Macronutrient metabolism plays an essential role in the overall health of an individual. Depending on a number of variables, for example, diet, fitness level, or metabolic disease state, protein, carbohydrate and fat have varying capacities to be oxidized and balanced. Further, when analyzing the oxidation of carbohydrate and fat in the skeletal muscle specifically, carbohydrate balance happens quite rapidly, while fat balance does not. The ability of skeletal muscle to adapt and respond to various nutrient states is critical to maintaining healthy metabolic function. Habitual high fat intake has been associated with reduced oxidative capacity, insulin resistance, increased gut permeability, inflammation, and other risk factors often preceding metabolic disease states. The disruption of gut function leads to gut permeability and increases endotoxins released into circulation. Endotoxins have been shown to play an important role in obesity-related whole body and tissue specific metabolic perturbations. Each of these disrupted metabolic processes is known to associate with obesity, metabolic syndrome and diabetes. To date, limited research has investigated the role of high fat diet on skeletal muscle substrate oxidation and its relationship to gut permeability and endotoxins. The purpose of this study was to determine the effects of an acute, five-day, isocaloric high fat diet (HFD) on skeletal muscle substrate metabolism in healthy non-obese humans. An additional purpose was to determine the effects of a HFD on gut permeability and blood endotoxins on healthy, non-obese, sedentary humans. Thirteen college age males were fed a control diet for two weeks, followed by five days of an isocaloric HFD. To assess the effects of a HFD on skeletal muscle metabolic adaptability and postprandial endotoxin levels, subjects underwent a high fat meal challenge before and after a HFD. Muscle biopsies were obtained; blood was collected; insulin sensitivity was assessed via intravenous glucose tolerance test; and intestinal permeability was assessed via the four-sugar probe test before and after the HFD. Postprandial glucose oxidation and fatty acid oxidation in skeletal muscle increased before the HFD intervention but was decreased after. Skeletal muscle in vitro assay of metabolic flexibility was significantly blunted following the HFD. Insulin sensitivity and intestinal permeability were not affected by HFD, but fasting endotoxin was significantly higher following the HFD. These findings demonstrate that in young, healthy males, following five days of an isocaloric high fat diet, skeletal muscle metabolic adaptation is robust. Additionally, increased fasting endotoxin independent of gut permeability changes are potentially a contributor to the inflammatory state that disrupts substrate oxidation. These findings suggest that even short-term changes in dietary fat consumption have profound effects on skeletal muscle substrate metabolism and fasting endotoxin levels, independent of positive energy balance and whole-body insulin sensitivity. / Ph. D. / Macronutrients, namely carbohydrates, fats and protein, and the way they are utilized play an important role in the overall health of an individual. Many variables come into play when considering the oxidization (or utilization) of each macronutrient, including, but not limited to diet, fitness level, and metabolic disease state. Skeletal muscle and its role in these processes is of special interest as it is the largest insulin sensitive organ in the body. Its ability to adapt and respond to various nutrient states is critical to maintaining healthy metabolic function. Habitual high fat intake has been associated with insulin resistance, increased gut permeability (increasing endotoxins, which are toxins released into circulation from the intestines), reduced oxidative capacity (ability to utilize macronutrients for energy), and inflammation, all of which are risk factors that precede metabolic disease states. To date, limited research has investigated the role of high fat diet on skeletal muscle oxidation of macronutrients and its relationship to what is going on in the gut, or intestines. The purpose of the study was to determine the effects of a short term high fat diet (five days) on skeletal muscle in healthy, non-obese humans, and to determine the effects of this diet on gut permeability and endotoxins. Thirteen college-age males were fed a control diet for two weeks followed by five days of a high fat diet. Each diet had the same caloric content. Subjects underwent a high fat meal challenge before and after the diet to assess the effects of the diet on skeletal muscle adaptability and post meal endotoxin levels. Before and after the high fat diet, muscle biopsies were obtained, blood was collected, insulin sensitivity was assessed and gut permeability was measured. We found that skeletal muscle metabolic adaptation is robust. Additionally, increased fasting endotoxin changes are a possible contributor to the inflammatory state that disrupts macronutrient oxidation. Therefore, even short-term changes in dietary fat consumption have profound effects on skeletal muscle metabolism and fasting endotoxin levels, independent of positive energy balance and whole-body insulin sensitivity.

