Chemists have known for more than a century about
Two main reasons explain why Gln has long been ignored in animal nutrition. First, there were technical difficulties in analyzing Gln in feeds and animal proteins because Gln is completely converted to glutamate under the standard conditions of acid hydrolysis (i.e., 6 M HCl, 110°C, and 24 h; Wu and Knabe, 1994). Second, generations of animal nutritionists had little knowledge about Gln because they had not been taught about this nutrient. Because of recent advances in analytical methods and biochemical research, Gln is now known to be particularly abundant in physiological fluids (e.g., plasma, cytoplasm, milk, fetal fluids; Wu, 2009) and both plant and animal proteins (Li et al., 2010). Indeed, Gln is a major metabolic fuel for rapidly dividing cells, including enterocytes and lymphocytes, as well as a key regulator of gene expression and cell signaling pathways (Rhoads and Wu, 2009).
The major objective of this article is to highlight 1) recent advances in understanding of the roles for Gln in improving swine nutrition, and 2) the molecular and cellular mechanisms responsible for the effects of Gln in animals.
ABUNDANCE OF GLN IN PHYSIOLOGICAL FLUIDS AND TISSUE PROTEINS
Although Gln was known in the 1980s to be the most abundant free α-AA in skeletal muscle (Maclennan et al., 1987), intensive interest in a potentially important role for Gln in swine nutrition arose from a seminal finding in the early 1990s that concentrations of free Gln in porcine milk increased progressively and markedly from 0.1 to 4 mM (i.e., 7 times the Gln concentration in maternal plasma) between d 1 and 28 of lactation (Wu and Knabe, 1994). Using a combination of proteases and peptidases to hydrolyze peptide bonds, Haynes et al. (2009) determined the content of Gln in porcine milk proteins. These authors found that Gln content was similar to glutamate content in the milk proteins of sows fed a 16% CP diet during the entire period of lactation. Notably, Gln plus glutamate accounted for approximately 20% of total peptide-bound AA in the colostrum and milk of sows fed an 18.4% CP diet (Table 1). The great abundance of both free and protein-bound Gln in porcine milk supports the rapid growth and maturation of the small intestine of the piglet (Wu, 1998). Note that the content of Gln plus glutamate in porcine colostrum and milk is greater than the maximal 12.4% of protein as a safety allowance for human consumption set by the Food and Drug Administration (Code of Federal Regulations: Title 21, Section 172.320, Subsection C.4).
The intriguing observation of the Gln abundance in milk led to extensive studies of Gln concentrations in the conceptus (i.e., embryo or fetus and associated membranes) and fetal fluids of pigs (Wu et al., 1995a, 1996a). We found that concentrations of Gln in the plasma of fetal pigs ranged from 0.8 to 1.4 mM during gestation (Wu et al., 1995a, 1996a), which were 2 to 3 times those in the plasma of all postnatal mammals studied (including pigs and sheep). Of particular note, concentrations of Gln in porcine amniotic and allantoic fluids increased from 0.64 to 1.3 and 0.69 to 3.5 mM, respectively, between d 30 and 40 of gestation (total duration of gestation = 114 d; Wu et al., 1996a, 1998), a critical period for placental growth and development (Bazer et al., 2009b). Similarly, concentrations of Gln in ovine allantoic fluid increased from 1.2 to 25 mM between d 30 and 60 of gestation (total duration of gestation = 147 d; Kwon et al., 2003). For comparison, concentrations of Lys (i.e., the reference AA in the “ideal protein”) in porcine amniotic fluid were relatively small (i.e., only 0.2 to 0.3 mM) during early pregnancy (Wu et al., 1996a). Recently, we observed that total recoverable amounts of Gln in porcine and ovine uterine flushings increased by 20-fold between d 10 and 15 of pregnancy (Bazer et al., 2009a; Gao et al., 2009). The unusual abundance of Gln in mammalian conceptuses implicates a key role for this AA in embryonic and fetal survival and growth.
