Hepatic glutaminase flux regulation of glutamine homeostasis. Studies in vivo

The role of hepatic glutaminase flux in regulating plasma glutamine homeostasis was studied in the intact rat. Interorgan glutamine flow during chronic metabolic acidosis was away from the splanchnic bed and to the kidneys. Hindquarter and hepatic glutamine release were the major sources of glutamine removed by the kidneys. Interorgan glutamate flow was from the liver to the hindquarters and kidneys. Chronic metabolic acidosis reduced arterial glutamine concentration 30%. Acute respiratory acidosis (pH 7.12 +/- 0.02) returned arterial glutamine concentration to normal values, increasing and decreasing hepatic glutamine and glutamate release respectively; renal and gut glutamine removal rates were not decreased. Hepatic unidirectional glutamine utilization measured isotopically was decreased 51% by acute acidosis; unidirectional glutamine production was unchanged. The results are consistent with the proposed role of ammonia-activated hepatic glutaminase in the regulation of glutamine homeostasis during acute acidosis.     

Acid-base balance and plasma glutamine concentration in man

Plasma glutamine concentrations were measured in chronic metabolic acidosis and alkalosis in healthy male volunteers. Metabolic acidosis resulted in a significant drop in glutamine concentration while metabolic alkalosis significantly elevated glutamine levels. These changes in glutamine concentration correlated with both the bicarbonate and PCO2 levels. To determine whether bicarbonate or PCO2 levels influence the glutamine concentrations, respectively acidosis was induced by respiring 5% CO2. This resulted in a significant elevation in both PCO2 and glutamine while bicarbonate levels remained unchanged. The results demonstrate an effect of acid-base alterations upon plasma glutamine concentration mediated by PCO2.     

Inhibition of glutamine synthetase in the mouse kidney: a novel mechanism of adaptation to metabolic acidosis

As part of a study on the regulation of renal ammoniagenesis in the mouse kidney, we investigated the effect of chronic metabolic acidosis on glutamine synthesis by isolated mouse renal proximal tubules. The results obtained reveal that, in tubules from control mice, glutamine synthesis occurred at high rates from glutamate and proline and, to a lesser extent, from ornithine, alanine, and aspartate. A 48 h, metabolic acidosis caused a marked inhibition of glutamine synthesis from near-physiological concentrations of both alanine and proline that were avidly metabolized by the tubules; metabolic acidosis also greatly stimulated glutamine utilization and metabolism. These effects were accompanied by a large increase (i) in alanine, proline, and glutamine gluconeogenesis and (ii) in ammonia accumulation from proline and glutamine. In the renal cortex of acidotic mice, the activity of phosphoenolpyruvate carboxykinase increased 4-fold, but that of glutamate dehydrogenase did not change; in contrast with what is known in the rat renal cortex, metabolic acidosis markedly diminished the glutamine synthetase activity and protein level, but not the glutamine synthetase mRNA level in the mouse renal cortex. These results strongly suggest that, in the mouse kidney, glutamine synthetase is an important regulatory component of the availability of the ammonium ions to be excreted for defending systemic acid-base balance. Furthermore, they show that, in rodents, the regulation of renal glutamine synthetase is species-specific.     

Role of hippurate in acidosis induced adaptation in renal gamma-glutamyltransferase

Metabolic acidosis results in an adaptation in renal γ-glutamyltransferase (γ-GT) and a doubling of hippurate excretion. The greater rate of γ-glutamohydroxamate, γ-GHA, formation from L-glutamine, but not from glutathione, by acidotic kidney homogenates suggest an increased γ-glutamyl-enzyme complex formation and a preference for glutamine as the γ-glutamyl donor in acidosis. Hippurate added $\text{in}$$\text{vitro}$ to cortical homogenates or microsomes mimics the affect of acidosis upon γ-GHA formation from glutamine. Acid extracts of urine stimulated ammonia formation from glutamine using cortical microsomes in agreement with the measured hippurate levels. Administering an exogenous hippurate load to fasting nonacidotic rats doubled ammonia excretion and the rate of γ-GHA formation by cortical homogenates. These results are consistent with the acidosis induced adaptation in renal γ-GT governed by hippurate.     

Muscle glutamine production in diabetic ketoacidotic rats.

