Levels Of Α-ketoglutarate Research Articles

Abstract 4963: Targeting glutamine metabolism as a means of enhancing antitumor T-cell responses

Abstract Tumors possess specialized metabolic programming that facilitates their rapid growth. We have developed a novel glutamine antagonist (JHU-083) that not only demonstrates single-agent efficacy in a variety of mouse tumor models but also synergizes with anti-PD-1, adoptive cellular therapy (ACT) and A2aR antagonism to overcome resistance to immunotherapy and promote durable cures. Interestingly, we have observed that JHU-083 markedly enhances endogenous antitumor T-cell responses even in the absence of additional immunotherapy. Flow cytometry analyses of tumor-infiltrating lymphocyte (TIL) reveal markedly increased infiltration of CD8+ T cells with increased proliferative index (Ki67) and the generation of robust memory phenotypes in JHU-083 treated mice. Interestingly, Gene Set Enrichment Analyses (GSEA) using RNA-sequencing data revealed that CD8+ TIL from vehicle treated mice showed significantly enhanced expression of apoptotic transcriptional programs compared with TIL from mice treated with JHU-083. Consistent with flow cytometry data, GSEA showed CD8+ TILs from mice treated with glutamine antagonism expressed transcriptional programs characteristic of long-lived memory cells. To this end, vaccination experiments in mice treated with JHU-083 demonstrate that many of the phenotypic changes observed in TIL can be generated outside the TME and are due to direct effects of glutamine blockade on effector T cells. Our studies show that these transcriptional changes are associated with profound remodeling of the histone epigenetic code and correlate with decreased intracellular levels of α-ketoglutarate in response to glutamine antagonism. Supplementation with dimethyl α-ketoglutarate, a cell-permeable form of α-ketoglutarate, partially reverses phenotypic and epigenetic changes in CD8 T cells undergoing activation in the setting of glutamine antagonism. These findings are consistent with inhibition of a family of enzymes known as α-ketoglutarate dependent dioxygenases, which are responsible for a broad range of demethylation reactions, including histone and DNA demethylation. Overall, our work demonstrates that glutamine antagonism can directly inhibit tumor growth and survival while also reprogramming maladaptive antitumor T-cell responses to enhance endogenous antitumor immunity. Such observations support the development of our novel glutamine antagonists as both monotherapy and in combination with immunotherapy. Citation Format: Robert Leone, Judson Englert, Im-Meng Sun, Min-Hee Oh, Jesse Alt, Im-Hong Sun, Ada J. Tam, Pavel Majer, Rana Rais, Barbara Slusher, Jonathan Powell. Targeting glutamine metabolism as a means of enhancing antitumor T-cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4963.

Metabolite profiling of Listeria innocua for unravelling the inactivation mechanism of electrolysed water by nuclear magnetic resonance spectroscopy

Bactericidal effects of low concentration electrolysed water (LcEW) on microorganisms are previously well reported; however, the inactivation mechanism of EW is not understood. The lethal and sublethal injuries of L. monocytogenes and L. innocua by EW treatments were determined and the metabolic profile changes for L. innocua were characterised using nuclear magnetic resonance (NMR). Microbial metabolomics approach combined with multivariate data analyses was used to interpret the cellular chemical fingerprints of L. innocua. The relative amount of intracellular reactive oxygen species (ROS) was assayed using 2′,7-dichlorodihydrofluorescein diacetate (H2DCFDA). The results showed that the proportion of the sublethally injured microbial cells L. monocytogenes and L. innocua increased from 40% to 70% and from 35% to 65%, respectively, when the free available chlorine (FAC) of LcEW increased from 2 to 8 mg/L. Overall, 36 low-molecular-weight metabolic compounds in L. innocua extracts were characterised by NMR spectroscopy. EW perturbation resulted in a drastic and multitude disruption across a wide range of biochemical process including peptidoglycan synthesis, nucleotides biosynthesis and amino acid metabolism. Elevated levels of α-ketoglutarate and succinate implicated the enhanced glutamate decarboxylase (GAD) system and γ-aminobutyric acid (GABA) shunt for the protection against oxidative stress. These findings provided the comprehensive insights into the metabolic response of Listeria to EW oxidative stress and can serve as a basis for better utilisation for sanitisation.

