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Diminished muscular activity is associated with alterations of protein metabolism. The aim of this study was to evaluate the effect of short-term muscle inactivity on regulation of whole-body protein deposition during amino acid infusion to simulate an experimental postprandial state. We studied nine healthy young volunteers at the end of 14 day periods of strict bed rest and of controlled ambulation using a cross-over design. Subjects received a weight-maintaining diet containing 1 g protein kg−1 day−1. l[1-13C]leucine was used as a marker of whole-body protein kinetics in the postabsorptive state and during a 3 h infusion of an amino acid mixture (0.13 g amino acid (kg lean body mass)−1 h−1). In the postabsorptive state, bed rest decreased (P < 0.05) the rate of leucine disposal (Rd) to protein synthesis and tended to decrease leucine rate of appearance (Ra) from proteolysis, whereas the rate of leucine oxidation did not change significantly. Amino acid infusion increased leucine Rd to protein synthesis and oxidation and decreased leucine Ra from proteolysis in both the bed rest and ambulatory conditions. Changes from basal in leucine Rd to protein synthesis were lower (P < 0.05) during bed rest than those in the ambulatory period, whereas changes in leucine Ra from proteolysis and oxidation were not significantly different. During amino acid infusion, net leucine deposition into body protein was 8 ± 3% lower during bed rest than during the ambulatory phase. In conclusion, short-term bed rest leads to reduced stimulation of whole-body protein synthesis by amino acid administration. Results of this study were, in part, presented at the meeting, Experimental Biology, 2004, Washington DC.
In the physiological postabsorptive state, the balance between whole-body protein synthesis and degradation is negative. Such protein loss is immediately compensated in the postprandial state by a protein gain mediated by nutrient intake (Tessari et al. 1987). Thus, the efficiency of the mechanisms responsible for the regulation of protein synthesis and degradation in the postabsorptive and fed states appears to be crucial for maintaining lean body mass throughout the day, thereby avoiding protein wasting. Meal-induced protein gain is largely mediated by the postprandial rise in plasma amino acid concentrations that acutely stimulates muscle and whole-body protein synthesis (Rennie et al. 2002). This stimulatory effect of amino acid administration on muscle protein synthesis is enhanced by previous performance of physical exercise (Biolo et al. 1997).
Diminished muscular activity is associated with impairment of muscle function and loss of lean body mass (Biolo et al. 2003; di Prampero & Narici, 2003). However, assessment of protein kinetics in the postabsorptive state during experimental bed rest by stable isotopes of amino acids failed to detect significant protein loss at the muscle and whole-body levels (Shangraw et al. 1988; Ferrando et al. 1996;Lovejoy et al. 1999; Stein et al. 1999). Nonetheless, despite the importance of assessing the regulation of protein kinetics in the postprandial state, no study of the effects of amino acid/protein administration on protein kinetics during muscle unloading in humans has been published.
Within the frame of the ‘Short-Term Bed Rest Study –Integrated Physiology’ (STBR-IP) set up by the German Aerospace Institute (DLR) under the auspices of the European Space Agency we tested the hypothesis that a reduced stimulation of whole-body protein synthesis by amino acid administration represents a major mechanism for the bed rest-induced loss of lean body mass. Subjects were studied at the end of 14 day periods of bed rest or ambulatory conditions using a cross-over experimental design. This approach allowed strict control and monitoring of nutrient intake and levels of physical activity both during the bed rest and ambulatory periods.