Both the neonate and fetus contain a relatively high content of Gln in tissue proteins, compared with many nutritionally essential and nonessential AA. For example, Gln represents approximately 5.2% of total AA in the body proteins of fetal pigs, 7-d-old pigs, and 30-d-old pigs that were weaned at 21 d of age (Wu et al., 1999, 2010a). Because skeletal muscle accounts for 40 to 45% of BW (Wu et al., 2006), this tissue is quantitatively the most important site for Gln storage in animals, including pigs (Li et al., 2009a). Thus, requirements of Gln for protein deposition are relatively large for gestating and rapidly growing neonatal pigs.
PHYSIOLOGICAL FUNCTIONS OF GLN
Physiological functions of Gln include its participation in multiple metabolic pathways (Figure 1) as well as its role in regulating gene expression and signal transduction.
In addition to its roles as a major substrate for multiple metabolic pathways, Gln has a plethora of key regulatory functions in animals (Figure 2). For example, Gln modulates the expression of genes that beneficially regulate nutrient metabolism and cell survival (Curi et al., 2005; Wang et al., 2008a; Brasse-Lagnel et al., 2009). These genes include those for ornithine decarboxylase (ODC), heat-shock proteins, and nitric oxide (NO) synthase (NOS) in multiple cell types (Kwon et al., 2004; Rhoads and Wu, 2009). Heat-shock proteins are crucial for protecting cells from death, whereas NOS catalyzes Arg oxygenation to form NO, a signaling molecule that regulates many cellular functions (Bryan et al., 2009). In activated macrophages, expression of inducible NOS is critical for the killing of pathogens (e.g., bacteria, fungi, viruses, and parasites) by macrophages, and this depends on the availability of Gln (Wu and Meininger, 2002). Notably, ODC is a key enzyme for converting ornithine into polyamines, which stimulate DNA and protein synthesis (Wu et al., 2009). Emerging evidence shows that Gln regulates the transcription of genes related to antioxidative reactions in multiple cell types. For example, results of microarray analysis revealed that Gln increased (120 to 124%) intestinal expression of genes that are necessary for cell growth and the removal of oxidants while reducing (34 to 75%) expression of genes that promote oxidative stress and immune activation (Wang et al., 2008a). The transcription of a gene to mRNA may be regulated by Gln through one or more of the following mechanisms: 1) alteration of the specificity of RNA polymerase for promoters; 2) binding of repressors to noncoding DNA sequences that are near or that overlap the promoter region; and 3) changes in the availability of transcription factors (e.g., upregulation and downregulation of coactivators and corepressors; Brasse-Lagnel et al., 2009; Palii et al., 2009).
Cell signaling pathways are also regulated by Gln. For example, in the presence of physiological concentrations of glucose, Gln activates the mammalian target of rapamycin (mTOR) in diverse cell types, including skeletal muscle, the small intestine, and placental cells (Curi et al., 2005; Kim et al., 2011; Xi et al., 2011) through the phosphorylation of this well-conserved protein kinase. Activated mTOR phosphorylates ribosomal protein S6 kinase-1 and eukaryotic translation initiation factor 4E-binding protein-1 (proteins critical to the initiation of mRNA translation) to stimulate protein synthesis (Curi et al., 2005). Activation of mTOR may also result in inhibition of protein degradation via autophagy (Meijer and Dubbelhuis, 2004). Thus, increasing extracellular concentrations of Gln stimulates protein synthesis and inhibits proteolysis in skeletal muscle (Maclennan et al., 1987; Wu and Thompson, 1990) and enterocytes (Xi et al., 2011), leading to an anabolic effect in organisms. Evidence also exists that Gln affects the activities of adenosine monophosphate-activated protein kinase, extracellular signal-related kinase, Jun kinase, and mitogen-activated protein kinase, thereby initiating a cascade of protein phosphorylation and a series of physiological responses (Curi et al., 2005; Wu et al., 2007). At present, it is not known whether Gln directly or indirectly phosphorylates mTOR and protein kinases. However, α-ketoglutarate, a major metabolite of Gln, promotes mTOR phosphorylation in the pig small intestine (Hou et al., 2010). Furthermore, Gln modulates the production of NO and CO in diverse cell types (e.g., endothelial cells), which are important gaseous signaling molecules in the body (Li et al., 2009c; Wu and Meininger, 2009). It is interesting that Pervin et al. (2007) demonstrated physiological concentrations of NO stimulate phosphorylation of mTOR in cells, leading to enhancement of their proliferation. Exquisite integration of these Gln-dependent regulatory networks affects cell proliferation, migration, differentiation, metabolism, homeostasis, survival, and function (Flynn et al., 2009; Jobgen et al., 2009).
