Life Sciences, Vol. 48, pp. 561-567 Printed in the U.S.A.
EFFECTS
OF K A I N A T E
ON GLUCOSE M E T A B O L I S I N G IN T H E B R A I N
Pergamon Pres;
ENZYMES
Sai Fan Leong Department of Physiology, National University of Singapore Kent Ridge, Singapore 0511 (Received in final form December 4, 1990)
Summary Hexokinase and g l u c o s e - 6 - p h o s p h a t e dehydrogenase activities were studied in brain regions after intraventricu l a r injection of kainic acid. Hexokinase activity was decreased by 10-15% in various regions while glucose-6phosphate dehydrogenase activity remained unaltered. Soluble hexokinase activity, which remained the smaller fraction of total hexokinase activity, showed slightly more dramatic decreases of 15-35% compared to normal activities in brain regions. This decrease of hexokinase activity in the cytosolic compartment could partly a c c o u n t for the k a i n a t e - i n d u c e d decreases s e e n in glucose metabolism. Brain h e x o k i n a s e (ATP: D - h e x o s e 6-phosphotransferase; E.C. 2.7.1.1) is the first enzyme involved in the metabolism of glucose, which is the normal obligatory energy source for the tissue. This enzyme has been regarded as the major regulatory point for glycolysis (13,19). Over 80% of hexokinase activity is known to be associated with the particulate fraction of brain homogenares (3,7,8). The e q u i l i b r i u m of hexokinase particulate activity with the soluble compartment is, however, dependent upon the energy status of the brain, as revealed in the in vivo studies of Knull et al (9). In unstressed rats, the basal energy metabolism varies as much as three fold in different brain regions (6). Basal energy metabolism in the brain regions have also been found to be correlated to the hexokinase a m o u n t s (20). G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (E.C. 1.1.1.49) c o n s t i t u t e s the first regulatory enzyme in the pentose phosphate pathway which u t i l i s e s g l u c o s e for g e n e r a t i o n of r e d u c i n g e q u i v a l e n t s for biosynthesis. The pathway, however, is a minor one compared to glycolysis in terms of glucose flux and enzyme activities in the adult brain (13,19). Both parenteral and intracerebral injections of kainic acid, an excitatory d i c a r b o x y l i c amino acid exhibiting potent n e u r o t o x i c i ty (18,14,2), increase local glucose u t i l i s a t i o n in brain areas 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
562
Kainate and Glucose Metabolising Enzymes
Vol. 48, No. 6, 1991
which are sensitive to the kainic acid neurotoxicity (21,11,4). Although studies have indicated that the effects on kainic acid injections into brain regions cause initially an increase during s e i z u r e and f o l l o w e d by a d e c r e a s e in g l u c o s e metabolism (21,11,1), the changes in the activities of the glucose metabolining enzymes have not been examined after such an insult.
NATERI&LS
AND METHODS
Male Sprague-Dawley rats weighing 250-300 g were anesthetised with sodium pentobarbitone (40 mg/kg body weight). Injections of kainic acid in i00 mM - sodium phosphate buffer, pH 7.4 or the buffer alone (for control) were carried out using a nanolitre pump attached with a glass microcapillary which was previously pulled to a fine tip. The tip was introduced into the brain through a burr hole in the skull. Bilateral injections into the ventricles of kainic acid (2.5 + 2.5 nmoles) were performed with the following c o o r d i n a t e s (15): AP 1.0 mm posterior to bregma, ML 1.4 mm, and DV 3.4 mm. The total volume injected per animal was 0.5 ul. The m i c r o c a p i l l a r y tip was slowly withdrawn three minutes after the completion of each injection. Animals were allowed to recover and to feed ad libitum for 7 days before use. Control animals were injected --wi~ I00 mM - sodium phosphate buffer at the same pH and treated in the same way as the kainic acid injected animals. On the 7th day, the animals were decapitated and brains dissected at 4°C according to the method previously described (5). Each region was homogenised (1%10 w / v ) i n a medium containing 250 mM sucrose, 0.5 mM -K+-EDTA and i 0 m M - - TrisCl, pH 7.4. An aliquot of each brain region homogenate was c e n t r i f u g e d at 16,000g for 10 min in a Kubota microfuge. The resultant supernatant was assayed for soluble cytosolic hexokinase activity. Hexokinase and gluc o s e - 6 - p h o s p h a t e dehydrogenase were determined by methods previously described (i0). Assays were monitored at 37°C.
