Effects of flooding amino acids on incorporation of labeled amino acids into human muscle protein

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1 Effects of flooding amino acids on incorporation of labeled amino acids into human muscle protein KENNETH SMITH, NIGEL REYNOLDS, SHAUN DOWNIE, AYYUB PATEL, AND MICHAEL J. RENNIE Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, Scotland, United Kingdom Smith, Kenneth, Nigel Reynolds, Shaun Downie, Ayyub Patel, and Michael J. Rennie. Effects of flooding amino acids on incorporation of labeled amino acids into human muscle protein. Am. J. Physiol. 275 (Endocrinol. Metab. 38): E73 E78, We investigated the effects of the nature of the flooding amino acid on the rate of incorporation of tracer leucine into human skeletal muscle sampled by biopsy. Twenty-three healthy young men ( yr, kg) were studied in groups of four or five. First, the effects of flooding with phenylalanine, threonine, or arginine (all at 0.05 g/kg body wt) on the incorporation of tracer [ 13 C]leucine were studied. Then the effects of flooding with labeled [ 13 C]glycine [0.1 g/kg body wt, 20 atoms percent excess ()] and [ 13 C]serine (0.05 g/kg body wt, 15 ) on the incorporation of simultaneously infused [ 13 C]leucine were investigated. When a large dose of phenylalanine or threonine was administered, incorporation of the tracer leucine was significantly increased (from to %/h and to %/h, respectively; each P 0.01). However, when arginine, glycine, or serine was administered as a flooding dose, no stimulation of tracer leucine incorporation could be observed. These results, together with those previously obtained, suggest that large doses of individual essential, but not nonessential, amino acids are able to stimulate incorporation of constantly infused tracer amino acids into human muscle protein. constant infusion; flooding dose; muscle protein synthesis; essential amino acids; nonessential amino acids THE MEASUREMENT of human tissue protein synthesis is a subject of considerable interest to those concerned with understanding the normal hormonal and nutritional control of the lean body mass and its derangement by disease and illness (16). We have previously demonstrated that when a large dose of leucine or valine was administered during the course of a constant tracer infusion of stable isotope-labeled amino acids, there was an apparent stimulation of the incorporation of the tracer amino acid (20, 21). We have also provided evidence that when the amino acid administered as a large dose is itself labeled, the concurrent synthesis rate (calculated from its rate of incorporation) is identical to the apparently stimulated synthesis rate calculated from incorporation of the constantly infused tracer. This result is interesting for a variety of reasons: it may explain why the flooding dose protocol consistently produces values for muscle protein synthetic rates that are higher than those obtained by the constant infusion protocol (6, 13, 20); it may also explain why it is more difficult to observe the effects of an increase in the protein synthetic rate in going from the fasted to the fed state (7, 14), when the expected anabolic effect of an increased availability of additional amino acids may be masked by the flooding dose effect. Last, it may point to the existence of a physiological mechanism whereby individual amino acids may stimulate muscle protein synthesis. To gain more insight into the phenomenon, we have designed a series of experiments to detect any possible stimulatory effects of flooding with a variety of amino acids on the incorporation of [ 13 C]leucine delivered at tracer doses. In particular, we attempted to distinguish between the effects of different essential amino acids (phenylalanine and threonine) and nonessential amino acids (arginine, glycine, and serine) delivered as flooding doses on the incorporation of tracer [ 13 C]leucine into skeletal muscle mixed protein. Naturally, we have considered the results in light of our previous results, in which essential amino acids (leucine and valine) were superimposed as floods on the tracer infusion of valine, leucine, and phenylalanine (20, 21). MATERIALS AND METHODS L-[1-13 C]leucine, [1-13 C]glycine, and L-[1-13 C]serine, 99 atoms percent, were obtained from Mass Trace, Woburn, MA. Subjects and experimental design. Twenty-three healthy male volunteers [ (SD) yr, kg] were given a primed (1 mg/kg body wt), constant infusion (1 mg kg 1 h 1 ) of [1-13 C]leucine over 7.5 h. After6hofinfusion, subjects