Query: Metabolic flow analysis of glutamine

title: Analysis of neuron astrocyte metabolic cooperation in the brain of db/db mice with cognitive decline using 13C NMR spectroscopy
abstract: Type 2 diabetes has been linked to cognitive impairment, but its potential metabolic mechanism is still unclear. The present study aimed to explore neuron astrocyte metabolic cooperation in the brain of diabetic (db/db, BKS.Cg m+ Leprdb/J) mice with cognitive decline using 13C NMR technique in combination with intravenous [2 13C] acetate and [3 13C] lactate infusions. We found that the 13C enrichment from [2 13C] acetate into tricarboxylic acid cycle intermediate, succinate, was significantly decreased in db/db mice with cognitive decline compared with wild type (WT, C57BLKS/J) mice, while an opposite result was obtained after [3 13C] lactate infusion. Relative to WT mice, db/db mice with cognitive decline had significantly lower 13C labeling percentages in neurotransmitters including glutamine, glutamate, and g aminobutyric acid after [2 13C] acetate infusion. However, [3 13C] lactate resulted in increased 13C enrichments in neurotransmitters in db/db mice with cognitive decline. This may indicate that the disturbance of neurotransmitter metabolism occurred during the development of cognitive decline. In addition, a reduction in 13C labeling of lactate and an increase in gluconeogenesis were found from both labeled infusions in db/db mice with cognitive decline. Therefore, our results suggest that the development of cognitive decline in type 2 diabetes may be implicated to an unbalanced metabolism in neuron astrocyte cooperation and an enhancement of gluconeogenesis.

title: 13C metabolic flux analysis of human adenovirus infection: Implications for viral vector production
abstract: Adenoviruses are human pathogens increasingly used as gene therapy and vaccination vectors. However, their impact on cell metabolism is poorly characterized. We performed carbon labeling experiments with [1,2 13C]glucose or [U 13C]glutamine to evaluate metabolic alterations in the amniocyte derived, E1 transformed 1G3 cell line during production of a human adenovirus type 5 vector (AdV5) Nonstationary 13C metabolic flux analysis revealed increased fluxes of glycolysis (17% and markedly PPP (over fourfold) and cytosolic AcCoA formation (nearly twofold) following infection of growing cells. Interestingly, infection of growth arrested cells increased overall carbon flow even more, including glutamine anaplerosis and TCA cycle activity (both over 1.5 fold) but was unable to stimulate the PPP and was associated with a steep drop in AdV5 replication (almost 80% Our results underscore the importance of nucleic and fatty acid biosynthesis for adenovirus replication. Overall, we portray a metabolic blueprint of human adenovirus infection, highlighting similarities with other viruses and cancer, and suggest strategies to improve AdV5 production. Biotechnol. Bioeng. 2017;114: 195 207.

title: Flow Injection Analysis of L Glutamine and Ammonia a New Approach with Gas Diffusion and Detection by ISFET Technology
abstract: This chapter discusses the flow injection analysis of l glutamine and ammonia. Two of the most important analytes for bioprocess control of mammalian cell cultivation are the amino acid L glutamine and ammonia. l glutamine is a nutrient supplemented to different media. Ammonia is a major waste product of glutamine metabolism released by metabolic deamination of glutamine to glutamate. The flow injection system described in the chapter allows the determination of both analytes l glutamine and ammonia. The sample is injected in a carrier stream, pH 12, to transform ammonium ions to ammonia that can diffuse through a gas diffusion membrane into an acceptor stream, pH 7. The resulting change in pH is monitored using an ISFET device. The original sample is transported to an enzyme reactor containing immobilized glutaminase, catalyzing the deamination of l glutamine to ammonia and l glutamate. Thus, simultaneous determination of ammonium ions and glutamine is possible using the same detection system.

