Effect of protein-free diet on the uptake of amino acids by the brain in vivo

EXPERIMENTAL

NEUROLOGY

68, 443-452

Effect of Protein-Free

(1980)

Diet on the Uptake of Amino Acids by the Brain in Vivo

JENOTOTH

AND ABEL

LAJTHA'

Diet that is low in essential amino acids results in lowered protein metabolism and protein content in the developing brain; adult brain is affected less. To learn more about the mechanisms responsible for these alterations, we studied cerebral amino acid uptake in malnourished mice. Protein-free diet resulted in changes in the concentration of several components in the free amino acid pool of the brain. Histidine and homocarnosine concentration greatly increased: other changes observed in young and adult brain were an increase in phenylalanine and some decreases, especially in valine. isoleucine. tyrosine. and lysine. In adult brain ornithine and arginine also decreased. The gross amino acid composition of proteins was not changed under these conditions. In young mice changes in amino acid uptake in the instances we measured were parallel to changes ofconcentrations of these amino acids in brain. Without amino acid administration histidine increased. lysine decreased. and leucine was unchanged in the malnourished animal. Similarly. histidine uptake increased, lysine uptake decreased, and leucine uptake was unchanged in the malnourished animals compared to controls; a-aminoisobutyrate uptake was also unaffected. We conclude that malnutrition affects cerebral transport processes in a complex way: uptake of some compounds is increased, of some others decreased. These changes in turn result in changes in the concentration of cerebral metabolites. and in the rate of their metabolism.

INTRODUCTION Diet low in proteins or in essential amino acids profoundly affects the protein content of organs. Changes in the brain are much less than in most other organs, and they affect the developing brain primarily (1, 4, 22). Although numerous studies investigated the vulnerability of the developing Abbreviations: TCA-trichloroacetic acid, AIB-cu-aminoisobutyric ’ This work was supported in part by U.S. Public Health Service York State Health Research Council award 1362.

grant

acid. NS 03226 and New

443

0014-4886/80/060443-lO$O?.OO/O Copyright ri; 1980 by Academic Press. Inc. All rights of reproduction in any form rexwed.

444

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AND

LAJTHA

brain and conditions of recovery, the reasons for the relative resistance of brain and the greater resistance of adult brain are not clear. Our previous results (2) indicated that in brain the protein breakdown decreased when malnutrition inhibited protein synthesis. This would indicate that control of breakdown rates participates in preserving cerebral proteins. An alternative mechanism might be the active transport of amino acid to the brain (17), which could maintain the level of amino acids in adult but may not be fully developed in the young (19). In the following, we present measurements of amino acid uptake in brain in severely undernourished mice (on a protein-free diet). For measuring changes that may be responsible for alterations of brain metabolism, uptake during a longer time period was measured, to estimate the end result of changes in capillary and cellular transport. MATERIALS AND METHODS Male Swiss mice bred in our colony were used. The adult mice were 8 weeks old and the young mice were 2 weeks old when the diet was started. The protein-free diet was purchased from Nutritional Biochemical Corporation: it was 85% corn starch, 10% vegetable oil, 4% salt mixture, and 1% cod liver oil with fat-soluble vitamins. B-complex vitamins were added to the drinking water. The preparation of brain samples for analysis of free amino acids was the same as reported previously (9). After decapitation the brains were rapidly excised and frozen in dry ice. Then the brains were weighed and homogenized in 4 vol of 1% perchloric acid. The homogenates were kept 20 min at 4°C and then centrifuged 20 min at 30,OOOg. The supernatants containing the free amino acids were stored at -20°C until analysis. Portions (0.2 ml) of the supernatant were analyzed directly in the Technicon TSM type autoanalyzer. The acidic and neutral amino acids were eluted with0.3 N lithium citrate atpH 3.2,2.7,3.3, and4.15. The basic amino acids were eluted with 0.5 and 0.8 N lithium citrate atpH 3.8, 5.15, and 6.0. For the analysis of protein-bound amino acids the pellets after centrifugation at 30,OOOg (as described above) were washed three times with 5% trichloroacetic acid (TCA). Nucleic acids were removed by heating the tissues in 5% TCA at 90°C for 15 min. Lipids were extracted by washing the pellets with chloroform:methanol(2: 1) mixture and three times with acetone and ether. The proteins were then dried, and 10 to 15 mg was hydrolyzed in 6 N HCI at 145°C (18). The hydrolyzed samples were dried over KOH pellets and P,O, powder under vacuum. The dried samples were dissolved in lithium citrate buffer pH 3.2 and analyzed for amino acid content.

