Response of rat cerebral glycolytic enzymes to hyperammonemic states

Neuroscience Letters, 161 (1993) 37-40 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00

37

NSL 09858

Response of rat cerebral glycolytic enzymes to hyperammonemic states L. R a t n a k u m a r i b a n d C h . R . K . M u r t h y a "School of Life Sciences, University of Hyderabad, Hyderabad (India) and bService of Medical Genetics, Hospital Sainte-Justine, Montreal, Que. (Canada) (Received 28 September 1992; Revised version received 12 April 1993; Accepted 30 June 1993)

Key words.: Brain; Glycolysis; Hyperammonemia Activity levels of enzymes of glycolytic pathway viz., hexokinase (EC.2.7.1.1), phosphofructokinase (EC.2.7.1.11), aldolase (EC.4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC.I.2.1.12), enolase (EC.4.2.1.11), pyruvate kinase (EC.2.7.1.40) and lactate dehydrogenase (EC.I.1.1.27) were estimated in cerebral cortex, cerebellum and brainstem of the rats treated with subacute and acute doses of ammonium acetate and compared with those of control animals. In general, the activities of all the enzymes except for hexokinase and lactate dehydrogenase, were elevated in all the three regions of the brain. The results suggets an enhanced rate of glycolysis in brain in hyperammonemic states and strengthens the role of ammonium ion in stimulating certain enzymes of the glycolytic pathway.

Evidences are accumulating in recent years that glucose metabolism is affecting in hyperammonemic states of different etiologies [2, 4]. It has been shown that the activities of enzymes of citric acid cycle are enhanced while those involved in the transport of reducing equivalents from cytosol to mitochondria are suppressed in hyperammonemic states [10-12]. It was also proposed that a part of the neurological toxicity of ammonia may be due to its interference with the normal transfer of reducing equivalents from cytoplasm to mitochondria [6]. The later effect of ammonia might have an influence on the activities of cerebral glycolytic enzymes. There are reports on the in vitro stimulation of phosphofructokinase by ammonium ions [7, 13] and an elevation in the levels of various glycolytic intermediates of brain [5], however, very little information is available on the activities of the enzymes of this pathway in brain in hyperammonemic states. Presently, we report an increase in the activities of the enzymes of glycolytic pathway in different regions of hyperammonemic rat brain. Hyperammonemia was induced in adult Wistar rats by intraperitoneal administration of 3.5 mmol/kg.b.wt. (subacute dose) or 25 mmol/kg.b.wt. (acute dose) of ammonium acetate. Animals in the acute group entered into a convulsive phase 20-25 min after the injection of ammonium acetate and were sacrificed during the convulCorrespondence: L. Ratnakumari, Service of Medical Genetics, Hospital Sainte-Justine, 3175 Cote Sainte Catherine, Montreal, Que., Canada, H3T 1C5. Fax: (l) (514) 345-4801.

sions. Animals in the subacute group showed no convulsions, but were decapitated 25 min after the administration of ammonium acetate to keep parity with the acute group. Control animals were treated with saline. The preparation of homogenates of different brain regions were similar to those described earlier [10, 11]. After decapitation, the brain was removed on to an ice-cooled glass plate, and cerebral cortex, cerebellum and brain stem were dissected out. These tissues were washed in 0.32 M sucrose to remove blood. A 1:10 (w/v) homogenate was then made in the 0.32 M sucrose by using Potter-Elvehjem homogenizer with a serrated teflon pestle. Triton X-100 was added to the homogenates at a final concentration of 0.2%. Enzyme activities were measured spectrophotometrically by monitering the change in absorbance of N A D H or N A D P H at 340 nm [1, 3, 7]. Protein content of the homogenates was determined by the method of Lowry et al. [8]. Statistical analysis of the data was made by Newman-Keul's multiple range analysis test. Administration of ammonium acetate resulted in behavioral changes in rats which were similar to those described earlier [11, 12]. There was a significant elevation in the ammonia content in blood and brain with this treatment (Table 1). Following the administration of subacute dose of ammonium acetate, there were no significant differences in the protein content of three different regions of brain. Under these conditions, activities of all glycolytic enzymes, excepting those of hexokinase, glyceraldehyde-3-

