Delayed appearance of liver growth hormone binding sites and of growth hormone-induced somatomedin production during rat development

Vol.

136,

No.

1, 1986

April

14,

1986

BIOCHEMICAL

RESEARCH

COMMUNICATIONS Pages

DELAYED AND

OF

APPEARANCE OF LIVER GROWTH HORMONE-INDUCED DURING RAT

B.

Departamento

Flandez,

de

Received

BIOPHYSICAL

AND

E.

Alvarez

Fisiologia,

February

10,

GROWTH HORMONE SOMATOMEDIN DEVELOPMENT

and

Facultad

de

E.

38-44

BINDING SITES PRODUCTION

Blazquez

Medicina,

Salamanca,

Spain

1986

SUMMARY: The present study was designed to determine whether the apparent paradox of high circulating growth hormone levels in the fetus and the minimal effect of this hormone on growth might reflect a diminished responsiveness of fetal target organs to GH. Specific uptake by rat liver of [ 12511 bGH was very low in fetuses as compared to suckling and adult rats. Also, liver uptake of the iodinated hormone decreased proportionally with the simultaneous injection of increasing amounts of growth hormone, but was not modified by the simultaneous injection of unlabelled chemically-related hormones. Since the water content is significantly greater in fetal than adult tissues, results were expressed by liver dry weight and again, [ 1251] bGH liver uptake continued to increase with age. After bovine growth hormone administration to adult rats, plasma somatomedin C concentrations increased significantly, while they had no effect in fetuses. These results suggest that reduced liver somatogenic binding sites in the fetus prevents growth hormone from inducing growth-promoting effects during intrauterine life. 0 1986 Academic

Press,

Inc.

Though gland

during

the

releases

greater

of

intra

and

hormone

to

fetal

growth

body

weight

gland

and

range

(2-4),

of

most

whether

the

levels

in

might

reflect

to

growth

&e/to

a

rat,

and

of

the

diminished

pituitary

than

the

of

the

birth

without

the

within and grow

thus

at

responsiveness

rates to

of

this fetal

until

determine

growth of

normal

monkey

normal

circulating

this

length pituitary

the

rhesus

designed

effect

other

contribution

Thus,

fetuses

in

hormone

hormone target

on

growth

organs

hormone.

Rats of the Wistar strain, used in our experiments.

0006-291X186 Copyright All rights

high

minimal

MATERIALS

were

(11, minimal.

to was

fetal

hormone

rabbit

continue study

paradox

fetus

be

the

hypopituitarismare of

present

apparent the

growth

human

with fetuses

The

pregnancy

life to

anencephalic

while

6).

of

seems

in

(5,

of

extrauterine

children

hypophysectomized birth

third

amounts

periods

and

last

with Female

AND METHODS free access to comnercial animals weighing 200-250

$1.50

Q 1986 by Academic Press, Inc. of reproduction in anv .form reserved.

38

rat chow and g were caged

water, with

Vol.

136,

No.

1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

males until copulation occurred. Vaginal smears were examined daily for spermatozoa early each morning. Pregnancy was dated from the first day on which spermatozoa were identified. The accuracy of this method of dating, estimated to have 6-12 hours error, was validated by the fact that all the rats delivered 22 days after the finding of spermatozoa. Suckling and adult rats, slightly anesthetized with ether, received a jugular vein injection of 20 ng/lOO g body weight of either [12?C] bGH or [1251] insulin, with or without unlabelled hormones dissolved in NaCl (0.9%) with 0.1% bovine serum albumin. Hormone administration in experiments with fetuses was done with a Hamilton microsyringe through the umbilical vein. Bovine growth hormone kindly provided by Drs. Dellacha (Buenos Aires, Argentina) and Sonemberg (New York, USA) was labelled with specific activities of 110-130 vCi/pg according to a procedure previously described (7). Mono [ 12511 insulin (280-350 pCi/pg) was obtained by the procedure of Freychet and Roth (8). The animals were maintained at room temperature. At the times indicated, blood samples were obtained from the inferior one from each lobe, were removed. In the case of fetuses vena cava, and liver slices, Radioactivity present in blood plasma and blood samples were obtained by decapitation. liver slices was measured with a gaauna-scintillation counter. Liver uptake was expressed as the liver to plasma ratio (L/P: counts per min/g liver cpm/g plasma), and as cpm/g dry liver weight, calculated as described by Retegui-Sardou et al. (9). Blood plasma determinations of somatomedin C were carried out at different times after the subcutaneous administrations of bGH (25 pg/lOO g body weight) dissolved in a solution of NaCl (0.9%) with 0.1% bovine serum albumin or of a placebo to 21 day-old fetuses and adult rats. In the case of the fetuses, the hormone or the placebo (containing NaCl and bovine serum albumin) were injected through the uterine wall. Somatomedin C determinations doassay (Nichol Institute Diagnosis, San Juan de Capistrano, were carried out by radio USA). The antibody used in this assay was of high specificity and affinity. Thus, IGF-II shows less than 2% cross reactivity with the antibody, and none of the known The sensitivity of the assay classical hormone cross react to any significant extent. was up to and including 0.1 U/ml and the percentages of intra and inter-assay variation were 5.2 and 9.4% respectively.

