Channel-mediated lactate release by K⁺-stimulated astrocytes.

Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K(+) or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

Fast and reversible stimulation of astrocytic glycolysis by K+ and a delayed and persistent effect of glutamate.

Synaptic activity is followed within seconds by a local surge in lactate concentration, a phenomenon that underlies functional magnetic resonance imaging and whose causal mechanisms are unclear, partly because of the limited spatiotemporal resolution of standard measurement techniques. Using a novel Förster resonance energy transfer-based method that allows real-time measurement of the glycolytic rate in single cells, we have studied mouse astrocytes in search for the mechanisms responsible for the lactate surge. Consistent with previous measurements with isotopic 2-deoxyglucose, glutamate was observed to stimulate glycolysis in cultured astrocytes, but the response appeared only after a lag period of several minutes. Na(+) overloads elicited by engagement of the Na(+)-glutamate cotransporter with d-aspartate or application of the Na(+) ionophore gramicidin also failed to stimulate glycolysis in the short term. In marked contrast, K(+) stimulated astrocytic glycolysis by fourfold within seconds, an effect that was observed at low millimolar concentrations and was also present in organotypic hippocampal slices. After removal of the agonists, the stimulation by K(+) ended immediately but the stimulation by glutamate persisted unabated for >20 min. Both stimulations required an active Na(+)/K(+) ATPase pump. By showing that small rises in extracellular K(+) mediate short-term, reversible modulation of astrocytic glycolysis and that glutamate plays a long-term effect and leaves a metabolic trace, these results support the view that astrocytes contribute to the lactate surge that accompanies synaptic activity and underscore the role of these cells in neurometabolic and neurovascular coupling.

Targeting of astrocytic glucose metabolism by beta-hydroxybutyrate.

The effectiveness of ketogenic diets and intermittent fasting against neurological disorders has brought interest to the effects of ketone bodies on brain cells. These compounds are known to modify the metabolism of neurons, but little is known about their effect on astrocytes, cells that control the supply of glucose to neurons and also modulate neuronal excitability through the glycolytic production of lactate. Here we have used genetically-encoded Förster Resonance Energy Transfer nanosensors for glucose, pyruvate and ATP to characterize astrocytic energy metabolism at cellular resolution. Our results show that the ketone body beta-hydroxybutyrate strongly inhibited astrocytic glucose consumption in mouse astrocytes in mixed cultures, in organotypic hippocampal slices and in acute hippocampal slices prepared from ketotic mice, while blunting the stimulation of glycolysis by physiological and pathophysiological stimuli. The inhibition of glycolysis was paralleled by an increased ability of astrocytic mitochondria to metabolize pyruvate. These results support the emerging notion that astrocytes contribute to the neuroprotective effect of ketone bodies.

Imaging mitochondrial flux in single cells with a FRET sensor for pyruvate.

Mitochondrial flux is currently accessible at low resolution. Here we introduce a genetically-encoded FRET sensor for pyruvate, and methods for quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution. In HEK293 cells, neurons and astrocytes, mitochondrial pyruvate uptake was saturated at physiological levels, showing that the metabolic rate is determined by intrinsic properties of the organelle and not by substrate availability. The potential of the sensor was further demonstrated in neurons, where mitochondrial flux was found to rise by 300% within seconds of a calcium transient triggered by a short theta burst, while glucose levels remained unaltered. In contrast, astrocytic mitochondria were insensitive to a similar calcium transient elicited by extracellular ATP. We expect the improved resolution provided by the pyruvate sensor will be of practical interest for basic and applied researchers interested in mitochondrial function.

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.

High resolution measurement of the glycolytic rate.

The glycolytic rate is sensitive to physiological activity, hormones, stress, aging, and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. Used in cultured astrocytes, the method showed for the first time that glycolysis can be activated within seconds by a combination of glutamate and K(+), supporting a role for astrocytes in neurometabolic and neurovascular coupling in the brain. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism. Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts, and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis.