Emerging evidence documents a key function for the forkhead transcription factor FoxO1 in cellular metabolism. Here, we investigate the role of FoxO1 in the regulation of fatty acid (FA) metabolism in muscle cells. C2C12 cells expressing an inducible construct with either wild type FoxO1 or a mutant form (FoxO1/TSS) refractory to the protein kinase B inhibitory effects were generated. FoxO1 activation after myotube formation altered the expression of several genes of FA metabolism. Acyl-CoA oxidase and peroxisome proliferator-activated receptor ␦ mRNA levels increased 2.2-fold and 1.4-fold, respectively, whereas mRNA for acetyl-CoA carboxylase decreased by 50%. Membrane uptake of oleate increased 3-fold, and oleate oxidation increased 2-fold. Cellular triglyceride content was also increased. The enhanced FA utilization induced by FoxO1 was mediated by a severalfold increase in plasma membrane level of the fatty acid translocase FAT/CD36 and eliminated by cell treatment with the CD36 inhibitor sulfo-Nsuccinimidyl-oleate. We conclude that FoxO1 activation induces coordinate increases in FA uptake and oxidation and that these effects are mediated, at least in part, by membrane enrichment in CD36. The data suggest that FoxO1 contributes to preparing the muscle cell for the increased reliance on FA metabolism that is characteristic of fasting. Dysregulation of FoxO1 in muscle could contribute to intramuscular lipid accumulation and insulin resistance by maintaining activation of FA uptake.Maintaining the appropriate balance between glucose and fatty acid (FA) 1 metabolism is essential for muscle cells as they undergo nutritional transitions from feeding to fasting. As circulating levels of glucose and insulin fall, the muscle cell increases its reliance on FA oxidation. The decline in muscle cell glucose utilization that is associated with fasting allows glucose sparing because glycogen body stores are limited and can be depleted in a short period of time. In contrast to glycogen, the amount of stored fat is substantial, and FA released by adipocytes is a more sustainable substrate. As a result, the ability of muscle cells to accomplish the substrate shift from glucose to FA is a physiologically important adaptation for surviving caloric restriction.FoxO1 is a member of the evolutionarily conserved FoxO subfamily of forkhead transcription factors (1, 2), which are thought to be important in mediating effects of insulin and growth factors on glucose homeostasis and processes such as apoptosis and the cell cycle (2-4). FoxO1 is expressed in tissues involved in energy metabolism such as liver, muscle, and adipose tissue, where its function is inhibited by insulin and insulin-like growth factor I (5). Phosphorylation of three highly conserved protein kinase B sites, corresponding to Thr 24 , Ser 256 , and Ser 319 in human FoxO1, suppresses transactivation and promotes nuclear exclusion of FoxO proteins by multiple mechanisms (6). In muscle, FoxO1 expression is increased with fasting (7), which induces the key regulatory ...

The purpose of this study was to identify the potential downstream functions associated with mammalian target of rapamycin (mTOR) signaling during myotube hypertrophy. Terminally differentiated myotubes were serum stimulated for 3, 6, 12, 24, and 48 h. This treatment resulted in significant myotube hypertrophy (protein/DNA) and increased RNA content (RNA/DNA) with no changes in DNA content or indices of cell proliferation. During myotube hypertrophy, the increase in RNA content was accompanied by an increase in tumor suppressor protein retinoblastoma (Rb) phosphorylation and a corresponding increase in the availability of the ribosomal DNA transcription factor upstream binding factor (UBF). Serum stimulation also induced an increase in cyclin D1 protein expression in the differentiated myotubes with a concomitant increase in cyclin D1-dependent cyclin-dependent kinase (CDK)-4 activity toward Rb. The increases in myotube hypertrophy and RNA content were blocked by rapamycin treatment, which also prevented the increase in cyclin D1 protein expression, CDK-4 activity, Rb phosphorylation, and the increase in UBF availability. Our findings demonstrate that activation of mTOR is necessary for myotube hypertrophy and suggest that the role of mTOR is in part to modulate cyclin D1-dependent CDK-4 activity in the regulation of Rb and ribosomal RNA synthesis. On the basis of these results, we propose that common molecular mechanisms contribute to the regulation of myotube hypertrophy and growth during the G1 phase of the cell cycle.