京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13611号 / 論医科博第12号 / 新制||医科||11(附属図書館) / 九州大学大学院薬学府創薬科学専攻 / (主査)教授 井上 治久, 教授 松田 秀一, 教授 萩原 正敏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

In many chronic inflammatory diseases, patients suffer from skeletal muscle weakness, exacerbating their symptoms. Serum levels of tumor necrosis factor-alpha (TNF) and sphingomyelinase are increased, suggesting their possible role in the progression of this weakness. This dissertation focuses on the role that reactive oxygen species (ROS) and nitric oxide (NO) play in mediating TNF-induced skeletal muscle weakness and to what extent sphingolipid signaling mediates cellular response to TNF. The first aim of this work was to identify which endogenous oxidant species stimulated by TNF contributes to skeletal muscle weakness. In C57BL/6 mice (n=38), intraperitoneal injection of TNF elicited a 25% depression of diaphragm contractile function. In separate experiments, diaphragm fiber bundles harvested from mice (n=39) and treated with TNF ex vivo showed a 38% depression of contractile function compared to untreated controls. Using ROS and NO-sensitive fluorescence microscopy in parallel with a genetic knockout animal model, TNF-induced contractile dysfunction was found to be mediated by NO generated by a specific isoform of nitric oxide synthase (NOS), nNOS. Basal levels of ROS were necessary co-mediators, but were not sufficient to elicit TNF-induced diaphragm weakness. The second aim of this dissertation was to investigate the extent to which sphingolipids could serve as a signaling cascade post-TNF stimulus leading to the generation of NO in skeletal muscle. The effects of TNF exposure in C2C12 skeletal muscle cells were studied in vitro using mass spectroscopy to measure sphingolipid metabolism and fluorescent microscopy to quantify oxidant production. TNF exposure was associated with significant mean increases in sphingosine (+52%), general oxidant activity (+33%), and NO production (+14%). These increases were due to specific modulation of nNOS as demonstrated by siRNA knockdown of neutral ceramidase and nNOS, and confirmed by pharmacologic inhibition using N-Oleoylethanolamine and di-methylsphingosine. In summary, these findings confirm NO as a major causative oxidant contributing to TNF’s deleterious phenotype in skeletal muscle. Moreover, the work suggests a new role for sphingosine in skeletal muscle and warrants further study of the enzymatic regulation of sphingosine to advance the discovery of new therapies for patients suffering from chronic inflammation.

<p>The main objective of the study was to investigate skeletal muscle force production during isometric contractions, active muscle stretches and shortenings. The motivation behind this work is to improve the dominant model of muscle contraction force generation based on the theories of Hill. The effect of force modification was observed after concentric and eccentric contractions and also stretch-shortening cycles. It has been shown that this force modification is not related to lengthening/shortening velocity, and the steady-state force after non-isometric contractions can be well described by initial isometric force and mechanical work performed by and on the muscle during length variations. The time constants calculated for isometric force redevelopment appeared to be in certain relations with those for initial isometric force development, an observation which extended our basis for ongoing muscle modeling. The main method of the project consists in two extensive series of experiments on mouse skeletal muscles. Analysis of the first series of experiments, concentric contractions, with an emphasis on the force depression has been presented in Paper 1. Paper 2 is based on contractions with various stretches and shortenings as well as their combination, force modification and its predictor are the quantities of interest. The third part of the project is also based on the second series of experiments. Timing aspects of the force production were calculated there.</p>

