Thomas J. McLoughlin

FoxO1 Stimulates Fatty Acid Uptake and Oxidation in Muscle Cells through CD36-dependent and -independent Mechanisms

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 δ 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-N-succinimidyl-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.

FOXO1 Regulates the Expression of 4E-BP1 and Inhibits mTOR Signaling in Mammalian Skeletal Muscle

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 [14C]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.

TNF induction of atrogin-1/MAFbx mRNA depends on Foxo4 expression but not AKT-Foxo1/3 signaling

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 (+125%). 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 (-87%) and increases atrogin mRNA (+340%). Activation with insulin-like growth factor (IGF) increases AKT phosphorylation (+126%) and reduces atrogin mRNA (-15%). Although AKT regulation is intact, our data suggest it does not mediate TNF effects on atrogin. TNF increases AKT phosphorylation (+50%) 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 (+55%). Small interfering RNA oligos targeted to two distinct regions of Foxo4 mRNA reduce the TNF-induced increase in atrogin mRNA (-34% and -32%). 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.

COX-2 inhibitor reduces skeletal muscle hypertrophy in mice

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.

FoxO1 induces apoptosis in skeletal myotubes in a DNA-binding-dependent manner.

Recent studies indicate that FoxO transcription factors play an important role in promoting muscle atrophy. To study mechanisms mediating effects of FoxO proteins on muscle wasting, FoxO1-estrogen receptor fusion proteins that are activated by treatment with 4-hydroxytamoxifen (4-OH-T) were stably transfected in C(2)C(12) skeletal myoblasts using the pBABE retroviral system and grown into multinucleated skeletal myotubes. Activation of FoxO1 resulted in significant muscle atrophy, which was accompanied by DNA fragmentation, evidenced by terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling. Cells expressing a DNA-binding-deficient form of FoxO1 also exhibited significant atrophy on FoxO1 activation but no hallmark signs of apoptosis. FoxO1 activation resulted in a significant increase in muscle atrophy F-box (MAFbx)/atrogin-1, muscle-specific RING finger protein 1 (MuRF-1), and Bcl-2-interacting mediator of cell death (Bim) gene expression, with no significant increase in Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNip3) gene expression. Although the ability of FoxO1 to induce MuRF-1 gene expression appeared to be independent of DNA binding, expression of MAFbx/atrogin-1 and Bim was significantly blunted in cells expressing DNA-binding-deficient FoxO1. BNip3 gene expression was significantly elevated in DNA-binding-deficient mutant cells. These findings indicate that FoxO1 promotes skeletal muscle atrophy through induction of proteolytic and apoptotic machinery via DNA-binding-dependent and -independent mechanisms.

Suppression of protein kinase C theta contributes to enhanced myogenesis In vitro via IRS1 and ERK1/2 phosphorylation

BackgroundDifferentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2.ResultsIn this study, PKCθ knockdown (PKCθshRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation.ConclusionCellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state.

Tax sugar, save muscle?

debates are currently raging in the United States over healthcare reform. Certain political circles are advocating the implementation of a tax on sugary products to reign in ballooning costs associated with treating the one in three individuals in the United States now classified as obese. Now, this

Surface electromyography of the forearm musculature during an overhead throwing rehabilitation progression program

OBJECTIVE The flexor carpi ulnaris (FCU) and flexor digitorum superficialis (FDS) provide dynamic stabilization to the medial elbow. It remains unclear how these muscles function during progressive throwing exercises. Our objective was to compare FCU and FDS surface electromyography (sEMG) during a throwing progression. DESIGN Crossover. SETTING Laboratory. PARTICIPANTS Sixteen healthy males. MAIN OUTCOME MEASURES Participants completed a plyometric throw (PLYO), long-toss 50% (LT50), long-toss 75% (LT75), and pitch (PITCH). sEMG was synchronized with three-dimensional kinematics to assess the acceleration phase of each exercise. Peak sEMG amplitude (%MVIC) and percentage change between progressive exercises was measured. Continuous sEMG data were assessed to determine when peak activation occurred during acceleration. RESULTS FCU activity was greater during PITCH than LT50, and during LT75 than LT50. Percentage change was greater from LT50-to-LT75 than PLYO-to-LT50 for both muscles. PLYO and PITCH increased most during late acceleration, whereas LT50 and LT75 increased most during mid-acceleration. CONCLUSIONS FCU activity did not increase in a stepwise manner, and FDS remained unchanged. Each muscle demonstrated a disproportionate increase in activation during the second exercise progression (LT50-to-LT75) compared to the first (PLYO-to-LT50), suggesting that additional exercises may be required to achieve a stepwise progression relative to forearm muscle activation.