Introduction: We previously reported glucose 6-phosphate dehydrogenase (G6PD) in pentose phosphate pathway (PPP) is activated via heat shock protein 27 (HSP27) phosphorylation by ataxia telangiectasia mutated (ATM) kinase during cerebral ischemia. However, it is unknown whether this potential endogenous anti-oxidative system also works during reperfusion. Thus, we performed a comparative metabolic analysis of the effects of reperfusion in rat cerebral cortex, using middle cerebral artery occlusion (MCAO).
Methods: After the desired period of reperfusion (0, 1, 3, 5, 24-h) following 1-h MCAO (n = 6/group), the rats were sacrificed. Ninety-six metabolites were measured by gas-chromatography/mass-spectrometry (GC/MS), and were analyzed using multivariate statistics. The associated specific metabolic enzymes were further investigated.
Results: GC/MS showed changes in metabolic states in reperfusion-time-dependent manner (Fig. 1A). Enrichment analysis showed PPP was most significantly upregulated during ischemia-reperfusion. In relation to PPP, ischemia-reperfusion induced increase of HSP27 mRNA (69-fold) over time of reperfusion. Immunoblotting showed a slow increase in HSP27 protein and a marked increase in HSP27 phosphorylation (4.5-fold) in reperfusion-time-dependent manner (Fig. 1B). G6PD activity was significantly elevated after 1-h MCAO (20%), reduced after 1-h reperfusion, thereafter gradually elevated. Increasing pattern of the NADPH/NADP+ ratio was similar. Furthermore, intracerebroventricular injection of ATM kinase inhibitor (KU-55933) significantly increased the infarct size (50%) after 24-h reperfusion following 90-min MCAO (Fig. 1CD).
Conclusions: G6PD activation via ischemia-induced HSP27 phosphorylation by ATM kinase may be part of an endogenous antioxidant defense neuroprotection mechanism during ischemia-reperfusion. These findings have important therapeutic implications for the treatment of stroke.