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Abstract

Interpretation of functional metabolic brain images requires understanding of metabolic shifts in working brain. Because the disproportionately higher uptake of glucose compared with oxygen (“aerobic glycolysis”) during sensory stimulation is not fully explained by changes in levels of lactate or glycogen, metabolic labeling by [6-¹⁴C]glucose was used to evaluate utilization of glucose during brief brain activation. Increased labeling of tricarboxylic acid cycle–derived amino acids, mainly glutamate but also γ-aminobutyric acid, reflects a rise in oxidative metabolism during aerobic glycolysis. The size of the glutamate, lactate, alanine, and aspartate pools changed during stimulation. Brain lactate was derived from blood-borne glucose and its specific activity was twice that of alanine, revealing pyruvate compartmentation. Glycogen labeling doubled during recovery compared with rest and activation; only 4% to 8% of the total ¹⁴C was recovered in lactate plus glycogen. Restoration of glycogen levels was slow, and diversion of glucose from oxidative pathways to restore its level could cause a prolonged reduction of the global O₂/glucose uptake ratio. The rise in the brain glucose–oxygen uptake ratio during activation does not simply reflect an upward shift of glycolysis under aerobic conditions; instead, it involves altered fluxes into various (oxidative and biosynthetic) pathways with different time courses.

Keywords

Aerobic glycolysis Sensory stimulation Metabolism Brain Astrocytes

Energy-dependent processes are the basis for metabolic imaging of functional and cognitive activities of the brain, and recent imaging studies of working human brain have renewed interest in the bioenergetics of mental activity, neuronal signaling, and neuron-astrocyte interactions required for neurotransmission. Reexamination of the long-held concept of close stoichiometric coupling of oxidative metabolism of glucose and functional activity in brain was stimulated by reports of 30% to 50% increases in the rates of cerebral glucose utilization (CMR_glc) and cerebral blood flow (CBF) with little or no change in oxygen consumption (CMRO₂) during stimulation (Fox and Raichle, 1986; Fox et al., 1988; Madsen et al., 1995a). Because glucose consumed in excess of oxygen is generally assumed to be converted to lactate, this phenomenon is sometimes called “aerobic glycolysis.” The low glycolytic yield of ATP led Fox et al. (1988) to conclude that energy required by working brain is much less than that inferred from oxidative metabolism. This apparent failure of CMRO₂ to rise during some but not all types or conditions of physiologic stimulation of normoxic subjects is unexpected and unexplained, and many issues related to glucose and lactate metabolism in working brain have been recently assessed in considerable detail (Attwell and Laughlin, 2001; Chih et al., 2001; Dienel and Hertz, 2001; Gjedde et al., 2002; Magistretti et al., 1999).

The brain clearly has the capacity to sustain large increases in CMRO₂ in vivo with heightened energy demand under normal and abnormal conditions. For example, visual stimulation increases CMRO₂ in humans (Hoge et al., 1999; Marrett et al., 1995; Vafaee et al., 1999). During seizures, CMRO₂ rises 1.5 to 2-fold in humans, and in animals the CMRO₂/CMR_glc ratio falls at seizure onset and increases thereafter, with rapid (<1-minute), persistent (up to 2-hour), and large (60% to 270%) increases in CMR_glc and CMRO₂ (Siesjö, 1978). Enhanced lactate production and its rapid efflux to blood from activated brain (Cruz et al., 1999; Hawkins et al., 1973) can contribute to both aerobic glycolysis and discrepant metabolic images obtained with [¹⁴C]glucose and [¹⁴C]deoxyglucose (Ackermann and Lear, 1989; Adachi et al., 1995; Collins et al., 1987). Increased lactate levels in stimulated brain are registered by in vivo microdialysis (Fellows et al., 1993; Kuhr and Korf, 1988) and may arise from astrocyte (e.g., Magistretti et al., 1999) and neuronal (Gjedde and Marrett, 2001) metabolism, but this remains to be established. The biochemical basis for aerobic glycolysis is not understood, and shifts in utilization of glucose underlying a disproportionate rise in uptake or utilization of glucose compared with oxygen in working brain in vivo are not known.

To investigate metabolic changes during aerobic glycolysis, we developed an in vivo animal model in which a high glucose-oxygen uptake ratio was induced by generalized sensory stimulation of the conscious rat (Madsen et al., 1995b), and showed that the excess uptake of glucose was not fully explained by lactate and glycogen accumulation in brain or lactate efflux to blood (Madsen et al., 1999). The present study tested the hypothesis that channeling of [6-¹⁴C]glucose into major metabolic pathways changes during and after sensory stimulation, and reports increased labeling of products of the oxidative pathways, altered metabolite pool sizes, and metabolic compartmentation. Slow restoration of glycogen level, which is mainly localized in astrocytes (reviewed by Swanson, 1992), suggests that astrocyte metabolism may influence the global glucose/oxygen uptake ratio, particularly after a stimulus.

MATERIALS AND METHODS

Chemicals

D-[6-¹⁴C]glucose (specific activity, 52 mCi/mmol) and 2-deoxy-D-[1-¹⁴C]glucose ([¹⁴C]DG; 51 mCi/mmol) were purchased from DuPont NEN (Boston, MA, U.S.A.). Radiochemical purity was assayed before use by phosphorylation with yeast hexokinase (Boehringer-Mannheim, Indianapolis, IN, U.S.A.) plus ATP, then purification by HPLC; the radiochemical purity of each tracer was >98%. ATP, D-glucose, and glutaminase were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.), and Dowex 50-H+ (AG50W-X8) was obtained from BioRad (Richmond, CA, U.S.A.).

Animals

Some rats (six per group) in which the levels of unlabeled glycogen, glucose, and lactate in ethanol, HCl, and perchloric acid extracts were reported in our accompanying article (i.e., in conjunction with extensive analysis of the effects of extraction procedure on measured levels of glycogen; Cruz and Dienel, 2002) were labeled in vivo with [6-¹⁴C]glucose during rest, activation, and recovery.

Preparative surgical procedures. Nonfasted male Sprague-Dawley rats (Zivic Laboratories, Pittsburgh, PA, U.S.A.) weighing about 350 to 400 g and that had been maintained on a 12-hour light-dark cycle with ad lib feeding were anesthetized between about 8:30 and 11:00 AM with halothane (1% to 2% to effect in air). Polyethylene catheters (PE 50) filled with physiologic saline containing 100 U/mL heparin were inserted into a femoral artery and vein. The surgical wounds were closed and infiltrated with lidocaine. Then a midline incision was made in the scalp skin of the anesthetized rat for placement of a funnel on the intact skull during the in situ freezing procedure (Pontén et al., 1973); tissue was cleared from the skull and anesthetic cream was applied to the wound. The rats were restrained by means of a plaster cast around the hind limbs and the cast was taped to an acrylic board. Rats were then placed in a shelter (constructed of five lead bricks and a cover) for 3 hours to allow recovery from anesthesia and the surgical procedure, the duration of which was about 30 minutes. The catheters were exteriorized between the bricks so that intravenous injections and blood sampling could be carried out without disturbing or alerting the animal. During recovery, the laboratory environment was very carefully controlled to minimize noise and other stimuli that might have activated brain metabolism.

Physiologic variables were determined just before the metabolic labeling (i.e., 3 hours after surgery). Rectal temperature was monitored with a thermistor (Yellow Springs Instrument Co. [YSI], Yellow Springs, OH, U.S.A.) and maintained at 37°C with a thermostatically controlled heating lamp. Arterial blood samples were drawn for determination of physiologic variables before beginning the experiment. Arterial blood PO₂, PCO₂, and pH were determined with a Model 170 pH/blood-gas analyzer (Corning Medical Scientific, Medfield, MA, U.S.A.). Arterial blood hematocrit was determined after centrifugation. Mean arterial blood pressure was measured with a Micro-Med Analyzer (Micro-Med, Louisville, KY, U.S.A.) that had been calibrated with an air-damped Hg manometer. Arterial plasma glucose and lactate concentrations were simultaneously measured in each sample by enzyme-sensor technology with a YSI 2700 Select Biochemistry Analyzer using glucose and lactate oxidases to produce H₂O₂ which is electrochemically oxidized at the respective platinum anodes; the instrument was calibrated with standards with each assay set, and standard curves were used to determine the linear range for each substrate.

Sensory stimulation. Generalized sensory stimulation was used to enhance glucose utilization in cerebral cortex of conscious rats using exactly the same procedures developed in our laboratory and used in our previous experiments (Madsen et al., 1995b, 1999), except that cerebral venous blood sampling was not carried out in the present study. Sensory stimulation consisted of removal of the bricks and top of the shelter and 5 minutes of gentle bilateral brushing of the head, whiskers, face, back, paws, and tail with a soft paintbrush to produce widespread changes in neural activity and metabolism throughout cerebral cortex. The rats moved around but did not vocalize during the brushing procedure. After stimulation, the rats were sequestered and placed back into the shelter for a 15-minute recovery interval.

