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Effect of undernutrition on cerebral protein metabolism
EXPERIMENTAL
NEUROLOGY
65, 157- 168 (1979)
Effect of Undernutrition M. BANAY-SCHWARTZ,
Center
for
Neurochemistry3 Received
on Cerebral
Protein
Metabolism
A. M. GIUFFRIDA, T. DE GUZMAN, AND A. LAJTHA’ Rockland
December
Il.
Research
Institute.
1978; revision
Ward’s
received
February
H. SERSHEN,
Island.
New? York
10035
7. 1979
Amino acid incorporation and protein breakdown were decreased in the brain of undernourished rats in early developmental stages (6 days old); at later ages the differences were less significant. Changes in the cerebral free amino acid pool were slight. The greater decrease in the rate of protein synthesis than in that of protein breakdown resulted in a decrease in protein deposition (growth) in the young brain. Changes were similar in the cerebellum and the cerebral hemispheres. The pattern of changes (decrease in both protein synthesis and breakdown) that occurs in brain during undernutrition especially during development differs from the pattern of changes in other organs such as liver and muscle, where protein synthesis and breakdown may change in opposite directions.
INTRODUCTION The effect of nutritional deficiencies on the nervous system has been studied at some detail. The resistance of the adult and the vulnerability of the developing brain is established, with the greatest vulnerability occurring in the rapid growth phase (1, 14, 35, 42, 43). The extent of changes and recovery depends on the extent and duration of malnutrition. Changes in the brain are considerably smaller than in most other organs studied. The comparable resistance, especially of the adult brain, is not well understood. Malnutrition results in reduced cell number and reduced protein content; therefore cerebral protein metabolism is likely to play an important role. Abbreviations: PCA-perchloric acid, OPT-o-phthaldialdehyde. ’ This work was supported in part by U.S. Public Health Service grant NS 03226 from the National Institute of Neurological and Communicative Disorders and Stroke. Dr. Giuffrida was a Visiting Professor; herpresent address is lstituto di Chimica Biologica, I’Universita di Catania, Viale Andrea Doria, 6, 95125 Catania, Italy. A travel grant from the Consiglio Nazionale delle Richerche, Rome, is gratefully acknowledged. 157
0014-4886/79/070157-12$02.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction I” any form reserved.
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The mechanisms of influencing cerebral protein synthesis are not known. Because amino acid pool changes may occur, pulse-labeling may be difficult to interpret; it might lead to findings of increased incorporation with one amino acid and decreased incorporation of another under similar circumstances (27, 28). We recently used a method of measuring brain protein turnover under conditions that permit labeling for a longer time during which the specific activity of the precursor amino acid is kept constant (21). Here we report our findings on the effect of undernutrition on cerebral protein turnover (synthesis and breakdown) with this method. MATERIALS
AND METHODS
Sprague-Dawley rats were kept undernourished from the 10th day of pregnancy through lactation, by providing them with approximately half their normal diet- 10 g food daily per rat before parturition and 15, 20, and 25 g daily in the lst, 2nd, and 3rd week of lactation, respectively (35). Control animals received the same food ad libitutn (Teklad 10% breeder diet, containing 16.5% crude protein, 10% crude fat, and 2.5% fiber). Water was freely available to all rats. Each rat, control or experimental, was nursing eight pups. Experiments were carried out at the following ages: 6,12,30, and 42 days old. The young rats were left with their mothers until the age of 30 days. The last age group (42 days old) were removed from the mother at the age of 30 days and fed 20 to 24 g food per day per group of eight experimental animals. On the morning of the experiment, the rats were injected i.p. with a tyrosine suspension (L-[U-14C]tyrosine) 0.01 ml/g body weight, containing 50 $Zi and 50 mg tyrosine per milliliter. After 2 h the brains were removed and dissected into three parts, (a) cerebrum, (b) cerebellum, and (c) midbrain, brain stem, and olfactory bulbs. The regions were frozen in dry ice, weighed, and homogenized in 2 ml 5% (w/v) perchloric acid (PCA). After centrifugation for 30 min at 10,000 rpm of the homogenates, the PCA supernatants were stored frozen for subsequent determination of the specific activity of the free tyrosine by the method described previously (22). The protein pellets were washed by repeated (3x) resuspension and centrifugation in 2.5 (w/v) PCA, followed by methanol, chlorofotmmethanol l:l, absolute ethanol, and ether extractions. The dry protein pellets were dissolved in 1 N NaOH, and on one portion protein determination was done (26); on another portion radioactivity was measured in Triton-toluene scintillation mixture (40); and a third portion was hydrolyzed 24 h in 6 N HCl at llO”C, followed by the enzymatic determination of the proteinbound tyrosine specific activity (22).
PROTEIN METABOLISM IN UNDERNUTRITION
159
On a number of samples the free tyrosine activity was determined also by column chromatography (2), using a modification of the method described by Neidle et al. (31). A column (75 x 0.6 cm) of Biorad Aminex A-6 resin was eluted with lithium citrate buffer gradients (37) from pH 2.8 to 6.1. A nine-chamber gradient was used, containing 37.5 ml in each chamber: pH 2.8 buffer in the first three, pH 4.3 in the next two, and pH 6.1 in the last four chambers. To facilitate separation of threonine and serine, 2.5 and 1.5 ml methanol were added to the first and second gradient chambers, respectively, replacing part of the pH 2.8 buffer. The column was kept at 45°C during the entire analysis, which required 9 h. During the amino acid analyses, buffers were pumped through the column at a rate of 0.6 mYmin. To remove traces of ammonia from the buffers, a small (10 x 1.5 cm) column containing DC-3 resin (Pierce Co., Rockford, Illinois) was connected to the system at the entrance to the Milton-Roy pump. This buffer-cleaning column was left in place permanently. After each run, both columns were regenerated with 0.3 N LiOH (1.5 h), followed by pH 2.8 lithium-citrate buffer for 3 h. The sample to be analyzed (in 3% sulfosalicylic acid) was loaded on top of the Aminex A-6 resin by use of a stream of nitrogen. The column effluent was passed through a splitstream connector, and part of it (0.23 mYmin) was passed through a peristaltic pump for the detection of the amino acid by fluorescence with o-phthaldialdehyde (OPT) using a Fluoromonitor. The rest of the effluent (0.37 ml/min) was collected on a fraction collector, directly into scintillation vials containing a toluene-T&on-based scintillation fluid (40) to form a gel, for radioactivity measurements in a scintillation counter. The OPT reagent was made up as follows: 80 mg OPT was dissolved in 1 ml methanol; 0.04 ml 2-mercaptoethanol was added, then 100 ml 0.4 M borate buffer, pH 10. The fluorescence intensity was recorded on a dual-channel recorder. The same method was used for the determination of the free amino acid composition of the brain regions of rats at several developmental stages. For the leucine experiments, the control and undernourished rats (12 and 30 days old) were injected i.p. with tracer amounts of L-(1-14C)leucine, 0.1 $X/g body weight. Total radioactivity of the free (precursor) leucine was determined in the PCA supernatants, the protein pellets were extracted and dried as above, and the incorporated radioactivity per milligram protein was determined. RESULTS Changes in Protein Synthesis Rates. As was shown previously (17), including in our laboratory (7, 20), the rate of cerebral protein synthesis is high at birth and declines during development. In mice, by 30 days of age amino acid incorporation rates are close to adult values. Incorporation
160
BANAY-SCHWARTZ TABLE
ET AL. 1
Changes in Protein Synthesis in Malnourished
Brain”
Protein synthesis in brain regions Synthesis rates in control brain percentage per hour
Age of animal (days)
Hemisphere
Cerebellum
Brain stem
6 12 30 42
1.81 0.94 0.68 0.70
2.20 1.03 0.77 0.76
1.54 0.83 0.64 0.78
Synthesis in malnourished percentage of controP Hemisphere 772 101 i85+ 101 +
7 10 9 6
Cerebellum 76 98 88 I05
+ c + 2
8 6 5 8
as
Brain stem 90 + lOOk 912 96?