were given a flooding dose of unlabeled amino acid, either phenylalanine, threonine, or arginine (0.05 g/kg, n 5 in each group), or [1-13 C]glycine [0.1 g/kg, 20 atoms percent (AP)] or L-[1-13 C]serine (0.05 g/kg, 15 AP, both n 4) into an antecubital vein. All subjects were in a normal nutritional state, were weight stable, and were not taking any medication. All were studied in a resting state after an overnight fast between 0800 and In all protocols blood samples were taken from a deep forearm vein at 0, 45, and 120 min and then approximately hourly before administration of the flood and 5, 10, 20, 30, 45, 60, and 90 min postflood. Plasma was separated from the blood and used for the determination of the concentration and enrichment of amino acids and the keto acid of leucine, -ketoisocaproate ( -KIC), by standard methods using gas chromatography-mass spectrometry (GC-MS) (19). Arginine concentration was measured by reverse-phase HPLC after precolumn derivatization with FMOC-Cl (GBC Scientific Equipment, Danderong, Victoria Australia). Muscle biopsies from the anterior tibialis muscle ( mg wet wt) were obtained under local anesthesia after 45 min, at 6 h before administration of the flood, and 90 min postflood; were frozen immediately in liquid nitrogen; and were stored at 80 C before analysis. Free amino acids were extracted from 100-mg muscle ground in liquid nitrogen into 0.2 M perchloric acid for the measurement of amino acid concentration and analysis of /98 $5.00 Copyright 1998 the American Physiological Society E73

2 E74 FLOODING AMINO ACIDS AND PROTEIN SYNTHESIS 13 C labeling as tert-butyldimethylsilyl (t-bdms) derivatives by GC-MS, as previously described (2). The remaining tissue was washed, and alkali-soluble protein was determined using the bicinchoninic acid method after solubilization of the protein pellet in 0.3 M NaOH (24). To measure the labeling of muscle protein-bound leucine, glycine, and serine, we used our routine methods involving acid hydrolysis of protein in 6 M HCl at 110 C for 15 h. The HCl was evaporated under nitrogen, the amino acids were purified by ion-exchange chromatography (Dowex, H, Aldrich, UK), and the t-bdms derivatives of the amino acids were separated by preparative gas chromatography and collected in U traps cooled in liquid nitrogen. The 13 C labeling was determined by isotope ratio mass spectrometry of the carboxyl-co 2 released from reaction of the isolated amino acid with ninhydrin (23). Plasma insulin was determined by radioimmunoassay with an antibody-coated tube method (Coat-a-count Insulin, Diagnostic Products, Los Angeles, CA). Calculations. The rates of muscle protein synthesis were calculated using standard equations appropriate to the constant infusion and flooding dose methods (6, 18). For the constant infusion, protein synthesis (k s, %/h) E m /E p 1/t 100, where E m is the change in enrichment in muscle, E p is the average enrichment of the precursor, and t is the time between biopsies. For the flooding dose, k s (%/h) E m 100/A 60, where A is the area under the curve for precursor enrichment. The changes in the measured enrichment of leucine in muscle protein between successive biopsies, i.e., 45 min to 6 h, and 6 to 7.5 h, were used to calculate the rates of muscle protein synthesis. When making the calculations according to the constant infusion method, we used the mean enrichment values over the periods between the biopsies of deep venous plasma -KIC as a surrogate for the labeling in the immediate precursor for protein synthesis, i.e., leucyl-trna (27). In the flooding dose protocol, the area under the curve of plasma enrichment during the flooding period was used to calculate the average enrichment of the precursor for protein synthesis (6). Values for muscle free amino acid enrichments were available only at the times of the biopsies, so when we calculated protein synthesis on the basis of the free tissue labeling of tracer amino acids, we assumed that the relationship of the labeling of the free muscle amino acids to the labeling of the venous plasma amino acids between the biopsies was the same as that observed at the time of the biopsies. Statistical analysis was Fig. 1. Labeling of plasma leucine and ketoisocaproate (KIC) before and during a flooding dose of arginine. Values are means SD (n 5). performed using Student s paired t-test, with significance being assigned at the 5% level. RESULTS Concentrations and labeling rates of tracer and flooding amino acids and ketoacids in the plasma free pool. As previously