Effect of chronic isoproterenol exposure on insulin binding and insulin-stimulated hexose transport in isolated rat adipocytes

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1093-1097

November 13, 1987

EFFECT OF CHRONIC ISOPROTERENOL EXPOSURE ON INSULIN BINDING AND INSULIN-STIMULATED HEXOSE TRANSPORT IN ISOLATED RAT ADIPOCYTES Alexander Sandra and Sandra J. Marshall University of Iowa, Department of Anatomy, Iowa City, IA

52242

Received September 15, 1987

SUMMARY: The effect of chronic exposure of isolated rat adipocytes t ° the ~-adrenergic agonist isoproterenol has been studied with respect to insulin binding and insulin-stimulated hexose uptake. Isoproterenol exposure led to a progressive decrease in both the number of surface insulin receptors and the stimulation of hexose uptake. The effect on insulin binding was reversible by removal of the ~-agonist within an hour of its addition. Later exposures of adipocytes to isoproterenol resulted in an irreversible cellular defect by leading to a progressive inability of the cells to regain their normal level of insulin-stimulated hexose uptake and insulin binding. ® 1987 Academic Press, Inc.

The effects of ~-adrenergic stimulation on isolated adipocytes results in a series of cellular alterations with respect to insulin action. include:

I) a marked decrease

These

in insulin binding to intact cells, an in-

direct effect mediated predominently by a reduction of the pH of the medium following ~-adrenergic stimulation of lipolysis and fatty acid release (I); 2) a decrease in insulin stimulated glucose uptake manifested at the post receptor

level

(2);

3)

an

alteration

in

the

disposition of the

insulin

receptor in the membrane as evidenced by a decreased sensitivity to trypsin (3); and 4)

a decrease of insulin receptor tyrosine

kinase activity (4).

All of these effects are apparent after brief (10-30 min) exposure of the cells to isoproterenol and, furthermore, are reversible upon removal of the ~-agonist.

In this report, the effect of chronic exposure of adipocytes to

isoproterenol was studied in a tissue culture system in order to assess the extent and reversibility of the ~-agonist effects under conditions of long term incubation. MATERIALS AND METHODS Adipocyte isolation and incubation: Rat adipocytes were isolated according to the basic procedure of Rodbell (5) by collagenase dissociation following aseptic removal of epididymal fat pads. The tissue was digested

Abbreviations: IlEPES, N-2-hydroxyethyl-piperazine-N'-2-ethane acid; KRH, Kreb's Ringer HEPES buffer; BSA, bovine serum albumin.

sulfonic

0006-291X/87 $1.50 1093

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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B I O C H E M I C AAND L BIOPHYSICAL RESEARCH COMMUNICATIONS

in sterile Krebs-Ringer-HEPES (I0 raM) buffer, pH 7.8 (KRH) containing 1% bovine serum albumin (BSA). Isolated cells were washed three times and cultured at 37°C in a water-saturated atmosphere of 5% CO 2 in air in Dulbecco's MEM containing I0 mM HEPES and I% BSA (6). Cultures were treated with isoproterenol (I0 ~M) for the times specified. To inhibit oxidation of the ~-agonist, superoxide dismustase and catalase (25 pg/ml each) were added to the culture medium (7). Control cultures were initiated with these agents, which had no effect on insulin binding or hexose uptake. Following isoproterenol treatment cells were centrifuged at low speed for 1 min over a cushion of silicone oil intermediate in density between the cells and medium. The cells were immediately resuspended in KRH-albumin buffer, pH 7.0 to remove any surface bound hormone and finally returned to KRH-albumin buffer, pH 7.8. Measurement of cell surface insulin binding: Adipocytes were incubated for I hr at 16°C with 0.2 ng/ml mono-A14-[aZ~l]-insulin alone or together with varying amount of native hormone. Non-specific binding was assessed by including a large excess (10 ~g/ml) of the native hormone and averaged 10-15% of total binding under these conditions. 2-Deoxyglucose uptake: Cultured adipocytes were washed three times and incubated for 45 min in the presence or absence of porcine insulin in KRB-albumin buffer saturated with a 95% 02 - 5% CO 2 gas mixture. The rate of 2-deoxyglucose uptake was determined by incubating 0.i ml aliquots of cells with 0.I mM 2-deoxy-D-glucose (2-deoxyglucose) containing 0.I pCi of 2-deoxy-D-[l-14C] glucose. Uptake was allowed to proceed for I min and terminated by the oil centrifugation method described above. The extracellular trapping and diffusion uptake of the labeled hexose was determined by the measurement of cell-associated radioactivity in the presence of I pg/ml cytochalasin B. RESULTS Isolated rat adipocytes have been shown to exhibit a 50% reduction in surface

