Brain Glucose Metabolism: Integration of Energetics with Function.

Glucose is the long-established, obligatory fuel for brain that fulfills many critical functions, including ATP production, oxidative stress management, and synthesis of neurotransmitters, neuromodulators, and structural components. Neuronal glucose oxidation exceeds that in astrocytes, but both rates increase in direct proportion to excitatory neurotransmission; signaling and metabolism are closely coupled at the local level. Exact details of neuron-astrocyte glutamate-glutamine cycling remain to be established, and the specific roles of glucose and lactate in the cellular energetics of these processes are debated. Glycolysis is preferentially upregulated during brain activation even though oxygen availability is sufficient (aerobic glycolysis). Three major pathways, glycolysis, pentose phosphate shunt, and glycogen turnover, contribute to utilization of glucose in excess of oxygen, and adrenergic regulation of aerobic glycolysis draws attention to astrocytic metabolism, particularly glycogen turnover, which has a high impact on the oxygen-carbohydrate mismatch. Aerobic glycolysis is proposed to be predominant in young children and specific brain regions, but re-evaluation of data is necessary. Shuttling of glucose- and glycogen-derived lactate from astrocytes to neurons during activation, neurotransmission, and memory consolidation are controversial topics for which alternative mechanisms are proposed. Nutritional therapy and vagus nerve stimulation are translational bridges from metabolism to clinical treatment of diverse brain disorders.

In vivo calibration of genetically encoded metabolite biosensors must account for metabolite metabolism during calibration and cellular volume.

Isotopic assays of brain glucose utilization rates have been used for more than four decades to establish relationships between energetics, functional activity, and neurotransmitter cycling. Limitations of these methods include the relatively long time (1-60 min) for the determination of labeled metabolite levels and the lack of cellular resolution. Identification and quantification of fuels for neurons and astrocytes that support activation and higher brain functions are a major, unresolved issues. Glycolysis is preferentially up-regulated during activation even though oxygen level and supply are adequate, causing lactate concentrations to quickly rise during alerting, sensory processing, cognitive tasks, and memory consolidation. However, the fate of lactate (rapid release from brain or cell-cell shuttling coupled with local oxidation) is long disputed. Genetically encoded biosensors can determine intracellular metabolite concentrations and report real-time lactate level responses to sensory, behavioral, and biochemical challenges at the cellular level. Kinetics and time courses of cellular lactate concentration changes are informative, but accurate biosensor calibration is required for quantitative comparisons of lactate levels in astrocytes and neurons. An in vivo calibration procedure for the Laconic lactate biosensor involves intracellular lactate depletion by intravenous pyruvate-mediated trans-acceleration of lactate efflux followed by sensor saturation by intravenous infusion of high doses of lactate plus ammonium chloride. In the present paper, the validity of this procedure is questioned because rapid lactate-pyruvate interconversion in blood, preferential neuronal oxidation of both monocarboxylates, on-going glycolytic metabolism, and cellular volumes were not taken into account. Calibration pitfalls for the Laconic lactate biosensor also apply to other metabolite biosensors that are standardized in vivo by infusion of substrates that can be metabolized in peripheral tissues. We discuss how technical shortcomings negate the conclusion that Laconic sensor calibrations support the existence of an in vivo astrocyte-neuron lactate concentration gradient linked to lactate shuttling from astrocytes to neurons to fuel neuronal activity.

A tribute to Leif Hertz: The historical context of his pioneering studies of the roles of astrocytes in brain energy metabolism, neurotransmission, cognitive functions, and pharmacology identifies important, unresolved topics for future studies.

