Developmental changes in the concentrations of glutamine and other amino acids in plasma and skeletal muscle of the Standardbred foal 1

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1 Published December 5, 2014 Developmental changes in the concentrations of glutamine and other amino acids in plasma and skeletal muscle of the Standardbred foal 1 H. C. Manso Filho,* K. H. McKeever,* M. E. Gordon,* H. E. Manso, W. S. Lagakos, G. Wu, and M. Watford 2 *Department of Animal Sciences, Rutgers the State University of New Jersey, New Brunswick 08901; Department of Animal Sciences, Federal Rural University of Pernambuco, Recife, Pernambuco, Brazil; Department of Nutritional Sciences, Rutgers the State University of New Jersey, New Brunswick 08901; and Department of Animal Science, Texas A & M University, College Station ABSTRACT: Glutamine is concentrated within skeletal muscle, where it has been proposed to play a regulatory role in maintaining protein homeostasis. The work presented here addressed the hypothesis that glutamine would be the most abundant free α-aa in plasma and skeletal muscle in the foal during the first year of life. Glycine, however, was the most abundant free α-aa in plasma at birth and between 3 and 12 mo of age. The concentration of glutamine, the second most abundant AA at birth, increased through the first 7 d (P < 0.05) and then returned to values similar to those at birth. This resulted in glutamine being the most abundant free α-aa in plasma from 1 d through 1 mo of age. The most abundant free α-aa in skeletal muscle at birth was glutamine, but the concentration fell by more than 50% by d 15 and continued to decrease, reaching about one-third of the original values by 1 yr of age (P < 0.05). Glutamine synthetase was barely detectable in skeletal muscle at birth, but the abundance increased rapidly within 15 d of birth. The concentration of glycine, the second most abundant α AA in muscle at birth, decreased by about 40% by d 15 (P < 0.05) and then stabilized at this value throughout the year. In contrast, glutamate, alanine, and serine concentrations, the third, fourth, and fifth most abundant free α-aa in muscle at birth, respectively, increased to new stable concentrations between 3 and 6 mo of age (P < 0.05). This resulted in alanine being the most abundant free α-aa in skeletal muscle at 12 mo of age, followed by glutamate, glutamine, and glycine. The decrease in intramuscular glutamine content, particularly during the first 2 wk after birth, is not compatible with a regulatory role for glutamine in muscle protein synthesis because it occurred at the time of maximum growth in these animals. The findings that, at certain times of development, glutamine was not the most abundant free α-aa in the foal is novel and signifies that intramuscular glutamine may have functions specific to muscle type and mammalian species. Key words: amino acid, body composition, glutamine, glutamine synthetase, horse, skeletal muscle 2009 American Society of Animal Science. All rights reserved. J. Anim. Sci : doi: /jas INTRODUCTION The first year of life for the foal is accompanied by high rates of growth, and during this period, AA homeostasis must be maintained to allow the required 1 Funding for this project was provided in part by Hatch Projects (MW) and (KHM) of the New Jersey Agricultural Experiment Station, Hatch Project 8200 (GW) of Texas AgriLife Research, and grants from the New Jersey State Equine Initiative (Rutgers University). 2 Corresponding author: Watford@aesop.rutgers.edu Received January 28, Accepted April 14, high rates of muscle protein synthesis. In most mammalian species studied to date, glutamine is the most abundant free α-aa in the body, being highly concentrated in skeletal muscle, where it has been proposed to play a regulatory role in maintaining net protein synthesis and accretion (Curthoys and Watford, 1995). Little is known about AA metabolism in the foal, but a few reports have indicated that glutamine and glycine are highly abundant in the circulation during the first months of life (Rogers et al., 1984; Zicker et al., 1991). Recently, we have shown that glutamine is highly abundant in plasma, skeletal muscle, and milk of adult Standardbred mares (Manso Filho et al., 2008b). In this work, we extended the study with mares to address the hypotheses that 1) glutamine is the most abundant 2528