Increased concentrations of extracellular Gln can influence secretion of insulin and GH by pancreatic β-cells and the anterior pituitary gland, respectively (Newsholme et al., 2005). Using established RIA (Kim and Wu, 2004), we found that supplementing 1% Gln to the diet of postweaning pigs (Wang et al., 2008a) did not affect concentrations of either insulin (76.3 ± 4.9 vs. 72.1 ± 5.5 pmol/L; means ± SEM, n = 8) or GH (368 ± 27 vs. 352 ± 31 pmol/L; means ± SEM, n = 8) in plasma on d 14 postweaning, compared with an isonitrogenous control group. However, concentrations of cortisol [the major glucocorticoid in swine (Wu et al., 2000)] in plasma on d 3 postweaning were 25% less in pigs supplemented with 1% Gln than in pigs supplemented with 1.22%
The interconversion of Gln and the closely related AA, glutamate, constitutes an intracellular, intercellular, and interorgan Gln-glutamate cycle in animals (Curi et al., 2005). In turn, glutamate can partially substitute for Gln in several pathways, including ATP production and syntheses of Arg, Ala, Pro, and aspartate (Reeds et al., 1997; Wu, 1998). In addition, glutamate inhibits Gln degradation by mitochondrial phosphate-activated glutaminase in extrahepatic tissues and cells (Curthoys and Watford, 1995), which has a sparing effect on the use of Gln as a metabolic fuel (Yin et al., 2010) and increases the availability of cellular Gln (Boutry et al., 2011). However, some key functions of Gln [syntheses of Gln-tRNA, aminosugars, carbamoylphosphate, NAD(P), as well as purines and pyrimidines; renal ammoniagenesis; and regulation of ODC expression] cannot be met by glutamate (Wu, 2009). Although both Gln and glutamate provided in the enteral diet are extensively catabolized by the small intestine, this organ takes up Gln, but not glutamate, from the arterial blood (Wu et al., 1994a). Finally, because of the complex compartmentalization of cellular metabolism, extracellular glutamate may channel preferentially into the cytoplasm rather than into mitochondria (Wu et al., 1994b) and, therefore, have different effects than the glutamate generated from Gln in mitochondria.
DIGESTION AND ABSORPTION OF PEPTIDE-BOUND AND SUPPLEMENTAL GLN IN DIETS
Dietary proteins are hydrolyzed by proteases in the luminal fluids of the stomach (i.e., pepsins A, B, and C and rennin; optimal pH 2 to 3) and of the small intestine (i.e., trypsin, chymotrypsin, elastase, carboxypeptidases A and B, and aminopeptidase; optimal pH 6 to 7.5) to form Gln-containing dipeptides, tripeptides, and free Gln (Figure 3). Our work with cannulated pigs found that free Gln is stable in the stomach and duodenum (Wu et al., 1996b). The alkaline medium in the lumen of the small intestine results from bile salts, pancreatic juice, and duodenal secretions. Mucosa-derived peptidases (or prolidases in the case of peptides containing both Gln and Pro) can hydrolyze the luminal small peptides to yield free Gln. Additionally, Gln-containing dipeptides or tripeptides in the lumen of the small intestine can be directly transported into the enterocytes (i.e., the absorptive epithelial cells) through their apical membrane by H+ gradient-driven peptide transporter 1 (PepT1; Daniel, 2004). Of particular interest, the transport of di- and tripeptides from the lumen into the enterocytes is associated with an influx of both Na+ and water. Neither free Gln nor peptides containing 4 or more AA residues are accepted as substrates for PepT1. The Gln-containing small peptides are hydrolyzed rapidly by intracellular peptidases to form free Gln (Haynes et al., 2009). It is possible that a small proportion of these peptides exit the enterocytes via their basolateral membrane into the bloodstream. Some published studies support the view that a peptide transporter is expressed in the basolateral membrane of the enterocytes for the movement of small peptides from inside the cell into the portal circulation (Gilbert et al., 2008). However, the identity of basolateral peptide transporters remains elusive.
