Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of 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.     

Preparation and properties of mitochondria derived from synaptosomes.

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

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.     

METABOLIC CONTROL MECHANISMS IN MAMMALIAN SYSTEMS

Abstract— The regulation by thyroid hormone of the activities of hexokinase (ATP: D-hexose 6-phosphotransferase; EC 2.7.1.1), phosphofructokinase (ATP: D-fructose-6- phosphate 1-phosphotransferase; EC 2.7.1.11) and pyruvate kinase (ATP: pyruvate phosphotransferase; EC 2.7.1.40) has been investigated in the soluble fractions of the cerebral cortex and cerebellum of the rat. Ontogenetic studies on these key glycolytic enzymes demonstrated marked increases in the normal cerebral cortex between 1 day and 1 yr of age; less pronounced increases in enzyme activities were noted in the normal cerebellum. Neonatal thyroidectomy, induced by treatment of 1-day-old rats with 100 μCi of ¹³¹I, ied to an impairment of body and brain growth and inhibited the developmental increases in hexokinase, phosphofructokinase and pyruvate kinase in both the cerebral cortex and cerebellum. Whereas 50 μCi of ¹³¹I had little or no effect on these brain enzymes, 200 μCi of the radioisotope markedly inhibited (35–65 per cent) the developmental increases of the various enzyme activities investigated. When administration of the radioisotope was delayed for 20 days after birth, little or no inhibition of the development of brain glycolytic enzymes was observed. Whereas treatment of normal neonatal animals with L-tri-iodothyronine had no significant effect on the activities of cerebro-cortical and cerebellar glycolytic enzymes, the hormone increased their activities in young cretinous rats. However, when the initiation of tri-iodothyronine treatment was delayed until neonatally thyroidectomized rats had reached adulthood, this hormone failed to produce any appreciable change in enzyme activity. Our results indicate that thyroid hormone exerts an important regulatory influence on the activities of hexokinase, phosphofructokinase and pyruvate kinase in the developing cerebral cortex and cerebellum.     

Characteristics of thiamine thiazolone diphosphate-induced inhibition of a pyruvate dehydrogenase complex in vitro and in intact mitochondria

Thiamine thiazolone diphosphate (TTPP) was capable of penetrating through the mitochondrial membrane and of inhibiting the pyruvate dehydrogenase complex (PDC) in intact mitochondria. TTPP depressed the activity of mammalian PDC in a mixed manner (Ki = 5.10(-8) M) and yeast pyruvate decarboxylase (Ki = 5.10(-6) M) via a competitive mechanism with respect to thiamine diphosphate. It was shown that decarboxylation of pyruvate in intact and disrupted mitochondria of rat liver and brain is less inhibited by TTPP than the overall activity of PDC determined by the formation of acetyl-CoA. It was assumed that TTPP as a transition state analog participates only in oxidative reactions (but not in simple decarboxylation of pyruvate).     

Influence of intermittent hypoxia and pyrimidinic nucleosides on cerebral enzymatic activities related to energy transduction

The effect of intermittent normobaric hypoxia and of biological pyrimidines (uridine and cytidine) on the specific activities of some enzymes related to cerebral energy metabolism were studied. Measurement were carried out on the following: (a) homogenate in toto; (b) purified mitochondrial fraction; (c) crude synaptosomal fraction, in different areas of rat brain: cerebral cortex, hippocampus, corpus striatum, hypothalamus, cerebellum, and medulla oblongata. Intermittent normobaric hypoxia (12 hours daily for 5 days) caused modifications of the enzyme activities in the homogenate in toto (decrease of hexokinase in cerebellum; increase of pyruvate kinase in medulla oblongata), in the purified mitochondrial fraction (increase of succinate dehydrogenase in the corpus striatum) and in the crude synaptosomal fraction (decrease of cytochrome oxidase activity in cerebral cortex, hippocampus, and cerebellum; decrease of malate dehydrogenase in hippocampus and cerebellum; decrease of lactate dehydrogenase in cerebellum). Daily treatment with cytidine or uridine altered some enzyme activities either affected or unaffected by intermittent hypoxia.     