RESULTS
The a c t i v i t i e s of hexokinase in the various brain regions of the normal animals (Table I) show that the cortex and hippocampus have the highest activities, followed by the cerebellum. The hypothalamus, striatum and midbrain cluster, have similar level of activities, while the medulla oblongata and pons region has the lowest activity. This ranking is similar to that reported in previous studies (i0). Similar ranking of activity is observed for both the phosphate buffer injected control and kainate injected animals in this study. The regional brain hexokinase activities did not appear to change in the sodium phosphate buffer injected controls when compared to c o r r e s p o n d i n g values in the normal rat brain (Table I). However, hexokinase activities in most regions showed decreases after kainic acid injection when compared to the corresponding activities in injected control or normal animals. These decreases, in the range of i0 to 15% below normal values (Table I and Fig I) are varied across the brain regions.
Vol. 48, No. 6, 1991
Kainate and Glucose Metabolislng Enzymes
TABLE Total Brain
Regions
Hexokinase
I
Activity
Normal
563
in B r a i n
Regions
Injected Control
Kainic Acid Injected
Cerebellum
34.26
+ 0.90
35.28
+ 1.05
31.67
+ 1.01 b
Medulla Oblongata & Pons Hypothalamus
20.42
+ 0.54
19.18
+ 0.19
16.58
+ 0.28 b
33.32
+ 0.91
33.94
+ 0.05
28.44
+ 1.73 a
Striatum
32.57
+ 0.44
29.28
+ 2.23
26.71
+ 0.76 n s
Midbrain
31.63
+ 1.00
30.37
+ 1.40
25.84
+ 0.65 b
Hippocampus
39.91
+ 0.94
37.21
+ 0.99
32.08
+ 0.81 b
Cortex
39.06
+ 1.27
41.21
+ 0.04
34.99
+ 0.29 b
Activity is e x p r e s s e d in u m o l / m i n / g wet weight of tissue (mean+SD). The r e s u l t s w e r e from 3 or m o r e d i s t i n c t e x p e r i m e n t s , w i t h ~ or m o r e a n i m a l s u s e d in e a c h e x p e r i m e n t . Enzyme activity was a s s a y e d in d u p l i c a t e at 2 d i f f e r e n t p r o t e i n c o n c e n t r a t i o n s in each experiment. The t - t e s t w a s used to e v a l u a t e d i f f e r e n c e s b e t w e e n the m e a n s of c o n t r o l a n d t r e a t m e n t g r o u p s : a = p
EFFECTS
OF K A I N A T E
ON GLUCOSE M E T A B O L I S I N G IN T H E B R A I N
Pergamon Pres;
ENZYMES
Sai Fan Leong Department of Physiology, National University of Singapore Kent Ridge, Singapore 0511 (Received in final form December 4, 1990)
Summary Hexokinase and g l u c o s e - 6 - p h o s p h a t e dehydrogenase activities were studied in brain regions after intraventricu l a r injection of kainic acid. Hexokinase activity was decreased by 10-15% in various regions while glucose-6phosphate dehydrogenase activity remained unaltered. Soluble hexokinase activity, which remained the smaller fraction of total hexokinase activity, showed slightly more dramatic decreases of 15-35% compared to normal activities in brain regions. This decrease of hexokinase activity in the cytosolic compartment could partly a c c o u n t for the k a i n a t e - i n d u c e d decreases s e e n in glucose metabolism. Brain h e x o k i n a s e (ATP: D - h e x o s e 6-phosphotransferase; E.C. 2.7.1.1) is the first enzyme involved in the metabolism of glucose, which is the normal obligatory energy source for the tissue. This enzyme has been regarded as the major regulatory point for glycolysis (13,19). Over 80% of hexokinase activity is known to be associated with the particulate fraction of brain homogenares (3,7,8). The e q u i l i b r i u m of hexokinase particulate activity with the soluble compartment is, however, dependent upon the energy status of the brain, as revealed in the in vivo studies of Knull et al (9). In unstressed rats, the basal energy metabolism varies as much as three fold in different brain regions (6). Basal energy metabolism in the brain regions have also been found to be correlated to the hexokinase a m o u n t s (20). G l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e (E.C. 1.1.1.49) c o n s t i t u t e s the first regulatory enzyme in the pentose phosphate pathway which u t i l i s e s g l u c o s e for g e n e r a t i o n of r e d u c i n g e q u i v a l e n t s for biosynthesis. The pathway, however, is a minor one compared to glycolysis in terms of glucose flux and enzyme activities in the adult brain (13,19). Both parenteral and intracerebral injections of kainic acid, an excitatory d i c a r b o x y l i c amino acid exhibiting potent n e u r o t o x i c i ty (18,14,2), increase local glucose u t i l i s a t i o n in brain areas 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
562
Kainate and Glucose Metabolising Enzymes
Vol. 48, No. 6, 1991
which are sensitive to the kainic acid neurotoxicity (21,11,4). Although studies have indicated that the effects on kainic acid injections into brain regions cause initially an increase during s e i z u r e and f o l l o w e d by a d e c r e a s e in g l u c o s e metabolism (21,11,1), the changes in the activities of the glucose metabolining enzymes have not been examined after such an insult.
