Consumption and Metabolism of Extracellular Pyruvate by Cultured Rat Brain Astrocytes

Abstract

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.

Endogenous Energy Stores Maintain a High ATP Concentration for Hours in Glucose-Depleted Cultured Primary Rat Astrocytes

“…Perchlorate lysates of cultured astrocytes were used to quantify cellular ATP contents as recently described [ 21 ] using a luciferine-luciferase-based [ 28 ] luminometric assay. Briefly, the cultures were washed twice with 1 mL ice-cold (4 °C) PBS and lysed in 200 µL of ice-cold 0.5 M HClO 4 on ice for 1 min.…”

“…However, astrocytes also possess an extensive mitochondrial metabolism [ 5 ], which is linked to efficient mitochondrial ATP regeneration. This is demonstrated by the rapid depletion of cellular ATP levels after application of inhibitors of mitochondrial oxidative phosphorylation to astrocytes that were co-exposed to a glycolysis inhibitor or that had been deprived of glucose [ 18 – 21 ]. Thus, despite the presence of many ATP consuming enzymes and transporters in astrocytes, a high cellular ATP level appears to be efficiently maintained by ATP regeneration via both cytosolic glycolysis and mitochondrial oxidative phosphorylation.…”

“…Glucose is considered as the main physiological substrate of astrocytes [ 8 ]. However, at least in culture these cells are able to also utilize a substantial number of other substrates that can be oxidized to provide energy via mitochondrial processes, including pyruvate, lactate, acetate, ketone bodies as well as amino acids and fatty acids [ 1 , 5 , 21 – 25 ].…”

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

“…Among the other substrates applied to test as potential glucose substitutes to supply electrons for NQO1-dependent WST1 reduction, only mannose but not the other sugars were found to be efficiently metabolised to provide electrons for WST1 reduction, consistent with literature data on the ability of cultured astrocytes to metabolise such extracellular substrates [ 6 , 36 , 39 , 44 – 46 ]. Also astrocytic substrates that are mainly metabolized in mitochondria such as lactate, pyruvate, ketone bodies and amino acids [ 47 , 48 ] were at best to a low extent able to provide electrons for NQO1-dependent WST1 reduction. Among those mitochondrial substrates, lactate appeared to be the best, enabling around 25% of the glucose-dependent WST1 reduction.…”

“…However, such side effect would not explain the absence of an inhibitory potential of these inhibitors on astrocytic WST1 reduction. Both inhibitors have been found to impair in the concentrations applied the mitochondrial membrane potential of astrocytes [ 48 ] (data not shown) and stimulate glycolytic lactate production in astrocytes [ 46 , 53 ], demonstrating their potential to impair mitochondrial respiration for the conditions used. Thus, the inability of rotenone and antimycin A to severely lower astrocytic WST1 reduction demonstrates that mitochondrial processes do not provide substantial amounts of electrons for astrocytic NQO1.…”