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    Least squares mean ± pooled SE abundance of gluteal muscle phosphorylation of translation initiation factors Akt at Ser473, Akt at Thr308, S6K1 at Thr389, rpS6 at Ser235/236 and Ser240/244, and 4EBP1 at Thr37/46 in mature (mean ± SD age, 11.0 ± 2.6 years; range, 7 to 14 years; n = 6; white bars) and aged (mean ± SD age, 23.5 years; range, 22 to 26 years; 6; black bars) horses following steady-state feeding, which consisted of a 50:50 mixture of a ration balancer pelleted feed and oats fed at a rate of 0.1% of body weight that was equally divided into 24 aliquots with 1 aliquot fed every 30 minutes for 7.5 hours. For each factor, the abundance was calculated as a ratio of the phosphorylated forms to the total forms, expressed in arbitrary units (AU), with the value for mature horses set at 1.0. *Within a translation initiation factor, the value for aged horses differs significantly (P < 0.05) from that for mature horses.

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

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  • 1 Department of Animal and Food Sciences, College of Agriculture, and the Center for Muscle Biology, College of Medicine, University of Kentucky, Lexington, KY 40546.
  • | 2 Department of Animal and Food Sciences, College of Agriculture, and the Center for Muscle Biology, College of Medicine, University of Kentucky, Lexington, KY 40546.
  • | 3 Department of Veterinary Science, University of Kentucky, Lexington, KY 40546.
  • | 4 Department of Veterinary Science, University of Kentucky, Lexington, KY 40546.
  • | 5 Department of Veterinary Science, University of Kentucky, Lexington, KY 40546.

Abstract

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 13C sodium bicarbonate, followed by a 4-hour primed constant infusion of 1-13C 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.

Abstract

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 13C sodium bicarbonate, followed by a 4-hour primed constant infusion of 1-13C 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.

Horses > 20 years old (ie, aged horses) represent approximately 7.6% of the equine population, and this proportion approaches 20% in some areas.1,2 Horses > 20 years old typically have obvious signs of aging,2 including loss of muscle mass.3 Sarcopenia is the involuntary loss of muscle mass and strength and, in aging humans, has been partially attributed to a multitude of factors, such as a decrease in physical activity and a less than optimal diet.4 In humans, studies5,6 that compared the protein requirements between elderly and nonelderly adults have yielded conflicting results. Findings of 1 study5 suggest that the protein requirement for humans increases as they age. Conversely, results of another study6 indicate that the protein requirements do not differ between nonelderly (approx 30 years old) and elderly (approx 73 years old) humans. Although there is anecdotal evidence to suggest that aging horses have a decrease in physical activity similar to that in aging humans, research regarding the protein requirements for aged horses is limited. Results of one7 of the few studies that have examined protein nutrition in aged horses suggest that those horses may have lower protein digestibility than their mature counterparts; however, those findings could not be replicated when that study protocol was repeated with a different cohort of aged horses.8 Thus, it remains to be determined whether protein metabolism and thereby protein requirements differ between aged and mature (< 20 years old) horses. Moreover, recently validated isotopic methodologies have improved our ability to understand protein metabolism in horses.9

Sarcopenia results from an imbalance of muscle protein synthesis and breakdown. Muscle protein synthesis is modulated by a series of intracellular signaling cascades that are associated with the mTOR pathway (formerly mammalian target of rapamycin pathway).10,11 The mTOR pathway regulates translation initiation and has been the subject of recent reviews.10,11 Briefly, insulin activates Akt through the activation of several intermediate signaling proteins.11,12 Activation of Akt inactivates the mTOR inhibitor tuberous sclerosis complex 2 through phosphorylation,11 which allows the activation of mTOR through phosphorylation. Amino acids and resistance exercise also result in mTOR phosphorylation through Akt-independent mechanisms.10,11 The mTOR then phosphorylates 2 downstream targets: S6K1 and 4EBP1. Phosphorylation of S6K1 results in the activation of rpS6.13 The phosphorylation of S6K1, rpS6, and 4EBP1 ultimately result in the activation of the translational equipment and subsequently protein synthesis.11 In humans and rodents, the increase in muscle protein synthesis in response to anabolic stimuli such as resistance exercise,14 aerobic exercise,15,16 amino acids,14,17 insulin,18 or meal consumption15,17 is reduced in elderly adults, compared with that in nonelderly adults, with a concurrent reduction in downstream mTOR signaling.14,19,20 Systemic inflammation associated with aging has also been associated with reduced rates of muscle protein synthesis in rats.21 Although mTOR signaling has been evaluated in the skeletal muscle of adolescent22 and mature22,23 horses, it has not been studied in aged horses. The objective of the study reported here was to determine the effects of aging on whole-body protein synthesis and the activation of mTOR signaling factors, specifically Akt, S6K1, rpS6, and 4EBP1, in the skeletal muscle of horses.

Materials and Methods

Animals and housing—The University of Kentucky Institutional Animal Care and Use Committee approved all study procedures. Twelve mixed-breed horses were obtained from the University of Kentucky Veterinary Sciences Farm and included 6 aged (3 geldings and 3 mares; mean ± SD age, 23.5 ± 2.6 years; range, 22 to 26 years) and 6 mature (3 geldings and 3 mares; mean ± SD age, 11 ± 2.6 years; range, 7 to 14 years) horses. To facilitate sampling procedures, the 12 study horses were divided into 3 blocks. Each block consisted of 4 horses with 2 from each age group (1 mare and 1 gelding). All horses were of moderate body condition (body condition score range, 5/9 to 7/9),24 healthy with no evidence of dental problems, accustomed to living outdoors in groups, fed a primarily pasture (spring, summer, and fall) or hay (winter) diet, regularly attended to by a farrier, and routinely administered anthelmintic, dental, and vaccine prophylaxis. The horses had not been enrolled in any formal exercise training programs for at least several months prior to the start of the study. Additionally, approximately 2 months prior to initiation of the study (late February to early March 2010), the plasma α-melanocyte stimulating hormone concentration, a marker of PPID (equine Cushing's disease),25 was determined for the pool of candidate study horses and was < 35 pmol/L (a plasma α-melanocyte stimulating hormone concentration ≥ 35 pmol/L is used as a cutoff for diagnosing PPID in horses) for all selected horses.25

