Creatine Kinase Activity from Rat Brain Is Inhibited by Branched-Chain Amino Acids in Vitro

Maple syrup urine disease (MSUD) is an inherited metabolic disorder biochemically characterized by the accumulation of branched-chain amino acids (BCAAs) and their branched-chain keto acids (BCKAs) in blood and other tissues. Neurological dysfunction is usually present in the affected patients, but the mechanisms of brain damage in this disease are not fully understood. Considering that brain energy metabolism seems to be altered in MSUD, the main objective of this study was to investigate the in vitro effect of BCAAs and BCKAs on creatine kinase activity, a key enzyme of energy homeostasis, in brain cortex of young rats. BCAAs, but not their BCKAs, significantly inhibited creatine kinase activity at concentrations similar to those found in the plasma of MSUD patients (0.5–5 mM). Considering the crucial role creatine kinase plays in energy homeostasis in brain, if this effect also occurs in the brain of MSUD patients, it is possible that inhibition of this enzyme activity may contribute to the brain damage found in this disease.     

Co-administration of Creatine Plus Pyruvate Prevents the Effects of Phenylalanine Administration to Female Rats During Pregnancy and Lactation on Enzymes Activity of Energy Metabolism in Cerebral Cortex and Hippocampus of the Offspring

Phenylketonuria (PKU) is the most frequent inborn error of metabolism. It is caused by deficiency in the activity of phenylalanine hydroxylase, leading to accumulation of phenylalanine and its metabolites. Untreated maternal PKU or hyperphenylalaninemia may result in nonphenylketonuric offspring with low birth weight and neonatal sequelae, especially microcephaly and intellectual disability. The mechanisms underlying the neuropathology of brain injury in maternal PKU syndrome are poorly understood. In the present study, we evaluated the possible preventive effect of the co-administration of creatine plus pyruvate on the effects elicited by phenylalanine administration to female Wistar rats during pregnancy and lactation on some enzymes involved in the phosphoryltransfer network in the brain cortex and hippocampus of the offspring at 21 days of age. Phenylalanine administration provoked diminution of body, brain cortex an hippocampus weight and decrease of adenylate kinase, mitochondrial and cytosolic creatine kinase activities. Co-administration of creatine plus pyruvate was effective in the prevention of those alterations provoked by phenylalanine, suggesting that altered energy metabolism may be important in the pathophysiology of maternal PKU. If these alterations also occur in maternal PKU, it is possible that pyruvate and creatine supplementation to the phenylalanine-restricted diet might be beneficial to phenylketonuric mothers.     

Pyruvate kinase and alanine synthesis in skeletal muscle

L-Phenylalanine is an allosteric inhibitor of M₁-type pyruvate kinase. Accordingly, the effects were studied of 20 mM phenylalanine on the metabolism of 5 mM [U-¹⁴C]glucose and 3 mM L-[U-¹⁴C]glutamate by isolated hemidiaphragms from starved rats. Phenylalanine inhibited lactate and¹⁴CO₂ production from both substrates and stimulated alanine release. It is concluded that pyruvate kinase may have a dual role in intermediary metabolism in skeletal muscle: the enzyme is a component of the lower glycolytic pathway and is implicated in a pathway of amino acid oxidation and alanine synthesis.     

