1 Department of Surgery, State University of New York, Stony Brook, New York 11794; and 2 Endocrine Research Unit, Division of Endocrinology and Metabolism, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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Muscle protein synthesis was measured by infusion of L-[2H5]phenylalanine in two groups of anesthetized dogs, before and during infusion of insulin with euaminoacidemia, and with differing concentrations of unlabeled phenylalanine (tracee). With the infusion of insulin, muscle protein synthesis increased 39 ± 12% based on phenylalanyl-tRNA. Calculation with plasma phenylalanine enrichment overestimated insulin stimulation by 40% (56 ± 12 vs. 39 ± 12%). Raising the concentration of plasma phenylalanine twofold during infusion of insulin further increased the apparent stimulation of muscle protein synthesis based on plasma relative to phenylalanyl-tRNA by 225% (65 ± 19 vs. 20 ± 14%, P < 0.001). In both experiments, the stimulation of synthesis rates calculated from phenylalanine enrichment within the muscle was closer to that from phenylalanyl-tRNA (48 ± 19%, experiment 1; 30 ± 14%, experiment 2). Results indicate that the enrichment of a labeled amino acid within plasma and tissue amino acid pools is affected by the concentration of tracee infused. Increasing the concentration of tracee overestimates the insulin-mediated stimulation of muscle protein synthesis when amino acid pools other than aminoacyl-tRNA are used as the precursor enrichment.
insulin; amino acids; aminoacyl-tRNA; L-[2H5]phenylalanine
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INTRODUCTION |
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DESPITE A NUMBER OF carefully designed studies, the fundamental question of the role of insulin in the regulation of protein synthesis in human muscle still remains unclear. Although insulin has been shown to stimulate muscle protein synthesis in growing rats (1, 12), similar experiments in humans have failed to show any change (10, 15, 20, 21). It has been suggested that the decrease in plasma amino acid concentrations after insulin administration might hinder the stimulation of muscle protein synthesis brought about by insulin (4). However, experiments in healthy volunteers in whom an amino acid mixture was infused with insulin (4, 10, 23) or insulin was infused locally to prevent hypoaminoacidemia (5, 20) have also given conflicting results.
Uncertainty about the effect of insulin on protein synthesis has also arisen from studies by Bennet et al. (4) and Newman et al. (23), in which muscle protein synthesis in volunteers was measured by the simultaneous infusion of two different tracers. Ambiguous results indicated a stimulation by insulin with one tracer but no stimulation with the other. Notably, muscle protein synthesis appeared to be stimulated when estimated by the tracer amino acid with the greater elevation in plasma concentration as a consequence of amino acid infusion. In both studies, the enrichment of the tracer amino acid in plasma was used to assess muscle protein synthesis. The possibility that the enrichment of the tracer in plasma might not reflect the true precursor, the aminoacyl-tRNA, under these circumstances was not addressed. This raises the possibility that the relationship between enrichment in plasma and the enrichment of aminoacyl-tRNA is not constant but alters with alterations in plasma amino acid concentration.
Measurement of the true rates of protein synthesis with labeled
amino acid tracers requires the assessment of isotopic enrichment of
the obligatory precursor pool for protein synthesis, that is, aminoacyl-tRNA. However, the low tRNA concentration in tissues and the
consequent need for large biopsies limit the routine measurement of
aminoacyl-tRNA. Protein synthesis rates have more usually been assessed
from the enrichment of surrogate pools, such as the free amino acid in
plasma or within the tissue (tissue fluid), or from a metabolite in the
plasma, such as -ketoisocaproate derived from intracellular leucine.
The assumption is made that these pools closely reflect the enrichment
of aminoacyl-tRNA. When the labeled amino acid is infused into the
plasma, aminoacyl-tRNA is charged from both the highly labeled amino
acid entering the cell from plasma and from unlabeled amino acid
derived from the degradation of protein (13). Moreover,
with intervention studies such as those with insulin and amino acid
infusion, the relationship of the enrichment of tracer in the surrogate
precursor to that in the true precursor may be altered.