The mechanism of activation of glutamine production by the hindlimb during diabetic ketoacidosis (DKA) was investigated in rats. Muscle glutamine production was estimated to account for over 90% of the total glutamine produced by the hindlimb. DKA produced significant increases in the concentrations of NH4+ and IMP in hindlimb muscles, suggesting that AMP deaminase is activated by DKA. NH4Cl- and HCl-induced acidosis did not produce these changes, indicating either that acidosis itself is not the stimulus for increased AMP deaminase activity or that the more severe degree of acidosis accompanying DKA is necessary for activation. Muscle glutamine concentrations were depressed in DKA. Experiments with isolated epitrochlearis muscle showed that the transport and permeability properties of the muscle cells (as judged by uptake and release of alpha-aminoisobutyrate and glutamine) were not altered by DKA. However, glutamine uptake by muscle cells was significantly inhibited by L-leucine, the concentration of which, along with other branched-chain amino acids, is markedly elevated in DKA.     

Brain glutamate metabolism during metabolic alkalosis and acidosis.

Glutamate modifies ventilation by altering neural excitability centrally. Metabolic acid-base perturbations may also alter cerebral glutamate metabolism locally and thus affect ventilation. Therefore, the effect of metabolic acid-base perturbations on central nervous system glutamate metabolism was studied in pentobarbital-anesthetized dogs under normal acid-base conditions and during isocapnic metabolic alkalosis and acidosis. Cerebrospinal fluid transfer rates of radiotracer [13N]ammonia and of [13N]glutamine synthesized de novo via the reaction glutamate+NH3-->glutamine in brain glia were measured during normal acid-base conditions and after 90 min of acute isocapnic metabolic alkalosis and acidosis. Cerebrospinal fluid [13N]ammonia and [13N]glutamine transfer rates decreased in metabolic acidosis. Maximal glial glutamine efflux rate jm equals 85.6 +/- 9.5 (SE) mumol.l-1 x min-1 in all animals. No difference in jm was observed in metabolic alkalosis or acidosis. Mean cerebral cortical glutamate concentration was significantly lower in acidosis [7.01 +/- 0.45 (SE) mumol/g brain tissue] and tended to be larger in alkalosis, compared with 7.97 +/- 0.89 mumol/g in normal acid-base conditions. There was a similar change in cerebral cortical gamma-aminobutyric acid concentration. Within the limits of the present method and measurements, the results suggest that acute metabolic acidosis but not alkalosis reduces glial glutamine efflux, corresponding to changes in cerebral cortical glutamate metabolism. These results suggest that glutamatergic mechanisms may contribute to central respiratory control in metabolic acidosis.     

Acidotic alterations in oxidative metabolism influencing rat renal slice ammoniagenesis

We believe that two findings are interconnected and help to comprehend a major mechanism behind the regulation of renal ammonia production during acidosis. First, slices from acidotic compared to control and alkalotic rats produce more ammonia from glutamine. Second, inhibition of renal oxidative metabolism at various points by metabolic inhibitors augments slice ammoniagenesis. Based on this, our purpose was to determine whether enhanced renal ammoniagenesis during acidosis could occur through the same mechanism as the metabolic inhibitors. However, metabolic inhibitors (malonate; arsenite; 2,4-dinitrophenol) usually decrease while acidosis increases slice gluconeogenesis. There is one known exception. Fluorocitrate, which blocks citrate metabolism, simulates the acidotic condition by enhancing both ammonia and glucose production. Accordingly, a block of oxidative metabolism if located prior to citrate oxidation in the tricarboxylic acid cycle could theoretically augment ammoniagenesis during acidosis. Lactate, is a major renal fuel whose oxidative metabolism would be blocked by fluorocitrate. There, we concentrated on the effects of acidosis on lactate as well as glutamine metabolism. Lactate decarboxylation decreases in the face of increased glucose production during acidosis, and lactate inhibition of glutamine decarboxylation decreases in slices from acidotic rats. Also, we found lesser oxygen consumption in the presence of lactate by kidney slices from acidotic rats compared to control and alkalotic rats. We postulate that relatively less incorporation of lactate into the TCA cycle, causing decreased citrate formation and citrate oxidation during acidosis, contributes, at least in part, to acidotic adaptation of ammoniagenesis.     

