Development of mitochondrial energy metabolism in rat brain

1. The development of pyruvate dehydrogenase and citrate synthase activity in rat brain mitochondria was studied. Whereas the citrate synthase activity starts to increase at about 8 days after birth, that of pyruvate dehydrogenase starts to increase at about 15 days. Measurements of the active proportion of pyruvate dehydrogenase during development were also made. 2. The ability of rat brain mitochondria to oxidize pyruvate follows a similar developmental pattern to that of the pyruvate dehydrogenase. However, the ability to oxidize 3-hydroxybutyrate shows a different developmental pattern (maximal at 20 days and declining by half in the adult), which is compatible with the developmental pattern of the ketone-body-utilizing enzymes. 3. The developmental pattern of both the soluble and the mitochondrially bound hexokinase of rat brain was studied. The total brain hexokinase activity increases markedly at about 15 days, which is mainly due to an increase in activity of the mitochondrially bound form, and reaches the adult situation (approx. 70% being mitochondrial) at about 30 days after birth. 4. The release of the mitochondrially bound hexokinase under different conditions by glucose 6-phosphate was studied. There was insignificant release of the bound hexokinase in media containing high KCl concentrations by glucose 6-phosphate, but in sucrose media half-maximal release of hexokinase was achieved by 70μm-glucose 6-phosphate 5. The production of glucose 6-phosphate by brain mitochondria in the presence of Mg²⁺+glucose was demonstrated, together with the inhibition of this by atractyloside. 6. The results are discussed with respect to the possible biological significance of the similar developmental patterns of pyruvate dehydrogenase and the mitochondrially bound kinases, particularly hexokinase, in the brain. It is suggested that this association may be a mechanism for maintaining an efficient and active aerobic glycolysis which is necessary for full neural expression.     

Energy-Metabolising Enzymes in Brain Regions of Adult and Aging Rats

Abstract: The regional enzyme activities of glucose metabolism in the rat brain were investigated. Hexokinase (EC 2.7.1.1) and pyruvate dehydrogenase (EC 1.2.4.1), key enzymes for glucose metabolism, showed no changes in activity in all the regions studied of the aging brain as compared with the adult brain. However, the activity of d -3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) is low throughout the adult brain and, in contrast with hexokinase and pyruvate dehydrogenase, its activity decreases significantly during aging. Other enzymes that showed significant decreases during aging are aldolase (EC 4.1.2.13), lactate dehydrogenase (EC 1.1.1.27), citrate synthase (EC 4.1.3.7), and NAD⁺-linked isocitrate dehydrogenase (EC 1.1.1.41). The catabolic enzyme in cholinergic metabolism, acetylcholinesterase (EC 3.1.1.7), selected as an example of a non-energy-metabolising enzyme, also showed significant decreases in all regions of the brain in aging, although its highest activity remained in the striatum. These results are discussed with respect to the energy metabolism in various brain regions and their status with aging.     

Activities of enzymes of ketone-body utilization in brain and other tissues of suckling rats

1. The activities of 3-hydroxybutyrate dehydrogenase and 3-oxo acid CoA-transferase in rat brain at birth were found to be about two-thirds of those of adult rat brain, expressed per g wet wt. The activities rose throughout the suckling period and at the time of weaning reached values about three times higher than those for adult brain. Later they gradually declined. 2. At birth the activity of acetoacetyl-CoA thiolase in rat brain was about 60% higher than in the adult. During the suckling period there was no significant change in activity. 3. In rat kidney the activities of the three enzymes at birth were less than one-third of those at maturity. They gradually rose and after 5 weeks approached the adult value. Similar results were obtained with rat heart. 4. The activity of glutamate dehydrogenase (a mitochondrial enzyme like 3-hydroxybutyrate dehydrogenase and 3-oxo acid CoA-transferase) also rose in brain and kidney during the suckling period, but at no stage did it exceed the adult value. 5. Throughout the suckling period the total ketone-body concentration in the blood was about six times higher than in adult fed rats, and the concentration of free fatty acids in the blood was three to four times higher. 6. It is concluded that the rate of ketone-body utilization in brains of suckling rats is determined by both the greater amounts of the key enzymes in the tissue and the high concentrations of ketone bodies in the blood. In addition, the low activities of the relevant enzymes in kidney and heart of suckling rats may make available more ketone bodies for the brain.     

Regional enzyme development in rat brain. Enzymes of energy metabolism.