Metabolic labeling. Metabolic activation induced by 5-minute generalized sensory stimulation was localized by autoradiography using [¹⁴C]DG (Adachi et al., 1995). [¹⁴C]DG (100 μCi/kg, 100 μCi/mL) was injected intravenously during rest, at the onset of sensory stimulation, or at 10 minutes after cessation of stimulation (n = 3 per group). Five minutes later the rats were killed with an overdose of pentobarbital (200 mg/kg, intravenously) and the brains were immediately removed and frozen in isopentane at about −40°C. Autoradiographs were prepared from 20-μm-thick coronal sections of brain and calibrated ¹⁴C standards, and local total ¹⁴C levels were determined by computer-assisted densitometry (Imaging Research, St. Catharines, Ontario, Canada). About 50% to 70% of the total ¹⁴C is recovered in metabolites of DG at 5 minutes after a pulse of [¹⁴C]DG (Adachi et al., 1995; Dienel and Cruz, 1993). A 30- to 45-minute experimental period is required for the fully quantitative [¹⁴C]DG method to minimize errors that can arise from estimates of rate constants used to calculate the integrated specific activity (ISA) of the precursor pool in brain and the unmetabolized [¹⁴C]DG in tissue at the end of the experiment; such errors can be high in 5- to 10-minute experiments, regardless of the labeled hexose (i.e., glucose or DG) used to assay CMR_glc (Adachi et al., 1995; Sokoloff, 1986; Sokoloff et al., 1977). For this reason, no correction was made for unmetabolized [¹⁴C]DG in tissue (which would be lower in activated structures), and label accumulation was calculated by dividing the total ¹⁴C in regions of interest by the plasma ISA for that animal.

To assess labeling of brain metabolic pools, an intravenous pulse of [6-¹⁴C]glucose (100 μCi/kg, 100 μCi/ml) was injected into conscious 350- to 400-g rats during rest, at onset of 5 minutes of generalized stimulation, or at 10 minutes after cessation of stimulation (n = 6 per group). Timed samples of arterial blood were drawn throughout the 5-minute labeling period; the first six samples were drawn sequentially during the initial 0.2 minutes, and subsequent samples were drawn at about 0.25, 0.5, 0.75, 1, 2, 3, and 5 minutes after the pulse. After centrifugation to obtain plasma, the ¹⁴C content of each sample was measured by liquid scintillation counting (Packard Model 2200, using external standardization) and the corresponding glucose level determined with a YSI Model 2700 Select Biochemistry Analyzer; [¹⁴C]glucose-specific activities were determined for each sample and used to calculate the ISA of the arterial plasma precursor pool for each animal.

Funnel freezing. In situ freezing (i.e., “funnel freezing”) was used to obtain brain samples for analysis of labeled and unlabeled metabolites (Pontén et al., 1973). At the end of the labeling period, the rats were briefly anesthetized by an intravenous injection of a fast-acting anesthetic (25-mg/kg thiopental) and immediately placed in a nose cone for delivery of 100% O₂ during the freezing process to minimize the possibility of hypoxia (Adachi et al., 1995). Within 30 seconds after injection of thiopental, a small plastic funnel with wet cotton on its base was placed onto the intact skull, and brains were frozen in situ by pouring liquid N₂ into the funnel (i.e., funnel frozen); the cotton freezes at the funnel-skull interface, thereby firmly adhering the funnel to the skull and preventing leakage of the liquid N₂, which might cause hypoxia–anoxia due to inadvertent inhalation of nitrogen or freezing of the neck muscles that can restrict blood flow. The dorsal cerebral cortex freezes rapidly, within about 40 seconds (Pontén et al., 1973; Siesjö, 1978), and liquid N₂ was continuously applied until the freezing front passed below the eyes (i.e., after about 3 or 4 minutes). Then the head was removed, immersed in liquid N₂ until completely frozen, and stored at −80°C.

Analytical procedures

Two tissue-extraction procedures were used in the present study. Labeled and unlabeled glucose, lactate, and glycogen were assayed after aqueous ethanol extraction (Dienel et al., 1990) to eliminate possible interference of perchlorate with the anion-exchange high-performance liquid chromatography (HPLC) purification of the carbohydrates. Perchloric acid extracts of parallel samples of frozen brain powders were used for amino acid analysis because this acid-extraction procedure is more rapid and less tedious than the quantitative ethanolextraction procedure. Perchlorate does not interfere with amino acid analysis because it was removed during the initial cationexchange chromatography step (see below) in which the amino acids were separated from neutral and acidic compounds to eliminate any potential interference by labeled glucose and its labeled metabolites (in the glycolytic pathway and tricarboxylic acid [TCA] cycle) that might co-elute with amino acids.

Assay of glucose, lactate, and glycogen. Details of the procedures for dissection of frozen tissue, powdering, weighing, extraction, and analysis of glucose, lactate, and glycogen are provided in the companion article (Cruz and Dienel, 2002). The tissue sampled for metabolite analysis corresponded as closely as possible to the structures drained by the venous system that was sampled when arteriovenous differences across the brain were determined to assess activity-dependent changes in the brain oxygen-glucose uptake ratio (Madsen et al., 1999). Metabolic responses to generalized sensory stimulation are expected to be heterogeneous throughout the cerebral cortex, but the values obtained for metabolite levels and labeling in these well-mixed powders are tissue averages weighted in proportion to the size, flow, and metabolism of the component brain structures in the sample.

Unlabeled glucose and lactate contents of each sample were determined with the Biochemistry Analyzer, by standard enzymatic fluorometric assays (Lowry and Passonneau, 1972), or by HPLC; all assay procedures gave similar values. Glycogen was assayed in the ethanol-insoluble fraction by determination of net release of labeled or unlabeled glucose after incubation in the presence minus that in the absence of amyloglucosidase. Washed amyloglucosidase-treated pellets (protein, DNA, RNA, lipid) were dissolved in 1N NaOH and assayed for their ¹⁴C contents. Total ¹⁴C in brain was determined by liquid scintillation counting after dissolving a separate weighed portion of frozen powder in 1N NaOH.

Purification of ¹⁴C-labeled glucose and lactate. ¹⁴C-labeled glucose and lactate were separated on a CarboPac PA1 anion exchange column with a Dionex BioLC HPLC System equipped with a pulsed amperometric detector (Dionex Corp., Sunnyvale, CA, U.S.A.) using an elution schedule that we designed to separate hexoses from their metabolites in brain extracts; glucose eluted at about 6 minutes and its acidic derivatives are recovered in later-eluting fractions (Adachi et al., 1995). Eluate samples were collected at 0.5- (between 4 to 8 minutes) or 1-minute intervals, acidified, and assayed for ¹⁴C content by liquid scintillation counting; recovery of ¹⁴C in tissue samples from the CarboPac column was approximately 100%. Unlabeled glucose in the extracts was detected with the pulsed amperometric detector, and its concentration in each sample was quantified by comparison of peak areas to those of standards; similar values were obtained for unlabeled glucose when assayed by HPLC, hexokinase and glucose-6-phosphate dehydrogenase (Lowry and Passonneau, 1972), or glucose oxidase (YSI Analyzer). Because tissue samples contain unlabeled and labeled compounds that may co-elute with glucose, portions of some samples were separated before and after incubation of the glucose with ATP and hexokinase. Virtually all of the ¹⁴C and the unlabeled compounds recovered in the glucose fraction were converted to glucose-6-phosphate and recovered in the appropriate fraction. We also previously calibrated (by means of ¹⁴C-labeled tracers) the elution times of some compounds (i.e., lactate, alanine, glutamine, glutamate, α-ketoglutarate, and pyruvate) that are not detectable by pulsed amperometry (Adachi et al., 1995). ¹⁴C-labeled alanine and glutamine eluted at 4 or 5 minutes, [¹⁴C]lactate eluted at 10 to 12 minutes, and ¹⁴C-labeled glutamate, pyruvate, and α-ketoglutarate, and unlabeled glucose-6-phosphate all eluted at 18 to 20 minutes. The ¹⁴C in extracts of brain that was labeled with [¹⁴C]glucose in vivo and recovered in the same HPLC fraction as the [¹⁴C]lactate standard was confirmed to be pure labeled lactate by incubation of samples with NAD, glutamate, lactate dehydrogenase, and glutamate-pyruvate transaminase (Lowry and Passonneau, 1972) to convert the labeled lactate sequentially to pyruvate, and then to alanine that could be recovered in the 4- or 5-minute HPLC fraction. Unlabeled lactate was determined with the previously described enzymatic assay and measurement of NADH fluorescence.

Amino acid analysis. Amino acids were first separated from labeled and unlabeled acidic and neutral compounds that would not be detectable but might co-elute with glucose-derived amino acids and interfere with analysis of metabolic labeling. Portions of perchloric acid extracts of frozen powders were applied to 1 mL Dowex-50-H+ columns and washed with ice-cold water; amino acids were then eluted with 10 mL 2-mol/L NH₄OH and lyophilized, dissolved in 500 μL distilled water, diluted in citrate sample buffer, and analyzed with a Model 7300 Beckman System Gold Amino Acid Analyzer using post column addition of o-phthalaldehyde and fluorometric detection. Baseline separations were obtained for most amino acids; elution times (in minutes) were as follows: aspartate, 9.3; serine plus glutamine, 12.0; glutamate, 15.8; glycine, 21.5; alanine, 23.3; and γ-aminobutyric acid (GABA), 51.2. Because glutamine and serine co-eluted, paired samples of the Dowex-50 eluates were incubated with or without 0.25 U glutaminase at 37°C in 50 mmol/L acetate buffer (pH 4.7) for 30 minutes to convert glutamine to glutamate (Lund, 1985), and glutamine and serine concentrations and ¹⁴C contents were calculated by difference; e.g., the glutamine was the average of the values obtained by difference from changes in glutamate level (i.e., [glutamate + glutamine] – glutamate) and serine level (i.e., [glutamine + serine] – serine) determined after incubation with and without glutaminase. Thus, four separate amino acid analyses were carried out for each brain sample to determine separately the tissue concentrations and the ¹⁴C contents of each amino acid; larger quantities of the tissue extract were required to obtain good count statistics. Effluent fractions were collected at 0.5- to 1-minute intervals at the elution times of glucose-derived amino acids and assayed for their ¹⁴C contents.

