Dietary glycine supplementation activates mechanistic target of rapamycin signaling pathway in tissues of pigs with intrauterine growth restriction.

The mechanistic target of rapamycin (mTOR) cell signaling pathway serves as the central mechanism for the regulation of tissue protein synthesis and growth. We recently reported that supplementing 1% glycine to corn- and soybean meal-based diets enhanced growth performance between weaning and market weights in pigs with intrauterine growth restriction (IUGR). Results of recent studies have revealed an important role for glycine in activating mTOR and protein synthesis in C2C12 muscle cells. Therefore, the present study tested the hypothesis that dietary glycine supplementation enhanced the mTOR cell signaling pathway in skeletal muscle and other tissues of IUGR pigs. At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of the two groups: supplementation with either 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Tissues were obtained from the pigs within 1 wk after the feeding trial ended at 188 d of age to determine the abundances of total and phosphorylated forms of mTOR and its two major downstream proteins: eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and ribosomal protein S6 kinase-1 (p70S6K). Results showed that IUGR decreased (P < 0.05) the abundances of both total and phosphorylated mTOR, 4EBP1, and p70S6K in the gastrocnemius muscle and jejunum. In the longissimus lumborum muscle of IUGR pigs, the abundances of total mTOR did not differ (P > 0.05) but those for phosphorylated mTOR and both total and phosphorylated 4EBP1 and p70S6K were downregulated (P < 0.05), when compared to NBW pigs. These adverse effects of IUGR in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum were prevented (P < 0.05) by dietary glycine supplementation. Interestingly, the abundances of total or phosphorylated mTOR, 4EBP1, and p70S6K in the liver were not affected (P > 0.05) by IUGR or glycine supplementation. Collectively, our findings indicate that IUGR impaired the mTOR cell signaling pathway in the tissues of pigs and that adequate glycine intake was crucial for maintaining active mTOR-dependent protein synthesis for the growth and development of skeletal muscle.

Soybean meal is the major source of dietary protein for growing pigs in many regions of the world, including North America, South America, and Asia. However, this conventional feedstuff is recognized to be severely deficient in glycine (the most abundant amino acid in the bodies of animals, including pigs). Compared to pigs with normal birth weights (NBW), pigs with intrauterine growth restriction (IUGR) have a lower ability to synthesize glycine and reduced growth performance between weaning and market weights. Results of recent studies with cultured muscle cells have revealed that glycine stimulates the mechanistic target of rapamycin (mTOR) cell signaling pathway (the master activator of initiation of protein synthesis in tissues) to promote protein synthesis and cell growth. There is also evidence that the mTOR pathway is impaired in the skeletal muscle of young IUGR pigs. Thus, dietary glycine supplementation may play an important role in maintaining an active mTOR cell signaling pathway for the growth of tissues, particularly skeletal muscle. Results of this study indicated that market-weight IUGR pigs had lower abundances of both total and phosphorylated mTOR, as well as its downstream target proteins in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum, when compared with NBW pigs. In contrast, neither IUGR nor glycine supplementation affected the mTOR cell signaling pathway in the liver of pigs. Taken together, these novel findings indicate that IUGR pigs have insufficient cell signaling via the mTOR cell pathway in a tissue-specific manner during their growing-finishing phases of development and that this negative impact of IUGR can be mitigated by supplementing corn- and soybean meal-based diets with 1% glycine.

Dietary glycine supplementation enhances glutathione availability in tissues of pigs with intrauterine growth restriction.

