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In JoVE (1)
Other Publications (24)
- American Journal of Physiology. Endocrinology and Metabolism
- The Journal of Biological Chemistry
- Epilepsy Research
- The Biochemical Journal
- The Journal of Biological Chemistry
- Prostaglandins, Leukotrienes, and Essential Fatty Acids
- The Biochemical Journal
- The Journal of Biological Chemistry
- Neurochemistry International
- The Journal of Nutrition
- The Journal of Biological Chemistry
- Neurochemistry International
- The Journal of Biological Chemistry
- Cancer Research
- Annual Review of Nutrition
- Proceedings of the National Academy of Sciences of the United States of America
- The Journal of Biological Chemistry
- Pediatric Research
- The Journal of Biological Chemistry
- Proceedings of the National Academy of Sciences of the United States of America
- Epilepsia
- Molecular Genetics and Metabolism
- Molecular Genetics and Metabolism
- The Journal of Biological Chemistry
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Articles by Ilana Nissim in JoVE
JoVE General
פרופיל איזוטופים יציבים של שטף מטבולית מתווך בשלב פיתוח למבוגרים Caenorhabditis elegans
1Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania
פרופיל איזוטופים יציבים ידי ניתוח כרומטוגרפיה מסת הגז spectrometric של השטף מטבולית מתווך מתואר נמטודות,
Other articles by Ilana Nissim on PubMed
Regulation of Urea Synthesis by Agmatine in the Perfused Liver: Studies with 15N
Administration of arginine or a high-protein diet increases the hepatic content of N-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of l-[guanidino-15N2]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with 15N-labeled glutamine and unlabeled ammonia or 15NH4Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-15N2]agmatine is formed in the liver from perfusate l-[guanidino-15N2]arginine ( approximately 90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of 15N-labeled NAG and [15N]urea from 15N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of 15N-labeled NAG and [15N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease.
Regulation of Leucine-stimulated Insulin Secretion and Glutamine Metabolism in Isolated Rat Islets
Glutamate dehydrogenase (GDH) is regulated by both positive (leucine and ADP) and negative (GTP and ATP) allosteric factors. We hypothesized that the phosphate potential of beta-cells regulates the sensitivity of leucine stimulation. These predictions were tested by measuring leucine-stimulated insulin secretion in perifused rat islets following glucose depletion and by tracing the nitrogen flux of [2-(15)N]glutamine using stable isotope techniques. The sensitivity of leucine stimulation was enhanced by long time (120-min) energy depletion and inhibited by glucose pretreatment. After limited 50-min glucose depletion, leucine, not alpha-ketoisocaproate, failed to stimulate insulin release. beta-Cells sensitivity to leucine is therefore proposed to be a function of GDH activation. Leucine increased the flux through GDH 3-fold compared with controls while causing insulin release. High glucose inhibited flux through both glutaminase and GDH, and leucine was unable to override this inhibition. These results clearly show that leucine induced the secretion of insulin by augmenting glutaminolysis through activating glutaminase and GDH. Glucose regulates beta-cell sensitivity to leucine by elevating the ratio of ATP and GTP to ADP and P(i) and thereby decreasing the flux through GDH and glutaminase. These mechanisms provide an explanation for hypoglycemia caused by mutations of GDH in children.
