AdvisorSchneider, Martin F.
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AbstractSkeletal muscle atrophy is regulated at the cellular level by several signaling pathways that modulate the balance between protein degradation and protein synthesis. A primary pathway regulating skeletal muscle atrophy is the IGF1 or Insulin/PI3K/Akt/Foxo1 pathway. IGF1/insulin lead to activation of a cascade of kinases, ultimately activating Akt which phosphorylates specific sites on Foxo1 and causes Foxo1 to translocate out of myofiber nuclei. When present in myofiber nuclei, Foxo1 functions as a transcription factor that activates transcription of several pro-atrophic genes, such as MurF1 and MAFbx/atrogin1. Here we use fluorescence time lapse imaging of the nuclear cytoplasmic distribution of Foxo1-GFP in adult isolated skeletal muscle fibers a to investigate (a) variability of FOXO1 nucleo-cytoplasmic distribution in mature muscle fibers as well as FOXO1 nuclear influx and efflux, and (b) the effects of IGF1 and insulin on Foxo1-GFP translocation in the presence of pharmacological inhibitors that target specific sections of the canonical IGF1 or insulin/PI3K/Akt/Foxo1 pathway. For our first aim, our results demonstrate that FOXO1 N/C and apparent rate coefficient (kI’) of FOXO1 nuclear influx are nearly identical for nuclei within the same fiber, they are highly varied for nuclei belonging to different fibers. These results indicate that variability in cellular factors, but not extracellular factors, determine FOXO1 distribution from fiber to fiber. Additionally, we show that application of IGF1 is able to reduce variation in kI’ and N/C in all fibers while AKT-inhibitor application does not, indicating other pathways are involved. For our second aim, our results demonstrate Akt is absolutely necessary for the dramatic IGF1 or insulin-induced rapid and near complete nuclear efflux of Foxo1, PI3K is not. Insulin induced movement of the PIP3 biosensor PH ARNO-GFP to transverse tubules is prevented by PI3K inhibitor BKM120, demonstrating that PI3K inhibition was effective. We have also found that two additional protein kinases, Ack1 and ATM, contribute to IGF1-induced Akt activation and Foxo1-GFP nuclear efflux. These results indicate the presence of PI3K-dependent -independent parallel pathways from IGF1 to Akt activation in adult muscle that are not well understood in Foxo1 regulation.
University of Maryland, Baltimore