Supplemental dietary leucine and the skeletal muscle anabolic response to essential amino acidsnure_

Emerging Science Supplemental dietary leucine and the skeletal muscle anabolic response to essential amino acidsnure_420 550..557 Stefan M Pasiakos and James P McClung Skeletal muscle protein synthesis (MPS) is regulated by a number of dietary factors, to include essential amino acids (EAAs). Leucine, a branched-chain amino acid, has been identified as a stimulator of MPS in many cell culture and animal studies. However, whether supplemental leucine exerts a unique stimulatory effect, as compared to other EAAs, on muscle anabolism in humans has not been clearly demonstrated. A recent study found no improvement in resting MPS in adults who consumed a 10 g EAA supplement providing added leucine (3.5 g leucine) when compared to a control 10 g EAA supplement (1.8 g leucine). These findings suggest that added leucine is unnecessary for the stimulation of MPS when sufficient EAAs are provided; however, the study of supplemental leucine during conditions such as endurance exercise, caloric deprivation, and ageing may be warranted. 2011 International Life Sciences Institute INTRODUCTION Protein turnover in skeletal muscle and other body tissues is the dynamic biological process that encompasses both the synthesis of new protein and the breakdown of existing protein. The rate of protein turnover varies between proteins and is specific to their individual function. Skeletal muscle proteins are particularly important because those proteins comprise approximately 50 75% of total body protein stores, and skeletal muscle protein turnover contributes to nearly 30 45% of wholebody protein metabolism. 1 Skeletal muscle protein turnover (SMPTO) is regulated in part by nutrition, as dietary energy intake and macronutrient distribution, especially the quantity and quality of dietary protein and amino acids, influence muscle protein synthesis (MPS) and breakdown (MPB). 2 4 The regulation of SMPTO has been well studied because alterations in SMPTO are one of the primary factors contributing to the loss of lean body mass in many pathophysiological conditions. 5 8 Availability of circulating amino acids regulates SMPTO. 9,10 Studies have reported increased MPS in response to dietary amino acid supplementation resulting in plasma levels exceeding normal concentrations. 11 13 Experimental evidence suggests that the anabolic response may be due to a combined effect of increased plasma and intramuscular amino acid availability. 10,14,15 Increasing plasma and intramuscular essential amino acid (EAA) levels has been shown to independently stimulate MPS both in vitro and in vivo. 12,16 19 Recent studies indicate that the unique anabolic stimulus caused by EAA administration may be attributed to the branched-chain amino acid (BCAA) leucine. 20,21 Although the specific mechanisms remain largely unknown, leucine likely functions as a cellular signal by modulating messenger ribonucleic acid (mrna) translation efficiency through the cell hypertrophy pathway that involves the mammalian target of rapamycin complex 1 (mtorc1). 22 24 However, the optimal dose of leucine and whether supplemental leucine is, in fact, necessary to promote human muscle anabolism under various circumstances in target populations consuming a range of total EAA remains unclear. As such, the purpose of this review is to examine the role of dietary leucine in the Affiliation: SM Pasiakos and JP McClung are with the Military Nutrition Division, US Army Research Institute of Environmental Medicine (USARIEM), Natick, Massachusetts, USA. Correspondence: JP McClung, Military Nutrition Division, US Army Research Institute of Environmental Medicine (USARIEM), Kansas St., Building Y2, Natick, MA 01760, USA. E-mail: james.mcclung@amedd.army.mil, Phone: +1-508-233-4979, Fax: +1-508-233-4869. Key words: branched-chain amino acid, intracellular signaling, leucine, protein synthesis 550 doi:10.1111/j.1753-4887.2011.00420.x Nutrition Reviews Vol. 69(9):550 557

context of human MPS and to highlight recent reports that provide new insight regarding the mechanisms by which supplemental leucine regulates muscle protein metabolism. LEUCINE AND MUSCLE PROTEIN SYNTHESIS For more than 30 years, leucine has been recognized as a potent independent stimulator of MPS in cells and animals (Table 1). 12,17,20 As early as 1975, Buse and Reid 20 observed that MPS increased in isolated rat diaphragms incubated with leucine. The stimulatory effect on MPS attributed to leucine was confirmed in other wellcontrolled in vitro studies. 12,17,25,26 Over the last decade, several in vivo animal studies have also demonstrated a positive effect of leucine supplementation on MPS, which was associated with alterations in mrna translation initiation. 