38

phosphate dehydrogenase and enolase, were elevated in the cerebral cortex (Table 2). In the cerebellum, but for hexokinase, activities of all the other glycolytic enzymes were elevated following administration of subacute doses of ammonium acetate (Table 3). In the brainstem of rats subjected to subacute ammonia toxicity, activities of glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase were unaltered while those of other enzymes were elevated (Table 4). Lactate dehydrogenase activity, when measured in the direction of lactate formation, was unaltered in the three regions of brain under these conditions (Table 2~4). However, in the reverse direction, its activity was unaltered in cerebral cortex (Table 2), elevated in cerebellum and brainstem (Tables 3 and 4). In the animals administered with an acute dose of ammonium acetate, there was an elevation in the activities of all the glycolytic enzymes in the three regions of brain (Tables 2 4 ) . Exceptions to this were observed in the case of hexokinase in cerebral cortex and cerebellum and lactate dehydrogenase (in the direction of pyruvate formation) in cerebral cortex and brainstem. Under these conditions, there was a fall in the hexokinase activity in cerebral cortex (Table 2) and cerebellum (Table 3). Lactate dehydrogenase activity (lactate to pyruvate) was suppressed in cerebral cortex (Table 2) and brainstem (Table 4). In general, the magnitude of changes observed in acute condition were greater than in subacute state. There were no statistically significant differences in the protein content in these three regions of brain even under acute hyperammonemic conditions. Elevation in the ammonia levels of blood and brain associated with several pathological states, are known to cause cerebral dysfunction [2]. In recent years it has been recognised that detoxification of excess ammonia (by way of glutamine formation) would result in the depletion of cytosolic pool of glutamate and this might adversely affect the transport of reducing equivalents from cytosol to mitochondria (for review see ref. 2). Derangement in the latter process might disrupt the flux of glucose carbon through glycolytic pathway, as N A D + is re-

TABLE 1 CEREBRAL A N D SERUM A M M O N I A LEVELS IN C O N T R O L A N D H Y P E R A M M O N E M I C RATS a,/~mol/g.wet wt.: b,/~mol/ml, n = 5 per each group. *P < 0.001 versus control.

Brain ~ Serum b

Control

Subacute group Acute group

0.42 ± 0.10 0.07 ± 0.01

1.31 + 0.4* 1.03 ± 0.1"

2.45 ± 0.3* 1.81 ± 0.2*

quired for the sustained activity of glyceraldehyde-3phosphate dehydrogenase. However, the reported elevation in the content of several glycolytic intermediates in acute hyperammonemia [5], the in vitro stimulation in the activity levels of phosphofructokinase by ammonium ions [7], and the unaltered rates of glucose oxidation in primary cell cultures in the presence of ammonium ions [9] are not supportive of the above concept. To understand the effects of ammonia on the cerebral glycolysis, we have measured the activities of enzymes involved in this pathway in different regions of brains of hyperammonemic rats and compared them with those of control animals. Administration of either subacute or acute doses of ammonium acetate suppressed the activity of hexokinase and elevated that of phosphofructokinase in cerebral cortex and cerebellum (Table 2 and 3). Such a condition might result in a marginal decrease in the production of glucose-6-phosphate but enhances its utilization in glycolytic pathway. This would lower the levels of glucose6-phosphate and its utilization in other pathways such as glycogen synthesis and hexose monophosphate shunt. As a result there might be an increase in the flux of glucose carbon into glycolytic pathway through the rate-limiting phosphofructokinase reaction. The reported decrease in the levels of glucose-6-phosphate and an increase in the levels of fructose-l,6-diphosphate in the supratentorial

TABLE II EFFECT OF A M M O N I A ON ENZYMES OF GLYCOLYT1C PATHWAY IN RAT CEREBRAL CORTEX Units, ,umol/mg protein/h. Values are mean + S.D. of five different experiments. Ammonium acetate was injected intraperitoneally at a dose of 3.5 mmol/kg b.wt. for subacute group and 25 mmol/kg b.wt. for acute group of animals. *P < 0.05 versus control group. Enzyme Hexokinase Phosphofructokinase Aldolase Glyceraldehyde-3phosphate dehydrogenase Enolase Pyruvatekinase Lactate dehydrogenase (Pyruvate Lactate~ Lactatedehydrogenase (Lactate Pyruvate)

