Interrelationships of sodium transport and carbon dioxide production by the toad bladder: response to changes in mucosal sodium concentration, to vasopressin and to availability of metabolic substrate.

Active sodium transport and CO2 production were measured simultaneously in toad bladders mounted in membrane chambers. The rate of sodium transport was varied by changing the concentration of sodium in the mucosal bath (substitution with choline), by adding vasopressin, by adding metabolic substrates and by adding malonate, and the ratio of the change of sodium transport and CO2 production was determined Mean values for deltaNa/deltaCO2 (equiv/mole) were: Na in equilibrium choline 18.3 +/- 1.1; vasopressin 15.5 +/- 2.8; and pyruvate (corrected for the increment in "nontransport" CO2) 15.4 +/- 3.5. Based on previously determined values for the respiratory quotient (R.Q.), calculated mean values for deltaNa/deltaO2 ranged between 15.5 and 18.5 equiv/mole. It appears that basal metabolism does not contribute to metabolism supporting sodium transport when the rate of sodium transport is varied. "Transport" metabolism appears much more responsive to changes in the availability of endogenous and exogenous substrates than does "nontransport" metabolism. We conclude that "transport" and "nontransport" metabolism are functionally separated in the toad bladder.     

Viability of some metabolic processes in the isolated toad brain adapted to two osmotic environments.

The viability of the isolated toad brain in an aerated Ringer-like medium has been evaluated by the following criteria: 1) amino acid content before and after incubation; 2) accumulation of amino acids in the incubation medium; 3) a comparison of glucose utilization and [U-14C]glucose metabolism with that occurring in vivo; 4) tissue swelling; and 5) tissue lactate contents. On the basis of these criteria, the isolated toad brain, from toads adapted to a fresh-water or a salt-water environment, retains considerable metabolic integrity for at least 2 hr of incubation at 25 degrees C. Specifically, there was no swelling of the tissue, no apparent accumulation of lactate in the tissue, glucose appeared to be utilized at a rate not too different from that calculated for the toad brain in vivo, and the distribution of label from [U-14C]glucose had an overall pattern which resembled that observed in vivo. The tissue levels of amino acids were generally stable in vitro; however, there was a marked decline in the content of aspartate. The accumulation of amino acids in the medium varied considerably from one amino acid to another. Thus, there was very little net efflux of aspartate, GABA, and glutamate from the tissue but considerable net efflux of glutamine. This efflux of amino acids was greater from brains of hyperosmotically adapted toads than from the brains of toads adapted to fresh water by amounts proportional to their initial tissue contents.     

Adenosine triphosphate utilization rates and metabolic pool sizes in intact cells measured by transfer of 18O from water.

The hydrolytic rates and metabolic pool sizes of ATP were determined in intact cells by monitoring the time courses of 18O incorporation from 18O-water into the gamma-phosphoryl of ATP and orthophosphate. To calculate the rate of ATP hydrolysis, a kinetic model is used to fit the time course of the 18O labeling. The size of the metabolic pool of ATP is calculated from the 18O distribution after isotopic equilibrium has been achieved. Metabolic pools have a binomial distribution of 18O whereas nonmetabolic pools exhibit negligible 18O labeling. The application and limitations of this approach are illustrated with data from isolated toad retinas and human platelets. At 22 degrees C, the time constant of ATP hydrolysis in the dark-adapted toad retina is about 30 s. Under these conditions, over 80% of the retinal ATP is involved in high-energy phosphate metabolism. It is calculated that when cGMP metabolic flux in the photoreceptors is maximally stimulated by light, it accounts for 10% of the ATP utilization by the entire retina. The time constant of ATP hydrolysis in human platelets at 37 degrees C is approximately 1 s, and 60% of the platelet ATP is involved in energy metabolism.     

Viability of some metabolic processes in the isolated toad brain adapted to two osmotic environments

The viability of the isolated toad brain in an aerated Ringer-like medium has been evaluated by the following criteria: 1) amino acid content before and after incubation; 2) accumulation of amino acids in the incubation medium; 3) a comparison of glucose utilization and [U-¹⁴C]glucose metabolism with that occurring in vivo; 4) tissue swelling; and 5) tissue lactate content. On the basis of these criteria, the isolated toad brain, from toads adapted to a fresh-water or a salt-water environment, retains considerable metabolic integrity for at least 2 hr of incubation at 25° C. Specifically, there was no swelling of the tissue, no apparent accumulation of lactate in the tissue, glucose appeared to be utilized at a rate not too different from that calculated for the toad brain in vivo, and the distribution of label from [U-¹⁴C]glucose had an overall pattern which resembled that observed in vivo. The tissue levels of amino acids were generally stable in vitro; however, there was a marked decline in the content of aspartate. The accumulation of amino acids in the medium varied considerably from one amino acid to another. Thus, there was very little net efflux of aspartate, GABA, and glutamate from the tissue but considerable net efflux of glutamine. This efflux of amino acids was greater from brains of hyperosmotically adapted toads than from the brains of toads adapted to fresh water by amounts proportional to their initial tissue contents.     

