In vivo 13C MRS in the mouse brain at 14.1 Tesla and metabolic flux quantification under infusion of [1,6-13C2]glucose

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 µmol/g/min) and neurons (VTCAn = 0.56 ± 0.03 µmol/g/min), pyruvate carboxylase activity (VPC = 0.041 ± 0.003 µmol/g/min) and neurotransmission rate (VNT = 0.084 ± 0.008 µmol/g/min), resulting in a cerebral metabolic rate of glucose (CMRglc) of 0.38 ± 0.02 µmol/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.

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In Vivo Metabolism of [1,6-13C2]Glucose Reveals Distinct Neuroenergetic Functionality between Mouse Hippocampus and Hypothalamus

Metabolites ◽

10.3390/metabo11010050 ◽

2021 ◽

Vol 11 (1) ◽

pp. 50

Author(s):

Antoine Cherix ◽

Rajesh Sonti ◽

Bernard Lanz ◽

Hongxia Lei

Keyword(s):

Metabolic Flux ◽

Aerobic Glycolysis ◽

Resonance Spectroscopy ◽

Flux Analysis ◽

Gamma Aminobutyric Acid ◽

Atp Production ◽

Functional Features ◽

Metabolic Dependence ◽

The Brain

Glucose is a major energy fuel for the brain, however, less is known about specificities of its metabolism in distinct cerebral areas. Here we examined the regional differences in glucose utilization between the hypothalamus and hippocampus using in vivo indirect 13C magnetic resonance spectroscopy (1H-[13C]-MRS) upon infusion of [1,6-13C2]glucose. Using a metabolic flux analysis with a 1-compartment mathematical model of brain metabolism, we report that compared to hippocampus, hypothalamus shows higher levels of aerobic glycolysis associated with a marked gamma-aminobutyric acid-ergic (GABAergic) and astrocytic metabolic dependence. In addition, our analysis suggests a higher rate of ATP production in hypothalamus that is accompanied by an excess of cytosolic nicotinamide adenine dinucleotide (NADH) production that does not fuel mitochondria via the malate-aspartate shuttle (MAS). In conclusion, our results reveal significant metabolic differences, which might be attributable to respective cell populations or functional features of both structures.

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Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping

The ISME Journal ◽

10.1038/s41396-020-00805-w ◽

2020 ◽

Author(s):

Christopher E. Lawson ◽

Guylaine H. L. Nuijten ◽

Rob M. de Graaf ◽

Tyler B. Jacobson ◽

Martin Pabst ◽

...

Keyword(s):

Carbon Metabolism ◽

Metabolic Networks ◽

Metabolic Flux ◽

Citrate Synthase ◽

Side Population ◽

Tricarboxylic Acid Cycle ◽

Anammox Bacteria ◽

Flux Analysis ◽

Anammox Bacterium

Abstract Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

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Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo13C and 2H metabolic network mapping

10.1101/835298 ◽

2019 ◽

Author(s):

Christopher E. Lawson ◽

Guylaine H.L. Nuijten ◽

Rob M. de Graaf ◽

Tyler B. Jacobson ◽

Martin Pabst ◽

...

Keyword(s):

Carbon Metabolism ◽

Metabolic Networks ◽

Metabolic Flux ◽

Citrate Synthase ◽

Side Population ◽

Tricarboxylic Acid Cycle ◽

Anammox Bacteria ◽

Flux Analysis ◽

Anammox Bacterium

AbstractAnaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and mixotrophy beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series 13C and 2H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis (INST-MFA). Our findings confirm predicted metabolic pathways used for CO2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of an oxidative tricarboxylic acid cycle, despite the genome not encoding a known citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO2 followed by reassimilation. In contrast, our data suggests that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

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An Untargeted Metabolomics Investigation of Milk from Dairy Cows with Clinical Mastitis by 1H-NMR

Foods ◽

10.3390/foods10081707 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1707

Author(s):

Chenglin Zhu ◽

Kaiwei Tang ◽

Xuan Lu ◽

Junni Tang ◽

Luca Laghi

Keyword(s):

Dairy Cows ◽

1H Nmr ◽

Tricarboxylic Acid Cycle ◽

Tca Cycle ◽

Quantitative Information ◽

Clinical Mastitis ◽

Economic Losses ◽

Resonance Spectroscopy ◽

Mastitic Milk ◽

Chemical Groups

Mastitis is one of the diseases with the highest incidence in dairy cows, causing huge economic losses to the dairy industry all over the world. The aim of the study was to characterize mastitic milk metabolome through untargeted nuclear magnetic resonance spectroscopy (1H-NMR). Taking advantage of the high reproducibility of 1H-NMR, we had the opportunity to provide quantitative information for all the metabolites identified. Fifty-four molecules were characterized, sorted mainly into the chemical groups, namely amino acids, peptides and analogues, carbohydrates and derivates, organic acids and derivates, nucleosides, nucleotides and analogues. Combined with serum metabolomic investigations, several pathways were addressed to explain the mechanisms of milk metabolome variation affected by clinical mastitis, such as tricarboxylic acid cycle (TCA cycle) and phenylalanine, tyrosine and tryptophan biosynthesis. These results provide a further understanding of milk metabolome altered by clinical mastitis, which can be used as a reference for the further milk metabolome investigations.

