%0 Thesis %A Turner, Mark C. %D 2015 %T Cell culture models of insulin signalling and glucose uptake %U https://repository.lboro.ac.uk/articles/thesis/Cell_culture_models_of_insulin_signalling_and_glucose_uptake/9609107 %2 https://repository.lboro.ac.uk/ndownloader/files/17255780 %K Skeletal muscle %K Tissue engineering %K Diabetes %K Cell biology %K Insulin %K Glucose %K Medical and Health Sciences not elsewhere classified %X 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. %I Loughborough University