Extracellular Concentrations Of Glutamine Research Articles

Cellular and computational models reveal environmental and metabolic interactions in MMUT-type methylmalonic aciduria.

Methylmalonyl-coenzyme A (CoA) mutase (MMUT)-type methylmalonic aciduria is a rare inherited metabolic disease caused by the loss of function of the MMUT enzyme. Patients develop symptoms resembling those of primary mitochondrial disorders, but the underlying causes of mitochondrial dysfunction remain unclear. Here, we examined environmental and genetic interactions in MMUT deficiency using a combination of computational modeling and cellular models to decipher pathways interacting with MMUT. Immortalized fibroblast (hTERT BJ5ta) MMUT-KO (MUTKO) clones displayed a mild mitochondrial impairment in standard glucose-based medium, but they did not to show increased reliance on respiratory metabolism nor reduced growth or viability. Consistently, our modeling predicted MUTKO specific growth phenotypes only for lower extracellular glutamine concentrations. Indeed, two of three MMUT-deficient BJ5ta cell lines showed a reduced viability in glutamine-free medium. Further, growth on 183 different carbon and nitrogen substrates identified increased NADH (nicotinamide adenine dinucleotide) metabolism of BJ5ta and HEK293 MUTKO cells compared with controls on purine- and glutamine-based substrates. With this knowledge, our modeling predicted 13 reactions interacting with MMUT that potentiate an effect on growth, primarily those of secondary oxidation of propionyl-CoA, oxidative phosphorylation and oxygen diffusion. Of these, we validated 3-hydroxyisobutytyl-CoA hydrolase (HIBCH) in the secondary propionyl-CoA oxidation pathway. Altogether, these results suggest compensation for the loss of MMUT function by increasing anaplerosis through glutamine or by diverting flux away from MMUT through the secondary propionyl-CoA oxidation pathway, which may have therapeutic relevance.

BI-10 * pH-WEIGHTED MRI IN HUMAN GLIOMAS

Regions of proliferating tumor are characterized by extracellular acidosis and hypoxia. Additionally, tumor cells utilize glutamine and other amino acids for fuel, resulting in elevated extracellular concentrations of glutamine in areas of metabolically active tumor. In the current study we explore the use of chemical exchange saturation transfer (CEST) MRI targeting exchangeable protons on the amine functional group in free amino acids as a pH-weighted imaging biomarker for non-invasively identifying regions of low pH, suggestive of regions with active tumor. The dependence of CEST contrast on pH was measured for three amino acids, and the dependence of CEST contrast on metabolite concentration was measured for glutamine. CEST data was collected in a cohort of twelve GBM patients undergoing radiochemotherapy. CEST data and corresponding histology were collected in a patient with suspected low grade glioma and a GBM patient at tumor recurrence. Results show a sigmoidal relationship between CEST contrast and pH within a range of 5.0-8.0. CEST contrast increased with both decreasing pH and increasing amino acid concentration, and the three amino acids produced similar CEST signatures within a physiologically relevant range of pH (6.0-7.0). Clinically, some patients who showed elevated CEST contrast in the tumor region appeared to show an increase in tumor volume at six months post-treatment, while a patient who did not show elevated CEST contrast in the tumor region appeared to show a slight decrease in tumor volume at six months post-treatment. In biopsy patients, regions of CEST contrast suggestive of low pH and active tumor correctly identified proliferative tumor, while areas suggestive of normal pH showed gliosis. In summary, results suggest CEST imaging of the amine functional group on free amino acids is sensitive to acidic pH and elevated amino acid concentration, allowing non-invasive identification of metabolically active tumor with a high specificity.

