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* Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033 and
Mead Johnson Nutritionals, Evansville, IN 47721
2To whom correspondence should be addressed. E-mail: jjefferson{at}psu.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • translation initiation • gastrocnemius muscle • mTOR
Ingestion of a mixed meal typically stimulates skeletal muscle protein synthetic rates in food-deprived animals (1,2). However, consumption of a protein-deficient meal does not elicit this response (3,4) and it is now clear that an adequate supply of amino acids is essential for feeding-induced changes in skeletal muscle protein synthesis. Moreover, recent studies suggest that it is the supply of branched-chain amino acids, and leucine in particular, that modulates the protein synthetic response in skeletal muscle to meal feeding (5,6).
Both in vitro and in vivo experiments have demonstrated that the mechanism(s) whereby leucine ingestion stimulates skeletal muscle protein synthesis involves the enhancement of mRNA translation initiation rates (7–9). However, leucine stimulates insulin secretion (10–12) and when administered in large doses, as is the case with the majority of studies performed to date, it causes a transient but significant increase in serum insulin concentrations (13). The stimulatory effects of insulin on mRNA translation initiation in skeletal muscle have been well documented (14–21) and as such, it has been difficult to characterize the direct contribution of leucine to the stimulation of skeletal muscle protein synthesis in vivo.
A recent study demonstrated that protein synthetic rates are significantly elevated in the gastrocnemius and plantaris muscles of diabetic rats 1 h following oral administration of a large dose of leucine (1.35 g/kg body wt) in comparison with diabetic controls (22). The change in protein synthesis was associated with alterations in phosphorylation or function of proteins associated with the regulation of mRNA translation initiation; however, the magnitude of the change in both protein synthetic rate and initiation factors was smaller than that observed in nondiabetic rats administered leucine. In an additional study, alterations in mRNA translation initiation factors were observed 30 min following oral leucine administration (1.35 g/kg body wt) when food-deprived rats were infused with somatostatin to inhibit insulin release and maintain serum insulin concentrations at fasting levels (13). There was not, however, a significant increase in protein synthetic rates in the gastrocnemius and plantaris muscles in rats administered somatostatin. Thus it appears that the oral administration of large doses of leucine can stimulate mRNA translation initiation in skeletal muscle of food-deprived rats independently of increased serum insulin concentrations. However, leucine-induced increases in circulating insulin appear to be necessary to elevate synthetic rates above values observed under food-deprived conditions.
The dose of leucine employed in the aforementioned studies is quite large, equivalent to that consumed in a 24-h period by age- and strain-matched rats when allowed free access to standard lab chow (9). Due to the relative insolubility of leucine, such a dose is unlikely to be compatible with human studies. Thus, the aim of the present study was to define the minimal dose of leucine required to stimulate protein synthesis in skeletal muscle and to identify the biomarkers of mRNA translation that mediate the response.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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200 g) were maintained on a 12-h light:dark cycle with a standard diet (Harlan-Teklad Rodent Chow 8604) and water provided ad libitum. Experimental design. Rats were food deprived for 18 h prior to experimentation. A suspension of 54.0 g L-leucine/L water was prepared and rats were administered measured volumes corresponding to 0.068 (5%, n = 12), 0.135 (10%, n = 12), 0.338 (25%, n = 8), 0.675 (50%, n = 10), and 1.35 g L-leucine/kg body wt (100%, n = 12) by oral gavage. Because the administered leucine was in the form of a suspension, rats were divided into their respective groups based on serum leucine concentrations. Rats in which serum leucine values differed significantly from the mean were removed from the study. The highest concentration of L-leucine employed is equivalent to that consumed in a 24-h period by age- and strain-matched rats when allowed free access to standard lab chow (9). Control rats (n = 12) were administered 0.155 mol/L NaCl at a volume of 2.5 mL/100 g body wt. This volume of saline is equivalent to the volume of leucine suspension administered to rats in the 100% leucine group and was chosen to control for any possible volume-induced effects of oral gavage, i.e., gastric expansion-induced signaling. There were fewer rats in the 25 and 50% groups because this study represents 2 separate experiments, wherein the first experiment did not include these groups.
