Tyrosine α ketoglutarate transaminase in the liver of rats with intra-uterine growth retardation

Life Sciences Vol. 10, Part II, pp . 1116-1123, 1971 . Printed in Great Britain

Pergamon Press

TYROSINE q. RETOGLUTARATE TRANSAMINASE IN THE LIVStt OF BATS WITH INTRA-UTERINE GROf~TfH 1lETARDATION C . DEGKEMONT, J . M . ROUB, E . SWIERCZEWSRI a~ C . TORDET~ARIDROTT t Contre de Recherches Biologiques Néonatales . Unité 29 . Hôpital Port-Royal, Paris . (Director : Prof . A . MINROiISRI)

(Received in final form 7 September 1971) Summary The postnatal inductioa .of TRT in the liver of rate submitted to intro-uterine growth retardation (I . U . G . R .) is much more greatez than in the liver of normal animals . This striking rise is activity, seams to ba related to the low blood glucoss levels found in the I . U . G . R . animale . Sowave r glucagon injections to I . U . G . R . fatusa s are not so efficient as is the case of the coatrole . Wa have shown in previous publications (1) that aawborn rate with azperimeatally induced iatra-uterine growth retardation (I . U . G . R .) have about half the blood glucose level of the normal controls . This hypoglycemia lasts at least for 10 days after birth . According to our results, total glycogen stores are significantly depressed in the liver of the stunted fatusas one day ante parfum (1) . However, after birth the glycogen pattern ie the soma in both groups of animals ; it seams likely that the glycogen mobilisation proceeds at the same rate in the ezparimeatal and control rats . Wa explored at the same time some key eaaymae of aeoglucogeaesis is the liver of I . U . G . R . and control rats . Fructose-l-6~iphosphatase (2) and

glutamic-ozaloacetic traneaminase (3) showed a slight delay in their incrnasiag steps, but that' reached their highest peak at the ease rate is both groups of animals . Tha activities of glucose-6-pho~phata dnhydrögenaea, lactico-

dashydrogeaaea and glucose-6-phoephataea are not modified is the developing liver of the stunted animals (2) . One consaquaace of the neonatal hypoglycemia is normal newborn rate ie the induction of the easyma tyroeiae,tkatoglutarata transamiaaea (E . C . 2 . 6 . 1 . 5 .) T .R .T, after birth (4, 5, 6) . Greangard and Deway assumed that the decrease is blood glucose stimulated the secretion of glucagoa which in turn triggers the synthesis of T .R .T . in the newborn rat liver (7) .

t Correspondence sad reprint request to C . Torlot-Caridroit .

1116

1118

~rrosine-a-I{etoglutarate Traneaminase

Vol . 10, No . 19

The question was rained whether the more severe hypoglycemia described in the I . U . G . R, rats, had an influence on the T . R . T . at birth . On the other hand we need to evaluate the possible effects of intra-uterine growth retardation on hormonal regulations . Studies on neonatal T . R . T . induction seemed to provide a good model for this . The present paper deals with the induction of liver T . R . T . and its regulation by glycemia and hormones in the I . U . G . R . fetuae e and newborn rats as compared to controls . Material and Methode 1 . Animals We induced iatra-uterine growth retardation in the pregnant rat by uterine artery clamping, originally described by Wigglesworth (8) . Female rate of Sherman strain were mated overnight . The uterine Vessels were clamped near the lower end of one horn at the 17th day after the mating . In this ezperimental horn fetuses of reduced weight were found at the and of the gestation . The opposite uterine horn was left untouched as a control . All the determinations were made oa 6 litters at least . Variability is greeted from one litter to another than between fetuses of the same litter . A reduction of weight of more than 20 p . cent is considered a good criterion of growth retardation . 2 . Glucose and hormones injections As shown by Greengard et al .

(5) enzyme TKT is particularly

responsive to glucagon 2 days before birth, whereas glucose inhibits the postnatal rise of enzyme activity . We injected, therefore, glucagon to fetuaee and glucose to neonatal rata . Insuline was injected to normal newborns to induce a severe neonatal hypoglycemia .

