Title: The Relationship between Moderate, Within Day Protein Intake and Energy Balance on Body Composition of Collegiate Sand Volleyball Players Background: Achieving an ideal body composition with relatively low fat mass and relatively high fat-free mass (FFM) is desirable for virtually all competitive athletes. Some studies suggest that protein intake, depending on quality, amount, and timing, may improve relative musculature by stimulating muscle protein synthesis, but some issues related to timing and amount of protein intake remain unclear. Current evidence suggests that frequent consumption of moderate amounts of protein is useful for muscle building. Purpose: The purpose of this study was to simultaneously assess energy balance and protein intake to determine if these factors are associated with body composition in a population of collegiate sand volleyball players. Methods: In a cross sectional, observational study, players completed a food intake and activity form for a 24-hour period to serve as the basis of energy balance and protein intake assessment. The assessment day was representative of a typical day during the regular training season. These data were entered into a software program providing total and hourly energy balance and nutrient content of the consumed foods. Athletes were measured for body composition via a multi-current bioelectrical impedance scale to predict weight, BMI, fat mass and fat free mass. Height was measured using a standard wall-mounted stadiometer. Data analyses included descriptive and frequency statistics, Spearman correlations and regression analyses. Results: Twelve women from the GSU sand volleyball team participated in the study using an IRB-approved protocol. The mean BMI was 22 kg/m2 (±3 kg/m2) and the mean body fat percentage was 18% (±7%). The mean protein intake for all participants was 132 grams (±52 g). Protein intake distribution was skewed, on average, toward the latter half of the day with approximately 19% of protein consumed in the morning and 34% consumed in the evening. The mean net energy balance at the end of the 24-hour assessment period was -404 (±385) kcal. Athletes, on average, spent 17 hours in a catabolic energy balance state (< 0 kcal). No significant correlation was found between energy balance per gram of protein consumption and body composition. However, regression analyses indicated that energy balance and protein variables explain a significant proportion (p=.037) of the variance in body fat percentage. Conclusions: Sand volleyball players in this study spent a high proportion of time in a negative energy balance, which may have compromised the potential benefit that frequent protein consumption may have had on FFM. Since both energy balance and protein explain a significant proportion of the variance in body composition, these athletes might benefit from improving within-day energy balance as a strategy for optimizing body composition.

An acute bout of resistance exercise (RE) and dietary protein consumption stimulate muscle protein synthesis (MPS). This anabolic effect is believed to be attenuated with resistance exercise training (RET), however, the mechanism for this plateau" is unknown. In addition, the ideal timing for protein consumption to optimize MPS is not well characterized. The central hypothesis of this research is that RE stimulates cumulative (measured over 24-36 h) MPS in rats and humans. Study one determined whether an acute bout of RE in rats enhances MPS when assessed with the traditional flooding dose (~ 25 min) and 2H2O (4 and 24 h measurements); thus a comparison of the two methodologies was made. An acute session of RE did not result in an elevation in MPS when quantified by either the flooding dose or 2H2O over 4 and 24 h (methods compared qualitatively). Therefore, an acute bout of RE in rats does not appear to be anabolic and adaptation resulting from multiple bouts is likely necessary. Study two determined if RET in rats results in attenuation in MPS (plateau effect) 16 h following the final RE session (peak anabolic window) and if it is due to an increase in 4E-BP1 (a key regulator of mRNA translation initiation) activity; or if the timing in anabolism changes, which could be detected with a cumulative assessment (2H2O). MPS at 16 h was unchanged following RE training. Consistent with this finding, there were no differences in 4E-BP1 activity. Conversely, cumulative MPS was significantly increased with RET, suggesting a temporal shift in anabolism. Study three determined if dietary protein consumed immediately following RE augments cumulative (24 h) MPS in young adult human males when energy and macronutrients are controlled. RE and post-RE protein had no effect on mixed MPS; however, myofibrillar MPS was significantly increased with RE suggesting specific changes within a heterogeneous protein pool. Collectively, these are the first studies to assess changes in cumulative MPS with RE in rats and humans. The long term goals of this research are to understand muscle protein anabolism in "free-living" mammals and the mechanisms that regulate this process.