Protein Kinase C Phosphorylates the System N Glutamine Transporter SN1 (Slc38a3) and Regulates Its Membrane Trafficking and Degradation

The system N transporter SN1 (also known as SNAT3) is enriched on perisynaptic astroglial cell membranes. SN1 mediates electroneutral and bidirectional glutamine transport, and regulates the intracellular as well as the extracellular concentrations of glutamine. We hypothesize that SN1 participates in the glutamate/γ-aminobutyric acid (GABA)-glutamine cycle and regulates the amount of glutamine supplied to the neurons for replenishment of the neurotransmitter pools of glutamate and GABA. We also hypothesize that its activity on the plasma membrane is regulated by protein kinase C (PKC)-mediated phosphorylation and that SN1 activity has an impact on synaptic plasticity. This review discusses reports on the regulation of SN1 by PKC and presents a consolidated model for regulation and degradation of SN1 and the subsequent functional implications. As SN1 function is likely also regulated by PKC-mediated phosphorylation in peripheral organs, the same mechanisms may, thus, have impact on e.g., pH regulation in the kidney, urea formation in the liver, and insulin secretion in the pancreas.

A metabolic perturbation by U0126 identifies a role for glutamine in resveratrol-induced cell death

Recent evidence has identified substantial overlap between metabolic and oncogenic biochemical pathways, suggesting novel approaches to cancer intervention. For example, cholesterol lowering statins and the antidiabetes medication metformin both act as chemopreventive agents in prostate and other cancers. The natural compound resveratrol has similar properties: increasing insulin sensitivity, suppressing adipogenesis, and inducing apoptotic death of cancer cells in vitro. However, in vivo tumor xenografts acquire resistance to resveratrol by an unknown mechanism, while mouse models of metabolic disorders respond more consistently to the compound. Here we demonstrate that castration-resistant human prostate cancer C4-2 cells are more sensitive to resveratrol-induced apoptosis than isogenic androgen-dependent LNCaP cells. The MEK inhibitor U0126 antagonized resveratrol-induced apoptosis in C4-2 cells, but this effect was not seen with other MEK inhibitors. U0126 was found to inhibit mitochondrial function and shift cells to aerobic glycolysis independently of MEK. Mitochondrial activity of U0126 arose through decomposition, producing both mitochondrial fluorescence and cyanide, a known inhibitor of complex IV. Applying U0126 mitochondrial inhibition to C4-2 cell apoptosis, we tested the possibility that glutamine supplementation of citric acid cycle intermediate α-ketoglutarate may be involved. Suppression of the conversion of glutamate to α-ketoglutarate antagonized resveratrol-induced death in C4-2 cells. A similar effect was also seen by reducing extracellular glutamine concentration in the culture medium, suggesting that resveratrol-induced death is dependent on glutamine metabolism, a process frequently dysregulated in cancer. Further work on resveratrol and metabolism in cancer is warranted to ascertain if the glutamine dependence has clinical implications.

Regulation of protein metabolism by glutamine: implications for nutrition and health

Glutamine is the most abundant free alpha-amino acid in plasma and skeletal muscle. This nutrient plays an important role in regulating gene expression, protein turnover, anti-oxidative function, nutrient metabolism, immunity, and acid-base balance. Interestingly, intracellular and extracellular concentrations of glutamine exhibit marked reductions in response to infection, sepsis, severe burn, cancer, and other pathological factors. This raised an important question of whether glutamine may be a key mediator of muscle loss and negative nitrogen balance in critically ill and injured patients. Therefore, since the initial reports in late 1980s that glutamine could stimulate protein synthesis and inhibit proteolysis in rat skeletal muscle, there has been growing interest in the use of this functional amino acid to improve protein balance under various physiological and disease conditions. Although inconsistent results have appeared in the literature regarding a therapeutic role of glutamine in clinical medicine, a majority of studies indicate that supplementing appropriate doses of glutamine to enteral diets or parenteral solutions is beneficial for improving nitrogen balance in animals or humans with glutamine deficiency.

