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The experimental work described in this thesis was conducted in the surgical research laboratories of Dr Francis D. Moore in the Peter Bent Brigham Hospital (now the Brigham & Women's Hospital) Boston, Massachusetts, USA, between 1978-1979. It formed part of an ongoing programme of research into protein metabolism in man; specifically to measure total body nitrogen turnover and hence protein synthesis and breakdown, initially in normal volunteers receiving various intravenous feeding regimens, and subsequently in patients. The previous year, 1977, had seen the publication of 'Substrate Interaction in Intravenous Feeding' by Bruce Wolfe et al., from the same laboratories. This was an extensive piece of work incorporating many studies and compared nitrogen balance data together with biochemical, hormonal and plasma amino acid data in normal men fed intravenously with a variety of regimens. Shortly afterwards a series of protein turnover studies was embarked upon, using the uN glycine method, and in collaboration with Dr Vernon Young of the Massachusetts Institute of Technology. The first experiments were essentially a repeat of the studies described by Wolfe et al. (vide supra) but in addition nitrogen turnover, protein synthesis and breakdown were estimated using a continuous 60 hour infusion of uN glycine and measuring enrichment of urinary urea with uN when a plateau was reached. Normal volunteers were studied firstly on normal oral diet and then on a iv succession of intravenous regimens:- amino acids alone (AA), amino acids plus 'high dose' glucose (AA + HOG), amino acids plus fat emulsion (AA + FE), amino acids plus 'low dose' glucose (AA + LOG), amino acids, fat emulsion and low dose glucose (AA + LDG+ FE), and finally 'low dose' glucose alone (LOG). The studies on normal diet, AA and AA+HOG were conducted by Andrew Sim (a Glasgow/Harvard exchange fellow) and Bruce Wolfe. The author took no practical role in these experiments, but was responsible for analysis of the data and the protein metabolism calculations, and was a co-author when the work was published in 1979 (Sim et al., Glucose Promotes Whole-Body Protein Synthesis from Infused Aminoacids in Fasting Man, Lancet i, 68-71). Subsequently, the author did the experiments using AA + LOG + FE, AA + FE, and AA + LOG and LOG. The results on these four regimens were incorporated in a paper presented in 1979 at the Tripartite Meeting of the Surgical Research Society at Oxford under the title 'Isotope Studies of substrate interaction in parenteral nutrition', and also at the 2nd European Congress on Parenteral and Enteral Nutrition at Newcastle upon Tyne in 1980, and later published as 'The Effect of Fat Infusion on Protein Metabolism' (Acta. Chir. Scand., Suppl. 507, 475-484, 1981). When the studies on the various intravenous feeding regimens were completed, attention was turned to the possible distorting effects of variables such as exercise and diet v on the behaviour of the isotope equilibrium curve and plateau. Such effects, if present, might have significance when studies were carried out on patients at a later stage in the research programme. Because each study lasted 48-60 hours, changes might occur either unintentionally or as a result of the needs of clinical management, and if they affected the plateau would alter the resultant calculations of turnover, synthesis and breakdown. Such a potential source of error clearly needed investigation. A pilot study was done in two subjects, later repeated on each, to observe any effects on the curve and plateau of both doubling protein intake and bicycle exercise. Subsequently, more extensive studies were done varying the protein and energy intakes, both orally and intravenously, allowing a detailed analysis of curve perturbation, and introducing the concept of basal catabolic rate. Finally, protein turnover, synthesis and breakdown were estimated seven times in four seriously ill patients. All of the studies mentioned above form the basis of the thesis. Unfortunately, the gestation period of this thesis has been long. There are two main reasons for this. Firstly, the work done was part of a five-year programme of research, with the intention of publishing a paper in a scientific journal at the completion of each stage. This was done vi with the first three regimens (normal diet, AA and AA + HDG) but not with the last four (AA + FE, AA + LOO+ FE, AA + LOG, LDG), although the results were presented at two scientific meetings. Shortly after returning to the United Kingdom the author was appointed a consultant surgeon and this career move assumed priority. Secondly, although it was intended to publish the perturbation studies, it proved impossible to reduce the size of the text to a manageable level suitable for publication in the form of a scientific paper. However, despite the long interval since the experiments were done, no similar work has been published. In particular, virtually no attention has been paid to intentional perturbation. Also, whereas there was a spate of interest in protein turnover studies in the late 1970s and early 1980s, virtually no publications have appeared since 1985. It seems that the potential applications of the method are considered exhausted, and interest has been lost, rather as it was in the 1950s following a short flurry of activity exploring the first cumbersome technique. Hence, it seemed all the more pertinent, even at this late stage, to publish the work in the form of a thesis which could describe in chronological order the continuum of studies as briefly mentioned above. In order to preserve such a progression, the following Introduction contains, with few exceptions, only references up to the time that the experimental studies were commenced, 1978, but the subsequent Discussion(s) in the various sections will attempt to include the relevant literature up to the present time.

