Peter J. Baab

Direct measurement of oxidative metabolism in the living brain by microdialysis: a review

This review summarizes microdialysis studies that address the question of which compounds serve as energy sources in the brain. Microdialysis was used to introduce 14C‐labeled glucose, lactate, pyruvate, glutamate, glutamine, and acetate into the interstitial fluid of the brain to observe their metabolism to 14CO2. Although glucose uptake from the systemic system supplies the carbon source for these compounds, compounds synthesized from glucose by the brain are subject to recycling including complete metabolism to CO2. Therefore, the brain utilizes multiple compounds in its domain to provide the energy needed to fulfill its function. The physiological conditions controlling metabolism and the contribution of compartmentation into different brain regions, cell types, and subcellular spaces are still unresolved. The aconitase inhibitor fluorocitrate, with a lower inhibition threshold in glial cells, was used to identify the proportion of lactate and glucose that was oxidized in glial cells versus neurons. The fluorocitrate data suggest that glial and neuronal cells are capable of utilizing both lactate and glucose for energy metabolism.

Elevation of Amino Acids in the Interstitial Space of the Rat Brain following Infusion of Large Neutral Amino and Keto Acids by Microdialysis: Leucine Infusion

A microenvironment similar to that found in maple syrup urine disease was created in the brain of free-moving, awake rats by the infusion of leucine into the brain using microdialysis. To determine the effects on amino acid homeostasis the eluate of the probe was analyzed. Perfusion with leucine elevated the interstitial levels of large neutral amino acids suggesting hetero exchange of large neutral amino acids from neuronal cells into the interstitial space. The data also demonstrated the inter relationship of leucine and glutamine, both of which may be nitrogen sinks in the brain. Elevation of large neutral amino acids in the interstitial space suggests a decreased concentration in neurons which might have an effect on the synthesis of serotonin and catecholamines and suggests a mechanism by which elevated leucine may affect neuronal function in maple syrup urine disease.

Neurochemical changes in murine trisomy 16: delay in cholinergic and catecholaminergic systems.

Abstract: Two strains of Mus musculus musculus, C57BL/6J and CD‐1, and Mus musculus poschiavinus, the tobacco mouse, were used to study the effects of increased gene dosage of mouse chromosome 16 (MMU 16). A developmental delay has been found in the brains of murine trisomy 16 (Ts 16) fetuses. Both the brain weight (in all three strains) and DNA content (in CD‐1) were reduced, while protein content was unchanged in Ts16 compared to normal littermates. The daily increments of weight and protein (except in M. m. poschiavinus) were significantly greater in Ts16. The activities of choline acetyltransferase and acetylcholinesterase and nuscarinic receptor binding were reduced. Their daily increments were also reduced to less than 56% that of littermates in Ts16 brains. The rate limiting enzymes of Catecholaminergic neurons, tyrosine hydroxylase and do‐pamine β‐hydroxylase, and the concentration of catecholamines in the brains of Ts16 animals were lower. The activities of three other Catecholaminergic enzymes, DOPA decarboxylase, catechol O‐methyltransferase, and monoamine oxidase, were generally elevated in Ts16 brain, as were their daily increments. These observations indicate a significant developmental alteration in the maturation of the trisomic brain and suggest future avenues for defining the effect of increased gene dosage of MMU 16 in the CNS.

Effect of fluorocitrate on cerebral oxidation of lactate and glucose in freely moving rats.

