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P1: GDW Metabolic Brain Disease [mebr]

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Style file version Nov. 19th, 1999

C 2003) Metabolic Brain Disease, Vol. 18, No. 1, March 2003 (°

Alanine Prevents the in Vitro Inhibition of Glycolysis Caused by Phenylalanine in Brain Cortex of Rats Maria da Gra¸ca Lutz, ¨ 1 Luciane Rosa Feksa,1 Angela Terezinha de Souza Wyse,1 Carlos Severo Dutra-Filho,1 Moacir Wajner,1 and Clóvis Milton Duval Wannmacher1,2 Received August 2, 2002; accepted August 20, 2002

Glycolysis is the main route that provides energy to brain functioning. In this study we investigated the in vitro effects of phenylalanine, the main metabolite known to accumulate in phenylketonuria, and/or alanine, on pyruvate kinase activity, glucose utilization, lactate release, and ADP concentration in brain cortex homogenates from 30-day-old Wistar rats. We found that phenylalanine decreased PK activity, glucose utilization, and lactate release, and increased ADP brain levels. We also verified that alanine per se did not modify these parameters, but prevented the effects of phenylalanine. Our data suggest that the inhibition of pyruvate kinase by phenylalanine decreases glycolysis and energy production, and that alanine, a known competitor of phenylalanine on the enzyme activity, prevents the reduction of glycolysis and energy production caused by phenylalanine, probably by preventing the enzyme inhibition provoked by the amino acid. These results suggest that inhibition of brain PK activity by phenylalanine may be related to the diminution of glucose metabolism observed in the brain of phenylketonuric patients and may be one of the mechanisms responsible for the neurological dysfunction found in these patients. Key words: Phenylalanine; alanine; phenylketonuria; pyruvate kinase.

INTRODUCTION Phenylketonuria (PKU) is an inborn error of metabolism caused by a severe deficiency of phenylalanine hydroxylase activity. This disease is biochemically characterized by the accumulation of phenylalanine (Phe) and its metabolites in blood and other tissues (Scriver and Kaufman, 2001). A prominent clinical characteristic in phenylketonuria is the low intelligence quotient in untreated patients (Berman et al., 1961; Fish et al., 1969). The mechanisms of brain damage in PKU seem to be multiple and are poorly understood. There are many lines of evidence suggesting that Phe is the main factor affecting brain development in PKU, but the exact role of Phe and its metabolites in relation to the mental deficiency manifested in PKU patients remains unclear (Scriver and Kaufman, 2001). It has been demonstrated that glucose metabolism is reduced in the brain of PKU patients (Hasselbach et al., 1996). Furthermore, it has been reported that Phe may alter 1 Departamento

de Bioqu´ımica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. 2 To whom correspondence should be addressed at Departamento de Bioqu´ımica, Instituto de Ciˆ encias Básicas da Saúde, UFRGS, Rua Ramiro Barcelos 2600, CEP 90.035-003, Porto Alegre, RS, Brazil. E-mail: [email protected] 87 C 2003 Plenum Publishing Corporation 0885-7490/03/0300-0087/0 °


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Lutz, ¨ Feksa, de Souza Wyse, Dutra-Filho, Wajner, and Wannmacher

glucose metabolism in rat brain slices during differentiation (Gimenez et al., 1974; Glazer and Weber, 1971; Weber et al., 1970). However, it has also been reported the accumulation of glucose and glycolytic intermediates in the brain of rats subjected to Phe administration, which might compensate the reduction of glucose uptake by brain leading to normal energy production (Miller et al., 1973). Pyruvate kinase (PK) is a crucial enzyme for glycolysis in the brain. It is well known that Phe inhibits the in vitro PK activity in human and rat brain by competition with the substrate phosphoenolpyruvate and that alanine, a nutritionally nonessential amino acid, prevents and reverses this inhibition (Chainy and Kanungo, 1978; Ibsen and Trippet, 1974; Schwark et al., 1971; Srivastava and Baquer, 1985; Vijayvargiya et al., 1969). However, it is unclear whether PK inhibition by Phe may reduce glucose utilization and energy production in the brain of PKU patients, and whether alanine may prevent such effects. The main objective of this study was to determine the in vitro effect of Phe on PK activity, on two parameters of glycolysis, namely glucose utilization and lactate release, and also on ADP levels, in brain cortex homogenates from young rats. We also investigated the role of alanine on the effects elicited by Phe.