Different behavior of polyphenols in energy metabolism of lipopolysaccharide-stimulated cells

Energy metabolism is one of the main sources of reactive oxygen species leading to oxidation and inflammation in pathophysiological processes. Lipopolysaccharide (LPS)-activated mouse embryonic fibroblast (MEF) cell lines from knock-out mice for paraoxonase-1 and from transgenic mice overexpressing monocyte chemoattractant protein-1 were obtained as model of pro-oxidant and pro-inflammatory scenarios. Theobroma cacao and Lippia citriodora (worldwide consumed and common ingredient of many food products) were tested in these cell models to assess the action of polyphenols in the energy management. Our metabolomics experiments show a different behavior of polyphenols: T. cacao extract partially reverts the effect of LPS in a pro-oxidant scenario through the antioxidant properties of theobromine, flavonols and procyanidins, while L. citriodora seems to act mainly in a pro-inflammatory cell model through the action of verbascoside decreasing the production of pro-inflammatory cytokines and MCP-1. Nevertheless, the action of polyphenols cannot be attributed only to a mechanism of action but the sum of different modulations in biological pathways. The capacity of both plant extracts to decrease α-ketoglutarate levels merits special attention due to the implications in future medicine. The action of polyphenols modulating oxidative stress, cytokine production and epigenetic changes make an interesting source of bioactive compounds for nutraceutical or functional food purposes.

Glutamate Dehydrogenase as a Neuroprotective Target Against Neurodegeneration.

Regulation of glutamate metabolism via glutamate dehydrogenase (GDH) might be the promising therapeutic approach for treating neurodegenerative disorders. In the central nervous system, glutamate functions both as a major excitatory neurotransmitter and as a key intermediate metabolite for neurons. GDH converts glutamate to α-ketoglutarate, which serves as a TCA cycle intermediate. Dysregulated GDH activity in the central nervous system is highly correlated with neurological disorders. Indeed, studies conducted with mutant mice and allosteric drugs have shown that deficient or overexpressed GDH activity in the brain can regulate whole body energy metabolism and affect early onset of Parkinson's disease, Alzheimer's disease, temporal lobe epilepsy, and spinocerebellar atrophy. Moreover, in strokes with excitotoxicity as the main pathophysiology, mice that overexpressed GDH exhibited smaller ischemic lesion than mice with normal GDH expression. In additions, GDH activators improve lesions in vivo by increasing α-ketoglutarate levels. In neurons exposed to an insult in vitro, enhanced GDH activity increases ATP levels. Thus, in an energy crisis, neuronal mitochondrial activity is improved and excitotoxic risk is reduced. Consequently, modulating GDH activity in energy-depleted conditions could be a sound strategy for maintaining the mitochondrial factory in neurons, and thus, protect against metabolic failure.

Diosmin Prevents Isoproterenol-Induced Heart Mitochondrial Oxidative Stress in Rats.

Cardiac mitochondrial oxidative stress causes mitochondrial damage that plays an important role in the pathology of myocardial infarction. The preventive effects of diosmin on cardiac mitochondrial oxidative stress in isoproterenol-induced myocardial infarcted rats were evaluated. Rats were pretreated with diosmin (10mg/kg body weight) daily for 10days. Myocardial infarction was induced in rats by isoproterenol (100mg/kg body weight) injection twice at an interval of 24h (on 11th and 12th day). Isoproterenol-induced myocardial infarcted rats showed a significant increase in the levels of cardiac diagnostic markers, heart mitochondrial lipid peroxidation, calcium ion, and a significant decrease in the levels of heart mitochondrial glutathione peroxidase, reduced glutathione, glutathione-S-transferase, isocitrate, malate, α-ketoglutarate, and succinate dehydrogenases. Transmission electron microscopic findings revealed damaged mitochondria with loss of cristae, swelling, and vacuolation in isoproterenol-induced rats' heart. Diosmin pretreatment showed significant preventive effects on all the biochemical parameters, and the structure of mitochondria was evaluated. Furthermore, the transmission electron microscopic study confirms the biochemical findings. The antioxidant and negative inotropic effects of diosmin inhibited cardiac mitochondrial oxidative stress and prevented mitochondrial damage in myocardial infarcted rats.

Mitochondrial H+-ATP synthase in human skeletal muscle: contribution to dyslipidaemia and insulin resistance.