10 7 3 7
o Incorporation of tyrosine in rat brain for 2 h was measured; averages of four experiments *SD are given. b Synthesis of the region as percentage of the control rate of synthesis of the same region at each age.
rates in cerebellum are higher than in the cerebrum in the immature brain (7). Similar results were found in our experiments, for control and for malnourished brain (Table 1). Protein synthesis rates were somewhat lower in malnourished brain: at 6 days of age amino acid incorporation was about 25% lower in the hemispheres and cerebellum; changes were small in the brain stem. From 12 days of age, incorporation in malnourished brain was close to control values, although slightly lower (about 10%) at 30 days. In these experiments we measured the incorporation of a large dose of labeled tyrosine administered intraperitoneally. The use of a tyrosine suspension, which assures continuing absorption of this relatively insoluble amino acid, results in a constant specific activity of the cerebral free tyrosine during the experimental period (21), as confirmed in the present study. This method also results in an increase in the cerebral free tyrosine levels without significantly affecting the other amino acids in the brain (see below). We also tried to measure the incorporation of tracer doses of labeled leucine at various times (15, 30, and 60 min). Only the 15-min experiments could be clearly interpreted, because by 30 min a major portion of the label in brain was present in compounds other than leucine. The short-term leucine experiments with 12- and 30-day-old animals gave results similar to those with tyrosine. Changes in Deposition and Breakdown of Brain Proteins. In the period of our study (from birth to 42 days) the brain undergoes rapid growth, including growth (net deposition) of brain proteins. The rate of growth is not uniform, but is high in the early stages of development (to 6 days), and growth is practically stopped by 42 days. The rate of deposition of
PROTEIN
METABOLISM
161
IN UNDERNUTRITION
proteins, calculated as percentage net increase per hour, was lower in the hemispheres of malnourished mice at 6 days; at later times growth was similar to controls. The decrease in the deposition of cerebellar proteins could be observed during a longer developmental period (Table 2). Because growth is most rapid at the early stages of development, the total protein content of hemispheres was lower at 30 days in the malnourished animals even though inhibition of growth could be clearly observed only in the 0- to 6-day period. As the rate of synthesis of proteins in the brain is greater than net deposition at all stages of development, continuous turnover (breakdown of proteins) occurs. In the (nongrowing) adult brain, breakdown is equal to synthesis; during development, growth is the result of greater synthesis than breakdown. The present experiments (Table 2) confirmed previous results (31) showing that the rate of breakdown is higher in the immature brain undergoing net growth than in the nongrowing adult brain. When growth stops, the rate of breakdown, like that of synthesis, reaches adult values. The changes of protein breakdown are similar to changes of synthesis: there was a significant (about 20%) inhibition of protein breakdown in the malnourished brain at 6 days, no change at 12 days, and a slight inhibition at 30 days (Table 2). Although breakdown was inhibited at 6 days, the fact that protein synthesis, which proceeds at a higher rate, was inhibited to a slightly greater degree resulted in the inhibition of net deposition of proteins at 6 days of age. TABLE
2
Changes in Protein Deposition and Breakdown Age of animal (days)
Rates in control brain percentage per hour
Brain”
Rates in malnourished as percentage of controP
Brain region
Deposition
6
Hemisphere Cerebellum
0.63 0.87
1.18 1.33
67 76
82 76
12
Hemisphere Cerebellum
0.36 0.52
0.58
0.51
83 90
112 106
Hemisphere Cerebellum
0.07 0.10
0.61 0.68
114 70
82 93
30
Breakdown
in Malnourished
Deposition
Breakdown
0 Deposition (net growth) of protein was estimated by measuring the total protein content of brain regions at various ages. Breakdown was calculated as the difference between total synthesis and deposition. b Synthesis of the region as percentage of the control rate of synthesis of the same region at each age.
162
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ET AL.
Changes in Levels of Cerebral Free Amino Acid. Because changes ir protein synthesis and breakdown may be related to changes in the composi, tion of the free amino acids in the tissue, we measured the levels of fret amino acids in the brain at various ages (Table 3). The developmenta pattern of increase in glutamate, glutamine, and aspartate levels and de, crease in glycine and alanine and in most of the essential amino acids with the quantitatively largest change in the decrease in taurine, confirmec numerous previous studies (15, 23)..Aspartate and glutamate levels wert decreased in the first 6 or 12 days of undernutrition, respectively, ant some decreases were observed in this period in the levels of valine, iso leucine, and leucine. In a separate set of experiments, we tested the in fluence of the administered tyrosine on the cerebral free amino acid pool The only change in brain was the increased level of tyrosine. This amine acid increased to 0.80 pmol/g from newborn to 12 days, to 0.60 at 30 days and to 0.48 at 42 days. The reduction with age of tyrosine uptake in these tyrosine-injected animals was similar to previous observations, indicating a maturation of cerebral barriers that decreased uptake of amino acids by the brain from the blood (38). The changes in the composition of the free amino acid pool of the cerebel lum during undernutrition were similar to the changes found in the cerebrum There was a decrease in glutamate and aspartate and a significant increase initially of taurine and alanine (Table 4). The changes in these amino acids indicate a retardation of developmental changes in at least some aminc acid values during the early phase of undernutrition.
