Wimmer RJ, Liu Y, Schachter TN, Stonko DP, Peercy BE, Schneider MF. Mathematical modeling reveals modulation of both nuclear influx and efflux of Foxo1 by the IGF-I/PI3K/Akt pathway in skeletal muscle fibers. Am J Physiol Cell Physiol 306: C570-C584, 2014. First published January 22, 2014; doi:10.1152/ajpcell.00338.2013.-Foxo family transcription factors contribute to muscle atrophy by promoting transcription of the ubiquitin ligases muscle-specific RING finger protein and muscle atrophy F-box/atrogin-1. Foxo transcriptional effectiveness is largely determined by its nuclear-cytoplasmic distribution, with unphosphorylated Foxo1 transported into nuclei and phosphorylated Foxo1 transported out of nuclei. We expressed the fluorescent fusion protein Foxo1-green fluorescent protein (GFP) in cultured adult mouse flexor digitorum brevis muscle fibers and tracked the time course of the nuclear-to-cytoplasmic Foxo1-GFP mean pixel fluorescence ratio (N/C) in living fibers by confocal imaging. We previously showed that IGF-I, which activates the Foxo kinase Akt/PKB, caused a rapid marked decline in N/C, whereas inhibition of Akt caused a modest increase in N/C. Here we develop a two-state mathematical model for Foxo1 nuclear-cytoplasmic redistribution, where Foxo phosphorylation/dephosphorylation is assumed to be fast compared with nuclear influx and efflux. Cytoplasmic Foxo1-GFP mean pixel fluorescence is constant due to the much larger cytoplasmic than nuclear volume. Analysis of N/C time courses reveals that IGF-I strongly increased unidirectional nuclear efflux, indicating similarly increased fractional phosphorylation of Foxo1 within nuclei, and decreased unidirectional nuclear influx, indicating increased cytoplasmic fractional phosphorylation of Foxo1. Inhibition of Akt increased Foxo1 unidirectional nuclear influx, consistent with block of Foxo1 cytoplasmic phosphorylation, but did not decrease Foxo1 unidirectional nuclear efflux, indicating that Akt may not be involved in Foxo1 nuclear efflux under control conditions. New media change experiments show that cultured fibers release IGF-I-like factors, which maintain low nuclear Foxo1 in the medium. This study demonstrates the power of quantitative modeling of observed nuclear fluxes.Akt1; Foxo1; IGF-I; skeletal muscle TRANSCRIPTION OF A GIVEN GENE is controlled by numerous regulatory proteins, including transcription factors, which are primary activators of transcription, as well as numerous other activators, coactivators, repressors, and corepressors (12, 15a). These factors combine to form macromolecular transcriptional regulatory complexes assembled on the promoter regions of the gene in question (16). To participate in such regulatory complexes, the regulatory molecules must be resident in the nucleus. Thus an important aspect of transcriptional regulation is control of the nuclear-cytoplasmic distribution of these regulatory molecules, and the mechanisms controlling nuclearcytoplasmic movements of transcriptional regulatory molecules can thus be important dete...

Prevention and slowing of skeletal muscle atrophy with nutritional approaches offers the potential to provide far reaching improvements in the quality of life for our increasingly aging population. Here we show polyphenol flavonoid Epigallocatechin 3-gallate (EGCG), found in the popular beverage green tea (Camellia sinensis), demonstrates similar effects as the endogenous hormones IGF-1 and insulin in the ability to suppress action of the atrophy-promoting transcription factor Foxo1 through a net translocation of Foxo1 out of the nucleus as monitored by nucleo-cytoplasmic movement of Foxo1-GFP in live skeletal muscle fibers. Foxo1-GFP nuclear efflux is rapid in IGF-1 or insulin, but delayed by an additional 30 min for EGCG. Once activated, kinetic analysis with a simple mathematical model shows EGCG, IGF-1 and insulin all produce similar apparent rate constants for Foxo1-GFP unidirectional nuclear influx and efflux. 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. Using the live fiber model system, we also determine the dose-response curve for both IGF-1 and insulin on Foxo1 nucleo-cytoplasmic distribution. The continued understanding of the activation mechanisms of EGCG could allow for nutritional promotion of Green Tea’s anti-atrophy skeletal muscle benefits and have implications in development of a clinically significant parallel pathway for new drugs to target muscle wasting and the reduced insulin receptor sensitivity which causes Type II Diabetes Mellitus.

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Environmental education focusing on water resource sustainability can be uniquely integrated into a community through the combined participation of public and private schools, industry, government and shared community resources. Over 4000 elementary, middle and high school students have participated in a community-supported environmental education program focusing on local water quality issues through the conversion and renovation of an 1893 boat house at a local museum. The students, along with teachers and parents, perform sample collection, lab analysis, and data collection to determine the general health of a local river. The learning experience includes the evaluation of physical, chemical, and microbial aspects of the river conditions. The instruction and program methods comply with required learning goals established by the State of Illinois. Program funding is provided by local business and grants from both local and state governments.

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