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
 |
ABSTRACT |
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 (24.5 ± 5.0 yr, 76.2 ± 8.3 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
[13C]leucine were
studied. Then the effects of flooding with labeled [13C]glycine
[0.1 g/kg body wt, 20 atoms percent excess (APE)] and [13C]serine (0.05 g/kg
body wt, 15 APE) on the incorporation of simultaneously infused
[13C]leucine were
investigated. When a large dose of phenylalanine or threonine was
administered, incorporation of the tracer leucine was significantly
increased (from 0.036 to 0.067 %/h and 0.037 to 0.070 %/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
 |
INTRODUCTION |
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
[13C]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
[13C]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-[1-13C]leucine,
[1-13C]glycine, and
L-[1-13C]serine,
99 atoms percent, were obtained from Mass Trace, Woburn, MA.
Subjects and experimental design.
Twenty-three healthy male volunteers [24.5 ± 5.0 (SD) yr,
76.2 ± 8.3 kg] were given a primed (1 mg/kg body wt),
constant infusion (1 mg · kg
1 · h
1)
of [1-13C]leucine over
7.5 h. After 6 h of infusion, 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
[1-13C]glycine
[0.1 g/kg, 20 atoms percent (AP)] or
L-[1-13C]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
1600.
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
(100-150 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
13C 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
13C labeling was determined by
isotope ratio mass spectrometry of the
carboxyl-CO2 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
(ks, %/h) =
Em/Ep × 1/t × 100, where
Em is the change in
enrichment in muscle, Ep is the
average enrichment of the precursor, and
t is the time between biopsies. For
the flooding dose,
ks (%/h) =
Em × 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 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
[13C]leucine and
-[13C]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).
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Table 1.
Protein synthetic rates before and after flooding calculated from
plasma leucine, -KIC, glycine, serine, and muscle amino
acid enrichment
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Fig. 1.
Labeling of plasma leucine and ketoisocaproate (KIC) before and during
a flooding dose of arginine. Values are means ± SD
(n = 5).
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Fig. 2.
Concentration of flooding amino acids in plasma before and throughout
period of flooding (A: , Phe; ,
Thr; , Arg; n = 5;
B: , Gly; , Ser;
n = 4). Error bars are within symbols
preflood and are omitted for clarity during flood.
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Fig. 3.
Labeling of
[13C]glycine and
[13C]serine in plasma
during flooding period (means ± SD,
n = 4 for both).
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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 two- to 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 13C
labeling of intramuscular leucine to
-KIC (0.93 ± 0.13, preflood) was not altered as a result of flooding with any of the amino acids (0.93 ± 0.15, postflood). At the end of the flooding period, the intramuscular-to-plasma labeling ratio for
[13C]glycine was 0.83 ± 0.09 (SD), suggesting that intramuscular flooding with labeled
glycine was effectively achieved. For serine the ratio was 1.03 ± 0.39. Ratios near 1.0 are commonly observed when leucine,
valine, or phenylalanine is used as the flooding amino acid (13).
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 0.067 %/h, n = 5; P < 0.05) and also in subjects given
threonine (+89%, to 0.070 %/h, n = 5; P < 0.05). However, the rates
observed during flooding with arginine, serine, and glycine were not
significantly different (0.050 ± 0.012, 0.048 ± 0.015, and
0.062 ± 0.019 %/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
[13C]glycine and
[13C]serine
incorporations, measured using the flooding technique, were 0.033 ± 0.001 and 0.049 ± 0.008 %/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 0.044 ± 0.008 %/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).

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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
[13C]glycine and
[13C]serine (open
bars, flooding amino acid, B).
Significantly different from preflood value
(P < 0.05); NS, not significant.
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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 6.5 ± 1.2 vs. 11.0 ± 2.3 peak, µIU/ml), Thr (6.9 ± 1.3 vs. 11.5 ± 3.4), Ser (10.3 vs. 17.7 n = 2),
and Gly (6.9 ± 0.7 vs. 13.1 ± 2.4) resulted in moderate 1.7- to
1.9-fold increases in plasma insulin at peak, whereas flooding with Arg
(5.4 ± 0.5 vs. 24.5 ± 4.1) caused a 4.5-fold increase in
insulin at peak. 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
[13C]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
[13C]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.
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Table 4.
Effect of flooding with various amino acids on muscle protein
synthesis rate calculated from a variety of constantly infused
tracer amino acids
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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 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
[13C]alanine for 8 h,
followed by a flooding dose of
[15N]proline over 90 min. Muscle was sampled after the proline flood. Muscle protein
synthetic rates for either alanine or proline use were similar at 0.046 %/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. Indeed, the intracellular concentrations of
leucine and threonine appear to fall slightly (~10%, not
significant) over this period, perhaps as a result of the short-term
increase in synthesis and or possibly a fall in proteolysis.
From the data presented here and also previous studies, it is apparent
that the choice of amino acid used to flood is critical. Consequently,
serine, which has a relatively small free pool and is relatively
abundant in tissue protein, would seem to be the preferred choice of
flooding amino acid for future clinical investigations.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Peter Watt for helpful advice.
 |
FOOTNOTES |
This work was supported by the Medical Research Council, the University
of Dundee, and Merz & Company.
Address for reprint requests: M. J. Rennie, Dept. of Anatomy and
Physiology, Univ. of Dundee, Dundee DD1 4HN, Scotland, UK.
Received 1 August 1997; accepted in final form 20 March 1998.
 |
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