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Body weight did not change significantly during the bed rest (−0.30 ± 0.28 kg) or ambulatory (−0.14 ± 0.34 kg) periods. Mean values of lean body mass were not significantly different at the beginning (59.2 ± 1.2 kg) and end (58.9 ± 1.2 kg) of the bed rest period, nor did they change significantly during the ambulatory period: 58.9 ± 1.1 kg at the beginning and 59.0 ± 1.1 kg at the end of the period. Table 1 shows plasma amino acid concentrations at the end of the ambulatory and bed rest periods in the postabsorptive state and during amino acid infusions. In the postabsorptive state, plasma concentrations of the branched chain amino acids leucine, valine and isoleucine were greater during bed rest than in the ambulatory condition. In contrast, the alanine concentration was lower during bed rest. Intravenous infusion of the amino acid mixture resulted in variable increments in the plasma concentrations of all the infused amino acids. Increments from the postabsorptive values varied according to the individual amino acid infusion rates and pool sizes but they were similar in the ambulatory and bed rest conditions. The plasma concentrations of amino acids that were not included in the infused mixture did not change significantly, with the exception of asparagine, which decreased by about 12%, and glutamine, which increased by about 5%. Following the infusion, the plasma amino acid concentrations were similar in the ambulatory and bed rest conditions. The results of indirect calorimetry indicated that, in the postabsorptive state, the values for oxygen consumption were not significantly different in the bed rest and ambulatory phases (86 ± 1 and 88 ± 2 μmol min−1 (kg LBM)−1, respectively). Postabsorptive rates of CO2 excretion were also similar in the bed rest and ambulatory phases (73 ± 1 and 75 ± 2 μmol min−1 (kg LBM)−1, respectively). Intravenous infusion of the amino acid mixture resulted in similar increments in oxygen consumption and CO2 production in the bed rest and ambulatory phases. During hyperaminoacidaemia, the rates of oxygen consumption were 96 ± 3 and 97 ± 4 μmol min−1 (kg LBM)−1, in the bed rest and ambulatory phases, respectively, whereas the rates of CO2 production were 79 ± 2 and 83 ± 2 μmol min−1 (kg LBM)−1 in the bed rest and ambulatory phases, respectively.
Table 1. Plasma amino acid concentrations at the end of the ambulatory and bed rest periods in the basal postabsorptive state and during amino acid (AA) infusion
| ||Ambulatory||Bed rest|
|Aspartate|| 4 ± 0.3|| 4 ± 0.2|| 4 ± 0.3|| 4 ± 0.2|
|Glutamate|| 54 ± 4||59 ± 5 ||52 ± 5||56 ± 5 |
|Asparagine|| 50 ± 3||43 ± 2*||50 ± 2||44 ± 2*|
|Serine||107 ± 5||223 ± 4* ||111 ± 4 ||234 ± 5* |
|Glutamine|| 636 ± 18||656 ± 20*||607 ± 12||650 ± 11*|
|Histidine|| 96 ± 4||143 ± 7* ||95 ± 4||146 ± 6* |
|Glycine||213 ± 7||556 ± 20*||220 ± 8 ||578 ± 19*|
|Threonine||142 ± 6||230 ± 6* ||140 ± 5 ||230 ± 5* |
|Arginine|| 83 ± 4||215 ± 14*||76 ± 5||207 ± 11*|
|Alanine|| 301 ± 21||423 ± 23*|| 264 ± 18†||414 ± 23*|
|Tyrosine|| 60 ± 2||55 ± 2*||60 ± 3||58 ± 2 |
|Methionine|| 24 ± 1||117 ± 5* ||25 ± 1||115 ± 5* |
|Valine||226 ± 9||476 ± 20*|| 240 ± 10†||510 ± 21*|
|Phenylalanine|| 65 ± 2||147 ± 4* ||70 ± 2||152 ± 6* |
|Isoleucine|| 58 ± 3||215 ± 11*|| 64 ± 4†||225 ± 10*|
|Leucine||140 ± 5||335 ± 15*||153 ± 6†||356 ± 15*|
|Lysine|| 209 ± 16||358 ± 27*|| 204 ± 17 ||356 ± 23*|
|Branched chain amino acids|| 424 ± 16||1026 ± 45* || 457 ± 20†||1091 ± 45* |
|Total amino acids||2511 ± 58||4302 ± 124*||2480 ± 57 ||4387 ± 114*|
Plasma [13C]KIC tracer/tracee ratios (Fig. 1) and breath 13CO2 tracer/tracee ratios were at steady state in the basal postabsorptive period and at the end of amino acid infusion. Table 2 shows the results of intracellular whole-body protein kinetics as determined by the l[1–13C]leucine tracer data and the reciprocal pool model. In the basal postabsorptive state, intracellular Ra from proteolysis tended to be lower (P < 0.10) during bed rest than in the ambulatory condition. Leucine oxidation was similar in the bed rest and ambulatory periods. In contrast, non-oxidative leucine disposal, an index of whole-body protein synthesis, was 6 ± 2% lower during bed rest. Following amino acid infusion, leucine Ra from proteolysis was significantly suppressed during bed rest and in the ambulatory condition. Leucine oxidation significantly increased in both conditions. Leucine utilization for protein synthesis increased by 35 ± 2% in the ambulatory condition, whereas it increased only by 30 ± 2% (P < 0.05) during bed rest. During amino acid infusion, the rate of net protein deposition, i.e. protein synthesis minus protein degradation, was 8 ± 3% lower during bed rest than in the ambulatory condition (Fig. 2).
