ENDOGENOUS SYNTHESIS AND NUTRITIONAL NEEDS OF GLYCINE IN SOW-REARED PIGLETS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1001768
• Grant No.: 2014-67015-21770
• Cumulative Award Amt.: $479,996.00
• Proposal No.: 2013-01941
• Project Start Date: Feb 15, 2014
• Project End Date: Feb 14, 2019
• Grant Year: 2014
• Program Code: [A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals

Project Director
Wu, G. W.

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Animal Science

Non Technical Summary
Low-birth-weight (LBW) piglets account for 25% of newborn pigs and exhibit impaired growth, high neonatal mortality, and inferior meat quality. Despite these significant problems in swine production, there are currently no effective means to improve growth or survival of the LBW piglets. We hypothesize that there is an insufficient quantity of glycine in sow's milk and that glycine is a major amino acid limiting maximal growth of LBW piglets. We will test this hypothesis by meeting specific aims to: (1) discover novel pathways for glycine synthesis in piglets; (2) identify metabolic defects in glycine synthesis in LBW piglets; (3) determine protein synthesis in milk-fed LBW piglets in response to either activation of glycine synthesis or dietary glycine supplementation; and (4) enhance pre-weaning and post-weaning growth of LBW pigs and their meat quality with improved glycine nutrition. We expect that increased glycine availability in LBW pigs will: (1) significantly decrease oxidative stress in the small intestine; (2) increase protein synthesis in skeletal muscle; (3) reduce death of neonatal piglets; and (4) improve postnatal growth and pork quality. These findings are expected to have an important impact on U.S. swine production by providing new knowledge about the nutrition and metabolism of LBW piglets that will lead to development of new practical means for managing LBW piglets to increase their survival and growth performance.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3510	1010	100%

Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

amino acids

metabolism

neonates

nutrition

pigs

pork meat

Goals / Objectives
The major goals of this project areto: (1) discover novel pathways for glycine synthesis in piglets; (2) identify metabolic defects in glycine synthesis in LBW piglets; (3) determine protein synthesis in milk-fed LBW piglets in response to either activation of glycine synthesis or dietary glycine supplementation; and (4) enhance pre-weaning and post-weaning growth of LBW pigs and their meat quality with improved glycine nutrition.

Project Methods
Tissues (including the kidneys) will be obtained from 0- to 21-day-old pigs to determine glycine synthesis from hydroxyproline proline using both enzymological and metabolic techniques. Protein synthesis in skeletal muscle and other tissues in piglets will be measured using a tracer approach. Finally, growth of pre-weaning and post-weaning pigs, as well as the pork quality of low-birth-weight pigs at market weight will be determined.