title: The heterocyclic compound Tempol inhibits the growth of cancer cells by interfering with glutamine metabolism
abstract: Tempol (4 hydroxy 2,2,6,6 Tetramethylpiperidine 1 oxyl, TPL) a nitroxide compound, inhibits proliferation and increases the vulnerability of cancer cells to apoptosis induced by cytotoxic agents. However, the molecular mechanism of TPL inhibiting cancer cell proliferation has not been fully understood. In this study, we evaluated the metabolic effect of TPL on cancer cells and explored its cancer therapeutic potential. Extracellular flow assays showed that TPL inhibited cellular basal and maximal oxygen consumption rates of mitochondrial. 13 C metabolic flux analysis showed that TPL treatment had minimal effect on glycolysis. However, we found that TPL inhibits glutamine metabolism by interfering with the oxidative tricarboxylic acid cycle (TCA) process and reductive glutamine process. We found that the inhibitory effect of TPL on metabolism occurs mainly on the step from citrate to a ketoglutarate or vice versa. We also found that activity of isocitrate dehydrogenase IDH1 and IDH2, the key enzymes in TCA, were inhibited by TPL treatment. In xenograft mouse model, TPL treatment reduced tumor growth by inhibiting cellular proliferation of xenograft tumors. Thus, we provided a mechanism of TPL inhibiting cancer cell proliferation by interfering with glutamine utilization that is important for survival and proliferation of cancer cells. The study may help the development of a therapeutic strategy of TPL combined with other anticancer medicines.

title: Metabolomics analysis of the potential anticancer mechanism of annonaceous acetogenins on a multidrug resistant mammary adenocarcinoma cell.
abstract: Although annonaceous acetogenins (ACGs) have been reported to have antitumor activity for over three decades, and many of the underlying mechanism of ACGs on cancer have been clarified, there are still outstanding issues. In particular, the changes of small metabolite in cancer cells, caused by ACGs intake, have been reported rarely. Recent research has showed that cellular metabolic profiling coupled with ultra flow liquid chromatography coupled to quadrupole time of flight mass spectrometry (UFLC Q TOF MS) and multivariable statistical analysis enables a good understanding of ACGs' effects on multidrug resistant human mammary adenocarcinoma (MCF 7/Adr) cells. As a result, 23 potential biomarkers (p 0.05, VIP >1) were identified, and 5 pathways (impact value 0.10) identified. The differential metabolites suggested that ACGs affected metabolomics pathways, including arginine and proline metabolism, glycerophospholipid metabolism, taurine and hypotaurine metabolism, alanine, aspartate and glutamate metabolism and D Glutamine and D glutamate metabolism.

title: Metabolic Alterations Induced by Kudingcha Lead to Cancer Cell Apoptosis and Metastasis Inhibition
abstract: Kudingcha is implicated in alleviating metabolic disorders in traditional Chinese medicine. However, the role of Kudingcha, one of the Ligustrum robustum species, in metabolic regulations and its antitumor activity in triple negative breast cancer (TNBC) remains to be determined. Two breast cancer cell lines and immunocompetent and immunodeficient mice were used to evaluate the therapeutic effects of Kudingcha treatment. The production of reactive oxygen species (ROS) and glucose uptake were examined by flow cytometry. Metabolic shift was examined by metabonomics and western blot analysis. In this study, we found that aqueous extract of Kudingcha dose dependently inhibited cell growth and induced apoptosis in vitro and in vivo. Moreover, Kudingcha supplementation significantly reduced cancer metastasis. Kudingcha significantly inhibited glycolysis and glutamine metabolism. In addition, we demonstrated that the antitumor effects of Kudingcha were dependent on ROS production, which was increased by b oxidation and oxidative phosphorylation. These findings provide a novel potential benefit of Kudingcha from targeting the cancer metabolism.

title: Defining Metabolic Rewiring in Lung Squamous Cell Carcinoma
abstract: Metabolomics based on untargeted flow infusion electrospray ionization high resolution mass spectrometry (FIE HRMS) can provide a snap shot of metabolism in living cells. Lung Squamous Cell Carcinoma (SCC) is one of the predominant subtypes of Non Small Cell Lung Cancers (NSCLCs) which usually shows a poor prognosis. We analysed lung SCC samples and matched histologically normal lung tissues from eight patients. Metabolites were profiled by FIE HRMS and assessed using t test and principal component analysis (PCA) Differentially accumulating metabolites were mapped to pathways using the mummichog algorithm in R, and biologically meaningful patterns were indicated by Metabolite Set Enrichment Analysis (MSEA) We identified metabolic rewiring networks, including the suppression of the oxidative pentose pathway and found that the normal tricarboxylic acid (TCA) cycle were decoupled from increases in glycolysis and glutamine reductive carboxylation. Well established associated effects on nucleotide, amino acid and thiol metabolism were also seen. Novel aspects in SCC tissue were increased in Vitamin B complex cofactors, serotonin and a reduction of g aminobutyric acid (GABA) Our results show the value of FIE HRMS as a high throughput screening method that could be exploited in clinical contexts.

