identify mechanisms involved. Analysis of variance followed by multiple comparison procedure will be used to assess effects of treatment. We expect that leucine supplementation of a protein and energy restricted diet will down-regulate protein degradation and reduce amino acid oxidation thereby improving amino acid utilization for growth. We also expect that the anabolic response will be greater when the diet is supplemented with all 3 branched-chain amino acids. The results will be reported at important scientific meetings, such as Experimental Biology and American Society of Animal Science, and submitted for publication to journals with high impact factors. The work is expected to have a positive impact by providing important information for the potential for using leucine supplementation to improve the efficiency of utilization of dietary nutrients for pork production.

Progress 01/15/13 to 01/14/19

Outputs
Target Audience:The target audience for the scientific information produced by these studies are animal nutritionists, particularly swine nutritionists. Health care professionals, including pediatric gastroenterologists and neonatologists will also benefit because the piglet is a good model for the human infant. The scientific information generated from this project is fundamental in nature and thus has broad application to nutrition in general. Changes/Problems:After the initiation of the grant, the herd of our pig supplier became infected with a respiratory virus. We had to locate a new pig herd in Texas with a similar genetic background and receive approval for their use in our studies from the Baylor College of Medicine Attending Veterinarian. Their supply has been much more limited. The animal facility at the Children's Nutrition Research Center was shut down for 4 months due to renovation of the facility. Two critical personnel that could not be replaced were out due to health issues for 5 months: a technician whose function was critical for a number of the required analyses was on maternity leave for 3 months and a Co-PI broke his leg and was unable to work for 2 months. The circumstances generated in Houston by Hurricane Harvey disrupted all activities for several weeks due to preparation for the event, inability to gain access to the facility during Hurricane Harvey, and lives being disrupted during the aftermath of the hurricane. These circumstances delayed our animal studies, laboratory and data analysis, and manuscript completion. What opportunities for training and professional development has the project provided?The PI has established an internationally recognized, research program providing leadership and mentorship for postdoctoral fellows. The PI recruited a postdoctoral fellow from the University of Guelph who conducted the studies with the assistance of others in the laboratory and gained valuable knowledge and skills in protein and amino acid metabolism and stable isotope methodology. Abstracts were presented at Experimental Biology, Pediatric Research Societies, International Life Sciences Institute North America Food, Nutrition & Safety Program, and American Society of Animal Science meetings, at FASEB Summer Research, Advances in Skeletal Muscle Biology, and Perinatal Biology conferences, at the National Animal Nutrition Program Summit, and at the 14th International Congress on Amino Acids, Peptides and Proteins. The PI lectured at the University of Illinois, University of Nebraska, and Texas A&M University and at the Emerging Role of Branched-chain Amino Acids in Human Diseases NIH Workshop. Manuscripts have been published in the American Journal of Physiology-Endocrinology and Metabolism, Amino Acids, Journal of Physiology, Journal of Animal Science and Biotechnology, The Journal of Nutrition, Pediatric Research, American Journal of Clinical Nutrition, and Current Opinions in Clinical Nutrition and Metabolic Care. How have the results been disseminated to communities of interest?Manuscripts were published in the American Journal of Physiology Endocrinology and Metabolism (5), Journal of Physiology (1), Current Developments in Clinical Nutrition and Metabolic Care (1), Journal of Animal Science and Biotechnology (1), Amino Acids (3), Pediatric Research (2), The Journal of Nutrition (1), Recent Research in Nutrition and Growth (1), Annual Reviews of Animal Biosciences, and the American Journal of Clinical Nutrition (1). Another manuscript has been submitted to The Journal of Nutrition and others are in preparation. Results were presented at a number of scientific meetings and invited lectures were presented at universities and scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The growth and survival of piglets is reduced when nutrient intake is suboptimal. Previously we showed that the branched-chain amino acid, leucine can acutely stimulate protein synthesis in skeletal muscle of young pigs but the long-term effects of leucine supplementation had not been explored. To test the impact of leucine supplementation on lean growth in piglets when dietary protein is marginal and to identify the mechanisms involved, piglets were meal-fed a milk protein-based diet with either 1) a high protein content, or 2) a marginal protein content, or 3) a marginal protein content supplemented with leucine to the same level as in the high protein diet for 21 days. Body weight, lean body mass, and skeletal muscle weight were lowest in those fed the marginal protein diet, highest in piglets fed the high protein diet, and intermediate in those fed the leucine supplemented marginal protein diet. Feeding increased muscle protein synthesis and the activation of the mammalian target of rapamycin complex 1 (mTORC1) intracellular signaling pathway that regulates protein synthesis and this was greatest in the leucine supplemented pigs. Protein degradation signaling was unaffected by protein intake or leucine supplementation. Thus, the results of the study showed that leucine supplementation can enhance muscle protein accretion but the capacity to do so in neonatal pigs that are meal-fed a milk protein-based diet that only marginally meets the protein needs for growth is modest. Early weaning of pigs frequently blunts their growth due to a reduction in total food intake that results from stress, environmental changes, and a shift from liquid to solid diet. To determine if leucine supplementation would up-regulate the mTORC1 signaling pathway leading to an increase in protein synthesis in muscle of neonatal piglets who consume less than their protein and energy requirements, neonatal piglets were meal-fed for 7 days either a 1) control diet, 2) marginally restricted protein and energy diet, or 3) a marginally restricted in protein and energy diet supplemented with leucine. We determined that leucine supplementation stimulated the activation of the mTORC1 signaling pathway, but it did not enhance skeletal muscle protein synthesis or improve short-term weight gain to control levels. This limited anabolic response to leucine supplementation may have been due to the redirection of amino acids towards energy production thereby limiting their availability as substrates for protein synthesis and/or to a specific imbalance in the level of the other branched-chain amino acids, isoleucine and valine thereby limiting their availability as substrates for protein synthesis. To determine if correction of an unbalanced ratio of branched chain amino acids would be beneficial, for muscle protein synthesis, degradation, and accretion in piglets in the context of suboptimal protein and energy intakes , neonatal pigs were fed for 21 d with milk replacer diets that met protein and energy requirement, were limiting in energy and protein, or limiting in energy and protein diet but supplemented with either leucine alone, or in combination with other branched-chain amino acids (isoleucine and valine) Supplementation with leucine alone increased plasma leucine and decreased isoleucine and valine levels, whereas branched-chain amino acid supplementation increased circulating leucine levels but maintained isoleucine and valine at control levels . Supplementation with either leucine alone or all branched-chain amino acids increased the activation of the mTORC1 signaling pathway that regulates translation initiation but did not enhance protein synthesis rates in skeletal muscle or lean gain. Lean gain, muscle protein synthesis rate, and mTORC1 signaling were highest in pigs fed the complete protein and energy diet. Protein degradation signaling, including the expression