The dentate gyrus differentially metabolizes glucose and alternative fuels during rest and stimulation.

The metabolic demands of neuronal activity are both temporally and spatially dynamic, and neurons are particularly sensitive to disruptions in fuel and oxygen supply. Glucose is considered an obligate fuel for supporting brain metabolism. Although alternative fuels are often available, the extent of their contribution to central carbon metabolism remains debated. Differential fuel metabolism likely depends on cell type, location, and activity state, complicating its study. While biosensors provide excellent spatial and temporal information, they are limited to observations of only a few metabolites. On the other hand, mass spectrometry is rich in chemical information, but traditionally relies on cell culture or homogenized tissue samples. Here, we use mass spectrometry imaging (MALDI-MSI) to focus on the fuel metabolism of the dentate granule cell (DGC) layer in murine hippocampal slices. Using stable isotopes, we explore labeling dynamics at baseline, as well as in response to brief stimulation or fuel competition. We find that at rest, glucose is the predominant fuel metabolized through glycolysis, with little to no measurable contribution from glycerol or fructose. However, lactate/pyruvate, ß-hydroxybutyrate (ßHB), octanoate, and glutamine can contribute to TCA metabolism to varying degrees. In response to brief depolarization with 50 mM KCl, glucose metabolism was preferentially increased relative to the metabolism of alternative fuels. With an increased supply of alternative fuels, both lactate/pyruvate and ßHB can outcompete glucose for TCA cycle entry. While lactate/pyruvate modestly reduced glucose contribution to glycolysis, ßHB caused little change in glycolysis. This approach achieves broad metabolite coverage from a spatially defined region of physiological tissue, in which metabolic states are rapidly preserved following experimental manipulation. Using this powerful methodology, we investigated metabolism within the dentate gyrus not only at rest, but also in response to the energetic demand of activation, and in states of fuel competition.

Spatially resolved metabolomics and isotope tracing reveal dynamic metabolic responses of dentate granule neurons with acute stimulation.

Neuronal activity creates an intense energy demand that must be met by rapid metabolic responses. To investigate metabolic adaptations in the neuron-enriched dentate granule cell (DGC) layer within its native tissue environment, we employed murine acute hippocampal brain slices, coupled with fast metabolite preservation and followed by mass spectrometry (MS) imaging, to generate spatially resolved metabolomics and isotope-tracing data. Here we show that membrane depolarization induces broad metabolic changes, including increased glycolytic activity in DGCs. Increased glucose metabolism in response to stimulation is accompanied by mobilization of endogenous inosine into pentose phosphates via the action of purine nucleotide phosphorylase (PNP). The PNP reaction is an integral part of the neuronal response to stimulation, because inhibition of PNP leaves DGCs energetically impaired during recovery from strong activation. Performing MS imaging on brain slices bridges the gap between live-cell physiology and the deep chemical analysis enabled by MS.

Spatially resolved metabolomics and isotope tracing reveal dynamic metabolic responses of dentate granule neurons with acute stimulation.

Neuronal activity creates an intense energy demand that must be met by rapid metabolic responses. To investigate metabolic adaptations in the neuron-enriched dentate granule cell (DGC) layer within its native tissue environment, we employed murine acute hippocampal brain slices coupled with fast metabolite preservation, followed by mass spectrometry imaging (MALDI-MSI) to generate spatially resolved metabolomics and isotope tracing data. Here we show that membrane depolarization induces broad metabolic changes, including increased glycolytic activity in DGCs. Increased glucose metabolism in response to stimulation is accompanied by mobilization of endogenous inosine into pentose phosphates, via the action of purine nucleotide phosphorylase (PNP). The PNP reaction is an integral part of the neuronal response to stimulation, as inhibiting PNP leaves DGCs energetically impaired during recovery from strong activation. Performing MSI on brain slices bridges the gap between live cell physiology and the deep chemical analysis enabled by mass spectrometry.

Metabolic regulation of species-specific developmental rates.

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.

The Na+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells.

