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Effect of food deprivation on glutathione and amino acid levels in brain and liver of young and aged rats
BRAIN RESEARCH ELSEVIER
Brain Research 678 (1995) 259-264
Research report
Effect of food deprivation on glutathione and amino acid levels in brain and liver of young and aged rats M. Benuck *, M. Banay-Schwartz, T. DeGuzman, A. Lajtha The Nathan S. Kline Institute for Psychiatric Research, Centerfor Neurochemistry, 140 Old Orangeburg Rd., Orangeburg, NY 10962, USA
Accepted 31 January 1995
Abstract
The effect of short-term food deprivation on glutathione (GSH) and amino acid levels in brain regions of young and aged rats was compared with changes observed in liver. Animals aged 3 months and 24 months were deprived of food for 48 h. GSH and amino acid levels from cerebral cortex, cerebellum, pons medulla, and liver were assayed and compared with levels in animals of the same age fed normal diets. In liver in both young and old rats, GSH levels fell 30%, from 13/zmol/g tissue to 8.7/xmol/g tissue. Significant changes were observed in other amino acids, including an increase of 30-50% in methionine, glycine, and glutamine, and a decrease of 30-50% in alanine in liver of both young and aged rats, and a 4-fold increase in taurine in young. In brain, little change was observed upon food deprivation. No decrease was observed in GSH, and only small changes were observed in other amino acids. In the aged animal aspartate, glutamate, and alanine levels were slightly lower; tyrosine in cerebellum was reduced by 30%, and both glycine and tyrosine in the pons medulla were reduced by 20-30%. In the brain areas examined, levels of GSH ranged from 1 - 2 / z m o l / g in young and 0.8-1.4/zmol/g in old; with levels in pons medulla being lower than those in cerebral cortex. In brain, in contrast to liver, levels were scarcely affected by short-term food deprivation. The relative stability of amino acids paralleled that of brain proteins, which did not significantly decrease even under conditions in which protein content of most other tissues was greatly reduced, indicating specific control of cerebral protein metabolism in protection in malnutrition. Keywords: Glutathione; Amino acid; Aging; Brain area; Liver; Malnutrition
Previous studies have shown that malnutrition and starvation result in loss of protein from various tissues, while brain proteins are preserved even under extreme conditions. Protease activity is also affected to a much greater extent in liver and muscle than in brain [10,24]. The latter effect may be altered by diet; protease activity was increased in brain and liver after feeding protein restricted diets supplemented with fatty acids and antioxidants [10]. We are interested in the mechanisms involved in protection of brain protein content in malnutrition. In a continuation of this series of studies, comparing effects of malnutrition on brain with its effects on other organs, we examined changes after brief starvation in the levels of glutathione ( G S H ) and amino acids in brain and liver of young and aged rats, since G S H level changes were observed in some organs after malnutrition.
* Corresponding author. Fax: (1) (914) 365-6107. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00204-9
Several studies have demonstrated a decrease in hepatic G S H after short-term food deprivation [13,36]. Glutathione is involved in various cellular functions, including neutralization of free radicals generated by oxidative stress. Oxidative stress affects proteolytic activity in brain [11,12], and oxidative mechanisms may be partially responsible for changes in protein metabolism in aging, with the production of free radicals contributing to the aging process [27,33]. Hence, the aging brain may be more sensitive to the effects of malnutrition.
1. Materials and methods Chemicals and reagents. 1-Napthylisocyanate was purchased from Aldrich Chemical Co. (Milwaukee, WI); G S H and amino acids were from Sigma Chemical Co. (St. Louis, MO); H P L C solvents were from Fisher Chemical Co. (Springfield, N J).
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Preparation o f tissue extracts. S p r a g u e - D a w l e y male rats, 3 m o n t h s a n d 24 m o n t h s of age, were used in this study. A n i m a l s were supplied with food a n d water at all times. T o e x a m i n e effects of short term starvation, food was removed for 48 h. A n i m a l s were killed by d e c a p i t a t i o n after prior sedation with CO 2, a n d liver a n d b r a i n were r e m o v e d rapidly a n d cleaned, a n d the b r a i n was divided into three sections, cerebral cortex, cerebellum, a n d ports medulla. A f t e r dissection a n d cleaning, tissue was frozen at - 8 0 ° C. Prior to assay, a section of tissue was removed, weighed, a n d homogenized in 3% perchloric acid ( 1 / 1 0 ) c o n t a i n i n g an internal s t a n d a r d of n o r l e u c i n e , 62.5 n m o l / m l . T h e hom o g e n a t e was microfuged, a n d the s u p e r n a t a n t was adjusted to n e u t r a l p H with sodium borate buffer a n d sodium hydroxide. N e u t r a l i z a t i o n was accomplished by a d d i n g 160 tzl of 1 M borate buffer, p H 6.2, 40 ~zl of 5N sodium hydroxide, a n d 200 Izl of water to 4 0 0 / z l of the s u p e r n a t a n t . Deriuatization. T h e p r o c e d u r e was similar to that described by Neidle et al. [30]. Briefly, a n aliquot of the n e u t r a l i z e d perchloric acid extract (800 ~1) was a d d e d to a n equal v o l u m e of the derivitizing agent 1-naphthylisocyanate (5 / z l / m l in dry a c e t o n e ) with i n s t a n t mixing. A f t e r 45 s, the reaction was stopped with the a d d i t i o n of 4 - 5 ml of cyclohexane. T h e mixture was stirred vigorously, and the u p p e r layer was discarded. Excess r e a g e n t was r e m o v e d by r e p e a t i n g the extraction 4 - 5 times. T h e derivatized sample was t h e n stored at - 20 ° C until analysis. For a m i n o acid analysis, a Vydac 201TP54 C18 reversed-phase H P L C c o l u m n was used with an auto-
