In vivo dynamics of galactose metabolism inSaccharomyces cerevisiae: Metabolic fluxes and metabolite levels

2001 ◽  
Vol 73 (5) ◽  
pp. 412-425 ◽  
Author(s):  
Simon Ostergaard ◽  
Lisbeth Olsson ◽  
Jens Nielsen
Keyword(s):  
Galactose Metabolism ◽  
Metabolic Fluxes

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

2015 ◽  
Vol 35 (5) ◽  
pp. 759-765 ◽  
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.

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

2005 ◽  
Vol 289 (1) ◽  
pp. E53-E61 ◽  
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.

scholarly journals Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80.

1989 ◽  
Vol 9 (7) ◽  
pp. 2950-2956 ◽  
Author(s):  
J M Salmeron ◽  
S D Langdon ◽  
S A Johnston
Keyword(s):  
Transcriptional Activation ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Wild Type ◽  
Galactose Metabolism ◽  
Metabolism Regulation ◽  
Transcriptional Activator Protein ◽  
The Difference ◽  
Carboxy Terminal

In Saccharomyces cerevisiae, transcriptional activation mediated by the GAL4 regulatory protein is repressed in the absence of galactose by the binding of the GAL80 protein, an interaction that requires the carboxy-terminal 28 amino acids of GAL4. The homolog of GAL4 from Kluyveromyces lactis, LAC9, activates transcription in S. cerevisiae and is highly similar to GAL4 in its carboxyl terminus but is not repressed by wild-type levels of GAL80 protein. Here we show that GAL80 does repress LAC9-activated transcription in S. cerevisiae if overproduced. We sought to determine the molecular basis for the difference in the responses of the LAC9 and GAL4 proteins to GAL80. Our results indicate that this difference is due primarily to the fact that under wild-type conditions, the level of LAC9 protein in S. cerevisiae is much higher than that of GAL4, which suggests that LAC9 escapes GAL80-mediated repression by titration of GAL80 protein in vivo. The difference in response to GAL80 is not due to amino acid sequence differences between the LAC9 and GAL4 carboxyl termini. We discuss the implications of these results for the mechanism of galactose metabolism regulation in S. cerevisiae and K. lactis.

scholarly journals Coarse-grained modeling of mitochondrial metabolism enables subcellular flux inference from fluorescence lifetime imaging microscopy

2020 ◽  
Author(s):  
Xingbo Yang ◽  
Daniel J. Needleman
Keyword(s):  
Fluorescence Lifetime ◽  
Metabolic Flux ◽  
Living Cells ◽  
Coarse Grained ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Metabolic Fluxes ◽  
Subcellular Resolution

AbstractMitochondria are central to metabolism and their dysfunctions are associated with many diseases1–9. Metabolic flux, the rate of turnover of molecules through a metabolic pathway, is one of the most important quantities in metabolism, but it remains a challenge to measure spatiotemporal variations in mitochondrial metabolic fluxes in living cells. Fluorescence lifetime imaging microscopy (FLIM) of NADH is a label-free technique that is widely used to characterize the metabolic state of mitochondria in vivo10–18. However, the utility of this technique has been limited by the inability to relate FLIM measurement to the underlying metabolic activities in mitochondria. Here we show that, if properly interpreted, FLIM of NADH can be used to quantitatively measure the flux through a major mitochondrial metabolic pathway, the electron transport chain (ETC), in vivo with subcellular resolution. This result is based on the use of a coarse-grained NADH redox model, which we test in mouse oocytes subject to a wide variety of perturbations by comparing predicted fluxes to direct biochemical measurements and by self-consistency criterion. Using this method, we discovered a subcellular spatial gradient of mitochondrial metabolic flux in mouse oocytes. We showed that this subcellular variation in mitochondrial flux correlates with a corresponding subcellular variation in mitochondrial membrane potential. The developed model, and the resulting procedure for analyzing FLIM of NADH, are valid under nearly all circumstances of biological interest. Thus, this approach is a general procedure to measure metabolic fluxes dynamically in living cells, with subcellular resolution.

scholarly journals Clickable Galactose Analogs for Imaging Glycans in Developing Zebrafish

2019 ◽  
Author(s):  
Jessica Daughtry ◽  
Wendy Cao ◽  
Johnny Ye ◽  
Jeremy Baskin
Keyword(s):  
Metabolic Flux ◽  
Lysosomal Storage Diseases ◽  
Underlying Disease ◽  
Uridine Diphosphate ◽  
Lysosomal Storage ◽  
Galactose Metabolism ◽  
New Class ◽  
Complex Glycans ◽  
Universal Donor

