Skeletal muscle protein anabolic response to resistance exercise and essential amino acids is delayed with aging

Subjects

We studied seven young and six old male subjects (age range: 24-77 yr). Subject characteristics are found in Table 1. The subjects were not engaged in any regular exercise training at the time of the enrollment; however, they were physically independent and overall healthy. Screening of subjects were performed with clinical history, physical exam, and laboratory tests including complete blood count with differential, liver, and kidney function tests, coagulation profile, fasting blood glucose and oral glucose tolerance test, hepatitis B and C screening, human immunodeficiency test, thyroid stimulating hormone test, urinalysis, drug screening, electrocardiogram, and stress test on a treadmill or bicycle. Subjects were also tested for maximal strength by performing a one-repetition maximum (1RM) on a leg extension machine (Cybex-VR2, Medway, MA) during the initial screening. A second 1RM testing was performed at least 1 wk prior to study participation. The starting weight used during the resistance exercise portion of this study was 70% of the subjects' predetermined 1RM. All subjects gave informed, written consent before participating in the study, which was approved by the Institutional Review Board of the University of Texas Medical Branch (which is in compliance with the Declaration of Helsinki).

Study design

All subjects were admitted to the General Clinical Research Center (GCRC) of the University of Texas Medical Branch the day prior to the exercise study, and a dual-energy X-ray absorptiometry scan (Hologic QDR 4500W, Bedford, MA) was performed to measure body composition and lean mass. The subjects were then fed a standard dinner and a snack at 2200. All subjects were studied following an overnight fast under basal conditions and refrained from exercise for 24 h prior to study participation. The morning of the infusion study, an 18-gauge polyethylene catheter was inserted into a forearm vein for tracer infusion and another catheter was inserted retrogradely in the forearm of the opposite arm, which was heated for arterialized blood sampling. After a background blood sample was drawn, a primed continuous infusion of L-[ring-²H₅] phenylalanine (Cambridge Isotope Laboratories, Andover, MA) was started (time = 0 h) and maintained at a constant rate until the end of the experiment (Fig. 1). The priming dose for the labeled phenylalanine was 2 μmol/kg and the infusion rate was 0.05 μmol·kg^-/-1·min^-1. All subjects were studied during the same time of day to avoid potential circadian changes.

A baseline muscle biopsy was taken 2 h following the initiation of the tracer infusion. The biopsy was obtained from the lateral portion of the vastus lateralis using a 5-mm Bergström biopsy needle, under sterile procedure and local anesthesia (1% lidocaine). Once harvested, the muscle tissue was immediately blotted and frozen in liquid nitrogen and stored at -80°C until analysis. Following the first biopsy, two blood samples were drawn 30 min apart from each other. At 105 min after the first biopsy, a second muscle biopsy was taken marking the end of the baseline period. However, under this circumstance, the biopsy needle was inclined at a different angle so that the second biopsy was taken approximately ~5 cm apart from the first.

Following the second biopsy, the subjects were escorted to the Cybex leg extension machine. There the subjects performed eight sets of ten repetitions of two-legged extension exercises set to 70% of their 1RM. The rest period between sets was 3 min. Blood samples were collected immediately after the third, sixth, and eighth sets. Following the last blood collection, the subjects were then transported back to their hospital bed for the remainder of the study. Blood samples were taken every 15 min during the first hour of recovery from exercise. At 1 h postexercise, a third muscle biopsy was obtained angling the needle once again so that the muscle biopsy was sampled ~5 cm from the second biopsy.

Immediately after the third biopsy, subjects ingested a solution (500 ml) that contained 20 g of EAAs. This EAA dosage is consistent with our previous publications (14, 18) and is optimal to stimulate protein synthesis and mTOR signaling. The composition of the EAA mixture was the following: histidine (8%), isoleucine (8%), leucine (35%), lysine (12%), methionine (3%), phenylalanine (14%), threonine (10%), and valine (10%) (Ajinomoto/Sigma Aldrich, Raleigh, NC). Therefore, our EAA mixture was highly enriched in leucine. To minimize changes in blood phenylalanine enrichment we added a small amount of L-[ring-²H₅] phenylalanine to the EAA mixture to increase the mixtures' L-[ring-²H₅] phenylalanine enrichment to the predicted blood L-[ring-²H₅] phenylalanine enrichment (i.e., 6.5%). Blood samples were collected every 15 min during the 2 h following the amino acid drink. At the end of the third hour postexercise, a fourth muscle biopsy was collected from a new incision site ~7 cm proximal to the first incision. Blood samples were drawn every 30 min for the next 3 h. A final biopsy was collected 6 h postexercise from the second incision site with the biopsy needle facing a new direction in the muscle than previously, ~5 cm from the fourth biopsy.