The development of several key enzymes of pyruvate and 3-hydroxybutyrate metabolism and of the tricarboxylic acid cycle was studied in six regions (cerebellum, medulla oblongata and pons, hypothalamus, striatum, mid-brain and cortex) of the neonatal, suckling and adult rat brain (2 days before birth to 60 days after birth). The enzymes whose developmental patterns were studied were: pyruvate dehydrogenase (EC 1.2.4.1), 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and fumarase (EC 4.2.1.2). Citrate synthase, isocitrate dehydrogenase and pyruvate dehydrogenase develop as a cluster in each region, although the pyruvate dehydrogenase appears to lag slightly behind the others. As with the glycolytic-enzyme cluster [Leong & Clark (1984) Biochem. J. 218, 131-138] the timing of the development of the activity of this group of enzymes varies from region to region; 50% of the adult activity developed first in the medulla oblongata, followed by the hypothalamus, striatum and mid-brain, and then in the cortex and cerebellum respectively. The 3-hydroxybutyrate dehydrogenase activity also develops earlier in the medulla oblongata than in the other regions. The results are discussed with respect to the neurophylogenetic development of the brain regions studied and the importance of the development of the enzymes of aerobic glycolysis in relationship to the development of neurological maturation.     

Effects of Acupuncture on Glycometabolic Enzymes in Multi-infarct Dementia Rats

Acupuncture has exhibited therapeutic effects on vascular dementia in our previous research. The mechanism of its anti-dementia effects involves energy metabolism. For brain cells, glucose metabolism is almost the only source of energy, and glucose metabolism disorders are early signs of dementia. In addition, glucose metabolism associates closely with glycometabolic enzymes, thereby maintains normal energy supply in brains and neurological and mental activities. In order to investigate its anti-dementia mechanism, we studied the effects of acupuncture on behavior of multi-infarct dementia (MID) rats and glycometabolic enzymes protein expression and activities in their brains. Results showed acupuncture improved the cognitive disorder, and increased the activities of hexokinase, pyruvate kinase, and glucose 6 phosphate dehydrogenase. Accordingly, it suggests that the anti-dementia effects of acupuncture may be mediated by up regulation of hexokinase, pyruvate kinase, and glucose 6 phosphate dehydrogenase activities, influencing energy metabolic system and thus overcoming the dysfunctional cognition of MID.     

Enzyme profiles in two wing polymorphic soapberry bug populations (Jadera haematoloma: Rhopalidae)

Wing-polymorphic insects are excellent models for asking questions about trade-offs in physiology and life-history because of the existence of multiple morphs exhibiting numerous strategies living in one environment. We measured activities of select key enzymes in oxidative metabolism (citrate synthase), glycolysis (hexokinase, pyruvate kinase) and fatty acid oxidation (?-hydroxyacyl CoA dehydrogenase, HOAD) in flying and non-flying long-winged bugs from two populations (ancestral and derived) of the wing-polymorphic soapberry bug (Jadera haematoloma). In the ancestral population, flyers had significantly greater activities of citrate synthase, hexokinase, pyruvate kinase, and HOAD than non-flyers. In contrast, in the derived population, flyers and non-flyers showed no significant differences in the activities of any of the enzymes tested. There were no significant differences in activities of citrate synthase as a function of adult age (3-20 d) for either population. Short-winged bugs in the derived population had significantly lower citrate synthase activities than either of the two derived, long-winged morphs, however, they were analogous to the ancestral long-winged non-flyer. These results suggest an evolution of alternative flight strategies between the two populations.     

Comparison of the Development of the Fatty Acid Content and Composition of the Brain of a Precocial Species (Guinea Pig) and a Non-Precocial Species (Rat)

Abstract: The fatty acid compositions of the brains of a precocial (guinea pig) and a non-precocial (rat) species have been studied as a function of development. In the rat brain the total fatty acid content expressed as mg g wet wt.^-1 increased more than fourfold during the period from 5 days after birth to adulthood. However, the percentage composition of this total fatty acid content when expressed per individual fatty acid remained fairly constant, with the exception of nervonic acid (C24:l) which also increased fourfold on a percentage basis. In the guinea pig brain, however, at birth the total fatty acid content, expressed as mg g wet wt.^-1, is the same as that of the adult, the concentration doubling during the period from 25 days before birth until birth. Again, if the fatty acid content is analysed and expressed on a percentage basis, the relative concentrations of the individual fatty acids remain fairly constant over the period from 25 days before birth until adulthood, with the exception of nervonic (C24:l) acid which increases about fivefold from 25 days before birth to birth and only marginally (20%) from birth to adulthood. These results are discussed in relationship to the onset of neurological competence in the two species. It is concluded that the increase in fatty acid content (both total and individually) of the brains of these species as a function of the foetal and neonatal development follows a pattern which is similar to the pattern of development of certain key enzymes of energy metabolism and of neurological competence.     

