The AMPK agonist 5‐aminoimidazole‐4‐carboxamide ribonucleotide (AICAR), but not metformin, prevents inflammation‐associated cachectic muscle wasting
2018; Springer Nature; Volume: 10; Issue: 7 Linguagem: Inglês
10.15252/emmm.201708307
ISSN1757-4684
AutoresDerek Hall, Takla Griss, F. Jennifer, Brenda Janice Sánchez, Jason Sadek, Anne Marie K Tremblay, Souad Mubaid, Amr Omer, Rebecca J. Ford, Nathalie Bédard, Arnim Pause, Simon S. Wing, Sergio Di Marco, Gregory R. Steinberg, Russell G. Jones, Imed‐Eddine Gallouzi,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
ResumoResearch Article29 May 2018Open Access Source DataTransparent process The AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), but not metformin, prevents inflammation-associated cachectic muscle wasting Derek T Hall Derek T Hall Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Takla Griss Takla Griss Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Department of Physiology, McGill University, Montreal, QC, Canada Search for more papers by this author Jennifer F Ma Jennifer F Ma Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Brenda Janice Sanchez Brenda Janice Sanchez Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Jason Sadek Jason Sadek Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Anne Marie K Tremblay Anne Marie K Tremblay Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Souad Mubaid Souad Mubaid Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Amr Omer Amr Omer Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Rebecca J Ford Rebecca J Ford Division of Endocrinology and Metabolism, Department of Medicine, McMaster University, Hamilton, ON, Canada Search for more papers by this author Nathalie Bedard Nathalie Bedard Department of Medicine, McGill University and the Research Institute of the McGill University Health Centre, Montreal, QC, Canada Search for more papers by this author Arnim Pause Arnim Pause Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Simon S Wing Simon S Wing Department of Medicine, McGill University and the Research Institute of the McGill University Health Centre, Montreal, QC, Canada Search for more papers by this author Sergio Di Marco Sergio Di Marco Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Gregory R Steinberg Gregory R Steinberg Division of Endocrinology and Metabolism, Department of Medicine, McMaster University, Hamilton, ON, Canada Search for more papers by this author Russell G Jones Russell G Jones Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Department of Physiology, McGill University, Montreal, QC, Canada Search for more papers by this author Imed-Eddine Gallouzi Corresponding Author Imed-Eddine Gallouzi [email protected] [email protected] orcid.org/0000-0003-4758-4835 Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Life Sciences Division, College of Sciences and Engineering, Hamad Bin Khalifa University (HBKU), Doha, Qatar Search for more papers by this author Derek T Hall Derek T Hall Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Takla Griss Takla Griss Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Department of Physiology, McGill University, Montreal, QC, Canada Search for more papers by this author Jennifer F Ma Jennifer F Ma Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Brenda Janice Sanchez Brenda Janice Sanchez Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Jason Sadek Jason Sadek Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Anne Marie K Tremblay Anne Marie K Tremblay Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Souad Mubaid Souad Mubaid Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Amr Omer Amr Omer Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Rebecca J Ford Rebecca J Ford Division of Endocrinology and Metabolism, Department of Medicine, McMaster University, Hamilton, ON, Canada Search for more papers by this author Nathalie Bedard Nathalie Bedard Department of Medicine, McGill University and the Research Institute of the McGill University Health Centre, Montreal, QC, Canada Search for more papers by this author Arnim Pause Arnim Pause Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Simon S Wing Simon S Wing Department of Medicine, McGill University and the Research Institute of the McGill University Health Centre, Montreal, QC, Canada Search for more papers by this author Sergio Di Marco Sergio Di Marco Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Search for more papers by this author Gregory R Steinberg Gregory R Steinberg Division of Endocrinology and Metabolism, Department