During the study, the diets fed were formulated to meet the nutrient requirements for idle mature horses,26 and all feeds were sent to a commercial laboratorya for proximate analysis. Horses were housed as a single group in a drylot (< 0.4 hectare) with unlimited access to a salt block and water, and grass hay (mean ± SD digestible energy, 0.91 ± 0.02 Mcal/kg; crude protein, 8.2 ± 0.8%; acid detergent fiber, 48.3 ± 0.3%; neutral detergent fiber, 76.5 ± 1.4%; crude fat, 2.0 ± 0.5%; and ash, 7.0 ± 0.5%) was provided at a rate of 2% of body weight/day on an as-fed basis. Horses were brought into 3.7 × 3.7-m stalls for approximately 1 hour twice daily at 8:00 am and 3:00 pm and individually fed a concentrate ration that consisted of a 50:50 mixture of a ration balancer pelleted feed and oats (Appendix) at a rate of 0.2% of body weight/day on an as fed basis, to ensure that their energy, protein, and micronutrient requirements were met. Horses were acclimated to the diet and housing for 2 weeks prior to initiation of experimental procedures. During the isotope infusions, horses were individually housed in 3.7 × 3.7-m stalls bedded with pine shavings and provided unlimited access to water and a salt block.

Study design and procedures—Following the 2-week acclimation period, horses were removed from the drylot at 8:00 am on day 0, weighed on a portable electronic scale,b put into individual stalls, and fed the morning concentrate ration in an amount equal to 0.1% of body weight and hay in an amount equal to 2% of body weight. After each horse had finished eating the concentrate ration, a blood sample (7 mL) was collected via jugular venipuncture into evacuated tubes that were designed specifically for the collection of blood samples for RNA analysisc for determination of gene expression of circulating inflammatory cytokines. At 3:00 pm, horses were fed the afternoon concentrate ration in an amount equal to 0.1% of body weight. Horses were individually housed in the stalls until the end of sampling procedures on day 3 and, unless otherwise noted, were fed as described for day 0.

After each horse had finished eating the morning concentrate ration on day 2, 2 indwelling catheters (14 gauge × 14.0 cm) were aseptically placed in each jugular vein as described.23 One catheter was used for the isotope infusions, and the other was used to obtain blood samples. Subcutaneous fat thickness in the tuber sacrale (croup) region near the proposed biopsy site was ultrasonographically measured, and the results were used to calculate percentage body fat as described.27

On day 3, whole-body phenylalanine kinetics were measured with primed, constant stable isotope infusions. During the course of the whole-body phenylalanine kinetic measurements, horses were fed the morning concentrate ration divided into 24 equal portions, with 1 portion fed every 30 minutes for 7.5 hours. The initial portion was fed 1.5 hours prior to the start of isotope administration. Hay was removed from the stall during the isotope infusions so that feed intake could be accurately quantified. This feeding regimen was used to bring all of the horses to a metabolic steady state, a requirement for the measurement of steady-state isotope kinetics. Each horse was IV administered a priming dose (14.4 μmol/kg) and a 2-hour constant-rate infusion (12 μmol/kg/h) of 13C sodium bicarbonate solutiond for determination of total CO2 production.28 This was followed by a priming dose (8.4 μmol/kg) and 4-hour constant-rate infusion (6 μmol/kg/h) of 1-13C phenylalanine solutiond for determination of whole-body phenylalanine oxidation and flux.29 The primed-to-constant ratios used for both 13C sodium bicarbonate and 1-13C phenylalanine solutions were the same as those used by investigators of other studies.9,30 The 13C sodium bicarbonate and 1-13C phenylalanine solutions were individually prepared by dissolving the isotope into sterile saline (0.9% NaCl) solution and filtering the solution through 0.22-m sterile filters into empty sterile ethylene vinyl acetate bags.e The isotope-filled ethylene vinyl acetate bags were attached to a surcingle that encircled the horse's thorax immediately behind the forelimbs and connected to the catheter with a primary IV set.e Each isotope was delivered into the catheter with a pressure-sensitive, cordless IV infusion pump,f which was also attached to the surcingle on each horse. This cordless pump-surcingle system permitted the horses to remain unrestrained in the stalls during the infusion period.

From each horse, baseline breath samples were obtained 30 minutes and immediately before initiation of the 13C sodium bicarbonate infusion and then at 30-minute intervals throughout both the 13C sodium bicarbonate and 1-13C phenylalanine infusions. Breath samples were collected with a maskg that enabled the collection of air through a 1-way valve into impermeable gas bags.h During each breath sample collection, horses wore the mask for approximately 1 minute to allow time for the air in the mask to equilibrate, and then bags were attached to the 1-way valve and remained there until full (approx 1 minute). Immediately following each sample collection, another portion of the morning concentrate ration was provided. A blood sample (10 mL) was collected at 30-minute intervals beginning 90 minutes after initiation of the 13C sodium bicarbonate infusion and continued until the end of the 1-13C phenylalanine infusion. All blood samples were collected into evacuated glass tubes that contained sodium heparin. The blood samples obtained 90 minutes after initiation of the 13C sodium bicarbonate infusion and immediately before initiation of the 1-13C phenylalanine infusion were used as baseline samples for measurement of the background enrichments of 1-13C phenylalanine in the blood prior to its infusion.

