Cold-water immersion (CWI) is a common recovery modality used to facilitate restoration of pre-exercise muscle force generation and soreness following high-intensity exercise. Although it is commonly used by athletes and commonly studied in sport science, evidence is equivocal regarding its efficacy. We compared 4 CWI protocols (10 or 30 minutes at 10 or 20°C) of different durations and temperatures with passive rest for their effects on drop jump and squat jump height, inflammation (IL-6, IL-10, IL-8, MPO, IL-1β, TNFα, IFNγ, GM-CSF, IL-2), and ratings of soreness/impairment following high-intensity intermittent sprint-exercise. CWI for 10 minutes at 10°C promoted restoration of force generation, while CWI for 30 minutes at 10°C was associated with lower ratings of soreness/impairment, but higher plasma IL-8 and MPO at 2 hours post-exercise. Overall, minor functional benefits of CWI for 10 minutes at 10°C were observed, while longer duration CWI protocols may increase post-exercise inflammation.

The aim of this Thesis was to further characterise and utilise an in vitro skeletal muscle (SkM) model, to investigate its potential use in further understanding the cellular and molecular adaptations to exercise in vivo. Candidate genes and proteins have been identified using in vivo, ex vivo and targeted in vitro experiments, however the complete picture of these molecular mechanisms are far from understood. Furthermore, the extent to which mechanical signals contribute to the intra-cellular mechanisms associated with exercise is also underinvesitgated. To this end, developing an in vitro model of SkM that can recapitulate in vivo SkM and respond to mechanical stimulation in a similar way to exercise will provide a means to begin to delineate the complex cellular and molecular regulation of SkM. The initial investigation (Chapter 3) characterised an optimal seeding density and culture period of C2C12 myoblasts within a 3 ml collagen gel. These data provided support for the use of collagen constructs seeded at 4 x 106 cells/ml, with no statistical differences observed in peak force, rate of force development and relative force compared to other seeding densities examined (table 3-2, all p > 0.05). However the use of 4 x 106 cells/ml supports previous data in a larger construct volume model, whilst the highest cell density possible in the system increases cell-cell contact required for fusion. Immunohistochemical and gene expression analyses provided evidence for the fusion of single seeded myoblasts into multinucleate myotubes, demonstrating an in vivo-like architecture. Chapter 4 presented data towards the characterisation and use of two distinct cyclical stretch regimens with respect to the acute biochemical and transcriptional responses. Data revealed increases in peak media lactate and reductions in peak media glucose, following cyclical stetch compared to control (p = 0.000 and p = 0.001 respectively, Fig. 4-2). Changes in mtDNA (Fig. 4-5) and associated mRNA transcriptional signals (Fig. 4-7) were mode dependent.

Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a global issue affecting about 24 million individuals. Because AD is a systemic pathology, dementia is not the only leading factor contributing to loss of independence in AD patients. AD may also impair skeletal muscle metabolism and function. Creatine (CR) supplementation may enhance skeletal muscle hypertrophy/mass and function in sarcopenia and muscular dystrophies, but has yet to be studied in AD. This study examined the effect of oral CR on muscle metabolism in a triple-transgenic (3xTg) AD mouse model. Twenty-four, 3×Tg AD mice (~8 month-old) were randomly assigned to control (CON) or CR (3% w/w) diet. Bodyweights and feed intakes were measured throughout the 8-week study. Lower limb (quadriceps muscle; QM and gastrocnemius; GM) and upper limb muscles (triceps; TM) were collected to analyze levels of CR, total protein, DNA, RNA, amino acids (AA), adenosine triphosphate (ATP), adenosine diphosphate (ADP), total and phosphorylated p70 ribosomal S6 kinase (p70S6K). Data (mean ± SEM) were assessed by analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) post hoc test. In comparison to the CON group, CR supplementation increased CR content in both GM (p=0.002) and QM (p=0.037), with higher (p=0.032) ATP/ADP ratio in CR in comparison with CON in QM. A higher protein concentration (p<0.0001) was notable in GM of CR supplemented group vs. CON. Total branched-chain AA levels in QM increased 2-fold (p< 0.0001) in CR groups. Additionally, CR resulted in a higher (p<0.05) protein/DNA ratio; an index of muscle cell size, in both QM and GM for CR groups. The index of cell capacity for protein synthesis (RNA/DNA ratio) in GM was also higher (p=0.001) in CR groups. However, phosphorylation (activation) level of p70S6K, an integral component in protein synthesis signalling pathway, did not show any significant differences in female (p=0.161) and male (p=0.292) CR supplemented groups compared with CON. To conclude, CR supplementation is capable of inducing muscle hypertrophy/growth parameters in the 3×Tg AD mouse model, thereby enhancing protein synthesis capacity in skeletal muscles, thus possibly promoting muscle function in AD. / May 2016

