The impact of metabolism on the adaptation of organisms to environmental change.

Since Jacob and Monod's discovery of the lac operon ∼1960, the explanations offered for most metabolic adaptations have been genetic. The focus has been on the adaptive changes in gene expression that occur, which are often referred to as "metabolic reprogramming." The contributions metabolism makes to adaptation have been largely ignored. Here we point out that metabolic adaptations, including the associated changes in gene expression, are highly dependent on the metabolic state of an organism prior to the environmental change to which it is adapting, and on the plasticity of that state. In support of this hypothesis, we examine the paradigmatic example of a genetically driven adaptation, the adaptation of E. coli to growth on lactose, and the paradigmatic example of a metabolic driven adaptation, the Crabtree effect in yeast. Using a framework based on metabolic control analysis, we have reevaluated what is known about both adaptations, and conclude that knowledge of the metabolic properties of these organisms prior to environmental change is critical for understanding not only how they survive long enough to adapt, but also how the ensuing changes in gene expression occur, and their phenotypes post-adaptation. It would be useful if future explanations for metabolic adaptations acknowledged the contributions made to them by metabolism, and described the complex interplay between metabolic systems and genetic systems that make these adaptations possible.

The early days of ex vivo 1 H, 13 C, and 31 P nuclear magnetic resonance in the laboratory of Dr. Robert G. Shulman from 1975 to 1995.

This paper provides a brief description of the early use of ex vivo nuclear magnetic resonance (NMR) studies of tissue and tissue extracts performed in the laboratory of Dr. Robert G. Shulman from 1975 through 1995 at Bell Laboratories, then later at Yale University. During that period, ex vivo NMR provided critical information in support of resonance assignments and the quantitation of concentrations for magnetic resonance spectroscopy studies. The period covered saw rapid advances in magnet technology, starting with studies of microorganisms in vertical bore high-resolution NMR studies, then by 1981 studies of small mammals in a horizontal bore magnet, and then studies of humans in 1984. Ex vivo NMR played a critical role in all these studies. A general strategy developed in the lab for using ex vivo NMR to support in vivo studies is presented, as well as illustrative examples.

Two transition states of the glycogen shunt and two steady states of gene expression support metabolic flexibility and the Warburg effect in cancer.

Previously we suggested that the early Warburg effect can be explained by the use by cancer cells the glycogen shunt during a rapid increase in glucose concentration. In analogy to the Crabtree effect in yeast, the shunt plays a critical role in maintaining homeostasis of glycolytic intermediate levels during these transitions. We extend this analysis here, and propose that the recently appreciated flexibility of cancer cell glucose and glycogen metabolism involves 4 metabolic states that we recently identified in metabolic control analysis studies of yeast. Under stable conditions of low glucose and normal O2 yeast, and by analogy cancer, cells are in the Respiration State in which through gene expression for oxidizing non glucose substrates. When their environment changes to high glucose with reduced O2 levels, such as occur in tumors, they transition to the Glycolysis State due to gene expression of new glycolytic enzyme isoforms such as PKM2. These isoforms optimize metabolism to sustain the Warburg effect. When the changes in glucose and O2 levels are rapid there may be insufficient time for gene expression to adapt. The metabolic flexibility conferred by 2 states of the glycogen shunt allow the cells to survive these transitions. The model explains experimental observations in cancer such as the function of the glycogen shunt and the frequent expression of PKM2 in cells undergoing the Warburg Effect. A surprising conclusion is that the function of PKM2 is to maintain glycolytic intermediate homeostasis rather than controlling the glycolytic flux. The glycogen shunt may also have an important role in cancer metabolic reprogramming by allowing cancer cells to survive large glucose and oxygen changes during the selection of mutations that lead to the Warburg phenotype.

Gene expression regulates metabolite homeostasis during the Crabtree effect: Implications for the adaptation and evolution of Metabolism.