At the end of the 1-13C phenylalanine infusion, the infusion pumps were turned off and the horses were placed in a set of equine stocks. Horses were lightly sedated with xylazine hydrochloride (0.3 mg/kg, IV), and a gluteal muscle biopsy specimen was obtained as described.23 Briefly, an area over a middle gluteal muscle (approx 100 cm2) was shaved, aseptically prepared, and desensitized with a local anesthetic (12 mL of a 2% lidocaine solution). A percutaneous needle biopsy technique31 was used to obtain an approximately 500-mg muscle specimen from a depth of 8 cm (ie, approx midway)22 within a middle gluteal muscle. From this specimen, approximately 100 mg was processed for western blot analysis and approximately 80 mg was stored in a solutioni for quantitative real-time PCR analysis of inflammatory cytokines. The remainder of the muscle specimen was flash frozen in liquid nitrogen and stored at −80°C until analysis. Both catheters were removed following muscle biopsy specimen collection.

After all samples had been collected, horses were provided with the remaining portions of the morning concentrate ration and their daily allotment of hay as a single feeding and then returned to the drylot. Each horse was administered phenylbutazone (2 g/d, PO) for the next 3 days to alleviate any discomfort associated with the sampling procedures.

Blood sample processing and storage—Blood samples collected for quantitative real-time PCR assayc were gradually frozen to −80°C in accordance with the assay manufacturer's instructions and were maintained at −80°C until analysis. The remaining blood samples were immediately centrifuged at 1,500 × g for 10 minutes at 4°C, and aliquots of plasma were frozen at −20°C until analysis for determination of glucose, insulin, and amino acid concentrations.

Plasma glucose and insulin concentrations—Plasma glucose concentrations were enzymatically assayed with an automated analyzer.j Plasma insulin concentrations were determined with a radioimmunoassay kit.k

Amino acid concentrations—The plasma free amino acid concentrations were measured with reverse-phase high-performance liquid chromatographyl of phenylisothiocyanate derivatives as described.23 Following a 24-hour acid hydrolysis in 6N HCl at 110°C, total amino acid concentrations of the pelleted feed, oats, and grass hay were also measured with reverse-phase high-performance liquid chromatography of phenylisothiocyanate derivatives as described.23

Plasma phenylalanine enrichment—The isotopic enrichment of phenylalanine (the amount of 1-13C phenylalanine relative to unlabeled phenylalanine) in the plasma samples collected on day 3 was determined by a commercial laboratorym by means of a previously described method,32 as modified by Matthews et al.33 Briefly, the isotopic enrichment of plasma samples was determined by negative chemical ionization gas chromatography–mass spectrometry analysis of a heptafluorobutyric n-propyl derivative. 1-13C phenylalanine enrichment was measured by methane negative chemical ionization gas chromatography–mass spectrometry,n with a capillary columno used to separate the derivatives of phenylalanine. Selected ion chromatograms were obtained by monitoring ions for L-phenylalanine (m/z, 383) and L-1-13C phenylalanine (m/z, 384).

Breath sample analysis—The 13CO2:12CO2 ratio in the breath samples was determined with an isotope selective nondispersive infrared absorption analyzer.h

Western blot analysis of muscle samples—The abundance of the total and phosphorylated forms of Akt, S6K1, rpS6, and 4EBP1 in the gluteal muscle homogenates was determined by means of electrophoresis followed by western blotting techniques, as described.22 For the primary antibody, individual rabbit polyclonal antibodiesp were used that recognized total, Ser473, and Thr308 Akt (1:2,000 dilution for each); total and Thr389 S6K1 (1:1,000 and 1:500 dilutions, respectively); and Ser235/236 and Ser240/244 rpS6 (1:2,000 dilutions for each). Rabbit monoclonal antibodiesp specific to total and Thr37/46 4EBP1 (1:1,000 dilution) and total rpS6 (1:10,000 dilution) were also used as primary antibodies. A goat anti-rabbit IgG conjugated with horseradish peroxidaseq (1:10,000 dilution) was used as the secondary antibody. All antibodies had been previously used to measure the equine isoforms of the proteins of interest.22,23 A chemiluminescence kitr was used to develop the membranes, and a film processors was used to visualize the membranes on x-ray film. With the aid of a computer software program,t band densities were quantified as the mean band intensity multiplied by the number of pixels. For each of the proteins evaluated, the abundance was calculated as a ratio of the phosphorylated forms to the total forms, expressed in arbitrary units, with the value for the mature horses set at 1.

Gels were assayed in duplicate. To reduce variation, all samples were run on a single gel for each protein and the membrane was first probed for the phosphorylated form of the protein, then stripped in accordance with described procedures22 and reprobed for the total form of the protein. Separate gels were run for each protein of interest.

RNA isolation—Total RNA was isolated with the aid of a kit.u Muscle samples (2 to 3 mm) that were stored in the solution for RNA analysis were homogenized in accordance with the bead beating technique34 with a commercially available solution.u Total RNA was then isolated by means of the phenol-chloroform extraction method, and RNA concentration was quantified with a spectrophotometer.v One microgram of each RNA sample was reverse transcribed with a reverse transcription master mixw and incubated for 15 minutes at 42°C, followed by 5 minutes at 95°C, as described.35,36 Complementary DNA samples were stored at −20°C until quantitative real-time PCR analysis was performed.

Real-time PCR assay—The mRNA expression of cytokines (IFN-γ, IL-1β, IL-6, TNF-α, and IL-10) was measured in cDNA samples with equine-specific intron-spanning primer and probe sets,37 as described.36 The PCR assays were prepared in 10-μL volumes, each of which contained 0.5 μL of 20× assay mixx for the primer-probe set of interest, 5 μL of master mix,y and 4.5 μL of cDNA. All PCR amplifications were run in duplicate under the following conditions: 95°C for 10 minutes followed by 45 cycles at 95°C for 15 seconds and 60°C for 60 seconds in a real-time PCR system.z Differences in RNA isolation and cDNA construction between samples were corrected with the use of an internal control (β-glucuronidase) for each sample.38

Fat-free mass—Body fat percentage was determined on the basis of the following equation39:

article image

Once determined, body fat percentage was used to calculate fat mass from total body mass:

article image

Fat-free mass was determined by the difference of body mass and fat mass.