Mitochondria are important regulators of the metabolic phenotype in type 2 diabetes. A key factor in mitochondrial physiology is the H+-ATP synthase. The expression and activity of its physiological inhibitor, ATPase inhibitory factor 1 (IF1), controls tissue homeostasis, metabolic reprogramming and signalling. We aimed to characterise the putative role of IF1 in mediating skeletal muscle metabolism in obesity and diabetes. We examined the 'mitochondrial signature' of obesity and type 2 diabetes in a cohort of 100 metabolically characterised human skeletal muscle biopsy samples. The expression and activity of H+-ATP synthase, IF1 and key mitochondrial proteins were characterised, including their association with BMI, fasting plasma insulin, fasting plasma glucose and HOMA-IR. IF1 was also overexpressed in primary cultures of human myotubes derived from the same biopsies to unveil the possible role played by the pathological inhibition of the H+-ATP synthase in skeletal muscle. The results indicate that type 2 diabetes and obesity act via different mechanisms to impair H+-ATP synthase activity in human skeletal muscle (76% reduction in its catalytic subunit vs 280% increase in IF1 expression, respectively) and unveil a new pathway by which IF1 influences lipid metabolism. Mechanistically, IF1 altered cellular levels of α-ketoglutarate and L-carnitine metabolism in the myotubes of obese (84% of control) and diabetic (76% of control) individuals, leading to limited β-oxidation of fatty acids (60% of control) and their cytosolic accumulation (164% of control). These events led to enhanced release of TNF-α (10±2pg/ml, 27±5pg/ml and 35±4pg/ml in control, obese and type 2 diabetic participants, respectively), which probably contributes to an insulin resistant phenotype. Overall, our data highlight IF1 as a novel regulator of lipid metabolism and metabolic disorders, and a possible target for therapeutic intervention.

Glutamate Metabolism in Brain Structures in Experimental Hemorrhagic Shock

Purpose. To study glutamate metabolism characteristics in phylogenetically different parts of the mammalian brain in experimentally induced hemorrhagic shock (HS) in cats. Material and methods. Experiments were performed on 76 cats. HS was induced by intermittent bloodletting from femoral artery at a rate of 10ml/kg•10 minutes, with the average volume of 24±0.8 ml/kg. The bloodletting was discontinued after arterial pressure (BP) drop to 60.0±1.5 mmHg. We studied ammonia, glutamate (Gt), and α-ketoglutarate (α-KG) levels and glutaminase (GS) and glutamate dehydrogenase (GDG) activity in specimens harvested from phylogenetically different parts of the brain (cortex, limbic system, diencephalon, and medulla oblongata). Results. In intact animals, the peak GDG activity was found in the medulla oblongata (phylogenetically the oldest part of the brain) and the peak GS activity was registered in the sensorimotor cortex (phylogenetically the youngest part of the brain); the glutaminase activity did not depend on the phylogenetic age of brain structures. In the case of HS, Gt metabolism changes began in the sensorimotor cortex manifested by decreased GS activity, which progresses by the 70th minute of the post%hemorrhagic period (PHP) accompanied by delayed increase in the GDG and glutaminase activity, as well as Gt accumulation. In the limbic system and diencephalon the Gt metabolism was changing (impaired glutamine synthesis, stimuled Gt synthesis with glutamine desamidization and α%KG amination) when developed by the 70th minute of the PHP. Similarly to sensorimotor cortex, changes were associated with Gt accumulation. During the agony, α%KG deficiency developed in all parts of the brain as a result of its increased contribution to Gt synthesis. At the same period of time, in the sensorimotor cortex, limbic system and diencephalon the Gt synthesis from glutamine was stimulated, however, the Gt contribution tothe formation of glutamine was decreased. The accumulation of ammonia regardless of the HS stage was detected only in the sensorimotor cortex, limbic system and diencephalon; in the medulla oblongata ammonium increase was found only during the agony. Сonclusion. HS creates conditions for glutamate accumulation in nerve cells by impairing the metabolism of glutamate in the brain structures. The nature and scope of these disorders depend both on the intensity of glutamate metabolism in phylogenetically different brain structures in acute blood loss and HS.

Circulating tricarboxylic acid cycle metabolite levels in citrin-deficient children with metabolic adaptation, with and without sodium pyruvate treatment.