DISCUSSION Effects of Undernutrition on Brain Proteins. It is well established that malnutrition during development results in a reduced number of cells in the brain (44), which upon longer duration can lead to a permanent deficit (42). The effects are complex and extensive. Cell numbers, also brain weight and brain protein content, are reduced, and myelin content is decreased (19). There is also a decrease in the rate of cell loss usually observed in the developing brain, and the length of the different phases of the cell cycle is altered (1). Depending on experimental conditions, cell acquisition rate can be reduced and degenerating cells can increase in number (24). Myelination and synaptogenesis may be affected (6). Regional Differences in Brain and Changes in Specific Protein Components. Our knowledge of the metabolic rates of well-characterized brain
proteins is meager, but the indications are that undernutrition affects the various proteins to a different degree and that there are regional differences not only in the effects of undernutrition on overall protein synthesis but in the effects on specific proteins as well. Myelin protein synthesis was
1.47 0.08 0.41 0.23 0.63
1.34 0.08 0.43 0.26 0.51
1.30
18.3 1.69 0.79 1.01 4.86 2.37 1.79 1.25 0.19 0.17 0.10 0.17 0.16 0.15
Experimental
6-day
1.88 0.05 0.29 0.17 0.44
16.0 2.68 0.93 1.31 7.19 3.01 0.78 1.56 0.22 0.10 0.12 0.25 0.26 0.12
Control
1.86 0.06 0.28 0.16 0.30
15.2 2.57 0.81 1.45 6.25 2.54 0.96 1.49 0.15 0.09 0.11 0.20 0.23 0.10
Experimental
12-day
3.51 0.03 0.45 0.26 0.62
9.88 4.83 0.92 1.60 11.3 5.14 1.22 1.28 0.23 0.15 0.17 0.34 0.20 0.15 3.07 0.04 0.37 0.17 0.47
1.64 11.7 4.86 1.22 1.25 0.22 0.10 0.15 0.30 0.21 0.14
10.1 5.51
Experimental
30-day Control
Cerebrum
of the Free Amino Acids in the Cerebrum”
3
2.48 0.04 0.16 0.14
8.73 3.78 0.93 1.00 12.7 5.21 0.84 0.93 0.14 0.06 0.09 0.10 0.08 0.08
Control
0.29 0.12
1.46
13.9 5.42 0.66 0.52 0.07 0.04 0.06 0.07 0.07 0.07
9.79 2.70
Experimental
42-day
I’ Micromoles amino acid per gram brain tissue. Averages of six experiments are given with standard deviations within 5% of the mean. ’ Whole brain in addition to cerebrum contained cerebellum, brain stem, and olfactory bulbs.
2.02 7.10 3.62 2.06 2.24 0.31 0.10 0.18 0.42 0.28 0.18 1.56
17.03 2.07 0.89 1.35 5.11 2.48 1.35 1.38 0.25 0.23 0.19 0.25 0.18 0.11
17.9 1.13 1.02 1.66 6.20 2.73 1.85 2.47 0.27 0.11 0.15 0.39 0.28 0.16
19.2 1.73
Taurine Aspartic acid Threonine Serine Glutamic acid Glutamine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine y-Aminobutyric acid Ornithine Lysine Histidine Arginine
Control
Control
Amino acid
Experimental
Whole brainb Newborn
Concentrations
TABLE
5
z
2 2 I% z E; P z
$ P z
2 3 m 2 g 3
1.56 0.06 0.32 0.18 0.30
1.71 0.09 0.36 0.19 0.33
19-day
2.97 0.10 0.63 0.22 0.50
0.39 0.49 0.19
1.44 4.41 0.31 0.14 0.17
15.1 1.92 1.39 1.84 7.87 4.16
Experimental
3.41 0.55 0.25 0.66
0.44 0.26 0.21
1.28 2.09 0.30 0.12 0.21
10.6 4.35 1.39 1.70 10.6 6.86
Control
30-day
3.59 0.55 0.20 0.70
0.53 0.30 0.23
1.39 2.13 0.28 0.11 0.27
8.43 4.73 1.77 10.0 5.94
Experimental
3.06 0.05 0.23 0.12 -
0.15 0.12 0.14
1.00 1.12 0.13 0.05 0.11
8.64 3.14 1.39 0.85 11.2 6.13
Control
42-day
1.32 0.38 0.11 -
0.08 0.12 0.09
0.63 0.53 0.08 0.11 0.08
8.12 2.22 0.59 12.8 6.27
Experimental
’ Micromoles amino acid per gram brain tissue. Averages of six experiments are given with standard deviations within 8% of the mean.