of ubiquitin ligases, was not impacted by the dietary treatments. Thus, when protein and energy are restricted, both leucine and branched-chain amino acid supplementation increase intracellular mTORC1 activation but this is insufficient to enhance protein synthesis or reduce protein degradation in skeletal muscle and thereby promote muscle growth in neonatal pigs. Because leucine supplementation can reduce the circulating concentrations of the other branched-chain amino acids, isoleucine and valine, these reductions have the potential to limit the ability of leucine to stimulate protein synthesis. Therefore, we conducted studies to investigate the impact of the leucine metabolite, beta-hydroxy-beta-methylbutyrate (HMB) on protein anabolism in the context of a suboptimal protein, but adequate energy intake, because it was postulated that it would not perturb circulating amino acids levels. Neonatal piglets were meal-fed for 24 hr a low protein formula, a high protein formula, or a low protein formula with 3 different levels of HMB. As anticipated, HMB supplementation did not alter circulating concentrations of isoleucine, valine, or other amino acids. HMB supplementation increased protein synthesis in skeletal muscle similar to that in the high protein-fed piglets and this increase was due to the activation of the mTORC1 signaling pathway that regulates translation initiation. Cell replication in muscle also was increased in both the HMB supplemented and high protein fed piglets. However, protein degradation signaling was unaffected by HMB supplementation or high protein feeding. The results suggest that HMB supplementation can increase skeletal muscle anabolism acutely in young piglets by stimulating protein synthesis and satellite cell proliferation but without any impact on protein degradation. To determine whether leucine supplementation will enhance lean growth when protein and energy intake are not restricted and circulating amino acid levels are maintained at constant levels, neonatal piglets were continuously fed formula via orogastric tube for 21 days with an additional pulse of leucine every 4 hours to mimic the rise that occurs after a meal. Alanine pulses were given to isonitrogenous controls. The pulsed leucine supplementation increased body weight and promoted lean growth while reducing fat gain. This positive effect was due to the leucine-induced increase in protein synthesis in skeletal muscle, which lead to an increase in muscle mass. We identified intracellular amino acid signaling proteins whose activation was associated with the leucine-induced increase in protein synthesis. There was no effect of leucine on ubiquitin-proteosome and autophagy-lysosome protein degradation pathways. RNAseq analysis identified novel genes involved in developmental processes, cell junction assembly, regulation of cellular processes, and organ development that were up-regulated by leucine pulses. Genes that are known negative regulators of muscle growth were also down-regulated by leucine pulses. The study demonstrated that pulses of leucine administered during continuous feeding of a formula that met protein and energy requirements stimulated intracellular amino acid signaling pathways that regulate protein synthesis but did not impact protein degradation in skeletal muscle. These responses elicited an increase in lean growth and a reduction in fat accretion. Thus, our studies demonstrated that when energy intake is adequate leucine supplementation can increase lean growth of young pigs by stimulating the intracellular signaling pathways that regulate protein synthesis but does not alter protein degradation in skeletal muscle. Whether meal feeding a complete diet that meets protein and energy requirements and supplemented with leucine enhances muscle accretion requires further study.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Suryawan A and Davis TA. Amino acid- and insulin-induced activation of mTORC1 in neonatal piglet skeletal muscle involves Sestrin2-GATOR2, Rag A/C-mTOR, and RHEB-mTOR complex formation. J Nutr 148: 825-833, 2018 doi: 10.1093/jn/nxy044 (PMID: 29796625 / PMCID: In progress).
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Manjar�n R, Columbus DA, Solis J, Hernandez-Garc�a A, Suryawan A, Nguyen HV, McGuckin MM, Jimenez RT, Fiorotto ML, and Davis TA. Short and long-term effect of leucine and BCAA inclusion in a reduced protein and energy diet on muscle protein metabolism in neonatal pigs. Amino Acids 50 (7): 943959 doi: 10.1007/s00726-018-2572-0 (PMID: 29728917 / PMCID: In progress).
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: El-Kadi SW, Boutry C, Suryawan A, Gazzaneo MC, Orellana RA, Srivastava N, Nguyen HV, Kimball SR, Fiorotto ML, and Davis TA. Long-term intermittent bolus feeding enhances lean growth more than continuous feeding by stimulating mTORC1-dependent translation initiation in neonatal pigs. Am J Clin Nutr 2018;108:830-841; doi: https://doi.org/10.1093/ajcn/nqy133 (PMID: 30239549 / PMCID: PMC6186212).
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Rudar M, Columbus DA, Steinhoff-Wagner J, Suryawan A, Nguyen HV, Fleischmann R, Davis TA, Fiorotto ML. Leucine supplementation does not restore diminished satellite cell abundance and myonuclear accretion induced by protein restriction in neonatal pig skeletal muscle. J Nutr (Submitted).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kao M, Columbus D, Suryawan A, Steinhoff-Wagner J, Hernandez-Garcia A, Nguyen HV, Davis SR, Fiorotto ML, and Davis TA. Oral supplementation with ?-hydroxy-?-methylbutyrate enhances protein synthesis in skeletal muscles of neonates. J Pediatr Gastroenterol Nutr 59:S201, 2014.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Fiorotto ML, Columbus DA, Steinhoff-Wagner J, Suryawan A, Nguyen HV, and Davis TA. Postnatal satellite cell proliferation demonstrates a specific requirement for dietary protein. Advances in Skeletal Muscle Biology in Health and Disease, January 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Davis TA, Kao M, Columbus DA, Suryawan A, Steinhoff-Wagner J, Hernandez-Garcia A, Nguyen H, Davis S, and Fiorotto, ML. Oral ?-hydroxy-?-methylbutyrate supplementation enhances protein synthesis in skeletal muscle of neonatal pigs. Advances in Skeletal Muscle Biology in Health and Disease, January 2016.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Suryawan A, Rudar M, Naberhuis J, Nguyen H, Fiorotto ML, Li Z, and Davis TA. Assessment of encapsulated leucine as a potential anabolic agent to enhance lean growth in neonates. Curr Dev Nutr 2(11): nzy037(OR09-04), 2018 https://doi.org/10.1093/cdn/nzy037.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Suryawan A, Nguyen H, Fiorotto ML, and Davis TA. Supplementation with the leucine metabolite, ?-hydroxy-?-methylbutyrate activates mTORC1 in skeletal muscle of the neonate via a Rag-independent process. Curr Dev Nutr 2(11):nzy038(OR10-06), 2018 https://doi.org/10.1093/cdn/nzy038.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Suryawan A, Fiorotto ML, and Davis TA. Supplementation with leucine and the leucine metabolite, ?-hydroxy-?-methylbutyrate stimulates muscle protein synthesis in neonatal pigs via different signaling mechanisms. ASAS meeting, Vancouver, CA, July 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Fiorotto, ML, Meadows JE, Fleischmann RE, Coarfa C, Davis TA. Postnatal undernutrition compromises proliferative capacity and alters gene expression of adult satellite cells in skeletal muscle from mice. E-PAS 2018, Toronto CA 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Fiorotto, ML, Meadows JE, Fleischmann RE, Coarfa C, Davis TA. Postnatal undernutrition compromises proliferative capacity and alters gene expression of adult satellite cells in skeletal muscle from mice. FASEB Summer Research Conference, Colorado Springs, July 2018.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Suryawan A and Davis TA. Enteral leucine and protein synthesis in skeletal and cardiac muscle. In: The Branched Chain Amino Acids in Health and Disease, edited by Rajendram R, Preedy VR, and Patel VB. Springer, 2014, p. 197-210.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Fiorotto ML and Davis TA. Critical windows for the programming effects of early-life nutrition on skeletal muscle mass. In: Recent Research in Nutrition and Growth, Nestl� Nutr Inst Workshop Ser, vol 89, edited by Colombo J, Koletzko B, Lampl M. Nestl� Nutrition Institute, Switzerland/S. Karger AG., Basel. pp 2535, 2018 (DOI: 10.1159/000486490).


