Whole-body phenylalanine kinetics and skeletal muscle protein signaling in horses with pituitary pars intermedia dysfunction Laurel M. Mastro, MS; Amanda A. Adams, PhD; Kristine L. Urschel, PhD

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 13C sodium bicarbonate followed by a priming dose and 4 hours of a constant rate infusion of 1-13C 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. (Am J Vet Res 2014;75:658–667)

P

ituitary pars intermedia dysfunction (also known as equine Cushing’s disease) is believed to affect 15% to 30% of aged horses.1,2 This disease results from the loss of dopaminergic inhibition of the pituitary pars intermedia leading to increased synthesis of proopiomelanocortin-derived peptides, α-melanocyte– Received November 27, 2013. Accepted February 14, 2014. From the Departments of Animal and Food Sciences (Mastro, Urschel) and Veterinary Science (Adams), College of Agriculture, Food and the Environment, University of Kentucky, Lexington, KY 40546. This manuscript represents a portion of a thesis submitted by Laurel Mastro to the University of Kentucky Department of Animal and Food Sciences as partial fulfillment of the requirements for a Master of Science degree. Supported by the Morris Animal Foundation First Award Grant (D09EQ-310). The authors thank Sara Tanner, Tammy Brewster-Barnes, and Nicole Holownia for assistance with care of the horses and sample collection. Dr. Macarena Sanz for placement of IV catheters in horses, and Dr. Jessica Suagee for assistance with statistical analysis. The information reported in this manuscript is part of a project of the Kentucky Agricultural Experiment Station and is published with the approval of the director. Address correspondence to Dr. Urschel ([email protected]). 658

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4E-BP1 ACTH Akt AMPK FoxO mTOR MuRF1 NF-κB PPID rpS6

ABBREVIATIONS

Eukaryotic initiation factor 4E–binding protein 1 Adrenocorticotrophic hormone Protein kinase B Adenosine monophosphate–activated protein kinase Forkhead box O Mechanistic target of rapamycin Muscle RING finger 1 Nuclear factor-kappa B Pituitary pars intermedia dysfunction Riboprotein S6

stimulating hormone, β-endorphin–related peptides, corticotropin-like intermediate lobe peptide, and ACTH.3 Clinical signs of PPID include hypertrichosis,4 laminitis,5 secondary infections,6 decreased peripheral tissue insulin sensitivity,7 and muscle atrophy.8 In humans, muscle atrophy is associated with decreased whole-body protein synthesis.9 In human patients with Cushing’s disease, whole-body protein metabolism is altered because of increased protein breakdown. These changes are presumably attributAJVR, Vol 75, No. 7, July 2014

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able to differences in body composition and high circulating concentrations of glucocorticoids associated with that disease, which increase protein oxidation.10 In horses with PPID, basal circulating concentrations of cortisol are typically within the reference range11,12; however, other authors have theorized that the circulating cortisol circadian rhythm in horses with PPID may be abolished, leading to increased cumulative cortisol exposure of tissues during a 24-hour period.13 Therefore, it is unclear whether similar alterations in wholebody protein metabolism would be detected in horses with PPID, compared with such metabolism in control horses without PPID. Isotope infusion techniques have been used for measurement of whole-body protein metabolism in humans14 and horses.15 Such methods have been used to determine the effects of advanced age on whole-body protein synthesis in horses16; however, no significant differences in whole-body phenylalanine kinetics were detected between healthy aged (approx 25 years) and mature (approx 11 years) horses in that study.16 Although age has no effect on whole-body phenylalanine kinetics, differences in protein metabolism may exist among aged horses, including horses with PPID, for which a loss of muscle mass has been identified.8 Muscle atrophy develops when the rate of protein degradation exceeds the rate of protein synthesis. In horses, PPID is associated with muscle loss attributable to atrophy of type 2A and 2B muscle fibers and loss of type 2B myofibers.8 The proposed13 overall increase in the amount of glucocorticoids in horses with PPID may lead to decreased protein synthesis and increased protein breakdown in muscles. Horses with PPID have increased expression of m-calpain, which is part of the nonlysosomal calcium protease-dependent system associated with protein breakdown.17 Results of that study suggest that calpains may be associated with muscle atrophy that develops in horses with PPID; however, whether the pathways of protein synthesis or other pathways of protein breakdown are involved is unknown, to the author’s knowledge. The pathways that regulate muscle protein synthesis and breakdown have been reviewed in other reports.18,19 Protein synthesis is regulated through the mTOR (formally mammalian target of rapamycin) signaling pathway.19 The factors Akt, 4E-BP1, and rpS6 in horses have been evaluated in another study20; phosphorylation of these factors leads to an increase in translation initiation and subsequently an increase in protein synthesis.20 Activation of Akt is also involved in protein degradation; Akt phosphorylates and inactivates nuclear transcription factor FoxO.18 Activation of FoxO increases the expression of genes expressed during proteosomal protein degradation (atrogin-1 and MuRF1).18 Muscle atrophy in horses may also be influenced by a decrease in protein synthesis because of upregulation of the negative regulators of protein synthesis, AMPK21 and myostatin.22 An objective of the study reported here was to compare whole-body phenylalanine kinetics in horses with PPID with that in age-matched control horses without PPID. Another objective was to characterize differences between horses with PPID and age-matched AJVR, Vol 75, No. 7, July 2014

