Higher Glutamine Research Articles

Effects of Dietary Glutamine Supplementation on Heat-Induced Oxidative Stress in Broiler Chickens: A Systematic Review and Meta-Analysis.

In avian species, heat stress (HS) is usually the result of being exposed to high ambient temperatures, whereas oxidative stress (OS) results from the overproduction of reactive oxygen species. The current literature suggests that HS often leads to OS. Therefore, this systematic review and meta-analysis was conducted to assess the effects of dietary supplementation of glutamine on the antioxidant status and growth performances in heat-stressed broilers. A total of 13 studies were deemed eligible after an exhaustive search of the literature from Google Scholar, PubMed, and Scopus. Briefly, the following criteria were used to select the studies: trials performed on broilers; publication in peer-review journals using English as the text language; and sufficient details about the design and inclusion of dietary glutamine as a treatment for HS. Two main categories of outcomes were extracted from the studies included in the review: growth parameters and OS markers. For the meta-analysis, a random effect model was used when the heterogeneity was higher than 50%, and a fixed effect model was applied otherwise. Pooled standardized mean differences (SMD), and mean differences (MD) with their confidence intervals (CI) from the studies revealed that dietary glutamine could increase body weight gain (SMD = 0.70, CI = 0.50 to 0.90, p < 0.05), and feed intake (FI) (SMD = 0.64, CI = 0.43 to 0.86, p < 0.05), and reduce the feed conversion ratio (MD = -0.05, CI = -0.07 to -0.02, p < 0.05) in heat-exposed birds. Additionally, higher glutamine (SMD = 1.21, CI = 1.00 to 1.43, p < 0.05), glutathione (SMD = 1.25, CI = 0.88 to 1.62, p < 0.05), superoxide dismutase (SOD) (SMD = 0.97, CI = 0.58 to 1.36, p < 0.05), and catalase (SMD = 0.94, CI = 0.72 to 1.16, p < 0.05) levels were recorded in the serum, breast, and thigh muscle after supplementation of glutamine. Furthermore, the subgroup analysis revealed that malondialdehydes levels were decreased only in the serum (SMD = -0.83, CI = -1.25 to -0.41, p < 0.001) and thigh muscle (SMD = -1.30, CI = -1.86 to -0.35, p < 0.001) while glutathione peroxidase (GPX) activity was increased in the breast (SMD = 1.32, CI = 0.95 to 1.68, p < 0.05) and thigh muscle (SMD = 1.53, CI = 1.06 to 1.99, p < 0.05). Meta-regression models indicated that longer periods of heat exposure were inversely associated with the effectiveness of dietary glutamine in increasing FI, GPX, and SOD (p < 0.05). Besides, increasing the dietary concentration of glutamine led to higher GPX and SOD levels (p < 0.05). Taken together, results suggest that dietary supplementation of glutamine can effectively mitigate the deleterious effects of HS by enhancing the antioxidant status and increasing growth performances in broilers.

Metabolic Adaptation as Potential Target in Papillary Renal Cell Carcinomas Based on Their In Situ Metabolic Characteristics.

Metabolic characteristics of kidney cancers have mainly been obtained from the most frequent clear cell renal cell carcinoma (CCRCC) studies. Moreover, the bioenergetic perturbances that affect metabolic adaptation possibilities of papillary renal cell carcinoma (PRCC) have not yet been detailed. Therefore, our study aimed to analyze the in situ metabolic features of PRCC vs. CCRCC tissues and compared the metabolic characteristics of PRCC, CCRCC, and normal tubular epithelial cell lines. The protein and mRNA expressions of the molecular elements in mammalian target of rapamycin (mTOR) and additional metabolic pathways were analyzed in human PRCC cases compared to CCRCC. The metabolic protein expression pattern, metabolite content, mTOR, and metabolic inhibitor sensitivity of renal carcinoma cell lines were also studied and compared with tubular epithelial cells, as “normal” control. We observed higher protein expressions of the “alternative bioenergetic pathway” elements, in correlation with the possible higher glutamine and acetate consumption in PRCC cells instead of higher glycolytic and mTOR activity in CCRCCs. Increased expression of certain metabolic pathway markers correlates with the detected differences in metabolite ratios, as well. The lower lactate/pyruvate, lactate/malate, and higher pyruvate/citrate intracellular metabolite ratios in PRCC compared to CCRCC cell lines suggest that ACHN (PRCC) have lower Warburg glycolytic capacity, less pronounced pyruvate to lactate producing activity and shifted OXPHOS phenotype. However, both studied renal carcinoma cell lines showed higher mTOR activity than tubular epithelial cells cultured in vitro, the metabolite ratio, the enzyme expression profiles, and the higher mitochondrial content also suggest increased importance of mitochondrial functions, including mitochondrial OXPHOS in PRCCs. Additionally, PRCC cells showed significant mTOR inhibitor sensitivity and the used metabolic inhibitors increased the effect of rapamycin in combined treatments. Our study revealed in situ metabolic differences in mTOR and metabolic protein expression patterns of human PRCC and CCRCC tissues as well as in cell lines. These underline the importance in the development of specific new treatment strategies, new mTOR inhibitors, and other anti-metabolic drug combinations in PRCC therapy.