Gender Differences of Brain Glucose Metabolic Networks Revealed by FDG-PET: Evidence from a Large Cohort of 400 Young Adults

Background

Gender differences of the human brain are an important issue in neuroscience research. In recent years, an increasing amount of evidence has been gathered from noninvasive neuroimaging studies supporting a sexual dimorphism of the human brain. However, there is a lack of imaging studies on gender differences of brain metabolic networks based on a large population sample.

Materials and Methods

FDG PET data of 400 right-handed, healthy subjects, including 200 females (age: 25∼45 years, mean age±SD: 40.9±3.9 years) and 200 age-matched males were obtained and analyzed in the present study. We first investigated the regional differences of brain glucose metabolism between genders using a voxel-based two-sample t-test analysis. Subsequently, we investigated the gender differences of the metabolic networks. Sixteen metabolic covariance networks using seed-based correlation were analyzed. Seven regions showing significant regional metabolic differences between genders, and nine regions conventionally used in the resting-state network studies were selected as regions-of-interest. Permutation tests were used for comparing within- and between-network connectivity between genders.

Results

Compared with the males, females showed higher metabolism in the posterior part and lower metabolism in the anterior part of the brain. Moreover, there were widely distributed patterns of the metabolic networks in the human brain. In addition, significant gender differences within and between brain glucose metabolic networks were revealed in the present study.

Conclusion

This study provides solid data that reveal gender differences in regional brain glucose metabolism and brain glucose metabolic networks. These observations might contribute to the better understanding of the gender differences in human brain functions, and suggest that gender should be included as a covariate when designing experiments and explaining results of brain glucose metabolic networks in the control and experimental individuals or patients.     

METABOLISM OF AMINO ACIDS IN INCUBATED SLICES OF MOUSE BRAIN1

• 1 Slices of mouse brain were incubated with [U-¹⁴C]alanine, valine, leucine, phenylalanine, proline, histidine, lysine, arginine or aspartic acid, and the extent of metabolism was estimated by analyses utilizing paper chromatography of the tissue extracts and with an amino acid analyser.

• 2 The metabolism of Ala and Asp was high; of Leu and Pro, moderate; and of Lys, Arg and Phe, low; the metabolism of Val and His was not significant. The time-course of metabolism in most cases showed varying rates, indicating heterogeneous metabolic compartments for the amino acids.

• 3 Production of CO₂ was high from Asp, moderate from Ala, and low from Leu; the other amino acids were not oxidized to CO₂ to any significant extent. A large portion of the metabolized label was trapped in the form of Glu or Asp.

• 4 Metabolism increased with increasing concentration of amino acid to some extent and was largely inhibited by omission of glucose, by anaerobic conditions, or by cyanide. Although these conditions also inhibit uptake, the time-course and extent of inhibition uptake and metabolism were different.

• 5 With Asp, Ala and Phe, metabolism was lowest in slices from pons-medulla; the brain area exhibiting the highest metabolism differed for each amino acid. The metabolism of Asp was lower in brain samples from newborn than in those from adults; the metabolism of Leu was higher in slices from newborn brain.

• 6 The results indicate that the majority of the amino acids can be metabolized in brain tissue and that the metabolic rates are influenced by a number of factors, among them the level of amino acids and the level of available energy.

     

Some neurochemical aspects of fluorocitrate intoxication

Abstract— Some metabolic and biochemical effects of fluorocitrate were studied in vivo in rat brain and cat spinal cord. During the preconvulsant and convulsant phases of fluorocitrate poisoning the contents of free glutamate, glutamine and aspartate declined progressively, while that of alanine increased. Incorporation of ¹⁴C from [U-¹⁴C]glucose into these amino acids also decreased, although somewhat more gradually. GABA exhibited a biphasic change, its content rising after an initial decrease while its relative specific activity rose initially and subsequently diminished. Incorporation of ¹⁴C from [U-¹⁴C]glucose and [U-¹⁴C]lysine into neural protein declined sharply. The citric acid content rose markedly in rat brain and cat spinal cord. In rat brain the glycogen content declined but ATP and ammonia contents were unchanged. The significance of these results with respect to energy metabolism and the possible mechanism of the convulsions during fluorocitrate poisoning is discussed.     