of Medicine, McMaster University, Hamilton, ON, Canada Search for more papers by this author Russell G Jones Russell G Jones Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Department of Physiology, McGill University, Montreal, QC, Canada Search for more papers by this author Imed-Eddine Gallouzi Corresponding Author Imed-Eddine Gallouzi [email protected] [email protected] orcid.org/0000-0003-4758-4835 Department of Biochemistry, McGill University, Montreal, QC, Canada Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada Life Sciences Division, College of Sciences and Engineering, Hamad Bin Khalifa University (HBKU), Doha, Qatar Search for more papers by this author Author Information Derek T Hall1,2, Takla Griss2,3, Jennifer F Ma1,2, Brenda Janice Sanchez1,2, Jason Sadek1,2, Anne Marie K Tremblay1,2, Souad Mubaid1,2, Amr Omer1,2, Rebecca J Ford4, Nathalie Bedard5, Arnim Pause1,2, Simon S Wing5, Sergio Di Marco1,2, Gregory R Steinberg4, Russell G Jones2,3 and Imed-Eddine Gallouzi *,*,1,2,6 1Department of Biochemistry, McGill University, Montreal, QC, Canada 2Rosalind and Morris Goodman Cancer Centre, Montreal, QC, Canada 3Department of Physiology, McGill University, Montreal, QC, Canada 4Division of Endocrinology and Metabolism, Department of Medicine, McMaster University, Hamilton, ON, Canada 5Department of Medicine, McGill University and the Research Institute of the McGill University Health Centre, Montreal, QC, Canada 6Life Sciences Division, College of Sciences and Engineering, Hamad Bin Khalifa University (HBKU), Doha, Qatar *Corresponding author. Tel: +1 514 398 4537; E-mails: [email protected]; [email protected] EMBO Mol Med (2018)10:e8307https://doi.org/10.15252/emmm.201708307 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Activation of AMPK has been associated with pro-atrophic signaling in muscle. However, AMPK also has anti-inflammatory effects, suggesting that in cachexia, a syndrome of inflammatory-driven muscle wasting, AMPK activation could be beneficial. Here we show that the AMPK agonist AICAR suppresses IFNγ/TNFα-induced atrophy, while the mitochondrial inhibitor metformin does not. IFNγ/TNFα impair mitochondrial oxidative respiration in myotubes and promote a metabolic shift to aerobic glycolysis, similarly to metformin. In contrast, AICAR partially restored metabolic function. The effects of AICAR were prevented by the AMPK inhibitor Compound C and were reproduced with A-769662, a specific AMPK activator. AICAR and A-769662 co-treatment was found to be synergistic, suggesting that the anti-cachectic effects of these drugs are mediated through AMPK activation. AICAR spared muscle mass in mouse models of cancer and LPS induced atrophy. Together, our findings suggest a dual function for AMPK during inflammation-driven atrophy, wherein it can play a protective role when activated exogenously early in disease progression, but may contribute to anabolic suppression and atrophy when activated later through mitochondrial dysfunction and subsequent metabolic stress. Synopsis Cachexia is a co-morbidity characterized by the loss of skeletal muscle that arises in patients with pro-inflammatory diseases, like cancer. Activators of AMPK were found to protect against inflammation-induced muscle atrophy, demonstrating the potential of targeting AMPK for therapy in cachexia. The AMPK activator AICAR, but not metformin, protected C2C12 myotubes from IFNγ/TNFα-driven atrophy. The differential effects of AICAR and metformin were associated with the ability to restore or inhibit mitochondrial function during inflammation, suggesting that the mechanism of AMPK activation affects the outcome of treatment. The anti-cachectic properties of AICAR were impaired by treatment with the AMPK inhibitor Compound C and were synergistic with the AMPK activator A-769662, suggesting that the effects of AICAR were mediated by AMPK activation. AICAR, but not metformin, was effective at preventing muscle mass loss in mice in both the C26 model of cancer cachexia and an endotoxin model of sepsis. Collectively, this study suggests that treatment with AMPK activators during the early stages of cachexia could be a novel avenue for the development of therapies. Introduction Cachexia is a wasting syndrome that often occurs as a comorbidity with chronic pro-inflammatory diseases, such as cancer, HIV infection, and sepsis (Fearon et al, 2011; Blum et al, 2014). Cachexia is primarily characterized by a progressive and extensive loss of skeletal muscle mass and strength, but can also present with loss of fat mass, anorexia, and cardiac atrophy and remodeling (Fearon et al, 2012; Groarke et al, 2013). The prevalence of cachexia in patients varies depending on the type of disease. For example, it is estimated that approximately half of all cancer patients experience cachexia (von Haehling & Anker, 2010, 2014). It is well established that onset of cachexia negatively impacts disease outcome, reducing the effectiveness of primary disease treatment and increasing patient morbidity and mortality (Andreyev et al, 1998; Prado et al, 2007; Utech et al, 2012; Vaughan et al, 2013; Vigano et al, 2017). While there are numerous symptoms of the cachectic state, one of the more debilitating aspects of this condition is the dramatic loss of skeletal muscle tissue (Fearon et al, 2011). Although the mechanisms behind cachectic muscle wasting are complex, it is believed that one of the primary triggers of muscle atrophy is the chronic elevation of pro-inflammatory cytokines (e.g., IL-1, IL-6, TNFα, IFNγ) (Morley et al, 2006; Argiles et al, 2009; Tisdale, 2009; Fearon et al, 2012). In keeping with this, induction of muscle atrophy can be recapitulated in culture and in vivo by exposure to different cytokine combinations [e.g., IFNγ and TNFα (Guttridge et al, 2000; Acharyya et al, 2004; Di Marco et al, 2005, 2012) or IL-6 (Bonetto et al, 2012; White et al, 2012)]. Extended cytokine exposure results in the continued activation of inflammatory signaling within muscle cells, leading to the expression of pro-cachectic genes (Guttridge et al, 2000; Bonetto et al, 2011; Hall et al, 2011; Fearon et al, 2012; Bonaldo & Sandri, 2013). For example, we and others have demonstrated that inducible nitric oxide synthase (iNOS) is dramatically upregulated during cytokine-driven muscle wasting and that the production of reactive nitrogen compounds, such as nitric oxide (NO), by this enzyme contributes to the pathogenesis of cachexia (Buck & Chojkier, 1996; Di Marco et al, 2005, 2012; Ramamoorthy et al, 2009; Hall et al, 2011). Recent evidence suggests that cytokine exposure also alters the metabolism of muscle. Indeed, it has been shown in several models of cachexia and inflammation-driven wasting that muscle undergoes a Warburg-like increase in aerobic glycolysis and mitochondrial abnormalities (Barreiro et al, 2005; Julienne et al, 2012; White et al, 2012; Der-Torossian et al, 2013; Fontes-Oliveira et al, 2013; McLean et al, 2014). The metabolic regulating enzyme, AMP-activated protein kinase (AMPK), has been associated with cytokine- and cancer-driven muscle wasting (White et al, 2011, 2013). AMPK is a heterotrimeric complex (composed of a catalytic α-subunit, linker β-subunit, and regulatory γ-subunit) that responds to cellular energy levels (Hardie et al, 2012). Activation of AMPK has been shown to suppress anabolic signaling through mTOR and has been found to increase the expression of muscle-specific E3-ligases (Krawiec et al, 2007; Nakashima & Yakabe, 2007; Shaw, 2009). However, AMPK has also been shown to have potent anti-inflammatory effects in a variety of cell types (Galic et al, 2011; Salminen et al, 2011; Mounier et al, 2013; O'Neill & Hardie, 2013). The anti-inflammatory function suggests that AMPK activation could be beneficial for muscle atrophy induced by chronic inflammation. Consistent with this concept, genetic deletion of skeletal muscle AMPK leads to the acceleration of aging-induced sarcopenia (Bujak et al, 2015). Further, an AMPK stabilizing peptide was recently shown to be effective at preventing adipose tissue wasting in cancer cachexia (Rohm et al, 2016). Therefore, there is an apparent contradiction for the role of AMPK during cachectic muscle wasting: While its association with atrophic signaling suggests AMPK can contribute to muscle wasting during cachexia, the anti-inflammatory functions of AMPK suggest that it could also prevent cytokine-driven atrophy. Here, we tested the hypothesis that compounds that activate AMPK could prevent cytokine-driven muscle wasting. To do so, we assessed the impact of two well-known AMPK activators—AICAR and metformin—on atrophy in cultured myotubes treated with the pro-inflammatory cytokines IFNγ and TNFα (Towler & Hardie, 2007; Viollet et al, 2012). These compounds activate AMPK through distinct mechanisms. AICAR is phosphorylated by cellular kinases to form ZMP, which acts as an AMP mimetic, binding directly to and activating AMPK (Towler & Hardie, 2007). In contrast, the biguanide metformin inhibits Complex I of the electron transport chain, leading to an