An acute bout of resistance exercise (RE) and dietary protein consumption stimulate muscle protein synthesis (MPS). This anabolic effect is believed to be attenuated with resistance exercise training (RET), however, the mechanism for this plateau" is unknown. In addition, the ideal timing for protein consumption to optimize MPS is not well characterized. The central hypothesis of this research is that RE stimulates cumulative (measured over 24-36 h) MPS in rats and humans. Study one determined whether an acute bout of RE in rats enhances MPS when assessed with the traditional flooding dose (~ 25 min) and 2H2O (4 and 24 h measurements); thus a comparison of the two methodologies was made. An acute session of RE did not result in an elevation in MPS when quantified by either the flooding dose or 2H2O over 4 and 24 h (methods compared qualitatively). Therefore, an acute bout of RE in rats does not appear to be anabolic and adaptation resulting from multiple bouts is likely necessary. Study two determined if RET in rats results in attenuation in MPS (plateau effect) 16 h following the final RE session (peak anabolic window) and if it is due to an increase in 4E-BP1 (a key regulator of mRNA translation initiation) activity; or if the timing in anabolism changes, which could be detected with a cumulative assessment (2H2O). MPS at 16 h was unchanged following RE training. Consistent with this finding, there were no differences in 4E-BP1 activity. Conversely, cumulative MPS was significantly increased with RET, suggesting a temporal shift in anabolism. Study three determined if dietary protein consumed immediately following RE augments cumulative (24 h) MPS in young adult human males when energy and macronutrients are controlled. RE and post-RE protein had no effect on mixed MPS; however, myofibrillar MPS was significantly increased with RE suggesting specific changes within a heterogeneous protein pool. Collectively, these are the first studies to assess changes in cumulative MPS with RE in rats and humans. The long term goals of this research are to understand muscle protein anabolism in "free-living" mammals and the mechanisms that regulate this process.

Is Supplemental Leucine Intake Associated with Enhanced Post Exercise Muscle Protein Synthesis and Attenuated Muscle Protein Breakdown? Knight AD, Benardot D, Thompson W, and Henes ST Introduction: The role of individual amino acids on protein synthesis and their impact on physical performance is of high importance to athletes and to those studying the science of sports nutrition. Leucine, one of three branched-chain amino acids, is a frequently researched amino acid because of its potential stimulatory effect on muscle protein synthesis (MPS) following exercise in humans. Purpose: Although there have been many studies conducted on leucine’s muscle stimulatory effect, questions remain as to the efficacy and feasibility of leucine as an MPS catalyst. Contributing to these questions are the widely varied dosing and timing strategies that different researchers have employed. It is the purpose of this thesis, therefore, to assess the differences in study protocols and shed light on the potential effectiveness on leucine as a MPS stimulator. Central to this issue is whether supplemental leucine intake is associated with enhanced post exercise MPS and, if so, what associated factors, including timing and level of intake, are most likely to influence this effect. Methods: A comprehensive review of the literature on leucine and its effect on MPS was performed. Studies were organized into similar topics, with an assessment and summary of effect produced for each topic area. A general conclusion was made that was based on the summary of each topic area. Results: Leucine is involved in protein metabolism regulation through its role in stimulating the mammalian target of rapamycin (mTOR) signaling cascade and by indicating energy and amino acid availability. It functions to initiate MPS and decrease muscle protein breakdown by downregulating the ubiquitin-proteasome system, lysosomal activity, and/or increasing circulating insulin. Conclusions: Supplementation with the amino acid leucine effectively enhances MPS and attenuates muscle protein degradation in humans following bouts of physical exertion. Leucine intake in amounts greater than that found in ~20g whole protein saturates MPS and increases leucine oxidation. For this reason, an upper limit of leucine intake should be established. While leucine successfully increases MPS, it remains unclear whether this translates to enhanced physical performance, an area that requires more studies to be conducted.

