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

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.

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

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.

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

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, ß-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.

Dissociation of ß2m from MHC class I triggers formation of noncovalent transient heavy chain dimers.

At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin (ß2m, encoded by B2M), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single-molecule co-tracking. We identify non-covalent MHC-I FHC dimers, with dimerization mediated by the α3 domain, as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single-molecule colocalization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to that seen for ß2m.