i DETERMINANTS OF THE MAGNITUDE OF TRAINING MEDIATED MUSCLE HYPERTROPHY ii DETERMINANTS OF THE MAGNITUDE OF RESISTANCE TRAINING MEDIATED MUSCLE HYPERTROPHY By CAMERON J. MITCHELL, M.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy McMaster University © Copyright by Cameron J. Mitchell, 2013 iii DOCTOR OF PHILOSOPHY (2013) McMaster University (Kinesiology) Hamilton, Ontario TITLE: Determinants of The Magnitude of Resistance Training Mediated Muscle Hypertrophy AUTHOR: Cameron J. Mitchell, M.Sc. (McMaster University) SUPERVISOR: Dr. Stuart M. Phillips SUPERVISORY COMMITTEE: Dr. Martin J. Gibala Dr. Gianni Parise NUMBER OF PAGES:118 iv ABSTRACT It is well established in the exercise science literature that chronic resistance training leads to muscle hypotrophy in a wide range of populations however most resistance training studies are relatively small in sample size. The few larger studies show a wide range in the magnitude of muscle growth among cohorts undergoing the same training regime. Three studies were conducted to better understand the sources of this variability. The first study employed a unilateral resistance training model to test the effects of relative training load and volume on the magnitude of hypertrophy and strength gains. This study showed that contrary to the recommendations of many professional organizations high relative training loads were no better than low training loads at inducing muscle hypertrophy provided that each set was performed to the point of muscular exhaustion. In agreement with previous finding, strength gains were greatest with the highest loads. The next two studies attempted to correlate various putative regulators of muscle hypertrophy with the magnitude of hypertrophy after 16 weeks of training in 23 subjects. Study two showed no association between the acute responses of testosterone, GH or IGF-1 and muscle hypertrophy but did show associations with androgen receptor content and acute phosphorylation of p70S6K. This suggests that local rather than systemic processes are the most important regulators of muscle hypertrophy. v The third study tested whether the acute post exercise protein synthetic response to a single bout of resistance exercise is related to the magnitude of hypertrophy following training in the same subjects. Although previous work has shown that acute post exercise protein synthetic response is qualitatively similar to the magnitude of hypertrophy after chronic training with similar manipulations in different subjects, we did not see any relationship. Based on the three studies in this thesis it appears that intrinsic factors rather than resistance training program variables are most important for regulating muscle hypertrophy. A large sample size and an ‘omic’ approach is the logical next step to explain greater variability in the magnitude of hypertrophy following training. vi ACKNOWLEDGEMENTS Firstly, I would like to thank Stu for taking a chance on me and always providing solid feedback and advice. I would like to thank the other EMRG faculty members Marty, Maureen and Gianni for helping to foster such a great atmosphere in the EMRG lab and constantly being available to answer questions in their areas of experiences. I want to acknowledge Burd and D. West for showing me the ropes and many great discussions around ‘the table’, as well as Leigh and Michaela for inviting me to partake in postdoc workout hour. I would also like to thank all the grad students who have made the EMRG lab and room 230 such a fun place to work. I want to say a huge thanks to all of the summer research and thesis students who helped with data collection and to organize my life; Sara, Devonna, Meg, Alex, Mel, Kim, Varsha, Dan, Chelsea and Jasmine. I would also like to say thanks to TCV for being there from start to finish and always being available to talk science. I would like to acknowledge Todd and Tracy for all the technique assistance and life skills advice. I would like to say a special thanks to the Phoenix and its staff for providing a refuge over the last 10+ years of my university education and a venue for some of the best discussion scientific and otherwise I have had during my PhD studies. Lastly I would like to thank my future wife Lindsay for always encouraging me to do what I love and allowing me to go 10 years without getting a real job. vii TABLE OF CONTENTS Abstract ............................................................................................................................ iv Acknowledgements ............................................................................................................. vi Table of contents ............................................................................................................... vii List of tables ........................................................................................................................ ix Format and organization of this thesis .............................................................................. xii Contribution to papers with multiple authors .................................................................. xiii CHAPTER 1: Introduction ................................................................................................ 16 1.1 Historical background......................................................................................... 16 1.2 Measurement of muscle hypertrophy ................................................................. 17 1.3 Variability in hypertrophic response .................................................................. 19 1.4 Resistance training load ...................................................................................... 24 1.5 Resistance training volume ................................................................................. 27 1.6 Protein turnover and hypertrophy ....................................................................... 28 1.7 Anabolic signaling pathways .............................................................................. 32 1.8 References .......................................................................................................... 35 CHAPTER 2: Resistance Exercise Load does not Determine Training-Mediated Hypertrophic Gains in Young Men ................................................................... 42 Abstract ..................................................................................................................... 43 Methods ................................................................................................................... 44 Results ...................................................................................................................... 45 Discussion ................................................................................................................. 46 References ................................................................................................................ 47 CHAPTER 3: Muscular and Systemic Correlates of Resistance Training-Induced Muscle Hypertrophy ....................................................................................................... 46 viii Abstract ..................................................................................................................... 51 Introduction ............................................................................................................. 51 Methods .................................................................................................................... 52 Results ...................................................................................................................... 53 Discussion ................................................................................................................. 54 References ............................................................................................................... 59 CHAPTER 4: Increases in Fed-State Post-exercise Myofibrillar Protein Synthesis are Unrelated to Resistance Training-Induced Muscle Hypertrophy ...................... 60 Key Points Summary ................................................................................................ 63 Abstract ..................................................................................................................... 64 Introduction .............................................................................................................. 65 Methods .................................................................................................................... 67 Results ...................................................................................................................... 72 Discussion ................................................................................................................. 73 References ................................................................................................................ 80 CHAPTER 5: Discussion and conclusions ........................................................................ 89 5.1 Optimal loading paradigms for muscle hypertrophy .......................................... 89 5.2 Considerations for future resistance training studies .......................................... 95 5.3 Local versus systemic control of muscle hypertrophy ....................................... 