Progress 01/15/17 to 01/14/18

Outputs
Target Audience:The target audience for the scientific information produced by these studies are animal nutritionists, particularly swine nutritionists. Health care professionals, including pediatric gastroenterologists and neonatologists will also benefit because the piglet is a good model for the human infant. The scientific information generated from this project is fundamental in nature and thus will have broad application to nutrition in general. Changes/Problems:After the initiation of the grant, the herd of our pig supplier became infected with a respiratory virus. We had to locate a new pig herd in Texas with a similar genetic background and receive approval for their use in our studies from the Baylor College of Medicine Attending Veterinarian. Their supply has been much more limited. The animal facility at the Children's Nutrition Research Center was shut down for 4 months due to renovation of the facility. Two critical personnel that could not be replaced were out due to health issues for 5 months. A technician whose function is critical for a number of the required analyses was on maternity leave for 3 months and a Co-PI broke his leg and was unable to work for 2 months. The circumstances generated in Houston by Hurricane Harvey disrupted all activities for several weeks due to preparation for the event, inability to gain access to the facility during Hurricane Harvey, and lives being disrupted during the aftermath of the hurricane. These circumstances have delayed our animal studies, laboratory and data analysis, and manuscript completion. What opportunities for training and professional development has the project provided?The PI has established an internationally recognized, research program providing leadership and mentorship for postdoctoral fellows. The PI recruited a postdoctoral fellow from the University of Guelph who is conducting the proposed studies with the assistance of others in the laboratory. Abstracts have been presented at Experimental Biology and American Society of Animal Science meetings. Manuscripts have been published in Am J Physiol Endocrinol, Amino Acids, J Physiol, J Anim Sci Biotech, Pediatr Res, and Curr Opin Clin Dev Metab Care. How have the results been disseminated to communities of interest?In previous reporting periods, manuscripts were published in the American Journal of Physiology Endocrinology and Metabolism (3), Journal of Physiology (1), Current Developments in Clinical Nutrition and Metabolic Care (1), Journal of Animal Science and Biotechnology (1), the journal, Amino Acids (2), and Pediatric Research (1). Another manuscript has been accepted for publication in The Journal of Nutrition (1), 2 have been submitted for review, and other manuscripts are in preparation. Results were also presented at the 2014, 2015, 2016, and 2017 Experimental Biology, American Society of Animal Science, and Pediatric Research Society meetings. Results also were presented at invited talks at the 14th International Congress on Amino Acids, Peptides and Proteins in Vienna, Austria, the Advances in Skeletal Muscle Biology Conference in Gainesville, FL, the Perinatal Biology Symposium in Snowmass, CO, and the International Life Sciences Institute North America Food, Nutrition & Safety Program meeting in Washington, D.C. What do you plan to do during the next reporting period to accomplish the goals?Studies are being completed to determine the impact of leucine supplementation on protein degradation in muscle and the mechanisms involved. Additional studies to identify the intracellular signaling components that regulate the response to the leucine metabolite, HMB are also being completed. The results will provide novel information on the potential for leucine supplementation to improve lean growth of piglets.

Impacts
What was accomplished under these goals? Early weaning of pigs frequently blunts their growth due to a reduction in food intake that results from stress, environmental changes, and a shift from liquid to solid diet. We have shown that the branched-chain amino acid, leucine stimulates protein synthesis in skeletal muscle on young pigs. To test the impact of leucine supplementation on lean growth in piglets when diet protein is marginal and to identify the mechanisms involved, piglets were meal-fed a whey-based diet with either 1) a high protein content, or 2) a marginal protein content, or 3) a marginal protein content supplemented with leucine to the same level as in the high protein diet for 21 days. Body weight, lean body mass, and skeletal muscle weight were lowest in those fed the marginal protein diet, highest in piglets fed the high protein diet, and intermediate in those fed the leucine supplemented marginal protein diet. Feeding increased muscle protein synthesis and the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway that regulates translation initiation and this was greatest in the leucine supplemented pigs. Protein degradation signaling was unaffected by treatment. Thus, leucine supplementation can enhance muscle protein accretion but the capacity to do so in neonatal pigs that are meal-fed a milk protein-based marginal protein diet is modest. To determine whether leucine supplementation will enhance lean growth when protein and energy intake are not restricted and circulating amino acid levels are maintained at constant levels, neonatal piglets were continuously fed formula via orogastric tube for 21 d with an additional pulse of either leucine or alanine (isonitrogenous control) every 4 h. Leucine supplementation increased body weight and this was due to an increase in lean gain. Muscle weight was enhanced in the leucine supplemented pigs due to an increase in protein synthesis. The increase in protein synthesis was associated with increased abundance of the leucine transporter, association of RagC with mTOR, phosphorylation of mTOR and downstream signaling proteins S6K1 and 4EBP1, and association of eIF4E with eIF4G that regulate mRNA binding to the ribosome. The study demonstrated that leucine supplement of a complete protein and energy formula stimulates the mTORC1-dependent translation initiation pathway leading to protein synthesis in skeletal muscle and promotes lean growth. The growth and survival of piglets is reduced when nutrient intake is suboptimal. To determine if leucine supplementation will up-regulate the mTORC1 signaling pathway leading to an increase in protein synthesis and greater anabolism in muscle of neonatal piglets who consume less than their protein and energy requirements, neonatal piglets were meal-fed for 7 days either a 1) control diet, 2) marginally restricted protein and energy diet, or 3) leucine supplemented diet that is marginally restricted in protein and energy. We found that leucine supplementation stimulated the activation of the mTORC1 signaling pathway, but it did not enhance skeletal muscle protein synthesis or improve short-term weight gain to control levels. This limited anabolic response to leucine supplementation may have been due to the reduction in isoleucine and valine levels which would have limited their availability as substrates for protein synthesis. Alternatively, the limited anabolic response to leucine supplementation may have been due to the consumption of a diet that is marginally restricted in protein and energy which could limit substrate availability because the amino acids could be redirected towards energy production instead of being used as substrates for protein synthesis. Leucine supplementation can reduce the circulating concentrations of the other branched-chain amino acids, isoleucine and valine, and these reductions have the potential to limit the ability of leucine to stimulate protein synthesis. Therefore, studies were conducted to investigate the impact of the leucine metabolite, beta-hydroxy-beta-methylbutyrate (HMB) on protein anabolism because it was postulated that these metabolites would not perturb circulating amino acids levels. Neonatal piglets were fed a low protein formula, a high protein formula, or a low protein formula with 3 different levels of HMB. HMB supplementation did not alter circulating concentrations of isoleucine, valine, or other amino acids. HMB supplementation increased protein synthesis in skeletal muscle similar to that in the high protein-fed piglets and this increase was due to the activation of the mTORC1 signaling pathway that regulates translation initiation. Cell replication in muscle also was increased in both the HMB supplemented and high protein fed piglets. However, protein degradation signaling was unaffected by HMB supplementation or high protein feeding. The results suggest that HMB supplementation increases skeletal muscle anabolism in young piglets by stimulating protein synthesis and satellite cell proliferation. Studies are currently underway to determine whether HMB stimulates mTORC1 activation in muscle via the same intracellular sensing components used by leucine. To test the impact of supplementation with all three branched-chain amino acids on muscle protein synthesis, degradation, and accretion in piglets who consume less than their protein and energy requirements for growth, neonatal pigs were fed milk replacement diets containing a complete protein and energy diet, a reduced energy and protein diet, or a reduced energy and protein diet supplemented with either leucine alone, or in combination with other branched-chain amino acids (isoleucine and valine) for 21 d. Supplementation with leucine increased plasma leucine and decreased isoleucine and valine levels; branched-chain amino acid supplementation increased circulating leucine levels alone. Supplementation with either leucine alone or all branched-chain amino acids increased the activation of the mTORC1 signaling pathway that regulates translation initiation. However, supplementation with leucine alone or all branched-chain amino acids did not enhance protein synthesis rates in skeletal muscle or lean gain. Lean gain, muscle protein synthesis rate, and mTORC1 signaling was highest in pigs fed the complete protein and energy diet. Protein degradation signaling, including the expression of ubiquitin ligases, was not impacted by dietary treatment. Thus, when protein and energy are restricted, both leucine and branched-chain amino acid supplementation increase mTORC1 activation but do not enhance skeletal muscle protein synthesis and muscle growth in neonatal pigs.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Naberhuis J, Suryawan A, Nguyen HV, Hernandez-Garcia AD, Fiorotto M, Stoll B, Burrin D, and Davis TA. Prematurity blunts feeding-induced stimulation of translation initiation signaling and protein synthesis in muscle of neonatal piglets. FASEB J 31:141.8, 2017.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Suryawan A, Nguyen HV, Parada RD, and Davis TA. Regulation of leucine-induced mTORC1 activation in skeletal muscle of neonatal pigs. FASEB J 31:652.8, 2017.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: McGuckin MM, Suryawan A, Davis TA, Peterson DG, and Manjarin R. In vitro supplementation of leucine increases murine HC11 cell proliferation. FASEB J 31:448.6, 2017.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Naberhuis J, Suryawan A, Nguyen H, Hernandez-Garcia A, Cruz S, Lau P, Olutoye O, Stoll B, Burrin D, Fiorotto M, and Davis TA. Prematurity blunts the feeding-induced stimulation of translation initiation signaling and protein synthesis in muscle of a neonatal piglet model. E-PAS2017: 2693224, May 2017.2.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Davis TA, El-Kadi SW, Boutry C, Suryawan A, and Fiorotto ML. Timing of nutrient delivery impacts muscle protein synthesis and lean growth. European Association of Animal Production. Publication No. 137, Skomial J, Lapierre H, eds. Wageningen Academic Publishers, Wageningen, The Netherlands. 2016, p. 25-26.8.