Leif Hertz, M.D., D.Sc. (honoris causa) (1930-2018), was one of the original and noteworthy participants in the International Conference on Brain Energy Metabolism (ICBEM) series since its inception in 1993. The biennial ICBEM conferences are organized by neuroscientists interested in energetics and metabolism underlying neural functions; they have had a high impact on conceptual and experimental advances in these fields and on promoting collaborative interactions among neuroscientists. Leif made major contributions to ICBEM discussions and understanding of metabolic and signaling characteristics of astrocytes and their roles in brain function. His studies ranged from uptake of K+ from extracellular fluid and its stimulation of astrocytic respiration, identification, and regulation of enzymes specifically or preferentially expressed in astrocytes in the glutamate-glutamine cycle of excitatory neurotransmission, a requirement for astrocytic glycogenolysis for fueling K+ uptake, involvement of glycogen in memory consolidation in the chick, and pharmacology of astrocytes. This tribute to Leif Hertz highlights his major discoveries, the high impact of his work on astrocyte-neuron interactions, and his unparalleled influence on understanding the cellular basis of brain energy metabolism. His work over six decades has helped integrate the roles of astrocytes into neurotransmission where oxidative and glycogenolytic metabolism during neurotransmitter glutamate turnover are key aspects of astrocytic energetics. Leif recognized that brain astrocytic metabolism is greatly underestimated unless the volume fraction of astrocytes is taken into account. Adjustment for pathway rates expressed per gram tissue for volume fraction indicates that astrocytes have much higher oxidative rates than neurons and astrocytic glycogen concentrations and glycogenolytic rates during sensory stimulation in vivo are similar to those in resting and exercising muscle, respectively. These novel insights are typical of Leif's astute contributions to the energy metabolism field, and his publications have identified unresolved topics that provide the neuroscience community with challenges and opportunities for future research.

Neurovascular coupling is optimized to compensate for the increase in proton production from nonoxidative glycolysis and glycogenolysis during brain activation and maintain homeostasis of pH, pCO2 , and pO2.

During transient brain activation cerebral blood flow (CBF) increases substantially more than cerebral metabolic rate of oxygen consumption (CMRO2 ) resulting in blood hyperoxygenation, the basis of BOLD fMRI contrast. Explanations for the high CBF vs. CMRO2 slope, termed neurovascular coupling (NVC) constant, focused on maintainenance of tissue oxygenation to support mitochondrial ATP production. However, paradoxically the brain has a 3-fold lower oxygen extraction fraction (OEF) than other organs with high energy requirements, like heart and muscle during exercise. Here, we hypothesize that the NVC constant and the capillary oxygen mass transfer coefficient (which in combination determine OEF) are co-regulated during activation to maintain simultaneous homeostasis of pH and partial pressure of CO2 and O2 (pCO2 and pO2 ). To test our hypothesis, we developed an arteriovenous flux balance model for calculating blood and brain pH, pCO2 , and pO2 as a function of baseline OEF (OEF0 ), CBF, CMRO2 , and proton production by nonoxidative metabolism coupled to ATP hydrolysis. Our model was validated against published brain arteriovenous difference studies and then used to calculate pH, pCO2, and pO2 in activated human cortex from published calibrated fMRI and PET measurements. In agreement with our hypothesis, calculated pH, pCO2, and pO2 remained close to constant independently of CMRO2 in correspondence to experimental measurements of NVC and OEF0 . We also found that the optimum values of the NVC constant and OEF0 that ensure simultaneous homeostasis of pH, pCO2, and pO2 were remarkably similar to their experimental values. Thus, the high NVC constant is overall determined by proton removal by CBF due to increases in nonoxidative glycolysis and glycogenolysis. These findings resolve the paradox of the brain's high CBF yet low OEF during activation, and may contribute to explaining the vulnerability of brain function to reductions in blood flow and capillary density with aging and neurovascular disease.

Potential new roles for glycogen in epilepsy.