Cerebral Glutamine Concentration and Lactate–Pyruvate Ratio in Patients with Acute Liver Failure

Hyperammonemia causes brain edema and high intracranial pressure (ICP) in acute liver failure (ALF) by accumulation of glutamine in brain. Since a high-level glutamine may compromise mitochondrial function, the aim of this study was to determine if the lactate-pyruvate ratio is associated with a rise in the glutamine concentration and ICP. In 13 patients with ALF (8F/5M; median age 46 (range 18-66) years) the cerebral extracellular concentrations of glutamine, lactate, and pyruvate were measured by in vivo brain microdialysis together with ICP and cerebral perfusion pressure (CPP). The cerebral glutamine concentration was 4,396 (1,011-9,712) microM, lactate 2.15 (1.1-4.45) mM, and pyruvate 101 (43-255) microM. The lactate-pyruvate ratio was 21 (16-40), ICP 20 (2-28) mmHg, and CPP 72 (56-115) mmHg. Cerebral glutamine concentration correlated with the lactate-pyruvate ratio (r = 0.89, P < 0.05). Also the ICP, but not CPP, correlated to the lactate-pyruvate ratio (r = 0.64, P < 0.05). ICP and the cerebral glutamine concentration in patients with ALF correlate to the lactate-pyruvate ratio. Since CPP was sufficient in all patients the rise in lactate-pyruvate ratio indicates that accumulation of glutamine compromises mitochondrial function and causes intracranial hypertension.

Quantitative determination of extracellular glutamine concentration in rat brain, and its elevation in vivo by system A transport inhibitor, alpha-(methylamino)isobutyrate.

The basal concentration of glutamine in the extracellular fluid, [GLN(ECF)], was determined to be 385 +/- 16 microm in the cortico-striatal region of awake rats. This in vivo concentration was determined by measuring glutamine concentrations in dialysates collected at several flow rates (0.2-4 microL/min), and extrapolating to the concentration at zero flow-rate. Dialysate glutamine concentrations in the somatosensory cortex, hippocampus and thalamus showed no statistically significant difference. In these brain regions, [GLN(ECF)] was elevated 1.5- to 1.8-fold upon perfusion of 50-250 mmalpha-(methylamino)isobutyrate (MeAIB), a competitive inhibitor of glutamine uptake by system A amino acid transporter. The results show, for the first time, that MeAIB causes elevation of brain GLN(ECF)in vivo. The MeAIB-induced elevation of [GLN(ECF)] provides additional support for the current view that system A GLN transporter (Gln T/SAT 1) is the major pathway for the uptake of GLN(ECF) by neurons, while GLN release from glia is mainly mediated by a system N transporter (SN1) which is not inhibitable by MeAIB. The steady-state GLN(ECF) concentration and the effectiveness of MeAIB in inhibiting neuronal GLN uptake in vivo, reported in this study, will be useful, when combined with the known in vitro kinetic properties of the GLN transporters, for study of GLN transport in the intact brain.

Regulative capacity of glutamine.

The amino acid glutamine plays a central role in nitrogen transport within the body and is a fuel for rapidly dividing cells, such as in the gut and the immune system. Plasma glutamine levels decline during critical illness, and therefore these cells suffer from glutamine starvation under these conditions. The present review summarizes data on the specific effect of extracellular glutamine on metabolism, function, stress response, and apoptosis of glutamine-utilizing cells. Glutamine starvation leads to an energy depletion that is associated with a reduced responsiveness to exogenous stimuli. In addition, glutamine-starving cells show a reduced expression of the 70000 M(r) heat shock protein, which is an important factor for cell survival, and contain a reduced level of the antioxidant glutathione. Recent findings show that the extracellular glutamine level affects the susceptibility of cells to different apoptosis triggers: whereas glutamine-starving cells are more sensitive to Fas ligand-mediated apoptosis, they are desensitized against the cytotoxic effects of TNF-alpha. In addition, this review summarizes current knowledge on the molecular mechanisms of glutamine sensing. It discusses the role of AMP-activated protein kinase, the cellular redox state, osmosignalling, the regulation of translation, and amino acyl transfer RNA synthetases. These data show that glutamine-utilizing cells possess molecular mechanisms to detect the availability of glutamine and to respond specifically to changes in the extracellular glutamine concentration.

