Kinetics of utilization in vivo of glucose carbon in the chick cerebral hemispheres during postnatal growth

Comp. Biochem. Physiol., 1976. Vol. 53B, pp. 215 to 224. Pergamon Pres.s. Pri, ted in Great Britain

KINETICS OF UTILIZATION IN VIVO OF GLUCOSE CARBON IN THE CHICK CEREBRAL HEMISPHERES D U R I N G POSTNATAL GROWTH PAUL R. LEHR* AND JACQUES GAYET Laboratoire de Physiologie G6n6rale, Universit6 de Nancy 1, 54 037, Nancy, France (Received 7 October 1974) Abstract--1. The incorporation of glucose carbon in vivo into glucose, amino acids and carboxylic acids in the cerebral hemispheres was studied during post-hatching growth of the chick after subcutaneous injection of [U-~4C]glucose, the animals being decapitated at different times after the injection. 2. The highest rate of incorporation of glucose carbon into the tissue free amino acids occurs between 10 hr and 4 days after hatching. 3. At hatching time, glutamic acid as well as neutral amino acids account for 40% of the whole radioactivity in the amino acid fraction. On the 30th day of postnatal growth, the % of radioactivity in glutamic acid (56%) is higher than that in the neutral amino acids (23%), which is a characteristic of the metabolism in cerebral hemispheres of adult chicks. 4. Studies of the evolution of cerebral glucose concentration during the postnatal growth show an increasing rate between 10 and 48 hr after hatching. 5. On the 2nd, 4th and 30th days of postnatal growth aspartate and glutamate have approximately the same specific radioactivity. On the other hand, at 10hr after hatching, the specific radioactivity of cerebral aspartate is always higher than that of glutamate. 6. At 10 hr after hatching, the specific radioactivity of cerebral ~-alanine is always considerably higher than that of other amino acids. On the 30th day of postnatal growth, and at 10 min after the injection of [U-14C]glucose, the specific radioactivity of ~-alanine is higher than that of other amino acids; afterwards, from 10 to 60 min after the injection of labelled glucose, the specific radioactivity of ~-alanine decreases and its value tends towards zero. INTRODUCTION THE METABOLISM of the adult mammalian brain is characterized by a rapid incorporation of the carbon of blood glucose into free amino acids. The rate of this incorporation was estimated in vivo, on the one hand in the cat (Gaitonde et al., 1964) and the rat (Vrba, 1962; Vrba et al., 1962; Cremer, 1964; Gaitonde et al., 1965; Lehr & Gayet, 1967) after the intravenous or the subcutaneous injection of FU-14C]glucose, on the other hand, in the rat (O'Neal & Koeppe, 1966) and the sheep (O'Neal et al., 1966) after the intravenous injection of [2-14C]glucose. Gaitonde & Richter (1966) had studied the metabolism of glucose in the rat brain during the postnataldevelopment, the animals being killed 20 min after the subcutaneous injection of [U-14C]glucose. In the brain tissue from newborn to 14-day-old rats, 2-13~o of the acid soluble ~4C were present in free amino acids, whereas this percentage reached 45-60 in brain tissue from 22-day-old or older rats. This rapid flux of the glucose carbon into cerebral amino acids appeared at the so-called critical period in the development located between 10 and 15 days after birth, when the cerebral cortex became functionally mature. Many investigations were performed on the morphology, the electrophysiology and the biochemistry of the avian brain during embryogenesis and posthatching maturation, but we had no data concerning

the rate of incorporation of glucose carbon into cerebral amino acids. In a preliminary communication (Lehr et al., 1971) we had followed the rate of utilization in vivo of labelled glucose in the cerebral hemispheres of the chick (Gallus domesticus) during the post-hatching growth: the high rate of incorporation of ~4C from subcutaneously injected [U-14C]glucose into cerebral amino acids took place during the 5 days following hatching. • In this paper, we extended our investigations by the determination of the specific radioactivity of glucose and of the amino acids associated with the tricarboxylic acid cycle (glutamic, aspartic and 7-aminobutyric acids, glutamine and c~-alanine) in the chick cerebral hemispheres at defined stages of the post-hatching maturation, the decapitation of the animals occurring from 5 to 60 min after the subcutaneous injection of [U-14C]glucose.

* Charg6 de recherche au Centre National de la Recherche Scientifique, to whom correspondence may be sent. 215

MATERIALS AND METHODS Animals The experiments were performed on chicks from crossbred flocks of Plymouth × Rhode Island Red at 1, 2, 4 and 30 days of age. The animals were injected subcutaneously with a solution containing 10#~i [U- 14C]glucose (Commissariat fi l'Energie Atomique, Saclay) and 1 mg carrier glucose in 0.5 ml water (approx 4/zCi/100 g body wt); they were decapitated at 5, 10, 20, 30 or 60min after the injection. The head was immediately, immersed into liquid nitrogen for 3 min, afterwards it was kept at -30°C; the blood was collected in a pre-heparinized glass dish.