Progress 01/15/16 to 01/14/17

Outputs
Target Audience:The target audience for the scientific information produced by these studies will be animal nutritionists, particularly swine nutritionists. Health care professionals, including pediatric gastroenterologists and neonatologists will also benefit because the piglet is a good model for the human infant. The scientific information generated from this project is fundamental in nature and thus will have broad application to nutrition in general. Changes/Problems:The herd of our pig supplier became infected with a respiratory virus shortly after the initiation of the grant. We identified a new pig herd in Texas with similar genetic background but their supply is limited. The postdoctoral fellow who was to participate in the studies was delayed a semester in completing his Ph.D. due to the illness and death of his doctoral mentor. My technician was on maternity leave for 3 months and a Co-PI, broke his leg and was unable to work for 2 months. This has delayed our animal studies and laboratory analysis. What opportunities for training and professional development has the project provided?The PI has established an internationally recognized, research program providing leadership and mentorship for postdoctoral fellows. The PI recruited a postdoctoral fellow from the University of Guelph who is conducting the proposed studies with the assistance of others in the laboratory. Abstracts have been presented at Experimental Biology and American Society of Animal Science meetings. Manuscripts have been published in Am J Physiol Endocrinol, Amino Acids, J Physiol, J Anim Sci Biotech, Pediatr Res, and Curr Opin Clin Dev Metab Care. How have the results been disseminated to communities of interest?In previous reporting periods, manuscripts were published in the American Journal of Physiology Endocrinology and Metabolism (3 ), Journal of Physiology (1), Current Developments in Clinical Nutrition and Metabolic Care (1), Journal of Animal Science and Biotechnology (1), the journal, Amino Acids (2), and Pediatric Research (1). This past year, manuscripts were published in American Journal of Physiology Endocrinology and Metabolism (2) and Pediatric Research (1). Other manuscripts are in preparation. Results were also presented at the 2014, 2015, and 2016 Experimental Biology, American Society of Animal Science, and Pediatric Research Society meetings. Results were also presented at invited talks at the 14th International Congress on Amino Acids, Peptides and Proteins in Vienna, Austria, the Advances in Skeletal Muscle Biology Conference in Gainesville, FL, the Perinatal Biology Symposium in Snowmass, CO, and the International Life Sciences Institute North America Food, Nutrition & Safety Program meeting in Washington, D.C. What do you plan to do during the next reporting period to accomplish the goals?Studies to determine the impact of leucine supplementation, with and without branched-chain amino acid supplement, of a diet that is marginally restricted in protein and energy on protein degradation in muscle will be completed. The analysis of the signaling proteins involved also will be completed. The results will provide novel information on the potential for leucine supplementation to improve lean growth of piglets.