Glutamate cycle changes in the putamen of patients with de novo Parkinson's disease using 1H MRS

IntroductionTo investigate glutamatergic metabolism changes in the putamen of patients with de novo Parkinson's Disease (PD) and test the hypothesis that glutamate (Glu) levels are abnormally elevated in the putamen contralateral to where the motor clinical signs predominate as expected from observations in animal models. Methods1H NMR spectra from 17 healthy control volunteers were compared with spectra from 17 de novo PD patients of who 14 were evaluated again after 2–3 years of disease progression. Statistical analysis used random-effects models. ResultsThe only significant difference between PD patients and controls was a higher glutamine (Gln) concentration in the putamen ipsilateral to the hemibody with predominant motor signs (Visit 1: 6.0 ± 0.4 mM vs. 5.2 ± 0.2 mM, p < 0.05; Visit 2: 6.2 ± 0.3 mM vs. 5.2 ± 0.2 mM, p < 0.05). At Visit 1, PD patients had higher Glu and Gln levels in the putamen ipsilateral versus contralateral to dominant clinical signs (Glu: 12.2 ± 0.6 mM vs. 10.4 ± 0.6 mM, p < 0.05; Gln: 6.0 ± 0.4 mM vs. 4.8 ± 0.4 mM, p < 0.05; Glu and Gln pool (Glx): 17.9 ± 0.8 mM vs. 14.7 ± 1.1 mM, p < 0.05). At Visit 2, the sum of the two metabolites remained significantly higher in the ipsilateral versus contralateral putamen (Glx: 18.3 ± 0.6 mM vs. 16.1 ± 0.9 mM, p < 0.05). ConclusionIn de novo PD patients, the putamen ipsilateral to the more affected hemibody showed elevated Gln versus controls and elevated Glu and Gln concentrations versus the contralateral side. Abnormalities in Glu metabolism therefore occur early in PD but unexpectedly in the putamen contralateral to the more damaged hemisphere, suggesting they are not dependent solely on dopamine loss.

Serum metabolomic signatures of gestational diabetes in South Asian and white European women

IntroductionThis study aimed to identify serum metabolomic signatures associated with gestational diabetes mellitus (GDM), and to examine if ethnic-specific differences exist between South Asian and white European women.Research design and...

The phosphorylated pathway of serine biosynthesis links plant growth with nitrogen metabolism.

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

Metabolic Pattern of Systemic Sclerosis: Association of Changes in Plasma Concentrations of Amino Acid-Related Compounds With Disease Presentation.

ObjectiveAmino acids (AA) and their derivatives play an integral role in the synthesis of structural and regulatory elements in human organisms and therefore pathologies such as systemic sclerosis that may alter the blood pattern of these compounds. This study aimed to evaluate changes in plasma concentrations of amino acid-related metabolites in systemic sclerosis in a search for potential biomarkers and mechanisms of the disease.MethodsPlasma samples from 42 patients diagnosed with systemic sclerosis (SSc) according to the 2013 American College of Rheumatology and European League Against Rheumatism ACR/EULAR classification criteria were compared to 27 matched healthy controls. Liquid chromatography/mass spectrometry was applied for the analysis of 36 amino acid-related metabolites.ResultsThe analysis of plasma AA metabolite patterns revealed the number of changes including an increase (20%) in concentrations of NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in SSc vs. healthy subjects. Furthermore, SSc patients had higher glutamine, proline, betaine, 1-methylhistidine, and methylnicotinamide levels, while the concentration of tryptophan was lower. The specific metabolic pattern was identified for several aspects of disease presentation. Most interestingly NOS inhibitor L-NAME was elevated in patients with diffuse systemic sclerosis or telangiectasia.ConclusionsThese results provide further evidence for the involvement of endothelium-dependent pathways in the mechanisms and presentation of SSc. Endothelial dysfunction biomarkers may be useful in the assessment of presentation and prognosis in SSc.

Association of high plasma glutamine levels with outcome in metastatic castration-resistant prostate (mCRPC) patients treated with taxanes.