m a t e d a m i n o acid analyzer a n d fluorescence detector m a n u f a c t u r e d by S h i m a d z u I n s t r u m e n t s , Columbia, MD. F o r analysis, 2 0 - 6 0 izl of sample e q u i v a l e n t to 0.625-1.8 n m o l of n o r l e u c i n e was injected. G S H a n d o t h e r a m i n o acids were d e t e c t e d using 238 n m as excitation m a x i m u m a n d 385 n m as emission m a x i m u m . For e s t i m a t i o n of relative fluorescence, a n d time of e l u t i o n of various a m i n o acids, a s t a n d a r d a m i n o acid mixture (Sigma AA-S-18) was used, to which GSH, glutamine, GABA, taurine, ornithine, and norleucine were added. This m e t h o d can detect as little as 0.5 nmol of G S H a n d picomolar a m o u n t s of o t h e r a m i n o acids. F o r c h r o m a t o g r a p h y of the a m i n o acid derivatives, two g r a d i e n t systems were used. F o r all a m i n o acids, except G S H , the c o l u m n was e q u i l i b r a t e d in buffer c o n t a i n i n g 2% 0.1 M N a H 2 P O 4 , 0.1 M sodium acetate ( p H 5.7), 2% m e t h a n o l , a n d 4% acetonitrile (buffer A). A f t e r injection of the sample, a n ±socratic g r a d i e n t in buffer A was applied for 30 m i n followed by a linear g r a d i e n t b e t w e e n buffer A a n d buffer B over the next 90 m i n (buffer B c o n t a i n s 50% acetonitrile a n d 10% methanol). T h e a m o u n t of buffer B r e a c h e d 70% of the total after 2 h. With this system, g l u t a t h i o n e is e l u t e d i m m e d i a t e l y after the a m m o n i a peak, but is often not well separated. F o r b e t t e r s e p a r a t i o n of glutathione, the e q u i l i b r a t i o n buffer (buffer A) was modified to c o n t a i n 12% of the 0.1 M N a H 2 P O 4 a n d 0.1 M sodium acetate solution ( p H 5.7), 10% m e t h a n o l , a n d 6% acetonitrile. A f t e r 30 min, a linear g r a d i e n t was applied, with buffer B increasing from 0 to 50%. T h e c o l u m n was r e g e n e r a t e d in buffer B. I n this sec-
Table 1 Amino acid levels in liver of young and aged rats fasted for 48 h compared to controls Amino acid
LIVER Young
Aspartate Glutamate Serine Glycine Glutamine Taurine Threonine Alanine GSH Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Ornithine
Aged
Control
Starved
Control
Starved
0.83 +_0.05 2.11 ± 0.18 0.76 ± 0.13 1.72 ± 0.07 3.78 _+0.17 1.11 ± 0.11 0.42 __+0.04 2.14 _+0.14 13.9 _+0.53 0.19 ± 0.01 0.30 _+0.02 0.08 _+0.01 0.18 _+0.01 0.38 + 0.02 0.15 ± 0.01 0.50 + 0.06
1.03 _+0.08 * 1.84 ± 0.15 0.70 _+0.07 2.47 _+0.06 * * * 5.31 ± 0.23 * * * 4.11 _+0.58 * * * 0.36 ± 0.03 1.06 ± 0,11 * * * 8.79 ± 0.33 * * * 0.21 _+0.02 0.33 ± 0.02 0.12 ± 0.01 * * * 0.19 ± 0.01 0.43 + 0.03 0.18 ± 0.01 * 0.56 _+0.05
1.12 + 0.10 2.04 _+0.12 0.96 _+0.18 2.21 ± 0.14 4.93 _+0.31 3.59 __+_0.92 0.70 _+0.07 2.92 ± 0.19 12.0 _+0.49 0.17 _+0.02 0.35 ± 0.03 0.07 ± 0.01 0.22 ± 0.02 0.44 ± 0.03 0.17 ± 0.02 0.53 ± 0.08
1.26 ± 0.12 1.68 +_0.15 1.01 + 0.15 2.94 ± 0.17 6.27 +_0.35 3.87 _+1.01 0.56 _+0.08 1.89 ± 0.22 8.63 ± 0.80 0.18 ± 0.02 0.36 + 0.02 0.11 ± 0.01 0.22 + 0.01 0.45 _+0.03 0.18 ± 0.01 0.52 __+__0.06
** **
*** ***
* P < 0.05; * * P < 0.01; * * * P < 0.002. Values shown are the averages _+S.E.M. (tzmol/g) from 4-8 animals, 3 months and 24 months of age. GSH and amino acid analyses are described in Materials and methods.
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M. Benuck et al. / Brain Research 678 (1995) 259-264 Table 2 Amino acid levels in cortex of young and aged rats fasted for 48 h compared to controls Amino acid
CORTEX Young
Aspartate Glutamate Serine Glycine Glutamine Taurine Threonine Alanine GABA GSH Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Ornithine
Aged
Control
Starved
Control
Starved
3.85 ± 0.06 10.3 ± 0.54 1.12 ± 0.10 1.02 + 0.08 5.19 ± 0.18 5.2 ± 0.42 0.65 ± 0.02 0.56 + 0.02 2.66 ± 0.22 2.14 + 0.20 0.088 + 0.006 0.095 ± 0.003 0.039 + 0.003 0.047 ± 0.002 0.095 + 0.004 0.059 ± 0.002 0.049 ± 0.003
3.98 ± 0.14 10.2 ± 0.39 1.23 ± 0.06 1.02 + 0.04 5.34 + 0.17 5.21 ± 0.14 0.51 + 0.02 * * 0.56 + 0.02 2.38 ± 0.20 1.99 + 0.19 0.081 + 0.004 0.101 + 0.003 0.042 + 0.003 0.049 ± 0.001 0.099 + 0.003 0.061 + 0.003 0.045 ± 0.002
4.08 ± 0.20 9.79 ± 0.19 1.46 ± 0.11 1.16 + 0.06 5.33 + 0.27 5.02 ± 0.21 0.62 +_ 0.03 0.74 ± 0.06 2.51 ± 0.13 1.36 ± 0.17 0.07 ± 0.01 0.089 ± 0.003 0.041 ± 0.004 0,045 ± 0.002 0.10 ± 0,005 0,058 ± 0.006 0,048 ± 0.005
3.49 ± 0.16 * 8.95 :t: 0.30 * 1.44 ± 0.12 1.08 ± 0.08 5.11 + 0.21 4.59 + 0.18 0.52 ± 0.03 * 0.57 _+ 0.04 * 2.60 ± 0.23 1.40 ± 0.12 0.05 ± 0.004 0.084 ± 0.003 0.043 ± 0.003 0.046 ± 0.002 0.093 ± 0.004 0.058 + 0.004 0.048 ± 0.004
* P < 0,05; * * P < 0.0002. Values shown are the averages ± S.E.M. (/~mol/g) from 4 - 8 animals, 3 months and 24 months of age. GSH and amino acid analyses are described in Materials and methods.
ond system, glutathione is well separated from other amino acids, being eluted between tyrosine and valine.
2. Results
The response to short-term food deprivation was sharply different in liver from that in brain. In the liver, glutathione levels decreased 30% upon starva-
tion, in both young and aged animals, while no change in GSH levels was observed in brain. Furthermore, significant changes were observed in several amino acids in the liver of both young and aged rats; in brain, relatively minor changes in several amino acids were seen mostly in the aged animal. Glutathione levels in liver of young and aged rats were 12-14 tzmol/g. Brain levels ranged from 1 to 2 /~mol/g. Levels in the brain decreased with age; GSH
Table 3 Amino acid levels in cerebellum of young and aged rats fasted for 48 h compared to controls Amino acid
CEREBELLUM Young
Aspartate Glutamate Serine Glycine Glutamine Taurine Threonine Alan±he GABA GSH Tyrosine Valine Methionine Isoleucine Leucine Phenyialanine Ornithine
Aged
Control
Starved
Control
Starved
2.96 + 0.11 8.89 + 0.23 0.60 ± 0,06 0.83 + 0,02 4.50 + 0.22 4.07 ± 0.09 0.67 ± 0,02 0.66 + 0.03 1.89 ± 0.08 1.03 ± 0.04 0,086 + 0.006 0.075 + 0.002 0.046 ± 0.002 0.040 ± 0.001 0.084 ± 0.003 0,065 ± 0.002 0.067 + 0.004
3.19 + 0.12 9.54 + 0.23 0.63 + 0.06 0.75 ± 0.04 4.76 ± 0.22 4.25 ± 0.10 0.53 ± 0.02 * 0.62 ± 0.03 1.89 + 0.05 1.19 ± 0.07 0.081 ± 0.004 0.077 ± 0,002 0.049 ± 0.001 0.040 ± 0.001 0.089 + 0.002 0.063 + 0.001 0.066 ± 0.003
2.98 + 0.15 9.38 + 0.35 0.80 + 0.08 1.11 + 0.08 4.58 ± 0.45 3.48 ± 0.20 0.61 + 0.06 0.69 + 0.06 2.03 ± 0.14 1.31 ± 0.17 0.072 + 0.008 0.073 ± 0.005 0.044 + 0.004 0.040 ± 0.002 0.090 ± 0.006 0.062 ± 0,005 0.081 + 0.005
2.88 + 0.20 9.04 ± 0.23 0.74 ± 0.09 1.06 + 0.08 4.10 ± 0,18 3.33 ± 0.11 0.50 ± 0.03 0.57 + 0.05 1.84 + 0.08 1.25 ± 0.13 0.051 ± 0.004 " 0.062 + 0.003 0.038 ± 0.002 0.035 ± 0.002 0.077 ± 0.004 0.052 + 0.003 0.071 ± 0.006
* P < 0.05. Values shown are the averages + S.E.M. ( # m o l / g ) from 5 - 9 animals, 3 months and 24 months of age. GSH and amino acid analyses are described in Materials and methods.