Calculations. The net incorporation of plasma [6-¹⁴C]glucose into the total brain metabolite pool per gram frozen weight tissue (i.e., μmol plasma glucose · (g wet wt.)⁻¹ · min⁻¹) during rest, activation, or recovery was calculated by difference (i.e., total label in brain minus that recovered in purified brain glucose). The calculation was as follows: for each animal, the difference ([total ¹⁴C][g brain powder]⁻¹ – [¹⁴C in purified brain glucose]/[g brain powder]⁻¹) = [nCi in metabolites]/[g brain powder]⁻¹); this value was then normalized to the plasma precursor pool for that rat by dividing by the ISA of glucose in arterial plasma (i.e., nCi · [μmol glucose]⁻¹·min). Similar calculations were made for each purified metabolite (i.e., ¹⁴C recovered in that metabolite fraction per gram brain powder divided by ISA). Dilution factors for the tissue extracts were calculated as described in Method 1 in the accompanying paper (Cruz and Dienel, 2002); for ethanol-soluble extracts, which were freeze dried, the dilution factor was the added volume of deionized water divided by the weight of frozen powder, whereas for the perchloric acid-soluble and ethanol-insoluble fractions the calculation factor was: ([volume of liquid added + weight of frozen powder]/weight of frozen powder). Differences in contributions of tissue water to the extract volume can introduce 2% to 8% variations in calculated values derived from soluble and particulate fractions (see Materials and Methods of Cruz and Dienel, 2002), but these do not affect activity-dependent changes in the level of a specific metabolite. Incorporation of [6-¹⁴C]glucose into purified metabolites was calculated by dividing the nCi recovered in the purified compound per gram frozen brain by the plasma glucose ISA. These estimates are minimal rates because the plasma glucose ISA exceeds that of the true precursor pool in brain owing to restricted entry of the tracer into brain; any loss of ¹⁴C-labeled products (e.g., CO₂, lactate) due to efflux from activated tissue would increase the magnitude of the underestimate. Specific activities of purified metabolites reflect labeling relative to any changes in pool size; specific activities in each rat were normalized to the respective plasma glucose ISA to minimize animal-to-animal variability arising from small differences in the injected dose and plasma glucose levels (i.e., [nCi · μmol metabolite⁻¹][ISA arterial plasma glucose]⁻¹ = μmol plasma glucose · μmol metabolite⁻¹ · min⁻¹).

RESULTS

Physiologic variables

Physiologic variables for all groups of rats were similar and in the normal range (Table 1). Blood O₂ levels were elevated during funnel freezing owing to use of 100% O₂ to minimize hypoxia.

TABLE 1.

Physiologic variables

			Arterial plasma		Arterial blood
Group	Experimental interval	Rectal temperature (°C)	Glucose (mmol/L)	Lactate (mmol/L)	Pressure (mm Hg)	Hematocrit (%)	pH	Po₂ (mm Hg)	PCO₂ (mm Hg)
Rest	Baseline	36.8 ± 0.4	9.2 ± 1.1	0.9 ± 0.3	108 ± 6	50 ±4	7.42 ± 0.01	88.1 ± 5.4	40.6 ± 2.6
	During freezing	ND	ND	ND	ND	ND	7.31 ± 0.04	429 ± 118	55.9 ± 5.0
Stimulation	Baseline	36.8 ± 0.4	9.3 ± 1.0	0.7 ± 0.3	109 ± 6	50 ± 2	7.41 ± 0.02	97.3 ± 16.9	39.6 ± 2.5
	During freezing	ND	ND	ND	ND	ND	7.33 ± 0.03	484 ± 78	47.8 ± 8.1
Recovery	Baseline	36.8 ± 0.4	9.3 ± 1.0	0.7 ± 0.2	111 ± 7	49 ± 2	7.42 ± 0.02	91.1 ± 6.4	39.1 ± 1.1
	During freezing	ND	ND	ND	ND	ND	7.35 ± 0.03	516 ± 53	49.2 ± 6.6

Values are means ± SD (n = 6 per group). Baseline values for the physiologic variables were determined just before the experiment, at about 3 hours after the surgical procedures; they were within the normal range and similar during rest, activation, and recovery. Blood gases and pH were also determined at about 1 minute after onset of the funnel-freezing procedure; oxygen levels are very high owing to use of 100% O₂ in the inspired air to minimize hypoxia during the freezing process (see Materials and Methods and Adachi et al., 1995).

ND, not determined.

Metabolic activation during brief generalized sensory stimulation

Qualitative [¹⁴C]DG autoradiographic assays were used to visualize shifts in cortical glucose use in a small number of animals (n = 3 per group) during brief generalized sensory stimulation compared with rest and recovery. Dark bands near the midlayer of cerebral cortex—presumably layer 4, which has high CMR_glc compared with other cortical layers (Zilles and Wree, 1995)—were more prominently labeled by [¹⁴C]DG during stimulation compared with rest and recovery (data not shown). Total tissue ¹⁴C levels in the caudate and dorsal cortex at the level of caudate/hippocampus were 20% higher (P < 0.05) during stimulation compared with rest; the magnitude of this increased labeling is underestimated due to the higher background contributed by a greater fraction of unmetabolized [¹⁴C]DG at 5 minutes in resting versus activated tissue (Dienel and Cruz, 1993; Adachi et al., 1995).

Labeling of major brain metabolites by [6-¹⁴C]glucose during and after brief stimulation

The specific activity of glucose in arterial plasma peaked within 0.1 or 0.2 minutes after the pulse injection and the first phase of clearance was very rapid and similar in all experimental groups (Fig 1). Between about 0.5 and 5 minutes, the fall in specific activity of plasma glucose was much more gradual, decreasing by approximately 50% during this interval in all three groups (Fig. 1, bottom panel). The specific activities of glucose in the last blood sample taken before funnel freezing and the ISAs of glucose in arterial plasma did not differ among the experimental groups (Fig. 1). The specific activities (nCi/μmol) of the last plasma glucose sample were slightly higher than those of brain glucose (P < 0.05 only for the stimulation group) owing to the lag (about 1 to 1.5 minutes) between drawing the last blood sample and freezing of the brain, during which time clearance of label from plasma would continue. Thus, with the exception of 2 of the 18 animals (bottom panel, Fig. 1), the areas under the plasma curves and temporal profiles of labeled precursor in blood were similar in all rats.

FIG. 1.

Temporal changes in specific activity of arterial plasma glucose after a pulse intravenous injection of [6-¹⁴C]glucose. Values are shown for all animals in which metabolic labeling was carried out during rest, during 5 minutes of generalized sensory stimulation, and during the 10- and 15-minute interval of recovery from stimulation. After the initial peak, which occurred within 0.2 minutes, the fall in glucose-specific activity was much more gradual (bottom panel). The linear regression lines determined for each data set between the interval 0.5 to 5 minutes (n = 41, 45, 40 pairs of values/group) are described as follows: rest, y = −6.3x + 71; activation, y = −8.0x + 77; recovery, y = −5.6x + 61. The standard errors of the estimates (slopes) are 1.3, 1.9, and 0.9, respectively, and the 95% confidence intervals of slopes overlapped. The specific activities of glucose (mean ± SD, n = 6 per group) during rest, activation, and recovery, respectively, were 40 ± 12, 34 ± 10, and 36 ± 10 in the last blood sample and 31 ± 4, 25 ± 3, and29±4inbrain; the blood- and brain-specific activities were not statistically significant except during stimulation (P < 0.05, t-test), presumably owing to continued clearance of label from blood during the freezing procedure and a lag between the last blood sample and freezing of the brain. The ISAs of [¹⁴C]glucose in arterial plasma were 374 ± 113, 324 ± 91, 301 ± 66 [nCi/μmol plasma glucose]min during rest, stimulation, and recovery, respectively, and did not differ among the three groups (ANOVA).

Incorporation of [6-¹⁴C]glucose into total labeled metabolites in dorsal cerebral cortex rose 26% during sensory stimulation compared with rest, and ¹⁴C recovered in the TCA cycle-derived amino acids accounted for most of this increase (Table 2). The total ¹⁴C in all labeled compounds (Table 2) determined in separate portions of the frozen brain powders from each animal agreed within 1% of the total ¹⁴C recovered in the ethanol extracts plus that in the insoluble fractions (glycogen, lipid, protein, DNA, and RNA), indicating that all labeled products of glucose in the tissue were quantitatively taken into account. At 5 minutes after the pulse of tracer, 15% to 25% of the total ¹⁴C remained as unmetabolized glucose, depending on the stage of activity, and identified compounds that were purified accounted for 50% to 60% of the total.

