Thesis

Silencing LPL Promotes Insulin Sensitivity in rat Skeletal Muscle Cells and Development of DMD-hiPSC Derived SMPCs for Regenerative and pre-Clinical Applications for DMD

Lipoprotein Lipase (LPL) has been proposed as a potential participant in the development of insulin resistance. LPL is the rate-limiting enzyme for the hydrolysis of triglycerides in lipoprotein particles, releasing free fatty acids (FFA) that are oxidized for energy. Skeletal muscle is a major site for LPL synthesis, and the major tissue responsible for whole body insulin-stimulated glucose uptake or disposal. Since FFA contribute to insulin resistance, down-regulation of muscle LPL expression and activity may result in increased insulin sensitivity. LPL-knock-out L6 cells were developed using siRNA technology and confirmed by RT-PCR methods. We examined the effects of silencing the LPL gene on insulin function using metabolic assays based on the use of radioactive metabolites, and measuring radioactive products in cultured L6 skeletal muscle cells. When cells were grown in glucose-supplemented medium, insulin-stimulated glycogen synthesis was greater in the LPL-KO cell line than the wild-type cells. Conversely, insulin-stimulated oxidation of radio-labeled glucose or oleic acid to CO2 declined in LPL-KO cells compared to wild-type cells. Our data suggests that silencing the LPL gene increases insulin sensitivity, promotes storage of excess glucose as glycogen, and depletes cellular fatty acids; forcing utilization of glucose as a fuel for muscle activity. Duchenne Muscular Dystrophy (DMD) is one of the most severe childhood muscular disorders, resulting in progressive muscle deterioration and ultimately death. This disease is caused by a lack of the protein dystrophin, often due to large genomic deletions that shift the reading frame. No effective treatment is currently available, but one potential therapy involves deriving skeletal muscle progenitor cells (SMPC) from human induced pluripotent stem cells (hiPSCs). Once expanded, these SMPCs could be corrected for proper dystrophin expression and then integrated into patient muscle. We have generated hiPSCs from healthy and DMD-patient fibroblasts to evaluate their use as a pre-clinical tool and in regenerative applications. It has been shown that SMPCs can be generated through the overexpression of specific genes - however, a method that does not require additional genetic manipulation would be required for clinical applications. To obtain a robust SMPC population, we tested 1) overexpression of key skeletal muscle transcription factors using non integrating platforms, 2) addition of growth factors known to induce skeletal muscle progenitor cell marker expression during embryogenesis in model organisms and 3) evaluation of the SMPC potential of genetically targeted cell lines with enhanced mesoderm potential. In addition we are developing a pre-clinical screening platform for evaluating combination therapies using SMPCs or terminally differentiated muscle as well as cardiomyocytes derived from DMD-hiPSCs. The refinement of these differentiation protocols for producing and maintaining SMPC populations will aid in the development of regenerative therapies for muscle disorders. This work will also lay the groundwork for future studies aimed at using combination therapies in muscular dystrophies in regenerative medicine.

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