Metabolic reorganization and signal transduction during estivation in the spadefoot toad

The maximal activities of 28 enzymes, representing multiple pathways of intermediary metabolism, were quantified in the brain, liver and skeletal muscle of spadefoot toads Scaphiopus couchii, comparing control toads with animals that had estivated for 2 months. Estivation-induced changes in brain enzyme activities were consistent with suppressed glycolysis and increased ketone body and amino acid catabolism. In liver, estivation resulted in reduced activities of eight enzymes representing carbohydrate, amino acid, ketone body and phosphagen metabolism, but the maximal activity of malic enzyme increased by 2.4-fold. Estivation led to a large-scale reorganization of skeletal muscle affecting most of the enzymes analyzed. Activities of enzymes of carbohydrate catabolism were generally elevated except for glycogen phosphorylase and hexokinase, whereas those of enzymes of fatty acid synthesis and ketone body metabolism were reduced. Increased glutamate dehydrogenase activities in both brain and muscle, as well as activities of other amino-acid-catabolizing enzymes in muscle, correlated with specific changes in the free amino acids pools in those tissues (reduced glutamine activity, increased glutamate, alanine and valine activities) that appear to be related to protein catabolism, for the purposes of elevating urea levels. The effects of estivation on signal transduction systems were also assessed. Total activities of protein kinases A and C (PKA and PKC) were largely unaltered in toad tissues during estivation (except for a 57% reduction in liver total PKC), but in seven organs there were strong reductions in the percentage of PKA present as the active catalytic subunit in estivating animals, and three contained a much lower percentage of membrane-bound active PKC during estivation. Activities of protein phosphatase types 1, 2A, 2B, and 2C were also frequently reduced during estivation. Overall, these results suggest that anuran estivation involves metabolic reorganization, including changing the maximal activities of key enzymes of intermediary metabolism as well as depressing the metabolic rate by suppressing signal transducing enzymes.     

The role of the autonomic nervous liver innervation in the control of energy metabolism

Despite a longstanding research interest ever since the early work by Claude Bernard, the functional significance of autonomic liver innervation, either sympathetic or parasympathetic, is still ill defined. This scarcity of information not only holds for the brain control of hepatic metabolism, but also for the metabolic sensing function of the liver and the way in which this metabolic information from the liver affects the brain. Clinical information from the bedside suggests that successful human liver transplantation (implying a complete autonomic liver denervation) causes no life threatening metabolic derangements, at least in the absence of severe metabolic challenges such as hypoglycemia. However, from the benchside, data are accumulating that interference with the neuronal brain–liver connection does cause pronounced changes in liver metabolism. This review provides an extensive overview on how metabolic information is sensed by the liver, and how this information is processed via neuronal pathways to the brain. With this information the brain controls liver metabolism and that of other organs and tissues. We will pay special attention to the hypothalamic pathways involved in these liver–brain–liver circuits. At this stage, we still do not know the final destination and processing of the metabolic information that is transferred from the liver to the brain. On the other hand, in recent years, there has been a considerable increase in the understanding which brain areas are involved in the control of liver metabolism via its autonomic innervation. However, in view of the ever rising prevalence of type 2 diabetes, this potentially highly relevant knowledge is still by far too limited. Thus the autonomic innervation of the liver and its role in the control of metabolism needs our continued and devoted attention.     

Effect of craniocerebral hypothermic perfusion on the metabolism of ischemic brain]

Intravascular perfusion of the brain by colloid-saline "extracellular" fluid was carried out in 58 experimental dogs against the background of acute hypoxia. Biochemical indices of metabolism in flowing and outflowing brain fluid as well as in the brain tissue were studied. It was shown that under conditions of acute hypoxia normo- and particularly hypothermic intravascular perfusion of the brain by colloid-saline fluid decreases the disturbances of metabolic homeostasis.     