216

PAUL R. LEHR AND JACQUESGAYET

Preparation of' the tissue extract The cerebral hemispheres were dissected out on a cold stage and weighed. The tissue was dispersed in 6% (w/v) chilled perchloric acid (approx 5 ml perchloric acid solution/g of tissue) (Gaitonde et al., 1965) with a Potter Elvehjem apparatus fitted with a Teflon pestle (Tissue grinder, size A, A. H. Thomas Co., Philadelphia). The suspension was centrifuged at 30000 for 15 rain at 2"C; the sediment was washed once with 5 ml, 6% chilled perchloric acid and centrifuged. The supernatant solution was combined with the main extract. The combined perchloric acid extract was neutralized with 2 M KOH and left 24 hr at 2~'C in order to precipitate all K-perchlorate. This latter was discarded by centrifugation at 3000 g for 15 rain. The supernatant solutions thus obtained were successively passed through a cation-exchange resin (Amberlite IR 120, H + form), then through an anion-exchange resin (Dowex2, formate form). The amino acids adsorbed on Amberlite IR 120 were eluted with 3 M NHgOH; the carboxylic acids adsorbed on Dowex-2 were eluted with 3 M HCOOH. The two eluates containing respectively the amino acids and the carboxylic acids, as the water washings from the column of Dowex-2 and containing the neutral fraction, were evaporated to dryness under reduced pressure at 40C. The residue were redissolved in determined volumes of 10% isopropanol for the amino acid fraction and of bidistilled water for the carboxylic acid and neutral fractions.

Preparation of the blood extract The proteins of 0.5 ml blood was precipitated with 5 ml perchloric acid solution (2.9 ml of 70% perchloric acid and bidistilled water to a vol of 100ml); the suspension was centrifuged at 3000g for 15 min and the supernatant was neutralized with 2 M KOH. The sediment of K-perchlorate was discarded by centrifugation. The extract was submitted to the above-described procedure in order to obtain three main blood fractions: the amino acid, carboxylic acid and neutral fractions.

Isolation of glucose Glucose was isolated from the other components in the neutral fraction by descending chromatography on Whatman No. 1 paper (57 c m x 46 cm) in a solvent having the following composition: n-butanol-acetic acid-water (4.1.5) (Partridge, 1948). The extract was applied at three points along a starting line perpendicular to the direction of displacement and located at 8 cm from the upper edge of the sheet. The chromatogram was dried at room temperature, the two lateral strips were impregnated with silver nitrate reagent (Trevelyan et al., 1950) in order to locate glucose on the central strip and to evaluate its radioactivity. The radioactivity of free glucose, after its separation by paper chromatography and its quantitative estimation in the neutral fraction of nervous tissue or blood, practically represented the total radioactivity evaluated in this fraction at all stages of postnatal growth. Therefore, the expression glucose fraction will be used instead of that of neutral fraction.

Estimation of' glucose The area containing glucose in the central strip of the chromatogram was eluted for 12 hr (Dent, 1947), and the eluate was collected and dried at 37°C. Glucose was redissolved in a definite vol of a perchloric acid solution (2.9 ml of 7050 perchloric acid and bidistilled water to a vol of 100 ml), and its estimation was made by the glucose oxidase-peroxidase method (Bergmeyer & Bernt, 1965).

Separation and estimation of amino acids The procedure of Biserte et al. (1958) and Biserte et al. (1960) which combines electropboretic and classical paper

chromatographic methods, was used. The optical density of the samples was estimated at 575nm in a Beckman spectrophotometer model DB.

Separation of amino acids Jbr measurement of radioactivity In order to evaluate the radioactivity in amino acids the simple electrophoretic separation, as previously described (Lehr & Gayet, 1967), was used. Oxidized glutathione and pyroglutamic acid were also localized on the electrophoregram; this latter molecule did not react with ninhydrin, but it was located at the site of an unknown ninhydrin positive compound. For the determination of the radioactivity in glutamine and ~-alanine within the whole of the neutral amino acids, the extract was first submitted to an electrophoresis at pH 3.9. The two lateral strips of the dried electrophoregram were treated with ninhydrin in order to permit comparatively the localization of the spot containing the individual neutral amino acids in the central strip. The neutral amino acids in the spot area were quantitatively eluted with bidistilled water on a sheet of Whatman No. 3 paper (57cm x 46cm), at a point located at 10 cm from the anodic edge (Biserte, 1957). After an electrophoresis in 1 M acetic acid pH 2.4, the two lateral strips in the dried paper were treated with ninhydrin permitting comparatively the localization of four spots corresponding to glutamine, serine, ~-alanine and glycine; these two latter amino acids being well located. The separation of glutamine was performed after an elution from the spot area on a sheet of Whatman No. 3 paper (57 cm × 46 cm), followed by a descending chromatography in n-butanol acetic acid--water (4.1.5).