Figure 1. Plasma tracer/tracee ratios at steady-state Plasma [13C]KIC tracer/tracee ratios during primed-continuous l[1-13C]leucine infusion in the basal postabsorptive period (min 160, 170 and 180) and at the end of amino acid infusion (min 340, 350 and 360) in the bed rest and ambulatory phases.
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Table 2. Effects of amino acid infusion (AA) on whole-body intracellular protein kinetics at the end of the ambulatory and bed rest periods
| ||Leucine Ra from proteolysis||Leucine Rd to oxidation||Leucine Rd to protein synthesis|
|Ambulatory||Basal|| 2.47 ± 0.05 ||0.23 ± 0.01 ||2.24 ± 0.05 |
| ||AA|| 2.11 ± 0.07* ||0.54 ± 0.02* ||3.03 ± 0.08* |
| ||Δ||−0.36 ± 0.05 ||0.31 ± 0.02 ||0.79 ± 0.05 |
|Bed rest||Basal|| 2.36 ± 0.05† ||0.25 ± 0.01 ||2.11 ± 0.04‡ |
| ||AA|| 1.89 ± 0.06*‡||0.64 ± 0.04*†||2.73 ± 0.07*‡|
| ||Δ||−0.47 ± 0.04 ||0.38 ± 0.04 ||0.63 ± 0.04‡ |
Figure 2. Leucine deposition into protein Rates of net leucine deposition (i.e. Rd to protein synthesis minus Ra from proteolysis) into body protein during amino acid infusion in ambulatory and bed rest conditions. *P < 0.05, bed rest versus ambulatory.
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We have assessed the response of whole-body protein kinetics to 14 days of bed rest in the postabsorptive state and during hyperaminoacidaemia in normal young volunteers using a cross-over experimental design. This approach allowed optimal control and monitoring of dietary intake and of levels of physical activity in both the ambulatory and the bed rest study phases. Subjects received a diet tailored for energy intake that maintained body weight at constant values in both study phases. Daily protein intake was fixed at 1 g (kg body weight−1). The results showed that an impaired amino acid-mediated stimulation of whole-body protein synthesis is a major catabolic mechanism for the effect of short-term bed rest on protein metabolism. Thus, the process of body protein synthesis become resistant to the anabolic stimulus of feeding during muscle inactivity, whereas protein balance is maintained in the fasted state, despite a reduction of whole-body protein turnover.
Our data in the postabsorptive state are in excellent agreement with previous human studies assessing changes in protein kinetics during bed rest using stable isotopes of amino acids. Ferrando et al. (1996) showed that after 14 days of bed rest postabsorptive values of muscle protein synthesis were decreased, while muscle protein balance did not significantly change. Also, at the whole-body level, short-term bed rest studies have demonstrated no change of protein balance in the postabsorptive state (Shangraw et al. 1988; Stuart et al. 1990; Lovejoy et al. 1999; Stein et al. 1999). We investigated for the first time the regulation of whole-body protein kinetics by hyperaminoacidaemia during bed rest. Amino acids were given intravenously for 3 h to simulate physiological postprandial conditions in the metabolic steady state. We have shown that, at the same level of hyperaminoacidaemia, net protein deposition was 8% lower during bed rest than in ambulatory conditions. This alteration was completely accounted for by less efficient stimulation of protein synthesis, while protein degradation was normally suppressed by amino acid infusion during bed rest.
