The Leloir Cycle in Glioblastoma: Galactose Scavenging and Metabolic Remodeling

Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of GBM to use galactose (Gal) as a fuel via the Leloir pathway is investigated. Methods: Gene transcript data were accessed to determine the association between expression of genes of the Leloir pathway and patient outcomes. Growth studies were performed on five primary patient-derived GBM cultures using Glc-free media supplemented with Gal. The role of Glut3/Glut14 in sugar import was investigated using antibody inhibition of hexose transport. A specific inhibitor of GALK1 (Cpd36) was used to inhibit Gal catabolism. Gal metabolism was examined using proton, carbon and phosphorous NMR spectroscopy, with 13C-labeled Glc and Gal as tracers. Results: Data analysis from published databases revealed that elevated levels of mRNA transcripts of SLC2A3 (Glut3), SLC2A14 (Glut14) and key Leloir pathway enzymes correlate with poor patient outcomes. GBM cultures proliferated when grown solely on Gal in Glc-free media and switching Glc-grown GBM cells into Gal-enriched/Glc-free media produced elevated levels of Glut3 and/or Glut14 enzymes. The 13C NMR-based metabolic flux analysis demonstrated a fully functional Leloir pathway and elevated pentose phosphate pathway activity for efficient Gal metabolism in GBM cells. Conclusion: Expression of Glut3 and/or Glut14 together with the enzymes of the Leloir pathway allows GBM to transport and metabolize Gal at physiological glucose concentrations, providing GBM cells with an alternate energy source. The presence of this pathway in GBM and its selective targeting may provide new treatment strategies.

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Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

Applied and Environmental Microbiology ◽

10.1128/aem.70.12.7277-7287.2004 ◽

2004 ◽

Vol 70 (12) ◽

pp. 7277-7287 ◽

Cited By ~ 71

Author(s):

Christoph Wittmann ◽

Patrick Kiefer ◽

Oskar Zelder

Keyword(s):

Carbon Source ◽

Corynebacterium Glutamicum ◽

Metabolic Flux ◽

Tca Cycle ◽

Pentose Phosphate ◽

Flux Analysis ◽

Phosphotransferase System ◽

Lysine Production ◽

Metabolic Fluxes ◽

Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

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68 Flux analysis of aerobic glycolysis in bovine blastocysts and CT1 cells

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab68 ◽

2019 ◽

Vol 31 (1) ◽

pp. 159

Author(s):

J. Chung ◽

R. Clifford ◽

G. Sriram ◽

C. Keefer

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Metabolic Flux ◽

Aerobic Glycolysis ◽

Carbon Sources ◽

Pentose Phosphate ◽

Gas Chromatography Mass Spectrometry ◽

Flux Analysis ◽

Metabolic Modelling ◽

Chromatography Mass Spectrometry

Embryo quality and maternal recognition are crucial for successful initiation of bovine pregnancy. Previous studies have proposed that better quality embryos use aerobic glycolysis to meet a high demand for biomass components. While hexoses are the principal carbon sources that provide energy to glycolysis, little is known about partitioning of hexoses into metabolic pathways or alteration of partitioning when different hexoses are simultaneously available. Specific metabolic utilisation of 13C-labelled substrates can be quantified by gas chromatography-mass spectrometry, an excellent noninvasive approach for studying cellular metabolism. To assess hexose flux through central metabolism, bovine blastocysts and CT1 cells (a bovine trophectoderm cell line) were cultured in SOF-based media supplemented with combinations of 50% uniformly labelled (U) and 50% naturally abundant (NA) glucose (Glc) or fructose (Fru) (U−13C Glc+NA Glc, U−13C Fru+NA Fru, U−13C Glc+NA Fru, and U−13C Fru+NA Glc), such that total hexose concentration was 1.5mM. Metabolites in spent media from 24-h cultures of single or 5 blastocysts (40-μL drops; 5% CO2, 5% O2, 90% N2) and 1-, 2-, 3-, 6-, 8-, and 24-h incubations of CT1 cells (150 μL; ~3×104 cells per well; 5% CO2, 95% air) were extracted with a MeOH-CHCl3 reagent, derivatized, and analysed by gas chromatography-mass spectrometry. Measurement of mass isotopomer distributions of metabolites, chiefly pyruvate, lactate, and amino acids, followed by correction for natural abundances and metabolic modelling, revealed several insights. For instance, five Day 7 or Day 8 blastocysts (Day 0=fertilization) supplied with U−13C Glc+NA Fru displayed 13C enrichments of 80.3%±1.4% for pyruvate and 71.6%±2.8% for lactate, whereas when supplied with U−13C Fru+NA Glc, they displayed lower 13C enrichments of 5.7%±2.4% for pyruvate and 2.8%±0.4% lactate (mean±standard deviation, n=3 to 4). Metabolic modelling revealed that when Glc and Fru are simultaneously available, the blastocysts used 2.5±0.2 moles of Fru per 100 moles of Glc used. Furthermore, 13C enrichment of pyruvate was 42.0±0.6% when U−13C Glc+NA Glc was supplied and 37.8±2.7% when U−13C Fru+NA Fru was supplied. Lactate enrichments followed a similar trend. This indicates that, individually, Glc and Fru were utilised majorly through aerobic glycolysis with some involvement of the pentose phosphate pathway. Alanine was negligibly labelled in all of the experiments, suggesting either a low TCA flux or that alanine is diluted by extra- or intracellular amino or fatty acids. Single blastocysts and CT1 cells showed a similar labelling pattern when hexoses were available. Following Glc depletion at 8h in CT1 cultures, the 13C enrichments of alanine and citrate in the media increased, suggesting a sharp alteration of metabolic state. These findings demonstrate that metabolic flux can be comprehensively analysed for single bovine blastocysts and CT1 cell metabolism models that of the blastocyst. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2015-67015-23237 from the USDA National Institute of Food and Agriculture.

