K.L. Urschel

Effect of feeding a high-protein diet following an 18-hour period of feed withholding on mammalian target of rapamycin-dependent signaling in skeletal muscle of mature horses

OBJECTIVE To determine the effect of refeeding following an 18-hour period of feed withholding on the phosphorylation of translation initiation factors in the skeletal muscle of mature horses. ANIMALS 8 adult horses. PROCEDURES Following an 18-hour period of feed withholding, horses either continued to have feed withheld (postabsorptive state) or were fed 2 g/kg of a high-protein feed (33% crude protein) at time 0 and 30 minutes (postprandial state). Blood samples were taken throughout the experimental period. At 90 minutes, a biopsy specimen was taken from the middle gluteal muscle to measure the phosphorylation of translation initiation factors and tissue amino acid concentrations. Plasma glucose, insulin, and amino acid concentrations were also measured. RESULTS Horses in the postprandial state had significantly higher plasma insulin, glucose, and amino acid concentrations than did those in the postabsorptive state at the time of biopsy. Refeeding significantly increased the phosphorylation state of riboprotein S6 and eukaryotic initiation factor 4E binding protein 1. CONCLUSIONS AND CLINICAL RELEVANCE In mature horses, feeding resulted in increased mammalian target of rapamycin signaling and the mechanism appeared to be independent of an increase in Akt phosphorylation at Ser⁴⁷³. Results indicate that adult horses may be able to increase rates of muscle protein synthesis in response to feeding and that dietary amino acids appear to be the main mediators of this effect.

Effects of leucine or whey protein addition to an oral glucose solution on serum insulin, plasma glucose and plasma amino acid responses in horses at rest and following exercise

REASONS FOR PERFORMING STUDY Providing protein or amino acid mixtures in combination with glucose to post exercise in man has resulted in increases in the post feeding insulin response and in muscle glycogen and protein synthesis rates. However, whether protein and/or amino acids can modify the post exercise insulin responses in horses remains to be fully elucidated. OBJECTIVES To determine whether whey protein or leucine addition to a glucose solution affects the post gavage plasma insulin, glucose and amino acid responses in horses and whether these responses are different following a period of exercise vs. rest. METHODS Six mature, conditioned Thoroughbreds received a nasogastric gavage containing either 1 g/kg bwt glucose (G), G + 0.3 g/kg bwt whey protein (GW) or G + 0.3 g/kg bwt leucine (GL), following a period of either rest (R) or an exercise test on a high speed treadmill (EX). Each horse was studied under all 6 treatment conditions, separated by 10 day intervals. Blood samples were collected pre-exercise/rest, pregavage and at regular intervals up to 300 min post gavage. Plasma was analysed for glucose and amino acid concentrations and serum insulin concentrations were determined. RESULTS There was a significantly (P < 0.05) greater insulin response in GL-R and GL-EX when compared to the other treatments. When compared to rest, post exercise plasma glucose responses were lower in G and GW but unchanged following GL administration. Plasma alanine concentrations were elevated post exercise in all EX treatments. With the exception of markedly elevated plasma leucine concentrations after GL-R and GL-EX, the plasma concentrations of all indispensable amino acids decreased during the post gavage period. CONCLUSIONS Leucine but not whey protein augmented the serum insulin response to an oral glucose load. Leucine supplementation warrants further investigation as a means to increase the rate of post exercise muscle glycogen synthesis in horses.

Amino Acid Supplementation Does Not Alter Whole-Body Phenylalanine Kinetics in Arabian Geldings

