Regulation of ornithine transcarbamylase activity in neonatal rat liver

Vol. 98, No. 1,198l January

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 51-57

15, 1981

REGULATION OF ORNITHINE ACTIVITY Claude Laboratoire

Received

November

Gautier

and Rene Vaillant

d'Endocrinologie,

UniversitE

TRANSCARBAMYLASE

IN NEONATAL RAT LIVER.

de Haute

ERA 891,

Normandie,

Facult6

76130

des Sciences,

Mont-Saint-Aignan.

3,198O

SUMMARY.

Postnatal increase in.ornithine transcarbamylase activity is abolished by repeatedly supplementing glucose after birth, while a single administration of actinomycin D does not affect this activity. When glucose and actinomycin D are administred in association at birth or 13 hours after birth, actinomycin D overcomes the inhibitory effect of glucose. Moreover, hepatic citrulline and urea concentrations remain low after glucose administration. The inhibitory effect of glucose on the postnatal rise in ornithine transcarbamylase activity might be due to the resulting insulin release. Ornithine enzymes

transcarbamylase

involved

of ureotelic activity late

in urea

animals appears

fetal

(1)

(EC. 2.1.3.3.)

synthesis.

(2-6)

is

and catalizes

on day 16.5

period

It

is

one of the five

located

in

the second

of gestation,

and presents

the mitochondrial

step

regularly

a marked

hepatic matrix

of the urea increases

increase

24 hours

cycle.

during

the

after

birth

Its

(7). It

is known

from previous

transcarbamylase

needs

rat.

Our recent

this

enzyme activity

roids

(2-3).

increased

have in

After

the rise

the appearance excretion.

fetus

were

that under

of actinomycin

in ornithine

in

the

D to

it

is known

that

later,

is

particularly birth

fetuses while

paradoxical

activity

the newborn,

of

of glucocorticoste-

24 hours this

and adult

and increase

18.5 days old

abolished

in

the neonate

control

transcarbamylase

of new fonctions

of ornithine

the appearance

as determined

of cortisol

In addition,

the activity

of the adrenals

of enzyme activity

administration

nitrogen

the

that

demonstrated

An administration

simultaneous

with

(8-10)

the presence

studies

the level

birth,

work

is

effect

a (11).

associated that

associated

of with

a

0006-291X/81/010051-07$01.00/0 51

Copyright 0 1981 by Academic Press, Inc. AN rights of reproducfion in any form reserved.

Vol. 98, No. 1,1981 major

endocrine

BIOCHEMICAL crisis,i.e.

ciated

with

a decrease

ratio.

This

hormonal

do not

correlate

itself

(7-12). We decided

rise

by altering

tration.

an abrupt in

insulin

and the

the

age of the

with

to investigate the hormonal

association

or not with

MATERIALS

AND METHODS.

increase

alters

in ornithine

rat,

but

This

the

at different

secretion,

fglucagon

with

activity birth

of the postnatal by a glucose

of actinomycin time

asso-

insulin

are associated

was attained

the effect

COMMUNICATIONS

transcarbamylase

the mechanisms

status.

glucose

RESEARCH

of glucagon

This

rise

further

we studied

and experimental

BIOPHYSICAL

release.

crisis

In addition,

Animals

AND

enzyme adminis-

D, administered

after

in

birth.

procedure.

All studies were carried out on pregnant Wistar rats fed "ad libitum"': Gestational age was calculated from the ovulation and fertilization dates which occurred around 1 a.m. Fertilization was confirmed by vaginal smear. Pregnancy in this strain lasts 21.5 days. On day 21.5 at 9 a.m. pregnant rats were killed by a blow on the head and the whole fetuses were removed from the uterus with the amniotic sac intact. The pups were then excised from the sac and the ombilical cord tied and cut. The entire litter was delivered within 5-10 min after the mother's death and the newborns were held in a humidicrib at 37°C without feeding for the duration of the experiments. Newborns delivered by caesarian section received immediately or 13 hours after birth a single administration of actinomycin D (3 pg in 0.05 ml i.p.) (Calbiochem, La Jolla, Ca) or 0.05 ml i.p. of a glucose solution (25 mg)repeahours. Control newborns received according to the same time tedly,every two schedule 0.05 ml of a 0.9 g/l NaCl solution. Ornithine transcarbamylase assay. At the choosen time, newborns were rapidly weighed, decapited and bled. Freshly excised livers were immediately weighed and homogenized in a solution of N-acetyl-N,N,N-trimethyl ammonium bromid (CTB) 0.1% in Tris-HCl buffer (0.05 M, pH = 7.4), (8 ml of CTB solution per gram of liver). After two successive centrifugations for 15 min at 4000 g at 4"C, the enzyme activity was assayed on the supematant solution according to the method of Brown and Cohen (13) with some modifications as previously described (II). A unit of enzyme was the amount which catalized the production of 1 pM citrulline/hour at 37°C under assay conditions. The activity was defined as units per milligram of proteins. Proteins were determined by mean of the biuret procedure (14) with bovine serum albumin as standard. Determination