Metabolism of Brain Amino Acids Following Pentylenetetrazole Treatment
We studied the effects of pentylenetetrazole (PTZ) on brain amino acid metabolism in mice. Administration of this convulsant did not change forebrain concentrations of amino acids, but when treated animals also received an injection of [15N]leucine, which served as a tracer of brain nitrogen metabolism, total (14N+15N) forebrain [leucine] exceeded control and [glutamate] and [aspartate] were less than control, as were forebrain concentrations of [15N]glutamate and [2-15N]glutamine. These data suggest greater uptake of [15N]leucine but diminished transamination of leucine to glutamate in experimental mice. In contrast to the [15N]leucine studies, which were associated with increased brain [leucine], the administration of [15N]alanine did not alter levels of alanine, glutamate or glutamine. However, label appeared in [2-15N]glutamine much more readily with [15N]alanine than with [15N]leucine as precursor and the ratio of enrichment in [2-15N]glutamine/[15N]alanine was much higher than that in [2-15N]glutamine/[15N]leucine, a finding that is compatible with preferential metabolism of alanine in astrocytes, which are the primary site of brain glutamine synthetase. We conclude that PTZ treatment favors the uptake of selected amino acids such as leucine but also diminishes transamination of leucine to yield glutamate via branched-chain amino acid transaminase. PTZ treatment may favor the "reverse" transamination of 2-keto-isocaproate (KIC), the ketoacid of leucine, to form leucine and to consume glutamate. A net result of these processes may be to enable the brain more readily to dispose of the glutamate that is released from neurons during convulsive activity.
Role of the Glutamate Dehydrogenase Reaction in Furnishing Aspartate Nitrogen for Urea Synthesis: Studies in Perfused Rat Liver with 15N
The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate.
A Signaling Role of Glutamine in Insulin Secretion
Children with hypoglycemia due to recessive loss of function mutations of the beta-cell ATP-sensitive potassium (K(ATP)) channel can develop hypoglycemia in response to protein feeding. We hypothesized that amino acids might stimulate insulin secretion by unknown mechanisms, because the K(ATP) channel-dependent pathway of insulin secretion is defective. We therefore investigated the effects of amino acids on insulin secretion and intracellular calcium in islets from normal and sulfonylurea receptor 1 knockout (SUR1-/-) mice. Even though SUR1-/- mice are euglycemic, their islets are considered a suitable model for studies of the human genetic defect. SUR1-/- islets, but not normal islets, released insulin in response to an amino acid mixture ramp. This response to amino acids was decreased by 60% when glutamine was omitted. Insulin release by SUR1-/- islets was also stimulated by a ramp of glutamine alone. Glutamine was more potent than leucine or dimethyl glutamate. Basal intracellular calcium was elevated in SUR1-/- islets and was increased further by glutamine. In normal islets, methionine sulfoximine, a glutamine synthetase inhibitor, suppressed insulin release in response to a glucose ramp. This inhibition was reversed by glutamine or by 6-diazo-5-oxo-l-norleucine, a non-metabolizable glutamine analogue. High glucose doubled glutamine levels of islets. Methionine sulfoximine inhibition of glucose stimulated insulin secretion was associated with accumulation of glutamate and aspartate. We hypothesize that glutamine plays a critical role as a signaling molecule in amino acid- and glucose-stimulated insulin secretion, and that beta-cell depolarization and subsequent intracellular calcium elevation are required for this glutamine effect to occur.
Ketogenic Diet, Brain Glutamate Metabolism and Seizure Control
We do not know the mode of action of the ketogenic diet in controlling epilepsy. One possibility is that the diet alters brain handling of glutamate, the major excitatory neurotransmitter and a probable factor in evoking and perpetuating a convulsion. We have found that brain metabolism of ketone bodies can furnish as much as 30% of glutamate and glutamine carbon. Ketone body metabolism also provides acetyl-CoA to the citrate synthetase reaction, in the process consuming oxaloacetate and thereby diminishing the transamination of glutamate to aspartate, a pathway in which oxaloacetate is a reactant. Relatively more glutamate then is available to the glutamate decarboxylase reaction, which increases brain [GABA]. Ketosis also increases brain [GABA] by increasing brain metabolism of acetate, which glia convert to glutamine. GABA-ergic neurons readily take up the latter amino acid and use it as a precursor to GABA. Ketosis also may be associated with altered amino acid transport at the blood-brain barrier. Specifically, ketosis may favor the release from brain of glutamine, which transporters at the blood-brain barrier exchange for blood leucine. Since brain glutamine is formed in astrocytes from glutamate, the overall effect will be to favor the release of glutamate from the nervous system.