164 Background: Cancer cells may metabolize glutamine to fulfill their metabolic needs. In prostate cancer it has been shown that androgen receptor signaling promotes glutamine metabolism by increasing the expression of the glutamine transporters, and that stromal glutamine may promote tumor growth. Methods: We retrospectively tested glutamine levels in frozen plasma samples from mCRPC patients treated with taxanes, which were included in a prospective biomarker study in our institution. Glutamine levels were determined by a bioluminescent assay. Optimal cut-offs for glutamine levels were assessed using maximally selected log-rank statistics to determine low and high level groups. Pre-treatment glutamine level was correlated with taxanes response and clinical outcome. Independent association with survival was evaluated by multivariate Cox modeling. Results: Seventy eight mCRPC patients treated with taxanes were included. Median age was 70.3 (55.8-83.5) years and median follow-up was 13.1 (0.2-53.9) months. Glutamine was tested in 88 plasma samples: 69 from pre-docetaxel and 19 pre-cabazitaxel treatment (10 patients had both samples). High glutamine levels significantly correlated with worst PSA-progression-free survival (PFS) (median 2.8 vs 4.7 months, hazard ratio [HR] 1.8, 95%CI 1.1-2.7, P= 0.012) and overall survival (OS) (median 12.4 vs 20.4, HR 2, 95%CI 1.2-3.3, P= 0.006). In a multivariate analysis, high plasma glutamine levels were independently associated with shorter PSA-PFS (HR 2.3, 95%CI 1.4-3.7, P&lt; 0.001) and OS (HR 2.2, 95%CI 1.3-3.7, P= 0.003). Patients with high glutamine levels were more likely to present PSA progression to taxanes than those with low levels (odds ratio [OR] 3, 95%CI 1.2-7.7, P= 0.016). Moreover, samples from patients treated with abiraterone or enzalutamide before taxanes (51 samples from 43 patients) had significantly higher glutamine levels (T-test, P= 0.014) than those without these prior therapies. Conclusions: Glutamine can be detected in plasma of mCRPC patients and higher levels are associated with adverse clinical outcome, supporting the relevance of metabolism in prostate cancer progression.

Abstract 3602: JHU395, a nervous tissue penetrant glutamine antagonist, restricts growth of malignant peripheral nerve sheath tumor with inhibition of nucleotide synthesis

Abstract Malignant peripheral nerve sheath tumor (MPNST) is a sarcoma that occurs in ~10% of patients with neurofibromatosis type I (NF1). Incomplete surgical resection at diagnosis leads to a 4-year event-free survival rate of &amp;lt;30%. Thus, improved treatments are needed. Recent evidence suggests that perturbing glutamine utilization warrants further exploration as a potential therapeutic strategy for MPNST. Competitive glutamine antagonists (GA) have been studied in several clinical trials for sarcoma previously, but clinical development was hampered by gastrointestinal (GI) toxicity. JHU395 is a broadly active nervous tissue penetrant GA. JHU395 delivers active GA preferentially to nervous tissue which may result in less GI toxicity than was observed in past trials. The primary goals of this study were to evaluate JHU395 in preclinical models of MPNST and to investigate tumor metabolic changes in response to JHU395. We investigated glutamine antagonism on growth of MPNST cells in culture and murine flank MPNST. JHU395 was administered orally for 14 days to mice bearing inoculated tumors derived from the NPcis (NF1+/-;p53+/-) genetically engineered model of MPNST. Tumors were measured every other day and tumor volume was calculated as the primary endpoint. Concurrent evaluations of GI and neurotoxicity were performed as secondary endpoints. GA levels in tumor versus plasma were compared using a previously validated bioanalytical method. In vivo stable isotope labeled glutamine (15N2- or 13C5-labeled) flux analysis and targeted liquid chromatography-mass spectrometry (LC-MS) metabolomics were performed on murine tumors. Compared to immortalized Schwann cells, growth of MPNST was preferentially inhibited by GA (IC50=8 micromolar versus &amp;gt;30 micromolar). JHU395 delivered GA to MPNST cells with &amp;gt;4-fold higher cell-to-plasma ratio compared to native GA and maintained ~2.5-fold higher tumor-to-plasma GA levels in vivo. Mice treated with JHU395 had &amp;gt;40% smaller mean tumor volume compared to controls with no overt toxicity. Quantitiative bioanalysis revealed that JHU395 treated tumors had &amp;gt;60% higher glutamine levels compared to controls 30 minutes after oral dosing. In vivo flux analysis demonstrated that JHU395 preferentially affects tumor glutamine utilization for nucleotide synthesis, while glutamine-derived substrates for the citric acid cycle appear unaffected. In summary, JHU395 inhibits growth of MPNST with inhibition of glutamine utilization in nucleotide synthesis and is well-tolerated. Nervous tissue penetrant glutamine antagonism is a feasible and effective therapeutic approach in preclinical models of MPNST. Future studies will investigate JHU395 in combination with nucleotide synthesis inhibitors in MPNST and investigate JHU395 efficacy in additional sarcoma models including patient-derived samples. Citation Format: Kathryn M. Lemberg, Liang Zhao, Ying Wu, Jesse Alt, Joanna Marie H. Aguilar, Jenny Lam, Alexandra J. Gadiano, Rana Rais, Pavel Majer, Jaishri Blakeley, Barbara S. Slusher. JHU395, a nervous tissue penetrant glutamine antagonist, restricts growth of malignant peripheral nerve sheath tumor with inhibition of nucleotide synthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3602.