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control horses without PPID regarding the activation of signaling pathways associated with muscle protein synthesis and breakdown in response to feeding. Materials and Methods Animals—All study procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee. Twelve horses (6 horses with PPID and 6 control horses of advanced age without PPID) were obtained from the Department of Veterinary Science Maine Chance Farm at the University of Kentucky. Horses in the 2 groups were of comparable ages (mean ± SD age of horses with PPID, 25.0 ± 2.5 years; mean ± SD age of control horses, 25.7 ± 2.0 years) and weights (mean ± SD weight of horses with PPID, 542 ± 35 kg; mean ± SD weight of control horses, 500 ± 50 kg). Both groups comprised 4 mares and 2 geldings, and all horses had moderate body condition scores (mean ± SD body condition score of horses with PPID, 5.5 ± 0.5; mean ± SD body condition score of control horses, 5.0 ± 0.7; scale, 1 to 923). Because the horses used in this study did not undergo postmortem examinations to determine the presence of pituitary gland lesions, 2 diagnostic methods were used to determine PPID status of horses with high accuracy.24 Horses were confirmed to have PPID on the basis of a resting circulating ACTH concentration of 50 pg/mL and serum cortisol concentration of 1 µg/dL 19 to 20 hours after administration of dexamethasone.24 Horses that were determined to have PPID on the basis of these methods had clinical signs consistent with that disease, such as hypertrichosis, hair coat abnormalities, hyperhidrosis, muscle atrophy, and a history of laminitis. In April 2012, blood samples (approx 10 mL) were collected from horses into tubes containing EDTA and analyzed to determine ACTH concentration (mean ± SD concentration for horses with PPID, 103.6 ± 73.7 pg/mL; mean ± SD concentration for control horses, 23.3 ± 8.2 pg/mL) with an assay performed by personnel in a laboratory.a In May 2012, dexamethasone suppression tests were performed in accordance with described procedures13; blood samples (approx 10 mL) were collected into evacuated glass tubes containing no additives before and 19 to 20 hours after IM administration of dexamethasoneb (0.04 mg/kg). Serum cortisol concentrations were determined by use of an assay performed by personnel in a laboratory.a Serum cortisol concentrations were suppressed in control horses (mean ± SD concentration, 0.46 ± 0.32 µg/dL) but not in horses with PPID (mean ± SD concentration, 2.27 ± 0.92 µg/dL); these findings were consistent with a diagnosis of PPID for horses in that group. Horses were acclimated to diets and housing for at least 2 weeks prior to isotope procedures. All horses were housed in a single paddock with free access to mixed grass hay (mean ± SD composition as fed: 10.1 ± 0.1% crude protein, 33.0 ± 0.6% acid detergent fiber, 50.9 ± 0.6% neutral detergent fiber, 2.7 ± 0.4% fat, and 6.5 ± 0.4% ash), water, and salt blocks. Twice daily (at 7:00 AM and 3:00 PM), horses were fed a commercially available concentrate feedc intended for geriatric horses (mean ± SD composition as fed, 14.1 ± 0.1% crude 659

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protein, 22.5 ± 0.7% acid detergent fiber, 37.2 ± 1.3% neutral detergent fiber, 6.2 ± 0.2% fat, and 7.4 ± 0.2% ash) individually in stalls (12 g/kg/d total), except on study procedure days during which the feeding procedure was modified. Analysis of the commercially available concentrate feed and hay was performed by personnel in a laboratory.d Procedures—On the morning prior to isotope administration procedures, horses were weighed and blood samples (approx 10 mL) were collected immediately before (0 minutes) and 90 minutes after the 7:00 AM meal (the amount fed was determined on the basis of weight of each horse) to determine postprandial changes in plasma glucose, insulin, and amino acid concentrations. Peak circulating amino acid concentrations in horses are attained 90 to 180 minutes following a meal.25 Following collection of the blood sample 90 minutes after feeding, a postprandial muscle biopsy sample was obtained from each horse. Horses were lightly sedated with xylazine hydrochloride (0.5 mg/kg, IV [concentration of xylazine, 100 mg/mL]). The muscle biopsy procedures were performed as described20; a Bergstrom needle was used to aseptically collect muscle samples (approx 500 mg) from a gluteus medius muscle. Following the biopsy procedure, horses received an NSAID (2 g of phenylbutazone, PO); additional doses were administered 24 and 48 hours later. After biopsies, horses were returned to their stalls and monitored. None of the horses developed biopsy site infections; minor swelling was detected at biopsy sites. The following morning, 1 catheter (14 gauge; length, 14.0 cm) was inserted into each jugular vein. One catheter was used for isotope administration, and the other catheter was used for collection of blood samples. To determine total CO2 production, each horse received a priming dose (7.1 µmol/kg) and 2-hour constant rate infusion (6.0 µmol/kg/h) of a 13C sodium bicarbonate solution IV by use of a cordless pumpe attached to a surcingle worn by the horse. The sodium bicarbonate solution was prepared by dissolving 13C sodium bicarbonatef in sterile saline (0.9% NaCl) solution, which was transferred into a sterile ethylene vinyl acetate bag.g Expired breath gas samples were obtained at 30, 60, and 0 minutes before and 30, 60, 75, 90, 105, and 120 minutes after 13C sodium bicarbonate administration. Breath samples were obtained by use of a modified maskh with a reversed 1-way valve that allowed expired gas to be trapped in gas-impermeable bags.i Horses wore the mask for 1 minute prior to sample collection to allow gas to equilibrate inside the mask. Then, the bag was attached to the 1-way valve; the bag was removed and capped after it was full. Baseline blood samples (10 mL) for determination of phenylalanine enrichment were obtained 90 and 120 minutes after the start of 13C sodium bicarbonate administration. After 2 hours, administration of 13C sodium bicarbonate was stopped, and administration of another isotope was started immediately. Each horse received a priming dose (10.2 µmol/kg) and a 4-hour constant rate infusion (7.2 µmol/kg/h) of 1-13C phenylalaninef for measurement of whole-body phenylalanine kinetics.26 A phenylalanine isotope infusion for measurement of whole-body protein metabolism has been used 660

