Kinetic constanis of asialoorosomucoid endocytosis comparison between hepatocytes from normal and streptozotocin-diabetic rats

Vol.

106,

June

30,

No.

4, 1982

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

1982

KINETIC

CONSTANTS

COMPARISON

ENDOCYTOSIS

OF ASIALODROSDMUCOID BETWEEN

HEPATOCYTES

FROM

AND STREPTOZOTOCIN-DIABETIC Michele

DODEUR,

Laboratoire

de

* Laboratoire

Jean-Pierre Jean

DUMONT, AGNERAY

de

Dominique and Jeanne

Neuro-Endocrinologie, 75014

May

NORMAL

RATS

Genevieve

DURANO*, FEGER

Biochimie - U.E.R. des Sciences Pharmaceutiques - E R A no 396, Universite Paris-Sud, Rue 92290 CHATENAY-MALABRY - FRANCE.

C.N.R.S.

Received

COMMUNICATIONS Pages 1169-1174

10,

PARIS

et Biologiques Clement,

J.B.

U 159, I.N.S.E.R.M., - FRANCE.

2

DURAND,

ter

rue

d’Alesia

1982

Summary : The initial mined for normal and

velocity of asialoorosomucoid internalization is deterdiabetic rat hepatocytes. The analysis of results according to Woolf-Hofstee’s method, showed no modification of the endocytosis constant. In contrast, the maximum velocity of asialoorosomucoid internalization is decreased by threefold in diabetic rat hepatocytes as compared to normal rat hepatocytes. No modification of internalization constant is observed between the two groups of rats. This suggests that the decrease of asialoorosomucoid total uptake, previously reported for diabetic rat hepatocytes is directly related to a decrease of total surface receptor number.

INTRODUCTION Among evident

in

in

sialyl

seromucoid

rats. is

metabolic

changes

variations of

other

cell-surface

in

-

5)

reported

fication

does

affect

its

decrease

of asialoorosomucoid

Recent

membranes

of that

from

hepatocytes not

plasma

of (4

mellitus

diabetes

glycoproteins.

content

glycoproteins

We have previously decreased

disturbances

(1 - 3) and in sugar from

the

binding

for

(7)

protein rats

asialoglycoprotein,

or asialotransferrin

by

some residues

streptozotocin-diabetic

streptozotocin-diabetic

affinity

accompanied

reported

studies

hepatocytes hepatic

is

(2)

total

activity

(7).

This

but

induces uptake

Abbreviations EDTA

: Ethylene

diamine

tretracetic

Na Cl

: Sodium

K Cl

: Potassium

chloride

K H2 PO4

: Potassium

dihydrogen

Pig so4

: Magnesium

Ca Cl2

: Calcium

chloride

Ra H CO3 : Sodium

hydrogen

acid

chloride

phosphate

sulphate

carbonate 0006-291X/82/121169-06$01.00/O

(6) modia by

Vol. 106, No. 4, 1982

the

diabetic

hepatocytes.

asialoglycoprotein

tor

BIOCHEMICAL

Howewer

total

we

uptake

was

did

not

RESEARCH COMMUNICATIONS

determine

whether

related

directly

the

to that

decrease

of surface

of

recep-

number. Since

thehepatic

total

uptake

(specific

loglycoprotein) tions

of

can cell

three

the and

be

requires

parameters

individually

ions

invol.ved

surface

non-specific

studied

calcium

(9

in

bound, -

the and

lo),

in

order

to bind

measurement

of

internalized

using

asia-

different

condi-

incubation.

In the experiments of asialoornsomucoid hepatocytes.

loglycoprotein

protein

binding

(8)

asialoglycoproteins

rat

AND BIOPHYSICAL

The

total

tornurrbervithout

reported

here

internalization

any

results

support

uptake

is

directly

change

in

the

we have for

normal

the

conclusion

related

apparent

determined and

initial

the

streptozotocin that

to the

decrease

the

variation

endocytosis

and

velocity diabetic

of

of

asia-

total

recep-

internalization

constants. MATERIALS

AND METHODS

orosomucoid, a gift from Professeur MONTREUIL (Universite des Techniques, Lille, France), was desialylated with Agaroseimmobilized neuraminidase type X-A (E.C. 3.2.1.18) (Sigma Chemical Co) 13Hl asialoorosomucoid was prepared by reductive methylation of Wilder (11) using tritiated sodium borohydride (16 Ci/mmol C.E.A. France) as described previously (7). Collagenase was obtained from Worthington. Male SpragueDavley rats (275-300 g) were obtained from Charles Rivers (France). Scintillation fluid (aqueous combining system) was from Amersham France. Other chemicals were reagent grade. Sciences

Human et

Treatment of rat and cell After an overnioht divided into two groups.