RESULTS

After liver of

the

uptake maximal

simultaneous Table

Figure

with

during

intrauterine

superior

to greater

of

corresponding (Fig.

2)

half-maximal

in

insulin

all

adult

growth same

either

as

the was

bGH

bGH

or

of insulin

experimental within

bioassays.

39

As

shown

in

rats

as

GH

uptake

50

pg/lOO

fetal

Interestingly, was

dramatically

postnatal

life

Doses

of

0.5

together a The

2

specific

in

concentration

kidney,

at

uptake

injected

lower

Both

1 and

liver. in

times

with

bound

minimal

produced groups.

the

and

(Figures

the

insulin.

hormones

rats.

liver

< 0.001).

while

1 bGH,

the

adult

are

was (p

[ lz51

significantly

by

insulin

that

the

insulin

rats

hormone,

radioactive

was

and

the

by

absorbed

liver

life,

or

weight

of

and

hormones

and

[ 125 I]

and

of

bGH

and

respectively;

decreased

unlabelled

simply

uptake weanling

that

cleared

of

insulin

minutes,

hormones

than

hepatic

1,

[ 125~1

20

suckling

rapidly

that

rather

and fetal,

iodinated

suggesting

sites

compared

were of

of

5 in

injection l),

binding

GH

uptake

uptake

the

body

at

studied

hormones

liver

and

was

injection

was hormonal

radioactive but

intravenous

to with

dose-response dose

at

the curve

which range

displacement used

for

g

Vol.

136,

No.

BIOCHEMICAL

1, 1986

AND

BIOPHYSICAL

5

RESEARCH

COMMUNICATIONS

0

i2.5 I - Insulin 12s

m

‘*f-bGH

a

‘?-bGH

I-Insulin

.2 :4 : J

plus

plus

unlabelled

lnwlin

unlabetled

bGH

3I.

2-

I-

21Day

10 Day-old Weaning

- o/d

Fetuses

It

might

fetus

be

could

(Fig.

2).

rats

were

given

for

that

the

rats

would

suggested

affect

that

by

However,

plasma 0.025

the

and liver

higher not

studies

Doy-old 30 Day-old

-

lower

Young

(1)

uptake.

in

GH levels uptake

pg/lOO

as

idea

fetuses lower

is

suggests with

supported

adult

those

This

compared

the bGH

and

than

g b.w.).

fetuses

in

[ 125 I]

of

21 day-old much

This

fetuses

by the liver of fetal, or presence of the pg/lOOg body weight).

liver

(l-50

21 day-old

Adults

circulating

respectively;

in

liver

c--o

high

GH levels

uptake

-21

2.0

the

pg/ml,

GH levels affect

the

dilution

0.10

2.5

p

[ 125I] insulin in the absence hormones (100

bGH or 1. Uptake of [ "'I] weanling and adult rats corresponding unlabelled

Fig.

Young

30 Oay-old Weonling

by

adult

the

fact

2.5

weonling adults

2.0 .o ‘i a

z I.5 : 4

1.5

: -J I.C

1.0

0.5

0.5

0

10 Untobelled

Fig.