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Table 4 Amino acid levels in pons medulla of young and aged rats fasted for 48 h compared to controls Amino acid PONS MEDULLA Young Aspartate Glutamate Serine Glycine Glutamine Taurine Threonine Alanine GABA GSH Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Ornithine
Aged
Control
Starved
Control
Starved
3.40 ± 0.05 5.44 _+0.18 0.97 _+0.02 3.33 + 0.09 3.17 _+0.10 1.45 ± 0.04 0.63 _+0.02 0.34 ± 0.01 1.49 _+0.06 1.08 ± 0.06 0.074 + 0.006 0.084 ± 0.002 0.031 ± 0.002 0.039 ± 0.001 0.085 ± 0.002 0.055 ± 0.001 0.086 ± 0.006
3.48 _+0.14 5.39 _+0.20 0.85 ± 0.09 3.18 + 0.08 3.14 ± 0.07 1.42 _+0.05 0.50 ± 0.02 * * 0.34 + 0.02 1.66 ± 0.09 1.04 _+0.12 0.071 + 0.005 0.085 ± 0.003 0.028 ± 0.001 0.040 ± 0.001 0.086 _+0.003 0.056 _+0.001 0.079 + 0.002
2.84 _+0.10 4.82 _+0.15 0.56 _+0.05 3.10 _+0.22 3.41 ± 0.15 1.43 ± 0.05 0.60 + 0.06 0.44 _+0.04 1.49 ± 0.14 0.81 + 0.18 0.065 ± 0.007 0.079 ± 0.007 0.036 + 0.004 0.042 ± 0.003 0.099 ± 0.010 0.060 ± 0.005 0.096 ± 0.005
2.71 ± 0.10 5.18 + 0.30 0.48 + 0.05 2.56 + 0.16 * 3.56 ± 0.22 1.72 ± 0.21 0.49 + 0.03 0.37 + 0.04 1.85 ± 0.28 0.66 + 0.12 0.047 ± 0.005 * 0.073 ± 0.004 0.037 ± 0.003 0.040 ± 0.002 0.084 ± 0.005 0.052 ± 0.003 0.085 ± 0.003
* P < 0.05; * * P < 0.002. Values shown are the averages _+S.E.M. (/~mol/g) from 4-6 animals, 3 months and 24 monthsof age. GSH and amino acid analyses are described in Materials and methods.
in c e r e b r a l cortex fell from 2.0 ~ m o l / g in the y o u n g a n i m a l to 1.4 / x m o i / g at 24 m o n t h s of age (Tables
1--4). Liuer. H e p a t i c G S H was r e d u c e d in b o t h young a n d a g e d rats after food d e p r i v a t i o n , d e c r e a s i n g from 1 2 - 1 4 / x m o l / g to 8 . 6 - 8 . 8 / z m o l / g . A m o n g c h a n g e s seen in o t h e r a m i n o acids were i n c r e a s e s in glycine, g l u t a m i n e , taurine, a n d m e t h i o n i n e a n d a d e c r e a s e in alanine. A l a n i n e levels in the starved animals were d e c r e a s e d 50 a n d 35% in the y o u n g a n d a g e d respectively. In contrast, levels o f glycine, g l u t a m i n e , a n d m e t h i o n i n e i n c r e a s e d 3 0 - 5 0 % in b o t h y o u n g a n d a g e d animals. T a u r i n e levels i n c r e a s e d fourfold in the y o u n g liver, but s h o w e d no c h a n g e after food d e p r i v a t i o n in the a g e d liver. All t h e s e c h a n g e s were statistically significant at the P = 0.002 level. Smaller, b u t significant, increases o c c u r r e d in levels o f p h e n y l a l a n i n e a n d asp a r t a t e in the y o u n g animal u p o n food d e p r i v a t i o n , b u t not in the aged. O f the r e m a i n i n g a m i n o acids m e a sured, little c h a n g e was o b s e r v e d in y o u n g o r old after food d e p r i v a t i o n ( T a b l e 1). Brain. In the brain, s h o r t - t e r m starvation h a d little effect on g l u t a t h i o n e o r a m i n o acid levels. G l u t a t h i o n e levels did not c h a n g e significantly in any of the t h r e e a r e a s e x a m i n e d , e i t h e r in the y o u n g or t h e a g e d animal. A m i n o acid levels, which w e r e significantly a l t e r e d in liver, w e r e not a f f e c t e d in the b r a i n o f the young animal. In t h e y o u n g a n i m a l only levels o f t h r e o n i n e were reduced (Tables 2-4). In t h e a g e d animal, small but significant c h a n g e s did occur in several a m i n o acids a f t e r food d e p r i v a t i o n . In
the c e r e b r a l cortex, d e c r e a s e s of 1 0 - 1 5 % in levels of aspartate, glutamate, and threonine and a decrease of 23% in alan±he levels w e r e n o t e d . N o significant c h a n g e s were n o t e d in o t h e r a m i n o acids. In the cerebellum, tyrosine d e c r e a s e d by 30%, while in the p o n s m e d u l l a , glycine a n d tyrosine showed a 2 0 - 3 0 % decrease. N o o t h e r significant d i f f e r e n c e s in a m i n o acid levels w e r e n o t e d ( T a b l e s 2 - 4 ) .
3. Discussion This study c o m p a r e s the effect o f short t e r m food d e p r i v a t i o n on levels of G S H a n d a m i n o acids in b r a i n a n d liver. In liver, several studies have shown that starvation o r d i e t a r y r e s t r i c t i o n r e d u c e s liver G S H levels [20,22,26,36]. V o g t a n d R i c h i e [36] f o u n d t h a t t h e G S H r e s p o n s e to 24 h o f fasting is m o r e severe in the liver o f a g e d mice t h a n in that of y o u n g e r mice. In o u r study, we f o u n d a d e p l e t i o n in h e p a t i c G S H o f 33% in b o t h a d u l t a n d a g e d rats after 48 h of fasting. O t h e r studies also s h o w e d that food d e p r i v a t i o n s t i m u l a t e s a c h a n g e in h e p a t i c G S H turnover, with a d e c r e a s e in h e p a t i c G S H a f t e r s h o r t - t e r m f o o d d e p r i v a t i o n [13]. It seems that r e d u c t i o n o f liver G S H levels is a sensitive early index of m a l n u t r i t i o n . Levels o f G S H in b r a i n a r e n o t r e d u c e d , e i t h e r in y o u n g or a g e d animals, by food d e p r i v a t i o n o f relatively short d u r a t i o n . O n l y small c h a n g e s in several a m i n o acids a r e found, p r i m a r i l y in the a g e d brain. In contrast, in the liver m o r e a m i n o acids a r e a l t e r e d
M. Benuck et al. / Brain Research 678 (1995) 259-264
significantly during starvation. Glutathione is decreased from 13 to 9 ~ m o l / g tissue. A decrease in liver GSH upon brief starvation may reflect an efflux of GSH from the liver to blood to maintain GSH levels in other tissues [22,23,28,36]. Our procedure for assay of glutathione does not separate the oxidized form of glutathione (GSSG) from reduced glutathione (GSH). Previous studies with rat or mouse brain have found GSSG to constitute only a few percent of total glutathione [14,16,32]. In liver, glutathione is also present mainly in the reduced form [23]. GSSG levels may change under conditions associated with oxidative stress; intracerebroventricular injection of tert-butylhydroperoxide, an oxidative stress inducing agent, significantly increased GSSG levels in mouse brain [1]. Other amino acid levels that are affected in liver include methionine, glycine, glutamine, taurine, and alanine. Methionine and glycine are precursors in GSH synthesis, and dietary deficiency of methionine prevents GSH synthesis [36]. Methionine has been used to restore GSH levels of starved animals [22]. Glutamine supplementation has been used to maintain liver GSH under various traumatic conditions such as hepatic injury [13]. Increase in the levels of the amino acids that are precursors of GSH (methionine, glycine, and glutamine) may be a response in liver to the enhanced GSH efflux during starvation. Taurine serves an essential role in development [34]. Rat liver has a high capacity for taurine biosynthesis, and taurine concentration in liver and brain is generally greater in the developing animal than in the mature animal [35]. In this study, taurine levels in the liver of young animals were lower than in the aged. The increase found in the young upon starvation may be