Seizures often originate in epileptogenic foci. Between seizures (interictally), these foci and some of the surrounding tissue often show low signals with 18 fluorodeoxyglucose (FDG) positron emission tomography (PET) in many epileptic patients, even when there are no radiologically detectable structural abnormalities. Low FDG-PET signals are thought to reflect glucose hypometabolism. Here, we review knowledge about metabolism of glucose and glycogen and oxidative stress in people with epilepsy and in acute and chronic rodent seizure models. Interictal brain glucose levels are normal and do not cause apparent glucose hypometabolism, which remains unexplained. During seizures, high amounts of fuel are needed to satisfy increased energy demands. Astrocytes consume glycogen as an additional emergency fuel to supplement glucose during high metabolic demand, such as during brain stimulation, stress, and seizures. In rodents, brain glycogen levels drop during induced seizures and increase to higher levels thereafter. Interictally, in people with epilepsy and in chronic epilepsy models, normal glucose but high glycogen levels have been found in the presumed brain areas involved in seizure generation. We present our new hypothesis that as an adaptive response to repeated episodes of high metabolic demand, high interictal glycogen levels in epileptogenic brain areas are used to support energy metabolism and potentially interictal neuronal activity. Glycogenolysis, which can be triggered by stress or oxidative stress, leads to decreased utilization of plasma glucose in epileptogenic brain areas, resulting in low FDG signals that are related to functional changes underlying seizure onset and propagation. This is (partially) reversible after successful surgery. Last, we propose that potential interictal glycogen depletion in epileptogenic and surrounding areas may cause energy shortages in astrocytes, which may impair potassium buffering and contribute to seizure generation. Based on these hypotheses, auxiliary fuels or treatments that support glycogen metabolism may be useful to treat epilepsy.

Mechanistic stoichiometric relationship between the rates of neurotransmission and neuronal glucose oxidation: Reevaluation of and alternatives to the pseudo-malate-aspartate shuttle model.

The ~1:1 stoichiometry between the rates of neuronal glucose oxidation (CMRglc-ox-N ) and glutamate (Glu)/γ-aminobutyric acid (GABA)-glutamine (Gln) neurotransmitter (NT) cycling between neurons and astrocytes (VNTcycle ) has been firmly established. However, the mechanistic basis for this relationship is not fully understood, and this knowledge is critical for the interpretation of metabolic and brain imaging studies in normal and diseased brain. The pseudo-malate-aspartate shuttle (pseudo-MAS) model established the requirement for glycolytic metabolism in cultured glutamatergic neurons to produce NADH that is shuttled into mitochondria to support conversion of extracellular Gln (i.e., astrocyte-derived Gln in vivo) into vesicular neurotransmitter Glu. The evaluation of this model revealed that it could explain half of the 1:1 stoichiometry and it has limitations. Modifications of the pseudo-MAS model were, therefore, devised to address major knowledge gaps, that is, submitochondrial glutaminase location, identities of mitochondrial carriers for Gln and other model components, alternative mechanisms to transaminate α-ketoglutarate to form Glu and shuttle glutamine-derived ammonia while maintaining mass balance. All modified models had a similar 0.5 to 1.0 predicted mechanistic stoichiometry between VNTcycle and the rate of glucose oxidation. Based on studies of brain ß-hydroxybutyrate oxidation, about half of CMRglc-ox-N may be linked to glutamatergic neurotransmission and localized in pre-synaptic structures that use pseudo-MAS type mechanisms for Glu-Gln cycling. In contrast, neuronal compartments that do not participate in transmitter cycling may use the MAS to sustain glucose oxidation. The evaluation of subcellular compartmentation of neuronal glucose metabolism in vivo is a critically important topic for future studies to understand glutamatergic and GABAergic neurotransmission.

Stop the rot. Enzyme inactivation at brain harvest prevents artifacts: A guide for preservation of the in vivo concentrations of brain constituents.

Post-mortem metabolism is widely recognized to cause rapid and prolonged changes in the concentrations of multiple classes of compounds in brain, that is, they are labile. Post-mortem changes from levels in living brain include components of pathways of metabolism of glucose and energy compounds, amino acids, lipids, signaling molecules, neuropeptides, phosphoproteins, and proteins. Methods that stop enzyme activity at brain harvest were developed almost 50 years ago and have been extensively used in studies of brain functions and diseases. Unfortunately, these methods are not commonly used to harvest brain tissue for mass spectrometry-based metabolomic studies or for imaging mass spectrometry studies (IMS, also called mass spectrometry imaging, MSI, or matrix-assisted laser desorption/ionization-MSI, MALDI-MSI). Instead these studies commonly kill animals, decapitate, dissect out brain and regions of interest if needed, then 'snap' freeze the tissue to stop enzymatic activity after harvest, with post-mortem intervals typically ranging from ~0.5 to 3 min. To increase awareness of the importance of stopping metabolism at harvest and preventing the unnecessary complications of not doing so, this commentary provides examples of labile metabolites and the magnitudes of their post-mortem changes in concentrations during brain harvest. Brain harvest methods that stop metabolism at harvest eliminate post-mortem enzymatic activities and can improve characterization of normal and diseased brain. In addition, metabolomic studies would be improved by reporting absolute units of concentration along with normalized peak areas or fold changes. Then reported values can be evaluated and compared with the extensive neurochemical literature to help prevent reporting of artifactual data.