Impacts
What was accomplished under these goals? Piglets that are early weaned frequently reduce their food intake due to stress, environmental changes, and a shift from liquid to solid diet, resulting in a reduction in growth. The branched-chain amino acid, leucine, has been shown to have anabolic effects on skeletal muscle and therefore has the potential to improve lean growth without increasing overall dietary intake or protein content. To test the impact of leucine supplementation on lean growth in piglets that consume a marginal protein diet and to identify the mechanisms involved, piglets were meal-fed a whey-based diet with either 1) a high protein content, or 2) a marginal protein content, or 3) a marginal restricted protein content supplemented with leucine to the same level as in the high protein diet for 21 days. Body weight, lean body mass, and skeletal muscle weight were highest in piglets fed the high protein diet, lowest in those fed the marginal protein diet and intermediate in those fed the leucine supplemented marginal protein diet. Feeding increased muscle protein synthesis and the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway that regulates translation initiation and this was greatest in the leucine supplemented pigs. Protein degradation signaling was unaffected by treatment. Thus, leucine supplementation can enhance muscle protein accretion but the capacity to do so in neonatal pigs that are meal-fed a milk protein-based marginal protein diet is modest. The growth and survival of piglets is reduced when nutrient intake is suboptimal. To determine if leucine supplementation will up-regulate the mTORC1 signaling pathway leading to an increase in protein synthesis and greater anabolism in muscle of neonatal piglets who consume less than their protein and energy requirements, neonatal piglets were meal-fed for 7 days either a 1) control diet, 2) marginally restricted protein and energy diet, or 3) leucine supplemented diet that is marginally restricted in protein and energy. Leucine supplementation stimulated the activation of the mTORC1 signaling pathway, but it did not enhance skeletal muscle protein synthesis or improve short-term weight gain to control levels. This limited anabolic response to leucine supplementation may have been due to the reduction in isoleucine and valine levels which would have limited their availability as substrates for protein synthesis. Therefore, studies are being conducted to test the impact of branched-chain amino acid supplementation on muscle protein degradation, synthesis, and accretion. Alternatively, the limited anabolic response to leucine supplementation may have been to the consumption of a diet that is marginally restricted in protein and energy which could limit substrate availability because the amino acids could be redirected towards energy production instead of being used as substrates for protein synthesis. To determine whether leucine supplementation will enhance lean growth when protein and energy intake are not restricted and circulating amino acid levels are maintained at constant levels, neonatal piglets were continuously fed formula via orogastric tube for 21 d with an additional pulse of either leucine or alanine (isonitrogenous control) every 4 h. Leucine supplementation increased body weight and this was due to an increase in lean gain. Muscle weight was enhanced in the leucine supplemented pigs due to an increase in protein synthesis. The increase in protein synthesis was associated with increased activation of the mTORC1 signaling pathway. The study demonstrated that leucine supplement of a complete protein and energy formulates stimulates the mTORC1-dependent translation initiation pathway leading to protein synthesis in skeletal muscle and promotes lean growth. Because leucine supplementation can reduce the circulating concentrations of other branched-chain amino acid (isoleucine and valine) and these reductions have the potential to limit the ability of leucine to stimulate protein synthesis, studies were conducted to investigate the impact of the leucine metabolite beta-hydroxy-beta-methylbutyrate (HMB) on protein anabolism. Neonatal piglets were fed a low protein formula, a high protein formula, or a low protein formula with 3 different levels of HMB. HMB supplementation did not alter circulating amino acid or insulin levels. HMB supplementation increased protein synthesis in muscle similar to that in the high protein fed piglets and this increase was due to the activation of the mTORC1 signaling pathway that regulates translation initiation. Cell replication in muscle was increased in both the HMB supplemented and high protein fed piglets. However, protein degradation signaling was unaffected by HMB supplementation or high protein feeding. The results suggest that HMB supplementation increases skeletal muscle anabolism in young piglets by stimulating protein synthesis and satellite cell proliferation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kao M, Columbus DA, Suryawan A, Steinhoff-Wagner J, Hernandez-Garcia A, Nguyen HV, Fiorotto ML, and Davis TA. Enteral ?-hydroxy-?-methylbutyrate increases protein synthesis in skeletal muscle of neonatal pigs. Am J Physiol Endocrinol Metab 310(11):E1072-84, 2016 (PMID: 27143558 / PMCID: PMC4935142)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Manjarin R, Suryawan A, Koo SJ, Wilson FA, Nguyen HV, Davis TA, and Orellana RA. Insulin modulates energy and substrate sensing and protein catabolism induced by chronic peritonitis in skeletal muscle of neonatal pigs. Pediatr Res 80(5): 744-752, 2016 (PMID: 2750897 / PMCID: Journal-In Progress).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hernandez-Garcia AD, Manjarin R, Columbus D, Nguyen HV, Suryawan A, Orellana R, and Davis TA. Leucine supplementation stimulates muscle protein synthesis in newborn pigs during acute endotoxemia. Am J Physiol Endocrinol Metab 311(4): E791-E801, 2016 (PMID: 27624100 / PMCID: PMC5241557).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Manjarin R, Columbus DA, Solis J, Suryawan A, Hern�ndez-Garc�a AD, Nguyen HV, Fiorotto ML, and Davis TA. Long-term leucine and BCAA inclusion in a protein and energy restricted diet increases mTORC1 signaling in skeletal muscle of neonatal pigs. FASEB J 30:124.3, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Suryawan A, Boutry C, Manjar�n R, C�novas A, Islas-Trejo A, Medrano JF, Nguyen HV, Fiorotto ML, and Davis TA. Intermittent leucine pulses during continuous feeding alter novel components involved in skeletal muscle growth of neonatal pigs. FASEB J 30:430.2, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Fiorotto ML, Columbus DA, Steinhoff-Wagner J, Suryawan A, Nguyen HV, and Davis TA. Postnatal muscle growth is dependent on satellite cell proliferation which demonstrates a specific requirement for dietary protein. FASEB J 30:1244.1. 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hernandez-Garcia AD, Suryawan A, Nguyen HV, Columbus DA, Manjarin R, Orellana RA, and Davis TA. Leucine ameliorates endotoxin-induced alterations in protein-protein interactions within mTORC1 complex in neonatal piglets. FASEB J 30:915.20, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Suryawan A, Boutry C, Manjar�n R, El-Kadi SW, Steinhoff-Wagner J, Nguyen HV, Fiorotto ML, and Davis TA. Long-term intermittent leucine pulses during continuous feeding impact the plasma metabolome of neonatal pigs. FASEB J 30:908.5, 2016.


Progress 01/15/15 to 01/14/16

Outputs
Target Audience:The target audience for the scientific information produced by these studies will be animal nutritionists, particularly swine nutritionists. Health care professionals, including pediatric gastroenterologists and neonatologists will also benefit because the piglet is a good model for the human infant. The scientific information generated from this project is fundamental in nature and thus will have broad application to nutrition in general. Changes/Problems:The herd of our pig supplier became infected with a respiratory virus shortly after the initiation of the grant. We identified a new pig herd in Texas with similar genetic background but their supply is limited. This has delayed our studies. What opportunities for training and professional development has the project provided?The PI has established an internationally recognized, research program providing leadership and mentorship for postdoctoral fellows. The PI recruited a postdoctoral fellow from the University of Guelph who is conducting the proposed studies with the assistance of others in the laboratory. Abstracts from initial studies have been presented at Experimental Biology and American Society of Animal Science meetings. Manuscripts have been published in Am J Physiol Endocrinol and Amino Acids. How have the results been disseminated to communities of interest?Results have been published in two manuscripts in the American Journal of Physiology Endocrinology and Metabolism and one manuscript in the journal, Amino Acids. Other manuscripts are in preparation. Results were also presented at the 2014 and 2015 Experimental Biology, American Society of Animal Science, and Pediatric Research Society meetings. Results were also presented at the 14th International Congress on Amino Acids, Peptides and Proteins in Vienna, Austria. What do you plan to do during the next reporting period to accomplish the goals?Studies are being completed to determine the effect of leucine supplementation as well as branched-chain amino acid supplementation of a diet that is restricted in both energy and protein on protein synthesis, degradation, and accretion of muscle and to identify the signaling mechanisms involved. The results will be important as they will provide novel information on the potential for leucine supplementation to improve lean growth of piglets.