97 5.4 The relationship between muscle protein synthesis and hypertrophy .............. 104 5.5 Conclusions and further directions ................................................................... 108 5.6 References ........................................................................................................ 112 APPENDIX A: The Relationship between Interleukin 6 and Hypertrophy Following Resistance Training ......................................................................................... 118 ix LIST OF TABLES CHAPTER 2 Table 1. Changes in knee extension performance and vastus lateralis fiber area following 10 wk of resistance training ................................................................... 45 CHAPTER 3 Table 1. Pre- and post-training resting hormone and cytokine concentrations, muscle fibre cross-sectional area, and strength. ................................................. 54 Table 2. Pre- and post-training area under the curve of acute hormonal and cytokine responses to a single bout of resistance exercise..................................... 54 Table 3. Pearson correlations between pre-training hormone, cytokine, and androgen receptor (AR) response and changes in muscle fibre cross-sectional area (CSA) following training. ...................................................................................... 57 Table 4. Stepwise multiple regression model. ..................................................... 58 x LIST OF FIGURES CHAPTER 2 Figure 1 Percentage change in quadriceps muscle volume following 10 wk of resistance training. ................................................................................................. 45 Figure 2. Phosphorylated Akt, mammalian target of rapamycin (mTOR), and p70S6K at rest and 1 h following resistance exercise. ........................................... 46 Figure 3. Knee extension strength........................................................................ 47 Figure 4. Maximal work and repetitions with 30 and 80% of 1RM before and after 10 wk of resistance training. .......................................................................... 47 CHAPTER 3 Figure 1. Correlation between the fold change in muscle AR protein content and changes in skeletal muscle fibre area following 16 weeks of resistance training. . 55 Figure 2. Phosphorylation of p70S6K following an acute bout of resistance exercise before 16 weeks of resistance training and the percentage change in skeletal muscle mean fibre area following the training ......................................... 56 Figure 3. Correlation between the AUC of the acute IL-6 response to resistance exercise before 16 weeks of resistance training and the percentage change in skeletal muscle mean muscle fibre area following 16 weeks of resistance training. .................................................................................................................. 58 CHAPTER 4 Figure 1. Muscle volume, muscle mass, and strength changes following resistance training. ................................................................................................. 85 Figure 2. Myofibrillar Protein synthesis. ............................................................. 86 Figure 3. Phosphorylation of anabolic signalizing proteins. ................................ 87 Figure 4. Relationship between muscle hypertrophy, MPS, and 4EBP-1 phosphorylation ...................................................................................................... 88 xi xii FORMAT AND ORGANIZATION OF THIS THESIS This thesis was prepared in the “sandwich” thesis format as outlined in the McMaster University School of Graduate Studies Thesis Preparation Guide. This thesis is comprised of a general introduction, three original research papers (Chapters 2-4) and a general conclusion. The papers presented in chapters two and three have been published in in peer reviewed journals with the candidate as first author. The paper presented in chapter four has been submitted for peer review prior to publication. xiii CONTRIBUTION TO PAPERS WITH MULTIPLE AUTHORS CHAPTER 2 Publication Mitchell CJ, Churchward-Venne TA, West DW, Burd NA, Breen L, Baker SK, & Phillips SM (2012). Resistance exercise load does not determine training-mediated hypertrophic gains in young men. J Appl Physiol. 113(1):71-7 Contributions CJ Mitchell, NA Burd, TA Churchward-Venne, DW West and SM Phillips planned the study. CJ Mitchell obtained ethics board approval. CJ Mitchell, NA Burd, TA Churchward-Venne, DWD West, SK Baker and SM Phillips collected the data. SM Phillips supervised the study and obtained muscle biopsies. CJ Mitchell performed the MRI and muscle fiber type analyses. Western blot analysis was performed by L Breen, NA Burd and CJ Mitchell. CJ Mitchell drafted the manuscript. All authors contributed to revising intellectual content of the manuscript and approved the final version. xiv CHAPTER 3 Publication CJ. Mitchell, TA. Churchward-Venne, L Bellamy , G Parise, SK. Baker, & SM. Phillips (2013). Muscular and Systemic Correlates of Resistance Training-Induced Muscle Hypertrophy. PLoS ONE 8(10): e78636. doi:10.1371/journal.pone.0078636 Contributions CJ Mitchell, G Parise and SM Phillips planned the study. CJ Mitchell obtained ethics board approval. CJ Mitchell, TA Churchward-Venne, L Bellamy, SM Phillips and many undergraduate research assistants collected the acute blood and muscle samples. The training and testing protocols were designed and supervised by CJ Mitchell and carried out by undergraduate research assistants. The hormonal and cytokine analysis was performed by staff at the McMaster Core Lab. L Bellamy performed the muscle fiber type analysis. TA Churchward-Venne and CJ Mitchell preformed the western blot analysis. CJ Mitchell drafted the manuscript. All authors contributed to revising intellectual content of the manuscript and approved the final version. xv CHAPTER 4 Publication CJ. Mitchell, TA. Churchward-Venne, G. Parise, L Bellamy , K. Baker, K. Smith, PJ Atherton , & SM. Phillips (2013) Increases in fed-state post-exercise myofibrillar protein synthesis are unrelated to resistance training-induced muscle hypertrophy. This article was submitted for peer review to PLoS ONE Contributions CJ Mitchell, SM Phillips, and G Parise conceived and designed the study. CJ Mitchell, L Bellamy, TA Churchward-Venne, and SM Phillips ran the experiments. CJ Mitchell, TA Churchward-Venne, K Smith, and PJ Atherton ran analyses. CJ Mitchell and SM Phillips prepared the manuscript and all authors provided feedback and input. All authors approved the final version of the manuscript. 16 CHAPTER 1: INTRODUCTION 1.1 HISTORICAL BACKGROUND Skeletal muscle mass is maintained through a balance in the magnitude and duration of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). The primary regulators of MPS and MPB in adult skeletal muscle are nutrition and activity. The term ‘activity’ is used as a generic term referring to increased loading in this thesis. Periodic feeding stimulates MPS and supresses MPB. Increased muscle activity generally increases MPS and MPB; however MPS contributes to a much greater extent. When activity is combined with nutrition MPS is stimulated to an even greater extent and MPB is supressed leading to a net new addition of muscle proteins. In young healthy adults receiving adequate nutrition muscle mass is relatively stable. However, muscle mass can be lost during periods of diminished activity such as step reduction or periods of immobilization (Glover et al., 2008; Krogh-Madsen et al., 2010). Small increases in whole body lean mass, presumably muscle mass, can also be brought about in addition to fat mass gains via a combination of energy intakes in excess of requirement and increased protein intakes (Bray et al., 2012). Exercise is the primary non-pharmacological means by which adults gain muscle mass, a process termed hypertrophy. There is evidence that traditional exercise modalities that are not strongly associated with high external loads, such as walking, running, and cycling may cause a small amount of muscle hypertrophy especially in untrained or older adults (Harber et al., 2012; Kubo et al., 2008; Ross et al., 2001). Nonetheless, resistance training is the mode of exercise most often employed to promote gains in strength and muscle mass. Resistance training is defined as a purposeful repeated body movement with a load greater than what would be encountered during normal activities of daily living (Folland et al., 17 2007). In this definition, resistance training is distinct from occupational activities or activities of daily living even if they are performed against a high level of resistance. Resistance training encompasses the use of free weight, weight machines, resistance bands as well as exercise with one’s own body weight. Olympic style weight lifting as well as strength training, which is training with weights with the specific goals of increasing strength, are both components of resistance training (ACSM, 2009). The main focus of this thesis will be resistance training with free weight or machines with the specific goal of increasing muscle mass. The idea of using resistance exercise to induce muscle hypertrophy first appeared in the literature in the 1940’s as a rehabilitation modality for soldiers injured during World War II (Coffey, 1946). At the time no reliable measurement was available to evaluate muscle hypertrophy so muscle strength was used as a proxy outcome measure for the effectiveness of resistance training in studies at the time. In the 1960’s resistance training gained popularity as a form of recreation and as a means of improving athletic performance. This lead to a series of studies by Berger that attempted to identify the optimal resistance training program design for the improvement of muscle strength however during this time hypertrophy following training was still not routinely measured (Berger, 1962; Carpinelli, 2002). As time progressed, more resistance training studies were conducted and studies began to measure changes in muscle size in addition to muscle strength (MacDougall et al., 1980). 