insulin binding upon brief

terenol.

This

immediately placed

effect

could be

exposure to ~-agonists such as isopro-

reversed by washing

following its incubation with cells.

in tissue

culture

out the isoproterenol

When rat adipocytes were

for 24 hr and challenged with isoproterenol,

the

time of exposure of the cells to the ~-agonist was inversely related to the reversibility

of

this

isoproterenol

effect.

Complete

reversibility

evident in adipocytes one hour after removal of isoproterenol.

was

With longer

time intervals between ~-agonist addition and removal, the recovery of cell surface insulin binding was progressively diminished to the point where less than 20% of the original binding was detected after 5 hrs. (Fig. I). The

decrease

chronic

treatment

Scatchard.

in

adipocyte

with

cell

isoproterenol

surface was

insulin

analyzed

binding by

the

following method

of

Consistent with the progressive decrease in hormone binding, a

time-dependent decrease in the high affinity binding component was detected (Fig. 2).

The number of total insulin binding sites did not significantly

change by long term exposure of cells to isoproterenol. The biological terenol

was

response

determined

of adipocytes

by an analysis

1094

chronically

exposed to isopro-

of 2-deoxyglucose

uptake.

Control

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8O

L C 0

5O

©

2O

i

i

i

,

i

1

2

3

4

5

Time

incubated

ten-fold

in

the

stimulation

exhibited

Isoproterenol

i

24 (hr)

Effect of the length of exposure of cultured rat adipocytes to isoproterenol (I0 NM) on subsequent 12Sl-insulin cell surface binding after removal of isoproterenol. Isolated adipocytes were cultured for 24 hr and incubated with isoproterenol for the times indicated. Cells were immediately washed of the ~-agonist, incubated with 1251-insulin as described in Methods and analyzed for cell surface insulin binding. Data is expressed at each time point relative to insulin binding in control cells, not exposed to isoproterenol.

Fig. I.

cells

in

__//

a

absence

in h e x o s e

progressive

of

the

uptake.

decrease

in

~-agonist Cells

showed

incubated

insulin

approximately

with

responsiveness

isoproterenol with

time

2.5

0 v-

X

•

1.5

Q) h

\

"IJ c"

0 m

•

0.5

Bound

Fig. 2.

i

i

1 O0

200

(fmol/10

i

300

5 cells)

Scatchard analysis of 12Sl-insulin cell surface binding to cultured adipocytes. (A) control cells cultured for 24 hr; (0) isoproterenol-treated cells cultured for 24 hr continuously with isoproterenol; (1) isoproterenol-treated cells cultured for 48 hr continuously with the ~-agonist. The control Scatchard plot for cells cultured for 48 hr was similar to the 24 hr plot.

1095

a

in

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/"

2{ 0 O_

D ~100 --

0

o

1\5o >, x Qc3

I

1

l

50

l

100

I

150

Insulin

Fig. 3.

culture. small

1

200

ff

1

250

1000

(uU/ml)

The biological response of cultured rat adipocytes incubated in the presence or absence (A) of isoproterenol as a function of time in culture. Cultures were treated continuously with the ~-agonist for 24 hr (e) or 48 hr (m) and assessed for insulin stimulated 2-deoxyglucose uptake as described in Methods.

At 24 hr only

degree

a two-fold stimulation

of stimulation

over

48 hr exposure to the ~-agonist

the basal

could be detected

level

and a very

could be detected after a

(Fig. 3).