2 Amino acid metabolism in foal 2529 free α-aa in the body of the foal, and that 2) plasma and intramuscular glutamine concentrations would be maintained during the first year of life, a time of net muscle protein synthesis and accretion. MATERIALS AND METHODS The Rutgers University Institutional Animal Care and Use Committee approved all methods and procedures used in this study. Animals and Management Eight clinically normal Standardbred foals (5 colts and 3 fillies), born inside with assistance at the Equine Science Center, Rutgers University, between late February and early April, 2003, were used in this study. Foals stayed with their mothers from birth until weaning at 6 mo. Mares and foals (sucklings and weanlings) were housed on pasture with free access to grass and hay and received twice daily supplementation with a commercially available pellet (15% CP and 3.00 Mcal/kg of DM), provided in individual stalls. The total ration was adjusted to provide energy and protein supplementation at 10% above the NRC (1989) recommendations for pregnant and lactating mares, and for weanlings. Approximately 55% of the energy and protein came from the pellets, with the remainder from the hay and grass (the pasture was classified as a dry lot and provided very little nutrition). All animals had free access to salt and water. Mares were not rebred after parturition, and full details of the mares have been published (Manso Filho et al., 2008b) Foals were checked for IgG concentrations at 24 and 48 h after birth, and mares and foals were given appropriate, periodic anthelmintics, vaccinations, and hoof-trimming, according to the standard practice used at the Rutgers University Equine Science Center. Body Composition Body weight, measured using an electronic scale, and estimation of body composition, from BW and rump fat thickness, were determined at birth, 24 h, 7 and 14 d, and at 1, 3, 6, 9, and 12 mo of age. Measurements of subcutaneous fat thickness at the rump were made by ultrasound (Aloka SSD-500, Tokyo, Japan) at the anatomical site described by Westervelt et al. (1976). The relationship between rump fat, determined by ultrasound (F, cm), and percentage of subcutaneous fat (Y) was determined by the equation Y = F. Biometric measurements (height at withers, girth, and cannon bone circumference) were made at birth and at 3, 6, 9, and 12 mo of age using appropriate metric scales and anatomical bone markers (McKeever et al., 1981; Manso Filho et al., 2000). Blood Collection Blood samples for metabolite determinations were collected from a vein directly into 10-mL Vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ) containing 143 US Pharmacopeia units of sodium heparin. Samples were taken immediately after birth (<30 min), 24 h later, at 7 and 14 d, and at 1, 3, 6, 9, and 12 mo of age. With the exception of the first 2 samples, all samples were taken at the same time of day (0700 h), 14 h after the last feeding. Plasma was isolated by centrifugation at 1,500 g for 15 min at 5 C, and aliquots were stored at 80 C for subsequent determination of lactate concentration. Additional aliquots of plasma were added to an equal volume of cold 10% (wt/vol) perchloric acid to precipitate proteins. Supernatants were isolated by centrifugation at 1,500 g for 15 min at 5 C, neutralized with potassium hydroxide, and stored at 80 C until analyzed for glucose and AA content. Muscle Biopsy Muscle biopsies were taken, after blood collection, immediately after birth (<30 min), and at 15 d and 3, 6, 9, and 12 mo of age. To reduce scar formation, biopsies were taken alternately from the right and left gluteus muscle at a fixed location near the first hind part of an imaginary line between the tuber coxae and the head of the tail, between 3 and 10 cm dorso-caudal to the rubber coxae. Samples were obtained at 50% of the total depth of muscle, as determined by ultrasound, for each horse. Samples were immediately frozen in liquid nitrogen and stored at 80 C until analysis. For AA analyses, samples of muscle were thawed and homogenized (Tissue Tearor, Biospec Products, Bartlesville, OK) directly in 10 volumes of ice-cold 10% (wt/vol) perchloric acid. Deproteinized supernatants were isolated and neutralized as described for plasma samples above. For Western blot analysis, additional muscle samples were extracted by homogenization in 5 to 10 volumes of Tris (100 mm), EDTA (1 mm), dithiothreitol (1 mm), ph 8.0 containing 0.05% (vol/vol) Protease Inhibitor Cocktail III (Calbiochem, San Diego, CA). Metabolite Analysis Muscle and plasma free AA in the neutralized extracts were determined after o-phthaldialdehyde derivatization, HPLC separation, and fluorometric detection (Wu and Knabe, 1994). Amino acids were quantified on the basis of authentic standards (Sigma-Aldrich, St. Louis, MO) using Millenium-32 Software (Waters, Milford, MA). Plasma lactate concentrations were determined using a YSI Sport 1500 lactate analyzer (YSI Inc., Yellow Springs, OH), and plasma glucose was measured in the neutralized, deproteinized extracts using a hexokinase-based method (Gordon and McK-