A key issue in both molecular and evolutionary biology has been to define the roles of genes and phenotypes in the adaptation of organisms to environmental changes. The dominant view has been that an organism's metabolic adaptations are driven by gene expression and that gene mutations, independent of the starting phenotype, are responsible for the evolution of new metabolic phenotypes. We propose an alternate hypothesis, in which the phenotype and genotype together determine metabolic adaptation both in the lifetime of the organism and in the evolutionary selection of adaptive metabolic traits. We tested this hypothesis by flux-balance and metabolic-control analysis of the relative roles of the starting phenotype and gene expression in regulating the metabolic adaptations during the Crabtree effect in yeast, when they are switched from a low- to high-glucose environment. Critical for successful short-term adaptation was the ability of the glycogen/trehalose shunt to balance the glycolytic pathway. The role of later gene expression of new isoforms of glycolytic enzymes, rather than flux control, was to provide additional homeostatic mechanisms allowing an increase in the amount and efficiency of adenosine triphosphate and product formation while maintaining glycolytic balance. We further showed that homeostatic mechanisms, by allowing increased phenotypic plasticity, could have played an important role in guiding the evolution of the Crabtree effect. Although our findings are specific to Crabtree yeast, they are likely to be broadly found because of the well-recognized similarities in glucose metabolism across kingdoms and phyla from yeast to humans.

A Non-cognitive Behavioral Model for Interpreting Functional Neuroimaging Studies.

The dominant model for interpreting brain imaging experiments, which we refer to as the Standard Cognitive Model (SCM), assumes that the brain is organized in support of mental processes that control behavior. However, functional neuroimaging experiments of cognitive tasks have not shown clear anatomic segregation between mental processes originally proposed by this model. This failing has been blamed on limitations in imaging technology and non-linearity in the brain's implementation of these processes. However, the validity of the underlying cognitive models used to describe the brain has rarely been questioned or directly tested against imaging results. We propose an alternative model of brain function, that we term the Non-cognitive Behavioral Model (NBM), which correlates observed human behavior directly with measured brain activity without making assumptions about intervening cognitive processes. Our model derives from behavioral psychology but is extended to include brain activity, in addition to behavior, as observables. A further extension is the role of neuroplasticity, as opposed to innate cognitive processes, in developing the brain's support of cognitive behavior. We present the theoretical basis with which the SCM maps cognitive processes onto functional magnetic resonance and positron emission tomography images and compare and contrast with the NBM. We also describe how the NBM can be used experimentally to study how the brain supports behavior. Two applications are presented that support the usefulness of the NBM. In one, the NBM use of the total functional imaging signal (not just the differences between states) provides a stronger correlation of neural activity with the behavioral state of consciousness than the SCM approach in both anesthesia and coma. The second example reviews studies of facial and object recognition that provide evidence for the NBM proposal that neuroplasticity and experience play key roles in the brain's support of recognition and other behaviors. The conclusions regarding neuroplasticity are then generalized to explain the incomplete functional segregation observed in the application of the SCM to neuroimaging.

The Glycogen Shunt Maintains Glycolytic Homeostasis and the Warburg Effect in Cancer.

Despite many decades of study there is a lack of a quantitative explanation for the Warburg effect in cancer. We propose that the glycogen shunt, a pathway recently shown to be critical for cancer cell survival, may explain the excess lactate generation under aerobic conditions characteristic of the Warburg effect. The proposal is based on research on yeast and mammalian muscle and brain that demonstrates that the glycogen shunt functions to maintain homeostasis of glycolytic intermediates and ATP during large shifts in glucose supply or demand. Loss of the glycogen shunt leads to cell death under substrate stress. Similarities between the glycogen shunt in yeast and cancer cells lead us here to propose a parallel explanation of the lactate produced by cancer cells in the Warburg effect. The model also explains the need for the active tetramer and inactive dimer forms of pyruvate kinase (PKM2) in cancer cells, similar to the two forms of Pyk2p in yeast, as critical for regulating the glycogen shunt flux. The novel role proposed for the glycogen shunt implicates the high activities of glycogen synthase and fructose bisphosphatase in tumors as potential targets for therapy.

Homeostasis and the glycogen shunt explains aerobic ethanol production in yeast.

