Regulation of skeletal muscle metabolism and development by small, non-coding RNAs : implications for insulin resistance and type 2 diabetes mellitus

microRNAs (miRNAs) are a class of epigenetic post-transcriptional regulators. These short (~22 nucleotides) non-coding RNAs can potently reduce protein abundance through direction of the RNA-induced silencing complex to targeted genes. Ultimately, miRNAs will reduce protein levels of target genes through mechanisms involving either inhibition of protein translation or destabilization and cleavage of target gene mRNA molecules. miRNAs are implicated in the regulation of several cellular processes, including growth, differentiation, and metabolism. The expression of miRNAs is altered in several clinical conditions including cancer, obesity, and diabetes. In this thesis, the aim was to elucidate how miRNAs are regulated in human skeletal muscle during three conditions, including in vitro skeletal muscle development, in type 2 diabetic patients, and following endurance exercise training. After identifying miRNAs that have altered expression in the aforementioned conditions, an additional aim was to characterize the functional effects of these miRNAs, including effects on target gene abundance and regulation of cellular metabolism. In Study I, miRNA expression was determined during human skeletal muscle cell differentiation with 48-hour resolution. Transcriptomic miRNA expression profiles of proliferative and differentiated cells were overlapped with gene expression alterations to identify reciprocal miRNA-mRNA expression patterns. Using this approach, miRNAcentered regulatory networks involved in in vitro human skeletal muscle development were predicted. miR-30b and miR-30c were among those differentially regulated miRNAs for which regulatory networks were modelled. In Study II, increased expression of miR-29a and miR-29c was identified in skeletal muscle from type 2 diabetic patients. Specifically, these miRNAs were expressed to a greater extent in type 2 diabetes. Thereafter these miRNAs were identified to induce insulin resistance and disturbances in glucose metabolism in human and mouse skeletal muscle. Several genes implicated in regulation of glucose and lipid metabolism were identified to be sensitive to altered miR-29 expression, including hexokinase 2. In Study III, miR-19b-3p and miR-107 were found to be induced in human skeletal muscle following 14 consecutive days of endurance exercise. The roles of these two miRNAs in the regulation of mRNA abundance and metabolic traits associated with skeletal muscle adaptation to endurance exercise training were determined. miR-19b-3p and miR-107 were identified to potently increase insulin sensitivity and glucose metabolism in human skeletal muscle cells, whereas functions of miR-19b-3p were also conserved in vivo and in in vitro models of mouse skeletal muscle. Together, these studies highlight roles of skeletal muscle miRNAs in posttranscriptional regulation of gene expression and modifications of insulin sensitivity in conditions such as type 2 diabetes and following endurance training. LIST OF SCIENTIFIC PAPERS I. Sjögren RJ, Egan B, Katayama M, Zierath JR, Krook A. Temporal analysis of reciprocal miRNA-mRNA expression patterns predicts regulatory networks during differentiation in human skeletal muscle cells. Physiol Genomics. 2015; 47(3):45-57. II. Massart J, Sjögren RJ, Lundell LS, Mudry JM, Franck N, O'Gorman DJ, Egan B, Zierath JR, Krook A. Altered miR-29 Expression in Type 2 Diabetes Influences Glucose and Lipid Metabolism in Skeletal Muscle. Diabetes. 2017; 66(7):1807-1818. III. Sjögren RJ, Massart J, Egan B, Garde C, Gu W, Lindgren M, Barrés R, O'Gorman DJ, Zierath JR, Krook A. Endurance exercise training-responsive miR-19b-3p and miR-107 improve glucose metabolism and insulin signaling in human skeletal muscle. Unpublished, in manuscript.