Injections of glucose, insulin and glucagon were made as follows

the needle was inserted at the dorsal base of the tail so that the tip of the needle extended up to the lumbar area . The solution was absorbed better than from iatraperitoneal injection . Animals were kept with the mother until decapitation . Ia all cases, animals were killed 6 hours after injection . Glucose iajactiona : Injections were carried on newborn animals, less than 1 hour after birth ; 0 .8 mg of glucose per g of animal was injected ae previously described (4, 5) .

Vol . 10, No. 19

Tyrosine-a-Ketoglutarate Transaminase

1117

Iaeulin_in~ectioan : 0.02 UI/g weight insulin was injected to the newborn rate (9), under the same conditions as above. Gluçagan -inieçtioas : 25 pg glucagon per animal (average weight : 3 .5 g) vas injected to fetal rate

(12 hours before birth)

(5) . The mothers were kept under

ether anesthesia . Fetuses were injected iatra-peritoneally with glucagon . After 5 hours, mothers ware killed, the fetuses removed from the uterus . Assay Procedures rosins .1.keto lutarata transaminase _~__~~-~--_~__r--_

The fetal or newborn animals ware decapitated,

the liver removed as

quickly ae possible, weighed, and homogenised in 10 volumes of iced water . Tyrosine ~katoglutarata traneaminaee was determined by a modification of the method described by Sereni et al . (10) . 0 .1 ml aliquots of homogenates ware added to beakers containing 1 .9 ml of 5 .5 mM tyrosine dissolved in 0 .05 ü Na phosphate buffer pH ~ 7 .6 . 0.1 ml mM dinthyldithiocarbamatn ; 0 .1 ml 3 mM pyridozal phosphate. After praiacubatioa of the mixture for 3 min . at 37 C the reaction was initiated by the addition of 0.3 ml of 0.2 M d-ketoglutarate (Na salt) . After a 10 minutes period at 37 C, the reaction was stopped by addition of 0.5 ml of 30 per cent trichloracetic acid . The mirturee were centrifuged to remove denaturated proteins and 2.0 ml aliquots of the clear supernatants were assayed for p . hydrozypheaylpyruvate by a modification of the Briggs reaction (11) . A black value was obtained is which water was put in place of .t,-ketoglutarate (Na salt) . Activity of TRT is azpressed in minute pst

mg

wet weight .

u$

of p . hydrozyphaaylpyruvate formed in one

G1 cr Y amia Blood was sampled on the living rate with hepariaiand pipettes and plasma separated by gentle centrifugation . Glycemia was determined by the glucose ozidaae method (12) . Results T1Cf activity in I. II . G. R.,

control and normal rats

Figure 1 represents the evolution of TRT activity is the liver of I. Ù. G. R., control and normal rate from fatal ago to

10 days poetnatally .

The normal increase 6 or 12 hours after birth described by other authors is

1118

Z~roeine-a~Setoglutarate Traneami>oaee

Vol . 10, No . 19

noted is all groups of aaimala . Nevertheless I . U . G . R. rate azhibit a strickiag peak of activity at 12 hours . The mean activity of I . U. G. R. rate at this stage, is about 4 limas greater thaa in the control sad normal animals . The level of TRT activity diminishes in all categories of animals, but I. II . G. B. naiatain a higher an:yme activity until 5 days . At ]0 days, there ie no more difference in the normal aad ezperimsatal rats . Table 1 shows the mean activity with etaadard deviation for all groups of animals, sad the number of litters utilised .

i

r 8"

11 11 11 11 I 1 I 1

IUGR Control Normal o~~

7. 6" S" 4" 3" 2~ 1~ 0

Fig. 1 Davelopmeat of TRf activity is normal, control and I. ü . G. H. rate (activities ezpreased in pg pH PP/mg wet weight/mia .)

Vol. 10, No. 19

Z~rrnsine-a-Setoglutarate Transaminase

1119

TABLE 1 Mesa values of T&T activities of I . U . G. R., control and normal Rata during liver development . (Activities eaprassed is hg pHPP/mg wt .waight/min. + Sta~ard deviations) Fetuses 6 Hours 12 Hours 24 $ours 48 Hours 3 Days 5 Daye 10 Days

Age Number of litters

6

15

6

10

11

8

9

8

I .U .G .R .

