Effects of colchicine on insulin binding to isolated rat hepatocytes

Effects of colchicine on insulin binding to isolated rat hepatocytes

Vol.103,No.3,1981 December BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1100-1106 15, 1981 EFFECTS OF COLCHICINE ON INSULIN BINDI...

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Vol.103,No.3,1981 December

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1100-1106

15, 1981

EFFECTS OF COLCHICINE ON INSULIN

BINDING

TO ISOLATED RAT BEPATOCYTES J. Whittaker,

V.A.

Hammond, and K.G.M.M.

Alberti

Department of Clinical Biochemistry and Metabolic University of Newcastle, Newcastle upon Tyne, Received

October

Medicine, U.K.

18,1981 SUMMAKY

The effects of colchicine, an inhibitor of microtubule polymerization, on the maintenance of steady_Ttate binding of insulin to isolated hepatocytes was studied. Colchicine (10 M) produced a 35-45X decrease in binding in presence of insulin (10e8 M) at 370C. This decrease in binding was time and temperature dependent. The decrease was also dependent on the amount of insulin bound to the cell. The results suggest that colchicine may prevent the maintenance of steady state binding of insulin by impairing transfer of newly synthesized or recycled receptor from within the cell to the plasma membrane. INTRODUCTION It

is now established

internalized

hormone

receptor

complex

state

binding

in

insulin

can be maintained

for

surface

receptors

probable known

known

that that

proteins

the whole

steady

about are

transported

and microfilament

in

of colchicine the role

There

of

in

is

(4).

shown

isolated

to the

the microtubule

cell

Colchicine,

to have marked

binding system

(5).

Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

1100

is

Thus cell

very that

during

little

is

plasma

membrane

the process

vesicles

being

by the microtubule

an inhibitor

of tubulin effects

therefore

hepatocytes turnover

also of

surface

We have

in the

(2).

secretory

inhibitory

in isolated

0006-291X/81/231100-07$01.00/0

suggested

for

It

is

concentrations

cells

vesicles

target

it

the cell.

At present

has been

of secretory

hepatocytes

on insulin

some target

now much evidence

systems

pinocytosis,

of high

replaced. It

the cytoplasm

,has been

processes

h

achieved.

from those

through

merization,

is

derived (3).

l-2

by many of its

enters

the presence

must be continually

how this

exocytosis

probably

is

by adsorptive

(1).

is

insulin achieved

cells

As this

that

poly-

on secretory examined

effects

to evaluate

of insulin

receptors.

of

BIOCHEMICAL

Vol. 103, No. 3,198l

AND

MATERIALS

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND METHODS

Hepatocytes were isolated from fed 150-180 g male Wistar rats by the All incubations were carried out in Krebs Ringer method of Le Cam (6). Hepes buffer, pH 7.4 (7), containing 30 mg/ml BSA and gentamycin 50 pg/ml. Immediately after isolation, prior to all experimental procedures cells were pre-incubated in buffer alone for 30 min at 37'C. Cell viability as determined by trypan blue exclusion was greater than 92% and did not change throughout the experimental procedures. Binding studies were performed at 20°C. Cells (0.5 to 1 x 106/m1) were incubated in Krebs Ringer Hepes 30 g/l BSA; pHl&4, containing gentamycin 50 ug/ml and bacitracin 0.8 mg/ml with insulin and I-tyr Al4 insulin in a total volume of 0.5 ml for 1 h. Incubations were terminated by adding 400 pl aliquots of cells to 3.5 ml ice cold Krebs Ringer Bicarbonate, pH 7.4, buffer and centrifuging. The supernatant was aspirated and the pellet was washed once more with Krebs Ringer Bicarbonate buffer. Details of individual experiments are given in the text and legends to figures. Hepatocytes (20 x 10s6) were solubilized by stirring for 1 h in 5 ml of O.lM phosphate b uffer, pH 7.4, containing triton X-100 1% (v/v) phenylmethanesulphonylfluoride 1 mol, aprotinin 4000 k.i.u./ml and bacitracin 1 mg/ml, and then centrifuged at 100,000 g for 1 h at 4OC. Insulin binding to the supernatant was determined by the method of Harrison (8). Collagenase (P-L Biochemicals) was purchased from International Enzymes Ltd, U.K., Centamycin, bacitracin, triton X-100 and Hepes from Sigma and colchicine was from Aldrich. Porcine monocomponent insulin (Actrapid) and 1251 tyr Al4 insulin (Sp. Act. 270 pCi/ pg) were gifts from Dr L Heding and Dr K Jorgensen respectively of the Novo Research Insitute, Copenhagen, Denmark. RESULTS When hepatocytes of 125 I tyr

binding 0.05%/106

cells

incubated

with

60 min to 0.68 unchanged. lo-+

10

insulin

effect

was observed 1).

vs 1.39 -+ 0.06%/106 1 shows the

of 10B5M colchicine

time

cells:

n = 4).

course

of the

at 37'C.