Plasma phenylalanine enrichment—Percentage excess isotope enrichment (mol) was calculated from peak area ratios at the isotopic steady state (samples obtained from 120 through 240 minutes of the phenylalanine infusion) and baseline (samples obtained 90 minutes after initiation of the 13C sodium bicarbonate infusion and immediately before initiation of the 1-13C phenylalanine infusion). The final value for all determinations was corrected on the basis of an enrichment calibration curve.

Breath CO2 enrichment and total CO2 production—The δ enrichment value obtained from the nondispersive infrared analyzer for each sample was converted to a percentage enrichment.40 Plateau CO2 enrichment, defined as a minimum of 3 enrichment measurements with a slope not significantly (P > 0.05) different from 0, as determined on the basis of simple linear regression analysis,aa was used to calculate total CO2 production.40

Relative quantity of inflammatory cytokines—Changes in mRNA expression were calculated by the comparative cycle threshold method,41 with the mean change in cycle threshold calculated for mature horses set as the calibrator for each individual cytokine. Results were expressed as the relative quantity calculated as 2−ΔΔCT, where CT is the cycle threshold.

Whole-body phenylalanine kinetics—The mean plasma enrichment of phenylalanine at the isotopic steady state (plateau) was used to calculate whole-body phenylalanine flux on the basis of an established formula.42

Flux (Q) includes the amount of amino acids entering the pool through dietary intake (I), de novo synthesis (N), and protein breakdown (B), or leaving the pool through protein synthesis (Z), oxidation (E), or the conversion to other metabolites (M):

article image

Phenylalanine intake was calculated on the basis of the formulas and assumptions described by Urschel et al,9 and phenylalanine oxidation was calculated on the basis of published formulas.42 The difference between phenylalanine flux and oxidation is termed nonoxidative phenylalanine disposal, and this can be used as an indicator of whole-body phenylalanine use for protein synthesis. Aside from protein synthesis, phenylalanine is used as a precursor of tyrosine.43 Because all horses were fed the same diet, phenylalanine conversion to tyrosine was assumed to be similar between the 2 age groups; therefore, any change in nonoxidative phenylalanine disposal was assumed to be indicative of changes in phenylalanine use for whole-body protein synthesis.

Statistical analysis—All analyses were performed with statistical software,bb and values of P < 0.05 were considered significant. All dependent variables were analyzed by means of ANOVA with mixed-effect models. For all models, age group (aged or mature) was included as a fixed effect and horse nested within age group and block was included as a random effect. When the effect of age group was significant, a pairwise difference procedure was used to calculate the least squares mean of that dependent variable for each age group. Data are presented as the respective least squares means ± pooled SEM, unless otherwise noted.

Results

Animals—Mean ± pooled SEM body weight (mature, 469 ± 30 kg; aged, 499 ± 30 kg) and fat-free mass (mature, 421 ± 26 kg; aged, 448 ± 26 kg) did not differ significantly between the 2 age groups. The mean fat-free mass indicated that the mature (age, 7 to 14 years) and aged horses (age, 22 to 26 years) were moderately lean. The α-melanocyte stimulating hormone concentration (mature, 10.1 ± 1.8 pmol/L; aged, 9.5 ± 1.8 pmol/L) did not differ between the 2 age groups and was < 35 pmol/L (the cutoff used to exclude horses with potential PPID)25 for all horses.

Plasma insulin, glucose, and amino acid concentrations—Plasma insulin, glucose, and amino acid concentrations were determined in the final blood sample obtained during the 1-13C phenylalanine infusion, immediately prior to the biopsy procedure. Plasma insulin (P = 0.53) and glucose (P = 0.43) concentrations did not differ between the 2 age groups (Table 1). The mean plasma concentrations of isoleucine (P = 0.05) and lysine (P = 0.04) were significantly higher for the aged horses, compared with those for the mature horses; otherwise, the plasma concentrations of the other amino acids did not differ significantly between the 2 age groups.

Table 1—

Least squares mean ± SD and pooled SE plasma metabolite concentrations for mature (mean ± SD age, 11.0 ± 2.6 years; range, 7 to 14 years; n = 6) and aged (mean ± SD age, 23.5 years; range, 22 to 26 years; 6) horses immediately after a 2-hour primed constant infusion of 13C sodium bicarbonate (priming dose, 14.4 μmol/kg; 2-hour infusion, 12 μmol/kg/h) followed by a 4-hour primed constant infusion of 1-13C phenylalanine (priming dose, 8.4 μmol/kg; 4-hour infusion, 6 μmol/kg/h) and just prior to performance of a gluteal muscle biopsy to obtain a specimen for determination of whole-body protein synthesis.