related to liver lipid metabolism and the function of taurine in the transport of bile acids. Short-term starvation results in a significant loss of protein in liver [21,24]. Alanine is one amino acid that modulates proteolytic activity in the liver during caloric deprivation [29]. The reduction in alanine levels in liver observed here in both young and adult during starvation may be a reflection of the role of alanine as modulator of proteolytic activity, its levels varying inversely with liver proteolysis [29]. The concentration of most amino acids in the protein-bound form is several hundred fold of that in the free amino acid pool. Therefore, the breakdown of even one percent of protein would increase free amino acid content several fold. The fact that there is a loss of proteins in the liver that is not accompanied by large changes in free amino acid levels illustrates the rapid metabolism a n d / o r exit of free amino acids in the liver. In the brain, little change is observed in amino acid levels. Nutritional status affects protein metabolism in nonneuronal tissues such as muscle and liver, while
263
protein loss in brain is minimal in starvation and changes in amino acid levels are also minimal. The major changes that do occur are in the aged brain: slight decreases in several amino acids in the cortex and in glycine and tyrosine in the pons medulla. In most sections of the brain there is a decrease in threonine in both young and old. The relationship between protein breakdown and amino acid levels in brain is not clear. As an example, brain protein breakdown in vivo decreases about 20 percent during development between 5 and 10 days of age [17], while tyrosine levels increase in this period [9]. In a study of distribution of amino acids in distinct areas of rat brain, tyrosine levels decreased with aging [4]. In this study, tyrosine levels decreased in pons medulla and cerebellum in aged animals with starvation. A reduction of tyrosine in areas such as the corpus striatum or nucleus accumbens may have an effect on dopamine synthesis [18]. Several studies have focused on changes in GSH with aging. In aging, levels of GSH in brain and other tissues are reported to decrease [14,19,31]. In this study, we also found lower GSH in the brain of aged rat than in that of the younger animal, with the largest decrease in cerebral cortex, where levels were reduced by almost 50%. Chen et al. [14] reported a decrease in GSH levels of about 30% with aging in mouse cortex, hippocampus and brain stem. Kudo et al. [25] also reported lower GSH levels in brain of 1-day-old rats than in that of 10-week-old rats. Ravindranath et al. [31] show that GSH levels in several areas of the brain are significantly lower in aged rats than in younger animals. In these studies, as in ours, levels of GSH are highest in the cerebrum and lowest in the brain stem. Hazelton and Lang [19] have shown that GSH decreases in the liver, kidney, and heart of the mouse with aging. Levels of GSSG do not change during aging in mouse brain [14]. GSSG contents of various organs in the mouse, including liver, kidney and heart, constituted less than 3% of the total glutathione and did not vary with age [19]. Fasting did not alter GSSG content of glutathione in the liver, blood and kidney of the aging mouse [22,36]. In a separate set of studies we have examined regional changes in brain amino acid with aging in the rat [2-5], and human brain [6-8], and numerous changes were found in the aging brain. The regional heterogeneity of distribution was large with 5-15 fold differences in levels in various structures. The heterogeneity of distribution was less in the human brain as compared to that in the rat brain. The relative stability in brain of GSH and amino acids parallels that of proteins, which remain stable even under conditions in which the protein content of most other tissues is greatly decreased [24]. Experiments on the effects of brief starvation on brain protein synthesis also indicate that brain proteins are spared in short-term food deprivation [15]. Malnutri-
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M. Benuck et al. /Brain Research 678 (1995) 259-264
t i o n a n d s t a r v a t i o n c a n r e s u l t in loss o f p r o t e i n f r o m various tissues, but brain proteins are preserved even under extreme conditions.
References [1] Adams Jr., J.D., Wang, B., Klaidman, L.K., LeBel, C.P., Odunze, I.N. and Shah, D., New aspects of brain oxidative stress induced by tert-butylhydroperoxide, Free Rad. Biol. Med., 15 (1993) 195-202. [2] Banay-Schwartz, M., Lajtha, A. and Palkovits, M., Changes with aging in the levels of amino acids in rat CNS structural elements. I. Glutamate and related amino acids, Neurochem. Res., 14 (1989) 555-562. [3] Banay-Schwartz, M., Lajtha, A. and Palkovits, M., Changes with aging in the levels of amino acids in rat CNS structural elements II. Taurine and small neutral amino acids, Neurochem. Res., 14 (1989) 563-570. [4] Banay-Schwartz, M., Lajtha, A. and Palkovits, M., Changes with aging in the levels of amino acids in rat CNS structural elements: III. Large neutral amino acids, J. Neurosci. Res., 26 (1990) 209-216. [5] Banay-Schwartz, M., Lajtha, A. and Palkovits, M., Changes with aging in the levels of amino acids in rat CNS structural elements: IV. Methionine and basic amino acids, J. Neurosci. Res., 26 (1990) 217-223. [6] Banay-Schwartz, M., Lajtha, A. and Palkovits, M., Regional distribution of glutamate and aspartate in adult and old human brain, Brain Res., 594 (1992) 343-346. [7] Banay-Schwartz, M., Palkovits, M. and Lajtha, A., Heterogeneous distribution of functionally important amino acids in brain areas of adult and aging humans, Neurochem. Res., 18 (1993) 417-423. [8] Banay-Schwartz, M., Palkovits, M. and Lajtha, A., Levels of amino acids in 52 discrete areas of postmortem brain of adult and aged humans, Amino Acids, 5 (1993) 273-287. [9] Bayer, S.M. and McMurray, W.C., The metabolism of amino acids in developing rat brain, J. Neurochem., 14 (1967) 695-706. [10] Benuck, M., Banay-Schwartz, M., DeGuzman, T., Vizi, E.S., Kekes-Szabo, A. and Lajtha, A., Effect of diet on tissue protease activity, J. Neurosci Res., in press. [11] Benuck, M., Banay-Schwartz, M. and Lajtha, A., Proteolytic activity is altered in brain tissue of rats upon chronic exposure to ozone, Life Sci., 52 (1993) 877-891. [12] Benuck, M., Banay-Schwartz, M., Ramacci, M.T. and Lajtha, A., Peroxidative stress effects on calpain activity in brain of young and adult rats, Brain Res., 596 (1992) 296-298. [13] Bray, T.M. and Taylor, C.G., Enhancement of tissue glutathione for antioxidant and immune functions in malnutrition, Biochem. Pharmacol., 47 (1994) 2113-2123. [14] Chen, T.W., Richie Jr., J.P. and Lang, C.A., The effect of aging on glutathione and cysteine levels in different regions of the mouse brain, Proc. Soc. Exp. Biol. Med., 190 (1989) 399-402. [15] Cherel, Y., Attaix, D., Rosolowska-Huszoz, D., Arnal, M. and Le Maho, Y., Brief fasting decreases protein synthesis in the brain of adult rats, Neurochem. Res., 16 (1991) 843-847. [16] Cooper, A.J.L., Pulsinelli, W.A. and Duffy, T.E., Glutathione