Biosynthesis of Agmatine in Isolated Mitochondria and Perfused Rat Liver: Studies with 15N-labelled Arginine
An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol x min(-1) x (mg of protein)(-1). Experiments with an increasing concentration (0- 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol x min(-1) x (mg of protein)(-1) for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol x min(-1) x g(-1)) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway.
The Role of Mitochondrially Bound Arginase in the Regulation of Urea Synthesis: Studies with [U-15N4]arginine, Isolated Mitochondria, and Perfused Rat Liver
The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-(15)N(4)]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of (15)N into various intermediate metabolites of the urea cycle. The results indicate that approximately 8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that approximately 60-70% of external [U-(15)N(4)]arginine catabolism was recovered as (15)N-labeled ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of (15)N-labeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U(m+1)) or two (U(m+2)) (15)N was generated from perfusate [U-(15)N(4)]arginine. The output of U(m+2) was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U(m+1) was formed following mitochondrial production of [(15)N]glutamate from [alpha,delta-(15)N(2)]ornithine and transamination of [(15)N]glutamate to [(15)N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic ornithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-(15)N(4)]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing ornithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.
Response of Brain Amino Acid Metabolism to Ketosis
Our objective was to study brain amino acid metabolism in response to ketosis. The underlying hypothesis is that ketosis is associated with a fundamental change of brain amino acid handling and that this alteration is a factor in the anti-epileptic effect of the ketogenic diet. Specifically, we hypothesize that brain converts ketone bodies to acetyl-CoA and that this results in increased flux through the citrate synthetase reaction. As a result, oxaloacetate is consumed and is less available to the aspartate aminotransferase reaction; therefore, less glutamate is converted to aspartate and relatively more glutamate becomes available to the glutamine synthetase and glutamate decarboxylase reactions. We found in a mouse model of ketosis that the concentration of forebrain aspartate was diminished but the concentration of acetyl-CoA was increased. Studies of the incorporation of 13C into glutamate and glutamine with either [1-(13)C]glucose or [2-(13)C]acetate as precursor showed that ketotic brain metabolized relatively less glucose and relatively more acetate. When the ketotic mice were administered both acetate and a nitrogen donor, such as alanine or leucine, they manifested an increased forebrain concentration of glutamine and GABA. These findings supported the hypothesis that in ketosis there is greater production of acetyl-CoA and a consequent alteration in the equilibrium of the aspartate aminotransferase reaction that results in diminished aspartate production and potentially enhanced synthesis of glutamine and GABA.
Brain Amino Acid Requirements and Toxicity: the Example of Leucine
Glutamic acid is an important excitatory neurotransmitter of the brain. Two key goals of brain amino acid handling are to maintain a very low intrasynaptic concentration of glutamic acid and also to provide the system with precursors from which to synthesize glutamate. The intrasynaptic glutamate level must be kept low to maximize the signal-to-noise ratio upon the release of glutamate from nerve terminals and to minimize the risk of excitotoxicity consequent to excessive glutamatergic stimulation of susceptible neurons. The brain must also provide neurons with a constant supply of glutamate, which both neurons and glia robustly oxidize. The branched-chain amino acids (BCAAs), particularly leucine, play an important role in this regard. Leucine enters the brain from the blood more rapidly than any other amino acid. Astrocytes, which are in close approximation to brain capillaries, probably are the initial site of metabolism of leucine. A mitochondrial branched-chain aminotransferase is very active in these cells. Indeed, from 30 to 50% of all alpha-amino groups of brain glutamate and glutamine are derived from leucine alone. Astrocytes release the cognate ketoacid [alpha-ketoisocaproate (KIC)] to neurons, which have a cytosolic branched-chain aminotransferase that reaminates the KIC to leucine, in the process consuming glutamate and providing a mechanism for the "buffering" of glutamate if concentrations become excessive. In maple syrup urine disease, or a congenital deficiency of branched-chain ketoacid dehydrogenase, the brain concentration of KIC and other branched-chain ketoacids can increase 10- to 20-fold. This leads to a depletion of glutamate and a consequent reduction in the concentration of brain glutamine, aspartate, alanine, and other amino acids. The result is a compromise of energy metabolism because of a failure of the malate-aspartate shuttle and a diminished rate of protein synthesis.