Adrenergic signalling is part of the sympathetic nervous system and is activated upon stimulation by the catecholamines epinephrine and norepinephrine. This regulates heart rate, energy mobilization, digestion and helps to divert blood flow to important organs. Insulin is released to regulate metabolism of carbohydrates, fats and proteins, mainly by taking up glucose from the blood. The insulin and the catecholamine hormone systems are normally working as opposing metabolic regulators and are therefore thought to antagonize each other. One of the major regulators involved in insulin signalling is the mechanistic target of rapamycin (mTOR). There are two different complexes of mTOR; mTORC1 and mTORC2, and they are essential in the control of cell growth, metabolism and energy homeostasis. Since mTOR is one of the major signalling nodes for anabolic actions of insulin it was thought that catecholamines might oppose this action by inhibiting the complexes. However, lately there are studies demonstrating that this may not be the case. mTOR is for instance part of the adrenergic signalling pathway resulting in hypertrophy of cardiac and skeletal muscle cells and inhibition of smooth muscle relaxation and helps to regulate browning in white adipose tissue and thermogenesis in brown adipose tissue (BAT). In this thesis I show that β-adrenergic signalling leading to glucose uptake occurs independently of insulin in skeletal muscle and BAT, and does not activate either Akt or mTORC1, but that the master regulator of this pathway is mTORC2. Further, my co-workers and I demonstrates that β-adrenergic stimulation in skeletal muscle and BAT utilizes different glucose transporters. In skeletal muscle, GLUT4 is translocated to the plasma membrane upon stimulation. However, in BAT, β-adrenergic stimulation results in glucose uptake through translocation of GLUT1. Importantly, in both skeletal muscle and BAT, the role of mTORC2 in β-adrenergic stimulated glucose uptake is to regulate GLUT-translocation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Skeletal muscle dysfunction in chronic obstructive pulmonary disease (COPD) is a condition in which peripheral skeletal muscle undergoes myopathic changes which impair muscle function, limit physical performance, and can lead to significant disability. While the etiology of the dysfunction is unknown, this study was conducted to test the hypothesis that chronic hypoxemia leads to alterations in oxygen transport and muscle function. A primary objective was to validate elastase-induced emphysema in rats as an animal model of skeletal muscle dysfunction in COPD.Arterial blood gases were used to determine the severity of hypoxemia and sodium dodecyl sulfate- polyacrylamide gel electrophoresis was used to determine the proportions of myosin heavy chain isoforms I, IIa, IIx, and IIb. Measures of microvascular oxygenation and blood flow in the spinotrapezius muscle allowed for determination of both convective and diffusive oxygen supply to the muscle, as well as calculation of muscle oxygen consumption at rest and during electrically stimulated three-minute muscle contractions. Muscle performance measures included peak force, force-time integral, and fatigue index. Due to a presumed rat respiratory virus, which likely resulted in the control group being nearly as hypoxemic as the elastase-induced emphysema group, this study was not able to definitively test the hypothesis that chronic hypoxemia leads to both a diminished supply and demand of oxygen in skeletal muscle. Although many of the results of the present study were not statistically significant, they exhibited consistent trends over time and are likely of physiological significance. All measures of muscle performance were lower in the emphysema group. In addition, spinotrapezius muscle oxygen consumption and blood flow were lower in the emphysema group. The addition of supplemental oxygen during isolated, small-muscle mass exercise did increase the force-time integral by ~18% in both groups, suggesting that muscle work in these hypoxemic animals may be limited by oxygen supply. Thus, the data on muscle fiber type, oxygen consumption and muscle performance suggest that elastase-induced emphysema in rats leads to a similar skeletal muscle dysfunction that is observed in humans with COPD, and indicates that it is a valid animal model of skeletal muscle dysfunction in COPD.