RNA isolation

Total RNA was isolated by homogenizing 30-40 mg tissue with a homogenizing dispenser (T10 Basic Ultra Turrax, IKA, Wilmington, NC) in a solution containing 1.0 ml of TRI reagent and 4 μl of polyacryl carrier (Molecular Research Center, Cincinnati, OH). The RNA was separated into an aqueous phase with 0.2 ml of chloroform and precipitated from the aqueous phase with 0.50 ml of isopropanol. Extracted RNA was washed with 1 ml of 75% ethanol, dried, and then suspended in a known amount (1.5 μl/mg tissue) of nuclease-free water. RNA was quantified spectrophotometrically (Bio-Rad, SmartSpec, Hercules, CA) at a wavelength of 260 nm. RNA concentration was calculated on the basis of total RNA yield. RNA quality was assessed by RNA agarose gel electrophoresis followed by visualization of the 18 and 28S ribosomal RNA bands under ultraviolet light. Total RNA was used to calculate the RNA-to protein ratio, an index of ribosomal capacity.

Muscle fractional synthetic rate

Muscle tissue samples were ground, and intracellular free amino acids and muscle proteins were extracted as previously described (13). Blood and muscle intracellular free concentration of leucine and phenylalanine and the enrichment of phenylalanine were determined by gas chromatography-mass spectrometry (GC-MS, 6890 Plus GC, 5973N MSD, 7683 autosampler, Agilent Technologies, Palo Alto, CA) using appropriate internal standards (60). Mixed muscle protein-bound phenylalanine enrichment was analyzed by GC-MS after protein hydrolysis and amino acid extraction (13), using the external standard curve approach (10). We calculated the fractional synthetic rate (FSR) of mixed muscle proteins by measuring the incorporation rate of the phenylalanine tracer into the proteins (ΔE_p/t) and using the precursor-product model to calculate the synthesis rate:

where ΔE_p is the increment in protein-bound phenylalanine enrichment between two sequential biopsies, t is the time between the two sequential biopsies, and E_M(1) and E_M(2) are the phenylalanine enrichments in the free intracellular pool in the two sequential biopsies. Data are expressed as percent per hour.

Immunoblot analysis

Details to immunoblotting can be found elsewhere (13). Briefly, ~70 mg of frozen tissue was homogenized and centrifuged for 10 min at 4°C, and supernatant was collected. Total protein concentrations were determined by the Bradford assay and quantified using the Smartspec Plus spectrophotometer (BioRad). The supernatant was diluted (1:1) in a sample buffer mixture containing 125 mM Tris, pH 6.8; 25% glycerol; 2.5% SDS 2.5% β-mercaptoethanol, and 0.002% bromophenol blue, then boiled for 3 min at 100°C. However, homogenate aliquots used to detect 4E-BP1 were initially boiled at 100°C for 10 min and spun for 30 min at 6,000 g before the supernatants were combined with the sample buffer. Equal amounts of total protein were loaded into each lane and the samples were separated by electrophoresis (150 V for 60 min) on a 7.5 or 15% polyacrylamide gel as determined by the size of the target protein (Criterion, Bio-Rad). Each sample was loaded in duplicate and each gel contained an internal loading control (rodent skeletal muscle) and molecular weight ladder (Precision Plus). Following electrophoresis, protein was transferred to a polyvinylidene difluoride membrane (Bio-Rad) at 50 V for 60 min. Blots were incubated in primary antibody overnight at 4°C (antibody concentrations are described below). The next morning, blots were incubated in secondary antibody for 1 h at room temperature. Chemiluminescent solution (ECL plus, Amersham BioSciences, Piscataway, NJ) was applied to each blot. After a 5-min incubation, optical density measurements were obtained with a phosphoimager (ChemiDoc, Bio-Rad) and densitometric analysis was performed with Quantity One 4.5.2 software (Bio-Rad). Membranes containing phospho-detected proteins were stripped of primary and secondary antibodies by use of Restore Western blot stripping buffer (Pierce Biotechnology). After incubation in stripping buffer, membranes were reprobed for total protein with specific antibody of interest. Phospho and total density values were normalized to an internal loading control (which was loaded on each respective blot) and the phospho-to-total protein ratios were determined. Immunoblot data was expressed as normalized phospho divided by normalized total protein (phospho/total). However, AMPKα phosphorylation was expressed as phospho normalized to internal loading control because AMPK is a heterotrimeric protein with differences in catalytic subunit activation due to exercise. AMPKα1 and α2 activity can both be altered with muscle contraction under certain circumstances (49); however, we chose to measure AMPKα2 phosphorylation on the basis of previous work showing that this isoform is predominantly activated during moderate to high-intensity muscle contraction in humans (4, 59).