Skeletal muscle metabolism in rats with low and high intrinsic aerobic capacity: Effect of aging and exercise training.

PurposeExercise training increases aerobic capacity and is beneficial for health, whereas low aerobic exercise capacity is a strong independent predictor of cardiovascular disease and premature death. The purpose of the present study was to determine the metabolic profiles in a rat model of inborn low versus high capacity runners (LCR/HCR) and to determine the effect of inborn aerobic capacity, aging, and exercise training on skeletal muscle metabolic profile.MethodsLCR/HCR rats were randomized to high intensity low volume interval treadmill training twice a week or sedentary control for 3 or 11 months before they were sacrificed, at 9 and 18 months of age, respectively. Magnetic resonance spectra were acquired from soleus muscle extracts, and partial least square discriminative analysis was used to determine the differences in metabolic profile.ResultsSedentary HCR rats had 54% and 30% higher VO2max compared to sedentary LCR rats at 9 months and 18 months, respectively. In HCR, exercise increased running speed significantly, and VO2max was higher at age of 9 months, compared to sedentary counterparts. In LCR, changes were small and did not reach the level of significance. The metabolic profile was significantly different in the LCR sedentary group compared to the HCR sedentary group at the age of 9 and 18 months, with higher glutamine and glutamate levels (9 months) and lower lactate level (18 months) in HCR. Irrespective of fitness level, aging was associated with increased soleus muscle concentrations of glycerophosphocholine and glucose. Interval training did not influence metabolic profiles in LCR or HCR rats at any age.ConclusionDifferences in inborn aerobic capacity gave the most marked contrasts in metabolic profile, there were also some changes with ageing. Low volume high intensity interval training twice a week had no detectable effect on metabolic profile.

Abstract 3482: Cachexia has profound metabolic consequences in the heart and skeletal muscle

Abstract Cachexia is an underexplored and yet devastating consequence of cancer that is the cause of 20% of all cancer related deaths [1]. Cachexia is associated with poor treatment outcome [3], fatigue, and extremely poor quality of life [2, 3]. Tissue wasting is one of the characteristics associated with cachexia syndrome. Here we have used 1H MRS to characterize the metabolic profile of skeletal muscle and heart muscle obtained from normal mice and noncachexia (Panc1) and cachexia inducing (Pa04C) human pancreatic cancer xenograft bearing mice to further understand this syndrome. Male severe combined immunodeficient (SCID) mice were inoculated in the right flank with cancer cells (2 × 106) and in the right hind leg muscle with cachexia reporter myoblasts (2×106) [4]. Mice were sacrificed once tumors were ~400 mm3, and the heart and skeletal muscle were harvested for dual phase solvent extraction. 1H magnetic resonance (MR) spectra of the water phase were acquired with a 750 MHz MR spectrometer. Quantitative changes in metabolite levels were obtained from 1H MR spectra. Leucine, creatine, lactate and glucose were significantly lower, and acetate and formate were significantly higher, in the muscle of cachectic mice compared to non-cachetic mice. Cachectic mice had significantly lower alanine, succinate, glycine, lipid and PUFA (poly unsaturated fatty acids), and significantly higher acetate, pyruvate, and formate compared to normal mice. Non-cachectic mice had significantly lower alanine, succinate, phosphocreatine, and glycine, and significantly higher leucine, isoleucine, valine, pyruvate, creatine, taurine and glucose compared to normal mice. In the heart, leucine, isoleucine, valine, aspartate, glucose, lipid and PUFA were significantly lower, and glutamine was significantly higher, in cachectic mice compared to non-cachetic mice. Cachectic mice had significantly lower leucine, isoleucine, valine, lactate, alanine, glutamate, and PUFA, and significantly higher glutamine and glucose compared to normal mice. Non-cachectic mice had significantly lower alanine, and significantly higher glucose and lipid compared to normal mice. These data highlight, for the first time, the profound metabolic changes that occur in skeletal muscle and the heart with cachexia, identifying potential in vivo 1H MRS indices to detect the onset of cachexia from changes in branched chain amino acids, glucose, and PUFAs. Our data also provide new insights into the effects of cachexia as well as noncachexia inducing tumors on skeletal muscle and heart metabolism that may lead to metabolic interventions in ‘metabolotheranostic' strategies to reduce the morbidity associated with cancer and cachexia. Supported by NIH R01CA193365, R35CA209960, and P30CA06973. Ref. 1. Argiles et al: Nat. Rev. Can 2014, 14(11):754 2. Fearon et al: HPB (Oxford) 2010, 12(5):323 3. Ozola et al: Pancreatology 2015, 15(1):19 4. Winnard et al: Can Res. 2016, 76(6):1441 Citation Format: Santosh Kumar Bharti, Paul Winnard, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. Cachexia has profound metabolic consequences in the heart and skeletal muscle [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 3482.