3 2530 eever, 2006). The within-assay CV for all metabolite assays was <5%. Manso Filho et al. Foal BW, fat-free mass, fat mass, and percentage body fat (Table 1) increased steadily throughout the first 12 mo of life (P < 0.05). The fastest gains in fatfree body mass (0.53 ± 0.02 kg/d) were apparent during the first 3 mo of life, with the slowest gains (0.18 ± 0.03 kg/d) being seen from 9 through 12 mo. Fat mass increased markedly between birth and 6 mo of age (the suckling period), but then increased more slowly up to 12 mo of age (the weaning period; P < 0.05). Girth and height increased throughout the study (P < 0.05); in contrast, cannon bone circumference increased markedly during the first 6 mo of life (P < 0.05), but remained stable after that time. Western Blot Analysis Muscle homogenates were centrifuged (8,000 g for 15 min at 5 C) and the supernatants were used for Western blotting to estimate the abundance of glutamine synthetase (Huang et al., 2007; Wang and Watford, 2007; Manso Filho et al., 2008a,b). The protein content of the supernatants was determined by the Bradford dye-binding method (Bio-Rad Laboratories, Hercules, CA) using BSA as the standard. Equal amounts (30 μg) of sample protein were solubilized and submitted to electrophoresis in 4 to 12% gradient resolving SDS gels (Invitrogen, Calsbad, CA) followed by electrotransfer to pure nitrocellulose membranes. Evenness of transfer was assessed by Ponceau staining. Membranes were blocked overnight with 5% nonfat milk in 20 mm Tris, 120 mm NaCl, 0.1% (vol/vol) Tween 20, ph 8.0, followed by washing and incubation with primary antibody (mouse anti-sheep glutamine synthetase; BD Laboratories, San Jose, CA) for 60 min. Glutamine synthetase bands were visualized using horseradish peroxidase-conjugated goat anti-mouse secondary antibody and the ECL detection system (Amersham Bioscience, Piscataway, NJ), followed by exposure to MR x-ray film (Kodak, Rochester, NY). Fluorographs were scanned and bands quantitated using the National Institutes of Health Image Software (SCION Image, Frederick, MD). Glutamine synthetase protein abundance is expressed as arbitrary densitometry units. Statistical Analysis Data were analyzed (SigmaStat, version 3, Jandel Scientific, San Rafael, CA) with differences between measurements made at different times compared using 1-way ANOVA for repeated measures. Posthoc differences were identified using the Tukey test with a significance value set at P < Correlations were performed using the Pearson product moment correlation. All data are reported as means ± SEM. RESULTS Growth and Body Composition Plasma Metabolites Glycine was the most abundant free α-aa (Table 2) in plasma at birth and between 3 and 12 mo of age. Plasma glycine content did not change with age, but the concentration of glutamine, the second most abundant AA at birth, increased through the first 7 d (P < 0.05) and then gradually decreased back to values similar to those seen at birth. This resulted in glutamine being the most abundant free α-aa in plasma from 1 d through 1 mo of age. The next most abundant AA in plasma were alanine, serine, valine, and leucine (Table 2). With the exceptions of the concentrations of glycine, lysine, and tryptophan (that did not change), and the concentration of taurine [that decreased consistently as the foal aged (P < 0.05)], the concentrations of all other AA tended to increase from birth through 15 to 30 d of age (P < 0.05) and then decrease back to birth values by 1 yr of age. Muscle AA The most abundant free α-aa in skeletal muscle (Table 3) at birth was glutamine, but the concentration declined by more than 50% by d 15 and continued to decrease, reaching about one-third of the original values by 1 yr of age (P < 0.05). Similarly the concentration of glycine, the second most abundant α-aa at birth, decreased by about 40% by d 15 (P < 0.05) and then stabilized at this value throughout the year. In contrast, glutamate, alanine, and serine concentrations, the third, fourth, and fifth most abundant free AA at birth, respectively, began to increase to new stable concentrations between 3 and 6 mo of age (P < 0.05). This resulted in alanine being the most abundant free α-aa in skeletal muscle at 12 mo of age, followed by glutamate, glutamine, and glycine (Table 3). Also notable was tyrosine content, which increased dramatically after 6 mo of age, reaching values 34 times greater at 9 mo of age than at birth (P < 