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Quantitative Glucose and ATP Sensing in Mammalian Cells

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ABSTRACT

The functioning and survival of mammalian cells requires an active energy metabolism. Metabolic dysfunction plays an important role in many human diseases, including diabetes, cancer, inherited mitochondrial disorders, and metabolic syndrome. The monosaccharide glucose constitutes a key source of cellular energy. Following its import across the plasma membrane, glucose is converted into pyruvate by the glycolysis pathway. Pyruvate oxidation supplies substrates for the ATP-generating mitochondrial oxidative phosphorylation (OXPHOS) system. To gain cell-biochemical knowledge about the operation and regulation of the cellular energy metabolism in the healthy and diseased state, quantitative knowledge is required about (changes in) metabolite concentrations under (non) steady-state conditions. This information can, for instance, be used to construct more realistic in silico models of cell metabolism, which facilitates understanding the consequences of metabolic dysfunction as well as on- and off-target effects of mitochondrial drugs. Here we review the current state-of-the-art live-cell quantification of two key cellular metabolites, glucose and ATP, using protein-based sensors. The latter apply the principle of FRET (fluorescence resonance energy transfer) and allow measurements in different cell compartments by fluorescence microscopy. We further summarize the properties and applications of the FRET-based sensors, their calibration, pitfalls, and future perspectives.

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Abbreviations

2-DG:

2-Deoxy-D-glucose

A:

fluorescence acceptor molecule

AcCoA:

acetyl-coenzyme A

ADP:

adenoside diphosphate

AMP:

adenosine monophosphate

AMPK:

AMP-activated protein kinase

ANT:

adenine nucleotide translocator

ATP:

adenoside triphosphate

D:

fluorescence donor molecule

DNP:

2,4-Dinitrophenol

ECFP:

enhanced cyan fluorescent protein

ER:

endoplasmic reticulum

EYFP:

enhanced yellow fluorescent protein

FCCP:

carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

FRET:

fluorescence resonance energy transfer

FS:

fractional saturation

G6P:

glucose-6-phosphate

GFP:

green fluorescent protein

GGBP:

glucose galactose-binding protein

GK:

glucokinase

GLUT:

glucose transporter

HK:

hexokinase

IAA:

iodoacetate

LDH:

lactate dehydrogenase

OFP:

orange fluorescent protein

OXPHOS:

oxidative phosphorylation

PBP:

periplasmic binding protein

PDH:

pyruvate dehydrogenase

PFK:

phosphofructokinase

PK:

pyruvate kinase

PKC:

protein kinase C

PM:

plasma membrane

PPP:

pentose phosphate pathway

ROS:

reactive oxygen species

SGLT:

sodium-dependent glucose cotransporters

SLO:

streptolysin O

SNR:

signal-to-noise ratio

TCA:

tricarboxylic acid

TPA:

phorbol 12-myristate 13-acetate

VDAC:

voltage-dependent anion channel

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ACKNOWLEDGMENTS & DISCLOSURES

This work was supported by an equipment grant of NWO (Netherlands Organization for Scientific Research, No: 911-02-008), the Dutch Ministry of Economic Affairs (Innovative Onderzoeks Projecten (IOP) Grant: #IGE05003), and the CSBR (Centres for Systems Biology Research) initiative from NWO (No: CSBR09/013V). We are grateful to Dr. J.J. Esseling & Mr. A. Klymov (Dept. of Biochemistry, NCMLS) for performing ATeam microscopy experiments. We apologize to those authors whose articles we were unable to cite because of space limitations.

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Authors and Affiliations

  1. Department of Biochemistry (286) Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, NL-6500 HB, Nijmegen, The Netherlands

    Dania C. Liemburg-Apers, Marleen Forkink, Marco Nooteboom, Herman G. Swarts, Roland Brock, Peter H. G. M. Willems & Werner J. H. Koopman

  2. Department of Pediatrics Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

    Dania C. Liemburg-Apers, Marco Nooteboom & Jan A. M. Smeitink

  3. Centre for Systems Biology and Bioenergetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

    Dania C. Liemburg-Apers, Marco Nooteboom, Jan A. M. Smeitink, Peter H. G. M. Willems & Werner J. H. Koopman

  4. Kyoto University, Kyoto, Japan

    Hiromi Imamura

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  1. Dania C. Liemburg-Apers
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Correspondence to Werner J. H. Koopman.

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Liemburg-Apers, D.C., Imamura, H., Forkink, M. et al. Quantitative Glucose and ATP Sensing in Mammalian Cells. Pharm Res 28, 2745–2757 (2011). https://doi.org/10.1007/s11095-011-0492-8

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  • DOI: https://doi.org/10.1007/s11095-011-0492-8

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