Changes in the insulin and glucagon receptors in the regenerating liver following intoxication with carbon tetrachloride

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

May 14,1W9

CHANGES

INSULIN

IN THE

LIVER

AND GLUCAGON

INTOXICATION

FOLLOWING Marisabel

Biochemistry

Mourelle*

Department,

Avanzados

de1

and

Boanerges de

April

Rubalcava+

Investigaci6n

Polit6cnico

y

National,

Mgxico Received

IN THE REGENERATING

RECEPTORS

WITH CARBON TETRACHLORIDE

Centro

Instituto

189-198

14,

D.

de

P.O.

Estudios

Box

14-740

F.

2, 1979

The response of rat liver plasma membrane adenylate cyclase was studied from one to 14 days after a single dose of carbon tetrachloride (Ccl,,). The response to glucagon was diminished to a greater extent than that of fluoride and was due to a deficiency in hormone binding. In contrast, insulin binding increased 300% over control; the change was due to increased number of binding sites. The "affinities" of receptors for either hormone were not altered. The tissue levels of adenosine 3':5'-monophosphate increased following Ccl,, poisoning reaching a peak precisely when the adenylate cyclase response to glucagon was at its lowest level.

These

studies

present

evidence

that

receptors

for

pancreatic

hor-

mones change differently when liver is damaged and during its regeneration following CCls intoxication. The change in pattern is remarkably similar to changes reported previously in fetal liver development or following partial hepatectomy of adult rat.

It ted

by

has

and

concluded

(7) of

tions

in

the

increased creased

be

of

a reversible

In

involved

and

receptors insulin

in

this

after

for

(8).

the

and

requests

suggests

pancreatic

We have

of

specific

blood

glucagon

healtera-

hormones,

We also

for

(2-6).

hepatectomy, by

while

regulathat

regulation

partial

partial fullfilment for M.Sc. of Centro de Investigation Politecnico National.

correspondence

is

process

be accompanied

receptors fashion

evidence

in

that, to

proliferation

vivo

participates

hormones

membrane

Presented as of Pharmacology de1 Instituto

+ To whom addressed.

liver

hepatocyte

(1).

appears

number in

may

that

these

regeneration

that

factors

glucagon

patic

*

proposed

blood-borne

insulin

levels

been

namely, receptors

reported

(9)

that

an de-

the

degree at Department y de Estudios Avanzados

reprints

should

be

0006-291X/79/090189-10$01.00/0 189

Copyright All rights

@ 1979

by Academic Press. Inc. in any form reserved.

of reproduction

Vol. 88, No. 1, 1979

fetal

liver

liver,

and

pacity

of

BIOCHEMICAL

is

less

that the

liver

but

the

nylate late

glucagon

action

cyclase.

Wicks

cyclase

can

be

and

bind

related

to

adult rather

These

has

to

of

period

response

of to

in

glucagon maximal

the

the

the

functional

was

same

suggested

in

ca-

glucagon

remained

findings

adult

a reduced

a defi-

levels

a relatively

catecholamines of

the

period

the

a change

reported

presence

due

fluoride

than

(10)

the

with

sodium

than

this

cyclase

life.

to

with

glucagon;

adenylate

response

action is

general

response

fetal

in

to

A reduced

glucagon

"resistance"

correspondsin

throughout cit

to

glucagon

hepatocyte

growth.

detected

sensitive

this

hyposensitivity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

ade-

large

adeny-

fetal

liver,

which

beta

adrenergic

receptors. It

is

well

tetrachloride (11).

et

formation.

causes

a marked

rise

higher

than

this

exposure

(12)

of

that

caused

polypeptide

is

animals

to

and

hepatocellular

liver

demonstrated

deoxyribonucleic

The in

of

a fatty

al.

synthesis

cell

of

new

the

produces

Leevy

injury,

ting

that

(Ccl\)

necrosis toxic

known

acute

serum by

acid

CCls

with

occurs, of

(13);

hepatic

liver indica-

concentration

hepatectomy

associated

following

intoxication

a-fetoprotein

partial

that

carbon

the

rat which

the

is

release

proliferation

(13,

14). The pattern seen

studies of

in

proliferation

fetal

reported

changes and

in

here the

were

pancreatic

proliferating

following

CCll,

designed hormones

livers

also

to

determine

receptors occurs

in

if

the

previously hepatic

intoxication.

