Iván Ruminot

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.

NBCe1 Mediates the Acute Stimulation of Astrocytic Glycolysis by Extracellular K

Excitatory synaptic transmission stimulates brain tissue glycolysis. This phenomenon is the signal detected in FDG-PET imaging and, through enhanced lactate production, is also thought to contribute to the fMRI signal. Using a method based on Förster resonance energy transfer in mouse astrocytes, we have recently observed that a small rise in extracellular K+ can stimulate glycolysis by >300% within seconds. The K+ response was blocked by ouabain, but intracellular engagement of the Na+/K+ ATPase pump with Na+ was ineffective, suggesting that the canonical feedback regulatory pathway involving the Na+ pump and ATP depletion is only permissive and that a second mechanism is involved. Because of their predominant K+ permeability and high expression of the electrogenic Na+/HCO3− cotransporter NBCe1, astrocytes respond to a rise in extracellular K+ with plasma membrane depolarization and intracellular alkalinization. In the present article, we show that a fast glycolytic response can be elicited independently of K+ by plasma membrane depolarization or by intracellular alkalinization. The glycolytic response to K+ was absent in astrocytes from NBCe1 null mice (Slc4a4) and was blocked by functional or pharmacological inhibition of the NBCe1. Hippocampal neurons acquired K+-sensitive glycolysis upon heterologous NBCe1 expression. The phenomenon could also be reconstituted in HEK293 cells by coexpression of the NBCe1 and a constitutively open K+ channel. We conclude that the NBCe1 is a key element in a feedforward mechanism linking excitatory synaptic transmission to fast modulation of glycolysis in astrocytes.

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.

The Electrogenic Sodium Bicarbonate Cotransporter NBCe1 Is a High-Affinity Bicarbonate Carrier in Cortical Astrocytes

The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) is a robust regulator of intracellular H+ and a significant base carrier in many cell types. Using wild-type (WT) and NBCe1-deficient (NBC-KO) mice, we have studied the role of NBCe1 in cortical astrocytes in culture and in situ by monitoring intracellular H+ using the H+-sensitive dye BCECF [2′,7′-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein] in wide-field and confocal microscopy. Adding 0.1–3 mm HCO3− to an O2-gassed, HEPES-buffered saline solution lowered the intracellular H+ concentration with a Km of 0.65 mm HCO3− in WT astrocytes, but slowly raised [H+]i in NBCe1-KO astrocytes. Human NBCe1 heterologously expressed in Xenopus oocytes could be activated by adding 1–3 mm HCO3−, and even by residual HCO3− in a nominally CO2/HCO3−-free saline solution. Our results demonstrate a surprisingly high apparent bicarbonate sensitivity mediated by NBCe1 in cortical astrocytes, suggesting that NBCe1 may operate over a wide bicarbonate concentration in these cells.

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.

Proton Fall or Bicarbonate Rise: Glycolytic Rate In Mouse Astrocytes Is Paved By Intracellular Alkalinization

Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H+], but those can also be stimulated by an increase in the intracellular [HCO3−]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H+] or [HCO3−]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H+] or a rise in intracellular [HCO3−], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H+] and [CO2]/[HCO3−]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H+], irrespective of a concomitant rise or fall in intracellular [HCO3−]. Transport of HCO3− into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3−], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types.

Near‐critical GLUT1 and Neurodegeneration

Recent articles have drawn renewed attention to the housekeeping glucose transporter GLUT1 and its possible involvement in neurodegenerative diseases. Here we provide an updated analysis of brain glucose transport and the cellular mechanisms involved in its acute modulation during synaptic activity. We discuss how the architecture of the blood‐brain barrier and the low concentration of glucose within neurons combine to make endothelial/glial GLUT1 the master controller of neuronal glucose utilization, while the regulatory role of the neuronal glucose transporter GLUT3 emerges as secondary. The near‐critical condition of glucose dynamics in the brain suggests that subtle deficits in GLUT1 function or its activity‐dependent control by neurons may contribute to neurodegeneration.