18 effects of TZDs are probably due to the remodeling of adipose tissue and increased adiponectin secretion. SUMMARY Studying the pathogenesis of insulin resistance and the role of PKC in insulin resistance In HHTg rats, elevated serum triglycerides and FFA were associated with the ectopic accumulation of triglycerides in tissues and reduced insulin sensitivity of peripheral tissues. Impaired glucose utilization in the peripheral tissues was associated with the reduced activity of GS in skeletal muscle. Decreased GS activity and glucose utilization in peripheral tissues indicate a possible defect in insulin signal transduction. In line with this, our results show that skeletal muscle IR was associated with the increased activation and translocation of PKC θ. Nutritionally induced obesity of HHTg rats resulted, in many cases, in the further deterioration of metabolic abnormalities associated with IR. We found that PKC θ, in particular, could contribute to the metabolic abnormalities associated with IR and obesity. The age-related increase in IR and deterioration of some parametres of carbohydrate and lipid metabolism, were not associated, in HHTg rats, with obesity but with increased serum levels of triglycerides and FFA. The age-related worsening of IR in HHTg rats was accompanied by increased...

Insulin maintains glucose homeostasis through its binding of the insulin receptor and activation of the insulin signalling cascade in insulin sensitive tissues. Skeletal muscle is a major endocrine organ, and is responsible for the majority of post-prandial glucose disposal. The maintenance of glucose homeostasis is a delicate balance and impairments in glucose disposal can have significant physiological effects, resulting in the onset of metabolic diseases such as diabetes mellitus. Insulin stimulated glucose uptake involves a number of signalling proteins to enable uptake to occur. In order to understand the complexities associated with the insulin signalling cascade, cell culture models have provided a controlled and easily manipulated environment in which to investigate insulin stimulated glucose uptake in skeletal muscle. While the majority of these experiments have been conducted in conventional monolayer cultures, the growing field of three-dimensional tissue engineering provides an alternative environment in which skeletal muscle cells can be grown to investigate their physiological function. The purpose of this thesis was to investigate the use of different cell culture models for investigating the effects of acute and chronic insulin exposure on skeletal muscle. Initial investigations aimed to establish glucose uptake in tissue engineering skeletal muscle constructs using tritium labelled (H3) 2-deoxy-d-glucose. Monolayer cultures were used to developed base line conditions. In these cultures, concentrations greater than 0.5 μCi for 15 minutes of insulin stimulation suggested an initial assay window for investigating insulin stimulated glucose uptake. However, the duration of insulin stimulation was not effective in measuring uptake in tissue engineered skeletal muscle constructs based upon western blot experiments of Akt phosphorylation, therefore insulin stimulation in skeletal muscle tissue engineered constructs was increased to 30 minutes. Glucose uptake is mediated via specific glucose transporter protein, GLUT1 and GLUT4. Therefore, the transcriptional profile of these transporters was elucidated in monolayer culture and tissue engineered skeletal muscle constructs. Time course experiments showed an increase in GLUT4 transcription in tissue engineered and monolayer culture systems which is associated with an increase in the transcription of skeletal muscle development and myogenic genes. In two dimensional culture, skeletal muscle cells were exposed to insulin during differentiation and in post-mitotic skeletal muscle myotubes to investigating the potential effects upon metabolic genes and proteins involved in insulin signalling. Chronic exposure to insulin during skeletal muscle differentiation reduced insulin signalling and resulted in an increase in basal glucose uptake and ablated insulin stimulated glucose uptake. In contrast, post-mitotic skeletal muscle myotubes did not shown similar changes and were not as responsive to acute insulin exposure. Therefore future experiments exposed skeletal muscle to insulin during differentiation. Using the previous findings as a basis for experimentation, the effects of chronic and acute insulin exposure upon three dimensional skeletal muscle constructs were investigated. Fibrin and collagen constructs were grown for a total period of 14 days. Constructs were exposed to insulin during differentiation and acutely stimulated for 30 minutes at day 14. Although there was a mean increase in Akt protein phosphorylation in both types of tissue-engineered constructs, these changes were not significant following acute insulin stimulation. In addition, glucose uptake in fibrin skeletal muscle constructs increased as a result of acute insulin stimulation however was not significantly difference to unstimulated constructs. The work presented in this thesis provides initial experimental data of the use of different skeletal muscle cell culture models for investigating insulin signalling and glucose uptake. Further research should further characterise these in vitro models for investigating skeletal muscle metabolism.

