Modulation of Cellular Levels of Adenosine Phosphates and Creatine Phosphate in Cultured Primary Astrocytes

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The glucose analogue 2-deoxyglucose (2DG) has frequently been used as a tool to study cellular glucose uptake and to inhibit glycolysis. Exposure of primary cultured astrocytes to 2DG caused a time- and concentration-dependent cellular accumulation of 2-deoxyglucose-6-phosphate (2DG6P) that was accompanied by a rapid initial decline in cellular ATP content. Inhibitors of mitochondrial respiration as well as inhibitors of mitochondrial uptake of pyruvate and activated fatty acids accelerated the ATP loss, demonstrating that mitochondrial ATP regeneration contributes to the partial maintenance of the ATP content in 2DG-treated astrocytes. After a 30 min exposure to 10 mM 2DG the specific content of cellular 2DG6P had accumulated to around 150 nmol/mg, while cellular ATP was lowered by 50% to around 16 nmol/mg. Following such a 2DG6P-loading of astrocytes, glycolytic lactate production from applied glucose was severely impaired during the initial 60 min of incubation, but was reestablished during longer incubation concomitant with a loss in cellular 2DG6P content. In contrast to glycolysis, the glucose-dependent NADPH regeneration via the pentose phosphate pathway (PPP) was only weakly affected in 2DG6P-loaded astrocytes and in cells that were coincubated with glucose in the presence of an excess of 2DG. Additionally, in the presence of 2DG PPP-dependent WST1 reduction was found to have doubled compared to hexose-free control incubations, indicating that cellular 2DG6P can serve as substrate for NADPH regeneration by the astrocytic PPP. The data presented provide new insights on the metabolic consequences of a 2DG exposure on the energy and glucose metabolism of astrocytes and demonstrate the reversibility of the inhibitory potential of a 2DG-treatment on the glucose metabolism of cultured astrocytes.

Section: Introductionmentioning

confidence: 83%

Section: Depletion Of Cellular Atp Contents In Cultured Astrocytes Af...mentioning

confidence: 99%

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

Harders,

Watermann,

Karger

et al. 2024

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“…The specific ATP contents that were maintained during a 24 h incubation with glucose (or other substrates) showed some variations between individual experiments and represented between 70 and 85% of the initial ATP contents. This lowered ATP contents compared to the initial values is most likely caused by the absence of serum during the 24 h incubation as serum has recently been reported to contain components which are required to maintain a maximal ATP content in cultured astrocytes [ 30 ].…”

Section: Discussionmentioning

confidence: 99%

“…The data presented here were obtained on cultures of an age between 14 and 28 days. In this range, the specific ATP content was not affected by the culture age [ 13 , 30 ]. Astrocyte-rich primary cultures contain mainly glial fibrillary acidic protein-positive astrocytes and only low numbers of contaminating other types of glial cells [ 29 , 31 , 32 ]…”

Section: Methodsmentioning

confidence: 99%

Exogenous Substrates Prevent the Decline in the Cellular ATP Content of Primary Rat Astrocytes During Glucose Deprivation

Harders,

Spellerberg,

Dringen

2024

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Brain astrocytes are well known for their broad metabolic potential. After glucose deprivation, cultured primary astrocytes maintain a high cellular ATP content for many hours by mobilizing endogenous substrates, but within 24 h the specific cellular ATP content was lowered to around 30% of the initial ATP content. This experimental setting was used to test for the potential of various exogenous substrates to prevent a loss in cellular ATP in glucose deprived astrocytes. The presence of various extracellular monocarboxylates, purine nucleosides or fatty acids prevented the loss of ATP from glucose-deprived astrocytes. Of the 20 proteinogenic amino acids, only alanine, aspartate, glutamate, glutamine, lysine or proline maintained high ATP levels in starved astrocytes. Among these amino acids, proline was found to be the most potent one to prevent the ATP loss. The astrocytic consumption of proline as well as the ability of proline to maintain a high cellular ATP content was prevented in a concentration-dependent manner by the proline dehydrogenase inhibitor tetrahydro-2-furoic acid. Analysis of the concentration-dependencies obtained by considering the different carbon content of the applied substrates revealed that fatty acids and proline are more potent than glucose and monocarboxylates as exogenous substrates to prevent ATP depletion in glucose-deprived astrocytes. These data demonstrate that cultured astrocytes can utilise a wide range of extracellular substrates as fuels to support mitochondrial ATP regeneration and identify proline as potent exogenous substrate for the energy metabolism of starved astrocytes.

“…Astrocytes play an important role in brain energy metabolism [ 1 – 4 ], but have also crucial functions in brain development [ 5 ], (ion) homeostasis [ 6 – 9 ], the regulation and modulation of neuronal signals [ 10 , 11 ], memory formation [ 12 ] and the protection against toxins and oxidative stress [ 13 – 15 ]. Although astrocytes are considered as a rather glycolytic cell type [ 16 ], also the oxidative metabolism plays an important role for astrocytic energy regeneration [ 17 – 19 ]. In this context, the α-ketoacid pyruvate is of high interest as it links cytosolic glycolysis and mitochondrial metabolism [ 17 , 20 , 21 ].…”

Section: Introductionmentioning

confidence: 99%

Modulation of Pyruvate Export and Extracellular Pyruvate Concentration in Primary Astrocyte Cultures

Denker,

Dringen

2024

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Astrocyte-derived pyruvate is considered to have neuroprotective functions. In order to investigate the processes that are involved in astrocytic pyruvate release, we used primary rat astrocyte cultures as model system. Depending on the incubation conditions and medium composition, astrocyte cultures established extracellular steady state pyruvate concentrations in the range between 150 µM and 300 µM. During incubations for up to 2 weeks in DMEM culture medium, the extracellular pyruvate concentration remained almost constant for days, while the extracellular lactate concentration increased continuously during the incubation into the millimolar concentration range as long as glucose was present. In an amino acid-free incubation buffer, glucose-fed astrocytes released pyruvate with an initial rate of around 60 nmol/(h × mg) and after around 5 h an almost constant extracellular pyruvate concentration was established that was maintained for several hours. Extracellular pyruvate accumulation was also observed, if glucose had been replaced by mannose, fructose, lactate or alanine. Glucose-fed astrocyte cultures established similar extracellular steady state concentrations of pyruvate by releasing pyruvate into pyruvate-free media or by consuming excess of extracellular pyruvate. Inhibition of the monocarboxylate transporter MCT1 by AR-C155858 lowered extracellular pyruvate accumulation, while inhibition of mitochondrial pyruvate uptake by UK5099 increased the extracellular pyruvate concentration. Finally, the presence of the uncoupler BAM15 or of the respiratory chain inhibitor antimycin A almost completely abolished extracellular pyruvate accumulation. The data presented demonstrate that cultured astrocytes establish a transient extracellular steady state concentration of pyruvate which is strongly affected by modulation of the mitochondrial pyruvate metabolism.