Therefore, in this study, we investigated systematically the question of whether or not the kinetics of the tracer amino acid were altered when the plasma concentration of the unlabeled analog of the same amino acid changed; specifically, whether the relationship of isotopic enrichment in the aminoacyl-tRNA to that of the same amino acid in surrogate precursor pools such as plasma and tissue fluid free amino acid was affected by elevated plasma amino acid concentrations. Muscle protein synthesis was measured with a constant infusion of L-[2H5]phenylalanine under basal conditions and then during the infusion of insulin plus glucose and an amino acid mixture to prevent the insulin-induced decline in the plasma levels of glucose and amino acids (9, 11). In addition to the infusion of amino acids to maintain euaminoacidemia, the study was also repeated with the infusion of unlabeled phenylalanine sufficient to raise plasma levels to twice the basal levels. The labeling of amino acid pools (plasma, tissue fluid, and aminoacyl-tRNA) was measured, and the rates of protein synthesis were calculated with aminoacyl-tRNA (the true precursor pool) and with surrogate precursor pools (free amino acid in plasma and tissue fluid).
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MATERIALS AND METHODS |
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Animals
Two groups of seven conditioned adult male dogs of mixed breed (20 ± 2 kg) were singly housed and fed a standard diet (Purina Lab Canine Chow, Purina Mills, Richmond, IN) forExperiments
Experiment 1: insulin and amino acid infusion with
euaminoacidemia.
Muscle protein synthesis was measured with a primed (7 µmol/kg) constant infusion of
L-[2H5]phenylalanine (12 µmol · kg1 · h
1 for
6 h; MassTrace, Woburn, MA) during the last 90 min of a basal period and during the last 90 min of insulin infusion with concomitant infusion of amino acids and glucose to maintain basal levels. The
protocol is illustrated in Fig. 1. In
addition to L-[2H5]phenylalanine,
two other tracers,
L-[1-13C]leucine and
L-[2-15N]lysine, given as a prime of 14 µmol/kg and then infused at 24 µmol · kg
1 · h
1
(MassTrace) for 6 h, were given to compare the labeling of
precursor pools of different amino acids to that of phenylalanine.
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Experiment 2: effect of insulin and amino acid infusion with
hyperphenylalaninemia.
The protocol was similar to that described in experiment 1 and depicted in Fig. 1.
L-[2H5]phenylalanine was infused
at a constant rate similar (but not identical) to that in
experiment 1 for 6 h, and muscle protein synthesis was
measured between 1.5 and 3 h (basal state) and between 4.5 and
6 h (during insulin and amino acid infusion). The only difference
from experiment 1 was that the amino acid infusion (TrophAmine), which was started at 3 h, was supplemented with additional phenylalanine (total phenylalanine infused 6.1 mg
kg1 · h
1). Phenylalanine was added
to the standard amino acid mixture to raise the plasma concentration of
phenylalanine during the infusion of insulin while maintaining the
concentrations of the other amino acids at basal levels.
Analytical Methods
Enrichment of tissue protein. Free and protein-bound amino acids were separated by precipitating tissue powdered in liquid N2 with cold perchloric acid (20g/l). Supernatants were used for analysis of free amino acid enrichment (tissue fluid), whereas muscle protein was extensively washed and hydrolyzed (21). The enrichment of L-[2H5]phenylalanine in protein was measured as previously described (22) with a MD800 gas chromatograph-mass spectrometer (Fisons Instruments, Beverly, MA) operated under electron impact condition and in splitless mode. The ions with mass-to-charge ratios (m/z) 106 (m+2) and m/z 109 (m+5) were monitored.
