Postexercise protein metabolism in older and younger men following moderate-intensity aerobic exercise
M. Sheffield-Moore,
C. W. Yeckel,
E. Volpi,
S. E. Wolf,
B. Morio,
D. L. Chinkes,
D. Paddon-Jones, and
R. R. Wolfe
Department of Surgery, University of Texas Medical Branch and Shriners Burns Hospital for Children, Galveston, Texas 77550
Submitted 18 July 2003
; accepted in final form 11 May 2004
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ABSTRACT
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Regular aerobic exercise strongly influences muscle metabolism in elderly and young; however, the acute effects of aerobic exercise on protein metabolism are not fully understood. We investigated the effect of a single bout of moderate walking (45 min at
40% of peak O2 consumption) on postexercise (POST-EX) muscle metabolism and synthesis of plasma proteins [albumin (ALB) and fibrinogen (FIB)] in untrained older (n = 6) and younger (n = 6) men. We measured muscle phenylalanine (Phe) kinetics before (REST) and POST-EX (10, 60, and 180 min) using L-[ring-2H5]phenylalanine infusion, femoral arteriovenous blood samples, and muscle biopsies. All data are presented as the difference from REST (at 10, 60, and 180 min POST-EX). Mixed muscle fractional synthesis rate (FSR) increased significantly at 10 min POST-EX in both the younger (0.0363%/h) and older men (0.0830%/h), with the younger men staying elevated through 60 min POST-EX (0.0253%/h). ALB FSR increased at 10 min POST-EX in the younger men only (2.30%/day), whereas FIB FSR was elevated in both groups through 180 min POST-EX (younger men = 4.149, older men = 4.107%/day). Muscle protein turnover was also increased, with increases in synthesis and breakdown in younger and older men. Phe rate of disappearance (synthesis) was increased in both groups at 10 min POST-EX and remained elevated through 60 min POST-EX in the older men. A bout of moderate-intensity aerobic exercise induces short-term increases in muscle and plasma protein synthesis in both younger and older men. Aging per se does not diminish the protein metabolic capacity of the elderly to respond to acute aerobic exercise.
muscle protein synthesis; moderate walking; plasma proteins
THE NORMAL AGING PROCESS in healthy humans is often accompanied by an involuntary decade-by-decade loss of physical capability. The inability to maintain physical function with advanced age is largely attributable to several key factors, including inadequate nutrition (10, 45), hormonal alterations (8, 22, 47, 49), reduced mitochondrial protein synthesis (2, 50), DNA alterations (43, 50), and physical inactivity (23, 38). Not unlike frail elderly, healthy elderly often display greater adiposity and lower fat-free mass than their younger counterparts. The mechanism(s) behind the apparent imbalance between protein synthesis and breakdown with aging has not been fully elucidated and remains the subject of much debate (53, 54, 62). Regardless of whether elderly do or do not have diminished whole body or muscle protein metabolism, the more important issue remains whether an intervention such as exercise can positively influence protein metabolism.
Considerable effort has been directed at studying the metabolic consequences and physiological significance of both acute and repeated bouts of aerobic and resistance exercise. Much of this research has focused on animals or young healthy humans and has been extensively reviewed (7). Aerobic and resistance training result in very different physiological effects. It is generally accepted that heavy-resistance exercise produces skeletal muscle hypertrophy (28) whereas aerobic exercise does not (29). In the past decade, studies examining the anabolic benefits of resistance exercise have dominated the literature, which has led to resistance exercise being the recommended mode of exercise for adults seeking to maintain or improve muscle size and strength. Numerous studies have supported this recommendation, demonstrating that resistance exercise clearly elevates muscle protein synthesis (4, 5, 13, 42, 56, 63, 64). Furthermore, in some cases resistance exercise-induced protein synthesis can remain elevated for a protracted period of time postexercise (
24 h) in both young (13, 42) and old (56).
The value of aerobic exercise has long been linked to its direct effects on cardiovascular fitness and endurance capacity, irrespective of age. Furthermore, aerobic exercise has been recommended as a means to decrease fat mass without thought of its potential effect on lean muscle mass. Until recently, our understanding of the benefits of aerobic training was strongly influenced by long-standing data indicating that aerobic exercise training increases mitochondrial volume (30), mitochondrial enzyme activity (48), capillary-to-muscle fiber ratio, capillary density, and the number of capillaries around a given muscle fiber (48).