August 13, 2008; doi:10.1152/ajpcell.00041.2008.-Murine models of starvation-induced muscle atrophy demonstrate that reduced protein kinase B (AKT) function upregulates the atrophy-related gene atrogin-1/ MAFbx (atrogin). The mechanism involves release of inhibition of Forkhead transcription factors, namely Foxo1 and Foxo3. Elevated atrogin mRNA also corresponds with elevated TNF in inflammatory catabolic states, including cancer and chronic heart failure. Exogenous tumor necrosis factor (TNF) increases atrogin mRNA in vivo and in vitro. We used TNF-treated C2C12 myotubes to test the hypothesis that AKT-Foxo1/3 signaling mediates TNF regulation of atrogin mRNA. Here we confirm that exposure to TNF increases atrogin mRNA (ϩ125%). We also confirm that canonical AKT-mediated regulation of atrogin is active in C2C12 myotubes. Inhibition of phosphoinositol-3 kinase (PI3K)/AKT signaling with wortmannin reduces AKT phosphorylation (Ϫ87%) and increases atrogin mRNA (ϩ340%). Activation with insulin-like growth factor (IGF) increases AKT phosphorylation (ϩ126%) and reduces atrogin mRNA (Ϫ15%). Although AKT regulation is intact, our data suggest it does not mediate TNF effects on atrogin. TNF increases AKT phosphorylation (ϩ50%) and stimulation of AKT with IGF does not prevent TNF induction of atrogin mRNA. Nor does TNF appear to signal through Foxo1/3 proteins. TNF has no effect on Foxo1/3 mRNA or Foxo1/3 nuclear localization. Instead, TNF increases nuclear Foxo4 protein (ϩ55%). Small interfering RNA oligos targeted to two distinct regions of Foxo4 mRNA reduce the TNF-induced increase in atrogin mRNA (Ϫ34% and Ϫ32%). We conclude that TNF increases atrogin mRNA independent of AKT via Foxo4. These results suggest a mechanism by which inflammatory catabolic states may persist in the presence of adequate growth factors and nutrition. skeletal muscle; cachexia; atrophy; ubiquitin; cytokines THE BULK OF MUSCLE PROTEIN lost during muscle atrophy is degraded by the ubiquitin-proteasome system (19,25,35). The specificity of this system is regulated by ubiquitin ligases (E3 proteins). Atrogin-1/MAFbx (atrogin) is a muscle-specific E3 protein that is upregulated in catabolic states that include humans under voluntary bed rest (37), or with spinal cord injury (46), amyotrophic lateral sclerosis (27), or chronic obstructive pulmonary disease (COPD) (9); and in animal models of cancer (4), sepsis (12, 49), aging (3), chronic heart failure (29), chronic kidney disease (10), diabetes (7, 24), starvation (20, 24), denervation (39), and unloading (17). The role of atrogin as a mediator of muscle atrophy is supported by studies that show overexpression in cell culture myotubes results in a fivefold reduction in myotube diameter (2). In addition, the absence of atrogin results in a 56% reduction in muscle atrophy after denervation in knockout mice (2).Atrogin gene expression is under the transcriptional control of Forkhead box O transcription factors (Foxo). Foxo1, Foxo3, and Foxo4 are related Foxo proteins thought to regulate atrogin unde...

