G6PDi-1 is a Potent Inhibitor of G6PDH and of Pentose Phosphate pathway-dependent Metabolic Processes in Cultured Primary Astrocytes

Abstract

Glucose-6-phosphate dehydrogenase (G6PDH) catalyses the rate limiting first step of the oxidative part of the pentose phosphate pathway (PPP), which has a crucial function in providing NADPH for antioxidative defence and reductive biosyntheses. To explore the potential of the new G6PDH inhibitor G6PDi-1 to affect astrocytic metabolism, we investigated the consequences of an application of G6PDi-1 to cultured primary rat astrocytes. G6PDi-1 efficiently inhibited G6PDH activity in lysates of astrocyte cultures. Half-maximal inhibition was observed for 100 nM G6PDi-1, while presence of almost 10 µM of the frequently used G6PDH inhibitor dehydroepiandrosterone was needed to inhibit G6PDH in cell lysates by 50%. Application of G6PDi-1 in concentrations of up to 100 µM to astrocytes in culture for up to 6 h did not affect cell viability nor cellular glucose consumption, lactate production, basal glutathione (GSH) export or the high basal cellular ratio of GSH to glutathione disulfide (GSSG). In contrast, G6PDi-1 drastically affected astrocytic pathways that depend on the PPP-mediated supply of NADPH, such as the NAD(P)H quinone oxidoreductase (NQO1)-mediated WST1 reduction and the glutathione reductase-mediated regeneration of GSH from GSSG. These metabolic pathways were lowered by G6PDi-1 in a concentration-dependent manner in viable astrocytes with half-maximal effects observed for concentrations between 3 and 6 µM. The data presented demonstrate that G6PDi-1 efficiently inhibits the activity of astrocytic G6PDH and impairs specifically those metabolic processes that depend on the PPP-mediated regeneration of NADPH in cultured astrocytes.

Consequences of a 2-Deoxyglucose Exposure on the ATP Content and the Cytosolic Glucose Metabolism of Cultured Primary Rat Astrocytes

“…An additional pathway that may contribute to the disappearance of 2DG6P from cultured astrocytes is the oxidation In panel (c), the significance of differences (t-test) between the data obtained for incubations in the absence and the presence of 2DG is indicated by # p < 0.05 and ## p < 0.01 of 2DG6P by G6PDH, as 6-phospho-2-deoxygluconate has been found in the brain of 2DG-exposed rats [4]. We have previously shown that cellular NADPH regeneration via the PPP can be studied by monitoring the NQO1-dependent reduction of extracellular WST1 to its formazan in the presence of the redox cycler β-lapachone [26,42]. In the presence of 2DG the glucose-independent WST1 reduction was almost doubled in a concentration-dependent manner by 2DG, suggesting that 2DG6P acts as substrate of G6PDH in living astrocytes, thereby providing electrons via NADPH for the WST1 reduction.…”

“…The WST1 reduction method that we have used to investigate the potential of accumulated 2DG6P to serve as substrate for the PPP or to inhibit the use of G6P by G6PDH is based on the NADPH-dependent reduction by NQO1 of the electron cycler β-lapachone that subsequently permeates in reduced form the cell membrane and reduces extracellular WST1 [26,42,48]. This indirect method allows conclusions on the electron flow through the PPP but gives no information on the cellular level of NADPH.…”

“…To test for the consequences of a high cellular concentration of 2DG6P on the NADPH regeneration by the PPP of astrocytes, the PPP-and NADPH-dependent WST1 reduction was investigated in the absence and the presence of glucose in the experimental paradigm previously described In panel (c), the significance of differences (t-test) between data from incubations in the absence or the presence of 2DG for the given incubation periods (black vs. green and red vs. yellow) is indicated by # p < 0.05, ## p < 0.01 and ### p < 0.001. Significant differences between data for the two different incubations periods of each condition (with or without 2DG preincubation, black vs. red and green vs. yellow) are indicated by + p < 0.05 and ++ p < 0.01 [26,42]. After the 30 min preincubation of astrocytes in the presence of 2DG, the cells contained a specific 2DG6P content of 130.6 ± 9.1 nmol/mg.…”

Analysis of the Effects of Pentose Phosphate Pathway Inhibition on the Generation of Reactive Oxygen Species and Epileptiform Activity in Hippocampal Slices

“…Results from a study on cultured astrocytes demonstrated that G6PDi−1 (10 µM) inhibited 60% of the supply of electrons for a NQO1-catalysed β-lapachone-mediated reduction in the water-soluble tetrazolium salt 1, suggesting that NADPH, derived from PPP, is the main source of electrons for cytosolic NAD(P)H quinone oxidoreductase 1 (NQO1) [43]. Importantly, another study from this group provided compelling evidence that G6PDi−1 efficiently and specifically inhibits astrocytic G6PDH without affecting other oxidoreductases, indicating that G6PDi−1 is a suitable and specific tool for inhibiting G6PDH and PPP-dependent processes [44].…”

“…Previous research on cultured astrocytes has shown that NADPH produced in the PPP is involved in several NADPH-dependent intracellular reactions, such as the function of cytosolic NAD(P)H-quinone oxidoreductase 1 (NQO1) [44].…”