DISCUSSION The

acute

isolated

effects

rat adipocytes

be due to a series

of

~-agonists

insulin

have been previously

binding

reported

effect

of ~-adrenergic

fication

of the medium.

buffered

and

Even

allowed

stimulation

of

the

receptor

(1,8,9)

on insulin binding

response and

or a change interaction

in

shown to The major

is an acidi-

in the case where the pH of the medium is well

to

fluctuate

insulin

binding

presumably because of such factors as an alteration

hormone-receptor

and

of both indirect and possibly direct effects.

indirect

not

on

is

still

in allosteric

in the stereostructure

reduced,

regulation

of the receptor.

That

itself may lead to a change in the conformation

of the insulin receptor is supported by the study of Pilch and Czech (I0) in which

the

occupied

trypsin proteolysis The ability incubation

was

tially useful resistance.

insulin

to study

intially model Under

receptor

demonstrated

than the unoccupied adipocytes

validated

for studying appropriate

greater

under conditions

by Marshall the chronic conditions,

levels of insulin binding and response

a

sesitivity

to

receptor. other than short term

(11) and represents aspects fat

cells

for several days.

1096

a poten-

of peripheral retain

near

However,

insulin normal

with time

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in culture,

insulin itself has been shown to induce a decrease in insulin

sensitivity

followed

by a reduction

in insulin

responsiveness

(12).

This

receptor down regulation appears to be a longer term regulatory mechansism of insulin resistance. cultured

adipocytes

receptor sites.

Insulin induced a progressive insulin resistance in

which

is sequentially manifested

at receptor and post

The effect of the ~-agonist noradrenaline on insulin bind-

ing to adipocytes

cultured

for 24 hr has

also been reported

(13). Again,

insulin binding is significantly reduced, the effect is completely blocked by B-antagonists and cannot be accounted for by trivial explanations such as ATP depletion. With glucose this

respect

transport,

functional

effect

to

the

effects

of

isoproterenol

on

insulin-stimulated

several studies have demonstrated a marked reduction of

parameter

on insulin binding.

in isolated This

adipocytes

effect was

(1,2)

lost upon

independent removal

of an

of the

6-

agonist unless the cells were treated with KCN immediately after incubation with

isoproterenol.

adipocyte dependent.

glucose

Therefore, transporter

changes brought

in the about

intrinsic by

activity

~-agonists

are

of the energy

Such an energy dependency could manifest itself in a variety of

ways including direct covalent modification or an indirect alteration of a regulatory protein(s).

ACKNOWLEDGEMENTS This

research

was

supported

by

NIH

grant

AM25295

and

the

American

Diabetes Association, Iowa Affiliate.

REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. i0. 11. 12. 13.

Arsenis, G. and Livingston, J.N. (1986) Endocrinology 119:50-57. Joost, H.G., Weber, T.M., Cushman, S.W. and Simpson, I.A. (1986) J. Biol. Chem. 261:10033-10036. Sandra, A. and Marshall, S.J. (1987) submitted. Haring, H., Kitsch, D., Obermaier, B., Ermel, B., and Machicao, F. (1986) Biochem. J. 234:59-66. Rodbell, M. (1964) J. Biol. Chem. 239:375-380. Marshall, S., Garvey, W.T. and Geller, M. (1984) J. Biol. Chem. 259: 6376-6384. Mahan, L.C. and Insel, P.A. (1984) Anal. Biochem. 136:208-216. Pessin, J.E., Gitomer, W., Oka, Y., Oppenheimer, C.L. and Czech, M.P. (1983) J. Biol. Chem. 258:7386-7394. Kitsch, D.M., Baumgarten, M., Dewfel, T., Rinninger, F., Kemmler, W., and Haring, H.V. (1983) Biochem. J. 216:737-745. Pilch, P.F. and Czech, M.P. (1980) Science 210:1152-1153. Marshall, S. (1983) Diabetes 32:319-325. Garvey, W.T., Olefsky, JiM. and Marshall, S. (1986) Diabetes 35:258267. Lonnroth, P. and Smith, V. (1983) Biochem. Biophys. Res. Comm. 112: 972-979.

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