Aerobic glycolysis in yeast and cancer cells produces pyruvate beyond oxidative needs, a paradox noted by Warburg almost a century ago. To address this question, we reanalyzed extensive measurements from (13)C magnetic resonance spectroscopy of yeast glycolysis and the coupled pathways of futile cycling and glycogen and trehalose synthesis (which we refer to as the glycogen shunt). When yeast are given a large glucose load under aerobic conditions, the fluxes of these pathways adapt to maintain homeostasis of glycolytic intermediates and ATP. The glycogen shunt uses glycolytic ATP to store glycolytic intermediates as glycogen and trehalose, generating pyruvate and ethanol as byproducts. This conclusion is supported by studies of yeast with a partial block in the glycogen shunt due to the cif mutation, which found that when challenged with glucose, the yeast cells accumulate glycolytic intermediates and ATP, which ultimately leads to cell death. The control of the relative fluxes, which is critical to maintain homeostasis, is most likely exerted by the enzymes pyruvate kinase and fructose bisphosphatase. The kinetic properties of yeast PK and mammalian PKM2, the isoform found in cancer, are similar, suggesting that the same mechanism may exist in cancer cells, which, under these conditions, could explain their excess lactate generation. The general principle that homeostasis of metabolite and ATP concentrations is a critical requirement for metabolic function suggests that enzymes and pathways that perform this critical role could be effective drug targets in cancer and other diseases.

Insights from neuroenergetics into the interpretation of functional neuroimaging: an alternative empirical model for studying the brain's support of behavior.

Functional neuroimaging measures quantitative changes in neurophysiological parameters coupled to neuronal activity during observable behavior. These results have usually been interpreted by assuming that mental causation of behavior arises from the simultaneous actions of distinct psychological mechanisms or modules. However, reproducible localization of these modules in the brain using functional magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging has been elusive other than for sensory systems. In this paper, we show that neuroenergetic studies using PET, calibrated functional magnetic resonance imaging (fMRI), (13)C magnetic resonance spectroscopy, and electrical recordings do not support the standard approach, which identifies the location of mental modules from changes in brain activity. Of importance in reaching this conclusion is that changes in neuronal activities underlying the fMRI signal are many times smaller than the high ubiquitous, baseline neuronal activity, or energy in resting, awake humans. Furthermore, the incremental signal depends on the baseline activity contradicting theoretical assumptions about linearity and insertion of mental modules. To avoid these problems, while making use of these valuable results, we propose that neuroimaging should be used to identify observable brain activities that are necessary for a person's observable behavior rather than being used to seek hypothesized mental processes.

Direct evidence for activity-dependent glucose phosphorylation in neurons with implications for the astrocyte-to-neuron lactate shuttle.

Previous (13)C magnetic resonance spectroscopy experiments have shown that over a wide range of neuronal activity, approximately one molecule of glucose is oxidized for every molecule of glutamate released by neurons and recycled through astrocytic glutamine. The measured kinetics were shown to agree with the stoichiometry of a hypothetical astrocyte-to-neuron lactate shuttle model, which predicted negligible functional neuronal uptake of glucose. To test this model, we measured the uptake and phosphorylation of glucose in nerve terminals isolated from rats infused with the glucose analog, 2-fluoro-2-deoxy-D-glucose (FDG) in vivo. The concentrations of phosphorylated FDG (FDG6P), normalized with respect to known neuronal metabolites, were compared in nerve terminals, homogenate, and cortex of anesthetized rats with and without bicuculline-induced seizures. The increase in FDG6P in nerve terminals agreed well with the increase in cortical neuronal glucose oxidation measured previously under the same conditions in vivo, indicating that direct uptake and oxidation of glucose in nerve terminals is substantial under resting and activated conditions. These results suggest that neuronal glucose-derived pyruvate is the major oxidative fuel for activated neurons, not lactate-derived from astrocytes, contradicting predictions of the original astrocyte-to-neuron lactate shuttle model under the range of study conditions.

Glutamatergic function in the resting awake human brain is supported by uniformly high oxidative energy.

Rodent (13)C magnetic resonance spectroscopy studies show that glutamatergic signaling requires high oxidative energy in the awake resting state and allowed calibration of functional magnetic resonance imaging (fMRI) signal in terms of energy relative to the resting energy. Here, we derived energy used for glutamatergic signaling in the awake resting human. We analyzed human data of electroencephalography (EEG), positron emission tomography (PET) maps of oxygen (CMR(O2)) and glucose (CMR(glc)) utilization, and calibrated fMRI from a variety of experimental conditions. CMR(glc) and EEG in the visual cortex were tightly coupled over several conditions, showing that the oxidative demand for signaling was four times greater than the demand for nonsignaling events in the awake state. Variations of CMR(O2) and CMR(glc) from gray-matter regions and networks were within ±10% of means, suggesting that most areas required similar energy for ubiquitously high resting activity. Human calibrated fMRI results suggest that changes of fMRI signal in cognitive studies contribute at most ±10% CMR(O2) changes from rest. The PET data of sleep, vegetative state, and anesthesia show metabolic reductions from rest, uniformly >20% across, indicating no region is selectively reduced when consciousness is lost. Future clinical investigations will benefit from using quantitative metabolic measures.

