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* Department of Surgery and ** Department of Medicine, Stony Brook University, Stony Brook, NY 11794; Department of Animal Sciences, University of Illinois, Urbana, IL 61801; and Department of Veterans Affairs Medical Center, Northport, NY 11768
2 To whom correspondence should be addressed. E-mail: giuseppe.caso{at}stonybrook.edu.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • feeding • L-[2H5]phenylalanine • translation initiation • p70S6K • 4E-BP1
Food intake is associated with an anabolic response of body protein metabolism to promote an accumulation of amino acids and minimize oxidative losses. Direct measurements of protein synthesis from incorporation of labeled amino acids into individual tissues in animals showed that feeding stimulates protein synthesis in muscle, although the magnitude of the effect may depend on age, developmental stage, and type of muscle (1–3). In young growing animals, muscle protein synthesis is particularly sensitive to feeding. Withdrawal of food is followed by a rapid inhibition of muscle protein synthesis, and refeeding induces an immediate stimulation (1,3,4). However, the stimulatory response of muscle protein synthesis to feeding is blunted in adult animals (2).
A stimulation of muscle protein synthesis was also observed in adult human subjects in response to food intake. Studies in human volunteers using a constant infusion of labeled L-[1-13C]leucine and measuring the direct incorporation of the labeled amino acid into muscle protein showed a 60–100% stimulation of muscle protein synthesis in response to feeding (5,6). A 50% stimulation was also observed in the synthesis rates of muscle myofibrillar proteins after feeding using a similar methodology (7). When the response of muscle protein synthesis to food intake was assessed with the flooding technique, a much smaller, nonsignificant stimulation of 
30% was reported (8). The reason for the discrepant results obtained with the 2 methods is not obvious, although the difference in methodology is a possibility.
The stimulatory effect of feeding on muscle protein synthesis in growing animals appears to be mediated in part by an upregulation of translation. Thus, feeding was shown to stimulate the phosphorylation of several proteins that regulate protein synthesis initiation, including the Mr = 70,000 ribosomal protein S6 kinase (p70S6K or S6K1)3 and the translation repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (9–11). Phosphorylation activates p70S6K, which then phosphorylates ribosomal protein S6. This event is thought to result in enhanced translation of mRNAs that encode ribosomal proteins and other components of the translational machinery (12,13). Phosphorylation of 4E-BP1 reduces its binding affinity for eukaryotic initiation factor 4E (eIF4E), thus increasing the availability of eIF4E for formation of the initiation complex that is necessary to start translation (12,14). Phosphorylation of both p70S6K and 4E-BP1 therefore plays an important role in the signaling pathway leading to the stimulation of muscle protein synthesis by feeding.
The main aim of this study was to determine whether the response of muscle protein synthesis to feeding is dependent on the technique used for measurement. To test differences in methodology only, we controlled the duration of fasting, nutrient composition, and size and timing of the meals. Muscle protein synthesis was measured in the postabsorptive state and after feeding in 2 groups of healthy volunteers, using 2 distinct protocols. In 1 protocol, muscle protein synthesis rates were assessed with the constant infusion technique; the other used the flooding method. Both measurements were carried out using the same stable isotope-labeled amino acid, L-[2H5]phenylalanine.
Measurement of muscle protein synthesis was also coupled with an assessment of the phosphorylation state of p70S6K and 4E-BP1. These measurements were carried out both to verify an increase in muscle protein synthesis using methodology that does not depend on isotopic tracers and to gain some insight into the molecular mechanisms by which feeding affects protein synthesis in human muscle.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Healthy nonsmoking volunteers (n = 16; 8 men, 8 women) took part in the study. After giving written informed consent, the subjects were admitted to the General Clinical Research Center (GCRC) at Stony Brook University Hospital. They were in good health as determined from a complete medical history, physical examination, and routine blood testing for hematologic, hepatic, renal, and general chemistry parameters.