0.05). Similarly, phenylalanine concentrations increased during this period to approximately 3 times those at birth (P < 0.05). Although the concentrations of other intramuscular AA (Table 3) did not show any distinct patterns, it is noteworthy that the sum concentration of the branched chain AA remained stable throughout the year. The concentration of taurine, a β-aa, was elevated during the first 6 mo of life, but then decreased to reduced values, approximately 6% of those at birth (Table 3), by 1 yr of age (P < 0.05). Muscle Glutamine Synthetase Expression The abundance of glutamine synthetase protein, detectable by Western blotting, in skeletal muscle was low at birth but increased considerably by d 15 (P < 0.05)

4 Amino acid metabolism in foal 2531 and then decreased slightly by 6 mo, but returned to the increased values by 9 and 12 mo (Figure 1). Glutamine synthetase abundance in skeletal muscle showed a negative correlation with the free glutamine content (R = 0.40, n = 47, P < 0.05). DISCUSSION This work represents the first report of a longitudinal study of AA metabolism in the foal during the first year of life, a period of rapid growth during which AA homeostasis is important for protein accretion. The increments of growth and hormonal changes seen in this study are similar to those reported by others in this and other horse breeds (Hintz and van Vleck, 1979; Mc- Keever et al., 1981; Jarrett et al., 1986; Manso Filho et al., 2000) and thus confirm that the foals were healthy and growing normally. Reports of AA metabolism in the developing foal are limited not only in number, but also in the tissues sampled and the spectrum of AA analyzed. Most studies, in which both glutamine and glycine are included, show that glycine is more abundant than glutamine in the circulation of various breeds of horses both at birth and in adulthood (Rogers et al., 1984; Zicker et al., 1991, 1994; Silver et al., 1994; Zicker and Rogers, 1994a,b; Hackl et al., 2006). An exception is between 18 and 28 h after birth, when Zicker et al. (1991) reported that glutamine was the most abundant AA in plasma from fed crossbred foals. This is similar to the pattern seen in the current study; in which glycine was the most abundant AA in plasma at birth, and also from 1 through 12 mo of age, but glutamine was more abundant from d 1 through 14 of age. It is also noteworthy that Zicker and Rogers (1994b) reported that plasma glycine concentrations doubled, whereas glutamine concentrations did not change, during starvation in the 2-d-old foal, with the result that glycine was by far the most abundant plasma AA in such animals. Thus, it is possible that whereas glycine and glutamine are clearly the most abundant AA in foal plasma, the precise concentrations are influenced by the sampling time relative to when the animal suckled. The other relatively abundant AA in foal plasma were serine, alanine, valine, and leucine, and this confirms several reports for both young and adult horses (Rogers et al., 1984; Zicker et al., 1991, 1994; Silver et al., 1994; Zicker and Rogers, 1994a,b; Hackl et al., 2006). Similarly, the concentrations of those AA in the plasma of our foals were well within the ranges reported in other studies (Rogers et al., 1984; Zicker et al., 1991, 1994; Silver et al., 1994; Zicker and Rogers, 1994a,b; Hackl et al., 2006). The range of plasma glutamine concentration in this study, 308 to 667 μmol/l, is greater than we found (consistently <300 μmol/l) for the mares associated with these foals sampled during the first few weeks of lactation (Manso Filho et al., 2008b). Interestingly, Rogers et al. (1984) reported that serum glutamine concentrations were greater in Quarterhorse foals (up to 3 wk of Table 1. Body weight and composition of foals during the first 12 mo of life 1 Age Birth 1 d 7 d 14 d 1 mo 3 mo 6 mo 9 mo 12 mo Item BW, kg 52.3 ± 2.2 a 53.0 ± 2.1 a 61.1 ± 3.3 ab 73.3 ± 3.2 bc 93.0 ± 3.6 c ± 5.5 d ± 7.4 e ± 10.4 f ± 11.3 g Fat free mass, kg 48.1 ± 2.0 a 48.1 ± 2.0 a 57.7 ± 3.1 ab 67.5 ± 3.0 bc 83.4 ± 2.9 c ± 4.3 d ± 6.6 e ± 6.7 f ± 10.0 g Fat mass, kg 4.55 ± 0.18 a 4.56 ± 0.18 a 5.45 ± 0.29 a 6.58 ± 0.32 ab 8.49 ± 0.44 b 15.5 ± 0.48 c 26.7 ± 1.00 d 29.8 ± 1.3 e 34.0 ± 1.4 f Fat, % BW 8.64 a 8.64 a 8.64 a 8.88 ± 0.18 a 9.20 ± 0.26 ab 9.82 ± 0.18 ab ± 0.29 d ± 0.22 cd ± 0.20 c Girth, cm 84.5 ± 1.1 a 120 ± 3 b 140 ± 4 c 153 ± 4 d 160 ± 4 e Height, cm 102 ± 2 a 123 ± 1 b 138 ± 2 c 143 ± 2 d 146 ± 2 d Cannon bone, cm 13.4 ± 0.2 a 16.1 ± 0.4 b 17.6 ± 0.3 c 18.1 ± 0.3 c 19.0 ± 0.4 c a g Means within a row with different superscript letters are significantly different (P < 0.05). 1 Values are means ± SEM where n = 8.