Life in the slow lane: molecular mechanisms of estivation

Estivation is a state of aerobic hypometabolism used by organisms to endure seasonally arid conditions, often in desert environments. Estivating species are often active for only a few weeks each year to feed and breed and then retreat to estivate in sheltered sites, often underground. In general, estivation includes a strong reduction in metabolic rate, a primary reliance on lipid oxidation to fuel metabolism, and methods of water retention, both physical (e.g. cocoons) and metabolic (e.g. urea accumulation). The present review focuses on several aspects of metabolic adaptation during estivation including changes in the activities of enzymes of intermediary metabolism and antioxidant defenses, the effects of urea on estivator enzymes, enzyme regulation by reversible protein phosphorylation, protein kinases and phosphatases involved in signal transduction mechanisms, and the role of gene expression in estivation. The focus is on two species: the spadefoot toad, Scaphiopus couchii, from the Arizona desert; and the land snail, Otala lactea, a native of the Mediterranean region. The mechanisms of metabolic depression in estivators are similar to those seen in hibernation and anaerobiosis, and contribute to the development of a unified set of biochemical principles for the control of metabolic arrest in nature.     

Principles of the 2-deoxyglucose method for the determination of the local cerebral glucose utilization

Sokoloff and co-workers developed the 2-deoxy-D-[1-14C]glucose (2DG) method in order to study the local cerebral glucose utilization (LCGU) of discrete brain regions in vivo. Energy metabolism of the adult mammalian brain is almost entirely dependent on glucose. The majority of the glucose taken up by the brain is needed for the maintenance of the membrane potentials and the electrical activity. The functional activity could thus be shown to be closely linked to energy metabolism. Consequently, examination of the energy metabolism by measuring the cerebral metabolic rate for glucose can provide information concerning functional activity in all of the neuroanatomically defined regions of the brain. Studying the fate of experimentally injected 2-deoxy-D-[1-14C]glucose, a radioactive labeled analogue of glucose, and, subsequently, employing quantitative autoradiographic techniques, it is possible to estimate the levels of the local cerebral glucose utilization in specific regions of the brain. According to Sokoloff (1982) the LCGU represents a "metabolic encephalography".     

The determination of the local cerebral glucose utilization with the 2-deoxyglucose method

Summary In the adult mammalian brain, the energy metabolism is almost entirely dependent on glucose. Furthermore, a close relationship between the energy metabolism and the functional activity could be shown. Thus, the functional activity of the brain or parts thereof can be quantified by measuring the cerebral metabolic rate for glucose. Studying in vivo the fate of a radioactive labeled analogue of glucose, the 2-deoxy-d-[1-¹⁴C]glucose, and using quantitative autoradiographic techniques, it is possible to estimate the cerebral glucose utilization of every discrete brain region. The advantage of the 2-deoxyglucose method is, that the local cerebral glucose utilization represents a metabolic encephalography (Sokoloff 1982).     

UPTAKE AND METABOLISM OF GLUTAMATE BY ISOLATED TOAD BRAINS CONTAINING DIFFERENT LEVELS OF ENDOGENOUS AMINO ACIDS

—The uptake of [U-¹⁴C]glutamate into the amphibian brain was studied in vitro using brains from toads (Bufo boreas) adapted either to a fresh water (FWA) or an hyperosmotic saline (HOA) environment. Initial rates of ¹⁴C-glutamate uptake showed a single apparent K_m of about 0·2 mm . Uptake by HOA brains was slower than that by FWA brains, reflecting perhaps a non-competitive type of inhibition by the higher content of glutamate in the HOA brains. Although the glutamate content of HOA brains was maintained during prolonged incubation at twice the level found in FWA toads, other metabolic parameters measured in the two types of brain preparations were surprisingly similar. Tissue to medium concentration ratios of greater than 3000:1 were generated by both FWA and HOA brains. In both brain systems the clearance of glutamate from the medium was accompanied by a rapid conversion of the amino acid to glutamine and its release into the medium. In both the FWA and HOA toad brain systems some [U-¹⁴C]glutamate was metabolized to aspartate and GABA; in both systems the specific radioactivity (SA) of glutamine in the tissue was from two to four times greater than that of glutamate; also the SA of glutamine released into the medium was higher by several orders of magnitude than the SA of glutamine in brain tissues. These and other findings support the concept that, in both the FWA and HOA toad brains, transport processes are instrumental in preserving low extracellular levels of glutamate but that mechanisms other than transport are responsible for the maintenance of different levels of glutamate in the FWA and HOA toad brains.     