Glutamine metabolism in the kidney during induction of, and recovery from, metabolic acidosis in the rat

Experiments were carried out on rats to evaluate the possible regulatory roles of renal glutaminase activity, mitochondrial permeability to glutamine, phosphoenolpyruvate carboxykinase activity and systemic acid–base changes in the control of renal ammonia (NH₃ plus NH₄⁺) production. Acidosis was induced by drinking NH₄Cl solution ad libitum. A pronounced metabolic acidosis without respiratory compensation [pH=7.25; HCO₃⁻=16.9mequiv./litre; pCO₂=40.7mmHg (5.41kPa)] was evident for the first 2 days, but thereafter acid–base status returned towards normal. This improvement in acid–base status was accompanied by the attainment of maximal rates of ammonia excretion (onset phase) after about 2 days. A steady rate of ammonia excretion was then maintained (plateau phase) until the rats were supplied with tap water in place of the NH₄Cl solution, whereupon pCO₂ and HCO₃⁻ became elevated [55.4mmHg (7.37kPa) and 35.5mequiv./litre] and renal ammonia excretion returned to control values within 1 day (recovery phase). Renal arteriovenous differences for glutamine always paralleled rates of ammonia excretion. Phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase activities and the rate of glutamine metabolism (NH₃ production and O₂ consumption) by isolated kidney mitochondria all increased during the onset phase. The increases in glutaminase and in mitochondrial metabolism continued into the plateau phase, whereas the increase in the carboxykinase reached a plateau at the same time as did ammonia excretion. During the recovery phase a rapid decrease in carboxykinase activity accompanied the decrease in ammonia excretion, whereas glutaminase and mitochondrial glutamine metabolism in vitro remained elevated. The metabolism of glutamine by kidney-cortex slices (ammonia, glutamate and glucose production) paralleled the metabolism of glutamine in vivo during recovery, i.e. it returned to control values. The results indicate that the adaptations in mitochondrial glutamine metabolism must be regulated by extra-mitochondrial factors, since glutamine metabolism in vivo and in slices returns to control values during recovery, whereas the mitochondrial metabolism of glutamine remains elevated.     

Acidosis and astrocyte amino acid metabolism

The relationship between acidosis and the metabolism of glutamine and glutamate was studied in cultured astrocytes. Acidification of the incubation medium was associated with an increased formation of aspartate from glutamate and glutamine. The rise of the intracellular content of aspartate was accompanied by a significant decline in the extracellular concentration of both lactate and citrate. Studies with either [2-(15)N]glutamine or [15N]glutamate indicated that there occurred in acidosis an increased transamination of glutamate to aspartate. Studies with L-[2,3,3,4,4-(2)H5]glutamine indicated that in acidosis glutamate carbon was more rapidly converted to aspartate via the tricarboxylic acid cycle. Acidosis appears to result in increased availability of oxaloacetate to the aspartate aminotransferase reaction and, consequently, increased transamination of glutamate. The expansion of the available pool of oxaloacetate probably reflects a combination of: (a) Restricted flux through glycolysis and less production from pyruvate of acetyl-CoA, which condenses with oxaloacetate in the citrate synthetase reaction; and (b) Increased oxidation of glutamate and glutamine through a portion of the tricarboxylic acid cycle and enhanced production of oxaloacetate from glutamate and glutamine carbon. The data point to the interplay of the metabolism of glucose and that of glutamate in these cells.     

Renal brush border glutamine transport: comparison between in situ and isolate membrane vesicle uptake

Glutamine uptake by renal cortical brush-border vesicles was compared to transport expressed by the functioning isolated kidney. Comparisons were made with regard to sodium dependency and the adaptive increase induced by chronic metabolic acidosis in the rat. The results show an absolute dependency upon a sodium gradient; sodium-independent glutamine uptake has no counterpart in situ. In addition, acidosis-induced adaptive increase in vesicle glutamine uptake has no counterpart in situ. Rather, the apparent adaptation reflects extravesicular gamma-glutamyltransferase-mediated conversion to glutamate and subsequent accumulation; acidosis-induced adaptation of this enzyme largely explains the apparent adaptation in glutamine uptake. Consequently the role of membrane transport in glutamine flux regulation can be assessed providing metabolic conversion is controlled.     

