Elaine E. Kaufman

Dichloroacetate effects on glucose and lactate oxidation by neurons and astroglia in vitro and on glucose utilization by brain in vivo

Neuronal cultures in vitro readily oxidized both D-[14C]glucose and l-[14C]lactate to 14CO2, whereas astroglial cultures oxidized both substrates sparingly and metabolized glucose predominantly to lactate and released it into the medium. [14C]Glucose oxidation to 14CO2 varied inversely with unlabeled lactate concentration in the medium, particularly in neurons, and increased progressively with decreasing lactate concentration. Adding unlabeled glucose to the medium inhibited [14C]lactate oxidation to 14CO2 only in astroglia but not in neurons, indicating a kinetic preference in neurons for oxidation of extracellular lactate over intracellular pyruvate/lactate produced by glycolysis. Protein kinase-catalyzed phosphorylation inactivates pyruvate dehydrogenase (PDH), which regulates pyruvate entry into the tricarboxylic acid cycle. Dichloroacetate inhibits this kinase, thus enhancing PDH activity. In vitro dichloroacetate stimulated glucose and lactate oxidation to CO2 and reduced lactate release mainly in astroglia, indicating that limitations in glucose and lactate oxidation by astroglia may be due to a greater balance of PDH toward the inactive form. To assess the significance of astroglial export of lactate to neurons in vivo, we attempted to diminish this traffic in rats by administering dichloroacetate (50 mg/kg) intravenously to stimulate astroglial lactate oxidation and then examined the effects on baseline and functionally activated local cerebral glucose utilization (lCMRglc). Dichloroacetate raised baseline lCMRglc throughout the brain and decreased the percent increases in lCMRglc evoked by functional activation. These studies provide evidence in support of the compartmentalization of glucose metabolism between astroglia and neurons but indicate that the compartmentalization may be neither complete nor entirely obligatory.

The extraneural distribution of gamma-hydroxybutyrate.

Abstract— γ‐Hydroxybutyrate has been found to be widely distributed in both neural and extraneural tissues in the rat. The kidney and brown fat have more than 10 times higher concentrations of y‐hydroxybutyrate than does the brain. This observation suggests that γ‐hydroxybutyrate may participate in the metabolism of many organs, and that GABA may not be the precursor in extraneural tissues.

An overview of γ-hydroxybutyrate catabolism: The role of the cytosolic NADP+-dependent oxidoreductase EC 1.1.1.19 and of a mitochondrial hydroxyacid-oxoacid transhydrogenase in the initial, rate-limiting step in this pathway

SummaryTwo enzymes have been found which catalyze the initial step in the catabolism of GHB. The oxidation of GHB to SSA, catalyzed by both of these enzymes, is coupled to the reduction of an oxoacid. In the case of the mitochondrial transhydrogenase, the coupling is obligatory. Although coupling is not obligatory for the GHB dehydrogenase, the stimulation provided by the coupled reaction, and the nature of the kinetics of the uncoupled reaction, may not only allow the reaction to proceed, but may provide a means of regulating the rate of the reaction under in vivo conditions. Since the oxidation of GHB to SSA is the rate limiting step in the overall catabolic pathway (the rate of conversion of GHB to SSA proceeds at approximately one one thousandth of the rate at which SSA is oxidized to succinate by SSA dehydrogenase (30)), factors which regulate the rate of either or both of these enzymes will, in turn, influence tissue levels of endogenous GHB as well as the duration and magnitude of the physiological effect of a dose of GHB.

PURIFICATION AND CHARACTERIZATION OF AN NADP + -LINKED ALCOHOL OXIDO-REDUCTASE WHICH CATALYZES THE INTERCONVERSION OF γ-HYDROXYBUTYRATE and SUCCINIC SEMIALDEHYDE1

Abstract— An NADP+ ‐linked enzyme, capable of interconverting γ‐hydroxybutyrate and succinic semialdehyde, has been isolated from hamster liver and brain. The enzyme which was isolated from liver has been purified 300‐fold and exhibits a single band by polyacrylamide gel electrophoresis. The molecular weight of the enzyme is ‐ 31,000 as estimated from gel filtration and 38,000 as estimated from sodium dodccyl sulfate gel electrophoresis. The enzyme is inhibited by amobarbital, diphenylhy‐dantoin, 2‐propylvalerate, and diethyldithiocarbamate, but not by pyrazole. The enzymes from brain and liver appear to be very similar with regard to their molecular weights and their kinetic constants for γ‐hydroxybutyrate and succinic semialdehyde.