Doctor of Philosophy / Department of Anatomy and Physiology / Timothy I. Musch / Nitric oxide (NO) is synthesized via distinct NO synthase (NOS) enzymes and constitutes an essential cardiovascular signaling molecule. Whereas important vasomotor contributions of endothelial NOS (eNOS) have been well-described, the specific vasomotor contributions of nNOS-derived NO in healthy subjects during exercise are unknown. The purpose of this dissertation is to test the global hypothesis that nNOS-derived NO is a critical regulator of exercising skeletal muscle vascular control. Specifically, we utilized the selective nNOS inhibitor S-methyl-L-thiocitrulline (SMTC) to investigate the effects of nNOS-derived NO on skeletal muscle vascular function within established rodent models of exercise performance. The first investigation (Chapter 2) identifies that nNOS inhibition with SMTC increases mean arterial pressure (MAP) and reduces rat hindlimb skeletal muscle blood flow at rest whereas there are no effects during low-speed (20 m/min) treadmill running. In Chapter 3 it is reported that nNOS inhibition with SMTC reduces blood flow during high-speed treadmill running (>50 m/min) with the greatest relative effects found in highly glycolytic fast-twitch muscles and muscle parts. Chapter 4 demonstrates that nNOS-derived NO modulates contracting skeletal muscle blood flow (increases), O2 consumption (VO2, increases), and force production (decreases) in the rat spinotrapezius muscle and thus impacts the microvascular O2 delivery-VO2 ratio (which sets the microvascular partial pressure of O2, PO2mv, and represents the pressure head that drives capillary-myocyte O2 diffusion). In Chapter 5 we report that systemic administration of the selective nNOS inhibitor SMTC does not impact lumbar sympathetic nerve discharge. This reveals that the SMTC-induced peripheral vascular effects described herein reflect peripheral nNOS-derived NO signaling as opposed to centrally-derived regulation. In conclusion, nNOS-derived NO exerts exercise-intensity and muscle fiber-type selective peripheral vascular effects during whole-body locomotor exercise. In addition, nNOS-derived NO modulates skeletal muscle contractile and metabolic function and, therefore, impacts the skeletal muscle PO2mv. These data identify novel integrated roles for nNOS-derived NO within healthy skeletal muscle and have important implications for populations associated with reduced NO bioavailability and/or impaired nNOS structure and/or function specifically (e.g., muscular dystrophy, chronic heart failure, advanced age, etc.).

