Figure 6.

Activity of glycolytic enzymes and lactate tracing with [¹³C₂-1,2]glucose. (A) Diagram depicting [¹³C₂-1,2]glucose metabolism through glycolysis and the pentose phosphate pathway (PPP) and the resulting labeling patterns of key intermediates and lactate. Glycolysis will result in the formation of one molecule each of [¹³C₂-2,3]lactate and [¹²C₃]lactate from one molecule of [¹³C₂-1,2]glucose, while the PPP will convert three molecules of [¹³C₂-1,2]glucose into one molecule each of [¹³C₂-1,3]lactate and [¹³C₁-3]lactate and three molecules of [¹²C3]lactate. The relative abundances of PPP-derived [¹³C₁-3]lactate (lactate m + 1) and glycolytic [¹³C₂-2,3]lactate (lactate m + 2) provide estimates of PPP and glycolytic activity, respectively. Key enzymes of glycolysis and the pentose phosphate pathway are depicted in the graph. G6P = glucose 6-phosphate; X5P = xylulose 5-phosphate; G6PDH = glucose-6-phosphate dehydrogenase; E4P = erythrose 4-phosphate; F6P = fructose 6-phosphate; F1,6BP = fructose 1,6 bis-phosphate; GAP= glyceraldehyde 3-phosphate. Red filled circles denote carbon 13, black unfilled circles denote carbon 12. (B) Hexokinase activity was significantly lower in proneural BTIC-8 and -18 than mesenchymal BTIC-11 and -13, while (C) the opposite applied to G6PDH activity. (D) Phosphofructosekinase activity corresponded partly to hexokinase activity. (E) The proneural BTIC-18 contained significantly more PPP-derived [¹³C₁-3]lactate (lactate m + 1, lactate derivative containing only C2 to C3) than the mesenchymal BTICs-11 and -13. Treatment with 1 mM metformin led to a significant drop in PPP-derived [¹³C₁-3]lactate in BTIC-11 and BTIC-13. (F) The relative content of glycolytic [¹³C₂-2,3]lactate (lactate m + 2, lactate derivative containing only C2 to C3) was increased in BTIC-13, albeit not significantly, as compared to BTIC-11 and -18. Treatment with 1 mM metformin resulted in a relative, albeit not significant increase in [¹³C₂-2,3]lactate in BTIC-11. Utilization of [¹³C₂-1,2]glucose via the PPP leads to the formation of (G) [¹³C₁-3]pyruvate and (I) [¹³C₁-3]lactate, whose m/z values increase by +1 unit in comparison to [¹²C₃]pyruvate and [¹²C₃-3]lactate. Utilization of [¹³C₂-1,2]glucose via glycolysis results in double labeled (m + 2) (H) [¹³C₂-2,3]pyruvate and (J) [¹³C₂-2,3]lactate. Hence, a higher fraction of m + 1 pyruvate or lactate isotopologues indicates increased PPP activity. All assays were performed in triplicate. Asterisks indicate * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.