Agmatine Stimulates Hepatic Fatty Acid Oxidation: a Possible Mechanism for Up-regulation of Ureagenesis
We demonstrated previously in a liver perfusion system that agmatine increases oxygen consumption as well as the synthesis of N-acetylglutamate and urea by an undefined mechanism. In this study our aim was to identify the mechanism(s) by which agmatine up-regulates ureagenesis. We hypothesized that increased oxygen consumption and N-acetylglutamate and urea synthesis are coupled to agmatine-induced stimulation of mitochondrial fatty acid oxidation. We used 13C-labeled fatty acid as a tracer in either a liver perfusion system or isolated mitochondria to monitor fatty acid oxidation and the incorporation of 13C-labeled acetyl-CoA into ketone bodies, tricarboxylic acid cycle intermediates, amino acids, and N-acetylglutamate. With [U-13C16] palmitate in the perfusate, agmatine significantly increased the output of 13C-labeled beta-hydroxybutyrate, acetoacetate, and CO2, indicating stimulated fatty acid oxidation. The stimulation of [U-13C16]palmitate oxidation was accompanied by greater production of urea and a higher 13C enrichment in glutamate, N-acetylglutamate, and aspartate. These observations suggest that agmatine leads to increased incorporation and flux of 13C-labeled acetyl-CoA in the tricarboxylic acid cycle and to increased utilization of 13C-labeled acetyl-CoA for synthesis of N-acetylglutamate. Experiments with isolated mitochondria and 13C-labeled octanoic acid also demonstrated that agmatine increased synthesis of 13C-labeled beta-hydroxybutyrate, acetoacetate, and N-acetylglutamate. The current data document that agmatine stimulates mitochondrial beta-oxidation and suggest a coupling between the stimulation of hepatic beta-oxidation and up-regulation of ureagenesis. This action of agmatine may be mediated via a second messenger such as cAMP, and the effects on ureagenesis and fatty acid oxidation may occur simultaneously and/or independently.
Short-term Fasting, Seizure Control and Brain Amino Acid Metabolism
The ketogenic diet is an effective treatment for seizures, but the mechanism of action is unknown. It is uncertain whether the anti-epileptic effect presupposes ketosis, or whether the restriction of calories and/or carbohydrate might be sufficient. We found that a relatively brief (24 h) period of low glucose and low calorie intake significantly attenuated the severity of seizures in young Sprague-Dawley rats (50-70 gms) in whom convulsions were induced by administration of pentylenetetrazole (PTZ). The blood glucose concentration was lower in animals that received less dietary glucose, but the brain glucose level did not differ from control blood [3-OH-butyrate] tended to be higher in blood, but not in brain, of animals on a low-glucose intake. The concentration in brain of glutamine increased and that of alanine declined significantly with low-glucose intake. The blood alanine level fell more than that of brain alanine, resulting in a marked increase ( approximately 50%) in the brain:blood ratio for alanine. In contrast, the brain:blood ratio for leucine declined by about 35% in the low-glucose group. When animals received [1-(13)C]glucose, a metabolic precursor of alanine, the appearance of (13)C in alanine and glutamine increased significantly relative to control. The brain:blood ratio for [(13)C]alanine exceeded 1, indicating that the alanine must have been formed in brain and not transported from blood. The elevated brain(alanine):blood(alanine) could mean that a component of the anti-epileptic effect of low carbohydrate intake is release of alanine from brain-to-blood, in the process abetting the disposal of glutamate, excess levels of which in the synaptic cleft would contribute to the development of seizures.