Control

Subacute

Acute

3.24 + 0.13 0.85 + 0,08

2.74 + 0.35* 1.50 + 0.5*

2.79 +- 0.21" 1.70 _+ 0.32*

1.00+ 0.16 1.31 +_ 0.11

1.31 +0.19 1.45 _+ 0.14

1.63+ 0.15" 1.65 _+ 0.22*

1.94 + 0.18 2.19 +- 0.38 10.7 _+ 1.28 24.3 +_3.l* 104 +_ 15 101 +_ 8.6

3.03 + 0.24* 19.3 +_ 5.4* 124 +_ 14.8

24

+_ 2.1

25.5 +_1.1

17.3 _+ (I.9"

39

structures of mouse brain following the administration of acute doses of ammonium salts [5], is in accordance with the present suggestion. In the brainstem, unlike cerebral cortex and cerebellum, there was an elevation in both hexokinase and phosphofructokinase in subacute and acute hyperammonemic states (Table 4) and such a change would not only enhance glucose uptake but also its utilization in glycolytic pathway in this region. Elevation in the activities of aldolase (Tables 2 4 ) would result in the enhanced production of triose phosphate, and this change is in accordance with the reported elevation of these compounds in hyperammonemic states [5]. The increase in the glyceraldehyde-3-phosphate dehydrogenase activity, though marginal as in cerebral cortex and brainstem in subacute states (Table 2 and 4), and in the subsequent enzymes (enolase and pyruvate kinase) might result in an enhanced production of pyruvate and this suggestion is supported by the reported increase in the levels of 2-phosphoglycerate and pyruvate under these conditions [5]. Lactate dehydrogenase activity (pyruvate to lactate) was not altered in subacute states and was elevated by a lesser magnitude when compared with the changes in the activities of other preceeding enzymes in acute states. Despite such meagre changes in the activity of this enzyme, lactate production may be enhanced due to (a) increased availability of pyruvate (elevated pyruvate kinase activity), (b) either normal or enhanced

TABLE III EFFECT OF AMMONIA ON ENZYMES OF GLYCOLYTIC PATHWAY IN RAT C E R E B E L L U M Units,/.tmol/mg protein/h. Values are m e a n + S.D. of five different experiments. A m m o n i u m acetate was injected intraperitoneally at a dose of 3.5 mmol/kg b.wt. for subacute group and 25 mmol/kg b.wt. for acute group of animals. *P < 0.05 versus control group. Enzyme Hexokinase Phosphofructokinase Aldolase Glyceraldehyde-3phosphate dehydrogenase Enolase Pyruvatekinase Lactate dehydrogenase (Pyruvate --~ Lactate) Lactatedehydrogenase (Lactate --* Pyruvate)

Control

Subacute

Acute

3.30 + 0.64 0 . 6 4 + 0.14

2.90 + 0.58 1.11 + 0.12"

2.63 + 0.49 1.24+ 0.20*

0.92 + 0.03 1.09 + 0.12

1.36 + 0.07* 1.28 + 0.07*

1.95 _+ 0.34* 1.48 _+ 0.21"

1.24 + 0.22 8.60+ 0.73 92.6 + 16

1.73 + 0.29* 12.7 + 2.6* 94.9 + 13.7

2.12 + 0.05* 19.5 + 2.7* 123 + 16"

15

24.0 +

+

1.1

1.8"

18.0 + 0.8*

TABLE IV EFFECT OF AMMONIA ON ENZYMES PATHWAY IN RAT B R A I N S T E M

OF G L Y C O L Y T I C

Units,/.tmol/mg protein/h. Values are mean + S.D. of five different experiments. A m m o n i u m acetate was injected intraperitoneally at a dose of 3.5 mmol/kg b.wt. for subacute group and 25 mmol/kg b.wt. for acute group of animals. *P < 0.05 versus control group. Enzyme Hexokinase Phosphofructokinase Aldolase Glyceraldehyde3-phosphate dehydrogenase Enolase Pyruvate kinase Lactate dehydrogenase (Pyruvate Lactate) Lactate dehydrogenase (Lactate Pyruvate)