20 bGff

30 fug/lOOg

40 body

50

0

IO Unlabulied

weight)

20

30 Insulin

2. Inhibition by unlabelled bGH or insulin of liver [1251] bGH and [I2511 insulin, respectively, in weanling and adult rats. Means + SEM. n-3-5. 40

40

50

Iw/lOOg

body

uptake fetal,

of

weight)

Vol.

136,

TABLE

No.

1.

21

BIOCHEMICAL

Specificity

[1X71]

of Rat

1, 1986

of

bGH in

liver

the

age

day-old fetuses

30 day-old weanlings

Adults

that in 19 [ 125 I] bGH studying

day-old

to

after

unlabelled L/P

administration

native

hormones

ratio

None bGH hPL Insulin

1.3420.28 0.54+0.15 1.37to.10 1.52+0.03

None bGH

1.55~0.08 0.55+0.01 were amount Means

used at of bGH + SEM.

plasma than

hGH

be

COMMUNICATIONS

Liver dry

(cpm/g

both

of

seen

radioactivity

various

hormones

lower

binding

was

of of

RESEARCH

0.39+0.03 0.21;0.04 0.31;0.05 0.3310.20

hormones for the weight.

were

BIOPHYSICAL

None bGH oPRL hCG

fetuses

uptake

the

binding

uptake

presence

Unlabelled administered

Unlabelled body weight, except of 100 pg/lOO g body

AND

to

in

adult

in

19.52k2.97 9.24k5.20 of

21

10 at

rats

levels

liver

lower

33.62t16.36 5.395 3.24 32.21111.04 29.63ilO.84

pg/lOO a dose

(0.014

animals;

isolated

4-fold

8.42+2.31 0.72TO.03 9.24T1.12 6.01;1.04

a concentration used in adult n=6-10.

hormone

wtxlO-4)

pg/ml)

and

additionally,

membranes, day-old

g

on

hormone

fetuses

than

in

adult

rats. Since our

changes

results,

stages

of

adult

(7l+2)

rat

in ratio

or

no the

displacement

any

time

somatomedin

0.001)

<

differences

of age

[

of

21

of

radioactivity

bGH

continued

(Fig.

3),

Also,

adult at

when

8

the rats

and

groups

bGH 13

administration.

41

(p

<

been

(Table

to

bGH

increase

was hormones

be

obtained. C

in

expressed these

1)

[ 12'1]

somatomedin than

be

(8824).

Using

not

in

to

related could

fetuses

between

in

content

found

weight.

circulating in

different

fetuses

functionally bGH

smaller

water was

has

125 I] and

modified at

day-old

liver

radioactive

found

in

animals.

have

liver rats

dry

of

0.001)

could determined

min/g

chemical

were

concentrations

uptake

uptake

However,

of (7224)

than

per

liver was

percentage

1 liver

tested (p

placebo.

<

counts

the

the organ

weanling

unlabelled

l),

of this

The day-old

as

with with

significantly and

of

(p

liver

significantly

At

content

weight

Table

and

calculations,

(Table

30

lower

injected

water

development.

Accordingly, L/P

dry

and

significantly

as

in

the

adult

levels

were

animals

injected

with

induced

an

increase

0.05)

hours

after

bGH of hormone

plasma

Vol.

136,

No.

BIOCHEMICAL

1, 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I

4

I

8 (hours)

Time

Fig.

3.

Effect of bovine growth hormone on somatomedin C of 21 day-old fetuses administered subcutaneously to the ( o ). Control fetuses (A ) and adult with a placebo. Means + SEM. n=8-13.

II

14

the circulating levels of and adult rats. bGH was fetuses ( A ) and adult rats rats (a ) were injected

DISCUSSION The

evidence

and

induces

(10)

and

could

the of

the

be

growth

a main

target

of

Thus

in

of

(12,

13).