2.58 0.06 0.45 0.23 0.53
0.35 0.28 0.17
0.21 0.34 0.12
0.24 0.30 0.13
Leucine Tyrosine Phenylalanine y-Aminobutyric acid Omithine Lysine Histidine Arginine
0.92 3.47 0.24 0.17 0.16
1.04 3.43 0.15 0.13 0.11
1.03 2.28 0.16 0.09 0.15
Glycine Alanine Vahne Methionine Isoleucine
Control 11.0 2.54 1.41 1.64 9.08 5.20
10.6 2.32 1.15 1.36 5.89 3.85
Taurine Aspartic acid Threonine Serine Glutamic acid Glutamine
Experimental
4
of the Free Amino Acids in the Cerebellum”
11.2 2.01 1.15 1.45 4.61 3.66
Control
Amino acid
12-day
Concentrations
TABLE
F
5 ; 4
3
2 k
g
PROTEIN
METABOLISM
IN UNDERNUTRITION
165
depressed more than other proteins; the depression in proteolipid protein was greater than in other myelin proteins (41). There were regional differences in the decreased levels of S-100 protein, whereas 14-3-2 did not change (30): S-100 decreased in the visual and increased in the sensory motor cortex (16). The type of changes we observed here-initial depression of synthesis with later recovery even during undernutrition-were observed in most regions, in measuring glutamate decarboxylase and choline acetyltransferase activity (36). Whereas choline acetyltransferase activity decreased, acetylcholinesterase activity in the brain stem increased (9). In general the effect of malnutrition was proportional to regional growth rates, the regions with the most rapid cell division being affected the most (12); but different cells in the cerebellum also showed different sensitivity to undernutrition (4). Changes in Amino Acids. Changes in cerebral amino acid content have been reported in several protein and calorie deficiency experiments. In rats, in general, amino acids decreased (29), although postnatal decreases in a number of amino acids were less marked or were delayed (10). Typtophan initially increased, then decreased, during malnutrition (18). In monkeys the greatest changes observed were increases in basic amino acids (histidine, homocamosine, methylhistidine, and lysine), with a smaller decrease in many others, and a decrease in acidic amino acids (11). The fact that growth hormone alleviated deficiency (43) indicated that amino acid changes were not the main cause of altered protein metabolism. Previously we did not find any changes in cellular amino acid transport in brain slices as a result of malnutrition (3). Changes in Protein Synthesis. Changes in protein turnover due to malnutrition have not been measured in the brain. Decreases in amino acid incorporation after prolonged undernutrition were observed (34), including in our laboratory (3). Not all proteins were affected in a similar manner; on a deficient diet, liver albumin synthesis and the concentration of albumin messenger RNA decreased, whereas much of general liver protein synthesis was not affected (33). Malic enzyme activity increased in liver under such conditions because rates of syhthesis were elevated, with no change in the breakdown of this enzyme (39). Tissue protein synthesis in adult animals on a protein-free diet was compared (13): in liver, protein synthesis was unaltered for 2 days, then increased; in muscle, synthesis immediately decreased a great deal; in heart, brain, and kidney, changes were small but significant decreases could be detected by 21 days. The decrease of protein synthesis by the 30th day is parallel to the termination of glia proliferation in the cerebral hemispheres and the termination of both neuronal and glial proliferation in the cerebellum. Our findings indicate a delayed growth in the undernourished brain as shown at 12 days. Undernutrition also delayed
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maturational changes in nucleic acid metabolism (Giuffrida and Hamberger, in preparation). Alterations of Protein Breakdown in Other Tissues. The effect of malnutrition on cerebral protein breakdown has not been measured previously. Protein synthesis and degradation represent independent mechanisms that often do not change in parallel fashion. Their relationship has been shown to vary in different organs and under different circumstances. During development, protein synthesis was increased and breakdown was decreased in the liver (5); in brain, both synthesis and breakdown were greater in the immature than in the mature organ, and growth was the result of the greater increase in synthesis than in breakdown (8). In malnutrition, protein synthesis and protein breakdown decreased in muscle, whereas in the liver both protein synthesis and protein breakdown increased; loss of proteins occurred in both tissues (13). In rat muscle protein synthesis and degradation were not affected in the soleus, whereas synthesis was decreased and breakdown was increased in the extensor digitorum longus (25). Measurements of breakdown utilizing the decrease in prelabeled proteins are difficult because changes in amino acid reincorporation could occur in malnutrition (32). In brain, changes in rates of synthesis and rates of breakdown appear to occur in parallel fashion during growth and during malnutriton. The increase during growth and the decrease during malnutrition in synthesis are greater than the corresponding changes in breakdown, resulting in normal growth in brain of young controls and decreased growth in brain of undernourished animals. REFERENCES 1. BALAZS, R., P. D. LEWIS, AND A. J. PATEL. 1975. Effects of metabolic factors on brain development. Pages 83-115 in M. A. B. BRAZIER, Ed., Growth and Development of the Brain. Raven Press, New York. 2. BANAY-SCHWARTZ, M., G. ZANCHIN, T. DEGUZMAN, AND A. LAJTHA. 1979. The effect of corticosteroids on amino acid content of brain tissue preparations. Psychoneuroendocrinology, in press. 3. BANAY-SCHWARTZ, M., G. ZANCHIN, T. DEGUZMAN, H. SERSHEN, AND A. LAJTHA. 1978. Decrease in cerebral protein synthesis on a low protein diet. In H. CH. BUNIATIAN, Ed., Problems of Brain Biochemistry, Vol. 13. Armenian Acad. Sci., U.S.S.R. 4. BARNES,‘D., AND J. ALTMAN. 1973. Effects ofdifferent schedules of early undernutrition on the preweaning growth of the rat cerebellum. Exp. Neural. 38: 406-419. 5. CONDE, R. D., AND 0. A. SCORNIK. 1977. Faster synthesis and slower degradation of liver protein during developmental growth. Biochem. J. 166: 115-121. 6. DEO, K., V. BIJLANI, AND M. G. DEO. 1978. Effects of malnutrition on cell genesis and migration in developing brain in rats. Exp. Neurol. 62: 80-92. 7. DUNLOP, D. S., W. VAN ELDEN, AND A. LAJTHA. 1977. Developmental effects on protein synthesis rates in regions of the CNS in vivo and in vifro. J. Neurochem. 29: 939-945.