Glucose is the primary carbon source to enter the adult brain for catabolic and anabolic reactions. Some studies suggest that astrocytes may metabolize glucose to lactate; the latter serving as a preferential substrate for neurons, especially during neuronal activation. The current study utilizes the aconitase inhibitor fluorocitrate to differentially inhibit oxidative metabolism in glial cells in vivo. Oxidative metabolism of 14C‐lactate and14C‐glucose was monitored in vivo using microdialysis and quantitating 14CO2 in the microdialysis eluate following pulse labeling of the interstitial glucose or lactate pool. After establishing a baseline oxidation rate, fluorocitrate was added to the perfusate. Neither lactate nor glucose oxidation was affected by 5 μmol/L fluorocitrate. However, 20 and 100 μmol/L fluorocitrate reduced lactate oxidation by 55 ± 20% and 68 ± 12%, respectively (p < 0.05 for both). Twenty and 100 μmol/L fluorocitrate reduced 14C‐glucose oxidation by 50 ± 14% (p < 0.05) and 24 ± 19% (ns), respectively. Addition of non‐radioactive lactate to 14C‐glucose plus fluorocitrate decreased 14C‐glucose oxidation by an additional 29% and 38%, respectively. These results indicate that astrocytes oxidize about 50% of the interstitial lactate and about 35% of the glucose. By subtraction, neurons metabolize a maximum of 50% of the interstitial lactate and 65% of the interstitial glucose.

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free‐moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min‐fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C‐amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U‐14C]glutamate and [U‐14C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.

Effect of α-Ketoisocaproate and Leucine on the in Vivo Oxidation of Glutamate and Glutamine in the Rat Brain

Leucine and α-ketoisocaproate (α-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects of elevated leucine or α-KIC on the oxidation of L-[U-14C]glutamate and L-[U-14C]glutamine in the brain were determined in the non-anesthetized rat. 14CO2 generated by the metabolic oxidation of [l4C]glutamate and [14C]glutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and α-KIC exhibited differential effects on 14CO2 generation from radioactive glutamate or glutamine. Infusion of 0.5 mM α-KIC increased [l4C]glutamate oxidation approximately 2-fold; higher concentrations of α-KIC did not further stimulate [14C]glutamate oxidation. The enhanced oxidation of [14C]glutamate may be attributed to the function of α-KIC as a nitrogen acceptor from [14C]glutamate yielding [14C]α-ketoglutarate, an intermediate of the tricarboxylic acid cycle. [14C-]glutamine oxidation was not stimulated as much as [14C-]glutamate oxidation and only increased at 10 mM α-KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either [14C]glutamate or [14C]glutamine. In maple syrup urine disease elevated α-KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.

In vivo glutamine hydrolysis in the formation of extracellular glutamate in the injured rat brain

Hydrolysis of extracellular glutamine as a potential source of increased extracellular glutamate in the quinolinic acid (QUIN)‐injured brain of the unanesthetized, free‐moving rat was examined by microdialysis and HPLC analysis. Injury was initiated by injection of 100 nmoles of QUIN into the hippocampus. Immediately postinjury or 24 hr postinjury, the injection site was perfused with artificial cerebrospinal fluid + 14C‐glutamine to measure its conversion to 14C‐glutamate. L‐trans‐pyrrolidine‐2,4‐dicarboxylate (L‐PDC), a glutamate uptake inhibitor, was added to the perfusate to enhance the detection of extracellular 14C‐glutamate. QUIN injury was followed by an immediate increase in extracellular glutamate that persisted 24 hr later. When 14C‐glutamine was added to the perfusate, a significant amount of 14C‐glutamate was recovered, and it was greater following QUIN injury than in control animals (P < 0.001). Up to 32% of the extracellular 14C‐glutamine was converted to 14C‐glutamate following QUIN injury. Considering the high concentration of glutamine normally present in the extracellular fluid, glutamine hydrolysis is a potential and important source for the increase in extracellular glutamate after neuronal injury in vivo. J. Neurosci. Res. 60:632–641, 2000

Oxidation of 14C-labeled compounds perfused by microdialysis in the brains of free-moving rats