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Combined Fluxomics and Transcriptomics Analysis of Glucose Catabolism via a Partially Cyclic Pentose Phosphate Pathway in Gluconobacter oxydans 621H

Applied and Environmental Microbiology ◽

10.1128/aem.03414-12 ◽

2013 ◽

Vol 79 (7) ◽

pp. 2336-2348 ◽

Cited By ~ 35

Author(s):

Tanja Hanke ◽

Katharina Nöh ◽

Stephan Noack ◽

Tino Polen ◽

Stephanie Bringer ◽

...

Keyword(s):

Phase Ii ◽

Growth Phase ◽

Pentose Phosphate Pathway ◽

Metabolic Flux ◽

Glucose Dehydrogenase ◽

Gluconobacter Oxydans ◽

Pentose Phosphate ◽

Flux Analysis ◽

Content Type ◽

Activity Measurements

ABSTRACTIn this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes inGluconobacter oxydans621H with glucose were studied by13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For13C-MFA, cells were cultivated with specifically13C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. SinceG. oxydanslacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP).13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.

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TAMI-80. CELLULAR METABOLISM IN DIFFUSE INTRINSIC PONTINE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.862 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi215-vi215

Author(s):

Omkar Ijare ◽

Jeanne Manalo ◽

Martyn Sharpe ◽

David Baskin ◽

Kumar Pichumani

Keyword(s):

Metabolic Flux ◽

Treatment Strategies ◽

Cellular Metabolism ◽

Tca Cycle ◽

Pediatric Brain Tumors ◽

Diffuse Intrinsic Pontine Glioma ◽

Glutamine Metabolism ◽

Flux Analysis ◽

Pontine Glioma ◽

Carboxylation Pathway

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of brain tumor in children, comprising >10% of all pediatric brain tumors. The median survival after diagnosis is < 1 year. Since DIPG tumors infiltrate brainstem and pons, they are inoperable. Currently radiotherapy is the mainstay of treatment, and there is a great need for novel therapies for the treatment of DIPG. Cellular metabolism plays a key role in carcinogenesis, unravelling active metabolic pathways in DIPG would help in developing targeted therapies. Glucose and glutamine are the two major nutrients necessary for the growth and proliferation of cancer cells. In this study, we have investigated the glucose and glutamine metabolism in SF8628 DIPG cells using 1H/13C NMR and GC-MS based metabolic flux analysis. SF8628 cells were grown in DMEM containing 11.0 mM glucose, supplemented with 10% FBS, and 2.0 mM glutamine at 37 °C under humidified air and 5% CO2. When cells reached confluency (replicates = 4), treated with 11.0 mM [U-13C]glucose or 4.0 mM glutamine in DMEM (supplemented with 10% FBS). After 24 h, cells were harvested for NMR/GC-MS analysis. The 13C-isotopomer analysis revealed that SF8628 cells produced 25.26 ± 10.63% acetyl-CoA from [U-13C]glucose which is ~3.7 times higher than that produced from GBM cells (6.83 ± 0.76%; our previous work), suggesting that DIPGs are metabolically very active. [U-13C]glutamine metabolism showed that DIPG cells also have an active TCA cycle metabolism (citrate M+4; 40.07 ± 1.06%) and moderately active reductive carboxylation pathway (citrate M+5; 10.59 ± 1.13%). Inhibition of both glycolytic and glutaminolysis pathways will be valuable in developing treatment strategies for DIPGs and these studies are in progress.