CEREBRAL

TRANSPORT

IN MALNUTRITION

445

In the uptake studies the mice were injected intraperitoneally with the solutions of the amino acids tested. After predetermined times the animals were killed by decapitation. Samples of plasma and whole brains were frozen in dry ice, weighed, and homogenized in 5 vol of 5% TCA. Homogenates were centrifuged 20 min at 30,OOOg and the supernatant was filtered on Whatman No. 1 filter paper. The pellets were resuspended in 5 vol of 5% TCA two times and centrifuged as before. The supernatants obtained were combined, and after removal of TCA on Dowex 2 the amino acid content was determined microbiologically (20). The uptake of a-aminoisobutyric acid (AIB) was measured by using labeled AIB for injection, and the uptake was calculated from the radioactivities of TCA extracts of plasma and brain samples, as AIB is not metabolized. RESULTS Changes in the Free Amino Acid Pool. Because malnutrition is known to affect protein content in young but not in adult brain, we tested changes of the cerebral free amino acid pool in young and adult animals. When young animals were maintained on a protein-free diet there were several changes in the free amino acid pool in the brain. Histidine and homocarnosine greatly increased; phenylalanine also increased significantly. Many amino acids decreased, especially valine. isoleucine, tyrosine, and lysine (Table 1). The changes after 2 weeks of protein deficiency were somewhat greater than after 4 weeks, but any adaptation seemed to be minimal. In the adult animals most changes were similar to those observed in the younger animals; the greatest change in the cerebral free amino acid pool was the increase in histidine and homocarnosine, and phenylalanine was also increased. The decrease of valine, isoleucine, tyrosine, and lysine could also be observed at both ages. Changes observed in adult but not in the younger brain were a decrease in ornithine and arginine and an increase in serine (Table 2). Changes in Proteins. To establish whether or not the protein composition in brain undergoes major changes we measured the amino acid composition of total brain proteins at two time points after the protein-free diet was started (Table 3). No significant change could be detected in the total protein-bound amino acid composition of brain protein hydrolysates. Uptake of Amino Acids. To study the relationship between changes in amino acid concentrations and amino acid uptake, we measured the uptake of some representative amino acids. We selected one time point @-week-old mice maintained on the protein-free diet for 30 days) and selected one amino acid that increased in the experimental animal (histidine), one that decreased (lysine), and one that did not change

446

TOTH AND LAJTHA

(leucine) under the experimental conditions. We compared uptake after 20 min in each case, and also measured shorter and longer periods when feasible. Uptake lasting 20 min represents the results of several processes-capillary transport, cellular transport (uptake and exit), and possibly some metabolism. We also measured the uptake of a nonmetabolizable amino acid (a-aminoisobutyric acid) in the same time periods. We adjusted the dose of intraperitoneally administered amino acid to greatly increase plasma concentrations (to 5 to 25 mM) and to yield a fairly constant plasma concentration in the 5 to 20-min period. The comparison of uptake in the three tests (Table 4) showed fairly good correlation between changes of concentration and of uptake as shown by the comparison of brain: plasma ratios of amino acids. This ratio was greater than one, indicating greater uptake in the experimental animals with histidine, where the concentration in the brain was also increased by the diet. Leucine concentrations and uptake were similar in the experimental and control animals, whereas those of lysine were decreased in the TABLE Effect of Protein-Free