Enrolled subjects were admitted to the GCRC for 2 separate overnight visits 1–3 wk apart, avoiding any strenuous exercise for at least 72 h before each visit. During each visit, the fractional synthesis rate (FSR) of muscle protein was measured in either the fasting (postabsorptive) or feeding state, in random order. Feeding involved the consumption of 12 small hourly meals, consisting of a drink of instant breakfast formula (Carnation Instant Breakfast, Nestle) with milk and cream providing 15% of energy as protein, 48% as carbohydrates, and 37% as fat. Each hourly meal provided one-twelfth of the individual's daily energy requirements. Daily energy requirements were estimated as 1.15 x basal metabolic rate, as predicted from equations using sex, age, weight, and height (15). The factor 1.15 was based on previous measurements of the increase in energy expenditure with feeding during bed rest (16).
The study included 2 experimental protocols (Experiments 1 and 2) (Figs. 1 and 2), with subjects matched for age, gender, and BMI, as illustrated in Table 1. The design of the 2 experiments was similar. The only difference between the 2 protocols was the technique used for the measurement of protein synthesis: in Experiment 1, muscle FSRs were measured with constant infusion of L-[2H5]phenylalanine, whereas in Experiment 2, the flooding method with the same labeled amino acid was employed. All of the study protocols were approved by the Committee on Research Involving Human Subjects at Stony Brook University.
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100 mg) was taken with a sterile procedure from the lateral portion of the quadriceps muscle (vastus lateralis) with a 6-mm Bergstrom biopsy needle. A second muscle biopsy was taken at 1045 (t = 9 h) to measure the incorporation of labeled phenylalanine into muscle protein and to assess translation initiation signaling (Fig. 1A). For the feeding protocol, small hourly meals were given over 12 h beginning at 0700 (Fig. 2A). Infusion of L-[2H5]phenylalanine was started at 1000 (t = 0 h), followed by a baseline muscle biopsy at 1300 (t = 3 h) and a second biopsy at 1900 (t = 9 h) (Fig. 2A). For measurements during both fasting and feeding, blood samples to monitor the enrichment of plasma free phenylalanine were drawn every hour from a venous catheter placed in a forearm vein on the arm opposite that of the infusion site. All muscle biopsies were immediately blotted to remove blood, frozen in liquid nitrogen, and stored at –70°C.
Experiment 2: flooding method. The design of this experiment was comparable to that of Experiment 1, except that muscle FSRs were measured using the flooding technique (18–20). For the fasting state, a solution containing L-[2H5]-phenylalanine and unlabeled phenylalanine (Ajinomoto; 43 mg/kg body weight) was injected at a constant rate over 10 min beginning at 0700. Blood samples for the measurement of isotopic enrichment of plasma free phenylalanine were then taken at intervals for 90 min. At 90 min, a muscle biopsy was taken from the lateral portion of the quadriceps muscle (vastus lateralis) with a trucut biopsy needle (Temno, Bauer Medicals), after local anesthesia of the biopsy site (Fig. 1B).
The feeding protocol involved an overnight fast, followed by hourly meals starting at 0700 for the duration of the protocol. At 1515, labeled phenylalanine (43 mg/kg body weight) was injected over 10 min, followed by sequential blood sampling and a muscle biopsy 90 min after injection of labeled phenylalanine (Fig. 2B).
As in Experiment 1, the sequential order of the feeding and fasting protocols was random. However, on study d 2, a baseline muscle biopsy was taken to assess the background enrichment of muscle protein from the previous measurement. In addition, the enrichment of the injected solution was increased from 10 to 20 mol% (MP).
Both the feeding and fasting protocols were designed to be as comparable as possible to those described in Experiment 1. Because the measurements with the flooding method take only 90 min compared with 360 min for the constant infusion method, the injection of the labeled amino acid was timed so that the median time of incorporation was the same in the 2 experiments (Figs. 1 and 2).
Analytical methods
Enrichment of phenylalanine in muscle protein. Frozen muscle tissue was powdered in liquid nitrogen, and protein was precipitated with cold perchloric acid (30 g/L). The resulting supernatant was used for measurement of enrichment of free phenylalanine in tissue fluid; the protein pellet was washed several times with perchloric acid, solubilized in 0.3 mol/L NaOH for 1 h at 37°C, reprecipitated and washed again with perchloric acid before hydrolysis with 6 mol/L HCl for 24 h at 110°C (20). Phenylalanine enrichment in the muscle hydrolysate was measured by MS using a MD800 GC-MS (Fisons Instruments) operated under electron impact conditions, as previously described (19,21,22).