<p>Galactose is one of only nine monosaccharide precursors used to build complex glycans in vertebrates. Defects in galactose metabolism cause galactosemia and lysosomal storage diseases, and the ability to visualize metabolic flux through these pathways would help to understand mechanisms underlying disease pathogenesis. Bioorthogonal metabolic reporters are widely used tools to image glycan biosynthesis, but to date, no galactose analogs have capitalized on this strategy. We demonstrate that the galactose salvage pathway is remarkably intolerant of unnatural galactose and galactose-1-phosphate analogs. Subtle modifications to uridine diphosphate galactose (UDP-Gal), the universal donor for galactosyltransferases, however, yielded effective metabolic probes for labeling glycans in vivo. We applied 6-alkynyl UDP-Gal, followed by click chemistry tagging, to visualize glycosylation during zebrafish development, revealing a striking accumulation into glycan-rich ridges within the organism’s enveloping layer. UDP-Gal analogs represent a new class of glycan metabolic probes for revealing physiological and pathological changes in glycosylation in vivo.</p>

scholarly journals Early detection of doxorubicin-induced cardiotoxicity in rats by its cardiac metabolic signature assessed with hyperpolarized MRI

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Kerstin N. Timm ◽  
Charith Perera ◽  
Vicky Ball ◽  
John A. Henry ◽  
Jack J. Miller ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Real Time ◽  
Chemotherapeutic Agent ◽  
Imaging Techniques ◽  
Functional Decline ◽  
Metabolic Fluxes ◽  
Key Factor ◽  
Hyperpolarized Mri ◽  
Magnetic Resonance Imaging Mri

AbstractDoxorubicin (DOX) is a widely used chemotherapeutic agent that can cause serious cardiotoxic side effects culminating in congestive heart failure (HF). There are currently no clinical imaging techniques or biomarkers available to detect DOX-cardiotoxicity before functional decline. Mitochondrial dysfunction is thought to be a key factor driving functional decline, though real-time metabolic fluxes have never been assessed in DOX-cardiotoxicity. Hyperpolarized magnetic resonance imaging (MRI) can assess real-time metabolic fluxes in vivo. Here we show that cardiac functional decline in a clinically relevant rat-model of DOX-HF is preceded by a change in oxidative mitochondrial carbohydrate metabolism, measured by hyperpolarized MRI. The decreased metabolic fluxes were predominantly due to mitochondrial loss and additional mitochondrial dysfunction, and not, as widely assumed hitherto, to oxidative stress. Since hyperpolarized MRI has been successfully translated into clinical trials this opens up the potential to test cancer patients receiving DOX for early signs of cardiotoxicity.

scholarly journals Regulation of Galactose Metabolism through the HisK:GalR Two-Component System in Thermoanaerobacter tengcongensis

2010 ◽  
Vol 192 (17) ◽  
pp. 4311-4316 ◽  
Author(s):  
Zhong Qian ◽  
Quanhui Wang ◽  
Wei Tong ◽  
Chuanqi Zhou ◽  
Qian Wang ◽  
...  
Keyword(s):  
Phosphoryl Group ◽  
Regulation Mechanism ◽  
Two Component System ◽  
Galactose Metabolism ◽  
Component System ◽  
Two Dimensional Electrophoresis ◽  
Thermoanaerobacter Tengcongensis ◽  
Two Component ◽  
Dimensional Electrophoresis

ABSTRACT Thermoanaerobacter tengcongensis could utilize galactose as a carbon source via the enzymes encoded by a novel gal operon, whose regulation mechanism has yet to be elucidated. We propose here that the gal operon in T. tengcongensis is regulated through a HisK:GalR two-component system. By using radioactive isotope assay and genetic analysis, we found that the kinase of this system, HisK, is phosphorylated by ATP, and the regulator, GalR, accepts a phosphoryl group during phosphorelay, in which the phosphoryl group at HisK-His-259 is transferred to GalR-Asp-56. Two-dimensional electrophoresis, followed by Western blotting, revealed that phosphorylation status of GalR is uniquely dependent on the galactose stimulus in vivo. Furthermore, DNA pulldown assays demonstrated that the phosphorylated GalR prefers binding to the operator DNA O2 , whereas the unphosphorylated GalR to O1 . A model of HisK:GalR is proposed to explain how galactose mediates the expression of the gal operon in T. tengcongensis.

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