Type I and II metabotropic glutamate receptors modulate periaqueductal grey glycine release: interaction between mGlu2/3 and A1 adenosine receptors

In this study we investigated the effects of type I and II mGlu receptors ligands in glycine extracellular concentrations at the periaqueductal gray (PAG) level by using in vivo microdialysis, in conscious rats. An agonist of type I mGlu receptors, (S)-3,5-DHPG (1 and 5 mM), but not a selective agonist for mGlu5 receptors, CHPG (3 and 5 mM), was noticed to increase the dialysate glycine levels in a concentration-dependent manner (60±15% and 136±13%, respectively). CPCCOEt (1 mM), a selective mGlu1 receptor antagonist, perfused in combination with (S)-3,5-DHPG, counteracted the effect induced by (S)-3,5-DHPG, but did not change per se the extracellular PAG glycine values, even at the highest dosage used (2 mM). MPEP (1 and 2 mM), a selective antagonist of mGlu5 receptor, did not modify extracellular glycine level. An agonist of type II mGlu receptors, 2R,4R-APDC (25 and 50 μM), decreased the dialysate glycine in a concentration-dependent manner (−26±4% and −54±6%, respectively). The 2R,4R-APDC-induced decrease in extracellular glycine was prevented by EGlu (0.5 mM), a selective type II mGlu receptors antagonist. EGlu (0.5 and 1 mM), per se, led to a significant decrease (–56±7% and –57±2%, respectively) in extracellular PAG glycine too. This effect was prevented by DPCPX (100 μM), a selective antagonist for A1 adenosine receptors, but was not affected by CPA (1 mM), a selective A1 adenosine receptors agonist. Intra-PAG perfusion of CPA (0.1–1 mM) decreased the extracellular PAG glycine values (–47±13%) with 1 mM concentration. The CPA-induced effect was prevented by DPCPX (100 μM), and resulted to be additive with the 2R,4R-APDC-induced decrease in glycine values. DPCPX (1 mM) increased per se extracellular glycine (48±7%) at the highest dose used. Dipyridamole (100 μM), an inhibitor of both adenosine reuptake and phosphodiesterases, decreased extracellular glycine (–28±7%). Extracellular concentrations of glutamine never changed throughout this study. These data show opposing effects of type I and II mGlu receptors in the regulation of PAG glycine values. Moreover, functional interaction between type II mGlu and adenosine A1 receptors, which possibly operate through a common transductional pathway, may be relevant in the physiological control of glycine release in awake, freely moving rats at the periaqueductal gray matter.

Why Is L-Glutamine Metabolism Important to Cells of the Immune System in Health, Postinjury, Surgery or Infection?

Glutamine is normally considered to be a nonessential amino acid. However, recent studies have provided evidence that glutamine may become "conditionally essential" during inflammatory conditions such as infection and injury. It is now well documented that under appropriate conditions, glutamine is essential for cell proliferation, that it can act as a respiratory fuel and that it can enhance the function of stimulated immune cells. Studies thus far have determined the effect of extracellular glutamine concentration on lymphocyte proliferation and cytokine production, macrophage phagocytic plus secretory activities and neutrophil bacterial killing. Other cells of the immune system remain to be studied. The high rate of glutamine utilization and its importance to the function of lymphocytes, macrophages and neutrophils have raised the question "why glutamine?" because these cells have access to a variety of metabolic fuels both in vivo and in vitro. I have attempted to answer this question in this article. Additionally, knowledge of the rate of utilization and the pathway of metabolism of glutamine by cells of the immune system raises some intriguing questions concerning therapeutic manipulation of utilization of this amino acid such that the proliferative, phagocytic and secretory capacities of cells of the defense system may be beneficially altered. Evidence to support the hypothesis that glutamine is beneficially immunomodulatory in animal models of infection and trauma, as well as trauma in humans, is provided.