The oxidative capacity of the brain for alternate substrates, glucose, lactate, pyruvate, acetate, glutamate, and glutamine was determined by using microdialysis to infuse 14C‐labeled compounds into the interstitial fluid of adult rat brain and by collecting the brain‐generated 14CO2 from the dialysis eluate. All compounds were readily oxidized. The recovery of 14CO2 was enhanced for those compounds metabolically close to entry into the TCA cycle or known to have a low interstitial concentration. Two compounds, pyruvate and lactate, demonstrated reciprocal competition when added as nonradioactive competitors. Oxidation of two amino acids, 14C‐glutamate and 14C‐glutamine, was stimulated by the addition of nonradioactive acetate and pyruvate. α‐Cyano‐4‐hydroxycinnamate decreased 14C‐lactate and 14C‐pyruvate oxidation, consistent with the transport of both compounds via a monocarboxylate transporter. The results of this in vivo study support the results of previous in vitro studies that showed that a wide range of compounds formed from glucose in the brain are also oxidized in the brain for energy production.

Hormonal modulation of amino acid neurotransmitter metabolism in the arcuate nucleus of the adult female rat: a novel action of estradiol.

Morphological plasticity in response to estradiol is a hallmark of astrocytes in the arcuate nucleus. The functional consequences of these morphological changes have remained relatively unexplored. Here we report that in the arcuate nucleus estradiol significantly increased the protein levels of the two enzymes in the glutamate-glutamine cycle, glutamine synthetase and glutaminase. We further demonstrate that these estradiol-mediated changes in the enzyme protein levels may underlie functional changes in neurotransmitter availability as: 1) total glutamate concentration in the arcuate nucleus was significantly increased and 2) microdialysis revealed a significant increase in extracellular glutamate levels after a synaptic challenge in the presence of estradiol. These data implicate the glutamate-glutamine cycle in the generation and/or maintenance of glutamate and suggest that the difference in extracellular glutamate between estradiol- and oil-treated animals may be related to an increased efficiency of the cycle enzymes. In vivo enzyme activity assays revealed that the estradiol mediated increase in glutamate-glutamine cycle enzymes in the arcuate nucleus led to an increase in gamma-aminobutyric acid and is likely not related to the increase in extracellular glutamate. Thus, we have observed two-independent effects of estradiol on amino acid neurotransmission in the arcuate nucleus. These data suggest a possible functional consequence of the well-established changes in glial morphology that occur in the arcuate nucleus in the presence of estradiol and suggest the importance of neuronal-glial cooperation in the regulation of hypothalamic functions such as food intake and body weight.

Ketone bodies, glucose and glutamine as lipogenic precursors in human diploid fibroblasts

Incorporation of [14C] from acetoacetate, D(-)- and L(+)-3-hydroxybutyrate, glucose, glutamine, acetate and palmitate in cellular lipids were studied in cultures in human diploid fibroblasts (HDF). The results showed that acetoacetate was 2–10 times more effective as a lipogenic precursor than was either D- or L-3-hydroxybutyrate. Its extent of incorporation into lipids was 2- to 8-fold more than the other precursors examined under conditions when the overall rates of nonsaponifiable and saponi-fiable lipogenesis as measured by3H2O incorporation were essentially unchanged. Acetoacetate supported both saponifiable and nonsaponifiable lipid syntheses with half-saturation values (Km app.) of 185 μM and 30 μM, respectively. Glucose stimulated acetoacetate incorporation into lipids whereas, conversely, acetoacetate inhibited [14C] glucose incorporation into lipids. The presence of low density lipoproteins (LDL) cholesterol (@40 μg cholesterol/mL) inhibited the incorporation of [14C] from acetoacetate 56% into nonsaponifiable lipids; the inhibition was consistently higher (75%) when [14C] glucose or glutamine were the precrusors. The loss of 3-hydroxy-3-methyl-glutaryl CoA (HMG CoA) reductase activity upon addition of LDL-cholesterol was greater than the suppression of [14C] incorporation from acetoacetate or glucose into nonsaponifiable lipids. In the presence of glucose, [14C] acetoacetate was incorporated into 3-βOH sterols (digitonin precipitable). 7.7±1.1 times more effectively than was [14C] glucose. The results suggest that HDF would be a suitable model to investigate the effects of various precrusors of HMG CoA on the rate of cholesterol biosynthesis.