Although previous research and long-standing beliefs have led us to conclude that aerobic exercise training primarily affects muscle quality, not quantity, the mere fact that mitochondrial volume and enzyme activity are enhanced following aerobic exercise training suggests that increased protein turnover is required. In fact, recent results from a study subjecting elderly and young to repeated bouts of aerobic exercise over a 4-mo period have demonstrated that aerobic exercise can enhance muscle protein synthesis irrespective of age (51). Previous studies conducted in young healthy subjects provided the first indication that acute aerobic exercise has the potential to increase mixed muscle protein synthesis (12, 52) and acute-phase liver proteins (12). These observations were made after intense (52) and lengthy (12) exercise bouts, neither of which represent a viable aerobic session for the elderly. The data from Carraro et al. (12) demonstrating exercise-induced increases in certain plasma proteins are compelling, as there is very little information available on the effects of exercise on the concentration or synthesis of acute-phase liver proteins. Moreover, quantification of any potential exercise-induced changes could provide insight into their preferential use by various tissues and organs. Equally compelling is the proposed age-associated alterations in acute-phase protein concentrations and synthesis rates in animals (41) and humans (24), which may be indicative of disease or age-related dysregulation of immune and inflammation functions (24, 41). Finally, although regular aerobic exercise training has recently been shown to enhance mixed muscle protein synthesis in elderly and young (51), it remains unclear whether a single bout of aerobic exercise is also capable of elevating muscle protein and acute-phase protein synthesis. Consequently, we sought to test the hypothesis that a single bout of moderate-intensity aerobic exercise would be sufficient to increase the postexercise synthesis rates of muscle and plasma proteins in young and older men with normal physical capabilities.
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METHODS
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Subjects.
Twelve healthy untrained men, six older [69 ± 1 (SE) yr] and six younger [29 ± 2 (SE) yr], were studied before (REST) and 10, 60, and 180 min postexercise (POST-EX), a single bout of moderate-intensity aerobic exercise. Older subjects were recruited through the Sealy Center on Aging of the University of Texas Medical Branch (UTMB). Young subjects were recruited from within the community by use of institutionally approved advertisements. Written informed consent was obtained from all subjects before any study-related procedures were performed. The study procedures and risks were explained in detail using a consent form approved by the UTMB Institutional Review Board, and all procedures conformed to both national and local ethics committee guidelines. Volunteers were considered to be eligible if they were found to be healthy on the basis of the following: clinical history, physical examination, electrocardiogram, ankle brachial index, exercise stress test, blood pressure, complete blood count, blood chemistries including blood glucose and liver and kidney function tests, hepatitis panel, human immunodeficiency virus test, and urinalysis. Exclusion criteria included the following: cardiac, liver, kidney, pulmonary, autoimmune or vascular disease; coagulation disorders; hypertension; diabetes; obesity; cancer; anemia; drug or alcohol abuse; being strength or aerobically trained; impairment of activities of daily living (ADLs); anabolic or corticosteroid use; or inability to discontinue anti-inflammatory or prophylactic aspirin therapy (e.g., 14 days for aspirin). Volunteers were queried regarding their ADLs, including their history of recent and past physical activity. The volunteers performed their regular activities and maintained their usual diet during the week preceding the study.
Prestudy testing.
Approximately 2 wk before the experimental protocol was conducted, subjects were admitted as outpatients to the General Clinical Research Center (GCRC) at UTMB. Total body fat, leg lean mass, and leg fat mass were determined using dual-energy X-ray absorptometry (DEXA). After the DEXA procedure, young subjects reported to the GCRC exercise lab for the determination of maximal oxygen consumption (
O2 peak).
O2 peak was determined on a treadmill by means of expired-gas analysis, as described previously (46). The test consisted of a progressive walk/run exercise test (i.e., Bruce protocol) (6), during which time the treadmill belt speed and elevation were incrementally increased every 3 min until volitional fatigue or
O2 peak was reached. Older subjects were escorted to the UTMB Heart Station to perform a medically supervised Bruce protocol maximal-exercise stress test to evaluate cardiovascular health and predict
O2 peak (6). After the tests, subjects were monitored, fed, and discharged.
Experimental protocol.
Subjects were admitted to the GCRC the evening before the study. Each subject was studied on one occasion after an overnight fast (from 2200 until completion of study protocol). At 0600, polyethylene catheters were inserted into a forearm vein for infusion of labeled phenylalanine, into a vein of the opposite hand for arterialized blood sampling, and into the femoral artery and vein of one leg for blood sampling. The femoral arterial catheter was also used for the infusion of indocyanine green (ICG, IC-Green; Akorn, Buffalo Grove, IL). Leg volume was obtained anthropometrically (35).