MATERIALS

AND METHODS

Glucagon and insulin were gifts from Lilly; [c(-~~P] ATP and cyclic AMP were sup lied by the International Chemical and Nuclear Corporation. ['H!cyclic AMP and [12'1] -iodoinsulin were purchased from New England Nuclear; creatine phosphate, creatine phosphokinase and bovine serum albumin from Sigma Chemical Co. Oxoid filters were obtained from Amersham Searle (Chica o) and 1125~1 iodoglucagon from Nuclear Medical Laboratories 9 Dallas). All other reagents were of analytical grade.

190

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Wistar male rats wighing 180-250 g fed ad libitum a Purina Chow were used for these studies. The treated animals received a single dose of 0.5 ml per 100 g body weight of CCll, in corn oil (1:l v/v) through an intragastric tube; the controls received the correspondent volume of corn oil. The rats were sacrificed at different periods (see results) to prepare both liver homogenates and partially purified plasma membranes; the homogenates were prepared using 50 mM Tris-HCl, pH 7.5 with 4 mM EDTA using fresh livers. The beaker with the homogenate was transferred to a bath of boiling water for 3 minutes and then centrifuged at 15,000 x g for 30 minutes; the final supernatant was frozen and stored at -80°C and used afterwards to measure the amount of cyclic AMP according to Pollard and Weingrand (15). Partially purified plasma membranes from the rat liver were prepared according to Neville's procedure (16) with a modification described by Pohl et al (17). The membranes were frozen and stored in liquid nitrogen and, for further use, were suspended in 20 mM TrisHCl, pH 7.5. Adenylate cyclase activity was measured by the method described by Salomon et al (18). the assay medium contained in 0.1 ml 1 mM EDTA, 3.2 -ca-32Pj mM ATP (20-40 cpm/pmole), 5 mM MgC12, 25 mM Tris-HCl pH 7.5, 1 mM cyclic AMP and an ATP regenerating system consisting of 20 mM creatine phosphate and 100 U/ml creatine phosphokinase. Either glucagon (1 PM.) or sodium fluoride (10 mM) was added to stimulate enzyme activity; incubations were carried out for 10 minutes at 30°C; the reaction was initiated by the addition of the membranes to give 30-40 pg of protein. The binding of [ 1251] iodoinsulin or [" I] iodoglucagon to liver membranes was carried out as described by Rubalcava and Rodbell (19); 25 mm diameter oxoid filters and 2-10 day old monoiodinated [1251] insulin (sp. activity90$i/ug) or glucagon activity 400 pCi/pg) were used. Greater than 90% of radio(sp. activity associated with each probe was precipitable with trichloNonspecific binding (radioactivity retained by roacetic acid. filters from parallel assay tubes incubated together with 10pM unlabelled hormone) was subtracted to obtain "specific binding". Liver membranes (30-50 ug of protein) were incubated at 30°C in a final volumen of 0.1 ml containing 20 mM Tris-HCl, pH 7.5, 1% bovine serum albumin and the radioactive hormone at different concentrations. Incubation terms were 20 minutes for glucagon binding and 30 minutes for insulin. Protein was measured by the procedure of Lowry et al (20) using bovine serum albumin as standard. RESULTS Fig.

1 illustrates

adenylate

cyclase

after

intoxication.

agent

the

ulated third de

day ion

all

stimulates

to

glucagon One

basal

either

the

activity by

glucagon

activities adenylate

day

effect and after

of

CCls

fluoride the

were to

cyclase

191

at

different

as well

fluoride

recovered

the

by

response

the

of as

those

diminished; control

a different

of periods

administration

(non-stimulated) or

on

at values. process

the

toxic stim-

the Fluorifrom

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

GlucOW

.

01 11

’ 0

1 2

1 4

I 6

DAYS

Fig.