5 2532 Manso Filho et al. Table 2. Plasma AA concentrations in foals during the first 12 mo of life 1 Age AA, μmol/l Birth 1 d 7 d 14 d 1 mo 3 mo 6 mo 9 mo 12 mo Alanine 141 ± 12 bcdefg 208 ± 23 gh 219 ± 11 h 194 ± 15 efgh 205 ± 17 fgh 190 ± 16 defgh 128 ± 14 abcde 144 ± 14 cdefg 121 ± 7 abcd Arginine 69 ± 4 abcdefg 90 ± 0 efgh 95 ± 3 fgh 86 ± 6 defgh 107 ± 8 h 96 ± 16 gh 74 ± 3 cdefgh 69 ± 1 abcedfg 70 ± 3 bcdefg Asparagine 28 ± 2 abcde 59 ± 4 fg 89 ± 4 h 74 ± 6 gh 56 ± 4 efg 42 ± 7 def 29 ± 2 bcd 31 ± 1 cd 28 ± 1 abcd Glutamate 34 ± 2 cdefgh 35 ± 2 defgh 50 ± 5 h 47 ± 4 fgh 48 ± 5 efgh 46 ± 7 efgh 30 ± 3 bcde 25 ± 2 abcd 22 ± 2 abcd Glutamine 402 ± 19 cdef 474 ± 45 efg 667 ± 38 h 592 ± 65 gh 544 ± 39 fgh 454 ± 49 defgh 308 ± 21 abcde 301 ± 17 abcde 323 ± 15 bcde Glycine 512 ± ± ± ± ± ± ± ± ± 18 Serine 86 ± 4 a 159 ± 22 bcdefgh 235 ± 21 gh 199 ± 15 efghi 249 ± 21 i 228 ± 32 fghi 174 ± 10 cdefghi 175 ± 7 defghi 210 ± 10 fghi Tyrosine 40 ± 2 abcde 81 ± 6 fgh 99 ± 9 h 88 ± 11 gh 67 ± 6 efgh 58 ± 11 defg 49 ± 4 cdef 44 ± 3 abcde 45 ± 2 bcde Aspartate 5.0 ± 0.4 abcde 7.0 ± 0.7 efgh 7.0 ± 0.7 cdefgh 6.3 ± 0.3 abcdef 7.5 ± 0.8 defgh 9.4 ± 0.5 gh 6.7 ± 0.5 bcdefg 8.6 ± 0.9 fgh 9.7 ± 1.2 h Histidine 47 ± 3 cdefg 94 ± 9 h 47 ± 12 defg 32 ± 3 abcdefg 34 ± 1 abcdefg 61 ± 11 g 45 ± 5 bcdefg 53 ± 4 efg 59 ± 3 fg Isoleucine 24 ± 2 abc 52 ± 7 defg 75 ± 4 gh 72 ± 4 fgh 94 ± 8 h 59 ± 7 efg 40 ± 1 cde 38 ± 2 abcde 39 ± 4 bcde Leucine 117 ± 5 defgh 142 ± 13 gh 149 ± 9 h 139 ± 9 fgh 135 ± 9 efgh 92 ± 12 cd 59 ± 4 abc 