Effect of insulin on glucagon binding to isolated rat epididymal adipocytes

Vol. 141, No. 2 , 1 9 8 6

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 8] 2-817

December15,1986

E F F E C T OF INSULIN ON G L U C A G O N B I N D I N G TO ISOLATED RAT E P I D I D Y M A L A D I P O C Y T E S Kiyoshi

HASHIZUME

and

Keishi

Y~4AUCHI

D e p a r t m e n t of Gerontology, E n d o c r i n o l o g y and Metabolism, Shinshu U n i v e r s i t y School of Medicine, Matsumoto, 390 JAPAN

Received October 31, 1986

Effect of insulin on g l u c a g o n b i n d i n g to rat e p i d i d y m a l adipocytes was studied in vitro. [ 1 2 5 I ] i o d o g l u c a g o n b i n d i n g to isolated adipocytes was increased by p r e i n c u b a t i o n of the cells w i t h insulin. M a x i m a l increase was o b s e r v e d w i t h 7 x l 0 - 1 0 M insulin. In S c a t c h a r d analysis, [ 1 2 5 I ] i o d o g l u c a g o n c o m p e t i t i o n data g e n e r a t e d one b i n d i n g site with a single a f f i n i t y for g l u c a g o n b i n d i n g in the cells p r e t r e a t e d with buffer alone. Pretreatm e n t of the cells w i t h insulin i n c r e a s e d the a f f i n i t y w i t h o u t changes in the n u m b e r of binding sites. [ 1 2 5 I ] i o d o g l u c a g o n binding to isolated adipocytes was not affected by p r e t r e a t m e n t of the cells w i t h l u t e i n i z i n g hormone, f o l l i c l e - s t i m u l a t i n g hormone, growth hormone, or with prolactin. These results suggest that insulin stimulates glucagon b i n d i n g to adipocytes.©i986Aoademie Press, Inc.

Glucagon 2,3)

is known

and to plasma

cagon

stimulates

and raises

to be bound

membranes

prepared

the adenylate

the c o n c e n t r a t i o n

specifically

of cyclic

that

pocytes

by p r e t r e a t m e n t

(7),

is inhibited

and we p o s t u l a t e d

ration

of cyclic

gon inhibition modification

insulin

is not certain,

thyrotropin(TSH)-

thyroid

binding

induces

0006-291 X/86 $1.50 Copy~ht © 1986 @ Aca~mk P ~ , Inc. All r~h~ of r~roduction in a ~ .~rm reserved.

structural

with glucagon

mechanism

kinase.

AMP-stimulated

rat adi-

is m e d i a t e d

to the

is induced

membranes,

812

Previously,

of the cells

which

by gene-

of glucastructural

by activa-

In in vitro

we have

shown

phosphorylation

changes

Glu-

ghosts(5),

to i s o l a t e d

it m a y relate

protein

plasma

or cyclic

membranes

~4P(6,7).

the p r e c i s e

membranes,

~4P-dependent

dies w i t h bovine

of fat cell

that the inhibition Although

of plasma

tion of cyclic

the plasma

~IP.

from adipocytes(4).

cyclase

we have d e m o n s t r a t e d

to adipocytes(1,

stuthat of

of the m e m b r a n e

Vol. 141, No. 2, 1986

(8,9

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and that results

of TSH binding(10). sphorylation tions,

hormones. can m o d i f y

Insulin

predict

the changes

In this

study,

glucagon

of the c h a r a c t e r i s t i c s

is also known

of its r e c e p t o r ( l l , 1 2 ) .

one m i g h t

may g e n e r a t e

in an a l t e r a t i o n

that

insulin

Based

to s t i m u l a t e on these

binding

binding

to examine

to isolated

MATERIALS

observa-

to its r e c e p t o r

in the c h a r a c t e r i s t i c s we aimed

the pho-

of b i n d i n g

whether

of

insulin

rat adipocytes

in vitro.