Tight coupling of astrocyte energy metabolism to synaptic activity revealed by genetically encoded FRET nanosensors in hippocampal tissue

The potassium ion, K+, a neuronal signal that is released during excitatory synaptic activity, produces acute activation of glucose consumption in cultured astrocytes, a phenomenon mediated by the sodium bicarbonate cotransporter NBCe1 (SLC4A4). We have explored here the relevance of this mechanism in brain tissue by imaging the effect of neuronal activity on pH, glucose, pyruvate and lactate dynamics in hippocampal astrocytes using BCECF and FRET nanosensors. Electrical stimulation of Schaffer collaterals produced fast activation of glucose consumption in astrocytes with a parallel increase in intracellular pyruvate and biphasic changes in lactate. These responses were blocked by TTX and were absent in tissue slices prepared from NBCe1-KO mice. Direct depolarization of astrocytes with elevated extracellular K+ or Ba2+ mimicked the metabolic effects of electrical stimulation. We conclude that the glycolytic pathway of astrocytes in situ is acutely sensitive to neuronal activity, and that extracellular K+ and the NBCe1 cotransporter are involved in metabolic crosstalk between neurons and astrocytes. Glycolytic activation of astrocytes in response to neuronal K+ helps to provide an adequate supply of lactate, a metabolite that is released by astrocytes and which acts as neuronal fuel and an intercellular signal.

Neuronal control of astrocytic respiration through a variant of the Crabtree effect

Significance Neuronal activity is immediately followed by aerobic glycolysis, the partial oxidation of glucose to lactate in the presence of oxygen, a phenomenon that seems paradoxical because full oxidation of glucose to CO2 would yield much more ATP. Here we show that short-term aerobic glycolysis involves a mechanism whereby neurons obtain oxygen by manipulating the glucose metabolism and mitochondrial respiration of neighboring astrocytes. Aerobic glycolysis is a phenomenon that in the long term contributes to synaptic formation and growth, is reduced by normal aging, and correlates with amyloid beta deposition. Aerobic glycolysis starts within seconds of neural activity and it is not obvious why energetic efficiency should be compromised precisely when energy demand is highest. Using genetically encoded FRET nanosensors and real-time oxygen measurements in culture and in hippocampal slices, we show here that astrocytes respond to physiological extracellular K+ with an acute rise in cytosolic ATP and a parallel inhibition of oxygen consumption, explained by glycolytic stimulation via the Na+-bicarbonate cotransporter NBCe1. This control of mitochondrial respiration via glycolysis modulation is reminiscent of a phenomenon previously described in proliferating cells, known as the Crabtree effect. Fast brain aerobic glycolysis may be interpreted as a strategy whereby neurons manipulate neighboring astrocytes to obtain oxygen, thus maximizing information processing.

Our hungry brain: Which role do glial cells play for the energy supply?

Abstract Our brain, which accounts for about 2 % of our body weight, uses up to 20 % of our total energy requirements. The supply with sufficient energetic substrates to all brain cells, which are very densely packed, in particular in the human brain, is a huge logistic challenge. The most important energy substrate for our brain is glucose, which reaches the brain via the blood circulation. Glucose is not only utilized by nerve cells directly, but to a large extent also taken up by glial cells, which then either store glucose after conversion to glycogen as energy reserve, or transfer it as lactate to nerve cells. Lactate in nerve cells can then be converted to pyruvate, which is efficiently utilized together with oxygen for the formation of chemical energy in form of ATP. The intermediate metabolic product lactate hence plays an important role as energetic substrate, which is exchanged between cells both under aerobic and non-aerobic conditions. Transport of lactate across the cell membrane is carried out in co-transport with protons (H+), which are crucial regulators of various metabolic processes and membrane transporters. In addition, the lactate carriers form a functional network with carbonic anhydrases, enzymes, which not only catalyze the equilibrium between carbon dioxide, hydrogen carbonate (bicarbonate) and protons, but also facilitate lactate transport. In this article, we focus on physiological processes of the energy metabolism in glial cells as well as on the transfer of energetic substrates to nerve cells, processes, which themselves are critically modulated by pH and its regulation in glial cells.