Myotube hypertrophy is associated with cancer-like metabolic reprogramming and limited by PHGDH

Muscle fiber size and oxidative metabolism are inversely related, suggesting that a glycolytic metabolism may offer a growth advantage in muscle fibers. However, the mechanisms underlying this advantage remains unknown. Nearly 100 years ago, Warburg reported that cancer cells take up more glucose to produce glycolytic intermediates for anabolic reactions such as amino acid-protein synthesis. The aim of this study was to test whether glycolysis contributes to anabolic signalling responses and hypertrophy in post-mitotic muscle cells. Skeletal muscle hypertrophy was induced in vitro by treating mouse C2C12 myotubes with IGF-1. 14C glucose was added to differentiation medium and radioactivity in isolated protein was measured. We exposed differentiated C2C12 and primary mouse myotubes, to 2-deoxyglucose (2DG) and PHGDH siRNA upon which we assessed myotube diameter and signaling pathways involved in the regulation of muscle fiber size. Here, we present evidence that, hypertrophying C2C12 myotubes undergo a cancer-like metabolic reprogramming. First, IGF-1-induced C2C12 myotube hypertrophy increases shunting of carbon from glucose into protein. Second, reduction of glycolysis through 2-deoxy-D-glucose (2DG) lowers C2C12 and primary myotube size 16-40%. Third, reducing the cancer metabolism-associated enzyme PHGDH decreases C2C12 and primary myotube size 25-52%, whereas PHGDH overexpression increases C2C12 myotube size ≈20%. Fourth, the muscle hypertrophy-promoting kinase AKT regulates PHGDH expression. Together these results suggest that glycolysis is important for hypertrophying C2C12 myotubes by reprograming their metabolism similar to cancer cells.


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Having sufficient muscle mass and strength is associated with low morbidity and mortality (Gabriel and Zierath, 83 2017; Wolfe, 2006). An individual's muscle mass and strength depend both on genetics (Arden and Spector, 1997;

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The main mechanism by which resistance exercise increases protein synthesis is the activation of the 90 serine/threonine kinase mTOR which is part of the mTORC1 complex (Goodman, 2019). In addition, hypertrophy-91 inducing stimuli such as synergist ablation (Chaillou et al., 2013) and acute resistance exercise (Pillon et al., 2020; 92 Vissing and Schjerling, 2014) extensively change gene expression. Here, one of the most robust changes is the 93 increased expression of the transcription factor Myc, whose expression increases >10-fold in synergist-ablated,   1927). In a key experiment, the Warburg group compared glucose uptake and lactate production of sarcomas with 101 that of healthy organs in rats. They noted that sarcomas took up more glucose and produced more lactate than other 102 organs such as the liver, kidney or brain (Warburg et al., 1927). This demonstrated that cancer cells take up more 103 glucose and have a higher glycolytic flux in the presence of oxygen. This phenomenon was termed Warburg effect 104 by Efraim Racker in contrast to anaerobic glycolysis or the Pasteur effect (Racker, 1972). The purpose of the 105 metabolic reprogramming of cancer cells was long poorly understood. Today we know that the pathways affected  (Semsarian et al., 1999), which is essentially the Warburg effect. Similarly, muscle activation of 120 AKT1, a known cancer metabolism regulator (Elstrom et al., 2004), not only causes muscle hypertrophy, but also 121 increases the expression of glycolytic enzymes (Izumiya et al., 2008). Similarly, mTORC1 activation through a 122 loss of its inhibitor NPRL2, results in muscle hypertrophy and induces aerobic glycolysis in mice (Dutchak et al., 123 2018). Finally, a loss of myostatin both induces muscle hypertrophy and promotes a shift to a more glycolytic 124 metabolism (Mouisel et al., 2014). Collectively, these data suggest that the stimulation of muscle hypertrophy -125 through increased IGF1-AKT1-mTORC1 or reduced myostatin signaling -is associated with increased glycolysis 126 and a metabolic reprogramming reminiscent to that which occurs in cancer cells (DeBerardinis and Chandel, 2016).

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A specific cancer metabolism-associated enzyme that may contribute to such metabolic reprogramming during 129 muscle hypertrophy is 3-phosphoglycerate dehydrogenase (PHGDH, E.C. 1.1.1.95). PHGDH channels 3-130 phosphoglycerate out of glycolysis, into serine biosynthesis and one-carbon metabolism, which is essential for reprograming is important for proliferation and cellular growth. Besides, muscle stem cells express more Phgdh 135 when they become activated and start to proliferate (supplementary data of Ryall et al., 2015)). In addition, Phgdh 136 mRNA expression also increases in terminally differentiated pig muscles when hypertrophy is stimulated with the 137 β2-agonist ractopamine (Brown et al., 2016). Together, this shows that PHGDH becomes activated and/or more 138 abundant in proliferating cells and in at least one type of skeletal muscle hypertrophy. 139 140 6 Currently, it is unclear whether a hypertrophying muscle reprograms its metabolism similar to cancer cells so that 141 glycolytic intermediates and other metabolites are shunted out of energy metabolism into anabolic reactions such 142 as serine biosynthesis and one-carbon metabolism. The aim of this study was therefore to investigate whether 143 hypertrophying C2C12 myotubes shunt more carbon from glucose into amino acid and nucleotides for protein and 144 RNA synthesis. We also investigated whether inhibition of glycolytic flux and Phgdh knockdown or 145 overexpression affected C2C12 myotube size, in untreated or IGF-1-treated myotubes. We found that 146 hypertrophying C2C12 and primary mouse myotubes indeed shunt more carbon from glucose into protein and            Table 1 for primer details). Experiments were   285

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following the manufacturer's instructions (see Table 3 for primer sequences).

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For retroviral infection, 3 x 10 4 C2C12 cells were seeded in a 6-well plate overnight at 37°C in a 5% CO2 incubator.

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1 h prior to infection, medium was changed to 1.5 ml of fresh growth medium and cells were infected by adding 308 retroviral particles in a ratio of 1:4. Cells were incubated until reaching 90% confluence and then differentiated as

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The inhibition of glycolytic flux through 2DG may not only affect the generation of glycolytic intermediates as 361 substrates for anabolic reactions but also energy-sensitive signalling mechanisms. To answer this, we measured 362 activity markers and the expression, phopho-AMPK and atrophy-associated E3 ubiquitin ligases. We observed no 363 effect of 2DG on AMPK phosphorylation (Figure 3a; two-way ANOVA, p=0.808), indicating that any potential 364 diminished energy state did not cause the reduced myotube size. On the other hand, we found that 2DG decreases 365 P70S6K phosphorylation (Figure 3b), repressing protein synthesis. While Trim63 remains unaffected by blocking 366 glycolysis (Figure 3e), the other protein degradation marker, Fbxo32, is attenuated by 2DG (Figure 3d). Together

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The second finding of this study is that the inhibition of glycolysis reduces C2C12 and primary myotube size

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In summary, this study provides evidence that glycolysis is important in hypertrophying C2C12 and primary mouse 537 myotubes, reminiscent of cancer-like metabolic reprogramming and that this limits typical myotube size and IGF-