AND METHODS

[125I]iodoglucagon(porcine, r e c e p t o r grade) (2200 Ci/mmol) and [125I]iodoinsulin(porcine, r e c e p t o r grade) (2200 Ci/mmol) w e r e p u r c h a s e d from New E n g l a n d N u c l e a r ( B o s t o n , M A ) . Insulin(bovine) (24 IU/mg) and g l u c a g o n ( e x t r a c t e d from a m i x t u r e of bovine and p o r c i n e pancreas) were p u r c h a s e d from Eli Lilly C o . ( I n d i a n a p o lis,IN) and from Sigma C h e m i c a l Co. (St. Louis,MO) r e s p e c t i v e l y . These peptides were p u r i f i e d by Sephacryl S - 2 0 0 ( P h a r m a c i a Fine C h e m i c a l Inc., Piscataway,NJ) column c h r o m a t o g r a p h y b e f o r e use. L u t e i n i z i n g hormone(human) (LH), f o l l i c l e - s t i m u l a t i n g hormone (human) (FSH) and prolactin(PRL) were all o b t a i n e d from Sigma. Growth hormone(human) (GH) was o b t a i n e d from S u m i t o m o P h a r m a c e u tical I n c . ( T o k y o , J a p a n ) . Collagenase(Clostridium histolyticum) was o b t a i n e d from W o r t h i n g t o n B i o c h e m i c a l Co. (Freehold,NJ). Dinonyl p h t h a l a t e was o b t a i n e d from Nakarai C h e m i c a l Co.(Tokyo, Japan). Isolated adipocytes were p r e p a r e d from whole e p i d i d y m a l fat pads of 130-g male W i s t a r r a t s ( S h i z u o k a E x p e r i m e n t a l A n i m a l s Co., Shizuoka, Japan) by c o l l a g e n a s e d i g e s t i o n a c c o r d i n g to the method of Rodbell(13). Before e v a l u a t i o n of g l u c a g o n b i n d i n g a c t i v i t y in adipocytes, the cells were p r e t r e a t e d w i t h insulin or other hormones. The incubation was p e r f o r m e d in a final volume of 500 91 K r e b s - R i n g e r - p h o s p h a t e buffer pH 7.6, c o n t a i n i n g 3% bovine serum albumin (BSA) and a p p r o x i m a t e l y l. Sx106 cells for 20 min. at 37°C. After washing, the cells were t r a n s f e r r e d to the fresh tubes which contained g l u c a g o n binding assay medium. [125I]iodoinsulin b i n d i n g was e s t i m a t e d as p r e v i o u s l y d e s c r i b e d (7). [ 1 2 5 I ] i o d o g l u c a g o n b i n d i n g was p e r f o r m e d in a final volume of 500 ~i K r e b s - R i n g e r - p h o s p h a t e buffer, pH 7.6, c o n t a i n i n g 3% BSA and a p p r o x i m a t e l y 1.0xl06 cells which were p r e v i o u s l y treated with insulin or buffer. The b i n d i n g assay was done at 17°C for 30 min. in the p r e s e n c e of 8x104 cpm r a d i o a c t i v e glucagon. After incubation, 300 ~i aliquots were t r a n s f e r r e d to a m i c r o c e n t r i f u g e t u b e ( B o e c k m a n Instruments, F u l l e r t o n , C A ) . The m i x t u r e was o v e r l a y e d by i00 ~i dinonyl phthalate. The cells were r e c o v e r e d by c e n t r i f u g a t i o n at i0,000 x g for 30 sec. The r a d i o a c t i v i t y in the cell pack s e p a r a t e d from the incubation m e d i u m was m e a s u r e d by A u t o - G a m m a s p e c t r o m e t e r ( P a c kard Instrument Co., Downers G r o v e , I L ; e f f i c i e n c y = 7 4 % ) . Nonspecific binding was m e a s u r e d w i t h 5.0 ~ M u n l a b e l e d g l u c a g o n in the binding medium. P r o t e i n was m e a s u r e d by the m e t h o d of Lowry et al. (14), w i t h BSA as the standard. S t a t i s t i c a l analysis of the s i g n i f i c a n c e b e t w e e n groups was done by means of S t u d e n t ' s t test. A P value less than 0.05 was c o n s i d e r e d s t a t i s t i c a l l y significant.

813

Vol. 1 4 1 , No. 2, 1 9 8 6

B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S

RESULTS

The

adipocytes

cific

binding

lated

adipocytes

sites

concentrations cells

were

prepared for

were

of

increased

the

with

increase

7xl0-10M

10-9M

to f r e s h m e d i u m

Pretreatment

concentration-dependent

manner,

or m o r e The

ding

observed

by 15 min.

of v a r i o u s

binding

binding

shown was

increase

binding

was

ob-

of the c e l l s increase

of g l u c a g o n

glucagon

of binThe

on

or i n s u l i n - t r e a t e d

×

x

in a

or m o r e ( F i g . 2 ) .

of u n l a b e l e d

to b u f f e r -

in F i g . l ,

observed

further

preincubation

radio-

significantly

pretreatment

insulin-induced

of v a r i o u s

contained

insulin As

spe-

The p r e t r e a t e d

and the m a x i m a l

concentrations

[125I]iodoglucagon

which

did not produce

binding.

effect

37°C.

binding.

