Development of a stable isotope method to measure protein fractional synthesis rate in equine skeletal muscle

Abstract

Muscle mass is determined by the balance between rates of muscle protein synthesis (MPS) and degradation. The mammalian target of rapamycin (mTOR) signaling pathway regulates the initiation of muscle protein synthesis, and previous work in horses has shown that the activation of mTOR signaling components responds in a dose-dependent manner to protein intake. Whether this translates to rates of muscle protein synthesis has not been explored in horses. The objective of this study was to develop an isotopic method to measure muscle protein fractional synthesis rate (FSR) in horses following the consumption of graded amounts of dietary protein. It was hypothesized that muscle protein FSR would increase in a dose-dependent manner with increasing protein intake. Eight mature Thoroughbred horses (n = 4 mares and n = 4 geldings; age 16.0 ± 3.0 yr old; weight 588 ± 43 kg BW; mean ± SD) were used in a repeated 4 × 4 Latin square design. Horses were studied before and following the consumption of a meal containing each of 4 levels (0 [NO], 0.125 [LOW], 0.25 [MED], and 0.5 [HIGH] g CP/kg BW) of protein, in a randomly determined order. A 4-h primed (8.4 μmol/kg BW), constant (6 μmol/kg BW per hour) intravenous infusion of [ring-²H₅] phenylalanine was used to measure muscle protein FSR. Muscle samples were collected at 1 h and 2 h post-feeding. The isotopic enrichment of the free and protein bound phenylalanine in muscle samples was measured using GC-MS analysis and used to calculate muscle protein FSR. Blood samples were collected from a jugular vein catheter before and every 30 min for 2 h following feeding for the measurement of plasma glucose, insulin, and amino acid concentrations. Data were analyzed using the mixed procedure of SAS. Muscle protein FSR was analyzed with a one-way ANOVA, with treatment as the fixed effect and period and square as random effects, and plasma metabolite concentrations were analyzed using repeated measures, with treatment, time, and their interaction as the fixed effects. Plasma glucose (P < 0.0001), insulin (P < 0.0001), and amino acid concentrations (P = 0.03 to <0.0001) increased following feeding, with higher concentrations in horses receiving LOW, MED, and HIGH compared with NO treatment. There was no effect of treatment on muscle protein FSR (P = 0.96). The developed methodologies allowed for the measurement of isotope incorporation into muscle protein and the calculation of muscle protein FSR. Despite horses clearly being in the postprandial state following meal consumption, higher levels of protein intake did not result in the expected increase in muscle protein FSR between 1 h and 2 h post-feeding.