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Flux profile and modularity analysis of time-dependent metabolic changes of de novo adipocyte formation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00670.2006 ◽

2007 ◽

Vol 292 (6) ◽

pp. E1637-E1646 ◽

Cited By ~ 22

Author(s):

Yaguang Si ◽

Jeongah Yoon ◽

Kyongbum Lee

Keyword(s):

Metabolic Flux ◽

Type I Collagen ◽

De Novo ◽

Cell Number ◽

Pentose Phosphate ◽

Gene Profiling ◽

Flux Analysis ◽

Type I ◽

White Adipocyte ◽

Modularity Analysis

White adipose tissue (WAT) mass is the main determinant of obesity and associated health risks. WAT expansion results from increases in white adipocyte cell number and size, which in turn reflect a series of shifts in the cellular metabolic state. To quantitatively profile the metabolic alterations occurring during de novo adipocyte formation, metabolic flux analysis (MFA) was used in conjunction with a novel modularity analysis algorithm on differentiating 3T3-L1 preadipocytes. Use of a type I collagen gel as an effective long-term culture substrate was also assessed. The calculated flux distributions predicted the sequential activation of several intracellular cross-compartmental pathways, including lipogenesis, the pentose phosphate pathway, and the malate cycle, in good agreement with earlier isotopic tracer experiments and gene profiling studies. Partition of the adipocyte metabolic network into highly interacting reaction subgroups suggested a functional reorganization of the major pathways consistent with the lipid-loading phenotype of the adipocyte. Flux and modularity analysis results together point to the flux distribution around pyruvate as a key indicator of adipocyte lipid accumulation.

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Comparative13C Metabolic Flux Analysis of Pyruvate Dehydrogenase Complex-Deficient, l-Valine-Producing Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.00575-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6644-6652 ◽

Cited By ~ 51

Author(s):

Tobias Bartek ◽

Bastian Blombach ◽

Siegmund Lang ◽

Bernhard J. Eikmanns ◽

Wolfgang Wiechert ◽

...

Keyword(s):

Pyruvate Dehydrogenase ◽

Metabolic Flux Analysis ◽

Metabolic Flux ◽

Pyruvate Dehydrogenase Complex ◽

Pentose Phosphate ◽

Strong Increase ◽

Flux Analysis ◽

Wild Type ◽

Content Type ◽

Split Ratio

ABSTRACTl-Valine can be formed successfully usingC. glutamicumstrains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-typeC. glutamicumand four PDHC-deficient strains were compared by13C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH forl-valine formation. In accordance, the introduction of theEscherichia colitranshydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into anl-valine-producingC. glutamicumstrain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated forl-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

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Amplified Expression of Fructose 1,6-Bisphosphatase in Corynebacterium glutamicum Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8587-8596.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8587-8596 ◽

Cited By ~ 140

Author(s):

Judith Becker ◽

Corinna Klopprogge ◽

Oskar Zelder ◽

Elmar Heinzle ◽

Christoph Wittmann

Keyword(s):

Corynebacterium Glutamicum ◽

Pentose Phosphate Pathway ◽

Metabolic Flux ◽

Carbon Sources ◽

Pentose Phosphate ◽

Metabolic Effects ◽

Flux Analysis ◽

Lysine Production ◽

Fructose 1,6 Bisphosphatase

ABSTRACT The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfb r . The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.

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13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis ofLactococcus lactisreveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses

PeerJ ◽

10.7717/peerj.3451 ◽

2017 ◽

Vol 5 ◽

pp. e3451 ◽

Cited By ~ 4

Author(s):

Kamalrul Azlan Azizan ◽

Habtom W. Ressom ◽

Eduardo R. Mendoza ◽

Syarul Nataqain Baharum

Keyword(s):

Amino Acids ◽

Malic Enzyme ◽

Metabolic Flux ◽

Pearson Correlation ◽

Starter Culture ◽

Flux Ratio ◽

Pentose Phosphate ◽

Flux Analysis ◽

Ratio Analysis ◽

Central Metabolism

Lactococcus lactissubsp.cremorisMG1363 is an important starter culture for dairy fermentation. During industrial fermentations,L. lactisis constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response ofL. lactisto several stresses has been described, the adaptation mechanisms at the level ofin vivofluxes have seldom been described. To gain insights into cellular metabolism,13C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism ofL. lactiswhen subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability ofL. lactis’central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) inL. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering inL. lactis. Overall, the integration of systematic analysis of amino acids and flux ratio analysis provides a systems-level understanding of howL. lactisregulates central metabolism under various conditions.