Role of glutamate receptors and glutamate transporters in the regulation of the glutamate-glutamine cycle in the awake rat.

In the present study we investigate the effects of a specific glutamate reuptake blocker, L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC), on extracellular concentrations of glutamine and glutamate in the striatum of the freely moving rat. Intracerebral infusions of PDC (1, 2 and 4 mM) produced a dose-related increase in extracellular concentrations of glutamate and a dose-related decrease in extracellular concentrations of glutamine. These increases in extracellular glutamate and decreases in extracellular glutamine were significantly correlated. To investigate the involvement of ionotropic glutamate receptors in the decreases of extracellular glutamine produced by PDC, N-methyl-D-aspartate (NMDA) receptor antagonist and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist were used. Perfusion of the NMDA receptor antagonist blocked the decrease of extracellular glutamine but had no effect on the increase of extracellular glutamate, both produced by PDC. Perfusion of the AMPA/kainate receptor antagonist attenuated the increase of extracellular glutamate and not only blocked the decrease of extracellular glutamine but also produced a significant increase of extracellular glutamine. The results reported in this study suggest that both NMDA and AMPA/kainate glutamatergic receptors are involved in the regulation of extracellular glutamine.

Macrophage-mediated lysis of a β-cell line, tumour necrosis factor-α release from bacillus Calmette–Gue´rin (BCG)-activated murine macrophages and interleukin-8 release from human monocytes are dependent on extracellular glutamine concentration and glutamine metabolism

Macrophages and monocytes are cells with a large capacity for cytokine production. Cytokines produced by these cells are not preformed and released upon stimulation, but must be transcribed and translated. Although much is known concerning the regulation of the latter processes at the molecular level, the role of exogenous amino acids in the secretory process has not been actively investigated. Glutamine is utilized by macrophages at a much faster rate than any other amino acid. The role for high rates of glutamine utilization in macrophages or monocytes is not fully understood. We demonstrate here that the rates of lipopolysaccharide-stimulated tumour necrosis factor-alpha secretion from bacillus Calmette-Guérin (BCG)-activated murine peritoneal macrophages and lipopolysaccharide-stimulated interleukin-8 production from human monocytes are dependent upon extracellular glutamine concentration. We also demonstrate that potent inhibition of cytokine production can be achieved by incubating macrophages or monocytes in the presence of the glutaminase inhibitor 6-diazo-5-oxo-norleucine. On co-culture of BCG-activated macrophages and the clonal pancreatic beta-cell line BRIN-BD11, macrophage-specific beta-cell death was significantly reduced on prior exposure of macrophages to 6-diazo-5-oxo-norleucine. Thus glutamine metabolism may be essential for generation of cytotoxic products from macrophages, including tumour necrosis factor-alpha.

Macrophage-mediated lysis of a β-cell line, tumour necrosis factor-α release from bacillus Calmette‒Guérin (BCG)-activated murine macrophages and interleukin-8 release from human monocytes are dependent on extracellular glutamine concentration and glutamine metabolism

Macrophage-mediated lysis of a β-cell line, tumour necrosis factor-α release from bacillus Calmette‒Guérin (BCG)-activated murine macrophages and interleukin-8 release from human monocytes are dependent on extracellular glutamine concentration and glutamine metabolism

Effect of extracellular glutamine concentration on primary and secondary metabolism of a murine hybridoma: An in vivo 13C nuclear magnetic resonance study

Effect of extracellular glutamine concentration on primary and secondary metabolism of a murine hybridoma: An in vivo 13C nuclear magnetic resonance study

Effect of extracellular glutamine concentration on primary and secondary metabolism of a murine hybridoma: An in vivo13C nuclear magnetic resonance study