Hypothesis: A Novel Neuroprotective Role for Glucose-6-phosphatase (G6PC3) in Brain-To Maintain Energy-Dependent Functions Including Cognitive Processes.

The isoform of glucose-6-phosphatase in liver, G6PC1, has a major role in whole-body glucose homeostasis, whereas G6PC3 is widely distributed among organs but has poorly-understood functions. A recent, elegant analysis of neutrophil dysfunction in G6PC3-deficient patients revealed G6PC3 is a neutrophil metabolite repair enzyme that hydrolyzes 1,5-anhydroglucitol-6-phosphate, a toxic metabolite derived from a glucose analog present in food. These patients exhibit a spectrum of phenotypic characteristics and some have learning disabilities, revealing a potential linkage between cognitive processes and G6PC3 activity. Previously-debated and discounted functions for brain G6PC3 include causing an ATP-consuming futile cycle that interferes with metabolic brain imaging assays and a nutritional role involving astrocyte-neuron glucose-lactate trafficking. Detailed analysis of the anhydroglucitol literature reveals that it competes with glucose for transport into brain, is present in human cerebrospinal fluid, and is phosphorylated by hexokinase. Anhydroglucitol-6-phosphate is present in rodent brain and other organs where its accumulation can inhibit hexokinase by competition with ATP. Calculated hexokinase inhibition indicates that energetics of brain and erythrocytes would be more adversely affected by anhydroglucitol-6-phosphate accumulation than heart. These findings strongly support the paradigm-shifting hypothesis that brain G6PC3 removes a toxic metabolite, thereby maintaining brain glucose metabolism- and ATP-dependent functions, including cognitive processes.

Metabolomic and Imaging Mass Spectrometric Assays of Labile Brain Metabolites: Critical Importance of Brain Harvest Procedures.

Metabolomic technologies including imaging mass spectrometry (IMS; also called mass spectrometry imaging, MSI, or matrix-assisted laser desorption/ionization-mass spectrometry imaging, MALDI MSI) are important methods to evaluate levels of many compounds in brain with high spatial resolution, characterize metabolic phenotypes of brain disorders, and identify disease biomarkers. ATP is central to brain energetics, and reports of its heterogeneous distribution in brain and regional differences in ATP/ADP ratios reported in IMS studies conflict with earlier studies. These discordant data were, therefore, analyzed and compared with biochemical literature that used rigorous methods to preserve labile metabolites. Unequal, very low regional ATP levels and low ATP/ADP ratios are explained by rapid metabolism during postmortem ischemia. A critical aspect of any analysis of brain components is their stability during and after tissue harvest so measured concentrations closely approximate their physiological levels in vivo. Unfortunately, the requirement for inactivation of brain enzymes by freezing or heating is not widely recognized outside the neurochemistry discipline, and procedures that do not prevent postmortem autolysis, including decapitation, brain removal/dissection, and 'snap freezing' are commonly used. Strong emphasis is placed on use of supplementary approaches to calibrate metabolite abundance in units of concentration in IMS studies and comparison of IMS results with biochemical data obtained by different methods to help identify potential artifacts.

Reevaluation of Astrocyte-Neuron Energy Metabolism with Astrocyte Volume Fraction Correction: Impact on Cellular Glucose Oxidation Rates, Glutamate-Glutamine Cycle Energetics, Glycogen Levels and Utilization Rates vs. Exercising Muscle, and Na+/K+ Pumping Rates.

