Brain slices from glutaminase-deficient mice metabolize less glutamine: a cellular metabolomic study with carbon 13 NMR

Abstract

In the brain, glutaminase is considered to have a key role in the provision of glutamate, a major excitatory neurotransmitter. Brain slices obtained from wild-type (control) and glutaminase-deficient (GLS1+/-) mice were incubated without glucose and with 5 or 1 mmol/L [3-(13)C]glutamine as substrate. At the end of the incubation, substrate removal and product formation were measured by both enzymatic and carbon 13 nuclear magnetic resonance ((13)C-NMR) techniques. Slices from GLS1+/- mice consumed less [3-(13)C]glutamine and accumulated less [3-(13)C]glutamate. They also produced less (13)CO(2) but accumulated amounts of (13)C-aspartate and (13)C-gamma-aminobutyric acid (GABA) that were similar to those found with brain slices from control mice. The newly formed glutamine observed in slices from control mice remained unchanged in slices from GLS1+/- mice. As expected, flux through glutaminase in slices from GLS1+/- mice was found diminished. Fluxes through all enzymes of the tricarboxylic acid cycle were also reduced in brain slices from GLS1+/- mice except through malate dehydrogenase with 5 mmol/L [3-(13)C]glutamine. The latter diminutions are consistent with the decreases in the production of (13)CO(2) also observed in the slices from these mice. It is concluded that the genetic approach used in this study confirms the key role of glutaminase for the provision of glutamate.

Figure 1

¹³C-NMR spectra of neutralized HClO₄ extracts obtained from rat brain slices incubated for 1 hour with 5 mmol/L [3-¹³C]glutamine from a control (A) and a glutaminase-deficient mice (B). For each peak, the corresponding ¹³C metabolite is indicated. The chemical shifts, in parts per million (p.p.m.), are referred to tetramethylsilane. The peaks of C3 glutamate and of C3 glutamine divided by 18 are also shown. Glu, glutamate; Asp, aspartate; Gln, glutamine; Gly, glycine; GABA, gamma-aminobutyric acid; Suc, succinate.

Figure 2

Scheme of glutamine metabolism in the brain. PAG, phosphate-activated glutaminase; GS, glutamine synthetase; GLDH, glutamate dehydrogenase; AlaAT, alanine aminotransferase; AspAT, aspartate aminotransferase; GAD, glutamic acid decarboxylase; GABA-T, gamma aminobutyric acid aminotransferase; αKGDH, α-ketoglutarate dehydrogenase; SucDH, succinate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; PDH, pyruvate dehydrogenase; PC, pyruvate carboxylase; CS, citrate synthase; ICDH, isocitrate dehydrogenase; TCA cycle, tricarboxylic acid cycle; Gln, glutamine; Glu, glutamate; αKG, α-ketoglutarate; Suc, succinate; Mal, malate; OAA, oxaloacetate; Cit, citrate; Asp, aspartate; Pyr, pyruvate; Ala, alanine; Ac-CoA, acetyl coenzyme A; GABA, gamma-aminobutyric acid.