MATERIALS AND METHODS Subjects and Reagents Thirty-day-old Wistar rats bred in the Animal House of the Department of Biochemistry, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, Brazil, were used in the experiments. Rats were kept with dams until 21st postpartum day. The animals had free access to water and to a standard commercial chow (Germani, Porto Alegre, RS, Brazil) containing 20.5% protein (predominantly soybean), 54% carbohydrate, 4.5% fiber, 4% lipids, 7% ash, and 10% moisture. Temperature was maintained at 24 ± 1◦ C, with a 12–12-h light–dark cycle. The “Principles of Laboratory Animal Care” (NIH publication no. 85-23, revised 1985) were followed in all the experiments, and the experimental protocol was approved by the Ethics Committee For Animal Research of the Federal University of Rio Grande do Sul. All chemicals were purchased from Sigma Chemical Co., St. Louis, MO.

Preparation of Brain Tissue The animals were sacrificed by decapitation without anesthesia. The brain was removed and dissected immediately on an ice-cooled glass plate. The cerebral cortex was dissected and washed in Ringer-bicarbonate buffer, pH 7.4, gassed with O2 /CO2 (19/1) mixture, and minced finely. The tissue was homogenized in the same buffer containing 5 mM glucose 1: 2 (w/v), with a Potter-Elvehjem glass homogenizer, and immediately incubated for 60 min at 37◦ C in a metabolic shaker (90 oscillations/min) and used for the determination of glucose, ADP, and lactate levels, and for PK activity assay. Phe and alanine were dissolved in Ringerbicarbonate buffer and added to the assay at 5.0 mM final concentration. Controls did not contain Phe or alanine.


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Enzyme Assay Pyruvate kinase activity was assayed essentially as described by Leong et al. (1981). The incubation medium consisted of 0.1 M Tris/HCl buffer, pH 7.5, 10 mM MgCl2 , 0.16 mM NADH, 75 mM KCl, 5.0 mM ADP, 7 units of L-lactate dehydrogenase, 0.1% (v/v) Triton X-100, and 1 µL of the homogenate in a final volume of 0.5 mL. The reaction was started by the addition of 1.0 mM phosphoenolpyruvate. All assays were performed in triplicate at 25◦ C. Results were expressed as mmol of pyruvate formed per hour per gram wet tissue.

Glucose Utilization Glucose was measured before and immediately after incubation by the glucose oxidase– peroxidase method (Trinder, 1969). The difference between the glucose amount measured before and after incubation was taken as glucose utilization. Results were expressed as µmol of glucose utilized per hour per gram wet tissue.

Lactate Release Two volumes of perchloric acid 0.3 N were immediately added to the brain homogenate and the excess of perchloric acid was precipitated as a potassium salt by the addition of one volume of a solution containing 0.5 N KOH, 0.1M imidazol, and 0.1 N KCl. After centrifugation by 5 min at 800 × g, lactate was measured in the supernatant before and after incubation by the lactase–peroxidase method (Shimojo et al., 1989). The difference between the lactate amount measured before and after incubation was taken as lactate released. Lactate levels before the incubation were practically nil. Results were expressed as µmol of lactate released per hour per gram wet tissue.

ADP Determination Two volumes of perchloric acid 0.3 N were immediately added to the samples and the excess of perchloric acid was precipitated as a potassium salt by the addition of one volume of a solution containing 0.5 N KOH, 0.1M imidazol, and 0.1 N KCl. After centrifugation by 5 min at 800 × g, ADP was measured in the supernatant before and after incubation by an adaptation of creatine kinase assay according to Hughes (1962). The difference between the ADP amount measured before and after incubation was taken as ADP formed. Briefly, ADP was assayed in medium containing 100 mM MgSO4-Trizma buffer, pH 7.5, 40 µL of the supernatant, and 1 unit pyruvate kinase, in a final volume of 0.1 mL. After 5 min of preincubation, the reaction was initiated by the addition of 5.9 mM creatine phosphate. The reaction was stopped after 10 min by the addition of 1 µmol of p-hydroxymercuribenzoic acid. The creatine liberated by reaction between creatine phosphate and ADP was estimated colorimetrically with 1-naphtol. Results were expressed as µmol of ADP formed per hour per gram wet tissue.