Accurate quantification of cellular contributions to rates of substrate utilization in resting, activated, and diseased brain is essential for interpretation of data from studies using [18F]fluorodeoxyglucose-positron-emission tomography (FDG-PET) and [13C]glucose/magnetic resonance spectroscopy (MRS). A generally-accepted dogma is that neurons have the highest energy demands of all brain cells, and calculated neuronal rates of glucose oxidation in awake, resting brain accounts for 70-80%, with astrocytes 20-30%. However, these proportions do not take cell type volume fractions into account. To evaluate the conclusion that neuron-astrocyte glucose oxidation rates are similar when adjusted for astrocytic volume fraction (Hertz, Magn Reson Imaging 2011; 29, 1319), the present study analyzed data from 31 studies. On average, astrocytes occupy 6.1, 9.6, and 15% of tissue volume in hippocampus, cerebral cortex, and cerebellum, respectively, and regional astrocytic metabolic rates are adjusted for volume fraction by multiplying by 17.6, 11.4, and 6.8, respectively. After adjustment, astrocytic glucose oxidation rates in resting awake rat brain are 4-10 fold higher than neuronal oxidation rates. Volume-fraction adjustment also increases brain glycogen concentrations and utilization rates to be similar to or exceed exercising muscle. Ion flux calculations to evaluate sodium/potassium homeostasis during neurotransmission are not correct if astrocyte-neuron volume fractions are assumed to be equal. High rates of glucose and glycogen utilization after adjustment for volume fraction indicate that astrocytic energy demands are much greater than recognized, with most of the ATP being used for functions other than glutamate processing in the glutamate-glutamine cycle, challenging the notion that astrocytes 'feed hungry neurons'.

Introduction to the Thematic Minireview Series: Brain glycogen metabolism.

The synthesis of glycogen allows for efficient intracellular storage of glucose molecules in a soluble form that can be rapidly released to enter glycolysis in response to energy demand. Intensive studies of glucose and glycogen metabolism, predominantly in skeletal muscle and liver, have produced innumerable insights into the mechanisms of hormone action, resulting in the award of several Nobel Prizes over the last one hundred years. Glycogen is actually present in all cells and tissues, albeit at much lower levels than found in muscle or liver. However, metabolic and physiological roles of glycogen in other tissues are poorly understood. This series of Minireviews summarizes what is known about the enzymes involved in brain glycogen metabolism and studies that have linked glycogen metabolism to multiple brain functions involving metabolic communication between astrocytes and neurons. Recent studies unexpectedly linking some forms of epilepsy to mutations in two poorly understood proteins involved in glycogen metabolism are also reviewed.

The "protected" glucose transport through the astrocytic endoplasmic reticulum is too slow to serve as a quantitatively-important highway for nutrient delivery.

A novel mechanism involving "protected glucose trafficking" through the lumen of the astrocytic endoplasmic reticulum (ER) was proposed by Müller et al. (Current Biology 28:3481, 2018) and highlighted by Pellerin (Current Biology 28:R1258, 2018) as a potential route for astrocyte-neuron lactate shuttling. In their model, glucose is taken up from blood into astrocytic endfeet, phosphorylated, and some glucose-6-phosphate (Glc-6-P) is transported into the ER lumen where it is hydrolyzed by glucose-6-phosphatase-ß to produce glucose. The glucose then diffuses within the ER lumen through peripheral astrocytic processes (PAPs) to regions in close proximity to synapses where it is released to cytoplasm and metabolized to lactate and neurotransmitter precursors that can be shuttled to neurons. Experimental evidence supports aspects of this model, but essential components were not established. This commentary critically evaluates the Müller et al. study and discusses its weaknesses and intriguing aspects. Most important, the rate-limiting step of the ER "protected highway," transport of Glc-6-P into the ER, is extremely slow, on the order of 550-3,700 times lower than glucose consumption by differentiated cultured astrocytes. Glucose diffusion through extracellular space to fuel neurons was not ruled out as an alternative mechanism. The ER glucose probes were not calibrated, and the ER luminal glucose reservoir size and flux of glucose through the ER are unknown. Effects of phosphatase knockdown are quite interesting and require further study. Glucose transport through a "protected intracellular highway" in the ER lumen is not quantitatively relevant to astrocytic glucose metabolism and is not energetically important.

