Abstract
Leucine is a nutrient regulator of muscle protein synthesis by activating mTOR and possibly other proteins in this pathway. The purpose of this study was to examine the role of leucine in the regulation of human myofibrillar protein synthesis (MPS). Twenty-four males completed an acute bout of unilateral resistance exercise prior to consuming either: a dose (25 g) of whey protein (WHEY); 6.25 g whey protein with total leucine equivalent to WHEY (LEU); or 6.25 g whey protein with total essential amino acids (EAAs) equivalent to WHEY for all EAAs except leucine (EAA-LEU). Measures of MPS, signalling through mTOR, and amino acid transporter (AAT) mRNA abundance were made while fasted (FAST), and following feeding under rested (FED) and post-exercise (EX-FED) conditions. Leucinaemia was equivalent between WHEY and LEU and elevated compared to EAA-LEU (P=0.001). MPS was increased above FAST at 1–3 h post-exercise in both FED (P <0.001) and EX-FED (P <0.001) conditions with no treatment effect.At 3–5 h, only WHEY remained significantly elevated above FAST in EX-FED(WHEY 184% vs. LEU 55% and EAA-LEU 35%; P =0.036). AAT mRNA abundance was increased above FAST after feeding and exercise with no effect of leucinaemia. In summary, a low dose of whey protein supplemented with leucine or all other essential amino acids was as effective as a complete protein (WHEY) in stimulating postprandial MPS; however only WHEY was able to sustain increased rates of MPS post-exercise and may therefore be most suited to increase exercise-induced muscle protein accretion.
Introduction
Ingestion or infusion of amino acids stimulates an increase in skeletal muscle protein synthesis (Bennet et al. 1989; Bohe et al. 2001, 2003; Atherton et al. 2010a), an effect
that is enhanced by prior resistance exercise (Tipton et al. 1999a; Wilkinson et al. 2007; Moore et al. 2009a,b; Tang et al. 2009; West et al. 2009). The essential amino acids (EAAs) are primarily responsible for this stimulation of muscle protein synthesis, with no apparent requirement for the non-essential amino acids (Smith et al. 1998;
Tipton et al. 1999b; Borsheim et al. 2002; Volpi et al. 2003). Several animal studies have demonstrated that leucine independently stimulatesmuscle protein synthesis by activating components of the mammalian target of rapamycin (mTOR) signalling cascade (Anthony et al. 2000a,b, 2002 ; Bolster et al. 2004; Crozier et al. 2005). This activation appears critical for both the contraction (Drummond et al. 2009), and EAA-mediated (Dickinson et al. 2011) increase in muscle protein synthesis. Thus,
leucine has been investigated as a pharmaconutrient with the potential to promote increases in muscle protein synthesis (Koopman et al. 2005, 2006, 2008; Katsanos et al.
2006; Rieu et al. 2006; Tipton et al. 2009; Glynn et al. 2010) and lean tissue mass (Verhoeven et al. 2009; Leenders et al. 2011). Nonetheless, while some studies indicate a role for leucine in the regulation of human muscle protein synthesis (Smith et al. 1992; Katsanos et al. 2006; Rieu et al. 2006), other studies have not found an enhanced
rate ofmuscle protein synthesis following leucine infusion (Nair et al. 1992), after increasing the amount of leucine within amixed EAA solution (Glynn et al. 2010), or by the
addition of free leucine to a protein containing supplement (Koopman et al. 2008; Tipton et al. 2009).
There is a dose-dependent relationship between amino acid (Bohe et al. 2003; Cuthbertson et al. 2005) and protein (Moore et al. 2009a) provision and muscle protein synthesis. We previously reported that ∼20 g of isolated egg protein (containing ∼8.6 g EAAs and ∼1.7 g leucine) stimulated muscle protein synthesis after resistance exercise above that observed with both 5 g and 10 g of protein but was not further stimulated with ingestion of 40 g of protein, indicating that 20 g of egg protein is saturating for muscle protein synthesis after resistance exercise (Moore et al. 2009a). These data are consistent with previous reports of a dose-dependent relationship between EAA ingestion and myofibrillar protein synthesis (MPS) up to a maximal stimulation at ∼10 g EAAs (containing∼2.1 g leucine; Cuthbertson et al. 2005). These dose–response data may provide insight into why other studies (Koopman et al. 2008; Tipton et al. 2009; Glynn et al. 2010) did not report a benefit of additional leucine on muscle protein synthesis when a sufficient amount of EAAs and/or leucine is provided.
Given what we know about the ingested protein dose–response of muscle protein synthesis (Bohe et al. 2003; Cuthbertson et al. 2005; Moore et al. 2009a), the aim of the present investigation was to examine the effects of supplementing a ‘suboptimal’ dose of whey protein (6.25 g whey containing ∼0.75 g of leucine) with additional leucine (LEU), or a mixture of EAAs with no leucine (EAA-LEU) on MPS at rest and following acute resistance exercise compared to a dose (25 g containing ∼3.0 g of leucine) of whey protein (WHEY) which is sufficient to induce a maximal stimulation of muscle protein synthesis after resistance exercise (Moore et al. 2009a). The suboptimal protein dose (6.25 g) was chosen to represent one-quarter of the 25 g dose in the WHEY treatment. We hypothesized that LEU would result in a stimulation of MPS equivalent to WHEY in both feeding (FED) and combined feeding and resistance exercise (EX-FED) conditions. Alternatively, we hypothesized that EAA-LEU would result in an increase in MPS in both the FED and EX-FED conditions, but the response would be significantly less than both LEU and WHEY due to the lower leucine content. In an attempt to gain insight into the mechanistic underpinnings of the response of MPS, we also examined changes in the phosphorylation status of protein targets of the Akt-mTOR pathway and in the mRNA abundance of select amino acid transporters (AATs) that have recently been shown to be regulated by EAAs (Drummond et al. 2010) and resistance exercise (Drummond et al. 2011)
Methods
Participants and ethical approval
Twenty-four recreationally active, young adult male participants (22±0.6 years; 1.80±0.02 m; 76.4±2.0 kg; BMI 24.3±0.6 kgm−2) voluntarily agreed to participate in the study. Participants were deemed healthy based on responses to a routine health screening questionnaire. Each participant was informed of the purpose of the study, the associated experimental procedures, and any potential risks prior to providing written consent. The study was approved by the Hamilton Health Sciences Research Ethics Board and conformed to the standards for the use of human subjects in research as outlined in the most recent update of the Declaration of Helsinki. The study also conformed to the standards established by the Canadian Tri-Council Policy on the ethical use of human subjects (2010)...
Author contributions
T.A.C.V. and S.M.P. contributed to the conception and the design of the experiment. All authors contributed to collection, analysis and interpretation of data. All authors contributed to drafting or revising intellectual content of the manuscript. All authors read, edited and approved the final version of the manuscript.
Acknowledgements
We thank Tracy Rerecich, Amy Hector, Leigh Breen and Todd Prior for their technical assistance and Randy Burd for his assistance in acquiring the whey protein used in this study. We thank the study participants for their time and effort. T.A.C.V. was supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship. This research was supported by a grant fromtheNatural Sciences and Engineering Research Council of Canada (NSERC) and Canadian Institutes of Health Research (CIHR) to S.M.P. This trial is registered at clinicaltrials.gov as: NCT01492010.