Vol. 91, No. 2, 1979 November

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 515-520

28, 1979

PRECOCIOUS

INDUCTION

OF GLUCOKINASE

CULTURES OF POSTNATAL Toshikazu

Nakamura,

Institute for Tokushima Received

October

Kazushi

IN PRIMARY

RAT HEPATOCYTESl Aoyama and Akira

Ichihara

Enzyme Research, School of Medicine, University, Tokushima 770, Japan

5, 1979

SUMMARY: Hepatocytes of 14-day-old rats have no detectable luco(lo- 3 El) in kinase activity but it was induced by insulin --in vivo, primary cultures of these hepatocytes. The glucokinase induced by insulin was separated by electrophoresis on a cellulose acetate membrane and identified by its low affinity for glucose. This precocious induction of glucokinase was completely prevented by the presence of either actinomycin D or cycloheximide. Glucagon also Dexamethasone and testosterone, inhibited its induction by insulin. strongly enhanced the inducwhich alone had no inductive effect, tion by jnsulin. When hepatocytes of ll-day-old rats were cultured with lo- M insulin, 10'6M dexamethasone and 10s7M testosterone for 48 hr, their glucokinase activity increased to the non-induced level in hepatocytes of adult rats. Estrogen, thyroxine or growth hormone did not induce glucokinase precociously. Testosterone did not enhance induction of glucokinase by insulin in cultured hepatocytes of adult rats. Glucokinase[EG in

the

(1).

liver

2.7.1.21

and it

The enzyme disappeared

insulin

plus

marker rat then

of

liver

terminal

rises

Exogenous

attempts

activity

glucose

first reaching

appears the

glucokinase

1. This work was supported the Ministry of Education,

could

liver

or alloxan prematurely

by

has been used

days

(7).

In

birth,and later for

is prevented

by infusion

as a

cells.

after

diabetes

rats

in primary

necessary it

adult

be induced

lo-12

are apparently

diet

rats

16 days

level

since

in

parenchymal

about

adult

development,

a carbohydrate-free

it

Glucokinase of

utilization

and glucose of adult

but

(5,6).

and insulin

during

to induce

(2-4),

differentiation

rapidly

of glucokinase ation,

rapidly

in glucose

by insulin

in hepatocytes

dexamethasone

its

a key enzyme

is induced

activity

culture

is

(7,S). increase

by starvHowever, of glucose

by Grants (Nos 337013 and 467052) Science and Culture of Japan.

from

0006-291X/79/220515-06$01.00/0 515

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved

Vol. 91, No. 2, 1979

or insulin, that

BIOCHEMICAL

or both,

corticoid

but

complexity

studies.

To avoid

this

old

in

of

immature

primary

stimuli

for

culture

paper postnatal

glucocorticoid

(2).

necessary

for

There

are

precocious

were ambiguous

(8-10).

This

of

system

used

the

complexity,

hepatocytes,

The present

are

findings

may be due to the

rats

RESEARCH COMMUNICATIONS

have been unsuccessful

and thyroxine

of glucokinase,

AND BIOPHYSICAL

--in vivo we used

to study

the

as reflected provides

terminal

that

the

of glucokinase

induction ambiguity in of

these ll-day-

differentiation

by synthesis

evidence

development

hepatocytes

reports

of glucokinase. physiological are

insulin,

and testosterone. MATERIALS

and METHODS

Materials --- The rats and materials used for cell isolation and culture were as reported previously (11). Yeast glucose-6phosphate dehydrogenase, NADP and ATP were obtained from Boehringer Mannheim, Mannheim; actinomycin D was from Schwazmann, New York; cycloheximide was from P-L Biochemicals, Milwaukeet and other chemicals were from Wako Pure Chemicals, Osaka. Isolation and monolayer culture of parenchymal cells from 14day-old rat liver --- Parenchymal hepatocytes were isolated from 14-day-old rats and adult rats and cultured as monolayers as reported previously (11). Suspensions of 5 x 10b cells were plated in 10 cm plastic dishes in 10 ml of Williams medium E supplemented with 10% fetal bovine serum and 10m6M dexamethasone, and cultured in a humidified incubator at 37OC under 45% 02, 5% CO2 and 50% N2. The medium was changed 24 hr after plating. were harvested with a rubber Assay of glucokinase --- The cells policeman and homogenized in 0.5 ml of 20 mM K-phosphate buffer (pH 7.5) containing 10 mM glucose, 0.1 mM dithiothreitol, 5 mM EDTA and 150 mM KC1 in a Polytron homogenizer for 0.5 min. The homogenate was centrifuged and the supernatant was used as the enzyme preparation, Glucokinase was assayed by the method of DiPietro and Weinhouse (12). Glucokinase activity was calculated by subtracting the hexokinase activity from the total activity of glucose-ATP phosphotransferase. One unit of glucokinase is defined as the amount forming 1 umole of product per min. Protein was measured by the method of Lowry et al. (13). Electrophoresis --- Electrophoresis was carried out on a cell1 x 6.7 in), in Verona1 buffer (pH ulose acetate membrane (Gelman, and 10 8.6, 1=0.05) containing 5 mM EDTA, 10 mM 2-mercaptoethanol mM glucose at 100 Volts for 90 min in a cold room. After electrophoresis the membrane was stained for hexokinase and glucokinase activity by the method of Sato et a1.(14). RESULTS and DISCUSSION No glucokinase hepatocytes