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Figure 1. In vitro effect of phenylalanine (Phe) and alanine (Ala) on pyruvate kinase activity of cerebral cortex from young rats. Data are expressed as mean ± SD for five independent experiments performed in triplicate. ∗∗ p < 0.01 compared to the other groups (Tukey test).

Statistical Analysis Data were analyzed by one-way ANOVA followed by the Tukey test when the F value was significant. All data were analyzed by the Statistical Package for the Social Sciences inserted in a PC. RESULTS First, we investigated the in vitro effect of Phe, alanine, or Phe plus alanine on PK activity in the brain cortex homogenates from rats. Phe significantly reduced PK activity by approximately 20% (F(3, 16) = 15.119; p < 0.001), whereas alanine per se did not affect this parameter, but fully prevented the reduction of the enzyme activity caused by Phe (Fig. 1). Next, we investigated whether glycolysis could be affected by Phe. This amino acid reduced glucose utilization by approximately 40% (F(3, 32) = 4.562; p < 0.001) (Fig. 2), and also reduced lactate release by approximately 33% (F(3, 16) = 8.455; p < 0.001)

Figure 2. In vitro effect of phenylalanine (Phe) and alanine (Ala) on glucose utilization by cerebral cortex from young rats. Data are expressed as mean ± SD for nine independent experiments performed in duplicate. ∗ p < 0.05 compared to the other groups (Tukey test).


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Figure 3. In vitro effect of phenylalanine (Phe) and alanine (Ala) on lactate release by cerebral cortex from young rats. Data are expressed as mean ± SD for five independent experiments performed in duplicate. ∗∗ p < 0.01 compared to the other groups (Tukey test).

(Fig. 3). Alanine per se did not modify glucose utilization or lactate release, but prevented the effect of Phe on these two parameters. In an attempt to evaluate whether the decrease of glycolysis affected energy metabolism, we determined the effect of Phe, alanine, and Phe plus alanine, on brain ADP content. Phe increased ADP levels by approximately 80% (F(3, 32) = 4.562; p < 0.05), whereas alanine did not alter these levels, but prevented the increase of ADP caused by Phe (Fig. 4). DISCUSSION In this study, we demonstrated that Phe alters the energy metabolism in brain cortex homogenates of young rats. We first observed that Phe inhibited PK activity around 20% and alanine prevented this inhibition. These results confirm other reports (Chainy and Kanungo, 1978; Ibsen and Trippet, 1974; Schwark et al., 1971; Srivastava and Baquer, 1985;

Figure 4. In vitro effect of phenylalanine (Phe) and alanine (Ala) on ADP formation by cerebral cortex from young rats. Data are expressed as mean ± SD for nine independent experiments performed in duplicate. ∗ p < 0.05 compared to the other groups (Tukey test).