59 ± 6 abd 64 ± 6 bcd Lysine 50 ± 7 98 ± ± 6 63 ± ± ± ± 3 96 ± ± 36 Methionine 15 ± 1 abcde 30 ± 3 def 52 ± 6 h 46 ± 6 gh 35 ± 3 fg 31 ± 4 efg 21 ± 1 cdef 20 ± 1 bcdfg 19 ± 1 abcde Phenylalanine 61 ± 2 efgh 62 ± 6 def 63 ± 4 h 61 ± 7 fgh 46 ± 4 cdefgh 47 ± 8 defgh 40 ± 4 bcdef 43 ± 1 bcdefgh 42 ± 1 abcdefgh Threonine 88 ± 7 cdefg 109 ± 9 efgh 162 ± 21 h 146 ± 21 gh 120 ± 15 fgh 97 ± 23 defgh 77 ± 12 bcdefg 53 ± 5 abcdef 61 ± 5 abcdef Tryptophan 49 ± 3 49 ± 3 55 ± 3 51 ± 4 54 ± 4 50 ± 7 36 ± 2 54 ± 7 62 ± 8 Valine 127 ± 6 cdef 248 ± 22 h 195 ± 10 gh 184 ± 13 fg 170 ± 10 efg 149 ± 22 defg 106 ± 6 abcd 107 ± 6 abcd 115 ± 6 bcde β-alanine 16 ± 2 defgh 17 ± 3 defh 27 ± 3 h 27 ± 3 gh 25 ± 4 fgh 11 ± 1 cde 4 ± 1 abcd 10 ± 1 abcde 11 ± 1 bcde Citrulline 60 ± 3 cde 100 ± 10 fg 154 ± 10 h 132 ± 12 gh 95 ± 9 ef 56 ± 8 bcd 38 ± 4 abcd 45 ± 3 abcd 61 ± 5 de Ornithine 46 ± 5 defgh 50 ± 5 gh 66 ± 7 h 49 ± 4 fgh 49 ± 5 efgh 34 ± 6 cdefg 23 ± 2 abcd 27 ± 4 abcdefg 32 ± 4 bcdefg Taurine 60 ± 8 gh 62 ± 3 h 28 ± 3 bcdef 23 ± 1 abcde 32 ± 4 def 43 ± 5 fgh 29 ± 2 cdef 36 ± 4 ef 23 ± 1 abcde BCAA ± 13 cd 443 ± 40 h 420 ± 22 gh 396 ± 26 efgh 400 ± 27 fgh 300 ± 43 def 205 ± 11 abcd 204 ± 14 abcd 210 ± 16 bcd Total AA 2,085 ± 78 cdefg 2,628 ± 175 efgh 3,058 ± 89 h 2,715 ± 194 gh 2,669 ± 197 fgh 2,449 ± 343 defgh 1,807 ± 109 abcd 1,799 ± 37 abcd 1,923 ± 79 bcdef a i Means within a row with different superscript letters are significantly different (P < 0.05). 1 Values are means ± SEM where n = 8. 2 BCAA = branched-chain AA.