1

Diet on the Free Amino Acids of Brains of Young Mice” Age

Amino

acid

start

control

2 Weeks

Taurine Glutathione Aspartic acid Glutamine + threonine Serine Glutamic acid Glycine Alanine Valine Methionine Cystathionine lsoleucine Leucine Tyrosine Phenylalanine GABA Ornithine Lysine Histidine Homocarnosine Arginine

Il.5 0.918 3.13

5 0.67 t 0.070 + 0.16

9.34 0.853 2.44

2.09 1.04 11.6 0.667 0.770 0.080 0.042 0.008 0.026 0.040 0.102 0.064 2.32 0.029 0.348 0.132 0.358 0.087

-+ -t 2 i +k + -t i k 5 t ? i t % + i

2.82 1.36 II.4 0.833 0.631 0.036 0.021 0.007 0.013 0.026 0.041 0. I18 2.29 0.024 0.181 0.347 0.656 0.108

” Twenty-day-old of free amino acids are presented.

male Swiss is described

0.58 0.05 0.95 0.027 0.045 0.003 0.009 0.001 0.001 0.001 0.003 0.002 0.20 0.009 0.020 0.002 0.001 0.008

diet

Age

control

1 Month

-c 0.22 +- 0.05 2 0.15

10.1 0.739 3.37

9.76 0.863 2.67

i 1.05 k 0.12 -t 0.86 -t 0.028 2 0.052 i 0.002 + 0.002 2 0.005 + 0.001 -t 0.002 t 0.004 2 0.007 ?O.ll z 0.004 k 0.010 t 0.020 zk 0.048 * 0.002

2.67 1.19 12.1 0.753 0.988 0.087 0.027 0.007 0.024 0.043 0.103 0.065 2.69 0.02 I 0.258 0.107 0.292 0.117

mice were put on a protein-free under Materials and Methods.

3.15 1.05 1 I.9 0.939 0.735

diet

control

? 0.030 i 0.04 t 0.049

10.9 0.944 2.46

t 0.530 t 0.170 i 0.246

i * * 2 F

1.99 1.03 11.8 0.741 0.765 0.075

2 i& 2 k 2

0.145 0.133 0.484 0.042 0.043 0.001

0.007 0.023 0.040 0.090 0.059 2.37 0.38 0.286 0.120 0.331 0.125

t k i-c k -t ? 2 * k 2

0.001 0.002 0.015 0.013 0.003 0.270 0.015 0.022 0.005 0.020 0.008

0.410 0.03 0.081 0.021 0.010

0.0505 0.001 0.007 0.016 0.030 0.038 0.069 2.67

-t if + 2 k

0.001 0.001 0.002 0.001 0.001 0.010

0.180 0.560 0.535 0.100

-+ i k *

0.001 0.003 0.063 0.007

diet for 2 or 4 weeks. The extraction The means of three experiments*so

448

TOTH AND LAJTHA

brain (5) showing large increases in histidine and homocarnosine and decreases in many essential amino acids (valine, isoleucine, and tyrosine; not phenylalanine, which increased). Some smaller changes (lysine, glutamate) seem to be in the opposite direction in the two species. Because in the monkey brain the changes showed considerable regional heterogeneity, it is not surprising to find some differences between species. Changes in Protein Metabolism. Changes of protein metabolism that were due to malnutrition were studied in muscle in some detail. In rat muscle, milder treatment such as undernutrition affected synthesis rates primarily: stronger treatment such as a prolonged protein-free diet resulted in decreased synthesis and also decreased breakdown, although the decrease in synthesis was greater. Complete starvation decreased synthesis but increased breakdown (14). The changes depended on the age and on the nutritional state of the animal. Fasting decreased protein synthesis in muscle of adult and young rats, but it increased protein degradation only in young animals (10). In liver, protein deprivation caused TABLE

