Experimental design.

Participants were randomly assigned to 1 of 2 experimental groups: control or LEU. Participant characteristics for each group are presented in Table 1. Each group completed an experimental trial over 2 consecutive days, which was identical for each group with the exception of the composition of the EAA solution that was ingested after exercise (Table 2). Participants were housed in the Institute for Translational Sciences Clinical Research Center (ITS-CRC) of the University of Texas Medical Branch for the entirety of the trial.

The evening before the experimental trial was initiated, participants were admitted to the ITS-CRC, and a dual-energy x-ray absorptiometry scan (QDR 4500W; Hologic) was performed after the bladder was voided to measure body composition and lean mass. Participants were then fed a standard dinner (12 kcal/kg body weight; 60% carbohydrate, 20% fat, and 20% protein) and a snack (5 kcal/kg body weight; 60% carbohydrate, 20% fat, and 20% protein) at 2200, both prepared by the Bionutrition Division of the ITS-CRC. Both days of the trial took place after an overnight fast, under basal conditions. All participants were studied during the same time of day (0600–1630) to avoid potential circadian changes, and participants were asked to refrain from exercise for at least 48 h before beginning the trial.

On morning 1 of the experimental trial, while the subjects were resting in a supine position, an 18-gauge polyethylene catheter was inserted into an antecubital vein for tracer infusion. Another 18-gauge polyethylene catheter was inserted retrograde in a hand vein of the contralateral arm, which was kept in a heated pad for arterialized blood sampling. After drawing a background blood sample, a primed continuous infusion of isotopically labeled l-[ring-¹³C₆]Phe and l-[1-¹³C]Leu tracers (Cambridge Isotope Laboratories) was initiated. Each tracer was dissolved in sterile 0.9% saline and passed through a 2-μm filter. The priming doses for l-[ring-¹³C₆]Phe and l-[1-¹³C]Leu were 2 and 4.8 μmol/kg, respectively. The constant infusion rates for l-[ring-¹³C₆]Phe and l-[1-¹³C]Leu were 0.05 and 0.08 μmol · kg⁻¹ · min⁻¹, respectively, and these rates were maintained throughout the trial day (13). Two hours after the initiation of the tracer infusion, muscle biopsy 1 was obtained from the lateral portion of the vastus lateralis of the leg with the biopsy site between 15 and 25 cm from the midpatella. The biopsy was performed using a 5-mm Bergström biopsy needle (23) with suction under sterile procedure and local anesthesia (1% lidocaine). At 2.5 h after biopsy 1, biopsy 2 was obtained from the same incision. The biopsy needle was inclined at a different angle so that biopsy 2 was obtained ~5 cm proximal to biopsy 1. After muscle biopsy 2, participants were seated on the leg-extension device to begin the exercise portion of the trial. Participants completed 8 sets of 10 repetitions at a mean intensity of 65% 1RM with 3 min of rest between each set. Total time for the exercise period was ~45 min. At 1 h after exercise, participants in the control group ingested 10 g of EAAs containing 1.85 g of Leu, whereas participants in the LEU group ingested 10 g of EAAs containing 3.5 g of Leu (Table 2) (24). Blood was obtained at 0, 0.5, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, and 5 h after exercise, and muscle biopsies were sampled at 2 h after exercise (1 h after the ingestion of the EAA solution) and 5 h after exercise. The biopsy obtained at 2 h after exercise was sampled from an incision on the opposite leg from the basal biopsies, and the 5-h postexercise biopsy was obtained from a new incision on the same leg made ~5 cm proximal to incision 2. Throughout the trial, subjects were instructed to remain in their hospital beds in a supine position, with the exception of the exercise portion and restroom visits. After collection of the 5-h postexercise muscle biopsy, day 1 was concluded, tracers were stopped, and participants were given a standard lunch. Participants remained in the ITS-CRC and were encouraged to move around the unit to compile between 1000 and 1500 steps (to avoid prolonged periods of inactivity or bed rest), which was monitored (but not directly measured) by a study nurse. Participants were fed a dinner and snack identical to those of the previous night before an overnight fast in preparation for day 2 of the trial.