MetaboliteMature horsesAged horsesPooled SEM
Insulin (μU/mL)5.8 ± 3.86.1 ± 2.80.8
Glucose (mmol/L)5.5 ± 0.35.3 ± 0.30.3
Alanine (μmol/L)203 ± 64218 ± 4024
Arginine (μmol/L)69 ± 1974 ± 106
Asparagine (μmol/L)38 ± 752 ± 115
Aspartate (μmol/L)19 ± 714 ± 52
Citrulline (μmol/L)83 ± 3091 ± 158
Glutamate (μmol/L)54 ± 1849 ± 86
Glutamine (μmol/L)273 ± 52306 ± 3927
Glycine (μmol/L)352 ± 51441 ± 8639
Histidine (μmol/L)58 ± 664 ± 63
Isoleucine (μmol/L)33 ± 946 ± 7*4
Leucine (μmol/L)68 ± 1587 ± 107
Lysine (μmol/L)69 ± 1792 ± 15*7
Methionine (μmol/L)26 ± 932 ± 33
Ornithine (μmol/L)34 ± 1044 ± 105
Phenylalanine (μmol/L)68 ± 883 ± 45
Proline (μmol/L)90 ± 1097 ± 128
Serine (μmol/L)232 ± 41288 ± 3823
Taurine (μmol/L)21 ± 528 ± 73
Threonine (μmol/L)94 ± 38112 ± 1111
Tryptophan (μmol/L)4.7 ± 0.45.3 ± 0.90.4
Tyrosine (μmol/L)50 ± 755 ± 75
Valine (μmol/L)121 ± 22148 ± 1612

Value differs significantly (P < 0.05) from that for mature horses.

Inflammatory cytokines—Age group had no effect on mRNA expression of the circulating inflammatory cytokines IL-1β (P = 0.92), IL-6 (P = 0.90), IL-10 (P = 0.20), IFN-γ (P = 0.30), and TNF-α (P = 0.38) or the muscle inflammatory cytokines IL-1β (P = 0.53), IL-10 (P = 0.23), IFN-γ (P = 0.49), and TNF-α (P = 0.58; Table 2). Muscle IL-6 mRNA expression was detected in only 3 mature horses and 1 aged horse; therefore, comparison of the mean expression of that cytokine between the 2 age groups was not performed.

Table 2—

Least squares mean ± SD and pooled SE relative quantities of circulating and gluteal muscle inflammatory cytokines for the mature and aged horses of Table 1.

Inflammatory cytokineMature horsesAged horsesPooled SE
Circulation
 IFN-γ1.23 ± 0.861.58 ± 0.560.22
 TNF-α1.11 ± 0.511.35 ± 0.550.18
 IL-61.63 ± 1.701.71 ± 1.260.77
 IL-1β1.16 ± 0.761.14 ± 0.360.13
 IL-101.21 ± 0.730.72 ± 0.390.22
Muscle
 IFN-γ1.02 ± 0.631.50 ± 0.800.43
 TNF-α0.43 ± 0.300.64 ± 0.310.23
 IL-1β2.61 ± 4.055.06 ± 6.242.53
 IL-100.24 ± 0.100.52 ± 0.240.17

Relative quantity was calculated as 2−ΔΔCT, where CT is the cycle threshold. Relative quantities of circulating inflammatory cytokines were determined in blood samples obtained the day prior to the isotope infusion procedures, following the consumption of the morning allocation of concentrate.

See Table 1 for remainder of key.

Activation of translation initiation factors—Age group had no effect on the activation of Akt at Ser473 (P = 0.33) and Thr308 (P = 0.83), rpS6 at Ser235/236 and Ser240/244 (P = 0.48), or 4EBP1 at Thr37/46 (P = 0.13; Figure 1). However, the activation of S6K1 at Thr389 was significantly (P = 0.03) lower in aged horses, compared with that for mature horses.

Figure 1—
Figure 1—

Least squares mean ± pooled SE abundance of gluteal muscle phosphorylation of translation initiation factors Akt at Ser473, Akt at Thr308, S6K1 at Thr389, rpS6 at Ser235/236 and Ser240/244, and 4EBP1 at Thr37/46 in mature (mean ± SD age, 11.0 ± 2.6 years; range, 7 to 14 years; n = 6; white bars) and aged (mean ± SD age, 23.5 years; range, 22 to 26 years; 6; black bars) horses following steady-state feeding, which consisted of a 50:50 mixture of a ration balancer pelleted feed and oats fed at a rate of 0.1% of body weight that was equally divided into 24 aliquots with 1 aliquot fed every 30 minutes for 7.5 hours. For each factor, the abundance was calculated as a ratio of the phosphorylated forms to the total forms, expressed in arbitrary units (AU), with the value for mature horses set at 1.0. *Within a translation initiation factor, the value for aged horses differs significantly (P < 0.05) from that for mature horses.

Citation: American Journal of Veterinary Research 74, 11; 10.2460/ajvr.74.11.1433

Whole-body phenylalanine kinetics—Age group had no effect on whole-body CO2 production (P = 0.51), phenylalanine flux (P = 0.84), phenylalanine oxidation (P = 0.15), phenylalanine release from protein breakdown (P = 0.99), or nonoxidative phenylalanine disposal (P = 0.48; Table 3).

Table 3—

Least squares mean ± SD and pooled SE whole-body phenylalanine kinetic parameters for the mature and aged horses of Table 1.

ParameterMature horsesAged horsesPooled SE
Phenylalanine flux (μmol/kg•h)41.5 ± 5.542.2 ± 6.92.3
Carbon dioxide production (μmol/kg•h)16,977 ± 5,26615,080 ± 4,5911,410
Phenylalanine entering the free phenylalanine pool*
 Phenylalanine from dietary intake (μmol/kg•h)4.0 ± 0.203.9 ± 0.200.08
 Phenylalanine from protein breakdown (μmol/kg•h)37.5 ± 5.537.5 ± 8.12.2
Phenylalanine leaving the free phenylalanine pool*
 Phenylalanine oxidation (μmol/kg•h)13.2 ± 4.016.7 ± 6.11.6
 Nonoxidative phenylalanine disposal (μmol/kg•h)28.3 ± 5.825.5 ± 10.42.7

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; and rate of phenylalanine leaving = phenylalanine oxidation + nonoxidative phenylalanine disposal.

See Table 1 for remainder of key.

