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.
Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in ...our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD
, as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research.
The mitochondrial calcium uniporter (MCU) is the main route of calcium (Ca
) entry into neuronal mitochondria. This channel has been linked to mitochondrial Ca
overload and cell death under ...neurotoxic conditions, but its physiologic roles for normal brain function remain poorly understood. Despite high expression of MCU in excitatory hippocampal neurons, it is unknown whether this channel is required for learning and memory. Here, we genetically down-regulated the Mcu gene in dentate granule cells (DGCs) of the hippocampus and found that this manipulation increases the overall respiratory activity of mitochondrial complexes I and II, augmenting the generation of reactive oxygen species in the context of impaired electron transport chain. The metabolic remodeling of MCU-deficient neurons also involved changes in the expression of enzymes that participate in glycolysis and the regulation of the tricarboxylic acid cycle, as well as the cellular antioxidant defenses. We found that MCU deficiency in DGCs does not change circadian rhythms, spontaneous exploratory behavior, or cognitive function in middle-aged mice (11-13 months old), when assessed with a food-motivated working memory test with three choices. DGC-targeted down-regulation of MCU significantly impairs reversal learning assessed with an 8-arm radial arm water maze but does not affect their ability to learn the task for the first time. Our results indicate that neuronal MCU plays an important physiologic role in memory formation and may be a potential therapeutic target to develop interventions aimed at improving cognitive function in aging, neurodegenerative diseases, and brain injury.
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.
Pancreatic beta cells, unique cells that secrete insulin in response to an increase in glucose levels, play a significant role in glucose homeostasis. Glucose-stimulated insulin secretion (GSIS) in ...pancreatic beta cells has been extensively explored. In this mechanism, glucose enters the cells and subsequently the metabolic cycle. During this process, the ATP/ADP ratio increases, leading to ATP-sensitive potassium (KATP) channel closure, which initiates depolarization that is also dependent on the activity of TRP nonselective ion channels. Depolarization leads to the opening of voltage-gated Na(+) channels (Nav) and subsequently voltage-dependent Ca(2+) channels (Cav). The increase in intracellular Ca(2+) triggers the exocytosis of insulin-containing vesicles. Thus, electrical activity of pancreatic beta cells plays a central role in GSIS. Moreover, many growth factors, incretins, neurotransmitters, and hormones can modulate GSIS, and the channels that participate in GSIS are highly regulated. In this review, we focus on the principal ionic channels (KATP, Nav, and Cav channels) involved in GSIS and how classic and new proteins, hormones, and drugs regulate it. Moreover, we also discuss advances on how metabolic disorders such as metabolic syndrome and diabetes mellitus change channel activity leading to changes in insulin secretion.
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 Ca
influx, but is not activated by feedforward Ca
signaling (Díaz-García et al., 2021a). Since ATP-powered Na
and Ca
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 Ca
could only increase NADH production if both forward Na
/Ca
exchange and Na
/K
pump activity were intact. Additionally, antidromic stimulation-induced intracellular Na
increases were reduced >50% by blocking Ca
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
/Ca
exchanger during extrusion of Ca
following stimulation.
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 Ca
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 Ca
uptake into the mitochondria is responsible for the buildup of mitochondrial NADH, probably through Ca
activation of dehydrogenases in the TCA cycle. In the cytosol, we do not observe a role for the Ca
/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 Ca
extrusion, and in mouse dentate granule cells, Ca
creates the majority of this demand.
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.
Pancreatic beta cells during the first month of development acquire functional maturity, allowing them to respond to variations in extracellular glucose concentration by secreting insulin. Changes in ...ionic channel activity are important for this maturation. Within the voltage-gated calcium channels (VGCC), the most studied channels are high-voltage-activated (HVA), principally L-type; while low-voltage-activated (LVA) channels have been poorly studied in native beta cells. We analyzed the changes in the expression and activity of VGCC during the postnatal development in rat beta cells. We observed that the percentage of detection of T-type current increased with the stage of development. T-type calcium current density in adult cells was higher than in neonatal and P20 beta cells. Mean HVA current density also increased with age. Calcium current behavior in P20 beta cells was heterogeneous; almost half of the cells had HVA current densities higher than the adult cells, and this was independent of the presence of T-type current. We detected the presence of α1G, α1H, and α1I subunits of LVA channels at all ages. The Cav 3.1 subunit (α1G) was the most expressed. T-type channel blockers mibefradil and TTA-A
significantly inhibited insulin secretion at 5.6 mM glucose, which suggests a physiological role for T-type channels at basal glucose conditions. Both, nifedipine and TTA-A
, drastically decreased the beta-cell subpopulation that secretes more insulin, in both basal and stimulating glucose conditions. We conclude that changes in expression and activity of VGCC during the development play an important role in physiological maturation of beta cells.
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.
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•Neuronal stimulation in slices or in vivo transiently increases cytosolic NADH:NAD+•Lactate shuttling into neurons is not required for the cytosolic NADH transients•Stimulation increases neuronal glycolysis rather than lactate metabolism•After stimulation, neurons are likely net exporters of lactate
Using fluorescent biosensors of metabolism, Díaz-García, Mongeon, Lahmann et al. observe the metabolic response of neurons to stimulation in brain slices and in vivo. Neurons increase their cytosolic NADH redox by increasing glycolysis, consistent with the mismatch between glucose and oxygen utilization that enables functional brain imaging.
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