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for humans14,27,28 and swine29,30; the method was recently validated for horses.15 The ratio (approx 1.5:1) of the priming dose to the constant rate infusion dose was determined on the basis of results of another study of horses.15 The preparation and administration of the 1-13C phenylalanine solution and the breath gas sample collection procedures were performed as described above for 13C sodium bicarbonate. Blood and breath samples were obtained, and feed was provided to horses every 30 minutes after the start of isotope administration. On the day of the isotope administration procedures, horses received 1/48 of the daily amount of concentrate feed every 30 minutes, beginning at least 90 minutes prior to the start of the 13C sodium bicarbonate infusion. To minimize changes in plasma phenylalanine concentration as a result of isotope administration, meals provided prior to the start of 1-13C phenylalanine isotope administration included L-phenylalanine powderj (0.6 mg/kg [3.6 µmol/kg]). Plasma sample analysis—The blood samples were transferred to evacuated glass tubes containing sodium heparin and refrigerated (4°C) immediately until centrifugation (1,000 X g at 4°C); aliquots of plasma were placed into tubes and stored at –20°C. The 2 plasma samples obtained before and after feeding were analyzed by use of an automated analyzerk for measurement of glucose concentrations and a commercially available kitl for measurement of insulin concentrations. That kit has been validated for analysis of equine plasma samples.31,32 Additionally, plasma free amino acid concentrations were measured for by use of HPLC analysism of phenyisothiocyanate derivatives, as described.20 Plasma samples collected during the isotope administration procedures were analyzed by personnel in a laboratoryn for the isotopic enrichment of a t-butyldimethylsilyl derivative of phenylalanine (m+1) by use of gas chromatography–mass spectrometry as described.16,33 Breath sample analysis—Breath samples were analyzed on the day of isotope administration. The 13 CO2:12CO2 ratio in exhaled breath gas samples was determined by use of an isotope-selective nondispersive infrared absorption analyzer.i Western blot analysis of muscle samples—Muscle sample homogenates were prepared, as described.20,34 Briefly, a portion of each muscle sample (approx 100 mg) was homogenized in lysis buffer. Homogenates were centrifuged (10,000 X g for 10 minutes at 4°C), and the supernatant was removed and frozen at –80°C until analysis. Protein content of the supernatant was determined with a Bradford assay; samples were diluted in Laemmli buffer to a concentration of 2 µg/µL and loaded into polyacrylamide gels. Proteins were separated by means of electrophoresis, transferred to polyvinylidene diflouride membranes, and incubated for 1 hour in a 5% fat-free milk solution (except for myostatin, which was incubated in a 5% bovine serum albumin solution). Membranes were incubated with the following primary antibodies: Akto (total and phosphorylated Ser473; 1:2,000 dilutions of each), rpS6° (total and phosphorylated Ser235/236 and Ser240/244; dilutions, 1:10,000 for total and 1:2,000 for phosphorylated anAJVR, Vol 75, No. 7, July 2014