Does shuttling of glycogen-derived lactate from astrocytes to neurons take place during neurotransmission and memory consolidation?

Glycogen levels in resting brain and its utilization rates during brain activation are high, but the functions fulfilled by glycogenolysis in living brain are poorly understood. Studies in cultured astrocytes have identified glycogen as the preferred fuel to provide ATP for Na+ ,K+ -ATPase for the uptake of extracellular K+ and for Ca2+ -ATPase to pump Ca2+ into the endoplasmic reticulum. Studies in astrocyte-neuron co-cultures led to the suggestion that glycogen-derived lactate is shuttled to neurons as oxidative fuel to support glutamatergic neurotransmission. Furthermore, both knockout of brain glycogen synthase and inhibition of glycogenolysis prior to a memory-evoking event impair memory consolidation, and shuttling of glycogen-derived lactate as neuronal fuel was postulated to be required for memory. However, lactate shuttling has not been measured in any of these studies, and procedures to inhibit glycogenolysis and neuronal lactate uptake are not specific. Testable alternative mechanisms to explain the observed findings are proposed: (i) disruption of K+ and Ca2+ homeostasis, (ii) release of gliotransmitters, (iii) imposition of an energy crisis on astrocytes and neurons by inhibition of mitochondrial pyruvate transport by compounds used to block neuronal monocarboxylic acid transporters, and (iv) inhibition of astrocytic filopodial movements that secondarily interfere with glutamate and K+ uptake from the synaptic cleft. Evidence that most pyruvate/lactate derived from glycogen is not oxidized and does not accumulate suggests predominant glycolytic metabolism of glycogen to support astrocytic energy demands. Sparing of blood-borne glucose for use by neurons is a reasonable explanation for the requirement for glycogenolysis in neurotransmission and memory processing.

Metabolomic Assays of Postmortem Brain Extracts: Pitfalls in Extrapolation of Concentrations of Glucose and Amino Acids to Metabolic Dysregulation In Vivo in Neurological Diseases.

Glucose utilization is reduced in vulnerable brain regions affected by neurological disorders, especially Alzheimer's disease (AD), but the basis for abnormal glucose homeostasis is unknown. Studies of brain-bank human tissue have made major contributions to understanding complex aspects of neurological, psychiatric, and neurodegenerative diseases, but they are not appropriate for metabolomic analysis of labile metabolites because postmortem intervals between death and tissue freezing are much too long. Recent reports of postmortem brain glucose levels led to suggestions that AD patients may be hyperglycemic and that elevated brain glucose levels along with reduced glycolytic activity reveal abnormal glucose homeostasis before clinical symptoms become manifest. These conclusions are, however, questioned because virtually all brain glucose is consumed within minutes after death, followed by progressive increases in glucose and amino acid levels, presumably due to autolytic changes. To illustrate pitfalls in use of autopsy material for metabolomic assays of labile metabolites, data from living human brain are compared with those from autopsy samples, and metabolism at the onset of postmortem ischemia is compared with calculated glycolytic enzyme activities. Postmortem glucose levels range from extremely low to unrealistically high, precluding their extrapolation to living brain. Indirect evaluation of glycolytic enzyme activities in postmortem AD brain is not valid because the glucose and amino acid concentrations used in the calculations are not stable after death, and reported values are unrealistically high. Specific recommendations are provided for non-invasive longitudinal monitoring of brain metabolism and metabolite levels in patients with neurological diseases.

Lack of appropriate stoichiometry: Strong evidence against an energetically important astrocyte-neuron lactate shuttle in brain.

