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Competitive HIF Prolyl Hydroxylase Inhibitors Show Protection against Oxidative Stress by a Mechanism Partially Dependent on Glycolysis.

Bergström AL, Fog K, Sager TN, Bruun AT, Thirstrup K - ISRN Neurosci (2013)

Bottom Line: In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12).Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress.In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurodegeneration, Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark.

ABSTRACT
The hypoxia inducible factor 1 (HIF-1) is a central transcription factor involved in the cellular and molecular adaptation to hypoxia and low glucose supply. The level of HIF-1 is to a large degree regulated by the HIF prolyl hydroxylase enzymes (HPHs) belonging to the Fe(II) and 2-oxoglutarate-dependent dioxygenase superfamily. In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12). Although the competitive HPH-inhibitor compounds were found to be pharmacologically more potent than the non-competitive compounds at inhibiting HPH2 and HPH1, this was not translated into the cellular effects of the compounds, where the non-competitive inhibitors were actually more potent than the competitive in stabilizing and translocatingHIF1 α to the nucleus (quantified with Cellomics ArrayScan technology). This could be explained by the high cellular concentrations of the cofactor 2-oxoglutarate (2-OG) as the competitive inhibitors act by binding to the 2-OG site of the HPH enzymes. Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress. In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

No MeSH data available.


Related in: MedlinePlus

Cpd A is partially protective even when glycolysis is inhibited. (a) ATP-levels in SH-SY5Y cells grown overnight in starvation media overnight, then treated with 10 mM 2-deoxyglucose for 3 hours (or nothing for control) followed by 3 hours of treatment with 6-OHDA at the indicated concentrations. (b) and (c) ATP-levels in SH-SY5Y cells grown over night in starvation media, then pretreated for 3 hours with 10 mM 2-deoxyglucose (or nothing for control) and Cpd A (25 or 50 μM or nothing for control) followed by 3 hours treatment with 100 μM 6-OHDA (b) or 300 μM 6-OHDA (c).
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fig5: Cpd A is partially protective even when glycolysis is inhibited. (a) ATP-levels in SH-SY5Y cells grown overnight in starvation media overnight, then treated with 10 mM 2-deoxyglucose for 3 hours (or nothing for control) followed by 3 hours of treatment with 6-OHDA at the indicated concentrations. (b) and (c) ATP-levels in SH-SY5Y cells grown over night in starvation media, then pretreated for 3 hours with 10 mM 2-deoxyglucose (or nothing for control) and Cpd A (25 or 50 μM or nothing for control) followed by 3 hours treatment with 100 μM 6-OHDA (b) or 300 μM 6-OHDA (c).

Mentions: It has been suggested that the primary mechanism of HIF-1-mediated cytoprotection could be ascribed to a metabolic shift towards glycolytic ATP-production [4]. This shift would make the cells less dependent on mitochondrial oxidative phosphorylation and more dependent on anaerobic glycolysis for ATP-production, which would be protective in a hypoxic setting: reviewed by [12]. To investigate this further, SH-SY5Y cells were grown in starvation media (with low glucose and serum levels) for 24 hours and furthermore the cells were treated with 2-deoxyglucose to block glycolysis. Treatment with 2-deoxyglucose significantly reduced both the basal and the 6-OHDA induced levels of ATP in the cells (Figure 5(a)). CpdA was however still able to protect the cells from 100 μM 6-OHDA induced ATP-loss despite the blockages of glycolysis by the combination of starvation media and 10 mM 2-deoxyglucose (Figure 5(b)). The protective effect was relatively similar to what was found when cells were grown without 2-deoxyglucose. At higher doses (300 μM 6-OHDA) the protective effect of CpdA was lost in the presence of 2-deoxyglucose (Figure 5(c)).


Competitive HIF Prolyl Hydroxylase Inhibitors Show Protection against Oxidative Stress by a Mechanism Partially Dependent on Glycolysis.

Bergström AL, Fog K, Sager TN, Bruun AT, Thirstrup K - ISRN Neurosci (2013)

Cpd A is partially protective even when glycolysis is inhibited. (a) ATP-levels in SH-SY5Y cells grown overnight in starvation media overnight, then treated with 10 mM 2-deoxyglucose for 3 hours (or nothing for control) followed by 3 hours of treatment with 6-OHDA at the indicated concentrations. (b) and (c) ATP-levels in SH-SY5Y cells grown over night in starvation media, then pretreated for 3 hours with 10 mM 2-deoxyglucose (or nothing for control) and Cpd A (25 or 50 μM or nothing for control) followed by 3 hours treatment with 100 μM 6-OHDA (b) or 300 μM 6-OHDA (c).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Cpd A is partially protective even when glycolysis is inhibited. (a) ATP-levels in SH-SY5Y cells grown overnight in starvation media overnight, then treated with 10 mM 2-deoxyglucose for 3 hours (or nothing for control) followed by 3 hours of treatment with 6-OHDA at the indicated concentrations. (b) and (c) ATP-levels in SH-SY5Y cells grown over night in starvation media, then pretreated for 3 hours with 10 mM 2-deoxyglucose (or nothing for control) and Cpd A (25 or 50 μM or nothing for control) followed by 3 hours treatment with 100 μM 6-OHDA (b) or 300 μM 6-OHDA (c).
Mentions: It has been suggested that the primary mechanism of HIF-1-mediated cytoprotection could be ascribed to a metabolic shift towards glycolytic ATP-production [4]. This shift would make the cells less dependent on mitochondrial oxidative phosphorylation and more dependent on anaerobic glycolysis for ATP-production, which would be protective in a hypoxic setting: reviewed by [12]. To investigate this further, SH-SY5Y cells were grown in starvation media (with low glucose and serum levels) for 24 hours and furthermore the cells were treated with 2-deoxyglucose to block glycolysis. Treatment with 2-deoxyglucose significantly reduced both the basal and the 6-OHDA induced levels of ATP in the cells (Figure 5(a)). CpdA was however still able to protect the cells from 100 μM 6-OHDA induced ATP-loss despite the blockages of glycolysis by the combination of starvation media and 10 mM 2-deoxyglucose (Figure 5(b)). The protective effect was relatively similar to what was found when cells were grown without 2-deoxyglucose. At higher doses (300 μM 6-OHDA) the protective effect of CpdA was lost in the presence of 2-deoxyglucose (Figure 5(c)).

Bottom Line: In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12).Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress.In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurodegeneration, Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark.

ABSTRACT
The hypoxia inducible factor 1 (HIF-1) is a central transcription factor involved in the cellular and molecular adaptation to hypoxia and low glucose supply. The level of HIF-1 is to a large degree regulated by the HIF prolyl hydroxylase enzymes (HPHs) belonging to the Fe(II) and 2-oxoglutarate-dependent dioxygenase superfamily. In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12). Although the competitive HPH-inhibitor compounds were found to be pharmacologically more potent than the non-competitive compounds at inhibiting HPH2 and HPH1, this was not translated into the cellular effects of the compounds, where the non-competitive inhibitors were actually more potent than the competitive in stabilizing and translocatingHIF1 α to the nucleus (quantified with Cellomics ArrayScan technology). This could be explained by the high cellular concentrations of the cofactor 2-oxoglutarate (2-OG) as the competitive inhibitors act by binding to the 2-OG site of the HPH enzymes. Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress. In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

No MeSH data available.


Related in: MedlinePlus