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tibodies [mixed together]), 4E-BP1° (dilutions for total and phosphorylated antibodies; 1:1,000), AMPKαo (total and phosphorylated Thr172; 1:1,000 dilutions of each), FoxO1p (total and phosphorylated Ser256; 1:4,000 and 1:5,000 dilutions, respectively), NF-κB p65° (total and phosphorylated Ser536; 1:4,000 and 1:1,000 dilutions, respectively), MuRF1q (total; 1:5,000 dilution), atrogin-1q (total; 1:1,000 dilution), myostatinr (total;1:10,000 dilution) and α-tubulino (total; 1:1,000 dilution). Membranes were washed and incubated with a goat anti-rabbit secondary antibody conjugated to horseradish peroxidases for 1 hour at 20°C. Membranes were developed with a chemiluminescent kitt and developed by use of a film processor.u After development, membranes were stripped and reprobed with a secondary antibody to determine the total amount of the protein of interest. For phosphorylated proteins, the antibody was bound to total amount of protein. Abundance of MuRF1, atrogin-1, and myostatin were standardized to α-tubulin, which has been used to standardize protein abundance in equine skeletal muscle samples35; this procedure was used because only the total abundance of protein was measured. Band densities were measured with densitometric computer software.v The abundance of phosphorylated forms of Akt, rps6, 4E-BP1, AMPK, NF-κB, and FoxO1 was corrected for the density of the total protein bands, and the value for the control horse group was set at 1.0 arbitrary units. The abundance of MURF1, atrogin-1, and myostatin was determined and compared with abundance of α-tubulin to correct for total protein. All gels were run in duplicate. Plasma enrichment of phenylalanine—Plasma enrichment of 1-13C phenylalanine was calculated with a formula, as described.36 Results were expressed in molecules percent excess. Breath CO2 enrichment and total CO2 production—For the breath samples collected during admin-

istration of 13C sodium bicarbonate, total CO2 production was calculated on the basis of breath CO2 enrichment by use of a formula.37 For all breath samples, the δ enrichment values determined with the nondispersive infrared absorption analyzer were converted to atoms percent excess values with a formula.37 Whole-body phenylalanine kinetics—During phenylalanine enrichment, a plateau, defined as at least 4 values with a slope not significantly (P > 0.05) different from 0, was determined with linear regression analysis.w The mean plasma enrichment at the isotopic steady state (plateau) was used to calculate whole-body phenylalanine kinetics. Then the plateau enrichment values were used to calculate the whole-body phenylalanine flux with formulas for horses.15 Briefly, flux (Q) is equal to the amount of phenylalanine entering the plasma pool because of dietary intake (I), de novo synthesis (N), and protein breakdown (B) or leaving the plasma pool because of protein synthesis (Z), oxidation (E), or conversion to other metabolites (M): Q=I+N+B=Z+E+M

Phenylalanine intake was determined by multiplication of dietary phenylalanine intake by 0.5 to account for prececal phenylalanine digestibility38 and the dietary phenylalanine that is extracted during first-pass splanchnic metabolism in other monogastric species.39,40 In horses, phenylalanine is an indispensable amino acid, and de novo synthesis does not occur. Therefore, protein breakdown was estimated with the following formula: B=Q–I

Phenylalanine oxidation was calculated with published41 equations. Nonoxidative phenylalanine metabolism can be calculated by determination of the difference between phenylalanine flux and oxidation; this value is an indication of whole-body protein syn-

Table 1—Glucose, insulin, and amino acids concentrations in plasma samples obtained from control horses (n = 6) and horses with PPID (6) immediately before (0 minutes) and 90 minutes after feeding. Control

PPID

0 minutes

90 minutes

0 minutes

Glucose (mmol/L) Insulin (µU/mL) Histidine (µmol/L) Isoleucine (µmol/L) Leucine (µmol/L) Lysine (µmol/L)

4.73 ± 0.27 9.0 (5.8–14.1) 79 ± 7 70 ± 4 126 ± 9 105 ± 12

5.31 ± 0.27 38.2 (18.2–81.2)* 78 ± 7 80 ± 4 131 ± 9 125 ± 12

5.35 ± 0.27 16.6 (8.8–31.6) 75 ± 7 69 ± 4 131 ± 9 109 ± 12

6.29 ± 0.27*† 57.6 (37.2–89.1)* 84 ± 7 71 ± 4 128 ± 9 119 ± 12

90 minutes

Methionine (µmol/L) Phenylalanine (µmol/L) Threonine (µmol/L) Tryptophan (µmol/L) Valine (µmol/L)

44 ± 4 68 ± 6 124 ± 12 13 ± 1 245 ± 18

47 ± 2 73 ± 6 129 ± 12 13 ± 1 261 ± 18

43 ± 4 59 ± 6 149 ± 12 12 ± 1 272 ± 18

41 ± 2 61 ± 6 152 ± 12 13 ± 1 267 ± 18

Data are least squares mean ± SE or geometric mean (95% confidence interval) values. For plasma glucose and insulin concentrations, the autoregression variance-covariance matrix was used for repeated-measures analysis. For repeated-measures analysis of plasma amino acids concentrations, the simple variancecovariance matrix was used to analyze histidine, isoleucine, leucine, lysine, and tryptophan concentrations; the autoregression variance-covariance matrix was used to analyze phenylalanine, threonine, and valine concentrations; and the heterogeneous autoregression variance-covariance matrix was used to analyze methionine concentrations. *Within a group of horses, the value 90 minutes after feeding is significantly (P < 0.05) different from the value before feeding (0 min). †Within a time, the value for horses with PPID is significantly different from the value for control horses. AJVR, Vol 75, No. 7, July 2014

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Figure 2—Representative western blots of phosphorylated (p) and total (t) forms of NF-κB p65 (an indicator of inflammatory status; A) and least squares mean ± pooled SE expression of the phosphorylated form of that protein (NF-κB p65 at Ser536; B) in gluteal muscle samples obtained from control horses (n = 6; white bars) and horses with PPID (6; black bars) after feeding. Expression of the phosphorylated form of the protein was expressed relative to total expression, and the values for control horses were expressed as an arbitrary value of 1.0.

