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Cited by 361Cancer, Hypoxia, and MetabolismAdvanced Fluorescence Microscopy TechniquesMolecular Sensors and Ion Detection

A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells

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San Martín, Alejandro, Ceballo, Sebastián, Ruminot, Iván, Lerchundi, Rodrigo, Frommer, Wolf B., Barros, Luis Felipe·2013·PLoS ONE
DOI 10.1371/journal.pone.0057712PMC PMC3582500
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Abstract

Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET)-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, α-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3-5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg effect in single mammalian cells.

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Cited by

Sorted by citations

Authors
San Martín, A., Ceballo, S., Ruminot, I., Lerchundi, R., Frommer, W. B., & Barros, L. F.
Affiliations
  • Austral University of Chile (CL)
  • Carnegie Institution for Science (US)
  • Centro de Estudios Científicos (CL)
Corresponding author
Barros, Luis Felipe
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-02-26
Volume
8
Issue
2
Pages
e57712
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0057712
PMC
PMC3582500
OpenAlex
W2055755990
Cited by
361
References
9
Citations by year
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FWCI
5.98
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Americanae (AECID Library) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion, other-oa)
Funders
  • National Institutes of Health
  • Comisión Nacional de Investigación Científica y Tecnológica
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK079109, 1RO1DK079109)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Physical Sciences › Chemistry › Spectroscopy › Molecular Sensors and Ion Detection
Keywords
lactate sensor, Warburg effect, monocarboxylate transporters, lactate flux, glioma cells, astrocytes, cancer metabolism, mitochondrial inhibition, sodium azide, lactic acid production
MeSH terms
Animals, Biological Transport, DNA-Binding Proteins, Glioma, Humans, Male, Models, Molecular, Protein Conformation, Transcription Factors, Biosensing Techniques, Lactic Acid, Escherichia coli Proteins, Fluorescence Resonance Energy Transfer, Mice, HEK293 Cells, Single-Cell Analysis, Spatio-Temporal Analysis

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