1:

hormonal is

has

for

been

with

and

showed

glucagon

but

not

cyclase

(19); of

the

for

therefore

I),

I 14

different

while

to

the

data

affect of

activities: The

the

in

ratio

enzyme

this

ion

possible

effects

of

by which

fluoride

affects

the

transport

phospholipids

fluoride the

process

Ccl,,

protein acidic

the the

the

that

carrier that

of

distinguishing

postulated

and glucagon/fluoride. (Table

I 12

CC14

response

versus

the

we

ratio

The

receptor-mediated

lipids

(11)

(21).

therefore,

It

acts. of

activation

on

FOLLOWING

1 IO

Effect of CCll, on response of adenylate cyclase to glucagon (1 uM) and fluoride ion (10 mM). Incubation conditions and procedures for assaying adenylate cyclase activity are described under methods. Each point represents the mean of 25 experiments t S.D.

useful,

Ccl,,

I 6

the

of required

rat

liver

glucagon/basal

and

192

the

lipids for

adenylate

calculated

fluoride/basal, fluoride/basal

interaction

are

1 were

Fig.

to

as

glucagon/basal remained

glucagon/fluoride

constant ratios

the

Vol. 88, No. 1, 1979

ADENYLATE

BIOCHEMICAL

TABLE

I

RATIOS

IN PLASMA MEMBRANES FROM

CYCLASE ACTIVITY LIVERS

OF RATS INTOXICATED

WITH Ccl4

DAYS FOLLOWING cc14

FLUORIDE BASAL

GLUCAGON BASAL

GLUCAGON FLUORIDE

0

3.35*

5.43

1.61

1

3.56

2.42

0.96

2

2.96

2.54

0.86

3

2.93

3.83

1.30

4

3.52

4.48

1.27

5

3.52

4.96

1.41

6-14

similar last

to value

* The ratios were calculated and represent the mean of

fell

24

and

48

control

values

mains

unchanged

affected

after

it the

hours

(Fig.

2).

but

then

precisely

when level.

changes To

adenylate

This

finding owing

determine

to

glucagon

was

of

glucagon

to

if the its

the

the

the

of

the

plotted

in Fig.

ratio due

the

amount

to

a peak

in

cyclase is to loss

of

nucleotide

Ccl,,

1,

returned

to

to

the

to

evidence

the

not

change

this

peak

glucagon that

more

AMP in

during

did

response

is

cyclic

livers

hours;

re-

glucagon

of

48

further

to the value

fluoride/basal

response

the

of of the

193

response

of

a difference binding

of

the

in occurred

was

glucagon

adenylate in

[ lZ51]

the

the

same

at

its

action

intoxication.

consequence receptors,

similar last

and

homogenates

Levels

the

differently

Since

measured

increased

to the value

intoxication

that

from

similar last

from the data 25 experiments.

later.

others;we

The

the

the

seems

prepared

periods

lowest

hours

72 hours

than

supernatant

24

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cyclase

binding iodoglucagon

EIOCHEMICAL

Vol. 88, No. 1, 1979

AND EIOPHYSICAL RESEARCH COMMUNICATIONS

0123456789 DAYS FOLLOWING Fig.

to

2:

liver

Effect of CCll, on the levels of CAMP determined in rat liver homogenates. Each bar represents the mean of 20 experiments + S.D. The procedure for assaying CAMP is described under methods. membranes

was

with

Ccl,,

intoxication decresed the

toxic

remained case

of

in

agent

(from

the

low

compared (Fig.

the

to

first

the

insulin

increase

to on

values

on there

two

days

(the

small

fall

the

the

third

day,

the

control

value;

the

initial

value.

0.63 the

the

second

binding

day of

are

not

apparent

affinity

194

of

protein)

and as

is

activity, On the

no

changes

at

the

first

in

the day

hand, binding

and

the

However, to

nearly

returned values

the

it other

significant).

binding

the

binding

finally,

day.

increased the

of

cyclase

were

after

administration

day;

third

insulin

thereafter The

the

points Glucagon

pmoles/mg

occurring

second

time II).

after

adenylate

first

seen

various Table

day

3.73 value

control

at 3 and

fluoride-stimulated

returned during

sharply

at

of

CC14

300%

on of

gradually of

the

peptides

to

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

k.-f‘y.d..‘..-b..I.a,

: :

f

_-. --___

: ‘. ,’

‘“I -

ti I

I

I

L

I

1

L

I

0

2

4

6

6

IO

12

14

DAYS

Fig.

for

for

C Cl4

Effect of Ccl,, on the binding of II’2511 iodoinsulin Incubation conditions and and fzz5~] iodoglucagon. procedures for assaying binding of labeled glucagon The results and insulin are described under methods. experiments + S.D. are expressed as mean value of 25

3:

their

change

FOLLOWING

receptors, (Table

obtained

II).