Glutamate transporters: the regulatory proteins for excitatory/excitotoxic glutamate in brain

Excitatory aminoacids (EAAs) are stored in glutamatergic neurons and related into synaptic cleft, where they can activate inotropic or metabotropic receptors. Their action ends due to transport mechanisms performed by EAAT transporters (EAAT1/GLAST, EAAT2/GLT1, EAAT3/EAAC1, and EAAT4 or EAAT5). Glutamate neurotoxicity has been described in several neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD) and amyotropic lateral sclerosis ALS). Some drugs, such as paclitaxel, are able to increase translation of microRNA and could be possible used as regulatory against glutamate neurotoxicity. Abbreviations: AD: Alzheimer’s disease; ALS: amyotrophic lateral sclerosis; AMPA: α-amino-3-hydroxy-5-methyl-isoxazole-4propionate; Asp: aspartate; CSF: cerebral spinal fluid; EAA: excitatory amino acid; EAAC1 (EAAT3): excitatory amino acid carrier; EAAT: excitatory amino acid transporter; GABA: gamma-aminobutyric acid; GDH: glutamate dehydrogenase; GLAST (EAAT1): glutamate – aspartate transporter; GLT1 (EAAT2): glutamate transporter; iGluR: ionotropic glutamate receptor; KA: kaninic acid; L-Glu: L-glutamate; mGluR: metabotropic glutamate receptor; miR: micro RNA; NMDA: N-methyl-D-aspartate; PD: Parkinson’s disease Introduction Some amino acids act as neurotransmitters in the nervous system, being glutamate and aspartate the common excitatory amino acids and GABA, glycine and taurine the inhibitory ones. Of these amino acids, glutamate and GABA are intimately associated, as their metabolism is associated through glutamic acid decarboxylase (E.C. 4.1.1.15.) (Figure 1). Furthermore, GABA and glutamic acid effects are antagonic and they are related with CO2 fixation (relevant to central ventilation). Glutamic acid metabolism is also related with NH3 detoxification (due to a reduction in α-ketoglutarate and glutamate contents and an increase in glutamine). The efflux of glutamate from brain across the hemato-encephalic barrier is much higher than the influx [1-3], meaning that metabolism of glutamate must play an important role in regulating the brain glutamate levels. Studies on metabolic generation of glutamate/glutamine by using radioactive substrates in brain shows that two pathways are involved. Glucose, glycerol, lactate, pyruvate, α-ketoglutarate and β-hydroxybutyrate seem to be metabolized to glutamate in neurons [4,5], as a low specific radioactivity of glutamine is obtained. In glial cells [6], where higher glutamine synthase is present [7,8], low radiolabelled glutamate and higher glutamine marked are obtained. This is the case of acetate, propionate, butyrate, citrate, leucine, GABA, aspartate and ammonia [9]. In order to decrease glutamic acid in synaptic cleft, excitatory amino acids transporters have an important effect. Therefore, in this paper we present some aspects of these proteins. Glutamatergic neurotransmission The excitatory amino acids (EAAs) are stored in synaptic vesicles in glutamatergic neurons and, upon an action potential, are released via exocytosis into the synaptic cleft where they can activate two different families of receptors: ionotropic (ligand-gated ion channels) and metabotropic (GTP-binding protein coupled) receptors. The ligandgated ion channels are further divided into three families: α-amino3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), kainate (KA) and N–methyl-D-aspartate (NMDA). While AMPA and kainate receptors mediate rapid depolarizing responses at most synapses in the mammalian central nervous system [10], the NMDA receptor participates in synaptic plasticity and synapse formation [11]. The family of metabotropic receptors consists of at least eight subtypes and are involved in the modulation of synaptic signaling by EAAs and other neurotransmitters [12]. The termination of the EAA action takes place by an uptake mechanism that uses the Na+, K+ and pH gradients as a driving force to translocate the neurotransmitters against their concentration gradients, keeping their concentration below the level that activates their receptors (~1 μM) [13-15] (Figure 2). Excessive activation of EAA receptors contributes to brain injury through a process known as excitotoxicity. Therefore, this transport mechanism is not only important for ensuring accurate synaptic signaling but also for limiting the EAA-mediated excitotoxicity Excitatory amino acid (EAA) transporters Three broad subtypes of EAA transport activities have been Correspondence to: Josep J Centelles, Departament de Bioquimica i Biologia Molecular (Biologia), Facultat de Biologia, Universitat de Barcelona, Avda Diagonal, 643. Edifici Prevosti, planta-2, 08028, Barcelona, Spain, Tel: 934021870; E-mail: josepcentelles@ub.edu Received: January 16, 2016; Accepted: January 27, 2016; Published: January 30, 2016 Centelles JJ (2016) Glutamate transporters: the regulatory proteins for excitatory/excitotoxic glutamate in brain J Transl Sci, 2016 doi: 10.15761/JTS.1000123 Volume 2(1): 92-99 extracellular concentrations of EAAs by reducing the driving force required to transport an EAA into the cytoplasm. The second activity is a chloride-dependent transport which exchanges amino acids identified in brain preparations. One type, which is directly coupled to ATP hydrolysis, introduces glutamate into vesicles for release upon depolarization of the synaptic terminal [16]. It indirectly ensures low Glutamine ADP + Pi Glutamine synthetase (GS) (E.C. 6.3.1.2.) Glutaminase (Gase) (E.C. 3.5.1.2.) NH4 H2O Glutamate GABA Glutamate decarboxylase (GD) (E.C. 4.1.1.15.) Oxalacetate Glutamate dehydrogenase (GDH) (E.C. 1.4.1.2.) Glutamate oxalacetate transaminase (GOT) (E.C. 2.6.1.1.) NH4 + NADH Aspartate α-Ketoglutarate CO2 NH4 + ATP