Measurement of radioactivity For each experiment we estimated, on the one hand the quantitative value of radioactivity which was administered to the animal, on the other hand the radiochemical purity of the injected [UJ4C]glucose and this one was about 95%. The radioactivity of [U-14]glucose and of the three fractions isolated from the perchloric acid extract of blood were expressed in counts~rain and in disintegrations per minute (dpm). The radioactivity of the three fractions isolated from the perchloric acid extract of brain tissue, that of brain glucose and that of the isolated amino acids were expressed in counts/min. Counting rates were determined in a Beckman liquid-scintillation spectrometer (LS 150 type).

(a) Estimation oj' the radioactivity in cpnl An aliquot of the extract to be estimated was deposited on a disc of Whatman No. 1 paper, and the sample, placed in a vial, was immersed in 20 ml of a scintillator having the following composition: 3 g PPO (2,5-diphenyloxazole), 100rag P O P O P [2,2'-paraphenylene-bis-(5-phenyl-oxazole)] dissolved in I000 ml toluene. The spot areas containing glucose or amino acids cut out from the central strip of chromatograms or electrophoregrams were immediately immersed into 20 ml of the same scintillator.

(b) Estimation of the radioactivity in dpm The scintillation counting mixture having the following composition was used: 4 g P P O , 100rag POPOP, dissolved in 1000 ml of toluene; 3 vol of the fluors were mixed with I vol of Triton X-100 (Rohm & Haas) (Lehr et al., 1971).

Estimation of blood glucose content Blood glucose was estimated by the glucose oxidase-peroxidase method (Bergmeyer & Bernt, 1965).

Glucose carbon in the chick cerebral hemispheres

217 "

Table 1. Distribution of radioactivity into the 3 fractions separated from the perchloric acid extract of the chick blood, 30 min after the subcutaneous injection of (U-14C) glucose Age of

Radioactivity in dpm/ml o f blood

Radioactivity in cpm/ml of blood

chicks

Glucose fraction

(days)

Amino acid fraction

Carboxylie acid fraction

Total

Glucose fraction

Am no a, id fra, tion

Carboxyllc acid fraction

Total

1

83 800

966

3 500

88 266

127 000

I 100

4 950

133 050

1

50 400

I I00

4 350

55 850

74 O00

I 900

5 150

81 050

2

46 200

I 224

3 300

50 724

71 O00

I 800

5 ooo

77 800

5

21 400

650

1 932

23 982

32 0OO

I 024

3 200

36 224

8

35 600

I 575

I00

41 275

52 0(30

2 350

5 900

60 250

15

50 400

1 IO0

4 350

55 8 5 0

77 400

2 250

5 300

84 950

30

27 800

I 124

2 832

31 756

37 600

I 000

3 I00

41 700

RESULTS

Blood The values for the total radioactivity of the glucose, amino acid and carboxylic acid fractions, expressed in cpm and in dpm per ml of blood, are collected in Table 1. The two modes of estimation of the radioactivity lead to different values. By immersing the area of paper containing the labelled compound in the scintillator, the compound does not enter into solution so that it is impossible to estimate its total radioactivity and we obtain relative values expressed in cpm. With the scintillation counting mixture containing Triton X-100 the labelled compound, quantitatively eluted, gives an homogeneous phase. The radioactivity values in cpm are therefore convertible, by using the quenching curve, into absolute radioactivity values, expressed either in dpm or in #Ci. With these two procedures we obtain a same distribution of the percentage in radioactivity between the three fractions. With the values contained in Table 1, we have followed the distribution of radioactivity between the

°~ o

75 2:5"

x x~ x ir--i~o

x

i !

!

HI

5

' I0

:15 D a y s 2'o

25

Fig. 1. Distribution of 14C between the three fractions separated from the perchloric acid extract of the chick

blood, during post-hatching growth, 30 min after the subcutaneous injection of [U-14C]glucose. Abscissae: age of the chicks, expressed in days. H =

hatching.

Ordinates: % of '4C in the three fractions. • free amino acid fraction. O glucose fraction, x carboxylic acid fraction.

three blood fractions during the postnatal development of the chick (Fig. 1). Between the first and the fifth day after hatching it occurs some small changes in the distribution of 14C: 93-89~o in the glucose fraction, 1.5-2'5~o in the amino acid fraction and 5.58'5~o in the carboxylic acid fraction. From the fifth to the thirtieth day, the distribution of the radioactivity between the three blood fractions does not show any significant change. The mean percentage of distribution of radioactivity is 89.5, 2.5 and 8, respectively for glucose, amino acids and carboxylic acids. The greater percentage of radioactivity in the amino acid fraction is attributable to neutral amino acids. The blood glucose content is practically constant between the first and the thirtieth day after hatching (Table 2).

