A Genetically Encoded FRET Lactate Sensor and Its Use To Detect the Warburg Effect in Single Cancer Cells

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.

NH4+ triggers the release of astrocytic lactate via mitochondrial pyruvate shunting

“…1J). Imaged in tissue slices, the sensor behaved as expected, with a decrease in signal on transacceleration of lactate export with the nonmetabolized MCT substrate monochloroacetate and an increase during exposure to 10 mM lactate (24). Addition of NH + 4 to the slice caused a rapid increase in lactate in protoplasmic astrocytes, a response that could be detected even at 0.05 mM NH + 4 ( Fig.…”

“…1A). To improve the sensitivity of detection, lactate was measured within cells using Laconic (24). All imaging of cultured astrocytes was performed on mixed cultures of astrocytes and neurons, in which astrocytes are better differentiated in terms of energy metabolism (25).…”

“…Cultured astrocytes are net lactate producers. To clarify whether the accumulation of lactate was due to increased production or impaired release, astrocytes were exposed to NH + 4 in the presence of the monocarboxylate transporter (MCT) blocker AR-C155858 (26), which in these cells abrogates lactate transport (24). As shown in Fig.…”

Nanomolar nitric oxide concentrations quickly and reversibly modulate astrocytic energy metabolism

“…A NOC-9-treated buffer that had been depleted of NO by 1 h of gassing with air/CO 2 was without apparent effect on the intracellular glucose level (data not shown). The small change in Laconic signal is consistent with the high concentration of lactate found in resting astrocytes (1.4 mM (17)) and with the doseresponse curve of this sensor (20).…”

“…1A), consistent with cytochrome oxidase inhibition and secondary glycolysis stimulation. To investigate the speed of the phenomenon, cytosolic glucose and lactate were measured using the genetically encoded FRET sensors FLII12Pglu700⌬6 (19) and Laconic (20), respectively. Astrocytes were studied in the presence of neurons, which foster their functional and metabolic differentiation (21).…”