1.2 MEASUREMENT OF MUSCLE HYPERTROPHY Muscle hypertrophy has been measured in a number of ways ranging from rudimentary muscle girths and anthropometry (O'Shea, 1966) to advanced imaging techniques (Mitchell et al., 2012). Today there are four main approaches used to quantify muscle hypertrophy. Firstly, the histological approach. Histochemical evaluation of hypertrophy involves the collection of 18 muscle biopsies which are then sectioned and mounted on slides, stained for cell borders and sometimes fibre type, and then photographed and quantified (West et al., 2009a). The cross- sections of the muscle cells in the resulting images are then measured and an average muscle fiber size can be obtained. The advantage of this technique is it allows for the direct quantification of different muscle fiber types and subtypes. However, since it is a random sampling of only a very small proportion of the muscle it can be confounded by samples with a low number of visible fibers or skewed mounting of the sample resulting in various degrees of oblique- rather than truly cross-sectional measurements. Another popular technique for the measurement of muscle hypertrophy is the use of Dual-energy X-ray absorptiometry (DXA). This method does not directly measure the mass or size of a single muscle, but rather quantifies fat- and bone-free (FBF) or lean mass which includes muscle as well as other protein-containing organs. It is generally assumed that visceral organs do not change in mass with resistance training and therefore changes in FBF mass are thought to solely reflect changes in lean or muscle mass. DXA can be used either to measure changes in whole-body lean mass or changes in a particular body segment such as a legs; however, it cannot be used to measure hypertrophy in a single muscle or muscle group (Nana et al., 2013). Ultrasound technology is sometime used as an inexpensive and non-invasive measure of muscle hypertrophy. Ultrasound can only provide a one dimensional measurement of muscle thickness or depth and because it is sensitive to the skill of the technician and not sensitive to changes in the three-dimensional size and shape of the muscle it is not considered an optimal method by which to measure muscle hypertrophy following resistance training (Abe et al., 2000). The other, and likely the most reliable, method that can be used to measure muscle hypertrophy with resistance training is imaging. The two major types of imaging that are commonly used are magnetic resonance imaging (MRI) 19 (Esmarck et al., 2001) and computerized tomography (CT) (Jones et al., 1987). Images are taken as coronal sections of a muscle and then an algorithm identifies fat, bone and muscle on the image to come up with the muscle cross sectional area (CSA). Many cross-sectional images which when taken serially from the muscle can be combined to calculate muscle volume which gives a more complete description of changes in muscle size with resistance training. Muscle CSA determination via CT is cheaper than MRI, however, MRI is higher resolution and is therefore considered the gold standard for measures of whole muscle hypertrophy. 1.3 VARIABILITY IN HYPERTROPHIC RESPONSE In recent years there have been a number of larger scale resistance training studies conducted. A consistent finding from these studies has been that there is a substantial degree of variability in the magnitude of muscle hypertrophy in participants completing the same resistance training program (Hubal et al., 2005; Kosek et al., 2006; Phillips et al., 2013; West et al., 2012). Like many biological processes, the degree of hypertrophy following resistance training is roughly normally distributed (Hubal, et al., 2005). Different statistical approaches such as Z-scores (West, et al., 2012) and k-means cluster analysis (Bamman et al., 2007) have been employed to group subjects according to the magnitude of muscle hypertrophy experienced by the participants. Regardless of the exact statistical method used, subjects are generally classified based on their hypertrophic response as low, moderate, or high-/extreme-responders. In most studies 15-20% of subjects are low or non-responders, 60-70% are moderate responders and 15-20% are high or extreme responders (Bamman, et al., 2007; West, et al., 2012). Body size is generally positively related to absolute gains in muscle mass following training; however, when gains are expressed relative to baseline body size or muscle mass this relationship disappears (Hubal, et al., 2005). Similarly it is often thought that sex is a major 20 factor in the magnitude of muscle hypertrophy following resistance training. While it is true that absolute gains in muscle mass are greater in men than in women, when these gains are normalized to body size these differences decrease drastically. Studies of hypertrophy with large sample sizes (i.e., >500) have shown significantly larger absolute and relative gains in muscle size in men than women following the same resistance training program. Nonetheless, the effect size of these differences was very small and the practical significance of sex differences in relative gains in muscle mass following resistance training is probably negligible (Hubal, et al., 2005). Interestingly, relative strength gains are much larger in women than in men (Hubal, et al., 2005). Differences in nutritional intake could possibly explain some differences in the magnitude of hypertrophy following resistance training. The influence of nutritional factors in hypertrophy have been studied in two ways: intentional manipulation via the addition of supplements to, or meal replacements in, the diet during training; and the analysis of habitual dietary intake during training, to examine if differences exist between high an lower responders. Dietary interventions during resistance training have focused mainly on the addition of protein to the diet (Hartman et al., 2007) and on supplements such as creatine monohydrate (Vandenberghe et al., 1997). The effects of protein timing (Esmarck, et al., 2001), protein source (Hartman, et al., 2007) and quantity (Josse et al., 2010) have been investigated. Some studies have shown slightly greater muscle hypertrophy on average in subjects when ingesting higher quality proteins such as milk or whey as they produce more rapid blood aminoacidemia (Hartman, et al., 2007). A recent meta-analysis has shown that over the course of a moderate duration (i.e., 12-16wk) training study consumption of supplemental protein resulted in the accrual of approximately one extra kilogram of lean mass (Cermak et al., 2012). Slightly larger increases in lean mass have 21 been observed after supplementation with creatine monohydrate during resistance training. However, strikingly within each supplementation regime the distribution of hypertrophic responses, low, moderate and high responders, is still evident (Nissen et al., 2003). The implication of this observation is thus that the supplement increases the group mean gain in muscle mass but not the variability in response. Theoretically, habitual dietary intake could have a large effect on hypertrophy with resistance training. For example, if a study participant were in a large energy deficit or were consuming very low amounts of protein and/or certain amino acids there would be limited potential for their accretion of muscle mass (Pasiakos et al., 2010). Conversely very high calorie diets with a normal macronutrient distributions have been shown the result in the accretion of muscle mass under controlled conditions (Bray, et al., 2012). It is possible that large caloric surplus during resistance training could result in an increased accretion of both fat and lean mass. To date studies that have reported habitual dietary intake in relation to hypertrophy have found that most people who volunteer for participation in studies involving a resistance training protocol are consuming sufficient energy and protein to support gains in muscle mass with training. These studies have also found that variations in energy or protein intake do not explain the response variability in resistance training induced muscle hypertrophy (Thalacker-Mercer et al., 2009). Variations in resistance training-mediated hypertrophy have been attributed to a variety of different physiological variables. For example, multiple attempts to find markers in the blood that can predict and individual’s training response as blood sampling is relatively easy and non- invasive (Kraemer et al., 2005; West, et al., 2012). Because of the potent anabolic effect of supraphysiological