3

Amino Acid Composition of the Cerebral Proteins of Young Mice on Control and Protein-Free Diets” Micromoles amino acid per gram protein Protein-free diet Amino acid Aspartic acid Threonine Setine Glutamic acid Proline Glycine Alanine Vahne Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

Control 548 + 316 2 335 k 713 + 320 2 470 2 509 k 439 + 337 k 568 k 191 + 261 + 413 2 141* 415 k

22 12 1 22 10 10 14 1 1 29 10 11 13 1 15

30-day 575 336 352 716 295 493 523 426 331 595 204 277 440 153 467

2 49 z!z36 2 38 2 60 -t 23 f 33 -+ 37 r 36 f 32 2 41 + 19 f 21 2 30 2 7 ? 8

45-day 521 + 312 2 336 2 654 f 256 2 444 2 458 + 373 t 228 r 516 ‘1852 253 f 374 ‘137-t 373 k

24 I1 40 19 8 10 5 4 7 14 6 3 22 3 27

Control:diet ratio 1.00 0.98 0.98 1.05 1.11 1.06 1.04 1.10 1.09 1.03 0.98 0.99 1.02 0.97 1.00

a The mice were fed a protein-free diet from 2 weeks of age. Brain proteins were prepared and hydrolyzed as described under Materials and Methods. The values presented are the means of three or more determinations.

CEREBRAL

TRANSPORT

449

IN MALNUTRITION

TABLE

4

Effect of Protein-free Diet on the Uptake of Amino Acids” Micromoles

amino

acid

per gram

fresh Diet

Control Amino

acid

Histidine

Leucine

Lysine

a-Aminoisobutyric

acid

” Eight-week-old intraperitoneally, 150 mM solution. Methods.

Time (min)

tissue

Plasma

Brain

Brain:plasma

Plasma

diet/brain: plasma control

Brain

0

0.09

-t 0.01

0.081

2 O.Oli

0.10

z? 0.01

0.31

? 0.03

3.4

20 60

9.99 9.59

2 I.21 k 0.95

0.98 1.90

i 0.12 k 0.21

9.34 9.15

2 1.04 -c 1.12

1.25 2.97

5 0.16 t 0.43

1.37 1.64

0

0.11

-t 0.01

0.036

i- 0.002

0.11

2 0.01

0.036

2 0.003

1.00

5 20

3.62 5.17

t 0.14 2 0.28

0.188 0.409

-t 0.023 -t 0.025

3.91 4.51

t 0.11 ‘*- 0.08

0.210 0.394

2 0.024 k 0.030

1.03 1.10

0 5

0.42 13.7

2 0.02 -t I.1

0.193 0.446

e 0.012 zk 0.034

0.42 14.8

I 0.03 2 1.1

0.143 0.3%

k 0.010 + 0.020

0.74 0.82

20

9.06

2 0.47

0.575

2 0.027

12.4

2 1.5

0.423

2 0.019

0.54

2 2.0 t 2.6

0.569 1.14

2 0.095 k 0.15

29.2 24.4

f 4.3 c 2.7

0.578 1.13

+ 0.096 -e 0.19

0.92 1 .O?

5 20

26.4 25.1

mice were were histidine-8

solution; lysine-13 For determination Averages of four

fed

control pmolig

Fmolig. of amino experiments

or protein-free body weight.

diets for 30 days. Amino acids, from a 260.mM solution; leucine-5

270 rn~ solution: and acids microbiologically ?SD are shown.

aminoisobutyrateor by label

see

injected pmol/g.

