Figure 3

AZ67 prevents the metabolic switch from PPP to glycolysis, redox and mitochondrial stress, and apoptosis in an in vitro model of ischemia/reperfusion in primary neurons. (a) Western immunoblotting showing that the treatment of neurons with OGD (3 h) followed by reoxygenation (plus glucose) (4 h), stabilized PFKFB3 protein levels. Incubation of neurons with AZ67 (10 nM) during the 4 h of reoxygenation did not alter PFKFB3 protein levels. (b) Incubation of neurons with AZ67 for 4 h revealed no effect on lactate release. Treatment of neurons with OGD (3 h) followed by reoxygenation (plus glucose) (4 h) increased lactate released. Incubation of neurons with AZ67 (10 nM) during the 4 h of reoxygenation prevented the increase in lactate release. (c) Incubation of neurons with AZ67 for 4 h revealed no effect on the glycolytic flux, as assessed by the formation of ³H₂O from [3-³H]glucose. However, treatment of neurons with OGD (3 h) followed by reoxygenation (plus glucose) (4 h) increased the glycolytic flux. Incubation of neurons with AZ67 (10 nM) during the 4 h of reoxygenation prevented the increase in the glycolytic flux. (d) Incubation of neurons with AZ67 for 4 h revealed no effect on the pentose-phosphate pathway (PPP) flux, as assessed by the difference in the formation of ¹⁴CO₂ from [1-¹⁴C]- and from [6-¹⁴C]glucose. However, treatment of neurons with OGD (3 h) followed by reoxygenation (plus glucose) (4 h) decreased the PPP flux. Incubation of neurons with AZ67 (10 nM) during the 4 h of reoxygenation prevented the decrease in the PPP flux. (e) Incubation of neurons with AZ67 for 4 h revealed no effect on H₂O₂ release, as assessed by the fluorescence of AmplexRed. However, treatment of neurons with OGD (3 h) followed by reoxygenation (plus glucose) (4 h) increased H₂O₂ release. Incubation of neurons with AZ67 (10 nM) during the 4 h of reoxygenation prevented the increase in H₂O₂ release. (f) Incubation of neurons with AZ67 for 4 h revealed no effect on mitochondrial ROS formation, as assessed by MitoSox fluorescence by flow cytometry. However, treatment of neurons with OGD (3 h) followed by reoxygenation (plus glucose) (4 h) increased mitochondrial ROS. Incubation of neurons with AZ67 (10 nM) during the 4 h of reoxygenation significantly prevented the increase in mitochondrial ROS formation. (g) Incubation of neurons with AZ67 for 4 h revealed no effect on pyruvate dehydrogenase (PDH) activity, as assessed by the conversion of [1-¹⁴C]pyruvate in ¹⁴CO₂. However, treatment of neurons with OGD (3 h) followed by reoxygenation (plus glucose) (4 h) decreased PDH activity that was not altered by incubation of neurons with AZ67 (10 nM) during the 4 h of reoxygenation. (h) Incubation of neurons with AZ67 for 4 h revealed no effect on mitochondrial membrane potential (∆ψ_m), as assessed by flow cytometry. However, treatment of neurons with OGD (3 h) followed by reoxygenation (plus glucose) (4 h) decreased ∆ψ_m. Incubation of neurons with AZ67 (10 nM) during the 4 h of reoxygenation prevented the decreased ∆ψ_m. (i) Incubation of neurons with AZ67 for 4 h revealed no effect on caspase-3 activity, a measure of apoptosis. However, treatment of neurons with OGD (3 h) followed by reoxygenation (plus glucose) (4 h) increased apoptosis. Incubation of neurons with AZ67 (10 nM) during the 4 h of reoxygenation prevented the increase in apoptosis. In all cases, data are mean ± S.E.M. values for n = 3 independent culture preparations. #p < 0.05 versus OGD at 0 nM AZ67; *p < 0.05 versus the corresponding normoxic condition (ANOVA followed by the least significant difference multiple range test). See also Supplementary Data 1 and Statistics Table 1.