Acute metabolic effects of taurine on the enzymes metabolizing glutamate and gaba

100 mg of taurine per kg body weight had been administered intraperitoneally and 30 min after the administration the animals were sacrificed. Glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, glutaminase, glutamine synthetase, glutamate decarboxylase and GABA aminotransferase along with the content of glutamate and GABA in cerebral cortex, cerebellum and brain stem were studied and compared with the same obtained in the rats treated with normal saline in place of taurine. The results indicated a significant decrease in the activity of glutamate dehydrogenase in cerebral cortex and cerebellum and a significant increase in brain stem. Glutaminase and glutamine synthetase were found to increase significantly both in cerebral cortex and cerebellum. The activities of glutamate decarboxylase was found to increase in all the three regions along with a significant decrease in GABA aminotransferase while the content of glutamate showed a decrease in all the three brain regions, the content of GABA was observed to increase significantly. The above effects of taurine on the metabolism of glutamate and GABA are discussed in relation to the functional role of GABA and glutamate. The results indicate that taurine administration would result in a state of inhibition in brain.     

Relationship Between Activation State of Pyruvate Dehydrogenase Complex and Rate of Pyruvate Oxidation in Isolated Cerebro-Cortical Mitochondria: Effects of Potassium Ions and Adenine Nucleotides

The relation between the activation (phosphorylation) state of pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) and the rate of pyruvate oxidation has been examined in isolated, metabolically active, and tightly coupled mitochondria from rat cerebral cortex. With pyruvate and malate as the substrates, the activation state of PDHC decreased on addition of ADP, while the rates of oxygen uptake and 14CO2 formation from [1-14C]pyruvate increased. The lack of correlation between the activation state of PDHC and rate of pyruvate oxidation was seen in media containing 5, 30, or 100 mM KCl. Both the activation state of PDHC and pyruvate oxidation increased, however, when KCl was increased from 5 to 100 mM. Although the PDHC is inactivated by an ATP-dependent kinase (EC 2.7.1.99), direct measurement of ATP and ADP failed to show a consistent relationship between the activation state of PDHC and either ATP levels or ATP/ADP ratios. Comparison of the activation state of PDHC in uncoupled or oligomycin-treated mitochondria also failed to correlate PDHC activation state to adenine nucleotides. In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.     

A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver.

The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.     

Effect of thyroid hormones on the activity of pyruvate kinase and lactate dehydrogenase in mature rat brain

The enzymatic activities of two "key" enzymes of the glycolytic pathway, pyruvate kinase and lactic dehydrogenase, were studied in seven areas of the brain in male adult rats in states of pharmacologically induced hyper and hypothyroidism. The brain areas were: anterior cortex, adenohypophysis, hypothalamus, amygdaline nucleus, septum, hippocampus and cerebellum. In T3 treated animals, pyruvate kinase activity showed significant increase in all the areas studied while lactic dehydrogenase activity decreased. In propyl-thiouracil treated animals these enzyme activities showed no significant variations from those in animals of the control group.     

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.     

THE CONTROL OF PYRUVATE DEHYDROGENASE IN ISOLATED BRAIN MITOCHONDRIA

Abstract— The activity and control of the pyruvate dehydrogenase complex in isolated rat brain mitochondria has been studied. The activity of this complex in mitochondria as isolated from normal fed rats was 78 ± 10nmol.min⁻¹ mg mitochondrial protein⁻¹ (n = 18) which represented 70% of the total pyruvate dehydrogenase activity. The pyruvate dehydrogenase in isolated brain mitochondria could be inactivated by incubation in the presence of ATP, oligomycin and NaF. The rate of inactivation was dependent upon the added ATP concentration but inactivation below approx 30% of the total pyruvate dehydrogenase activity could not be achieved. The inactivation of pyruvate dehydrogenase in brain mitochondria was inhibited by pre-incubation with pyruvate. Reactivation of inactivated pyruvate dehydrogenase in rat brain mitochondria was incomplete in the incubation medium unless 10mM-Mg²⁺+ 1 mM-Ca²⁺ were added; NaF, however, prevented any reactivation (Fig. 4). It is concluded that the pyruvate dehydrogenase complex in rat brain mitochondria is controlled in a manner similar to that in other tissues, and that pyruvate protection of pyruvate dehydrogenase activity may be important in maintaining brain energy metabolism.     

Activation of pyruvate dehydrogenase complex (PDC) of rat heart mitochondria by glyburide

The effects of the second generation sulfonylurea, glyburide, on the pyruvate dehydrogenase multienzyme complex (PDC) of rat myocardial tissue were examined using rat ventricular slices and isolated mitochondria. Therapeutic concentrations (10(-7) to 10(-6)M) of glyburide produced a 30% increase in the decarboxylation of [1(-14)C] pyruvate by the PDC of ventricular tissue. Addition of glyburide to intact rat heart mitochondria stimulated activity of the PDC in a time- and concentration-dependent manner. Half-maximal stimulation of the enzyme occurred with 6 X 10(-5)M glyburide and maximal activation of the enzyme was achieved with 1 X 10(-4)M glyburide. At the height of stimulation, PDC activities were 6-fold greater than those observed under control conditions with succinate alone. When mitochondria were disrupted by sonication or freeze-thawing, glyburide produced no stimulation of pyruvate decarboxylation. We conclude that glyburide directly stimulates the decarboxylation of pyruvate by the PDC of the myocardium. Furthermore, the presence of intact mitochondria is necessary for the stimulatory action of glyburide on the PDC.     

