Extensive astrocyte metabolism of γ‐aminobutyric acid (GABA) sustains glutamine synthesis in the mammalian cerebral cortex

Abstract

Synaptic transmission is closely linked to brain energy and neurotransmitter metabolism. However, the extent of brain metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the relative metabolic contributions of neurons and astrocytes, are yet unknown. The present study was designed to investigate the functional significance of brain GABA metabolism using isolated mouse cerebral cortical slices and slices of neurosurgically resected neocortical human tissue of the temporal lobe. By using dynamic isotope labeling, with [ 15 N]GABA and [U-13 C]GABA as metabolic substrates, we show that both mouse and human brain slices exhibit a large capacity for GABA metabolism. Both the nitrogen and the carbon backbone of GABA strongly support glutamine synthesis, particularly in the human cerebral cortex, indicative of active astrocytic GABA metabolism. This was further substantiated by pharmacological inhibition of the primary astrocytic GABA transporter subtype 3 (GAT3), by (S)-SNAP-5114 or 1-benzyl-5-chloro-2,3-dihydro-1H-indole-2,3-dione (compound 34), leading to significant reductions in oxidative GABA carbon metabolism. Interestingly, this was not the case when tiagabine was used to specifically inhibit GAT1, which is predominantly found on neurons. Finally, we show that acute GABA exposure does not directly stimulate glycolytic activity nor oxidative metabolism in cultured astrocytes, but can be used as an additional substrate to enhance uncoupled respiration. These results clearly show that GABA is actively metabolized in astrocytes, particularly for the synthesis of glutamine, and challenge the current view that synaptic GABA homeostasis is maintained primarily by presynaptic recycling.

Astrocyte metabolism of the medium-chain fatty acids octanoic acid and decanoic acid promotes GABA synthesis in neurons via elevated glutamine supply

“…Medium was exchanged twice a week and serum concentration reduced to 15% in the second week and 10% in the third week and 0.25 mM dibutyryl-cAMP (final concentration) was added to the medium for the last week of culture. The oxygen consumption rate (OCR) of the cultured astrocytes were determined using a Seahorse XFe96 analyzer as previously described [ 37 ]. Briefly, the astrocytes were washed twice and the media changed to Seahorse assay medium (DMEM with 2.5 mM glucose and 3 mg/L phenol red, pH 7.4) and kept at 37 °C (non-CO 2 incubator) for 1 h. When indicated the assay medium was further supplemented with C8, C10 or the combination (all 200 µM).…”

Functional Metabolic Mapping Reveals Highly Active Branched-Chain Amino Acid Metabolism in Human Astrocytes, Which Is Impaired in iPSC-Derived Astrocytes in Alzheimer's Disease

“…Human neocortical tissue was obtained from six patients (four females, two males) aged 25-52 years. Additional information on patient cohort characteristics can be found in Andersen et al (2020). The use of human neocortical tissue was approved by the local Ethics Committee in Copenhagen (H-2-2011-104) with written informed consent from all the patients prior to surgery.…”

“…Histopathological examination of the neocortical tissue revealed no abnormal pathological features for any of the patients. The metabolic integrity of resected cerebral cortical human tissue has been established previously (Andersen et al, 2020).…”

“…Incubation of acutely isolated cerebral cortical brain slices of mice and humans was performed as described previously (Andersen et al, 2019(Andersen et al, , 2020. For mouse brain slices, the experiments were performed one mouse at a time.…”

Pharmacological inhibition of mitochondrial soluble adenylyl cyclase in astrocytes causes activation of AMP‐activated protein kinase and induces breakdown of glycogen

“…Oxygen consumption rate (OCR) of cultured astrocytes was monitored using a Seahorse XFe96 instrument (Agilent, Santa Clara, USA) as previously described (Andersen et al, 2020). Briefly, the cells were pre‐incubated in assay medium (DMEM with 3 mg/L phenol red, 2.5 mM glucose, pH adjusted to 7.4) for 1 h at 37°C before the experiment.…”