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29. MOUREK, J., H. C. AGRAWAL, J. M. DAVIS, AND W. A. HIMWICH. 1970. The effects of short-term starvation on amino acid content in rat brain during ontogeny. Bruin Res. 19: 229-237. 30. MOORE, B. W., R. MENKE, A. L. PRENSKY, M. A. FISHMAN, ANDH. C. AGRAWAL. 1977. Selective decreases of S-100 in discrete anatomical ares of undernourished rat brain. Neurochem. Res. 2: 549-553. 31. NEIDLE, A., J. KANDERA, AND A. LAJTHA. 1975. Compartmentation and exchangeability of brain amino acids: Evidence from studies of transport into tissue slices. Arch. Biochem. Biophys. 169: 397-405. 32. NETTLETON, J. A., AND D. M. HEGSTED. 1974. The effects of protein and calorie restriction on tissue nitrogen content and protein catabolism. Nurr. Metab. 17: 166180. 33. PAIN, V. M., M. J. CLEMENS, AND P. J. GARLICK. 1978. The effect of dietary protein deficiency on albumin synthesis and on the concentration of albumin messenger ribonucleic acid in rat liver. Biochem. J. 172: 129- 135. 34. PATEL, A. J., D. J. ATKINSON, AND R. BAL.&ZS. 1975. Effect of undernutrition on metabolic compartmentation of glutamate and on the incorporation of (14C)leucine into protein in the developing rat brain. Develop. Psychobiol. 8: 453-464. 35. PATEL, A. J., R. BALAZS, AND A. L. JOHNSON. 1973. Effect of undernutrition on cell formation in the rat brain. J. Neurochem. 20: 1151- 1165. 36. PATEL, A. J., M. DEL VECCHIO, AND D. J. ATKINSON. 1978. Effect of undernutrition on the regional development of transmitter enzymes: glutamate decarhoxylase and choline acetyltransferase. Dev. Neurosci. 1: 41-53. 37. PERRY, T. L., D. STEDMAN, AND S. HANSEN. 1968. A versatile lithium buffer elution system for single column automatic amino acid chromatography. J. Chromatogr. 38: 460-466. 38. SETA, K., H. SERSHEN, AND A. LAJTHA. 1972. Cerebral amino acid uptake in vivo in newborn mice. Brain Res. 47: 415-425. 39. STARK, M. J., AND I. FRENKEL. 1978. Turnover ofrat liver malic enzyme during induction by protein deprivation. Biochim. Biophys. Acta 520: 452-459. AND A. LAJTHA. 1974. The mode of morphine uptake into 40. TELLER, D. N., T. DEGIJZMAN, brain slices. Bruin Res. 77: 121-136. 41. WIGGINS, R. C., J. A. BENJAMINS, M. R. KRIGMAN, AND P. MORRELL. 1974. Synthesis of myelin proteins during starvation. Brain Res. 80: 345-349. 42. WINICK, M. 1974. Malnutrition and the developing brain. Pages 253-261 in F. PLUM, Eds., Res. Publ. Assoc. Nerv. Merit. Dis.. Vol. 53. Raven Press, New York. 43. ZAMENHOF, S., E. VAN MARTHENS, AND L. GRAUEL. 1971. Prenatal cerebral development: Effect of restricted diet, reversal by growth hormone. Science 174: 954-955. 44. ZAMENHOF, S., E. VAN MARTHENS, AND L. GRAUEL. 1971. DNA (cell number) and protein in neonatal rat brain: Alteration by timing of maternal dietary protein restriction. J. Nutr. 101: 1265-1270.
NEUROLOGY
65, 157- 168 (1979)
Effect of Undernutrition M. BANAY-SCHWARTZ,
Center
for
Neurochemistry3 Received
on Cerebral
Protein
Metabolism
A. M. GIUFFRIDA, T. DE GUZMAN, AND A. LAJTHA’ Rockland
December
Il.
Research
Institute.
1978; revision
Ward’s
received
February
H. SERSHEN,
Island.
New? York
10035
7. 1979
Amino acid incorporation and protein breakdown were decreased in the brain of undernourished rats in early developmental stages (6 days old); at later ages the differences were less significant. Changes in the cerebral free amino acid pool were slight. The greater decrease in the rate of protein synthesis than in that of protein breakdown resulted in a decrease in protein deposition (growth) in the young brain. Changes were similar in the cerebellum and the cerebral hemispheres. The pattern of changes (decrease in both protein synthesis and breakdown) that occurs in brain during undernutrition especially during development differs from the pattern of changes in other organs such as liver and muscle, where protein synthesis and breakdown may change in opposite directions.
INTRODUCTION The effect of nutritional deficiencies on the nervous system has been studied at some detail. The resistance of the adult and the vulnerability of the developing brain is established, with the greatest vulnerability occurring in the rapid growth phase (1, 14, 35, 42, 43). The extent of changes and recovery depends on the extent and duration of malnutrition. Changes in the brain are considerably smaller than in most other organs studied. The comparable resistance, especially of the adult brain, is not well understood. Malnutrition results in reduced cell number and reduced protein content; therefore cerebral protein metabolism is likely to play an important role. Abbreviations: PCA-perchloric acid, OPT-o-phthaldialdehyde. ’ This work was supported in part by U.S. Public Health Service grant NS 03226 from the National Institute of Neurological and Communicative Disorders and Stroke. Dr. Giuffrida was a Visiting Professor; herpresent address is lstituto di Chimica Biologica, I’Universita di Catania, Viale Andrea Doria, 6, 95125 Catania, Italy. A travel grant from the Consiglio Nazionale delle Richerche, Rome, is gratefully acknowledged. 157
0014-4886/79/070157-12$02.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction I” any form reserved.
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ET AL.
The mechanisms of influencing cerebral protein synthesis are not known. Because amino acid pool changes may occur, pulse-labeling may be difficult to interpret; it might lead to findings of increased incorporation with one amino acid and decreased incorporation of another under similar circumstances (27, 28). We recently used a method of measuring brain protein turnover under conditions that permit labeling for a longer time during which the specific activity of the precursor amino acid is kept constant (21). Here we report our findings on the effect of undernutrition on cerebral protein turnover (synthesis and breakdown) with this method. MATERIALS
AND METHODS
Sprague-Dawley rats were kept undernourished from the 10th day of pregnancy through lactation, by providing them with approximately half their normal diet- 10 g food daily per rat before parturition and 15, 20, and 25 g daily in the lst, 2nd, and 3rd week of lactation, respectively (35). Control animals received the same food ad libitutn (Teklad 10% breeder diet, containing 16.5% crude protein, 10% crude fat, and 2.5% fiber). Water was freely available to all rats. Each rat, control or experimental, was nursing eight pups. Experiments were carried out at the following ages: 6,12,30, and 42 days old. The young rats were left with their mothers until the age of 30 days. The last age group (42 days old) were removed from the mother at the age of 30 days and fed 20 to 24 g food per day per group of eight experimental animals. On the morning of the experiment, the rats were injected i.p. with a tyrosine suspension (L-[U-14C]tyrosine) 0.01 ml/g body weight, containing 50 $Zi and 50 mg tyrosine per milliliter. After 2 h the brains were removed and dissected into three parts, (a) cerebrum, (b) cerebellum, and (c) midbrain, brain stem, and olfactory bulbs. The regions were frozen in dry ice, weighed, and homogenized in 2 ml 5% (w/v) perchloric acid (PCA). After centrifugation for 30 min at 10,000 rpm of the homogenates, the PCA supernatants were stored frozen for subsequent determination of the specific activity of the free tyrosine by the method described previously (22). The protein pellets were washed by repeated (3x) resuspension and centrifugation in 2.5 (w/v) PCA, followed by methanol, chlorofotmmethanol l:l, absolute ethanol, and ether extractions. The dry protein pellets were dissolved in 1 N NaOH, and on one portion protein determination was done (26); on another portion radioactivity was measured in Triton-toluene scintillation mixture (40); and a third portion was hydrolyzed 24 h in 6 N HCl at llO”C, followed by the enzymatic determination of the proteinbound tyrosine specific activity (22).