27 32 Bolster et al. 30 demonstrated increased MPS in rat skeletal muscle perfused with leucine; this was coupled with increased formation and phosphorylation of key factors associated with mrna translation. In another study, rats were orally administered 270 mg of valine, isoleucine, leucine, or saline. 29 Leucine administration increased MPS by 65% in comparison to the saline group; neither valine nor isoleucine stimulated MPS. The administration of leucine also increased the phosphorylation of various anabolic intracellular signaling proteins, whereas neither valine nor isoleucine influenced the molecular regulation of MPS. 29 The discovery that leucine is a potent stimulator of MPS in animals has led to a number of investigations examining the influence of leucine supplementation on MPS in humans. Katsanos et al. 33 compared the effects of two isonitrogenous EAA solutions with differing concentrations of leucine on SMPTO in young and older adults. Ingestion of both leucine concentrations increased rates of MPS in young adults, but in older adults, MPS was increased only when the EAA supplement with the greater concentration of leucine was consumed. Similar findings were reported by Rieu et al. 34 These observations suggest that the mechanism accounting for the benefit of added leucine consumption on resting MPS may be age dependent, which highlights the unique ability of leucine to overcome the blunted MPS response to protein or EAA consumption in ageing skeletal muscle. Recent studies have attempted to identify the complex biochemical mechanisms by which supplemental leucine stimulates MPS. Several investigators have approached this problem by providing leucine as the principal component of an enriched solution containing EAA and carbohydrate (35% leucine of total 20 g EAA), and observed effects on MPS in young, physically active adults. 35 38 Increased MPS and enhanced intracellular signaling through the mtorc1 pathway have been observed in response to leucine-enriched EAA supplementation provided after resistance exercise. 37 However, conflicting results have been reported, with some indicating that leucine supplementation may not provide an added benefit on skeletal muscle anabolism during recovery. 39 Tipton et al. 38 found enhanced net protein balance in untrained adults who consumed 16.6 g of whey protein supplemented with 3.4 g of additional leucine compared to a non-nitrogenous placebo before a bout of resistance exercise. Although enhanced net protein balance was observed in individuals consuming whey protein with additional leucine in the study by Tipton et al., 38 the postexercise skeletal muscle response was not different from that observed in other studies providing 20 g of whey protein without added leucine. 40 Based on these studies, the authors concluded that additional leucine is unnecessary when sufficient high-quality protein (e.g., whey protein) is consumed during recovery from resistance exercise. Recent findings from Koopman et al. 39 support this assertion, as co-ingestion of leucine combined with whey protein (69 g total protein containing 17.6 g leucine) failed to enhance MPS when compared to an isonitrogenous whey protein drink (69 g total protein containing 4.7 g leucine) consumed over a 6-hour period after performing a 30 min bout of moderate exercise (combined cycling and resistance exercise) in older adults. Differences between study populations (e.g., age), the mode and intensity of exercise, and, most importantly, the dose and timing of supplemental leucine may contribute to the inconsistencies among studies. LEUCINE AND REGULATION OF mtorc1 INTRACELLULAR SIGNALING The molecular regulation of MPS occurs primarily through the modulation of mrna translation. 22 24,41 This regulatory mechanism is comprised of a series of intracellular signaling events involving several initiation and elongation factors; it can be altered by energy status, growth factors (e.g., insulin and IGF-1), and nutrient (e.g., amino acid) availability. 4,21 Perhaps the most important nutritionally regulated signaling component affecting mrna translation is a multiunit protein complex termed mtorc1, which includes the mammalian target of rapamycin (mtor) kinase. 42,43 mtorc1 is the central component of the protein synthetic insulin signaling cascade (e.g., insulin/igf-pi3k pathway; Figure 1) that regulates mrna translation through two primary mechanisms: 1) inactivation of the repressor of mrna translation, eukaryotic initiation factor 4E-binding protein (4E- BP1); and 2) the activation of p70 S6 kinase (p70 S6K ). Together, changes in the phosphorylation state of these critical intracellular signaling proteins affect mrna Nutrition Reviews Vol. 69(9):550 557 551

