Abstract
Muscle atrophy is regulated by the balance between protein degradation and synthesis. FOXO1, a transcription factor, helps to determine this balance by activating pro-atrophic gene transcription when present in muscle fiber nuclei. Foxo1 nuclear efflux is promoted by AKT-mediated Foxo1 phosphorylation, eliminating FOXO1's atrophy-promoting effect. AKT activation can be promoted by insulin-like growth factor 1 (IGF1) or insulin via a pathway including IGF1 or insulin, phosphatidylinositol 3-kinase, and AKT. We used confocal fluorescence time-lapse imaging of FOXO1-GFP in adult isolated living muscle fibers maintained in culture to explore the effects of IGF1 and insulin on FOXO1-GFP nuclear efflux with and without pharmacological inhibitors. We observed that although AKT inhibitor blocks the IGF1- or insulin-induced effect on FOXO1 nuclear efflux, phosphatidylinositol 3-kinase inhibitors, which we show to be effective in these fibers, do not. We also found that inhibition of the protein kinase ACK1 or ATM contributes to the suppression of FOXO1 nuclear efflux after IGF1. These results indicate a novel pathway that has been unexplored in the IGF1- or insulin-induced regulation of FOXO1 and present information useful both for therapeutic interventions for muscle atrophy and for further investigative areas into insulin insensitivity and type 2 diabetes.
Highlights
Skeletal muscle is essential for all voluntary and reflex movement and is one of the primary insulin sensitive tissues
When the same addition of insulin-like growth factor 1 (IGF1) to another compartment was preceded by addition of AKT-I VIII, the loss of nuclear FOXO1–GFP caused by IGF1 addition was completely eliminated (Fig. 1, A, bottom row, and B, red squares and red line), and the time course was essentially the same as that observed when only AKT-I VIII was added by itself, without any IGF1 (Fig. 1B, black line)
Using intact individual skeletal muscle fibers isolated from young adult mice, we find that the robust marked effect of IGF1 or insulin of promoting rapid and pronounced nuclear efflux of FOXO1–GFP is almost fully insensitive to the addition of any of the three phosphatidylinositol 3-kinase (PI3K) inhibitors
Summary
Skeletal muscle is essential for all voluntary and reflex movement and is one of the primary insulin sensitive tissues. We investigate a major signaling pathway involved in muscle atrophy that is regulated by both insulin and IGF1 Both insulin and IGF1 play large and important roles in skeletal muscle. Inactivation of insulin and IGF1 receptors in skeletal muscle has been shown to cause type 2 diabetes in mice [14] These growth hormones play roles in preventing muscle atrophy. AKT (protein kinase B) is a serine/threonine specific protein kinase that, when activated, phosphorylates FOXO1 on three conserved sites (Thr, Ser256, and Ser319) [21] in both cytoplasm and nuclei [22] This phosphorylation causes the FOXO1 nuclear localization signal to be obscured, preventing nuclear entry of FOXO1. The canonical pathway for activating AKT kinase activity by IGF1/insulin begins with IGF1/insulin activating the IGF1/ insulin receptors, causing receptor autophosphorylation and phosphorylation of Insulin Receptor Substrate 1 (IRS1), resulting
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