Details of the experimental protocol including blood and muscle sampling are outlined in Fig. 1. Briefly, after a blood sample for the measurement of background phenylalanine enrichment and concentration and ICG concentration were obtained, a primed (2 µmol/kg) continuous infusion of L-[ring-2H5]phenylalanine (0.05 µmol·kg1·min1) was started (time 0) and maintained for the duration of the experiment (
405 min). Subjects remained supine in bed during the REST and POST-EX periods to prevent shifts in plasma volume and alter plasma proteins. After the REST period, subjects were transferred to the treadmill from the bed. Aerobic exercise consisted of a 45-min walk on a treadmill at 40% of
O2 peak. Table 1 outlines the exercise parameters for the younger and older men, respectively. After exercise, subjects were transferred back to bed and remained supine for the entire POST-EX period. Muscle biopsies (
80100 mg of tissue) were taken at the time points indicated in Fig. 1. The first POST-EX biopsy sample was taken at 10 min to allow for transfer of the subject to the bed from the treadmill. Biopsy samples were taken from the lateral portion of the vastus lateralis of the leg,
20 cm above the knee, using a 5-mm Bergström needle. Two separate biopsy sites were prepared for the collection of the four biopsies. Each biopsy site was used to obtain a biopsy both distal and proximal (i.e., superior) to the incision site. The tissue was rinsed, blotted, and immediately frozen in liquid nitrogen and stored at 80°C until analysis. Leg blood flow was measured during REST (130150 min), exercise (EX; 3545 min), and twice during the POST-EX (4050 and 150160 min) period using the ICG dye dilution method (33). Femoral arterial and venous blood samples were taken throughout REST and POST-EX to measure phenylalanine concentration and enrichment. Upon completion of the study, the tracer infusion was stopped, and all catheters were removed. Subjects were fed and monitored for 2 h before discharge.

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Fig. 1. Experimental protocol. Details of the stable isotope infusion protocol and the time points for collection of arteriovenous samples, muscle biopsies, and measurement of leg blood flow. AV, arteriovenous; O2 peak, peak O2 consumption.
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Analytical methods.
Phenylalanine enrichments and concentrations in arterial and venous blood samples were determined after the addition of an internal standard, deproteinization with sulfosalicylic acid, extraction with cation exchange chromatography, and tert-butyldimethylsilyl (t-BDMS) derivatization using gas chromatography-mass spectrometry (GC-MS) in electron impact mode (GC HP 5890, MSD HP 5989; Hewlett-Packard, Palo Alto, CA).
Muscle samples were weighed, and the proteins were precipitated with 450 µl of 10% sulfosalicylic acid. An internal standard containing phenylalanine was added to measure intracellular concentration. Tissue homogenization and centrifugation were performed on three separate occasions, and the supernatant was collected. The enrichment and concentration of free tissue phenylalanine were determined on its t-BDMS derivative by GC-MS (60). Calculation of phenylalanine intracellular concentration was then possible by accounting for the tissue value in addition to the ratio of intracellular to extracellular water (0.16) (3). The remaining pellet containing mixed muscle proteins was repeatedly washed and then dried at 110°C for 24 h in 6 N HCl. Amino acids in the hydrolysate were extracted and derivatized, as described above for the plasma samples, before the measurement of phenylalanine enrichment using GC-MS (GC 8000 series, MD 800; Fisons Instruments, Manchester, UK), monitoring the ions 237 and 239, and using the standard curve approach as described by Calder et al. (9).
Albumin concentration was measured spectrophotometrically at
= 628 nm (Sigma Diagnostics, St. Louis, MO), and fibrinogen concentration was determined at the UTMB Clinical Laboratory by the previously described Clauss method (15) using the MDA Fibriquik assay (Biomerieux, Durham, NC). The fractional synthetic rates (FSR) of albumin and fibrinogen were determined by GC-MC using protein-bound phenylalanine. First, plasma albumin and fibrinogen were purified from 1 ml of plasma, as previously described (17). Subsequently, plasma proteins were hydrolyzed in 6 N HCl at 110°C, and the amino acids derived from the hydrolyzed proteins were processed, extracted, and derivatized, as described above for the plasma and muscle samples, before the measurement of phenylalanine enrichment using GC-MS.
ICG concentration in infusate and serum samples was measured spectrophotometrically at
= 805 nm.
Calculations.
Kinetic parameters were calculated for phenylalanine using both the arteriovenous (a-v) balance method (60) and the three-pool model (3). Phenylalanine was used because it is an essential amino acid and is not oxidized in the muscle tissue. Thus phenylalanine utilization in the muscle is a direct index of muscle protein synthesis, and its release from the muscle is a measure of muscle proteolysis.