Effects of a GTP-insensitive Mutation of Glutamate Dehydrogenase on Insulin Secretion in Transgenic Mice
Glutamate dehydrogenase (GDH) plays an important role in insulin secretion as evidenced in children by gain of function mutations of this enzyme that cause a hyperinsulinism-hyperammonemia syndrome (GDH-HI) and sensitize beta-cells to leucine stimulation. GDH transgenic mice were generated to express the human GDH-HI H454Y mutation and human wild-type GDH in islets driven by the rat insulin promoter. H454Y transgene expression was confirmed by increased GDH enzyme activity in islets and decreased sensitivity to GTP inhibition. The H454Y GDH transgenic mice had hypoglycemia with normal growth rates. H454Y GDH transgenic islets were more sensitive to leucine- and glutamine-stimulated insulin secretion but had decreased response to glucose stimulation. The fluxes via GDH and glutaminase were measured by tracing 15N flux from [2-15N]glutamine. The H454Y transgene in islets had higher insulin secretion in response to glutamine alone and had 2-fold greater GDH flux. High glucose inhibited both glutaminase and GDH flux, and leucine could not override this inhibition. 15NH4Cl tracing studies showed 15N was not incorporated into glutamate in either H454Y transgenic or normal islets. In conclusion, we generated a GDH-HI disease mouse model that has a hypoglycemia phenotype and confirmed that the mutation of H454Y is disease causing. Stimulation of insulin release by the H454Y GDH mutation or by leucine activation is associated with increased oxidative deamination of glutamate via GDH. This study suggests that GDH functions predominantly in the direction of glutamate oxidation rather than glutamate synthesis in mouse islets and that this flux is tightly controlled by glucose.
Ifosfamide-induced Nephrotoxicity: Mechanism and Prevention
The efficacy of ifosfamide (IFO), an antineoplastic drug, is severely limited by a high incidence of nephrotoxicity of unknown etiology. We hypothesized that inhibition of complex I (C-I) by chloroacetaldehyde (CAA), a metabolite of IFO, is the chief cause of nephrotoxicity, and that agmatine (AGM), which we found to augment mitochondrial oxidative phosphorylation and beta-oxidation, would prevent nephrotoxicity. Our model system was isolated mitochondria obtained from the kidney cortex of rats treated with IFO or IFO + AGM. Oxidative phosphorylation was determined with electron donors specific to complexes I, II, III, or IV (C-I, C-II, C-III, or C-IV, respectively). A parallel study was done with (13)C-labeled pyruvate to assess metabolic dysfunction. Ifosfamide treatment significantly inhibited oxidative phosphorylation with only C-I substrates. Inhibition of C-I was associated with a significant elevation of [NADH], depletion of [NAD], and decreased flux through pyruvate dehydrogenase and the TCA cycle. However, administration of AGM with IFO increased [cyclic AMP (cAMP)] and prevented IFO-induced inhibition of C-I. In vitro studies with various metabolites of IFO showed that only CAA inhibited C-I, even with supplementation with 2-mercaptoethane sulfonic acid. Following IFO treatment daily for 5 days with 50 mg/kg, the level of CAA in the renal cortex was approximately 15 micromol/L. Taken together, these observations support the hypothesis that CAA is accumulated in renal cortex and is responsible for nephrotoxicity. AGM may be protective by increasing tissue [cAMP], which phosphorylates NADH:oxidoreductase. The current findings may have an important implication for the prevention of IFO-induced nephrotoxicity and/or mitochondrial diseases secondary to defective C-I.