Stable isotope infusion methods have not been extensively used in horses to study protein metabolism. The objectives were to develop infusion and sampling methodologies for [1-(13)C] phenylalanine and apply these methods to determine whether the addition of supplemental amino acids to a control diet affected whole-body phenylalanine kinetics in mature horses. Arabian geldings were studied using a 6-h primed (9 μmol/kg), constant (6 μmol · kg(-1) · h(-1)) i.v. infusion of L-[1-(13)C] phenylalanine, with blood and breath sampled every 30 min, to measure whole-body phenylalanine kinetics in response to receiving the control diet (n = 12) or the control diet supplemented with equimolar amounts of glutamate (+Glu; 55 mg · kg(-1) · d(-1); n = 5), leucine (+Leu; 49 mg · kg(-1) · d(-1); n = 5), lysine (+Lys; 55 mg · kg(-1) · d(-1); n = 5), or phenylalanine (+Phe; 62 mg · kg(-1) · d(-1); n = 6). The plasma concentrations of the supplemented amino acid in horses receiving the +Leu, +Lys, and +Phe diets were 58, 53, and 36% greater, respectively, than for the control treatment (P < 0.05). Isotopic plateau was attained in blood [1-(13)C] phenylalanine and breath (13)CO(2) enrichments by 60 and 270 min, respectively. Phenylalanine flux (+20%) and oxidation (+110%) were greater (P < 0.05) in horses receiving the +Phe treatment than in those fed the control diet. There was no effect of treatment diet on nonoxidative phenylalanine disposal or phenylalanine release from protein breakdown. The developed methods are a valuable way to study protein metabolism and assess dietary amino acid adequacy in horses and will provide a useful tool for studying amino acid requirements in the future.

Effects of advanced age on whole-body protein synthesis and skeletal muscle mechanistic target of rapamycin signaling in horses

OBJECTIVE To determine the effects of advanced age on whole-body protein synthesis and activation of the mechanistic target of rapamycin (mTOR) signaling pathway in skeletal muscle of horses. ANIMALS Six 22- to 26-year-old (aged) and six 7- to 14-year-old (mature) horses. PROCEDURES Whole-body protein synthesis was measured with a 2-hour primed constant infusion of (13)C sodium bicarbonate, followed by a 4-hour primed constant infusion of 1-(13)C phenylalanine. After the infusions, a biopsy specimen was obtained from a gluteus medius muscle and activation of protein kinase B (Akt), p70 riboprotein S6 kinase (S6K1), riboprotein S6 (rpS6), and eukaryotic initiation factor 4E binding protein 1 (4EBP1) was determined with western immunoblot analysis. For all horses, inflammatory cytokine expression in muscle and blood samples was measured with quantitative real-time PCR analysis. RESULTS Advanced age had no effect on whole-body protein synthesis or the phosphorylation of Akt, rpS6, and 4EBP1; however, muscle specimens of aged horses had 42% lower phosphorylation of S6K1 than did those of mature horses. Aged and mature horses had similar inflammatory cytokine expression in muscle and blood samples. CONCLUSIONS AND CLINICAL RELEVANCE The lower S6K1 activation for aged horses, compared with that for mature horses, could be indicative of low rates of muscle protein synthesis in aged horses. However, advanced age had no effect on any other indicators of whole-body or muscle protein synthesis or on measures of systemic or muscle inflammation, which suggested that protein metabolism and subsequently requirements may not differ between healthy mature and aged horses.

Developmental regulation of the activation of translation initiation factors of skeletal muscle in response to feeding in horses

OBJECTIVE To determine whether feeding-induced activation of translation initiation factors, specifically protein kinase B, ribosomal protein S6 kinase (S6K1), ribosomal protein S6 (rpS6), and eukaryotic initiation factor 4E binding protein 1, in horses is affected by age. ANIMALS 6 yearlings, six 2-year-old horses, and 6 mature horses. PROCEDURES After an 18-hour period of feed withholding, horses consumed a high-protein meal (2 g/kg) at time 0 and 30 minutes (postprandial state) or continued to have feed withheld (postabsorptive state). Blood samples were collected for the duration of the experimental procedures and used to determine plasma concentrations of glucose, insulin, and amino acids. At 90 minutes, biopsy specimens were collected from a gluteal muscle and used to measure phosphorylation of translation initiation factors. RESULTS Plasma glucose, insulin, and amino acid concentrations were elevated for the postprandial state, compared with results for the postabsorptive state, regardless of age. Phosphorylation of protein kinase B, S6K1, rpS6, and eukaryotic initiation factor 4E binding protein 1 was increased for the postprandial state. There was an effect of age with increased phosphorylation of S6K1 at Thr(389) and rpS6 at Ser(235/236) in the yearlings and mature horses, compared with results for the 2-year-old horses. CONCLUSIONS AND CLINICAL RELEVANCE Food consumption resulted in an increase in the activation of translation initiation factors, with the highest degree of responsiveness in the yearlings. This indicated that increased muscle accretion seen during growth could be a result of increased rates of muscle protein synthesis in response to a meal stimulus.