[Résumé traduit automatiquement par le site web Reverso : Un problème majeur dans la biologie de cellule souche pour la recherche de base et clinique est l'accessibilité aux cellules souches de sain ou des individus de malade et le maintien(la maintenance) de leur puissance pour l'expérimentation, des écrans de médicament(drogue) thérapeutiques, ou des transplantations. Ici nous rapportons des conditions pour l'isolement de cellules myogenic squelettiques viables et fonctionnelles de l'homme jusqu'à 17 jours et la souris jusqu'à 14 jours post-mortem, significativement au-delà des rapports précédents. Les cellules souches de muscle ont été enrichies dans le mouchoir en papier(le tissu) post-mortem suggérant un avantage de survie sélectif comparé à d'autres types cellulaires. Les transplantations de 4 jour le muscle de souris post-mortem et des cellules souches haematopoietic ont régénéré des mouchoirs en papier(des tissus) solidement. La quiétude cellulaire contribue à cette viabilité de cellule souche de muscle où les cellules adoptent un état inerte réversible caractérisé par l'activité métabolique réduite, une phase de décalage prolongée avant la première division cellulaire et un statut transcriptional moins prêt pour l'engagement. Plus loin(De plus), nous montrons que la réponse de stress(d'accent) de cellules souches à l'environnement post-mortem est NF-? B-independent et que des cellules souches de muscle post-mortem sont caractérisées par les niveaux élevés de ROS, plus haut mitochondrial le contenu d'ADN et l'activité inférieure d'oxyde super dismutases, pourtant ils ne montrent(n'affichent) pas de changements(monnaies) de niveaux de redox. Finalement, l'hypoxie sévère(grave), ou l'anoxie sont critiques pour maintenir(entretenir) la viabilité de cellule souche et la capacité régénératrice robuste. Ces découvertes ont des implications majeures pour des études fondamentales et cliniques sur des cellules souches et ils peuvent aussi être prolongés(étendus) à d'autres systèmes de cellule souche de normal et des patients de malade (comme un exemple que nous avons aussi montré que des cellules souches hematopoietic survivent pendant une période prolongée(étendue) après la mort dans des mouchoirs en papier(des tissus) et restent fonctionnelles in vivo).] / A major issue in stem cell biology for basic and clinical research is the accessibility to stem cells from healthy or diseased individuals, and the maintenance of their potency for experimentation, therapeutic drug screens, or transplantations. Here we report conditions for the isolation of viable and functional skeletal myogenic cells from human up to 17 days, and mouse up to 14 days post mortem, significantly beyond previous reports. Muscle stem cells were enriched in post mortem tissue suggesting a selective survival advantage compared to other cell types. Transplantations of 4 day post mortem mouse muscle and haematopoietic stem cells regenerated tissues robustly. Cellular quiescence contributes to this muscle stem cell viability where cells adopt a reversible dormant state characterised by reduced metabolic activity, a prolonged lag phase before the first cell division, and a transcriptional status less primed for commitment. Further, we show that the stress response of stem cells to the post mortem environment is NF-κB-independent, and that post mortem muscle stem cells are characterised by elevated levels of ROS, higher mitochondrial DNA content, and lower activity of super oxide dismutases, yet they do not display changes in redox levels. Finally, severe hypoxia, or anoxia is critical for maintaining stem cell viability and robust regenerative capacity. Ces découvertes ont des implications majeures pour des études fondamentales et cliniques sur des cellules souches et ils peuvent aussi être prolongés(étendus) à d'autres systèmes de cellule souche de normal et des patients de malade (comme un exemple que nous avons aussi montré que des cellules souches hematopoietic survivent pendant une période prolongée(étendue) après la mort dans des mouchoirs en papier(des tissus) et restent fonctionnelles in vivo).

As the age increases, the physical fitness of seniors decreases. This shows the fragility of their body. Managing everyday activities becomes increasingly more difficult for them. Result of this is reduced self-sufficiency of the seniors, which leads to reduced mobility and to the greater loss of muscle mass and higher dependence on professional care. Aging is accompanied by loss of muscle mass and muscle strength - sarcopenia. This is one of the main causes of geriatric fragility. Sarcopenia presents a serious health problem with both social and economic consequences. The term sarcopenia (from Greek words sarx - meaning flesh referring to muscle and penia - loss) was first used in 1989 by Irwin Rosenberg to describe the loss of muscle mass accompanying aging. Exactly defining the term sarcopenia has helped explain this gradual loss of muscle mass. Three objectives were set in the thesis. The first objective was to find out whether sarcopenia reduces the quality of life in the elderly. The second goal was to determine, which quality of life tests are suitable for testing sarcopenia and the last one was whether the SARC - F questionnaire predicts sarcopenia.Quantitative research was used for the empirical part of this work. Data collection was performed using a method of a questionnaire. These were standardized questionnaires aimed at assessing sarcopenia and quality of life. The research was carried out with a total of 77 respondents with sarcopenia and respondents without sarcopenia. The quantitative part of the research was statistically processed using the MS Excel computer program.