Both resistance and endurance exercise elicit an increase in muscle protein synthesis during recovery from exercise. Ingestion of amino acids augments the exercise-induced stimulation of muscle protein synthesis following resistance exercise. Our work showed that 8 wk of unilateral resistance training induced muscle hypertrophy only in the exercised limb. Importantly, using this unilateral model we showed that muscle hypertrophy was confined to the exercised leg and occurred without measurable changes in circulating anabolic hormones. We then went on to use the unilateral leg resistance exercise model to study how animal-derived (milk) and plant-derived (soy) proteins impacted acute post-exercise protein turnover. We observed that ingestion of soy or milk protein resulted in a positive net protein balance following resistance exercise. Moreover, milk promoted a greater net protein balance and muscle protein synthesis than soy protein. In the final study, a key finding was that acute endurance and resistance exercise differentially stimulated myofibrillar and mitochondrial protein synthesis and also differentially affected cellular signaling proteins involved in the regulation of the protein synthetic response. Specifically, the acute, untrained state response showed that resistance exercise stimulated myofibrillar and mitochondrial protein synthesis while endurance exercise stimulated mitochondrial protein synthesis. Following resistance training only myofibrillar protein synthesis increased after exercise, while mitochondrial protein synthesis was unchanged. Endurance exercise training did not affect the acute protein synthetic response and so following training mitochondrial protein synthesis was stimulated as it was acutely, prior to training. In conclusion, the studies within this thesis provided novel insights on the impact of intact dietary proteins and differing modes of exercise on the control skeletal muscle protein metabolism. / Thesis / Doctor of Philosophy (PhD)

Pacientes com diabetes mellitus apresentam perda de massa e força muscular esquelética. O treinamento de força é prescrito aos pacientes diabéticos como parte do tratamento, pois melhora o controle glicêmico além de promover aumento da massa muscular. Foram investigados os mecanismos envolvidos na hipertrofia muscular induzida por sobrecarga mecânica durante o estabelecimento do estado diabético do tipo I induzido por estreptozotocina em ratos. Os experimentos foram realizados nos músculos com predominância de fibras oxidativas (sóleo) ou glicolíticas (extensor digital longo - EDL). Avaliou-se a modulação da via de síntese de proteínas PI3K-AKT-mTOR sete dias após indução de hipertrofia dos músculos sóleo por tenotomia do músculo gastrocnêmio e do EDL pela ablação do músculo tibial. Determinou-se também a expressão de mRNA de outras vias de sinalização que controlam a hipertrofia muscular: mecanotransdução (FAK), Wnt/&beta;-catenina e miostatina e folistatina. Os músculos sóleo e EDL quando submetidos à sobrecarga funcional sofreram hipertrofia semelhante em animais controles e diabéticos. O aumento das forças tetânica e isotônica, absolutas e específicas, ocorreu na mesma magnitude que a hipertrofia muscular. A hipertrofia do músculo EDL nos animais diabéticos envolveu principalmente a via PI3K-AKT-mTOR além da redução no conteúdo de AMPK e diminuição da expressão de miostatina. No músculo sóleo, a hipertrofia foi mais pronunciada nos animais diabéticos por ativação mais intensa da via pelas proteínas rpS6 e aumento na expressão de mRNA de IGF-1, MGF e folistatina além de diminuição nos conteúdos de miostatina, MuRF-1 e atrogina-1. As modificações relacionadas à sinalização permitiram ao músculo sóleo alcançar valores de força e massa muscular similares ao grupo controle. / Patients with diabetes mellitus have reduction in skeletal muscle mass and strength. Strength training is prescribed to diabetic patients as part of the treatment since it improves glycemic control and promotes an increase of skeletal muscle mass. The mechanisms involved in the overload-induced muscle hypertrophy during the establishment of the type I diabetic state, induced by streptozotocin, were investigated in rats. The experiments were performed in muscles with predominance of oxidative (soleus) or glycolytic (EDL) fibers. PI3K/AKT/mTOR protein synthesis pathway was evaluated seven days after the overload-induced hypertrophy of the soleus muscle by tenotomy of the gastrocnemius muscle and of the EDL muscle by tibialis anterior muscle ablation. The mRNA expression of genes associated with different signaling pathways that control muscle hypertrophy was also evaluated: mechanotransduction (FAK) signaling, Wnt/&beta;-catenin, myostatin and follistatin. The soleus and EDL muscles when submitted to overload had similar hypertrophic responses in control and diabetic animals. The increase of twitch and tetanic, absolute and specific, forces had the same magnitude as the muscle hypertrophic response. Hypertrophy of the EDL muscle from diabetic animals mostly involved mechanical loading-stimulated PI3K/AKT/mTOR pathway in addition to the reduced activation of AMPK and decrease of myostatin expression. Hypertrophy was more pronounced in the soleus muscle of diabetic animals due to a more potent activation of rpS6 and increased mRNA expression of IGF-1, MGF and follistatin, and decrease of the myostatin, MuRF-1 and atrogin-1 contents. The activated signaling pathways enabled the soleus muscle mass and force of the diabetic rats to reach the values of the control group.