Kinetic properties of pyruvate kinase of rabbit brain

Summary The kinetic properties of rabbit brain pyruvate kinase have been studied to determine its role in the regulation of glycolysis. One of the substrates of the enzyme, phosphoenolpyruvate, exhibits homotropic cooperativity (Hill coeff. of 1.45); thus, it is a moderate activator of the enzyme. The other substrate, ADP, shows normal Michaelis-Menton kinetics. Fructose-6-phosphate and glucose-6-phosphate activate the enzyme only slightly at the 1mm level and inhibit slightly at higher levels, and hence have no metabolic influence on the enzyme activity. Fructose-1, 6-diphosphate also has a slight activation up to 0.5 mm but no inhibition at higher level; therefore, it has no influence either. ATP, 2-phosphoglycerate, and phenylalanine are inhibitors of the enzyme. ATP, being the energy reservoir derived from glycolysis as well as a product of the reaction catalyzed by the enzyme, is a significant feedback inhibitor of the enzyme. These kinetic properties suggest a key role for pyruvate kinase in the regulation of glycolysis. Phenylalanine inhibition of the enzyme has been reported to be a possible mechanism of damage to the developing brain in phenylketonuria. The inhibition by phenylalanine at 10 mm in the assay mixture is reversed by alanine, cysteine, or serine at 0.2 mm level. Furthermore, the effect of these amino acids in reversing the phenylalanine inhibition are mutually enhancing. Consequently phenylalanine cannot have a significant inhibition on the activity of pyruvate kinase in brain.A preliminary report has been presented at the American Society of Biological Chemists Meeting at Atlanta, Georgia, June 1978.     

Effect of thyroid hormones on the glycolytic enzyme activity in brain areas of the rat

The glycolytic metabolism through the key enzymes, hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase, have been studied in the brain areas: anterior cortex, amygdala, hypothalamus, septum and hippocampus in adult rats with pharmacologically induced hyperthyroidism. The oxidative metabolism of glucose is accelerated in most brain areas by treatment with high doses of T3, as is shown by the increase in HK activity, approaching normality on reducing the dose. This decrease can also by observed in the PFK activity through the effect of assayed doses of thyroxine. The anterior cortex is the only brain area that does not show significant variations of PK activity through the effects of treatment with thyroid hormones. On the other hand, a general inhibition of the glycolytic anaerobic pathway by treatment with T3 was observed.     

Localization and role of pyruvate kinase isoenzymes in the regulation of carbohydrate metabolism and pyruvate recycling in rat kidney cortex

This work was performed to gain more information on the role of pyruvate kinase isoenzymes in the regulation of renal carbohydrate metabolism. Immunohistochemically, pyruvate kinase type L is shown to be localized in the proximal tubule of the nephron and pyruvate kinase type M2 in the distal tubule and the collecting duct. a tight relationship between gluconeogenesis and pyruvate recycling was found. The rate of gluconeogenesis (8 mumol/g wet wt. per 30 min) was of the same order of magnitude as the rate of pyruvate recycling (10.92 mumol/g wet wt. per 30 min). Stimulation of gluconeogenesis from 20 mM lactate in kidney cortex slices of 24-h-starved rats by dibutyryl-cAMP, alanine and parathyroid hormone was connected with a decrease in pyruvate recycling; inhibition of gluconeogenesis due to a lack of Ca2+ in the incubation medium was linked with an increase in pyruvate recycling. The degradation of [6-14C]glucose to lactate, pyruvate, ketone bodies and CO2 and of [2-14C]lactate was unaffected by dibutyryl-cAMP, alanine, epinephrine, vasopressin or the omission of Ca2+ from the incubation medium. 1 mM dibutyryl-cAMP or 5 mM alanine did not alter the activities of oxaloacetate decarboxylase, 'malic' enzyme and malate dehydrogenase from rat kidney cortex. Since aerobic glycolysis in the distal tubules and the collecting ducts is not influenced by hormones, dibutyryl-cAMP and Ca2+, pyruvate kinase type M2 residing in this tissue is unlikely to be a control point of glycolysis. Since this tissue degrades only one-seventh of the glucose formed via gluconeogenesis, it does not contribute significantly to pyruvate recycling. Therefore, the decrease of pyruvate recycling in the presence of dibutyryl-cAMP and alanine in rat kidney cortex slices, leading to increased renal gluconeogenesis, has to be ascribed to the regulation of pyruvate kinase type L.     