Administration of metabolic tracer and sample collection. Twenty minutes following oral gavage, a flooding dose (1.0 mL/100 g body wt) of L-[2,3,4,5,6-3H]phenylalanine (150 mmol/L, containing 3.70 GBq/L) was administered via tail vein injection for the measurement of protein synthesis (23). Rats were killed by decapitation 10 min later. Serum was obtained from trunk blood by centrifugation at 1800 x g for 10 min at 4°C. The right gastrocnemius and plantaris muscles were quickly excised as 1 unit (hereafter referred to as gastrocnemius) and homogenized in 7 vol of buffer consisting of 20 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), 100 mmol/L KCl, 0.2 mmol/L EDTA, 2 mmol/L ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mmol/L dithiothreitol, 50 mmol/L sodium fluoride, 50 mmol/L ß-glycerophosphate, 0.1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, and 0.5 mmol/L sodium vanadate. An aliquot (0.5 mL) of the homogenate was used for the measurement of muscle protein synthesis as described under Measurement of muscle protein synthesis. The remainder of the homogenate was immediately centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was used for analysis of mRNA translation initiation factors as described under Analysis of initiation factor phosphorylation state and Analysis of eIF4E complexes. The remaining tissue was used to assess eIF2B activity as described under Analysis of eIF2B activity.
Serum measurements. Serum insulin concentrations were analyzed using a commercial RIA kit for rat insulin (Linco Research). Serum leucine concentrations were analyzed by derivatization with phenylisothiocyanate and HPLC analysis as described previously (24).
Measurement of muscle protein synthesis. Fractional rates of protein synthesis were assessed from the rate of incorporation of radioactive phenylalanine into total mixed muscle protein as described previously (25). The time from injection of the metabolic tracer until homogenization of the muscle was recorded as the actual time for radiolabeled phenylalanine incorporation.
Analysis of initiation factor phosphorylation state. Phosphorylation of eukaryotic initiation factor (eIF)3 4G, 4E-binding protein 1 (4E-BP1), and ribosomal protein S6 kinase (S6K1) at Thr389 was evaluated in 10,000 x g supernatants of muscle homogenates by protein immunoblot analysis as described previously (26–28).
Analysis of eIF4E complexes. eIF4E was immunoprecipitated from 10,000 x g supernatants using a monoclonal eIF4E antibody (28). Samples were subjected to immunoblot analysis using polyclonal antibodies to either 4E-BP1 or eIF4G to determine the association of 4E-BP1 and eIF4G with eIF4E, respectively (28).
Analysis of eIF2B activity. The guanine nucleotide exchange activity of eIF2B was assessed as the rate of exchange of [3H]GDP bound to eIF2 for nonradioactively labeled GDP as described previously (15).
Statistical analysis. Data are means ± SEM. Statistical outliers within each treatment group were identified using a Grubbs test (GraphPad Software) and removed. All remaining data were analyzed by the InStat Version 3 statistical software package (GraphPad Software). Correlation coefficients were determined by Pearson correlation test. Means were compared using a one-way ANOVA. When ANOVA indicated a significant overall effect, differences among individual means were assessed using the Sidak test for multiple comparisons as described previously (29). Differences with P values < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Translation initiation may also be regulated through the binding of mRNA to a protein complex, composed of eIF4A, eIF4E, and eIF4G and referred to as eIF4F, which facilitates transport of mRNA to the 40S ribosomal subunit (32). The formation of eIF4F can be limited by sequestration of one of the component proteins of the eIF4F complex, i.e., eIF4E, by the eIF4E binding proteins (4E-BPs) (33). To evaluate the effect of oral leucine administration on eIF4F assembly in the gastrocnemius, coimmunoprecipitation experiments were performed. As demonstrated in Fig. 3A, the disassociation of 4E-BP1 from eIF4E 30 min following leucine administration preceded the observed changes in protein synthetic rates, with significant differences existing between controls and rats administered doses of leucine as low as 5%. The ability of 4E-BPs to sequester eIF4E is demarcated by their phosphorylation state, because hyperphosphorylation of the 4E-BPs results in a decreased binding affinity for eIF4E (34). Accordingly, SDS-PAGE analysis revealed that 4E-BP1 phosphorylation was increased in the gastrocnemius of rats fed leucine compared to controls (Fig. 3B), with significant differences from controls observed at all doses of leucine aside from the 5% dose.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Similar to what has been reported previously (13), the synthesis rate of total mixed protein in the gastrocnemius of 18-h food-deprived rates was 43% greater than that of controls 30 min after oral administration of leucine at the 100% dose. Interestingly, protein synthesis rates were also enhanced in rats administered the 10% dose. It has been hypothesized (13) that a transient increase in serum insulin concentrations is necessary for the enhancement of protein synthetic rates in the gastrocnemius of food-deprived rats following oral administration of leucine. In this study, however, synthetic rates in the gastrocnemius were significantly greater than control values 30 min following leucine administration even with low doses of leucine that did not induce a significant insulin release. The results therefore suggest that leucine can stimulate skeletal muscle protein synthesis in food-deprived rats without concomitant increases in circulating insulin. Interestingly, similar results were attained in a recent study on the regulation of skeletal muscle protein synthesis in neonatal pigs infused with low doses of leucine (40).