observed (18), administration of a primed, constant infusion of leucine resulted in the attainment of a steady state of [ 13 C]leucine and -[ 13 C]KIC labeling (Table 1, Fig. 1) and of leucine and -KIC concentration in plasma (results not shown). On the application of every flooding dose, no matter of which amino acid, leucine and -KIC enrichments and concentrations remained steady; thus the ratio of labeling of plasma -KIC to that of plasma leucine remained constant throughout the study (Table 2). The concentration of all flooding amino acids rose rapidly to a peak in excess of 1 mm at 5 min (glycine concentration exceeded 3 mm) before falling exponentially over the succeeding 90 min to values of about two to three times basal (preflood, P 0.001; Fig. 2). Both glycine and serine enrichments peaked at 5 min and then fell over the following 90 min (Fig. 3). Table 1. Protein synthetic rates before and after flooding calculated from plasma leucine, -KIC, glycine, serine, and muscle amino acid enrichment Preflood Postflood Postflood Flooding Amino Acid Phe Thr Arg [ 13 C]Ser [ 13 C]Gly KIC, Muscle Protein synthesis, %/h KIC, Muscle Protein Synthesis, %/h * * Precursor (AUC), min , Muscle labeling, Protein synthesis, %/h * Values are means SD throughout pre- and postflood periods. AUC, area under the curve of enrichment for serine and glycine floods;, atoms percent excess; KIC, ketoisocaproate. Protein synthesis rate calculations are based on actual times between biopsies for each individual; these range from5h5minto5h20min. *Significantly different from preflood value (P 0.05).

3 FLOODING AMINO ACIDS AND PROTEIN SYNTHESIS E75 Table 2. Relationship of labeling of plasma KIC to leucine before and as a result of flooding KIC:Leu Flooding Amino Acid Preflood Postflood Phenylalanine Threonine Arginine [ 13 C]glycine [ 13 C]serine Values are means SD. A constant infusion of [ 13 C]leucine was given throughout (see MATERIALS AND METHODS). Concentrations and rates of labeling of tracer and flooding amino acids in the intramuscular free pool. The sizes of intramuscular free amino acid pools of those amino acids measured were unaffected to any significant extent by any of the flooding doses (Table 3). The concentration of all flooding amino acids was twoto threefold higher in muscle at the end of the flooding period (P 0.05) with the exception of glycine, which did not change significantly. Free arginine concentration in muscle was not determined. The ratio of 13 C labeling of intramuscular leucine to -KIC ( , preflood) was not altered as a result of flooding with any of the amino acids ( , postflood). At the end of the flooding period, the intramuscular-to-plasma labeling ratio for [ 13 C]glycine was (SD), suggesting that intramuscular flooding with labeled glycine was effectively achieved. For serine the ratio was Ratios near 1.0 are commonly observed when leucine, valine, or phenylalanine is used as the flooding amino acid (13). Fig. 2. Concentration of flooding amino acids in plasma before and throughout period of flooding (A: l, Phe; r, Thr; o, Arg; n 5; B: s, Gly; m, Ser; n 4). Error bars are within symbols preflood and are omitted for clarity during flood. Fig. 3. Labeling of [ 13 C]glycine and [ 13 C]serine in plasma during flooding period (means SD, n 4 for both). Effect of the nature of the flooding amino acid on the calculated rate of muscle protein synthesis. The apparent rates of muscle protein synthesis for the preflood infusion period, calculated using venous plasma -KIC labeling to represent the labeling of precursor pool, were similar for all subjects (Table 3). These values are similar to those observed routinely by us and others (22). During the flooding period, the calculated muscle protein synthetic rates increased significantly in subjects given phenylalanine ( 86%, to %/h, n 5; P 0.05) and also in subjects given threonine ( 89%, to %/h, n 5; P 0.05). However, the rates observed during flooding with arginine, serine, and glycine were not significantly different ( , , and %/h, respectively; NS; Fig. 3) from those observed from tracer incorporation before flooding. Protein synthetic rates, calculated using the labeling of muscle free leucine to represent the precursor, showed the same pattern, but the values were 10 15% higher (results not shown). Thus, in the groups receiving phenylalanine and threonine, almost a doubling in the calculated rate of protein synthesis was observed (each P 0.05), whereas in the groups receiving nonessential amino acids, there were no statistically