Free Gln in the lumen of the small intestine is taken up by enterocytes primarily via the Na+-dependent system N transporters (SN1 and SN2) and, to a lesser extent, Na+-dependent transporters B, B0,+, and A, Na+-independent transporter b0,+ and system L, and system y+L (requiring Na+ to transport neutral AA efficiently; Bode, 2001). The Gln-containing small peptides and free Gln can also be taken up by luminal bacteria via specific transporters (Ling and Armstead, 1995). In bacteria, Gln-containing dipeptides or tripeptides are rapidly hydrolyzed into free Gln, which is utilized via multiple metabolic pathways (Dai et al., 2010). The jejunum is the major site for absorption of dietary Gln, followed by the ileum and duodenum. There is evidence that Gln dipeptides and supplemental (i.e., free) Gln are metabolized similarly in porcine enterocytes (Haynes et al., 2009).
Approximately 67% of dietary Gln is utilized by the small intestine (i.e., mucosal cells plus luminal microorganisms) in swine, with the remaining Gln entering the portal circulation (Figure 2). For comparison, 95 to 97% of dietary glutamate is extracted by the pig small intestine in the first pass (Stoll and Burrin, 2006; Wu et al., 2010a). These findings question the traditional textbook view that almost all AA released from protein and peptide digestion in the lumen of the small intestine are absorbed into the portal circulation (Maynard et al., 1979; Bondi, 1987) and represent a paradigm shift in our understanding of swine protein nutrition.
UTILIZATION OF DIETARY AND ARTERIAL BLOOD GLN IN PIGS
All tissues and cells in fetal, neonatal, postweaning, finishing, gestating, and lactating pigs utilize Gln (Wu et al., 2010a). As noted previously, large amounts of dietary Gln are utilized by the small intestine in swine. The total flux of Gln in arterial plasma (i.e., entry of Gln into plasma from the diet, endogenous synthesis, and protein degradation), as determined after the infusion of 13C-Gln, is large (e.g., 3.27 g/kg of BW per day in young pigs; Bertolo and Burrin, 2008). Isotopic studies have also revealed that large amounts of Gln in arterial plasma (0.47 g/kg of BW per day) are oxidized to CO2 in young pigs (Stoll et al., 1999). Available evidence shows that the small intestine, nonintestinal portal-drained viscera (e.g., stomach, spleen, and pancreas), kidneys, skeletal muscle, lymphoid organs, and vascular endothelia are the major users of arterial Gln in healthy young pigs (Figure 4). Of particular interest, Gln is the only AA in arterial blood that is taken up by the small intestine of swine (Wu et al., 1994a). Furthermore, the small intestine represents only 3 to 4% of the BW, but utilizes 30% of total arterial Gln in pigs (Wu, 1998). Thus, the gut derives Gln from both the diet and blood.
Less than 10% of Gln utilized by the pig gastrointestinal tract is used for protein synthesis (Stoll et al., 1998). At physiological concentrations of Gln in plasma, net oxidation of Gln by the liver is virtually absent because of the intercellular Gln-glutamate cycle (Curthoys and Watford, 1995). Therefore, the Gln that is not extracted by the small intestine is effectively available to nonsplanchnic bed organs (e.g., kidneys and skeletal muscle). Based on published results, quantitative data on the utilization of dietary and arterial Gln by young pigs are summarized in Figure 5. Note that rates of Gln utilization by the small intestine and extraintestinal tissues of the enterally fed young swine are 965 and 970 mg/kg of BW per day, respectively, with a combined rate of 1.94 g/kg of BW per day. Protein deposition, which requires 0.41 g of Gln/kg of BW per day (Figure 4), accounts for approximately 21% of the Gln utilized by the growing pig (Wu et al., 2010a).