indirect activation of AMPK by increasing cytoplasmic AMP levels (Viollet et al, 2012). Surprisingly, we found that while AICAR, metformin, and IFNγ/TNFα treatment activated AMPK, only AICAR prevented IFNγ/TNFα-induced atrophy. In addition, AICAR, but not metformin, was found to partially restore normal metabolic function and inhibit the pro-cachectic iNOS/NO pathway. The effects of AICAR were blocked by co-treatment with the AMPK inhibitor Compound C and recapitulated with the more specific AMPK activator A-769662 (Cool et al, 2006; Goransson et al, 2007). In addition, A-769662 and AICAR were found to synergistically prevent wasting, suggesting that the effects of these compounds are through AMPK. Finally, AICAR was able to restore muscle mass in multiple murine models of cachectic muscle wasting. Results Activation of AMPK by AICAR but not metformin prevents muscle wasting To assess whether AMPK activation could prevent cytokine-induced muscle atrophy, we performed studies in C2C12 myotubes treated with IFNγ and TNFα. These pro-cachectic cytokines are a well-established model to induce a muscle wasting-like phenotype in vitro that begins with signaling events occurring within the first 24 h, followed by atrophy detectable by 48 h and culminating in loss of integrity by 72 h (Di Marco et al, 2005, 2012). To activate AMPK, we used two AMPK activators, AICAR and metformin. As expected, both AICAR and metformin showed increased AMPK phosphorylation at Thr172 (pAMPK), a post-translational modification that is required for AMPK activity, 24 h after treatment (Hardie et al, 2012; Fig 1A). AICAR and metformin treatment also led to the increased phosphorylation of acetyl-CoA carboxylase (ACC) at Ser79 (pACC) (Fig 1A). Acetyl-CoA carboxylase is a well-established downstream target of AMPK and is often used to demonstrate increased AMPK activity within cells (Munday, 2002). Interestingly, IFNγ/TNFα treatment alone also increased pAMPK and pACC levels at 24 h, corroborating previous reports that AMPK phosphorylation increases during the progression of cachexia-induced muscle wasting (Fig 1A; Penna et al, 2010; White et al, 2011, 2013). To further understand the dynamics of AMPK activation in this model, we tested the phosphorylation status of AMPK and ACC over a time course of the first 24 h of cytokine treatment. We observed that while cytokine treatment alone resulted in detectable phosphorylation of ACC at 6–12 h, respectively, co-treatment with AICAR resulted in detectable levels by 30 min (Fig EV1). Metformin treatment, in turn, induced detectable ACC phosphorylation by 1 h (Fig EV1). Thus, while AMPK does seem to be activated by cytokine treatment, eventually, treatment with the AMPK agonists increased AMPK activity at a time when it was not normally induced by cytokine treatment. Figure 1. AICAR but not metformin prevents IFNγ/TNFα-induced myotube atrophy A. Western blotting for phospho-Thr172-AMPK (pAMPK), total AMPK, phospho-Ser79-ACC (pACC), and total ACC 24h after treatment. Quantification represents the pAMPK/AMPK and pACC/ACC ratios relative to the non-treated (NT) control. B. Phase contrast images of fibers 72 h after treatment. Scale bars represent 0.25 mm. C. Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48 h after treatment. Scale bars represent 50 μm. Quantification represents the average myotube width. D, E. RT–qPCR analysis of the mRNA levels of MyoD (D) and myogenin (E) 24 h after treatment relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance P-values were calculated using Fisher's LSD. *P < 0.05; **P < 0.01 from NT controls; ††P < 0.01 from IFNγ/TNFα-treated controls. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Time course of AMPK activation during cytokine and AICAR or metformin co-treatment Western blotting for phospho-Thr172 AMPKα (pAMPK), total AMPKα (AMPK), phospho-Ser79-ACC (pACC), and total ACC during the first 24 h of treatment. Download figure Download PowerPoint Having established the AMPK activation status across the different treatment regimes, we next sought to determine what would be the effect of AMPK agonists on the progression of muscle wasting. Surprisingly, we found that AICAR treatment, but not metformin, was able to prevent the loss of integrity in myotubes exposed to IFNγ/TNFα over a 72-h period (Fig 1B). To determine whether the AICAR treatment also protected myotubes from the initial atrophying that precedes myotube collapse, we measured myotube widths 48 h after cytokine treatment. Again, only AICAR treatment prevented the cytokine-induced