Cellular ATP that is consumed to perform energetically expensive tasks must be replenished by new ATP through the activation of metabolism. Neuronal stimulation, an energetically demanding process, transiently activates aerobic glycolysis, but the precise mechanism underlying this glycolysis activation has not been determined. We previously showed that neuronal glycolysis is correlated with Ca2+ influx, but is not activated by feedforward Ca2+ signaling (Díaz-García et al., 2021a). Since ATP-powered Na+ and Ca2+ pumping activities are increased following stimulation to restore ion gradients and are estimated to consume most neuronal ATP, we aimed to determine if they are coupled to neuronal glycolysis activation. By using two-photon imaging of fluorescent biosensors and dyes in dentate granule cell somas of acute mouse hippocampal slices, we observed that production of cytoplasmic NADH, a byproduct of glycolysis, is strongly coupled to changes in intracellular Na+, while intracellular Ca2+ could only increase NADH production if both forward Na+/Ca2+ exchange and Na+/K+ pump activity were intact. Additionally, antidromic stimulation-induced intracellular [Na+] increases were reduced >50% by blocking Ca2+ entry. These results indicate that neuronal glycolysis activation is predominantly a response to an increase in activity of the Na+/K+ pump, which is strongly potentiated by Na+ influx through the Na+/Ca2+ exchanger during extrusion of Ca2+ following stimulation.

A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors.

Genetically encoded fluorescent biosensors are powerful tools used to track chemical processes in intact biological systems. However, the development and optimization of biosensors remains a challenging and labor-intensive process, primarily due to technical limitations of methods for screening candidate biosensors. Here we describe a screening modality that combines droplet microfluidics and automated fluorescence imaging to provide an order of magnitude increase in screening throughput. Moreover, unlike current techniques that are limited to screening for a single biosensor feature at a time (e.g. brightness), our method enables evaluation of multiple features (e.g. contrast, affinity, specificity) in parallel. Because biosensor features can covary, this capability is essential for rapid optimization. We use this system to generate a high-performance biosensor for lactate that can be used to quantify intracellular lactate concentrations. This biosensor, named LiLac, constitutes a significant advance in metabolite sensing and demonstrates the power of our screening approach.

Metabolism-based therapies for epilepsy: new directions for future cures.

OBJECTIVE: Thousands of years after dietary therapy was proposed to treat seizures, how alterations in metabolism relates to epilepsy remains unclear, and metabolism-based therapies are not always effective. METHODS: We consider the state of the science in metabolism-based therapies for epilepsy across the research lifecycle from basic to translational to clinical studies. RESULTS: This analysis creates a conceptual framework for creative, rigorous, and transparent research to benefit people with epilepsy through the understanding and modification of metabolism. INTERPRETATION: Despite intensive past efforts to evaluate metabolism-based therapies for epilepsy, distinct ways of framing a problem offer the chance to engage different mindsets and new (or newly applied) technologies. A comprehensive, creative, and inclusive problem-directed research agenda is needed, with a renewed and stringent adherence to rigor and transparency across all levels of investigation.

The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle.

When neurons engage in intense periods of activity, the consequent increase in energy demand can be met by the coordinated activation of glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. However, the trigger for glycolytic activation is unknown and the role for Ca2+ in the mitochondrial responses has been debated. Using genetically encoded fluorescent biosensors and NAD(P)H autofluorescence imaging in acute hippocampal slices, here we find that Ca2+ uptake into the mitochondria is responsible for the buildup of mitochondrial NADH, probably through Ca2+ activation of dehydrogenases in the TCA cycle. In the cytosol, we do not observe a role for the Ca2+/calmodulin signaling pathway, or AMPK, in mediating the rise in glycolytic NADH in response to acute stimulation. Aerobic glycolysis in neurons is triggered mainly by the energy demand resulting from either Na+ or Ca2+ extrusion, and in mouse dentate granule cells, Ca2+ creates the majority of this demand.

Delivery of AAV for Expression of Fluorescent Biosensors in Juvenile Mouse Hippocampus.