Progress 02/15/14 to 02/14/19

Outputs
Target Audience:animal scientists, swine producers, and livestock producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students received training from this project. They are Shengdi Hu, Erin Posey, Wenliang He, and Chandler Steele. How have the results been disseminated to communities of interest?Results of our work was presented at the 2014-2018 annual meeting of the American Society of Animal Science. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Glycine is severely deficient in sow's milk and must be synthesized de novo to support protein synthesis and other glycine-dependent metabolic pathways (e.g., glutathione and heme synthesis) in piglets. Our results indicated the presence of a large amount of hydroxyproline as a tripeptide in sow's milk and piglet plasma for endogenous synthesis of glycine in piglet tissues. The specific activities of hydroxyproline oxidase (OH-POX), alanine:glyoxylate transaminase (AGT), and 4-hydroxy-2-oxoglutarate aldolase (HOA) decreased (P < 0.05) in the liver and kidneys between d 1 and 21, increased (P < 0.05) in the pancreas and small intestine over time. Similar results were obtained for the mRNA levels of these enzymes and proline oxidase (POX). For the serine- and threonine-dependent glycine pathways, the specific activities or mRNA levels of serine hydroxymethyl transferase (SHMT) and threonine dehydrogenase (TDH) increased (P < 0.05) between d 1 and 21 in most tissues. Based on Immunohistochemical (IHC) results, the localization of OH-POX and POX in the liver switched from periportal to perivenous hepatocytes with age, which indicated a change in hepatic catabolism of hydroxyproline and proline. In the kidneys, the abundance of OH-POX and POX proteins appeared to decrease with age. Besides the liver and kidneys, IHC results revealed the presence of these two proteins in the pancreas, small intestine, stomach, skeletal muscle, and gallbladder. These findings indicate the presence of the hydroxyproline-glycine pathway for the synthesis of glycine from milk- and collagen-derived hydroxyproline via inter-organ metabolism in neonatal pigs to compensate for a severe deficiency of glycine in sow's milk. We found that glycine is a nutritionally essential amino acid for maximum growth of milk-fed young pigs. When glycine was provided from a milk protein-based diet withoutsupplementation, piglets grew at a suboptimal rate. Glycine supplementation increasedamino acid transport by the small intestine and whole-body growth in young pigs. These results indicate that, when fed a regular sow's milk, young pigs cannot synthesize sufficient glycine to support protein synthesis possibly because of limited provision of precursors (e.g., serine, threonine and glucose) for glycine synthesis. Furthermore, we found that glycine can stimulateprotein synthesis and ameliorate oxidative stress in intestinal epithelial cells. Our work led to the notion that glycine is a functional amino acid with regulatory roles in pig nutrition. Further studies indicate that glycine promotes protein synthesis in skeletal muscle cells through the activation of the mechanistic target of rapamycin (mTOR) signaling pathway and the inhibition of expression of genes involved in protein degradation. Specifically, compared with controls, 0.25 and 0.5 mmol/L glycine enhanced skeletal muscle C2C12 cell growth by 22% and 15%, respectively, at 24 h post-treatment. Cells incubated with glycine (0.25 mmol/L) led to increased DNA replication. This effect of glycine was associated with enhanced mTOR phosphorylation and decreased mRNA levels of atrogin-1 and MuRF1, which were blocked by PI3K/AKT inhibitor LY294002. Moreover, glycine incubation resulted in decreased AMPK phosphorylation, a negative regulator of mTOR signaling. Importantly, we found that glycine addition abrogated serum starvation induced autophagy activation in muscle cells. Furthermore, glycine has an anti-oxidative function by augmenting glutathione synthesis. Thus, glycine has an important regulatory role in promoting muscle protein synthesis and inhibiting muscle proteolysis. Finally, we determined the effects of dietary supplementation with glycine to low-birth-weight pigs (IUGR; birth weight = 0.98 ± 0.03 kg, mean ± SEM) or normal-birth-weight pigs (NBW; birth weight = 1.44 ± 0.02 kg, mean ± SEM) between weaning and market-weight (~125 kg) on growth performance, meat quality, and body composition. At weaning (21 d of age), pigs within each birth weight group were assigned randomly into corn- and soybean meal-based diets supplemented with 1% glycine plus 0.19% corn starch or 1.19% alanine (isonitrogenous control). Crude protein content in basal diets was 20% between d 21 and 64, 18% between d 65 and 108, and 16% between d 109 and 120 of age. During the 100-d period of feeding, feed intake per kg body weight did not differ between IUGR and NBW pigs or between control and glycine groups. Growth rates of NBW pigs supplemented with 1% glycine did not differ from those for NBW pigs without glycine supplementation. In contrast, growth rates of IUGR pigs supplemented with 1% glycine were 28%, 15%, and 10% greater (P ? 0.05) than those for IUGR pigs without glycine supplementation during d 21-35, d 35-64, and d 65-120 of age, respectively. Growth rates of NBW pigs were greater (P ? 0.05) than those for IUGR pigs without glycine supplementation during any experimental period. By d 120 of age, the body weight of IUGR pigs with glycine supplementation did not differ from that of NBW pigs. Collectively, our results indicate that dietary supplementation with 1% glycine (a low-cost supplement) beneficially improves their growth rate and economic returns. At market weight (~120 to 125 kg), dressing percentage of the carcass (%) in IUGR pigs was 2.1% lower (P < 0.05) than that in NBW pigs, but did not differ between alanine- and glycine-supplemented pigs. Backfat thickness at the 10th rib tended to be lower (P = 0.081) in glycine-supplemented pigs, compared with alanne-supplemented pigs. Birth weight or dietary glycine supplementation did not affect loineye area, muscle score, muscle pH, muscle drip loss, or bone & cartilage. In summary, glycine is a crucial amino acid to increase the survival and growth performance of IUGR pigs without negatively affecting meat quality.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Wang, W.W., Z.L. Wu, G. Lin, S.D. Hu, B. Wang, Z.L. Dai, and G. Wu. 2014. Glycine stimulates protein synthesis and inhibits oxidative stress in pig small-intestinal epithelial cells. J. Nutr. 144:1540-1548.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hou, Y.Q., Y.L. Yin, and G. Wu. 2015. Dietary essentiality of "nutritionally nonessential amino acids" for animals and humans. Exp. Biol. Med. 240:997-1007.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Sun, K.J., Z.L. Wu, Y. Ji, and G. Wu. 2016. Glycine regulates protein turnover by activating Akt/mTOR and inhibiting expression of genes involved in protein degradation in C2C12 myoblasts. J. Nutr. 146:2461-2467.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wu, Z.L., Y.Q. Hou, Z.L. Dai, C.A. Hu, and G. Wu. 2019. Metabolism, nutrition and redox signaling of hydroxyproline. Antioxid. Redox Signal. 30:674-682.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Fan, X.X., S. Li, Z.L. Wu, Z.L. Dai, J. Li, X.L. Wang, and G. Wu. 2019. Glycine supplementation to breast-fed piglets attenuates postweaning jejunal epithelial apoptosis: a functional role of CHOP signaling. Amino Acids 51:463-473.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hou, Y.Q. and G. Wu. 2017. Nutritionally nonessential amino acids: A misnomer in nutritional sciences. Adv. Nutr. 8:137-139.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Li, P. and G. Wu. 2018. Roles of dietary glycine, proline and hydroxyproline in collagen synthesis and animal growth. Amino Acids 50:29-38.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Le Floch, N., A. Wessels, E. Corrent, G. Wu, and P. Bosi. 2018. The relevance of functional amino acids to support the health of growing pigs. Anim. Feed Sci. Technol. 245:104-116.