Baseline brain energy supports the state of consciousness.

An individual, human or animal, is defined to be in a conscious state empirically by the behavioral ability to respond meaningfully to stimuli, whereas the loss of consciousness is defined by unresponsiveness. PET measurements of glucose or oxygen consumption show a widespread approximately 45% reduction in cerebral energy consumption with anesthesia-induced loss of consciousness. Because baseline brain energy consumption has been shown by (13)C magnetic resonance spectroscopy to be almost exclusively dedicated to neuronal signaling, we propose that the high level of brain energy is a necessary property of the conscious state. Two additional neuronal properties of the conscious state change with anesthesia. The delocalized fMRI activity patterns in rat brain during sensory stimulation at a higher energy state (close to the awake) collapse to a contralateral somatosensory response at lower energy state (deep anesthesia). Firing rates of an ensemble of neurons in the rat somatosensory cortex shift from the gamma-band range (20-40 Hz) at higher energy state to <10 Hz at lower energy state. With the conscious state defined by the individual's behavior and maintained by high cerebral energy, measurable properties of that state are the widespread fMRI patterns and high frequency neuronal activity, both of which support the extensive interregional communication characteristic of consciousness. This usage of high brain energies when the person is in the "state" of consciousness differs from most studies, which attend the smaller energy increments observed during the stimulations that form the "contents" of that state.

Neurophysiology of functional imaging.

The successes of PET and fMRI in non-invasively localizing sensory functions had encouraged efforts to transform the subjective concepts of cognitive psychology into objective physical measures. The assumption was that mental functions could be decomposed into non-overlapping, context-independent modules that are operated on by separable areas of a computer-like brain. The failures of cognitive modularity and of a very localized phrenology are generally, but not universally, accepted; but in their place, and usually not distinguished from the original revolutionary hopes of clarification, experimental results are being interpreted in terms of rather flexible definitions of both cognitive concepts and the degree of localization. In an alternative approach, we have connected fMRI, (13)C MRS, and electrophysiology measurements of brain energy to connect with observable properties of mental life (i.e., awareness). We illustrate this approach with a sensory stimulation experiment; the degree of localization found in BOLD signals was related to the global energy of the brain which, when manipulated by anesthetics, affected the degree of awareness. The influence of brain energy upon functional imaging maps is changing the interpretations of neuroimaging experiments, from psychological concepts generating computer-like responses to empirical responses dominated by the high brain energy and signaling at rest. In our view "baseline" is an operational term, an adjective that defines a property of a state of the system before it is perturbed by a stimulus. Given the dependence of observable psychological properties upon the "baseline" energy, we believe that it is unnecessarily limiting to define a particular state as the baseline.

Energetics of neuronal signaling and fMRI activity.

Energetics of resting and evoked fMRI signals were related to localized ensemble firing rates (nu) measured by electrophysiology in rats. Two different unstimulated, or baseline, states were established by anesthesia. Halothane and alpha-chloralose established baseline states of high and low energy, respectively, in which forepaw stimulation excited the contralateral primary somatosensory cortex (S1). With alpha-chloralose, forepaw stimulation induced strong and reproducible fMRI activations in the contralateral S1, where the ensemble firing was dominated by slow signaling neurons (SSN; nu range of 1-13 Hz). Under halothane, weaker and less reproducible fMRI activations were observed in the contralateral S1 and elsewhere in the cortex, but ensemble activity in S1 was dominated by rapid signaling neurons (RSN; nu range of 13-40 Hz). For both baseline states, the RSN activity (i.e., higher frequencies, including the gamma band) did not vary upon stimulation, whereas the SSN activity (i.e., alpha band and lower frequencies) did change. In the high energy baseline state, a large majority of total oxidative energy [cerebral metabolic rate of oxygen consumption (CMR(O2))] was devoted to RSN activity, whereas in the low energy baseline state, it was roughly divided between SSN and RSN activities. We hypothesize that in the high energy baseline state, the evoked changes in fMRI activation in areas beyond S1 are supported by rich intracortical interactions represented by RSN. We discuss implications for interpreting fMRI data where stimulus-specific DeltaCMR(O2) is generally small compared with baseline CMR(O2).