and ascorbate during ischemia and postischemic reperfusion in rat brain, 3. Neurochem., 35 (1980) 1242-1245. [17] Dunlop, D.S., Elden, W.V. and Lajtha, A., Protein degradation rates in regions of the central nervous system in vivo during development, Biochem. J., 170 (1978) 637-642. [18] During, M.J., Acworth, I.N. and Wurtman, R.J., Effects of systemic L-tyrosine on dopamine release from rat corpus striatum and nucleus accumbens, Brain Res., 452 (1988) 378-380. [19] Hazelton, G.A. and Lang, C.A., Glutathione contents of tissues in the aging mouse, Biochem. J., 188 (1980) 25-30. [20] Hum, S., Robitaille, L. and Hoffer, L.J., Plasma glutathione turnover in the rat: effect of fasting and buthionine sulfoximine, Can. J. Physiol. Pharmacol., 69 (1991) 581-587. [21] Hutson, N.J. and Mortimore, G.E., Suppression of cytoplasmic protein uptake by lysosomes as the mechanism of protein regain in livers of starved-refed mice, J. Biol. Chem., 257 (1982) 95489554. [22] Jaeschke, H. and Wendel, A., Diurnal fluctuation and pharmacological alteration of mouse organ glutathione content, Biochem. Pharmacol., 34 (1985) 1029-1033. [23] Kaplowitz, N., Aw, T.Y. and Ookhtens, M., The regulation of hepatic glutathione, Annu. Rev. Pharmacol. Toxicol., 25 (1985) 715-744. [24] Kenessey, A., Banay-Schwartz, M., DeGuzman, T. and Lajtha, A., Effects of brief starvation on brain protease activity, Neurochem. Res., 16 (1991) 1001-1007. [25] Kudo, H., Kokunai, T., Kondoh, T., Tamaki N. and Matsumoto, S., Quantitative analysis of glutahione in rat central nervous system: comparison of GSH in infant brain with that in adult brain, Brain Res., 511 (1990) 326-328. [26] Langley, S.C. and Kelly, F.J., Differing response of the glutathione system to fasting in neonatal and adult guinea pigs, Biochem. Pharmacol., 44 (1992) 1489-1494. [27] LeBel, C.P. and Bondy, S.B., Persistent protein damage despite reduced oxygen radical formation in the aging rat brain, Int. J. Dev. Neurosci., 9 (1991) 139-146. [28] Meister, A., On the antioxidant effects of ascorbic acid and glutathione, Biochem. Pharmacol., 44 (1992) 1905-1915. [29] Mortimore, G.E., Wert Jr., J.J. and Adams, C.E., Modulation of the amino acid control of hepatic protein degradation by caloric deprivation, J. Biol. Chem., 263 (1988) 19545-19551. [30] Neidle, A., Banay-Schwartz, M., Sacks, S. and Dunlop, D.S., Amino acid analysis using 1-napthylisocyanate as a precolumn high performance liquid chromatography derivitization reagent, Anal Biochem., 180 (1989) 291-297. [31] R a v i n d r a n a t h , V., S h i v a k u m a r , B.R. and A n a n datheerthavarada, H.K., Neurosci. Lett., 101 (1989) 187-190. [32] Slivka, A., Spina, M.B. and Cohen, G., Reduced and oxidized glutathione in human and monkey brain, Neurosci. Lett., 74 (1987) 112-118. [33] Stadtman E.R., Protein oxidation and aging, Science, 257 (1992) 1220-1224. [34] Sturman, J.A. Taurine in development, Physiol. Rev., 73 (1993) 119-147. [35] Sturman, J.A. and Gaull, G.E., Taurine in the brain and liver of the developing human and monkey, J. Neurochem., 25 (1975) 831-835. [361 Vogt, B.L. and Richie Jr., J.P., Fasting-induced depletion of glutathione in the aging mouse, Biochem. Pharmacol., 46 (1993) 257-263.
Brain Research 678 (1995) 259-264
Research report
Effect of food deprivation on glutathione and amino acid levels in brain and liver of young and aged rats M. Benuck *, M. Banay-Schwartz, T. DeGuzman, A. Lajtha The Nathan S. Kline Institute for Psychiatric Research, Centerfor Neurochemistry, 140 Old Orangeburg Rd., Orangeburg, NY 10962, USA
Accepted 31 January 1995
Abstract
The effect of short-term food deprivation on glutathione (GSH) and amino acid levels in brain regions of young and aged rats was compared with changes observed in liver. Animals aged 3 months and 24 months were deprived of food for 48 h. GSH and amino acid levels from cerebral cortex, cerebellum, pons medulla, and liver were assayed and compared with levels in animals of the same age fed normal diets. In liver in both young and old rats, GSH levels fell 30%, from 13/zmol/g tissue to 8.7/xmol/g tissue. Significant changes were observed in other amino acids, including an increase of 30-50% in methionine, glycine, and glutamine, and a decrease of 30-50% in alanine in liver of both young and aged rats, and a 4-fold increase in taurine in young. In brain, little change was observed upon food deprivation. No decrease was observed in GSH, and only small changes were observed in other amino acids. In the aged animal aspartate, glutamate, and alanine levels were slightly lower; tyrosine in cerebellum was reduced by 30%, and both glycine and tyrosine in the pons medulla were reduced by 20-30%. In the brain areas examined, levels of GSH ranged from 1 - 2 / z m o l / g in young and 0.8-1.4/zmol/g in old; with levels in pons medulla being lower than those in cerebral cortex. In brain, in contrast to liver, levels were scarcely affected by short-term food deprivation. The relative stability of amino acids paralleled that of brain proteins, which did not significantly decrease even under conditions in which protein content of most other tissues was greatly reduced, indicating specific control of cerebral protein metabolism in protection in malnutrition. Keywords: Glutathione; Amino acid; Aging; Brain area; Liver; Malnutrition
Previous studies have shown that malnutrition and starvation result in loss of protein from various tissues, while brain proteins are preserved even under extreme conditions. Protease activity is also affected to a much greater extent in liver and muscle than in brain [10,24]. The latter effect may be altered by diet; protease activity was increased in brain and liver after feeding protein restricted diets supplemented with fatty acids and antioxidants [10]. We are interested in the mechanisms involved in protection of brain protein content in malnutrition. In a continuation of this series of studies, comparing effects of malnutrition on brain with its effects on other organs, we examined changes after brief starvation in the levels of glutathione ( G S H ) and amino acids in brain and liver of young and aged rats, since G S H level changes were observed in some organs after malnutrition.
* Corresponding author. Fax: (1) (914) 365-6107. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00204-9
Several studies have demonstrated a decrease in hepatic G S H after short-term food deprivation [13,36]. Glutathione is involved in various cellular functions, including neutralization of free radicals generated by oxidative stress. Oxidative stress affects proteolytic activity in brain [11,12], and oxidative mechanisms may be partially responsible for changes in protein metabolism in aging, with the production of free radicals contributing to the aging process [27,33]. Hence, the aging brain may be more sensitive to the effects of malnutrition.
1. Materials and methods Chemicals and reagents. 1-Napthylisocyanate was purchased from Aldrich Chemical Co. (Milwaukee, WI); G S H and amino acids were from Sigma Chemical Co. (St. Louis, MO); H P L C solvents were from Fisher Chemical Co. (Springfield, N J).