Enrichment of amino acids in plasma and tissue fluid. Amino acids from plasma and tissue fluid samples were purified by application to a cation exchange column (AG 50W Resin, 100-200 mesh, hydrogen form; Bio-Rad Laboratories, Richmond, CA) and eluted with 4 M NH4OH (6). L-[2H5]phenylalanine enrichment was measured by monitoring the ions at m/z 336 and 341 of the tertiary butyldimethylsilyl derivative on a MD800 gas chromatograph-mass spectrometer (Fisons Instruments) operated under electron impact conditions (22). Leucine and lysine were derivatized as their N-trifluoroacetyl isopropyl esters, and their enrichment was measured by gas chromatography-mass spectrometry (GC-MS; Hewlett-Packard, Avondale, PA) under positive ion chemical ionization conditions by monitoring the ions at m/z 288/287 for leucine and at m/z 399/398 for lysine (19).
Enrichment of aminoacyl-tRNA. Aminoacyl-tRNA was isolated by the method of Kelley et al. (18), based on phenol extraction and ethanol precipitation, as modified by Baumann et al. (3) and Ljungqvist et al. (19). Aminoacyl-tRNA was deacylated by addition of 50 mM NaHCO3 with incubation at 37°C for 60 min. After acidification and removal of insoluble material, the amino acid solution was evaporated and resuspended in 0.01 M HCl before derivatization for mass spectrometry. Amino acids were derivatized to their N-trifluoroacetyl isopropyl esters, and their isotopic enrichments were determined by GC-MS, operated under selected ion monitoring and positive ion chemical ionization conditions (19).
Substrate and hormone analyses. Plasma amino acid concentrations were measured after acetonitrile precipitation of plasma protein with 6-amino-n-caproic acid as internal standard on a Waters 2690 HPLC system (Waters, Milford, MA) with o-phthaldehyde derivatization and fluorescence detection. Plasma insulin was determined by radioimmunoassay (Diagnostic Products, Los Angeles, CA), and plasma glucose was monitored with a Beckman Glucose Analyzer 2 (Beckman Instruments, Brea, CA).
Calculations
Protein fractional synthesis rates (FSR), corresponding to the percentage of the muscle protein pool synthesized per day, were calculated with the following formula (14)
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Statistics
All data are expressed as means ± SE. Comparisons of the basal period and the period of insulin and amino acid infusion were made with paired t-test analysis. Comparisons of experiments 1 and 2 were made with the t-test for unpaired data, and comparisons of three or more groups (e.g., phenylalanine vs. leucine and lysine) were made by ANOVA with Student-Newman-Keuls correction for multiple comparisons. The stimulation of muscle protein synthesis in the combined data of experiments 1 and 2 was also analyzed by two-way ANOVA. A value of P < 0.05 was taken as statistically significant. ![]() |
RESULTS |
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Plasma Insulin, Glucose, and Amino Acids
Both experiment 1 and experiment 2 involved a 3-h basal period followed by a period of insulin, glucose, and amino acid infusion, as depicted in Fig. 1. The plasma concentrations of insulin, glucose, and phenylalanine in experiment 1, with phenylalanine concentrations maintained at basal levels, and in experiment 2, with phenylalanine at twice basal levels during insulin infusion, are shown in Fig. 2. Plasma insulin concentrations in the basal state were similar in experiments 1 and 2 (59 ± 10 vs. 49 ± 7 pmol/l). When insulin was infused, plasma levels of insulin increased to 253 ± 7 and 277 ± 7 pmol/l (Fig. 2A, basal vs. insulin, P < 0.001), with the levels in experiment 2 ~10% higher than in experiment 1 (P < 0.05). During the infusion of insulin, glucose was infused at variable rates to maintain plasma glucose concentration at the basal level for each dog. The mean glucose concentration was somewhat higher in experiment 2 (average 5.4 ± 0.2 vs. 4.9 ± 0.3 mmol/l; Fig. 2B), but this difference did not reach statistical significance. The lower glucose levels in experiment 1 resulted from the inclusion of one dog with a low level of plasma glucose (3.8 mmol/l). The plasma levels of amino acids in the basal state and during the infusion of insulin plus the amino acid mixture are shown in Table 1. In the basal state, plasma levels of several amino acids (leucine, lysine, valine, methionine, and arginine) were lower in experiment 2 than in experiment 1, although this difference is not reflected in differences in muscle protein synthesis in the basal state (Table 3). With the infusion of insulin and the commercial amino acid mixture (TrophAmine), the decline in plasma concentration of most amino acids was prevented or minimized (Table 1). However, the commercially available mixture did not completely replace all amino acids; plasma concentrations of tyrosine and asparagine decreased in both experiments during the infusion of insulin. Lysine and threonine concentrations also decreased in both experiments, although the decline was not statistically significant in experiment 2. Although there was not complete euaminoacidemia for all amino acids during the infusion of insulin, the two experiments exhibited the same change in plasma levels between the basal state and during insulin plus amino acid infusion, except for phenylalanine, which was significantly elevated during the infusion of insulin plus amino acids in experiment 2 (P < 0.001). This was the result of phenylalanine supplementation of the amino acid solution in experiment 2, when the average plasma concentration of phenylalanine increased by 87% over the basal levels in the last 3 h (Table 1 and Fig. 1C).