Genetically encoded fluorescent biosensors are versatile tools for studying brain metabolism and function in live tissue. The genetic information for these biosensors can be delivered into the brain by stereotaxic injection of engineered adeno-associated viruses (AAVs), which can selectively target different cell types depending on the capsid serotype and/or the viral promoter. Here, we describe a protocol for intracranial injections of two viral vectors encoding the metabolic biosensor Peredox and the calcium biosensor RCaMP1h. When combined with 2-photon microscopy and fluorescence lifetime imaging, this protocol allows the simultaneous quantitative assessment of changes in the cytosolic NADH/NAD+ ratio and the intracellular Ca2+ levels in individual dentate granule cells from acute hippocampal slices. Graphic abstract: Workflow diagram for biosensor expression in the mouse hippocampus using intracranial injections of adeno-associated viruses.

Fluorescent Biosensors for Neuronal Metabolism and the Challenges of Quantitation.

Over the past decade, genetically encoded fluorescent biosensors that report metabolic changes have become valuable tools for understanding brain metabolism. These sensors have been targeted to specific brain regions and cell types in different organisms to track multiple metabolic processes at single cell (and subcellular) resolution. Here, we review genetically encoded biosensors used to study metabolism in the brain. We particularly focus on the principles needed to use these sensors quantitatively while avoiding false inferences from variations in sensor fluorescence that arise from differences in expression level or environmental influences such as pH or temperature.

Hepatic NADH reductive stress underlies common variation in metabolic traits.

The cellular NADH/NAD+ ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX1, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD+ ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD+ ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance2, insulin resistance3 and mitochondrial disease4, and are associated with a common genetic variant in GCKR5, which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD+ ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of 'causal metabolism'.

Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor.

Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR-TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose-binding protein with a circularly permuted variant of the monomeric fluorescent protein T-Sapphire. Here, we provide more details on iGlucoSnFR-TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2-10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4-2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7-2.5 mM in cortical neurons from awake mice.

Neurons rely on glucose rather than astrocytic lactate during stimulation.

Brain metabolism increases during stimulation, but this increase does not affect all energy metabolism equally. Briefly after stimulation, there is a local increase in cerebral blood flow and in glucose uptake, but a smaller increase in oxygen uptake. This indicates that temporarily the rate of glycolysis is faster than the rate of oxidative metabolism, with a corresponding temporary increase in lactate production. This minireview discusses the long-standing controversy about which cell type, neurons or astrocytes, are involved in this increased aerobic glycolysis. Recent biosensor studies measuring metabolic changes in neurons, in acute brain slices or in vivo, are placed in the context of other data bearing on this question. The most direct measurements indicate that, although both neurons and astrocytes may increase glycolysis after stimulation, neurons do not rely on import of astrocytic-produced lactate, and instead they increase their own glycolytic rate and become net exporters of lactate. This temporary increase in neuronal glycolysis may provide rapid energy to meet the acute energy demands of neurons.

Fueling thought: Management of glycolysis and oxidative phosphorylation in neuronal metabolism.

The brain's energy demands are remarkable both in their intensity and in their moment-to-moment dynamic range. This perspective considers the evidence for Warburg-like aerobic glycolysis during the transient metabolic response of the brain to acute activation, and it particularly addresses the cellular mechanisms that underlie this metabolic response. The temporary uncoupling between glycolysis and oxidative phosphorylation led to the proposal of an astrocyte-to-neuron lactate shuttle whereby during stimulation, lactate produced by increased glycolysis in astrocytes is taken up by neurons as their primary energy source. However, direct evidence for this idea is lacking, and evidence rather supports that neurons have the capacity to increase their own glycolysis in response to stimulation; furthermore, neurons may export rather than import lactate in response to stimulation. The possible cellular mechanisms for invoking metabolic resupply of energy in neurons are also discussed, in particular the roles of feedback signaling via adenosine diphosphate and feedforward signaling by calcium ions.

BAD and KATP channels regulate neuron excitability and epileptiform activity.