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Preparation o f tissue extracts. S p r a g u e - D a w l e y male rats, 3 m o n t h s a n d 24 m o n t h s of age, were used in this study. A n i m a l s were supplied with food a n d water at all times. T o e x a m i n e effects of short term starvation, food was removed for 48 h. A n i m a l s were killed by d e c a p i t a t i o n after prior sedation with CO 2, a n d liver a n d b r a i n were r e m o v e d rapidly a n d cleaned, a n d the b r a i n was divided into three sections, cerebral cortex, cerebellum, a n d ports medulla. A f t e r dissection a n d cleaning, tissue was frozen at - 8 0 ° C. Prior to assay, a section of tissue was removed, weighed, a n d homogenized in 3% perchloric acid ( 1 / 1 0 ) c o n t a i n i n g an internal s t a n d a r d of n o r l e u c i n e , 62.5 n m o l / m l . T h e hom o g e n a t e was microfuged, a n d the s u p e r n a t a n t was adjusted to n e u t r a l p H with sodium borate buffer a n d sodium hydroxide. N e u t r a l i z a t i o n was accomplished by a d d i n g 160 tzl of 1 M borate buffer, p H 6.2, 40 ~zl of 5N sodium hydroxide, a n d 200 Izl of water to 4 0 0 / z l of the s u p e r n a t a n t . Deriuatization. T h e p r o c e d u r e was similar to that described by Neidle et al. [30]. Briefly, a n aliquot of the n e u t r a l i z e d perchloric acid extract (800 ~1) was a d d e d to a n equal v o l u m e of the derivitizing agent 1-naphthylisocyanate (5 / z l / m l in dry a c e t o n e ) with i n s t a n t mixing. A f t e r 45 s, the reaction was stopped with the a d d i t i o n of 4 - 5 ml of cyclohexane. T h e mixture was stirred vigorously, and the u p p e r layer was discarded. Excess r e a g e n t was r e m o v e d by r e p e a t i n g the extraction 4 - 5 times. T h e derivatized sample was t h e n stored at - 20 ° C until analysis. For a m i n o acid analysis, a Vydac 201TP54 C18 reversed-phase H P L C c o l u m n was used with an auto-
m a t e d a m i n o acid analyzer a n d fluorescence detector m a n u f a c t u r e d by S h i m a d z u I n s t r u m e n t s , Columbia, MD. F o r analysis, 2 0 - 6 0 izl of sample e q u i v a l e n t to 0.625-1.8 n m o l of n o r l e u c i n e was injected. G S H a n d o t h e r a m i n o acids were d e t e c t e d using 238 n m as excitation m a x i m u m a n d 385 n m as emission m a x i m u m . For e s t i m a t i o n of relative fluorescence, a n d time of e l u t i o n of various a m i n o acids, a s t a n d a r d a m i n o acid mixture (Sigma AA-S-18) was used, to which GSH, glutamine, GABA, taurine, ornithine, and norleucine were added. This m e t h o d can detect as little as 0.5 nmol of G S H a n d picomolar a m o u n t s of o t h e r a m i n o acids. F o r c h r o m a t o g r a p h y of the a m i n o acid derivatives, two g r a d i e n t systems were used. F o r all a m i n o acids, except G S H , the c o l u m n was e q u i l i b r a t e d in buffer c o n t a i n i n g 2% 0.1 M N a H 2 P O 4 , 0.1 M sodium acetate ( p H 5.7), 2% m e t h a n o l , a n d 4% acetonitrile (buffer A). A f t e r injection of the sample, a n ±socratic g r a d i e n t in buffer A was applied for 30 m i n followed by a linear g r a d i e n t b e t w e e n buffer A a n d buffer B over the next 90 m i n (buffer B c o n t a i n s 50% acetonitrile a n d 10% methanol). T h e a m o u n t of buffer B r e a c h e d 70% of the total after 2 h. With this system, g l u t a t h i o n e is e l u t e d i m m e d i a t e l y after the a m m o n i a peak, but is often not well separated. F o r b e t t e r s e p a r a t i o n of glutathione, the e q u i l i b r a t i o n buffer (buffer A) was modified to c o n t a i n 12% of the 0.1 M N a H 2 P O 4 a n d 0.1 M sodium acetate solution ( p H 5.7), 10% m e t h a n o l , a n d 6% acetonitrile. A f t e r 30 min, a linear g r a d i e n t was applied, with buffer B increasing from 0 to 50%. T h e c o l u m n was r e g e n e r a t e d in buffer B. I n this sec-
Table 1 Amino acid levels in liver of young and aged rats fasted for 48 h compared to controls Amino acid
LIVER Young
Aspartate Glutamate Serine Glycine Glutamine Taurine Threonine Alanine GSH Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Ornithine
Aged
Control
Starved
Control
Starved
0.83 +_0.05 2.11 ± 0.18 0.76 ± 0.13 1.72 ± 0.07 3.78 _+0.17 1.11 ± 0.11 0.42 __+0.04 2.14 _+0.14 13.9 _+0.53 0.19 ± 0.01 0.30 _+0.02 0.08 _+0.01 0.18 _+0.01 0.38 + 0.02 0.15 ± 0.01 0.50 + 0.06
1.03 _+0.08 * 1.84 ± 0.15 0.70 _+0.07 2.47 _+0.06 * * * 5.31 ± 0.23 * * * 4.11 _+0.58 * * * 0.36 ± 0.03 1.06 ± 0,11 * * * 8.79 ± 0.33 * * * 0.21 _+0.02 0.33 ± 0.02 0.12 ± 0.01 * * * 0.19 ± 0.01 0.43 + 0.03 0.18 ± 0.01 * 0.56 _+0.05
1.12 + 0.10 2.04 _+0.12 0.96 _+0.18 2.21 ± 0.14 4.93 _+0.31 3.59 __+_0.92 0.70 _+0.07 2.92 ± 0.19 12.0 _+0.49 0.17 _+0.02 0.35 ± 0.03 0.07 ± 0.01 0.22 ± 0.02 0.44 ± 0.03 0.17 ± 0.02 0.53 ± 0.08
1.26 ± 0.12 1.68 +_0.15 1.01 + 0.15 2.94 ± 0.17 6.27 +_0.35 3.87 _+1.01 0.56 _+0.08 1.89 ± 0.22 8.63 ± 0.80 0.18 ± 0.02 0.36 + 0.02 0.11 ± 0.01 0.22 + 0.01 0.45 _+0.03 0.18 ± 0.01 0.52 __+__0.06
** **
*** ***
* P < 0.05; * * P < 0.01; * * * P < 0.002. Values shown are the averages _+S.E.M. (tzmol/g) from 4-8 animals, 3 months and 24 months of age. GSH and amino acid analyses are described in Materials and methods.
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M. Benuck et al. / Brain Research 678 (1995) 259-264 Table 2 Amino acid levels in cortex of young and aged rats fasted for 48 h compared to controls Amino acid
CORTEX Young
Aspartate Glutamate Serine Glycine Glutamine Taurine Threonine Alanine GABA GSH Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Ornithine
Aged
Control
Starved
Control
Starved
3.85 ± 0.06 10.3 ± 0.54 1.12 ± 0.10 1.02 + 0.08 5.19 ± 0.18 5.2 ± 0.42 0.65 ± 0.02 0.56 + 0.02 2.66 ± 0.22 2.14 + 0.20 0.088 + 0.006 0.095 ± 0.003 0.039 + 0.003 0.047 ± 0.002 0.095 + 0.004 0.059 ± 0.002 0.049 ± 0.003
3.98 ± 0.14 10.2 ± 0.39 1.23 ± 0.06 1.02 + 0.04 5.34 + 0.17 5.21 ± 0.14 0.51 + 0.02 * * 0.56 + 0.02 2.38 ± 0.20 1.99 + 0.19 0.081 + 0.004 0.101 + 0.003 0.042 + 0.003 0.049 ± 0.001 0.099 + 0.003 0.061 + 0.003 0.045 ± 0.002
4.08 ± 0.20 9.79 ± 0.19 1.46 ± 0.11 1.16 + 0.06 5.33 + 0.27 5.02 ± 0.21 0.62 +_ 0.03 0.74 ± 0.06 2.51 ± 0.13 1.36 ± 0.17 0.07 ± 0.01 0.089 ± 0.003 0.041 ± 0.004 0,045 ± 0.002 0.10 ± 0,005 0,058 ± 0.006 0,048 ± 0.005
3.49 ± 0.16 * 8.95 :t: 0.30 * 1.44 ± 0.12 1.08 ± 0.08 5.11 + 0.21 4.59 + 0.18 0.52 ± 0.03 * 0.57 _+ 0.04 * 2.60 ± 0.23 1.40 ± 0.12 0.05 ± 0.004 0.084 ± 0.003 0.043 ± 0.003 0.046 ± 0.002 0.093 ± 0.004 0.058 + 0.004 0.048 ± 0.004
* P < 0,05; * * P < 0.0002. Values shown are the averages ± S.E.M. (/~mol/g) from 4 - 8 animals, 3 months and 24 months of age. GSH and amino acid analyses are described in Materials and methods.
ond system, glutathione is well separated from other amino acids, being eluted between tyrosine and valine.