Using Bioconductor Package BiGGR for Metabolic Flux Estimation Based on Gene Expression Changes in Brain

Predicting the distribution of metabolic fluxes in biochemical networks is of major interest in systems biology. Several databases provide metabolic reconstructions for different organisms. Software to analyze flux distributions exists, among others for the proprietary MATLAB environment. Given the large user community for the R computing environment, a simple implementation of flux analysis in R appears desirable and will facilitate easy interaction with computational tools to handle gene expression data. We extended the R software package BiGGR, an implementation of metabolic flux analysis in R. BiGGR makes use of public metabolic reconstruction databases, and contains the BiGG database and the reconstruction of human metabolism Recon2 as Systems Biology Markup Language (SBML) objects. Models can be assembled by querying the databases for pathways, genes or reactions of interest. Fluxes can then be estimated by maximization or minimization of an objective function using linear inverse modeling algorithms. Furthermore, BiGGR provides functionality to quantify the uncertainty in flux estimates by sampling the constrained multidimensional flux space. As a result, ensembles of possible flux configurations are constructed that agree with measured data within precision limits. BiGGR also features automatic visualization of selected parts of metabolic networks using hypergraphs, with hyperedge widths proportional to estimated flux values. BiGGR supports import and export of models encoded in SBML and is therefore interoperable with different modeling and analysis tools. As an application example, we calculated the flux distribution in healthy human brain using a model of central carbon metabolism. We introduce a new algorithm termed Least-squares with equalities and inequalities Flux Balance Analysis (Lsei-FBA) to predict flux changes from gene expression changes, for instance during disease. Our estimates of brain metabolic flux pattern with Lsei-FBA for Alzheimer’s disease agree with independent measurements of cerebral metabolism in patients. This second version of BiGGR is available from Bioconductor.     

Free fatty acids, diacyl- and triacylglycerols and total phospholipids in vertebrate retina: comparison with brain, choroid and plasma

Abstract— A comparative study of the concentration and fatty acid distribution in diacyl- and triacylgly-cerols. free fatty acids and total phospholipids from rabbit, cattle and toad retina is presented. With respect to the toad, a comparison is made with brain, choroid and plasma lipids. Marked differences in diacylglycerol composition and levels between mammalian and toad retina are found: in the mammal arachidonate predominates (25 per cent), in the toad docosahexaenoate is the main fatty acid (42 per cent). The total phospholipid composition parallels that of the diacylglycerols only in the toad, whereas in the mammalian retina the phospholipids are richer in docosahexaenoate than are the diacylglycerols. It is suggested that there is a relationship between diacylglycerols and phosphoglyceride metabolism in the toad; in the retinas of other species the diacylglycerols may be related to specific phosphatides. In the three species, triacylglycerols show high levels of unsaturation; however, marked differences are found in the distribution of polyunsaturated acyl groups: in the cattle and toad retina docosahexaenoate predominates. whereas in the rabbit a higher proportion of 22:4 is found. Retina free fatty acid pools also show different features in the three species: the cattle retina contains the highest proportion of free 20:4 and 22:6. The triacylglycerol concentration is much higher in the toad choroid than in the retina, although the fatty acid compositions are similar. A possible relationship between these choroid lipids and those of the retina is suggested.     

Reconstruction and flux analysis of coupling between metabolic pathways of astrocytes and neurons: application to cerebral hypoxia

Background

It is a daunting task to identify all the metabolic pathways of brain energy metabolism and develop a dynamic simulation environment that will cover a time scale ranging from seconds to hours. To simplify this task and make it more practicable, we undertook stoichiometric modeling of brain energy metabolism with the major aim of including the main interacting pathways in and between astrocytes and neurons.

Model

The constructed model includes central metabolism (glycolysis, pentose phosphate pathway, TCA cycle), lipid metabolism, reactive oxygen species (ROS) detoxification, amino acid metabolism (synthesis and catabolism), the well-known glutamate-glutamine cycle, other coupling reactions between astrocytes and neurons, and neurotransmitter metabolism. This is, to our knowledge, the most comprehensive attempt at stoichiometric modeling of brain metabolism to date in terms of its coverage of a wide range of metabolic pathways. We then attempted to model the basal physiological behaviour and hypoxic behaviour of the brain cells where astrocytes and neurons are tightly coupled.

Results

The reconstructed stoichiometric reaction model included 217 reactions (184 internal, 33 exchange) and 216 metabolites (183 internal, 33 external) distributed in and between astrocytes and neurons. Flux balance analysis (FBA) techniques were applied to the reconstructed model to elucidate the underlying cellular principles of neuron-astrocyte coupling. Simulation of resting conditions under the constraints of maximization of glutamate/glutamine/GABA cycle fluxes between the two cell types with subsequent minimization of Euclidean norm of fluxes resulted in a flux distribution in accordance with literature-based findings. As a further validation of our model, the effect of oxygen deprivation (hypoxia) on fluxes was simulated using an FBA-derivative approach, known as minimization of metabolic adjustment (MOMA). The results show the power of the constructed model to simulate disease behaviour on the flux level, and its potential to analyze cellular metabolic behaviour in silico.