doses of testosterone on hypertrophy (Sinha-Hikim et al., 2002); responses of 22 circulating testosterone have been viewed as candidate regulators of training-mediated muscle hypertrophy and have been hypothesized as being causative, and thus explaining variability, in the training-induced hypertrophic response (Kraemer, et al., 2005). Because their serum concentrations increase acutely following resistance exercise, testosterone and other hormones such as growth hormone and insulin-like growth factor-1 (IGF-1) have been proposed to be anabolic and thus hypertrophy promoting. Thus, the response of these anabolic hormones has been measured acutely after resistance exercise with the thesis that the magnitude of this rise is indicative of, or at least related to, the magnitude of muscle hypertrophy which would occur with chronic training (Kraemer, et al., 2005). Previous work from our lab has shown no relationship between the magnitude of the acute rise of testosterone and subsequent muscle hypertrophy after training (West, et al., 2012). Other potentially anabolic hormones such as IGF-1 show the same lack of relationship as is observed with testosterone and hypertrophy (West, et al., 2012). A weak relationship has been observed between type I fiber hypertrophy and growth hormone response to an acute bout of resistance exercise (West, et al., 2012). Because supraphysiological doses of growth hormone do not stimulate muscle protein synthesis (Doessing et al., 2010) the mechanism of action by which the acute increase in growth hormone concentration could induce muscle hypertrophy is unclear (Burd et al., 2010b). It is likely that this isolated finding of a correlation between acute serum rise in growth hormone and type I fiber hypertrophy (West, et al., 2012) is a result of type I error due to correlations which were not corrected for multiple comparisons. A number of mechanisms have been investigated that are hypothesized to be able to explain the variability in the resistance training-mediated hypertrophic response. The commonality linking all of these mechanisms is that they are intrinsic to the muscle and 23 measured through the use of muscle biopsies. Satellite cells (SC) are muscle-specific progenitor cells that reside between the basal lamina and sarcolemma of human muscle cells. Satellite cells are activated in response to muscle injury in order to facilitate repair and remodelling. The function of these cells is to donate their nuclei to existing myofibers to either replace nuclei that may have undergone apoptosis (Hawke et al., 2001) or to maintain a relatively constant nuclei to cytoplasmic ratio (Allen et al., 1999). Extreme hypertrophic ‘responders’ to resistance training appear to demonstrate a greater ability to expand their SC pool following training in comparison to either moderate or non-responders (Petrella et al., 2008). These results indicate that SC may not be an important regulatory factor in modest hypertrophy, however the ability to mobilize a robust SC response may partially separate extreme from moderate hypertrophic responders to resistance training. Micro RNA (miRNA) are small non-coding fragments of RNA that act as post- transcriptional regulators of gene translation. Expression of certain miRNAs has been shown to impact muscle hypertrophy (Davidsen et al., 2011). The mechanisms through which miRNA modulate hypertrophy is unknown. However, there is evidence that specific miRNAs modulate both anabolic signaling (Small et al., 2010) and MPB in animal models (Xu et al., 2012). Even without a full mechanistic understanding, the differential expression of miRNA has been used to explain the variance in resistance training mediated hypertrophy (Timmons, 2011). Four miRNA transcripts have been found to be differentially regulated in high and low responders to resistance exercise. High responders to training did not exhibit any change in miRNA expression over 12 weeks of resistance training whereas low responders showed a down-regulation of miRNA-378, miRNA-29a, and miRNA-26a and an up-regulation of miRNA-451 (Davidsen, et al., 2011). Changes in expression of miR-378 alone were able to explain 51% of the variance in muscle 24 hypertrophy in a cohort of high and low responders (Davidsen, et al., 2011). The amount of variance explained in the population as a whole is, however, likely significantly less as the sample only included the upper and lower quartiles from a single study population. The authors of the above study suggested that the miRNA changes within the low responder group may be reflected of a failure to up-regulate growth or remodelling genes and may be compensatory in nature (Davidsen, et al., 2011). There have been other attempts to explain variability in resistance training-mediated muscle hypertrophy such as using signalling protein phosphorylation (Terzis et al., 2008) and genome-wide transcriptome profiling (Phillips, et al., 2013) which will be discussed later in this document. Although several studies have been successful in explaining some of the variance in resistance training mediated hypertrophy, no single biomarker has been validated and shown to explain a large proportion of the variance. The main common finding between the few large scale studies which have attempted to explain variability in training induced muscle hypertrophy has been a consistent distribution of low, moderate and high responders to training (Bamman, et al., 2007; Davidsen, et al., 2011; Hubal, et al., 2005; Phillips, et al., 2013) 1.4 RESISTANCE TRAINING LOAD The external load used during resistance exercise is one of the major program variables that can be modulated in exercise prescription (ACSM, 2009) and is thought to have a profound effect on hypertrophy (Holm et al., 2008) . Load can be reported as an absolute mass but relative load is most often reported in the literature (ACSM, 2009). Relative load can be reported as a percentage of the heaviest load an individual can lift once for a given exercise (single repetition maximum – 1RM) or as load which can only be lifted a set number of times but no more such as a ten repetition maximum or 10RM. Relative load is often referred to in the literature as 25 ‘intensity’. This terminology may be appropriate when discussing a single repetition, however, when many repetitions are performed a low relative load can still be quite ‘intense’ at least at the point of fatigue (Steele, 2013). For this reason, relative load will be used in place of intensity throughout this document. DeLorme published one of the earliest studies on the benefits of higher relative loads in promoting resistance exercise-induced strength gains (Delorme, 1945). However, data produced by Berger has had a heavy influence on the prescription of resistance exercise to this day (Berger, 1962). Berger suggested that training between 4 and 8 repetitions would maximize strength gains in the bench press exercise (Berger, 1962). The study he conducted was underpowered and not well controlled by today’s standards, but that doctrine still stands today. There have been other studies which have confirmed that heavier relative loads are superior at producing strength gains yet there have been very few studies that have addressed the effects of relative load on muscle hypertrophy(ACSM, 2009). A single study by Campos and colleagues is often cited as evidence that a heavy load must be employed to produce hypertrophy (Campos et al., 2002). This study demonstrated that training with 3-5RM or 9-11RM for 8 weeks resulted in hypertrophy in the vastus lateralis whereas training with 28-30 RM did not result in detectible hypertrophy. The training program employed by Campos et al (Campos, et al., 2002) has been used by another research group who showed hypertrophy in both the high and low repetition training groups but did not show any difference in hypertrophy between the high and low repetition groups after 8 weeks of training (Leger et al., 2006). The Campos et al. (Campos, et al., 2002) study along with numerous anecdotal reports, have led to the popularization of training in a so-called ‘hypertrophy zone’ between 6 and 12RM as the optimal method to achieve resistance training induced muscle hypertrophy (Baechle et al., 26 2000; Kraemer et al., 2004; Schoenfeld, 2010). This concept is an extension of the strength- endurance continuum model, which states that training with a high relative load and low repetitions maximizes strength gains whereas training with a light relative load and higher repetitions will maximize gains in muscle endurance (Campos, et al., 2002; Robinson et al., 2009). The strength-endurance continuum has strong empirical support and is largely a function of neural adaptations induced by heavy relative loads and metabolic adaptations induced by lighter relative loads (ACSM, 2009). Although the concept of a ‘hypertrophy zone’ has little empirical support, proponents argue that the size principle of motor unit recruitment implies that light relative loads do not result in recruitment of the large type II motor units. Type II motor units innervate type II muscle fibres that are particularly sensitive to hypertrophy with resistance training (Carpinelli, 2008). The size principal states that motor units are recruited in an orderly fashion from smallest to largest based on motor neuron diameter in order, to generate the required force for a given task (Henneman, 1957). Type I motor units have the smallest motor neuron diameter and type IIx motor units have the largest motor neuron diameter. Based on this theory maximal contractions would require activation of all the motor units in a muscle whereas contractions requiring a low relative load should only activate a portion of the motor unit pool and thus primarily rely on force generation from type I fibers (Henneman et al., 1965). This is taken as evidence that lifting relatively light loads cannot induce hypertrophy in type II fibers because these fibers are not activated. What is missed by promoters of this view is that when light relative loads are lifted repeatedly type I fibers that are originally activated fatigue and therefore to continue to exert a given amount of force the muscle must recruit more fibers. This results in the use of fibers from the type II pool (Carpinelli, 2008). If a load is lifted until the point of failure then theoretically all muscle fibers should be activated; thus, there should be a 27 potential for all muscle fibers to experience hypertrophy when light relative loads are lifted to the point of failure. 