On morning 2 of the experimental trial, an infusion protocol identical to that described above was initiated, which was preceded by a blood draw. Muscle biopsies were obtained from a single incision at 2 and 4.5 h after initiation of the tracer infusion. The biopsy at 4.5 h corresponded to 24 h after exercise. After collection of the 24-h postexercise biopsy, participants were given a lunch and discharged from the unit.

Muscle tissue from all biopsies was immediately blotted, frozen in liquid nitrogen, and stored at −80°C until analysis.

EAA composition.

The composition of the EAA solution for each group is presented in Table 2. The Leu quantities were chosen based on previous research indicating a Leu threshold of ~2 g in older adults (22, 25), and the use of an isonitrogenous mixture of EAAs was used to control for any influence of EAA total content (8). To minimize tracer dilution with the addition of the EAAs, we added l-[ring-¹³C₆]Phe and l-[1-¹³C]Leu tracer to the oral EAA solution at 7.5% and 6.0% of the Phe and Leu total content, respectively. EAAs (Sigma-Aldrich) for the two groups were individually weighed and fully dissolved by continuous overnight stirring at room temperature in a noncaloric and caffeine-free flavored beverage (350 mL) to increase palatability (24).

Tissue processing for protein fractions.

Muscle samples were processed to obtain general protein and myofibrillar protein fractions as described previously (26). Briefly, ~30–50 mg of frozen muscle tissue was placed in buffer (27), homogenized (1:9, wt:v), and centrifuged at 3400 × g for 10 min at 4°C. The resulting supernatant was collected and used for general cytosolic protein immunoblotting as described below. The resulting pellet was then suspended in isolation buffer (1 mol/L sucrose, 1 mol/L Tris-HCl, 1 mol/L KCl, and 0.5 mol/L EDTA, pH 7.4) containing protease and phosphatase inhibitors and centrifuged for 10 min at 4°C and 700 × g. After a series of 3 PBS buffer suspensions and 5-min centrifugations of 15,000 × g at 4°C, the pellet was resuspended and agitated on ice 2 times for 20 min and in a 4°C sonication bath in high-salt buffer (1:4, wt:v). The slurry was centrifuged at 15,000 × g for 10 min at 4°C. The resulting pellet was fully suspended in double-distilled water and centrifuged at 15,000 × g for 5 min at 4°C. To precipitate the myofibrillar proteins, 1 mL of 0.3 mol/L NaOH was added to resuspend the pellet and heated at 50°C for 30 min with frequent mixing by vortex. After centrifugation at 10,000 × g for 5 min at 4°C, the supernatant was collected, and an additional 1 mL of 0.3 mol/L NaOH was added to resuspend the pellet and heated at 37°C for 10 min with frequent mixing by vortex. After centrifugation at 10,000 × g for 5 min at 4°C, the supernatant was collected as the myofibrillar protein fraction and the collagen pellet was discarded. Precipitate was created by addition of 1 mL of perchloric acid to the collected supernatant and pelleted at 805 × g for 10 min at 4°C. This pellet was washed 2 times with 70% ethanol and then hydrolyzed overnight in 1.5 mL of 6 mol/L HCl for determination of myofibrillar protein–bound enrichment.

Amino acid enrichments and concentrations.

A separate piece of muscle (~20–30 mg) was homogenized and used to obtain intracellular free amino acids as described previously (27). Myofibrillar protein–bound l-[ring-¹³C₆]Phe enrichment (26) and muscle intracellular fluid l-[ring-¹³C₆]Phe and l-[1-¹³C]Leu enrichment were determined via GC-MS (6890 Plus GC, 5973N MSD, 7683 autosampler; Agilent Technologies). Myofibrillar protein–bound l-[ring-¹³C₆]Phe enrichment was determined by GC-MS in triplicate after protein hydrolysis and amino acid extraction (27) using the m+6:m+4 ratio and an external standard curve of known m+6:m+0 ratios (28, 29). Muscle intracellular free l-[ring-¹³C₆]Phe and l-[1-¹³C]Leu enrichments were determined by GC-MS in triplicate using the m+6:m+0 and m+1:m+0 ratios, respectively. Blood l-[ring-¹³C₆]Phe and l-[1-¹³C]Leu enrichments were determined from deproteinized blood samples in duplicate using the m+6:m+0 and m+1:m+0 ratios, respectively. All tracer enrichments were determined using tert-butyldimethylsilyl amino acid derivatives.