The a-v balance method is dependent on the measurement of phenylalanine enrichments and concentrations in the femoral artery and vein to estimate muscle protein synthesis, breakdown, and net balance. These parameters are based on the extraction of labeled phenylalanine from the femoral artery, the appearance of unlabeled phenylalanine from the muscle in the femoral vein, and the net a-v difference in phenylalanine concentrations, respectively (60). The three-pool model is an expansion of the a-v balance method and relies not only on the measurement of phenylalanine enrichments and concentrations in the femoral artery and vein but also on the direct measurement of phenylalanine enrichment in the free tissue water. Thus the direct measurement of phenylalanine intracellular utilization for protein synthesis and release from protein breakdown is possible. In addition, it is possible to calculate the rate of phenylalanine transport from the artery into the muscle tissue and from the muscle tissue into the venous blood. The a-v balance method and the three-pool model are well established and have been presented elsewhere (60). Data are presented per 100 ml of leg volume.
Leg plasma flow was calculated using the dye dilution technique as previously described (33). Leg blood flow was calculated by correcting the plasma flow by the hematocrit.
The FSR of mixed muscle proteins was calculated from the incorporation rate of L-[ring-2H5]phenylalanine into the proteins and the free-tissue phenylalanine enrichment using the precursor-product model previously described (60)
where
EP is the increment of protein-bound phenylalanine enrichment between two sequential biopsies, and t is the time interval between the two sequential biopsies; EM(1) and EM(2) are the phenylalanine enrichments (tracer-to-tracee ratio) in the free muscle pool in two subsequent biopsies. The results are presented as percent per hour.
Albumin and fibrinogen FSR are calculated in a similar manner as described above
EP is the increment of protein-bound phenylalanine enrichment between two sequential blood draws, and t is the time interval between the two sequential blood draws; EB(1) and EB(2) are the phenylalanine enrichments (tracer-to-tracee ratio) in the blood pool from two subsequent draws. The results are presented as percent per day.
Statistics.
Statistical analyses were performed with Dunnett's one-tailed simultaneous 95% confidence limits using 90% upper and lower limits on each of the following primary and secondary parameters. Primary parameters include mixed muscle, albumin, and fibrinogen FSR. Secondary parameters include phenylalanine net balance, three-pool model-derived protein synthesis (Fom) and breakdown (Fmo), and two-pool model-derived protein synthesis [rate of disappearance (Rd)] and protein breakdown [rate of appearance (Ra)]. Statistical analysis was carried out using NCSS (2004). All primary and secondary data are presented as the difference from REST (at 10, 60, and 180 min POST-EX). Tertiary parameters and descriptive data are presented as means ± SE.
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RESULTS
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Subjects' characteristics.
Subjects' characteristics are given in Table 1. Height, weight, total leg volume, and total leg lean mass were similar in the younger and older men. However, percent body fat and total leg fat mass were significantly higher in the older men. There was no difference in exercise intensity between the older (43 ± 3% of predicted
O2 peak) and younger men (38 ± 1% of measured
O2 peak). Finally, there was no difference in any of the exercise parameters, which included exercising
O2, treadmill speed, or treadmill grade between the young and older men.
Mixed muscle and plasma protein FSR.
The FSR of mixed muscle protein increased significantly at 10 min POST-EX in the younger [0.0363%/h, 90% confidence interval (CI) = 0.0141 to 0.0584] and older men (0.0830%/h, 90% CI = 0.0394 to 0.1266), with the younger men staying elevated through 60 min POST-EX (0.0253%/h, 90% CI = 0.0031 to 0.0475; Fig. 2). The FSR of albumin and fibrinogen are presented in Figs. 3 and 4, respectively. The FSR of albumin increased significantly at 10 min POST-EX in the younger men only (2.30%/day, 90% CI = 1.131 to 3.478). Conversely, fibrinogen FSR was significantly increased at 10 and 180 min POST-EX in both the younger and older men (10 min: younger men = 4.102%/day, 90% CI = 2.586 to 5.619; older men = 4.178%/day, 90% CI = 2.723 to 5.634; and 180 min: younger men = 4.149%/day, 90% CI = 2.632 to 5.665; older men = 4.107%/day, 90% CI = 2.652 to 5.563). No measurements were made for albumin or fibrinogen FSR at 60 min.

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Fig. 2. Fractional synthesis rate (FSR) of mixed muscle. The difference in FSR of mixed muscle in a group of healthy younger and older men measured from before exercise (REST) to 10 min (empty bars), 60 min (hatched bars), and 180 min postexercise (POST-EX; gray bars). *Significant difference from REST to 10 and 60 min POST-EX in younger men, and 10 min POST-EX in older men. Values are expressed as %/h.