<p> Resistance exercise and amino acids independently and synergistically stimulate muscle protein synthesis. Unloading of skeletal muscle depresses fasted state muscle protein synthesis, but the effect on the fed state response is unknown. Elucidation of the signaling pathways underlying the regulation of these processes in humans is in its infancy. Therefore, the purpose of this thesis was to determine how resistance exercise, feeding, and unloading interact to affect muscle protein turnover and its markers. In study 1 young men (N=9) underwent an acute bout of unilateral leg resistance exercise with or without feeding, with biopsies 6 h post exercise. Exercise dephosphorylated eiF2Bε and together with feeding potentiated the increase in phosphorylation of p70s6k and rps6. In study 2, 12 young people received primed constant infusions of 13C6-Phe in the fasted state and at one of two i.v. AA infusion rates (low, 42.5 mg/kg/h AA; high: 261 mg/kg/h AA) after 14 d of knee-brace mediated immobilization. Immobilization decreased fasted and fed state myofibrillar protein synthesis at both doses without obviously affecting translational signaling proteins. In study 3, two markers of muscle protein breakdown and oxidative damage were measured in 21 subjects (men, N=13, women, N=8) after 2 d and 14 d of knee-brace mediated immobilization. Protein ubiquitination was elevated after 2 d of immobilization but there was no sustained elevation in ubiquination at 14 d or increases in the 14kDa actin fragment or protein carbonyls and 4-hydroxy-2-nonenal. These studies support the concept that the responses of human muscle to changes in loading are primarily at the level of protein synthesis, and the p70 pathway appears to play a role in mediating the hypertrophic response. The currently known static markers of translational signaling and protein breakdown, however, are not very informative when attempting to account for an underlying molecular mechanism for disuse atrophy. </p> / Thesis / Doctor of Philosophy (PhD)

O metabolismo protéico em idosos, analisado pela medida da velocidade de reciclagem, é um importante fator para a análise da manutenção da massa muscular e das atividades de vida diária. Dados coletados em idosos apontam uma redução da síntese protéica com o envelhecimento. Outros relatam ser esta mantida e a degradação aumentada. Esta investigação teve por objetivo avaliar o metabolismo protéico de idosos saudáveis e independentes utilizando a glicina, marcada com o isótopo 15N. Sete idosos saudáveis foram estudados.Foram feitas avaliações clínica, nutricional e bioquímica em todos os voluntários, sendo excluídos aqueles portadores de doenças ou usuários de medicamentos que interferissem no metabolismo protéico. Foi oferecida uma dose oral de 200 mg de 15N-Glicina e coletadas amostras de sangue e urina (basal, antes do consumo da glicina, quatro horas e meia e nove horas após a ingestão da glicina). Foram quantificados amônia, uréia e nitrogênio total e as amostras analisadas por espectrometria de massa, para a determinação do enriquecimento isotópico (15N). Os voluntários tinham 65,4 ± 2,8 anos (média ± desvio padrão), quatro mulheres e três homens, com IMC de 22,73 ± 2,4 Kg/m2. Total de nitrogênio excretado de 3,31 ± 0,7 gN/9horas e a ingestão de 7,76 ± 1,0 gN/9horas, o fluxo de nitrogênio 15 foi de 30,36 ± 6,3 gN/9horas, o balanço nitrogenado de 4,46 ± 1,0 g/N. Os valores encontrados nesta pesquisa foram similares aos da literatura para idosos e menores que os referidos para jovens. Este estudo estabeleceu os valores do metabolismo protéico em idosos saudáveis, ingerindo alimentação típica (arroz e carne moída), o que permitirá posteriores estudos de intervenção. / Protein metabolism in the elderly, analyzed though the protein turnover rate, is of great importance to muscle trophysm and maintenance of activities of daily living. Some studies in the elderly have shown a reduction of protein synthesis with aging, while others found that it is maintained and degradation increased. Methods using stable isotopes are of great relevance in the research of protein metabolism, being non-invasive and safe. This investigation aimed to study protein turnover in healthy independent elderly through the method of glycine labeled with 15N. Seven healthy elderly persons were studied. All volunteers were assessed by clinic, nutritional and biochemical evaluation, with the exclusion of diseases and medications that could affect protein metabolism. A 200 mg oral dose of 15N-Glycine was administered and urine and blood samples were collected (basal sample before isotope intake, four hours after isotope intake and the last sample after 9 hours). Ammonium, urea and total nitrogen were quantified and analyzed by mass spectrometry, with the determination of isotope enrichment (15N). Volunteers were aged 65.4 ± 2.8 years (mean ± SE), four women and three men, with BMI 22.73 ± 2.4 Kg/m2. Total nitrogen output was 3.31 ± 0.7 gN/9hours and intake 7.76 ± 1.0 g/N; 15N nitrogen flux was 30.36 ± 6.3 gN/9hours, so the nitrogen balance was 4.46 ± 1.0 g/N. These findings were similar to those of others studies with old persons in the literature and lower than those for younger persons. This research established the values of protein metabolism in healthy old persons during the ingestion of typical food (rice and meat) and will allow the development of further intervention studies.