from

activity 14-day-old

was detectable rats,

516

and it

in did

freshly not

isolated

appear

in

hepa-

BIOCHEMICAL

Vol. 91, No. 2, 1979

O

AND BIOPHYSICAL

20

IO Time

30 in culture.

RESEARCH COii4MJNlCATlONS

50

40 h

Fig. 1. Precocious induction of glucokinase by insulin in 14-dayDexamethasone was added from start of ratpatocytes in culture. culture and insulin was added 10 hr later. Values are means + S.D. for 4 experiments. o, with insulin; o, without insulin, tocytes

cultured

in organ

without

culture

increased

of

insulin

fetal

moderately

without

ared

cells

20 hr. ly

impaired

freshly cells

isolated

insulin

attain

(4).

cells

(unpublished

reduced to

functions

is

data). receptors

and then

freshly

isolated

about

However,

the

start

The glucokinase and identified as shown in Fig.

of

induced

half

their

are restored

the maximum induction

10 hr after

was reported

glucokinase (15).

activity

As shown in Fig.

increased

that

of glucokinase,

for

have great-

receptors

on

on intact

impaired during

appe-

linearly

hepatocytes

The number of insulin only

It

10m7M insulin,glucokinase

10 hr,

We have shown that

that

added hormone

in medium containing within

up to 48 hr.

mouse liver

1, however, in the

for

liver

functions

culture.

and

Therefore,

insulin

was added

culture. in this

by electrophoresis

way was extracted on a cellulose

2. The band with

517

the highest

from the

acetate mobility

cells

membrane, toward

the

Vol. 91, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

cathode

anode

B

t origin Fig. 2. Electrophoretic identification of glucokinase in lrl-day-rat hepatocvtes in culture. The enzyme extract, urepared from hepatocytes cultured as shown in Fig. l-for 48 hr,'was concentrated in a small dialysis bag using Sephadex G-200. A sample of 3 ~1 of the concentrated enzyme extract was placed on the cathodeside of a cellulose acetate membrane. Bands of enzyme were stained with 0.5 mM(A) or 100 mM glucose(B).

anode was identified

as glucokinase,

with

but

100 mM glucose,

glucokinase entration with

by insulin of

The precocious

(data

not

alone

hepatocytes,

of conc-

activity

increased

insulin. was completely

inhibited

or actinomycin

D (0.3

glucokinase

in cultures

induce

but

it

Dexamethasone

I).

(11).

by insulin,

Induction

The induced

(10 -5M)

did not

may stimulate

previously

3).

be stained

ug/ml)

shown).

(Table

61, or it

could

a physiological

of glucokinase

of cycloheximide

rat

insulin

with

up to lo-*M

induction

Dexamethasone ll-day-old

(Fig.

insulin

it

0.5 mM glucose,

was detectable

10cgM insulin

the dose of

by addition

not with

because

induced

by insulin

mature

hepatocytes,

plus

activity

inhibited

as shown -in vivo

induction

(8).

Since

examined

518

in general

the

dexamethasone

we next

the

may have a permissive

cellular

Glucagon

enhanced

induction

of

by effect

(5,

as shown

of glucokinase

the enzyme activity

was only the effects

about

half

of other

that

of

Vol. 91, No. 2, 1979

BIOCHEMICAL

0bql 0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

’

10-g Insulin,

10-r M@

Fig. !. Dose-response curve for the induction of glucokinase by insulin in ll-day-old rat hepatocytes in culture. Culture conditions were as for Fig. 1. Values are means + S.D. for 4 experiments.

Table

I.