α-Ketoglutarate regulation of glutamine transport and deamidation by renal mitochondria

α-ketoglutarate was found to be a potent inhibitor of glutamine transport and deamidation in mitochondria isolated from rat kidney; physiological concentrations of the ketoacid (～0.3mM) reduced transport and deamidation 45–60 percent. The observed concentration-inhibition relationship between α-ketoglutarate and mitochondrial glutamine transport and deamidation indicated that changes in renal concentration of the ketoacid occurring during conditions associated with an increase in glutamine deamidation (e.g. metabolic acidosis) would have significant effects on glutamine transport and deamidation by renal mitochondria in vivo. The inhibitory effect of α-ketoglutarate was specific; several of the other major organic acids found in renal cells stimulated rather than inhibited mitochondrial glutamine transport.     

Regulation of flux through glutaminase and glutamine synthetase in isolated perfused rat liver

1. Glutaminase and glutamine synthetase are simultaneously active in the intact liver, resulting in an energy consuming cycling of glutamine at a rate up to 0.2 mumol per g per min. 2. An increase in portal glutamine concentration was followed by an increased flux through glutaminase, but flux through glutamine synthetase remained unchanged. Glutaminase flux was also increased by ammonium ions or glucagon; these effects were additive. 3. Glutamine synthetase flux was increased by ammonium ions, but this activation was partly overcome by increasing portal glutamine concentrations. Glutamine synthetase flux was slightly increased by glucagon at portal glutamine concentrations of about 0.2-0.3 mM, but was strongly inhibited above 0.6 mMs. 4. During experimental metabolic acidosis there was an increased net release of glutamine by the liver, being due to opposing changes of flux through glutaminase and glutamine synthetase. Conversely, an increased glutamine uptake by the liver during metabolic alkalosis was observed due to an inhibition of glutamine synthetase and an activation of glutaminase. However, the two enzyme activities respond differently depending on whether glucagon or ammonium ions are present.     

Altered Glutamatergic Metabolism Associated with Punctate White Matter Lesions in Preterm Infants

Preterm infants (∼10% of all births) are at high-risk for long-term neurodevelopmental disabilities, most often resulting from white matter injury sustained during the neonatal period. Glutamate excitotoxicity is hypothesized to be a key mechanism in the pathogenesis of white matter injury; however, there has been no in vivo demonstration of glutamate excitotoxicity in preterm infants. Using magnetic resonance spectroscopy (MRS), we tested the hypothesis that glutamate and glutamine, i.e., markers of glutamatergic metabolism, are altered in association with punctate white matter lesions and “diffuse excessive high signal intensity” (DEHSI), the predominant patterns of preterm white matter injury. We reviewed all clinically-indicated MRS studies conducted on preterm infants at a single institution during a six-year period and determined the absolute concentration of glutamate, glutamine, and four other key metabolites in the parietal white matter in 108 of those infants after two investigators independently evaluated the studies for punctate white matter lesions and DEHSI. Punctate white matter lesions were associated with a 29% increase in glutamine concentration (p = 0.002). In contrast, there were no differences in glutamatergic metabolism in association with DEHSI. Severe DEHSI, however, was associated with increased lactate concentration (p = 0.001), a marker of tissue acidosis. Findings from this study support glutamate excitotoxicity in the pathogenesis of punctate white matter lesions, but not necessarily in DEHSI, and suggest that MRS provides a useful biomarker for determining the pathogenesis of white matter injury in preterm infants during a period when neuroprotective agents may be especially effective.     

Metabolic fate of glutamate carbon in rat renal tubules. Studies with 13C nuclear magnetic resonance and gas chromatography-mass spectrometry.