Glutamate-stimulated aerobic glycolysis in astrocytes coupled with lactate shuttling to neurons where it can be oxidized was proposed as a mechanism to couple excitatory neuronal activity with glucose utilization (CMRglc ) during brain activation. From the outset, this model was not viable because it did not fulfill critical stoichiometric requirements: (i) Calculated glycolytic rates and measured lactate release rates were discordant in cultured astrocytes. (ii) Lactate oxidation requires oxygen consumption, but the oxygen-glucose index (OGI, calculated as CMRO2 /CMRglc ) fell during activation in human brain, and the small rise in CMRO2 could not fully support oxidation of lactate produced by disproportionate increases in CMRglc . (iii) Labeled products of glucose metabolism are not retained in activated rat brain, indicating rapid release of a highly labeled, diffusible metabolite identified as lactate, thereby explaining the CMRglc -CMRO2 mismatch. Additional independent lines of evidence against lactate shuttling include the following: astrocytic oxidation of glutamate after its uptake can help "pay" for its uptake without stimulating glycolysis; blockade of glutamate receptors during activation in vivo prevents upregulation of metabolism and lactate release without impairing glutamate uptake; blockade of ß-adrenergic receptors prevents the fall in OGI in activated human and rat brain while allowing glutamate uptake; and neurons upregulate glucose utilization in vivo and in vitro under many stimulatory conditions. Studies in immature cultured cells are not appropriate models for lactate shuttling in adult brain because of their incomplete development of metabolic capability and astrocyte-neuron interactions. Astrocyte-neuron lactate shuttling does not make large, metabolically significant contributions to energetics of brain activation. © 2017 Wiley Periodicals, Inc.

Determination of Glucose Utilization Rates in Cultured Astrocytes and Neurons with [14C]deoxyglucose: Progress, Pitfalls, and Discovery of Intracellular Glucose Compartmentation.

2-Deoxy-D-[14C]glucose ([14C]DG) is commonly used to determine local glucose utilization rates (CMRglc) in living brain and to estimate CMRglc in cultured brain cells as rates of [14C]DG phosphorylation. Phosphorylation rates of [14C]DG and its metabolizable fluorescent analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), however, do not take into account differences in the kinetics of transport and metabolism of [14C]DG or 2-NBDG and glucose in neuronal and astrocytic cells in cultures or in single cells in brain tissue, and conclusions drawn from these data may, therefore, not be correct. As a first step toward the goal of quantitative determination of CMRglc in astrocytes and neurons in cultures, the steady-state intracellular-to-extracellular concentration ratios (distribution spaces) for glucose and [14C]DG were determined in cultured striatal neurons and astrocytes as functions of extracellular glucose concentration. Unexpectedly, the glucose distribution spaces rose during extreme hypoglycemia, exceeding 1.0 in astrocytes, whereas the [14C]DG distribution space fell at the lowest glucose levels. Calculated CMRglc was greatly overestimated in hypoglycemic and normoglycemic cells because the intracellular glucose concentrations were too high. Determination of the distribution space for [14C]glucose revealed compartmentation of intracellular glucose in astrocytes, and probably, also in neurons. A smaller metabolic pool is readily accessible to hexokinase and communicates with extracellular glucose, whereas the larger pool is sequestered from hexokinase activity. A new experimental approach using double-labeled assays with DG and glucose is suggested to avoid the limitations imposed by glucose compartmentation on metabolic assays.

Organ Distribution of 13N Following Intravenous Injection of [13N]Ammonia into Portacaval-Shunted Rats.

Ammonia is neurotoxic, and chronic hyperammonemia is thought to be a major contributing factor to hepatic encephalopathy in patients with liver disease. Portacaval shunting of rats is used as an animal model to study the detrimental metabolic effects of elevated ammonia levels on body tissues, particularly brain and testes that are deleteriously targeted by high blood ammonia. In normal adult rats, the initial uptake of label (expressed as relative concentration) in these organs was relatively low following a bolus intravenous injection of [13N]ammonia compared with lungs, kidneys, liver, and some other organs. The objective of the present study was to determine the distribution of label following intravenous administration of [13N]ammonia among 14 organs in portacaval-shunted rats at 12 weeks after shunt construction. At an early time point (12 s) following administration of [13N]ammonia the relative concentration of label was highest in lung with lower, but still appreciable relative concentrations in kidney and heart. Clearance of 13N from blood and kidney tended to be slower in portacaval-shunted rats versus normal rats during the 2-10 min interval after the injection. At later times post injection, brain and testes tended to have higher-than-normal 13N levels, whereas many other tissues had similar levels in both groups. Thus, reduced removal of ammonia from circulating blood by the liver diverts more ammonia to extrahepatic tissues, including brain and testes, and alters the nitrogen homeostasis in these tissues. These results emphasize the importance of treatment paradigms designed to reduce blood ammonia levels in patients with liver disease.