Table 1 An abridged review of in vitro and in vivo (animal) studies demonstrating an effect of leucine on muscle protein synthesis*. Reference In vitro/in vivo MPS assessment Treatment D MPS Conclusion (methodology) Li et al. (1978) 12 Rat 14 C-phenylalanine No AA Leucine MPS Hemicorpus incorporation 1 AA perfusion Gastrocnemius 5 AA 5 AA (no BCAA) 5 BCAA 10 BCAA 10 leucine Hong et al. Rat 14 C-tyrosine incorporation 0.5 mm leucine Leucine MPS (1984) 17 Isolated AA Soleus/EDL incubation Buse et al. Rat 14 C-lysine incorporation 0.3 mm BCAA Leucine MPS (1975) 20 Isolated AA Diaphragm 0.5 mm leucine incubation 0.5 mm isoleucine 0.5 mm valine Buse et al. Rat 3 H-tyrosine incorporation 0.5 mm leucine / Leucine MPS in healthy (1977) 25 Isolated AA Diaphragm 0.5 mm isoleucine / and diabetic rat muscle incubation 0.5 mm valine / at same rate of 0.5 mm BCAA / combined BCAA Anthony et al. Rat 3 H-isoleucine incorporation Exercise food deprived Leucine alone or with CHO (1999) 27 Oral gavage Gastrocnemius/plantaris restores MPS following Exercise CHO meal exercise Exercise leucine meal / Exercise CHO+ leucine / Anthony et al. Rat 3 H-phenylalanine (2000) 28 incorporation meal** Saline Leucine alone or with CHO stimulates MPS with mtorc1 signaling Oral gavage Gastrocnemius/plantaris CHO mtorc1 signaling CHO+ leucine meal** / Leucine meal / Anthony et al. Rat 3 H-phenylalanine (2000) 29 incorporation 270 mg leucine Leucine MPS and mtorc1 signaling Oral gavage Gastrocnemius/plantaris 270 mg valine mtorc1 signaling 270 mg isoleucine Bolster et al. Rat 3 H-phenylalanine (2004) 30 incorporation Hindlimb perfusion Gastrocnemius/soleus mtorc1 signaling Crozier et al. Rat 3 H-phenylalanine (2005) 31 incorporation 10X leucine Leucine MPS and mtorc1 signaling 0.068 g leucine/kg Low to high leucine doses MPS and mtorc1 signaling Oral gavage Gastrocnemius/plantaris 0.135 g leucine/kg mtorc1 signaling 0.338 g leucine/kg 0.675 g leucine/kg 1.35 g leucine/kg * Studies with corresponding reference numbers in parentheses (table excludes studies of similar design and results). Skeletal muscle perfused with amino acids (AA) at 1,5,and10 normal plasma concentrations. Change in MPS compared to non-treated control skeletal muscle. Skeletal muscle incubated with equal concentrations of leucine, isoleucine, and valine. Change in MPS compared to healthy/diabetic control rats. ** Meals provided 270 mg leucine. Change in MPS compared to sedentary fed control rats. Change in MPS for exercise CHO+ leucine and exercise leucine compared to sedentary fed control/exercise food deprived and exercise CHO fed rats. Change in MPS for CHO+ leucine and leucine compared to sedentary fed control/saline and CHO fed rats. Change in MPS compared to saline incubated skeletal muscle or saline fed rats. Abbreviations and symbols: DMPS:,, or, indicate statistical increase, no statistical difference, or statistical reduction in MPS, respectively; BCAA, branched-chain amino acid; CHO, carbohydrate; MPS, muscle protein synthesis; mtorc1, mammalian target of rapamycin complex 1. translation initiation and elongation, which in turn directly influence the rate of MPS. It is well established that leucine acts as a nutrient signal to stimulate MPS through the mtorc1 pathway. The MPS stimulatory effect of leucine is independent of the effects of insulin and other hormones and growth factors, which stimulate mtor indirectly through upstream phosphorylation of phosphatidylinositol 552 Nutrition Reviews Vol. 69(9):550 557

Essential Amino Acids Growth Factors (Insulin) Leucine? P13K IRS Akt hvps34 MAP4K3 TSC1 TSC2 Contraction Rheb AMPK Energy mtorc1 p70s6k1 4E-BP1 eef2k rps6 eef2 mrna translation initiation Muscle protein synthesis (MPS) Figure 1 Essential amino acid (EAA), growth factor, and contraction-induced mtorc1 intracellular signaling. Proteins that stimulate muscle protein synthesis (MPS) are depicted in grey, whereas inhibitory proteins are depicted in black. Abbreviations: IRS1, insulin receptor substrate 1; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; TSC1/TSC2, tuberous sclerosis complex; Rheb, ras homolog enriched in brain; hvps34, vacuolar protein sorting 34; MAP4K3, mitogen-activated protein kinase; AMPK, AMP-activated protein kinase; mtorc1, mammalian target of rapamycin complex 1; 4E-BP1, eukaryotic initiation factor 4E-binding protein; p70 S6K, 70 kda S6 kinase; rps6, ribosomal protein S6; eef2k, eukaryotic elongation factor 2 kinase; eef2, eukaryotic elongation factor 2. Figure adapted and modified from Drummond et al. 41 3-kinase (PI3K) and protein kinase B (PKB/Akt). 28,44,45 Under that regulatory control, Akt then activates mtor by phosphorylation of the tuberous sclerosis complex (TSC2) and subsequent activation of Rheb. In contrast, leucine seems to stimulate mtor directly. Although the direct regulatory mechanism remains undefined, recent evidence suggests that a novel class 3 PI3K, hvps34 (vacuolar protein sorting 34), may be responsible for leucineinduced activation of mtor signaling. 46,47 In addition, the mitogen-activated protein kinase (MAP4K3) recently identified in Drosophila may be an amino acid sensing mechanismthataffectsmtoractivation. 