Hormones and glucose/lactate

Serum concentrations of cortisol, growth hormone, insulin, and total testosterone were determined by chemiluminescent enzyme immunoassay using the Immulite 2000. Plasma glucose and lactate concentration was measured using an automated glucose and lactate analyzer (YSI, Yellow Springs, OH).

Antibodies

The phospho primary antibodies used were purchased from Cell Signaling (Beverly, MA) unless otherwise noted: phospho-Akt (Ser473; 1:1,000), phospho-AMPKα (Thr172; 1:1,000), phosphomTOR (Ser2448; 1:1,000), phospho-p70 S6K1 (Thr389; 1:500), phospho-4E-BP1 (Thr37/46; 1:1,000), phospho-eukaryotic elongation factor 2 (eEF2) (Thr56; 1:1,000), phospho-glycogen synthase kinase β (GSK3β) (Ser9; 1:1,000), phospho-eukaryotic initiation factor α (eIF2α) (Ser52; 1:1,000; Invitrogen, Carlsbad, CA), phospho-ERK1/2 (Thr202/Tyr204; 1:1,000) and phospho-MNK1 (Thr197; 1:1,000). Total protein for each of these molecules [including total neuronal nitric oxide synthase (nNOS)] was purchased from Cell Signaling and detected by using an antibody dilution of 1:1,000. Anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody was purchased from Amersham Bioscience (1:2,000).

Statistical analysis

Data are reported as means ± SE. Betweenand within-group differences were tested by a repeated-measures ANOVA. Simple contrasts were used to assess main and interaction effects between particular time points. Post hoc paired t-tests were done to identify specific intragroup differences when the ANOVA models produced significant main or interaction effects. Alpha was set to <0.05. All analyses were done with SPSS v14 software.

Baseline measurements

As expected, the ages of the two groups were significantly different from each other (P < 0.05; Table 1). In addition, young subjects had a trend for higher leg lean mass (Leg LM) (P = 0.087) compared with the old. However, when the 1RM data were arranged to account for Leg LM [an indirect measure of muscle quality; Relative 1RM (1RM/Leg LM)] these strength differences were minimized (P > 0.05). Young subjects had a significantly greater absolute 1RM and lifted a greater average weight per set (P < 0.05). Total work per set in young subjects was greater than the old subjects (2.54 ± 0.01 vs. 1.68 ± 0.01 kJ, respectively) (P < 0.05).

To further determine skeletal muscle growth regulation differences between young and old, we identified the total protein expression of nNOS. nNOS appears to be a regulator of force production and therefore has been shown to be less in rats undergoing atrophy (hindlimb suspension) (47). Our data indicate that there were no differences in total nNOS at baseline (young: 0.85 ± 0.14 and old: 0.77 ± 0.20 arbitrary units) or at 6 h postexercise between young and old subjects (young: 0.77 ± 0.13 and old: 0.57 ± 0.11 arbitrary units) (P > 0.05).