2. Results
The response to short-term food deprivation was sharply different in liver from that in brain. In the liver, glutathione levels decreased 30% upon starva-
tion, in both young and aged animals, while no change in GSH levels was observed in brain. Furthermore, significant changes were observed in several amino acids in the liver of both young and aged rats; in brain, relatively minor changes in several amino acids were seen mostly in the aged animal. Glutathione levels in liver of young and aged rats were 12-14 tzmol/g. Brain levels ranged from 1 to 2 /~mol/g. Levels in the brain decreased with age; GSH
Table 3 Amino acid levels in cerebellum of young and aged rats fasted for 48 h compared to controls Amino acid
CEREBELLUM Young
Aspartate Glutamate Serine Glycine Glutamine Taurine Threonine Alan±he GABA GSH Tyrosine Valine Methionine Isoleucine Leucine Phenyialanine Ornithine
Aged
Control
Starved
Control
Starved
2.96 + 0.11 8.89 + 0.23 0.60 ± 0,06 0.83 + 0,02 4.50 + 0.22 4.07 ± 0.09 0.67 ± 0,02 0.66 + 0.03 1.89 ± 0.08 1.03 ± 0.04 0,086 + 0.006 0.075 + 0.002 0.046 ± 0.002 0.040 ± 0.001 0.084 ± 0.003 0,065 ± 0.002 0.067 + 0.004
3.19 + 0.12 9.54 + 0.23 0.63 + 0.06 0.75 ± 0.04 4.76 ± 0.22 4.25 ± 0.10 0.53 ± 0.02 * 0.62 ± 0.03 1.89 + 0.05 1.19 ± 0.07 0.081 ± 0.004 0.077 ± 0,002 0.049 ± 0.001 0.040 ± 0.001 0.089 + 0.002 0.063 + 0.001 0.066 ± 0.003
2.98 + 0.15 9.38 + 0.35 0.80 + 0.08 1.11 + 0.08 4.58 ± 0.45 3.48 ± 0.20 0.61 + 0.06 0.69 + 0.06 2.03 ± 0.14 1.31 ± 0.17 0.072 + 0.008 0.073 ± 0.005 0.044 + 0.004 0.040 ± 0.002 0.090 ± 0.006 0.062 ± 0,005 0.081 + 0.005
2.88 + 0.20 9.04 ± 0.23 0.74 ± 0.09 1.06 + 0.08 4.10 ± 0,18 3.33 ± 0.11 0.50 ± 0.03 0.57 + 0.05 1.84 + 0.08 1.25 ± 0.13 0.051 ± 0.004 " 0.062 + 0.003 0.038 ± 0.002 0.035 ± 0.002 0.077 ± 0.004 0.052 + 0.003 0.071 ± 0.006
* P < 0.05. Values shown are the averages + S.E.M. ( # m o l / g ) from 5 - 9 animals, 3 months and 24 months of age. GSH and amino acid analyses are described in Materials and methods.
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Table 4 Amino acid levels in pons medulla of young and aged rats fasted for 48 h compared to controls Amino acid PONS MEDULLA Young Aspartate Glutamate Serine Glycine Glutamine Taurine Threonine Alanine GABA GSH Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Ornithine
Aged
Control
Starved
Control
Starved
3.40 ± 0.05 5.44 _+0.18 0.97 _+0.02 3.33 + 0.09 3.17 _+0.10 1.45 ± 0.04 0.63 _+0.02 0.34 ± 0.01 1.49 _+0.06 1.08 ± 0.06 0.074 + 0.006 0.084 ± 0.002 0.031 ± 0.002 0.039 ± 0.001 0.085 ± 0.002 0.055 ± 0.001 0.086 ± 0.006
3.48 _+0.14 5.39 _+0.20 0.85 ± 0.09 3.18 + 0.08 3.14 ± 0.07 1.42 _+0.05 0.50 ± 0.02 * * 0.34 + 0.02 1.66 ± 0.09 1.04 _+0.12 0.071 + 0.005 0.085 ± 0.003 0.028 ± 0.001 0.040 ± 0.001 0.086 _+0.003 0.056 _+0.001 0.079 + 0.002
2.84 _+0.10 4.82 _+0.15 0.56 _+0.05 3.10 _+0.22 3.41 ± 0.15 1.43 ± 0.05 0.60 + 0.06 0.44 _+0.04 1.49 ± 0.14 0.81 + 0.18 0.065 ± 0.007 0.079 ± 0.007 0.036 + 0.004 0.042 ± 0.003 0.099 ± 0.010 0.060 ± 0.005 0.096 ± 0.005
2.71 ± 0.10 5.18 + 0.30 0.48 + 0.05 2.56 + 0.16 * 3.56 ± 0.22 1.72 ± 0.21 0.49 + 0.03 0.37 + 0.04 1.85 ± 0.28 0.66 + 0.12 0.047 ± 0.005 * 0.073 ± 0.004 0.037 ± 0.003 0.040 ± 0.002 0.084 ± 0.005 0.052 ± 0.003 0.085 ± 0.003
* P < 0.05; * * P < 0.002. Values shown are the averages _+S.E.M. (/~mol/g) from 4-6 animals, 3 months and 24 monthsof age. GSH and amino acid analyses are described in Materials and methods.
in c e r e b r a l cortex fell from 2.0 ~ m o l / g in the y o u n g a n i m a l to 1.4 / x m o i / g at 24 m o n t h s of age (Tables
1--4). Liuer. H e p a t i c G S H was r e d u c e d in b o t h young a n d a g e d rats after food d e p r i v a t i o n , d e c r e a s i n g from 1 2 - 1 4 / x m o l / g to 8 . 6 - 8 . 8 / z m o l / g . A m o n g c h a n g e s seen in o t h e r a m i n o acids were i n c r e a s e s in glycine, g l u t a m i n e , taurine, a n d m e t h i o n i n e a n d a d e c r e a s e in alanine. A l a n i n e levels in the starved animals were d e c r e a s e d 50 a n d 35% in the y o u n g a n d a g e d respectively. In contrast, levels o f glycine, g l u t a m i n e , a n d m e t h i o n i n e i n c r e a s e d 3 0 - 5 0 % in b o t h y o u n g a n d a g e d animals. T a u r i n e levels i n c r e a s e d fourfold in the y o u n g liver, but s h o w e d no c h a n g e after food d e p r i v a t i o n in the a g e d liver. All t h e s e c h a n g e s were statistically significant at the P = 0.002 level. Smaller, b u t significant, increases o c c u r r e d in levels o f p h e n y l a l a n i n e a n d asp a r t a t e in the y o u n g animal u p o n food d e p r i v a t i o n , b u t not in the aged. O f the r e m a i n i n g a m i n o acids m e a sured, little c h a n g e was o b s e r v e d in y o u n g o r old after food d e p r i v a t i o n ( T a b l e 1). Brain. In the brain, s h o r t - t e r m starvation h a d little effect on g l u t a t h i o n e o r a m i n o acid levels. G l u t a t h i o n e levels did not c h a n g e significantly in any of the t h r e e a r e a s e x a m i n e d , e i t h e r in the y o u n g or t h e a g e d animal. A m i n o acid levels, which w e r e significantly a l t e r e d in liver, w e r e not a f f e c t e d in the b r a i n o f the young animal. In t h e y o u n g a n i m a l only levels o f t h r e o n i n e were reduced (Tables 2-4). In t h e a g e d animal, small but significant c h a n g e s did occur in several a m i n o acids a f t e r food d e p r i v a t i o n . In
the c e r e b r a l cortex, d e c r e a s e s of 1 0 - 1 5 % in levels of aspartate, glutamate, and threonine and a decrease of 23% in alan±he levels w e r e n o t e d . N o significant c h a n g e s were n o t e d in o t h e r a m i n o acids. In the cerebellum, tyrosine d e c r e a s e d by 30%, while in the p o n s m e d u l l a , glycine a n d tyrosine showed a 2 0 - 3 0 % decrease. N o o t h e r significant d i f f e r e n c e s in a m i n o acid levels w e r e n o t e d ( T a b l e s 2 - 4 ) .