Cerebral hemispheres Distribution of radioactivity between the threeJract ions. Figure 2 (a,b,c,d) illustrates the distribution of 14C between the glucose, amino acid and carboxylic acid fractions extracted from the chick cerebral hemispheres during the postnatal maturation, the animals being decapitated at 5, 10, 20, 30 and 60min after the injection of [U-14C]glucose. In the 10-hr old chick (Fig. 2a), the percentage of radioactivity in the glucose fraction is high, decreasing from 81.5 to 51.4 between 5 and 60 min; at the second day after hatching (Fig. 2b) this same percentage, decreases from 83.9 to 25.6 between 5 and 60 min. In the 4-day and 30-day old chicks the percentage of radioactivity in the glucose fraction decreases as early as the first 5 min being respectively 73'3 and 57.7. At the fourth day (Fig. 2c) this percentage decreases abruptly between 5 and 30 min, then it keeps a constant and relatively high value of about 22; on the other hand, at the thirtieth day (Fig. 2d), after a rapid decrease during the first 20min, the percentage of radioactivity in glucose reaches the low values of 9 and 6, respectively at 30 and 60 min. The evolution of radioactivity in the free amino acid fraction is similar. In the 10-hr old chick (Fig. 2a) the percentage of labelling slowly increases between 5 rain (4"9~o) and 60 min (33'6~o); in the 2-day old chick (Fig. 2b) this percentage steadily increases from 7 and 5min to 67'8 at 60min. On the other hand, at the fourth day after hatching (Fig. 2c), after

218

PAUL R. LEHR A N D JACQUESGAYET Table 2. Blood glucose content of the chick during post-hatching growth Age of the chicks

about I0 hours

glucose

content

(mg glucose/100 ml of blood)

17

158

2 days

9

156

+- 4

4 days

]4

157

+

30 days

12

159

-+ 7

a rapid increase between 5 and 30 min, the percentage of radioactivity keeps a constant value of about 67. In the 30-day old chick (Fig. 2d) it also appears a clear-cut increase during the first 30 min, but the values obtained were higher since the percentage of radioactivity was 77 and 81.5, respectively at 30 and 60 rain. It is important to note that 5 min after the injection of [U-IgC]glucose it appears an increase of the percentage of radioactivity in the free amino acids during the postnatal development of the chick: from 4"9 at 10 hr after hatching this percentage reaches 10.7 at 4 days and 14.1 at 30 days after hatching. The percentage of radioactivity in the carboxylic acids is relatively constant between 5 and 60 rain in 10-hr, 2 and 4-day old chicks (Fig. 2a,b,c), but it de-

-+ 9

8

creases in the 30-day old chick (Fig. 2d) starting from 28"2~o at 5 min it reaches 12.4% at 60 rain. Distribution of radioactivity between amino acids. The sum of the values of radioactivity determined in the individual amino acids (aspartic, glutamic, 7aminobutyric and pyroglutamic acids, basic and neutral amino acids, oxidized glutathione) after an electrophoretic separation, gives a total value which is close to that in the whole amino acid fraction; therefore the recovery of the radioactivity after this treatment is quantitative. The oxidized glutathione and the group of basic amino acids are poorly labelled, since they contain less than 1~ of the whole radioactivity in the free amino acid fraction, henceforth they will not be taken (c)

IOC

(a)

iO0

,,

Blood

Number of estimations

o

75

o~

o

50

5(3

o

__o 25

x- - o ~

o/| i

iO

~

= i

20

i

i

40

30

x

x

x

6

5O

_

60

x

~

x ×

i

i

dO

20

i

~

I

i

i

40

50

60

rain

rain

(b)

I

-x x ~

(d) ioo

I00I o

. f |

75

50

25 x

-x o

,;

2'o

~'o rain

~o

go

~'o

i

i

l

#

i

i

I0

20

30

40

5O

60

min

Fig. 2. Distribution of ~4C between the three fractions separated from the perchloric acid extract of the chick cerebral hemispheres. Abscissae: time, expressed in min, between the subcutaneous injection of [U-14C]glueose and the decapitation of the chick. I = injection of I-U-14C]glncose. Ordinates: % of l'~C in the three fractions. • free amino acid fraction. O glucose fraction. × carboxylic acid fraction. (a) about 10-hr old chicks. (b) 2-day old chicks. (c) 4-day old chicks. (d) 30-day old chicks.

Glucose carbon in the chick cerebral hemispheres I00

(a) 75

50

e~----~

.