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Precursor Enrichment and Muscle Protein Synthesis
Experiment 1: insulin and amino acid infusion with euaminoacidemia.
In experiment 1, three labeled tracers,
L-[2H5]phenylalanine,
L-[1-13C]leucine, and
L-[2-15N]lysine, were given at a constant
rate throughout the 6-h protocol. The enrichment [in moles
percent excess (MPE)] of free
L-[2H5]phenylalanine from plasma,
tissue fluid, and phenylalanyl-tRNA is shown in Fig.
3A. Due to the priming of the
amino acid pools and the 1.5 h of infusion, by the time of the
first biopsy at 1.5 h, the enrichment of plasma phenylalanine had
reached a constant value; however, the enrichment of tissue fluid and
phenylalanyl-tRNA increased between 1.5 and 3 h (Fig.
3A). The enrichment of phenylalanine in plasma was much
higher than that of either phenylalanyl-tRNA or free phenylalanine
in the tissue fluid. In the basal state, phenylalanyl-tRNA enrichment
reached a value of only 53 ± 2% that of plasma (8.29 ± 0.56 vs. 15.52 ± 0.74 MPE, P < 0.001) but was 89 ± 3% of the enrichment of tissue fluid (8.29 ± 0.56 vs.
9.43 ± 0.74 MPE, P = 0.01).
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Experiment 2: insulin and amino acids with hyperphenylalaninemia. In the basal state, the protocol for experiment 2 was the same as for experiment 1. As in experiment 1, the average enrichment of phenylalanyl-tRNA in the basal state was only 55 ± 2% that of the phenylalanine in plasma (9.74 ± 0.18 vs. 17.66 ± 0.50 MPE, P < 0.001) and 88 ± 3% that of the free phenylalanine in tissue fluid (11.10 ± 0.42 MPE, P < 0.01), as shown in Fig. 3B. During the infusion of insulin in experiment 2, the increase in plasma phenylalanine concentration was associated with a substantial (33%) decrease in plasma phenylalanine enrichment (11.78 ± 0.30 vs. 17.66 ± 0.50 MPE, P < 0.001) due to the supplementation of the infused amino acid mixture with additional unlabeled phenylalanine. This was accompanied by a parallel decrease (16%) in phenylalanine enrichment in tissue fluid (9.28 ± 0.42 vs. 11.10 ± 0.42 MPE, P < 0.05). However, the labeling of phenylalanyl-tRNA was not significantly altered by the additional phenylalanine infused into the plasma (9.74 ± 0.18 vs. 8.94 ± 0.34 MPE).