preparation

rats were randomly streptozotocin (65 mg/ kg body weight, dissolved in isotonic saline acidified to pH 4.5 with citric acid) through the tail vein (diabetic rats). Rats from control group were injected with the medium alone (normal rats). After 11 days, animals with a blood sugar level superior to 250 mg/lOO ml were considered as diabetic and used for cell preparation. Hepatocytes were prepared using the collagenase perfusion procedure of Berry and Friend (12). Final cell-pellets were suspended in Krebs buffer containing 0.15 M Na C1,0.005 MKCl, 0.001 M K H2 P04, 0.001 M Mg SO4, gassing viable

lactate rats.

fast

(with

Animals

free access from one group

water) received

to

0.003 Ca C12, 0.025 M Na H CO , maintained at cf 02/C02. (95/5). Routinely 7 8 -85 X of single cells, as Judged by either trypan blue exclusion deshydrogenase (EC 11.1.28) (13) were obtained

pH 7.4 with continuous cells and 80-90 % of or the release of from normal or diabetic

Study

ofasialoorosomucoidendocytosis The cells were suspended to a final concentration of 1 x lo6 cells/ml Krebs buffer, pH 7.4,continuouslygassed with 02!CO2 (95/5), and kept in Experiments in duplicate were carried qiratory shaking bath (60 rev/min). at 37O C in flat-bottomed tubes by incubation of 1 x lo6 cells with different (0.9 - 11 x 10-Y M) at different concentrations of \ 3H\ asialoorosomucoid

Determination

of

total

of

a out times.

uptake

After

incubation at 37O C. the reaction was stopped by adding 3 ml of iceThe cells were harvested and washed four ticold Krebs buffer into the tubes. The final pellet was mes in Krebs buffer by centrifugation at 50 g, 120 sec. dissolved in 10 ml of scintillation f1ui.d and counted in a liquid scintillation spectrometer (Intertechnique SL 30). 1170

Vol.

106,

No.

BIOCHEMICAL

4, 1982

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Determination of internalized asialoorosomucoid After incubation at 37O C, the reaction was stopped by adding 3 ml of icecold Krebs buffer containing 20 rnp of EDTA sodium-salt. The cells were harvesThe cell pellets were resuspended in ted by centrifugation at 50 g, 120 sec. the same buffer and incubated for 15 min at 4' C. Then the cells were washed three times still in the same buffer. This incubation released specific surfaceJ3 (9 -

H ] asialoorosomucoid 10).

into

the

medium

without

any

change

in

cell

viability

Determination of non-specific surface-bound 1 3H/ asialoorosomucoid coprotein as described above, The cells were incubated with IjHlasialogl either in presence of 20 mM EDTA before adding 1 3 HI asialoorosomucoid or in absence of calcium ions in the medium. The cells were washed as des ribed for determination of internalized asialoorosomucoid. The non-specific 1 5 HI incorporation was subtracted from the values obtained for total uptake and internalisation of asialoorosomucoid. Determination Specific 3Hl cpm total

of

specific surface uptake -

surface bound asialoorosomucoid bound asialoorosomucoid was estimated internalized 13Hl cpm.

RESULTS The normal

time

and

centrations

from

diabetic to To

ty

or

(vi)

tions

or

of for

ted

of

normal

rats.

to

diabetic

city

was

and

2 A). 2 A)

about

cells/set)

as appears

is

due

maximum

total receptors

number

With

linear

to

The

at

hepatocytes

from all

between

normal

rats

decreased of

maximum

the

maximum

initial

veloci-

velocities

were

normal

to and

1 8)

Woolf-

diabetic

No modification (3.5

diabetic 2 5 x 10 uptake

velocity

plot-

(fig.

according

normal

(53

total

of

concentra-

contrast,

of

decreased

concentration

both

least

was

all

concentrations.

In

at

up

the

initial

hepatocytes

to

a decrease

for

In

ratio

hepatocytes. in

the

time

both,

molar

observed

rat

compared

to

concentration

was

with

a decrease

or

The

1 A).

was calculatedat

hepatocytes

were

that

to

from con-

analysis.

taken

asialoorosomucoid

threefold

it

endocytosis The

the

the

constant -9 x 10 M)

+ 0.3

:

hepatocytes

kinetic

(fig.

due

hepatocytes.

molar

(fig.

hepatocytes

surface

of

(fig.

M/IO6

by

linearly rats

constant,

rat

curves

(3

was

endocytosis

method

decreased

of

decrease

diabetic

difference

asialoorosomucoid

asialoorosomucoid

i

endocytosis

a function

Therefore

to

altered

H

I

asialoorosomucoid

endocytosis

and

apparent

this

group 3

of

vi/asialoorosomucoid

apparent

rat

increased any

amount

whether an

as

the

the

lo-l7

the

normal

Hofstee's rats,

11

rats,

1 3H[

either of

-

determine

velocity

endocytosis

was studied at different 1 3Hl -12 x 10 M) used in the subsequent

I H I asialoorosomucoid concentrations for

all

compared

1 3 H 1 asialoorosomucoid

3

of

at

of

the

AND DISCUSSION

rats

(0.9

endocytosis 5 min

course

diabetic

by

f

0.5

the

maximum

rats -17

(16

without

2 x

cells). in

any

velof

M/lo6

observed

x 10-9H)

diabetic change

of

constant. velocity of were

is occupied estimated

proportional

to

cell-surface at

the

internalization

receptor.

different II71

concentrations

The

constant occupied

cell-

of 1 3l-l 1 asialooro-

and

Vol. 106, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MINUTES

Fig.