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Vijayvargiya et al., 1969; Weber, 1969). It is possible that alanine prevents the inhibition caused by Phe through competition with this amino acid for a critical site in the enzyme, since it has been reported a competition between these two amino acids in the binding to PK from rabbit muscle (Kayne and Price, 1973). Next, we investigated the effect of these amino acids on two parameters of glycolysis, namely glucose utilization, since glucose is the major substrate used for neural cell metabolism, and lactate release, since this metabolite is produced in considerable amounts by the brain, and more specifically by glial cells. We observed that Phe decreased glucose utilization by around 40%, and lactate release by approximately 33%, strongly indicating that the inhibition caused by Phe on PK activity reduced glycolysis in the cerebral cortex homogenate. It is interesting to observe that the 20% reduction of PK activity caused 40% reduction of glycolysis, probably because the accumulation of glycolytic intermediates reduces the activities of other enzymes of the glycolytic pathway. In fact, glucose and intermediate substances of glycolytic pathway, such as triose phosphates, accumulate in the brain of rats subjected to Phe administration (Miller et al., 1973). We also observed that alanine addition to the incubation medium prevented the inhibitory effect caused by Phe on glucose utilization and lactate release, suggesting that the reduction of glycolysis was provoked by the inhibitory action of Phe on PK activity. We also observed that Phe nearly doubled brain ADP levels, and again alanine was able to prevent this increase. The data reinforce the hypothesis that PK inhibition by Phe is probably responsible for the reduction of glycolysis and the consequent increase of brain ADP content. We measured ADP concentration because this nucleotide has been considered a more sensible parameter than has ATP to assess energy metabolism. ADP concentrations in brain (0.2–0.4 mM) are 10 times lower than those of ATP (2–4 mM) (Nakai et al., 2000; Plaschke et al., 1999) and depends on the balance between production and utilization of ATP. Therefore, small variations in ATP levels provokes great variations in those of ADP. Therefore, we presume a sequence of events provoked by Phe: inhibition of PK activity and consequently of glycolysis, which leads to a reduction of ATP synthesis increasing ADP levels. Our data suggest that the increase in glycolytic intermediates do not overcome PK inhibition, as proposed by Miller et al. (1973). In summary, our findings indicate that Phe provokes a deficit of brain energy production, possibly secondary to PK inhibition, and that alanine prevents Phe effects. PK catalyses a key regulatory step in the glycolytic pathway, the main route that provides energy for brain function. There are at least four known isozymic forms of PK in vertebrates, designed as L, M1, M2 (or A), and R (Hall and Cottam, 1978). The PK M1 is the brain isozyme and has been isolated and studied from rat (Srivastava and Baquer, 1985), bovine (Terlecki, 1989), and pig brain (Farrar and Farrar, 1995). All studied M1 isoenzymes present a hyperbolic kinetics for PEP as the substrate, and are inhibited by Phe. Furthermore, since this inhibition is prevented or reversed by alanine, It has been suggested that the antagonism between Phe and Ala on PK activity might be a physiologic mechanism of modulation of this enzyme activity and consequently a modulation of glycolysis (Chainy and Kanungo, 1978; Schwark et al., 1971; Vijayvargyia et al., 1969). Patients affected by PKU develop a variable degree of brain dysfunction, whose pathophysiology is still unclear. Phe is considered the main neurotoxic metabolite accumulating in this disease, but the mechanisms of neurotoxicity seem to be multiple. The inhibition of brain PK by Phe causing a reduction of brain energy metabolism, as observed in the


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present investigation, might represent one of such mechanisms. Although it is difficult to extrapolate our findings to the human PKU, they agree with the observation of a reduced glucose metabolism in the brain of PKU patients, mainly in white matter (Hasselbach et al., 1996). On the other hand, it is possible that the in vitro effect of alanine preventing the inhibitory effect of Phe on PK activity may also occur in vivo. It has been reported in hyperphenylalaninemic rats an inhibition of exodus of alanine from nonbrain tissues caused by an inhibitory effect of Phe on the L carrier system, the carrier system responsible for the release of alanine from nonbrain tissues. As a consequence, plasma alanine levels decrease, reducing the brain uptake of this amino acid (Cespedes et al., 1989). So, if this also occurs in PKU patients, it is possible that reduced brain alanine levels could enhance Phe neurotoxicity in this disorder. If this is the case, it is conceivable that dietary alanine supplementation to PKU patients might be useful as an adjuvant in their treatment, as well as a carbohydraterich diet, because it might enhance the biosynthesis of alanine and provide more glycolytic substrates to overcome PK inhibition. Further studies are however necessary to evaluate the potential benefit of carbohydrate and/or alanine supplementation to the diet of PKU patients.

ACKNOWLEDGMENTS This work was supported in part by grants from FAPERGS, RS-Brazil, PROPESQ/ UFRGS, RS-Brazil, and CNPq, DF-Brazil.