3n have

been

described the

the in

primate

vivo,

GHs

in sites,

binding

successfully and

interactions

of

The

has

difficult

studies

hGH

to for

test

15);

with

42

the

has

and

because to

body used

somatotropic

growth to

recognize

receptors.

proteins and

recognize

and

induce been

of

hepatocytes.

somatogenic

Turyn

of

promoting

only

liver

because

species

procedure

specific

that

isolated

different

(14,

transport

sites,

growth GHs

acid

characterization

different or

both

bGH

been binding

experimental GHs

suggests

receptors,

amino

hormone.

primate

most

simple

(11)

membranes

techniques

applied

as

from

display

hepatocyte

such

lactogenic hormone

non

and

a

rapid

liver

liver

while

Accordingly, binding

this

rat and

of

activity,

lactogenic

for

in

receptors

rodents,

lactogenic

somatomedins

organ

ability

possesses events

somatogenic

varying types

hormone

of

receptors

presence the

growth biological

production

hormone

both

vivo

that different

or

hormones

Dellacha that

(16) detects and

lactogenic

have in

Vol.

136,

No.

binding

1, 1986

sites

either

in

[ 1251]

the

dose

the

insulin

of

liver

bGH

of

the

liver.

or

of

obtained validation Our

with

results

with

animals,

sites

could

high

circulating

life

(1). stimulates in

19).

membranes that

the

amino

tissues

of

In

addition,

of

fetal

life.

progressively

to

in its

Clinical

in In

as

the

postnatal

fetal

presence is

of

is

target

organs Since

or growth cells

through

to

factor

This

and

findings.

decapitated

23,

(2-4,

maximal

linear

seem

to

the

growth,

depend

fact

of

on

that life

which

24).

control

intrauterine 27),

Thus,

pituitary

not

numbers

suckling

(21-22).

birth

under

despite

(26,

increased

from

hypopituitarism

until

true

the

animals

of

during

6 day-old

these

is

liver indicating

with

supports

during

in

hormone, in

does

somatomedin has

well growth

rats

period

is that

that

activity

rate

a

fetuses

reduced

experimental

activity

growth be

and 30,

its

in

of

weanling

growth

that

minimal,

with

fetal

decarboxylase

those found

children

normal

of

the

there skeletal

circulates

in

receptors

in

its

(28).

would

(29,

a

suggest by

somatic

hormones

at skeletal

hormone.

amounts

also

and

somatomedin

growth

reduced

the

days reported

significantly

day-old

most

However,

stimulated

significant

and

mammal,

evidence

growth

28

pattern

(25). life,

strong

its

in

despite

ornithine

very

lack

binding

last

are

of

a

compared

liver

been

in

hGH

correlate

and

grow

has

and

as

GH

have

action

the

represents

bGH

rats

the

for

evidence

to

somatomedin in

fetus

125 I]

the

not

(20)

also

results

growth

continue

al.

biological

[

fetal

and

are

fetuses

normal

of

it

but

with

which

activity,

sites

sites

activity

human a

ufea2o

via

the

(17),

during

rats et

experimental

anencephalic present

transport

These

full and

acid

binding

increased

absence

rats

hypothesis,

Kelly

binding

intrauterine

this

bGH

development

agreement

decreased

hormone

neonatal

rats

lactogenic

its

of

this

by

us.

in the

not

radioactive rapid

in

vit/to

hormone-promoting of

support

is

uptake

that

growth

that

by

C production

levels In

liver

suggesting

prevent

activity

reduced

somatomedin

adult

(18,

a

in used

of to

but

the

here

procedure

uptake decreased

Also,

membranes,

the

show

bGH-induced

sites. reported

liver of

liver

respectively, suggesting

specific sites

the

COMMUNICATIONS

proportionally

insulin,

hormones,

to

binding

another

was or

RESEARCH

procedure,

insulin

other

BIOPHYSICAL

this

bGH

bound

insulin

AND

With

[ 125 I]

unlabelled

were

results

GH

rat

administration

or

of

BIOCHEMICAL

high

is

Besides, affinity

multifactorial In

involved.

insulin

31).

a

increase

fact

placental

may sites

43

several lactogen,

somatomedin

insulin binding

process,

epidermal

production play

(32)

a which

role

factors

by in

increase

fetal

fetal

growth (33)

in

Vol.

136,

No.

with

parallel

days

1, 1986

of

factor

pregnancy receptors

the

BIOCHEMICAL

circulating and

indirectly

AND

levels

BIOPHYSICAL

of

by

insulin

cross-reacting

RESEARCH

(34)

COMMUNICATIONS

during with

the

the

last

growth

(35). ACKNOWLEDGEMENTS

This Cientifica

work has been supported by grants y Ticnica and from the Instituto

from the National

Comision Asesora de Investigation de la Salud, Spain.