3. Discussion This study c o m p a r e s the effect o f short t e r m food d e p r i v a t i o n on levels of G S H a n d a m i n o acids in b r a i n a n d liver. In liver, several studies have shown that starvation o r d i e t a r y r e s t r i c t i o n r e d u c e s liver G S H levels [20,22,26,36]. V o g t a n d R i c h i e [36] f o u n d t h a t t h e G S H r e s p o n s e to 24 h o f fasting is m o r e severe in the liver o f a g e d mice t h a n in that of y o u n g e r mice. In o u r study, we f o u n d a d e p l e t i o n in h e p a t i c G S H o f 33% in b o t h a d u l t a n d a g e d rats after 48 h of fasting. O t h e r studies also s h o w e d that food d e p r i v a t i o n s t i m u l a t e s a c h a n g e in h e p a t i c G S H turnover, with a d e c r e a s e in h e p a t i c G S H a f t e r s h o r t - t e r m f o o d d e p r i v a t i o n [13]. It seems that r e d u c t i o n o f liver G S H levels is a sensitive early index of m a l n u t r i t i o n . Levels o f G S H in b r a i n a r e n o t r e d u c e d , e i t h e r in y o u n g or a g e d animals, by food d e p r i v a t i o n o f relatively short d u r a t i o n . O n l y small c h a n g e s in several a m i n o acids a r e found, p r i m a r i l y in the a g e d brain. In contrast, in the liver m o r e a m i n o acids a r e a l t e r e d
M. Benuck et al. / Brain Research 678 (1995) 259-264
significantly during starvation. Glutathione is decreased from 13 to 9 ~ m o l / g tissue. A decrease in liver GSH upon brief starvation may reflect an efflux of GSH from the liver to blood to maintain GSH levels in other tissues [22,23,28,36]. Our procedure for assay of glutathione does not separate the oxidized form of glutathione (GSSG) from reduced glutathione (GSH). Previous studies with rat or mouse brain have found GSSG to constitute only a few percent of total glutathione [14,16,32]. In liver, glutathione is also present mainly in the reduced form [23]. GSSG levels may change under conditions associated with oxidative stress; intracerebroventricular injection of tert-butylhydroperoxide, an oxidative stress inducing agent, significantly increased GSSG levels in mouse brain [1]. Other amino acid levels that are affected in liver include methionine, glycine, glutamine, taurine, and alanine. Methionine and glycine are precursors in GSH synthesis, and dietary deficiency of methionine prevents GSH synthesis [36]. Methionine has been used to restore GSH levels of starved animals [22]. Glutamine supplementation has been used to maintain liver GSH under various traumatic conditions such as hepatic injury [13]. Increase in the levels of the amino acids that are precursors of GSH (methionine, glycine, and glutamine) may be a response in liver to the enhanced GSH efflux during starvation. Taurine serves an essential role in development [34]. Rat liver has a high capacity for taurine biosynthesis, and taurine concentration in liver and brain is generally greater in the developing animal than in the mature animal [35]. In this study, taurine levels in the liver of young animals were lower than in the aged. The increase found in the young upon starvation may be related to liver lipid metabolism and the function of taurine in the transport of bile acids. Short-term starvation results in a significant loss of protein in liver [21,24]. Alanine is one amino acid that modulates proteolytic activity in the liver during caloric deprivation [29]. The reduction in alanine levels in liver observed here in both young and adult during starvation may be a reflection of the role of alanine as modulator of proteolytic activity, its levels varying inversely with liver proteolysis [29]. The concentration of most amino acids in the protein-bound form is several hundred fold of that in the free amino acid pool. Therefore, the breakdown of even one percent of protein would increase free amino acid content several fold. The fact that there is a loss of proteins in the liver that is not accompanied by large changes in free amino acid levels illustrates the rapid metabolism a n d / o r exit of free amino acids in the liver. In the brain, little change is observed in amino acid levels. Nutritional status affects protein metabolism in nonneuronal tissues such as muscle and liver, while
263
protein loss in brain is minimal in starvation and changes in amino acid levels are also minimal. The major changes that do occur are in the aged brain: slight decreases in several amino acids in the cortex and in glycine and tyrosine in the pons medulla. In most sections of the brain there is a decrease in threonine in both young and old. The relationship between protein breakdown and amino acid levels in brain is not clear. As an example, brain protein breakdown in vivo decreases about 20 percent during development between 5 and 10 days of age [17], while tyrosine levels increase in this period [9]. In a study of distribution of amino acids in distinct areas of rat brain, tyrosine levels decreased with aging [4]. In this study, tyrosine levels decreased in pons medulla and cerebellum in aged animals with starvation. A reduction of tyrosine in areas such as the corpus striatum or nucleus accumbens may have an effect on dopamine synthesis [18]. Several studies have focused on changes in GSH with aging. In aging, levels of GSH in brain and other tissues are reported to decrease [14,19,31]. In this study, we also found lower GSH in the brain of aged rat than in that of the younger animal, with the largest decrease in cerebral cortex, where levels were reduced by almost 50%. Chen et al. [14] reported a decrease in GSH levels of about 30% with aging in mouse cortex, hippocampus and brain stem. Kudo et al. [25] also reported lower GSH levels in brain of 1-day-old rats than in that of 10-week-old rats. Ravindranath et al. [31] show that GSH levels in several areas of the brain are significantly lower in aged rats than in younger animals. In these studies, as in ours, levels of GSH are highest in the cerebrum and lowest in the brain stem. Hazelton and Lang [19] have shown that GSH decreases in the liver, kidney, and heart of the mouse with aging. Levels of GSSG do not change during aging in mouse brain [14]. GSSG contents of various organs in the mouse, including liver, kidney and heart, constituted less than 3% of the total glutathione and did not vary with age [19]. Fasting did not alter GSSG content of glutathione in the liver, blood and kidney of the aging mouse [22,36]. In a separate set of studies we have examined regional changes in brain amino acid with aging in the rat [2-5], and human brain [6-8], and numerous changes were found in the aging brain. The regional heterogeneity of distribution was large with 5-15 fold differences in levels in various structures. The heterogeneity of distribution was less in the human brain as compared to that in the rat brain. The relative stability in brain of GSH and amino acids parallels that of proteins, which remain stable even under conditions in which the protein content of most other tissues is greatly decreased [24]. Experiments on the effects of brief starvation on brain protein synthesis also indicate that brain proteins are spared in short-term food deprivation [15]. Malnutri-
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t i o n a n d s t a r v a t i o n c a n r e s u l t in loss o f p r o t e i n f r o m various tissues, but brain proteins are preserved even under extreme conditions.
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