significant changes (Fig. 4). Protein synthetic rates calculated from [ 13 C]glycine and [ 13 C]serine incorporations, measured using the flooding technique, were and %/h, respectively. Correction of the synthesis rates obtained with glycine by use of intramuscular glycine labeling to account for the dilution of label in the muscle free pool gave a rate of %/h. This value is identical to that routinely obtained by use of leucine and valine tracers given as a constant infusion and significantly lower than values observed previously when the flooding approach was used with leucine, valine, or phenylalanine (6, 14, 21). Effect of flooding on plasma insulin concentrations. In all floods, plasma insulin was transiently increased above basal after administration of the flooding dose, peaking at 5 or 10 min, and returned to basal levels at 90 min. Flooding with Phe (basal vs peak, µiu/ml), Thr ( vs ), Ser (10.3 vs n 2), and Gly ( vs ) resulted in moderate 1.7- to 1.9-fold increases in plasma insulin at peak, whereas flooding with Arg ( vs ) caused a 4.5-fold increase in insulin at peak.

4 E76 FLOODING AMINO ACIDS AND PROTEIN SYNTHESIS Table 3. Intramuscular amino acid concentrations before and after flooding Flooding AA Leucine Phenylalanine Threonine Preflood Postflood Preflood Postflood Preflood Postflood Phe * Thr * Arg Glycine Serine Preflood Postflood Preflood Postflood Gly Ser ND ND Amino acid (AA) values are means SE and are in µmol/mg protein; n 5 for all except Gly (n 4). ND, not determined. Arginine was not measured in muscle samples, and two sets of serine flood concentrations were not determined for technical reasons; these values therefore represent the mean of 2 determinations. *Significantly different from preflood value (P 0.05). Similar changes have been observed previously with leucine, valine, and phenylalanine floods (13, 20). DISCUSSION We have demonstrated clearly in this study that the essential amino acids phenylalanine and threonine, when administered as a flooding dose, increase the incorporation of [ 13 C]leucine tracer amino acids into human skeletal muscle protein. We have previously shown that leucine and valine have similar effects (20, 21). Furthermore, we show here that flooding with the nonessential amino acids glycine, serine, and arginine does not increase the rate of [ 13 C]leucine tracer incorporation into skeletal muscle. For the purposes of discussion, a summary of the effect of flooding with a variety of amino acids on muscle protein synthesis, calculated using incorporation of a number of different constantly infused tracers, is presented in Table 4. Whether the apparent difference in the effects of the essential and nonessential amino acids is real or artifactual is a question of some importance. Those who use the flooding dose method have argued that the major problem with the constant infusion approach is the uncertainty surrounding the use of either the plasma -KIC labeling or the free intramuscular amino acid labeling as an index of the true precursor for protein synthesis, i.e., aminoacyl-trna. However, the evidence available suggests that this error is likely to be small ( 15%) (1, 27) and therefore unlikely to account for the twofold increase in incorporation observed as a result of flooding with essential amino acids. The lack of any change during the transition from the preflooding to the intraflood period in the ratio of labeling of intracellular leucine to plasma KIC supports this. The twofold increase in incorporation seen on flooding with essential amino acids would require a halving in the precursor pool labeling during flooding; such changes are not observed. Furthermore, in all situations in which both constant infusion and flooding approaches are used simultaneously, the methods give similar synthesis Table 4. Effect of flooding with various amino acids on muscle protein synthesis rate calculated from a variety of constantly infused tracer amino acids Flooding AA Constantly Infused AA Effect on k a Reference No. Fig. 4. Muscle protein synthesis rates before and after flooding by use of constant infusion approach (hatched bars, preflood; solid bars, postflood, A and B), and values obtained from flooding with [ 13 C]glycine and [ 13 C]serine (open bars, flooding amino acid, B). Significantly different from preflood value (P 0.05); NS, not significant. Previous studies Leu Val >1.9 20, 21 Leu Phe > Leu Gly > Val Leu > Val Phe > Present studies Phe Leu >1.9 Thr Leu >1.9 Arg Leu & Ser Leu & Gly Leu & k s, muscle protein synthesis rate.

