Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, gamma-aminobutyrate (GABA), glutamine, and aspartate. D-[1-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for 13C-NMR analysis and amino acid analysis. The absolute amount of 13C present within each carbonatom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance 13C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates.(ABSTRACT TRUNCATED AT 250 WORDS)
Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM-1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10(-7)-10(-5) M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.
Leucine and alpha-ketoisocaproate (alpha-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects of elevated leucine or alpha-KIC on the oxidation of L-[U-14C]glutamate and L-[U-14C]glutamine in the brain were determined in the non-anesthetized rat. 14CO2 generated by the metabolic oxidation of [14C]glutamate and [14C]glutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and alpha-KIC exhibited differential effects on 14CO2 generation from radioactive glutamate on glutamine. Infusion of 0.5 mM alpha-KIC increased [14C]glutamate oxidation approximately 2-fold; higher concentrations of alpha-KIC did not further stimulate [14C]glutamate oxidation. The enhanced oxidation of [14C]glutamate may be attributed to the function of alpha-KIC as a nitrogen acceptor from [14C]glutamate yielding [14C]alpha-ketoglutarate, an intermediate of the tricarboxylic acid cycle. [14C]glutamine oxidation was not stimulated as much as [14C]glutamate oxidation and only increased at 10 mM alpha-KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either [14C]glutamate or [14C]glutamine. In maple syrup urine disease elevated alpha-KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.
The activity of glutaminase per milligram of protein increased threefold after cultured human diploid fibroblasts were subcultured in fresh medium. The maximum activity was reached after 2 days of growth and decreased once the cells reached confluency. The increase of glutaminase activity was independent of the glutamine concentration between 0.2 and 2.0 mmol/1. In contrast, the specific activity of glutamate dehydrogenase was independent both of the glutamine concentration and the growth phase of the cultured cells. These results indicate that glutaminase, the first enzyme involved in the utilization of glutamine as an energy source, is elevated in rapidly dividing human diploid fibroblasts.
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