Enrichment of phenylalanine in plasma and tissue fluid. Free amino acids from plasma and tissue fluid were purified by cation-exchange chromatography. L-[2H5]phenylalanine enrichment was then measured by monitoring the ions with mass:charge ratio (m/z) 336 and 341 of the tertiary butyldimethylsilyl derivative on a GC-MS (MD800; Fisons Instruments) (19,21,22).
Assessment of the phosphorylation status of p70S6K and 4E-BP1.
The phosphorylation state of p70S6K and 4E-BP1 was determined on portions of muscle biopsies taken at t = 9 h in Experiment 1 (23–26). Tissue was homogenized in cold buffer containing 50 mmol/L HEPES, 50 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, 0.2 mmol/L phenylmethylsulfonyl fluoride, 0.05 mmol/L microcystin LR, 1 mg/L pepstatin A, 2 mg/L leupeptin, and 1.7 mg/L aprotinin (pH 7.5). The homogenate was centrifuged at 10,000 x g for 15 min at 4°C. The resulting supernatant was assayed for protein using a Bradford assay (Bio-Rad Laboratories). Equal amounts of supernatant protein were either combined directly with SDS sample buffer (for p70S6K) or first boiled for 10 min and centrifuged at 10,000 x g for 15 min before combining the supernatant with SDS sample buffer (for 4E-BP1). Samples were then subjected to immunoblot analysis. Signals were visualized using primary rabbit polyclonal antibodies to p70S6K and 4E-BP1 (Bethyl Laboratories), followed by IRDye800-conjugated secondary antibody (Rockland Immunochemicals). Signals were quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences). Changes in phosphorylation of p70S6K and 4E-BP1 were assessed from a change in electrophoretic mobility (24–26). Phosphorylation is expressed as the ratio of the density of the more phosphorylated, slower migrating species (i.e., ß and 
bands) to that of the total protein (i.e., 
, ß, and bands) (24–26). Phosphorylation of p70S6K was also assessed using a primary antibody that recognizes p70S6K only when phosphorylated on Thr389 (Cell Signaling Technology) and an Alexa Fluor 680-linked secondary antibody (Molecular Probes).
Amino acid and insulin analysis. Plasma amino acid and insulin levels were assessed on blood samples collected during the feeding and fasting protocols 5.25 h after the beginning of the tracer infusion in Experiment 1 and at a corresponding time, before the injection of labeled phenylalanine, in Experiment 2, as previously described (21,22).
Calculations
Protein FSRs were calculated from the incorporation of labeled phenylalanine into muscle protein and from the enrichment of the precursor pool for protein synthesis using the formula (21,22):
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where Ep0 and Ep1 represent the enrichment of the protein-bound phenylalanine at the start and end of the incorporation periods, respectively, A indicates the area under the curve of precursor enrichment and time, and t is the time of incorporation expressed in days. In Experiment 1 (constant infusion), incorporation was determined between 3 and 9 h, and the mean enrichment of free phenylalanine in tissue fluid (3 and 6 h) or in plasma was used to estimate the enrichment of the precursor pool for protein synthesis. In Experiment 2 (flooding method), the incorporation time was 90 min, and plasma free phenylalanine enrichment measured throughout the 90-min period was used to estimate the enrichment of phenylalanine used for protein synthesis.
Statistics
All data are expressed as means ± SE. Comparisons within each experiment between measurements in the fasting and feeding states or between tissue fluid and plasma were analyzed with a t test for paired data. Comparisons of results between experiments were performed by using a t test for unpaired data. A P-value 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasma amino acid concentrations were elevated during the feeding compared with the fasting protocols (Table 2). The proportional increases with feeding did not differ between Experiments 1 and 2 for nonessential (14 ± 3 vs. 27 ± 12%), essential (16 ± 6 vs. 30 ± 11%), and branched-chain amino acids (20 ± 7 vs. 30 ± 10%) (Table 2). The apparently higher increases in Experiment 2 (P = NS) resulted from one subject showing a much greater elevation of plasma amino acid concentration during feeding.
