The Menadione-Mediated WST1 Reduction by Cultured Astrocytes Depends on NQO1 Activity and Cytosolic Glucose Metabolism

Ehrke, Eric; Steinmeier, Johann; Stapelfeldt, Karsten; Dringen, Ralf

doi:10.1007/s11064-019-02930-1

Cited by 7 publications

(14 citation statements)

References 62 publications

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“…In contrast, the electrons required for pyruvate reduction to lactate via LDH are unlikely to be only derived from initially present cytosolic NADH. The cellular NADH pool in cultured astrocytes accounts for only 0.70 ± 0.03 nmol/mg [ 68 ] and, even if the entire amount would be present as cytosolic NADH, it would have to be recycled more than 400 times by cytosolic processes to allow the formation of the pyruvate-derived lactate that was found extracellularly (around 300 nmol/mg). This appears highly unlikely for glucose-depleted astrocytes, as they are unable to sustain their NADH levels under glucose-depletion [ 19 , 68 ].…”

Section: Discussionmentioning

confidence: 99%

“…The cellular NADH pool in cultured astrocytes accounts for only 0.70 ± 0.03 nmol/mg [ 68 ] and, even if the entire amount would be present as cytosolic NADH, it would have to be recycled more than 400 times by cytosolic processes to allow the formation of the pyruvate-derived lactate that was found extracellularly (around 300 nmol/mg). This appears highly unlikely for glucose-depleted astrocytes, as they are unable to sustain their NADH levels under glucose-depletion [ 19 , 68 ]. It appears more likely that the large amounts of NADH needed for cytosolic pyruvate reduction under the conditions studied are derived from mitochondria, where NADH is continuously produced by mitochondrial pyruvate oxidation via the PDH and the citric acid cycle dehydrogenases.…”

Section: Discussionmentioning

confidence: 99%

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Consumption and Metabolism of Extracellular Pyruvate by Cultured Rat Brain Astrocytes

et al. 2022

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Brain astrocytes are considered as glycolytic cell type, but these cells also produce ATP via mitochondrial oxidative phosphorylation. Exposure of cultured primary astrocytes in a glucose-free medium to extracellular substrates that are known to be metabolised by mitochondrial pathways, including pyruvate, lactate, beta-hydroxybutyrate, alanine and acetate, revealed that among the substrates investigated extracellular pyruvate was most efficiently consumed by astrocytes. Extracellular pyruvate was consumed by the cells almost proportional to time over hours in a concentration-dependent manner with apparent Michaelis–Menten kinetics [Km = 0.6 ± 0.1 mM, Vmax = 5.1 ± 0.8 nmol/(min × mg protein)]. The astrocytic consumption of pyruvate was strongly impaired in the presence of the monocarboxylate transporter 1 (MCT1) inhibitor AR-C155858 or by application of a 10-times excess of the MCT1 substrates lactate or beta-hydroxybutyrate. Pyruvate consumption by viable astrocytes was inhibited in the presence of UK5099, an inhibitor of the mitochondrial pyruvate carrier, or after application of the respiratory chain inhibitor antimycin A. In contrast, the mitochondrial uncoupler BAM15 strongly accelerated cellular pyruvate consumption. Lactate and alanine accounted after 3 h of incubation with pyruvate for around 60% and 10%, respectively, of the pyruvate consumed by the cells. These results demonstrate that consumption of extracellular pyruvate by astrocytes involves uptake via MCT1 and that the velocity of pyruvate consumption is strongly modified by substances that affect the entry of pyruvate into mitochondria or the activity of mitochondrial respiration.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Consumption and Metabolism of Extracellular Pyruvate by Cultured Rat Brain Astrocytes

et al. 2022

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“…For cell lysates of astrocytes dicoumarol has been shown to inhibit NQO1 activity with half-maximal inhibition in the nM range [39]. However, for intact astrocytes even at a concentration of 1 µM extracellular dicoumarol was unable to completely inhibit NQO1-dependent β-lap-mediated GSH oxidation, as evident by the small but significant increase in the GSSG to GSH ratio of astrocytes that had been exposed to β-lap plus dicoumarol for 5 min.…”

Section: Discussionmentioning

confidence: 99%

“…Unexpectedly, the extracellular lactate accumulation in the presence of β-lap and 30 µM dicoumarol was found elevated compared to the values obtained for control cells. Previous work from our group suggests that in cultured astrocytes NADPH may be the preferred electron donor to deliver electrons for NQO1-dependent reactions [ 39 ]. Assuming that the consumption of NADPH is lowered by dicoumarol-mediated inhibition of cytosolic NQO1, less glucose-6-phosphate would be needed as substrate for NADPH regeneration by the PPP [ 56 ] and more glucose-6-phosphate could be used for glycolytic lactate production.…”