Loss of function mutation of the Slc38a3 glutamine transporter reveals its critical role for amino acid metabolism in the liver, brain, and kidney.

Glutamine, the most abundant amino acid in mammals, is critical for cell and organ functions. Its metabolism depends on the ability of cells to take up or release glutamine by transporters located in the plasma membrane. Several solute carrier (SLC) families transport glutamine, but the SLC38 family has been thought to be mostly responsible for glutamine transport. We demonstrate that despite the large number of glutamine transporters, the loss of Snat3/Slc38a3 glutamine transporter has a major impact on the function of organs expressing it. Snat3 mutant mice were generated by N-ethyl-N-nitrosurea (ENU) mutagenesis and showed stunted growth, altered amino acid levels, hypoglycemia, and died around 20 days after birth. Hepatic concentrations of glutamine, glutamate, leucine, phenylalanine, and tryptophan were highly reduced paralleled by downregulation of the mTOR pathway possibly linking reduced amino acid availability to impaired growth and glucose homeostasis. Snat3-deficient mice had altered urea levels paralleled by dysregulation of the urea cycle, gluconeogenesis, and glutamine synthesis. Mice were ataxic with higher glutamine but reduced glutamate and gamma-aminobutyric acid (GABA) levels in brain consistent with a major role of Snat3 in the glutamine-glutamate cycle. Renal ammonium excretion was lower, and the expression of enzymes and amino acid transporters involved in ammoniagenesis were altered. Thus, SNAT3 is a glutamine transporter required for amino acid homeostasis and determines critical functions in various organs. Despite the large number of glutamine transporters, loss of Snat3 cannot be compensated, suggesting that this transporter is a major route of glutamine transport in the liver, brain, and kidney.

Markers of glutamate signaling in cerebrospinal fluid and serum from patients with bipolar disorder and healthy controls

Glutamate is the major excitatory neurotransmitter in the brain. Aberrations in glutamate signaling have been linked to the pathophysiology of mood disorders. Increased plasma levels of glutamate as well as higher glutamine+glutamate levels in the brain have been demonstrated in patients with bipolar disorder as compared to healthy controls. In this study, we explored the glutamate hypothesis of bipolar disorder by examining peripheral and central levels of amino acids related to glutamate signaling. A total of 215 patients with bipolar disorder and 112 healthy controls from the Swedish St. Göran bipolar project were included in this study. Glutamate, glutamine, glycine, l-serine and d-serine levels were determined in serum and in cerebrospinal fluid using high performance liquid chromatography with fluorescence detection. Serum levels of glutamine, glycine and d-serine were significantly higher whereas l-serine levels were lower in patients with bipolar disorder as compared to controls. No differences between the patient and control group in amino acid levels were observed in cerebrospinal fluid. The observed differences in serum amino acid levels may be interpreted as a systemic aberration in amino acid metabolism that affects several amino acids related to glutamate signaling.

