Monitoring and Modeling Effects of IGF1, Insulin and Green Tea Compound EGCG on Nuclear-Cytoplasmic Distribution of Foxo1-GFP in Skeletal Muscle Fibers

Abstract

The transcription factor Foxo1 promotes transcription of the ubiquitin ligases MuRF1 and MAFbx/atrogin1, which contribute to muscle fiber protein breakdown, leading to muscle atrophy and wasting. Foxo1 phosphorylated at its protein kinase B (Akt) sites is transported out of nuclei, and Foxo1 dephosphorylated at these sites is transported into nuclei. Foxo1 net nuclear efflux decreases Foxo1 transcriptional activity, thus opposing muscle protein breakdown and atrophy. Here we use quantitative fluorescence confocal imaging of the time course of Foxo1-GFP net nuclear fluxes together with mathematical modeling of Foxo1-GFP nuclear movements to examine the effects of upstream signaling on nuclear Foxo1. IGF1 and insulin, which activate Akt via the PI3k/PDK1/Akt signaling pathway, caused concentration dependent decreases of nuclear Foxo1-GFP. High IGF1 or insulin caused similar rapid and marked Foxo1-GFP net nuclear efflux. ECGC caused a more delayed net loss of nuclear Foxo1-GFP, possibly indicative of need for EGCG to enter the fiber to initiate its effect, whereas IGF1 and insulin work at cell surface receptors. Excluding the delay, the final rate and extent of decay of nuclear Foxo1-GFP was similar for EGCG, IGF1 and insulin. Analysis using our recently presented reduced (2 state)model for Foxo nuclear fluxes (Wimmer et al, 2014) showed that IGF1, insulin and EGCG all markedly and similarly increased the apparent unidirectional rate constant for Foxo1-GFP nuclear efflux, as well as markedly decreasing the apparent unidirectional rate constant for nuclear influx, indicative of increased Foxo1 phosphorylation in both cytoplasm and nuclei, both of which contribute to the observed decrease in nuclear Foxo1-GFP. Thus, IGF1, insulin and EGCG all activate Akt, both in the cytoplasm and within the nuclei. Supported by NIH R01AR056477 and by UMB-UMBC Seed Grant