The Activities of Some Energy-Metabolising Enzymes in Nonsynaptic (Free) and Synaptic Mitochondria Derived from Selected Brain Regions

Abstract: The enzyme complement of two different mitochondrial preparations from adult rat brain has been studied. One population of mitochondria (synaptic) is prepared by the lysis of synaptosomes, the other (nonsynaptic or free) by separation from homogenates. These populations have been prepared from distinct regions of the brain: cortex, striatum, and pons and medulla oblongata. The following enzymes have been measured: pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), fumarase (EC 4.2.1.2), NAD-linked malate dehydrogenase (EC 1.1.1.37), D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and mitochondrially bound hexokinase (EC 2.7.1.1) and creatine kinase (EC 2.7.3.2). The nonsynaptic (free) mitochondria show higher enzyme specific activities in the regions studied than the corresponding values recorded for the synaptic mitochondria. The significance of these observations is discussed in the light of the different metabolic activities of the two populations of mitochondria and the compartmentation of the metabolic activities of the brain.     

Changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of ATP citrate lyase, ;malic' enzyme, glucose 6-phosphate dehydrogenase, pyruvate kinase and fructose 1,6-diphosphatase, and in the ability to incorporate [1-(14)C]acetate into lipid have been measured in the livers of developing rats between late foetal life and maturity. 2. In male rats the activities of those systems directly or indirectly concerned in lipogenesis (acetate incorporation into lipid, ATP citrate lyase and glucose 6-phosphate dehydrogenase) fall after birth and are maintained at a low value until weaning. After weaning these activities rise to a maximum between 30 and 40 days and then decline, reaching adult values at about 60 days. ;Malic' enzyme activity follows a similar course, except that none could be detected in the foetal liver. Pyruvate kinase activity is lower in foetal than in adult livers and rises to slightly higher than the adult value in the post-weaning period. Fructose 1,6-diphosphatase activity rises from a very low foetal value to reach a maximum at about 10 days but falls rapidly after weaning to reach adult values at about 30 days. 3. Weaning rats on to a high-fat diet caused the low activities of acetate incorporation, ATP citrate lyase, glucose 6-phosphate dehydrogenase and pyruvate kinase, characteristic of the suckling period, to persist. ;Malic' enzyme and fructose 1,6-diphosphatase activities were not altered appreciably. 4. No differences could be detected in hepatic enzyme activities between males and females up to 35 days, but after this time female rats gave higher values for acetate incorporation, glucose 6-phosphate dehydrogenase activity and ;malic' enzyme activity. 5. The results are discussed in relation to changes in alimentation and hormonal influences.     

Changes in various links of metabolism and the antioxidant system in the bone marrow erythroid cells of the pig during the neonatal period]

The age dynamics of activities of enzymes which catalysis several stages of metabolism (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, 2,3-diphosphoglycerate mutase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase and cytochrome oxidase) and antioxidant system (superoxide dismutase, glutathione peroxidase and glutathione reductase) was studied in the bone marrow erythroid cells of pig during the 10-day period after birth as well as in the cells of 30 days old animals. It was established that in the neonatal period of development the reorganization of energy metabolism in pig bone marrow erythrokaryocytes took place. It consisted in the intensification of oxidative processes and in a great measure was directed on the activation of 2,3-diphosphoglycerate mutase formation in the nature red cells. During the early period after birth the activation of antioxidant system in erythroid cells of pig bone marrow was observed.     

The Pyruvate Dehydrogenase Complex: Cloning of the Rat Somatic El α Subunit and Its Coordinate Expression with the mRNAs for the E1β E2, and E3 Catalytic Subunits in Developing Rat Brain

Abstract: We report the isolation of cDNA clones encoding the somatic form of the E1α subunit of the pyruvate dehydrogenase complex of rat. The deduced amino acid sequence has 99.5, 98, and 97% identity, respectively, with the orthologous proteins of mouse, human, and pig and 98.5% identity with a rat E1α sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1α subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1α cDNA, in conjunction with cDNA probes to the E1β, E2, and E3 catalytic subunits of rat pyruvate dehydrogenase complex and also to rat citrate synthase, to perform RNase protection assays of developing rat whole brain RNA. The results show a 2.5-fold increase in the concentration of each of the subunit mRNAs and a 1.2-fold increase in citrate synthase mRNA from late foetal stage to 5 days post partum. Thereafter, the mRNA levels remained constant. These data indicate that the respective six-and threefold increases in the amounts of pyruvate dehydrogenase complex and citrate synthase found to occur in rat brain between birth and adulthood are mediated principally by translational and/or posttranslational mechanisms.     

