Real Time Pulse Chase (RTPC) In-Cell NMR Spectroscopy Reveals Critical Metabolites in Subminute Metabolism of...

Neurons undergo extensive metabolic reprogramming during differentiation; this reprogramming leads to specific changes in the kinetics and concentrations of metabolites. We developed an in-cell NMR-based method that monitors this metabolic transformation with subminute time resolution. Undifferentiated SH-SY5Y human neuronal precursor cells were encapsulated into alginate gel beads and differentiated inside the gel. Real time pulse chase (RTPC) in-cell NMR was used to measure relative steady state concentrations of glycolysis and TCA cycle metabolites and the kinetics of metabolite production and clearance in differentiated and undifferentiated cells. Neuronal differentiation slowed glycolysis, increased TCA cycle activity and glutamate production. Fructose 1,6 bisphosphate and glutathione were identified as major biomarkers of undifferentiated and differentiated cells, respectively. The results demonstrate that RTPC-NMR analysis of neuronal cells is an effective method for studying changes in metabolite profiles induced by stress and drug-induced stimuli.