Evaluation of the importance of transamination versus deamination in astrocytic metabolism of [U‐ 13C] glutamate

Abstract

Glutamate metabolism was studied in primary cultures of cerebral cortical astrocytes to determine the significance of transamination for the oxidative metabolism of glutamate. Cultures were incubated with [U-13C]glutamate (0.5 mM) in the presence and absence of the transaminase inhibitor aminooxyacetic acid (AOAA) and in some cases with methionine sulfoximine, an inhibitor of glutamine synthetase. Perchloric acid extracts of the cells as well as redissolved lyophilized incubation media were subjected to nuclear magnetic resonance spectroscopy to identify 13C-labeled metabolites. Additionally, biochemical analyses were performed to quantify amino acids, lactate, citrate, and ammonia. Glutamine released into the medium and intracellular glutamate were labeled uniformly to a large extent, but the C-3 position showed not only the expected apparent triplet but also a doublet due to 12C incorporation into the C-4 and C-5 positions. Incorporation of 12C into the C-4 and C-5 positions of glutamate and glutamine as well as labeling of lactate, citrate, malate, and aspartate could only arise via metabolism of [U-13C]glutamate through the tricarboxylic acid (TCA) cycle. Entry of the carbon skeleton of glutamate into the TCA cycle must proceed via 2-oxoglutarate. This conversion can occur as a transamination or an oxidative deamination. After blocking transamination with AOAA, metabolism of glutamate through the TCA cycle was still taking place since lactate labeling was only slightly reduced. Glutamate and glutamine synthesis from 2-oxoglutarate could, however, not be detected under this condition. It therefore appears that while glutamate dehydrogenase is important for glutamate degradation, glutamate biosynthesis occurs mainly as a transamination.

Astrocytic Control of Biosynthesis and Turnover of the Neurotransmitters Glutamate and GABA

“…According to this model glutamate formed by the action of PAG located in the mitochondrial membrane (17) gets access to the mitochondrial matrix where it is transaminated by AAT to form α-ketoglutarate which is translocated to the cytoplasm where it undergoes a second transamination via AAT to produce glutamate. That this mechanism is indeed working is supported by the demonstration that while oxidation of the carbon skeleton of glutamate in the TCA cycle is dependent on activity of GDH (18), the formation of glutamate from α-ketoglutarate is catalyzed by an aminotransferase which is most likely mitochondrial AAT (19). This glutamate is subsequently used for vesicular release (16).…”

The Glutamate–Glutamine (GABA) Cycle: Importance of Late Postnatal Development and Potential Reciprocal Interactions between Biosynthesis and Degradation

“…In cultured cerebellar astrocytes conversion of glutamate to α-ketoglutarate at least mainly occurs via a transamination (Westergaard et al, 1996). This is consistent with a recent in vivo study by Pardo et al (2011), which established that the contents of glutamate and glutamine in cultured astrocytes increase by ∼50% in the presence of aspartate at a concentration of ≥100 μM, but not in the presence of alanine or leucine.…”

Glutamate Pays Its Own Way in Astrocytes

“…Transamination occurs primarily by aspartate aminotransferase (AAT), but also readily takes place via either branched-chain amino acid aminotransferase (BCAT) or alanine aminotransferase (ALAT) (3, 38–40). Studies from our group and others demonstrate that the oxidative metabolism of exogenous glutamate taken up from the extracellular milieu proceeds primarily via GDH in astrocytes from rat brain [since it is relatively unaffected by the transaminase inhibitor aminooxyacetic acid, AOAA] (30, 41). The α-ketoglutarate formed from glutamate is metabolized for energy in the sequential reactions of the TCA cycle to the four carbon compound oxaloacetate (Figures 1A,B) and yielding the equivalent of nine ATP molecules in this process.…”