Control

Subacute

Acute

1.49 + 0.24 0.44 + 0.07

1.80 + 0.15" 0.61 + 0.07*

1.93 + 0.20* 0.70 + 0.07*

0.85 + 0.03 1.28 + 0.15

1.23 + 0.18" 1.42 + 0.25

1.71 + 0.15" 1.51 + 0.14"

1.27 + 0.08 6.20 + 1.4 122 + 8.8

1.74 _+ 0.39* 6.95 + 1.2 124 + 4.2

1.75 + 0.23* 10.8 + 1.7" 141 + 12.7"

14.9 + 1.2

26.0 + 2 . 0 *

12.7 + 0.4*

availability of NADH (in glyceraldehyde-3-phosphate dehydrogenase reaction), and (c) very high (10-fold) activity of this enzyme as a result of which, it can cope up with changes in the contents of pyruvate and NADH. Suppression in the activity of this enzyme in reverse direction, especially in cerebral cortex (Table 2) and brainstem (Table 4), would prevent the reutilization of regenerated NAD ÷ and makes this compound available for glyceraldehyde-3-phosphate dehydrogenase reaction. Thus the present results, when considered along with those reported for citric acid cycle enzymes [11, 12], suggest an increased utilization of glucose in cerebral energy metabolism in hyperammonemic states. Moreover, these results also indicate that the ammonium ion stimulates certain enzymes of glycolysis, such as phosphofructokinase and pyruvate kinase, not only in in vitro conditions but also under in vivo conditions. Financial assistance was provided by University Grants Commission through a senior research fellowship to L R K during the tenure of this work. 1 Bergmeyer, H.U. and Bernt, E. In: H.U. Bergrneyer (Ed.), Methods of Enzymatic Analysis, Vol. XIII, Academic Press, New York, 1974, pp .430, 449, 473,509-510, 574~579. 2 Cooper, A.J.L. and Plum, F., Biochemistry and physiology of brain ammonia, Physiol. Rev., 67 (1987) 440-519. 3 Dagher, S.M. and Deal Jr., W.C., Glyceraldehyde-3-phosphate dehydrogenase from pig liver. In W.A. Wood (Ed.), Methods in Enzymology, Vol. 89, Academic Press, NY, pp. 310-316.

40 4 Duffy, T.E. and Plum, F., Hepatic encephalopathy. In 1. Arias, H. Popper, D. Schachter and D.A. Shafritz (Eds.), The Liver: Biology and Pathobiology, Raven, New York, 1982, pp. 693-715. 5 Hawkins, R.A., Miller, A.L., Nielson, R.C. and Veech, R.L., The acute action of ammonia on rat brain metabolism in vivo, Biochem. J., 134 (1973) 1001-1008. 6 Hindfelt, B., Plum, F. and Duffy, T.E., Effect of acute ammonia intoxication on cerebral metabolism in rats with portacaval shunts, J. Clin. Invest., 59 (1977) 386-396. 7 Lowry, O.H. and Passonneau, J.V., Kinetic evidence for multiple binding sites on phosphofructokinase, J. Biol. Chem., 241 (1966) 2268-2279. 8 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J.. Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265 275. 9 Murthy, Ch.R.K. and Hertz, L., Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia, Neurochem. Res., 13 (1988) 57 61.

10 Ratnakumari, L., Subbalakshmi, G.Y.C.V. and Murthy, Ch.R.K., Cerebral citric acid cycle enzymes in methionine sulphoximine toxicity, J. Neurosci. Res., 14 (1985) 449 459. 11 Ratnakumari, k., Subbalakshmi, G.Y.C.V. and Murthy, Ch.R.K., Acute effects of ammonia on the enzymes of citric acid cycle in rat brain, Neurochem. Int., 8 (1986) 115 120. 12 Ratnakumari, L. and Murthy, Ch.R.K., Activities of pyruvate dehydrogenase, enzymes of citric acid cycle and aminotransferases in the subcellular fractions of cerebral cortex in normal and hyperammonemic rats, Neurochem. Res., 14 (1989) 221 228. 13 Sugden, P.H. and Newsholme, E.A., The effects of ammonium, inorganic phosphate and potassium ions on the activity of phosphofructokinase from muscle and nervous tissues of vertebrates and invertebrates, Biochem. J., 150 (1975) 113 122.