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The Entner-Doudoroff and Nonoxidative Pentose Phosphate Pathways Bypass Glycolysis and the Oxidative Pentose Phosphate Pathway in Ralstonia solanacearum

mSystems ◽

10.1128/msystems.00091-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Poonam Jyoti ◽

Manu Shree ◽

Chandrakant Joshi ◽

Tulika Prakash ◽

Suvendra Kumar Ray ◽

...

Keyword(s):

Network Analysis ◽

Metabolic Network ◽

Pentose Phosphate Pathway ◽

Ralstonia Solanacearum ◽

Metabolic Flux ◽

Pentose Phosphate ◽

Flux Analysis ◽

Content Type ◽

Metabolic Network Analysis ◽

Balance Analysis

ABSTRACT In Ralstonia solanacearum, a devastating phytopathogen whose metabolism is poorly understood, we observed that the Entner-Doudoroff (ED) pathway and nonoxidative pentose phosphate pathway (non-OxPPP) bypass glycolysis and OxPPP under glucose oxidation. Evidence derived from 13C stable isotope feeding and genome annotation-based comparative metabolic network analysis supported the observations. Comparative metabolic network analysis derived from the currently available 53 annotated R. solanacearum strains, including a recently reported strain (F1C1), representing the four phylotypes, confirmed the lack of key genes coding for phosphofructokinase (pfk-1) and phosphogluconate dehydrogenase (gnd) enzymes that are relevant for glycolysis and OxPPP, respectively. R. solanacearum F1C1 cells fed with [13C]glucose (99% [1-13C]glucose or 99% [1,2-13C]glucose or 40% [13C6]glucose) followed by gas chromatography-mass spectrometry (GC-MS)-based labeling analysis of fragments from amino acids, glycerol, and ribose provided clear evidence that rather than glycolysis and the OxPPP, the ED pathway and non-OxPPP are the main routes sustaining metabolism in R. solanacearum. The 13C incorporation in the mass ions of alanine (m/z 260 and m/z 232), valine (m/z 288 and m/z 260), glycine (m/z 218), serine (m/z 390 and m/z 362), histidine (m/z 440 and m/z 412), tyrosine (m/z 466 and m/z 438), phenylalanine (m/z 336 and m/z 308), glycerol (m/z 377), and ribose (m/z 160) mapped the pathways supporting the observations. The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. IMPORTANCE Understanding the metabolic versatility of Ralstonia solanacearum is important, as it regulates the trade-off between virulence and metabolism (1, 2) in a wide range of plant hosts. Due to a lack of clear evidence until this work, several published research papers reported on the potential roles of glycolysis and the oxidative pentose phosphate pathway (OxPPP) in R. solanacearum (3, 4). This work provided evidence from 13C stable isotope feeding and genome annotation-based comparative metabolic network analysis that the Entner-Doudoroff pathway and non-OxPPP bypass glycolysis and OxPPP during the oxidation of glucose, a component of the host xylem pool that serves as a potential carbon source (5). The outcomes help better define the central carbon metabolic network of R. solanacearum that can be integrated with 13C metabolic flux analysis as well as flux balance analysis studies for defining the metabolic phenotypes. The study highlights the need to critically examine phytopathogens whose metabolism is poorly understood.

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13C Metabolic Flux Analysis Indicates Endothelial Cells Attenuate Metabolic Perturbations by Modulating TCA Activity

Metabolites ◽

10.3390/metabo11040226 ◽

2021 ◽

Vol 11 (4) ◽

pp. 226

Author(s):

Bilal Moiz ◽

Jonathan Garcia ◽

Sarah Basehore ◽

Angela Sun ◽

Andrew Li ◽

...

Keyword(s):

Pentose Phosphate Pathway ◽

Metabolic Flux Analysis ◽

Metabolic Flux ◽

Isotope Labeling ◽

Umbilical Vein ◽

Pentose Phosphate ◽

Metabolic Inhibitors ◽

Flux Analysis ◽

Therapeutic Effects ◽

13C Metabolic Flux Analysis

Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.

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