A possible explanation for the leucine-induced insulin-independent changes in skeletal muscle protein synthesis observed in this study, but not in the study by Anthony et al. (13), is that the stimulation of skeletal muscle protein synthesis by leucine is transient (13). As such, significant changes in protein synthesis may be observable 30 min following leucine administration, as in the present study, but not at 1 h, as in the prior study. This suggestion is supported by the recent observation that 30 min following the administration of the leucine analogue norleucine, protein synthesis in the gastrocnemius of food-deprived rats is enhanced to the same extent as in rats administered leucine, but without an increase in serum insulin (41). A cautionary note should be made, however, because it is possible that insulin secretion induced by small doses of leucine may occur more rapidly than with large doses of leucine and, therefore, that a transient leucine-induced increase in circulating insulin may contribute to these results.
Independent of dose, leucine appears to mediate its effect on skeletal muscle protein synthesis, at least in part, via the regulation of mRNA translation initiation. Of the biomarkers of mRNA translation examined in the present study, the one that correlated best with the observed changes in protein synthesis was the amount of eIF4G associated with eIF4E. Both protein synthesis and eIF4G binding to eIF4E were slightly, but not significantly (P = 0.67 and 0.25), increased at the lowest leucine dose tested and both were maximally changed at 10% of the highest tested dose of the amino acid, i.e., when there is a relatively small increase in serum leucine concentrations, similar to that observed following intake of a complete meal. The other biomarkers examined were also affected by leucine concentrations in the 5–25% range, but were further altered in response to higher doses. Although changes in the other biomarkers did not correlate directly with altered rates of global protein synthesis or eIF4G association with eIF4E, a role for such changes in the regulation of mRNA translation cannot be eliminated. Indeed, it is likely that the observed changes in eIF4G phosphorylation and 4E-BP1 association with eIF4E contribute to the enhanced binding of eIF4G to eIF4E observed at lower leucine doses. Moreover, and as with changes in circulating insulin concentrations, it should be noted that the measurements in this study represent but a snapshot of possible effects induced by leucine administration. Thus it is possible that changes in some biomarkers occurred soon after leucine administration, but returned to control levels by 30 min. These changes would have also contributed to the observed changes in protein synthetic rate. A more thorough assessment of the contribution of translational control mechanisms to leucine-stimulated protein synthesis will therefore require earlier time course data.
Unlike protein synthesis and eIF4G binding to eIF4E, hyperphosphorylation of 4E-BP1 and phosphorylation of S6K1 on Thr389 were directly correlated with serum leucine concentration. However, the serum leucine concentration measured at the highest dose is several-fold greater than that observed after a complete meal and is thus supraphysiological. The finding that supraphysiological serum leucine concentrations directly correlate with enhanced phosphorylation of 4E-BP1 and S6K1 would argue against the effects being mediated through a receptor-based mechanism, which would be expected to exhibit maximal activation at leucine concentrations near, or slightly above, physiological levels. This is particularly relevant for 4E-BP1 phosphorylation because its only known function is to promote release of the protein from the 4E-BP1 · eIF4E complex to allow assembly of the eIF4G · eIF4E complex. In contrast, the finding that protein synthesis and eIF4G association with eIF4E are maximally stimulated at a physiological serum leucine concentration would be consistent with these events being regulated through a receptor-mediated process.
