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

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)

This study determined the effect of milk or carbohydrate-electrolyte supplementation immediately after resistance exercise on muscle protein breakdown before and after a 10-week resistance training program. Nineteen untrained males, 18-25 years of age, consumed either a carbohydrate-electrolyte (CHO) or milk (MILK) beverage immediately after a strenuous leg resistance exercise bout, both before and after training. Muscle protein breakdown, as estimated by 3-methylhistidine-to-creatinine ratio, was significantly reduced after resistance exercise for both groups, as the ratio was decreased by 19.9% from baseline on the day of resistance exercise. A trend was present for a training effect for 3-methylhistidine-to-creatinine ratio (p<0.07), as the reduction from before to after resistance exercise was greater after training. There was no difference in muscle protein breakdown between the groups. One hour after exercise, serum concentrations of amino acids were significantly elevated for MILK and significantly reduced for CHO. Serum glucose was significantly higher for both groups 30 minutes post-exercise than baseline, and serum insulin was greater than baseline 30 minutes and 1 hour after exercise. Serum insulin was significantly greater for CHO than MILK 1 hour after resistance exercise. No effect of training was observed for the response of serum amino acids, glucose, or insulin to resistance exercise with beverage ingestion. In conclusion, although the type of beverage ingested post-exercise affected serum insulin and amino acid concentrations, it did not influence the reduction in muscle protein breakdown observed after resistance exercise. A trend was present for a greater reduction in protein breakdown after training. / Master of Science

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.

This diploma thesis deals with questions of the present meat products market, meat products quality and the relevant legislation with focus on steamed ham. According to the present legislation ham is divided into three categories based on the content of pure muscle protein. The categories are as follows: top-grade quality ham, choice ham and standard ham. Use of starch, food colouring ingredients, fibrous additives, vegetable protein and different animal protein is not allowed with top-grade quality ham and choice ham. Within the practical part of the diploma work twenty samples of ham from all categories were examined. The tests showed that starch was not added to any top-grade quality ham or choice ham. Starch was found only in two samples of standard ham, in which starch addition is allowed by legislation.

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.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24782号 / 医博第4974号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 濵﨑 洋子, 教授 湊谷 謙司, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Skeletal muscle is a plastic tissue capable of responding to environmental perturbations. Increased loading via resistance exercise (RE) activates muscle protein synthesis (MPS) and, to a lesser extent, muscle protein breakdown (MPB). The ingestion of protein further stimulates MPS and suppresses MPB, inducing a positive net protein balance and protein accretion – i.e., muscle hypertrophy. In contrast, muscle unloading reduces MPS, which is thought to be the key driver underpinning skeletal muscle atrophy. The degree of muscle hypertrophy and atrophy in response to loading and unloading varies significantly between individuals and provides an opportunity to investigate the molecular regulators of skeletal muscle remodelling. To that end, we developed a novel unilateral model in which one leg was subjected to RE to induce hypertrophy (Hyp) and the contralateral limb was immobilized to induce atrophy (At). In study 1, we characterized the morphological changes induced by our HypAt model and validated the use of ultrasonography to measure changes in muscle size in both limbs. We discovered that by assessing the differential change in muscle size between legs we reduced the coefficient of variation between subjects. This enabled a more in-depth means-based characterization of the molecular regulators of skeletal muscle remodelling. Indeed, we discovered significantly more genes regulated by muscle remodelling than similarly-sized studies. We also identified a transcriptional signature that scaled with lean mass gains in three independent cohorts and included RNA species that were only modulated at their untranslated regions. Finally, in study 3 we simultaneously measured MPS and MPB in response to short-term immobilization (4 days) and demonstrated for the first time that MPB is statistically unchanged by unloading. Taken together, these studies contribute significantly to our understanding of skeletal muscle remodelling under different loading states and provide a valuable hypothesis-generating resource for future research in the field. / Thesis / Doctor of Philosophy (PhD) / Adaptations of skeletal muscle to loading and unloading are variable between individuals. Herein, we employed a unilateral approach to better understand the drivers of this variability by assessing the influence of resistance training (RT) and disuse on muscle protein turnover and gene expression. First, we validated the use of ultrasound for measuring changes in muscle size in response to loading and unloading. We then identified thousands of genes regulated by loading status and discovered many that were correlated with lean mass gain – some of which would not have been detected without our model. We also demonstrated that RT-induced increases in muscle protein synthesis were not associated with changes in muscle size; however, reductions in muscle protein synthesis were associated with the degree of muscle atrophy observed in response to disuse. Together, these studies contribute significantly to our understanding of how skeletal muscle size is regulated by muscle loading and unloading.