Figure 1—Representative western blots of phosphorylated (p) and total (t) forms of proteins involved in the upregulation of protein synthesis (A) and least squares mean ± pooled SE expression of phosphorylated forms of those proteins (Akt at Ser473, rpS6 at Ser235/236 and Ser240/244, and 4E-BP1 at Thr 37/46; B) in gluteal muscle samples obtained from control horses (n = 6; white bars) and horses with PPID (6; black bars) after feeding. Expression of the phosphorylated forms of proteins was expressed relative to total expression, and the values for control horses were expressed as an arbitrary value of 1.0.

thesis. The primary non-CO2 product of phenylalanine metabolism is tyrosine; however, the only source of dietary tyrosine for horses in the present study was pelleted complete feed, which all horses received in the same amount corrected for weight. Therefore, detection of a reduction in whole-body phenylalanine oxidation would suggest an increase in use of phenylalanine for whole-body protein synthesis. Statistical analysis—All data were analyzed with statistical software.x Values of P < 0.05 were considered significant. Metabolite concentrations in plasma samples obtained before and after feeding were assessed by use of a repeated-measures analysis with group, time, and the interaction between group and time as the fixed effects, time as the repeated variable, and horse nested in group as the subject. The variance-covariance matrix was chosen for each repeated-measures analysis on the basis of the lowest value for the corrected Akaike information criterion. Prior to analysis, plasma glucose and insulin concentrations were assessed for normality with Studentized residuals.x Because plasma insulin concentrations were not normally distributed, values were logarithmically transformed before performance of statistical analysis. Following statistical analysis, the least squares means were back-transformed to geometric means. Standard error values cannot be reported for 662

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back-transformed geometric means; therefore, variations in plasma insulin concentrations were indicated with 95% confidence intervals for each geometric mean. Phenylalanine kinetic parameters and western blot analysis data were analyzed with a 1-way ANOVA with PPID status (group) as the fixed effect and horse nested in group as the random effect. All data were reported as least squares mean and pooled SE values. Results Plasma insulin, glucose, and amino acids concentrations—Plasma insulin, glucose, and amino acids concentrations were measured prior to and 90 minutes after feeding of horses. Plasma insulin concentrations were significantly (P < 0.01) higher 90 minutes after feeding than they were before feeding for control horses and horses with PPID (Table 1). Plasma glucose concentrations were significantly (P < 0.01) higher 90 minutes after feeding than they were before feeding for horses with PPID. Plasma glucose concentrations for horses with PPID were significantly (P = 0.02) higher than they were for control horses 90 minutes after feeding. Plasma insulin concentrations for horses with PPID were higher than they were for control horses 90 minutes after feeding; however, results were not significant (P = 0.10). No significant effect of the interaction between time and PPID status was detected for plasma glucose or insulin concentrations. Plasma amino acids concentrations were not significantly affected by time, PPID status, or the interaction between time and PPID status. Western blot analysis—Western blot analysis results for horses with PPID and control horses after feeding were compared. The PPID status of horses had no significant effect on the activation of Akt at Ser473 (P = 0.93), rpS6 at Ser235/236 and Ser240/244 (P = 0.20), and 4E-BP1 at Thr37/46 (P = 0.36 ; Figure 1); NF-κB p65 at AJVR, Vol 75, No. 7, July 2014

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Ser536 (P = 0.48; Figure 2); AMPKα at Thr172 (P = 0.54) and myostatin (P = 0.27; Figure 3); and FoxO1 (P = 0.85), MuRF1 (P = 0.67), and atrogin-1 (P = 0.53; Figure 4) after feeding. Whole-body phenylalanine kinetics—Phenylalanine kinetics was determined for control horses and horses with PPID. No significant effect of PPID status was detected for CO2 production, phenylalanine flux, phenylalanine intake, phenylalanine oxidation, phenylalanine release attributable to protein breakdown, or nonoxidative phenylalanine disposal (Table 2).

Figure 3—Representative western blots of proteins involved in negative regulation of protein synthesis (AMPKα and myostatin; A) and least squares mean ± pooled SE expression of the phosphorylated form (AMPKα at Thr172) or relative abundance (myostatin; B) of those proteins in gluteal muscle samples obtained from control horses (n = 6; white bars) and horses with PPID (6; black bars) after feeding. Expression of the phosphorylated form (p) of AMPKα was expressed relative to total expression (t), expression of myostatin was expressed relative to α-tubulin expression, and the values for control horses were expressed as an arbitrary value of 1.0.