of hepatic

citrulline

and urea

concentrations.

Hepatic urea concentration is determined by mean of urease of the supernatant fraction of the homogenate (25-16). Citrulline tion is determined spectrophotometrically after the whithdrawal interfering urea (15-17). --Statistical Statistical

analysis. analysis

was performed

using

52

Fisher's

t test.

on an aliquot concentraof all the

Vol. 98, No. 1,198l

O.TC.ase 6.000

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Activity

(PM citrulline

/h/g

Liver)

/

/

l control

7,000

newborns

025 mg Glucose

every

d +Actinomycina

0 at birth

0 +*ctinomycine l tt*r birth

D 13 how*

2h /

/

/

/

/

/

/

,

/

/

/

/

, .

/

1’ 1’

/ / /

6.000

/

/

/ / i,

5,DOO 9 a.m. 1 0

6

ga.m + 24

I 13

930 Postnatal

Hours

Fig. I : Time course of ornithine transcarbamylase activity. Effects of a repeated postnatal administration of glucose, with or without an administration of actinomycin D. (Values are expressed as the mean T SEM. The numbers indicate how many enzyme assays were performed on the pooled livers of the same litter).

RESULTS Effect

of a glucose Glucose

administration

administration

caesarian

section

ornithine

transcarbamylase

(Fig.

1 and Table

21.5

days old

urea

(Fig.

24th

hour

vely. urea

(25 mg every

on day 21.5

1).

fetuses.

Glucose

birth, supply

concentrations

two hours)

prevented

activity

for

which

The enzyme activity In control

2B) concentrations after

at birth.

attaining at birth increased

24 hours

occurs

remained

comparable

about prevented

about

liver

this

in coordination

transcarbamylase.

53

fold

with

this

with (Fig.

between

Hepatic the

activity

of period

that

of

2A) and the

I1 gM/g of liver

increase.

by

increase

during

citrulline

three

1.5 PM and

delivered

the marked

normally

neonates,

increased

to neonates

13th and

respecti-

citrulline of omithine

and

Vol. 98, No. 1,1981

BIOCHEMICAL

AND

Table 1 : Ornithine transcarbamylase Influence of a repeated glucose administration Activity /g liver

TFZATMENT

Control at 21.5 pregnancy

fetuses days

Glucose

at

are

96.8229.4 (7)

9430-C1117e (8)

76.7

8848k

81.5f

expressed of

A single

Effect

the

the

same

mean

litter

administration

effect

On the other

at the level

109.6

5.53

+0.42* (18)

*

0.3ico.20

delivered

increase

0.72

5.80+1.03'

;‘3)

k0.67 (9)

The number of TV. Statistically

parentheses.

4.10+2.47' (9)

enzyme

assays different.

at birth. D (3 pg)

at birth

and liver

did

urea

not

prevent

concentration,

1). of glucose at birth

and actinomycin

was abolished

at birth

hepatic

or

citrulline

13 hours and urea

D at birth.

by a single after

birth

(Fig.l

concentrations

the neonates.

by caesarian

in omithine section

fO.94" (20)

(6)

DISCUSSION.

The main

1.33* (15)

8.83?1.17' (7)

+ 10.2 (9)

activity

D either

in

0.57

122.5t20.3

in

of glucose

found

10.503

11.9

interval.

given

hand,

(22) 0.48*

(8)

administration

of actinomycin 1).

9.3

14.7

(Table

4.0+0.5(!