Human studies clearly indicate that decreased protein synthesis is the main protein catabolic mechanism associated with muscle inactivity during bed rest (Ferrando et al. 1996) and in a microgravity environment (Stein et al. 1999). Stein et al. (1999) demonstrated that a 3 month space flight was associated with a 45% decrease in whole-body protein synthesis when compared with pre-flight rates. In contrast to protein synthesis, the rates of proteolysis appeared to be mostly unaffected or even slightly decreased by muscle unloading both at muscle and whole-body levels (Shangraw et al. 1988; Stuart et al. 1990; Ferrando et al. 1996; Lovejoy et al. 1999; Stein et al. 1999). Such decreased protein turnover associated with muscle unloading could contribute to the negative effects of immobility by delaying removal of defective proteins and impairing regulation of enzymatic systems and other metabolic processes. When acceleration of proteolysis was observed during short-term space flights (Stein & Schluter, 1997), this alteration was associated with the effect of stress mediators, e.g. cortisol (Ferrando et al. 1999).
We have recently shown that the protein anabolic effect of hyperaminoacidaemia was enhanced by previous performance of resistance exercise (Biolo et al. 1997). Taking this observation together with the results of the present study, we suggest that amino acid administration and, possibly, protein intake may interact with any level of physical activity by increasing stimulation of tissue protein synthesis in the case of exercise or by decreasing stimulation of tissue protein synthesis in the case of muscle inactivity, possibly at the level of skeletal muscle. The mechanisms of such an interaction between amino acid availability and levels of physical activity are unclear. They may include modulation of insulin sensitivity, peripheral blood flow and rates of amino acid delivery to tissues by exercise or muscle inactivity (Ferrando et al. 1996; Biolo et al. 1997).
In agreement with data obtained during short-term human space flight (Stein & Schluter, 1999), in our study we observed a selective increase in the plasma concentrations of the branched chain amino acids leucine, valine and isoleucine in the postabsorptive state during bed rest. Mechanisms leading to such an altered amino acid pattern following muscle unloading are unclear because the rate of leucine oxidation was not decreased during bed rest in our study. Nonetheless, due to the potential role of leucine in the regulation of tissue protein synthesis (Kimball & Jefferson, 2002), the bed rest-induced inhibition of protein synthesis appeared even greater when normalized for the prevailing value of plasma leucine concentration.
In the present study energy intake was carefully tailored to the resting energy expenditure of individual subjects and to the level of physical activity. Achievement of an energy balance throughout the experimental periods was demonstrated by the fact that the body weight and fat mass of the subjects did not change significantly during either the bed rest or the ambulatory periods. Despite the fact that energy balance was achieved both in the bed rest and the ambulatory phases, the difference in absolute energy content between the two groups (i.e. 30% of the basal metabolic rate) could have contributed to decreasing protein turnover rates during bed rest without affecting protein balance.
In agreement with the results of previous, diet-controlled, short-term bed rest studies (Ferrando et al. 1999; Lovejoy et al. 1999; Blanc et al. 2000), the whole-body lean mass of our subjects, as determined by DEXA, did not change significantly following 14 days of bed rest. Unfortunately, as a result of technical problems, we could not determine regional changes in lean body mass by DEXA. Thus, we cannot rule out the possibility that small changes in volume of selected muscles were not detected by our whole-body DEXA determinations, as previously shown in short-term bed rest using different techniques (Stuart et al. 1990). Nonetheless, on the basis of our leucine kinetic data we may predict a loss of lean body mass during the 14 days of bed rest of our study. We found that during amino acid infusion 57 ± 3 or 63 ± 2% of the administered leucine was incorporated into body protein during bed rest or the ambulatory phase, respectively. When these figures are applied to the level of daily protein intake adopted in our study (i.e. 1 g (kg body weight)−1 for both study phases) we may predict a negative difference between cumulative postprandial protein anabolism during the 14 days of bed rest and the ambulatory phase of approximately 67 ± 27 g of body protein, which was not compensated for in the postabsorptive state. Such a change is not easily detectable at the whole-body level using DEXA.
