Growth of Paracoccus denitrificans on [2, 3- 13 C]succinate and [1, 4- 13 C]succinate. I. The flux of carbon in energy metabolism and the operation of the TCA cycle

1987 ◽  
Vol 231 (1264) ◽  
pp. 339-347 ◽  
Keyword(s):  
Mass Spectrometry ◽  
Energy Metabolism ◽  
Malic Enzyme ◽  
Paracoccus Denitrificans ◽  
Tca Cycle ◽  
Labelling Pattern ◽  
Carboxyl Groups ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

The metabolism of Paracoccus denitrificans , grown on either [2, 3- 13 C]- succinate or [1, 4- 13 C]succinate, was investigated by using gas chromato­graphy-mass spectrometry. The distribution of label in a group of metabolites closely related to the TCA-cycle intermediates showed that the flux of carbon from succinate in energy metabolism in vivo was via pyruvate (malic enzyme) and acetyl CoA. The labelling pattern of the carboxyl groups showed that one fifth of the succinate pool was formed by the regeneration of succinate via the TCA cycle, and four fifths was supplied externally as substrate from the medium.

Growth of Paracoccus denitrificans on [2, 3- 13 C]succinate and [1, 4- 13 C]succinate IIl. Biosynthetic pathways

1988 ◽  
Vol 233 (1270) ◽  
pp. 1-15 ◽  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Gas Chromatography ◽  
Paracoccus Denitrificans ◽  
Tca Cycle ◽  
Gas Chromatography Mass Spectrometry ◽  
Biosynthetic Pathways ◽  
The Tca Cycle ◽  
Phosphoenol Pyruvate

The biosynthesis in vivo of a number of amino acids, sugars, and purines in Paracoccus denitrificans grown on either [2, 3- 13 C]succinate or [1, 4- 13 C]succinate was investigated by using gas chromatography–mass spectrometry. The distribution of label in the TCA-cycle-related amino acids indicated that carbon intermediates of energy metabolism were utilized as precursors for the biosynthesis of these amino acids in vivo . The biosynthesis of glycine, serine, phenylalanine and glycerol from labelled succinate in vivo were consistent with phosphoenol pyruvate as an intermediate. A mechanism for the formation of C 4 , C 5 and C 6 sugars without the use of fructose-1, 6-bisphosphate aldolase (which has not been detected in P. denitrificans ) is proposed. The 13 C-enrichments of ribose in the bacterium indicate that there are at least three routes of ribose biosynthesis operating during growth on labelled succinate. The probability distribution of labelled purine molecules was successfully predicted for adenine, guanine and adenosine, thus confirming their generally accepted route of biosynthesis in vivo .

scholarly journals Ammonia inhibits energy metabolism in astrocytes in a rapid and glutamate dehydrogenase 2-dependent manner

2020 ◽  
Vol 13 (10) ◽  
pp. dmm047134
Author(s):  
Leonie Drews ◽  
Marcel Zimmermann ◽  
Philipp Westhoff ◽  
Dominik Brilhaus ◽  
Rebecca E. Poss ◽  
...  
Keyword(s):  
Amino Acids ◽  
Energy Metabolism ◽  
Glutamate Dehydrogenase ◽  
Mitochondrial Respiration ◽  
Tca Cycle ◽  
Dependent Manner ◽  
Independent Manner ◽  
The Tca Cycle ◽  
Branched Chain ◽  
Tca Cycle Intermediates

ABSTRACTAstrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.

scholarly journals Evidence for reverse flux through pyruvate kinase in skeletal muscle

2009 ◽  
Vol 296 (4) ◽  
pp. E748-E757 ◽  
Author(s):  
Eunsook S. Jin ◽  
A. Dean Sherry ◽  
Craig R. Malloy
Keyword(s):  
Skeletal Muscle ◽  
Glucose Production ◽  
Tca Cycle ◽  
Hepatic Glycogen ◽  
Direct Transfer ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates ◽  
Minced Muscle ◽  
Labeling Pattern