Progress 02/15/17 to 02/14/18

Outputs
Target Audience:animal scientists, swine producers, and livestock producersanimal scientists, swine producers, and livestock producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student and one undergraduate student received training from this project. How have the results been disseminated to communities of interest?Results of our work was presented at the 2017 American Society of Animal Science. What do you plan to do during the next reporting period to accomplish the goals?We will determine the quality of meat from low-birth-weight piglets supplemented with glycine.

Impacts
What was accomplished under these goals? Our results indicated the presence of a large amount of hydroxyproline as a tripeptide in sow's milk and piglet plasma. The specific activities of hydroxyproline oxidase (OH-POX), alanine:glyoxylate transaminase (AGT), and 4-hydroxy-2-oxoglutarate aldolase (HOA) decreased (P < 0.05) in the liver and kidneys between d 1 and 21, increased (P < 0.05) in the pancreas and small intestine over time. Similar results were obtained for the mRNA levels of these enzymes and proline oxidase (POX). For the serine- and threonine-dependent glycine pathways, the specific activities or mRNA levels of serine hydroxymethyl transferase (SHMT) and threonine dehydrogenase (TDH) increased (P < 0.05) between d 1 and 21 in most tissues. Based on Immunohistochemical (IHC) results, the localization of OH-POX and POX in the liver switched from periportal to perivenous hepatocytes with age, which indicated a change in hepatic catabolism of hydroxyproline and proline. In the kidneys, the abundance of OH-POX and POX proteins appeared to decrease with age. Besides the liver and kidneys, IHC results revealed the presence of these two proteins in the pancreas, small intestine, stomach, skeletal muscle, and gallbladder. These findings indicate the presence of the hydroxyproline-glycine pathway for the synthesis of glycine from milk- and collagen-derived hydroxyproline via inter-organ metabolism in neonatal pigs to compensate for a severe deficiency of glycine in sow's milk.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hou, Y.Q. and G. Wu. 2017. Nutritionally nonessential amino acids: A misnomer in nutritional sciences. Adv. Nutr. 8:137-139. Ji, Y., Z.L. Wu, Z.L. Dai, X.L. Wang, J. Li, B.G. Wang, and G. Wu. 2017. Fetal and neonatal programming of postnatal growth and feed efficiency in swine. J. Anim. Sci. Biotechnol. 8:42. Li, N., W. Wang, G. Wu, and J. Wang. 2017. Nutritional support for low birth weight infants: insights from animal studies. Br. J. Nutr. 117:1390-1402.