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.

Saccharomyces cerevisiae is confronted with continually changing and diverse nutrient conditions. As such, it has adapted to utilize both a wide variety of nutrient sources and to preferentially use the most nutrient-rich source to obtain a competitive advantage. The transcription, intracellular trafficking and protein turnover of nutrient transporters, including the hexose transporter proteins (Hxts), are strictly regulated in response to nutrient conditions. The low affinity hexose transporter Hxt3p is highly expressed and localizes to the plasma membrane during growth in abundant glucose where it plays a major role in the transport of this sugar. However, following a shift to ethanol as a sole carbon source, Hxt3p is endocytosed and targeted to the vacuole for degradation while its expression is also repressed. In contrast, the high affinity hexose transporter Hxt7p is actively expressed and functional in the plasma membrane when glucose is limiting and nitrogen is abundant. Upon nitrogen starvation or rapamycin treatment, HXT7 transcription decreases and the protein is targeted for degradation. The mechanisms that govern these regulatory steps are poorly understood. The Vid and Gid proteins, several of which compose the Vid30 complex (Vid30c), facilitate the nutrient-dependent degradation of the gluconeogenic enzymes FBPase and Mdh2p when glucose-starved cells are replenished with glucose. Here we show that components of the Vid30c are needed for the ethanol-induced turnover of Hxt3p and the rapamycin or nitrogen starvation-induced degradation of Hxt7p. In addition, we demonstrate that the signals for the ethanol-induced turnover of Hxt3p and the rapamycin-induced turnover of Hxt7p converge on the Vid30c upstream of the Ras/cAMP/PKA pathway, ultimately controlling the degradation of both these hexose transporters. Finally, we provide evidence that the Vid30c controls the localization of the Ras GEF Cdc25p and may therefore directly regulate the activity of the Ras/cAMP/PKA pathway.