6 Amino acid metabolism in foal 2533 Figure 1. Skeletal muscle glutamine synthetase in the foal. Results are expressed as arbitrary densitometry units and are means ± SEM of 8 animals per time point. A E Values with different letters are significantly (P < 0.05) different. A representative Western blot is shown in the insert. Table 3. Skeletal muscle AA concentrations during the first 12 mo of life 1 Age AA, μmol/kg Birth 14 d 3 mo 6 mo 9 mo 12 mo Alanine 1,279 ± 172 abcd 979 ± 80 ab 1,875 ± 224 bcd 3,687 ± 1,016 cde 3,983 ± 712 de 5,117 ± 891 e Arginine 563 ± 71 e 227 ± 13 abcd 385 ± 52 cde 386 ± 49 de 303 ± 50 c 219 ± 22 abcd Asparagine 179 ± 27 e 150 ± 10 de 75 ± 12 bc 64 ± 3 abc 79 ± 8 c 69 ± 10 abc Glutamate 1,627 ± 134 abcd 1,645 ± 116 abcd 2,580 ± 403 bcde 3,473 ± 252 e 2,814 ± 329 de 2,624 ± 427 cde Glutamine 4,025 ± 613 e 1,851 ± 138 cd 2,350 ± 389 d 1,799 ± 140 bcd 1,336 ± 138 abcd 1,342 ± 268 abcd Glycine 2,186 ± 197 e 1,395 ± 85 abcd 1,571 ± 241 bcde 1,820 ± 106 de 1,609 ± 136 abcd 1,441 ± 203 abcd Serine 534 ± 64 abc 791 ± 38 cde 772 ± 97 bcde 740 ± 40 abcde 955 ± 124 de 957 ± 167 e Tyrosine 80 ± ± ± ± 282 2,749 ± 1,924 1,147 ± 242 Aspartate 397 ± ± ± ± ± ± 67 Histidine 53 ± 9 cd 10 ± 1 abc 49 ± 9 abcd 51 ± 9 bcd 63 ± 15 de 98 ± 14 e Isoleucine 45 ± 12 abc 91 ± 6 abcd 106 ± 16 bcde 162 ± 8 e 123 ± 17 de 108 ± 26 cde Leucine 287 ± 47 cde 186 ± 13 abcd 201 ± 23 abcde 227 ± 12 bcde 364 ± 42 e 310 ± 55 de Lysine 348 ± 54 e 128 ± 10 abc 271 ± 50 abcde 277 ± 59 bcde 301 ± 35 de 289 ± 56 cde Methionine 67 ± ± 9 31 ± 7 41 ± 7 41 ± 3 42 ± 4 Phenylalanine 82 ± 9 abc 54 ± 4 ab 85 ± 13 bcd 168 ± 28 e 167 ± 16 de 137 ± 26 cde Threonine 470 ± ± ± ± ± ± 51 Tryptophan 19 ± 4 abcd 14 ± 2 abc 25 ± 4 bcd 27 ± 2 cd 33 ± 2 de 42 ± 4 e Valine 208 ± ± ± ± ± ± 40 β-alanine 275 ± 50 ab 867 ± 58 e 622 ± 72 cde 673 ± 79 de 448 ± 149 bcd 270 ± 51 ab Citrulline 213 ± 31 de 223 ± 12 e 156 ± 21 cde 83 ± 24 abc 116 ± 11 abc 148 ± 45 bcde Ornithine 507 ± 108 e 142 ± 18 abcd 170 ± 35 bcd 125 ± 21 abcd 270 ± 44 d 228 ± 39 cd Taurine 6,195 ± 747 de 3,033 ± 396 bcd 6,714 ± 1,229 e 4,074 ± 1,162 cde 1,137 ± 763 abc 354 ± 59 a BCAA ± ± ± ± ± ± 123 Total AA 19,648 ± 1,474 12,841 ± ,260 ± 2,673 19,586 ± ,878 ± 2,484 16,926 ± 2,089 a e Means within a row with different superscript letters are significantly different (P < 0.05). 1 Values are means ± SEM where n = 8. 2 BCAA = branched-chain AA.