Discussion

To our knowledge, the present study was the first in which whole-body protein kinetics and mTOR signaling were evaluated in aged horses. Results indicated that the whole-body protein kinetics of aged horses (age, 22 to 26 years) did not differ from those of mature horses (age, 7 to 14 years); however, the phosphorylation of S6K1, a downstream mTOR signaling factor, in the gluteal muscle of aged horses was less than that in the gluteal muscle of mature horses following 7.5 hours of feeding designed to achieve steady-state protein synthesis.

All of the aged horses in the present study were clinically normal, were mobile, had no evidence of dental abnormalities, and were thriving in group housing in an outdoor environment, which suggested that these horses were healthy. Studies19,44 conducted to assess the effect of age on protein anabolic response in humans have used aged populations that had significantly greater body weight and fat mass than did the mature populations used. In this study, the physical characteristics of the aged horses did not differ from those of the mature horses, with the exception of age. Although a loss of muscle mass has been described as a characteristic of aging in horses,3 data to support this claim in healthy aged horses are lacking. Results of a study27 that measured body composition in healthy horses from 3 to 29 years old indicate no correlation between age and body weight or percentage body fat; therefore, it is unknown whether healthy aged horses do in fact have a loss in muscle mass. Although aging in horses and rodents has been associated with chronic low-grade inflammation,21,45,46 in the present study, the mRNA expression of the circulating and muscle inflammatory cytokines evaluated did not differ significantly between mature and aged horses. Investigators of the study45 that identified age-related differences in circulating inflammatory cytokine gene expression in horses used a mature population that was substantially younger (mean age, 4.5 years), compared with the age of the mature horses (mean age, 11 years) of the present study, and this may at least partially explain the conflicting findings between that study45 and the present one. Findings of another study35 suggest that inflammatory cytokine production in aged horses is associated with body condition; horses with a body condition score ≥ 7 have greater cytokine production than do horses with a body condition score of 5 or 6. In the present study, none of the horses were obese and the aged horses had a median body condition score that was similar to that of the mature horses, which may be the reason that no differences in systemic and muscle inflammatory gene expression were identified between the 2 age groups.

Markers of whole-body protein kinetics did not differ between the aged and mature horses of this study. This was an unexpected finding, which might have been caused by the similar body condition and health status of the mature and aged horses evaluated. Results of another study16 indicate that up to 87% of the variance observed in whole-body protein kinetic parameters can be attributed to differences in fat-free mass. In humans, muscle protein fractional synthesis rates for lean aged (75-year-old) men did not differ from that of lean young adult (20-year-old) men following the ingestion of a protein and leucine supplement,47 and leucine oxidation and fractional synthesis rates of sarcoplasmic proteins did not differ between middle-aged (52-year-old) and aged (77-year-old) men and women.48 The results from those studies47,48 support the findings of the present study in that the narrow age range and similar physical characteristics of the study subjects likely attributed to the lack of significant differences in phenylalanine kinetics between the aged and mature horses.

Ribosomal protein S6 kinase is a downstream mTOR effector involved in the phosphorylation of other proteins, including rpS6 and eukaryotic initiation factor 4B, which are involved in the regulation of ribosomal assembly and protein synthesis.49,50 Of the translation initiation factors studied, S6K1 was the only factor that was significantly associated with age group. Compared with the phosphorylation of S6K1 at Thr389 in the gluteal muscle of mature horses, that in the gluteal muscle of aged horses was reduced by 42%, a finding that is similar to when the phosphorylation of S6K1 at Thr389 in the soleus muscle of very aged (36 months) male rodents was compared with that in the soleus muscle of aged (30 months) male rodents.51 Phosphorylation of S6K1 in response to availability of amino acids and insulin does not increase from the basal rate in aging humans19 or following ingestion of a meal in aged rats with and without chronic low-grade inflammation.21 For the aged horses of the present study, the significant reduction in the amount of S6K1 P-Thr389 without concurrent increases in the muscle gene expression of the inflammatory cytokines IL-1β, TNF-α, and IFN-γ may indicate that age affects S6K1 through some other mechanism; however, this requires further elucidation. It is possible that the lack of an age effect on Akt, rpS6, and 4EBP1 phosphorylation can be attributed to the small difference (8 years) in the age ranges between the mature and aged horses of the present study, and would be consistent with the findings of the study51 in which no age-related differences in the activation of these factors were identified between the middle-aged and aged rodents.51

The association of age group with S6K1 phosphorylation in the absence of an association between age group and whole-body protein synthesis may indicate that examination of protein synthesis at the whole-body level obscured the changes at the skeletal-muscle level. Although skeletal muscle comprises a substantial portion of body weight (approx 50% of body weight in horses52), the rate of muscle protein turnover is much slower than that in tissues such as the gastrointestinal tract or liver53; therefore, measureable changes in whole-body protein synthesis might not be detected without drastic changes in skeletal muscle protein synthesis. Alternatively, it is possible that reduced S6K1 phosphorylation in aged horses does not limit the rate of muscle protein synthesis. The importance of the observed reduction in S6K1 P-Thr389 without a similar reduction in the whole-body protein synthesis rate in aged versus mature horses requires further study.