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Experiment 1 (constant infusion). The enrichment of plasma phenylalanine rose to a plateau by the time of the first muscle biopsy and remained stable between 3 and 9 h, with no detectable differences between the fasted and feeding protocols (Fig. 3). However, enrichment of free phenylalanine in plasma was consistently higher than in the tissue fluid. The enrichment of phenylalanine in plasma was 44 ± 8% higher than in tissue fluid during fasting (6.06 ± 0.24 vs. 4.23 ± 0.30 MP; P < 0.001) and 39 ± 8% higher during feeding (5.68 ± 0.50 vs. 4.05 ± 0.19 MP; P < 0.005). As a consequence, the FSR calculated using plasma as the precursor pool was significantly lower than that calculated using the tissue fluid (P < 0.002) (Table 3).
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Experiment 2 (flooding method).
The enrichment of plasma free phenylalanine increased rapidly after injection of the isotope solution, reaching a value 85% of that of the injected solution at 10 min and declining thereafter (data not shown). The curves of the enrichment of plasma phenylalanine vs. time were similar during the fasting and feeding protocols. As in Experiment 1, the rates of muscle protein synthesis during feeding were significantly higher than during fasting (Table 3). Feeding resulted in a stimulation of muscle FSR by 22 ± 5% relative to the fasting FSR (P < 0.01).
FSRs measured with the flooding technique during both fasting and feeding were not different from those obtained with the constant infusion technique (Experiment 1) when the enrichment of free phenylalanine in the tissue fluid was used as the estimate of precursor enrichment (Table 3).
Effect of feeding on p70S6K and 4E-BP1 phosphorylation
Immunoblot analysis of muscle samples from Experiment 1 showed a decrease in the electrophoretic mobility of p70S6K, indicating that feeding stimulated the phosphorylation of p70S6K. The ratio of the intensity of the slower species to that of total protein (ß+/+ß+ bands) was increased (P < 0.02) in the feeding state (Fig. 4A). Enhanced phosphorylation of p70S6K in the feeding state was also confirmed by the direct observation of higher amounts of p70S6K phosphorylated on Thr389 (P < 0.03; Fig. 4B). In contrast to the changes in p70S6K, the electrophoretic mobility and phosphorylation state of 4E-BP1 did not differ between the fasting and feeding states (data not shown).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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15–22% after oral feeding compared with the postabsorptive state. Not only was the degree of stimulation similar, but the rates of synthesis of muscle protein in the postabsorptive state were also comparable with the 2 techniques. In the past, there has been some controversy about the optimal technique to use for measuring protein synthesis rates in human muscle (27,28) because values obtained with the flooding method were found to be higher than those measured with the constant infusion technique [e.g., (6,8)]. Two possible explanations were given for the apparent discrepancies: either basal protein synthesis is stimulated with the flooding technique, or an error is introduced with the continuous infusion technique when surrogate measures (e.g., plasma amino acid or ketoacid) are used in place of aminoacyl-tRNA as the precursor for protein synthesis (27,28).
Measurements of FSR employing the enrichment of aminoacyl-tRNA, the direct precursor for amino acids incorporated into protein, show that the constant infusion and the flooding techniques yield comparable values in both animals (21,22) and human muscle (see Table 4). However, the direct assessment of enrichment of aminoacyl-tRNA is not always practical for routine human studies because relatively large (0.5-g) muscle samples are required. Calculation of muscle FSR therefore relies on estimates of the enrichment of the precursor amino acid from more accessible pools, i.e., amino acid or corresponding ketoacid in plasma or tissue fluid. Several studies (21,29,30) examined the enrichment of tracer amino acids in aminoacyl-tRNA and other surrogate precursor pools during an infusion of a tracer amino acid. The results indicated that the free amino acid in the tissue fluid most closely reflects the enrichment of aminoacyl-tRNA in muscle (21,22,29,30). Enrichments of amino acid or ketoacid in plasma are consistently higher than aminoacyl-tRNA and their use as surrogate pools leads to an underestimation of calculated FSR using the constant infusion technique [i.e., (21,22,29,30)]. When the enrichment of amino acid in the tissue fluid is used as a surrogate for the enrichment of the precursor for protein synthesis with the constant infusion technique, the estimates of muscle protein synthesis are much closer to those determined with the flooding technique (Table 4). These observations are confirmed in the present study in human muscle (Table 3). The results of the present study are also in line with our previous studies in different animal species demonstrating that constant infusion produces rates of protein synthesis in muscle comparable to those obtained with the flooding method when an appropriate precursor pool is chosen (22,31).