Impacts
What was accomplished under these goals? Food intake is frequently reduced in early weaned pigs due to stress, environmental changes, and a shift from liquid to solid diet, and this can limit growth. The branched-chain amino acid, leucine, has been shown to have anabolic effects on skeletal muscle and therefore has the potential to improve lean growth without increasing overall dietary intake or protein content. To determine if prolonged enteral leucine supplementation improves lean growth in piglets fed a diet that is restricted in protein and to identify the mechanisms involved, neonatal pigs were fed a whey-based, milk replacement diet with either 1) a high protein content, or 2) a restricted protein content, or 3) a restricted protein content supplemented with leucine to the same level as in the high protein diet. Pigs were meal fed for 21 days. As reported in the previous progress report, feeding the high protein diet resulted in greater body weight, lean body mass, and skeletal muscle weight compared to restricted protein-fed pigs and these responses were intermediate in pigs fed the leucine supplemented restricted protein diet. Protein synthesis and the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway were increased with feeding and this was greatest in the longissimus dorsi muscle of the leucine supplemented pigs. There was no effect of dietary treatment on protein degradation signaling. Our results suggest that leucine supplementation can enhance muscle protein accretion but the capacity to do so in neonatal pigs that are meal-fed milk protein-based diets that are restricted in protein is modest. Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight piglets. To determine if enteral leucine supplementation will up-regulate the mTORC1 signaling pathway leading to an increase in protein synthesis and greater anabolism in muscle of neonatal piglets who consume less than their protein and energy requirements, neonatal piglets were meal fed for 7 days either a control diet, restricted protein and energy diet, or a leucine supplemented restricted protein and energy diet. As we recently reported, circulating insulin, isoleucine, and valine concentrations were reduced by leucine supplementation of a restricted diet. Although leucine supplementation stimulated the activation of the mTORC1 signaling pathway, it did not enhance skeletal muscle protein synthesis or improve short-term weight gain to control levels. This limited anabolic response to leucine supplementation of a protein and energy restricted diet may be due to substrate limitation, as amino acids may have been redirected towards energy production instead of being used as substrates for protein synthesis. Studies are being conducted to test the impact of branched-chain amino acid supplementation on muscle protein degradation, synthesis, and accretion. To determine whether leucine supplementation will enhance lean growth when protein and energy intake are not restricted and circulating insulin and amino acid levels are maintained at constant levels, neonatal piglets (n=11-12/group) were continuously fed formula via orogastric tube for 21 d with an additional pulse of of either leucine or alanine (isonitrogenous control) every 4 h. The results show that body and muscle weights and lean gain were ~25% greater and fat gain was 48% lower in leucine supplemented than alanine supplemented pigs. Muscle protein synthesis rates were 30% higher in leucine supplemented pigs and this was associated with increased activation of the mTORC1 signaling pathway. Moreover, we identified Rag proteins as critical leucine sensors upstream of mTOR. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 d of continuous enteral feeding of a complete protein and energy formula enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway leading to protein synthesis in skeletal muscle of neonates. These results suggest that leucine supplementation can enhance the efficiency with which nutrients are metabolized to improve lean growth.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Columbus DA, Steinhoff-Wagner J, Suryawan A, Nguyen HV, Hernandez-Garcia A, Fiorotto ML, and Davis TA. Impact of prolonged leucine supplementation on protein synthesis and lean growth in neonatal pigs. Am J Physiol 309: E601-E610, 2015 (PMID: 26374843 / PMC4572453 PMCID: Journal-In Process).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Manjarin R, Columbus DA, Fiorotto ML, Suryawan A, Nguyen HV, Hernandez-Garcia AD, Hoang N-M, and Davis TA. Leucine supplementation of a protein and energy restricted diet enhances mTORC1 activation but not muscle protein synthesis in neonatal pigs. Amino Acids 48(1):257-67, 2016 (PMID: 26334346 / PMCID: PMC4713246).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Boutry C, El-Kadi SW, Suryawan A, Steinhoff-Wagner J, Stoll B, Orellana RA, Nguyen HV, Kimball SR, Fiorotto ML, and Davis TA. Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs. Am J Physiol 2016 Feb 16:ajpendo.00479.2015. doi: 10.1152/ajpendo.00479.2015.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Hernandez-Garc�a A, Manjar�n R, Suryawan A, Nguyen HV, Davis TA, and Orellana RA. Amino acids, independent of insulin, attenuate skeletal muscle autophagy in neonatal pigs during endotoxemia. Pediatr Res In press.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Suryawan A, Boutry C, El-Kadi S, Steinhoff-Wagner J, Nguyen H, Parada R, Stoll B, Orellana R, and Davis TA. Leucine pulses during long-term continuous feeding enhance mTORC1 and mTORC2 activation in skeletal muscle of neonatal pigs. FASEB J, 29:123.6, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kao M, Columbus D, Suryawan A, Steinhoff-Wagner J, Hernandez-Garcia A, Nguyen H, Davis S, Fiorotto M, and Davis TA. Enteral supplementation with ?-hydroxy-?-methylbutyrate increases muscle protein synthesis in neonatal pigs. FASEB J, 29:742.6, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hernandez-Garcia A, Orellana R, Suryawan A, Columbus D, Manjarin R, Nguyen H, and Davis TA. Leucine antagonizes protein degradation induced by endotoxin in skeletal muscle of neonatal pigs. FASEB J, 29:755.3, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Orellana R, Manjarin R, Koo SJ, Suryawan A, Nguyen H, Wilson F, Fiorotto M and Davis TA. Insulin antagonizes SIRT1 and NF-?B signaling in skeletal muscle of neonatal pigs during chronic infection. FASEB J, 29:742.13, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Columbus D, Steinhoff-Wagner J, Suryawan A, Nguyen H, Hernandez-Garcia A, Fiorotto M, and Davis TA. Effect of prolonged enteral leucine supplementation on muscle protein synthesis and mTOR activation in neonatal pigs fed a restricted protein \diet. FASEB J, 29:129.2, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hernandez-Garcia A, Columbus D, Manjarin R, Suryawan A, Nguyen H, Almonaci R, Orellana R, and Davis TA. Leucine attenuates the endotoxin-induced reduction in skeletal muscle protein synthesis in neonatal pigs. FASEB J, 29:742.1, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Manjarin R, Columbus D, Suryawan A, Hernandez-Garcia A, Nguyen H, Fiorotto M, NM Hoang, and Davis TA. Leucine supplementation of a protein and energy restricted diet enhances mTORC1 activation but not protein synthesis in muscle of neonatal pigs. FASEB J, 29:123.8, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Manjarin R, Koo SJ, Suryawan A, Nguyen H, CossBu JA, Fiorotto M, Davis TA, and Orellana RA. Insulin ameliorates neonatal skeletal muscle NF-?B and SIRT1 signaling during chronic infection in pigs. E-PAS2015:4103.25. May 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kao M, Columbus DA, Suryawan A, Steinhoff-Wagner J, Hernandez-Garcia A, Nguyen HV, Davis SR, Fiorotto ML, and Davis TA. Protein synthesis in skeletal muscle of neonatal pigs is increased by enteral ?-hydroxy-?-methylbutyrate supplementation. E-PAS2015:2190.4. May 2015.
  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Hernandez-Garcia A, Orellana RA, Suryawan A, Columbus DA, Manjarin R, Nguyen H, and Davis TA. Leucine antagonizes endotoxin-induced skeletal muscle catabolism of neonatal pigs. E-PAS2015:455-.8. May 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Manjarin R, Columbus DA, Suryawan A, Nguyen HV, Hernandez-Garcia A, Parada RD, Fiorotto ML, and Davis TA. Long-term leucine and branched-chain amino acid supplementation in a protein and energy deficient diet increases muscle mTORC1 activation in neonatal pigs. J Anim Sci 93:979, E-Suppl. 2, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Davis TA, Columbus DA, Steinhoff-Wagner J, Suryawan A, Fiorotto ML, and Nguyen HV. Impact of prolonged leucine supplementation on protein synthesis and lean growth in neonatal pigs. Amino Acids 47(8):1642, 2015.


