The Essentiality of Serine and Glycine for Skeletal Muscle Regeneration
Authors: Thalacker‐Mercer Anna , Blum Jamie , Gheller Brandon 
Published: June 2021

Non‐essential amino acids (NEAA), serine (ser) and glycine (gly), are recognized as essential to support proliferation of some cell types, including skeletal muscle (SkM) progenitor cells (MPCs). MPCs and a well‐orchestrated myogenic program are essential for SkM regeneration following injury; impaired regeneration underlies pathological tissue remodeling and functional decline. It is appreciated that the metabolic microenvironment and nutrient availability influence the myogenic process. The objective of this research was to investigate the metabolic and cellular requirements of ser and gly for the regenerative process (MPC expansion, differentiation, and myotube formation) in vitro. Human primary MPCs (hMPCs) were cultured with and without ser/gly and markers of proliferation (live and dead staining, BrdU incorporation, Propidium Iodide cell‐cycle analysis) were assessed; markers of differentiation, and myotube formation (gene and protein expression of select myogenic regulatory factors [MRFs], myosin staining) are being investigated. Additionally, the regulation of the de novo ser synthesis pathway (SSP), ser/gly interconversion (SHMT1, SHMT2), and changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined with/without ser/gly using RNAseq, qPCR, immunoblotting, and a Seahorse bioanalyzer. Ser/gly depletion caused hMPCs to reside in a non‐proliferative state without inducing apoptosis and decreased OCR (p<0.05) but not ECAR (p=0.07), supporting a quiescent metabolic state. BrdU incorporation and cell‐cycle sorting with flow cytometry suggested that depletion of ser/gly inhibits hMPCs from leaving G1 phase. Intriguingly, proliferating hMPCs expressed all SSP enzymes (PHGDH, PSAT1, PSPH). The expression of SSP enzymes, SHMT2, ser transporters, and ATF4 (transcription factor involved in nutrient sensing) increased during ser/gly depletion suggesting ser/gly depletion is sensed by hMPCs and mechanisms to increase ser and gly availability are upregulated. Ser and gly are substrates for multiple metabolic pathways; building blocks for lipids, proteins, and nucleotides; one‐carbon donors; and help maintain cellular (epi)genetic status and redox state. Providing formate (a one‐carbon source), nucleotides, or additional glucose did not rescue proliferation. RNAseq analysis revealed an upregulation of genes involved in the glutathione metabolism pathway. In support of these findings a cell permeable version of glutathione provided a modest rescue to hMPC cell number and intracellular levels of total glutathione and the ratio of reduced to oxidized glutathione were diminished after the ser/gly depletion; glutathione may be a necessary product of ser/gly metabolism. Ongoing RNAseq analysis will allow us to identify potential mechanisms underlying the quiescent‐like state. Ongoing culture experiments will determine the effects of ser/gly depletion on differentiation and myotube formation. This research builds upon recent findings that clearly identify an exogenous requirement of ser and gly for hMPC expansion and challenges the notion that ser and gly are strictly NEAA, particularly in states of tissue regeneration.Support or Funding InformationPresident's Council of Cornell Women and Cornell Division of Nutritional SciencesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.