We were

by

Scatchard

unable

to

plots

detect

(22),

high

did

not

affinity

sites

insulin. DISCUSSION The

effect

administration glucagon the

that

on

In

was and

Thus

fact,

of

the

effect the the

adenylate

cyclase

a different

one

basal of

Ccl,,

catalytic rise

and on

the

for

fluoride hormone

component in

activity

tissue

the

the

levels

195

activity

stimulated action

of

following

seems

Ccl,, stimulated

activities to

(Table

be greater

adenylate

cyclase

of

AMP during

cyclic

by I).

than

system. the

fall

labeled

nM

of

0.5

to

binding

of

1 MM.

hormones

25

capacity

x

0.13

0.13

0.10

0.05

f

f

f

f

f

and

are

described

0.11

0.12

0.26

0.14

0.15

+ 0.20

*

f

f

+ 0.12

of 0.10

t

Ka

M-l

+ 0.15

BINDING

0.00

0.00

0.32

0.34

0.42

0.51

0.36

0.71

0.54

0.32

+ 0.43

f

f

+ 0.25

f

f

f

f

f

f

+ 0.21

f

f

+ 0.64

under

from methods

Incubation

obtained

3.77

4.21

3.92

3.88

4.40

4.39

4.63

3.62

4.69

3.16

3.96

0.06

+ S.D.

were

3.73 0.06

PLASMA

[lz51]

CAPACITY

LIVER

OF

(pmol/wl

BINDING

GLUCAGON

ON

+ O,ll*

lOa

Ccl4

experiments

1.36

14

value

1.52

12

*The

1.07

11

mean

1.16

1.43

9

10

1.54

1.11

7

1.38

6

8

1.50

1.41

1.46

3

5

1,lO

2

4

1.38

1.26

1

Ka

OF

0

ccl4

FOLLOWING

DAYS

EFFECT

TABLE

x

0.13

0.06

0.07

0.20

0.15

0.04

0.20 0.10

and

plots,

+ 0.13

f

+ 0.10

f

+ 0.17

f

2 0.11

+ 0.20

f

f

f

f

f

+ 0.05

INSULIN

Each were

procedures for

RAT

0.95

1.07

for

f

f

f

f

f

f

f

f

*

f

f

hormone

assaying

represents

0.98

1.05

0.70

3.00

1,35

1.40

2,20

1.40

1.27

2.26

1.27

+ 1.66

f

f

CAPACITY

TO

either

value

4.30

4.80

4.20

13.00

10.30

12.67

11.64

12.63

0,57

10.12

9.89

5,55

2.92

3.85

(pmul/mg)

BINDING

INSULIN M-1

AND[1251]

lo7

concentrations

conditions

Scatchard

1.41

1.14

1.10

I,00

0.95

0.96

0.90

1,06

I,12

0.92

0.99

1.11

1.16

1,25

Ka

MEMBRANES

GLUCAGON

II

the from

binding

Vol. 88, No. 1, 1979

BIOCHEMICAL

in

glucagon

suggests

by

CC14

response treatment.

intoxication

high

(23).

it

the

in

We are

in

and

receptors.

regenerating

which to

with

has

the

same

[-I]

is

altered

could

be

after

the

inactivation

been

of

case

were

were are

CCls the

same

reduced

of

of

not

for of

damaged

markedly,

selective

pattern

liver

of

in

of

number

the

glucagon

receptor

following

insulin, of

effect

conditions

changes

hepatoctomy

in

to

inhibit in

Agustin

for

in

The

coincides

synthesis

the

synthesis

of

progress

using

number

of

the

the

hepatectomy

mitomycin

this insulin

of

C (13)

RNA in

addition

antibiotic

to

receptors

under

here.

indebted

superb

that

with

the

involve

the peptide

of

either

partial

suppresed

alterations

interesting seen

is

reported

We are

receptors

Ccl4

on

these

unmasking is

a-fetoprotein.