TABLE 2.

Labeling of major cortical glucose carbon pools by [6-¹⁴C]glucose before, during, and after 5 minutes of generalized sensory stimulation

	Rest	Sensory stimulation	Recovery
Metabolite	Normalized incorporation of [6-¹⁴C]glucose into brain metabolite pools 10² × (μmol plasma glucose[g wet wt.]⁻¹ [min⁻¹]
All labeled compounds (precursor + products)	93 ± 19	111 ± 13*	97 ± 15
Purified, unmetabolized [¹⁴C]glucose	19 ± 6	18 ± 4	25 ± 4
Total ¹⁴C-labeled metabolites	74 ± 14	93 ± 10*	73 ± 12
Purified metabolites
Lactate	2.9 ± 1.1	7.5 ± 1.7*	2.9 ± 0.7
Glycogen	0.3 ± 0.1	0.2 ± 0.0	0.5 ± 0.2*
Ethanol-insoluble pellet after amyloglucosidase	0.5 ± 0.1	0.6 ± 0.1	0.5 ± 0.1
Glutamine	3.0 ± 1.7	3.9 ± 2.0	3.2 ± 1.8
GABA	2.5 ± 1.3	4.0 ± 0.6*	2.9 ± 0.4
Glutamate	22.9 ± 11.5	33.8 ± 3.5*	27.5 ± 3.9
Aspartate	6.1 ± 0.9	6.8 ± 0.8	5.8 ± 0.8
Alanine	0.5 ± 0.3	1.1 ±0.2*	0.7 ± 0.1
Serine	0.2 ± 0.1	0.1 ± 0.1	0.1 ± 0.1
Total labeling of identified metabolites	38	58	44
Total labeling of unidentified metabolites	36	35	29

Rats were given an intravenous pulseof[6-¹⁴C]glucose during rest, at onset of 5-minute sensory stimulation, or at 10 minutes after cessation of stimulation, and brains were frozen in situ 5 minutes after tracer injection; metabolite labeling was assayed in compounds solublized by ethanol or perchloric acid extraction of dorsal cerebral cortex and in the ethanol-insoluble fraction (i.e., glycogen and the sum of the total labeling of the remaining particulate matter [protein, DNA, RNA, and lipid] that remained after enzymatic removal of glycogen). Total ¹⁴C in all labeled compounds was determined in separate portions of the frozen brain powders from each animal. Calculated fluxes [6-¹⁴C]glucose into metabolites (see Materials and Methods) were multiplied by 100. Values are mean ± SD (n = 6 per group). The total labeled metabolites and unindentified labeled metabolites in each animal were calculated by difference (i.e., total tissue ¹⁴C minus that recovered in purified glucose and total ¹⁴C in metabolites minus that in identified compounds, respectively)

P < 0.05 versus rest (ANOVA and Dunnett test).

Lactate and glycogen are products of nonoxidative pathways and together accounted for only a small fraction (i.e., 4% to 8%) of the ¹⁴C recovered in metabolites at any stage of activity (Table 2). The percent increase in labeling of lactate and alanine during stimulation was, however, higher than that of other metabolites. Labeling of brain glycogen was low and did not change during stimulation, but doubled during recovery. Labeling of all pools except glycogen normalized within 15 minutes after cessation of the stimulus.

The largest quantity of ¹⁴C was incorporated into glutamate (i.e., 31% to 38%) at all stages of activity, exceeding that incorporated into alanine, glutamine, GABA, or aspartate. Labeling of glutamate rose by nearly 50% during sensory stimulation, reflecting increased oxidative metabolism; that is, for [6-¹⁴C]glucose-derived label to be trapped in glutamate via the transaminase exchange reaction, the labeled carbon must first pass through the mitochondrial pyruvate and isocitrate dehydrogenase steps, thereby producing NADH, which is oxidized via the respiratory chain. Increased labeling of glutamate by [6-¹⁴C]glucose agrees with previous work showing (1) rapid, preferential incorporation of [¹⁴C]glucose into glutamate (incorporation of labeled glucose into a “large” glutamate compartment has been interpreted as mainly neuronal on the basis of a low [about 0.3 to 0.5; Table 5] glutamine-to-glutamate-specific activity ratio compared with that obtained with acetate or other precursors [>1.0; Balázs and Cremer, 1972; Berl et al., 1975], but glucose is probably also incorporated into the glial glutamate pool); and (2) a lag before glucose label incorporation into glutamine, presumably via glutamate-glutamine cycling between astrocytes and neurons (Sibson et al., 1997). Most of the intermediates in the glycolytic, pentose phosphate shunt, and TCA cycle pathways are present in brain in small quantities compared with the major pools of glucose-derived compounds (Bachelard, 1989; Siesjö, 1978). Because these compounds were not individually purified, labeling of the composite pool of unidentified ethanol-soluble metabolites by [6-¹⁴C]glucose, which was determined by difference, accounted for 40% to 50% of the total ¹⁴C. Labeling of this heterogeneous pool did not change during stimulation, and fell somewhat during recovery (Table 2). To summarize, the fraction of the additional [6-¹⁴C]glucose metabolized during stimulation over and above that in the resting state was 73% for the oxidative pathway (i.e., TCA cycle-derived amino acids) and 25% for the lactate remaining in tissue at sampling time. Any efflux of labeled lactate from the activated tissue (to blood or other brain regions [Cruz et al., 1999; Hawkins et al., 1973]) would, however, alter these proportions.

Levels and specific activities of purified metabolites

Glucose, lactate, and glycogen. The levels of these three unlabeled metabolites were included in the pooled values shown in Table 1 in the accompanying report (Cruz and Dienel, 2002); data from the animals used in the ¹⁴C-labeling studies are included in Table 3 to facilitate direct comparison of their concentrations to their corresponding specific activities, as well as to the levels of amino acids. Glucose levels and normalized specific activities were constant during rest, activation, and recovery (Table 3), indicating that glucose demand was matched by its supply to brain. The lactate-specific activity was also constant with stage of activity even though its level rose almost threefold after 5 minutes of stimulation (Table 3); the increased amount was cleared within 15 minutes after stimulation ceased, in agreement with our earlier observations (Madsen et al., 1999). The net increase in the quantity of unlabeled lactate at the end of the stimulation period (i.e., 1.1 μmol/g) corresponded to only about 12% of the lactate that could have been produced from the glucose consumed during the 5-minute period of activation; that is, the minimum rate of glucose utilization (see Materials and Methods) during sensory stimulation, 0.93 μmol · g⁻¹ · min⁻¹ (Table 2), is equivalent to at least 9.3 μmol lactate produced during the 5-minute period (i.e., 2 moles lactate/mole glucose × 5 minutes).

TABLE 3.

Levels and specific activities of major glucose carbon pools before, during, and after 5 minutes of generalized sensory stimulation

Metabolite	Rest	Sensory stimulation	Recovery
Concentration in dorsal cerebral cortex (μmol/g wet wt.)
Glucose	1.9 ± 0.2	2.0 ± 0.3	2.3 ± 0.2
Lactate	0.6 ± 0.1	1.7 ± 0.4	0.6 ± 0.1
Glycogen	12.3 ± 3.9	9.6 ± 4.3	8.2 ± 0.7
Normalized specific activity 10³ × (μmol plasma glucose/[μmol metabolite · min])
Glucose	88 ± 24	78 ± 16	99 ± 17
Lactate	46 ± 11	46 ± 8	46 ± 8
Glycogen	0.29 ± 0.11	0.27 ± 0.14	0.68 ± 0.23*

Values (mean ± SD, n = 6 per group) are derived from ethanol extract fractions of the same animals as Table 2. Specific activity was calculated as nCi/μmol metabolite, then normalized to the integrated specific activity of plasma glucose in each rat to minimize animal-to-animal variability arising from differences in injected dose, plasma glucose level, and clearance of label from blood.

P < 0.05 compared with rest (ANOVA and Dunnett test).

The mean glycogen level tended to fall between rest and 15 minutes of recovery. Owing to high variability, the lower concentration did not become statistically significantly different from rest until data from two experiments were pooled to increase animal number from 6 to 9–11 per group (Cruz and Dienel, 2002); the magnitude of change, about 4 μmol/g, was similar in the individual and pooled groups. The normalized specific activity of glycogen did not change during stimulation but doubled during recovery (Table 3), reflecting increased label incorporation (Table 2) and a lower concentration (Table 3). The overall apparent rate of glucose utilization normalized well before normal glycogen levels were reestablished, in spite of increased glycogen labeling during recovery (Tables 2 and 3). Thus, return to a resting rate of glucose utilization does not signify normalization of the partitioning of glucose carbon into various pathways.