The horses of the present study were fed half their daily concentrate allocation (0.1% of body weight) in 1/24 aliquots every 30 minutes for 7.5 hours prior to the gluteal muscle biopsy to maintain a metabolic steady state for isotope measurements. Each of these small concentrate meals provided only 0.5% and 0.37% of the daily crude protein and digestible energy requirements, respectively, and might not have provided an adequate anabolic stimulus for age-related differences in the phosphorylation of Akt, rpS6, and 4EBP1 to be detected. The plasma glucose, insulin, and total indispensable amino acid concentrations immediately prior to the gluteal muscle biopsy more closely resembled those concentrations for mature horses in the postabsorptive state (glucose concentration, 5.5 mmol/L; insulin concentration, 5 mU/L; total indispensable amino acids concentration, 898 μmol/L)23 than did those in the postprandial state (glucose concentration, 6.5 mmol/L; insulin concentration, 36 mU/L; total indispensable amino acids concentration, 1,245 μmol/L).23 Additionally, nonoxidative disposal, the indirect measure of protein synthesis, was less than phenylalanine release from protein breakdown, which is characteristic of the postabsorptive state.44 Investigators of a study54 conducted with neonatal piglets found no increase in the phosphorylation of S6K1 and 4EBP1 and only a modest increase in muscle protein synthesis in skeletal muscle following a 25.5-hour period of continuous feeding, compared with those prior to feeding. However, when neonatal piglets were fed the same total amount of nutrients as a meal every 4 hours instead of as a continuous feeding, the phosphorylation of S6K1 and 4EBP1 and rate of muscle protein synthesis in skeletal muscle samples obtained 1.5 hours after each meal were substantially increased, compared with those prior to feeding.54 In growing horses, the skeletal muscle of yearlings has a greater postprandial increase in the phosphorylation of rpS6 and 4EBP1 than does the skeletal muscle of 2-year-old horses, despite a similar amount of phosphorylation of these factors in the postabsorptive state.22 If this decrease in the postprandial phosphorylation of rpS6 and 4EBP1 continues as horses age, an association between age group and mTOR signalling might have been identified in the present study had the horses been fed 1 large protein meal instead of several small protein meals. In humans, age has no effect on the skeletal muscle phosphorylation of translation initiation factors or protein synthesis during the postabsorptive state, even when differences exist in a stimulated state.14,19,20 Additional research involving horses is necessary to determine whether advanced age results in changes in the activation of muscle mTOR signaling or whole-body protein synthesis during a true postprandial state.

Although differences in whole-body protein synthesis or breakdown were not detected in horses during the continuously fed state in the present study, there may have been differences in the molecular markers of muscle protein degradation, which were not measured. Increases in factors associated with skeletal muscle protein degradation (eg, atrogin-1, muscle-RING-finger protein 1, or forkhead box proteins) have been described in aged rodents55,56 and may contribute to the loss of muscle observed in aged horses. In the present study, plasma concentrations of lysine and isoleucine in aged horses were increased, compared with those for mature horses, which might indicate that aged horses had a greater rate of release of lysine and isoleucine from body proteins than did mature horses, although additional research is necessary to determine whether muscle proteolysis increases with age in healthy horses.

For the present study, aged horses were defined as those > 20 years old, which is consistent with the definition for aged horses used in other studies.1,57 Results of a survey57 of horse owners suggest that most owners describe horses as old or aged by 22 to 23 years of age. Findings of the present study suggested that the management practices required for healthy nonobese aged horses are similar to those required for healthy nonobese mature horses. These findings appear to contradict those of another study7 in which aged horses had significantly lower protein digestibility than did mature horses. However, when the same investigators repeated that study7 with a different population of study horses, the protein digestibility did not differ between aged and mature horses.8 The investigators attributed the differences in the results between the 2 studies7,8 to the fact that the aged horses in the first study7 might have sustained intestinal damage during maturation because they were born before the routine use of anthelmintics in horses was accepted as a best management practice. Regardless, the protein requirements for mature and aged horses do not differ in the 6th edition of the National Research Council's Nutrient Requirements of Horses.26 Results of the present study indicated that whole-body protein metabolism did not differ between aged and mature horses, which implies that the protein requirements do not differ between healthy aged and mature horses, a finding that is similar to that between healthy elderly and mature humans.6 However, extrapolation of the findings of the present study to extremely geriatric horses or horses with diseases such as PPID should be done with caution. In elderly humans, the incidence of sarcopenia is greater in those > 80 years old (11% to 50%), compared with that in those 60 to 70 years old (5% to 13%).58 In rats, sarcopenia is associated with sex; females reach a minimum muscle-to-body weight ratio by 26 months of age, whereas males continue to lose muscle mass until 30 and 36 months of age.51 Horses with PPID frequently have muscle atrophy,25 which might be caused, at least in part, by enhanced protein degradation. Results from 1 study59 indicate that horses with PPID have increased protein degradation via the calpain system of proteolysis, compared with that of age-matched horses without PPID, although the pathways involved in muscle protein synthesis were not examined. Horses > 26 years old and horses with PPID likely differ from healthy mature horses in terms of body composition and whole-body and muscle protein metabolism, and these horses may require specific management practices to retain muscle mass. Protein metabolism in extremely aged and diseased horses requires further elucidation.

Results of the present study indicated that activation of S6K1 in the skeletal muscle of horses decreases with age in a manner similar to that in the skeletal muscle of humans and rodents. Decreased S6K1 activation could impair muscle protein synthesis, although evidence of this was not observed in the present study. Additionally, circulating and muscle inflammatory cytokine mRNA expression did not differ between the aged (22 to 26 years) and mature (7 to 14 years) horses of this study, which suggested that these 2 groups of horses had similar whole-body protein synthesis rates. Although further research is necessary to determine age-related effects on muscle protein metabolism in response to a large anabolic stimulus, these findings indicated that healthy nonobese aged horses had whole-body protein metabolism and requirements similar to those of mature horses.

ABBREVIATIONS

4EBP1

Eukaryotic initiation factor 4E binding protein 1

Akt

Protein kinase B

IFN

Interferon

IL

Interleukin

mTOR

Mechanistic target of rapamycin

PPID

Pituitary pars intermedia dysfunction

rpS6

Ribosomal protein S6

S6K1

Ribosomal protein S6 kinase

TNF

Tumor necrosis factor

a.

Dairy One Forage Laboratory, Ithaca, NY.

b.

Model 700, Tru Test Inc, Mineral Wells, Tex.

c.