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Assessment of Metabolic Fluxes in the Mouse Brain in Vivo Using 1H-[13C] NMR Spectroscopy at 14.1 Tesla

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2014.251 ◽

2015 ◽

Vol 35 (5) ◽

pp. 759-765 ◽

Cited By ~ 15

Author(s):

Lijing Xin ◽

Bernard Lanz ◽

gxia Lei ◽

Rolf Gruetter

Keyword(s):

Mouse Models ◽

Mouse Brain ◽

Conversion Rate ◽

Tca Cycle ◽

Compartment Model ◽

13C Nmr Spectroscopy ◽

Resonance Spectroscopy ◽

Metabolic Fluxes ◽

Short Echo Time

13C magnetic resonance spectroscopy (MRS) combined with the administration of 13C labeled substrates uniquely allows to measure metabolic fluxes in vivo in the brain of humans and rats. The extension to mouse models may provide exclusive prospect for the investigation of models of human diseases. In the present study, the short-echo-time (TE) full-sensitivity 1H-[13C] MRS sequence combined with high magnetic field (14.1 T) and infusion of [U-13C6] glucose was used to enhance the experimental sensitivity in vivo in the mouse brain and the 13C turnover curves of glutamate C4, glutamine C4, glutamate+glutamine C3, aspartate C2, lactate C3, alanine C3, γ-aminobutyric acid C2, C3 and C4 were obtained. A one-compartment model was used to fit 13C turnover curves and resulted in values of metabolic fluxes including the tricarboxylic acid (TCA) cycle flux VTCA (1.05 ± 0.04 μmol/g per minute), the exchange flux between 2-oxoglutarate and glutamate Vx (0.48 ± 0.02 μmol/g per minute), the glutamate-glutamine exchange rate Vgln (0.20 ± 0.02 μmol/g per minute), the pyruvate dilution factor Kdil (0.82 ± 0.01), and the ratio for the lactate conversion rate and the alanine conversion rate VLac/ VAla (10 ± 2). This study opens the prospect of studying transgenic mouse models of brain pathologies.

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Metabolic control of nitrogen fixation in rhizobium-legume symbioses

Science Advances ◽

10.1126/sciadv.abh2433 ◽

2021 ◽

Vol 7 (31) ◽

pp. eabh2433

Author(s):

Carolin C. M. Schulte ◽

Khushboo Borah ◽

Rachel M. Wheatley ◽

Jason J. Terpolilli ◽

Gerhard Saalbach ◽

...

Keyword(s):

Metabolic Flux ◽

Tricarboxylic Acid Cycle ◽

Oxygen Demand ◽

Metabolic Model ◽

Redox Balance ◽

Ammonia Assimilation ◽

Nodule Formation ◽

Flux Analysis ◽

Carbon And Nitrogen ◽

Genome Scale

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD+ ratio than sugars. Modeling and 13C metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing dicarboxylates as the energy and electron source donors for N2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses.

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Fast Isotopic Exchange between Mitochondria and Cytosol in Brain Revealed by Relayed 13C Magnetization Transfer Spectroscopy

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2008.170 ◽

2009 ◽

Vol 29 (4) ◽

pp. 661-669 ◽

Cited By ~ 12

Author(s):

Jehoon Yang ◽

Su Xu ◽

Jun Shen

Keyword(s):

Isotopic Exchange ◽

Tricarboxylic Acid Cycle ◽

Magnetization Transfer ◽

Tricarboxylic Acid ◽

Transfer Effect ◽

Resonance Spectroscopy ◽

Exchange Reactions ◽

Acid Cycle ◽

Longitudinal Relaxation

In vivo13C magnetic resonance spectroscopy has been applied to studying brain metabolic processes by measuring 13C label incorporation into cytosolic pools such as glutamate and aspartate. However, the rate of exchange between mitochondrial α-ketoglutarate/oxaloacetate and cytosolic glutamate/aspartate ( Vx) extracted from metabolic modeling has been controversial. Because brain fumarase is exclusively located in the mitochondria, and mitochondrial fumarate is connected to cytosolic aspartate through a chain of fast exchange reactions, it is possible to directly measure Vx from the four-carbon side of the tricarboxylic acid cycle by magnetization transfer. In isoflurane-anesthetized adult rat brain, a relayed 13C magnetization transfer effect on cytosolic aspartate C2 at 53.2ppm was detected after extensive signal averaging with fumarate C2 at 136.1ppm irradiated using selective radiofrequency pulses. Quantitative analysis using Bloch–McConnell equations and a four-site exchange model found that VxE13–19 µmol per g per min (≫ VTCA, the tricarboxylic acid cycle rate) when the longitudinal relaxation time of malate C2 was assumed to be within ±33% of that of aspartate C2. If VxE VTCA, the isotopic exchange between mitochondria and cytosol would be too slow on the time scale of 13C longitudinal relaxation to cause a detectable magnetization transfer effect.

