• FOXO1 Nucleo-Cytoplasmic Distribution and Regulation

      Russell, Sarah; Schneider, Martin F. (2020)
      Skeletal 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.
    • Green Tea (EGCG), Insulin, IGF-1 Suppression of Atrophy Associated Transcription Factor Foxo1 Activity in Skeletal Muscle and Mathematical Modeling of Nuclear Influx Efflux of Foxo1 in Skeletal Muscle

      Wimmer, Robert; Schneider, Martin F. (2014)
      Preventing, slowing and reversing skeletal muscle atrophy offers the potential to substantially improve the quality of life for our increasingly aging population. In addition to the primary function of force generation for movement, skeletal muscle plays an equally important role in maintenance of homeostasis through regulation of metabolic critical organic compounds such as amino acids, growth hormones, fatty acids, and glucose. Disruption of the body's homeostasis during diseases such as cancer, renal failure, sepsis, or diabetes leads to significant skeletal muscle atrophy. Our study utilizes over-expression of a skeletal muscle atrophy associated transcription factor, Foxo1, tagged with a green fluorescent protein (GFP) to provide a visual indication where within the muscle fiber (Nucleus or Cytoplasm) Foxo1 is located. This provides an indication of the activation status of the Foxo1 by comparing nuclear to cytoplasmic ratios with time. A two state mathematical model was created to account for the observed Foxo1-GFP nuclear concentration time course and presented here in Chapter 2. This model made two interesting observations, one implying that the kinase Akt not the predominant enzyme that phosphorylates Foxo1 in the nucleus prior to Foxo1 nuclear efflux. The second indicates that in the presence of IGF, intra-nuclear Akt activity, or possibly the activity of another kinase, has become strongly activated with the presence of IGF within the muscle fiber nuclei. EGCG is found in green tea, the most popularly consumed beverage in the world after water. Presented in Chapter 3, we show results of reduced Foxo1 activation induced by EGCG which are similar to the effects of well established endogenous growth hormones, IGF-1 and insulin. Interestingly EGCG appears to have its effect at least partially via parallel signaling pathways that are independent of IGF-1's (and insulin's) downstream PI3K/Akt/Foxo1 signaling axis. Future work to further understand EGCG's parallel signaling pathways could have implications both in slowing muscle atrophy as it relates to Foxo1 or, more broadly in providing a clinical parallel pathway to IGF-1 and/or insulin.