Abstract 3377: Glutamine mediated aggressiveness and drug sensitivity in ovarian cancer cells

Abstract Platinum-based drugs such as cisplatin, carboplatin and xoaliplatin hav been introduced into clinical trials and their outcomes has progressed the treatment on several tumors, including ovarian cancer. However, cancer cells acquire chemo-resistance in the long term incubation of drugs, which turns out to be the major limitation for cancer therapy. Recent studies have shown that cisplatin can accumulate in mitochondria and induce cytochrome C release, thereby resulting in apoptosis. We found that glutamine plays a vital role in maintaining mitochondrial integrity and function by regulating NAD+ and NADH. Using bioenergetics and isotopomer analysis we show that chemodrugs in presence of glutamine affect oxidative phosphorylation in drug sensitive parental ovarian cancer cells. Addition of NAD+ substrates increased NAD/NADH ratio abrogating cisplatin's effect on cell death and mitochondrial dysfunction. Furthermore, drug resistant cancer cells had relatively higher glutamine dependence when compared with parental cells. Importantly, we found that when glutamine's entry into TCA cycle was inhibited ovarian cancer cells became more sensitive to chemotherapy. Our findings are based on the quantification of metabolites, related to glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acids and nucleotide metabolism, and comparison of these pathways fluxes in drug sensitive and drug resistant ovarian cancer. The insights obtained present a unique opportunity for overcoming the drug resistance limitation in clinical trials in ovarian cancers. Citation Format: Lifeng Yang, Tyler J. Moss, Juan C. Marini, Selanere Mangala, Stephen Wahlig, Julia Win, Dan Su, Anil K. Sood, Prahlad T. Ram, Deepak Nagrath. Glutamine mediated aggressiveness and drug sensitivity in ovarian cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3377. doi:10.1158/1538-7445.AM2014-3377

Abstract 1248: Enhanced glutamine metabolism contributes to docetaxel resistance in DU145 prostate cancer cells

Abstract Chemotherapeutic resistance is one of the major factors contributing to cancer mortality. Although evidence supports a mechanistic link between metabolic enzyme polymorphisms and drug sensitivity, little is known about the metabolic changes tumor cells undergo when treated with a chemotherapeutic agent. Moreover, the metabolic consequences of exposure to these agents may provide essential adaptive functions contributing to chemotherapeutic resistance. Here we used metabolic flux analysis to identify metabolic changes accompanying and potentially accounting for docetaxel resistance in DU145 prostate cancer cells. We focused on the utilization of glucose and glutamine, the two most abundant extracellular nutrients, which together account for the bulk of carbon and nitrogen metabolism in tumor cells. Docetaxel resistant (DOCETR) cells consumed glutamine more avidly and secreted glutamate and ammonia at a greater rate than sensitive parental cells. Surprisingly, contribution of glutamine carbon into the TCA cycle was unchanged in DOCETR cells despite the higher glutamine utilization. Transfer of glutamine carbon into both the intracellular and extracellular glutamate pools, on the other hand, was higher in DOCETR cells, indicating that the DOCETR cells had an increased ability to convert glutamine to glutamate and then to secrete the glutamate. Accordingly, these cells also had increased expression and activity of glutaminase, the major enzyme responsible for glutamine deamidation. Because glutaminase activity does not yield energy unless the resulting glutamate is further oxidized, we hypothesized that this metabolic transition served a non-bioenergetic role, possibly supporting enhanced rates of glutathione synthesis for modification and secretion of docetaxel. Consistent with this idea, DOCETR cells displayed an increase in de novo synthesis of glycine, which is required for the production of glutathione. Importantly, a glutaminase inhibitor re-sensitized DOCETR cells to docetaxel, strongly suggesting that their enhanced glutamine metabolism was involved in resistance to the drug. We speculate that alterations in glutamine metabolism provide a general and targetable mechanism for xenobiotic resistance in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1248. doi:10.1158/1538-7445.AM2011-1248

1H MR spectroscopy in Friedreich's ataxia and ataxia with oculomotor apraxia type 2

Background and aimFriedreich's ataxia (FRDA) and ataxia with oculomotor apraxia type 2 (AOA2) are the two most frequent forms of autosomal recessive cerebellar ataxias. However, brain metabolism in these disorders is poorly characterized and biomarkers of the disease progression are lacking. We aimed at assessing the neurochemical profile of the pons, the cerebellar hemisphere and the vermis in patients with FRDA and AOA2 to identify potential biomarkers of these diseases. MethodsShort-echo, single-voxel proton (1H) magnetic resonance spectroscopy data were acquired from 8 volunteers with FRDA, 9 volunteers with AOA2, and 38 control volunteers at 4T. Disease severity was assessed by the Friedreich's Ataxia Rating Scale (FARS). ResultsNeuronal loss/dysfunction was indicated in the cerebellar vermis and hemispheres in both diseases by lower total N-acetylaspartate levels than controls. The putative gliosis marker myo-inositol was higher than controls in the vermis and pons in AOA2 and in the vermis in FRDA. Total creatine, another potential gliosis marker, was higher in the cerebellar hemispheres in FRDA relative to controls. Higher glutamine in FRDA and lower glutamate in AOA2 than controls were observed in the vermis, indicating different mechanisms possibly leading to altered glutamatergic neurotransmission. In AOA2, total N-acetylaspartate levels in the cerebellum strongly correlated with the FARS score (p<0.01). ConclusionDistinct neurochemical patterns were observed in the two patient populations, warranting further studies with larger patient populations to determine if the alterations in metabolite levels observed here may be utilized to monitor disease progression and treatment.