DEVELOPMENT OF MITOCHONDRIAL PYRUVATE METABOLISM IN RAT BRAIN

The activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1-¹⁴C]pyruvate to ¹⁴CO₂ via pyruvate dehydrogenase and the fixation of H¹⁴CO₃^? in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four-fold and 15-fold respectively during 10–35 postnatal days. The rates of the fixation of H¹⁴CO₃^? by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD-malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol-pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three- to 10-fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.     

Inhibition of Brain Glycolysis by Aluminum

Abstract: Aluminum inhibited both the cytosolic and mitochondrial hexokinase activities in rat brain. The IC₅₀ values were between 4 and 9 μ M . Aluminum was effective at mildly acidic (pH 6.8) or slightly alkaline (pH 7.2–7.5) pH, in the presence of a physiological level of magnesium (0.5 m M ). However, saturating (8 m M ) magnesium antagonized the effect of aluminum on both forms of hexokinase activity. Other enzymes examined were considerably less sensitive to inhibition by aluminum. The IC₅₀ of aluminum for phosphofructokinase was 1.8 m M and for lactate dehydrogenase 0.4 m M . At 10–600 μ M , aluminum actually stimulated pyruvate kinase. Aluminum also inhibited lactate production by rat brain extracts: this effect was much more marked with glucose as substrate than with glucose-6-phosphate. However, the IC₅₀ for inhibiting lactate production using glucose as substrate was 280 μ M , higher than that required to inhibit hexokinase. This concentration of aluminum is comparable to those reportedly found in the brains of patients who had died with dialysis dementia and in the brains of some of the patients who had died with Alzheimer disease. Inhibition of carbohydrate utilization may be one of the mechanisms by which aluminum can act as a neurotoxin.     

A comparison of the properties of fructose 1,6-diphosphatase, and the activities of other key enzymes of carbohydrate metabolism, in the livers of embryonic and adult rat, sheep and domestic fowl

1. The activities of some key enzymes of glycolysis and gluconeogenesis were measured in embryonic chick, sheep and rat livers. 2. In chicken the activities of hexokinase, phosphofructokinase and pyruvate kinase are low, but those of glucose 6-phosphatase and fructose diphosphatase are very high; the converse situation exists in the rat (Burch et al. 1963), but in sheep the activities of both phosphofructokinase and fructose diphosphatase are high, and the activities of hexokinase and glucose 6-phosphatase are low. These findings are discussed in relation to carbohydrate metabolism in these embryonic livers. 3. The regulatory properties of fructose diphosphatase from the embryonic livers of these three species were compared with the properties of the enzymes from adult animals. The inhibitions by AMP and fructose diphosphate and the effects of Mg(2+) and pH on the activities of adult and foetal fructose diphosphatase are almost identical. 4. It is concluded that regulatory properties are characteristic of fructose diphosphatase from embryonic and adult tissue, and the importance of this in relation to enzyme development is discussed.     

Differences in the distribution of energy-metabolizing enzymes in rat brain regions

The activities of several enzymes of glucose metabolism (glycolytic and tricarboxylic acid pathways) in four different regions of rat brain (cerebellum, medulla oblongata and pons, cerebral cortex and diencephalon) have been studied. Statistical differences were found in the activities of all the enzymes analyzed in the four regions, except in the case of the soluble hexokinase and pyruvate kinase. The changes observed in citrate synthase activity may account for physiological differences in those areas related to myelin formation and energy metabolism. Cerebral cortex and diencephalon showed enzyme activities which were generally greater than those of the cerebellum and medulla oblongata and pons. The results obtained lend support to the concept of a differential energy metabolism in brain regions.     

Enzyme Activities in Regions of the Hypothalamus

Abstract: The activities of certain key enzymes have been measured in the ventral medial and ventral lateral areas of the hypothalamus, which are implicated in feeding behaviour, and compared with enzyme activities in the cortex and brainstem. The enzymes measured are concerned with glucose metabolism [hexokinase (EC 2.7.1.1) and glucosesphosphate dehydrogenase (EC 1.1.1.49)], ketone body metabolism [3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)], fatty acid utilisation [carnitine palmitoyl transferase (EC 2.3.1.7)], citric acid cycle activity [pyruvate dehydrogenase (EC 1.2.4.2) and citrate synthase (EC 4.1.3.7)] and neurotransmitter synthesis [glutamate dehydrogenase (EC 1.4.1.3)].     