Progress 01/15/14 to 01/14/15

Outputs
Target Audience: The target audience for the scientific information produced by these studies will be animal nutritionists, particularly swine nutritionists. Health care professionals, including pediatric gastroenterologists and neonatologists will also benefit because the piglet is a good model for the human infant. The scientific information generated from this project is fundamental in nature and thus will have broad application to nutrition in general. Changes/Problems: Changes/Problems The herd of our pig supplier became infected with a respiratory virus shortly after the initiation of the grant. We identified a new pig herd in Texas with similar genetic background but their supply is more limited. This has delayed our studies. What opportunities for training and professional development has the project provided? The PI has established an internationally recognized, research program providing leadership and mentorship for postdoctoral fellows. The PI recruited a postdoctoral fellow from the University of Guelph who is conducting the proposed studies with the assistance of others in the laboratory. Abstracts from initial studies have been presented at Experimental Biology and American Society of Animal Science meetings. How have the results been disseminated to communities of interest? Results were presented at the 2014 and 2015 Experimental Biology and 2014 American Society of Animal Science meetings. Manuscripts will be submitted in a timely manner. What do you plan to do during the next reporting period to accomplish the goals? Studies are underway to determine the effect of leucine supplementation of an energy and protein restricted diet on protein degradation in the hindlimb and to identify the signaling mechanisisms involved. The results will be important as they will provide novel information on the potential for leucine supplementation to improve lean growth of piglets.

Impacts
What was accomplished under these goals? Early weaning of neonatal pigs frequently results in a reduction in food intake due to stress, environmental changes, and a shift from liquid to solid diet. The branched-chain amino acid, leucine, has been shown to have anabolic effects on skeletal muscle and therefore has the potential to improve lean growth without increasing overall dietary intake or protein content. The objective of the study was to determine if prolonged enteral leucine supplementation improves lean growth in piglets fed a restricted protein diet and to identify the mechanisms involved. To test that hypothesis that prolonged enteral leucine supplementation improves lean growth in neonatal pigs by upregulating protein synthesis and decreasing protein degradation, neonatal pigs (n = 14-16/diet; 5-day-old; 1.8 ± 0.3 kg body) were fed by gastric catheter a whey-based, milk replacement diet with either a high protein or restricted protein content or restricted protein supplemented with leucine to the same level as in the HP diet. Pigs were fed 40 mL?kg BW-1 per meal every 4 hours for 21 days. Feeding the high protein diet resulted in greater total body weight and lean body mass compared to restricted protein-fed pigs (P < 0.05). Masses of the longissimus dorsi muscle, heart, and kidneys were greater in the high protein than restricted protein fed pigs (P < 0.05). Body weight, lean body mass, and masses of the longissimus dorsi, heart, and kidneys in pigs fed the leucine supplemented restricted protein diet were intermediate to the restricted protein and high protein fed pigs. Protein synthesis and mTOR signaling, including 4EBP1 and S6K1 phosphorylation, and eIF4G-eIF4E abundance, were increased in all muscles with feeding (P < 0.05). Leucine supplementation also increased mTOR signaling and protein synthesis rate in the longissimus dorsi (P < 0.05). There was no effect of dietary treatment on the abundance of the ubiquitin ligases, Atrogin-1 and MuRF -1, indices of ubiquitin-proteasome mediated protein degradation, and there was no effect on LC3 activation, an indicator of the autophagy-lysosome system, although feeding reduced LC3 activation. Our results suggest that leucine supplementation can enhance muscle protein accretion but the capacity to do so in neonatal pigs that are meal-fed milk protein-based diets that are restricted in protein is modest. Thus, prolonged leucine supplementation of a restricted protein diet has the potential to improve muscle growth in young piglets. Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70% of their protein and energy requirements, enteral leucine supplementation will up-regulate the mammalian target of rapamycin complex 1 (mTORC1) pathway in skeletal muscle, leading to an increase in protein synthesis and greater muscle anabolism. Nineteen 4-d-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight-1·d-1) either 1) 16 g protein and 190 kcal (control diet), 2) 10.9 g protein and 132 kcal (restricted protein and energy diet), or 3) 10.8 g protein + 0.2 % leucine and 136 kcal (leucine supplemented, restricted protein and energy diet) at 4-h intervals for 7 d. On d 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, whereas muscle protein synthesis rate and mTORC1 activation were determined at 120 min post-feeding. At 120 min, leucine was highest in leucine supplemented restricted group (P < 0.001), whereas insulin, isoleucine and valine were lower in the restricted group and the leucine supplemented restricted group compared to control piglets (P < 0.001). Compared to the restricted and the leucine supplemented restricted groups, the control diet increased (P < 0.01) body weight, protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E·eIF4G). Leucine supplementation of a restricted diet increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E·eIF4G compared to the restricted diet alone. In conclusion, when both protein and energy intakes are restricted, leucine supplementation increases muscle mTORC1 activation but does not improve short-term body weight gain or skeletal muscle protein synthesis in neonatal pigs.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 1. Suryawan A, Orellana RA, Nguyen HV, Almonaci RD, and Davis TA. Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs. J Anim Sci Biotech 5: 8-11, 2014 (PMID: 24438646 / PMCID: PMC3901752).
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: 2. Wheatley SM, El-Kadi SW, Suryawan A, Boutry C, Orellana RA, Nguyen HV, Davis SR, and Davis TA. Protein synthesis in skeletal muscle of neonates is enhanced by administration of ?-hydroxy-?-methylbutyrate. Am J Physiol Endocrinol Metab 306: E91-9, 2014 (PMID: 24192287 / PMCID: Journal-In Process).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 3. Fiorotto ML, Davis TA, Sosa H, Villegas-Montoya C, and Fleischman R. Ribosome abundance regulates the recovery of skeletal muscle protein mass upon recuperation from postnatal undernutrition in mice. J Physiol 592.23:5269-5286, 2014 (PMID: 25239457 / PMCID: PMC4262338).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 4. Davis TA, Fiorotto ML, and Suryawan A. Bolus versus continuous feeding to optimize anabolism in neonates. Cur Opin Clin Nutr Metab Care 18: 102-108. 2015 (PMID: 25474017).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 5. Columbus DA, Fiorotto ML, and Davis TA. Leucine is a primary regulator of muscle protein synthesis in neonates. Amino Acids 47: 259-270 (PMID: 25408462 / PMCID: PMC4304911).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 6. Steinhoff-Wagner J, Boutry C, Columbus DA, Kao M, Suryawan A, Nguyen HV, Almonaci RD, Fiorotto ML, and Davis TA. Prolonged stimulation of protein synthesis and growth by leucine alone or BCAA in neonatal piglets. Proc Soc Nutr Physiol 23: 132, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 7. Davis TA, Boutry C, El-Kadi SW, Suryawan A, Steinhoff-Wagner J, Stoll B, Orellana RA, Nguyen HV, Fiorotto ML. Lean gain is enhanced by administration of leucine pulses during long-term continuous feeding. Advances in Skeletal Muscle Biology in Health and Disease Conference, University of Florida, March 5-7, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 8. Suryawan A, Nguyen H, Almonaci R, and Davis T. Regulation of the protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs. FASEB J April 2014 28:137.1.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 9. Columbus D, Steinhoff-Wagner J, Suryawan A, Kao M, Hernandez-Garcia A, Boutry C, Nguyen H, Almonaci R, Scull B, Fiorotto M, and Davis T. Prolonged enteral leucine supplementation improves lean growth in neonatal pigs fed a restricted protein diet. FASEB J April 2014 28:258.6.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 10. El-Kadi S, Boutry C, Suryawan A, Gazzaneo M, Orellana R, Srivastava N, Nguyen H, Kimball S, Fiorotto M, and Davis T. Intermittent bolus feeding enhances vital organs growth compared with continuous feeding in neonates. FASEB J April 2014 28:258.7.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 11. El-Kadi S, Boutry C, Suryawan A, Gazzaneo M, Orellana R, Srivastava N, Nguyen H, Kimball S, Fiorotto M, and Davis T. Vital organ growth is enhanced by intermittent bolus compared with continuous feeding in neonatal pigs. E-PAS2014: 2927.419. May 2014.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 12. El-Kadi SW, Boutry B, Suryawan A, Gazzaneo MC, Orellana RA, Srivastava N, Nguyen HV, Kimball SR, Fiorotto ML, and Davis TA. Enhanced protein accretion and vital organ growth with intermittent bolus compared with continuous feeding in neonatal pigs. J Anim Sci 92:188, E-Suppl. 2, 2014.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 13. Columbus DA, Steinhoff-Wagner J, Suryawan A, Kao M, Hernandez-Garcia A, Boutry C, Nguyen HV, Fiorotto HV, and Davis TA. Leucine supplementation of a restricted protein diet improves lean growth in neonatal pigs. J Anim Sci 92:219, E-Suppl. 2, 2014.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 14. Davis TA, Fiorotto ML, Suryawan A, and Columbus D. Leucine: A potent nutrient signal for protein synthesis in neonates. J Anim Sci 92:229, E-Suppl. 2, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 15. Surywan A and Davis TA. The role of insulin and amino acids in the regulation of protein degradation pathways in skeletal muscle of neonatal pigs. FASEB Science Research Conference, Nutrient Sensing and Metabolic Signaling, FASEB Science Research Conference, August 10-15, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 16. Davis TA, Boutry C, El-Kadi SW, Suryawan A, and Nguyen HV. Leucine pulses during long-term continuous feeding enhance lean gain by stimulating translation initiation. FASEB Science Research Conference, Nutrient Sensing and Metabolic Signaling, FASEB Science Research Conference, August 10-15, 2014.