Daily protein intake was kept constant at the level of 1 g (kg body weight)−1 in both the bed rest and ambulatory phases. In a previous study, Stuart et al. showed that increasing the protein content in the diet from 0.6 to 1.0 g protein kg−1 day−1 prevented the nitrogen loss and the decrease in whole-body protein turnover associated with 7 days of bed rest at the low protein intake (Stuart et al. 1990). In the present study, 14 days of bed rest at a protein intake level of 1.0 g kg−1 day−1 depressed whole-body protein turnover in the postabsorptive state and impaired the anabolic effect of amino acid administration. Taken together, these results suggest the hypothesis that during short-term inactivity a greater than normal protein intake could be required to maintain normal protein turnover in the postabsorptive state and to achieve the same postprandial anabolic effect as are observed in subjects undertaking a normal level of physical activity.
The calculation of leucine oxidation requires a correction for the fact that not all labelled CO2 produced from [1-13C]leucine at the cellular level is excreted in the breath. Recovery in the breath of labelled CO2 increases during feeding and exercise proportionally to oxygen consumption (Wolfe, 1992; Leijssen & Elia, 1996). Labelled CO2 can be stored in bone or contribute to various metabolic pathways, including isotopic exchange in the trycarboxylic acid pool. The general experimental design of the STBR-IP did not allow us to determine the effect of bed rest and amino acid infusion on labelled CO2 recovery during labelled bicarbonate infusion on separate study days. Thus, we have assumed constant correction factors in the ambulatory and bed rest phases of 0.74 in the postabsorptive state and of 0.84 during amino acid infusion (Leijssen & Elia, 1996). Several lines of evidence indicate that bed rest should not have significantly affected the bicarbonate recovery factor. First, it has been shown that there is a direct relationship between metabolic rate and bicarbonate recovery in both physiological and pathological conditions (Wolfe, 1992). In our study, values of oxygen consumption in the postabsorptive state and during amino acid infusion were not significantly different in the ambulatory and bed rest phases and exhibited similar increments during hyperaminoacidaemia in both phases. We may predict therefore similar bicarbonate recovery in the two conditions. Second, values of CO2 production in the postabsorptive state and during amino acid infusion were also not significantly different in the ambulatory and bed rest phases, suggesting that the bicarbonate pool was similar in the two conditions. Third, evidence indicates that labelled CO2 recovery increases during exercise. In our study, assessment of leucine kinetics was performed with subjects lying in bed, even during the ambulatory phase. Nonetheless, in the case of changes in bicarbonate recovery in the bed rest phase, such changes would be expected to be in the opposite direction to changes during exercise. In the case of a decrease in bicarbonate recovery during bed rest, amino acid-mediated stimulation of whole-body leucine Rd to protein synthesis would have been even greater in the ambulatory phase than during bed rest. Finally, we have calculated the impact of a 15% increase in bicarbonate recovery during bed rest both in the postabsorptive state (i.e. from 0.74 to 0.87) and during hyperaminoacidaemia (i.e. from 0.84 to 0.97) on amino acid-mediated stimulation of whole-body leucine Rd to protein synthesis. Even assuming such large difference in bicarbonate kinetics between the two conditions, the amino acid-mediated stimulation of whole-body leucine Rd to protein synthesis would still have been 13 ± 5% greater (P= 0.04) in the ambulatory phase than during bed rest.
In conclusion, we have investigated the regulation of whole-body protein kinetics in the postabsorptive state and in an experimentally controlled postprandial state during short-term bed rest. Results indicated that, despite a reduction of whole-body protein turnover, protein balance was maintained in the fasted state. In contrast, a blunted amino acid-induced stimulation of protein synthesis appeared to be the main catabolic mechanism associated with short-term inactivity.