Role of FoxO1 in skeletal muscle metabolism and growth.
Creators: name: Southgate, Robert
Publisher: name: RMIT University
Year: 2024

description: There are multiple binding domains on the promoter region of the peroxisome proliferator activator receptor gamma (γ) coactivator-1 α (PGC-1α) gene, including a trio of insulin responsive elements that are activated by the Forkhead box class-O (FoxO1) winged helix transcription factor, which is known to be regulated by acute transforming retrovirus thymoma (Akt). The first two experimental chapters (Chapters 2 and 3) of this thesis indicate that in skeletal muscle biopsy specimens from healthy humans and cultured human skeletal myotubes, insulin phosphorylates Akt (Ser473) and FoxO1 (Thr24, Ser256), leading to reduced nuclear abundance of FoxO1 total protein. This is associated with an insulin-mediated repression of the mRNA expression of PGC-1α and downstream genes associated with oxidative phosphorylation. In contrast, in muscle taken from insulin resistant humans or in palmitate treated insulin resistant myotubes, neither Akt nor FoxO1 was phosphorylated by insulin resulting in a failure for nuclear exclusion of FoxO1 total protein, and an inability for insulin to repress the mRNA expression of PGC-1α and downstream genes. To determine whether the regulation of FoxO1 was Akt dependent, the work that constitutes Chapter 4 describes experiments where Akt2 −/− and wild type mice were treated with or without insulin. Insulin phosphorylated Akt and FoxO1 (Thr24, Ser256) resulted in a reduced nuclear expression of FoxO1 total protein in wild type but not Akt2 −/− skeletal muscle. <br><br>The mammalian target of rapamycin (mTOR) is regulated by growth factors to promote protein synthesis. In mammalian skeletal muscle, the Forkhead-O1 transcription factor (FoxO1) promotes protein catabolism by activating ubiquitin-protein ligases. Using both C2C12 mouse myoblasts stably expressing FoxO1-ER fusion proteins that are rapidly activated by 4- OH tamoxifen and transgenic mice that specifically overexpress constitutively active FoxO1 in skeletal muscle (FoxO++/+), this work provides evidence that FoxO1 inhibits mTOR signaling and protein synthesis. Activation of constitutively active FoxO1 induced the expression of eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) mRNA and protein, but reduced eIF4E-BP1 phosphorylation (Thr37/46). The reduction in eIF4E-BP1 phosphorylation was associated with a reduction in the abundance of Raptor and mTOR, Raptor-associated mTOR, reduced phosphorylation of the downstream protein p70S6 kinase and attenuated protein synthesis. The FoxO++/+ mice, characterized by severe skeletal muscle atrophy, displayed increased nuclear FoxO1 abundance and eIF4E-BP1 protein expression which was associated with reduced eIF4E-BP1 phosphorylation, Raptor, and mTOR protein abundance. These results provide the first evidence that FoxO1 per se inhibits protein synthesis via increased expression of the translational protein eIF4E-BP1 and reduced signaling through mTOR/Raptor complexes. <br> <br><br>In conclusion, insulin decreases the expression of genes involved in oxidative metabolism in healthy but not insulin resistant muscle, due to a decrease in FoxO1 phosphorylation and nuclear exclusion secondary to reduced Akt activity. FoxO1 may be an important therapeutic target in human disease where catabolism of muscle is observed, as FoxO1 appears to inhibit protein synthesis and translation/initiation pathways.<br>