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Human Nutrition Research Centre of Clermont-Ferrand and Institut National de la Recherche Agronomique, Unité de Nutrition et Métabolisme Protéique, 63122 Ceyrat, France
1To whom correspondence should be addressed. E-mail: irieu{at}clermont.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • aging • muscle protein synthesis • leucine • chronic • rats
During aging, a decline in skeletal muscle mass occurs in both humans (1 ,2 ) and rodents (3 ). This atrophy is associated with a loss of muscle strength, which directly affects the mobility and health of elderly people. The mechanisms leading to sarcopenia are still unclear and result from an imbalance between rates of protein synthesis and degradation. This imbalance is not obvious when basal rates of protein turnover are measured, (4 –6 ) but is detected in the postprandial state. An apparent defect in the stimulation of muscle protein synthesis has been shown in old rats (4 ) and elderly humans (7 ) after the ingestion of a normal protein meal; this defect results in a daily small muscle protein loss, leading in the long term to muscle wasting. This reflects a progressive decrease in the response to feeding that is high during early life and decreases with development and aging (8 –10 ).
Amino acids, especially leucine, play an important role in the regulation of the postprandial stimulation of muscle protein synthesis (11 –13 ). We and others have demonstrated that leucine alone, at levels within the range of postprandial concentrations, is able to stimulate protein synthesis in incubated epitrochlearis muscles from young rats to the same extent as incubations with all amino acids (14 –15 ). Furthermore, leucine acts as a real mediator, i.e., it is able to stimulate specifically intracellular pathways linked to the translation of proteins. The "leucine signal" stimulates the mammalian target of rapamycin (mTOR) pathways, 70-kDa ribosomal protein S6 kinase (p70S6kinase) activity, and enhances eIF4E-binding protein (4EBP1) phosphorylation and the association of eukaryotic initiation factor (eIF)4E with eIF4G, both in vitro and in vivo (15 –19 ). In old rats, in vitro muscle protein synthesis becomes resistant to the stimulatory effect of leucine in the range of physiologic postprandial concentrations (15 ). This resistance has been correlated with a defect in the stimulation of p70S6K activity by this amino acid. However, this stimulation can be restored if leucine concentration is raised to supraphysiologic concentrations (15 ). On the basis of these observations, we recently examined the effect of a leucine-supplemented meal on the postprandial stimulation of muscle protein synthesis in old rats. We showed that a single leucine-supplemented meal corrected the defect of postprandial muscle protein synthesis stimulation in old rats, whereas it had no further beneficial effect in adult rats (20 ). The effect was due only to the increase in leucine availability to peripheral tissues because plasma leucine was increased twofold compared with a nonsupplemented meal, whereas all other amino acids as well as insulin levels did not differ from those of rats fed a control meal.
Taken together, these findings suggested that increasing leucine availability would be beneficial for maintaining muscle protein mass in elderly persons. However, the effect of leucine has to persist for a long time to contribute to muscle protein gain. This is questionable because adaptive processes may occur with time. Indeed, increasing dietary leucine intake has the following effects: 1) depresses food intake and limits growth in various species (21
–23
), 2) competes with valine and isoleucine for the L-carrier system (24
) and 3) stimulates catabolism of the three branched-chain amino acids (BCAA). The BCAA antagonism has been well described (25
–28
). The first reaction in the catabolism of BCAA is a reversible transamination catalyzed by a BCAA aminotransferase (EC 2.6.1.42). The branched chain 
-keto acids (BCKA) formed are then irreversibly decarboxylated by BCKA dehydrogenase (EC 1.2.4.4) (29
). Both enzyme activities are stimulated by an excess of leucine (30
,31
). Thus, an excess of leucine decreases plasma valine and isoleucine concentrations. Moreover, because leucine can modulate the expression of several genes (e.g., CCAAT/enhancer binding protein homologous protein and insulin-like growth factor binding protein 1) and metabolic pathways (e.g., proteolysis) (32
,33
), a deleterious effect of chronic high leucine intake has not been excluded.
To determine whether leucine could be used to prevent muscle protein loss in old subjects, we tested the effect of a chronic leucine-rich meal on postabsorptive and postprandial protein synthesis in adult and old rats in both gastrocnemius and soleus muscles. Maintaining normal levels of postprandial plasma valine and isoleucine concentrations presented a challenge.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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L-[1-13C] phenylalanine (99%) was purchased from Cambridge Isotope Laboratory (Andover, MA). Rabbit polyclonal anti-total p70S6K and goat polyclonal anti-4EBP1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-eIF4E was purchased from Transduction Laboratories (Lexington, KY). Anti-rabbit, anti-goat and anti-mouse horseradish peroxidase (HRP) were from Santa Cruz Biotechnology.