Brain α-Ketoglutarate Dehydrogenase Complex: Kinetic Properties, Regional Distribution, and Effects of Inhibitors

The substrate and cofactor requirements and some kinetic properties of the alpha-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for alpha-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex greater than cerebellum greater than or equal to midbrain greater than striatum = hippocampus greater than hypothalamus greater than pons and medulla greater than olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2-2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both beta-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 microM, calcium slightly stimulated (by 10-15%) the brain mitochondrial KGDHC. At concentrations above 100 microM, calcium (IC50 = 1 mM) inhibited both brain mitochondrial and purified heart KGDHC. The present results suggest that some of the kinetic properties of the rat brain mitochondrial KGDHC differ from those of the purified bovine heart and rat heart mitochondrial enzyme complexes. They also suggest that the inhibition of KGDHC by ammonia and the consequent effect on the citric acid cycle fluxes may be of pathophysiological and/or pathogenetic importance in hyperammonemia and in diseases (e.g., hepatic encephalopathy, inborn errors of urea metabolism, Reye's syndrome) where hyperammonemia is a consistent feature. Brain accumulation of calcium occurs in a number of pathological conditions. Therefore, it is possible that such a calcium accumulation may have a deleterious effect on KGDHC activity.     

Regulation of plant pyruvate dehydrogenase complex by phosphorylation

The ATP-dependent inactivation of the pyruvate dehydrogenase complex (PDC) was examined using ruptured mitochondria and partially purified pyruvate dehydrogenase complex isolated from broccoli and cauliflower (Brassica oleracea) bud mitochondria. The ATP-dependent inactivation was temperature- and pH-dependent. [(32)P]ATP experiments show a specific transphosphorylation of the gamma-PO(4) of ATP to the complex. The phosphate attached to the PDC was labile under mild alkaline but not under mild acidic conditions. The inactivated-phosphorylated PDC was not reactivated by 20 mm MgCl(2), dialysis, Sephadex G-25 treatment, apyrase action, or potato acid phosphatase action. However, partially purified bovine heart PDC phosphatase catalyzed the reactivation and dephosphorylation of the isolated plant PDC. The ATP-dependent inactivation-phosphorylation of the PDC was inhibited by pyruvate. It is concluded that the ATP-dependent inactivation-phosphorylation of broccoli and cauliflower mitochondrial PDC is catalyzed by a PDC kinase. It is further concluded that the PDC from broccoli and cauliflower mitochondria is capable of interconversion between an active (dephosphorylated) and an inactive (phosphorylated) form.     

Changes of pyruvate dehydrogenase in brain during alloxan diabetes

Alloxan diabetes causes a decrease in the active form of pyruvate dehydrogenase in rat brain. The effect is severe in the cerebellum and brain stem compared to cerebral hemispheres. The changes observed in the total form are not as significant as those found in the active (dephosphorylated) form. The effects are reversed after administration of insulin to diabetic animals. The severity of diabetes was also found to affect the activity of pyruvate dehydrogenase with inverse correlation. There was a gradual increase in the proportion of active (dephosphorylated) form with increase of time after the onset of diabetes.     

Regulation of steady state pyruvate dehydrogenase complex activity in plant mitochondria : reactivation constraints

The requirements for reactivation (dephosphorylation) of the pea (Pisum sativum L.) leaf mitochondrial pyruvate dehydrogenase complex (PDC) were studied in terms of magnesium and ATP effects with intact and permeabilized mitochondria. The requirement for high concentrations of magnesium for reactivation previously reported with partially purified PDC is shown to affect inactivation rather than reactivation. The observed rate of inactivation catalyzed by pyruvate dehydrogenase (PDH) kinase is always greater than the reactivation rate catalyzed by PDH-P phosphatase. Thus, reactivation would only occur if ATP becomes limiting. However, pyruvate which is a potent inhibitor of inactivation in the presence of thiamine pyrophosphate, results in increased PDC activity. Analysis of the dynamics of the phosphorylation-dephosphorylation cycle indicated that the covalent modification was under steady state control. The steady state activity of PDC was increased by addition of pyruvate. PDH kinase activity increased threefold during storage of mitochondria suggesting that there may be an unknown level of regulation exerted on the enzyme complex.