0 .17 + 0 .06

2 .42 + 0 .58

+

10 .60 0.52

4 .82 + 0.88

4.18 + 0.42

3 .60 2 .18 + 0 .36 + 0 .52

1 .42 + 0 .11

CONTROL

0 .16 + 0 .10

2 .72 + 0.56

+

2 .34 0 .62

1 .94 + 0 .28

2 .08 + 0.30

2 .34 1 .24 + 0 .20 + 0 .10

1 .36 + 0 .08

NORMAL

0 .10 + 0 .14

2 .56 + 0 .41

+

2 .0 0 .50

0 .98 + 0 .25

2 .10 + 0 .43

1 .64 1 .32 + 0 .10 + 0 .11

0.72 + 0 .10

Effect of Slucose level on TRT açtiviti We represented in figure 2 the T&T activity of I. U. G. R . and control rats plotted agaiaat the reciprocal values of glycemia . As seen on this graph there ie a linear relation between these data . We calculated the regression line (elope ~ 5 .67) and found a significant correlation between the ezperimental values . (Correlation coefficient : 0 .90 - P ~ 0 .01) . After glucose injection (table 2) birth,

to newborn rats, 1 hour after

there ie a significant decrease of the TRT activity in both I. U . G. R.

and controls of the same, litter . In the case of I . U. G. R. rate, the fall of activity seems to be lass effective. Effect of-Bluca~oa ia~ection to fetal ra te These results are summarised in figure 3 . The values of TRT activity rise significantly is both I. U. G. R. and control rate . Neverthaleea the percent average increase of activity as compared to eon treated animals, is 44 p. cant for I . U. G. R . and 62 p . cent for control rate .

Tyrosine-0c-Setoglutarate Transaminase

1120

F Y F 15

>

ûc

0 e 0 ~

Vol. 10, No . 19

6h 12 h 3d 5d

Op~n symtwl~: Control~ Black symbol~: I U G R

10

~1,27

0 0 ~,2

FIG. 2 1/G : inverse values of glycemia ezprasead in mg per ml . Relationship between liver TRT activity and blood glycamia in I . U . G. R. and control rata aged from 6 hours to 5 days . TABLE 2 Appearance of a lower liver TRT activity (azpreesad is pg pHPP/~ . wet rt/min .) in I . U . G. R. and control newborns after glucose injection lees than l h after birth. Each assay was carried on a pool 'of 3 livers at leant . Control

Control + glucose

Reduction p . cent

1

2 .089

1 .380

34

2

1 .584

0 .758

52

3

0 .56

0 .020

96

4

2 .052

1 .496

27

5

4 .767

1 .431

70

6

2 .580

1 .644

36

Eaperimeat n°

7 8

I .U .G .R . 1 .936 5 .622

L U .G .R . + glucose 1 .657 3 .405

14 40

Vol . 10, No. 19

Tgrosine-a.-Ketoglutarate Traneamina.se

1121

FIG . 3 TRT induction in fetal liver by glucagon injection to I . U . G . R . rate and their controls of the same litter . Each bar represents the mean activity + S . D . obtained from 8 to 12 animals . (activities ezpreaeed in Fag pHPP/mg wet weight/min .) Insulin administration to normal newborn rats lese than 1 hour after birth The positive response of TKT to glucagon in fetal I . U . G . R, and TABLE 3 Appearaace . of a higher liver TKT activity (expressed in hg pHPP/wet weight/min) in normal newborn rats after injection of insulin, 1 hour after birth . Each assay was carried on a pool of 3 livers at least . Litter n °

Controls

1

1 .325

2 .257

41

2

2 .025

2 .601

22

3

4 .767

5 .388

11

4

2 .580

3 .180

13

Injected

Increase p . cent

1122

Zgrosine-a-Ketoglutarate Traneaminase

Vol. 10, No . 19

control rate lad us to induce eaperimantally an increased hypoglycemia in normal newborn rate by insulin injections . Tha results of these ezperimeata era represented in table 3. Increased valwa of TRT activity are found in injected animals ae compared to non treated ones . Diecuesion As reported for normal newborn rate, by Greangard and Dewey (5) and others