REFERENCES M. and Foh, P.P. (1974) Proc. Sot. Exp. 1. Blizquez, E., Simon, F.A., Blazquez, Biol. Med. 147, 780-783 2. Nanagas, J.C. (1925) Am. J. Anat. 35, 445-460. 3. Mosier, H.D. (1956) J. Pediatrics 48, 633-639. 4. Reid, J.D. (1960) J. Pediatrics 56, 658-664. 5. Jost, A. and Picon, L. (1970) Adv. Metab. Dis. 4, 123-184. 6. Eguchi, Y. (1961) Endocrinology, 68, 716-718. 7. Roth, J. (1975) Methods of Enzymology 37, 223-228. 8. Freychet, P., Roth, J. and Neville, D.M. (1971) Biochem. Biophys. Res. Conmnu~. 43, 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20.

400-408.

Retegui-Sardou, L.A., Scaramal, L.M., Dellacha, J.M. and Paladini, A. (1977) Mol. Cell. Biochem. 16, 87-96. Riggs, R.T. and Walker, L.M. (1960) J. Biol. Chem. 235, 3603-3607. MC Connaghey, P. and Sledge, C.B. (1970) Nature 225, 1249-1250. Kleinberg, D.L. and Frantz, A.G. (1971) J. Clin. Invest. 50, 1557-1568. Li, CH. (1972) in Recent knowledge of the chemistry of lactogenic hormones, Wolstenholme, G.E.W. and Knight, J. (eds.), Churchill, Livingstone, London, pp. 7-22. Kamnerman, S. and Canfield, R.E. (1972) Endocrinology 90, 384-389. Boylan, E.S. and Wittliff, J.L. (1973) Cancer Res. 33, 2903-2908. Turyn, D. and Dellacha, J.M. (1978) Endocrinology 103, 1190-1195. Blazquez, E., Rubalcava, B., Montesano, R., Orci, L. and Unger, R.H. (1976) Endocrinology 98, 1014-1024. Freemark, M. and Handwerger, S. (1983) Endocrinology 112, 402-404. Hurley, T.W., Kuhn, C.M., Shanberg, S.M. and Handwerger, S. (1980) Life. Sci. 27, 2269-2275. Kelly, P.A., Posner, B.I., Tsushima, T. and Friesen, H.G. (1974) Endocrinology 95,

532-539.

21. Heggestad, C.B. (1955) Anat. Record 121, 399-400. 22. Heggestad, C.B. and Well, L.M. (1965) Acta Anatomica 60, 348-361. 23. Chez, R.A., Hutchinson, D.L., Salazar, H. and Mintz, D.H. (1970) Am. J. Obst. Gynecol. 108, 643-650. 24. Liggins, G.C. and Kennedy, P.C. (1968) J. Endocrinol. 40, 371-381. 25. Van Wyk, J.J. and Underwood, L.E. (1975) Ann. Rev. Med. 26, 427-441. 26. Gluckman, P.D. and Brinsmead, M.W. (1976) J. Clin. Endocrinol. Metab. 43, 1378-1381.

27. Ashton, J.K. and Francis, M.J.O. (1978) J. Endocrinol. 76, 473-477. 28. Rosenfeldt, R., Thorsson, A.V. and Hintz, R.L. (1979) J. Clin. Endocrinol. Metab. 48, 456-461. 29. Adams, S.O., Nissley, S.P., Handwerger, S. and Rechler, M.M. (1983) Nature 302, 150-153. 30. Richman, R.A., Benedict, M.R., Florini, J.L. and Toly, B.A. (1985) Endocrinology 116, 180188. 31. Hill, D.J. and Milner, R.D.G. (1980) Diabetologia 19, 143-147. 32. Massague, J., Blinderman, L.A. and Czech (1982) J. Biol. Chem 257, 13958-13963. 33. Caliendo, A.M. and Patel, M.S. (1983) Arch. Biochem. Biophys. 227, 552-561. 34. Blazquez, E. Montoya, E. and Lopez Quijada, C. (1970) J. Endocrinol. 48, 453-461. 35. Roth, J. (1970) in Endocrinology, De Groot, L. (ed.) Grunne and Stratton, New York pp. 2037-2054. 44