This study tested the hypothesis that dietary supplementation with glycine enhances the synthesis and concentrations of glutathione (GSH, a major antioxidant) in tissues of pigs with intrauterine growth restriction (IUGR). At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of two groups, representing supplementation with 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Blood and other tissues were obtained from the pigs within 1 wk after the feeding trial ended at 188 d of age to determine GSH, oxidized GSH (GSSG), and activities of GSH-metabolic enzymes. Results indicated that concentrations of GSH + GSSG or GSH in plasma, liver, and jejunum (P < 0.001) and concentrations of GSH in longissimus lumborum and gastrocnemius muscles (P < 0.05) were lower in IUGR pigs than in NBW pigs. In contrast, IUGR increased GSSG/GSH ratios (an indicator of oxidative stress) in plasma (P < 0.001), jejunum (P < 0.001), both muscles (P < 0.05), and pancreas (P = 0.001), while decreasing activities of γ-glutamylcysteine synthetase and GSH synthetase in liver (P < 0.001) and jejunum (P < 0.01); and GSH reductase in jejunum (P < 0.01), longissimus lumborum muscle (P < 0.01), gastrocnemius muscle (P < 0.05), and pancreas (P < 0.01). In addition, IUGR pigs had greater (P < 0.001) concentrations of thiobarbituric acid reactive substances (TBARS; an indicator of lipid peroxidation) in plasma, jejunum, muscles, and pancreas than NBW pigs. Compared with isonitrogenous controls, dietary glycine supplementation increased concentrations of GSH plus GSSG and GSH in plasma (P < 0.01), liver (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P = 0.001), and gastrocnemius muscle (P < 0.05); activities of GSH-synthetic enzymes in liver (P < 0.01) and jejunum (P < 0.05), while reducing GSSG/GSH ratios in plasma (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P < 0.001), gastrocnemius muscle (P = 0.01), pancreas (P < 0.05), and kidneys (P < 0.01). Concentrations of GSH plus GSSG, GSH, and GSSG/GSH ratios in kidneys were not affected (P > 0.05) by IUGR. Furthermore, glycine supplementation reduced (P < 0.001) TBARS concentrations in plasma, jejunum, muscles, and pancreas. Collectively, IUGR reduced GSH availability and induced oxidative stress in pig tissues, and these abnormalities were prevented by dietary glycine supplementation in a tissue-specific manner.

Pigs have the highest rate of intrauterine growth restriction (IUGR) among livestock species. These pigs, which have low birth weights (<1.1 kg) and account for ~15% to 20% of newborn pigs, are often culled after birth because they have lower growth performance and feed efficiency due to multiple factors (including oxidative stress in tissues), when compared with litter mates with normal birth weights (NBW). Much evidence shows that glutathione, which is a tripeptide synthesized from glutamate, glycine, and cysteine via enzymes (biological catalysts, γ-glutamylcysteine synthetase, and glutathione synthetase), is a major low-molecular-weight antioxidant in animal cells. Based on the findings of our recent study that dietary glycine supplementation enhanced the growth performance of IUGR pigs from weaning to market weight, the current study tested the hypothesis that this nutritional strategy increased the synthesis and availability of glutathione in their tissues. Our results indicated that the key organs of the digestive system (the small intestine, liver, and pancreas) as well as both longissimus lumborum and gastrocnemius muscles of IUGR pigs had lower concentrations of glutathione as compared with NBW pigs, due to reductions in both the activities of glutathione-synthetic enzymes and the availability of glycine. Dietary supplementation with 1% glycine prevented these metabolic deficiencies in tissues of IUGR pigs. Our findings support the notion that IUGR pigs fed conventional corn- and soybean meal-based diets do not synthesize adequate glutathione and that dietary glycine supplementation plays an important role in enhancing the availability of glutathione and mitigating oxidative stress to improve health and growth in these compromised animals.

Dietary glycine supplementation enhances postweaning growth and meat quality of pigs with intrauterine growth restriction.

Pigs with intrauterine growth restriction (IUGR) have suboptimum growth performance and impaired synthesis of glycine (the most abundant amino acid in the body). Conventional corn- and soybean meal-based diets for postweaning pigs contain relatively low amounts of glycine and may not provide sufficient glycine to meet requirements for IUGR pigs. This hypothesis was tested using 52 IUGR pigs and 52 litter mates with normal birth weights (NBW). At weaning (21 d of age), IUGR or NBW pigs were assigned randomly to one of two nutritional groups: supplementation of a corn-soybean meal-based diet with either 1% glycine plus 0.19% cornstarch or 1.19% L-alanine (isonitrogenous control). Feed consumption and body weight (BW) of pigs were recorded daily and every 2 or 4 wks, respectively. All pigs had free access to their respective diets and clean drinking water. Within 1 wk after the feeding trial ended at 188 d of age, blood and other tissue samples were obtained from pigs to determine concentrations of amino acids and meat quality. Neither IUGR nor glycine supplementation affected (P > 0.05) feed intakes of pigs per kg BW. The final BW, gain:feed ratio, carcass dressing percentages, and four-lean-cuts percentages of IUGR pigs were 13.4 kg, 4.4%, 2%, and 15% lower (P < 0.05) for IUGR pigs than NBW pigs, respectively. Compared with pigs in the alanine group, dietary glycine supplementation increased (P < 0.05) final BW, gain:feed ratio, and meat a* value (a redness score) by 3.8 kg, 11%, and 10%, respectively, while reducing (P < 0.05) backfat thickness by 18%. IUGR pigs had lower (P < 0.05) concentrations of glycine in plasma (-45%), liver (-25%), jejunum (-19%), longissimus dorsi muscle (-23%), gastrocnemius muscle (-26%), kidney (-15%), and pancreas (-6%), as compared to NBW pigs. In addition, dietary glycine supplementation increased (P < 0.05) concentrations of glycine in plasma and all analyzed tissues. Thus, supplementing 1% of glycine to corn-soybean meal-based diets improves the growth performance, feed efficiency, and meat quality of IUGR pigs.

