Figure 4. Investigation of reversed glycolytic reactions.

Fructose-6-phosphate was used to investigate reversal of glycolytic reactions as it appears before the block at GAPDH. Representative 2D- ¹H, ¹³C-HSQC spectra were used to show ¹³C label incorporation in both control and FK866. ( A) Carbon 1 shows peaks, therefore ¹³C label, in both 48-hour FK866 samples; ( B) carbon 5 shows peaks in the FK866 samples, with no presence of ¹³C label in control or 24-hour FK866 samples. ( C) Labelling pattern analysis shows how ¹³C _1,2-glucose is metabolised to 1,2-labelled fructose-6-phosphate during glycolysis, explaining the label incorporation in carbon 1 in both control and FK866 samples. The route to ¹³C label in carbon 5 of fructose-6-phosphate is via 1,2-labelled glyceraldehyde-3-phosphate taking the backward reaction to fructose-1,6-bisphosphate. The presence of label incorporation in carbon 5 of FK866 samples is evidence of reversal of these reactions as a result of an NAD ⁺-dependent build-up of G3P. ( D) Glycogen levels of C2C12s. ( E) m+6 portion of fructose-1,6-bisphosphate as shown through LCMS analysis. * p<0.05, One-way ANOVA performed on raw data; Dunnett’s multiple comparison test, treatment compared to control. All data are the mean ±SEM, n=3.