O metabolismo protéico em idosos, analisado pela medida da velocidade de reciclagem, é um importante fator para a análise da manutenção da massa muscular e das atividades de vida diária. Dados coletados em idosos apontam uma redução da síntese protéica com o envelhecimento. Outros relatam ser esta mantida e a degradação aumentada. Esta investigação teve por objetivo avaliar o metabolismo protéico de idosos saudáveis e independentes utilizando a glicina, marcada com o isótopo 15N. Sete idosos saudáveis foram estudados.Foram feitas avaliações clínica, nutricional e bioquímica em todos os voluntários, sendo excluídos aqueles portadores de doenças ou usuários de medicamentos que interferissem no metabolismo protéico. Foi oferecida uma dose oral de 200 mg de 15N-Glicina e coletadas amostras de sangue e urina (basal, antes do consumo da glicina, quatro horas e meia e nove horas após a ingestão da glicina). Foram quantificados amônia, uréia e nitrogênio total e as amostras analisadas por espectrometria de massa, para a determinação do enriquecimento isotópico (15N). Os voluntários tinham 65,4 ± 2,8 anos (média ± desvio padrão), quatro mulheres e três homens, com IMC de 22,73 ± 2,4 Kg/m2. Total de nitrogênio excretado de 3,31 ± 0,7 gN/9horas e a ingestão de 7,76 ± 1,0 gN/9horas, o fluxo de nitrogênio 15 foi de 30,36 ± 6,3 gN/9horas, o balanço nitrogenado de 4,46 ± 1,0 g/N. Os valores encontrados nesta pesquisa foram similares aos da literatura para idosos e menores que os referidos para jovens. Este estudo estabeleceu os valores do metabolismo protéico em idosos saudáveis, ingerindo alimentação típica (arroz e carne moída), o que permitirá posteriores estudos de intervenção. / Protein metabolism in the elderly, analyzed though the protein turnover rate, is of great importance to muscle trophysm and maintenance of activities of daily living. Some studies in the elderly have shown a reduction of protein synthesis with aging, while others found that it is maintained and degradation increased. Methods using stable isotopes are of great relevance in the research of protein metabolism, being non-invasive and safe. This investigation aimed to study protein turnover in healthy independent elderly through the method of glycine labeled with 15N. Seven healthy elderly persons were studied. All volunteers were assessed by clinic, nutritional and biochemical evaluation, with the exclusion of diseases and medications that could affect protein metabolism. A 200 mg oral dose of 15N-Glycine was administered and urine and blood samples were collected (basal sample before isotope intake, four hours after isotope intake and the last sample after 9 hours). Ammonium, urea and total nitrogen were quantified and analyzed by mass spectrometry, with the determination of isotope enrichment (15N). Volunteers were aged 65.4 ± 2.8 years (mean ± SE), four women and three men, with BMI 22.73 ± 2.4 Kg/m2. Total nitrogen output was 3.31 ± 0.7 gN/9hours and intake 7.76 ± 1.0 g/N; 15N nitrogen flux was 30.36 ± 6.3 gN/9hours, so the nitrogen balance was 4.46 ± 1.0 g/N. These findings were similar to those of others studies with old persons in the literature and lower than those for younger persons. This research established the values of protein metabolism in healthy old persons during the ingestion of typical food (rice and meat) and will allow the development of further intervention studies.

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.

Every cell consists of carefully orchestrated biomolecules such as lipids, carbohydrates, and proteins. To maintain internal stability (homeostasis), cells maintain the right amount of these molecules at the right time and at the right place. This process is especially true for proteins since they are the foundation functional units within the cell. Proteins form structures and perform chemistry that bestows cells overarching functional roles. Cells maintain protein homeostasis (proteostasis) by modulating synthesis, folding, and degradation processes (turnover) to maintain the abundance levels for all proteins. This is the foundational kinetic model of proteostasis that is covered in this work, and it comprises protein abundance and turnover essential for protein homeostasis. When proteostasis is lost, cells may also fail to perform their internal cellular functions which will impact their external role. The sustained loss of proteostasis leads to disease. In the area of proteomics, we seek out the mechanisms of proteome change that result in the loss of normal proteostasis that are associated with disease states. As biochemists we explore the role of different proteins within biological systems and disease states. Predominantly, these studies involve isolating proteins (generally one at time) to measure abundance levels, function, and structure. In more recent years, technological advances in liquid chromatography and mass spectrometry (LC-MS) ushered in the golden age of proteomics. With LC-MS we can explore thousands of proteins in a single experiment to measure their expression levels. This work covers the fundamentals of this process as well as examples of LC-MS based proteomics for biomarker discovery and individual protein dynamics. In a sense, these experiments are like taking a snapshot of what proteins are found within a biological system at a given moment. However, cells are not static systems, rather they are dynamic systems in which proteins are being created and destroyed to maintain proteostasis. In this regard, LC-MS has recently become a powerful tool to explore protein turnover for thousands of proteins. Combined with protein abundance measurements, protein turnover yields a dynamic image of the internal state of the cell. This work applies the ideas within the kinetic model of proteostasis to explore the changes in protein homeostasis associated with Apolipoprotein E (ApoE) isoforms. ApoE isoforms are a genetic risk factor of ongoing research because of their role in disease and longevity. This work reviews some of the proposed mechanisms associated with ApoE genotype, and the LC-MS experiment we created to measure both proteome wide abundance and turnover changes associated with ApoE genotype. Our findings not only provide evidence that unifies previous ApoE studies, and it provides a benchmark for how to incorporate both quantitative and kinetic proteomics to monitor proteostasis.