ENDOGENOUS SYNTHESIS OF GLN IN PIGS
Because of extensive catabolism of Gln by the small intestine and increased rates of Gln utilization by the whole body, the provision of Gln from the milk of sows and a corn- and soybean meal-based diet is inadequate to meet the needs of suckling pigs and postweaning pigs, respectively (Wu et al., 2010a). Thus, Gln must be synthesized endogenously in swine to meet its needs. It is now known that most of the circulating Gln is derived from de novo synthesis from glutamate and ammonia by Gln synthetase in multiple tissues, with skeletal muscle, the lactating mammary gland, and adipose tissue being the major sites of synthesis of Gln (Li et al., 2009b; Wu, 2009). For example, endogenous synthesis of Gln provides 58% of total Gln utilized daily in milk-fed piglets (Figure 5), and 55% of Gln in porcine milk is supplied by its synthesis from branched-chain AA (BCAA) and α-ketoglutarate in the lactating mammary glands (Li et al., 2009a). Thus, Gln synthesis is an elegant example of interorgan metabolism of AA in animals. The N and C skeleton of glutamate originate primarily from AA (i.e., mainly BCAA via BCAA transaminase and other AA via glutamate dehydrogenase) and glucose (i.e., the major source of α-ketoglutarate), respectively (Wu et al., 2005; Li et al., 2009b). Ammonia used for Gln synthesis is produced primarily from catabolism of nutritionally essential AA, whose intake from the diet greatly exceeds their deposition in tissue proteins of growing pigs (Wu et al., 2010a). Results of Gln balance studies indicate that the rate of Gln synthesis in the whole body of milk-fed young pigs is 1.15 g/kg of BW per day (Figure 5; Wu et al., 2010a).
Despite extensive utilization of Gln by the gut, little synthesis of Gln occurs in the small intestine of neonatal, growing, and lactating swine because of the negligible activity of Gln synthetase in mucosal cells (Chen et al., 2009; Haynes et al., 2009; Li et al., 2009b). Likewise, Gln synthetase activity (means ± SEM, n = 6) is minimal in the small intestine (0.43 ± 0.04 nmol/mg of protein per minute) and enterocytes (0.51 ± 0.06 nmol/mg of protein per minute) of gestating gilts (d 60 of gestation). Thus, the growth, integrity, and health of the small intestine in swine are critically dependent on enteral provision and endogenous synthesis of Gln.
Most mammalian tissues (e.g., skeletal muscle, adipose tissue, liver, and heart) contain both Gln synthetase and phosphate-activated glutaminase (i.e., the enzyme to hydrolyze Gln in cells with mitochondria). However, the lactating porcine mammary gland has increased Gln synthetase activity but lacks phosphate-activated glutaminase activity (O’Quinn et al., 2002). The absence of Gln degradation via the glutaminase pathway maximizes the availability of newly synthesized Gln for the production of milk proteins. Glutamine made in the alveolar cells is secreted into the mammary alveolar lumen and duct system and then released to suckling piglets after an oxytocin surge (Kim and Wu, 2009). The presence of the Gln-synthetic pathway in the absence of Gln degradation by glutaminase is consistent with the greater abundance of Gln in the milk of mammals, including pigs (Table 1).
REQUIREMENTS OF GLN BY PIGS
Little is known about dietary requirements for “nutritionally nonessential AA” by livestock species (Wu, 2009). However, like nutritionally essential AA (Maynard et al., 1979), requirements of Gln by a tissue (e.g., the small intestine) or the whole body can be estimated on the basis of factorial analysis, namely, the sum of the needs for Gln for metabolic pathways. The physiological needs of Gln by the small intestine can also be determined from the utilization of dietary Gln by the gut in the first-pass metabolism plus the flux of Gln into the small intestine from arterial blood. As for other AA (Baker, 2009), digestibility of protein-bound Gln must be taken into consideration in recommending its dietary requirement for swine. Thus, dietary requirements of Gln by swine (the whole body) for growth, reproduction, or lactation can be estimated as follows: dietary requirement = (MNSI + MNEIT − ESEIT)/RE, where MNSI and MNEIT are the metabolic needs for Gln by the small intestine and extraintestinal tissues, respectively, ESEIT is endogenous synthesis of Gln in extraintestinal tissues, and RE is the true digestibility of protein-bound Gln (release of dietary protein-bound Gln into the lumen of the small intestine). As illustrated for young pigs fed a milk-protein diet (Figure 1), the metabolic needs of Gln include 1) oxidation as a metabolic fuel; 2) protein synthesis; 3) generation of biologically active substances, and 4) formation of tissue- or sex-specific products (e.g., mucin, embryo or fetus, and milk). Calculated values of dietary Gln requirements, as for other AA (Tan et al., 2009a,b), should be verified by feeding trials, N-balance experiments, or studies of functional outcome (e.g., fertility, health, or survival). At this time, we are unable to estimate dietary Gln requirements by gestating or lactating sows because of the lack of data on whole-body Gln oxidation.