The relationship between mitochondrial heterogeneity and gluconeogenesis in liver mitochondria of the rat, pigeon and guinea pig.

Isolated mitochondria of pigeon and guinea pig liver were subjected to zonal centrifugation. With pigeon liver mitochondria there was uniform distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, aspartate aminotransferase and glutamate dehydrogenase activities. Guinea pig liver mitochondria demonstrated two pyruvate carboxylase and phosphoenolpyruvate carboxykinase maxima but only one maximum with aspartate aminotransferase, malate dehydrogenase and glutamate dehydrogenase. Mitochondrial enzyme levels in rat, pigeon and guinea pig indicate different roles of certain gluconeogenic enzymes in the transport of carbon and hydrogen in and out of mitochondria.     

Inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in developing rat and human brain

1. The effect of the branched-chain amino acids, namely leucine, isoleucine and valine and their corresponding 2-oxo acids on the metabolism of 2-oxoglutarate by developing rat and human brain preparations was investigated. 2. The decarboxylation of 2-oxo[1-(14)C]glutarate to (14)CO(2) by mitochondria from adult rat brain was inhibited by the branched-chain 2-oxo acids whereas the branched-chain amino acids had no inhibitory effect on this process. 3. The activity of 2-oxoglutarate dehydrogenase complex was about 0.2unit/g of brain from 2-day-old rats and increased by about fourfold reaching an adult value by the end of the third postnatal week. 4. The K(m) value for 2-oxoglutarate of the 2-oxoglutarate dehydrogenase complex in rat and human brain was 100 and 83mum respectively. 5. The branched-chain 2-oxo acids competitively inhibited this enzyme from suckling and adult rats brains as well as from foetal and adult human brains, whereas the branched-chain amino acids had no effect on this enzyme. 6. Approximate K(i) values for the branched-chain 2-oxo acids found for this enzyme were in the range found for these 2-oxo acids in plasma from patients with maple-syrup-urine disease. 7. The possible significance of the inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in brains of untreated patients with maple-syrup-urine disease is discussed in relation to the energy metabolism and the biosynthesis of lipids from ketone bodies.     

Regional and Subcellular Distribution of ATP-Citrate Lyase and Other Enzymes of Acetyl-CoA Metabolism in Rat Brain

The activities of ATP-citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 μmol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 μmol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP-citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and B_p. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl-CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP-citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP-citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.     

Differential Lowering by Manganese Treatment of Activities of Glycolytic and Tricarboxylic Acid (TCA) Cycle Enzymes Investigated in Neuroblastoma and Astrocytoma Cells Is Associated with Manganese-Induced Cell Death

Manganese (Mn) is a trace metal required for normal growth and development. Manganese neurotoxicity is rare and usually associated with occupational exposures. However, the cellular and molecular mechanisms underlying Mn toxicity are still elusive. In rats chronically exposed to Mn, their brain regional Mn levels increase in a dose-related manner. Brain Mn preferentially accumulates in mitochondria; this accumulation is further enhanced with Mn treatment in vivo. Exposure of mitochondria to Mn in vitro leads to uncoupling of oxidative phosphorylation. These observations prompted us to investigate the hypothesis that Mn induces alterations in energy metabolism in neural cells by interfering with the activities of various glycolytic and TCA cycle enzymes using human neuroblastoma (SK-N-SH) and astrocytoma (U87) cells. Treatments of SK-N-SH and U87 cells with MnCl2 induced cell death in these cells, in a concentration- and time-dependent manner, as determined by MTT assays. In parallel with the Mn-induced, dose-dependent decrease in cell survival, treatment of these cells with 0.01 to 4.0 mM MnCl2 for 48 h also induced dose-related decreases in their activities of hexokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, and malate dehydrogenase. Hexokinase in SK-N-SH cells was the most affected by Mn treatments, even at the lower range of concentrations. Mn treatment of SK-N-SH cells affected pyruvate kinase and citrate synthase to a lesser extent as compared to its effect on other enzymes investigated. However, citrate synthase and pyruvate kinase in U87 cells were more vulnerable than other enzymes investigated to the effects of Mn. The results suggest the two cell types exhibited differential susceptibility toward the Mn-induced effects. Additionally, the results may have significant implications in flux control because HK is the first and highly regulated enzyme in brain glycolysis. Thus these results are consistent with our hypothesis and may have pathophysiological implications in the mechanisms underlying Mn neurotoxicity.