Fig. 3: Circadian regulation of glycolysis and pentose phosphate pathways (PPP) fluxes in human red blood cells (RBCs).
From: Rhythmic glucose metabolism regulates the redox circadian clockwork in human red blood cells
A Schematic showing experimental protocol used to collect samples. RBCs from n = 3 human subjects were incubated with either 11 mM 2-13C1-glucose or 1,2-13C2-glucose and kept under constant conditions (37 °C in continuous darkness) and sampling was performed every 4 h over 3 days period. B Schematic showing fate of 2-13C1-Glucose metabolized through glycolysis and the pentose phosphate pathway (PPP). Metabolic fluxes for glycolysis and PPP were quantified by 13C-NMR (nuclear magnetic resonance) spectroscopy. When 2-13C1-glucose is used as a tracer, it is metabolized via glycolysis and produces singly labeled 13C1-2-lactate with chemical shift, δ = 71.2. The same 2-13C1-glucose metabolized through the PPP produces singly labeled 13C1-3-lactate with chemical shift δ = 22.8. C Rhythmic glycolytic and PPP fluxes (P = 0.009) in RBCs measured by NMR. P-values were obtained from rhythmicity analysis using RAIN algorithm. Graph bars present mean ± s.e.m (n = 3 biological replicates). D Rhythmic PRDX oxidation used as a control for circadian rhythmicity in RBCs for measuring metabolic fluxes by NMR experiment. P-values were obtained from rhythmicity analysis using RAIN algorithm. Data are presented mean ± s.e.m (n = 3 biological replicates). E Schematic showing fate of 1,2-13C2-Glucose metabolized by glycolysis and the pentose phosphate pathway (PPP). Metabolic fluxes were measured with GC-MS. When 1,2-13C2-glucose is used as a tracer, it is metabolized via glycolysis and produces doubly labeled 2,3-13C2-lactate. The same 1,2-13C2-glucose metabolized through the PPP produces singly labeled 13C1-lactate. The glycolytic m + 2 isotopomer of 13C2-lactate and the pentose phosphate pathway m + 1 isotopomer of 13C1-lactate were thus used for flux measurements (see Methods). f Rhythmic regulation of glycolysis and PPP fluxes in RBCs measured by GC-MS. P-values were obtained from rhythmicity analysis using RAIN algorithm. Data are presented mean ± s.e.m (n = 3 biological replicates). G Rhythmic PRDX oxidation used as a control for circadian rhythmicity in RBCs for measuring metabolic fluxes by GC-MS experiment. P-values were obtained from rhythmicity analysis using RAIN algorithm. Data are presented mean ± s.e.m (n = 3 biological replicates). H Comparison of glycolytic flux measurements in RBCs measured by NMR and GC-MS methods. This correlation figure is obtained from Fig. 3C, F. Data are presented mean ± s.e.m (n = 3 biological replicates).