A BOLD search for baseline.

While we occasionally observe negative BOLD signals, its physiological basis has remained uncertain. This is in part due to the qualitative use of fMRI where the baseline is conveniently differenced away to reveal focal area(s) of interest. Recently, however, there has been a noticeable trend towards quantitative neuroimaging where changes in oxidative energetics (CMR(O2)) are quantified by calibrated fMRI. Pasley et al. [Pasley, B.N., Inglis, B.A., Freeman, R.D., 2007. Analysis of oxygen metabolism implies a neural origin for the negative BOLD response in human visual cortex. NeuroImage] used calibrated fMRI in conjunction with a novel stimulus paradigm to investigate the neural basis of the negative BOLD signal in awake humans. They hypothesized - based on prior results - that if the baseline was lowered then DeltaCMR(O2) would have to be larger. While their main findings point to an energetic basis for the negative BOLD signal, their results have far reaching implications for the present definition of baseline as well as for future research investigating the neural and/or energetic basis of baseline.

Neuronal-glial glucose oxidation and glutamatergic-GABAergic function.

Prior 13C magnetic resonance spectroscopy (MRS) experiments, which simultaneously measured in vivo rates of total glutamate-glutamine cycling (V(cyc(tot))) and neuronal glucose oxidation (CMR(glc(ox), N)), revealed a linear relationship between these fluxes above isoelectricity, with a slope of approximately 1. In vitro glial culture studies examining glutamate uptake indicated that glutamate, which is cotransported with Na+, stimulated glial uptake of glucose and release of lactate. These in vivo and in vitro results were consolidated into a model: recycling of one molecule of neurotransmitter between glia and neurons was associated with oxidation of one glucose molecule in neurons; however, the glucose was taken up only by glia and all the lactate (pyruvate) generated by glial glycolysis was transferred to neurons for oxidation. The model was consistent with the 1:1 relationship between DeltaCMR(glc(ox), N) and DeltaV(cyc(tot)) measured by 13C MRS. However, the model could not specify the energetics of glia and gamma-amino butyric acid (GABA) neurons because quantitative values for these pathways were not available. Here, we review recent 13C and 14C tracer studies that enable us to include these fluxes in a more comprehensive model. The revised model shows that glia produce at least 8% of total oxidative ATP and GABAergic neurons generate approximately 18% of total oxidative ATP in neurons. Neurons produce at least 88% of total oxidative ATP, and take up approximately 26% of the total glucose oxidized. Glial lactate (pyruvate) still makes the major contribution to neuronal oxidation, but approximately 30% less than predicted by the prior model. The relationship observed between DeltaCMR(glc(ox), N) and DeltaV(cyc(tot)) is determined by glial glycolytic ATP as before. Quantitative aspects of the model, which can be tested by experimentation, are discussed.

Glycogen turnover forms lactate during exercise.

The power needs of muscle are supplied by rapid anaerobic glycogenolytic ATP generation. Lactate is built up by the mismatch between steady state energy needs and short-term power demands and the increased concentration drives the lactate shuttle. In this model, contributions to fatigue should be looked for in the flux of glucose through glycogen rather than in the concentrations of fuel.

The contribution of GABA to glutamate/glutamine cycling and energy metabolism in the rat cortex in vivo.

Previous studies have shown that the glutamate/glutamine (Glu/Gln) neurotransmitter cycle and neuronal glucose oxidation are proportional (1:1), with increasing neuronal activity above isoelectricity. GABA, a product of Glu metabolism, is synthesized from astroglial Gln and contributes to total Glu/Gln neurotransmitter cycling, although the fraction contributed by GABA is unknown. In the present study, we used (13)C NMR spectroscopy together with i.v. infusions of [1,6-(13)C(2)]glucose and [2-(13)C]acetate to separately determine rates of Glu/Gln and GABA/Gln cycling and their respective tricarboxylic acid cycles in the rat cortex under conditions of halothane anesthesia and pentobarbital-induced isoelectricity. Under 1% halothane anesthesia, GABA/Gln cycle flux comprised 23% of total (Glu plus GABA) neurotransmitter cycling and 18% of total neuronal tricarboxylic acid cycle flux. In isoelectric cortex, glucose oxidation was reduced >3-fold in glutamatergic and GABAergic neurons, and neurotransmitter cycling was below detection. Hence, in both cell types, the primary energetic costs are associated with neurotransmission, which increase together as cortical activity is increased. The contribution of GABAergic neurons and inhibition to cortical energy metabolism has broad implications for the interpretation of functional imaging signals.