PROTEIN METABOLISM IN UNDERNUTRITION
159
On a number of samples the free tyrosine activity was determined also by column chromatography (2), using a modification of the method described by Neidle et al. (31). A column (75 x 0.6 cm) of Biorad Aminex A-6 resin was eluted with lithium citrate buffer gradients (37) from pH 2.8 to 6.1. A nine-chamber gradient was used, containing 37.5 ml in each chamber: pH 2.8 buffer in the first three, pH 4.3 in the next two, and pH 6.1 in the last four chambers. To facilitate separation of threonine and serine, 2.5 and 1.5 ml methanol were added to the first and second gradient chambers, respectively, replacing part of the pH 2.8 buffer. The column was kept at 45°C during the entire analysis, which required 9 h. During the amino acid analyses, buffers were pumped through the column at a rate of 0.6 mYmin. To remove traces of ammonia from the buffers, a small (10 x 1.5 cm) column containing DC-3 resin (Pierce Co., Rockford, Illinois) was connected to the system at the entrance to the Milton-Roy pump. This buffer-cleaning column was left in place permanently. After each run, both columns were regenerated with 0.3 N LiOH (1.5 h), followed by pH 2.8 lithium-citrate buffer for 3 h. The sample to be analyzed (in 3% sulfosalicylic acid) was loaded on top of the Aminex A-6 resin by use of a stream of nitrogen. The column effluent was passed through a splitstream connector, and part of it (0.23 mYmin) was passed through a peristaltic pump for the detection of the amino acid by fluorescence with o-phthaldialdehyde (OPT) using a Fluoromonitor. The rest of the effluent (0.37 ml/min) was collected on a fraction collector, directly into scintillation vials containing a toluene-T&on-based scintillation fluid (40) to form a gel, for radioactivity measurements in a scintillation counter. The OPT reagent was made up as follows: 80 mg OPT was dissolved in 1 ml methanol; 0.04 ml 2-mercaptoethanol was added, then 100 ml 0.4 M borate buffer, pH 10. The fluorescence intensity was recorded on a dual-channel recorder. The same method was used for the determination of the free amino acid composition of the brain regions of rats at several developmental stages. For the leucine experiments, the control and undernourished rats (12 and 30 days old) were injected i.p. with tracer amounts of L-(1-14C)leucine, 0.1 $X/g body weight. Total radioactivity of the free (precursor) leucine was determined in the PCA supernatants, the protein pellets were extracted and dried as above, and the incorporated radioactivity per milligram protein was determined. RESULTS Changes in Protein Synthesis Rates. As was shown previously (17), including in our laboratory (7, 20), the rate of cerebral protein synthesis is high at birth and declines during development. In mice, by 30 days of age amino acid incorporation rates are close to adult values. Incorporation
160
BANAY-SCHWARTZ TABLE
ET AL. 1
Changes in Protein Synthesis in Malnourished
Brain”
Protein synthesis in brain regions Synthesis rates in control brain percentage per hour
Age of animal (days)
Hemisphere
Cerebellum
Brain stem
6 12 30 42
1.81 0.94 0.68 0.70
2.20 1.03 0.77 0.76
1.54 0.83 0.64 0.78
Synthesis in malnourished percentage of controP Hemisphere 772 101 i85+ 101 +
7 10 9 6
Cerebellum 76 98 88 I05
+ c + 2
8 6 5 8
as
Brain stem 90 + lOOk 912 96?
10 7 3 7
o Incorporation of tyrosine in rat brain for 2 h was measured; averages of four experiments *SD are given. b Synthesis of the region as percentage of the control rate of synthesis of the same region at each age.
rates in cerebellum are higher than in the cerebrum in the immature brain (7). Similar results were found in our experiments, for control and for malnourished brain (Table 1). Protein synthesis rates were somewhat lower in malnourished brain: at 6 days of age amino acid incorporation was about 25% lower in the hemispheres and cerebellum; changes were small in the brain stem. From 12 days of age, incorporation in malnourished brain was close to control values, although slightly lower (about 10%) at 30 days. In these experiments we measured the incorporation of a large dose of labeled tyrosine administered intraperitoneally. The use of a tyrosine suspension, which assures continuing absorption of this relatively insoluble amino acid, results in a constant specific activity of the cerebral free tyrosine during the experimental period (21), as confirmed in the present study. This method also results in an increase in the cerebral free tyrosine levels without significantly affecting the other amino acids in the brain (see below). We also tried to measure the incorporation of tracer doses of labeled leucine at various times (15, 30, and 60 min). Only the 15-min experiments could be clearly interpreted, because by 30 min a major portion of the label in brain was present in compounds other than leucine. The short-term leucine experiments with 12- and 30-day-old animals gave results similar to those with tyrosine. Changes in Deposition and Breakdown of Brain Proteins. In the period of our study (from birth to 42 days) the brain undergoes rapid growth, including growth (net deposition) of brain proteins. The rate of growth is not uniform, but is high in the early stages of development (to 6 days), and growth is practically stopped by 42 days. The rate of deposition of
PROTEIN
METABOLISM
161
IN UNDERNUTRITION
proteins, calculated as percentage net increase per hour, was lower in the hemispheres of malnourished mice at 6 days; at later times growth was similar to controls. The decrease in the deposition of cerebellar proteins could be observed during a longer developmental period (Table 2). Because growth is most rapid at the early stages of development, the total protein content of hemispheres was lower at 30 days in the malnourished animals even though inhibition of growth could be clearly observed only in the 0- to 6-day period. As the rate of synthesis of proteins in the brain is greater than net deposition at all stages of development, continuous turnover (breakdown of proteins) occurs. In the (nongrowing) adult brain, breakdown is equal to synthesis; during development, growth is the result of greater synthesis than breakdown. The present experiments (Table 2) confirmed previous results (31) showing that the rate of breakdown is higher in the immature brain undergoing net growth than in the nongrowing adult brain. When growth stops, the rate of breakdown, like that of synthesis, reaches adult values. The changes of protein breakdown are similar to changes of synthesis: there was a significant (about 20%) inhibition of protein breakdown in the malnourished brain at 6 days, no change at 12 days, and a slight inhibition at 30 days (Table 2). Although breakdown was inhibited at 6 days, the fact that protein synthesis, which proceeds at a higher rate, was inhibited to a slightly greater degree resulted in the inhibition of net deposition of proteins at 6 days of age. TABLE
2
Changes in Protein Deposition and Breakdown Age of animal (days)
Rates in control brain percentage per hour
Brain”
Rates in malnourished as percentage of controP
Brain region
Deposition
6
Hemisphere Cerebellum
0.63 0.87
1.18 1.33
67 76
82 76
12
Hemisphere Cerebellum
0.36 0.52
0.58
0.51
83 90
112 106
Hemisphere Cerebellum
0.07 0.10
0.61 0.68
114 70
82 93
30
Breakdown
in Malnourished
Deposition
Breakdown
0 Deposition (net growth) of protein was estimated by measuring the total protein content of brain regions at various ages. Breakdown was calculated as the difference between total synthesis and deposition. b Synthesis of the region as percentage of the control rate of synthesis of the same region at each age.