Dietary crude protein intake influences rates of whole-body protein synthesis in weanling horses

The objective of this study was to measure whole-body protein kinetics in weanling horses receiving forage and one of two different concentrates: (1) commercial crude protein (CCP) concentrate, which with the forage provided 4.1 g CP/kg bodyweight (BW)/day (189 mg lysine (Lys)/kg BW/day), and (2) recommended crude protein (RCP) concentrate which, with the same forage, provided 3.1 g CP/kg BW/day (194 mg Lys/kg BW/day). Blood samples were taken to determine the response of plasma amino acid concentrations to half the daily concentrate allocation. The next day, a 2 h-primed, constant infusion of [(13)C]sodium bicarbonate and a 4 h-primed, constant infusion of [1-(13)C]phenylalanine were used with breath and blood sampling to measure breath (13)CO2 and blood [(13)C]phenylalanine enrichment. Horses on the CCP diet showed an increase from baseline in plasma isoleucine, leucine, lysine, threonine, valine, alanine, arginine, asparagine, glutamine, ornithine, proline, serine, and tyrosine at 120 min post-feeding. Baseline plasma amino acid concentrations were greater with the CCP diet for histidine, isoleucine, leucine, threonine, valine, asparagine, proline, and serine. Phenylalanine, lysine, and methionine were greater in the plasma of horses receiving the RCP treatment at 0 and 120 min. Phenylalanine intake was standardized between groups; however, horses receiving the RCP diet had greater rates of phenylalanine oxidation (P = 0.02) and lower rates of non-oxidative phenylalanine disposal (P = 0.04). Lower whole-body protein synthesis indicates a limiting amino acid in the RCP diet.

Splanchnic Extraction of Phenylalanine in Mature Mares Was Not Affected by Threonine Supplementation

This study determined splanchnic extraction of phenylalanine at two intakes of threonine. Six Thoroughbred mares were supplemented with isonitrogenous amounts of either threonine or glutamate. Dietary threonine intakes were 119 (+Thr) and 58 (Basal) mg/kg/day, respectively. Each horse received each diet twice and each was studied once with an oral and once with an intravenous (IV) infusion of [1-(13)C]phenylalanine. A 2-h primed, constant IV infusion of [(13)C]sodium bicarbonate and a 4-h primed, constant infusion of [1-(13)C]phenylalanine, either orally or IV, were used to measure isotopic enrichments. Phenylalanine kinetics were not affected by diet (P > 0.05). Values for the splanchnic extraction of phenylalanine were 26 ± 5% and 27 ± 3% for the +Thr and Basal supplemented diets, respectively. These values will improve the accuracy of future equine indicator amino acid oxidation studies.

Controlled trial of whole body protein synthesis and plasma amino acid concentrations in yearling horses fed graded amounts of lysine

Lysine has been reported as the first limiting amino acid in typical equine diets. Indicator amino acid oxidation (IAAO) has become the standard method for determining amino acid requirements in other species, but prior to this study, it has not been used to determine equine requirements. The aim of this study was to evaluate whole body protein synthesis and plasma and muscle amino acid concentrations in response to graded levels of lysine intake in yearling horses. Six Thoroughbred colts (358 ± 5 kg) were fed each of six treatment lysine intakes ranging from 76 to 136 mg/kg body weight/day. Blood samples were taken before and 90 min after the morning concentrate meal. Gluteal muscle biopsies were taken ~100 min after the morning concentrate meal. The next day, whole body phenylalanine kinetics were determined using a 2 h primed, constant infusion of [(13)C] sodium bicarbonate followed by a 6 h primed, constant infusion of [1-(13)C] phenylalanine. Plasma lysine concentrations increased linearly (P <0.05) at both the 0 and 90 min time points with increasing lysine intakes. Free muscle asparagine, aspartate, arginine, glutamine, lysine, taurine and tryptophan concentrations responded quadratically to lysine intake (P <0.05). Phenylalanine kinetics did not differ between treatment intakes (P > 0.10). A broken line analysis of lysine intake and phenylalanine oxidation failed to yield a breakpoint from which to determine a lysine requirement. These diets may have been limiting in an amino acid other than lysine, underscoring the lack of data concerning amino acid requirements and bioavailability data in the horse.