Shifts in level and labeling of glucose-derived amino acid pools during brain activation. During sensory stimulation the levels of glutamate and alanine rose by small but statistically significant amounts (6% and 100%, respectively), whereas the level of aspartate fell by 18% and those of the neurotransmitter GABA, glutamine, and serine were unchanged (Table 4). The concentrations of all amino acids except alanine normalized within 15 minutes after termination of stimulation. Although the percent increase in the size of the glutamate pool was small, the rise in the amount of carbon (i.e., 0.8 μmol/g × 5 carbon atoms/μmol = 4) was equal to that of the net increase of two pools derived from pyruvate; i.e., lactate (1.1 μmol/g) + alanine (0.2 μmol/g) (total = 1.3 μmol/g × 3 carbon atoms/μmol = 3.9). Loss of carbon from the aspartate pool (i.e., 0.7 μmol/g × 4 = 2.8) during activation was equivalent to 35% of the net gain in carbon in glutamate, alanine, and lactate pools. The specific activities of glutamate, aspartate, and GABA rose 14%, 40%, and 24%, respectively, during activation compared with rest and recovery, whereas those of glutamine, alanine, and serine did not change significantly with activity phase (Table 4). The high magnitude of relative incorporation of ¹⁴C from glucose into the glutamate pool is underscored by the fact that the glutamate pool size is >60 times that of alanine, whereas the specific activities of the two amino acids are similar at 5 minutes after the intravenous pulse of tracer.

TABLE 4.

Levels and specific activities of glucose-derived amino acids before, during, and after 5 minutes of generalized sensory stimulation

Metabolite	Rest	Sensory stimulation	Recovery
Concentration in dorsal cerebral cortex (μmol/g wet wt.)
Glutamine	5.5 ± 1.4	5.8 ± 2.1	5.8 ± 1.2
GABA	1.2 ± 0.1	1.3 ± 0.1	1.2 ± 0.1
Glutamate	12.5 ± 0.6	13.3 ± 0.3*	12.9 ± 0.4
Aspartate	4.0 ± 0.3	3.3 ± 0.3*	4.2 ± 0.3
Alanine	0.2 ± 0.0	0.4 ± 0.0*	0.3 ± 0.0*
Serine	1.2 ± 0.3	0.9 ± 0.4	1.0 ± 0.3
Normalized specific activity (10³ × (μmol plasma glucose/[μmol metabolite · min]))
Glutamine	7 ± 2	8 ± 2	7 ± 1
GABA	25 ± 2	31 ± 4*	24 ± 4
Glutamate	22 ± 1	25 ± 2*	21 ± 3
Aspartate	15 ± 1	21 ± 2*	14 ± 2
Alanine	25 ± 2	27 ± 4	22 ± 3
Serine	1 ± 0.6	1 ± 0.6	1.6 ±0.4

Values (mean ± SD, n = 6 per group) are derived from perchloric acid extracts of powders from the same animals as Table 3. Specific activities were normalized to the integrated specific activity of plasma glucose of each animal.

P < 0.05 compared with rest (ANOVA and Dunnett test).

GABA, γ-aminobutyric acid.

Ratios of specific activities. Compartmentation of metabolism can be revealed by differences in the specific activities of products relative to that of a common precursor. Because only 1 of the 2 moles of the trioses in the glycolytic pathway would be labeled by [6-¹⁴C]glucose, the theoretical maximum specific activity for pyruvate, lactate, and alanine relative to that of glucose is 0.5. The lactate/glucose-specific activity ratio was approximately equal to 0.5, indicating that at 5 minutes after pulse labeling, brain lactate must be derived mainly from blood-borne glucose during all stages of activity (Table 5); we obtained similar results in both hemispheres in rats subjected to unilateral spreading cortical depression (Adachi et al., 1995). Both alanine and lactate are derived from pyruvate, but synthesis of these compounds appears to be compartmentalized because the alanine-glucose-specific activity ratios were about half those of lactate–glucose during rest, activation, and recovery (Table 5), even though the concentration of alanine is only 30% to 50% that of lactate (Tables 3 and 4). A lower specific activity pyruvate pool that serves as the precursor for alanine could be derived from various sources, such as amino acids or glycogen (Hamprecht and Dringen, 1994; Hertz et al., 2000; Sonnewald et al., 1996) or different cell types (see Discussion), and its maintenance would not require much carbon since the levels of pyruvate (0.050–0.1 mmol/L; Siesjö, 1978) and alanine (0.2–0.4 mmol/L) are low compared with glutamate, aspartate, glucose, and glycogen (Tables 3 and 4).

TABLE 5.

Ratios of specific activities before, during, and after brain activation

Specific activity ratio	Rest	Sensory stimulation	Recovery
lactate–glucose	0.55 ± 0.15	0.59 ± 0.03	0.48 ± 0.06
alanine–glucose	0.26 ± 0.03‡	0.36 ± 0.05*†	0.23 ± 0.04‡
glutamine–glutamate	0.31 ± 0.07	0.26 ± 0.14	0.26 ± 0.14
GABA–glutamate	1.12 ± 0.02§	1.22 ± 0.08*§	1.14 ± 0.07§

Values (means ± SD, n = 6/group) were calculated for each animal from values in Tables 3 and 4.

P < 0.05 compared with rest (ANOVA and Dunnett test)

†

P < 0.002

‡

P < 0.001 compared with lactate-glucose ratio (t-test)

P < 0.001 compared with glutamine–glutamate ratio (t-test).

GABA, γ-aminobutyric acid.

The lactate–glucose and alanine–glucose normalized specific activity ratios did not fall during stimulation and recovery compared with rest (Table 5), and this stability in conjunction with a concomitant decrease in glycogen level (Cruz and Dienel, 2002) suggests the possibility of compartmentation of metabolism of blood-borne glucose and glycogen. In cultured astrocytes, glycogen is converted mainly to lactate and exported to the medium (Dringen et al., 1993) and brain glycogen has a very low specific activity compared with glucose, lactate, and alanine (i.e., 70- to 300-fold, Tables 3 and 4). During the initial 5-minute phase of the 20-minute activation-recovery interval, the mean glycogen level decreased by 2.7 μmol/g (i.e., about 5 and 25 times the carbon equivalent gain in lactate and alanine contents, respectively; Tables 3 and 4), and it fell another 1.4 μmol/g during the next 15 minutes; the average net losses of glucosyl units from glycogen during these intervals are calculated to be 0.54 and 0.09 μmol/min, respectively. These values are about 60% and 13%, respectively, of the corresponding estimated minimal rates of metabolism of blood-borne glucose (i.e., 0.93 μmol/min during stimulation and 0.73 μmol/min during recovery; Table 3), and would increase the true rate of energy metabolism substantially above that determined by tracer labeling. Mixing of low-specific-activity products derived from mainly unlabeled glycogen with the high-specific-activity compounds derived from blood glucose should greatly reduce the specific activity of pyruvate and its products, lactate and alanine, particularly during activation, but this did not occur (Table 5). The very low net incorporation of [¹⁴C]glucose into glycogen during the 5-minute labeling period (Table 2) and larger net loss of glucose carbon from glycogen (Table 3; note that the glycogen level at 15 minutes of recovery from stimulation was statistically significantly lower than at rest in the pooled data set [Cruz and Dienel, 2002]) render it less likely that “laston-first-off” metabolic cycling of highly labeled glucose in glycogen during the 5-minute labeling with activation could produce the equivalent high specific activity glycogen-derived lactate compared with blood glucose-derived lactate. These results suggest an intriguing hypothesis, that glycolytic and transamination products of blood-borne glucose are segregated from metabolites of glycogen.

Glutamine and GABA are both synthesized from glutamate in astrocytes and neurons, respectively (Hertz et al., 1999, 2000). The glutamine–glutamate-specific activity ratio is much lower than the GABA–glutamate ratio, reflecting the similar magnitude of labeling of these two compounds by glucose (Table 2) but a fivefold larger pool size for glutamine (Table 4). During brain activation the GABA–glutamate-specific activity ratio rose (i.e., the relative labeling of GABA increased more than that of glutamate), whereas that for glutamine was stable (Table 5).

DISCUSSION

Aerobic glycolysis and increased oxidative metabolism are not mutually exclusive

The biochemical and cellular basis for the phenomenon of aerobic glycolysis has not been elucidated since the initial reports of apparent uncoupling of glucose and oxygen metabolism in working brain (Fox and Raichle, 1986; Fox et al., 1988). The notion that increased brain uptake and utilization of glucose compared with oxygen simply reflects a shift from oxidative to glycolytic metabolism was directly examined in the rat model developed in the NIH laboratory (Madsen et al., 1995b), and the glucose uptake into brain in excess of oxygen was not fully explained by accumulation of lactate or glycogen in brain or of lactate efflux from brain to blood (Madsen et al., 1999). Major findings of the present study are that oxidative metabolism of glucose rises (Table 2) during the same interval of brain activation when glucose is taken up into brain in excess of oxygen (Madsen et al., 1999) and there are small increases and reductions in the sizes of carbohydrate and amino acid pools during activation. Pyruvate compartmentation was evident in vivo, and has been previously detected in cultured neurons and astrocytes (Cruz et al., 2001; Zwingmann et al., 2000, 2001). Taken together, these data indicate that the energetics of working brain are complex and cannot be adequately evaluated on the basis of global measures of fuel uptake, assays of selected metabolic steps, or changes in the brain levels of only a few end-product metabolites such as lactate.