BENEFICIAL EFFECTS OF GLN SUPPLEMENTATION ON SWINE GROWTH AND PRODUCTION PERFORMANCE
l-Gln Supplementation to Healthy and Stressed Piglets Reared by Sows
Although milk contains large amounts of Gln, the provision of supplemental Gln to sow-reared healthy piglets enhances intestinal and whole-body growth. Of note, 32% of the dietary Gln enters the portal vein in 14-d-old pigs nursed by sows (Wu et al., 2010a), and this rate does not change in the piglets receiving oral administration of supplemental Gln (0.5 g/kg of BW twice daily). Haynes et al. (2009) reported that oral administration of Gln (0.5 g/kg of BW twice daily) to 7- to 21-d-old suckling piglets did not affect their milk intake but increased concentrations of Gln in plasma by 42% over the control group (732 ± 64 vs. 516 ± 39 nmol/mL; means ± SEM, n = 8; P < 0.01) when blood samples were obtained at 1 h postadministration. It is important to note that Gln supplementation stimulated the growth of sow-reared piglets by 12% (Haynes et al., 2009), indicating that augmenting available Gln beyond that from milk is beneficial for improving efficiency in the utilization of dietary protein and other nutrients by the neonates.
Infection, which results in a stressful condition associated with reduced concentrations of Gln in plasma (Haynes et al., 2009), is a significant cause of death in neonatal pigs (Rhoads et al., 2007). The lipopolysaccharide (LPS)-challenged piglet provides a good animal model to study the response of the intestine to endotoxins from inflammatory pathogens. The endotoxin binds to Toll-like receptor 4 on the plasma membrane, which triggers myeloid differentiation factor 88-dependent and independent pathways, resulting in activation of nuclear factor kappa-light-chain enhancer of activator B cells and apoptosis (Gribar et al., 2008). Oral administration of Gln (0.5 g/kg of BW; twice daily) to sow-reared septic piglets ameliorated the Gln deficiency, reduced fever, prevented intestinal injury, and enhanced intestinal and whole-body growth (Haynes et al., 2009). Three mechanisms may be coordinately responsible for the anti-apoptotic effects of Gln in enterocytes. First, Gln inhibits intestinal expression of Toll-like receptor 4 (Haynes et al., 2009) and myeloid differentiation factor 88 (Kessel et al., 2008), as well as nuclear factor kappa-light-chain enhancer of activator B cell activation (Haynes et al., 2009) in LPS-challenged mucosal cells. Second, Gln attenuates the production of active caspase-3 through a cytochrome C-dependent mechanism and inhibits NO synthesis by inducible NOS (Umeda et al., 2009), thereby preventing DNA damage as well as protein oxidation and S-nitrosylation. Third, Gln increases intestinal expression of antioxidative proteins, including glutathione S-transferase-ω (Wang et al., 2008a), heme oxygenase-1 (i.e., a master regulator of oxidative defense and cell homeostasis), and heat-shock proteins (i.e., cytoprotective proteins) to provide a cytoprotective effect (Madden et al., 2008).
Fetal growth restriction is another stressful condition that constrains swine production. Interestingly, pigs exhibit the most severe naturally occurring intrauterine growth retardation (IUGR) among livestock species (Wu et al., 2006), which is exacerbated further by the current widespread practice in the swine industry of restricted-feeding programs to prevent excessive BW gain by gilts and sows during gestation (Mateo et al., 2007; Kim et al., 2009). Approximately 25% of newborn piglets have a birth weight less than 1.1 kg (Wu et al., 2010b), with runt piglets weighing only one-half or even one-third as much as the heaviest littermates at birth (Vallet et al., 2002). Notably, in IUGR neonates, the key organs involved in nutrient digestion and utilization (e.g., the small intestine and skeletal muscle) suffer oxidative stress and are disproportionately smaller than those of the larger littermates (Wang et al., 2008b). Low-birth-weight piglets represent 76% of preweaning deaths (Wu et al., 2010b) and they have reduced concentrations of Gln in plasma (e.g., 0.53 ± 0.04 vs. 0.36 ± 0.03 mM in 1-d-old normal and IUGR pigs, respectively; means ± SEM, n = 8; P < 0.01). It is important to note that oral administration of Gln (0.5 g/kg of BW twice daily; 1 g/kg of BW per day) between d 0 and 21 of age enhances the growth of IUGR piglets by 16% while reducing their preweaning mortality by 48% (Table 2). Additionally, concentrations of ammonia in plasma were 19% less (P < 0.05) in the Gln-supplemented IUGR piglets than in the control group (Table 2), indicating that Gln administration resulted in an overall reduction in whole-body AA oxidation. These findings have important implications for the nutritional management of compromised neonates in the livestock industry.