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Fig. 3. FSR of albumin. The difference in FSR of albumin in a group of healthy younger and older men measured from REST to 10 min (empty bars) and 180 min POST-EX (gray bars). *Significant difference from REST to 10 min POST-EX in younger men. Values are expressed as %/day.
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Fig. 4. FSR of fibrinogen. The difference in FSR of fibrinogen in a group of healthy younger and older men measured from REST to 10 min (empty bars) and 180 min POST-EX (gray bars). *Significant difference from REST to 10 min and REST to 180 min POST-EX in younger and older men. Values are expressed as %/day.
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Leg blood flow.
Figure 5 displays the difference in leg blood flow obtained at REST and again at 10, 60, and 180 min POST-EX. Leg blood flow was significantly elevated at 10 min POST-EX in the younger men (17.937 ml·min1·100 ml leg volume1, 90% CI = 13.699 to 22.175). Leg blood flow in the older men was significantly elevated throughout 180 min of the POST-EX period (10 min: 17.203 ml·min1·100 ml leg volume1, 90% CI = 15.231 to 19.174; 60 min: 5.302 ml·min1·100 ml leg volume1, 90% CI = 3.331 to 7.274; 180 min: 2.862 ml·min1·100 ml leg volume1, 90% CI = 0.8904 to 4.833).

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Fig. 5. Leg blood flow. Leg blood flow in a group of healthy younger and older men measured from REST to 10 min (empty bars), 60 min (hatched bars), and 180 min POST-EX (gray bars). *Significant difference from REST to 10 min POST-EX in younger men and from REST to 10, 60, and 180 min POST-EX in the older men. Values are expressed as ml·min1·100 ml leg volume1.
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Leg phenylalanine kinetics.
Phenylalanine Rd, an index of muscle protein synthesis, was significantly increased at 10 and 60 min POST-EX in the older men (10 min: 71.195 nmol·min1·100 ml leg 1, 90% CI = 30.592 to 111.797; 60 min: 50.047 nmol·min1·100 ml leg 1, 90% CI = 9.445 to 90.650) and 10 min POST-EX in the younger men (71.555 nmol·min1·100 ml leg 1, 90% CI = 15.215 to 127.895; Fig. 6). Phenylalanine Ra, an index of protein breakdown, was elevated at 10 min POST-EX in both groups (10 min: younger men: 82.722 nmol·min1·100 ml leg 1, 90% CI = 10.120 to 155.325; older men: 78.089 nmol·min1·100 ml leg 1, 90% CI = 15.883 to 140.296; Fig. 7). Net balance of phenylalanine was negative throughout REST and all POST-EX periods (Fig. 8). Finally, phenylalanine delivery to the leg and release from the leg appeared to be elevated above REST at 10 min POST-EX in both groups (Table 2). The differences from REST in Fom and Fmo derived from the three-pool model are displayed in Figs. 9 and 10, respectively. Both groups of men displayed increases in protein synthesis at 10 min (younger men: 100.381 nmol·min1·100 ml leg 1, 90% CI = 0.6112 to 200.151; older men: 86.085 nmol·min1·100 ml leg 1, 90% CI = 18.900 to 153.270), whereas only the older men displayed a significant increase in protein breakdown at any POST-EX period (10 min: 92.980 nmol·min1·100 ml leg 1, 90% CI = 6.213 to 179.746). Table 3 displays the transport of phenylalanine from the artery into the muscle. Arterial phenylalanine transport appeared to be increased by 10 min POST-EX in both groups, with transport in the older men remaining elevated through 180 min POST-EX. In the older men, transport from the muscle and a-v shunting appeared elevated from REST to 10 min POST-EX. a-v shunting of phenylalanine appeared to be increased by 10 min POST-EX in the younger men, with the younger men shunting approximately twofold more than the older men.

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Fig. 6. Protein synthesis (rate of disappearance, Rd). The difference ( ) in 2-pool model-derived protein synthesis in a group of healthy younger and older men measured from REST to 10 min (empty bars), 60 min (hatched bars), and 180 min POST-EX (gray bars). *Significant difference from REST to 10 min POST-EX in younger men and from REST to 10 and 60 min POST-EX in the older men. Values are expressed as nmol·min1·100 ml leg1.
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Fig. 7. Protein breakdown (rate of appearance, Ra). The difference in 2-pool model-derived protein breakdown in a group of healthy younger and older men measured from REST to 10 min (empty bars), 60 min (hatched bars), and 180 min POST-EX (gray bars). *Significant difference from REST to 10 min POST-EX in both younger and older men. Values are expressed as nmol·min1·100 ml leg1.