2-Deoxyglucose Incorporation into Rat Brain Glycogen During Measurement of Local Cerebral Glucose Utilization by the 2-Deoxyglucose Method

Abstract: The incorporation of 14C into glycogen in rat brain has been measured under the same conditions that exist during the measurement of local cerebral glucose utilization by the autoradiographic 2‐[14C]deoxyglucose method. The results demonstrate that approximately 2% of the total 14C in brain 45 min after the pulse of 2‐[14C]deoxyglucose is contained in the glycogen portion, and, in fact, incorporated into α‐1‐4 and α‐1‐6 deoxyglucosyl linkages. When the brain is removed by dissection, as is routinely done in the course of the procedure of the 2‐[14C]deoxyglucose method to preserve the structure of the brain for autoradiography, the portion of total brain 14C contained in glycogen falls to less than 1%, presumably because of postmortem glycogenolysis which restores much of the label to deoxyglucose‐phosphates. In any case, the incorporation of the 14C into glycogen is of no consequence to the validity of the autoradiographic deoxyglucose method, not because of its small magnitude, but because 2‐[14C]deoxyglucose is incorporated into glycogen via [14C]deoxyglucose‐6‐phosphate, and the label in glycogen represents, therefore, an additional “trapped” product of deoxyglucose phosphorylation by hexokinase. With the autoradiographic 2‐[14C]deoxyglucose method, in which only total 14C concentration in the brain tissue is measured by quantitative autoradiography, it is essential that all the labeled products derived directly or indirectly from [14C]deoxyglucose phosphorylation by hexokinase be retained in the tissue; their chemical identity is of no significance.

Pyretic action of low doses of γ-hydroxybutyrate in rats

Abstract γ-Hydroxybutyrate (GHB) has been found to have a biphasic effect on body temperature with increased body temperature after low doses (5–10 mg/kg) and decreased body temperature after high doses (300–500 mg/kg). Brain levels of GHB between 30 and 60 min post-injection of GHB were not altered by the low doses (5–10 mg/kg), although a dose of 200 mg/kg produced a large increase in the brain concentration.

Evidence for the Participation of a Cytosolic NADP+‐Dependent Oxidoreductase in the Catabolism of γ‐Hydroxybutyrate In Vivo

Abstract: The concentration of γ‐hydroxybutyrate (GHB) in brain, kidney, and muscle as well as the clearance of [1‐14C]GHB in plasma have been found to be altered by the administration of a number of metabolic intermediates and drugs that inhibit the NADP+‐dependent oxidoreductase, “GHB dehydrogenase,” an enzyme that catalyzes the oxidation of GHB to succinic semialdehyde. Administration of valproate, salicylate, and phenylacetate, all inhibitors of GHB dehydrogenase, significantly increased the concentration of GHB in brain; salicylate increased GHB concentration in kidney, and α‐ketoisocaproate increased GHB levels in kidney and muscle. The half‐life of [1‐14C]GHB in plasma was decreased by D‐glucuronate, a compound that stimulates the oxidation of GHB by this enzyme and was increased by a competitive substrate of the enzyme, L‐gulonate. The results of these experiments suggest a role for GHB dehydrogenase in the regulation of tissue levels of endogenous GHB.

Regulation and Properties of an NADP+ Oxidoreductase Which Functions as a γ‐Hydroxybutyrate Dehydrogenase

A number of naturally occurring biological intermediates have been found to inhibit competitively the activity of a highly purified NADP+‐dependent oxidore‐ductase which catalyzes the simultaneous oxidation of γ‐hydroxybutyrate to succinic semialdehyde, and the reduction of D‐glucuronate to L‐gulonate. Of the inhibitors studied, those with the lowest Ki are the α‐keto analogues of the branched chain or aromatic amino acids. The Vmax and Km for this enzyme are affected by pH; consequently, changes in substrate concentration can markedly alter the pH optimum. The enzyme has been found to be inhibited by reducing agents such as dithiothreitol and mercapto‐ethanol, protected against this inhibition by oxidizing agents such as oxidized glutathione or H2O2, and finally, protected against heat inactivation by the presence of either NADP+ or NADPH.

Oxidation of γ‐Hydroxybutyrate to Succinic Semialdehyde by a Mitochondrial Pyridine Nucleotide‐Independent Enzyme

Abstract: An antibody that inhibits over 95% of the cytosolic NADP+‐dependent 7‐hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1–14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1–14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADPT and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+‐depen‐dent cytosolic oxidoreductase which catalyzes this reaction.

Evidence for Cooperativity between Neurons and Astroglia in the Regulation of CO2 Fixation in vitro

Increases in the extracellular potassium concentration which correspond to the increased potassium concentration seen with both normal and abnormal neuronal stimulation produce marked increases in the rate of CO2 fixation in astroglial cells in primary culture. This increase in CO2 fixation is seen only in astroglial cells; the low rate of CO2 fixation seen in the neurons did not respond to the increased potassium concentration. Cultures of astroglia and mixed astroglia-neurons were labeled with NaH[14C]O3 for 20 h to obtain material for preliminary identification of the labeled products of CO2 fixation in the extracellular and intracellular compartments. While both cultures had similar intracellular levels of labeled products (mainly amino acids), astroglial cultures released 4-fold more labeled products into the extracellular fluid. While labeled glutamine is a prominent product of astroglia, the bulk of the released labeled products are nonamino acids. These are presumably the products available to be recycled back to surrounding neurons (which cannot fix CO2) to replenish intermediates of the TCA cycle lost due to release of the neurotransmitters glutamate, aspartate and GABA.