Brain metabolism can profoundly influence neuronal excitability. Mice with genetic deletion or alteration of Bad (BCL-2 agonist of cell death) exhibit altered brain-cell fuel metabolism, accompanied by resistance to acutely induced epileptic seizures; this seizure protection is mediated by ATP-sensitive potassium (KATP) channels. Here we investigated the effect of BAD manipulation on KATP channel activity and excitability in acute brain slices. We found that BAD's influence on neuronal KATP channels was cell-autonomous and directly affected dentate granule neuron (DGN) excitability. To investigate the role of neuronal KATP channels in the anticonvulsant effects of BAD, we imaged calcium during picrotoxin-induced epileptiform activity in entorhinal-hippocampal slices. BAD knockout reduced epileptiform activity, and this effect was lost upon knockout or pharmacological inhibition of KATP channels. Targeted BAD knockout in DGNs alone was sufficient for the antiseizure effect in slices, consistent with a 'dentate gate' function that is reinforced by increased KATP channel activity.

BAD knockout provides metabolic seizure resistance in a genetic model of epilepsy with sudden unexplained death in epilepsy.

Metabolic alteration, either through the ketogenic diet (KD) or by genetic alteration of the BAD protein, can produce seizure protection in acute chemoconvulsant models of epilepsy. To assess the seizure-protective role of knocking out (KO) the Bad gene in a chronic epilepsy model, we used the Kcna1-/- model of epilepsy, which displays progressively increased seizure severity and recapitulates the early death seen in sudden unexplained death in epilepsy (SUDEP). Beginning on postnatal day 24 (P24), we continuously video monitored Kcna1-/- and Kcna1-/- Bad-/- double knockout mice to assess survival and seizure severity. We found that Kcna1-/- Bad-/- mice outlived Kcna1-/- mice by approximately 2 weeks. Kcna1-/- Bad-/- mice also spent significantly less time in seizure than Kcna1-/- mice on P24 and the day of death, showing that BadKO provides seizure resistance in a genetic model of chronic epilepsy.

Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD+ ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD+ ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD+ ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD+ ratio, and this increase is mitigated by the presence of NAD+-generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD+ ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD+ ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real-time measurements of the ratio in living, functioning liver cells, overcoming many limitations of previous methods for measuring this important redox parameter. The feasibility of using Peredox in liver slices is particularly attractive because slices allow preservation of hepatic microanatomy and metabolic zonation of hepatocytes.

Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells.

Cells use multiple feedback controls to regulate metabolism in response to nutrient and signaling inputs. However, feedback creates the potential for unstable network responses. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox. Across various conditions, including glycolytic or mitochondrial inhibition or cell proliferation, we observed distinct patterns of AMPK activity, including both stable adaptation and highly dynamic behaviors such as periodic oscillations and irregular fluctuations that indicate a failure to reach a steady state. Fluctuations in AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox state were temporally linked in individual cells adapting to metabolic perturbations. By monitoring single-cell dynamics in each of these contexts, we identified PI3K/Akt regulation of glycolysis as a multifaceted modulator of single-cell metabolic dynamics that is required to maintain metabolic stability in proliferating cells.

Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake.

Proper brain function requires a substantial energy supply, up to 20% of whole-body energy in humans, and brain activation produces large dynamic variations in energy demand. While local increases in cerebral blood flow are well known, the cellular responses to energy demand are controversial. During brain excitation, glycolysis of glucose to lactate temporarily exceeds the rate of mitochondrial fuel oxidation; although the increased energy demand occurs mainly within neurons, some have suggested this glycolysis occurs mainly in astrocytes, which then shuttle lactate to neurons as their primary fuel. Using metabolic biosensors in acute hippocampal slices and brains of awake mice, we find that neuronal metabolic responses to stimulation do not depend on astrocytic stimulation by glutamate release, nor do they require neuronal uptake of lactate; instead they reflect increased direct glucose consumption by neurons. Neuronal glycolysis temporarily outstrips oxidative metabolism, and provides a rapid response to increased energy demand.