This Month in Gastroenterology

This Month in Gastroenterology

Glutamate Dysfunction in People with Prodromal Symptoms of Psychosis: Relationship to Gray Matter Volume

The glutamate model of schizophrenia proposes that altered glutamatergic neurotransmission is fundamental to the development of the disorder. In addition, its potential to mediate neurotoxicity raises the possibility that glutamate dysfunction could underlie neuroanatomic changes in schizophrenia. Here we determine whether changes in brain glutamate are present in subjects at ultra high risk of developing psychosis and whether these changes are related to reductions in cortical gray matter volume. Twenty-seven individuals with an at-risk mental state and a group of 27 healthy volunteers underwent proton magnetic resonance spectroscopy and volumetric proton magnetic resonance imaging using a 3-Tesla scanner. Glutamate and glutamine levels were measured in anterior cingulate, left hippocampus, and left thalamus. These measures were then related to cortical gray matter volume. At-risk mental state (ARMS) subjects had significantly lower levels of glutamate than control subjects in the thalamus (p < .05) but higher glutamine in the anterior cingulate (p < .05). Within the ARMS group, the level of thalamic glutamate was directly correlated with gray matter volume in the medial temporal cortex and insula (p < .01). This study provides the first evidence that brain glutamate function is perturbed in people with prodromal signs of schizophrenia and that glutamatergic dysfunction is associated with a reduction in gray matter volume in brain regions thought to be critical to the pathogenesis of the disorder. These findings support the hypothesis that drugs affecting the glutamate system may be of benefit in the early stages of psychotic illness.

Effects of PII Deficiency on Expression of the Genes Involved in Ammonium Utilization in the Cyanobacterium Synechocystis sp. Strain PCC 6803

The Synechocystis sp. strain PCC 6803 mutant deficient in PII protein (the glnB gene product) was found to express glutamine synthetase activity at levels several times higher than the wild-type strain. There was no significant difference in nitrate reductase activity levels between the two strains, and the nitrite reductase levels were somewhat lower in the mutant than in the wild-type strain. The higher glutamine synthetase activity in the mutant was ascribed to higher expression levels of the glutamine synthetase genes (glnA and glnN), which belong to the regulon controlled by NtcA, a Crp-family transcription regulator. Examination of the effects of PII deficiency on other NtcA-regulated genes revealed that the transcript levels of amt1 (encoding an ammonium permease) and gifB (encoding an inhibitor of glutamine synthetase) were increased, whereas that of gifA (a homolog of gifB, encoding another glutamine synthetase inhibitor) was decreased, with those of nirA, nrtC, icd, sigE (rpoD2-V), nblA and ntcA being unaffected. Unlike the Synechococcus elongatus strain PCC 7942, induction or repression of the NtcA-regulated genes proceeded normally in the PII-deficient mutant upon nitrogen depletion. The altered steady-state expression levels of glnA, glnN, amt1, gifA and gifB in the PII-deficient mutant suggested that Synechocystis sp. strain PCC 6803 has a mechanism for regulation of the subset of the NtcA-regulated genes related directly to ammonium assimilation.

Accumulation of ammonium ion in cadmium tolerant and sensitive cultivars of Oryza sativa

Cd-tolerant and Cd-sensitive rice cultivars were used to study the role of NH4+ accumulation in Cd-induced toxicity. NH4+ accumulation seems to be involved in regulating the toxicity of rice seedlings caused by CdCl2. This conclusion was based on the observations that (a) on treatment with CdCl2, NH4+ content increased rapidly in the leaves of the Cd-sensitive cultivar (cv. Taichung Native 1, TN1) but not in the Cd-tolerant cultivar (cv. Tainumg 67, TNG67), (b) pretreatment with abscisic acid (ABA) enhanced Cd tolerance and reduced Cd-induced NH4+ accumulation in TN1 seedlings, (c) exogenous application of the ABA biosynthesis inhibitor, fluridone, decreased Cd tolerance and increased NH4+ content in leaves of TNG67, (d) exogenous application of phosphinothricin, an inhibitor of glutamine synthetase (GS), which resulted in NH4+ accumulation in the leaves, also induced toxicity similar to Cd in TN1 seedlings. Evidence is presented to show that Cd-induced NH4+ accumulation in TN1 leaves is attributable to a decrease in GS activity. Since Cd-treated TN1 leaves had higher glutamine and glutamate contents than control leaves, it is unlikely that glutamine (or glutamate) depletion is the mechanism which regulates Cd-induced toxicity.