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.
Highlights
Lactate is an organic anion that participates in the intermediate metabolism of eukaryotic and prokaryotic cells
Genetically-encoded Forster Resonance Energy Transfer (FRET) nanosensors have been developed for measuring the dynamic changes in concentration of several molecules of biological interest with improved spatiotemporal resolution
FRET sensors are fusion proteins composed of a ligand-binding moiety, the recognition element, and a fluorescent pair with overlapping emission and excitation spectra, typically CFP and YFP
Summary
Lactate is an organic anion that participates in the intermediate metabolism of eukaryotic and prokaryotic cells. Lactate is produced from pyruvate by the cytosolic enzyme lactate dehydrogenase (LDH) and is exchanged with the interstitial space and between subcellular compartments via monocarboxylate transporters (MCTs). Intercellular and subcellular exchanges of lactate, termed lactate shuttles, are an integral part of the normal energy metabolism of muscle and brain [1,2]. Despite normal or elevated oxygen tissue levels, neural activity is accompanied by an acute rise in tissue lactate. Whether and when neurons produce or consume lactate during neural activity remains a controversial issue [3,4,5,6,7], which would greatly benefit from lactate measurements in individual cells. Pathophysiological roles for lactate include inflammation, wound healing, microbial infection, neurodegeneration and cancer [14,15,16,17,18]
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