Repeated Glucose Deprivation/Reperfusion Induced PC-12 Cell Death through the Involvement of FOXO Transcription Factor

Background: Cognitive impairment and brain damage in diabetes is suggested to be associated with hypoglycemia. The mecha￢nisms of hypoglycemia-induced neural death and apoptosis are not clear and reperfusion injury may be involved. Recent studies show that glucose deprivation/reperfusion induced more neuronal cell death than glucose deprivation itself. The forkhead box O (FOXO) transcription factors are implicated in the regulation of cell apoptosis and survival, but their role in neuronal cells re￢mains unclear. We examined the role of FOXO transcription factors and the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt and apoptosis-related signaling pathways in PC-12 cells exposed to repeated glucose deprivation/reperfusion. Methods: PC-12 cells were exposed to control (Dulbecco’s Modified Eagle Medium [DMEM] containing 25 mM glucose) or glu￢cose deprivation/reperfusion (DMEM with 0 mM glucose for 6 hours and then DMEM with 25 mM glucose for 18 hours) for 5 days. MTT assay and Western blot analysis were performed for cell viability, apoptosis, and the expression of survival signaling pathways. FOXO3/4’,6-diamidino-2-phenylindole staining was done to ascertain the involvement of FOXO transcription factors in glucose deprivation/reperfusion conditions. Results: Compared to PC-12 cells not exposed to hypoglycemia, cells exposed to glucose deprivation/reperfusion showed a re￢duction of cell viability, decreased expression of phosphorylated Akt and Bcl-2, and an increase of cleaved caspase-3 expression. Of note, FOXO3 protein was localized in the nuclei of glucose deprivation/reperfusion cells but not in the control cells. Conclusion: Repeated glucose deprivation/reperfusion caused the neuronal cell death. Activated FOXO3 via the PI3K/Akt path￢way in repeated glucose deprivation/reperfusion was involved in genes related to apoptosis.