nhibiting serine synthesis pathway by PHGDH inhibition reprograms skeletal muscle cell metabolism and impairs anabolic processes

Introduction: Skeletal muscle mass maintenance and growth require amino acids, nucleotides, and lipids for macromolecule and cell organelle synthesis. Some of these essential building blocks can be de novo synthesized from intermediates of the glycolysis in serine synthesis and pentose phosphate pathways. However, the knowledge of the importance of these metabolic pathways in muscle size regulation is poorly understood. The aim of this study was to identify enzymes from these metabolic pathways that limit myotube size, growth, and proliferation in cultured muscle cells. Methods: We first tested the effect of inhibition of key enzymes of glycolysis, pentose phosphate pathway, and serine synthesis pathway on muscle cell protein synthesis. This small-scale screen led us to focus on studying the importance and mechanisms of the serine synthesis pathway using two different inhibitors of an essential enzyme phosphoglycerate dehydrogenase (PHGDH). We investigated murine C2C12 and human primary skeletal muscle cells by using a wide variety of analysis methods including EdU and radiolabelling, western blotting, confocal microscopy, qPCR, and nuclear magnetic resonance (NMR) spectroscopy and liquid chromatographymass spectrometry (LC-MS) metabolomics. In addition, we screened several potential exogenous compounds that could reverse the effects of PHGDH inhibition. Results: We found that the inhibition of the serine synthesis pathway through PHGDH inhibition decreased protein synthesis in a dose-responsive manner and decreased myoblast proliferation and myotube size without affecting cell viability. The inhibition of PHGDH decreased glucose-derived carbon incorporation into proteins, RNA, and lipids in myotubes and myoblasts. Further, we found that PHGDH inhibition accelerated glycolysis and altered amino acid, nucleotide, and lipid metabolism without a major effect on mitochondrial parameters. In addition, PHGDH inhibition increased adenosine monophosphate (AMP) content and AMPK signalling, and unfolded protein response accompanied by decreased mTORC1 signalling in myotubes, which was associated with decreased muscle protein synthesis. Lastly, we found that antioxidant/redox modulator N-acetylcysteine supplementation partially rescued the decreased protein synthesis and mTORC1 signalling induced by PHGDH inhibition. Conclusions: The results suggest that the serine synthesis pathway and especially its first enzyme PHGDH is essential for the maintenance of biomass in skeletal muscle cells and that this is connected to mTORC1 signalling and changes in oxidative stress and redox balance. In further studies, the role of the serine synthesis pathway and its mechanisms in vivo should be studied in sarcopenia and muscle-wasting diseases.     «

Introduction: Skeletal muscle mass maintenance and growth require amino acids, nucleotides, and lipids for macromolecule and cell organelle synthesis. Some of these essential building blocks can be de novo synthesized from intermediates of the glycolysis in serine synthesis and pentose phosphate pathways. However, the knowledge of the importance of these metabolic pathways in muscle size regulation is poorly understood. The aim of this study was to identify enzymes from these metabolic pathways...     »