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Fig. 8. Net balance of phenylalanine. The difference in the net balance of phenylalanine in a group of healthy younger and older men measured from REST to 10 min (empty bars), 60 min (hatched bars), and 180 min POST-EX (gray bars). Values are expressed as nmol·min1·100 ml leg1.
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Table 2. Phenylalanine kinetics in healthy younger and older men at rest and 10, 60, and 180 min postexercise using the a-v balance (2-pool) model
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Fig. 9. Protein synthesis (Fom). The difference in 3-pool model-derived protein synthesis in a group of healthy younger and older men measured from REST to 10 min (empty bars), 60 min (hatched bars), and 180 min POST-EX (gray bars). *Significant difference from REST to 10 min POST-EX in younger and older men. Values are expressed as nmol·min1·100 ml leg1.
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Fig. 10. Protein breakdown (Fmo). The difference in 3-pool model-derived protein breakdown in a group of healthy younger and older men measured from REST to 10 min (empty bars), 60 min (hatched bars), and 180 min POST-EX (gray bars). *Significant difference from REST to 10 min POST-EX in the older men. Values are expressed as nmol·min1·100 ml leg1.
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Table 3. Phenylalanine kinetics in healthy younger and older men at rest and 10, 60, and 180 min postexercise using a three-pool model
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Plasma albumin and fibrinogen concentrations.
Plasma albumin concentrations in the artery and vein remained unchanged in both younger and older men during the POST-EX period (Table 4). Arterial and venous fibrinogen concentrations remained constant in both groups, except for an apparent decrease in venous concentrations of fibrinogen seen at the 180 min POST-EX time point in the older men (Table 4).
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Table 4. Plasma concentrations of albumin and fibrinogen in femoral artery and vein in healthy younger and older men at rest and 10, 60, and 180 min postexercise
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Phenylalanine concentrations and enrichments.
Table 5 displays the phenylalanine concentrations and enrichments in the femoral artery, vein, and muscle of the younger and older men. Arterial phenylalanine concentrations and enrichments appeared to be increased in the younger men in the immediate POST-EX period (10 min). In contrast, arterial concentrations in the older men remained relatively constant throughout the POST-EX period. Furthermore, phenylalanine enrichments in the vein appeared elevated in the older men from 60 through 180 min POST-EX. Muscle enrichments also appeared elevated in the older men at 10 and 60 min POST-EX, with no apparent increase in the younger men until 180 min POST-EX (Table 5).
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Table 5. Phenylalanine concentrations and enrichments in femoral artery, femoral vein and muscle tissue in healthy younger and older men at rest and 10, 60, and 180 min postexercise
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DISCUSSION
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The acute effects of a single bout of moderate-intensity aerobic exercise on protein metabolism are not well established. Given the long-standing beliefs that regular or acute aerobic exercise lacks the potential to be anabolic, we opted to examine the effect of a single bout of moderate-intensity walking on postexercise protein metabolism in younger and older men. Despite new evidence that regular aerobic exercise training enhances muscle protein synthesis in older and younger individuals, no similar data exist regarding the protein metabolic influence of acute aerobic exercise without the confounding variables often found in long-term outcome studies. Therefore, our study was intentionally designed to mimic a "typical" exercise session performed by many older individuals (i.e., walking on the beach). To date, only two studies have addressed the acute effects of aerobic exercise, neither of which examined older individuals, and each occurred at an intensity [4,600-m swim at 8590% of maximum age-predicted heart rate (52)] and duration [4 h of treadmill walking at 40% of maximal
O2 (12)] far greater than what was used in the present study (45 min of treadmill walking at 40% of
O2 peak). This investigation was the first to 1) examine and compare mixed muscle protein synthesis following a single bout of aerobic exercise in younger and older men and 2) examine and compare albumin and fibrinogen plasma protein synthesis following a single bout of aerobic exercise in younger and older men.
The results of the present study demonstrate that moderate-intensity walking induces short-term increases in postexercise muscle and plasma protein synthesis in both postabsorptive younger and older men, although the increase in muscle protein synthesis is balanced by an increase in breakdown. Furthermore, central to our hypothesis that acute moderate-intensity aerobic exercise would stimulate protein synthesis across the leg was our finding that it also stimulates plasma protein synthesis, specifically of fibrinogen, an essential liver protein capable of rapid turnover and previously found to increase in younger men in response to aerobic exercise (12). More importantly, our data confirm those of a recent study showing that age does not impair the leg blood flow response to moderate-intensity aerobic exercise (44).
Aerobic exercise and muscle protein synthesis in young.