Brief tracer studies can be used to evaluate activity-dependent shifts in glucose utilization by assaying the initial labeling of brain metabolic pools in conscious animals under different experimental conditions without altering tissue metabolite levels. Labeled deoxyglucose has the advantage that metabolic products are trapped and activation can be qualitatively detected in brief experiments and quantitatively assessed in 30- to 45-minute studies; its limited metabolism does not, however, permit analysis of the fate of glucose. Although local metabolic balance assays in short nonsteady state experiments with tracer levels of [¹⁴C]glucose can identify some shifts in its metabolism, quantitative assays are especially difficult to carry out for many reasons. For example, precursor ISA in brain cannot be directly determined in each rat, quantitative product trapping is required to assess metabolic rates, efflux of labeled products cannot be prevented or easily evaluated (Ackermann and Lear, 1989; Adachi et al., 1995; Collins et al., 1987), time-course studies require different experimental groups, and laborintensive purification of many labeled products must be quantitative.

The intravenous pulse of [6-¹⁴C]glucose quickly mixes with blood, enters the brain more slowly due to restrictions imposed by the blood–brain barrier, and labels the various metabolic pools. Brain glucose has a half-life of about 1 minute in resting brain (Savaki et al., 1980), and within about 2 minutes after intravenous pulse labeling with [2-¹⁴C]glucose the specific activity of brain glucose approximates that in arterial plasma (Cremer et al., 1978; Hawkins et al., 1974). In the present study, the ISAs of plasma glucose were the same in each group, but it is possible that differences in the actual timing of exposure of tissue to precursor might influence the relative labeling of pools more proximal to the precursor compared with those more distal. We have not modeled these effects, but, for example, an early pulse peak with rapid clearance of precursor might preferentially label the distal pools compared with tissue labeling with the same quantity of precursor given in a slowly increasing ramp with a later pulse peak to enhance labeling of the most proximal pools. Any such effects in the present study cannot be ruled out because the time courses of labeling of the various metabolites could not be determined in each animal. The similarity of the temporal profiles of plasma glucose-specific activity in all animals throughout most of the labeling period (i.e., the 0.5- to 5-minute interval; Fig. 1) should, however, minimize the differences in precursor pool labeling arising from timing of precursor delivery.

Analyzing the metabolism of labeled glucose mainly relies on label trapping and filling of sequential metabolic pools with labeled material. The rate of equilibration of the specific activity of a given metabolite with that of its precursor influences the time span during which the pools can be used to identify changes in metabolic fluxes. Pool filling with tracer continues until constant specific activity is achieved and changes in metabolite fluxes are no longer tracked. (Changes in pool size during the experimental period will influence the total amount of label trapped.) This limitation is apparent in the present study, that is, specific activity of lactate reached its theoretical maximum, 0.5 that of [6-¹⁴C]glucose, within 5 minutes after introduction of the pulse label (Adachi et al., 1995, Table 5). Thus, the total labeling of lactate (and alanine) is less than that of glutamate (Table 2), even though glucose-derived carbon must flow through the pyruvate (lactate-alanine) pool to reach the much larger glutamate pool where label is more effectively trapped. Furthermore, the metabolites in the glycolytic pathway between glucose and lactate–pyruvate, all of which are present in brain in relatively low concentrations (Bachelard, 1989; Siesjö, 1978), must also be as rapidly labeled and have achieved their maximal specific activities within the 5-minute interval; this may also be true for TCA cycle intermediates, and could account for the constancy of the label in unidentified metabolites with stage of activity (Table 2).

Overall glucose utilization in the dorsal cerebral cortex increased by at least 25% during stimulation (Table 2), a value smaller than the 44% rise calculated from arteriovenous differences and blood flow (Madsen et al., 1999). This underestimation arises, in part, from calculations using plasma ISA (see Materials and Methods) and, perhaps, also from some loss of [¹⁴C]metabolites. Adult rat brain has the capacity to rapidly clear lactate to blood (13% to 20% of glucose entering brain; Cruz et al., 1999; Hawkins et al., 1973) or other brain regions, and efflux of glucose-derived lactate would have the greatest impact owing to its high specific activity compared with other metabolites (Tables 3 and 4). Although there was no net efflux of unlabeled lactate to blood during stimulation (Madsen et al., 1999), there could have been some exchange of labeled and unlabeled lactate across the endothelium, which was not assessed. Loss of ¹⁴CO₂ from [6-¹⁴C]glucose via the TCA cycle is expected to be minimized by trapping of ¹⁴C in large unlabeled amino acid pools via exchange reactions (Hawkins and Mans, 1989; Hawkins et al., 1985), although there could be small compartments that are metabolically activated, do not quickly mix with the total metabolite pools, and rapidly release labeled CO₂. However, efflux of labeled lactate to blood greatly exceeds that of labeled CO₂ in normal human brain (Blomqvist et al., 1990), as well as in conscious rats during spreading depression (Cruz et al., 1999). Synaptosomes increase oxidative metabolism of pyruvate during depolarization (Schaffer and Olson, 1980), have tight coupling between glycolytic rate and respiration, and also have a high glycolytic capacity (Kauppinen and Nicholls, 1986a,b); if the synaptosomal amino acid pools do not quickly mix with other cellular amino acid pools, these structures might be a source for loss of labeled CO₂ and, perhaps, a source of transient lactate production during brain activation if glycolytic activity temporarily exceeds the capacity of the malateaspartate shuttle to transfer reducing equivalents into mitochondria and pyruvate dehydrogenase activity. To summarize, the equivalent of about half of the additional labeled glucose consumed during brain activation that remained trapped in brain during the interval when the brain glucose-oxygen uptake ratio was elevated was recovered in products of the oxidative pathway.

Lactate accumulation, pyruvate compartmentation, and metabolite trafficking

The net twofold increase in lactate content during activation represents only a small fraction, 12%, of the minimal amount of glucose consumed, and, on the basis of the lactate/glucose-specific activity ratios, blood-borne glucose is identified as the major source of brain lactate at all stages of activity. The stability of this ratio plus downward shifts in glycogen content during activation led to our working hypothesis that metabolism of blood-borne glucose and astrocytic glycogen stores may be segregated. This is an important but technically difficult area for future studies because it suggests rapid, active turnover and different functional uses for metabolites derived from the incoming and stored energy sources, as exemplified by studies of Bouzier et al. (1998, 2000).

A shuttle system involving lactate and alanine has been proposed to transport nitrogen between neurons and astrocytes, and trafficking of lactate, alanine, glutamine, or other compounds provides an opportunity for rapid egress of [¹⁴C]metabolites from activated tissue via extracellular space and paravascular flow (Rennels et al., 1985) or other mechanisms. Pyruvate-alanine-lactate compartmentation is evident in cultured brain cells (Cruz et al., 2001; Zwingmann et al., 2000, 2001), and the cells that predominately synthesize alanine from lactate appear to differ with brain region; that is, astrocytes in cortical cultures (Sonnewald et al., 1991; Westergaard et al., 1993; Waagepetersen et al., 1998a,b) and neurons in cerebellar cultures (Waagepetersen et al., 2000). If a glucose-derived pyruvate-lactate pool were the major source for alanine in brain of conscious rats in vivo, the specific activities of lactate and alanine relative to that of glucose should be similar, but this was not the case (Table 5). Alanine-specific activity in cerebral cortex was about half that of lactate (even with an increase during activation) and, as observed for lactate, both the incorporation of label from [¹⁴C]glucose into alanine and the alanine level rose during stimulation (Tables 2 –4). These data suggest either two segregated pyruvate pools or extremely slow pyruvate transamination relative to its conversion to lactate. Synthesis of pyruvate from lower specific activity (Table 4) TCA cycle derivatives (Cerdan et al., 1990; Hassel and Sonnewald, 1995; Sonnewald et al., 1993, 1996) might supply an alanine precursor pool, but metabolism of neurotransmitter-derived glutamate is a less-likely source because labeled glutamate is not incorporated into alanine in cultured astrocytes (McKenna et al., 1996). Thus, use of lactate- or alanine-specific activity (which differs by almost twofold; Tables 3 and 4) to estimate the specific activity of pyruvate for calculation of TCA cycle fluxes might not be appropriate, depending on which pool reflects the true pyruvate precursor pool(s) for the TCA cycle(s) of interest.

Potential influence of glycogen turnover on oxygen–glucose uptake ratio

The high brain glycogen levels (Cruz and Dienel, 2002) suggest large energy stores and the capability for “rapid burst” metabolic responses to high ATP demand in working astrocytes. Pulse labeling with [¹⁴C]glucose, which preferentially labels outer tiers of glycogen (Strang and Bachelard, 1971; Watanabe and Passonneau, 1973), indicates that the glycogen is being synthesized in vivo during rest, activation, and recovery even though there is a progressive downward trend in glycogen level during and after brain activation (Table 3; Madsen et al., 1999; Swanson et al., 1992); restoration of glycogen content after the brief stimulus was slow compared with normalization of other compounds and overall glucose utilization (Tables 2 –4). The estimate of stimulus-induced, glycogen-derived glucose utilization (0.54 μmol · g⁻¹ · min⁻¹, calculated from the 2.7 μmol/g mean loss of glycogen after 5 min stimulation) is about half that of blood-borne glucose in the activated dorsal cerebral cortex (0.93 μmol/g/min, Table 2), and is 1.4 times the incremental increase in CMR_glc estimated by arteriovenous differences and blood flow during activation in the same animal model (i.e., 0.38 μmol · g⁻¹ · min⁻¹; Madsen et al., 1999). Thus, astrocytic glycogen could make a substantial contribution to energy metabolism during the initial phase of brain activation, and use of this energy source is not detectable by determination of metabolic rates by arteriovenous differences or tracer labeling assays of glucose metabolism.