Effects of Various Hormones on Induction of Glucokinase in Primary Cultures of Hepatocytes from Adult and 14-Day-Old Rats Glucokinase

Hormones

(mU/mg protein)

ll-day-old hepatocytes

Adult hepatocytes

Dexamethasone(10-6M)

0.11

+ 0.07

7.28

+ 0.72

Insulin(10-7M)

2.66

+ 0.25

12.30

+ 0.95

4.27

+- 0.14

17.91

_+ 1.01

11.32

+ 1.34

8.78

f 1.21

15.14

+ 1.52

Dexamethasone insulin

and

Dexamethasone,insu and glucagon(l0

-3 M) in 1 . 1o +- o . 1o

Dexamethasone and testosterone(10-7M)

o*56

Dexamethasone,insulin and testosterone

8 . 2y --+ o 58

+_ o:22

Dexamethasone was present from the start of except in experiments on addition of insulin Ten hr later various hormones were added and kinase was assayed 48 hr after the start of Values are means t S.D. for 2-4 experiments.

519

culture, alone. glucoculture.

Vol. 91, No. 2, 1979

hormones

on its

asone

did

these

hormones

to the

not

precocious induce

induction.

the

highest

level

in

, .or enhanced

induction

not

shown).

counteract

Estrogen

the

interesting

effect

that

induction (Table

I).

The reason

liver

but

not

in

adult

activity

rat (0.1

did

pg/ml)

did

not

not

adult

is

(data enhance

hepatocytes

did

not

change

I). not

the not

to

Thyroxine induce

enzyme, shown).

in primary

It at all

or It

is

insulin-dependent

is effective

unknown.

insulin

and dexamethasone

induce

why testosterone liver

of

dexameth-

was comparable (Table

by insulin

did in

addition

liver

of testosterone

testosterone

of glucokinase

hexokinase

also

but

plus

induction,which

adult

hormone

RESEARCH COMMUNICATIONS

Testosterone

much,

and human growth

glucokinase

that

AND BIOPHYSICAL

glucokinase

caused

non-induced

(1O"'M)

(data

BIOCHEMICAL

should

culture

in neonatal be mentioned

in these

experim-

ents. REFERENCES 1. 2.

3.

4. 5. 6. 7. 8,

9. 10. 11. 12. 13. 14. 15.

Weinhouse,S.(1976) Curr. Top. Cell. Regul. 11, l-50. Pariza,M.W.,Yager,J.D.,Goldfarb,S.,Gurr,J.A.,Yanagi,S.,Grossmann,S.H., Becker,J.E. & Potter, V.R. (1975) in Gene Expression and Carcinogenesis in Cultured Liver (Gerschenson,L.E. & Thompson,E.B.,eds) pp 137-167, Academic Press, New York. Junge,U.,Nagamori,S.,Brunner,G. & Sbling,H.D.(1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (Tager,J.M.,Sdling,H.D. & Williams,J.R.,eds) pp 201-206,NorthHolland Publ, Co., Amsterdam. Nakamura,T.,Aoyama,K.,Rato,S.,Tomita,Y. & Ichihara,A. (1979) in Carcino-Embryonic Proteins (Lehmann,F.G.,ed) Vol. II, pp 723-728, North-Holland Publ. Co., Amsterdam. Schudt,C.(1979) Eur. J. Biochem. 98, 77-141. Katz,N.R.,Nauck,M.A. & Wilson,P.T.(1979) Biochem. Biophys. Res. Commun. 88, 23-29. Walker,D.G. & Holland,G. (1965) Biochem. J. 97, 845-854. Jamdar,S.C. & Greengard,O. (1970) J. Biol. Chem. 245, 2779-2783. & Freeman,C.(1972) Endocrinology 90, 1551-1560. Adelman,R.C. Partridge,N.C., Hoh,C.H.,Weaver,P.K. & Oliver,I.T.(1975) Eur. J. Biochem. 51, 49-54. & Ichihara,A.(1978) J. Biochem. 84, Tanaka,K..Sato,M.,Tomita,Y. 937-946. . . DiPietr0,D.L. & Weinhouse,S.(1960) J. Biol. Chem. 235,254; !-2545. Rosebrough,N.J.,Farr,A.L. & Randall,R.J.(1951) J. Lowry,O.H., Biol. Chem. 193, 265-275. & Sugimura,T.(1969) Cancer Res. 29, 1437Sato,S., Matsushima,T. 1446; Nakamura,T. & Kumegawa,M.(1973) Biochem. Biophys. Res. COnUnun. 51, 474-479.