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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Adenosine triphosphate (ATP) is the central energy currency of all cells. Cultured primary rat astrocytes contain a specific cellular ATP content of 27.9 ± 4.7 nmol/mg. During incubation in a glucose- and amino acid-free incubation buffer, this high cellular ATP content was maintained for at least 6 h, while within 24 h the levels of ATP declined to around 30% of the initial value without compromising cell viability. In contrast, cells exposed to 1 mM and 5 mM glucose maintained the initial high cellular ATP content for 24 and 72 h, respectively. The loss in cellular ATP content observed during a 24 h glucose-deprivation was fully prevented by the presence of glucose, fructose or mannose as well as by the mitochondrial substrates lactate, pyruvate, β-hydroxybutyrate or acetate. The high initial specific ATP content in glucose-starved astrocytes, was almost completely abolished within 30 min after application of the respiratory chain inhibitor antimycin A or the mitochondrial uncoupler BAM-15, while these inhibitors lowered in glucose-fed cells the ATP content only to 60% (BAM-15) and 40% (antimycin A) within 5 h. Inhibition of the mitochondrial pyruvate carrier by UK5099 alone or of mitochondrial fatty acid uptake by etomoxir alone hardly affected the high ATP content of glucose-deprived astrocytes during an incubation for 8 h, while the co-application of both inhibitors depleted cellular ATP levels almost completely within 5 h. These data underline the importance of mitochondrial metabolism for the ATP regeneration of astrocytes and demonstrate that the mitochondrial oxidation of pyruvate and fatty acids strongly contributes to the maintenance of a high ATP concentration in glucose-deprived astrocytes.

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The pyruvate analogue 3-bromopyruvate (3-BP) is an electrophilic alkylator that is considered a promising anticancer drug because it has been shown to kill cancer cells efficiently while having little toxic effect on nontumor cells. To test for potential adverse effects of 3-BP on brain cells, we exposed cultured primary rat astrocytes to 3-BP and investigated the effects of this compound on cell viability, glucose metabolism, and glutathione (GSH) content. The presence of 3-BP severely compromised cell viability and slowed cellular glucose consumption and lactate production in a time-and concentrationdependent manner, with half-maximal effects observed at about 100 mM 3-BP after 4 hr of incubation. The cellular hexokinase activity was not affected in 3-BP-treated astrocytes, whereas within 30 min after application of 3-BP the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inhibited, and cellular GSH content was depleted in a concentration-dependent manner, with half-maximal effects observed at about 30 mM 3-BP. The depletion of cellular GSH after exposure to 100 mM 3-BP was not prevented by the presence of 10 mM of the monocarboxylates lactate or pyruvate, suggesting that 3-BP is not taken up into astrocytes predominantly by monocarboxylate transporters. The data suggest that inhibition of glycolysis by inactivation of GAPDH and GSH depletion contributes to the toxicity that was observed for 3-BP-treated cultured astrocytes. V C 2014Wiley Periodicals, Inc.

Antiretroviral protease inhibitors are a class of important drugs that are used for the treatment of human immunodeficiency virus infections. Among those compounds, ritonavir is applied frequently in combination with other antiretroviral protease inhibitors, as it has been reported to boost their therapeutic efficiency. To test whether ritonavir affects the viability and the glutathione (GSH) metabolism of brain cells, we have exposed primary astrocyte cultures to this protease inhibitor. Application of ritonavir in low micromolar concentrations did not compromise cell viability, but caused a time- and concentration-dependent loss of GSH from the cells which was accompanied by a matching increase in the extracellular GSH content. Half-maximal effects were observed for ritonavir in a concentration of 3 μM. The ritonavir-induced stimulated GSH export from astrocytes was completely prevented by MK571, an inhibitor of the multidrug resistance protein 1. In addition, continuous presence of ritonavir was essential to maintain the stimulated GSH export, since removal of ritonavir terminated the stimulated GSH export. Ritonavir was more potent to stimulate GSH export from astrocytes than the antiretroviral protease inhibitors indinavir and nelfinavir, but combinations of ritonavir with indinavir or nelfinavir did not further stimulate astrocytic GSH export compared to a treatment with ritonavir alone. The strong effects of ritonavir and other antiretroviral protease inhibitors on the GSH metabolism of astrocytes suggest that a chronic treatment of patients with such compounds may affect their brain GSH metabolism.

Efavirenz is a widely prescribed non-nucleoside reverse transcriptase inhibitor for the treatment of HIV infections. To test for potential long-term consequences of efavirenz on brain cells, cultured primary astrocytes were incubated with this substance or with its primary metabolite 8-hydroxy efavirenz for up to 7 days. Both, efavirenz and 8-hydroxy efavirenz caused time- and concentration-dependent cell toxicity and stimulated in subtoxic concentrations the glycolytic flux (glucose consumption and lactate release) in astrocytes. As 8-hydroxy efavirenz was less toxic than efavirenz and stimulated glycolysis in lower concentrations we tested for a potential hydroxylation of efavirenz to 8-hydroxy efavirenz in astrocytes. Analysis of media and cell lysates by HPLC-UV and mass spectrometry revealed that after 3 days of incubation viable astrocytes had accumulated about 17 and 7 % of the applied efavirenz and 8-hydroxy efavirenz, respectively. However, in cultures treated with efavirenz neither 8-hydroxy efavirenz nor any other known metabolite of efavirenz was detectable. These data demonstrate that cultured rat astrocytes efficiently accumulate, but not metabolize, efavirenz and 8-hydroxy efavirenz and that the observed chronic stimulation of glycolysis is mediated by both efavirenz and 8-hydroxy efavirenz.

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.