Imaging of the Lactate/Pyruvate Ratio Using a Genetically Encoded Förster Resonance Energy Transfer Indicator.

The ratio between the cytosolic concentrations of lactate and pyruvate is a direct readout of the balance between glycolysis and mitochondrial oxidative metabolism. Current approaches do not allow detection of the lactate/pyruvate ratio in a single readout with high spatial/temporal resolution in living systems. Using a Förster resonance energy transfer (FRET)-based screening strategy, we found that the orphan transcriptional factor LutR from Bacillus licheniformis is an endogenous sensor of the lactate/pyruvate ratio, suitable for use as a binding moiety to develop a lactate/pyruvate ratio FRET-based genetically encoded indicator, Lapronic. The sensitivity of the indicator to lactate and pyruvate was characterized through changes in the fluorescence FRET ratio and validated with isothermal titration calorimetry. Lapronic was insensitive to physiological pH and temperature and did not respond to structurally related molecules acetate and ß-hydroxybutyrate or cofactors NAD+ and NADH. Lapronic was expressed in HEK 293 cells showing a homogeneous cytosolic localization and was also targeted to the mitochondrial matrix. A calibration protocol was designed to quantitatively assess the lactate/pyruvate ratio in intact mammalian cells. Purified protein from Escherichia coli showed robust stability over time and was found suitable for lactate/pyruvate ratio detection in biological samples. We envision that Lapronic will be of practical interest for basic and applied research.

In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons.

Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons.

Single-cell imaging tools for brain energy metabolism: a review.

Neurophotonics comes to light at a time in which advances in microscopy and improved calcium reporters are paving the way toward high-resolution functional mapping of the brain. This review relates to a parallel revolution in metabolism. We argue that metabolism needs to be approached both in vitro and in vivo, and that it does not just exist as a low-level platform but is also a relevant player in information processing. In recent years, genetically encoded fluorescent nanosensors have been introduced to measure glucose, glutamate, ATP, NADH, lactate, and pyruvate in mammalian cells. Reporting relative metabolite levels, absolute concentrations, and metabolic fluxes, these sensors are instrumental for the discovery of new molecular mechanisms. Sensors continue to be developed, which together with a continued improvement in protein expression strategies and new imaging technologies, herald an exciting era of high-resolution characterization of metabolism in the brain and other organs.