(7)

of actinomycin

later

Urea uWg liver

(22)

9.0

91.7k

transcarbamylase

24 hours

1.41f

(21)

'confidence is

administration

The inhibitory

remained

83.3+

D administration

of a simultaneous

Table

f (9)

9.2

(16)

(9)

as

in ornithine

as determined

and

1661'

of an actinoqcin

rise

0.39io.34 (5)

4.4.

95.52

9.3%

(9)

neonates

Effects

79.2i (18)

f 17.3* (17)

8872k2301e (7)

24 hours after birth. with or without an

Citrulline uM/g liver

(22)

Glucose at birth + Actinomycin D 13 h later

the

93.5

69.7t

D

values

11.8

COMMUNICATIONS

Proteins mgfg liver

(25)

8449k 1229* (17)

D

Glucose + Actinomycin at birth

pooled

67.0?

birth

Actinomycin at birth

All

948

(25)

RESEARCH

activity attained postnatal administration of actinomycin D.

Specific Activity

540gr

Control newborns 24 hours('post partum"

BIOPHYSICAL

transcarbamylase

on day 21.5

54

takesplace

activity between

in newborns the 6th

and

on the

Vol. 98, No. 1,1981

AND

BIOCHEMICAL

Cltrullino pM/g

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

liver A

8

1

6

0

13

21

11-

10.

6.

6.

22 4.

;/

+----4---~

*.a

---___ -i==

2-

, 0

6

13

postnatal

Hours

Fig. 2 : Time course of citrulline and urea liver concentrationsafter birth. Effects of a repeated postnatal administration of glucose during the first 24 hours of life. (Values are expressed as the mean t SEM. The numbers indicate how many assays were performed on the pooled livers of the same litter).

the

13th

hour

supplementing tion

(10

birth.

This

actinomycin pg)

blished

data).

mylase

activity

a protein

after

to

D at

newborns These is

birth.

partially results

not

postnatal

associated

increase In

suggest

that with

this the

a new

synthesis.

55

not

abolished

by

cycloheximide

addition,

overcomes

is

rise RNA

postnatal in

administraincrease

ornithine

synthesis,

(unpu-

transcarbabut

might

involve

Vol. 98, No. 1,1981

BIOCHEMICAL

The postnatal

glucose

ses the insulin does not of glucose effect

of insulin

be explained

of enzyme

. If

to the

this

D to the

and this

lead

would

The postnatal is

known

hypoglycemia) This

might

trations brought

be regulated Insulinemia

control

by the

insulinemia

and blood

and a high

with

the

WC

insulinemia

3-mercaptopicolinate) AMPc to the

was found low.

of glucose,

however, occurs

the urea of

Omithine

the

are

concentrations

correlated. (23)

inhibitors and the effect

work,

II.

effects.

the marked priming

metabolites.

liver

concen-

of gluconeogenesis

glucagon

of supplementing

to specify

at birth insulin

might

metabolites.

to be associated

we intend

has been

activity

An important

It

postnatal

enzyme activity

of ureogenesis

are known

In further

has other

transcarbamylase

or by the supply

glucagon

secretion

and urea

although

to be high

in reference

of its

low citrulline

could

The administration

after

cycle

D might

explanation

insulin

described

level.

decrease.

of administring

insulin

D injection,

to the

of actinomycin

fetal

effect

by the inhibitory

in which

(which

the occurrence

administration

This

the

increa-

The inhibitory

be caused

effect

depriving

actinomycin

(22)

effects

thus

glucose

(19).

effect

: it

release.

inhibit

gluconeogenesis

explain

back

might

consequent

this

transcarbamylase

administration

(21),

after

fetus

the

COMMUNICATIONS

insulin

to the paradoxical

to inhibit

might

case,

of the

fetus

that

of glucagon

activity

of ornithine

of actinomycin

is known

was the

case of the

and the activity

It

RESEARCH

has a major

secretion

by an inhibition

be extended (20)

(18).

the postnatal

on the rise

BIOPHYSICAL

administration

secretion

alter

AND

(for

release at birth the instant

, glucagon

and

fetus.

ACKNOWLEDGEMENTS. the

This work Scientifique.

was supported

by a grant

of the

Centre

National

de la Recher-

REFERENCES I. 2. 3.

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56

Vol. 98, No. 1,198l

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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