Citrin deficiency causes adult-onset type II citrullinemia (CTLN-2), which later manifests as severe liver steatosis and life-threatening encephalopathy. Long-standing energy deficit of the liver and brain may predispose ones to CTLN-2. Here, we compared the energy-driving tricarboxylic acid (TCA) cycle and fatty acid β-oxidation cycle between 22 citrin-deficient children (age, 3-13years) with normal liver functions and 37 healthy controls (age, 5-13years). TCA cycle analysis showed that basal plasma citrate and α-ketoglutarate levels were significantly higher in the affected than the control group (p<0.01). Conversely, basal plasma fumarate and malate levels were significantly lower than those for the control (p<0.001). The plasma level of 3-OH-butyrate derived from fatty acid β-oxidation was significantly higher in the affected group (p<0.01). Ten patients underwent sodium pyruvate therapy. However, this therapy did not correct or attenuate such deviations in both cycles. Sodium pyruvate therapy significantly increased fasting insulin secretion (p<0.01); the fasting sugar level remained unchanged. Our results suggest that citrin-deficient children show considerable deviations of TCA cycle metabolite profiles that are resistant to sodium pyruvate treatment. Thus, long-standing and considerable TCA cycle dysfunction might be a pivotal metabolic background of CTLN-2 development.

The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid composition.

The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L-/-) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L-/- mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.

Regional tumour glutamine supply affects chromatin and cell identity

Limited perfusion of solid tumours produces a nutrient-deprived tumour core microenvironment. Low glutamine levels in the tumour core are now shown to lead to reduced levels of α-ketoglutarate and decreased histone demethylase activity, thereby promoting a less differentiated and more therapy-resistant state of the tumour cells.

AMPK/α-Ketoglutarate Axis Dynamically Mediates DNA Demethylation in the Prdm16 Promoter and Brown Adipogenesis

Promoting brown adipose tissue (BAT) development is an attractive strategy for the treatment of obesity, as activated BAT dissipates energy through thermogenesis; however, the mechanisms controlling BAT formation are not fully understood. We hypothesized that as a master regulator of energy metabolism, AMP-activated protein kinase (AMPK) may play a direct role in the process and found that AMPKα1 (PRKAA1) ablation reduced Prdm16 expression and impaired BAT development. During early brown adipogenesis, the cellular levels of α-ketoglutarate (αKG), a key metabolite required for TET-mediated DNA demethylation, were profoundly increased and required for active DNA demethylation of the Prdm16 promoter. AMPKα1 ablation reduced isocitrate dehydrogenase 2 activity and cellular αKG levels. Remarkably, postnatal AMPK activation with AICAR or metformin rescued obesity-induced suppression of brown adipogenesis and thermogenesis. In summary, AMPK is essential for the epigenetic control of BAT development through αKG, thus linking a metabolite to progenitor cell differentiation and thermogenesis.

Regional glutamine deficiency in tumours promotes dedifferentiation through inhibition of histonedemethylation.

Poorly organized tumour vasculature often results in areas of limited nutrient supply and hypoxia. Despite our understanding of solid tumour responses to hypoxia, how nutrient deprivation regionally affects tumour growth and therapeutic response is poorly understood. Here, we show that the core region of solid tumours displayed glutamine deficiency compared with other amino acids. Low glutamine in tumour core regions led to dramatic histone hypermethylation due to decreased α-ketoglutarate levels, a key cofactor for the Jumonji-domain-containing histone demethylases. Using patient-derived (V600E)BRAF melanoma cells, we found that low-glutamine-induced histone hypermethylation resulted in cancer cell dedifferentiation and resistance to BRAF inhibitor treatment, which was largely mediated by methylation on H3K27, as knockdown of the H3K27-specific demethylase KDM6B and the methyltransferase EZH2 respectively reproduced and attenuated the low-glutamine effects in vitro and in vivo. Thus, intratumoral regional variation in the nutritional microenvironment contributes to tumour heterogeneity and therapeutic response.

Cancer-associated isocitrate dehydrogenase mutations induce mitochondrial DNA instability.