However,

glucagon was

with

of g l u c a g o n

insulin.

insulin

at

had

reported(7) . Iso-

in the p r e s e n c e

20 min.

[125I]iodoglucagon

insulin-induced

by

for

digestion

as p r e v i o u s l y

preincubated

transferred glucagon.

tained

insulin

insulin

iodinated

by c o l l a g e n a s e

cells

2

a z

2

z O

1

(D

t)

3 0

J

i

~

(D

0

L4I

,'~.

I

I

,'~16 169 1~;8

®

INSULIN (M)

o

lo

20

TIME IN MINUTES

Fig.l. Effect of insulin on [125I]iodoglucagon binding to isolated adipocytes. Prepared adipocytes were preincubated with various concentrations of insulin for 20 min. at 37°C. After incubation, the cells(ixl06 cells) were incubated with radioactive glucagon(Sxl04 cpm). The binding assay was performed at 17°C for 30 min. Points and vertical brackets indicate mean ± SEM of five determinations. Fig.2. Effect of insulin on [125I]iodoglucagon binding to isolated adipocytes. The prepared cells were preincubated with buffer(e) or insulin(o) (Ix~Q~9M) for various times at 37°C. After preincubation, [±2bI]iodoglucagon binding was assessed as described in Materials and Methods. Points and vertical brackets indicate mean ± SE~I of five determinations.

814

V01. 1 4 1 , No. 2, 1 9 8 6

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

•

/I

.

QO~

\ × F u

,,=,

E3 Z

n-

=o z o o<[

°

2

U-

Z

1

0.01

3 O r

0 • 1 11690 1 168 0

®


167

GLUCAGUN ADDED (M)

0

I

2

3

4

5

10

GLUCAGON BOUND{M x '1(~11)

Fig.3. Effect of various concentrations of unlabeled glucagon on [125I]iodoglucagon binding to isolated rat adi~ocytes. The cells were pretreated with buffer(O) or Ixl0- M insulin(o) for 20 min. at 37°C. After pretreatment [125I]iodoglucagon binding was determined in the presence of various concentrations of unlabeled glucagon. Points and vertical brackets indicate mean ± SEM of four determinations. Fig.4. Scatchard analysis of glucagon binding to buffer-treated (Q) and insulin-treated(o) adipocytes. Each point is calculated from the results obtained in Fig.3.

is shown in Fig.3. iodoglucagon

binding

the cells w i t h gon

without

As shown

b i n d i n g was

lin, p r e t r e a t m e n t duce

an i n c r e a s e

in Table

glucagon

analysis,

in the cells

pretreated insulin

in the n u m b e r

by p r e t r e a t m e n t PRL,

a single w i t h buf-

increased

of b i n d i n g

the

sites

125 [ I]iododglucagon

of the cells w i t h

insu-

or w i t h GH did not pro-

[125I]iodo-glucagon

binding.

DISCUSSION In this r e p o r t we p r o v i d e d glucagon

binding

to i s o l a t e d

evidence

that

rat a d i p o c y t e s .

815

of

[125I]iodogluca-

site w i t h

i, a l t h o u g h

[125I]-

by p r e i n c u b a t i o n

one b i n d i n g

w i t h LH, FSH, of

increased

of the cells w i t h

changes

increased

of u n l a b e l e d

In S c a t c h a r d

binding

Pretreatment

the a f f i n i t y

clearly

data g e n e r a t e d

for g l u c a g o n

fer alone.

(Fig.4).

was

insulin.

competition

affinity

In the a b s e n c e

insulin

enhances

Previously,

we

Vol. 141,No. 2 , 1 9 8 6

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table i. Effect of peptide hormones on [125I]iodoglucagon binding to isolated rat adipocytes Groups

Pretreatment of cells

Determi[125I]iodoglucagon nations binding(cpm/106 cells)

P values --

A.

Buffer

5

1260 ~

B.

Insulin(10-9M)

5

2240 ~ 160

A and B~0.05

C.

LH (i00 mU/ml)

5

1340 ~ 140

A and C:N.S.

D.

FSH (i00 mU/ml )

5

1090 ~

60

A and D:N.S.

E. F.

PRL(100 ng/ml) GH (10-9M)

5 5

1080 ~ 140

A and E:N.S.