The Ketogenic Diet and Brain Metabolism of Amino Acids: Relationship to the Anticonvulsant Effect
In many epileptic patients, anticonvulsant drugs either fail adequately to control seizures or they cause serious side effects. An important adjunct to pharmacologic therapy is the ketogenic diet, which often improves seizure control, even in patients who respond poorly to medications. The mechanisms that explain the therapeutic effect are incompletely understood. Evidence points to an effect on brain handling of amino acids, especially glutamic acid, the major excitatory neurotransmitter of the central nervous system. The diet may limit the availability of oxaloacetate to the aspartate aminotransferase reaction, an important route of brain glutamate handling. As a result, more glutamate becomes accessible to the glutamate decarboxylase reaction to yield gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter and an important antiseizure agent. In addition, the ketogenic diet appears to favor the synthesis of glutamine, an essential precursor to GABA. This occurs both because ketone body carbon is metabolized to glutamine and because in ketosis there is increased consumption of acetate, which astrocytes in the brain quickly convert to glutamine. The ketogenic diet also may facilitate mechanisms by which the brain exports to blood compounds such as glutamine and alanine, in the process favoring the removal of glutamate carbon and nitrogen.
Beyond Aerobic Glycolysis: Transformed Cells Can Engage in Glutamine Metabolism That Exceeds the Requirement for Protein and Nucleotide Synthesis
Tumor cell proliferation requires rapid synthesis of macromolecules including lipids, proteins, and nucleotides. Many tumor cells exhibit rapid glucose consumption, with most of the glucose-derived carbon being secreted as lactate despite abundant oxygen availability (the Warburg effect). Here, we used 13C NMR spectroscopy to examine the metabolism of glioblastoma cells exhibiting aerobic glycolysis. In these cells, the tricarboxylic acid (TCA) cycle was active but was characterized by an efflux of substrates for use in biosynthetic pathways, particularly fatty acid synthesis. The success of this synthetic activity depends on activation of pathways to generate reductive power (NADPH) and to restore oxaloacetate for continued TCA cycle function (anaplerosis). Surprisingly, both these needs were met by a high rate of glutamine metabolism. First, conversion of glutamine to lactate (glutaminolysis) was rapid enough to produce sufficient NADPH to support fatty acid synthesis. Second, despite substantial mitochondrial pyruvate metabolism, pyruvate carboxylation was suppressed, and anaplerotic oxaloacetate was derived from glutamine. Glutamine catabolism was accompanied by secretion of alanine and ammonia, such that most of the amino groups from glutamine were lost from the cell rather than incorporated into other molecules. These data demonstrate that transformed cells exhibit a high rate of glutamine consumption that cannot be explained by the nitrogen demand imposed by nucleotide synthesis or maintenance of nonessential amino acid pools. Rather, glutamine metabolism provides a carbon source that facilitates the cell's ability to use glucose-derived carbon and TCA cycle intermediates as biosynthetic precursors.
3-isobutylmethylxanthine Inhibits Hepatic Urea Synthesis: Protection by Agmatine
We previously showed that agmatine stimulated hepatic ureagenesis. In this study, we sought to determine whether the action of agmatine is mediated via cAMP signaling. A pilot experiment demonstrated that the phosphodiesterase inhibitor, 3-isobutylmethylxanthine (IBMX), inhibited urea synthesis albeit increased [cAMP]. Thus, we hypothesized that IBMX inhibits hepatic urea synthesis independent of [cAMP]. We further theorized that agmatine would negate the IBMX action and improve ureagenesis. Experiments were carried out with isolated mitochondria and (15)NH(4)Cl to trace [(15)N]citrulline production or [5-(15)N]glutamine and a rat liver perfusion system to trace ureagenesis. The results demonstrate that IBMX induced the following: (i) inhibition of the mitochondrial respiratory chain and diminished O(2) consumption during liver perfusion; (ii) depletion of the phosphorylation potential and overall hepatic energetic capacity; (iii) inhibition of [(15)N]citrulline synthesis; and (iv) inhibition of urea output in liver perfusion with little effect on [N-acetylglutamate]. The results indicate that IBMX directly and specifically inhibited complex I of the respiratory chain and carbamoyl-phosphate synthase-I (CPS-I), with an EC(50) about 0.6 mm despite a significant elevation of hepatic [cAMP]. Perfusion of agmatine with IBMX stimulated O(2) consumption, restored hepatic phosphorylation potential, and significantly stimulated ureagenesis. The action of agmatine may signify a cascade effect initiated by increased oxidative phosphorylation and greater ATP synthesis. In addition, agmatine may prevent IBMX from binding to one or more active site(s) of CPS-I and thus protect against inhibition of CPS-I. Together, the data may suggest a new experimental application of IBMX in studies of CPS-I malfunction and the use of agmatine as intervention therapy.