.

.

.

~'o

25

rain (b)

0 "-'----'------0- - - - - _ _ _ _ _ . 0 ~0

m

~

m

~

/

rain

(c)

75

•

m

~

~

m

•

rain

(d) 75

@ 50

25

i--

• ....

D

Q

i I0

20

i

m

•

0 i

30

~

i

50

i

60

rnin

Fig. 3. Distribution of 14C between amino acids in the amino acid fraction isolated from the chick cerebral hemispheres. Abscissae: time, expressed in min, between the subcutaneous injection of [U-14C]glucose and the decapitation of the chick. I = injection of [U-14C]glucose. Ordinates: % of ~4C in the amino acids. O glutamic acid. • aspartic acid. [] ?-aminobutyric acid. • neutral amino acids. (a) about lO-hr old chicks. (b) 2-day old chicks. (c) 4-day old chicks. (d) 30-day old chicks.

219

into account for the evaluation of the distribution of radioactivity. Pyroglutamic acid, the metabolism of which is under investigation in our laboratory (Lehr et al. unpublished results), seems to arise from a cyclisation of glutamic acid during the treatment of brain tissue with perchloric acid. The radioactivity in pyroglutamic acid therefore will be added to that of gluramie acid in the evaluation of the distribution of radioactivity. The distribution of ~4C between aspartic, glutamic and 7-aminobutyric acid and the group of neutral amino acids in the cerebral hemispheres of the developing chick is represented in Fig. 3 (a,b,c,d); the values obtained at 5 min after the injection of labelled glucose are not indicated since they are too low. In the 10-hr old chick (Fig. 3a), it appears that the percentages of radioactivity in glutamic acid and in the group of neutral amino acids are approximately the same (about 40Vo). Aspartic and 7-aminobutyric acids are clearly less labelled, the radioactivity in this first amino acid being 2-fold that in this latter one. In the 30-day old chick (Fig. 3d), if the distribution of radioactivity between aspartic and 7-aminobutyric acid is the same as that evaluated in the 10-hr old chick, it is not the same for glutamic acid and neutral amino acids. Between 10 and 60 min after the injection of [U-14C]glucose, the percentage of radioactivity in glutamic acid (56Vo) is more than 2-fold that in neutral amino acids (23%). At 2 and 4 days after hatching (Fig. 3b and c), the distribution of 14C between the amino acids bears a resemblance to that evaluated in the 30-day old chick. Whatever the stage of the postnatal maturation may be, glycine and serine too are poorly labelled. After the chromatographic separation of glutamine, the radioactivity is totally located in the spot area containing it, therefore we assume that practically all the radioactivity determined in this area is attributable to glutamine. Glucose and amino acid contents in cerebral hemispheres. In Table 3 are collected the values for the concentrations of glucose, aspartic, glutamic, and 7aminobutyric acids, glutamine and ~-alanine in the cerebral hemispheres of the chick during postnatal maturation. A 67~/o increase in glucose concentration occurs between 10 hr and 2 days after hatching, then this concentration decreases by 12.7~o between 2 and 4 days, finally, at 30 days, it reaches the value initially obtained at the hatching-time. If the concentrations of glutamic acid and ~-alanine steadily decrease between 10 hr and 30 days after hatching, for aspartic and 7-aminobutyric acids and glutamine it occurs a transient increase of the values between 10hr and 2 days. It is difficult to compare our values with those previously obtained by Levi & Morisi (1971) since these latter concern the whole chick brain. Specific radioactivity of glucose and amino acids in cerebral hemispheres. In Tables 4, 5, 6 and 7 are collected the values of the specific radioactivities of glucose and of the amino acids associated with the tricarboxylic acid cycle in the cerebral hemispheres of the chick during postnatal maturation. In 10-hr old chicks (Table 4), the specific radioactivities of glucose and amino acids steadily increase between 10 and 60 min; the labelling of ~-alanine is higher than that of the other amino acids. Among

220

P A U L R. LEHR AND JACQUES G A Y E T

Table 3. Concentration of glucose and of the amino acids associated with the tricarboxylic acid cycle in the chick cerebral hemispheres, during post-hatching growth (expressed in iLmoles/g of chilled cerebral tissue). The number of estimations is indicated in brackets Age of the chicks Compounds estimated About I0 hours

2 days

30 days

4 days

1,33 ~ 0,09 (9)

2,20 ! 0,29 (12)

1,92 ~ 0,15 (12)

1,23 Z 0,03 (8)

glutamic acid

10,47 ~ 0,40 (10)

9,53 ~ 0,50 (12)

9,28 ~ 0,39 (8)

9,24 ~ 0,35 (lO)

aspartic acid

2,04 ~ 0~15 (9)

2,57 ~ 0,26 (II)