Discussion Although characteristics of some pathways associated with muscle protein breakdown in horses with PPID were compared with those in age-matched control horses in another study,17 the present study was the first in which whole-body phenylalanine kinetics and mTOR signaling were compared between horses with PPID and age-matched horses without PPID, to the author’s knowledge. Despite detection of an increase in plasma insulin and glucose concentrations after feeding, no concurrent increase in the plasma concentrations of any indispensable amino acids was detected 90 minutes after feeding. No significant differences in whole-body

Figure 4—Representative western blots of FoxO1, MuRF1, and atrogin-1 (A) and least squares mean ± pooled SE expression of the phosphorylated form (FoxO1) or relative abundance (MuRF1 and atrogin-1; B) of those proteins in gluteal muscle samples obtained from control horses (n = 6; white bars) and horses with PPID (6; black bars) after feeding. Expression of the phosphorylated form (p) of FoxO1 was expressed relative to total expression (t), expression of MuRF1 and atrogin-1 was expressed relative to α-tubulin expression, and the values for control horses were expressed as an arbitrary value of 1.0.

Table 2—Least squares mean whole-body phenylalanine kinetics variables for control horses (n = 6) and horses with PPID (6) and pooled SEs of those values.

Phenylalanine flux (µmol•kg •h ) CO2 production (µmol•kg•1•h•1) Phenylalanine entering the free phenylalanine pool Phenylalanine from dietary intake (µmol•kg•1•h•1) Phenylalanine from protein breakdown (µmol•kg•1•h•1) Phenylalanine leaving the free phenylalanine pool Phenylalanine oxidation (µmol•kg•1•h•1) Nonoxidative phenylalanine disposal (µmol•kg•1•h•1) •1

•1

Control

PPID

Pooled SE

38 14

39 15

1 1

7 30

7 31

0 1

9 29

8 31

1 1

The following stochastic model of phenylalanine kinetics was used: flux = rate of phenylalanine entry = rate of phenylalanine leaving; rate of phenylalanine entry = phenylalanine intake + phenylalanine release from protein breakdown; rate of phenylalanine leaving = phenylalanine oxidation + nonoxidative phenylalanine disposal. Values were not significantly (P > 0.05) different between horses with PPID and control horses.

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phenylalanine kinetics were detected between horses with PPID and control horses; in addition, significant differences in the abundance or activation of the evaluated signaling factors associated with protein synthesis and degradation were not detected between groups of horses. These findings suggested that, although PPID status did not affect whole-body phenylalanine kinetics or the evaluated signaling pathways, advanced age may lead to a decreased ability to release amino acids into the circulation after feeding. Horses with PPID had significantly higher plasma glucose concentrations than control horses in the present study. Horses with PPID also had numerically higher plasma insulin concentrations than control horses, but the results were not significant. Therefore, despite the finding of similar or slightly higher plasma insulin concentrations in the horses with PPID versus control horses, horses with PPID had higher plasma glucose concentrations than control horses; these findings suggested that the horses with PPID may have had some dysregulation of insulin-mediated glucose uptake. Results of other studies7,42 suggest that horses with PPID have higher insulin resistance than horses without PPID, and such horses have a high incidence of hyperinsulinemia1; however, control horses in those other studies were substantially younger than horses with PPID. Associations between muscle atrophy and PPID may be partially attributable to differences in insulin sensitivity between groups of horses; additional research is warranted. In the present study, plasma glucose and insulin concentrations 90 minutes after feeding of horses were higher than concentrations before feeding; however, feeding did not result in an increase in plasma concentrations of any indispensable amino acids. Results of another study25 indicate plasma amino acids reach peak concentrations approximately 90 minutes after feeding of horses; therefore, the lack of detection of a postprandial increase in plasma amino acids concentrations in the present study was unexpected. In the present study, horses consumed 0.86 g of protein/kg during each meal, which was lower than the amount of protein consumed by horses in other studies of mTOR signaling.20,34 However, the amount was higher than the amount consumed by mature horses in another study15 (0.55 g of protein/kg); in that study, plasma amino acids concentrations were higher 120 minutes after feeding than they were before feeding. Therefore, it seemed unlikely that the timing of collection of postprandial plasma samples in the present study was not appropriate or that the amount of protein fed was inadequate for detection of an expected postprandial increase in plasma amino acids concentrations. The lack of a significant postprandial increase in plasma amino acids concentrations in the present study may have been attributable to the advanced age (approx 25 years) of horses in both groups. This would have been consistent with results of studies43 of rats of advanced age indicating that age alters splanchnic extraction of amino acids. In aged rats, arterial plasma amino acids concentrations are approximately 20% to 30% lower than concentrations in adult rats to which amino acids have been administered.43 Protein diges664

