Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that activates the cellular response to hypoxia. The HIF1␣ subunit is constantly synthesized and degraded under normoxia, but degradation is rapidly inhibited when oxygen levels drop. Oxygen-dependent hydroxylation by prolyl-4-hydroxylases (PHD) mediates HIF1␣ proteasome degradation. Brain ischemia limits the availability not only of oxygen but also of glucose. We hypothesized that this circumstance could have a modulating effect on HIF. We assessed the separate involvement of oxygen and glucose in HIF1␣ regulation in differentiated neuroblastoma cells subjected to ischemia. We report higher transcriptional activity and HIF1␣ expression under oxygen deprivation in the presence of glucose (OD), than in its absence (oxygen and glucose deprivation, OGD). Unexpectedly, HIF1␣ was not degraded at reoxygenation after an episode of OGD. This was not due to impairment of proteasome function, but was associated with lower HIF1␣ hydroxylation. Krebs cycle metabolites fumarate and succinate are known inhibitors of PHD, while ␣-ketoglutarate is a co-substrate of the reaction. Lack of HIF1␣ degradation in the presence of oxygen was accompanied by a very low ␣-ketoglutarate/fumarate ratio. Furthermore, treatment with a fumarate analogue prevented HIF1␣ degradation under normoxia. In all, our data suggest that postischemic metabolic alterations in Krebs cycle metabolites impair HIF1␣ degradation in the presence of oxygen by decreasing its hydroxylation, and highlight the involvement of metabolic pathways in HIF1␣ regulation besides the well known effects of oxygen.The hypoxia-inducible transcription factor (HIF) 5 is expressed at very low levels in cells under normal oxygen tension, but is rapidly induced upon exposure to hypoxia (1), triggering the activation of a genetic program that enables the metabolic adaptation of cells (2). HIF is a heterodimeric factor composed of a hypoxia-regulated ␣-subunit (HIF1␣ or HIF2␣) and constitutively expressed HIF1␤ (also known as aryl hydrocarbon receptor nuclear translocator, ARNT) (2). Although the ␣-subunit is constantly transcribed and translated, it is also degraded in an oxygen-dependent mechanism. It is only with dwindling oxygen levels that HIF1␣ or HF2␣ expression is readily detected (3). In the presence of oxygen, HIF prolyl-hydroxylases (PHD) hydroxylate two proline residues (positions 402 and 564 in human HIF1␣), in a reaction that requires molecular oxygen and ␣-ketoglutarate as co-substrates (4). These hydroxyproline residues are recognized by the Von Hippel-Lindau tumor suppressor protein (pVHL), one of the components of a E3 ubiquitin-ligase complex that also contains elongins B and C, cullin2, and Rbx, which conjugates ubiquitin to HIF␣ (4, 5). This results in the oxygen-dependent targeting of HIF␣ to the proteasome. Decreased oxygen concentration results in impaired prolyl-hydroxylation, reduced targeting of HIF␣ to the proteasome and the accumulation of HIF in the nucleus, where it activates a pleth...

Brain ischemia causes neuronal cell death by several mechanisms involving necrotic and apoptotic processes. The contributions of each process depend on conditions such as the severity and duration of ischemia, and the availability of ATP. We examined whether glucose affected the development of apoptosis after transient ischemia, and whether this was sensitive to caspase inhibition. Retinoic acid-differentiated SH-SY5Y human neuroblastoma cells were subjected to oxygen and glucose deprivation for 15 h followed by various periods of reoxygenation in either the presence or absence of glucose. Oxygen and glucose deprivation induced cell death in the hours following reoxygenation, as detected by propidium iodide staining. At the end of the period of oxygen and glucose deprivation, both cytochrome c and apoptosis-inducing factor translocated from mitochondria to cytosol. Reoxygenation in the presence of glucose accelerated cell death, and enhanced caspase-3 activity and apoptosis. The glucosedependent increase in apoptosis was prevented by treatment with the caspase inhibitor zVAD-fmk, but not with calpeptin, a calpain inhibitor. Nevertheless, both zVAD-fmk and calpeptin decreased cell death in the glucose-treated group. ATP levels dropped dramatically after oxygen and glucose deprivation, but recovered steadily thereafter, and were significantly higher at 6 h of reoxygenation in the glucose-treated group. This indicates that energy recovery may promote the glucosedependent cell death. We conclude that glucose favours the development of caspase-dependent apoptosis during reoxygenation following oxygen and glucose deprivation. Cao et al. 2003;Plesnila et al. 2004;Culmsee et al. 2005). Apoptosis has also been identified in peri-infarction areas of the human brain after stroke (Sairanen et al. 2006). Apoptosis is an energy-dependent process that needs the development and execution of a cell death programme (Alison and Sarraf 1992). For this reason, the availability of energy substrates might condition the type of cell death (Nicotera et al. 1998). Indeed, high glucose induces a switch from necrosis to apoptosis (Fujita and Ueda 2003;Ueda and Fujita 2004), whereas ATP depletion has the opposite effect (Eguchi et al. 1997). Here we sought to evaluate whether glucose availability and ATP level after a transient period of ischemia affected the development of apoptotic cell death, and whether this type of death could be blocked by caspase inhibition. We developed a model of delayed cell death in differentiated SH-SY5Y neuroblastoma cells transiently exposed to a period of oxygen and glucose deprivation (OGD) and then reoxygenated either in the presence or in the absence of glucose. Our results show that glucose accelerates cell death after ischemia by promoting caspase-dependent apoptosis. Materials and Methods MaterialsMost chemicals used were from SIGMA. All-trans-retinoic acid (RA) was purchased from Calbiochem/Bionova (Madrid, Spain). zVAD-fmk and calpeptin were from Bachem (Weil am Rhein, Germany), and were diss...