title: Determination of the Glutamate Glutamine Cycling Flux Using Two Compartment Dynamic Metabolic Modeling is Sensitive to Astroglial Dilution
abstract: Over the last decade 13C magnetic resonance spectroscopy (13C MRS) combined with the infusion of [1 13C]glucose has been used to measure the cerebral rate of the glutamate glutamine cycle (Vcyc) However, the effect of the astroglial label dilution pathways on the accuracy and precision of the C MRS measurement of Vcyc has not been evaluated or realized. In this report, we use the numerical Monte Carlo method to study the effect of astroglial dilution on the reliability of extracting Vcyc using the neuronal astroglial two compartment metabolic model and [1 13C]glucose infusion. The results show that omission of the astroglial dilution flux leads to a large loss in the sensitivity of the glutamine turnover curve to Vcyc. When the measured isotopic dilution of cerebral glutamine is accounted for in the analysis, the value of Vcyc can be precisely and accurately determined.

title: In vivo 13C MRS in the mouse brain at 14.1 Tesla and metabolic flux quantification under infusion of [1,6 13C2]glucose
abstract: In vivo 13C magnetic resonance spectroscopy (MRS) enables the investigation of cerebral metabolic compartmentation while, e.g. infusing 13C labeled glucose. Metabolic flux analysis of 13C turnover previously yielded quantitative information of glutamate and glutamine metabolism in humans and rats, while the application to in vivo mouse brain remains exceedingly challenging. In the present study, 13C direct detection at 14.1 T provided highly resolved in vivo spectra of the mouse brain while infusing [1,6 13C2]glucose for up to 5 h. 13C incorporation to glutamate and glutamine C4, C3, and C2 and aspartate C3 were detected dynamically and fitted to a two compartment model: flux estimation of neuron glial metabolism included tricarboxylic acid cycle (TCA) flux in astrocytes (Vg 0.16 0.03 umol/g/min) and neurons (VTCAn 0.56 0.03 umol/g/min) pyruvate carboxylase activity (VPC 0.041 0.003 umol/g/min) and neurotransmission rate (VNT 0.084 0.008 umol/g/min) resulting in a cerebral metabolic rate of glucose (CMRglc) of 0.38 0.02 umol/g/min, in excellent agreement with that determined with concomitant 18F fluorodeoxyglucose positron emission tomography (18FDG PET).We conclude that modeling of neuron glial metabolism in vivo is accessible in the mouse brain from 13C direct detection with an unprecedented spatial resolution under [1,6 13C2]glucose infusion.

title: Metabolic characterization of cultured mammalian cells by mass balance analysis, tracer labeling experiments and computer aided simulations.
abstract: Studying metabolic directions and flow rates in cultured mammalian cells can provide key information for understanding metabolic function in the fields of cancer research, drug discovery, stem cell biology, and antibody production. In this work, metabolic engineering methodologies including medium component analysis, (13)C labeling experiments, and computer aided simulation analysis were applied to characterize the metabolic phenotype of soft tissue sarcoma cells derived from p53 null mice. Cells were cultured in medium containing [1 (13)C] glutamine to assess the level of reductive glutamine metabolism via the reverse reaction of isocitrate dehydrogenase (IDH) The specific uptake and production rates of glucose, organic acids, and the 20 amino acids were determined by time course analysis of cultured media. Gas chromatography mass spectrometry analysis of the (13)C labeling of citrate, succinate, fumarate, malate, and aspartate confirmed an isotopically steady state of the cultured cells. After removing the effect of naturally occurring isotopes, the direction of the IDH reaction was determined by computer aided analysis. The results validated that metabolic engineering methodologies are applicable to soft tissue sarcoma cells derived from p53 null mice, and also demonstrated that reductive glutamine metabolism is active in p53 null soft tissue sarcoma cells under normoxia.