7 2534 age) than in mares. Although Zicker et al. (1991) found that plasma glutamine concentrations were greater in foal plasma than in mare plasma at from d 2 through 27 after birth, they also reported that the converse was true at birth, before suckling. Again, such results indicate a pronounced effect of dietary intake on circulating AA concentrations. Given the low expression of muscle glutamine synthetase at birth, it is likely that much of the circulating glutamine in the newborn foal arises from the mother in utero. Due to the increased concentration of glutamine in skeletal muscle, and the large mass of this tissue, glutamine is the most abundant free α-aa in the body of other mammalian species (mouse, rat, dog, human) studied to date (Curthoys and Watford, 1995; Watford and Wu, 2005). This is probably true in the foal up to 3 mo of age, but after that time, alanine, glutamate, and at times, glycine, would be more abundant than glutamine because their concentrations in skeletal muscle are greater than that of glutamine. The identity of the most abundant AA in adult horse muscle is not known. We found skeletal muscle glutamine concentrations of 4 to 7 μmol/g of wet weight in adult Standardbred mares (Manso Filho et al., 2008a,b) and Poso et al. (1991) reported muscle glutamine content in the 15 to 20 μmol/g of dry weight range (about 4 to 8 μmol/g of wet weight) for adult Standardbreds before exercise. Thus, glutamine is certainly abundant in the muscle of adult horses, but in the absence of complete AA profile data, the possibility exists for other AA to present at equal or greater concentrations. The significance of very abundant intramuscular glutamine concentrations is not definitively known. It has been proposed that the maintenance of increased intramuscular glutamine concentrations is important to maintain increased rates of skeletal muscle protein synthesis, decreased rates of proteolysis, and hence net protein accretion (Curthoys and Watford, 1995; Watford and Wu, 2005). In the foal, the very abundant intramuscular glutamine concentrations seen at birth would agree with a role in maintaining protein accretion during the ensuing period of rapid growth. The significant decrease in glutamine concentration by d 14, and maintenance of lesser concentrations throughout the first year of life, however, would appear to negate any such role for glutamine in the growing foal. A major function of skeletal muscle AA metabolism is the release of glutamine into the circulation to be utilized by a variety of tissues such as the liver, kidney, small intestine, and cells of the immune system (Curthoys and Watford, 1995). Given that, in most species, all dietary glutamine is metabolized during absorption in the intestine, the large glutamine pool in the body is essentially synthesized de novo through the action of glutamine synthetase (Watford and Reeds, 2003). Glutamine synthetase activity is absent before birth in rat skeletal muscle but increases dramatically during the first 5 d after birth, after which it deceases Manso Filho et al. to a nadir around the time of weaning (20 to 21 d after birth), before increasing slightly to adult concentrations by 1 mo of age (Lie-Venema et al., 1998). Although the absolute time frame differs, a similar pattern is seen in foal skeletal muscle, in which we found very low expression at birth, a rapid rise to peak at 14 d, followed by a decrease over the next few months. Similar to the rat, the least expression in the foal was seen at the end of weaning (6 mo in the horse) before the abundance increased to a new steady state by 9 mo of age. The findings that the least intramuscular glutamine concentrations were seen at times of relatively high glutamine synthetase expression imply that muscle glutamine synthesis is accompanied by increased rates of glutamine release at this time, but definitive evidence requires detailed analysis of interorgan glutamine flux. The results of this study represent the first report, in any mammalian species, of AA (glutamate, alanine, glycine) other than glutamine being concentrated within skeletal muscle to the same, or greater, values as glutamine. The transient nature of AA concentrations in skeletal muscle through the first year of life may represent changes in the expression of key enzymes of AA synthesis and degradation in this and other tissues, but whether there is any functional significance to the concentrations cannot be assessed from the data presented here. The finding of a decrease in intramuscular glutamine content at a time of increased rates of net muscle protein synthesis and accretion does not support a role for this AA in the regulation of muscle protein turnover. Similarly, the finding that, at times, glutamine is not the most abundant free α-aa in foal skeletal muscle is not only novel, but it could also indicate that glutamine does not have a unique role in this tissue. LITERATURE CITED Curthoys, N. P., and M. Watford Regulation of glutaminase activity and glutamine metabolism. Annu. Rev. Nutr. 15: Gordon, M. E., and K. H. McKeever Oral and intravenous carbohydrate challenges decrease active ghrelin concentrations and alter hormones related to control of energy metabolism in horses. J. Anim. Sci. 84: Hackl, S., R. van de Hoven, M. Zickl, J. Spona, and J. Zentek Individual differences and repeatability of post-prandial changes of plasma free amino acid in young horses. J. Vet. Med. 53: Hintz, R. L., and D. van Vleck Growth rate in Thoroughbreds. Effect of age of dam, year and month of birth, and sex of foal. J. Anim. Sci. 48: Huang, Y.-F., X. Wang, and M. Watford Glutamine directly downregulates glutamine synthetase protein in mouse C2C12 skeletal muscle myotubes. J. Nutr. 137: Jarrett, S. H., K. H. McKeever, B. L. Reid, and W. A. Schurg Nitrogenous constituents in plasma of foals weaned at three, four, and six months. J. Equine Vet. Sci. 6: Lie-Venema, H. W., T. B. Harkvoort, F. J. van Hemert, A. F. Moorman, and W. H. Lamers Regulation of the spatiotemporal pattern of expression of the glutamine synthetase gene. Prog. Nucleic Acid Res. Mol. Biol. 61:

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