The effect of changes in extracellular glutamine level on metabolism of a murine hybridoma was examined with in vivo nuclear magnetic resonance (NMR) spectroscopy. Cells were cultured in a hollow-fiber bioreactor at high cell density to allow intracellular metabolite levels to be determined on a metabolically relevant time scale. Steady infusions of [1-13C] glucose were used to label glycolytic and tricarboxylic acid cycle intermediates, which permitted continuous monitoring with NMR spectroscopy during changes in environmental glutamine level. Samples of the extracellular medium were also analyzed to determine the effect of glutamine on other metabolites associated with primary and secondary metabolism. The changes in glutamine concentration had several effects on primary and secondary metabolism, depending on the rate the changes were made. For a brief reduction in feed glutamine concentration from 4 to 0 mM (which produced a rapid change from 0.67 to approximately 0 mM in residual glutamine), large changes were observed in the rate of consumption of metabolites normally associated with energy production. Antibody synthesis was strongly stimulated and nitrogen metabolism was significantly altered. For a more prolonged reduction from 2.4 to 1.2 mM (which produced a slower reduction from 0.30 to 0. 08 mM in residual glutamine), much smaller changes were observed even though the concentration of glutamine at the reduced feed level was very low. Energy metabolism did not appear to be limited by glutamine at 0.08 mM, which suggests that significant futile cycling may occur in energy producing pathways when excess glucose and glutamine are available. However, this concentration of extracellular glutamine appeared to affect some anabolic pathways, which require amino groups from glutamine.

Amphetamine increases extracellular concentrations of glutamate in the prefrontal cortex of the awake rat: a microdialysis study.

Using microdialysis, the effect was investigated of intracerebral infusions of different doses of amphetamine (1.25, 2.5, 5, 10, and 20 microg/microl) on the extracellular concentrations of glutamate in the medial prefrontal cortex of the rat. Amphetamine produced a dose-related increase in extracellular concentrations of glutamate. At the highest dose, amphetamine increased extracellular glutamate by 445% of baseline as well as extracellular concentrations of taurine, and reduced extracellular concentrations of glutamine. Amphetamine did not modify other amino acids such as arginine. Increases in extracellular concentrations of glutamate and taurine were independent of calcium in the perfusion medium. This is the first study showing that amphetamine produces a calcium-independent increase in extracellular concentrations of glutamate and taurine in the medial prefrontal cortex of the rat.

Stimulatory effect of glutamine on human monocyteactivation as measured by interleukin-6 and soluble interleukin-6 receptor release

The activation state of murine peritoneal macrophages is shown to depend on extracellular glutamine concentration. However, there are no studies on the effect of glutamine concentration on the activation state of human monocytes. We studied the effect of extracellular glutamine concentration on interleukin (IL-6) and soluble IL-6 receptor (sIL-6R) production by activated monocytes. Monocytes separated by density gradient centrifugation and adherence to glass from buffy coats obtained from healthy donors were activated by 1 pg/ml or 1 ng/ml Iipopolysaccharide (LIPS) and incubated for 48 h in 0, 0.1, 0.2, 0.6 and 2.0 mM glutamine. Levels of IL-6 and sIL-6R in culture medium were measured by immunoassay. The extracellular glutamine concentration had a significant effect on IL-6 secretion by activated human monocytes. The mean levels of IL-6 in 0.1 mM glutamine were only marginally higher ( P = 0.54) compared to those in the absence of glutamine. A minimum of 0.2 mM glutamine was required to reach the maximal production of IL-6. At all glutamine concentrations the higher concentration of LPS (1 ng/ml) induced higher mean levels of IL-6 than the lower one (1 pg/ml). Glutamine concentration did not affect sIL-6R production. Our results indicate that very low levels of glutamine in plasma may impair the activation of human monocytes as measured by IL-6 secretion.

Inhibition of pulmonary microvascular endothelial glutamine transport by glucocorticoids and endotoxin.