About 15­20% of pigs are born naturally with low birth weights (<1.1 kg) due to intrauterine growth restriction (IUGR). These pigs are often culled after birth because they have lower growth performance and feed efficiency during the production period from weaning to market weight, compared with litter mates with normal birth weights (NBW). In many countries and regions (including North America, South America, and Asia), postweaning pigs are generally fed corn- and soybean meal-based diets that contain relatively a low amount of glycine. Glycine is the most abundant amino acid in the plasma and tissue proteins of pigs but may not be formed adequately from other amino acids in the body, particularly IUGR pigs that are now known to have an impaired ability for glycine synthesis. Results of the present study indicate that IUGR pigs fed conventional corn-SBM-based diets had lower concentrations of glycine in plasma and tissues (including skeletal muscle), compared with NBW litter mates. Dietary supplementation with 1% glycine improved the growth performance, feed efficiency, and meat quality of IUGR pigs. This simple nutritional means is expected to enhance the productivity of the global swine industry.

Equine enterocytes actively oxidize l-glutamine, but do not synthesize l-citrulline or l-arginine from l-glutamine or l-proline in vitro.

In livestock species, the enterocytes of the small intestine are responsible for the synthesis of citrulline and arginine from glutamine and proline. At present, little is known about de novo synthesis of citrulline and arginine in horses. To test the hypothesis that horses of different age groups can utilize glutamine and proline for the de novo synthesis of citrulline and arginine, jejunal enterocytes from 19 horses of three different age groups: neonates (n = 4; 7.54 ± 2.36 d of age), adults (n = 9; 6.4 ± 0.35 yr), and aged (n = 6; 22.9 ± 1.0 yr) with healthy gastrointestinal tracts were used in the present study. Enterocytes were isolated from the jejunum and incubated at 37 °C for 30 min in oxygenated (95% O2/5% CO2) Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and 0 mM, 2-mM L-[U-14C]glutamine, or 2 mM L-[U-14C]proline plus 2 mM L-glutamine. Concentrations of arginine, citrulline, and ornithine in cells plus medium were determined using high-performance liquid chromatography. Results indicate that the rate of oxidation of glutamine to CO2 was high in enterocytes from neonatal horses, but low in cells from adult and aged horses. Enterocytes from all age groups of horses did not degrade proline into CO2. Regardless of age, equine enterocytes formed ornithine from glutamine and proline, but failed to convert ornithine into citrulline and arginine. Because arginine is an essential substrate for the synthesis of not only proteins, but also nitrogenous metabolites (e.g., nitric oxide, polyamines, and creatine), our novel findings have important implications for the nutrition, performance, and health of horses.

The amino acid arginine (Arg) is a precursor for the synthesis of multiple biological molecules including nitric oxide, polyamines, and creatine that are involved in cell proliferation, cellular remodeling, dilation of blood vessels, and phosphocreatine production for a readily available source of energy. Multipurpose capabilities of Arg have increased the interest in its effects in other species and must be evaluated in the horse. Levels of Arg are deficient in the milk of mammals such as humans, cows, sheep, and pigs, but their neonates are capable of synthesizing citrulline and Arg from glutamine and proline in the small intestine. High concentrations of Arg in milk have been observed in the horse, warranting investigation in case that the foal cannot synthesize Arg to support growth and thus rely on milk as the sole source of Arg. To date, no research has determined the endogenous production of Arg in horses to support metabolic and physiological processes; therefore, our experiment quantifies the synthesis of Arg in enterocytes of the small intestine of neonatal, adult, and aged horses. Data collected from this study serve as the necessary first step to determine the Arg requirement in the horse that has over-reaching implications to improve the growth, performance, reproductive efficiency, and to enhance longevity of the horse.

L-Arginine Nutrition and Metabolism in Ruminants.