REFERENCES Berman, P.W., Graham, F.K., Eichman, P.L., and Waisman, H.A. (1961). Psychologic and neurologic status of diet-treated phenylketonuric children and their siblings. Pediatrics 28:924–934. Cespedes, C., Thoene, J.G., Lower, K., and Christensen, H.N. (1989). Evidence of inhibition of exodus of small neutral amino acids from non-brain tissues in hyperphenylalaninemic rats. J. Inher. Metab. Dis. 12:166–180. Chainy, G.B.N., and Kanungo, M.S. (1978). Induction and properties of pyruvate kinase of the cerebral hemisphere of rats of various ages. J. Neurochem. 30:419—427. Farrar, G., and Farrar, W.W. (1995). Purification and properties of the pyruvate kinase isozyme M1 from the pig brain. Int. J. Biochem. Cell Biol. 27:1145–1151. Fish, O.R., Torres, F., Graven, H.J., Greenwood, C.S., and Anderson, J.A. (1969). Twelve years of clinical experience with phenylketonuria. Neurology 19:659–666. Gimenez, C., Valdivieso, F., and Major, F. (1974). Glycolysis in the brain and liver of rats with experimentally induced phenylketonuria. Biochem. Med. 11:81–86. Glazer, R.I., and Weber, G. (1971). The effects of L-phenylalanine and phenylpyruvate on glycolysis in rat cerebral cortex. Brain Res. 33:439–450. Hall, E.R., and Cottam, G.L. (1978). Isoenzymes of pyruvate kinase in vertebrates: Their physical, chemical, kinetic and immunological properties. Int. J. Biochem. 9:785–793. Hasselbach, S., Knudsen, G.M., Toft, P.B., Hogh, P., Tedeschi, E., Holm, S., Videbaek, C., Henriksen, O., Lou, H. C., and Paulson, O.B. (1996). Cerebral glucose metabolism is decreased in white matter changes in patients with phenylketonuria. Pediatr. Res. 40:21–24. Hughes, B.P. (1962). A method for estimation of serum creatine kinase and its use in comparing creatine kinase and aldolase activity in normal and pathological sera. Cin. Chim. Acta 7:597–603. Ibsen, K.H., and Trippet, P. (1974). Effects of amino acids on the kinetic properties of three non-interconvertible rat pyruvate kinases. Arch. Biochem. Biophys. 163:570–580. Kayne, F.J., and Price, N.C. (1973). Amino acid effector binding to rabbit muscle pyruvate kinase. Arch. Biochem. Biophys. 159:292–296.


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Leong, S.F., Lai, J.C.K., Lim, L., and Clark, J.B. (1981). Energy-metabolising enzymes in brain regions of adult and aging rats. J. Neurochem. 37:1548–1556. Miller, A.L., Hawkins, R.A., and Veech, R.L. (1973). Phenylketonuria: Phenylalanine inhibits brain pyruvate kinase in vivo. Science 179:904–906. Nakai, A., Taniuchi, Y., Asakura, H., Oya, A., Yokota, A., Koshino, T., and Araki, T. (2000). Developmental changes in mitochondrial activity and energy metabolism in fetal and neonetal rat brain. Brain Res. Dev. 121:67–72. Plaschke, K., Yun, S.W., Martin, E., Hoyer, S., and Bardenheur, H.J. (1999). Interrelation between cerebral energy metabolism and behaviour in a rat model of permanent brain vessel occlusion. Brain Res. 830:320–329. Schwark, W.S., Singhal, R.L., and Ling, G.M. (1971). Regulation of pyruvate kinase in the rat cerebral cortex. J. Neurochem. 18:123–134. Scriver, C.R., and Kaufman, S. (2001). Hyperphenylalaninemia: Phenylalanine hydroxylase deficiency. In (C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.), The Metabolic & Molecular Bases of Inherited Diseases, 8th edn., McGraw-Hill, New York, pp. 1667–1724. Shimojo, N., Naka, K., Nakajima, C., Yoshikawa, C., Okuda, K., and Okada, K. (1989). Test-strip method for measuring lactate in whole blood. Clin. Chem. 35:1992–1994. Srivastava, L.K., and Baquer, N.Z. (1985). Purification and properties of rat brain pyruvate kinase. Arch. Biochem. Biophys. 236:703–713. Terlecki, G. (1989). Purification and properties of pyruvate kinase M1 from bovine brain. Int. J. Biochem. 21:1053– 1060. Trinder, P. (1969). Determination of blood glucose using an oxidase–peroxidase system with a non-carcinogenic chromogen. J. Clin. Pathol. 22:158–161. Vijayvargiya, R., Schwark, W.S., and Singhal, R.L. (1969). Pyruvate kinase: Modulation by L-phenylalanine and L-alanine. Can. J. Biochem. 47:895–898. Weber, G. (1969). Inhibition of human brain pyruvate kinase and hexokinase by phenylalanine and phenylpyruvate: Possible relevance to phenylketonuric brain damage. Proc. Nat. Acad. Sci. (USA) 63:1365–1369. Weber, G., Glazer, R.I., and Ross, R.A (1970). Regulation of human and rat brain metabolism: Inhibitory action of phenylalanine and phenylpyruvate on glycolysis, protein, lipid, DNA and RNA metabolism. Adv. Enzyme Reg. 8:13–36.