l-Gln Supplementation to Postweaning Pigs
In the US swine industry, pigs are usually weaned at 3 wk of age to increase the productivity of the sow (NRC, 1998). However, early-weaned pigs naturally experience reduced feed intake and intestinal epithelial damage within 1 wk postweaning, which is associated with inadequate intake of Gln from the diet and reduced synthesis of Gln from glucose plus BCAA and other AA (Wu et al., 1996b; Wang et al., 2008a). Clearly, the diet for weanling pigs must be supplemented with Gln solely based on a consideration of meeting the necessary physiological needs of the small intestine. The Gln requirement of the small intestine (965 mg/kg of BW per day) in the 21- to 35-d-old sow-reared piglet can be used as an ideal reference for that in the age-matched postweaning pigs. However, the provision of Gln from the diet plus arterial blood is only 618 mg/kg of BW per day (Wu et al., 2010a). Thus, the need for supplemental Gln in the diet is 347 mg/kg of BW per day. Based on feed intake (33 g/kg of BW per day) of 21- to 35-d-old postweaning pigs (Wu et al., 1996b), the required percentage of supplemental Gln in diet is 1.05% (0.347/33 × 100 = 1.05%).
Although increasing dietary protein intake appears to be a simple way for providing weanling pigs with additional Gln, this approach has 2 major problems. First, increased intake of dietary protein is known to cause diarrhea in weanling pigs because of a reduced ability to digest complex proteins (Lalles et al., 2007) and to histamine-mediated secretion of water into the intestinal lumen (Ou et al., 2007). Second, increased intake of protein results in an additional N load in the diet and, therefore, an excessive amount of N excretion into the environment solely based on the principle of N balance. In contrast, dietary Gln supplementation has 2 important advantages for weanling pigs. First, supplemental Gln directly meets the physiological needs of the piglet small intestine for Gln. Second, Gln supplementation can spare the conversion of nutritionally essential AA and other AA for Gln synthesis by extraintestinal tissues, thereby improving efficiency in the utilization of dietary AA for protein synthesis and other physiological needs.
As noted previously, Gln is stable in the lumen of the stomach and small intestine (Wu et al., 1996b). Thus, supplemental Gln is effective in increasing its availability to the small intestine. It is important to note that dietary supplementation with 1% Gln can prevent jejunal atrophy (a major problem in swine production) during the first week postweaning (Wu et al., 1996b; Wang et al., 2008a) and increase feed efficiency (i.e., G:F) by 25% during the second week postweaning (Wu et al., 1996b). Similarly, dietary supplementation with Gln or glycyl-Gln dipeptide improved growth performance, small intestinal morphology, and immune responses in Escherichia coli K88+- or LPS-challenged weanling piglets (Yi et al., 2005; Jiang et al., 2009). These findings indicate that Gln is a nutritionally essential AA for postweaning pigs.
l-Gln Supplementation to Gestating Gilts
l-Gln Supplementation to Lactating Sows
Lactating sows have an increased requirement for Gln (Kim and Wu, 2009), a nutrient that is essential for the growth, development, and function of the small intestine in neonatal piglets (Rhoads and Wu, 2009). There is evidence that the uptake of Gln by porcine mammary glands is inadequate for the synthesis of milk proteins (Li et al., 2009a). For example, on d 10 of lactation, the lactating porcine mammary gland takes up 16 g of Gln/d from the arterial circulation (Trottier et al., 1997) but secretes 36 g of Gln/d in milk (Haynes et al., 2009). The synthesis of Gln by the lactating mammary gland must take place to support milk production. After extensive research, Li et al. (2009a) recently demonstrated that catabolism of BCAA played an important role in Gln synthesis by the lactating porcine mammary glands. However, this biochemical event occurs at the expense of BCAA and, therefore, reduced efficiency in the utilization of dietary AA.