Effect of phenylalanine and its metabolites on ATP diphosphohydrolase activity in synaptosomes from rat cerebral cortex

The in vitro effects of phenylalanine and some of its metabolites on ATP diphosphohydrolase (apyrase, EC 3.6.1.5) activity in synaptosomes from rat cerebral cortex were investigated. The enzyme activity in synaptosomes from rats subjected to experimental hyperphenylalaninemia (-methylphenylalanine plus phenylalanine) was also studied. In the in vitro studies, a biphasic effect of phenylalanine on both enzyme substrates (ATP and ADP) was observed, with maximal inhibition at 2.0 mM and maximal activation at 5.0 mM. Inhibition of the enzyme activity was not due to calcium chelation. Moreover, phenylpyruvate, when compared with phenylalanine showed opposite effects on the enzyme activity, suggesting that phenylalanine and phenylpyruvate bind to two different sites on the enzyme. The other tested phenylalanine metabolites (phenyllactate, phenylacetate and phenylethylamine) had no effect on ATP diphosphohydrolase activity. In addition, we found that ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats with chemically induced hyperphenylalaninemia was significantly enhanced by acute or chronic treatment. Since it is conceivable that ATPase-ADPase activities play an important role in neurotransmitter (ATP) metabolism, it is tempting to speculate that our results on the deleterious effects of phenylalanine and phenylpyruvate on ATP diphosphohydrolase activity may be related to the neurological dysfunction characteristics of naturally and chemically induced hyperphenylalaninemia.     

Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.     

Tyrosine impairs enzymes of energy metabolism in cerebral cortex of rats

Tyrosine levels are abnormally elevated in tissues and physiological fluids of patients with inborn errors of tyrosine catabolism, especially in tyrosinemia type II, which is caused by deficiency of tyrosine aminotransferase and provokes eyes, skin, and central nervous system disturbances. Considering that the mechanisms of brain damage in these disorders are poorly known, in this study, we investigated the in vivo and in vitro effects of tyrosine on some parameters of energy metabolism in cerebral cortex of 14-day-old Wistar rats. We observed that 2 mM tyrosine inhibited in vitro the pyruvate kinase (PK) activity and that this inhibition was prevented by 1 mM reduced glutathione with 30, 60, and 90 min of preincubation. Moreover, administration of tyrosine methyl ester (TME) (0.5 mg/g of body weight) decreased the activity of PK and this reduction was prevented by pre-treatment with creatine (Cr). On the other hand, tyrosine did not alter adenylate kinase (AK) activity in vitro, but administration of TME enhanced AK activity not prevented by Cr pre-treatment. Finally, TME administration decreased the activity of CK from cytosolic and mitochondrial fractions and this diminution was prevented by Cr pre-treatment. The results suggest that tyrosine alters essential sulfhydryl groups necessary for CK and PK functions, possibly through oxidative stress. In case this also occurs in the patients, it is possible that energy metabolism alterations may contribute, along with other mechanisms, to the neurological dysfunction of hypertyrosinemias.     

Proline Reduces Creatine Kinase Activity in the Brain Cortex of Rats

Type II hyperprolinemia is an inherited disorder caused by a deficiency of ¹-pyrroline-5-carboxilic acid dehydrogenase, whose biochemical hallmark is proline accumulation in plasma and tissues. Although neurological symptoms occur in most patients, the neurotoxicity of proline is still controversial. The main objective of the present study was to investigate the effect of acute and chronic administration of proline on creatine kinase activity of brain cortex of Wistar rats. Acute treatment was performed by subcutaneous administration of one injection of proline to 22-day-old rats. For chronic treatment, proline was administered twice a day from the 6th to the 21st postpartum day. The results showed that creatine kinase activity was significantly inhibited in the brain cortex of rats subjected to acute proline administration. In contrast, this activity was increased in animals subjected to chronic administration. We also measured the in vitro effect of proline on creatine kinase activity in cerebral cortex of 22-day-old nontreated rats. Proline significantly inhibited creatine kinase activity. Considering the importance of creatine kinase forthe maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which proline might be neurotoxic.     

Acute regulation of pyruvate kinase activity in rat epididymal adipose tissue by insulin.