Conclusion

The predictive power of the constructed model for the key flux distributions, especially central carbon metabolism and glutamate-glutamine cycle fluxes, and its application to hypoxia is promising. The resultant acceptable predictions strengthen the power of such stoichiometric models in the analysis of mammalian cell metabolism.     

Interrelationships of sodium transport and carbon dioxide production by the toad bladder: Response to changes in mucosal sodium concentration,to vasopressin and to availability of metabolic substrate

Summary Active sodium transport and CO₂ production were measured simultaneously in toad bladders mounted in membrane chambers. The rate of sodium transport was varied by changing the concentration of sodium in the mucosal bath (substitution with choline), by adding vasopressin, by adding metabolic substrates and by adding malonate, and the ratio of the change of sodium transport and CO₂ production was determined Mean values for Na/CO₂ (equiv/mole) were: Nacholine 18.3±1.1; vasopressin 15.5±2.8; and pyruvate (corrected for the increment in nontransport CO₂) 15.4±3.5. Based on previously determined values for the respiratory quotient (R.Q.), calculated mean values for Na/O₂ ranged between 15.5 and 18.5 equiv/mole. It appears that basal metabolism does not contribute to metabolism supporting sodium transport when the rate of sodium transport is varied. Transport metabolism appears much more responsive to changes in the availability of endogenous and exogenous substrates than does nontransport metabolism. We conclude that transport and nontransport metabolism are functionally separated in the toad bladder.These results were presented in part at the Annual Meeting of the American Society of Nephrology, November 1973.     

Metabolic compensation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> catalase-deficient mutants

Multigenic families are widely represented in the genomes of higher plants, and are required for the reliability of cellular functions. Damage of individual genes can be compensated by diverse metabolic alterations, but the exact mechanisms of such compensations still remain not fully understood. Here we present novel data regarding the mechanisms of metabolic compensation in photorespiratory knock-out mutants cat2, cat3 and cat2cat3 of Arabidopsis thaliana, which are lacking activity of catalase isoforms CAT2 and CAT3. It was found that cultivation of the mutants under low light at optimal or increased temperature did not result in any morphological, physiological or biochemical signs of oxidative stress. Each of the mutant lines shows specific features of the compensatory mechanisms. Increased activity of CAT3 isoenzyme was found in the cat2 mutant, whereas cat3 and cat2cat3 demonstrate induction of CAT1, an isoform normally absent in young leaves, as well as activation of peroxidases, namely APX and POD. Comparison of these results and earlier published data revealed that the lack of CAT2 and CAT3 isoforms is compensated by preferential activation of non-enzymatic and enzymatic protection mechanisms, respectively.     

Astrocyte activation in working brain: energy supplied by minor substrates

Glucose delivered to brain by the cerebral circulation is the major and obligatory fuel for all brain cells, and assays of functional activity in working brain routinely focus on glucose utilization. However, these assays do not take into account the contributions of minor substrates or endogenous fuel consumed by astrocytes during brain activation, and emerging evidence suggests that glycogen, acetate, and, perhaps, glutamate, are metabolized by working astrocytes in vivo to provide physiologically significant amounts of energy in addition to that derived from glucose. Rates of glycogenolysis during sensory stimulation of normal, conscious rats are high enough to support the notion that glycogen can contribute substantially to astrocytic glucose utilization during activation. Oxidative metabolism of glucose provides most of the ATP for cultured astrocytes, and a substantial contribution of respiration to astrocyte energetics is supported by recent in vivo studies. Astrocytes preferentially oxidize acetate taken up into brain from blood, and calculated local rates of acetate utilization in vivo are within the range of calculated rates of glucose oxidation in astrocytes. Glutamate may also serve as an energy source for activated astrocytes in vivo because astrocytes in tissue culture and in adult brain tissue readily oxidize glutamate. Taken together, contributions of minor metabolites derived from endogenous and exogenous sources add substantially to the energy obtained by astrocytes from blood-borne glucose. Because energy-generating reactions from minor substrates are not taken into account by routine assays of functional metabolism, they reflect a "hidden cost" of astrocyte work in vivo.