However,

cells:

initial

binding

at was

concentrations were was not n = 4). (1.37

decrease

in binding

a maximum of 42% at 60 min.

when

had decreased

of insulin

A 20% decrease

1101

n = 4).

incubations

in binding

+ 0.06%/106

in the absence

(0 min 1.38 -+

colchicine

when all

decrease

vs 1.44

had no effect

with

constant

binding

(n = 4) although

However induced

cells

cells:

in addition,

cells

10 -8 M insulin,

1 h at 37'C with

(100 PM) remained

M colchicine

colchicine

attained

for

2 0.04%/106

+ 0.09%/106

Fig

which

-5

to 10m4M (Table

colchicine cells

Al4

incubated

vs 60 min 1.38 + 0.01%/106

This

at 20°C the (1.42

were

in binding

carried

from out

observed In addition

+- 0.05%/106

in the presence

was observed

at 5 min

Vol. 103, No. 3,198l

8lOCHEMlCAL

AND

BIOPHYSICAL

Table Concentration

effects

Colchicine

of

RESEARCH COMMUNICATIONS

1

Colchicine

on Insulin

Concentration

Binding

X Initial Binding (mean + SD, n = 4)

Of) lo-’

95.2

)

5 x lo-’

83.2

+ 3.4

10-6

66.7

+

1o-5

67.0

+ 5.6

Hepatocyte

(2 x 106/m1)

indicated

binding

insulin

(lOsaM)

of

colchicine

for

concentrations Cells

37Oc.

with

of

were

then

insulin

(10

separated -a

M)

from 125

and

was measured in the presence

the

60 min

at

and

Al4

of colchicine

1.6

and

medium I-tyr

6.0

insulin

as described

in the text.

Fig

shows

can be seen

trations

studied.

binding

competitive

10 -8 M insulin

with It

2A

at 37'C that

in

the presence

colchicine Scatchard

OL,

0

studies

causes plots

I 10

(9)

I 20

after or absence

a decrease of this

1 30 Time (min)

pre-incubation

of 10

-5 M colchicine.

in binding data

I 40

a 1 hour

(Fig

I 50

at all

2B) are

concen-

parallel,

1 50

Figure 1. Time course of the effect of cobchicine on insulin binding to were incubated at hegatocytes. Isolate_dahepatocytes (2 x 10 ce Is/ml) f M). At the indicated 37 C with insulin (10 M) and colchicine (lotime points cells were separated from medium b centrifugation at 50 g with insulin at a concentration of 0.5 x 10 x cells/ml I tyr A 4 insulin (100 PM). Binding studies were then carried out as describe a in “methods”.

1102

BIOCHEMICAL

Vol. 103, No. 3,198l

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

% 12s l-insulin bound (100 cells/l) 0.05

5r

lo”0

10-Q

10-a

r

1 2 Insulin bound fmol x 1O’O)

1o-7

Insulin (mol/l)

Figure 2A. Equilibrium binding studies aftgr preincubation of hepatocytes with insulin and colchicine. Cells (2 x 10 /ml) were incubated with insulin (10e8 M) at 37'C for 1 h in the presence or absence of colchicine (1O-5 M). Cells were then separated from the medium by centrifugation at 50 E and then resuspended for 30 min at 220C in Krebs Ringer Hepe3 3% BSA, pH 7.4, in the presence (-o-) or absence (-*-) of colchicine (10 M). The cells were then separated by centrifugation and the procedure repeated 4 times. Binding studies were then performed by incubating cells for 90 min at 10°C in the presence and absence of colchicine (lo-5 M) at the indicated concentrations of insulin with 1251 tyr Al4 insulin (33 PM). The results represent the mean and standard deviation of 6 separate cell preparations. Scatchard Analysis. Colchicine (-o-).

%%%$s-)

suggesting

that

the decrease

Scatchard

in binding

plot

is

of data from Fig 2A.

due to a decrease

in receptor

concentration.

which

Competitive

binding

had been

incubated

or absence

studies for

could

colchicine

in binding

were

and varying

appeared

surface

(see Fig carried

to be dependent

confirmed

can be maintained

out

on the

and a half

10 -8 M insulin

in

the presence

1103

hepatocytes

in the

difference

presence

in receptor

in the presence of insulin

maximal

of high

or absence

(Fig

concentration

DISCUSSION receptor concentrations

that

on solubilized

3).

concentrations

maximum of 47% at 10m8M insulin

We have

performed

No significant

be detected

When pre-incubations

also

1 h at 37'C with

of 10 -5 M colchicine.