After glycogen is consumed, its level must be restored and the nonoxidative biosynthetic activity will contribute to disproportionate uptake of glucose compared with oxygen. For example, the levels of glycogen at 15 minutes after sensory stimulation were about 4 μmol/g below normal (Cruz and Dienel, 2002; Table 3). If CMR_glc were 0.7 μmol · g⁻¹ · min⁻¹ during recovery (Table 2) and 10% of this glucose (equivalent to reducing the oxygen-glucose uptake ratio from 5.9 to 5.5, the magnitude observed in rat or human brain [Madsen et al., 1995a,b; 1999]) were diverted to astrocytes to restore glycogen, then a disproportionate uptake of glucose would occur for an additional 14 to 57 minutes during the recovery period after only 5 minutes of gentle stimulation of the rat. The duration of this “restoration period” would be extended considerably if glycogen levels were further reduced by more intense or more prolonged activation from 8 μmol/g to the generally reported levels of 2 or 3 μmol/g. Thus, the recent history of the subject (activation or stress) and astrocytic metabolic activities are critical, previously unrecognized factors that can influence the global oxygen-glucose uptake ratio.

To summarize, manifestation of aerobic glycolysis during physiologic activation of the normal, conscious rat includes shifts of glucose labeling of oxidative and biosynthetic pathways in neurons and astrocytes with different temporal profiles. The “mystery of the extra glucose” (Hertz and Fillenz, 1999) consumed in excess of oxygen during activation is probably a summation of many changes that vary with the intensity and duration of the stimulus and time after the stimulus ceases; lactate release from activated tissue is an important but confounding factor due to the difficulty in its detection and quantification, as well as its potential for rapid efflux. The increase in oxidative metabolism of glucose during the same interval when there is an excess of glucose taken up relative to oxygen suggests loss of glucose metabolites from the activated tissue, which is reflected by incomplete trapping of labeled products of glucose (Collins et al., 1987). Astrocyte energy stores can be very high, and glycogenolysis begins shortly after stimulus onset and is quite slow to recover. During stimulation, glycogenolysis in astrocytes can substantially raise (by >50%) the initial rate of total glucose utilization over and above that taken up from blood for use by all brain cells (i.e., CMR_glc assayed with labeled glucose metabolism tracers). On the other hand, replenishment of glycogen in astrocytes could (1) depress the oxygen–glucose uptake ratio for at least 3 to 10 times the duration of stimuli that triggered activation, depending on the magnitude of glycogen depletion; and (2) be the sole or major contributor to the manifestation of prolonged “aerobic glycolysis” during that time frame. These findings plus segregated pyruvate-lactate pools suggest the possibility of separate glycolytic pathways to extract energy from blood-borne glucose and stored glucose (glycogen), and raise provocative, challenging questions for future in vivo studies regarding temporally selective enhancement of metabolic activity in specific cellular compartments of working brain, especially the contributions of astrocytes and the functional interactions of astrocytes and neurons.

Footnotes

Acknowledgments:

The authors thank Dr. Louis Sokoloff for his support of this project and Tom Burlin for help with amino acid analysis.

References

Ackermann

Lear

(1989) Glycolysis-induced discordance between glucose metabolic rates measured with radiolabeled fluorodeoxyglucose and glucose. J Cereb Blood Flow Metab 9:774–785

Adachi

Cruz

Sokoloff

Dienel

(1995) Labeling of metabolic pools by [6–14C]glucose during K+-induced stimulation of glucose utilization in rat brain. J Cereb Blood Flow Metab 15 97–110

Attwell

Laughlin

(2001) An energy budget for signaling in the gray matter of the brain. J Cereb Blood Flow Metab 21 1133–1145

Bachelard

(1989) Measurement of carbohydrates and their derivatives in neural tissues. In: Neuromethods, vol 11: Carbohydrates and energy metabolism ( Boulton

Baker

Butterworth

eds), Clifton, NJ: Humana Press, pp 133–154

Balázs

Cremer

(1972) Metabolic compartmentation in the brain. New York: John Wiley

Berl

Clarke

Schneider

(1975) Metabolic compartmentation and Neurotransmission: Relation to brain structure and function. New York: Plenum Press

Blomqvist

Stone-Elander

Halldin

Roland

Widen

Lindqvist

Swahn

C-G

Langstrom

Wiesel

(1990) Positron emission tomographic measurements of cerebral glucose utilization using [1–11C]glucose. J Cereb Blood Flow Metab 10:467–483

Bouzier

Goodwin

de Gannes

Valeins

Voisin

Canioni

Merle

(1998) Compartmentation of lactate and glucose metabolism in C6 glioma cells: A 13C and 1H NMR study. J Biol Chem 273:27162–27169

Bouzier

Thiaudiere

Biran

Rouland

Canioni

Merle

(2000) The metabolism of [3–13C]lactate in the rat brain is specific of a pyruvate carboxylase-deprived compartment. J Neurochem 75:480–486

10.

Cerdan

Künnecke

Seelig

(1990) Cerebral metabolism of [1,2–13C2] acetate as detected by in vivo and in vitro with 13C NMR. J Biol Chem 265:12916–12926

11.

Chih

C-P

Lipton

Roberts

Jr (2001) Do active cerebral neurons really use lactate rather than glucose? Trends Neurosci 24:573–578

12.

Collins

McCandless

Wagman

(1987) Cerebral glucose utilization: Comparison of [14C]deoxyglucose and [6–14C]glucose quantitative autoradiography. J Neurochem 49:1564–1570

13.

Cremer

Sarna

Teal

Cunningham

(1978) Amino acid precursors: Their transport into brain and initial metabolism. In: Amino acids as chemical transmitters (NATO Advanced Study Institute Series: Series A, Life Sci, vol. 16, Fonnum

, ed.) New York: Plenum Press, pp 669–689

14.

Cruz

Villalba

Garcia-Espinosa

Ballesteros

Bogonez

Satrustegui

Cerdan

(2001) Intracellular compartmentation of pyruvate in primary cultures of cortical neurons as detected by 13C NMR spectroscopy with multiple 13C labels. J Neurosci Res 66:771–781

15.

Cruz

Dienel

(2002) High glycogen levels in brains of rats with minimal environmental stimuli: Implications for metabolic contributions of working astrocytes. J Cereb Blood Flow Metab 22:1476–1489

16.

Cruz

Adachi

Dienel

(1999) Metabolite trafficking during K+-induced spreading cortical depression: Rapid efflux of lactate from cerebral cortex. J Cereb Blood Flow Metab 19:380–392

17.

Dienel

Cruz

(1993) Synthesis of deoxyglucose-1-phosphate, deoxyglucose-1,6-phosphate, and other metabolites of deoxyglucose-6-phosphate in rat brain in vivo: Influence of time and tissue glucose level. J Neurochem 60:2217–2231

18.

Dienel

Hertz

(2001) Glucose and lactate metabolism during brain activation. J Neurosci Res 66:824–838

19.

Dienel

Cruz

Mori

Sokoloff

(1990) Acid liability of metabolites of 2-deoxyglucose in rat brain: Implications for estimates of kinetic parameters of deoxyglucose phosphorylation and transport between blood and brain. J Neurochem 54:1140–1448

20.

Dringen

Gebhardt

Hamprecht

(1993) Glycogen in astrocytes: Possible function as lactate supply for neighboring cells. Brain Res 623:208–214

21.

Fellows

Boutelle

Fillenz

(1993) Physiological stimulation increases nonoxidative glucose metabolism in the brain of the freely moving rat. J Neurochem 60:1258–1263

22.

Fox

Raichle

(1986) Focal physiological uncoupling of cerebral blood flow and oxidative metabolism during somatosensory stimulation in human subjects. Proc Natl Acad Sci U S A 83:1140–1144

23.

Fox

Raichle

Mintun

Dence

(1988) Nonoxidative glucose consumption during focal physiologic neural activation. Science 241:462–464

24.

Gjedde

Marrett

(2001) Glycolysis in neurons, not astrocytes, delays oxidative metabolism of human visual cortex during sustained checkerboard stimulation in vivo. J Cereb Blood Flow Metab 21:1384–1392

25.

Gjedde

Marrett

Vafaee

(2002) Oxidative and nonoxidative metabolism of excited neurons and astrocytes. J Cereb Blood Flow Metab 22:1–14

26.

Hamprecht

Dringen

(1994) On the role of glycogen and pyruvate uptake in astroglial-neuronal interaction. In: Pharmacology of cerebral ischemia ( Krieglstein

Oberpichler-Schwenk

, eds), Stuttgart: Medpharm Scientific Publishers, pp 191–202

27.

Hassel

Sonnewald

(1995) Glial formation of pyruvate and lactate from TCA cycle intermediates: Implications for the inactivation of transmitter amino acids? J Neurochem 65:2227–2234

28.

Hawkins

Miller

Nielsen

Veech

(1973) The acute action of ammonia on rat brain metabolism in vivo. Biochem J 134:1001–1008

29.