A potential explanation for the lack of correlation between leucine signaling to 4E-BP1 and changes in eIF4G association with eIF4E is that the amount of eIF4G available for formation of the eIF4G · eIF4E complex is limiting (42) and that release of a fraction of total eIF4E from the 4E-BP1 · eIF4E complex provides enough eIF4E to promote maximal formation of the eIF4G · eIF4E complex. Alternatively, eIF4G binding to eIF4E may be differentially regulated. For example, administration of the drug rapamycin, a highly specific inhibitor of the kinase mammalian target of rapamycin (mTOR), to rats in vivo completely abrogates the leucine-induced dissociation of 4E-BP1 from eIF4E in skeletal muscle, whereas the leucine-induced association of eIF4G with eIF4E is only partially inhibited (43). Thus whereas an mTOR-dependent pathway regulates the association of eIF4E with 4E-BP1, eIF4E’s association with eIF4G is regulated by both mTOR-dependent and -independent pathways and a differential response in the association of eIF4G and 4E-BP1 with eIF4E in this study is not totally unexpected. Several biomarkers examined in the present study appear to exhibit a biphasic response to leucine administration, reaching a plateau at low to moderate doses of the amino acid and increasing further as the leucine dose is increased. For example, 4E-BP1 association with eIF4E is significantly decreased at the 5% leucine dose and then remains constant until 50%, when it decreases further. Phosphorylation of eIF4G on Ser1108 exhibits a similar pattern of change. Interestingly, for both biomarkers the dose of leucine that elicits the second change (i.e., 50%) also results in a significant increase in serum insulin concentration. Thus one explanation for the apparent biphasic response of eIF4G phosphorylation and 4E-BP1 association with eIF4E is that insulin released in response to provision of higher leucine concentrations mediates the changes observed at 50 and 100% doses. In support of this idea, leucine and insulin act synergistically to enhance S6 phosphorylation in skeletal muscle of neonatal pigs (44) and humans (45).
Although leucine-induced insulin secretion was not associated with significant increases in skeletal muscle protein synthetic rates in the current study, this rise in circulating insulin may still affect mRNA translation. For example, the translational control mechanisms initiated by leucine-induced insulin secretion may affect the translation of a subset of mRNAs, whose increased synthesis alone would not be detectable by the methods employed in this study. For example, mRNAs having highly structured 5'-untranslated regions appear to be preferentially translated under conditions of enhanced eIF4F assembly (49–51). Pertinently, several mRNAs encoding proteins that regulate cell growth and development have such untranslated structures.
In conclusion, the oral administration of low doses of leucine effectively stimulates skeletal muscle protein synthesis in food-deprived mature rats. Because proteins that facilitate both the transportation of mRNA to the 40S ribosomal subunit and the synthesis of the translational apparatus itself are affected by low-dose leucine administration, changes in the control of mRNA translation initiation likely contribute to this stimulation of protein synthesis. Although the study is limited in its ability to assess which pathways mediate the observed changes in translational control and protein synthesis, it does demonstrate that activation, or perhaps more correctly the degree of activation, of these pathways is dependent upon the dose of leucine administered. These results also suggest that the small increases in circulating insulin levels arising from the administration of high doses of leucine may further stimulate mRNA translation in the skeletal muscle of food-deprived mature rats. That the oral administration of small doses of leucine can stimulate skeletal muscle protein synthesis in food-deprived rats suggests that future research on the regulation of skeletal muscle protein synthesis by orally administered leucine will be feasible in humans. It has been suggested previously that leucine administration may prove to be an effective therapy for conditions such as type 2 diabetes, trauma, and infection that are characterized by both insulin-resistant and skeletal muscle wasting (45). The results of this study support this proposal, but further studies will be required to elucidate how long-term leucine administration affects protein turnover and skeletal muscle mass in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: eIF, eukaryotic initiation factor; 4E-BP1, 4E-binding protein 1; mTOR, mammalian target of rapamycin; S6K1, ribosomal protein S6 kinase; SAL, saline-treated controls; TOP mRNA, terminal oligopyrimidine sequence.