16 pmolig, 240 under Materials

mM and

an increase in protein synthesis and in protein breakdown rates (6). In all these cases, when the changes resulted in greater breakdown than synthesis, there was a decrease in tissue protein content. The decrease of breakdown when synthesis is decreased would serve to minimize protein losses, whereas the increased breakdown during drastic starvation would make amino acids available to other tissues. Temporary transient changes in breakdown such as was observed with dietary changes in rat liver ornithine aminotransferase (3) could serve to establish new equilibrium levels of enzymes or metabolism. Fewer studies have centered on changes in brain protein metabolism during malnutrition. In one study a decrease in phenylalanine incorporation, but an increase in tryptophan incorporation, was found (12, 13). This may have been the result of changes in the specific activity of precursor pools that might have been caused by a decrease in the uptake of one but an increase in the uptake of another amino acid. In protein concentration changes caused by malnutrition, no change in one 114-3-21 but a decrease in another IS-1001 protein was found (15); the change in S-100 was heterogeneous, increasing in one region and decreasing in another (8). In our studies (2) the changes in brain were smaller than those for other

450

TOTH

AND

LAJTHA

organs, but amino acid incorporation was decreased in brain, with some regional variations. It seemed in our studies that extreme nutritional deficiency in brain, unlike in liver and muscle, causes only a decrease in protein breakdown. This may be because muscle serves in emergency as a protein depot, whereas brain proteins need to be protected and preserved. Changes in protein metabolism do not seem to explain the changes in the free amino acid pool. Because in the young brain there is a decrease in proteins, a net breakdown would liberate amino acids, and the more slowly metabolized essential amino acids, such as valine, would be expected to be at relatively higher concentrations instead of the lower concentrations we found (Tables 1 and 2). Our results indicate that changes in membrane properties and transport are responsible for the alterations. Changes in Vulnerability in the Developing Brain. A number of studies showed that malnutrition primarily affects metabolism of the immature brain, and that the mature brain is fairly resistant. During active cell division the production of cells is inhibited, and if malnutrition continues for a longer period this results in permanent reduction of cell numbers (23). During a later developmental phase, when DNA does not increase but protein does (cell enlargement period), the increase in protein is restricted; but this is reversed after refeeding (21). These results show that the more severe and more prolonged the malnutrition, and the earlier it is started, the greater is its effect and the longer it lasts (4). Because the cerebellum at birth is more immature (cell division is more active) it is more affected by malnutrition (16), although some adaptation occurs (7). Changes in Amino Acid Pools. The young brain is more vulnerable to malnutrition than the adult; low protein diets decrease protein content only in the developing brain. Because the blood-brain barrier and cerebral amino acid transport are not fully developed, such developmental differences in vulnerability of protein metabolism may be caused by changes in the amino acid pools. Our results indicate that this is not so: the changes in amino acid content are similar in the young and adult brain; furthermore, mild undernutrition in the young affects protein content without concomitant alterations in the amino acid pool. Thus the greater vulnerability of protein metabolism in the young is related more to cell division than to changes in amino acids. Our results show that transport processes are also affected by malnutrition and that severe protein deficiency alters the transport of several (but not all) compounds in a complex manner, the uptake of one compound being increased while that of another is decreased. The parallel changes in uptake and concentration indicate that transport may be the system primarily affected, which then results in the changes in the cerebral levels of these compounds.

CEREBRAL

TRANSPORT

IN MALNUTRITION

451

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K., H. SERSHEN, AND A. LAJTHA. 1972. Cerebral amino acid uptake in r,ivo in newborn mice. Bruin Res. 47: 415-425. 21. WINICK, M. 1974. Malnutritionand the developing brain. Pages 253-261 in F. PLUM, Ed., Brain Dysfunction in Metabolic Disorders. Res. Publ. Assoc. Nerv. Ment. Dis., Vol. 53. Raven Press, New York. 22. 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. Nufr. 101: 1265- 1270. 23. ZAMENHOF, S., AND D. GUTHRIE. 1977. Differential responses to prenatal malnutrition among neonatal rats. Biol. Neonate 32: 205-210. 20. SETA,