N-carbamylglutamate Markedly Enhances Ureagenesis in N-acetylglutamate Deficiency and Propionic Acidemia As Measured by Isotopic Incorporation and Blood Biomarkers
N-acetylglutamate (NAG) is an endogenous essential cofactor for conversion of ammonia to urea in the liver. Deficiency of NAG causes hyperammonemia and occurs because of inherited deficiency of its producing enzyme, NAG synthase (NAGS), or interference with its function by short fatty acid derivatives. N-carbamylglutamate (NCG) can ameliorate hyperammonemia from NAGS deficiency and propionic and methylmalonic acidemia. We developed a stable isotope (13)C tracer method to measure ureagenesis and to evaluate the effect of NCG in humans. Seventeen healthy adults were investigated for the incorporation of (13)C label into urea. [(13)C]urea appeared in the blood within minutes, reaching maximum by 100 min, whereas breath (13)CO(2) reached a maximum by 60 min. A patient with NAGS deficiency showed very little urea labeling before treatment with NCG and normal labeling thereafter. Correspondingly, plasma levels of ammonia and glutamine decreased markedly and urea tripled after NCG treatment. Similarly, in a patient with propionic acidemia, NCG treatment resulted in a marked increase in urea labeling and decrease in glutamine, alanine, and glycine. These results provide a reliable method for measuring the effect of NCG on nitrogen metabolism and strongly suggest that NCG could be an effective treatment for inherited and secondary NAGS deficiency.
Elimination of KATP Channels in Mouse Islets Results in Elevated [U-13C]glucose Metabolism, Glutaminolysis, and Pyruvate Cycling but a Decreased Gamma-aminobutyric Acid Shunt
Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1(-/-)) and control mouse islets with d-[U-(13)C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1(-/-) islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-beta-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1(-/-) and glyburide-treated SUR1(+/+) islets. Glucose oxidation and pyruvate cycling was increased in SUR1(-/-) islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1(-/-) islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1(-/-) islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.
Myc Regulates a Transcriptional Program That Stimulates Mitochondrial Glutaminolysis and Leads to Glutamine Addiction
Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Most of the remaining metabolites taken up by proliferating cells are not catabolized, but instead are used as building blocks during anabolic macromolecular synthesis. Investigations of phosphoinositol 3-kinase (PI3K) and its downstream effector AKT have confirmed that these oncogenes play a direct role in stimulating glucose uptake and metabolism, rendering the transformed cell addicted to glucose for the maintenance of survival. In contrast, less is known about the regulation of glutamine uptake and metabolism. Here, we report that the transcriptional regulatory properties of the oncogene Myc coordinate the expression of genes necessary for cells to engage in glutamine catabolism that exceeds the cellular requirement for protein and nucleotide biosynthesis. A consequence of this Myc-dependent glutaminolysis is the reprogramming of mitochondrial metabolism to depend on glutamine catabolism to sustain cellular viability and TCA cycle anapleurosis. The ability of Myc-expressing cells to engage in glutaminolysis does not depend on concomitant activation of PI3K or AKT. The stimulation of mitochondrial glutamine metabolism resulted in reduced glucose carbon entering the TCA cycle and a decreased contribution of glucose to the mitochondrial-dependent synthesis of phospholipids. These data suggest that oncogenic levels of Myc induce a transcriptional program that promotes glutaminolysis and triggers cellular addiction to glutamine as a bioenergetic substrate.