Conversion of lactate to glucose was examined in myotubes, minced muscle tissue, and rats exposed to 2H2O or 13C-enriched substrates. Myotubes or minced skeletal muscle incubated with [U-13C3]lactate released small amounts of [1,2,3-13C3]- or [4,5,6-13C3]glucose. This labeling pattern is consistent with direct transfer from lactate to glucose without randomization in the tricarboxylic acid (TCA) cycle. After exposure of incubated muscle to 2H2O, [U-13C3]lactate, glucose, and glutamine, there was minimal release of synthesized glucose to the medium based on a low level of 2H enrichment in medium glucose but 50- to 100-fold greater 2H enrichment in glucosyl units from glycogen. The 13C enrichment pattern in glycogen from incubated skeletal muscle was consistent only with direct transfer of lactate to glucose without exchange in TCA cycle intermediates. 13C nuclear magnetic resonance (NMR) spectra of glutamate from the same tissue showed flux from lactate through pyruvate dehydrogenase but not flux through pyruvate carboxylase into the TCA cycle. Carbon from an alternative substrate for glucose production that requires metabolism through the TCA cycle, propionate, did not enter glycogen, suggesting that TCA cycle intermediates do not exchange with phospho enolpyruvate. In vivo, the 13C labeling patterns in hepatic glycogen and plasma glucose after administration of [U-13C3]lactate did not differ significantly. However, skeletal muscle glycogen was substantially enriched in [1,2,3-13C3]- and [4,5,6-13C3]glucose units that could only occur through skeletal muscle glyconeogenesis rather than glycogenesis. Lactate serves as a substrate for glyconeogenesis in vivo without exchange into symmetric intermediates of the TCA cycle.

scholarly journals NMR Determination of the TCA Cycle Rate and α-Ketoglutarate/Glutamate Exchange Rate in Rat Brain

1992 ◽  
Vol 12 (3) ◽  
pp. 434-447 ◽  
Author(s):  
Graeme F. Mason ◽  
Douglas L. Rothman ◽  
Kevin L. Behar ◽  
Robert G. Shulman
Keyword(s):  
Exchange Rate ◽  
Rat Brain ◽  
Tca Cycle ◽  
Isotopic Labeling ◽  
Cycle Rate ◽  
The Tca Cycle ◽  
The Difference ◽  
Tca Cycle Intermediates

A mathematical model of cerebral glucose metabolism was developed to analyze the isotopic labeling of carbon atoms C4 and C3 of glutamate following an intravenous infusion of [1-13C]glucose. The model consists of a series of coupled metabolic pools representing glucose, glycolytic intermediates, tricarboxylic acid (TCA) cycle intermediates, glutamate, aspartate, and glutamine. Based on the rate of 13C isotopic labeling of glutamate C4 measured in a previous study, the TCA cycle rate in rat brain was determined to be 1.58 ± 0.41 μmol min−1 g−1 (mean ± SD, n = 5). Analysis of the difference between the rates of isotopic enrichment of glutamate C4 and C3 permitted the rate of exchange between α-ketoglutarate (α-KG) and glutamate to be assessed in vivo. In rat brain, the exchange rate between α-KG and glutamate is between 89 ± 35 and 126 ± 22 times faster than the TCA cycle rate (mean ± SD, n = 4). The sensitivity of the calculated value of the TCA cycle rate to other metabolic fluxes and to concentrations of glycolytic and TCA cycle intermediates was tested and found to be small.

scholarly journals Colorectal cancers utilize glutamine as an anaplerotic substrate of the TCA cycle in vivo

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yiqing Zhao ◽  
Xuan Zhao ◽  
Vanessa Chen ◽  
Ying Feng ◽  
Lan Wang ◽  
...  
Keyword(s):  
Tca Cycle ◽  
Genetically Engineered ◽  
Glutamine Metabolism ◽  
Colorectal Cancers ◽  
Wild Type ◽  
Colon Tumors ◽  
The Tca Cycle ◽  
Genetically Engineered Mice ◽  
Tca Cycle Intermediates

AbstractCancer cells in culture rely on glutamine as an anaplerotic substrate to replenish tricarboxylic acid (TCA) cycle intermediates that have been consumed. but it is uncertain whether cancers in vivo depend on glutamine for anaplerosis. Here, following in vivo infusions of [13C5]-glutamine in mice bearing subcutaneous colon cancer xenografts, we showed substantial amounts of infused [13C5]-glutamine enters the TCA cycle in the tumors. Consistent with our prior observation that colorectal cancers (CRCs) with oncogenic mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic (PIK3CA) subunit are more dependent on glutamine than CRCs with wild type PIK3CA, labeling from glutamine to most TCA cycle intermediates was higher in PIK3CA-mutant subcutaneous xenograft tumors than in wild type PIK3CA tumors. Moreover, using orthotopic mouse colon tumors estalished from human CRC cells or patient-derived xenografts, we demonstrated substantial amounts of infused [13C5]-glutamine enters the TCA cycle in the tumors and tumors utilize anaplerotic glutamine to a greater extent than adjacent normal colon tissues. Similar results were seen in spontaneous colon tumors arising in genetically engineered mice. Our studies provide compelling evidence CRCs utilizes glutamine to replenish the TCA cycle in vivo, suggesting that targeting glutamine metabolism could be a therapeutic approach for CRCs, especially for PIK3CA-mutant CRCs.

scholarly journals Glutamate-glutamine homeostasis is perturbed in neurons and astrocytes derived from patient iPSC models of frontotemporal dementia