PAXgene Blood RNA Tube Qiagen Inc, Santa Clarita, Calif.

d.

Isotec, Miamisburg, Ohio.

e.

Baxter Healthcare Corp, Deerfield, Ill.

f.

VetPro Infusion Pump, Jorgensen Laboratories Inc, Loveland, Colo.

g.

Equine Aeromask, Trudell Medical International, London, ON, Canada.

h.

Wagner Analysen Technik Vetriebs GmbH, Bremen, Germany.

i.

RNAlater, Qiagen Inc, Santa Clarita, Calif.

j.

YSI 2700 SELECT Biochemistry Analyzer, YSI Inc, Life Sciences, Yellow Springs, Ohio.

k.

Coat-A-Count RIA, Siemens Healthcare Diagnostics Inc, Deerfield, Ill.

l.

3.9 × 300-mm PICO-TAG reverse-phase column, Waters Corp, Milford, Mass.

m.

Metabolic Solutions Inc, Nashua, NH.

n.

Agilent 5973 EI/CI MSD with an Agilent 6890 GC, Agilent Technologies, Santa Clara, Calif.

o.

Phenomenex ZB-1ms, Phenomenex, Torrance, Calif.

p.

Cell Signaling Technology Inc, Boston, Mass.

q.

Bio-Rad, Hercules, Calif.

r.

Amersham ECL Plus western blotting detection reagents, GE Healthcare, Piscataway, NJ.

s.

Kodak X-OMAT film processor, Kodak Health Imaging Division, Rochester, NY.

t.

Adobe Photoshop Elements, version 8.0, Alpha Innotech, San Leandro, Calif.

u.

RNA-Stat 60, Tel-Test, Friendswood, Tex.

v.

BioPhotometer, Eppendorf, Hamburg, Germany.

w.

Promega, Madison, Wis.

x.

Applied Biosystems, Foster City, Calif.

y.

Taqman Gene Expression Master Mix, Applied Biosystems, Foster City, Calif.

z.

7900HT, Applied Biosystems, Foster City, Calif.

aa.

GraphPad Prism, version 4, GraphPad Software Inc, La Jolla, Calif.

bb.

SAS, version 9.2, SAS Institute Inc, Cary, NC.

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Appendix

Mean ± SD as-fed nutrient composition of ration balancer pellet and oats that comprised (50:50) the concentrate ration fed to mature (mean ± SD age, 11.0 ± 2.6 years; range, 7 to 14 years; n = 6) and aged (mean ± SD age, 23.5 ± 2.6 years; range, 22 to 26 years old; 6) horses twice daily and during the 7.5-hour steady-state feeding period during infusion of 13C sodium bicarbonate and 1-13C phenylalanine for determination of whole-body protein synthesis and immediately prior to performance of a gluteal muscle biopsy to obtain a specimen for determination muscle mTOR signaling.

 Concentrate component
NutrientRation balancer pelletOats
Moisture9.5 ± 0.211.2 ± 0.3
Digestible energy (Mcal/kg)2.76 ± 0.013.15 ± 0.06
Crude protein17.0 ± 0.313.4 ± 1.9
Alanine0.51 ± 0.030.35 ± 0.04
Arginine0.56 ± 0.030.49 ± 0.07
Aspartate and asparagine0.81 ± 0.040.58 ± 0.04
Glutamate and glutamine1.50 ± 0.061.43 ± 0.14
Glycine0.44 ± 0.020.35 ± 0.05
Histidine0.24 ± 0.010.16 ± 0.03
Isoleucine0.37 ± 0.020.25 ± 0.04
Leucine0.75 ± 0.020.54 ± 0.08
Lysine0.41 ± 0.020.27 ± 0.04
Methionine0.09 ± 0.010.09 ± 0.01
Proline0.71 ± 0.040.40 ± 0.06
Phenylalanine0.47 ± 0.020.37 ± 0.06
Serine0.47 ± 0.010.37 ± 0.05
Threonine0.35 ± 0.020.23 ± 0.05
Tyrosine0.29 ± 0.020.21 ± 0.04
Valine0.47 ± 0.030.32 ± 0.05
Acid detergent fiber18.4 ± 1.111.0 ± 0.9
Neutral detergent fiber32.5 ± 2.823.3 ± 2.2
Nonfiber carbohydrates45.1 ± 2.463.8 ± 2.7
Starch29.7 ± 3.056.0 ± 3.8
Water-soluble carbohydrates7.1 ± 0.54.2 ± 0.8
Crude fat6.2 ± 0.28.4 ± 0.7
Ash9.6 ± 0.13.6 ± 0.9
Calcium1.15 ± 0.030.24 ± 0.20
Phosphorus0.64 ± 0.040.32 ± 0.06
Potassium1.81 ± 0.010.62 ± 0.06
Sodium0.33 ± 0.040.12 ± 0.15
Iron (mg/kg)509 ± 13157 ± 47
Zinc (mg/kg)62 ± 535 ± 5

Unless otherwise indicated, values represent the percentage of the nutrient present as a portion of the total diet on an as-fed basis. Proximate analysis of each component was conducted by a commercial forage analysis laboratory. Amino acid content of each component was determined by means of high-performance liquid chromatography analysis following an acid hydrolysis of the feed sample. Each horse was fed 0.1% of body weight of the concentrate ration twice daily.

Contributor Notes

Dr. Wagners present address is Cooperative Research Farms, 1919 Huguenot Rd, Richmond, VA 23235.

This manuscript represents a portion of a dissertation submitted by Dr. Wagner to the University of Kentucky Graduate School as partial fulfillment of the requirements for a Doctor of Philosophy degree.

Supported by the Morris Animal Foundation First Award Grant (D09EQ-310).

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.

The authors thank Rosalyn B. Ennis for technical assistance.

Address correspondence to Dr. Urschel (klur222@uky.edu).