Animals and experimental design.
The animal facilities and protocol were approved by the local animal ethics committee. Male Wistar rats (Iffa-Credo, Lyon, France) aged 9 mo (adult) and 21 mo (old) were housed individually under controlled environmental conditions (temperature 22°C; 12-h dark period starting at 0800 h) and fed a semiliquid 18.2 g/100 g protein standard diet (Table 1 ); water was provided freely. Rats had free access to food from 0900 to 1700 h. They were acclimated for 1 mo to their surroundings and food intake was measured daily. Food consumption during the adaptation period was stable; adult and old rats ate 18–20 g dry matter daily, including 7–9 g during the first hour of feeding.
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The "leucine meal" was supplemented with leucine to increase plasma leucine concentration 100% relative to the normal postprandial level. To prevent the fall of plasma valine and isoleucine concentrations induced by leucine supplementation, the "leucine meal" also was supplemented with appropriate amounts of these amino acids (20 ). However, these supplementations were valid only for d 1 of the experiment. A pilot experiment was then performed for 18 d to determine how to adjust the BCAA supplements over time. Plasma leucine and isoleucine remained stable at the expected levels for 18 d, whereas valine increased significantly on d 5 (Fig. 1 ). Therefore, the amount of valine was reduced by 10% from d 5 to 10 in the present experiment. Alanine, an amino acid that has no apparent effect on muscle protein metabolism, was included in the control meal to render the meals isonitrogenous (34 ).
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On d 10, protein synthesis rates were assessed in gastrocnemius and soleus muscles from rats in the postabsorptive state (groups PA and PA leu) or within 150–180 min after the beginning of the experimental meals (groups PP and PP leu). Protein synthesis rates were measured using the flooding-dose method (35 ,36 ). As described and validated by Dardevet et al. (20 ), each rat was injected intravenously with L-[1-13C] phenylalanine (99%) (50 µmol/100 g body), 40 min before killing (i.e., 110–140 min after the beginning of the experimental meals), to flood the precursor pools with L-[1-13C] phenylalanine. Rats were then anesthetized with pentobarbital sodium; blood was rapidly collected and centrifuged at 3000 x g for 10 min. Gastrocnemius and soleus muscles were excised, weighed, frozen in liquid nitrogen and stored at -80°C until analysis. Free and bound phenylalanine enrichments were determined and measured as previously described by Dardevet et al. (20 ).
Plasma insulin and amino acid measurements.
On d 8, plasma insulin was measured in the postabsorptive state (groups PA and PA leu) or within 45–60 min after the beginning of the meal consumption (groups PP and PP leu). Under similar conditions, a previous experiment on the time course of plasma insulin secretion showed that a 45- to 60-min period yielded a better indication of postprandial plasma insulin levels (peak value at that time) than did the time of killing (150–180 min) when levels had returned to postabsorptive values (20 ). Blood samples were collected from the tail vein and plasma insulin concentration was measured by homologous RIA using a rat insulin standard (Rat Insulin RIA Kit, Linco Research, St. Charles, MO). The assay is 100% cross-reactive with rat insulin. The lowest limit of detection was 0.017 pmol/L. Intra- and interassay CV were 2.7 and 3.1%, respectively. Plasma amino acid concentrations were determined at the time of killing in each group as previously described by Dardevet et al. (20 ).
Calculations.
Protein fractional synthesis rate (FSR, in %/d) was calculated from the formula (35
):
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where Sb is the enrichment at time t (minus natural basal enrichment of protein) of the protein-bound phenylalanine, t is the incorporation time in d, and Sa is the mean enrichment of free tissue phenylalanine between time 0 and time t. The mean Sa enrichment was the Sa (t1/2) value calculated from the linear regression obtained in tissue between time 0 and time t. The absolute synthesis rate (ASR) was calculated from the product of FSR and the protein content of the tissue and expressed in mg/h. Protein synthetic capacity was estimated as the ratio of RNA to protein (mg RNA/g protein) because most of the RNA in tissues is ribosomal.
Analysis of eukaryotic initiation factors.
