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

Abstract

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.

Application of the Hydrophilic Interaction Liquid Chromatography (HILIC-MS) Novel Protocol to Study the Metabolic Heterogeneity of Glioblastoma Cells

“…The amount of proline has almost not changed in the medium of T98G unlike in the culture medium from the other cell line. It was shown, that proline metabolism is associated with cancer metastasis and during glucose deprivation of astrocytes, it compensates as a substrate for ATP production, which refers to an important anaplerotic role [ 48 , 49 ]. In contrast, 5-MTA was released into culture medium of the U118 cell line but not into the culture medium of the T98G cell line and the intracellular amount was also higher in U118.…”

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

“…However, cellular ATP contents in 2DG-treated cells were even after 24 h of incubation above the critical threshold level of ATP (around 25% of the normal ATP content) that is needed to prevent toxicity of cultured astrocytes [18,43]. Despite the initial consumption of ATP for 2DG phosphorylation, astrocytes will continuously consume ATP for various additional cellular processes [18,20,31,44]. Therefore, efficient and continuous ATP regeneration is required in 2DG-treated astrocytes.…”

“…Glycolysis and mitochondrial respiration contribute to the ATP regeneration in cultured astrocytes [18,20,31]. To investigate the consequences of an application of the glycolysis inhibitor 2DG and of inhibitors of mitochondrial oxidative phosphorylation on the cellular ATP content, astrocyte cultures were incubated for up to 60 min in glucose-free IB without (0 mM) or with 5 mM 2DG in the absence or the presence of mitochondrial inhibitors.…”