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tion may decrease in horses with advanced age,44 although that is uncertain.45 The lack of a postprandial increase in plasma amino acids concentrations in old horses is of interest because it could affect the amount of anabolic stimuli for stimulation of a postprandial increase in muscle protein accretion; the causes of such findings warrant further research. Muscle atrophy in horses with PPID may be attributable to high circulating concentrations of inflammatory cytokines, which stimulate protein breakdown.46 In horses, inflammation increases with increasing age.47 Interestingly, horses with PPID have lower48 or similar17 expression of various inflammatory cytokines versus healthy aged-matched control horses. In the present study, inflammation was not measured directly; however, no significant differences were found in the relative abundance of phosphorylated NF-κB between horses with PPID and control horses. The phosphorylation and activation of NF-κB is stimulated by inflammatory cytokines49; therefore, it was unlikely that there was a difference in inflammatory signaling in skeletal muscle between aged horses in those 2 groups in the present study. Results of a study14 of humans indicates that the rate of whole body muscle protein synthesis decreases with increasing age.14 However, results of another study50 indicate that aging does not alter the rate of whole-body muscle protein synthesis. The authors of that study50 theorized that the lack of difference in protein anabolic responses among humans of various ages may have been attributable to similar muscle mass index values and levels of physical activity between the evaluated elderly and young groups.50 Up to 87% of the variation in whole-body protein kinetics is attributable to differences in fat-free mass.14 Fat-free mass was not measured directly in the present study, but both groups of horses had moderate body condition scores; this suggested that differences in fat-free mass between the two groups were minimal. This was consistent with results of another study16 in which no differences in whole-body phenylalanine kinetics were detected between aged and mature horses with similar body condition scores. In the present study, no significant differences were detected between horses with PPID and control horses for any measures of whole-body protein kinetics. It was hypothesized that there would be lower rates of whole-body protein synthesis and greater rates of whole-body protein degradation in horses with PPID, compared with control horses, because of previous reports of increased muscle atrophy8 and increased expression of a factor associated with protein breakdown pathway nonlysosomal calcium proteasedependent systems in horses with PPID.17 Results of the present study also differed from results of a study10 that indicate protein breakdown is increased in human patients with Cushing’s disease.10 These findings suggest differences between humans with Cushing’s disease and horses with PPID. No significant differences in whole-body protein kinetics may have been detected in this study because of characteristics of the horses that were included. Although histories regarding the time of onset of PPID symptoms in the horses were not available, all the aged AJVR, Vol 75, No. 7, July 2014

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horses in the present study were otherwise healthy, were adapted to management in a group setting, and did not have visible signs of extreme losses in fat-free mass, which can develop as horses age. If horses with more advanced PPID had been included in the study, differences in whole-body phenylalanine kinetics may have been detected between groups. Furthermore, measures of whole-body phenylalanine kinetics only indicate information about the overall rates of protein synthesis and breakdown in animals; such measures do not indicate information regarding the contributions of each tissue type to those processes. Therefore, although no significant differences in whole-body protein synthesis or breakdown were detected between groups of horses in this study, there may be an effect of PPID on protein synthesis and degradation in muscle. Future studies including old horses should be conducted to directly measure fractional rates of muscle protein synthesis with isotopic tracers, as has been performed for aged animals of other species.50–52 However, we did evaluate the activation of signaling pathways of muscle protein synthesis and degradation in the present study; such data may be an indicator of the effects of PPID status on the rates of muscle protein synthesis and degradation. Protein synthesis is regulated by the mTOR pathway. Briefly, Akt is activated by insulin and subsequently activates factors in mTOR signaling (rps6 and 4E-BP1), leading to protein synthesis. Glucocorticoids, which may have high circulating concentrations in horses with PPID during a 24-hour period, decrease Akt phosphorylation in horses.35 No significant differences were detected between horses with PPID and control horses regarding the phosphorylation of any of the mTOR signaling factors evaluated in this study; this suggested that PPID status did not alter muscle protein synthesis in response to feeding of horses. Protein synthesis is stimulated by insulin and negatively affected by glucocorticoid excess; this suggests that insulin sensitivity and cumulative daily glucocorticoid concentrations may not have been different between the 2 groups of horses in this study, although these factors were not measured. Myostatin is a negative regulator of skeletal muscle mass by means of inhibition of activation of factors associated with mTOR signaling.22 In the present study, no significant differences were detected in muscle abundance of myostatin; this finding was consistent with results of another study17 in which data for horses with PPID horses were compared with those for agematched control horses. Another negative regulator of protein synthesis is AMPK (a sensor of cellular energy) that is activated by high AMP concentrations as a result of energy starvation; this results in mTOR inhibition.53 Because of differences in plasma glucose concentrations between the 2 groups of horses in this study, there may have been differences in AMPK phosphorylation between those groups. However, no significant differences were detected between horses with PPID and control horses regarding phosphorylation of AMPK; this indicated there were no differences in cellular energy status between the groups. In addition to activation of protein synthesis, Akt AJVR, Vol 75, No. 7, July 2014