1250 ~ 140

A and F:N.S.

80

The prepared cells were preincubated with peptides for 20 min. at 37°C. After incubation the [125I]iodoglucagon binding was assessed as described in Materials and 24ethods. Each value indicates mean ± SEM. N.S. indicates not significant.

have d e m o n s t r a t e d

that p r e t r e a t m e n t

inhibits

the i n s u l i n

binding,

hibition

is m e d i a t e d

by g e n e r a t i o n

the c o n s i d e r a t i o n , of g l u c a g o n

insulin

to its r e c e p t o r

lin binding,

which

indicates

to its r e c e p t o r

ment

of g l u c a g o n

gesting ding

insulin

was

indicated

closely

mechanism

increased

regulation

mechanisms

by p r e t r e a t m e n t

to the

receptor

structure

of the c h a n g e s

of insubin-

the e n h a n c e -

preincubated

with

than

sug-

10-9M),

of insulin bin-

constant

of

for g l u -

of the cells w i t h function,

of receptor,

to its receptor.

the of

of r e g u l a t i o n

that a s s o c i a t i o n

that the g l u c a g o n

binding

We o b s e r v e d

of i n s u l i n ( l e s s

finding

be r e l a t e d

fied by i n s u l i n

binding

The

binding

of g l u c a g o n

the cells were

is one of the p h y s i o l o g i c a l

Based on

increases

an a u t o r e g u l a t i o n

in the m e m b r a n e .

concentrations

~4P(7).

that i n s u l i n

by i n c r e a s e

that the g l u c a g o n - m e d i a t e d

cagon b i n d i n g

may

introduces

binding when

i n s u l i n binding.

of cyclic

that the in-

that the p r e c e d i n g

is m e d i a t e d

ding

physiological

and we p o s t u l a t e d

the o b s e r v a t i o n

binding

of the cells w i t h g l u c a g o n

However,

in the c h a r a c t e r i s t i c s

which

is m o d i -

the p r e c i s e

of g l u c a g o n

is not certain.

A l t h o u g h we did not stimulation

of g l u c a g o n

show the d i r e c t binding

evidences

and g l u c a g o n

816

for insulin

inhibition

of in-

Vol. 141, No. 2, 1986

sulin b i n d i n g munication patients

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in yiv__0o, the o b s e r v a t i o n s

m a y relate

to the e t i o l o g y

reported of insulin

in this

com-

resistance

in

with hyperglucagonemia(15). REFERENCES

1 2 3 4 5 6 7. 8. 9. I0. ii. 12. 13. 14. 15.

Sonne, O., Glieman, J. and Gammeltoff, S. (1973) A c t a Endocrinol. (Copenhagen) Supple. 17, 274 Livingston, J.N., Cuatrecasas, P. and Lockwood, D.H. (1974) Lipid Res. 15, 26-32 Sonne, O. and Glieman, J. (1977) Biochim. Biophys. Acta 499, 259-272 Desbuquois, B. and Laudat, M.H. (1974) Mol. Cell. E n d o c r i nol. i, 355-370 Harwood, J.P., L~w, H. and Rodbell, M. (1973) J. Biol. Chem. 248, 6239-6245 Butcher, R.W., Barid, C.E. and Sutherland, E.W. (1968) J. Biol. Chem. 243, 1705-1712 Yamauchi, K. and Hashizume, K. (1986) E n d o c r i n o l o g y 119, 218-223 IIashizume, K. and DeGroot, L.J. (1980) E n d o c r i n o l o g y 106, 1463-1468 IIashizume, K. and DeGroot, L.J. (1985) Endocrinol. Japon. 32, 569-575 Hashizume, K., Yamauchi, K., Miyamoto, T., Ichikawa, K. and Kobayashi, M. (1986) Endocrinol. Japon. 33, 81-88 Kasuga, M., Zick, Y., Blithe, D.L., Crettaz, M. and Kahn, C.R. (1982) Nature 298, 667-669 Kohanski, R.A., Frost, S.C. and Lane, D. (1986) J. Biol. Chem. 261, 12272-12281 Rodbell, M. (1964) J. Biol. Chem. 239, 375-380 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Unger, R.H. and Orci, L. (1979) in E N D O C R I N O L O G Y , e d i t e d by DeGroot, L.J., Odell, W.D., Martini, L., Potts, J.T.Jr., Nelson, D.H., Steinberger, E. and Wine grad, A.I. Grune & Stratton, N e w York, pages 959-980

817