1. Evidence is presented that exposure of epididymal fat-pads from fed rats to insulin leads to a marked diminution in the Km for phosphoenolpyruvate of pyruvate kinase. Effects of insulin may be readily demonstrated in experiments both in vivo and in vitro and are not secondary to the activation by the hormone of glucose transport. No effect of insulin is apparent in tissues from 48 h-starved animals. 2. The mechanism of the effect of insulin on pyruvate kinase was not established. The observed changes in Km do not appear to be the result of alterations in the amounts of bound effectors such as fructose 1,6-bisphosphate and alanine. Rather, as the effect persists in incubated extracts, it appears that a change in the degree of phosphorylation or some other covalent modification of the enzyme may be involved.     

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.     

Stimulation of tumor-cell respiration by inhibitors of pyruvate kinase.

In a model system consisting of highly coupled rat liver mitochondria respiring in the presence of substrate, pyruvate kinase, phosphoenolpyruvate, ATP, hexokinase and glucose, the increase in the mitochondrial concentration results in a progressive decrease in the activity of pyruvate kinase. These results are in accord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with mitochondrial oxidative phosphorylation for the available ADP. The addition of adequate amounts of the amino acids, cysteine, alanine and phenylalanine, known as inhibitors of pyruvate kinase, to living Ehrlich ascites tumor cell suspensions results in a stimulation of the respiratory rate and in a decrease of the glycolytic rate of the cells. Concomitant with these changes, there is an accumulation of intracellular phosphoenolpyruvate and ADP, and a decrease in pyruvate and ATP. These results provide additional evidence for paying attention to pyruvate kinase as another key enzyme whose properties and activities may be major determinants for the control of glycolysis and the Crabtree and Pasteur effects of tumor cells.     

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.     

PYRUVATE METABOLISM BY HOMOGENATES OF HUMAN BRAIN: EFFECTS OF PHENYLPYRUVATE AND IMPLICATIONS FOR THE ETIOLOGY OF THE MENTAL RETARDATION IN PHENYLKETONURIA

Abstract— The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by homogenates of human brain was investigated. In the presence of 5 mM pyruvate as substrate homogenates of human cerebral cortex fixed about 1 μmol of H¹⁴CO₃^-_- per g of tissue in 30 min. Phenylpyruvate at a concentration of 5 raw inhibited the fixation of H¹⁴ CO₃^-_- by homogenates of human brain by approximately 50 per cent, whereas 5 mM phenylalanine had no effect. The inhibition of pyruvate carboxylation by phenylpyruvate was dependent upon the concentration of the inhibitor. The activity of pyruvate carboxylase (EC 6.4.1.1) in human cerebral cortex was 02–0.4 units, with a K_m for pyruvate of about 0.2 mM. Homogenates of human cerebral cortex decarboxylated [1-¹⁴C]pyruvate to ¹⁴CO₂ at a rate of about 5 μmol per g of tissue per 15 min, with a 20–50 per cent reduction in the presence of 5 mM phenylpyruvate; phenylalanine at the same concentration had no effect. The possible toxic effect of phenylpyruvate on the metabolism of pyruvate in the brains of untreated phenylketonuric patients is discussed.     

The effect of phenylpyruvate on pyruvate metabolism in rat brain

1. The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by isolated mitochondria from rat brain was investigated. 2. Phenylpyruvate inhibited the fixation of H(14)CO(3) (-) in the presence of pyruvate by intact rat brain mitochondria, whereas phenylalanine and other metabolites of this amino acid had no inhibitory effect on this process. 3. Pyruvate carboxylase activity in freeze-dried rat brain mitochondrial preparations was also inhibited only by phenylpyruvate, and a ;mixed type' inhibition was observed. 4. The K(m) for pyruvate of rat brain pyruvate carboxylase was about 0.2mm. 5. The concentration of phenylpyruvate required for a 50% inhibition of H(14)CO(3) (-) fixation by the intact mitochondria and of pyruvate carboxylase activity was dependent on the concentration of pyruvate used in the incubation medium. 6. The possible significance of inhibition of pyruvate carboxylase activity by phenylpyruvate in the brains of phenylketonuric patients is discussed.     

Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro

D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.     

Inhibition of the Mitochondrial Respiratory Chain by Phenylalanine in Rat Cerebral Cortex

Phenylketonuria (PKU) is biochemically characterized by the accumulation of phenylalanine (Phe) and its metabolites in tissues of affected children. Neurological damage is the clinical hallmark of PKU, and Phe is considered the main neurotoxic metabolite in this disorder. However, the mechanisms of neurotoxicity are poorly known. The main objective of the present work was to measure the activities of the mitochondrial respiratory chain complexes (RCC) and succinate dehydrogenase (SDH) in brain cortex of Wistar rats subjected to chemically induced hyperphenylalaninemia (HPA). We also investigated the in vitro effect of Phe on SDH and RCC activities in the cerebral cortex of 22-day-old rats. HPA was induced by subcutaneous administration of 2.4 mol/g body weight -methylphenylalanine, a phenylalanine hydroxylase inhibitor, once a day, plus 5.2 M/g body weight phenylalanine, twice a day, from the 6th-21st postnatal day. The results showed a reduction of SDH and complex I + III activity in brain cortex of rats subjected to HPA. We also verified that Phe inhibited the in vitro activity of complexes I + III, possibly by competition with NADH. Considering the importance of SDH and RCC for the maintenance of energy supply to brain, our results suggest that energy deficit may contribute to the Phe neurotoxicity in PKU.     

In-silico and in-vitro investigation on the phenylalanine metabolites’ interactions with hexokinase of Rat’s brain mitochondria

Hexokinase (HK) is the first enzyme of glycolysis pathway. In brain, most dominant form of HK, HK-I, binds reversibly to the outer mitochondria membrane. Those metabolites that affect binding or releasing of the enzyme from the mitochondria have regulatory effect on glucose consumption of the cell. In this study destructive effect of phenylalanine and its metabolites in relation to glucose metabolism in brain have been studied. The results show that phenylpyruvic acid decreases the activity of enzyme in the presence and absence of glucose-6-phosphate (G6P) and increases the release of the enzyme from mitochondria, whereas phenylalanine and phenyllactic acid have no such effects. Obtained Interactions and elicited binding energies of docking and MD simulations also showed more affinity for phenylpyruvic acid compared with the other potent inhibitors for hexokinase after the natural product of G6P. It is possible that phenylpyruvic acid is the cause of the reduction of glucose consumption by decreasing hexokinase activity and the higher inhibitory function. Therefore, production of ATP declines in brain cells.     

Pyruvate Metabolism in the Brain of Young Rats Intoxicated with Organic and Inorganic Lead

Abstract: The activities of lipoyl dehydrogenase, aspartate transaminase, and alanine transaminase, and levels of lactate were estimated in cerebral cortex, cerebellum, and brainstem of rats intoxicated acutely with tetraethyl lead and chronically with lead acetate. A significant inhibition of lipoyl dehydrogenase was observed in both groups of animals, whereas transaminase activities were increased in inorganic lead toxicity. Oxidative decarboxylation and anaplerosis of pyruvate was assessed in brain slices using [l-¹⁴C]pyruvate. Pyruvate dehydrogenase activity was decreased in both organic and inorganic lead toxicity, whereas labelling of aspartate and alanine was increased in inorganic lead toxicity. In studies in vitro , lead acetate showed a more significant effect than tetraethyl lead. The higher anaerobic metabolism in inorganic lead toxicity, as evidenced by increased anaerobic lactate production by brain slices, could either be an adaptive mechanism or be due to the delayed maturation of brain in the developing rat. Such a mechanism does not occur in acute organic lead toxicity, as the compound brings about massive and rapid degenerative changes in brain, resulting in convulsive seizures and death of the animals.