Anti-inflammatory strategies are often used to reduce muscle pain and soreness that can result from high-intensity muscular activity. However, studies indicate that components of the acute inflammatory response may be required for muscle repair and growth. The hypothesis of this study was that cyclooxygenase (COX)-2 activity is required for compensatory hypertrophy of skeletal muscle. We used the synergist ablation model of skeletal muscle hypertrophy, along with the specific COX-2 inhibitor NS-398, to investigate the role of COX-2 in overload-induced muscle growth in mice. COX-2 was expressed in plantaris muscles during compensatory hypertrophy and was localized mainly in or near muscle cell nuclei. Treatment with NS-398 blunted the increases in mass and protein content in overloaded muscles compared with vehicle-treated controls. Additionally, the COX-2 inhibitor decreased activity of the urokinase type plasminogen activator, macrophage accumulation, and cell proliferation, all of which are required for hypertrophy after synergist ablation. Expression of insulin-like growth factor-1 and phosphorylation of Akt, mammalian target of rapamycin, and p70S6K were increased following synergist ablation, but were not affected by NS-398. Additionally, expression of atrogin-1 was reduced during hypertrophy, but was also not affected by NS-398. These results demonstrate that COX-2 activity is required for skeletal muscle hypertrophy, possibly through facilitation of extracellular protease activity, macrophage accumulation, and cell proliferation.

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 catabolism by activating ubiquitin-protein ligases. Using C2C12 mouse myoblasts that stably express inducible FOXO1-ER fusion proteins and transgenic mice that specifically overexpress constitutively active FOXO1 in skeletal muscle (FOXO ؉؉/؉ ), we show that FOXO1 inhibits mTOR signaling and protein synthesis. Activation of constitutively active FOXO1 induced the expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) mRNA via binding to the promoter. This resulted in an increased total 4E-BP1 abundance and a reduced 4E-BP1 (Thr-37/46) phosphorylation. The reduction in 4E-BP1 phosphorylation was associated with a reduction in the abundance of Raptor and mTOR proteins, Raptor-associated mTOR, reduced phosphorylation of the downstream protein p70S6 kinase, and attenuated incorporation of [ 14 C]phenylalanine into protein. The FOXO ؉؉/؉ mice, characterized by severe skeletal muscle atrophy, displayed similar patterns of mRNA expression and protein abundance to those observed in the constitutively active FOXO1 C2C12 myotubes. These data suggest that FOXO1 may be an important therapeutic target for human diseases where anabolism is impaired.Loss of skeletal muscle tissue is a clinical manifestation of many conditions and diseases, including aging, cancer, sepsis, human immunodeficiency virus, and diabetes (1), and important progress has been made in understanding the molecular regulation of both catabolism and anabolism within skeletal muscle (2). MAFbx (atrogin-1), an F-box protein, promotes skeletal muscle protein degradation in response to glucocorticoids (3, 4) and contributes toward muscle atrophy by targeting proteins for ubiquitination and proteasomal degradation (5). MAFbx is a target gene of FOXO transcription factors (3, 6), which are members of the Forkhead box-"Other" (or FOXO) 6 subfamily of Forkhead transcription factors and mammalian orthologs of DAF-16, the Caenorhabditis elegans FOXO protein. FOXO1 is expressed in insulin-sensitive tissues such as skeletal muscle, liver, and adipose tissue (7) and is an important target of insulin signaling downstream of acute transforming retrovirus thymoma (Akt) protein. In this context, insulin negatively regulates FOXO1 transactivation of target genes, in such a way that, upon insulin stimulation, Akt phosphorylates FOXO1, resulting in its exclusion from the nucleus (8 -12) and subsequent proteasomal degradation (13) following ubiquitination by Skp2 (14).The decreased protein synthesis observed in skeletal muscle in various catabolic conditions is associated with defects in mRNA translation initiation (15, 16). The translation of mRNA is divided into three steps, initiation, elongation, and termination (17), and is facilitated and regulated by eukaryotic initiation factors (18). The stable, heterotrimeric complex consisting of eIF4E, eIF4G, and eIF4A, which form t...