A major advance in understanding the progression and prognostic outcome of certain cancers, such as low-grade gliomas, acute myeloid leukaemia, and chondrosarcomas, has been the identification of early-occurring mutations in the NADP+-dependent isocitrate dehydrogenase genes IDH1 and IDH2 These mutations result in the production of the onco-metabolite D-2-hydroxyglutarate (2HG), thought to contribute to disease progression. To better understand the mechanisms of 2HG pathophysiology, we introduced the analogous glioma-associated mutations into the NADP+ isocitrate dehydrogenase genes (IDP1, IDP2, IDP3) in Saccharomyces cerevisiae Intriguingly, expression of the mitochondrial IDP1R148H mutant allele results in high levels of 2HG production as well as extensive mtDNA loss and respiration defects. We find no evidence for a reactive oxygen-mediated mechanism mediating this mtDNA loss. Instead, we show that 2HG production perturbs the iron sensing mechanisms as indicated by upregulation of the Aft1-controlled iron regulon and a concomitant increase in iron levels. Accordingly, iron chelation, or overexpression of a truncated AFT1 allele that dampens transcription of the iron regulon, suppresses the loss of respirative capacity. Additional suppressing factors include overexpression of the mitochondrial aldehyde dehydrogenase gene ALD5 or disruption of the retrograde response transcription factor RTG1 Furthermore, elevated α-ketoglutarate levels also suppress 2HG-mediated respiration loss; consistent with a mechanism by which 2HG contributes to mtDNA loss by acting as a toxic α-ketoglutarate analog. Our findings provide insight into the mechanisms that may contribute to 2HG oncogenicity in glioma and acute myeloid leukaemia progression, with the promise for innovative diagnostic and prognostic strategies and novel therapeutic modalities.

Mitophagy-driven mitochondrial rejuvenation regulates stem cell fate.

Our understanding on how selective mitochondrial autophagy, or mitophagy, can sustain the archetypal properties of stem cells is incomplete. PTEN-induced putative kinase 1 (PINK1) plays a key role in the maintenance of mitochondrial morphology and function and in the selective degradation of damaged mitochondria by mitophagy. Here, using embryonic fibroblasts from PINK1 gene-knockout (KO) mice, we evaluated whether mitophagy is a causal mechanism for the control of cell-fate plasticity and maintenance of pluripotency. Loss of PINK1-dependent mitophagy was sufficient to dramatically decrease the speed and efficiency of induced pluripotent stem cell (iPSC) reprogramming. Mitophagy-deficient iPSC colonies, which were characterized by a mixture of mature and immature mitochondria, seemed unstable, with a strong tendency to spontaneously differentiate and form heterogeneous populations of cells. Although mitophagy-deficient iPSC colonies normally expressed pluripotent markers, functional monitoring of cellular bioenergetics revealed an attenuated glycolysis in mitophagy-deficient iPSC cells. Targeted metabolomics showed a notable alteration in numerous glycolysis- and TCA-related metabolites in mitophagy-deficient iPSC cells, including a significant decrease in the intracellular levels of α-ketoglutarate -a key suppressor of the differentiation path in stem cells. Mitophagy-deficient iPSC colonies exhibited a notably reduced teratoma-initiating capacity, but fully retained their pluripotency and multi-germ layer differentiation capacity in vivo. PINK1-dependent mitophagy pathway is an important mitochondrial switch that determines the efficiency and quality of somatic reprogramming. Mitophagy-driven mitochondrial rejuvenation might contribute to the ability of iPSCs to suppress differentiation by directing bioenergetic transition and metabolome remodeling traits. These findings provide new insights into how mitophagy might influence the stem cell decisions to retain pluripotency or differentiate in tissue regeneration and aging, tumor growth, and regenerative medicine.

Formation of volatile compounds, peptidolysis and carbohydrate fermentation by mesophilic lactobacilli and streptoccocci cultures in a cheese extract

Many studies about the influence of Lactococcus lactis on cheese flavour have been reported, while the impact of mesophilic lactobacilli and Streptococcus thermophilus, either as single or mixed cultures, have been less studied. The ability of Lactobacillus paracasei 90, Lb. casei 72 (INLAIN collection) and S. thermophilus 2 (commercial strain) to produce flavour-related biochemical changes, as single or mixed (lactobacilli + streptococci) cultures, was assessed in a cheese model. These three strains, with different activities of glutamate dehydrogenase (GDH) and aminotransferases (AT), were incubated (14d/37 °C) alone or in mixed cultures in a cheese model consisting of a sterile extract of a fresh cheese. Mesophilic lactobacilli showed fermentation of carbohydrates, an increase of peptidolysis and production of volatile compounds that were correlated with their AT activities. In addition, these strains also produced completely different profiles of the amino acceptor compounds: pyruvate and α-ketoglutarate. S. thermophilus 2 increased the level of α-ketoglutarate due to its high GDH activity, but its contribution to flavour compound production was negligible; small additional changes existed when S. thermophilus 2 was mixed with the lactobacilli. Biochemical changes leading to flavour formation were mainly due to the activity of lactobacilli. Our results suggest that the AT profile and the peptidolytic activity of each strain influenced the volatilome of the extracts during incubation. So, both lactobacilli strains could be used as adjunct cultures in cheeses to increase/diversify the flavour, but more studies are needed to deepen the knowledge about the potential of S. thermophilus 2 for the production of flavour compounds.