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Interactions between Pyruvate and Lactate Metabolism in Propionibacterium freudenreichii subsp.shermanii: In Vivo 13C Nuclear Magnetic Resonance Studies

Applied and Environmental Microbiology ◽

10.1128/aem.66.5.2012-2020.2000 ◽

2000 ◽

Vol 66 (5) ◽

pp. 2012-2020 ◽

Cited By ~ 16

Author(s):

Catherine Deborde ◽

Patrick Boyaval

Keyword(s):

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

Tricarboxylic Acid Cycle ◽

Dry Weight ◽

Resonance Spectroscopy ◽

Propionibacterium Freudenreichii ◽

Pyruvate Metabolism ◽

13C Nuclear Magnetic Resonance ◽

Nuclear Magnetic

ABSTRACT In vivo 13C nuclear magnetic resonance spectroscopy was used to elucidate the pathways and the regulation of pyruvate metabolism and pyruvate-lactate cometabolism noninvasively in living-cell suspensions of Propionibacterium freudenreichiisubsp. shermanii. The most important result of this work concerns the modification of fluxes of pyruvate metabolism induced by the presence of lactate. Pyruvate was temporarily converted to lactate and alanine; the flux to acetate synthesis was maintained, but the flux to propionate synthesis was increased; and the reverse flux of the first part of the Wood-Werkman cycle, up to acetate synthesis, was decreased. Pyruvate was consumed at apparent initial rates of 148 and 90 μmol · min−1 · g−1 (cell dry weight) when it was the sole substrate or cometabolized with lactate, respectively. Lactate was consumed at an apparent initial rate of 157 μmol · min−1 · g−1when it was cometabolized with pyruvate. P. shermanii used several pathways, namely, the Wood-Werkman cycle, synthesis of acetate and CO2, succinate synthesis, gluconeogenesis, the tricarboxylic acid cycle, and alanine synthesis, to manage its pyruvate pool sharply. In both types of experiments, acetate synthesis and the Wood-Werkman cycle were the metabolic pathways used most.

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Cortical Substrate Oxidation during Hyperketonemia in the Fasted Anesthetized Rat in Vivo

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2011.91 ◽

2011 ◽

Vol 31 (12) ◽

pp. 2313-2323 ◽

Cited By ~ 19

Author(s):

Lihong Jiang ◽

Graeme F Mason ◽

Douglas L Rothman ◽

Robin A de Graaf ◽

Kevin L Behar

Keyword(s):

Time Course ◽

Ketone Bodies ◽

Tricarboxylic Acid Cycle ◽

Metabolic Model ◽

Substrate Oxidation ◽

Resonance Spectroscopy ◽

Neural Function ◽

Anesthetized Rats ◽

Time Courses

Ketone bodies are important alternate brain fuels, but their capacity to replace glucose and support neural function is unclear. In this study, the contributions of ketone bodies and glucose to cerebral cortical metabolism were measured in vivo in halothane-anesthetized rats fasted for 36 hours ( n=6) and receiving intravenous [2,4-13C2]-d- β-hydroxybutyrate (BHB). Time courses of 13C-enriched brain amino acids (glutamate-C4, glutamine-C4, and glutamate and glutamine-C3) were measured at 9.4 Tesla using spatially localized 1H-[13C]-nuclear magnetic resonance spectroscopy. Metabolic rates were estimated by fitting a constrained, two-compartment (neuron–astrocyte) metabolic model to the 13C time-course data. We found that ketone body oxidation was substantial, accounting for 40% of total substrate oxidation (glucose plus ketone bodies) by neurons and astrocytes. d- β-Hydroxybutyrate was oxidized to a greater extent in neurons than in astrocytes (∼70:30), and followed a pattern closely similar to the metabolism of [1-13C]glucose reported in previous studies. Total neuronal tricarboxylic acid cycle (TCA) flux in hyperketonemic rats was similar to values reported for normal (nonketotic) anesthetized rats infused with [1-13C]glucose, but neuronal glucose oxidation was 40% to 50% lower, indicating that ketone bodies had compensated for the reduction in glucose use.

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