1.5 RESISTANCE TRAINING VOLUME The volume used during a session of resistance training is often defined as the product of the load used. The number of repetitions preformed and the number of sets completed is often reported as total kilograms lifted (load X reps X sets) (Burd et al., 2010a). Changes in the number of sets which are performed is the most common way to manipulate the volume of resistance training. It is generally agreed that larger volumes of resistance exercise produce larger gains in muscle hypertrophy to a certain point after which additional volume is no longer beneficial for hypertrophy (ACSM, 2009). There is debate over what the exact optimal volume to induce to maximize hypertrophy is. Some researchers believe that a single set to failure is all that is necessary (Carpinelli et al., 1998) and others believe that 4-6 sets or more per muscle group is optimal for maximizing muscle hypertrophy with resistance training (Peterson et al., 2005). Most individual studies comparing single and multiple sets have failed to show a significant difference in hypertrophy gains over studies ranging from 4 to 25 weeks (Carpinelli, et al., 1998; McBride et al., 2003; Ostrowski et al., 1997). Although, a recent meta-analysis lead to the conclusion that more sets (i.e., 3 > 1) are beneficial for maximizing muscle hypertrophy (Krieger, 2010). Multiple sets were recommended; however, no recommendations on the optimal number of sets to maximize muscle hypertrophy could be made. This discrepancy probably arises due to the large individual variation in the magnitude of resistance training induced muscle hypertrophy and relatively small sample sizes used in the majority of studies (Carpinelli, et al., 1998; Timmons, 2011). In fact, only a single study included in the Krieger (2010) meta-analysis independently found greater lean mass or muscle cross-sectional area gains with the performance 28 of multiple sets rather than a single set (Ronnestad et al., 2007). If this single study is removed from the Krieger (2010) meta-analysis then there is no longer a statically significant benefit of performing multiple rather than a single set for inducing muscle hypertrophy (Ronnestad, et al., 2007). It appears that there are likely small benefits to increased training volumes for resistance training induced hypertrophy and that these benefits are probably more pronounced in more experienced resistance trainers. 1.6 PROTEIN TURNOVER AND HYPERTROPHY Muscle mass is regulated by the dynamic balance between muscle protein synthesis (MPS) and muscle protein break down (MPB) (Millward et al., 1976). Both processes are ongoing and in constant opposition to each other, however, consumption of protein and/or carbohydrate as well as performance of exercise can alter the balance and shift between MPS and MPB (Bennet et al., 1989; Phillips et al., 1997). In the post-absorptive state, MPB is greater than MPS putting the muscle in a state of net catabolism. When a meal containing amino acids is ingested MPS is up-regulated and MPB is down-regulated resulting in a period of net protein accretion (Glynn et al., 2010). In young healthy individuals who are properly nourished (i.e., in energy balance with protein at least as high as the protein RDA – 0.8g/kg/d), the magnitude of post-absorptive muscle catabolism and postprandial muscle anabolism are roughly equal throughout the day, and thus muscle mass is maintained. When resistance exercise is performed in the post-absorptive state, MPS is increased and MPB is also increased but to a smaller extent. Thus net protein balance (MPS minus MPB) becomes less negative and there is an overall increase in the rate of protein turnover, however the muscle remains in a state of net catabolism (Phillips, et al., 1997). It is only when amino acids are consumed in close temporal proximity after resistance exercise that net protein accretion occurs. Amino acids and resistance exercise 29 act in a synergistic fashion when the two stimuli are combined which promotes larger increases in MPS and net protein balance than when either stimulus is applied independently (Biolo et al., 1997). When resistance exercise is performed chronically over time (training) and in combination with adequate protein intake, the periods of net protein anabolism are larger than the periods of net protein catabolism and muscle undergoes hypertrophy (Phillips, 2000). If both MPS and MPB could be measured over the entire course of a resistance training study then it would be theoretically possible to calculate the exact percentage increase in muscle size. MPS is most often measured with acute stable isotope infusion methodology; this technique is both expensive and invasive and is therefore only used for periods of a few hours in duration. MPB can also be calculated using stable isotope methodology, however, it is more technically challenging and cannot be calculated at the same time with the same tracer (Kumar et al., 2009a). Because MPS appears to be more responsive to exercise and nutritional manipulations and because of the technical challenges associated with MPB, may studies measure MPS in the hours after a manipulation as a surrogate for long term muscle protein accretion (Glynn, et al., 2010). In addition, changes in MPS are more than 2-3 times as great as those in MPB (Phillips, 2004), pointing to the fact that a more obvious loci of regulation in determining net muscle protein balance is by regulation of MPS and not MPB. It is possible that changes in MPB are playing some kind of regulatory role, however, in healthy young men or women who are receiving an adequate energy and protein intake it is unlikely that proteolysis is substantially elevated and unlikely that it is adaptively regulated to control muscle protein mass. Previous work from our lab has shown that acute manipulations of exercise-based or nutrition-related variables result in a qualitatively similar pattern of outcomes in acute MPS response and hypertrophy in different groups of subjects. An acute study showed that consumption of both soy and milk protein after 30 resistance exercise lead to an increase in MPS but that the magnitude of the increases was greater in the group who ingested milk protein (Wilkinson et al., 2007). A 12 week resistance training study showed an identical pattern of results where subjects who were fed either soy or milk protein after their workout gained lean mass but that magnitude of the gain was greater in the subjects who consumed milk protein post exercise (Hartman, et al., 2007). Similarly an acute study by West et al. showed that there was no difference in biceps MPS when subjects preformed the same workout in isolation or with elevated endogenous hormones induced by an intense lower body workout (West et al., 2009b). This study was followed with a training study by the same author who showed that when one bicep was trained in isolation and the other was trained under elevated endogenous hormones induced by an intense lower body workout, there was identical biceps hypertrophy in both conditions (West, et al., 2009a). Together this pair of studies shows a similar pattern of results between acute MPS studies and training studies in different subjects which involved the same manipulated variables. Acute measurements of MPS have shown a dose-response relationship between relative load and MPS where lower relative loads result in lower post exercise FSR measurements (Kumar et al., 2009b). Holm and colleagues showed when equated for total volume, contractions at 70% of 1RM resulted in a greater stimulation of muscle FSR than contractions at 16% of 1RM (Holm et al., 2010). Similarly, Kumar and coworkers conducted a study testing the MPS responses to loads ranging from 20 to 90% of 1RM where the number of repetitions preformed in each condition was controlled to equate total volume. They found that loads of 60% of 1RM or greater were required to maximize the post exercise FSR response (Kumar, et al., 2009b). Taken together, these studies suggest that heavier relative training loads may be required to maximize post exercise anabolism. Because volume was equated in these studies the higher relative load 31 conditions resulted in the accumulation of fatigue