1,95 ~ 0,19 (7)

2,40 ~ 0,14 (IO)

gamma-aminobutyric acid

1,88 ~ 0,20 (9)

2,03 ~ 0,18 (15)

2,09 ~ 0,10 (7)

1,81 ± 0,09 (11)

glucose

glutsmine

4,30 ~ 0,29 (9)

5,47 Z 0,50 (7)

4,15 ~ 0,22 (12)

4,71 ~ 0,33 (13)

a-alanine

0,67 Z 0,13 (12)

0,56 ~ 0,09 (1o)

0,40 ~ 0,09 (17)

0,49 ~ 0,08 (14)

Table 4. Specific radioactivities of glucose and amino acids in the cerebral hemispheres of about 10-hr old chicks, after the subcutaneous injection of (UJ4C) glucose Time between the injection of (U-14C) glucose and the decapitation of the chicks

Body weight (g) Cerebral hemispheres wet weight (g)

Total injected r a d i o a c t i v i t y (~Ci)

IO minutes (expt. n ° 7)

20 minutes (expt. n* I0)

38

36

40

36

0,395

0.378

0,390

0,330

1.14

1,15

1,48

1,15

30minutes (expt. n* 24 D)

60 minutes (expt. n* 12)

Specific radioactivity (cpm/~mole) : 2784

7907

3759

8772

glutamic acid

64

63

II0

338

a s p a r t i c acid

103

108

189

421

67

117

119

269

a-alanine

425

1116

624

2261

glutamine

15

51

134

505

glucose

8a~ma-aminobutyric acid

Table 5. Specific radioactivities of glucose and amino acids in the cerebral hemispheres of 2-day old chicks, after the subcutaneous injection of (UJ4C) glucose Time between the injection of (U-14C) glucose and the decapitation of the chicks

Body weight (g) Cerebral hemispheres wet weight (g) Total injected radioactivity (~Ci)

10 minutes (expt. n ° 33A)

20 minutes (expt. n ° 33B)

30 minutes (expt. n ° 33C)

36

40

38

0,400

0,250

0,344

0,378

1,O5

1,05

1,05

1,05

60 minutes (expt. n ° 33D)

40

Specific radioactivity (cpm/~mole) : 3523

12 636

4658

1684

g l u t a ~ c acid

IOI

934

1159

472

a s p a r t i c acid

49

973

1075

360

gal~a-aminobutyric acid

62

345

537

358

223

593

1037

80

69

426

585

355

glucose

a-alanine glut~ne

221

Glucose carbon in the chick cerebral hemispheres Table 6. Specific radioactivities of glucose and amino acids in the cerebral hemispheres of 4-day old chicks, after the subcutaneous injection of (U-14C) glucose Time between the injection of (U-14C) glucose and the decapitation of the chicks lOminutes ( e ~ c . n ° 14B)

Body weight (8)

35

Cerebral hemispheres wet weight (g) Total injected r a d i o a c t i v i t y

(~Ci)

20 minutes (expt. n* 14D) 37

30 minutes (expt. n ° |6A) 35

60minutes ( e x p t . n ° 16B)

40

0,420

0,460

0,470

0,510

1,09

1,09

1,08

1,08

Specific radioactivity (cpmlumole) :

glucose

3317

4812

2881

3012

81utamlc acid

215

996

1089

1051

aspartic acid

138

975

1200

I006

71

494

610

821

a-alanine

525

1357

1862

1347

81utamine

29

655

833

945

8amma-aminobutyrlc acid

these other amino acids, aspartic acid has the highest specific radioactivity. The specific radioactivity of 7aminobutyric acid is equal to or higher than that of glutamic acid during the first 30min. The specific radioactivity of glutamine, which is low at 10min, steadily increases and is higher than that of aspartic acid at 60 min. In the 2-day and 4-day old chicks (Tables 5 and 6), the specific radioactivities of the amino acids reach maximal values at 30min after the injection of labelled glucose. At 10 min, the specific radioactivity of ~-alanine is clearly higher than that of the other amino acids; on the other hand, between 20 and 60 min it approaches those of glutamic and aspartic acids which have approximately the same value. The specific radioactivities of 7-aminobutyric acid and glutamine are lower. It will be noted that the specific radioactivities of the amino acids are higher at 4 days than at 2 days after hatching.

In the 30-day old chick (Table 7) the specific radioactivity of glucose steadily decreases between 10 and 60min after the administration of [U-14C-lglu cose; conversely, the specific radioactivities of glutamic, aspartic, 7-aminobutyric acids and glutamine steadily increase during this same period. The specific radioactivities of glutamic and aspartic acids are approximately the same, those of 7-aminobutyric acid and glutamine are lower. The specific radioactivity of 7-alanine, which is higher than those of the other amino acids at 10min, afterwards steadily decreases and approaches zero at 60 min. DISCUSSION

In the chick, between the fifth and the thirtieth day of the post-hatching growth, 30 min after the subcutaneous injection of [U-14C]glucose, the blood free amino acids contain 2-5% of the acid soluble 14C.