NATERI&LS
AND METHODS
Male Sprague-Dawley rats weighing 250-300 g were anesthetised with sodium pentobarbitone (40 mg/kg body weight). Injections of kainic acid in i00 mM - sodium phosphate buffer, pH 7.4 or the buffer alone (for control) were carried out using a nanolitre pump attached with a glass microcapillary which was previously pulled to a fine tip. The tip was introduced into the brain through a burr hole in the skull. Bilateral injections into the ventricles of kainic acid (2.5 + 2.5 nmoles) were performed with the following c o o r d i n a t e s (15): AP 1.0 mm posterior to bregma, ML 1.4 mm, and DV 3.4 mm. The total volume injected per animal was 0.5 ul. The m i c r o c a p i l l a r y tip was slowly withdrawn three minutes after the completion of each injection. Animals were allowed to recover and to feed ad libitum for 7 days before use. Control animals were injected --wi~ I00 mM - sodium phosphate buffer at the same pH and treated in the same way as the kainic acid injected animals. On the 7th day, the animals were decapitated and brains dissected at 4°C according to the method previously described (5). Each region was homogenised (1%10 w / v ) i n a medium containing 250 mM sucrose, 0.5 mM -K+-EDTA and i 0 m M - - TrisCl, pH 7.4. An aliquot of each brain region homogenate was c e n t r i f u g e d at 16,000g for 10 min in a Kubota microfuge. The resultant supernatant was assayed for soluble cytosolic hexokinase activity. Hexokinase and gluc o s e - 6 - p h o s p h a t e dehydrogenase were determined by methods previously described (i0). Assays were monitored at 37°C.
RESULTS
The a c t i v i t i e s of hexokinase in the various brain regions of the normal animals (Table I) show that the cortex and hippocampus have the highest activities, followed by the cerebellum. The hypothalamus, striatum and midbrain cluster, have similar level of activities, while the medulla oblongata and pons region has the lowest activity. This ranking is similar to that reported in previous studies (i0). Similar ranking of activity is observed for both the phosphate buffer injected control and kainate injected animals in this study. The regional brain hexokinase activities did not appear to change in the sodium phosphate buffer injected controls when compared to c o r r e s p o n d i n g values in the normal rat brain (Table I). However, hexokinase activities in most regions showed decreases after kainic acid injection when compared to the corresponding activities in injected control or normal animals. These decreases, in the range of i0 to 15% below normal values (Table I and Fig I) are varied across the brain regions.
Vol. 48, No. 6, 1991
Kainate and Glucose Metabolislng Enzymes
TABLE Total Brain
Regions
Hexokinase
I
Activity
Normal
563
in B r a i n
Regions
Injected Control
Kainic Acid Injected
Cerebellum
34.26
+ 0.90
35.28
+ 1.05
31.67
+ 1.01 b
Medulla Oblongata & Pons Hypothalamus
20.42
+ 0.54
19.18
+ 0.19
16.58
+ 0.28 b
33.32
+ 0.91
33.94
+ 0.05
28.44
+ 1.73 a
Striatum
32.57
+ 0.44
29.28
+ 2.23
26.71
+ 0.76 n s
Midbrain
31.63
+ 1.00
30.37
+ 1.40
25.84
+ 0.65 b
Hippocampus
39.91
+ 0.94
37.21
+ 0.99
32.08
+ 0.81 b
Cortex
39.06
+ 1.27
41.21
+ 0.04
34.99
+ 0.29 b
Activity is e x p r e s s e d in u m o l / m i n / g wet weight of tissue (mean+SD). The r e s u l t s w e r e from 3 or m o r e d i s t i n c t e x p e r i m e n t s , w i t h ~ or m o r e a n i m a l s u s e d in e a c h e x p e r i m e n t . Enzyme activity was a s s a y e d in d u p l i c a t e at 2 d i f f e r e n t p r o t e i n c o n c e n t r a t i o n s in each experiment. The t - t e s t w a s used to e v a l u a t e d i f f e r e n c e s b e t w e e n the m e a n s of c o n t r o l a n d t r e a t m e n t g r o u p s : a = p