whereas the low relative load conditions resulted in lower levels of fatigue. In contrast, Burd et al. tested both the effects of an equal volume at a lower relative load and the same low load lifted to fatigue by conducting a study with three conditions; 90% 1RM load preformed to the point of failure, 30% 1RM load preformed to the point of failure and 30% 1RM load with the total work matched to the 90% 1RM condition. The results from this study showed that when both the 30% and 90% loads were lifted to the point of failure there were large and similar increases in acute post exercise FSR. However, in the 30% work matched group there were only small and transient increases in post exercise FSR (Burd et al., 2010c). These results indicate that training to failure regardless of load may be a potent stimulus for hypertrophy. The optimal number of sets, to maximize the post exercise MPS response, has been investigated in both young and older subjects. Previous work from our lab has shown that in young men, 3 sets of knee extensions resulted in an FSR response that was both greater in magnitude and duration compared to a single set of knee extensions (Burd, et al., 2010a). In this study each set was performed to the point of muscular failure. A study conducted by Kumar and colleagues looked at the effects of higher exercise volumes on post exercise MPS and compared the FSR response of 3 and 6 sets of knee extensions in both young and older men (Kumar et al., 2012). They found that 6 sets produced a larger FSR response than 3 sets in older men, but not in young men. There was, however, a trend towards a larger MPS response with 6 sets in young men. Other than the small sample size used in this study there are two major confounding factors. First, volume was strictly controlled so that none of the sets were performed to the point of failure and second, all MPS measurements were made in the fasted state. In the Kumar et al. (2012) study MPS returned to resting levels 24 hours post exercise in all conditions whereas in 32 the study by Burd et al. MPS was elevated 24 hours after the performance of 3 sets in the fed state (Burd, et al., 2010a). It is possible that greater exercise volume prolongs a sensitization to feeding but not an elevation in resting MPS (Burd et al., 2011) which may be an underlying mechanism for the greater observed hypertrophy with higher training volumes (Krieger, 2010). 1.7 ANABOLIC SIGNALING PATHWAYS Protein synthesis is regulated by multiple overlapping pathways that regulate both the initiation of protein translation and the elongation of proteins (Drummond et al., 2009a). The pathway involved in protein synthesis that has received the most attention is the Akt-mammalian target of rapamycin (mTOR) pathway. mTOR is centrally important in this pathway and exists in two complexes (1 and 2) (Drummond, et al., 2009a). mTOR complex 1 (mTORC1) is thought to be the main site that integrates signals from contraction, growth factors, and nutrition, serving to regulate the phosphorylation of downstream proteins such as p70S6K1 and 4EBP-1 (Bodine et al., 2001). One of the most investigated upstream regulatory proteins of mTOR is Akt, also known as protein kinase B (PKB), which can be activated by insulin and other growth factors (Bodine, et al., 2001). Although Akt phosphorylation has been found to increase after many diet and exercise manipulations, there are also reports of mTOR phosphorylation and elevations of MPS in an Akt-independent fashion. This suggests a low degree of concordance between Akt phosphorylation and MPS (Liu et al., 2002). Nutrients can also regulate mTOR in a number of different ways. First, the muscle cell is unable to mount a large protein synthetic response in the face of low energy availability. The inability to activate mTOR in low energy is likely due to activation of Adeonsine monophosphate kinase (AMPK), which appears to act as an energy sensor and activates tuberous sclerosis complex-2 (TSC2) in the face of low cellular energy status, which serves to inhibit 33 mTOR (Hardie et al., 2012). The essential amino acid leucine is thought to be a primary positive nutritional regulator of mTOR, possibly through direct action of the amino acid on the protein itself, however, the exact mechanism by which leucine interacts with mTOR is yet to be fully elucidated. It also appears that leucine likely acts through multiple redundant mechanisms (Kimball et al., 2001). Leucine’s main transporter LAT1 (Drummond et al., 2010) may be involved in the process as well as Vps34 and other endosomal proteins (MacKenzie et al., 2009). Contraction is known to be a potent activator of mTOR and MPS. However, the mechanism by which the contraction signal is detected is not well understood. Recent evidence suggests that phosphorylation (inactivation) of the mTOR inhibitor TSC2 may be involved in the mechanotransduction process. Recent work by Jacobs and colleagues showed that contraction phosphorylates TSC2 at a different site than Akt (Jacobs et al., 2013) but the upstream contraction sensor that transduces the contractile signal is still unknown. Two potential candidates for a contraction sensor are phospholipase D which catalyzes the formation of the second messenger phosphatidic acids and Focal adhesion kinase (FAK). Phospholipase D inhibition has been shown to prevent the mechanical activation of mTOR signalling (Hornberger, 2011). FAK knockdown C2C12 cells are unable to respond to IGF-1 by increases in protein synthesis (Crossland et al., 2013). More work is needed to determine the exact physiological roles of each of the putative contraction sensors. Multiple lines of evidence from animal (Bodine, et al., 2001) and cell culture models (Crossland, et al., 2013) to acute protein synthetic measurements in humans (Drummond et al., 2009b) to large scale transcriptomic studies (Phillips, et al., 2013) have clearly implicated the mTOR pathway as important in muscle hypertrophy. For this reason many authors have speculated that downstream targets of this pathway could be used as a proxy measure for protein 34 synthesis or muscle hypertrophy. The earliest demonstration of this concept is work by Keith Baar which showed a correlation between the acute phosphorylation status of p70S6K after an acute bout of electrical muscle stimulation and the magnitude of hypertrophy after repeated electrical stimulation in a rodent model (Baar et al., 1999). The first human study to show a correlation between p70S6K phosphorylation after an acute bout of resistance exercise and training induced hypertrophy was conducted by Terzis et al. (2008) however, this study only included 8 subjects. A weak correlation was also found between p70S6K phosphorylation after an acute bout of resistance exercise and training mediated hypertrophy by a second group (Mayhew et al., 2009). There have also been other studies including one with a very large sample size, which have not shown any relationship between acute phosphorylation of downstream mTOR targets and training mediated hypertrophy (Fernandez-Gonzalo et al., 2013; Phillips, et al., 2013). There are also multiple reports in the literature of weak correlations between both 4EBP-1 (Burd, et al., 2010c) and p70S6K (Kumar, et al., 2009b) and the acute elevation in MPS after resistance exercise. Because negative or non-significant results are less likely to be published there may be additional findings of no relationship between the acute phosphorylation of downstream mTOR targets and hypertrophy which are not included in the literature (Easterbrook et al., 1991). More work investigating both the time course of phosphorylation of mTOR targets and employing larger samples sizes will be required to better understand how acute signalling might relate to long term adaptation. 35 1.8 REFERENCES 1. Abe, T., DeHoyos, D. V., Pollock, M. L., & Garzarella, L. Time course for strength and muscle thickness changes following upper and lower body resistance training in men and women. Eur J Appl Physiol, 81(3), 174-180. 2000 2. ACSM. American College of Sports Medicine position stand. 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Kidney Int, 82(4), 401-411. 2012 42 CHAPTER 2 TITLE: Resistance exercise load does not determine training-mediated hypertrophic gains in young men AUTHORS: Mitchell CJ, Churchward-Venne TA, West DW, Burd NA, Breen L, Baker SK, & Phillips SM PUBLICATION: Journal of Applied Physiology. 2012 Jul;113(1):71-7 43 44 45 46 47 48 49 50 CHAPTER 3 TITLE: Muscular and Systemic Correlates of Resistance Training-Induced Muscle Hypertrophy AUTHORS: Mitchell CJ, Churchward-Venne TA, Bellamy L, Parise G, Bake SK, & Phillips SM PUBLICATION: PLoS ONE. 8(10): e78636. 2013. 51 52 53 54 55 56 57 58 59 60 61 CHAPTER 4 TITLE: Increases in fed-state post-exercise myofibrillar protein synthesis are unrelated to resistance training-induced muscle hypertrophy AUTHORS: Mitchell CJ., Churchward-Venne TA., Parise G., Bellamy L., Baker SK., Smith K., Atherton PJ., and. Phillips SM. PUBLICATION: Submitted to PLoS ONE 62 Increases in fed-state post-exercise myofibrillar protein synthesis are unrelated to resistance training-induced muscle hypertrophy Cameron J. Mitchell 1 , Tyler A. Churchward-Venne, 1 , Gianni Parise 1 , Leeann Bellamy 1 , Steven K. Baker 2 , Kenneth Smith 3 , Philip J. Atherton 3 , and Stuart M. Phillips 1 1 Exercise Metabolism Research Group, Department of Kinesiology, McMaster University. Hamilton, Ontario, Canada. 