Concentrations of Phe and Leu were determined in blood and muscle intracellular fluid using tracer enrichments and l-[¹⁵N]Phe and l-[5,5,5-²H₃]Leu as internal standards for Phe and Leu, respectively, as described previously (30). Basal intracellular amino acid concentrations were taken as the mean from the 2 pre-exercise muscle biopsies.

Calculation of muscle protein fractional synthesis rate.

The fractional synthesis rate of myofibrillar protein was determined by examining the rate of l-[ring-¹³C₆]Phe incorporated into myofibrillar protein using following the precursor product model:

where FSR is the fractional synthesis rate, Ep is the increment in myofibrillar protein–bound l-[ring-¹³C₆]Phe enrichment between 2 muscle biopsies, t is the time between the 2 muscle biopsies, and EM(1) + EM(2) are the l-[ring-¹³C₆]Phe enrichments in the free intracellular pool in the 2 muscle biopsies. Data are expressed as percentage per hour.

Immunoblot analysis.

Immunoblot analysis on the general cytosolic protein fractions was performed as detailed previously (27). To maintain consistency with our previous publications most relevant to this study (6, 24), immunoblot data are presented as phosphorylation status relative to an internal control sample (rat soleus muscle) that was loaded on every gel for comparison across blots and then expressed as a fold change from basal.

Antibodies.

The phosphorylated and total antibodies used for immunoblotting were purchased from Cell Signaling Technologies: phosphorylated mTOR (Ser²⁴⁴⁸; 1:250), phosphorylated S6K1 (Thr³⁸⁹; 1:500), phosphorylated 4E-BP1 (Thr^37/46; 1:1000), phosphorylated eukaryotic elongation factor 2 (eEF2; Thr⁵⁶; 1:5000), and total eEF2 (1:2000). mTOR, S6K1, and 4E-BP1 total protein were detected using an antibody dilution of 1:1000. Anti-rabbit IgG HRP-conjugated secondary antibody was purchased from GE Healthcare (1:2000).

RNA extraction and semiquantitative real-time PCR.

RNA isolation, cDNA synthesis, and real-time qPCR were performed as we described previously (31, 32). Real-time qPCR was performed with an iQ5 Multicolor Real-Time PCR cycler (Bio-Rad). cDNA was analyzed with SYBR green fluorescence (iQ SYBR green supermix; Bio-Rad). Primer sequences for the current investigation were published previously (31, 33). β2-Microglobulin was used as a normalization/housekeeping gene. Relative fold changes were determined from the cycle threshold values using the 2^−ΔΔCt method (34).

Blood and intracellular amino acid concentrations.

There were no differences between groups in blood Leu or Phe concentrations during the basal, exercise, or immediate 1 h postexercise periods. Blood Leu concentrations increased above basal values (mean of pre-RE time points) for the 2 groups after EAA ingestion at 1 h after exercise (Fig. 1A). Blood Leu concentrations remained elevated in the control group for 1.5 h after EAA ingestion (2.5 h after exercise), whereas blood Leu concentrations remained elevated in the LEU group for 2 h after EAA ingestion [3 h after exercise (P < 0.05)] (Fig. 1A). Furthermore, blood Leu concentrations were higher in the LEU group than in the control group for 1.5 h after EAA ingestion (2.5 h after exercise) (P < 0.05) (Fig. 1A). Blood Phe concentrations were elevated in each group for 2 h after EAA ingestion (3 h after exercise) (P < 0.05), and no group differences were observed for blood Phe concentrations at any time point (Fig. 1B).

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FIGURE 1

Time course of arterialized blood Leu (A) and Phe (B) concentrations in older men after the combination of RE and postexercise (1 h) ingestion of 10 g of EAAs containing 1.85 g of Leu (CTRL; n = 7) or 3.5 g of Leu (LEU; n = 8). Data are means ± SEMs. *Different from basal (−2 h), P < 0.05; ^†group difference, P < 0.05. CTRL, control group; EAA, essential amino acid; LEU, Leu group; RE, resistance exercise.