Two studies have provided preliminary evidence of the potential anabolic benefit of intense aerobic exercise in the postabsorptive state. In the study conducted by Tipton et al. (52) in trained female swimmers, FSR of mixed muscle increased
41% above resting levels but did not reach statistical significance. Moreover, no change in whole body phenylalanine Ra, an index of whole body protein breakdown, during the recovery period was noted. Carraro et al. (12) reported a significant increase in mixed muscle FSR in the 4-h period following treadmill exercise in untrained men. However, 3-methylhistidine excretion also increased during the postexercise recovery period, indicating increased muscle protein breakdown. Despite the differences in training status of subjects, and with regard to the mode, intensity, and duration of the exercise in these two studies, it appears that acute aerobic exercise initiates moderate increases in muscle protein synthesis in the absence of nutrition. Conversely, the effects of aerobic exercise on muscle protein breakdown are less clear, given the different findings of these studies. Although these studies in the young provide valuable preliminary evidence supporting the anabolic potential of aerobic exercise, they were designed to examine high-intensity and long-duration exercise. Our FSR data in the younger men after moderate-intensity aerobic exercise showed a statistically significant 45% increase in the incorporation of phenylalanine into bound protein from rest to the initial postexercise period (10 min POST-EX). This is similar to the percent change (41% greater than REST; P > 0.05) seen in FSR during recovery following high-intensity swimming (52). In the present study, model-derived Fom significantly increased (
150%) from rest to 10 min postexercise in both the younger and the older men. Two- and three-pool model-derived protein breakdown data are also consistent with previous research (52), as our younger subjects had returned to resting levels of protein breakdown by
60 min postexercise. Overall, the results indicate that a single bout of aerobic exercise was capable of inducing short-term increases in muscle protein synthesis and breakdown, indicating that muscle protein turnover is acutely elevated. The mechanism for the increased turnover is multifactorial, with cellular repair and maintenance likely one component of the response (11). However, given the recent finding that repeated bouts of moderate-intensity aerobic exercise increase muscle protein synthesis in both young and elderly (51), this study should be repeated with sufficient amino acid supplementation to determine whether there is an interactive effect of exercise and amino acids in the postprandial period.
Aerobic exercise and muscle protein synthesis in elderly.
This is the first study in which the acute postexercise effects of a single bout of aerobic exercise on muscle protein synthesis have been quantified in older men. It is widely accepted that aging affects the metabolic capacities of skeletal muscle. In particular, unfavorable changes are known to occur in levels of enzymatic activities and protein turnover [i.e., synthesis, breakdown, and repair capacities (11)]. Whether these alterations affect the metabolic capacity of the aged to respond to acute aerobic exercise remains an important issue.
Our results indicate that the muscle of older men undergoes significant increases in muscle protein turnover in response to aerobic exercise to a similar extent to that of younger men. For example, protein synthesis in the older men increased 112% (FSR), 158% (Rd), and 110% (3-pool model) from rest to 10 min postexercise, respectively. These data are similar to the protein synthetic response seen in the younger men in the present study (45%, FSR; 153%, Rd; and 106%, 3-pool model). When postexercise variables such as protein synthesis, protein breakdown, and leg blood flow between younger and older men are compared, it becomes apparent that the older men tend to have a more prolonged response to the exercise. One the one hand, it could be argued that prolonged elevation of muscle protein breakdown following exercise may be attributed to some underlying "stress" that slows the reversal of catabolism in older muscle (i.e., stress/damage response genes), thereby diminishing the overall positive effect of exercise-induced muscle protein synthesis in older individuals. This may be due to a differential expression of genes typical of stress or damage (34) and proteins (i.e., mitogen-activated proteins) associated with the cellular signaling cascade linking exercise or contractile activity in humans to biochemical responses and ultimately gene expression in contracting skeletal muscle (1, 5759). Recent results from a study comparing skeletal muscle gene expression in young and elderly confirm that the elderly have elevated expression of genes typical of a stress or damage response at rest, with a reduced activation of the stress/damage response genes after a single bout of resistance exercise (34). However, it is important to note that the net balance of phenylalanine remains relatively stable throughout the postexercise period in both groups of men; yet muscle protein turnover in the older men appears to remain active longer postexercise than in the younger men. Alternatively, one could argue that, if protein turnover is important for altering protein expression or protein quality, the prolonged turnover demonstrated by the older men could perhaps afford them a greater short-term protein metabolic benefit from the exercise bout. Finally, although the resting stress status of elderly muscle is likely a contributing factor to the sarcopenic process that accompanies aging, our data clearly indicate that older muscle is capable of initiating short-term increases in muscle protein synthesis in response to a single bout of moderate-intensity aerobic exercise despite being fasted.