Blood metabolomics analysis identifies abnormalities in the citric acid cycle, urea cycle, and amino acid metabolism in bipolar disorder

BackgroundBipolar disorder (BD) is a severe and debilitating psychiatric disorder. However, the precise biological basis remains unknown, hampering the search for novel biomarkers. We performed a metabolomics analysis to discover novel peripheral biomarkers for BD. MethodsWe quantified serum levels of 116 metabolites in mood-stabilized male BD patients (n=54) and age-matched male healthy controls (n=39). ResultsAfter multivariate logistic regression, serum levels of pyruvate, N-acetylglutamic acid, α-ketoglutarate, and arginine were significantly higher in BD patients than in healthy controls. Conversely, serum levels of β-alanine, and serine were significantly lower in BD patients than in healthy controls. Chronic (4-weeks) administration of lithium or valproic acid to adult male rats did not alter serum levels of pyruvate, N-acetylglutamic acid, β-alanine, serine, or arginine, but lithium administration significantly increased serum levels of α-ketoglutarate. ConclusionsThe metabolomics analysis demonstrated altered serum levels of pyruvate, N-acetylglutamic acid, β-alanine, serine, and arginine in BD patients. General significanceThe present findings suggest that abnormalities in the citric acid cycle, urea cycle, and amino acid metabolism play a role in the pathogenesis of BD.

Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain.

Glutamate is present in the brain at an average concentration—typically 10–12 mM—far in excess of those of other amino acids. In glutamate-containing vesicles in the brain, the concentration of glutamate may even exceed 100 mM. Yet because glutamate is a major excitatory neurotransmitter, the concentration of this amino acid in the cerebral extracellular fluid must be kept low—typically µM. The remarkable gradient of glutamate in the different cerebral compartments: vesicles > cytosol/mitochondria > extracellular fluid attests to the extraordinary effectiveness of glutamate transporters and the strict control of enzymes of glutamate catabolism and synthesis in well-defined cellular and subcellular compartments in the brain. A major route for glutamate and ammonia removal is via the glutamine synthetase (glutamate ammonia ligase) reaction. Glutamate is also removed by conversion to the inhibitory neurotransmitter γ-aminobutyrate (GABA) via the action of glutamate decarboxylase. On the other hand, cerebral glutamate levels are maintained by the action of glutaminase and by various α-ketoglutarate-linked aminotransferases (especially aspartate aminotransferase and the mitochondrial and cytosolic forms of the branched-chain aminotransferases). Although the glutamate dehydrogenase reaction is freely reversible, owing to rapid removal of ammonia as glutamine amide, the direction of the glutamate dehydrogenase reaction in the brain in vivo is mainly toward glutamate catabolism rather than toward the net synthesis of glutamate, even under hyperammonemia conditions. During hyperammonemia, there is a large increase in cerebral glutamine content, but only small changes in the levels of glutamate and α-ketoglutarate. Thus, the channeling of glutamate toward glutamine during hyperammonemia results in the net synthesis of 5-carbon units. This increase in 5-carbon units is accomplished in part by the ammonia-induced stimulation of the anaplerotic enzyme pyruvate carboxylase. Here, we suggest that glutamate may constitute a buffer or bulwark against changes in cerebral amine and ammonia nitrogen. Although the glutamate transporters are briefly discussed, the major emphasis of the present review is on the enzymology contributing to the maintenance of glutamate levels under normal and hyperammonemic conditions. Emphasis will also be placed on the central role of glutamate in the glutamine-glutamate and glutamine-GABA neurotransmitter cycles between neurons and astrocytes. Finally, we provide a brief and selective discussion of neuropathology associated with altered cerebral glutamate levels.