Estudos têm mostrado que o treinamento de força de baixa intensidade com oclusão vascular (TFOV) tem apresentado resultados similares nos ganhos de força e hipertrofia comparado ao treinamento de força (TF) de alta intensidade. O objetivo deste estudo foi comparar os efeitos de três diferentes programas de TF nos ganhos de força e hipertrofia musculares e na expressão da miostatina (MSTN) e seus antagonistas. Para isso, vinte e nove jovens do sexo masculino, sem experiência em TF, foram recrutados e divididos randomicamente nos grupos: treinamento de força de baixa intensidade sem oclusão (BI), treinamento de força de baixa intensidade com oclusão (BIO) e treinamento de força de alta intensidade sem oclusão (AI). Os grupos BIO e BI treinaram com intensidade de 20% 1RM, enquanto o grupo AI treinou com intensidade de 80% 1RM. A ANOVA one way foi utilizada para testar as diferenças percentuais nos ganhos de força (1RM) e na área de secção transversa (AST) do músculo quadríceps femoral. O modelo misto para análise das medidas repetidas foi utilizado para testar as diferenças nas variáveis miostatina (MSTN), folistatina-3 (FLST-3), SMAD-7 e GASP-1 nos grupos BI, BIO e AI nas condições pré e pós-treinamento. Os resultados mostraram que os aumentos de força e hipertrofia musculares nos grupos BIO e AI foram similares, entretanto superiores ao grupo BI. Esses resultados podem ser atribuídos a maior diminuição na expressão da MSTN nos grupos BIO (45%) e AI (41%) comparados com o grupo BI (16%) e o aumento na expressão dos genes que antagonizam sua atividade (SMAD-7, FLST-3 e GASP-1). Podemos concluir que a inibição na atividade da MSTN dos grupos BIO e AI podem responder em parte a similaridade nos ganhos de força e hipertrofia entre os grupos e a diferença para o grupo BI / It has been demonstrated that low intensity training associated to vascular occlusion (LIO) promotes similar gains in strength and muscle mass when compared to high intensity strength training (HI). The aim of the present study was to evaluate the effect of three different training programs on skeletal muscle hypertrophy and atrophy related gene expression. Twenty nine young male, with no previous experience in strength training were randomly allocated in three groups: low intensity strength training (i.e. 20% - 1-RM) (LI); low intensity strength training associated to vascular occlusion (i.e. 20% - 1-RM) (LIO); high intensity strength training (HI) (i.e. 80% - 1-RM). One-way ANOVA was used to assess differences in % delta change values of 1-RM and cross sectional area (CSA) of the quadriceps femoris. Mixed model analysis was used to compare myostatin (MSTN), folistatyn-3 (FLST-3), SMAD-7 e GASP-1 changes between groups pre and post training. Results demonstrated similar increases in strength and muscle hypertrophy for LIO and HI groups. Moreover, such increases were significantly greater when compared to LI. These results may be, at least in part, explained by a significant decrease in MSTN mRNA expression in LIO (45%) and HI (41%) when compared to LI (16%); additionally, SMAD-7; FLST-3 and GASP-1 mRNA expression were significantly increased. In conclusion, LIO training promotes similar gains than HI training. The results may be explained by changes in MSTN and related genes mRNA expression