CCll,

Acknowledgments.

it

of

are

of

if and

CC1 Ir or

appearance

reported

examining

masking

following

Experiments

Martinez

iodoinsulin

receptors

or

following

the

minimally

nucleotide

possibility

for

process

the

the

DNA (24).

examine

catecholamines

is

liver.

liver

protein

this

of

induces

the

increase

time

levels

degradation In

apparent

plasma

proliferation

adult

synthesis

the

effects

Ccl,,

fetal

regenerating

in

the

Thus,

observed

of

iodoglucagon

that

receptor.

levels

receptors

[-I]

appears

high

enzyme

enzymes.

Since the

the

An alternative

phosphodiesterase

while

that

The

by the

explained

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to

Miss

technical

Eunice

Zavala

and

Mr.

assistance.

REFERENCES 1. 2. 3. 4.

Fisher, B., Szuch, P. and Fischer, E.R. (1971) Cancer Res. 3J, 322-331 Rabes, H.M. and Brandle, H. (1969) Cancer Res. 29, 817-822 Echave Llanos, J.M. Gomez Dumm, C.L. and Surur, J.M. (1971) Experientia 27, 574-575 Rixon, R.H. and Whifield, J.F. (1972) Proc. Sot. Exp. Biol. Med. 141, 93-97

197

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Leffert, H. (1974) J. Cell Biol. 62, 792-801 Bucher, N.L.R. and Seaffield, M.N. (1975) Proc. Nat. Acad. Sci. USA 72, 1157-1160 H., Alexander, N.M., Faloona, G., Rubalcava, B. and 7. Leffert, Unger, R. (1975) Proc. Nat. Acad. Sci. USA 72, 4033-4036 H.L., Koch, K.S. Rubalcava, B., Sell, S., Moran, T. 8. Leffert, and Boorstein, R. (1978) J. Natl. Cancer Inst. Monogr. 48 87-101 9. Blazquez, E., Rubalcava, B., Montesano, R., Orci, L. and Unger, R. (1976) Endocrinology 98-, 1014-1024 Wicks, J. (1969) J. Biol. Chem. 244, 3941-3950 ;: : Recknagel, R.O. (1967) Pharmacol. Rev. 19, 145-208 C.M., Hollister, R.M., Schid, R., MacDonald, R.A. 12. Leevy, and Davidson, C.S. (1959) Proc. Sot. Exp. Biol. and Med. 672-675 102, 13. Taketa, K., Watanabe, A. and Kosaka, K. (1975) Ann, N.Y. Acad. Sci. 259, 80-89 E.A., Kiplitz, M. and Sell, S. (1976) Cancer Res. 14. Smuckler, 36, 4558-4561 H.B. and Weingrand, D. (1976) Anal. Biochem. 76, 15. Pollard, 382-384 16. Neville, D.R. (1968) Biochem. Biophys. Acta 154, 540-552 S.L., Birnbaumer, L. and Rodbell, M. (ml) J. Biol. 17. Pohl, Chem. 246, 1849-1856 18. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541-548 B., and Rodbell M. (1973) J. Biol. Chem. 248, 19. Rubalcava, 3831-3837 20. Lowry, O.H., Rosenbrough, N.Y., Farr, A.L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 21. Rodbell, M. (1972) in Glucagon: Molecular Physiology, Clinical and Therapeutic Implications (Lefevbre, P.S. and Unger, R.H., eds.) pp. 61-75 Per amon Press, New York. G. (1949 Y Ann. N.Y. Acad. Sci. 51: 660-675 22. Scatchard, P.H., and Brody, T.M. (1963) J. Phazacol. Exp. Ther. 23. Stern, 65-73 141, J.J.T., Lo, M. and Webster, D.A. (1971) Cancer Res. 24. McCann,

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31,

1573-1579.

198