13C-n.m.r. spectroscopy and g.c.-m.s. were used to determine the metabolic fate of glutamate carbon in rat kidney. The main purpose was to characterize the effect of chronic metabolic acidosis on the utilization of glutamate carbon. Renal tubules obtained from normal and chronically acidotic rats were incubated in Krebs buffer, pH 7.4, in the presence of 2.5 mM-[3-13C]glutamate. During the course of incubation the concentrations of total glucose and NH3 were significantly (P less than 0.05) higher in tissue from acidotic rats. The levels of some tricarboxylic-acid-cycle intermediates were higher (P less than 0.05) in control tissue. In control tissue, 13C-n.m.r. spectra demonstrated a significantly higher rate of 13C appearance of aspartate, glutamine and [2,4-13C]glutamate. However, in acidosis the resonances of [13C]glucose carbon atoms were significantly higher. In the control, approx. 15% of glutamate carbon was accounted for by [13C]glucose formation as against 30% in chronic acidosis. However, in control tissue, 44% of glutamate carbon utilization was accounted for by recycling to glutamate and formation of aspartate, glutamine and GABA. In acidosis, only 11% was so recovered. Analysis of 15NH3 formation during the course of incubation with 2.5 mM-[15N]glutamate demonstrated a positive association between the appearance of [13C]glucose and 15NH3 both in the control and in acidosis. The data suggest that the control of gluconeogenesis and ammoniagenesis in acidosis is, in part, referable to a diminution in the rate of the reductive amination of alpha-oxoglutarate, that of the transamination reaction and that of glutamine synthesis.     

Metabolism of glutamine and glutamate by rat renal tubules. Study with 15N and gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry was utilized to study the metabolism of [15N]glutamate, [2-15N]glutamine, and [5-15N]glutamine in isolated renal tubules prepared from control and chronically acidotic rats. The main purpose was to determine the nitrogen sources utilized by the kidney in various acid-base states for ammoniagenesis. Incubations were performed in the presence of 2.5 mM 15N-labeled glutamine or glutamate. Experiments with [5-15N]glutamine showed that in control animals approximately 90% of ammonia nitrogen was derived from 5-N of glutamine versus 60% in renal tubules from acidotic rats. Experiments with [2-15N]glutamine or [15N]glutamate indicated that in chronic acidosis approximately 30% of ammonia nitrogen was derived either from 2-N of glutamine or glutamate-N by the activity of glutamate dehydrogenase. Flux through glutamate dehydrogenase was 6-fold higher in chronic acidosis versus control. No 15NH3 could be detected in renal tubules from control rats when [2-15N]glutamine was the substrate. The rates of 15N transfer to other amino acids and to the 6-amino groups of the adenine nucleotides were significantly higher in normal renal tubules versus those from chronically acidotic rats. In tubules from chronically acidotic rats, 15N abundance in 15NH3 and the rate of 15NH3 appearance were significantly higher than that of either the 6-amino group of adenine nucleotides or the 15N-amino acids studied. The data indicate that glutamate dehydrogenase activity rather than glutamate transamination is primarily responsible for augmented ammoniagenesis in chronic acidosis. The contribution of the purine nucleotide cycle to ammonia formation appears to be unimportant in renal tubules from chronically acidotic rats.     

Arteriovenous differences for amino acids and lactate across kidneys of normal and acidotic rats.

1. Arteriovenous differences fro amino acids across kidneys of normal and chronically acidotic rats were measured. Glutamine was the only amino acid extracted in increased amounts in acidosis. There was a considerable production of serine by kidneys from both normal and acidotic rats. 2. The arterial blood concentration of glutamine was significantly decreased in acidotic animals. 3. The glutamine extracted by kidneys of acidotic rats was largely and probably exclusively derived from the plasma. 4. The blood lactate concentration was unchanged in acidosis, as was the uptake of lactate by the kidney.     

Glutamine release from hindlimb and uptake by kidney in the acutely acidotic rat.

The arteriovenous difference (release) for glutamine across the hindlimb increases significantly during acute HCl-induced acidosis. This additional amount release by muscle tissue can account for the extra glutamine taken up by the kidney.     

Characteristics of glutamine metabolism by rat kidney tubules: a carbon and nitrogen balance.