A portion of gastrocnemius stored at -80°C was homogenized in 7 volumes of 20 mmol/L Hepes, 100 mmol/L KCl, 0.2 mmol/L EGTA, 1 mmol/L dithiothreitol, 50 mmol/L NaF, 50 mmol/L ß-glycerophosphate, 0.1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine and 0.5 mmol/L Na3VO4, pH 7.4, buffer using a Polytron homogenizer. The homogenate was centrifuged at 10,000 x g for 10 min at 4°C. The protein content in the resultant supernatant was measured by the Bradford reaction.
Aliquots containing 50 µg (for 4EBP1 and eIF4E) or 20 µg (for total p70S6K) of total proteins were resolved by SDS-PAGE (15% for 4EBP1 and eIF4E or 7.5% for total p70S6K) and transferred to polyvinylidene difluoride (immobilon) membrane (1 h, 75V, at 4°C). Membranes were blocked with TBST buffer (20 mmol/L Tris-base, 500 mmol/L NaCl, 0.05% Tween 20) supplemented with 50 g/L nonfat powdered milk for 1 h and incubated overnight at 4°C with appropriate primary antibody. After extensive washing in TBST, membranes were exposed for 1.5 h at room temperature to the appropriate secondary antibody conjugated with HRP. The blots were developed using the enhanced chemiluminescence detection system (Amersham, Aylesbury, Bucks, UK) according to the manufacturer’s directions.
Statistical analysis.
Values are presented as mean ± SEM. Statistical evaluation of the synthesis capacity, FSR, ASR and plasma insulin and amino acid concentration data was performed by three-way ANOVA to analyze the effects of age, supplementation state (leucine or alanine) and nutritional state (postprandial and postabsorptive). A two-way ANOVA was used to analyze the effects of age and supplementation state on characteristics of the rats. When a significant overall effect was detected, differences among individual means were assessed with Student’s t tests. Paired t tests were used to analyze the plasma BCAA concentration changes during the leucine supplementation period (pilot experiment). The level of significance was set at P < 0.05 for all statistical tests.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Body weights of adult and old rats were similar at the beginning of the experiment (561 ± 10 and 589 ± 16 g, respectively) and did not vary during the experiment. Daily intakes of the experimental and control diets were similar in both groups of age for the first 9 d (Table 2 ). On the day of killing (d 10), rats in the postabsorptive state did not receive any food, whereas rats in the postprandial state consumed the same amount of food as on the previous days.
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The total protein content in gastrocnemius muscle was lower in old than in adult rats due to the atrophy of this muscle because no modification of protein concentration occurred (Table 2) . Leucine supplementation did not affect muscle protein concentration or total protein content in either age group.
Plasma amino acid concentrations.
In rats of both ages, postabsorptive plasma free amino acid concentrations did not differ between diet groups (PA leu vs. PA) (Table 3 ). Compared with the postabsorptive state, the alanine and leucine meals both significantly increased the plasma BCAA (leucine, valine, isoleucine) and methionine (PP vs. PA and PP leu vs. PA leu) concentrations in old and adult rats. Moreover, the alanine meal significantly increased plasma free threonine only in old rats, and the leucine meal decreased plasma free histidine and threonine in rats of both ages. However, in the postprandial state, only plasma free leucine differed significantly between diet groups. Indeed, leucine was markedly higher in the leucine groups than the alanine groups (+202 ± 28 and +157 ± 22 µmol/L in old and adult rats, respectively).
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Plasma insulin concentrations.
Postabsorptive plasma insulin concentrations did not differ among the four groups (Fig. 2 ). Feeding similarly increased plasma insulin in all groups. As a result, postprandial plasma insulin also did not differ among the groups.
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There was a significant interaction among age of rats, supplementation state and nutritional state (P < 0.014) (Table 4 ). Feeding adult rats the alanine meal significantly stimulated FSR in both gastrocnemius and soleus muscles (22 ± 2.4 and 19 ± 1.6%, respectively, compared with the postabsorptive value). Feeding adult rats the leucine meal also significantly increased the FSR in soleus (PP leu vs. PA leu) but not in the gastrocnemius (Table 4) . After 9 d of leucine supplementation, the postabsorptive FSR (PA leu) in gastrocnemius muscle had already reached the postprandial level and was not stimulated further. The ASR in gastrocnemius muscle yielded roughly the same results as the FSR (Fig. 3 ). In soleus muscle, no significant stimulation of the postprandial ASR was detected after the leucine meal. In adult rats, protein synthesis capacity was not involved in the changes in muscle protein synthesis because none of the groups differed (Table 4) .
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Western blot analysis.