Progress 01/15/13 to 01/14/14

Outputs
Target Audience: We are in the initial stages of the studies, and therefore the intended target audience for these studies has not yet been reached. The target audience for the scientific information produced by these studies will be animal nutritionists, particularly swine nutritionists. Health care professionals, including pediatric gastroenterologists and neonatologists will also benefit because the piglet is a good model for the human infant. The scientific information generated from this project is fundamental in nature and thus will have broad application to nutrition in general. Changes/Problems: The herd of our pig supplier became infected with a respiratory virus shortly after the initiation of the grant. We identified a new pig herd in Texas with similar genetic background but their supply is more limited. This has delayed our studies. What opportunities for training and professional development has the project provided? The PI has established an internationally recognized, research program providing leadership and mentorship for postdoctoral fellows. The PI recruited a postdoctoral fellow from the University of Guelph who is conducting the proposed studies with the assistance of others in the laboratory. Abstracts from initial studies have been accepted for oral presentation at Experimental Biology and the American Society of Animal Science meetings. How have the results been disseminated to communities of interest? Preliminary results will be presented at the 2014 Experimental Biology and 2014 American Society of Animal Science meetings. Manuscripts will be submitted in a timely manner. What do you plan to do during the next reporting period to accomplish the goals? Analyses are ongoing to determine the abundance and activation of signaling proteins in the next year. The results will be important as they will provide novel information on the potential for leucine supplementation to improve lean growth of piglets.

Impacts
What was accomplished under these goals? Early weaning of neonatal pigs frequently results in a reduction in food intake due to stress, environmental changes, and a shift from liquid to solid diet. The branched-chain amino acid, leucine, has been shown to have anabolic effects on skeletal muscle and therefore has the potential to improve lean growth without increasing overall dietary intake or protein content. The objective of the study was to determine if prolonged enteral leucine supplementation improves lean growth in piglets fed a restricted protein diet and to identify the mechanisms involved.Preliminary studies were performed to determine whether the response to leucine required supplementation with the other branched-chain amino acids, isoleucine and valine, as well as leucine. Neonatal pigs were fed balanced diets containing marginal protein, marginal protein supplemented with leucine, and marginal protein supplemented with leucine, isoleucine, and valine. The results suggested that supplementation with the three branched-chain amino acids was not required. To test our hypothesis that leucine supplementation will improve lean growth by reducing protein degradation and increasing protein synthesis, piglets (n = 14-16/group) were fed either a control protein (22.5% protein, 2.4% leucine) or restricted protein (11.2% protein, 1.2% leucine) milk replacement diet orrestricted proteinsupplemented with leucine to the same level as in the control protein diet. Pigs were fed every 4 h for 21 d. Body composition was determined by dual-energy x-ray absorptiometry on d 0 and 20. The activation and abundance of key indicators of protein degradation and synthesis signaling pathways are currently being determined. The preliminary results suggest that feeding the controlprotein diet resulted in greater total body weight and lean body mass after 21 d compared to restricted protein fed pigs (P < 0.05). The mass of the longissimus dorsi muscle, heart, liver, and kidney were also greater in the controlprotein than the restricted proteinfed pigs (P < 0.05). Body weight, lean body mass, and mass of the longissimus dorsi muscle, heart, liver, and kidney in pigs fed the leucine supplemented restricted protein diet were intermediate to the restricted protein and control protein fed pigs.These preliminary results suggest that prolonged leucine supplementation of a restricted protein diet has the potential to improve overall growth in neonatal pigs through an increase in lean tissue growth.

Publications