Manuscript received 19 November 2004. Initial review completed 5 December 2004. Revision accepted 31 December 2004.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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  P. J. Morrison, D. Hara, Z. Ding, and J. L. Ivy Adding protein to a carbohydrate supplement provided after endurance exercise enhances 4E-BP1 and RPS6 signaling in skeletal muscle J Appl Physiol, April 1, 2008; 104(4): 1029 - 1036. [Abstract] [Full Text] [PDF]
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 T. C. Vary Acute Oral Leucine Administration Stimulates Protein Synthesis during Chronic Sepsis through Enhanced Association of Eukaryotic Initiation Factor 4G with Eukaryotic Initiation Factor 4E in Rats J. Nutr., September 1, 2007; 137(9): 2074 - 2079. [Abstract] [Full Text] [PDF]
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 R. A. Frost, G. J. Nystrom, L. S. Jefferson, and C. H. Lang Hormone, cytokine, and nutritional regulation of sepsis-induced increases in atrogin-1 and MuRF1 in skeletal muscle Am J Physiol Endocrinol Metab, February 1, 2007; 292(2): E501 - E512. [Abstract] [Full Text] [PDF]
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T. C. Vary and C. J. Lynch Meal Feeding Stimulates Phosphorylation of Multiple Effector Proteins Regulating Protein Synthetic Processes in Rat Hearts J. Nutr., September 1, 2006; 136(9): 2284 - 2290. [Abstract] [Full Text] [PDF]
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 I. Rieu, M. Balage, C. Sornet, C. Giraudet, E. Pujos, J. Grizard, L. Mosoni, and D. Dardevet Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia J. Physiol., August 15, 2006; 575(1): 305 - 315. [Abstract] [Full Text] [PDF]
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T. C. Vary and C. J. Lynch Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation Am J Physiol Endocrinol Metab, April 1, 2006; 290(4): E631 - E642. [Abstract] [Full Text] [PDF]
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J. Escobar, J. W. Frank, A. Suryawan, H. V. Nguyen, S. R. Kimball, L. S. Jefferson, and T. A. Davis Regulation of cardiac and skeletal muscle protein synthesis by individual branched-chain amino acids in neonatal pigs Am J Physiol Endocrinol Metab, April 1, 2006; 290(4): E612 - E621. [Abstract] [Full Text] [PDF]
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 S. R Kimball and L. S Jefferson New functions for amino acids: effects on gene transcription and translation Am. J. Clinical Nutrition, February 1, 2006; 83(2): 500S - 507S. [Abstract] [Full Text] [PDF]
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L. E. Norton and D. K. Layman Leucine Regulates Translation Initiation of Protein Synthesis in Skeletal Muscle after Exercise J. Nutr., February 1, 2006; 136(2): 533S - 537S. [Abstract] [Full Text] [PDF]
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S. R. Kimball and L. S. Jefferson Signaling Pathways and Molecular Mechanisms through which Branched-Chain Amino Acids Mediate Translational Control of Protein Synthesis J. Nutr., January 1, 2006; 136(1): 227S - 231S. [Abstract] [Full Text] [PDF]
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H. Kobayashi, H. Kato, Y. Hirabayashi, H. Murakami, and H. Suzuki Modulations of Muscle Protein Metabolism by Branched-Chain Amino Acids in Normal and Muscle-Atrophying Rats J. Nutr., January 1, 2006; 136(1): 234S - 236S. [Abstract] [Full Text] [PDF]
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D. K. Layman and D. A. Walker Potential Importance of Leucine in Treatment of Obesity and the Metabolic Syndrome J. Nutr., January 1, 2006; 136(1): 319S - 323S. [Abstract] [Full Text] [PDF]
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-form, exhibits the slowest mobility. Phosphorylation of 4E-BP1 was assessed by Western blot analysis 30 min following administration of saline or leucine at doses ranging from 0.068 to 1.35 g leucine/kg body wt using a polycolonal antibody that recognizes phosphorylated and unphosphorylated forms of 4E-BP1 and expressed as the percentage of protein in the 
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