The relationships of phenylalanine enrichment among plasma, tissue fluid, and tRNA pools were altered by the infusion of insulin plus amino acids supplemented with additional phenylalanine. The ratio of the enrichment of phenylalanyl-tRNA to that of plasma phenylalanine was 0.55 ± 0.02 in the basal state but increased to 0.76 ± 0.02 (P < 0.001) with insulin plus amino acid infusion. The ratio of the enrichment of phenylalanyl-tRNA to that of phenylalanine in tissue fluid also increased during the infusion period (0.88 ± 0.03 vs. 0.97 ± 0.04, P = 0.05). For leucine and lysine, the infusion of insulin and amino acids was not accompanied by a dramatic change in plasma concentration as occurred with phenylalanine. In the presence of elevated plasma phenylalanine (experiment 2), the ratios of the enrichment of leucyl-tRNA enrichment to that of plasma (0.54 ± 0.03 basal vs. 0.59 ± 0.02 with insulin and amino acids) were similar to those observed in experiment 1 (0.57 ± 0.02, 0.63 ± 0.03). Similarly, the ratios of the enrichment of lysyl-tRNA to those of plasma lysine in experiment 2 (0.31 ± 0.04 basal, 0.26 ± 0.01 with insulin) were also similar to those in experiment 1 (0.39 ± 0.06, 0.38 ± 0.07) and were not significantly altered from the basal level by the infusion of insulin plus amino acids. The alterations in relative enrichment for L-[2H5]phenylalanine in plasma, tissue fluid, and phenylalanyl-tRNA are also reflected in the estimates of muscle FSR calculated with the enrichment in these different pools (Table 3). With infusion of insulin plus phenylalanine-supplemented amino acids, the rate calculated from any of the three potential precursor pools demonstrated an increase in the rate of protein synthesis, as in experiment 1; however, the magnitude of the increase was not the same for each precursor (Table 3 and Fig. 4B). When FSR was calculated from phenylalanyl-tRNA, the stimulation of muscle protein synthesis with insulin and amino acids was 20 ± 14% (Table 3 and Fig. 4B), but when plasma phenylalanine was used to represent precursor enrichment, the apparent stimulation was 65 ± 19% (P < 0.01). This apparent stimulation in muscle protein synthesis (65 ± 19%) was significantly higher (P < 0.01) than that (20 ± 14%) calculated using the true precursor, phenylalanyl-tRNA (Fig. 4B). When the synthesis rate of muscle protein was estimated from the enrichment of tissue fluid phenylalanine, the stimulation with insulin and amino acids (30 ± 14%) was significantly lower than that based on plasma (P < 0.01) and not significantly different from that calculated from phenylalanyl-tRNA enrichment (P = 0.07; Fig. 4B). ![]() |
DISCUSSION |
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Because exogenous insulin alters plasma amino acid concentrations (9, 11), studies of the regulation of muscle protein synthesis by insulin have involved the concomitant infusion of amino acids (4, 23, 24). This present study demonstrates that an alteration in the concentration of the unlabeled amino acid corresponding to the tracer used to estimate protein synthesis can alter the labeling of that amino acid in plasma and tissue such that, if these are assumed to be precursor pools for protein synthesis, erroneous estimates of protein synthesis are made. Infusion of insulin with euglycemia and euaminoacidemia (experiment 1) stimulated muscle protein synthesis in biceps femoris muscle; this stimulation, calculated from phenylalanyl-tRNA enrichment, was 39 ± 12%. When muscle protein synthesis was estimated from the enrichment of plasma phenylalanine, the stimulation was somewhat greater (56 ± 22%; Table 3 and Fig. 4A). However, when the infusion of insulin with euglycemia and euaminoacidemia was accompanied by an increase in the plasma concentration of phenylalanine to twice that in experiment 1, the apparent stimulation by insulin of muscle protein synthesis, if calculated from the enrichment of plasma phenylalanine, was 225% higher than the value from phenylalanyl-tRNA (Table 3 and Fig. 4B). This overestimation of the stimulation of muscle protein synthesis was the result of alterations in the enrichment of plasma phenylalanine that did not accurately reflect the enrichment of phenylalanyl-tRNA, the true precursor for protein synthesis (Fig. 3).