1 A : Timg course of asialoorosomucoid endocytosis. Hepatocytes cells/ml) isolated f om normal and diabetic rats were ikkbated at 37O C urithl ‘H] asialoorosomucoid (40-500 rig/ml). At the indicated time points, cells were harvested and washed as described under “Materials and Methods”. Each point gives the average value obtained from six separate experiments using six rats of each class. o---J, normal rat diabetic rat

Fig.

1 B :

somucoid

by

a steady

state

face

bound

and

Methods”.

0.24

incubating

I

cells

ted

previously bound

of

I The

values

I

HI It

at and

and

M and

about

5 min,

a time

receptors

rat

2 0.01

(10).

needed The

as described

hepatocytes

x lo-l2

bound

M of

I 3H1

to

obtain

specific

sur-

under

“Materials

a maximum

of

asialoorosomucoid/

2 B). about

values

the

asialoorosomucoid, had

C for

occupied

diabetic

(fig. From

37O

was calculated

0.08

represented

(7). 3

free

normal

respectively

These

cells

asialoorosomucoid

x lo-l2

lo6

face

the

number 3H

+ 0.025

be estimated.

initial velocity of asisloorosomucoid endocytosis as a function of asialoorosomucoid molar concentration. The initial velocity was calculated from the linear part of the curve (fig. 1 A) at all asialoorosomucoid concentration. U normal rat n disbetic rat The

the

90

apparent

the same

% of

obtained

value 1172

for

the

total

for

the

site

number

maximum

internalization normal

and

as

velocity

constant diabetic

estimaand

rat

surcould

hepatocy-

BIOCHEMICAL

Vol. 106, No. 4, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

50

1

40

I

0 a

2

f:

30

2

‘s \i

(0 P

2 E

20

‘_

k ‘0

>

1C

,f$ % 6

T

i

r ‘:

5

10 Y

[asiaioorosomucoid

Fig.

2 A :

Fig.

2B

Initial velocity of asialoorosomucoid endocytosis as a function of the initial velocity/asialoorosomucoid molar concentration ratio accordingto Woolf-Hofstee’s method. u normal rat b--o diabetic rat

: Specific

surface

aaialoorosomucoid. cells/ml) iso ated from normal and diabetic rats were incubated at 37O C with 1 3HI asialoorosomucoid (40-500 rig/ml) for 5 min. Specific surface bound asialoorosomucoid was calculated as described under method, Each point gives the average value obtained from six separate experiments. &--A normal rat w diabetic rat Hepatocytes

tes :

200

results

+ 25

showed

ved

in

the

cell

x that

surface

I

3h

The

that

surface

either.

estimation

A

of

unaltered

similar

result

-1

respectively.

had

obser-

to a lowering

internalization

association normal been

These

endocytosis

related

apparent

apparent from

the

receptors

the

at

diabetic

previously

of and

constant

and

value and

rat

close al

(15)

hepatocytes,

the

time

37O

hepatocytes) found

normal to

for

that

normal

and

to

diabetic

reported

rat

as expressed

by

1173

to

by

total

the

hepatocytes

Weigel.

(lo),

molecules

The

showed

internalize

rat

hepatocytes. in

constant

needed

quasi-equivalent

from

is

internalization

apparent

conditions

asialoorosomucoid,

1

cell-surface

Bridges

+ 15 x lob5 set 3H of I I asialoorosomucoid rats can be directly

number.

suggest

our experimental

This

normal

decrease

diabetic

220

studying

at 4s C (7).

The

the

the

with

modified

and

from

constants

not

under of

set -1

receptor

(asialoorosomucoid

interaction

bound

(lo6

1o-5

hepatocytes

endocytosis

was

I#]

receptor was

internalized

velocity

amount

number

about

Tolleshaug maximum

of

that

an

on

8 min. (14)

and

for asialoo-

C

Vol. 106, No. 4, 1982

BIOCHEMICAL

rosomucoid/cell/sec that

was

found

by

These ted

a

This

Weigel

new of can

latter

The found

might

Recherche

previous

was

similar

in

which

results

to a decrease

the

to

uptake

accumulation

of

by

diabetic

hepatocytes.

cell-surface

of

receptors.

glycoproteins

some

repor-

we

serum

in

(4).

authors

The

work

This

our total

:

assistance.

confirm

It

asialoorosomucoid

authors

Acknowledgments

molecules/cell/set.

now be attributed explain

other

by

318

RESEARCH COMMUNICATIONS

(TO).

observations

decrease decrease

about

AND BIOPHYSICAL

wish

to

was supported

Scientifique

(E

R A

thank

grant

by

Lorinquer

F.

for

from Centre

her

technical

National

de

la

396).

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