Because it has been demonstrated that oral administration of leucine enhances the phosphorylation of 4EBP1 and p70S6K (18 ), we examined the phosphorylation of these two factors in each group of rats (Fig. 4 ). Irrespective of the composition of the meal, feeding increased the phosphorylation of 4EBP1 in both adult and old rats (PA vs. PP and PA leu vs. PP leu). Leucine supplementation appeared to increase the phosphorylation of 4EBP1 more than alanine (control meal) (PP leu vs. PP, in each group). Unfortunately, the quantification of the data was not possible. Feeding also increased the phosphorylation state of p70S6K in both adult and old rats. However, the effects of the leucine and alanine meals did not appear to differ in either age group. The eIF4E content did not differ among the four groups (Fig. 4) .
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2 times in both age groups after feeding the leucine meal, whereas this increase was only 30% after feeding the alanine meal. The pilot experiment indicated that postprandial leucine concentrations were constantly elevated during the 10 d of the study without obvious modification of the other BCAA (Fig. 1)
. The differences between adult and old rats in the response of muscle protein synthesis to leucine may be explained by the dose-response curves of leucine action on protein synthesis that we recorded in a previous study in incubated epitrochlearis muscles (15 ). We found that muscle protein synthesis in vitro still responded to leucine in old rats but at leucine concentrations one or two times greater than in young or adult rats. This indicated that at postprandial leucine levels, muscle protein synthesis was maximally stimulated in adult rats but poorly stimulated in old rats. This observation suggested that a leucine supplement was therefore needed to increase postprandial muscle protein synthesis in old rats in vivo (15 ,20 ).
Surprisingly, the chronic feeding study further revealed an improvement in postabsorptive skeletal muscle protein synthesis in adult rats fed the leucine-supplemented meal. This effect did not occur in old rats in which the postabsorptive muscle protein synthesis rate was similar in both diet groups. At that point, muscles of old rats were resistant to this specific effect of leucine. However, because an increase of leucine availability restored the response of muscle protein synthesis in the fed state in old rats, we hypothesize that the modifications induced by leucine during the postprandial period (i.e., enhancement of muscle protein translation machinery) were more transient than in adults and disappeared before the intake of the following leucine meal. This lack of a long-lasting effect of leucine may be a reflection of a defect in the regulation of muscle metabolism. Dysregulation of proteolysis and protein synthesis in muscles of old rats has been shown to occur due to glucocorticoid excess, food deprivation and exercise (4 ,37 ,38 ).
Chronic consumption of the leucine meal did not alter food intake in our study. Many published studies have shown that high leucine diets depress food intake, limit appetite and stop growth (21 –23 ). A deleterious effect of leucine on food intake was observed in rats only when they were fed a low protein diet (22 ,26 ). This was not the case in our experiment because we used a normal protein diet. Furthermore, in our study, rats were fed a leucine-rich meal for only 1 h and were fed normally during the rest of the day. Thus, our rats were hyperleucinemic for only a short period each day. Adjusting BCAA supplementation during the period of experimental feeding was a challenge. Indeed, the BCAA antagonism was visible as soon as the first leucine-supplemented meal was given (20 ). To prevent the fall of plasma valine and isoleucine concentrations induced by leucine excess, the "leucine meal" was also supplemented with valine and isoleucine (0.57 and 0.98 g/100 g dry matter, respectively) (39 ,40 ). Due to the significant increase in plasma free valine after 5 d, valine supplementation was slightly decreased thereafter (0.51 g/100 g dry matter). The origin of this phenomenon is not known.
Although leucine has been shown to stimulate insulin secretion, it does not seem likely that insulin could mediate the effect of leucine-supplemented meals in either old (stimulation of postprandial muscle protein synthesis) or adult rats (increase in postabsorptive muscle protein synthesis). Indeed, neither postabsorptive nor postprandial plasma insulin was influenced by the type of diet or the age of the rats. A similar conclusion was drawn from postprandial insulin kinetics in acute experiments (20 ). Moreover, it has been shown that leucine stimulates muscle protein synthesis through a signaling mechanism independent of insulin (19 ). However, it is important to emphasize that the presence of insulin seems to be necessary for the postprandial stimulation of muscle protein synthesis by amino acids (41 ,42 ).