Hawkins

Miller

Cremer

Veech

(1974) Measurement of the rate of glucose utilization by rat brain in vivo. J Neurochem 23:917–923

30.

Hawkins

Mans

Davis

Vina

Hibbard

(1985) Cerebral glucose use measured with [14C]glucose labeled in the 1, 2, or 6 position. Am J Physiol 248:C170–C176

31.

Hawkins

Mans

(1989) Determination of cerebral glucose use in rats using [14C]glucose. In: Neuromethods, Vol 11: Carbohydrates and energy metabolism ( Boulton

Baker

Butterworth

, eds) Clifton, NJ: Humana Press, pp 195–232

32.

Hertz

Fillenz

(1999) Does the “mystery of the extra glucose” during CNS activation reflect glutamate synthesis? Neurochem Int 34:71–75

33.

Hertz

Dringen

Schousboe

Robinson

(1999) Astrocytes: Glutamate producers for neurons. J Neurosci Res 57:417–428

34.

Hertz

ACH

Kala

Schousboe

(2000) Neuronal-astrocyte and cytosolic-mitochondrial metabolite trafficking during brain activation, hyperammonemia, and energy deprivation. Neurochem Int 37:83–102

35.

Hoge

Atkinson

Gill

Crelier

Marrett

Pike

(1999) Linear coupling between cerebral blood flow and oxygen consumption in activated human cortex. Proc Natl Acad Sci U S A 96:9403–9408

36.

Kauppinen

Nicholls

(1986a) Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia. Eur J Biochem 158:159–165

37.

Kauppinen

Nicholls

(1986b) Failure to maintain glycolysis in anoxic nerve terminals. J Neurochem 47:1864–1869

38.

Kuhr

Korf

(1988) Extracellular lactic acid as an indicator of brain metabolism: Continuous on-line measurement in conscious, freely moving rats with intrastriatal dialysis. J Cereb Blood Flow Metab 8:130–137

39.

Lowry

Passonneau

(1972) A flexible system of enzymatic analysis. New York: Academic Press

40.

Lund

(1985) L-Glutamine and L-glutamate. In: Methods of enzymatic analysis, Vol VIII, 3rd ed ( Bergmeyer

, ed), Deerfield Beach, FL: VCH Publishers, pp 357–363

41.

Madsen

Hasselbalch

SAG

Hangman

Olsen

Bülow

Holm

Wildschøidtz

Paulson

Lassen

(1995a) Persistent resetting of the cerebral oxygen/glucose uptake ratio by brain activation: evidence obtained by the Kety-Schmidt technique. J Cereb Blood Flow Metab 15:485–491

42.

Madsen

Cruz

Dienel

(1995b) Metabolite flux to and from brain and levels of metabolites in brain during rest, sensory stimulation, and recovery. J Cereb Blood Flow Metab 15(suppl 1):S77

43.

Madsen

Cruz

Sokoloff

Dienel

(1999) Cerebral oxygen/glucose ratio is low during sensory stimulation and rises above normal during recovery: Excess glucose consumption during stimulation is not accounted for by lactate efflux from or accumulation in brain tissue. J Cereb Blood Flow Metab 19:393–400

44.

Magistretti

Pellerin

Rothman

Shulman

(1999) Energy on demand. Science 283:496–497

45.

Marrett

Meyer

Kuwabara

Evans

Gjedde

(1995) Differential increases of oxygen metabolism in visual cortex. J Cereb Blood Flow Metab 15(suppl 1):S80

46.

McKenna

Sonnewald

Huang

Stevenson

Zielke

(1996) Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes. J Neurochem 66:386–393

47.

Pontén

Ratcheson

Salford

Siesjö

(1973) Optimal freezing conditions for cerebral metabolites in rats. J Neurochem 21:1127–1138

48.

Rennels

Gregory

Blaumanis

Fujimoto

Grady

(1985) Evidence for a “paravascular” fluid circulation in the mammalian central nervous system, provided by the rapid distribution of tracer protein throughout the brain from the subarachnoid space. Brain Res 326:47–63

49.

Savaki

Davidson

Smith

Sokoloff

(1980) Measurement of free glucose turnover in brain. J Neurochem 35:495–502

50.

Schaffer

Olson

(1980) The regulation of pyruvate oxidation during membrane depolarization of rat brain synaptosomes. Biochem J 192:741–751

51.

Sibson

Dhankhar

Mason

Behar

Rothman

Shulman

(1997) In vivo 13C NMR measurements of cerebral glutamine synthesis as evidence for glutamate-glutamine cycling. Proc Nat Acad Sci U S A 94:2699–2704

52.

Siesjö

(1978) Brain energy metabolism, New York: John Wiley and Sons

53.

Sokoloff

Reivich

Kennedy

Des Rosiers

Patlak

Pettigrew

Sakurada

Shinohara

(1977) The [14C]deoxyglucose method for the measurement of local glucose utilization: Theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neurochem 28:897–916

54.

Sokoloff

(1986) Cerebral circulation, energy metabolism, and protein synthesis: General characteristics and principles of measurement. In: Positron emission tomography and autoradiography: Principles and applications for the brain and heart ( Phelps

Mazziotta

Schelbert

, eds), New York: Raven Press, pp 1–71

55.

Sonnewald

Westergaard

Krane

Unsgard

Petersen

Schousboe

(1991) First direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes. Neurosci Lett 128:235–239

56.

Sonnewald

Westergaard

Petersen

Unsgard

Schousboe

(1993) Metabolism of [U-13C]glutamate in astrocytes studied by 13C NMR spectroscopy: Incorporation of more label into lactate than into glutamine demonstrates the importance of the tricarboxylic acid cycle. J Neurochem 61:1179–1182

57.

Sonnewald

Westergaard

Jones

Taylor

Bachelard

Schousboe

(1996) Metabolism of [U-13C5] glutamine in cultured astrocytes studied by NMR spectroscopy: First evidence of astrocytic pyruvate recycling. J Neurochem 67:2566–2572

58.

Strang

RHC

Bachelard

(1971) Extraction, purification and turnover of rat brain glycogen. J Neurochem 18:1067–1076

59.

Swanson

(1992) Physiologic coupling of glial glycogen metabolism to neuronal activity in brain. Can J Physiol Pharmacol 70(suppl):S138–S144

60.

Swanson

Morton

Sagar

Sharp

(1992) Sensory stimulation induces local cerebral glycogenolysis: Demonstration by autoradiography. Neurosci 51:451–461

61.

Vafaee

Meyer

Marrett

Paus

Evans

Gjedde

(1999) Frequency-dependent changes in cerebral metabolic rate of oxygen during activation of human visual cortex. J Cereb Blood Flow Metab 19:272–277

62.

Waagepetersen

Bakken

Larsson

Sonnewald

Schousboe

(1998a) Metabolism of lactate in cultured GABAergic neurons studied by 13C nuclear magnetic resonance spectroscopy. J Cereb Blood Flow Metab 18:109–117

63.

Waagepetersen

Bakken

Larsson

Sonnewald

Schousboe

(1998b) Comparison of lactate and glucose metabolism in cultured neocortical neurons and astrocytes using 13C-NMR spectroscopy. Dev Neurosci 20:310–320

64.

Waagepetersen

Sonnewald

Larsson

Schousboe

(2000) A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons. J Neurochem 75:471–479

65.

Westergaard

Varming

Peng

Sonnewald

Hertz

Schousboe

(1993) Uptake, release, and metabolism of alanine in neurons and astrocytes in primary cultures. J Neurosci Res 35:540–545

66.

Watanabe

Passonneau

(1973) Factors affecting the turnover of cerebral glycogen and limit dextran in vivo. J Neurochem 20:1543–1554

67.

Zilles

Wree

(1995) Cortex: Areal and laminar structures. In: The rat nervous system, 2nd ed ( Paxinos

, ed), San Diego: Academic Press, pp 649–685

68.

Zwingmann

Richter-Landsberg

Brand

Leibfritz

(2000) NMR spectroscopic study on the metabolic fate of [3–13C]alanine in astrocytes, neurons, and cocultures: Implications for glia-neuron interactions in neurotransmitter metabolism. Glia 32:286–303

69.

Zwingmann

Richter-Landsberg

Leibfritz

(2001) 13C isotopomer analysis of glucose and alanine metabolism reveals cytosolic pyruvate compartmentation as part of energy metabolism in astrocytes. Glia 34:200–212

Generalized Sensory Stimulation of Conscious Rats Increases Labeling of Oxidative Pathways of Glucose Metabolism When the Brain Glucose–Oxygen Uptake Ratio Rises

Abstract

Keywords

MATERIALS AND METHODS

Chemicals

Animals

Analytical procedures

RESULTS

Physiologic variables

Metabolic activation during brief generalized sensory stimulation

Labeling of major brain metabolites by [6-14C]glucose during and after brief stimulation

Levels and specific activities of purified metabolites

DISCUSSION

Aerobic glycolysis and increased oxidative metabolism are not mutually exclusive

Lactate accumulation, pyruvate compartmentation, and metabolite trafficking

Potential influence of glycogen turnover on oxygen–glucose uptake ratio

Footnotes

Acknowledgments:

References

Labeling of major brain metabolites by [6-¹⁴C]glucose during and after brief stimulation