atrophy of muscle myotubes (Fig 1C). In contrast, metformin had no effect on cytokine-induced atrophy and showed a trend toward smaller widths when used alone (Fig 1C). Finally, we assessed the mRNA expression levels of the pro-myogenic transcription factors MyoD and myogenin, which are down-regulated within the first 24 h of IFNγ/TNFα treatment (Fig 1D and E). We observed that AICAR treatment, but not metformin, significantly increased the mRNA levels of MyoD in cytokine-treated myotubes compared to cytokine treatment alone, though they were not restored to non-treated control levels (Fig 1D). However, AICAR treatment did restore Myogenin mRNA to basal levels during cytokine treatment (Fig 1E). Taken together, these results clearly demonstrate that, despite both compounds activating AMPK, only co-treatment with AICAR prevents the progression of cytokine-induced myotube wasting. AICAR-mediated activation of AMPK restores normal muscle metabolism In recent years, it has been demonstrated that muscle undergoing cachectic wasting exhibit altered metabolic profiles compared to healthy fibers (Julienne et al, 2012; Der-Torossian et al, 2013; Fontes-Oliveira et al, 2013). Further, given that AMPK is intimately involved in metabolism, we predicted that cytokine treatment would significantly alter metabolic activity in myotube cells, and that the treatment with the AMPK agonists may affect these changes (Hardie et al, 2012). It has been shown that tumor-bearing mice with cachexia exhibit a metabolic signature in muscle characterized by a Warburg-like increase in glycolysis (Der-Torossian et al, 2013). To assess whether this was also the case in our model, we measured the rate of glucose consumption and lactate production, which are indicative of the rate of glycolytic flux. We observed a significant increase in lactate production and glucose consumption in myotubes treated with IFNγ/TNFα, indicative of elevated glycolysis induced by this treatment (Fig 2A and B). Metformin, a known inhibitor of mitochondrial respiration that induces a compensatory increase in glycolysis, also increased lactate production and glucose consumption, as expected, with no additional increase when co-treated with inflammatory cytokines (Fig 2A and B; Viollet et al, 2012). AICAR, on the other hand, did not affect glycolysis on its own and reduced the increase in glycolysis caused by cytokine treatment (Fig 2A and B). Therefore, in keeping with their ability to protect or not against IFNγ/TNFα-induced wasting, AICAR but not metformin was able to prevent the increased glycolytic rate induced by these cytokines. Figure 2. AICAR corrects cellular metabolic changes in cytokine-treated myotubes A, B. Rates of glucose consumption (A) and lactate production (B) measured in the media 24 h after treatment relative to the non-treated (NT) control from three independent experiments (n = 3). Error bars represent the SEM. C–F. Seahorse XF extracellular flux analysis performed on cells 24 h after treatment. Sequential injections of oligomycin (Oligo.), FCCP, and a combination of rotenone and antimycin A (Rot. + AmA) were performed to assess mitochondrial fitness. Flux was normalized to relative protein units (RPU) measured after the run with an SRB assay. Data are representative of three independent experiment (n = 3). Error bars represent the standard deviation of biological replicates (SD). (C) Oxygen consumption rates (OCR). (D) Extracellular acidification rates (ECAR). (E) Coordinate plot of OCR and ECAR showing the cellular metabolic profile. (F) Measurements of uncoupled (oligomycin-resistant) and coupled (oligomycin-sensitive) respiration. Coupling efficiency was calculated as the percentage of basal respiration associated with coupled respiration. Data information: Significance between means was first determined using ANOVA. Significance P-values were calculated using Fisher's LSD. *P < 0.05; **P < 0.01 from NT controls. Download figure Download PowerPoint Changes in glycolytic activity are often, though not always, associated with changes in oxidative respiration in the mitochondria. Cytokine exposure has been associated with impaired mitochondrial function and reduced oxidative capacity. In addition, several reports have found evidence of mitochondrial dysfunction in pre-clinical models of cachexia (Constantinou et al, 2011; Julienne et al, 2012; Tzika et al, 2013). Therefore, we assessed mitochondrial respiration in C2C12 myotubes using the Seahorse XF extracellular flux system. We found that cytokine treatment significantly