48 To date, only one study has successfully characterized the effects of leucine-enriched EAA supplementation on skeletal muscle hvps34 and MAP4K3 mrna expression in young and older adults at rest and in response to resistance exercise. 49 Gene expression of these purported leucinesensing regulators of mtorc1 was not influenced by the combined effects of increased leucine availability and resistance exercise in either young or older adults. However, Drummond et al. 49 did report elevated hvps34 mrna expression in older adults before consuming a leucine-enriched EAA supplement at rest. If hvps34 expression is indeed greater in older than younger persons, that adaptation could account for the agedependent anabolic effect of leucine under resting conditions, thereby providing a mechanism enabling ageing skeletal muscle to compensate for reduced amino acid Nutrition Reviews Vol. 69(9):550 557 553

availability. 33 35,49 Although the results of recent studies are intriguing, the finite mechanisms by which leucine, hvps34, and MAP4K3 activate mtorc1 and affect human MPS has not been elucidated. LEUCINE FOR THE STIMULATION OF MUSCLE PROTEIN SYNTHESIS IN HUMANS: A CRITICAL NEW FINDING As previously discussed, not all studies observed greater human MPS in response to the provision of supplemental leucine. Many of the inconsistencies observed across human studies are attributable to variations in the dose of leucine, the total amount of EAA consumed, and the use of appropriate study controls (e.g., non-nitrogenous placebos). Two recently published studies that did not have these limitations demonstrated a dose-dependent relationship between dietary protein and EAA supplementation on human MPS. 50,51 Moore et al. 51 measured MPS after providing 0, 5, 10, 20, or 40 g of egg protein, a quality source of EAA, to healthy adults immediately after a bout of intense resistance exercise. Changes in post-exercise MPS were dose dependent, reaching maximal stimulation at 20 g (~9 g EAA), with increased leucine oxidation observed when protein intake exceeded 20 g. Cuthbertson et al. 50 also demonstrated a dose-dependent relationship, as 10 g EAA maximally stimulated MPS at rest. These data suggest that approximately 10 g of EAA with an amino acid profile consistent with a 20 g serving of high-quality protein is sufficient to maximize MPS at rest and following resistance exercise in young adults. Future studies assessing acute changes in human MPS should use this optimal dose of EAA in their design. Whether the anabolic response can be further enhanced by altering the concentration of leucine within an EAA supplement containing this amount of total EAA remained unclear until publication of a recent report by Glynn et al. 52 Glynn et al. 52 compared the effects of isonitrogenous (10 g) EAA supplementation at two different leucine concentrations in healthy young adults. The amino acid profile of the control EAA supplement was similar to commercially available dietary supplements containing high-quality protein, and provided approximately 1.8 g of leucine, whereas the experimental supplement was leucine-enriched and provided 3.5 g of leucine. The amino acid mixtures were void of other nutrients (e.g., carbohydrates) eliminating potential confounding factors affecting the findings. A comprehensive approach, including whole-body and intramuscular leucine kinetics, and repeat assessments of MPS and mtorc1 signaling (e.g., 60, 120, and 180 min postingestion), were used to characterize the effect of leucine-enriched EAA supplementation on resting muscle protein metabolism. The major finding from this study was that consumption of the EAA supplement containing a higher concentration of leucine did not provide an added benefit to MPS, 52 which was increased above baseline similarly between dietary treatment groups. The authors hypothesized that intramuscular, rather than plasma, leucine concentration was the major determinant of the MPS response and that as plasma leucine concentration increased, transport of leucine into muscle cells displayed saturation-type kinetics such that intramuscular leucine transport and availability were not different between groups. That finding is consistent with other observations and suggests that intramuscular amino acid transport is an important rate-limiting step affecting intramuscular amino acid (e.g., leucine) availability, ultimately influencing the regulation of human MPS. 53,54 Neither of the EAA supplements affected insulin signaling through Akt despite a transient increase in plasma insulin. Amino acid supplementation increased mtor and p70 S6K phosphorylation, with minor exceptions at various time points where mtor and p70 S6K phosphorylation were not elevated in the EAA group. Phosphorylation of 4E-BP1 was increased and eukaryotic elongation factor (eef2) was decreased in the leucine-enriched EAA group, but not for those consuming the non-enriched EAA supplement. These findings suggest that leucine enrichment is necessary to stimulate maximal protein synthetic intracellular signaling; however, consumption of the supplement with a higher concentration of leucine did not promote a greater MPS response. Dissociations between intracellular signaling events and MPS have been documented previously, 4,11,55 indicating that resting MPS may be maximally stimulated when sufficient EAA is consumed in young adults, regardless of the potentially stimulating effect of leucine on the mtorc1 intracellular signaling cascade. The study by Glynn et al. 52 represents a major advance in our understanding of the regulation of protein metabolism for several reasons. This is the first study designed to assess resting MPS by using an optimal dose of EAA (10 g) provided to healthy adults with two different amounts of leucine (3.5 versus 1.85 g). Other studies have documented that resting MPS was stimulated to a greater degree as the amount of EAA increased; although that approach provides higher concentrations of leucine, one cannot delineate whether excess EAA in general or increased leucine, specifically, was responsible for the added MPS stimulation. 56 In other experiments, EAA supplements with varying leucine concentrations have been studied, but suboptimal doses of total EAA were used such that the MPS response was limited. 33 Furthermore, the design used by Glynn et al. 52 eliminated numerous confounding variables present in other investigations, including exercise, age, EAA dose, carbohydrate, or the use of proteincontaining whole-foods. 554 Nutrition Reviews Vol. 69(9):550 557

Further study is certainly required to examine whether similar EAA supplementation regimens affect skeletal muscle anabolism in response to other metabolic stressors (e.g., endurance-type exercise) known to affect leucine metabolism, SMPTO, and intracellular signaling. 57 61 Most noteworthy, the new findings reported by Glynn et al. 52 clearly indicate that the MPS response to an optimal dose of EAA under resting conditions in young adults is not affected by added leucine intake. The failure of added leucine to affect intramuscular transport despite increased plasma leucine concentrations emphasizes an important aspect of intramuscular leucine metabolism: the MPS response to EAA appears to exhibit saturation-type kinetics, probably attributable to the rate-limiting effects of amino acid transport into muscle under resting conditions. 11,14,50 Whether the apparent sparing of plasma leucine availability affects MPS in response to suboptimal doses of EAA that provide added leucine or during situations of increased metabolic demand cannot be determined from this study. Certainly, the leucine and total EAA requirement necessary to maximize MPS for conditions generally associated with suppressed MPS with increased proteolysis and leucine oxidation, such as ageing, endurance exercise, and caloric restriction may differ and should be the focus of future research. CONCLUSION The benefit of leucine on MPS in well-controlled cell culture and animal studies has been well established; however, reports observing a stimulatory effect of leucine on human MPS have been inconsistent. In a recent study, Glynn et al. 52 reported no added benefit for resting human MPS in response to an optimal dose of EAA providing a higher concentration of leucine. Findings from Glynn et al. 52 are important because they demonstrate that an EAA profile consistent with high-quality proteins is sufficient for the stimulation of MPS in humans at rest,and that added leucine provides little added benefit to MPS. However, the potential role of leucine in stimulating muscle anabolism in populations susceptible to muscle loss, to include proteolytic conditions, such as ageing, cachexia, calorie deprivation, and sustained endurancetype exercise should be the focus of future studies. Acknowledgment The authors acknowledge Dr Andrew J. Young for his critical review in support of the development of this manuscript. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the United States Army or the Department of Defense. Any citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement of approval of the products or services of these organizations. Declaration of interest.the authors have no relevant interests to declare. REFERENCES 1. Rennie MJ, Tipton KD. Protein and amino acid metabolism during and after exercise and the effects of nutrition. Annu Rev Nutr. 2000;20:457 483. 2. Bolster DR, Pikosky MA, Gaine PC, et al. Dietary protein intake impacts human skeletal muscle protein fractional synthetic rates after endurance exercise. Am J Physiol. 2005;289:E678 E683. 3. Fujita S, Dreyer HC, Drummond MJ, et al. 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