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

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.

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Simultaneous Determination of the Rates of the TCA Cycle, Glucose Utilization, α-Ketoglutarate/Glutamate Exchange, and Glutamine Synthesis in Human Brain by NMR

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1995.2 ◽

1995 ◽

Vol 15 (1) ◽

pp. 12-25 ◽

Cited By ~ 244

Author(s):

Graeme F. Mason ◽

Rolf Gruetter ◽

Douglas L. Rothman ◽

Kevin L. Behar ◽

Robert G. Shulman ◽

...

Keyword(s):

Human Brain ◽

Glucose Consumption ◽

Tca Cycle ◽

High Rate ◽

Cycle Rate ◽

The Tca Cycle ◽

Glutamine Synthesis ◽

Physiological Values

13C isotopic tracer data previously obtained by 13C nuclear magnetic resonance in the human brain in vivo were analyzed using a mathematical model to determine metabolic rates in a region of the human neocortex. The tricarboxylic acid (TCA) cycle rate was 0.73 ± 0.19 μmol min−1 g−1 (mean ± SD; n = 4). The standard deviation reflects primarily intersubject variation, since individual uncertainties were low. The rate of α-ketoglutarate/glutamate exchange was 57 ± 26 μmol min−1 g−1 ( n = 3), which is much greater than the TCA cycle rate; the high rate indicates that α-ketoglutarate and glutamate are in rapid exchange and can be treated as a single combined kinetic pool. The rate of synthesis of glutamine from glutamate was 0.47 μmol min−1 g−1 ( n = 4), with 95% confidence limits of 0.139 and 3.094 μmol min−1 g−1; individual uncertainties were biased heavily toward high synthesis rates. From the TCA cycle rate the brain oxygen consumption was estimated to be 2.14 ± 0.48 μmol min−1 g−1 (5.07 ± 1.14 ml 100 g−1 min−1; n = 4), and the rate of brain glucose consumption was calculated to be 0.37 ± 0.08 μmol min−1 g−1 ( n = 4). The sensitivity of the model to the assumptions made was evaluated, and the calculated values were found to be unchanged as long as the assumptions remained near reported physiological values.

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Evidence for reverse flux through pyruvate kinase in skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90935.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. E748-E757 ◽

Cited By ~ 8

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.

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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

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1987.0047 ◽

1987 ◽

Vol 231 (1264) ◽

pp. 339-347 ◽

Cited By ~ 4

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.

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

Scientific Reports ◽

10.1038/s41598-019-55718-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

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.

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

Biochemical Journal ◽

10.1042/bj2540593 ◽

1988 ◽

Vol 254 (2) ◽

pp. 593-598 ◽

Cited By ~ 47

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22052746 ◽

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).

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

Plants ◽

10.3390/plants10030444 ◽

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.

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Sensitive and accurate determination of neurotransmitters from in vivo rat brain microdialysate of Parkinson's disease using in situ ultrasound-assisted derivatization dispersive liquid–liquid microextraction by UHPLC-MS/MS

RSC Advances ◽

10.1039/c6ra23808d ◽

2016 ◽

Vol 6 (110) ◽

pp. 108635-108644 ◽

Cited By ~ 20

Author(s):

Xian-En Zhao ◽

Yongrui He ◽

Ping Yan ◽

Na Wei ◽

Renjun Wang ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Rat Brain ◽

Accurate Determination ◽

Ultrasound Assisted ◽

Dispersive Liquid Liquid Microextraction ◽

Liquid Microextraction

In situ UA-DDLLME coupled with UHPLC-MS/MS has been developed for simultaneous determination of neurotransmitters and baicalein from Parkinson's disease rats.

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

Scientific Reports ◽

10.1038/s41598-021-95590-7 ◽

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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TCA cycle flux estimates from NMR- and GC-MS-determined [13C]glutamate isotopomers in liver

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.6.c2049 ◽

1997 ◽

Vol 272 (6) ◽

pp. C2049-C2062 ◽

Cited By ~ 17

Author(s):

J. A. Vogt ◽

D. M. Yarmush ◽

Y. M. Yu ◽

C. Zupke ◽

A. J. Fischman ◽

...

Keyword(s):

Tca Cycle ◽

Gas Chromatography Mass Spectrometry ◽

Flow Rates ◽

13C Nuclear Magnetic Resonance ◽

Two Factors ◽

Subsequent Measurement ◽

System 1 ◽

Relative Flow

Infusion of 13C-labeled lactate into rabbits and the subsequent measurement of glutamate isotopomers by 13C nuclear magnetic resonance (NMR) spectroscopy enables one to calculate relative flow rates associated with the tricarboxylic acid (TCA) cycle, albeit with a lower precision than one would obtain using a perfused organ. Two factors contribute to the lower precision in the determination of relative flow rates for the in vivo system: 1) a poorly defined pyruvate input and 2) low levels of 13C-enriched oxaloacetate and acetyl-CoA isotopomers, which give rise to weaker glutamate isotopomer NMR signals. To help overcome these limitations, we introduce a procedure to 1) include experimental data from gas chromatography-mass spectrometry (GC-MS) and 2) account for the uncertainty in the labeling of the input to pyruvate by creating the labeling as a measurement that is subject to measurement error. The effects of the uncertainties in the input labeling, NMR data, and MS data are evaluated via a Monte Carlo method. The change in the precision of the relative fluxes for the cases of high/low NMR and high/low MS precision is given. An uncertainty in the lactate measurements of up to 10% does not add significantly to the imprecision of the relative flow rates.

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