Manso et al. (2007) reported that supplementing 1% Gln to the diet of lactating sows increased concentrations of Gln in milk. Extending this preliminary work, we found that dietary supplementation with 1% Gln between d 0 and 21 of lactation enhanced concentrations of Gln in the plasma, skeletal muscle, and whole milk of sows, as well as piglet growth and survival (Table 4). Several mechanisms may be responsible for the beneficial effects of Gln on lactation. First, Gln supplementation directly provides Gln for tRNA-Gln formation and spares BCAA for metabolic utilization, including the synthesis of milk proteins, by serving as both substrates and activators of the mTOR signaling pathway (Curi et al., 2005). Second, Gln may stimulate expression of ODC in lactating mammary tissue, as reported for the small intestine (Rhoads and Wu, 2009), thereby promoting the formation of polyamines that are essential for lactogenesis. Third, Gln modulates cell signaling via extracellular signal-related kinase, Jun kinase, mitogen-activated protein kinase, and NO, which in turn regulates milk production (e.g., syntheses of protein, lipids, and lactose) in the lactating mammary gland (Wu et al., 2007).
SAFETY OF GLN SUPPLEMENTATION TO SWINE
SUMMARY AND CONCLUSIONS
Over the past 20 yr, our extensive and systematic research, which originated from a seminal study of AA composition in porcine milk (Wu and Knabe, 1994), has transformed Gln from a substance not described in classical animal nutrition textbooks to a major nutrient that is now recognized to improve health, survival, growth, development, lactation, and reproduction effectively in swine (Wu, 2010b; Wu et al., 2010b). Results of biochemical studies indicate that Gln is a physiologically essential AA for the synthesis of proteins and other nitrogenous substances, with key metabolic functions in the body. Compelling evidence shows that Gln is a nutritionally essential AA for weanling pigs to maintain normal intestinal physiology and enhance efficiency in the utilization of dietary nutrients for gut and whole-body growth. Additionally, our recent findings indicate that adequate amounts of dietary Gln are necessary to support the maximum lactation and reproduction performance of pigs. All this new knowledge should be taken into consideration in revising the current version of NRC (1998)-recommended requirements of AA for swine to formulate balanced diets for all ages of swine.
In the early 1990s, a conceivable limitation of our research on Gln biochemistry and nutrition was the exceedingly high cost of Gln, which may prohibit its practical use in swine production. Although we were justifiably challenged by our colleagues, we were constantly encouraged by the historic development of cost-effective Lys manufacturing through microbial fermentation (Hirose and Shibai, 1980) and were optimistic that inexpensive Gln could become commercially available in the future. Fortunately, this dream came true in 2005 when Ajinomoto Co. Inc. successfully produced low-cost feed-grade Gln (i.e., AminoGut) for swine and poultry diets (Ajinomoto, 2005). This significant advance in animal nutrition exemplifies the power of basic research in discovering new knowledge and solving major practical problems in livestock production.
With development of high-output approaches (e.g., genomics, transcriptomics, epigenomics, proteomics, and metabolomics) over the past decade (He et al., 2009; Wang et al., 2009a,b), pioneering research has been conducted to elucidate the cellular and molecular mechanisms responsible for the beneficial actions of Gln in the small intestine and skeletal muscle of animals. However, it remains unknown how Gln supplementation can improve lactational performance and fetal growth in pigs (Bazer et al., 2008, 2009b). The underlying mechanisms are likely complex but may include multiple and interacting signal transduction pathways in mammary tissue and the conceptus. Future work in this exciting area of investigation can be greatly facilitated by using not only traditional approaches (e.g., analysis of metabolites, reverse-transcription PCR, and Western blot analysis; Bergen and Wu, 2009; Chen et al., 2009), but also powerful “omic” tools and bioinformatics (Fu et al., 2010).