Progress 02/15/16 to 02/14/17

Outputs
Target Audience:animal scientists, swine producers, and livestock producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student and one undergraduate student received training from this project. How have the results been disseminated to communities of interest?Results of our work was presented at the 2016 International Swine Nutrition Conference organized by the American Society of Animal Science and China Swine Industry. What do you plan to do during the next reporting period to accomplish the goals?We will determine protein synthesis in skeletal muscle of low-birth-weight piglets supplemented with glycine.

Impacts
What was accomplished under these goals? We found that glycine promotes protein synthesis in skeletal muscle cells through the activation of the mechanistic target of rapamycin (mTOR) signaling pathway and the inhibition of expression of genes involved in protein degradation. Specifically, compared with controls, 0.25 and 0.5 mmol/L glycine enhanced skeletal muscle C2C12 cell growth by 22% and 15%, respectively, at 24 h post-treatment. Cells incubated with glycine (0.25 mmol/L) led to increased DNA replication. This effect of glycine was associated with enhanced mTOR phosphorylation and decreased mRNA levels of atrogin-1 and MuRF1, which were blocked by PI3K/AKT inhibitor LY294002. Moreover, glycine incubation resulted in decreased AMPK phosphorylation, a negative regulator of mTOR signaling. Importantly, we found that glycine addition abrogated serum starvation induced autophagy activation in muscle cells. Furthermore, glycine has an anti-oxidative function by augmenting glutathione synthesis. Thus, glycine has an important regulatory role in promoting muscle protein synthesis and inhibiting muscle proteolysis.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Yi, D., Y.Q. Hou, L. Wang, M.H. Long, S.D. Hu, H.M. Mei, L.Q. Yan, C.A. Hu, and G. Wu. 2016. N-Acetylcysteine stimulates protein synthesis in enterocytes independently of glutathione synthesis. Amino Acids 48:523-533. Hou, Y.Q., K. Yao, Y.L. Yin, and G. Wu. 2016. Endogenous synthesis of amino acids limits growth, lactation and reproduction of animals. Adv. Nutr. 7:331-342. Sun, K.J., Z.L. Wu, Y. Ji, and G. Wu. 2016. Glycine regulates protein turnover by activating Akt/mTOR and inhibiting expression of genes involved in protein degradation in C2C12 myoblasts. J. Nutr. 146:2461-2467.

Progress 02/15/15 to 02/14/16

Outputs
Target Audience:animal scientists, swine producers, and livestock producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student received training from this project. How have the results been disseminated to communities of interest?Results of our work was presented at the 2015 International Conference of Proteins, Peptides and Amino Acids at Vienna, Austria. What do you plan to do during the next reporting period to accomplish the goals?We will determine glycine synthesis from hydroxyproline in renal tubules, skeletal muscle, and hepatocytes of pigs.