162
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ET AL.
Changes in Levels of Cerebral Free Amino Acid. Because changes ir protein synthesis and breakdown may be related to changes in the composi, tion of the free amino acids in the tissue, we measured the levels of fret amino acids in the brain at various ages (Table 3). The developmenta pattern of increase in glutamate, glutamine, and aspartate levels and de, crease in glycine and alanine and in most of the essential amino acids with the quantitatively largest change in the decrease in taurine, confirmec numerous previous studies (15, 23)..Aspartate and glutamate levels wert decreased in the first 6 or 12 days of undernutrition, respectively, ant some decreases were observed in this period in the levels of valine, iso leucine, and leucine. In a separate set of experiments, we tested the in fluence of the administered tyrosine on the cerebral free amino acid pool The only change in brain was the increased level of tyrosine. This amine acid increased to 0.80 pmol/g from newborn to 12 days, to 0.60 at 30 days and to 0.48 at 42 days. The reduction with age of tyrosine uptake in these tyrosine-injected animals was similar to previous observations, indicating a maturation of cerebral barriers that decreased uptake of amino acids by the brain from the blood (38). The changes in the composition of the free amino acid pool of the cerebel lum during undernutrition were similar to the changes found in the cerebrum There was a decrease in glutamate and aspartate and a significant increase initially of taurine and alanine (Table 4). The changes in these amino acids indicate a retardation of developmental changes in at least some aminc acid values during the early phase of undernutrition.
DISCUSSION Effects of Undernutrition on Brain Proteins. It is well established that malnutrition during development results in a reduced number of cells in the brain (44), which upon longer duration can lead to a permanent deficit (42). The effects are complex and extensive. Cell numbers, also brain weight and brain protein content, are reduced, and myelin content is decreased (19). There is also a decrease in the rate of cell loss usually observed in the developing brain, and the length of the different phases of the cell cycle is altered (1). Depending on experimental conditions, cell acquisition rate can be reduced and degenerating cells can increase in number (24). Myelination and synaptogenesis may be affected (6). Regional Differences in Brain and Changes in Specific Protein Components. Our knowledge of the metabolic rates of well-characterized brain
proteins is meager, but the indications are that undernutrition affects the various proteins to a different degree and that there are regional differences not only in the effects of undernutrition on overall protein synthesis but in the effects on specific proteins as well. Myelin protein synthesis was
1.47 0.08 0.41 0.23 0.63
1.34 0.08 0.43 0.26 0.51
1.30
18.3 1.69 0.79 1.01 4.86 2.37 1.79 1.25 0.19 0.17 0.10 0.17 0.16 0.15
Experimental
6-day
1.88 0.05 0.29 0.17 0.44
16.0 2.68 0.93 1.31 7.19 3.01 0.78 1.56 0.22 0.10 0.12 0.25 0.26 0.12
Control
1.86 0.06 0.28 0.16 0.30
15.2 2.57 0.81 1.45 6.25 2.54 0.96 1.49 0.15 0.09 0.11 0.20 0.23 0.10
Experimental
12-day
3.51 0.03 0.45 0.26 0.62
9.88 4.83 0.92 1.60 11.3 5.14 1.22 1.28 0.23 0.15 0.17 0.34 0.20 0.15 3.07 0.04 0.37 0.17 0.47
1.64 11.7 4.86 1.22 1.25 0.22 0.10 0.15 0.30 0.21 0.14
10.1 5.51
Experimental
30-day Control
Cerebrum
of the Free Amino Acids in the Cerebrum”
3
2.48 0.04 0.16 0.14
8.73 3.78 0.93 1.00 12.7 5.21 0.84 0.93 0.14 0.06 0.09 0.10 0.08 0.08
Control
0.29 0.12
1.46
13.9 5.42 0.66 0.52 0.07 0.04 0.06 0.07 0.07 0.07
9.79 2.70
Experimental
42-day
I’ Micromoles amino acid per gram brain tissue. Averages of six experiments are given with standard deviations within 5% of the mean. ’ Whole brain in addition to cerebrum contained cerebellum, brain stem, and olfactory bulbs.
2.02 7.10 3.62 2.06 2.24 0.31 0.10 0.18 0.42 0.28 0.18 1.56
17.03 2.07 0.89 1.35 5.11 2.48 1.35 1.38 0.25 0.23 0.19 0.25 0.18 0.11
17.9 1.13 1.02 1.66 6.20 2.73 1.85 2.47 0.27 0.11 0.15 0.39 0.28 0.16
19.2 1.73
Taurine Aspartic acid Threonine Serine Glutamic acid Glutamine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine y-Aminobutyric acid Ornithine Lysine Histidine Arginine
Control
Control
Amino acid
Experimental
Whole brainb Newborn
Concentrations
TABLE
5
z
2 2 I% z E; P z
$ P z
2 3 m 2 g 3
1.56 0.06 0.32 0.18 0.30
1.71 0.09 0.36 0.19 0.33
19-day
2.97 0.10 0.63 0.22 0.50
0.39 0.49 0.19
1.44 4.41 0.31 0.14 0.17
15.1 1.92 1.39 1.84 7.87 4.16
Experimental
3.41 0.55 0.25 0.66
0.44 0.26 0.21
1.28 2.09 0.30 0.12 0.21
10.6 4.35 1.39 1.70 10.6 6.86
Control
30-day
3.59 0.55 0.20 0.70
0.53 0.30 0.23
1.39 2.13 0.28 0.11 0.27
8.43 4.73 1.77 10.0 5.94
Experimental
3.06 0.05 0.23 0.12 -
0.15 0.12 0.14
1.00 1.12 0.13 0.05 0.11
8.64 3.14 1.39 0.85 11.2 6.13
Control
42-day
1.32 0.38 0.11 -
0.08 0.12 0.09
0.63 0.53 0.08 0.11 0.08
8.12 2.22 0.59 12.8 6.27
Experimental
’ Micromoles amino acid per gram brain tissue. Averages of six experiments are given with standard deviations within 8% of the mean.
