The Mediator Complex has an essential function in regulating the transcription of several genes, working as a scaffold between transcription factors and polymerase II. It is composed of 26 subunits, with the kinase, or CDK subunit, being one of the most important for the mediator complex activation. In fact, when the kinase subunit is bound to the complex, binding between the mediator complex and polymerase II is hindered and therefore transcription is repressed. This suggests that the kinase complex and its components are essential for the expression of polymerase II transcripts. MED13 is part of the kinase subunit and high-throughput transcriptomic analysis showed MED13 as being upregulated in human skeletal muscle biopsies following an oral glucose tolerance test. The aim of this project is to understand the function of MED13 in human skeletal muscle metabolism and the relationship between MED13 knockdown and AMPK (AMP-activated protein kinase) function. Primary human skeletal muscles cells obtained from three healthy donors were cultured and transfection with MED13 siRNA was performed on differentiated myotubes. Metabolic assays such as fatty acid and glucose oxidation were performed as well as mitochondrial function. Quantitative PCR and western blot techniques were used to assess the pathways altered by MED13. Glucose uptake and fatty acid transporters were significantly increased upon MED13 silencing. Reduction in fatty acid oxidation and unchanged glucose oxidation suggest that MED13 favors substrate storage. Western blot analysis hinted on an increase in AMPK phosphorylation and in mitochondrial biogenesis. The production of ATP by mitochondria was also increased. In conclusion, MED13 plays an important role in human skeletal muscle substrate utilization for energy production.

Mitochondria is no longer viewed as merely a powerhouse of the cell. It is now apparentthat mitochondria play a central role in signaling, maintaining cellular homeostasis and cell fate.Mitochondrial dysfunction has been linked to many human diseases caused by cellular metabolicderegulation, such as obesity, diabetes, neurodegenerative disease, cardiovascular disease andcancer. Eukaryotic organisms have evolved an efficient way in sensing, communicating andresponding to cellular stress and regulating mitochondrial activity correspondingly through acomplex network of intercommunicating protein kinases and their downstream effectors. Thisdissertation focuses on the interplay of two of the master metabolic regulators in the cell: AMPKand PASK, and characterization of the functions of their downstream substrates: OSBP andMED13. AMPK is an energy sensing kinase that maintains energy homeostasis in the cell,whereas PASK is a nutrient sensing kinase that regulates glucose partitioning and respiration inthe cell. Both kinases play important roles in mitochondrial function and regulation, anddeficiency in either kinase has been found to associate with various human pathologies. Furthercharacterization of the cross-talk and molecular mechanisms of both kinases in controllingmitochondrial health and function may aid in the identification of new targets for treatingmetabolic diseases.