FIG. 6.

Targeted metabolomics of the Krebs cycle intermediates, acetoacetate, and β-OHB and evidence for the isocitrate/pyruvate redox shuttle. Total cellular metabolite levels (A), their selected ratios (B), and AA and β-OHB and their ratios as indicated (C) were quantified in INS-1E cells preincubated with 3 mM glucose for 1 h; then, glucose was raised by zero, 8, and 17 mM surplus to reach a final concentration of 3 mM (black bars), 11 mM (dark gray bars), and 20 mM (gray bars) glucose and subsequently incubated for another 30 min. ANOVA for (A, B) yielded (n = 5) ***p < 0.001; whereas Student's t-test for (A, B) yielded (n = 5): ^@@p < 0.05; ^@p < 0.1; and for (C) (all estimates from two independent experiments are shown with averages and SDs); p < 0.001 for all combinations between the two compounds. The difference between the β-OHB/AA ratios was not significant. Notably, the significant β-OHB increase suggests also the increase in mitochondrial matrix NAD⁺, since β-OHB dehydrogenase, which exists only in the mitochondrial matrix, produces β-OHB from AA at the expense of NADH, thus forming NAD⁺ (46). The fact that AA does not proportionally decrease reflects other reactions (43, 46) and penetration of AA into the cytosol during the sample preparation (Supplementary Fig. S2Fa). (D) ¹³C incorporation from 1-¹³C-l-glutamine into citrate, malate, and 2OG is expressed for normalized data for 25 mM glucose (2-h incubations) in relation to average values obtained after a 2-h incubation with 3 mM glucose. Data were first calculated in % of ¹³C accumulated amounts versus total (¹³C+¹²C) amount of a given compound when accounted for the natural ¹³C content. Evidence for the isocitrate/pyruvate redox shuttle is suggested by the existence of the ¹³C-accumulation, as such. This is because the ¹³C-accumulation into citrate from 1-¹³C-glutamine cannot exist on the forward Krebs cycle, since ¹³C-CO₂ is formed and eliminated from the sample; hence any ¹³C-labeled citrate or malate molecule (when subtracting those with naturally occurring ¹³C) must originate from the reverse Krebs cycle direction, given by the IDH2-mediated NADPH-driven reductive carboxylation of 2OG. ANOVA for (D) (n = 6): ^**p < 0.05. Also, insignificantly (“ns”) increased ¹³C incorporation into malate and 2OG is indicated. β-OHB, β-hydroxybutyrate; AA, acetoacetate; ns, nonsignificant; SDs, standard deviations.