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phosphorylation also affects protein breakdown through phosphorylation and subsequent inactivation of FoxO,18 leading to an increase in protein breakdown through the ubiquitin-proteosomal pathway. Phosphorylation of FoxO increases the expression of MuRF1 and atrogin-1, which are associated with muscle atrophy.54 Because of the muscle atrophy associated with PPID,17 an increase in expression of factors associated with the ubiquitin-proteosomal protein breakdown pathway would be expected in horses with PPID, compared with expression in control horses. However, such changes in expression were not detected in the present study. No differences were detected between horses with PPID and control horses regarding the phosphorylation of FoxO1; this finding was consistent with the lack of significant differences in expression of MuRF1 and atrogin-1 between groups of horses. In another study17 of protein breakdown in horses with PPID, compared with that in age-matched control horses, no significant differences between groups were detected regarding the expression of factors in major proteolytic systems (with the exception of m-calpain, which had higher expression in horses with PPID). Therefore, results of the present study generally supported results of the other study17 that the ubiquitin-proteosomal pathway of protein breakdown is not affected by PPID status in aged horses. Alternatively, collection of muscle biopsy samples from horses 90 minutes after feeding may not have allowed detection of differences in protein synthesis and breakdown between groups in this study. Furthermore, only the abundance or phosphorylation of such factors was evaluated. Responses to other anabolic stimuli, such as exercise, may be different between such groups of horses. In the present study, muscle biopsy samples were obtained 90 minutes after feeding; the time of muscle sample collection was determined on the basis of results of other studies20,34 of young horses. However, no significant changes in plasma amino acids concentrations were detected after feeding of horses in this study; this finding suggested that 90 minutes may not have been an appropriate time for collection of muscle biopsy samples after feeding of the aged horses in the study. Because amino acids stimulate mTOR signaling,55 differences in signaling pathways between the 2 groups of horses may not have been detected. In addition, muscle biopsy samples were not collected before feeding of horses in this study; therefore, it was not possible to determine whether feeding stimulated an increase in mTOR signaling, as has been detected in other studies.20,34 In humans, aging decreases the responsiveness of mTOR signaling in response to anabolic stimuli; however, differences in mTOR signaling may not be detected between young and old humans in a basal state.52 The inability of feeding to stimulate mTOR signaling or suppress protein breakdown could be a potential mechanism to explain muscle loss as seen in aged horses and in particular horses with PPID, although additional research is necessary to confirm this in horses.8 Results of the present study indicated the importance of determination of amino acid requirements and digestibility for aged horses because none of the horses in the study had an increase in plasma amino acids 665

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concentration in response to feeding. Although muscle atrophy is associated with PPID in horses, no significant differences in whole-body protein kinetics were detected between the 2 groups of aged horses in this study. Additionally, although insulin sensitivity of tissues may be lower in horses with PPID versus other horses, it did not seem to affect abundance or phosphorylation of factors associated with protein synthesis and breakdown in muscle. None of the evaluated factors associated with protein synthesis and breakdown in muscle had significant differences in expression between the 2 groups of horses. That finding suggested whole-body phenylalanine kinetics were not affected by PPID status. Identification of the mechanisms of the loss of muscle mass in horses with PPID would require performance of additional studies. a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p. q. r. s. t. u. v. w. x.

Animal Health Diagnostic Center, Ithaca, NY. DexaJect, Butler Schein Animal Health, Dublin, Ohio. Equine Senior, Purina Mills, Gray Summit, Mo. Dairy One Forage Laboratory, Ithaca, NY. VetPro Infusion Pump, Jorgensen Laboratories Inc, Loveland, Colo. Isotec, Miamisburg, Ohio. Baxter Healthcare, Deerfield, Ill. Equine Aeromask, Trudell Medical International, London ON, Canada. Wagner Analysen Technik Vertriebs GmbH, Bremen, Germany. L-phenylalanine, Sigma-Aldrich, St Louis, Mo. YSI 2700 SELECTTM Biochemistry Analyzer, YSI Inc, Life Sciences, Yellow Springs, Ohio. Coat-A-Count RIA, Siemens Healthcare Diagnostics Inc, Deerfield, Ill. 3.9 X 300-mm PICO-TAG reverse-phase column, Waters Corp, Milford, Mass. Metabolic Solutions, Inc, Nashua, NH. Cell Signaling Technology, Beverly, Mass. Santa Cruz Biotechnology, Dallas, Tex. ECM Biosciences, Versailles, Ky. Abcam, Cambridge, Mass. Bio-Rad, Hercules, Calif. Abersham ECL Plus Western Blotting Detection System, GE Healthcare, Piscataway, NJ. Kodak X-OMAT film processor, Kodak Health Imaging Division, Rochester, NY. ImageJ, version 1.46r, National Institutes of Mental Health, Bethesda, Md. GraphPad Prism, version 4, GraphPad Software Inc, La Jolla, Calif. Mixed procedure, SAS, version 9.3, SAS Institute Inc, Cary, NC.

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