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Blanca I. Aldana ◽  
Yu Zhang ◽  
Pia Jensen ◽  
Abinaya Chandrasekaran ◽  
Sofie K. Christensen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Stem Cells ◽  
Energy Metabolism ◽  
Frontotemporal Dementia ◽  
Pluripotent Stem Cells ◽  
Quantitative Proteomics ◽  
Tca Cycle ◽  
Glutamate Metabolism ◽  
The Tca Cycle ◽  
Molecular Determinants

Abstract Frontotemporal dementia (FTD) is amongst the most prevalent early onset dementias and even though it is clinically, pathologically and genetically heterogeneous, a crucial involvement of metabolic perturbations in FTD pathology is being recognized. However, changes in metabolism at the cellular level, implicated in FTD and in neurodegeneration in general, are still poorly understood. Here we generate induced human pluripotent stem cells (hiPSCs) from patients carrying mutations in CHMP2B (FTD3) and isogenic controls generated via CRISPR/Cas9 gene editing with subsequent neuronal and glial differentiation and characterization. FTD3 neurons show a dysregulation of glutamate-glutamine related metabolic pathways mapped by 13C-labelling coupled to mass spectrometry. FTD3 astrocytes show increased uptake of glutamate whilst glutamate metabolism is largely maintained. Using quantitative proteomics and live-cell metabolic analyses, we elucidate molecular determinants and functional alterations of neuronal and glial energy metabolism in FTD3. Importantly, correction of the mutations rescues such pathological phenotypes. Notably, these findings implicate dysregulation of key enzymes crucial for glutamate-glutamine homeostasis in FTD3 pathogenesis which may underlie vulnerability to neurodegeneration. Graphical abstract Neurons derived from human induced pluripotent stem cells (hiPSCs) of patients carrying mutations in CHMP2B (FTD3) display major metabolic alterations compared to CRISPR/Cas9 generated isogenic controls. Using quantitative proteomics, 13C-labelling coupled to mass spectrometry metabolic mapping and seahorse analyses, molecular determinants and functional alterations of neuronal and astrocytic energy metabolism in FTD3 were characterized. Our findings implicate dysregulation of glutamate-glutamine homeostasis in FTD3 pathogenesis. In addition, FTD3 neurons recapitulate glucose hypometabolism observed in FTD patient brains. The impaired mitochondria function found here is concordant with disturbed TCA cycle activity and decreased glycolysis in FTD3 neurons. FTD3 neuronal glutamine hypermetabolism is associated with up-regulation of PAG expression and, possibly, ROS production. Distinct compartments of glutamate metabolism can be suggested for the FTD3 neurons. Endogenous glutamate generated from glutamine via PAG may enter the TCA cycle via AAT (left side of neuron) while exogenous glutamate taken up from the extracellular space may be incorporated into the TCA cycle via GDH (right side of the neuron) FTD3 astrocytic glutamate uptake is upregulated whilst glutamate metabolism is largely maintained. Finally, pharmacological reversal of glutamate hypometabolism manifesting from decreased GDH expression should be explored as a novel therapeutic intervention for treating FTD3.

scholarly journals Propionate metabolism in the rat heart by 13C n.m.r. spectroscopy

1988 ◽  
Vol 254 (2) ◽  
pp. 593-598 ◽  
Author(s):  
A D Sherry ◽  
C R Malloy ◽  
R E Roby ◽  
A Rajagopal ◽  
F M Jeffrey
Keyword(s):  
Rat Heart ◽  
Tca Cycle ◽  
Methylmalonic Aciduria ◽  
Non Invasive ◽  
Propionate Metabolism ◽  
The Tca Cycle ◽  
Exogenous Substrate ◽  
Oxidative Pathway ◽  
Tca Cycle Intermediates

High-resolution 13C n.m.r. spectroscopy has been used to examine propionate metabolism in the perfused rat heart. A number of tricarboxylic acid (TCA) cycle intermediates are observable by 13C n.m.r. in hearts perfused with mixtures of pyruvate and propionate. When the enriched 13C-labelled nucleus originates with pyruvate, the resonances of the intermediates appear as multiplets due to formation of multiply-enriched 13C-labelled isotopomers, whereas when the 13C-labelled nucleus originates with propionate, these same intermediates appear as singlets in the 13C spectrum since entry of propionate into the TCA cycle occurs via succinyl-CoA. An analysis of the isotopomer populations in hearts perfused with [3-13C]pyruvate plus unlabelled propionate indicates that about 27% of the total pyruvate pool available to the heart is derived directly from unlabelled propionate. This was substantiated by perfusing a heart for 2 h with [3-13C]propionate as the only available exogenous substrate. Under these conditions, all of the propionate consumed by the heart, as measured by conventional chemical analysis, ultimately entered the oxidative pathway as [2-13C] or [3-13C]pyruvate. This is consistent with entry of propionate into the TCA cycle intermediate pools as succinyl-CoA and concomitant disposal of malate to pyruvate via the malic enzyme. 13C resonances arising from enriched methylmalonate and propionylcarnitine are also detected in hearts perfused with [3-13C] or [1-13C]propionate which suggests that 13C n.m.r. may be useful as a non-invasive probe in vivo of metabolic abnormalities involving the propionate pathway, such as methylmalonic aciduria or propionic acidaemia.