Serum hormones and blood glucose/lactate

Cortisol levels were significantly reduced in the young at 4 and 5 h postexercise compared with baseline (P < 0.05; Table 2). Insulin was significantly increased immediately after exercise and at 1, 2, and 3 h postexercise in the young compared with baseline (P < 0.05). Testosterone was significantly increased immediately after exercise but was lower at 3 h postexercise in the young compared with baseline (P < 0.05). There were no changes in old for cortisol, insulin, or testosterone compared with baseline over the course of study (P > 0.05). Blood glucose was significantly lower during 2- 6 h postexercise for young and old subjects compared with baseline (P < 0.05). Blood lactate was significantly elevated for young and old subjects during and 1 h after exercise compared with baseline. Blood lactate was higher during exercise in the young compared with old (P < 0.05). Blood lactate levels significantly decreased below baseline levels between 4-6 h postexercise in the old and by 6 h postexercise in the young (P < 0.05).

Phenylalanine and leucine concentrations

Blood phenylalanine and leucine concentrations were significantly elevated in the young and old subjects at 2, 3, and 4 h post exercise compared with baseline (P < 0.05; Table 3); however, the increase was greater in the old than young at 4 h postexercise (P < 0.05). Blood phenylalanine and leucine concentrations continued to remain elevated in the old at 5 and 6 h postexercise (P < 0.05) whereas blood phenylalanine values returned to baseline in the young. Muscle intracellular concentrations of phenylalanine and leucine were significantly elevated in the young and old at 3 h postexercise compared with baseline (P < 0.05). Muscle intracellular concentrations of leucine continued to stay elevated in the young and old at 6 h postexercise compared with baseline (P < 0.05).

Muscle protein synthesis

Mixed muscle protein synthesis (i.e., FSR) was significantly elevated in the young 1-3 h postexercise compared with baseline and was significantly higher than the old (Fig. 2A; P < 0.05). Muscle protein synthesis at 3- 6 h postexercise was significantly elevated to a similar extent in both age groups compared with baseline (Fig. 2A; P < 0.05). Muscle protein synthesis over the entire 5-h post-EAA ingestion period (1- 6 h postexercise) was increased above baseline and similar between young and old (Fig. 2B; P < 0.05).

Upstream mTOR regulators

The phosphorylation status of Akt at Ser473 was significantly increased for the young compared with baseline at 3 h postexercise (P < 0.05) whereas there was no difference in the old (Fig. 3A). At 1 and 3 h postexercise, AMPKα phosphorylation at Thr 172 was higher in the old at 1 and 3 h (Fig. 3B; P < 0.05), and this increase was significantly greater than the young at 3 h postexercise (P < 0.05).

mTOR signaling

mTOR phosphorylation at Ser2448 was increased to a similar extent at all time points compared with baseline for both young and old subjects (Fig. 4A; P < 0.05). S6K1 phosphorylation at Thr389 was significantly increased at all time points postexercise compared with baseline for both age groups although there was a tendency (P = 0.07) for S6K1 phosphorylation to be increased in the young at 6 h postexercise (Fig. 4B; P < 0.05). However, phospho-S6K1 tended to be higher in young than the old subjects at 1 h postexercise prior to EAA ingestion (P = 0.09). 4E-BP1 phosphorylation at Thr37/46 was significantly increased at 3 h postexercise for both the young and old subjects but only remained elevated at 6 h postexercise in the young (Fig. 4C; P < 0.05). eEF2 phosphorylation at Thr56 tended to be lower at 1 h postexercise in both age groups (P = 0.07) and was significantly lower at 3 and 6 h postexercise for young and old subjects (Fig. 4D; P < 0.05).

Eukaryotic initiation factor 2B regulators

GSK3β phosphorylation at Ser9 did not change in either group postexercise, although it was slightly lower in the young at 6 h postexercise compared with baseline (Fig. 5A; P < 0.05). eIF2α phosphorylation was significantly lower at 1 h postexercise in the young compared with baseline (P < 0.05). but unchanged in the old (Fig. 5B).

ERK signaling

ERK1/2 phosphorylation was significantly elevated only in the young at 1 h postexercise (P < 0.05)., which tended to be greater than the old (Fig. 6A; P = 0.07). MNK1 phosphorylation was significantly elevated at 1 h postexercise only in the young, and these values were significantly greater than the old (Fig. 6B; P < 0.05). Furthermore, MNK1 phosphorylation was higher in young compared with the old at 3 h postexercise (P < 0.05).