2 Department of Neurology, School of Medicine, McMaster University. Hamilton, Ontario, Canada. 3 Metabolic and Molecular Physiology Research Group, MRC-ARUK Centre of Excellence for Musculoskeletal Ageing Research, School of Graduate Entry Medicine and Health, Derby, UK Running Title: Myofibrillar Protein Synthesis and Hypertrophy Key Words: Stable isotope, Protein turnover, Exercise, Variability Address for correspondence: Dr. Stuart Phillips Department of Kinesiology, McMaster University 1280 Main Street West Hamilton, Ontario L8S4L8 Canada Phone: 905 525 9140 ext. 24465 E-mail: phillis@mcmaster.ca 63 KEY POINTS SUMMARY  Skeletal muscle hypertrophy is observable following regular resistance exercise (training) and involves the expansion of the myofibrillar protein pool.  The degree of muscle hypertrophy after a resistance exercise training program is, however, highly variable between individuals and may be related to an individual’s capacity to activate the process of myofibrillar protein synthesis.  We measured signalling protein phosphorylation as well as myofibrillar protein synthesis (MPS) after an acute bout of resistance exercise in the untrained state and muscle hypertrophy following 16 weeks of resistance training; however, there was no relationship between acute MPS and hypertrophy.  There was a significant, albeit weak, correlation between phosphorylation of the signaling protein 4EBP-1 (Thr37/46) one hour after an acute bout of resistance exercise and muscle hypertrophy after 16 weeks of training.  Acute measures of either MPS and/or signalling protein phosphorylation are unrelated, or only weakly related, to measures of hypertrophy 64 ABSTRACT Muscle hypertrophy following resistance training (RT) involves activation of myofibrillar protein synthesis (MPS) to contribute new proteins to the myofibrillar protein pool. The degree of hypertrophy following RT is, however, highly variable. We sought to determine the relationship between the acute activation of MPS and RT-induced hypertrophy. We measured MPS and signalling protein activation after the first session of resistance exercise (RE) in untrained men (n=23) and then examined the relation between these variables with magnetic resonance image-measured hypertrophy. To measure MPS, young men (24 ± 1yr; body mass index = 26.4 ± 0.9 kg • m -2 ) received a primed constant infusion of L-[ring- 13 C6] phenylalanine and MPS was measured at rest and for 6 h, in the fed state, following their first bout of RE prior to 16wk of RT. Rates of MPS were increased 235 ± 38% (P < 0.001) above rest 1-3 h post-exercise and 184 ± 28% (P = 0.037) 3-6 h post exercise. Quadriceps volume increased 7.9 ± 1.6% (range: -1.9-24.7%; P < 0.001) with RT. There was no correlation between changes in quadriceps muscle volume and rates of MPS measured over 1-3 h (r = 0.02), 3-6 h (r = 0.16) or the aggregate 1-6 h post-exercise period (r = 0.10). Hypertrophy with RT was correlated (r = 0.42, P = 0.05) with phosphorylation of 4EBP-1 Thr37/46 at 1 h post RE. We conclude that acute measures of MPS following an initial exposure to RE in untrained novices are not correlated with muscle hypertrophy following chronic RT. Abbreviations: 1RM, one repetition maximum; DXA, dual-energy x-ray absorptiometry; FSR, fractional synthetic rate; MPB, myofibrillar protein breakdown; MPS, myofibrillar 65 protein synthesis; FSR, fractional synthesis rate; MRI, magnetic resonance image; RT, resistance training; RE, resistance exercise; and mTOR, mammalian target of rapamycin. INTRODUCTION Skeletal muscle hypertrophy with resistance training (RT) requires the net addition of new myofibrillar proteins; thus, myofibrillar protein synthesis (MPS) must exceed myofibrillar protein breakdown (MPB). Using phosphorylation as a proxy for activation and activity, signalling pathway proteins in the Akt (PKB)-mTOR pathway have been measured in humans and some (Terzis et al., 2008), but not all (Mitchell et al., 2012), have reported correlations between the phosphorylation state of certain proteins and hypertrophy. In larger samples, measures of protein phosphorylation in multiple signaling proteins are unrelated to the hypertrophic response seen with RT (Phillips et al., 2013); however, it is unknown if the same is true for a relationship between MPS and hypertrophy. Rates of MPS in the fed-state, in combination with resistance exercise (RE), have been used assess potential for induction of muscle hypertrophy with RT (Wilkinson et al., 2007; Tang et al., 2009). Responses of post-exercise MPS with ingestion of milk or soy protein (Wilkinson et al., 2007) or carbohydrate (Tang et al., 2007) are congruent with RT-induced hypertrophy seen, in different groups of subjects, with RT utilizing similar post-exercise nutrition (Hartman et al., 2007). Similarly, the MPS response with fatiguing heavier or lighter load RE (Burd et al., 2010b), or with differing volumes of RE (Burd et al., 2010a) are aligned with muscle hypertrophy seen in different subjects following RT with similar RE programs (Mitchell et al., 2012). Taken together, the relative congruence 66 between MPS responses and chronic RT-induced hypertrophy suggests that measures of post-exercise MPS appear to vary similarly and thus may be correlated with muscle hypertrophy; however, such a possibility has not been tested. There is a high degree of variability in the hypertrophic response to RT. Typical coefficients of variation of the hypertrophic response measured using muscle fibre size changes in young and old men and women can exceed 100% (Hartman et al., 2007; Petrella et al., 2008; Mitchell et al., 2012; Phillips et al., 2013). There have been attempts to explain the variability in hypertrophy using gene expression (Davidsen et al., 2011; Phillips et al., 2013), satellite cell enumeration (Petrella et al., 2008), measurement of signaling protein phosphorylation (Terzis et al., 2008; Mitchell et al., 2013), and measures of systemic hormonal responses to RE (West & Phillips, 2012). Both gene expression (Davidsen et al., 2011; Phillips et al., 2013) and satellite cell continent (Petrella et al., 2008) appear related to hypertrophy whereas acute post-exercise systemic hormonal responses show no relationship to RT-induced hypertrophy (West & Phillips, 2012). In humans, protein signaling appears aligned but only weakly to hypertrophy (Mayhew et al., 2009). In small samples stronger correlations between phosphorylation of p70S6K and hypertrophy have been observed (Terzis et al., 2008), but this is not consistently seen (Mitchell et al., 2012; Mitchell et al., 2013). To date, however, there have been no attempts to assess the relationship between acute measures of MPS and hypertrophy following RT, in the same subjects. Thus, the purpose of this study was to determine if MPS measured in training-naïve subjects after their first bout of RE, with 67 protein consumption, was related to muscle hypertrophy following 16 weeks of RT. Our hypothesis was that these measures would be related. METHODS Subjects. Twenty-three healthy young men (177 ± 2 cm; 84.1 ± 3.5 kg; body mass index = 26.4 ± 1.0 kg•m -2 ; 24 ± 1 yr, means ± SD) participated in the experiment. Subjects were recreationally active but had not engaged in RT within the last year. The protocol was approved by the Research Ethics Board of Hamilton Health Sciences and McMaster University and complied with all ethical standards for research involving human participants set by the Declaration of Helsinki and by the Canadian Tri-Council statement on ethics in human research (http://www.ethics.gc.ca/eng/policy- politique/initiatives/tcps2-eptc2/Default/). Experimental Design. Participants underwent a magnetic resonance imagining (MRI) scan of their right thigh to determine muscle volume and a dual-energy x-ray absorptiometry (DXA) scan to assess whole body fat- and bone-free mass (lean mass). Subjects were then strength tested to determine their maximal isotonic strength, traditionally termed one repetition maximum (1RM) for all training exercises. At least 5 d following strength testing participants reported to the lab after a 10 h overnight fast for stable isotope infusion to measure MPS using measures we have described extensively previously. Resting MPS was measured, subjects then completed four sets of 8 repetitions of leg press, leg extension, leg curl and calf press. They then ingested a protein beverage containing 30 g of milk protein, 25.9 g of carbohydrates and 3.4 g of fat (Musahi P30, Notting Hill, Australia). Muscle biopsies were then taken at 1, 3 and 6 http://www.ethics.gc.ca/eng/policy-politique/initiatives/tcps2-eptc2/Default/ http://www.ethics.gc.ca/eng/policy-politique/initiatives/tcps2-eptc2/Default/ 68 hours post exercise to measure MPS. Subjects then completed 16 weeks of RT while ingesting the protein beverage immediately after their exercise session and with breakfast on non-training days, as previously described (Mitchell et al., 2013). Briefly, participants trained four times weekly with two upper and two lower body workouts. Lower body exercises are described above in the acute exercise session. Upper body exercises consisted of chest press, shoulder press, seated row, seated pulldown, bicep curl and tricep extension. The program was progressive in linear manner moving from 3 sets of 12 