Table 7. Specific radioactivities of glucose and amino acids in the cerebral hemispheres of 30-day old chicks, after the subcutaneous injection of (U-~4C) glucose Time between the i n j e c t i o n of (U-14C) glucose and the d e c a p i t a t i o n of the c h i c k s I0 mlnuces (egpc. n ° 21)

20 minutes ( e x p t . u ° 22)

30 minutes ( e x p t . n ° 19)

( e x p t . n ° 23)

575

585

306

470

Cerebral hemispheres wet weight (g)

0,865

0,737

0,870

0,800

Total injected radioactlvity (¢Ci)

25,1

25,1

12,6

23,4

6955

3199

2990

1906

glutanL~C acid

534

1674

1953

2638

asparcic acid

546

1357

1964

2292

8amma-am£nobutyric a c i d

362

900

1207

1865

865

692

704

255

213

792

927

1645

Body weight (g)

Specific radioactivity (cpm/~mole) : glucose

a-alanlne

glutamine

222

PAUL R. LEHR AND JACQUES GAYET

This result confirms that labelled a-amino acids are amino acids (23~o); the percentage of radioactivity in formed from glucose present in the brain and that ~-alanine is very low and tends to zero at 60min they are not transported from the blood stream into after the injection of labelled glucose. In the cerebral the brain. Vrba (1962) had previously made the same hemispheres of the adult rat, after a subcutaneous inobservation in the adult rat: 30 min after the injection jection of [U-14C]glucose, we have obtained a perof [U-14C]glucose into the caudal vein, the blood free centage of radioactivity in glutamic acid (63.5~o) amino acids contained less than l~o of the total which is more than 3-fold that in the neutral amino radioactivity in the acid soluble extract. The blood free acids (18~). This result appears to be a metabolic amino acids in the chick are more labelled than those feature of the adult mammalian and avian cerebral in the rat. This is probably due to the proper metabo- hemispheres. lism of chick erythrocytes, the labelled amino acids In the 30-day old chick (Table 7) the specific and carboxylic acids being released during the break- radioactivity of brain glucose rapidly increases during down of the erythrocytes by perchloric acid. The the first 10min following the injection of labelled glucose erythrocyte metabolism seems to be influenced by (1016 cpm/#mole at 5 min), then it steadily decreases starvation (Lehr, unpublished results). up to 60 min. In the adult rat brain, Gaitonde (1965) The blood glucose being the main substrate from had shown that the specific radioactivity of glucose which the amino acids in the central nervous system reached its maximal value 30 min after the subcuare synthesized, it follows that the content of glucose taneous injection of [U-14C]glucose. The difference in the blood is an important factor in the supplying between our results and those above-mentioned may of brain glucose. We have shown that the blood glu- be due to the isotopic dilution of glucose; the glucose cose concentration is constant between the first and content in the chick cerebral hemispheres is indeed the thirtieth days after hatching, therefore it does not higher than in the rat brain (0.22 innole/g tissue wet take a part in the metabolic changes which occur in wt, according to Gaitonde, 1965). Nevertheless, in the the central nervous system during the post-hatching two types of experiments, the rate of utilization of growth of the chick. injected [U-lgC]glucose is extremely fast: this result The remaining blood volume in the chick brain is is illustrated in Fig. 2(d) where the percentage of very small, and it represents 2'5-3"3~o of the whole radioactivity in carboxylic acids is relatively high brain wet wt (Seifter et al., 1970). Taking into between 5 and 20min. In the 10-hr old chick, the account this small content of remaining brain blood rate of glucose utilization is very slow; indeed, the and the constancy of blood glucose concentration, the percentage of radioactivity in the glucose fraction changes in the glucose content of the cerebral hemis- remains high (Fig. 2a) and the specific radioactivity pheres, determined during the post-hatching growth of glucose steadily increases between 10 and 60 min of the chick, are specific to the cerebral tissue studied. after the injection of labelled glucose (Table 4). In The abrupt increase in the tissue glucose between the 2 and 4-day old chicks, on the other hand, the specific first and the second day after hatching may be radioactivity of glucose reaches a maximal value explained by the important increase in the functional about 20min after the injection. This last result activity of the neurones, which requires an enhanced accounts, on the one hand, for the isotopic dilution utilization of the energetic stores, especially glycogen. due to the high increase in the glucose concentration, Edwards & Rogers (1972) indeed have shown an im- on the other hand, for the enhanced rate of glucose portant decrease in the glycogen content of the chick utilization particularly towards amino acid synthesis. brain, between the first and the second days after In the cerebral hemispheres of 2, 4 and 30-day old hatching. This enhanced glycogenolysis leads to a chicks, also in the adult rat brain (Gaitonde et al., transient increase in the glucose content of the cere- 1965), the specific radioactivities of glutamic and bral hemispheres, which is contemporary with the set- aspartic acid are rather similar. It is not the same ting of the metabolic pathways governing the rapid situation in the 10-hr old chick where the specific incorporation of glucose carbon into amino acids, radioactivity of aspartic acid is always higher than expressing the enhanced synthesis of brain amino that of glutamic acid (Table 4). By subcutaneously acids. This rapid incorporation of glucose carbon into injecting [U-14C]glucose to adult rats, Gaitonde, the amino acids of the chick cerebral hemispheres Dahl & Elliott (1965) had found that the specific takes place between the first and the fourth days fol- radioactivity of aspartic acid was higher than that lowing hatching, as is evident in Figs. 2(a and c). The ofglutamic acid in the liver tissue; according to thc~ setting of this process is specific to the central nervous authors, in this ease labelled aspartic acid would orisystem which has kept its structural integrity, since ginate for a large part from the direct fixation of in cerebral hemispheres slices prepared from 30-day 14COz on pyruvic acid molecule. It would be interestold chick it does not appear a high rate of incorpor- ing to know if this hypothesis is applicable to the ation of glucose carbon into amino acids (Nehlig et immature chick brain. al., to be published). In the adult rat (Gaitonde et al., 1965) and mouse As related to the radioactivity in the whole amino brain (Shimada et al., 1973), the specific radioactivity acid fraction, the percentage of labelling of glutamic of 7-alanine is higher than that of the other amino acid and of the neutral amino acids are the same acids during the first 20 min following the injection in the 10-hr old chick (approx 40~). The greater part of [U-~4C]glucose. In the case of the cerebral hemisof the radioactivity in the neutral amino acids is attri- pheres of the 30-day old chick (Table 7) we do not butable to c~-alanine during the first 30 min following obtain the same result: the specific radioactivity of the injection of [U-14C]glucose. In the 30-day old ~-alanine, which is higher than that of the other chick, conversely, the percentage of radioactivity in amino acids at 10min, afterwards decreases towards glutamic acid (56~) is almost 3-fold that of neutral zero at 60 min. In the 10-hr old chick (Table 4), the

