|Effects of obesity on the transcriptional regulation of protein degradation in skeletal muscle by Lance M. Bollinger November, 2013 Director of Thesis/Dissertation: Jeffrey J. Brault Major Department: Kinesiology Protein degradation is a major cause of skeletal muscle atrophy and is, at least in part, regulated at the level of gene transcription. While obesity is characterized by impaired skeletal muscle carbohydrate and lipid metabolism, the effects of obesity on skeletal muscle protein metabolism, specifically protein degradation, have not been thoroughly examined. Despite increased skeletal muscle mass, skeletal muscle of the severely obese overexpresses protein degradation genes, most notably, myostatin, which increases protein degradation by activating the transcription factor SMAD3, a mediator of transforming growth factor-[beta] family signaling. GOALS: The aims of the present project were: 1) to determine how obesity affects skeletal muscle protein degradation mediated by the ubiquitin-proteasome system and the autophagic/lysosomal pathway, particularly at the level of gene expression and 2) to determine the role of SMAD3 in regulating gene transcription of MuRF-1, a gene involved in degradation of contractile proteins. METHODS: In order to accomplish these goals, primary human skeletal muscle (HSkMC) myotubes from lean and severely obese women were subjected to various atrophic stimuli (100nM dexamethasone or starved of serum and amino acids) and analyzed for gene expression and flux through the major protein degradation pathways. Additionally, MuRF-1 promoter activity and RNA content were measured following co-expression of SMAD3 and FoxO3, a transcription factor known to induce MuRF-1 gene expression and cause muscle atrophy. RESULTS: Myotubes from the obese significantly overexpressed FoxO3, had a decreased rate of flux through the autophagic/lysosomal pathway, and increased proteasome capacity compared to lean. Despite these differences, myotubes from lean and obese women atrophied at similar rates and displayed similar total protein degradation rates under basal and starved conditions. Additionally, SMAD3 overexpression was insufficient to induce MuRF-1 promoter activity or gene expression, but synergistically augmented FoxO3-induced MuRF-1 gene expression by increasing FoxO3 protein content and enhancing FoxO3 binding to the proximal MuRF-1 promoter region. Furthermore, knockdown of SMAD3, via overexpression of a dominant-negative SMAD3, significantly attenuated FoxO3-induced MuRF-1 promoter activity. Mutation of the SMAD Binding Element (SBE) within the MuRF-1 promoter region attenuated FoxO3 binding to the FoxO Response Element (FRE), indicating SMAD3 is required for optimal FoxO3-induced MuRF-1 gene transcription. CONCLUSIONS: Skeletal muscle of the severely obese displays altered flux through the major protein degradation pathways, which may contribute to some of the metabolic impairments associated with obesity. However, severe obesity does not affect the total protein degradation rate or rate of muscle atrophy. Furthermore, SMAD3 regulates MuRF-1 gene transcription through dual mechanisms: 1) increasing FoxO3 protein content and 2) increasing FoxO3 binding within the proximal MuRF-1 promoter region. Atrophic conditions that increase SMAD3 transcriptional activity may augment FoxO3-induced transcription of protein degradation genes and accelerate muscle atrophy. Due to the observed overexpression of FoxO3, skeletal muscle of the severely obese may be more susceptible than that of lean persons to atrophy under conditions that increase expression and/or activity of SMAD3.