Section: Discussionmentioning

confidence: 99%

“…Astrocytes have been reported to contain substantial activity of NQO1 [35,39] and should therefore encounter NQO1-mediated ROS production and oxidative stress after application of β-lap. Here we report that acute exposure of cultured astrocytes to low micromolar concentrations of β-lap caused rapid cellular ROS formation and GSH oxidation which is followed by GSSG export and a delayed impairment in cell metabolism and cell viability.…”

Section: Introductionmentioning

confidence: 99%

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β-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol

et al. 2020

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β-lapachone (β-lap) is reduced in tumor cells by the enzyme NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) to a labile hydroquinone which spontaneously reoxidises to β-lap, thereby generating reactive oxygen species (ROS) and oxidative stress. To test for the consequences of an acute exposure of brain cells to β-lap, cultured primary rat astrocytes were incubated with β-lap for up to 4 h. The presence of β-lap in concentrations of up to 10 µM had no detectable adverse consequences, while higher concentrations of β-lap compromised the cell viability and the metabolism of astrocytes in a concentration- and time-dependent manner with half-maximal effects observed for around 15 µM β-lap after a 4 h incubation. Exposure of astrocytes to β-lap caused already within 5 min a severe increase in the cellular production of ROS as well as a rapid oxidation of glutathione (GSH) to glutathione disulfide (GSSG). The transient cellular accumulation of GSSG was followed by GSSG export. The β-lap-induced ROS production and GSSG accumulation were completely prevented in the presence of the NQO1 inhibitor dicoumarol. In addition, application of dicoumarol to β-lap-exposed astrocytes caused rapid regeneration of the normal high cellular GSH to GSSG ratio. These results demonstrate that application of β-lap to cultured astrocytes causes acute oxidative stress that depends on the activity of NQO1. The sequential application of β-lap and dicoumarol to rapidly induce and terminate oxidative stress, respectively, is a suitable experimental paradigm to study consequences of a defined period of acute oxidative stress in NQO1-expressing cells.

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β-lapachone-mediated WST1 Reduction as Indicator for the Cytosolic Redox Metabolism of Cultured Primary Astrocytes

Watermann

Dringen

2023

Neurochem Res

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Electron cycler-mediated extracellular reduction of the water-soluble tetrazolium salt 1 (WST1) is frequently used as tool for the determination of cell viability. We have adapted this method to monitor by determining the extracellular WST1 formazan accumulation the cellular redox metabolism of cultured primary astrocytes via the NAD(P)H-dependent reduction of the electron cycler β-lapachone by cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1). Cultured astrocytes that had been exposed to β-lapachone in concentrations of up to 3 µM remained viable and showed an almost linear extracellular accumulation of WST1 formazan for the first 60 min, while higher concentrations of β-lapachone caused oxidative stress and impaired cell metabolism. β-lapachone-mediated WST1 reduction was inhibited by the NQO1 inhibitors ES936 and dicoumarol in a concentration-dependent manner, with half-maximal inhibition observed at inhibitor concentrations of about 0.3 µM. β-lapachone-mediated WST1 reduction depended strongly on glucose availability, while mitochondrial substrates such as lactate, pyruvate or ketone bodies allowed only residual β-lapachone-mediated WST1 reduction. Accordingly, the mitochondrial respiratory chain inhibitors antimycin A and rotenone hardly affected astrocytic WST1 reduction. Both NADH and NADPH are known to supply electrons for reactions catalysed by cytosolic NQO1. Around 60% of the glucose-dependent β-lapachone-mediated WST1 reduction was prevented by the presence of the glucose-6-phosphate dehydrogenase inhibitor G6PDi-1, while the glyceraldehyde-3-phosphate dehydrogenase inhibitor iodoacetate had only little inhibitory potential. These data suggest that pentose phosphate pathway-generated NADPH, and not glycolysis-derived NADH, is the preferred electron source for cytosolic NQO1-catalysed reductions in cultured astrocytes.

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The Menadione-Mediated WST1 Reduction by Cultured Astrocytes Depends on NQO1 Activity and Cytosolic Glucose Metabolism

Cited by 7 publications

References 62 publications

Consumption and Metabolism of Extracellular Pyruvate by Cultured Rat Brain Astrocytes

Consumption and Metabolism of Extracellular Pyruvate by Cultured Rat Brain Astrocytes

β-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol

β-lapachone-mediated WST1 Reduction as Indicator for the Cytosolic Redox Metabolism of Cultured Primary Astrocytes

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