In vivo ¹³ C NMR measurements of cerebral glutamine synthesis as evidence for glutamate–glutamine cycling

Abstract

The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measured in rats in vivo under normal physiological and hyperammonemic conditions using 13 C NMR spectroscopy. In the hyperammonemic animals, blood ammonia levels were raised from control values of Ϸ0.05 mM to Ϸ0.35 mM by an intravenous ammonium acetate infusion. Once a steady-state of cerebral metabolites was established, a [1-13 C]glucose infusion was initiated, and 13 C NMR spectra acquired continuously on a 7-tesla spectrometer to monitor 13 C labeling of cerebral metabolites. The time courses of glutamate and glutamine C-4 labeling were fitted to a mathematical model to yield TCA cycle rate (V TCA ) and the f lux from glutamate to glutamine through the glutamine synthetase pathway (V gln ). Under hyperammonemia the value of V TCA was 0.57 ؎ 0.16 mol͞min per g (mean ؎ SD, n ‫؍‬ 6) and was not significantly different (unpaired t test; P > 0.10) from that measured in the control animals (0.46 ؎ 0.12 mol͞min per g, n ‫؍‬ 5). Therefore, the TCA cycle rate was not significantly altered by hyperammonemia. The measured rate of glutamine synthesis under hyperammonemia was 0.43 ؎ 0.14 mol͞min per g (mean ؎ SD, n ‫؍‬ 6), which was significantly higher (unpaired t test; P < 0.01) than that measured in the control group (0.21 ؎ 0.04 mol͞ min per g, n ‫؍‬ 5). We propose that the majority of the glutamine synthetase f lux under normal physiological conditions results from neurotransmitter substrate cycling between neurons and glia. Under hyperammonemia the observed increase in glutamine synthesis is comparable to the expected increase in ammonia transport into the brain and reported measurements of glutamine eff lux under such conditions. Thus, under conditions of elevated plasma ammonia an increase in the rate of glutamine synthesis occurs as a means of ammonia detoxification, and this is superimposed on the constant rate of neurotransmitter cycling through glutamine synthetase.Glutamine synthetase in the brain is predominantly an astrocytic enzyme that catalyzes the formation of glutamine from glutamate and ammonia. It has been suggested that this pathway forms part of a neurotransmitter cycle between neurons and glia (1). In this cycle, astrocytes take up neurotransmitter glutamate from the synaptic cleft, convert it to glutamine via glutamine synthetase, and release this glutamine into the extracellular space for uptake by neurons, where it is converted back to glutamate primarily by glutaminase. ␥-Aminobutyric acid (GABA) released from inhibitory neurons also may be taken up by astrocytes, converted to glutamate, and recycled to neruons in the same way. The function of such a cycle would be to remove the synaptically active metabolite from the synaptic cleft and to recycle the carbon skeletons back to the neuronal pool by a synaptically inactive form.While ammonia is a normal constituent of tissue, elevated concentrations of blood and brain ammonia have been found to interfere with cerebral energy metabolism and may redu...

Energetics of Inhibition: Insights with a Computational Model of the Human GABAergic Neuron–Astrocyte Cellular Complex

“…The high oxidative activity seems to support this interpretation, but its further investigation requires a kinetic model. Whereas extensive studies of the glutamate-glutamine cycle (Shen et al, 1999) and measurements of glutamine flux from astrocytes to neurons in glutamatergic transmission (Sibson et al, 1997;Su et al, 1997) have been reported, in GABAergic neurotransmission, the difficulty in separating relative contributions of exogenous glutamate and glutamine to GABA synthesis has resulted in large differences in the quantification of the GABA-glutamine flux: a very small glutamine flux from astrocytes to GABAergic neurons, recently reported by Yang et al (2007), emphasized a lower contribution of glutamine to GABA synthesis than previously hypothesized, which is qualitatively in line with our findings. The present model comprises the astrocyte and GABAergic neuron only, and the omission of the glutamatergic neuron compartment is being taken into account only by reducing the availability of substrates.…”

“…Exogeneous glutamine, formed in astrocytes through the astrocyte-specific enzyme glutamine synthetase, is the most prominent candidate for GABA precursor. In the context of energetics of excitation, glutamine is generally accepted as a metabolic precursor for glutamate synthesis in glutamatergic neurons in which its influx from astrocytes has been experimentally identified and quantified (Sibson et al, 1997;Shen et al, 1999). In GABAergic neurons, in which the machinery for synthesis of GABA from glutamine is more complex and may vary across the different populations of GABAergic neurons (Waagepetersen et al, 1998), the role of glutamine is not yet fully understood.…”

Brain lactate by magnetic resonance spectroscopy during fulminant hepatic failure in the dog

“…42 Of note, in vivo 13 C MRS studies suggest that the rate of the tricarboxylic acid cycle is not significantly altered during ammonia infusion. 43 The anesthetized model of FHF, first described by Sielaff et al, 14 was used in this study. This dog model provided a large brain for intraventricular catheter placement and MRS data acquisition.…”

Studies of metabolic compartmentation and glucose transport using in vivo MRS

“…Using mass balance analysis Sibson and coworkers 29,30 showed that the only net flux from astrocytic pyruvate dehydrogenase into glutamine is via anaplerosis, which is also localized to the glia. A schematic of the glutamate/glutamine cycle (see text for description).…”

“…18,27 However the rate of the glutamate/glutamine cycle was not uniquely determined from these first experiments due to the inability to distinguish the glutamate/glutamine cycle from other sources of glutamine labeling in the glia. The studies in rats by Sibson and coworkers 29,30 allowed a constraint to be placed on the maximum flow of label from glial pyruvate dehydrogenase into glutamine as the rate of anaplerosis. Using this constraint in the isotopic analysis, Shen and colleagues 33 were able to assess whether a high level of glutamatergic activity is present Figure 2.…”