O ATP é a moeda corrente de energia no organismo, sendo indispensável para a manutenção da homeostase celular. A fosfocreatina é a fonte mais rápida de regeneração do ATP, através da ação da enzima creatina quinase (CK). O presente estudo tem como objetivo avaliar os efeitos da suplementação de curto prazo com creatina (Cr) sobre parâmetros da função muscular em ratos, além de alterações celulares e moleculares que auxiliem na compreensão de tais efeitos. A suplementação com Cr promoveu uma atenuação da fadiga em todos os músculos analisados. A expressão proteica do GH na hipófise dos animais suplementados encontrou-se reduzida, assim como a expressão gênica do IGF-I hepático. No músculo, O IGF-I teve sua expressão aumentada. Houve hipertrofia das fibras musculares tipo I e aumento na proporção de fibras tipo II no sóleo. O conteúdo muscular de espécies reativas de oxigênio mostrou-se reduzido no grupo Cr. Ensaios cell free apontam para uma ação direta da molécula de Cr sobre o radical superóxido. Assim, os efeitos observados podem se dever a uma ação antioxidante da Cr, que muito se assemelha à resposta observada quando animais são tratados com N-acetil-cisteína, um potente antioxidante. / The ATP is the energy unit of currency in the body and is indispensable for the maintenance of cellular homeostasis. Phosphocreatine is the fastest source for ATP regeneration, catalyzed by creatine kinase (CK). This study aims to evaluate the effects of short-term creatine (Cr) supplementation upon muscular functional parameters and cellular and molecular alterations that could explain such effects. Cr supplementation promoted an attenuation of muscle fatigue. GH protein expression was reduced in supplemented animals, as well as IGF-I mRNA in the liver, but not in muscle, where its expression was stimulated by Cr. Muscle fibers of soleus were hypertrophied and the type II fiber content was augmented. The muscular content of oxygen reactive species was lower in Cr group. Cell free experiments show a direct scavenger action of Cr on superoxide anion. Thus, the effects could result from a antioxidant properties of Cr. These effects was similar to that observed when N-Acetyl-Cysteine (a potent antioxidant) was administered to adult rats prior to intense contractile activity.

A interleucina-6 (IL-6) é uma citocina com dupla função na sensibilidade à insulina. O objetivo desse estudo foi investigar o efeito da IL-6 durante 1 e 24 horas no metabolismo de células musculares resistentes à insulina. Em células de indivíduos saudáveis, 1 hora de exposição à IL-6 aumentou a síntese de glicogênio e a captação de glicose além de pJAK2 e pSTAT3, mas não em células de diabéticos tipo 2. Diferentemente, a IL-6 aumentou a oxidação de ácido graxo em ambos os grupos. Por outro lado, em células musculares da linhagem L6, a IL-6, em 24 horas, reduziu a síntese de glicogênio e a captação de glicose em condições normais, mas não em células resistentes à insulina. Além de reduzir pJak2, pStat3 e pAkt. A IL-6 aumentou oxidação de ácido graxo e pACC em ambos os grupos. Sendo assim, células musculares resistentes à insulina apresentam uma resposta alterada ao efeito da IL-6. Nessas células, o efeito dessa citocina está abolido no metabolismo de carboidratos mas permanece efetivo no lipídico. / Interleukin-6 (IL-6) is a cytokine with a dual role in modulating insulin sensitivity. The aim of this study was to investigate the effect of IL-6 exposure for 1 hour and 24 hours on metabolism of insulin-resistant skeletal muscle cells. In muscle cells from healthy subjects, 1 hour of exposure to IL-6 increased glycogen synthesis, glucose uptake, pJAK2 and pSTAT3, which were not observed in muscle cells of type 2 diabetic subjects. In contrast, IL-6 increased fatty acid oxidation in cells from both groups. On the other hand, in skeletal muscle cell line L6, IL-6 for 24 hours reduced glycogen synthesis and glucose uptake in normal conditions, which were not observed in insulin-resistant L6 cells. In addition, pJak2, pStat3 and pAkt were reduced in insulin-resistant cells. In contrast, IL-6 increased fatty acid oxidation and pACC in both groups. Therefore, insulin resistant skeletal muscle cells have an altered response to the effect of IL-6. On these cells, the effect of IL-6 is abolished on carbohydrate metabolism, while remaining effective on lipid metabolism.