Glucose carbon in the chick cerebral hemispheres specific radioactivity of ~t-alanine is much higher than that of the other amino acids and it steadily increases between 10 and 60 min. The differences between the specific radioactivity and the concentration values in ~-alanine in the cerebral hemispheres of the 10-hr old and of the 30-day old chick are related to the changes in the activity of glutamic-pyruvic transaminase (E.C. 2.6.1.2), which catalyzes the synthesis of ct-alanine. Johnson (1972) had shown, in the adult cat brain, that the rate of activity of this enzyme was clearly lower than that of glutamic-oxaloacetic transaminase (E.C. 2.6.1.1). It would be expected that the activity of the glutamic-pyruvic transaminase, which is low in the cerebral hemispheres of the 30-day old chick, would be much higher in the cerebral hemispheres of the 10-hr old chick; the immature cerebral tissue would then be metabolically closer to the liver tissue. According to Balazs (1965), the activity of the glutamic-pyruvic transaminase is clearly higher in the liver than in the adult brain. The metabolic changes which occur in the chick during the "critical period" located between the first and the fourth day after hatching, are attributable to changes in the enzyme systems, particularly in those related to the amino acid metabolism. Thus, the activity of the glutamic-oxaloacetic transaminase in the cerebral hemispheres increases from 810 to 3640 units from the fourteenth day of embryogenesis to the fourth day after hatching (Vos et al., 1967). During this same period of development it appears also an important increase of the activity of the glutamate decarboxylase (E.C. 4.1.1.15) (Sisken et al., 1960). If the enhanced activity of these enzymes explains the setting of the rapid incorporation of the glucose carbon into amino acids during the first four days following hatching in the chick, we do not know the exact mechanism of regulation which initiates this new metabolic pathway. Would this mechanism be under a hormonal control? Cocks et al. (1970) had indeed shown that the rate of incorporation of the glucose carbon into amino acids in the rat brain during postnatal growth was slowed down or enhanced according to the concentration of thyroid hormone in the blood stream. Acknowledoements--Thanks are due to Mr. Daniel Moncotel for his excellent technical assistance and to Mrs Christiane Math for illustrating and typing the manuscript. This research was supported by grants from the Centre National de la Recherche Scientifique (E.R.A. 331) and from the Fondation pour la Recherche M6dicale Franqaise. REFERENCES

223

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