Cerebral pyruvate carboxylase flux is unaltered during bicuculline-seizures.

Glutamine synthesis in the astroglia reflects the sum of neurotransmitter cycling (glutamate and gamma-aminobutyric acid [GABA]) and de novo synthesis (anaplerosis), the latter catalyzed by pyruvate carboxylase. Previous studies have shown that the glutamate plus GABA cycling flux is correlated strongly with neuronal activity; however, the relationship between pyruvate carboxylase flux and neuronal activity is not known. In this study, pyruvate carboxylase flux was assessed during intravenous infusion of [2-(13)C]glucose using localized (1)H-[(13)C] NMR spectroscopy at 7 Tesla in vivo in halothane-anesthetized and ventilated adult Wistar rats during 85 min of bicuculline-induced seizures (1 mg/kg, intravenously) and in nontreated controls. During seizures, concentrations of lactate, alanine, glutamine, GABA, and succinate increased whereas glutamate and aspartate decreased such that the decrease in glutamate plus aspartate equaled the increase in glutamine plus GABA. Pyruvate carboxylase flux was assessed by the sum of [2-(13)C] and [3-(13)C] of glutamine and glutamate (Glx(2+3)) labeling during [2-(13)C]glucose infusion. During seizures the initial rate of Glx(2+3) synthesis (0.069 +/- 0.013 micromol/g/min) was not significantly different (P = 0.68) from that of the controls (0.059 +/- 0.010 micromol/g/min), indicating that anaplerotic flow through pyruvate carboxylase was unaltered. Intense neuronal activation of seizures did not seem to increase anaplerosis through pyruvate carboxylase, despite the substantial increase in neuronal activity and glutamate/glutamine cycling shown in a previous study (Patel et al., 2004b).

Glutamatergic neurotransmission and neuronal glucose oxidation are coupled during intense neuronal activation.

13C nuclear magnetic resonance (NMR) experiments have previously shown that glutamatergic neurotransmitter flux (Vcycle(Glu/Gln)) changes proportionately with neuronal glucose oxidation (CMRglc(ox)N) in the nonactivated cortex of anesthetized rats. Positron Emission Tomography measurements of glucose and oxygen uptake during sensory stimulation had shown that the incremental glucose utilization is greater than oxygen leading to the suggestion that the energy required for stimulated neuronal activity arises from nonoxidative glucose metabolism. In this study, the authors used spatially localized 1H-observed, 13C-edited NMR spectroscopy during an infusion of [1,6-13C2]glucose to assess the relationship between changes in Vcycle(Glu/Gln) and glucose utilization (CMRglc(ox)N and CMRglc(nonox)) during the intense cortical activity associated with bicuculline-induced seizures. Metabolic fluxes were determined by model-based analysis of the 13C-enrichment time courses of glutamate-C4 and glutamine-C4 (CMRglc(ox)N, Vcycle(Glu/Gln)) and lactate-C3 (CMRglc(nonox)). The exchange rate between alpha-ketoglutarate and glutamate was found to be significantly faster than TCA cycle flux both for control (41 micromol.g(-1).min(-1); 95% CI, 5 to 109 micromol.g(-1).min(-1)) and during seizures (21 micromol.g(-1).min(-1); 95% CI, 4.4 to 51.8 micromol.g(-1).min(-1)). During seizures, total glucose utilization (CMRglc(ox+nonox)) increased substantially (466% between 0 and 6 minutes; 277% between 6 and 55 minutes). Glucose oxidation (CMRglc(ox)N) also increased (214%; from 0.26 +/- 0.02 to 0.57 +/- 0.07 micromol.g(-1).min(-1)) but to a lesser degree, resulting in a large increase in cortical lactate concentration. Vcycle(Glu/Gln) increased 233% (from 0.22 +/- 0.04 to 0.52 +/- 0.07 micromol.g(-1).min(-1)), which was similar to the increase in glucose oxidation. The value of Vcycle(Glu/Gln) and CMRglc(ox)N obtained here lie on the line predicted in a previous study. These results indicate that neuronal glucose oxidation and not total glucose utilization is coupled to the glutamate/glutamine cycle during intense cortical activation.