Aerobic glycolysis during brain activation: adrenergic regulation and influence of norepinephrine on astrocytic metabolism.

Aerobic glycolysis occurs during brain activation and is characterized by preferential up-regulation of glucose utilization compared with oxygen consumption even though oxygen level and delivery are adequate. Aerobic glycolysis is a widespread phenomenon that underlies energetics of diverse brain activities, such as alerting, sensory processing, cognition, memory, and pathophysiological conditions, but specific cellular functions fulfilled by aerobic glycolysis are poorly understood. Evaluation of evidence derived from different disciplines reveals that aerobic glycolysis is a complex, regulated phenomenon that is prevented by propranolol, a non-specific ß-adrenoceptor antagonist. The metabolic pathways that contribute to excess utilization of glucose compared with oxygen include glycolysis, the pentose phosphate shunt pathway, the malate-aspartate shuttle, and astrocytic glycogen turnover. Increased lactate production by unidentified cells, and lactate dispersal from activated cells and lactate release from the brain, both facilitated by astrocytes, are major factors underlying aerobic glycolysis in subjects with low blood lactate levels. Astrocyte-neuron lactate shuttling with local oxidation is minor. Blockade of aerobic glycolysis by propranolol implicates adrenergic regulatory processes including adrenal release of epinephrine, signaling to brain via the vagus nerve, and increased norepinephrine release from the locus coeruleus. Norepinephrine has a powerful influence on astrocytic metabolism and glycogen turnover that can stimulate carbohydrate utilization more than oxygen consumption, whereas ß-receptor blockade 're-balances' the stoichiometry of oxygen-glucose or -carbohydrate metabolism by suppressing glucose and glycogen utilization more than oxygen consumption. This conceptual framework may be helpful for design of future studies to elucidate functional roles of preferential non-oxidative glucose utilization and glycogen turnover during brain activation. Aerobic glycolysis, the preferential up-regulation of glucose utilization (CMRglc ) compared with oxygen consumption (CMRO2 ) during brain activation, is blocked by propranolol. Epinephrine release from the adrenal gland stimulates vagus nerve signaling to the locus coeruleus, enhancing norepinephrine release in the brain, and regulation of astrocytic and neuronal metabolism to stimulate CMRglc more than CMRO2 . Propranolol suppresses CMRglc more than CMRO2 .

Biochemical, Metabolic, and Behavioral Characteristics of Immature Chronic Hyperphenylalanemic Rats.

Phenylketonuria and hyperphenylalanemia are inborn errors in metabolism of phenylalanine arising from defects in steps to convert phenylalanine to tyrosine. Phe accumulation causes severe mental retardation that can be prevented by timely identification of affected individuals and their placement on a Phe-restricted diet. In spite of many studies in patients and animal models, the basis for acquisition of mental retardation during the critical period of brain development is not adequately understood. All animal models for human disease have advantages and limitations, and characteristics common to different models are most likely to correspond to the disorder. This study established similar levels of Phe exposure in developing rats between 3 and 16 days of age using three models to produce chronic hyperphenylalanemia, and identified changes in brain amino acid levels common to all models that persist for ~16 h of each day. In a representative model, local rates of glucose utilization (CMRglc) were determined at 25-27 days of age, and only selective changes that appeared to depend on Phe exposure were observed. CMRglc was reduced in frontal cortex and thalamus and increased in hippocampus and globus pallidus. Behavioral testing to evaluate neuromuscular competence revealed poor performance in chronically-hyperphenylalanemic rats that persisted for at least 3 weeks after cessation of Phe injections and did not occur with mild or acute hyperphenylalanemia. Thus, the abnormal amino acid environment, including hyperglycinemia, in developing rat brain is associated with selective regional changes in glucose utilization and behavioral abnormalities that are not readily reversed after they are acquired.