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.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第22671号 / 人博第957号 / 新制||人||227(附属図書館) / 2020||人博||957(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 川本 卓男, 教授 宮下 英明, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

Nesta tese é proposto um modelo fenomenológico eletromecânico com relação constitutiva variacional para representar numericamente a resposta ativa/passiva do tecido muscular esquelético, a fadiga muscular de diferentes tipos de fibras e a combinação de contrações. Na literatura não são encontrados modelos que representem esses comportamentos combinados. Para atingir este objetivo são definidos conjuntos de variáveis auxiliares e variáveis internas para desencadear e mapear a evolução das principais características do tecido muscular esquelético. É considerado que o tecido se contrai localmente, através da propagação de um potencial de ação elétrico advindo do sistema nervoso através de uma ou mais unidades motoras. A proposta de propagação do potencial de ação é construída com a criação de critérios de malha e discretização temporal, eliminando o erro dentro desses limites. Com a propagação do potencial de ação é informado ao modelo constitutivo se localmente o tecido está ativo, através de uma variável de acoplamento binária. A relação constitutiva mecânica utiliza este parâmetro para iniciar a contração muscular, que depende da evolução de uma variável interna. A evolução dessa variável substitui a função de ativação encontrada em outros modelos. Também é utilizado um segundo potencial dissipativo para representar a eficiência metabólica do tecido resultando na representação da fadiga muscular. Um conjunto de potenciais elásticos e dissipativos são utilizados para representar diferentes níveis de forças após combinações de contração do tipo isométrica-concêntrica-isométrica e isométrica-excêntrica-isométrica para diferentes velocidades e alterações de comprimento. A proposta é verificada frente ao ajuste de parâmetros de experimentos reais, obtendo boa representação. Através da implementação no método de elementos finitos é possível observar o comportamento do modelo proposto em geometrias tridimensionais. / In this thesis an electromechanical phenomenological model with variational constitutive relation is proposed to numerically represent the active/passive response of skeletal muscle tissue, muscle fatigue of different types of fibers and contraction combination. In the literature there are no models that represent these combined behaviors. To achieve this goal, sets of auxiliary variables and internal variables are defined to trigger and map the evolution of the main characteristics of skeletal muscle tissue. The tissue is considered to contract locally by propagating an electrical action potential from the nervous system through one or more motor units. The proposed propagation of the action potential is defined with the creation of mesh criteria and temporal discretization, eliminating the error within these limits. With the propagation of the action potential it is informed to the constitutive model if the tissue is locally active, through a binary coupling variable. The mechanical constitutive relation uses this parameter to initiate muscle contraction, which depends on the evolution of an internal variable. The evolution of this variable replaces the activation function found in other models. A second dissipative potential is also used to represent the metabolic efficiency of the tissue resulting in the representation of muscle fatigue. A set of elastic and dissipative potentials are used to represent different levels of forces after isometric-concentric-isometric and isometric-eccentric-isometric contraction combinations for different velocities and length changes. The proposed model capabilities are verified with the parameters adjustment to reproduce real experiments, obtaining good representation. The implementation in the finite element method allows to observe the behavior of the proposed model in three-dimensional geometries.

Skeletal muscle injuries and muscle degenerative diseases pose significant challenges to the healthcare. Surgical interventions are restricted due to tissue availability, donor site morbidity and alterations to tissue biomechanics. Current cell-based therapies are hindered by low survival and long-term engraftment for the transplanted cells due to the lack of appropriate supportive microenvironment (cell niche) in the injured muscle. Therefore, there is a critical need for developing strategies that provide cellular and structural support in the regeneration of functional muscle. In the present work, a bioengineered cell niche mimicking the native skeletal muscle microenvironment has been developed for skeletal muscle regenerative engineering. It is hypothesized that the bioengineered scaffolds with appropriate structural and cell instructive properties will support myoblast alignment and function, as well as promote the myogenic responses in clinically relevant skeletal muscle injuries. The current work utilized a three-pronged approach to design biomaterial scaffolds to aid in skeletal muscle regeneration. In the first part, aligned poly(lactide-co-glycolide) (PLGA) fiber scaffolds mimicking the oriented muscle fiber microenvironment with fiber diameters of 335±154 nm (nanoscale), 1352±225 nm (microscale) and 3013±531 nm (microscale) were fabricated and characterized. Myoblasts were found to respond to fiber diameter as observed from the differences in cell alignment, cell elongation, cell spreading area, proliferation and differentiation. <i>In vivo</i> study demonstrated the potential of using microscale fiber scaffolds to improve myogenic potential in the <i>mdx</i> mouse model. In the second part, we designed, synthesized, and characterized an implantable glycosaminoglycan-based composite hydrogel consisting of hyaluronic acid, chondroitin sulfate and polyethylene glycol (HA-CS) with tailored structural and mechanical properties for skeletal muscle regeneration applications. We demonstrated that HA-CS hydrogels provided a suitable microenvironment for <i>in vitro</i> myoblast proliferation and differentiation. Furthermore, <i>in vivo</i> studies using a volumetric muscle loss model in the mouse quadriceps showed that HA-CS hydrogels integrated with the surrounding host tissue and facilitated <i>de novo</i> myofiber generation, angiogenesis, nerve innervation and minimized scar tissue formation. In the third part, we investigated the effects of PC12 secreted signaling factors in modulating C2C12 myoblast behavior. We showed that PC12 conditioned media modulated myoblast proliferation and differentiation in both 2D culture and 3D aligned electrospun fiber scaffold system in a dose dependent manner. We also demonstrated the biomimetic HA-CS hydrogel system enabled 3D encapsulation of PC12 cells secreting signaling factors and promoted survival and proliferation of myoblasts in co-culture. Further proteomics analysis identified a total of 2088 protein/peptides from the secretome of the encapsulated PC12 cells and revealed the biological role and overlapping functions of nerve secreted proteins for skeletal muscle regeneration, potentially through regulating myoblast behavior, nerve function, and angiogenesis. These set of experiments not only provide critical insight on exploiting the interactions between muscle cells and their microenvironment, but they also open new avenues for developing advanced bioengineered scaffolds for regenerative engineering of skeletal muscle tissues.<br>