Effect of Insulin on Precursor Labeling
The enrichment of L-[2H5]phenylalanine in plasma, within the tissue, and in phenylalanyl-tRNA is a function of both the amount of labeled phenylalanine infused and the amount of unlabeled phenylalanine entering each pool. In experiment 1, the enrichment of plasma phenylalanine was not altered during the infusion of insulin and exogenous unlabeled phenylalanine. Because protein degradation would be suppressed by insulin (10, 15, 17, 20), the exogenously supplied phenylalanine balanced the decrement in unlabeled phenylalanine entering the plasma from the degradation of body protein. Within the tissue, the contribution of a reduced supply of unlabeled phenylalanine from protein degradation and an increased uptake of labeled phenylalanine from the plasma, as reported for other amino acids (5), resulted in an ~10% increase in the enrichment of phenylalanine in both tissue fluid (10.20 ± 0.73 vs. 9.43 ± 0.74 MPE, P < 0.01) and phenylalanyl-tRNA (9.31 ± 0.63 vs. 8.29 ± 0.56 MPE, P < 0.05) relative to the basal state.The contribution of plasma phenylalanine to phenylalanyl-tRNA rose from 53 ± 2 in the basal state to 59 ± 3% during the infusion of insulin and amino acids (P < 0.05; Fig. 3A). There was also a suggestion that the contribution of plasma leucine to the enrichment of leucyl-tRNA was increased by insulin and amino acid infusion (from 57 ± 2 to 63 ± 3%, P = 0.06). Plasma lysine, however, made a significantly smaller contribution to lysyl-tRNA enrichment than was observed with either phenylalanine or leucine, and this was not altered by the infusion of insulin and amino acids (39 ± 6 vs. 38 ± 7%).
Because the estimate of muscle protein synthesis is inversely related to the enrichment of the precursor, the rate of muscle protein synthesis calculated with plasma phenylalanine enrichment as the precursor is only 54% of the value with phenylalanyl-tRNA as precursor (1.64 ± 0.19 vs. 3.02 ± 0.35%/day, P < 0.001; Table 2). Although calculation with plasma phenylalanine enrichment underestimated the rate of muscle protein synthesis, the proportional stimulation in muscle protein synthesis due to insulin and amino acid infusion was similar to that determined with phenylalanyl-tRNA (56 ± 22% vs. 39 ± 12%) and that observed with the enrichment of phenylalanine in tissue fluid (48 ± 19%), as shown in Table 2 and Fig. 4A.
This consistency of the proportional stimulation in muscle protein synthesis observed with all three phenylalanine pools in experiment 1 was not observed in experiment 2. Experiment 2 was designed to potentiate the alterations in the labeling of free amino acid pools by altering the concentration of tracee while the rate of infusion of labeled phenylalanine was unaltered. An amino acid mixture was infused with the insulin to prevent the insulin-induced drop in plasma amino acid concentration, as in experiment 1; however, phenylalanine was given at twice the rate needed to maintain the basal concentration. In experiment 2, the enrichment of phenylalanine in plasma declined by 33% during the infusion of insulin with the phenylalanine-supplemented amino acid mixture (Fig. 3B). This increased concentration of phenylalanine in the plasma stimulated uptake of phenylalanine by the tissue, as can be seen in the enhanced contribution of the plasma amino acid pool to the enrichment of phenylalanyl-tRNA. The ratio of phenylalanyl-tRNA to plasma phenylalanine enrichment was 0.76 ± 0.02 in experiment 2 compared with 0.59 ± 0.03 in experiment 1 (P < 0.001; Fig. 3). Because the contribution of plasma phenylalanine to the enrichment of phenylalanyl-tRNA was altered between the basal state and during the infusion of insulin and amino acids, the apparent stimulation of muscle protein synthesis assessed from the plasma phenylalanine enrichment was significantly greater than that observed with phenylalanyl-tRNA enrichment as the precursor (65 ± 19 vs. 20 ± 14%, P < 0.001; Fig. 4B). Because the relationship of the enrichment of plasma phenylalanine to that of the true precursor was altered between the basal state and during the infusion of insulin and amino acids, the conclusion about the effect of insulin and amino acids on protein synthesis based on plasma is not correct. However, the enhanced contribution of plasma phenylalanine to phenylalanyl-tRNA was specific to phenylalanine, the amino acid with increased plasma concentration. Neither of the other tracers, leucine or lysine, was associated with increased plasma concentration and did not exhibit enhanced contribution of the plasma to aminoacyl-tRNA (Table 2), suggesting that the high phenylalanine concentration in the plasma did not substantially alter the intramuscular levels of either leucine or lysine, even though leucine shares the L system for amino acid transport with phenylalanine (16).