repetitions to 4 sets of 6 repetitions. At the end of the training period, MRI, DXA scans, and strength testing were repeated. Infusion protocol. On the trial day, participants reported to the lab after an overnight fast having refrained from any strenuous physical activity for at least 3 days. A 20-gauge plastic catheter was inserted into an antecubital vein and a baseline blood sample was obtained. Following the start of a primed constant infusion of L-[ring- 13 C6] phenylalanine (prime: 2 μmol kg −1 ; infusion: 0.05 μmol kg −1 min −1 ), participants rested for 3 h before a muscle biopsy was obtained to determine their resting (basal) rate of MPS. Subjects then completed the lower body exercise protocol described above and ingested their protein beverage (described above). They then rested in bed for the next 6 h while biopsies (vastus lateralis) were taken 1, 3 and 6 h after cessation of the exercise bout. The drink containing 30 g of milk-based protein was enriched to 6% of the protein-contained phenylalanine content with free [ 13 C6] phenylalanine tracer to minimize disruptions in isotopic steady state, which is an approach we have used numerous times before with good maintenance of isotopic steady-state (Burd et al., 2012c; Churchward- 69 Venne et al., 2012). Biopsies were obtained with a 5 mm Bergström biopsy needle modified for manual suction under local anaesthesia (2% xylocaine). Biopsy samples were blotted and freed of any visible fat and connective tissue, frozen in liquid nitrogen (within ∼20 s of being taken from the muscle) and stored at −80°C until further analysis. Imaging. After arriving at the site of the MRI scanner, subjects rested in a supine position for 1 h prior to scanning to prevent fluid shifts from influencing measurements of muscle volume. Subjects were instructed not to engage in any strenuous activity for at least 24 h prior to the scan. MRI scans were performed in a 3-T HD scanner (Signa MRI System; GE Medical, Milwaukee, WI) at the Brain-Body Institute, Imaging Research Centre, St. Joseph's Healthcare (Hamilton, Ontario). Image acquisition was carried out using T1 fluid attenuation inversion recovery in the axial plane with the following parameters: repetition time/echo time = 2,100 ms/23.8 ms; field of view = 25–30 cm; matrix size = 512/512 slice thickness = 5 mm. Thigh image acquisitions utilized an eight-channel torso coil with two excitations. There was a 10 mm gap between slices. Quadriceps volume was calculated by multiplying the slice area by the distance between slices. Volume was measured from the first slice where the rectus femoris was visible to the first slice where the gluteus maximus was visible. ImageJ software (U. S. National Institutes of Health, Bethesda, MD) was used to determine the area of each slice. Pre- and post-scans were performed at the same time of day and joint angle and leg compression was controlled using a custom built foot frame to suspend the heel of the subject. 70 Whole-body DXA scans (QDR-4500A; Hologic, software version 12.31) were carried out pre and post training to determine total body weight, fat mass, and (fat and bone free) lean mass. Western Blotting. Muscle samples (~40-50 mg) were homogenized on ice in buffer (10 μl mg −1 25mM Tris 0.5% v/v Triton X-100 and protease/phosphatase inhibitor cocktail tablets (Complete Protease Inhibitor Mini-Tabs, Roche, Indianapolis, IN; PhosSTOP, Roche Applied Science, Mannhein, Germany). Samples were then centrifuged at 15,000 g for 10 minutes 4°C. The supernatant was removed and protein concentrations were determined via BCA protein assay (Thermo Scientific, Rockford, IL). Working samples of equal concentration were prepared in Laemmli buffer. Equal amounts (20 µg) of protein were loaded onto 10% or gradient precast gels (BIO-RAD Mini-PROTEAN TGX Gels, Bio-Rad Laboratories, Hercules, CA) for separation by electrophoresis. Proteins were then transferred to a polyvinylidene fluoride membrane, blocked (5% skim milk) and incubated overnight at 4°C in primary antibody: phospho-Akt Ser473 (1:1000, Cell Signalling Technology, #4058) phospho-mTOR Ser2448 (1:1000, Cell Signalling Technology, #2971), phospho-4E-BP1 Thr37/46 (1:1000, Cell Signalling Technology, #2855), Phospho-S6 Ser240/244 Ribosomal protein (1:2000, Cell Signalling Technology, #2215). Membranes were then washed and incubated in secondary antibody (1 h at room temperature) before detection with chemiluminescence (SuperSignalWest Dura Extended Duration Substrate, ThermoScientific, #34075) on a FluorChem SP Imaging system (Alpha Innotech, Santa Clara, CA). Phosphorylation status was expressed relative to α- 71 tubulin (1:2000, Cell Signalling Technology, #2125). Images were quantified by spot densitometry using ImageJ software (US National Institutes of Health). Isotopic analyses. As described previously (Burd et al., 2010b) approximately 20 mg (wet weight) of muscle was used to isolate free intracellular amino acids. A separate piece of muscle (∼30 mg) was used to isolate, hydrolyse, purify, derivatize and analyse the myofibrillar protein fraction enrichment. The rate of myofibrillar protein synthesis was calculated using the standard precursor–product method as previously described (Burd et al., 2010b): FSR (% • h -1 ) = [(Ep2 - Ep1) / (Eic • t -1 )] • 100 Where, FSR is the fractional synthetic rate, Ep2 and Ep1 are the protein bound enrichments from muscle biopsies at time 2 (Ep2) and plasma proteins or the previous muscle biopsy at time 1 (Ep1) and thus their difference is the change in bound protein enrichment between two time points; Eic is the mean intracellular phenylalanine enrichment from biopsies at time 2 and time 1; and t is the tracer incorporation time. The utilization of “tracer naïve” subjects allowed us to use the pre-infusion blood sample (i.e., mixed plasma protein fraction) as the baseline enrichment (Ep1) for the calculation of resting MPS. This approach makes the assumption that the baseline 13 C enrichment (δ 13 CPDB) in the blood reflects that of muscle protein; this is an assumption that has been previously (West et al., 2009) and shown to be valid in allowing calculation of a reliable rate of MPS in the fasted state (Burd et al., 2012a; Burd et al., 2012b). Statistics. Differences in means from pre to post training were compared with paired Student’s t-tests. Temporal differences in the phosphorylation of signalling proteins and 72 FSR were compared with one-way repeated measures ANOVA. Relationships between variables were assessed using the Pearson’s product moment correlation. All analyses were conducted using SPSS version 20 (IBM Armonk, New York, USA). Alpha was set at P ≤ 0.05. Means are reported ± SE unless otherwise indicated. RESULTS Plasma and muscle intracellular free phenylalanine enrichment. Intracellular free- phenylalanine precursor enrichments were 0.046 ± 0.003 at rest and 0.066 ± 0.004 throughout the fed-state post exercise incorporation period. The slope of a linear regression lines fit through the intracellular enrichments was not significantly different from zero during the post-exercise period (P > 0.05). Plasma enrichments at 60, 180 and 360 min were 0.070 ± 0.002, 0.075 ± 0.003 and 0.076 ± 0.003, respectively. Linear regression analysis indicated that the slopes of the plasma enrichments were not significantly different from zero (P > 0.05) and thus an isotopic plateau was achieved and that the use of the steady-state precursor product equation was appropriate. Muscle Size and Strength. Quadriceps muscle volume increased from 1837 ± 195 to 1970 ± 71 cm 3 (Figure 1A), while whole body fat- and bone-free mass increased from 62.6 ± 2.0 to 64.8 ± 2.1 kg (Figure 1B). Maximal isotonic strength, expressed as 1 RM, increased from 236 ± 15 to 380 ± 15 kg and from 77 ± 4 to 96 ± 4 kg in the leg press (Figure 1C) and chest press (Figure 1D) exercises respectively. Western Blotting. Phosphorylation of mTOR Ser2448 was increased above rest at 1 and 3 h post-exercise but had returned to baseline by 6 h post-exercise (Figure 3A). Phosphorylation of Akt Ser473 was increased above resting at 1 h post-exercise then 73 returned to baseline by 3 h post-exercise (Figure 3B). Phosphorylation of 4EBP-1 Thr37/46 was not significantly increased at any time post-exercise (P=0.142; Figure 3C). Phosphorylation of rpS6 Ser240/244 was elevated above rest at 1,3 and 6 h post-exercise; however, at 6 h post exercise the phosphorylation was reduced compared to 1 and 3 h (Figure 3D). There was a significant correlation between the phosphorylation of 4EBP-1 phosphorylation at 1 h post exercise and the RT-induced change in muscle volume (r = 0.42, P=0.047, Figure 4B). Myofibrillar Protein Synthesis. Rates of MPS following RE were increased compared to rest from 1 to 3 h post-exercise (P< 0.005) and from 3 to 6 h post-exercise (P=0.034; Figure 2). There was no statistically significant difference between the 1-3 h and the 3-6 h rates (P=0.159). The aggregate MPS response over the entire post-exercise period (1-6 h) was 0.052 ± 0.04 %•h -1 , which was significantly greater than resting MPS rates. There was no correlation between MPS at any of the time periods measured and the change in muscle volume as measured by MRI. Figure 4a shows the correlation between MPS measured over the full post exercise infusion period and change in muscle volume (r = 0.01). This comparison is highlighted becaus