(4, 6), TRT activity reaches its highest peak at

12 hours after birth

this is also the case of rats submitted to experimental hypotrophy . But is this latter group,

the values era 4 to 5 times greater than in normal newborns . This

difference is not due to the stress of the operation ; as a matter of fact the ezparimeatal control rata azhibit the same values ae the normal ones . On the other hand, Rnoz and Auerbach (13), Shapiro et al . (!4) have shown that TRT ie inducible by stress only two weeks poetaatally, when the pituitary-adrnnoaortical azis is wall established . We assumed that the increased stimulus of T&T described in I.U .G .R . rate is related to a lower blood glucose level . As saes is our results, there is as inverse relationship between the TRf values and the blood glucose . The administration of glucose to newborn I . U. G. R. inhibite d this postnatal rise . Our data confirm the hypothesis

of

Dawkins (15), Greangard and Dewey (5), that

the postnatal appearance of TRT ie stimulated by neonatal hypoglycemia . The capacity to raise TRf activity is the normal fatal liver whey eaposad to glucagon (7) ezists also for I. U. G. R. fetuse s 12 hours before birth. However the response of TRf activity to glucagon administration is lower in the case of I. U. G. R . fetuses . Thes e latter animals could be enabled to respond to hormonal stimuli with good efficiency . Navertheleas our results found with I. U. G. R. nsruborrte point out that neonatal hypoglycemia provokes the appearance of a higher pack of TRT as compared to normal animals . Soma hormonal determinations (glucagoa and insulin)

is the plasma and pancreas of

I. II . G. R. fete ses and newborns era now carried on is our laboratory . Ia the last azparimants we tried to induce a more important neonatal hypoglycemia in normal newborn rate by insulin injections . The aim of this study was to verify if the lower blood glucose level in the I . U. G. R. newborns was the single physiological factor which stimulated higher TRT synthesis at . birth. As seen is our results,

insulin injections increased TRT

response in normal newborns . However in no case did the TRT activity reach the levels found in the liver of the stunted animals . As described by Sareai et al . (10), T&T becomes iaducible by

Tyrosine-a'-Ketoglutarate Transaminase

Vol. 10, No . 19

1123

hydro-cortisone one day postnatally in adreaalectomiaed newborns . We found in former studies (16) that the adrenals weight of I . U . G. R. rate is diminished as compared to controls . However, at the present time, we lack data upon the development of the corticoids, is the hypotrophic rate . These questions are now under study is our laboratory . Rafarences 1 . C . CHANEZ, J . M. ROUE, C. TORDET-CARIDROIT, Comptes Rand . Soc . Biol .,

16^,

2272, (1969) 2 . C . CHANEZ, C . TORDET-CARIDROTT and J . M. ROU%, Biol . Neonate, 17, 355, (1971) 3 . C . TORDET~:ARIDROIT, Ann. Biol . animale, Biochimie, Bioph . in press, 4. P . G. HOLT, I. T . OLIVER, Biochem. J ., ~08, 333, 5 . 0 . GRESNGARD,

J. %. DEWEY, J . Biol . Chem .,

6. D . YEUNG, I. T . OLIVER, Biochem. J ., 7 . 0 . (RlEENGARD, Science, 163,

891,

242,

1~, 325,

2986, (1967) (1968)

(1969)

8. J . 3 . WIGGLESWORTH, J . Path . Bact ., 88, 1,

(1964)

9. C . B. BROWN and M. CIVEN, Endocrinology, 84, 381, 10 . F. SERENI, F. T. RENNEY sad N . RRETCHMER, il . G. MBDES, Biochem. J .,

26, 917,

(1969)

J. Biol . Chem ., 234,

609,

(1959)

(1932)

12 . A. G. NUGGET and D . A. NI%ON, Biochem. J .,

66, 12 P (1956)

13 . W. E . &NO% and V . H. AUERBACH, J. Biol . Chem .,

214, 307,

14 . S. SHAPIRO, A. YUWILLER and E . GELLER, Science, ~52, 15 . M. J. R. DAWKINS, Ana. N. Y . Aced . Sci.,

wI,

203,

(1955)

1642, (1966)

(1963)

16 . C . TORDET-CARIDROIT, J. M. ROU% sad C . CHANEZ, C . R. Soc . Biol ., 1321, (1969) .

(1971)

(1968)

163,