Effects of Threonine Supplementation on Whole-Body Protein Synthesis and Plasma Metabolites in Growing and Mature Horses

Current equine threonine requirement estimates do not account for probable use of threonine to maintain gut health and mucin synthesis. The objective of this study was to determine if threonine supplementation (+Thr) would increase whole-body protein synthesis (WBPS) in weanling colts (Study 1) and adult mares (Study 2). Both studies used a crossover design, where each of six animals was studied twice while receiving the isonitrogenous diets. The basal diets contained lower threonine levels (Basal) than the threonine (+Thr) supplemented diets. Threonine intakes in mg/kg BW/day were as follows: 79 (Basal) and 162 (+Thr) for Study 1 and 58 (Basal) and 119 (+Thr) for Study 2, in comparison to the NRC estimated requirements of 81 and 33 mg/kg BW/day for weanling and mature horses, respectively. Following 5 days of adaptation, blood samples were taken before and 90 min after the morning concentrate meal. The next day, whole-body phenylalanine kinetics were determined using a 2 h primed, constant infusion of [(13)C]sodium bicarbonate followed by a 4 h primed, constant infusion of [1-(13)C]phenylalanine. Most plasma amino acid (AA) concentrations were elevated post-feeding (P < 0.01). Lysine and valine plasma concentrations were lower (P <0.10), while methionine, threonine, and glycine plasma concentrations were greater (P <0.10) 90 min post concentrate meal feeding with +Thr in both studies. Phenylalanine flux, intake, oxidation and non-oxidative disposal were similar between treatments (P > 0.05). These findings suggest that supplementation of a single AA can affect the metabolism of several AAs and threonine was not a limiting AA in these diets.

Whole-body phenylalanine kinetics and skeletal muscle protein signaling in horses with pituitary pars intermedia dysfunction

OBJECTIVE To compare whole-body phenylalanine kinetics and the abundance of factors in signaling pathways associated with skeletal muscle protein synthesis and protein breakdown between horses with pituitary pars intermedia dysfunction (PPID) and age-matched control horses without PPID. ANIMALS 12 aged horses (6 horses with PPID and 6 control horses; mean age, 25.0 and 25.7 years, respectively). PROCEDURES Plasma glucose, insulin, and amino acids concentrations were determined before and 90 minutes after feeding. Gluteal muscle biopsy samples were obtained from horses 90 minutes after feeding, and the abundance and activation of factors involved in signaling pathways of muscle protein synthesis and breakdown were determined. The next day, horses received a priming dose and 2 hours of a constant rate infusion of (13)C sodium bicarbonate followed by a priming dose and 4 hours of a constant rate infusion of 1-(13)C phenylalanine IV; whole-body protein synthesis was determined. RESULTS Plasma glucose and insulin concentrations were higher after feeding than they were before feeding for both groups of horses; however, no significant postprandial increase in plasma amino acids concentrations was detected for either group. Phenylalanine flux, oxidation, release from protein breakdown, and nonoxidative disposal were not significantly different between groups. No significant effect of PPID status was detected on the abundance or activation of positive or negative regulators of protein synthesis or positive regulators of protein breakdown. CONCLUSIONS AND CLINICAL RELEVANCE Results of this study suggested that whole-body phenylalanine kinetics and the postprandial activation of signaling pathways that regulate protein synthesis and breakdown in muscles were not affected by PPID status alone in aged horses.