The main aim of this thesis was to increase our understanding of the metabolic responses associated with short-term high-fat overfeeding. To this end, four separate studies are described in this thesis; each of which involved the provision of a high-fat, high-energy diet to young, healthy, lean individuals. The first of these experimental chapters (Chapter 2) determined the effects of a 7-day, high-fat (65%), high-energy (+50%) diet on postprandial metabolic and endocrine responses to a mixed meal challenge. This chapter demonstrates that 7-days of overfeeding impaired glycaemic control in our subject cohort but did not influence the response of selected gut hormones (acylated ghrelin, GLP-1 and GIP). In a mechanistic follow up study utilising stable isotope tracer methodology we then demonstrate that overfeeding-induced impairments in glycaemic control are attributable to subtle alterations in plasma glucose flux, rather than the overt tissue-specific adaptations (e.g. increased EGP, or reduced glucose disposal) that have previously been reported (Chapter 3). In an attempt to delineate the time-course of diet-induced impairments in glycaemic control, we then investigated the effects of 1-day of overfeeding (+80% energy with 73% of total energy coming as fat) (Chapter 4). Results demonstrate that a single day of overfeeding elicits responses which are comparable to 7-days of high-fat overfeeding; highlighting the rapidity with which excessive high-fat food intake can negatively influence glucose metabolism. In chapter 5 we utilised stable isotope tracer and muscle biopsy techniques to demonstrate that 7-days of high-fat overfeeding impairs glycaemic control but does not influence the fed-state mixed muscle protein fractional synthesis rate (FSR). In conclusion, the findings of this thesis demonstrate that while short-term high-fat overfeeding negatively influences whole-body glucose metabolism, skeletal muscle protein metabolism appears to be relatively unaffected in young, lean, healthy humans.

The development of lean body mass (LBM) is closely linked to protein feeding. Along with resistance exercise protein feeding, or amino acid provision, stimulate muscle protein synthesis (MPS). Repeated stimulation of MPS above protein breakdown results in lean mass accretion. Many athletes aim to build or maintain LBM. The aim of this thesis was to better understand the relationship between LBM and protein feeding in trained individuals. This aim was studied in the applied setting and at whole body, muscle and molecular level. Chapter 2 revealed differences in total body mass and LBM between young rugby union players competing at different playing standards. Protein consumption was higher in players that played at a higher standard. The protein consumption of players at both playing standards was higher than current protein recommendations for athletes. The Under 20 (U20) rugby union players in Chapter 3 also consumed more protein than current recommendations state. Their dietary habits changed depending on their environment and they consumed more protein while in Six Nations (6N) camp compared with out of camp. Also, there were changes in dietary habits for individuals, however, those changes did not occur at the group level. Using the camp as an education tool for good nutrition habits could be advantageous. As a group, rugby union players’ body composition did not change from pre to post a 6N tournament. However, there was individual variation, which could be meaningful for the individual players. We provide evidence suggesting that in elite sport, athletes should be considered as individuals as well as part of a group if appropriate. The protein ribosomal protein S6 kinase 1 (p70S6K1) is part of the mammalian target of rapamycin complex 1 (mTORC1) pathway, which regulates MPS. The response of p70S6K1 activity was 62% greater following resistance exercise coupled with protein feeding compared with protein feeding alone in Chapter 3. P70S6K1 activity explained a small amount of the variation in previously published MPS data. The activity of the signalling protein p70S6K1 was unchanged in response to different doses of whey protein in Chapter 4 and 5. These data suggest that resistance exercise is a larger stimulus of p70S6K1 activity and when manipulating aspects of protein feeding p70S6K1 activation may be a limited measure. Consumption of 40 g of whey protein stimulated myofibrillar MPS to a greater extent than 20 g after a bout of whole body resistance exercise. The amount of LBM that the trained individual possessed did not influence this observed response. These data suggest that the amount of muscle mass exercised may influence the amount of protein required to increase MPS stimulation. For those engaging in whole body resistance exercise 20 g of protein is not sufficient to maximally stimulate MPS. The athletes in Chapters 2 and 3 of this thesis consumed more protein than current recommendations that do not take into account whole body exercise. Current post-exercise protein recommendations may no longer be optimal given this new information. Future work should directly investigate the MPS response to protein ingestion following resistance exercise engaging different amounts of muscle mass in well trained and elite populations. Identifying the protein dose required for maximal stimulation of MPS following whole body exercise would be an informative area of future research.