The metabolism of glutamine by a suspension of rat kidney tubules was studied in vitro. The influence of duration of incubation, glutamine concentration, and metabolic state of the donor animals was investigated. The relative importance of glucose synthesis, amino acid production, and oxidation to CO2 was estimated by drawing a complete balance of the nitrogens and the carbon chains of the extracted glutamine. It was found that the initial (first 15 min) rate of glutamine utilization was significantly greater than the subsequent rate due to an initial, but transient, extracellular accumulation of glutamate. This phenomenon was suppressed when a small amount of glutamate was added to the incubation medium. Glucose production constitutes the major fate for glutamine metabolism. No net oxidation of glutamine could be detected with 1 mM glutamine during the first 30 min. However, glutamine oxidation becomes significant after prolonged incubation (16% at 120 min). The metabolic fate of glutamine differs when 5 or 10 mM are presented to the tubules, glutamate production and oxidation to CO2 becoming more important. Metabolic acidosis or a 48-h fast increases glutamine extraction and enhances its utilization glucose synthesis while they depress glutamate accumulation and oxidation to CO2. Metabolic alkalosis has the opposite effect. It is concluded that the metabolism of glutamine in vitro is dependent on the conditions of the study. Furthermore, total oxidation to CO2 is not a major fate for glutamine metabolism at physiological concentration and is not enhanced by acidosis in the rat kidney in vitro.     

The interrelationship of the concentration of hydrogen ions, bicarbonate ions, carbon dioxide and calcium ions in the regulation of renal gluconeogenesis in the rat

1. The interrelationship of acidosis and Ca(2+) on the stimulation of gluconeogenesis by rat kidney-cortex slices was studied. 2. Ca(2+) stimulated gluconeogenesis from glutamine, glutamate, 2-oxoglutarate, succinate, malate, pyruvate, lactate and fructose, but not from galactose. 3. The [Ca(2+)] needed for optimum gluconeogenesis was about 2mm, but at this concentration, acidosis, produced in vitro by a decrease of [HCO(3) (-)] in the medium at constant pCO(2) or by an increase in pCO(2) at constant [HCO(3) (-)], did not stimulate gluconeogenesis. 4. In the absence of Ca(2+), acidosis (low [HCO(3) (-)]) stimulated gluconeogenesis from glutamine, glutamate, 2-oxoglutarate, succinate, malate, pyruvate and lactate but not from fructose or galactose. With succinate as substrate, the stimulatory effect of acidosis (low [HCO(3) (-)]) disappeared at Ca(2+) concentrations above 1.0mm. 5. The [HCO(3) (-)] was the most important determinant of the acidosis effect since a decrease of pH caused by an increase in pCO(2) did not uniformly stimulate gluconeogenesis, whereas a decrease in [HCO(3) (-)] without a change in pH consistently stimulated glucose formation in a way similar to the stimulation produced by acidosis (low [HCO(3) (-)]) in the absence of Ca(2+). 6. Acidosis in vitro inhibited the rate of decrease of activity of phosphoenolpyruvate carboxylase in slices, and Ca(2+) caused an increase in the activity of fructose 1-phosphate aldolase. 7. Respiratory acidosis in vitro caused an increase in the activity of phosphoenolpyruvate carboxylase in kidney cortex and an increase in gluconeogenesis from glutamine. 8. Possible points of interaction between Ca(2+), H(+) and HCO(3) (-) with the gluconeogenic sequence are discussed.     

Effect of the blood lactate concentration on renal glutamine metabolism in dogs with chronic metabolic acidosis

It appears that glutamine and lactate are the principal substrates for the kidney in dogs with chronic metabolic acidosis. Accordingly, the purpose of this study was to determine if a higher or lower rate of renal lactate extraction would influence the rate of glutamine extraction at a constant rate of renal ATP turnover. The blood lactate concentration was 0.9 +/- 0.01 mM in 15 acidotic dogs. However, eight dogs with chronic metabolic acidosis had a spontaneous blood lactate concentration of 0.5 mM or lower. The kidneys of these dogs extracted considerably less lactate from the arterial blood (19 vs. 62 mumol/100 mL glomerular filtration rate (GFR]. Nevertheless, glutamine, alanine, citrate, and ammonium metabolism were not significantly different in these two groups of dogs. Renal ATP balance in acidotic dogs with a low blood lactate could only be achieved if a substrate other than additional glutamine were oxidized in that segment of the nephron which normally oxidized lactate; presumably a fat-derived substrate and (or) lactate derived from glucose was now the metabolic fuel at these more distal sites. When the blood lactate concentration was greater than 1.9 mM, lactate extraction rose to 219 mumol/100 mL GFR. Glutamine, alanine, citrate, and ammonium metabolism were again unchanged; in this case, ATP balance required substrate flux to products other than carbon dioxide, presumably, gluconeogenesis. It appears that renal ammoniagenesis is a proximal event and is independent of the rate of renal lactate extraction.