Basal intracellular Leu and Phe concentrations were similar between groups (Table 3). Intracellular Leu concentrations increased only in the LEU group at 2 h after exercise (1 h after EAA ingestion) (P < 0.05), and intracellular Leu concentration was higher in the LEU group than in the control group at this time point (P < 0.05) (Table 3). Intracellular Leu concentrations were similar to basal values in the 2 groups at 5 and 24 h after exercise (P > 0.05). Intracellular Phe concentrations increased in the 2 groups at 2 h after exercise (1 h after EAA ingestion) (P < 0.05) and were similar to basal values in each group at 5 and 24 h after exercise (P > 0.05) (Table 3). There were no group differences in intracellular Phe concentrations at any time point during the experimental trial.

MyoPS rate.

The basal MyoPS rate was similar between groups (Fig. 2). After the combination of RE and EAA ingestion (1 h after exercise), the MyoPS rate was increased by ~90% in the 2 groups from 2 to 5 h after exercise (P < 0.05), with no difference between groups (P > 0.05) (Fig. 2). In contrast, the MyoPS rate was elevated above basal values only in the LEU group at 24 h after exercise (P < 0.05) (Fig. 2).

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FIGURE 2

Time course of myofibrillar protein FSR in older men after the combination of resistance exercise and postexercise (1 h) ingestion of 10 g of essential amino acids containing 1.85 g of Leu (CTRL; n = 7) or 3.5 g of Leu (LEU; n = 8). Data are means ± SEMs. *Different from basal, P < 0.05. CTRL, control group; FSR, fractional synthesis rate; LEU, Leu group.

Cell signaling.

Total protein content did not change during the time course for any signaling protein (P > 0.05). Phosphorylation of mTOR (Ser²⁴⁴⁸) was increased above basal in the control group only at 2 h after exercise (1 h after EAA ingestion) (P < 0.05), whereas phosphorylation of mTOR was increased above basal in the LEU group at 2 and 5 h after exercise (P < 0.05) (Fig. 3A). Phosphorylation of mTOR was similar to basal in the 2 groups at 24 h after exercise (P > 0.05), and no group differences were observed in mTOR phosphorylation at any time point. Phosphorylation of S6K1 (Thr³⁸⁹) was increased above basal in the 2 groups at 2 h (P < 0.05) but was similar to basal at 5 and 24 h after exercise (P > 0.05) (Fig. 3B). No group differences were observed in S6K1 phosphorylation at any time point. Phosphorylation of 4E-BP1 (Thr^37/46) was similar to basal at all postexercise time points in the control group (P > 0.05) (Fig. 3C). In contrast, phosphorylation of 4E-BP1 was increased above basal in the LEU group at 2 and 5 h after exercise (P < 0.05) but not at 24 h after exercise (P > 0.05) (Fig. 3C). No group differences were observed in 4E-BP1 phosphorylation at any time point. Phosphorylation of eEF2 (Thr⁵⁶) was decreased below basal in the control group at 2 h after exercise (P < 0.05) but not at 5 or 24 h after exercise (P > 0.05) (Fig. 3D). Phosphorylation of eEF2 was similar to basal at all postexercise time points in the LEU group (P > 0.05) (Fig. 3D).

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FIGURE 3

Time course of phosphorylation of mTOR at Ser²⁴⁴⁸ (A), S6K1 at Thr³⁸⁹ (B), 4E-BP1 at Thr^37/46 (C), and eEF2 at Thr⁵⁶ (D) in older men after the combination of resistance exercise and postexercise (1 h) ingestion of 10 g of essential amino acids containing 1.85 g of Leu (CTRL; n = 7) or 3.5 g of Leu (LEU; n = 8). Immunoblot data were normalized to an internal control sample, and data are adjusted to represent fold change from basal in phosphorylated protein. Data are expressed as means ± SEMs. *Different from basal, P < 0.05. Representative blots are provided in Supplemental Figure 1. CTRL, control group; eEF2, eukaryotic elongation factor 2; LEU, Leu group; mTOR, mammalian/mechanistic target of rapamycin; S6K1, p70 S6 kinase 1; 4E-BP1, 4E binding protein 1.