Bidirectionality and Compartmentation of Metabolic Fluxes Are Revealed in the Dynamics of Isotopomer Networks

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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13C-NMR isotopomer distribution analysis: a method for measuring metabolic fluxes in condensation biosynthesis

NMR in Biomedicine ◽

10.1002/nbm.795 ◽

2002 ◽

Vol 15 (6) ◽

pp. 404-415 ◽

Cited By ~ 4

Author(s):

Caterina Puccetti ◽

Tommaso Aureli ◽

Cesare Manetti ◽

Filippo Conti

Keyword(s):

13C Nmr ◽

Distribution Analysis ◽

Isotopomer Distribution ◽

Metabolic Fluxes

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Effect of murine strain on metabolic pathways of glucose production after brief or prolonged fasting

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00601.2004 ◽

2005 ◽

Vol 289 (1) ◽

pp. E53-E61 ◽

Cited By ~ 46

Author(s):

Shawn C. Burgess ◽

F. Mark H. Jeffrey ◽

Charles Storey ◽

Angela Milde ◽

Natasha Hausler ◽

...

Keyword(s):

Genetic Manipulation ◽

Glucose Production ◽

Tca Cycle ◽

Inbred Strains ◽

Mouse Strains ◽

Background Strain ◽

Metabolic Fluxes ◽

Inbred Strains Of Mice ◽

Total Glucose

Background strain is known to influence the way a genetic manipulation affects mouse phenotypes. Despite data that demonstrate variations in the primary phenotype of basic inbred strains of mice, there is limited data available about specific metabolic fluxes in vivo that may be responsible for the differences in strain phenotypes. In this study, a simple stable isotope tracer/NMR spectroscopic protocol has been used to compare metabolic fluxes in ICR, FVB/N (FVB), C57BL/6J (B6), and 129S1/SvImJ (129) mouse strains. After a short-term fast in these mice, there were no detectable differences in the pathway fluxes that contribute to glucose synthesis. However, after a 24-h fast, B6 mice retain some residual glycogenolysis compared with other strains. FVB mice also had a 30% higher in vivo phospho enolpyruvate carboxykinase flux and total glucose production from the level of the TCA cycle compared with B6 and 129 strains, while total body glucose production in the 129 strain was ∼30% lower than in either FVB or B6 mice. These data indicate that there are inherent differences in several pathways involving glucose metabolism of inbred strains of mice that may contribute to a phenotype after genetic manipulation in these animals. The techniques used here are amenable to use as a secondary or tertiary tool for studying mouse models with disruptions of intermediary metabolism.

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DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1202392109 ◽

2012 ◽

Vol 109 (13) ◽

pp. 4968-4973 ◽

Cited By ~ 216

Author(s):

S. Foret ◽

R. Kucharski ◽

M. Pellegrini ◽

S. Feng ◽

S. E. Jacobsen ◽

...

Keyword(s):

Dna Methylation ◽

Honey Bees ◽

Gene Splicing ◽

Metabolic Fluxes ◽

Alternative Phenotypes

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From Metabolomics to Fluxomics: A Computational Procedure to Translate Metabolite Profiles into Metabolic Fluxes

Biophysical Journal ◽

10.1016/j.bpj.2014.11.1857 ◽

2015 ◽

Vol 108 (1) ◽

pp. 163-172 ◽

Cited By ~ 40

Author(s):

Sonia Cortassa ◽

Viviane Caceres ◽

Lauren N. Bell ◽

Brian O’Rourke ◽

Nazareno Paolocci ◽

...

Keyword(s):

Computational Procedure ◽

Metabolic Fluxes ◽

Metabolite Profiles

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Determination of the steady-state turnover rates of the metabolically active pools of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in human erythrocytes

Biochemical Journal ◽

10.1042/bj2590893 ◽

1989 ◽

Vol 259 (3) ◽

pp. 893-896 ◽

Cited By ~ 17

Author(s):

C E King ◽

P T Hawkins ◽

L R Stephens ◽

R H Michell

Keyword(s):

Steady State ◽

Rate Constants ◽

Human Erythrocytes ◽

Turnover Rates ◽

Metabolic Fluxes ◽

Metabolic Pool ◽

Phosphatidylinositol 4 ◽

Kinetics Of ◽

Phosphate Groups

When intact human erythrocytes are incubated at metabolic steady state in a chloride-free medium containing [32P]Pi, there is rapid labelling of the gamma-phosphate of ATP, followed by a slower labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] [King, Stephens, Hawkins, Guy & Michell (1987) Biochem. J. 244, 209-217]. We have analysed the early kinetics of the labelling of these phosphate groups, in order to determine: (a) the steady-state rates of the interconversions of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2; and (b) the fractions of the total cellular complement of PtdIns4P and PtdIns(4,5)P2 that participate in this steady-state turnover. The experimental data most closely fit a pattern of PtdIns4P and PtdIns(4,5)P2 turnover in which one-quarter of the total cellular complement of each lipid is in the metabolic pool that participates in rapid metabolic turnover, with rate constants of 0.028 min-1 for the interconversion of PtdIns and PtdIns4P, and of 0.010 min-1 for the PtdIns4P/PtdIns(4,5)P2 cycle. These rate constants represent metabolic fluxes of approx. 2.1 nmol of lipid/h per ml of packed erythrocytes between PtdIns and PtdIns4P and of approx. 5.7 nmol/h per ml of cells between PtdIns4P and PtdIns(4,5)P2.

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Regulation and control of metabolic fluxes in microbes

Current Opinion in Biotechnology ◽

10.1016/j.copbio.2011.04.016 ◽

2011 ◽

Vol 22 (4) ◽

pp. 566-575 ◽

Cited By ~ 95

Author(s):

Luca Gerosa ◽

Uwe Sauer

Keyword(s):

Metabolic Fluxes ◽

And Control

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Properties of alternative microbial hosts used in synthetic biology: towards the design of a modular chassis

Essays in Biochemistry ◽

10.1042/ebc20160015 ◽

2016 ◽

Vol 60 (4) ◽

pp. 303-313 ◽

Cited By ~ 30

Author(s):

Juhyun Kim ◽

Manuel Salvador ◽

Elizabeth Saunders ◽

Jaime González ◽

Claudio Avignone-Rossa ◽

...

Keyword(s):

Synthetic Biology ◽

Genetic Information ◽

Essential Element ◽

Model Organisms ◽

Biotechnological Applications ◽

Cell Models ◽

Genetic Circuits ◽

Metabolic Diversity ◽

Translational Machinery ◽

Metabolic Fluxes

The chassis is the cellular host used as a recipient of engineered biological systems in synthetic biology. They are required to propagate the genetic information and to express the genes encoded in it. Despite being an essential element for the appropriate function of genetic circuits, the chassis is rarely considered in their design phase. Consequently, the circuits are transferred to model organisms commonly used in the laboratory, such as Escherichia coli, that may be suboptimal for a required function. In this review, we discuss some of the properties desirable in a versatile chassis and summarize some examples of alternative hosts for synthetic biology amenable for engineering. These properties include a suitable life style, a robust cell wall, good knowledge of its regulatory network as well as of the interplay of the host components with the exogenous circuits, and the possibility of developing whole-cell models and tuneable metabolic fluxes that could allow a better distribution of cellular resources (metabolites, ATP, nucleotides, amino acids, transcriptional and translational machinery). We highlight Pseudomonas putida, widely used in many different biotechnological applications as a prominent organism for synthetic biology due to its metabolic diversity, robustness and ease of manipulation.

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