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The uncertainties about the choice of a suitable precursor pool for protein synthesis are minimized with the flooding technique. Because the labeled tracer is administered together with a large amount of unlabeled amino acid, all of the amino acid pools reach comparable enrichment (18,22,32), and the enrichment of the precursor pool can be determined accurately from sampling the more easily accessible amino acid plasma pool. Determining the enrichment of the precursor pool from plasma results in a practical advantage in that fewer and smaller biopsies (10 mg) are required to make measurements of protein synthesis. The smaller biopsies in Experiment 2 did not increase the variability of the measurements, as suggested by comparing the within-group CV of the rates of protein synthesis in the 2 experiments (see Table 3). Further, comparison of the protein enrichment in several (n = 9) separate small samples (10 mg) taken from the same muscle area in one of the subjects at the end of Experiment 1, also confirmed that variability due to muscle sampling is minimal (CV = 0.45%). Therefore, in human vastus lateralis muscle, smaller biopsies are as representative of the whole muscle as are larger ones.
Other practical advantages of the flooding technique make it very suitable for clinical studies. These include a shorter duration required for taking measurements (90 min) and the possibility of investigating acute changes in the rates of protein synthesis, particularly in clinical studies, in which a metabolic steady state cannot always be maintained. The concern about using the flooding method is that the large amount of amino acid administered may itself alter the rate of protein synthesis (28). However, this possibility is not supported by the results of the current study nor by our previous findings in animals (22,31).
The effect of feeding observed in Experiments 1 and 2 (Table 3) is smaller than that reported previously by other authors (5–7). The present study confirms that this does not arise from inherent methodological problems because the constant infusion and flooding techniques give comparable FRS values. The smaller stimulation of muscle FSR in the present study compared with others (5–7) may be related in part to differences in the experimental protocols among studies, in particular the feeding regimen and the size and composition of meals provided. Because administration of amino acids to elevate plasma concentrations was shown to stimulate muscle protein synthesis (33,34), a greater stimulation is to be expected when feeding regimens result in higher plasma amino acid concentrations than those achieved in the present study (i.e., after the ingestion of a single larger meal). However, the feeding regimen of small hourly meals was used in this study to maintain a metabolic steady state, required by the constant infusion technique (35).
The stimulation of FSR by feeding was associated with increased phosphorylation of p70S6K, suggesting that the effect may be mediated by an upregulation of translation initiation. However, in contrast to the results obtained in animal studies [e.g., (9,36,37)], no effect could be detected on the phosphorylation of 4E-BP1. In animal studies, the effect of feeding is generally investigated after a brief period of refeeding after food deprivation and is therefore acute, resulting in a larger postprandial hormonal response and higher plasma amino acid concentrations than observed in the present study. It is therefore possible that the increase in amino acid and insulin concentrations in the current study were sufficient to stimulate the phosphorylation of p70S6K but not of 4E-BP1. Alternatively, 4E-BP1 phosphorylation may have been stimulated at earlier time points after starting the feeding regimen, subsequently becoming undetectable after 12 h, when the analyses were done.
In summary, the present study demonstrates that FSRs in human muscle obtained with the constant infusion method are comparable to those obtained with the flooding technique when the enrichment of amino acid within the tissue fluid is taken to represent the precursor enrichment. Moreover, with comparable feeding regimens, these 2 techniques also give comparable estimates of the stimulation of FSR by feeding. Therefore, the investigator can choose to use whichever method is technically easier or more suitable for the experimental conditions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; eIF4E, eukaryotic initiation factor 4E; FSR, fractional synthesis rate; MP, mol%; P70S6K, S6 kinase.
Manuscript received 18 January 2006. Initial review completed 21 February 2006. Revision accepted 27 March 2006.
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