Purpose: To determine the effect of four postexercise beverages, differing in macronutrient content, on metabolic response to an acute resistance exercise bout. Methods: Forty male subjects performed five sets of eight repetitions at 80% 1RM for leg press and leg extension, and then consumed one of four postexercise beverages (Placebo, PL: a carbohydrate-electrolyte beverage, CE; or one of two milk-based beverages, MILK 1: 1% chocolate milk; MILK 2: a high protein milk beverage). Indicators of muscle protein synthesis (MPS) were assessed before and 1-hr after consuming a postexercise beverage. Muscle protein degradation (MPD) was examined the day before and the day of exercise. Results: No significant differences were found among groups in MPS. The resistance exercise bout increased the amount of eIF4E-eIF4G by 4.5% 1-hr postexercise (p<0.05) without affecting the amount of eIF4E-4E-BP1. One hour after beverage consumption, serum total amino acid concentration increased for MILK 1 (p=0.003) and MILK 2 (p<0.001) but decreased for CE (p=0.028) and PL (p=0.276). Consumption of MILK 1, MILK 2, and CE significantly increased circulating levels of serum insulin (p<0.001). Serum growth hormone increased 3-fold as a result of the exercise bout but fell to baseline for all groups by 60 min (p<0.001). Conclusion: The resistance exercise bout was anabolic as shown by the increase in the active eIF4E-eIF4G complex and serum growth hormone. Consumption of MILK 2 led to the most optimal environment for muscle anabolism; however, none of the experimental beverages influenced the measured indicators of muscle protein translation 1-hr after ingestion. / Master of Science

<p>There is a prevalent belief that acute hormone responses to resistance exercise mediate adaptations in skeletal muscle hypertrophy; however, there is little supporting evidence. We conducted studies to examine the relationship between acute hormonal increases after resistance exercises and subsequent changes in muscle anabolism.</p> <p>We tested the hypothesis that exercise-induced responses of anabolic hormones—growth hormone (GH) and testosterone—would enhance rates of myofibrillar protein synthesis (MPS) after an acute bout of resistance exercise, and would augment muscle hypertrophy after training. We concluded, however, that resistance exercise-induced increases in putative anabolic hormones do not enhance MPS or hypertrophy.</p> <p>We also examined whether rates of MPS would be attenuated in women (compared with men) after resistance exercise, due to their lack of post-exercise testosteronemia. We reported similar increases in MPS in men and women; post-exercise testosterone responses in women, which were 45-fold lower than men, did not attenuate elevations in MPS.</p> <p>Collectively, our work leads to the conclusion that the acute rise in hormones such as testosterone and GH has very little bearing on MPS and hypertrophy responses to resistance exercise. Instead, the rise in these hormones appears to be a non-specific response to exercise stress rather than a response that is important for muscle anabolism. Contrary to widely used principles, our data suggests that exercise programs should not be designed based on nuances in the post-exercise hormonal milieu. Alternatively, understanding local mechanotransduction, which is directly linked to muscle fibre loading, will reveal the processes that drive human exercise-mediated muscle hypertrophy.</p> / Doctor of Philosophy (PhD)