During septic states, the lungs produce increased amounts of glutamine, an event that is mediated by both endotoxin and glucocorticoid hormones and is presumed to be due to accelerated intracellular glutamine biosynthesis. Because enhanced net glutamine release in vivo could also be due to a decrease in cellular uptake, we assayed glutamine transport in cultured rat microvascular pulmonary endothelial cells. The effect of Escherichia coli endotoxin (LPS, 1 microgram/mL), various cytokines, and dexamethasone (DEX, 0.1 mumol/L) on glutamine transport activity was studied in rat lung microvascular endothelial cells grown in varying glutamine concentrations (0, 0.1, 0.5, and 2 mmol/L). Experiments were also performed in cells treated with cycloheximide, actinomycin D, or chelerythrine chloride. More than 90% of glutamine transport was mediated by the Na+ -dependent transport system ASC. DEX and LPS inhibited endothelial glutamine uptake in a time- and dose-dependent manner, a response that was only observed with incubation medium contained the lower concentrations of glutamine. Neither DEX nor LPS altered transport activity in cells cultured in medium containing 2 mmol glutamine/L. There was no synergistic or additive effect when both compounds were added together. The cytokines tumor necrosis factor alpha, interleukin (IL) 1, IL-2, and IL-6 did not alter glutamine transport. both DEX and LPS inhibited glutamine transport by decreasing transporter maximal transport velocity (Vmax) without affecting transporter affinity (Km). Cycloheximide and actinomycin D abrogated the inhibition of transport activity that was observed in DEX- or LPS-treated cells, whereas the protein kinase C inhibitor chelerythrine chloride had no effect on either control or stimulated glutamine transport. These data suggest that DEX and LPS "down-regulate" glutamine uptake by lung microvascular endothelial cells by inducing the synthesis of an inhibitory protein that modulates the activity of the system ASC protein. This response in vitro appears to be influenced by the extracellular glutamine concentration. This decrease in microvascular endothelial glutamine transport may be one mechanism by which net lung glutamine release is enhanced during critical illness.

Suppression of Anionic Amino Acid Transport Impairs the Maintenance of Intracellular Glutamate in Ha-ras-Expressing Cells

When the expression of a Ha-ras oncogene is triggered in NIH3T3 cells, a progressive inhibition of sodium dependent transport of anionic amino acids through system X−AG is observed. After 48h of ras expression the transport activity of system X−AG is almost abolished, while other transport systems involved in anionic amino acid transport are unaffected or even stimulated. In the presence of high extracellular concentrations of glutamine, the intracellular concentration of glutamate is comparable in ras expressing and non-expressing cells. On the contrary, when the extracellular pool of glutamine is depleted by the enzyme L-asparaginase, intracellular glutamate decreases at a much faster rate in ras expressing, low-transport cells. These results suggest that transport system X−AG significantly contributes to the homeostasis of intracellular glutamate under conditions of glutamine deprivation.

Production of glutamine and utilization of glutamate by rat subcutaneous adipose tissue in vivo.

Information about adipose tissue amino acid metabolism is limited, with most data derived from studies in vitro. The purpose of this study was to further characterize the role of adipose tissue in glutamine metabolism in the rat in vivo. The extracellular concentrations of glutamine, glutamate, alanine, and ammonia were measured in the rat inguinal fat pad using a microdialysis sampling technique. A calibration method was used to accurately assess the extracellular levels of metabolites, and a comparison of these concentrations with those in arterial blood allowed determination of the net flux of each compound. The adipose tissue-arterial blood concentration differences were 122 +/- 19, 54 +/- 37, -61 +/- 21, and -28 +/- 13 microM for glutamine, alanine, glutamate, and ammonia, respectively, indicating a production of glutamine and an uptake of glutamate by subcutaneous adipose tissue. The magnitude of glutamine production suggests that adipose tissue may play a significant role in whole body glutamine homeostasis.