scholarly journals Mitochondrial Mistranslation in Brain Provokes a Metabolic Response Which Mitigates the Age-Associated Decline in Mitochondrial Gene Expression

2021 ◽  
Vol 22 (5) ◽  
pp. 2746
Author(s):  
Dimitri Shcherbakov ◽  
Reda Juskeviciene ◽  
Adrián Cortés Sanchón ◽  
Margarita Brilkova ◽  
Hubert Rehrauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Response ◽  
Mitochondrial Gene ◽  
Tca Cycle ◽  
Brain Mitochondria ◽  
Mitochondrial Gene Expression ◽  
Rna Seq ◽  
The Tca Cycle ◽  
Neurological Phenotype

Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).

scholarly journals Cytochrome c Deficiency Differentially Affects the In Vivo Mitochondrial Electron Partitioning and Primary Metabolism Depending on the Photoperiod

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 444
Author(s):  
Igor Florez-Sarasa ◽  
Elina Welchen ◽  
Sofia Racca ◽  
Daniel H. Gonzalez ◽  
José G. Vallarino ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Tca Cycle ◽  
Day Length ◽  
Primary Metabolism ◽  
Stress Signaling ◽  
Alternative Pathways ◽  
Tca Cycle Intermediates ◽  
Growth Adaptation ◽  
Plant Respiration

Plant respiration provides metabolic flexibility under changing environmental conditions by modulating the activity of the nonphosphorylating alternative pathways from the mitochondrial electron transport chain, which bypass the main energy-producing components of the cytochrome oxidase pathway (COP). While adjustments in leaf primary metabolism induced by changes in day length are well studied, possible differences in the in vivo contribution of the COP and the alternative oxidase pathway (AOP) between different photoperiods remain unknown. In our study, in vivo electron partitioning between AOP and COP and expression analysis of respiratory components, photosynthesis, and the levels of primary metabolites were studied in leaves of wild-type (WT) plants and cytochrome c (CYTc) mutants, with reduced levels of COP components, under short- and long-day photoperiods. Our results clearly show that differences in AOP and COP in vivo activities between WT and cytc mutants depend on the photoperiod likely due to energy and stress signaling constraints. Parallel responses observed between in vivo respiratory activities, TCA cycle intermediates, amino acids, and stress signaling metabolites indicate the coordination of different pathways of primary metabolism to support growth adaptation under different photoperiods.

scholarly journals Differential and shared effects of eicosapentaenoic acid and docosahexaenoic acid on serum metabolome in subjects with chronic inflammation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wan-Chi Chang ◽  
Jisun So ◽  
Stefania Lamon-Fava
Keyword(s):  
Metabolic Syndrome ◽  
Docosahexaenoic Acid ◽  
Eicosapentaenoic Acid ◽  
Chronic Inflammation ◽  
Cell Function ◽  
Tca Cycle ◽  
Differential Effects ◽  
Epa And Dha ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

AbstractThe omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affect cell function and metabolism, but the differential effects of EPA and DHA are not known. In a randomized, controlled, double-blind, crossover study, we assessed the effects of 10-week supplementation with EPA-only and DHA-only (3 g/d), relative to a 4-week lead-in phase of high oleic acid sunflower oil (3 g/day, defined as baseline), on fasting serum metabolites in 21 subjects (9 men and 12 post-menopausal women) with chronic inflammation and some characteristics of metabolic syndrome. Relative to baseline, EPA significantly lowered the tricarboxylic acid (TCA) cycle intermediates fumarate and α-ketoglutarate and increased glucuronate, UDP-glucuronate, and non-esterified DHA. DHA significantly lowered the TCA cycle intermediates pyruvate, citrate, isocitrate, fumarate, α-ketoglutarate, and malate, and increased succinate and glucuronate. Pathway analysis showed that both EPA and DHA significantly affected the TCA cycle, the interconversion of pentose and glucuronate, and alanine, and aspartate and glutamate pathways (FDR < 0.05) and that DHA had a significantly greater effect on the TCA cycle than EPA. Our results indicate that EPA and DHA exhibit both common and differential effects on cell metabolism in subjects with chronic inflammation and some key aspects of metabolic syndrome.

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