Functions for diverse metabolic activities in heterochromatin

Growing evidence demonstrates that metabolism and chromatin dynamics are not separate processes but that they functionally intersect in many ways. For example, the lysine biosynthetic enzyme homocitrate synthase was recently shown to have unexpected functions in DNA damage repair, raising the question of whether other amino acid metabolic enzymes participate in chromatin regulation. Using an in silico screen combined with reporter assays, we discovered that a diverse range of metabolic enzymes function in heterochromatin regulation. Extended analysis of the glutamate dehydrogenase 1 (Gdh1) revealed that it regulates silent information regulator complex recruitment to telomeres and ribosomal DNA. Enhanced N-terminal histone H3 proteolysis is observed in GDH1 mutants, consistent with telomeric silencing defects. A conserved catalytic Asp residue is required for Gdh1's functions in telomeric silencing and H3 clipping. Genetic modulation of α-ketoglutarate levels demonstrates a key regulatory role for this metabolite in telomeric silencing. The metabolic activity of glutamate dehydrogenase thus has important and previously unsuspected roles in regulating chromatin-related processes.

Changes in the metabolome of rats after exposure to arginine and N-carbamylglutamate in combination with diquat, a compound that causes oxidative stress, assessed by 1H NMR spectroscopy.

Numerous factors can induce oxidative stress in animal production and lead to growth retardation, disease, and even death. Arginine and N-carbamylglutamate can alleviate the effects of oxidative stress. However, the systematic changes in metabolic biochemistry linked to oxidative stress and arginine and N-carbamylglutamate treatment remain largely unknown. This study aims to examine the effects of arginine and N-carbamylglutamate on rat metabolism under oxidative stress. Thirty rats were randomly divided into three dietary groups (n = 10 each). The rats were fed a basal diet supplemented with 0 (control), 1% arginine, or 0.1% N-carbamylglutamate for 30 days. On day 28, the rats in each treatment were intraperitoneally injected with diquat at 12 mg per kg body weight or sterile solution. Urine and plasma samples were analyzed by metabolomics. Compared with the diquat group, the arginine + diquat group had significantly lower levels of acetamide, alanine, lysine, pyruvate, tyrosine, α-glucose, and β-glucose in plasma; N-carbamylglutamate + diquat had higher levels of 3-hydroxybutyrate, 3-methylhistidine, acetone, allantoin, asparagine, citrate, phenylalanine, trimethylamine-N-oxide, and tyrosine, and lower levels of low density lipoprotein, lipid, lysine, threonine, unsaturated lipid, urea, and very low density lipoprotein (P < 0.05) in plasma. Compared with the diquat group, the arginine + diquat group had significantly higher levels of citrate, creatinine, homogentisate, and α-ketoglutarate while lower levels of acetamide, citrulline, ethanol, glycine, isobutyrate, lactate, malonate, methymalonate, N-acetylglutamate, N-methylnicotinamide, propionate, and β-glucose (P < 0.05) in urine. Compared with the diquat group, the N-carbamylglutamate + diquat group had significantly higher levels of allantoin, citrate, homogentisate, phenylacetylglycine, α-ketoglutarate, and β-glucose while lower levels of acetamide, acetate, acetone, benzoate, citrulline, ethanol, hippurate, lactate, N-acetylglutamate, nicotinamide, ornithine, and trigonelline (P < 0.05) in urine. Overall, these results suggest that arginine and N-carbamylglutamate can alter the metabolome associated with energy metabolism, amino acid metabolism, and gut microbiota metabolism under oxidative stress.

Abstract B32: FOXOs support the metabolic requirements of normal and tumor cells by regulating IDH1 expression

Abstract FOXO transcription factors are considered bona fide tumor suppressors, however recent studies showed FOXOs are also required for tumor survival. Here we identify FOXOs as transcriptional regulators of IDH1. FOXOs regulate IDH1 expression and thereby maintain the cytosolic levels of α-ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise regulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation likely by repressing differentiation. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion. Furthermore these data suggest a role for FOXOs in maintaining the de-differentiated state by regulating α-ketoglutarate and/or 2-hydroxyglutarate levels. Indeed, in models of differentiation (e.g. 3T3-L1 differentiation towards adipocytes) loss of FOXO, as well as loss of IDH1 expression repress differentiation and we are currently further exploring the molecular underpinnings. Note: This abstract was not presented at the conference. Citation Format: Paraskevi Charitou, Maria Rodriguez-Colman, Johan Gerrits, Nanda Verhoeven-Duif, Boudewijn Burgering. FOXOs support the metabolic requirements of normal and tumor cells by regulating IDH1 expression. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B32.