Aerobic exercise and plasma protein synthesis.
Several studies have examined the response of plasma proteins in young following prolonged (12) and high intensity aerobic exercise (12, 31, 40, 61). Carraro et al. (12) reported an increase in fibrinogen synthesis following a prolonged moderate-intensity exercise bout. A recent study examining the effect of high altitude and exercise on rates of synthesis of albumin and fibrinogen found both to be elevated, with exercise being the principle stimulus (31). In our study, the exercise resulted in an increased synthesis rate for fibrinogen in younger and older men, but albumin synthesis increased only modestly during exercise in younger men. These results are similar to the results in the study of prolonged moderate-intensity aerobic exercise (12). It should be noted, however, that, although fibrinogen synthesis rate increased in the present study, fibrinogen concentration did not change or was slightly lower than starting values. This implies that breakdown or clearance was also increased to a similar or possibly greater amount as synthesis. Clearly albumin synthesis is more strongly coupled to high-intensity exercise when plasma volume and total albumin content increase (25, 36), which did not occur in this study. On the other hand, the increase in fibrinogen synthesis during recovery from exercise may be a compensatory response to the exercise (i.e., stress, contraction) initiated efflux of N2 from the muscle resulting from accelerated protein breakdown, especially considering that fibrinogen synthesis in the present study and in Carraro et al. persists for a minimum of 3 h postexercise.
Aging and blood flow.
It has been proposed that age-associated declines in aerobic capacity are a function not only of changes in central cardiovascular control mechanisms but also of reductions in skeletal muscle blood flow capacity (39). Previous work in animal models has shown similar exercise-induced increases in muscle blood flow when younger and senescent animals were compared (18, 26, 27, 32). Studies in aging humans, however, have been equivocal. There is evidence showing that basal (1921, 37) and exercise (33, 55) whole limb blood flows are lower in older individuals than in young healthy individuals. In contrast, we have not previously detected differences in leg blood flow between healthy young and elderly at rest with the dye dilution technique (54), nor did we find differences at rest in the present study. Furthermore, the older men demonstrated the same increase in leg blood flow in response to the exercise bout in the present study, and, unlike in the younger men, blood flow remained elevated for a large part of the postexercise period in the older men. We believe that the different findings are primarily a function of methodological differences [dye dilution (54) vs. plethysmography (37) vs. Doppler (20)], with each technique measuring different components of leg blood flow. We suggest that a novel measurement of muscle tissue perfusion [i.e., nutritive vs. nonnutritive blood flow (14, 16)] using contrast-enhanced ultrasound may be a more appropriate measure of skeletal muscle vascular dynamics and metabolism. Nevertheless, regardless of methodological differences, our data clearly show that healthy older men are capable of inducing "functional" hyperemia, as indicated by the stimulus of muscle protein synthesis in response to moderate-intensity aerobic exercise.
In conclusion, this investigation provides clear evidence that a single bout of moderate-intensity aerobic exercise is sufficient to produce significant increases in muscle protein turnover in younger and older men. Both younger and older men demonstrated short-term increases in mixed muscle and plasma protein synthesis following aerobic exercise despite the normal elevation in protein breakdown associated with the postabsorptive condition. It is possible that increases in muscle protein turnover are the metabolic basis for cellular repair and maintenance in aging skeletal muscle.
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GRANTS
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This project was supported by the National Institutes of Health/National Institute on Aging Claude D. Pepper Older Americans Independence Center Grants P60-AG-17231-01 (PIs J. S. Goodwin and R. R. Wolfe) and R01-AG-21539 (to M. Sheffield-Moore). Studies were conducted at the General Clinical Research Center of UTMB at Galveston and funded by Grant M01-RR-00073 from the National Center for Research Resources, National Institutes of Health, United States Public Health Service.
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ACKNOWLEDGMENTS
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We thank the volunteers for their time and effort, Dan Creson, Dessa Gemar, Stephanie Blase, Melissa Bailey, Chris Danesi, Guarang Jariwala, and Aaron Matlock for technical assistance, and Judah I. Rosenblatt for considerable statistical expertise. We further thank James S. Goodwin MD and the Pepper Center Volunteer Registry at The University of Texas Medical Branch (UTMB) for assistance in recruiting.
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FOOTNOTES
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Address for reprint requests and other correspondence: M. Sheffield-Moore, Univ. of Texas Medical Branch, Dept. of Surgery/Metabolism Unit, 815 Market St., Galveston, TX 77550 (E-mail: melmoore{at}utmb.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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