Figure 5

Pyruvate starvation inhibits glycolytic flux and GAPDH and hexokinase activity under high-glucose conditions. (A) Time course of ECAR in the [Glc 5 mM/Pyr (+)] (blue), [Glc 5 mM/Pyr (−)] (yellow), [Glc 100 mM/Pyr (+)] (brown), [Glc 100 mM/Pyr (−)] (green), and [Glc 100 mM/Pyr (−)/2-OG (+)] (red) groups measured using an Extracellular Flux Analyzer. (B) Time course of ECAR in the groups described as (A) measured using an Extracellular Flux Analyzer and XF Glycolytic test. Glucose, pyruvate and 2-OG-induced glycolysis (C) and maximum glycolytic capacity (D) under these five conditions was estimated by changes in ECAR through sequential addition of glucose, pyruvate and 2-OG, and oligomycin. (E) The amount of glucose uptake into IMS32 cells under the five conditions described in (A). GAPDH (F, G) and hexokinase (H, I) activity in IMS32 cells at 30 min (C, E) or 60 min (D, F) in the groups described in (A). Values represent mean + SD from 13–14 (A), 9–10 (B-D), six (E), and three (F-I) experiments (individual values are depicted as circles, triangles, pluses, crosses and asterisks). (A) [Glc 100 mM/Pyr (−)] compared with (a1) [Glc 5 mM/Pyr (+)], [Glc 5 mM/Pyr (−)], or [Glc 100 mM/Pyr (−)/2-OG (+)] (P < 0.01), (a2) [Glc 5 mM/Pyr (+)] or [Glc 5 mM/Pyr (−)] (P < 0.01), and (a3) [Glc 5 mM/Pyr (+)] (P < 0.01). (B) [Glc 100 mM/Pyr (−)] compared with (b1) [Glc 5 mM/Pyr (+)], [Glc 5 mM/Pyr (−)], [Glc 100 mM/Pyr (+)], or [Glc 100 mM/Pyr (−)/2-OG (+)] (P < 0.01), (b2) [Glc 5 mM/Pyr (+)], or [Glc 5 mM/Pyr (−)] (P < 0.01), (b3) [Glc 5 mM/Pyr (+)], [Glc 100 mM/Pyr (+)], or [Glc 100 mM/Pyr (−)/2-OG (+)] (P < 0.01), (b4) [Glc 5 mM/Pyr (+)], [Glc 100 mM/Pyr (+)], or [Glc 100 mM/Pyr (−)/2-OG (+)] (P < 0.01), and [Glc 5 mM/Pyr (−)] (P < 0.05), (b5) [Glc 100 mM/Pyr (+)] (P < 0.05), (b6) [Glc 100 mM/Pyr (+)], or [Glc 100 mM/Pyr (−)/2-OG (+)] (P < 0.01). (C-I) * P < 0.05, ** P < 0.01.