The results of this present study may explain some of the discrepancies
observed in studies of the effect of insulin on protein metabolism in
humans determined from the enrichment of tracers in plasma. For
example, Bennet et al. (4) simultaneously infused two
different tracers, phenylalanine and leucine, and showed that protein
synthesis was stimulated by insulin plus amino acid infusion when
assessed with labeled phenylalanine but not when assessed with labeled
leucine. Similarly, in another multitracer study, Newman et al.
(23) found that insulin and amino acid infusion stimulated
muscle protein synthesis when measured with phenylalanine but not with
leucine. Contrasting results between different tracers were also shown
by Tessari et al. (24), investigating the effect of
insulin infusion on whole body protein synthesis. In all of these
studies, discrepancies in the conclusions about the effect of insulin
and amino acids drawn from the different tracers can be related to
alterations in the concentration of the tracee of the amino acid tracer
used to determine protein synthesis. In particular, in agreement with
the conclusion of the present study, an apparently greater stimulation
of protein synthesis was found with those tracers of which the tracee
concentration was elevated (Table 4).
Where the tracee concentration was less elevated (leucine in Refs.
4 and 23 and phenylalanine in Ref.
24), there was no significant stimulation in the apparent
rate of protein synthesis by insulin. The results of the present study
suggest that, in studies in which labeled amino acids are used to
assess protein synthesis, alterations in the relationship of the
enrichment in measured precursor pools to the true precursor can have a
major impact on the conclusions.
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Unlike the problems encountered when plasma phenylalanine is used to represent the precursor for protein synthesis, the enrichment of phenylalanyl-tRNA is much closer to that of phenylalanine within the muscle (tissue fluid) both in the basal state and during the infusion of insulin and amino acids (phenylalanyl-tRNA/tissue fluid enrichment 0.89 ± 0.03 vs. 0.91 ± 0.03, experiment 1). When the plasma phenylalanine concentration was increased (experiment 2), the ratio of enrichment of phenylalanyl-tRNA to phenylalanine in the tissue fluid was significantly increased (0.88 ± 0.03 vs. 0.97 ± 0.04, P = 0.05), albeit to a much smaller extent than the alteration in the relationship of phenylalanyl-tRNA to plasma enrichment (Fig. 3B). As a consequence, the rate of muscle protein synthesis estimated from tissue fluid shows a stimulation brought about by insulin and amino acid infusion that is closer to the value obtained when protein synthesis is calculated from the phenylalanyl-tRNA (Fig. 4B). This suggests that free phenylalanine within the tissue is a better surrogate precursor pool for protein synthesis than plasma phenylalanine.