L-Arginine (Arg) plays a central role in the nitrogen metabolism (e.g., syntheses of protein, nitric oxide, polyamines, and creatine), blood flow, nutrient utilization, and health of ruminants. This amino acid is produced by ruminal bacteria and is also synthesized from L-glutamine, L-glutamate, and L-proline via the formation of L-citrulline (Cit) in the enterocytes of young and adult ruminants. In pre-weaning ruminants, most of the Cit formed de novo by the enterocytes is used locally for Arg production. In post-weaning ruminants, the small intestine-derived Cit is converted into Arg primarily in the kidneys and, to a lesser extent, in endothelial cells, macrophages, and other cell types. Under normal feeding conditions, Arg synthesis contributes 65% and 68% of total Arg requirements for nonpregnant and late pregnany ewes fed a diet with ~12% crude protein, respectively, whereas creatine production requires 40% and 36% of Arg utilized by nonpregnant and late pregnant ewes, respectively. Arg has not traditionally been considered a limiting nutrient in diets for post-weaning, gestating, or lactating ruminants because it has been assumed that these animals can synthesize sufficient Arg to meet their nutritional and physiological needs. This lack of a full understanding of Arg nutrition and metabolism has contributed to suboptimal efficiencies for milk production, reproductive performance, and growth in ruminants. There is now considerable evidence that dietary supplementation with rumen-protected Arg (e.g., 0.25-0.5% of dietary dry matter) can improve all these production indices without adverse effects on metabolism or health. Because extracellular Cit is not degraded by microbes in the rumen due to the lack of uptake, Cit can be used without any encapsulation as an effective dietary source for the synthesis of Arg in ruminants, including dairy and beef cows, as well as sheep and goats. Thus, an adequate amount of supplemental rumen-protected Arg or unencapsulated Cit is necessary to support maximum survival, growth, lactation, reproductive performance, and feed efficiency, as well as optimum health and well-being in all ruminants.

Amino Acids and Their Metabolites for Improving Human Exercising Performance.

Achieving adequate nutrition for exercising humans is especially important for improving both muscle mass and metabolic health. One of the most common misunderstandings in the fitness industry is that the human body has requirements for dietary whole protein and that exercising individuals must consume only whole protein to meet their physiological needs. This view, however, is incorrect. Instead, humans at rest or during exercise have requirements for dietary amino acids (AAs), and dietary protein is a source of AAs in the body. The requirements for AAs must be met each day to avoid a negative nitrogen balance in individuals with moderate or intense physical activity. By properly meeting increased requirements for AAs through increased intake of high-quality protein (the source of AAs) plus supplemental AAs, athletes can improve their overall athletic performance. AAs or metabolites that are of special importance for exercising individuals include arginine, branched-chain AAs, creatine, glycine, taurine, and glutamine. The AAs play vital roles as both substrates for protein synthesis and molecules for regulating blood flow and nutrient metabolism. The functional roles of AAs include the maintenance of cell and tissue integrity; stimulation of mechanistic target of rapamycin and AMP-activated protein kinase cell signaling pathways; energy sources for the small intestine, cells of the immune system, and skeletal muscle; antioxidant and anti-inflammatory reactions; production of neurotransmitters; modulation of acid-base balance in the body. All of those roles are crucial for the overall goal of improving exercise performance. Therefore, adequate intakes of proteinogenic AAs and their functional metabolites, especially those noted in this review, are essential for optimal human health (including optimum muscle mass and function) and should be a primary goal of exercising individuals.

Amino Acids in Swine Nutrition and Production.

Amino acids are the building blocks of proteins in animals, including swine. With the development of new analytical methods and biochemical research, there is a growing interest in fundamental and applied studies to reexamine the roles and usage of amino acids (AAs) in swine production. In animal nutrition, AAs have been traditionally classified as nutritionally essential (EAAs) or nutritionally nonessential (NEAAs). AAs that are not synthesized de novo must be provided in diets. However, NEAAs synthesized by cells of animals are more abundant than EAAs in the body, but are not synthesized de novo in sufficient amounts for the maximal productivity or optimal health (including resistance to infectious diseases) of swine. This underscores the conceptual limitations of NEAAs in swine protein nutrition. Notably, the National Research Council (NRC 2012) has recognized both arginine and glutamine as conditionally essential AAs for pigs to improve their growth, development, reproduction, and lactation. Results of recent work have also provided compelling evidence for the nutritional essentiality of glutamate, glycine, and proline for young pigs. The inclusion of so-called NEAAs in diets can help balance AAs in diets, reduce the dietary levels of EAAs, and protect the small intestine from oxidative stress, while enhancing the growth performance, feed efficiency, and health of pigs. Thus, both EAAs and NEAAs are needed in diets to meet the requirements of pigs. This notion represents a new paradigm shift in our understanding of swine protein nutrition and is transforming pork production worldwide.