Impacts
What was accomplished under these goals? Results of our study indicated that dietary glycine supplementation is needed for optimal intestinal development in neonatal pigs. Because concentrations of glycine in the lumen of the small intestine are reduced in low-birth-weight piglets, we conducted experiments to determine whether augmenting glycine provision may beneficially improve intestinal mucosal barrier in piglet enterocytes (absorptive epithelial cells of the small intestine). Compared with 0.05 mM glycine (control; concentration of glycine in the small intestinal lumen of low-birth-weight piglets), piglet enterocytes cultured with 0.25 or 1.0 mmol glycine/L exhibited an increase in transepithelial electrical resistance (TEER) by 46-53% and 80-111%, respectively. Consistently, paracellular permeability was reduced by 6-21% and 18-27%, respectively, for 0.25 or 1.0 mM glycine treatment. Compared with controls, protein abundances for claudin-3, claudin-7, and zonula occludens (ZO)-3 were enhanced (25-33%) by 0.25 and 1.0 mM, while those for occludin, claudin-1, claudin-4, and ZO-2 were not affected. A glycine concentration of 0.25 mM sustained the localization of claudin-7, and ZO-3 to the interface between enterocytes. Interestingly, 1 mM glycine promoted the distribution of claudin-4 and claudin-7 to the cytosol and nucleus, and the localization of ZO-3 to the plasma membranes. Collectively, these results indicate that physiological concentrations of glycine support intestinal mucosal barrier function by regulating the abundance and distribution of claudin-7 and ZO-3 in enterocytes. Supplementation with glycine may provide an effective nutritional strategy to improve intestinal integrity in low-birth-weight piglets.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wang, H., C. Zhang, G. Wu, Y.L. Sun, B. Wang, B.B. He, Z.L. Dai, and Z.L. Wu. 2015. Glutamine enhances tight-junction protein expression and modulates CRF signaling in the jejunum of weanling piglets. J. Nutr. 145:25-31.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Li, W., K.J. Sun, Y. Ji, Z.L. Wu, W. Wang, Z. Dai and G. Wu. 2016. Glycine regulates expression and distribution of claudin-7 and ZO-3 proteins in porcine intestinal epithelial cells. J. Nutr. doi:10.3945/jn.115.228312
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hou, Y.Q., Y.L. Yin, and G. Wu. 2015. Dietary essentiality of "nutritionally nonessential amino acids" for animals and humans. Exp. Biol. Med. 240:997-1007.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Jiao, N., Z.L. Wu, Y. Ji, B. Wang, Z.L. Dai, and G. Wu. 2015. L-Glutamate enhances barrier and anti-oxidative functions in intestinal porcine epithelial cells. J. Nutr. 145:2258-2264.

Progress 02/15/14 to 02/14/15

Outputs
Target Audience: animal scientists, swine producers, and livestock producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One graduate student received training from this project. How have the results been disseminated to communities of interest? The results were published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? We will determine glycine flux in the kidney and the whole-body of young pigs.

Impacts
What was accomplished under these goals? We found that glycine is a nutritionally essential amino acid for maximum growth of milk-fed young pigs. When glycine was provided from a milk protein-based diet withoutsupplementation, piglets grew at a suboptimal rate. Glycine supplementation increasedamino acid transport by the small intestine and whole-body growth in young pigs. These results indicate that, when fed a regular sow's milk, young pigs cannot synthesize sufficient glycine to support protein synthesis possibly because of limited provision of precursors (e.g., serine, threonine and glucose) for glycine synthesis. Furthermore, we found that glycine can stimulateprotein synthesis and ameliorate oxidative stress in intestinal epithelial cells. Our work led to the notion that glycine is a functional amino acid with regulatory roles in pig nutrition.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Wang, W.W., Z.L. Dai, Z.L. Wu, G. Lin, S.C. Jia, S.D. Hu, S. Dahanayaka, and G. Wu. 2014. Glycine is a nutritionally essential amino acid for maximal growth of milk-fed young pigs. Amino Acids 46:2037-2045.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Wu, G., F.W. Bazer, and H.R. Cross. 2014. Land-based production of animal protein: impacts, efficiency, and sustainability. Ann. N.Y. Acad. Sci. 1328:18-28.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Wang, W.W., Z.L. Wu, G. Lin, S.D. Hu, B. Wang, Z.L. Dai, and G. Wu. 2014. Glycine stimulates protein synthesis and inhibits oxidative stress in pig small-intestinal epithelial cells. J. Nutr. 144:1540-1548.