Effect of Insulin on Muscle Protein Synthesis
Experiment 2 demonstrates that increasing the plasma concentration of unlabeled phenylalanine altered the relationship of the enrichment of plasma phenylalanine to the enrichment of phenylalanyl-tRNA. However, when the rate of muscle protein synthesis is calculated from the enrichment of phenylalanyl-tRNA, the rates should give comparable estimates of the effect of insulin on the rate of muscle protein synthesis regardless of any changes in plasma labeling. The stimulation of muscle protein synthesis from phenylalanyl-tRNA was 39 ± 12% in experiment 1 and 20 ± 14% in experiment 2. Although the stimulation observed in experiment 2 failed to reach statistical significance, a two-way analysis of variance for the combined data indicates a highly significant effect of insulin treatment (P = 0.008) but no significant effect of experiment, suggesting that the results of the two experiments are not different from each other. The smaller stimulation by insulin in experiment 2 is unlikely to be due to differences in insulin concentrations that were higher in experiment 2 than in experiment 1. It is also unlikely to be due to inhibition of the effect of insulin by the higher concentration of plasma phenylalanine in experiment 2. Measurement of muscle protein synthesis obtained with a comparable protocol but with a flooding amount of phenylalanine indicated an increase in muscle protein synthesis of 54 ± 10% over basal rates during an infusion of insulin plus amino acids (2.33 ± 0.22 vs. 3.56 ± 0.34%/day, P < 0.001) (7). With the flooding amount of phenylalanine, the plasma phenylalanine concentration was increased more than threefold over basal levels, and the stimulation in muscle protein synthesis was not significantly different from that observed in either experiment 1 or experiment 2. Moreover, the rate of muscle protein synthesis in the basal state assessed with the flooding amount of phenylalanine in the presence of a threefold elevation in phenylalanine concentration was not significantly different from the rate of muscle protein synthesis determined with a constant infusion of labeled phenylalanine, with phenylalanyl-tRNA enrichment used to represent the enrichment of the precursor for protein synthesis (Table 3). Although insulin and amino acid infusion stimulated muscle protein synthesis in the adult dog, there was some variability in the responsiveness of muscle protein synthesis to insulin in different groups of dogs. However, there is no evidence that the ability of insulin to stimulate muscle protein synthesis was inhibited at elevated concentrations of phenylalanine.The stimulation of muscle protein synthesis by insulin in the adult dog contrasts with the observation that muscle protein synthesis was not stimulated in humans (10, 15, 20, 21). The contrasting conclusions of the effect of insulin between the dogs and the humans may be species related. There is also a possibility that the 20-kg dogs used in the present study were developmentally more immature than adult humans [age range 18-32 yr (10, 15, 20, 21)], and therefore more responsive to the effect of insulin, as has been reported for immature vs. mature rats (1, 2). More significant, however, is the fact that, in some of the human studies, exogenous amino acids were not provided, and Wolfe (25) has suggested that the provision of exogenous amino acids may be necessary for the enhancement of protein synthesis by insulin; thus the observed difference between the results with dogs and those in humans may result from differences in amino acid levels, although the present study highlights the need for careful determination of precursor enrichment in studies where tracee concentrations increase.
In summary, this study shows that muscle protein synthesis in the adult dog was increased by simultaneous infusion of insulin and amino acids. However, the study also highlights the fact that the labeling of amino acid pools such as plasma amino acids, tissue free amino acids, and aminoacyl-tRNAs is affected by the plasma concentration of the amino acid infused as the tracer. The relationship between the enrichment of the free amino acid in plasma and aminoacyl-tRNA was altered when the plasma concentration of tracee was increased. Therefore, if the enrichment of the amino acid in plasma is used as a measure of precursor labeling, then the estimates of changes in muscle protein synthesis may be in error. In particular, increased concentration of the unlabeled amino acid leads to an overestimate of the stimulation of muscle protein synthesis brought about by insulin and amino acid infusion. Although the amino acid within the tissue fluid represents a better surrogate precursor than plasma for protein synthesis, results obtained when the concentration of the tracer amino acid is altered should be interpreted with caution.
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ACKNOWLEDGEMENTS |
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The expert and impeccable technical assistance of G. Casella, Y. Hong, D. Sasvary, and B. Tyndall is gratefully acknowledged.
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FOOTNOTES |
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This study was supported in part by National Institutes of Health Grants R01 DK-4878602, RR-10710, and RR-00585.
Address for reprint requests and other correspondence: G. Caso, Dept. of Surgery, HSC T19-048, State Univ. of New York at Stony Brook, Stony Brook, NY 11794-8191.
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.
Received 20 June 2000; accepted in final form 14 February 2001.
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