2.58 0.06 0.45 0.23 0.53
0.35 0.28 0.17
0.21 0.34 0.12
0.24 0.30 0.13
Leucine Tyrosine Phenylalanine y-Aminobutyric acid Omithine Lysine Histidine Arginine
0.92 3.47 0.24 0.17 0.16
1.04 3.43 0.15 0.13 0.11
1.03 2.28 0.16 0.09 0.15
Glycine Alanine Vahne Methionine Isoleucine
Control 11.0 2.54 1.41 1.64 9.08 5.20
10.6 2.32 1.15 1.36 5.89 3.85
Taurine Aspartic acid Threonine Serine Glutamic acid Glutamine
Experimental
4
of the Free Amino Acids in the Cerebellum”
11.2 2.01 1.15 1.45 4.61 3.66
Control
Amino acid
12-day
Concentrations
TABLE
F
5 ; 4
3
2 k
g
PROTEIN
METABOLISM
IN UNDERNUTRITION
165
depressed more than other proteins; the depression in proteolipid protein was greater than in other myelin proteins (41). There were regional differences in the decreased levels of S-100 protein, whereas 14-3-2 did not change (30): S-100 decreased in the visual and increased in the sensory motor cortex (16). The type of changes we observed here-initial depression of synthesis with later recovery even during undernutrition-were observed in most regions, in measuring glutamate decarboxylase and choline acetyltransferase activity (36). Whereas choline acetyltransferase activity decreased, acetylcholinesterase activity in the brain stem increased (9). In general the effect of malnutrition was proportional to regional growth rates, the regions with the most rapid cell division being affected the most (12); but different cells in the cerebellum also showed different sensitivity to undernutrition (4). Changes in Amino Acids. Changes in cerebral amino acid content have been reported in several protein and calorie deficiency experiments. In rats, in general, amino acids decreased (29), although postnatal decreases in a number of amino acids were less marked or were delayed (10). Typtophan initially increased, then decreased, during malnutrition (18). In monkeys the greatest changes observed were increases in basic amino acids (histidine, homocamosine, methylhistidine, and lysine), with a smaller decrease in many others, and a decrease in acidic amino acids (11). The fact that growth hormone alleviated deficiency (43) indicated that amino acid changes were not the main cause of altered protein metabolism. Previously we did not find any changes in cellular amino acid transport in brain slices as a result of malnutrition (3). Changes in Protein Synthesis. Changes in protein turnover due to malnutrition have not been measured in the brain. Decreases in amino acid incorporation after prolonged undernutrition were observed (34), including in our laboratory (3). Not all proteins were affected in a similar manner; on a deficient diet, liver albumin synthesis and the concentration of albumin messenger RNA decreased, whereas much of general liver protein synthesis was not affected (33). Malic enzyme activity increased in liver under such conditions because rates of syhthesis were elevated, with no change in the breakdown of this enzyme (39). Tissue protein synthesis in adult animals on a protein-free diet was compared (13): in liver, protein synthesis was unaltered for 2 days, then increased; in muscle, synthesis immediately decreased a great deal; in heart, brain, and kidney, changes were small but significant decreases could be detected by 21 days. The decrease of protein synthesis by the 30th day is parallel to the termination of glia proliferation in the cerebral hemispheres and the termination of both neuronal and glial proliferation in the cerebellum. Our findings indicate a delayed growth in the undernourished brain as shown at 12 days. Undernutrition also delayed
166
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ET AL.
maturational changes in nucleic acid metabolism (Giuffrida and Hamberger, in preparation). Alterations of Protein Breakdown in Other Tissues. The effect of malnutrition on cerebral protein breakdown has not been measured previously. Protein synthesis and degradation represent independent mechanisms that often do not change in parallel fashion. Their relationship has been shown to vary in different organs and under different circumstances. During development, protein synthesis was increased and breakdown was decreased in the liver (5); in brain, both synthesis and breakdown were greater in the immature than in the mature organ, and growth was the result of the greater increase in synthesis than in breakdown (8). In malnutrition, protein synthesis and protein breakdown decreased in muscle, whereas in the liver both protein synthesis and protein breakdown increased; loss of proteins occurred in both tissues (13). In rat muscle protein synthesis and degradation were not affected in the soleus, whereas synthesis was decreased and breakdown was increased in the extensor digitorum longus (25). Measurements of breakdown utilizing the decrease in prelabeled proteins are difficult because changes in amino acid reincorporation could occur in malnutrition (32). In brain, changes in rates of synthesis and rates of breakdown appear to occur in parallel fashion during growth and during malnutriton. The increase during growth and the decrease during malnutrition in synthesis are greater than the corresponding changes in breakdown, resulting in normal growth in brain of young controls and decreased growth in brain of undernourished animals. REFERENCES 1. BALAZS, R., P. D. LEWIS, AND A. J. PATEL. 1975. Effects of metabolic factors on brain development. Pages 83-115 in M. A. B. BRAZIER, Ed., Growth and Development of the Brain. Raven Press, New York. 2. BANAY-SCHWARTZ, M., G. ZANCHIN, T. DEGUZMAN, AND A. LAJTHA. 1979. The effect of corticosteroids on amino acid content of brain tissue preparations. Psychoneuroendocrinology, in press. 3. BANAY-SCHWARTZ, M., G. ZANCHIN, T. DEGUZMAN, H. SERSHEN, AND A. LAJTHA. 1978. Decrease in cerebral protein synthesis on a low protein diet. In H. CH. BUNIATIAN, Ed., Problems of Brain Biochemistry, Vol. 13. Armenian Acad. Sci., U.S.S.R. 4. BARNES,‘D., AND J. ALTMAN. 1973. Effects ofdifferent schedules of early undernutrition on the preweaning growth of the rat cerebellum. Exp. Neural. 38: 406-419. 5. CONDE, R. D., AND 0. A. SCORNIK. 1977. Faster synthesis and slower degradation of liver protein during developmental growth. Biochem. J. 166: 115-121. 6. DEO, K., V. BIJLANI, AND M. G. DEO. 1978. Effects of malnutrition on cell genesis and migration in developing brain in rats. Exp. Neurol. 62: 80-92. 7. DUNLOP, D. S., W. VAN ELDEN, AND A. LAJTHA. 1977. Developmental effects on protein synthesis rates in regions of the CNS in vivo and in vifro. J. Neurochem. 29: 939-945.
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