Ketosis and Brain Handling of Glutamate, Glutamine, and GABA
We hypothesize that one mechanism of the anti-epileptic effect of the ketogenic diet is to alter brain handling of glutamate. According to this formulation, in ketotic brain astrocyte metabolism is more active, resulting in enhanced conversion of glutamate to glutamine. This allows for: (a) more efficient removal of glutamate, the most important excitatory neurotransmitter; and (b) more efficient conversion of glutamine to GABA, the major inhibitory neurotransmitter.
Effects of a Single Dose of N-carbamylglutamate on the Rate of Ureagenesis
We studied the effect on ureagenesis of a single dose of N-carbamylglutamate (NCG) in healthy young adults who received a constant infusion (300 min) of NaH(13)CO(3). Isotope ratio-mass spectrometry was used to measure the appearance of label in [(13)C]urea. At 90 min after initiating the H(13)CO3-infusion each subject took a single dose of NCG (50 mg/kg). In 5/6 studies the administration of NCG increased the formation of [(13)C]urea. Treatment with NCG significantly diminished the concentration of blood alanine, but not that of glutamine or arginine. The blood glucose concentration was unaffected by NCG administration. No untoward side effects were observed. The data indicate that treatment with NCG stimulates ureagenesis and could be useful in clinical settings of acute hyperammonemia of various etiologies.
Measuring in Vivo Ureagenesis with Stable Isotopes
Stable isotopes have been an invaluable adjunct to biomedical research for more than 70years. Indeed, the isotopic approach has revolutionized our understanding of metabolism, revealing it to be an intensely dynamic process characterized by an unending cycle of synthesis and degradation. Isotopic studies have taught us that the urea cycle is intrinsic to such dynamism, since it affords a capacious mechanism by which to eliminate waste nitrogen when rates of protein degradation (or dietary protein intake) are especially high. Isotopes have enabled an appreciation of the degree to which ureagenesis is compromised in patients with urea cycle defects. Indeed, isotopic studies of urea cycle flux correlate well with the severity of cognitive impairment in these patients. Finally, the use of isotopes affords an ideal tool with which to gauge the efficacy of therapeutic interventions to augment residual flux through the cycle.
Down-regulation of Hepatic Urea Synthesis by Oxypurines: Xanthine and Uric Acid Inhibit N-acetylglutamate Synthase
We previously reported that isobutylmethylxanthine (IBMX), a derivative of oxypurine, inhibits citrulline synthesis by an as yet unknown mechanism. Here, we demonstrate that IBMX and other oxypurines containing a 2,6-dione group interfere with the binding of glutamate to the active site of N-acetylglutamate synthetase (NAGS), thereby decreasing synthesis of N-acetylglutamate, the obligatory activator of carbamoyl phosphate synthase-1 (CPS1). The result is reduction of citrulline and urea synthesis. Experiments were performed with (15)N-labeled substrates, purified hepatic CPS1, and recombinant mouse NAGS as well as isolated mitochondria. We also used isolated hepatocytes to examine the action of various oxypurines on ureagenesis and to assess the ameliorating affect of N-carbamylglutamate and/or l-arginine on NAGS inhibition. Among various oxypurines tested, only IBMX, xanthine, or uric acid significantly increased the apparent K(m) for glutamate and decreased velocity of NAGS, with little effect on CPS1. The inhibition of NAGS is time- and dose-dependent and leads to decreased formation of the CPS1-N-acetylglutamate complex and consequent inhibition of citrulline and urea synthesis. However, such inhibition was reversed by supplementation with N-carbamylglutamate. The data demonstrate that xanthine and uric acid, both physiologically occurring oxypurines, inhibit the hepatic synthesis of N-acetylglutamate. An important and novel concept emerging from this study is that xanthine and/or uric acid may have a role in the regulation of ureagenesis and, thus, nitrogen homeostasis in normal and disease states.