Orientador: Maria Alice Rostom de Mello / Banca: Eliete Luciano / Banca: Carlos Alberto da Silva / Banca: Maria Esméria C. do Amaral. / Banca: Silvia Cristina Crepaldi Alves / Resumo: Este estudo visou analisar o metabolismo da glicose no músculo sóleo de ratos submetidos a desnervação bem como a resposta do animal como um todo à insulina exógena e ao exercício. Os animais foram tratados de acordo com o Guide for care use of laboratory animals. (National Research Council, 1996). Para isso foram realizados 3 experimentos. No primeiro, ratos adultos (3 a 5 meses) foram submetidos à secção do nervo isquiático da pata direita. Observou-se redução da captação de glicose pelo músculo sóleo isolado da pata desnervada após 3 e 7 dias mas não após 28 dias em relação a animais controles íntegros. Não houve diferença após 3 e 7 dias na captação de glicose pelo músculo sóleo da pata contralateral em relação ao animal controle. Houve aumento da captação de glicose na pata contralateral 28 dias após a desnervação. Verificou-se que após 28 dias de desnervação, não houve reinervação do nervo isquiático. A taxa de remoção da glicose em resposta à insulina exógena após 28 dias de desnervação foi superior à de animais controle íntegros e aquela observada após 3 e 7 dias de desnervação. No segundo, ratos nas mesmas condições, após 48 h de desnervação iniciaram programa de natação 1h/dia, 5dias/semana, durante 28 dias. Ratos desnervados submetidos ao exercício mostraram maior taxa de remoção da glicose sangüínea em resposta à insulina exógena comparados aos sedentários e a animais controle íntegros. O exercício elevou a captação e a oxidação da glicose pelo músculo contralateral e pelo desnervado assim como pelo músculo de animais controles íntegros. Esses resultados sugerem que alterações restritas do metabolismo glicídico muscular influenciam a homeostase glicêmica do animal. Além disso, o exercício melhora o aporte e a utilização da glicose no músculo desnervado... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to analyze the metabolism of glucose in the soleus muscle of rats submitted to denervation, as well as the response of the animal as a whole to exogenous insulin and to exercise. To do this, 3 experiments were carried out. In the first, adult rats (3 to 5 months old) were submitted to section of the sciatic nerve in the right paw. A reduction was observed in the uptake of glucose by the isolated soleum muscle of the denervated paw after 3 and 7 days, but not after 28 days in relation to the whole control animals. There was no difference after 3 and 7 days in glucose uptake by the soleum muscle of the opposite paw in relation to the control animal. There was increased glucose uptake in this same paw 28 days after denervation. The rate of glucose removal in response to exogenous insulin after 28 days of denervation was higher than that of whole control animals and those observed after 3 and 7 days of denervation. In the second experiment, rats in the same conditions, after 48 h of denervation started a swimming program 1h/day, 5day/week, for 28 days. The denerved rats submitted to exercise showed a higher blood glucose removal rate in response to exogenous insulin compared to the sedentary and whole control animals. Exercise raised the uptake and oxidation of glucose by the opposite muscle and by the denervated one, as well as by the muscles of the whole control animals. These results suggest that restricted alterations of the muscular glucose metabolism influenced the glycemic homeostasis of the animal. Furthermore, exercise improved the uptake and use of glucose in the denerved muscle. In the third experiment, adult rats in the same conditions as in the first study, had an assessment made of their insulin secretion by isolated pancreatic islets, incubated in the presence of growing concentrations of glucose (2.8; 5.6; 8.3 and 16.7 mM)... (Complete abstract, access undermentioned electronic address) / Doutor

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder characterized by insufficient SMN protein, resulting in motoneuron death. Initially, it was thought that motoneuronal death is followed by muscle atrophy; however, recent research is beginning to reveal possible muscle intrinsic defects, independent of motoneuron defects, in SMA. Previous studies have elucidated the cooperative involvement of CARM1, HuD and SMN in motoneurons, revealing HuD as a possible key player in the SMA phenotype. In this study, we focus on HuR, a ubiquitous family member of HuD, and the possibility that it plays a similar key role with CARM1 and SMN in skeletal muscle. Through the use of an shCARM1 stable line of C2C12s, we show that CARM1 is necessary for HuR functionality during differentiation. We further show that the methylation of HuR is necessary for its capability to translocate cytoplasmically during differentiation. We confirm an interaction between HuR and SMN, suggestive of a similar mechanism as was shown previously with HuD. In light of these findings, we next progressed to determine whether HuR is misregulated in an SMA mouse model. We report increased CARM1 levels in skeletal muscles of these mice. We further discovered that a deficiency in SMN protein impairs HuR upregulation and cytoplasmic translocation in response to HuR activation through sciatic nerve denervation. These findings were correlated with aberrant mRNA expression of HuR targets upon denervation. Taken together, these results show that HuR methylation is essential for proper myogenesis, and that the mechanism by which it acts likely requires sufficient SMN protein levels. In a deficiency of SMN, HuR shows signs of misregulation that may play a role in the inability to maintain or repair muscle in SMA.

No description available.