reduced both the basal and maximal oxygen consumption rates (OCR; Fig 2C). This was associated with a dramatic increase in the extracellular acidification rate (ECAR), in keeping with our findings that cytokine treatment increases glycolytic flux (Fig 2D). Together, the shifts in OCR and ECAR show a dramatic shift in C2C12 myotubes treated with IFNγ/TNFα from an aerobic to glycolytic metabolism (Fig 2E). As expected, metformin, a known mitochondrial inhibitor, induced a similar inhibition of OCR and elevation of ECAR, though not to the same magnitude as cytokine treatment (Fig 2C–E). Metformin co-treatment with IFNγ/TNFα had no additional effects (Fig 2C–E). In contrast, AICAR co-treatment partially restored both basal and maximal respiration and reduced the ECAR (Fig 2C–E). To assess mitochondrial coupling, we compared the respiration rates before and after injection of the ATP-synthase inhibitor oligomycin (Brand & Nicholls, 2011). We found that the decreases in basal respiration during cytokine and metformin treatment were the result of reductions in both coupled and uncoupled respiration (Fig 2F). However, while metformin did not affect the coupling efficiency, IFNγ/TNFα significantly reduced it (Fig 2F). Interestingly, although AICAR co-treatment did not restore basal respiration to its non-treated levels, it did fully recover the coupling efficiency (Fig 2F). Therefore, in cytokine-treated cells co-treated with AICAR, but not metformin, there is a recovery of ATP synthesis-dependent mitochondrial respiration. In contrast, metformin alone impairs mitochondrial respiration and has no recovery effect during cytokine co-treatment. Collectively, the metabolomics analysis shows that cytokines induce a shift toward glycolysis associated with severely impaired mitochondrial oxidative respiration that is blunted by co-treatment with AICAR. In contrast, metformin treatment alone impairs mitochondrial respiration and has no additive effect during co-treatment with cytokines. This suggests that the inability of metformin to recover atrophy during cytokine treatment, unlike AICAR, could be due to a lack of recovery of mitochondrial function. One potential consequence of altered metabolism is reduced anabolism. Indeed, the inhibition of anabolic signaling is considered to be a key mechanism underlying atrophy in a variety of overlying pro-inflammatory conditions (Rennie et al, 1983; Smith & Tisdale, 1993). Furthermore, AMPK activation has been implicated in suppressing anabolic signaling in cancer cachexia by inhibiting mTOR (White et al, 2013). To assess how AICAR and metformin treatments affect anabolic signaling in cytokine-treated myotubes, we determined the phosphorylation status of the ribosomal protein S6 kinase (S6K) at Thr389 and its target ribosomal protein S6 (S6) at Ser235/236, a downstream target of signaling mTOR (Hornberger et al, 2007; Roux et al, 2007). As expected, cytokine treatment resulted in hypo-phosphorylation of S6K and S6 48 h after treatment, which is indicative of reduced translation initiation (Fig 3A; Roux et al, 2007). AICAR treatment, but not metformin, was able to prevent this decrease (Fig 3A). It is important to note that, while previous reports have demonstrated that exogenous activation of AMPK leads to mTOR suppression, the dosage and timing of AICAR and metformin used here had no significant effect on phosphorylation of S6K or S6 on its own (Fig 3A; Williamson et al, 2006; Xu et al, 2012). Figure 3. AICAR restores anabolic signaling and de novo protein synthesis in cytokine-treated myotubes Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6), and total S6. Quantification represents the pS6K/S6K (n = 3) and pS6/S6 (n = 4) ratios relative to the non-treated (NT) control. Radiographic analysis of de novo protein synthesis using 35S-labeling. Quantification represents whole lane radiation signal density standardized to Coomassie staining and relative to NT control levels (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance P-values were calculated using Fisher's LSD. *P < 0.05; **P < 0.01 from NT controls; ††P < 0.01 from IFNγ/TNFα-treated controls. Source data are available online for this figure. Download figure Download PowerPoint We next sought to directly determine whether these effects on anabolic signaling correlated with changes in general protein biosynthesis. To do so, we performed radio-labeling experiments in which myotubes treated with IFNγ/TNFα in combination with AICAR or metformin were i
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