5 FLOODING AMINO ACIDS AND PROTEIN SYNTHESIS E77 rates for muscle, suggesting that both approaches are measuring the same process, and therefore arguments depending on changes in precursor labeling are probably invalid. It would appear that the existence of any difference between the rates of protein synthesis observed with the two methods is due mainly, and perhaps solely, to the choice of amino acid used to flood. The lack of a stimulatory effect when nonessential amino acids are used supports this conclusion, because the synthesis rates obtained from the incorporation of the labeled nonessential amino acids are identical to those obtained by the constant infusion of tracer leucine both before and during flooding. Further supportive evidence of a differential effect of essential vs. nonessential amino acids comes from a recent study carried out by our group for another purpose, i.e., the measurement of bone collagen synthetic rate, although muscle was also biopsied (17). Patients were given a constant infusion of [ 13 C]alanine for 8 h, followed by a flooding dose of [ 15 N]proline over 90 min. Muscle was sampled after the proline flood. Muscle protein synthetic rates for either alanine or proline use were similar at %/h (unpublished observations, M. J. Rennie and J. N. A. Gibson). The amino acids we have used to date are transported into muscle by a variety of systems with different characteristics [e.g., insulin sensitivity and transstimulation (4, 10)], making it unlikely that the changes we see are due to increased activity of any single transporter system in response to a single amino acid or other stimulus, e.g., insulin. For example, threonine, which causes a stimulation of protein synthesis, is transported by the same system, system ASC, as serine, which does not. Leucine and valine, which are transported by system L, both increase incorporation of constantly infused nonessential tracer glycine (26), which enters the cell by a different transporter, i.e., system Gly (4). Arginine, transported by two distinctive cationic amino acid transporters, i.e., system y and y L, has no stimulatory effect on leucine incorporation. Most of the flooding amino acids we used caused a small, transient (i.e., 20 min) increase (5 20 µiu/ml) in the levels of circulating insulin, but the existence (or lack) of a stimulation seemed unrelated to this; indeed, flooding with arginine caused the biggest insulin response, yet no stimulation of synthesis was observed. What other possible explanations are there for the flooding stimulation? The branched-chain amino acids (BCAA) (5, 9, 12), and leucine (3, 25) in particular, were reported some years ago to stimulate muscle protein synthesis in vivo and in vitro. However, there are also a number of reports in which the BCAA elicit no stimulation (8, 12, 15). However, no report exists of a stimulatory effect of BCAA on tissue protein synthesis in humans. Because we have also demonstrated that threonine stimulates leucine tracer incorporation, it would appear that the effect is not limited to the BCAA and may be a property of all essential amino acids. We are unaware of any reports suggesting that nonessential amino acids other than glutamine (11), which may be conditionally essential, stimulate protein synthesis. In our studies, the flooding period is relatively short, only 90 min, and it is conceivable that the stimulation we see is limited to that period, enabling substrate supply for protein synthesis to be maintained by endogenous levels of amino acids without causing a drain on the intracellular pool. 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