The chronic feeding pattern with the leucine-supplemented meal restored stimulation of muscle protein synthesis during the feeding period in old rats. In agreement with our results, Arnal et al. showed that skeletal muscle protein synthesis was restored in old rats fed 4 meals containing 11, 66, 11 and 11% of dietary protein, respectively, instead of 4 equal meals containing 25% protein (43 ). In that experiment, a high portion of the daily leucine intake was consumed during the experimental meal (67% with the protein pulse pattern vs. 25% with the spread control pattern, instead of 66% with the leucine-supplemented meal vs. 30% with the alanine control meal in our experiment), suggesting that leucine availability might be the key to the restoration of postprandial muscle protein synthesis. Plasma free leucine showed higher incremental increases after the leucine-supplemented meal in our experiment than after the pulse-protein feeding because dietary leucine intakes were greater (0.69 g/d vs. 0.23 g/d). This suggests that smaller leucine supplements than those used in the present experiment would be sufficient to improve postprandial protein synthesis in old rats.
Leucine has been shown to act as a real mediator by stimulating intracellular signaling pathways involved in the efficiency of protein translation (i.e., mTOR pathways, p70S6K activity) (15 –19 ). In the present study, p70S6K was hyperphosphorylated after the meal (PP states) in both adult and old rats, but we did not detect a significant effect of leucine supplementation, suggesting that the beneficial effect of leucine supplementation did not occur through p70S6K activation. This discrepancy with earlier investigations may be explained by differences in nutritional conditions. In most published studies, leucine was given alone (without any other amino acids), inducing a dramatic increase in plasma leucine concentration (10 x basal level) which is undoubtedly not representative of results after a normal meal (17 ,18 ). To our knowledge, the present study is the first that considered the specific effect of leucine excess as part of a normal meal. By contrast, the increase in the phosphorylated form of 4EBP1 observed after feeding was more pronounced with leucine- than with alanine-supplemented meals, in agreement with previous data concerning the mechanism involved in the stimulation of protein synthesis by leucine (16 –19 ). However, this was associated with an increase in protein synthesis only in old rats, probably because basal protein synthesis (PA leu) was already stimulated in adults. 4EBP1 and p70S6K phosphorylation would not be the key step in the muscle protein synthesis modulation by leucine. Accordingly, chronic leucine supplementation of drinking water increased protein synthesis in muscle of fed young rats without any changes in signaling proteins (i.e., 4EBP1 and S6K1) (44 ). Further investigations will be required to determine exactly the mechanism by which leucine restores muscle protein synthesis after feeding in old rats, especially at the level of the eIF4E-eIF4G interaction, a key step in the stimulation of translation. Alternatively, we hypothesize that other steps may be involved. Kimball et al. (45 ) showed in L6 myoblasts that leucine regulated protein synthesis through modulation of eIF2B activity.
The beneficial effect of leucine-supplemented meals on protein synthesis in gastrocnemius did not limit loss of muscle proteins and muscle atrophy in old rats. Based on the data obtained (Table 4) , the gastrocnemius of leucine-supplemented rats was expected to synthesize an additional 0.2 mg protein/h. Assuming that the improvement of protein synthesis persisted over 4 h, gastrocnemius would have synthesized 8 mg protein/10 d, without taking into account protein degradation. This amount is too low to be detectable. If we consider that the proteolysis rate is 50%, a significant variation of muscle mass would be visible only after 3 mo of leucine supplementation. For the proteolysis pathways, it has been recently shown that oral administration of leucine rapidly suppressed protein degradation in skeletal muscle of young rats (46
) but no information was given in vivo in adult and old rats.
In conclusion, we have shown that the ability of a leucine-supplemented meal to restore postprandial muscle protein synthesis in old rats [previously described (20 )] persisted after a 10-d period. However, further experiments are warranted to determine the duration and level of leucine supplementation required to obtain protein gain in muscle without negative effects. Then, leucine supplementation may be considered to be a good alternative to high protein diets [which could have deleterious effects on renal function in the elderly (47 )] to limit muscle protein losses during aging. Moreover, we have shown that in adult rats, chronic daily leucine-supplemented meals enhance postabsorptive muscle protein synthesis to postprandial levels. If this process persists for a long time, feeding adult subjects a leucine-rich diet would be another way to prevent the progressive muscle loss.
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FOOTNOTES |
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-keto acids; 4EBP1, eIF4E-binding protein; eIF, eukaryotic initiation factor; FSR, fractional synthesis rate; HRP, horseradish peroxidase; mTOR, mammalian target of rapamycin kinase; p70S6K, 70-kDa ribosomal protein S6 kinase; PA, rats fed alanine meals and killed in the postabsorptive state; PA leu, rats fed leucine meals and killed in the postabsorptive state; PP, rats fed alanine meals and killed in the postprandial state; PP leu, rats fed leucine meals and killed in the postprandial state. 
Manuscript received 30 September 2002. Initial review completed 9 November 2002. Revision accepted 23 December 2002.
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