concentration

lo-'M

were

effect

4).

of

The decrease

of insulin

with

at lo-'M

insulin.

on the hepatocyte concentrations

of insulin

a

Vol. 103, No. 3,1981

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

04 to solubilized hepatocytes. Figure 3. Effect of colchicine on insulin l$nding M) in the presence (-o-) and Repatocytes were incubated with insulin (10 absence (-*-> of colchicine and then washed as described in the legend to Fig 2A. The cells were then solubilized and insulin binding te the solubilized cells was determined as described in “Methods”. The results are presented as Scatchard plots. Each point represents the mean of six separate hepatocyte preparations. Figure 4. Dose response hepatocytes in the presence studies were carried out the legent to Fig 1 except concentration of 50 pM. insulin 10m8 M. Results in binding.

for

up to 60 tin

can cause these

(1).

a marked

In addition

reduction

conditions.

This

that

this

receptors

therefore

propose

receptors

in the plasma

of van Obberghen on insulin

binding

in cell

surface

on the amount is

that

colchicine

to cultured

bound

These

who found

that

the results

did not

of

the cell.

replacement

of insulin

are in contrast was without However

occur

to the cell,

within

colchicine

human lymphocytes.

1104

under

on the internalization

of receptors impairs

membrane.

et al(lO),

of insulin

colchicine

concentration

concentration

dependent

and redistribution

that

receptor

in receptor

phenomenon

occupied

we have demonstrated

decrease

at 20°C and was dependent suggesting

effect of insulin on insulin binding to Pre-incubations and binding of colchicine. with the indicated concentrations of insulin in 125 I tyr that Al4 insulin was present in a Maximal decrease in binding (47X) occurred with are therefore expressed as % maximal decrease

these

We

to those effect workers

BIOCHEMICAL

Vol. 103, No. 3,198l only

examined

under

the effects

conditions

for

plasma

secretory

of transfer

membrane.

impairs

the

membrane.

cyte

membrane

transfer

molecules

164% of cell

surface

present

study,

of colchicine.

replaced

by denovo as complete

results

pool

(2).

internalization

receptorslhr

in

protein

the plasma receptor

This

finding

suggests

the cell,

35-472

of replacement studies

for

at 30°C insulin

a turnover

We acknowledge Health Authority

was observed

Gorden, P., Diabetologia

are

to the membrane

by another

by cyclohexiside

only

under

these

experimental

that

there

may be a pre-formed

receptors

for

are

recycled

the asialoglycoprotein

the LDL receptor

the British Diabetic Association for financial support.

Carpentier, J-L, 18, 263-274.

Freychet,

1105

P.,

In

in the

receptors

in fibroblasts

and the Newcastle

REFERENCES 1.

of

(2).

ACKNOWLEDGEMENTS Area

of

between

the remaining

or internalized demonstrated

was

secretion.

method

synthesis

binding

suggested

effect

This

of lOsaM insulin

and exported

insulin

as has been

and also

that

of protein in

membrane.

of degradation

of only

of hepatothe

has calculated

unlikely

synthesis

latter

within

membrane (12)

is

sole

the presence

a decrease It

the

to the

shown to retard

a one to one stoichiometry

during

suppression

of receptors

is

to the

synthesis

of glycoprotein

on the basis

assuming

in a 10% decrease

conditions

to the cell

effect this

Terris

however,

presence

route

to its

hepatocytes,

and receptor

Golgi

the

colchicine

apparatus

of the

has been

being

apparatus

manner

the Golgi study

(5),

Golgi

in an analogous

in vivo

from the

that

i.e.

shown to be essential

hepatocyte

from the

colchicine

however

COMMUNICATIONS

of insulin,

has been

receptor..from

glycoproteins(l1)

receptors.

isolated

vesicles

in a recent

proportional

internalized

system

that

of insulin

is unlikely

the absence

of the isolated

is possible

of glycoprotien

It

the

function

Indeed,

quantitatively

in

It

RESEARCH

turnover.

of secretory

transfer

cell

receptor

in

of the microtubular

the normal

mechanism

BIOPHYSICAL

of colchicine

of basal

The integrity

AND

and Orci,

L.

(1960)

(13).

to

Vol. 103, No. 3,198l

2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13.

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

Terris, S., and Steiner, D.F. (1980) in "Insulin; Chemistry, Structure and Function of Insulin and Related Hormones". (D. Brandenburg and A. Wollmer. Eds) 277-284. Walter De Gruyter, Borling, New York. Palade, G.E. (1975) Science 175, 347-358. Malaisse, W.J., Malaisse-Lage, F., Walker, M.O., and Lacey, P.E. (1971) Diabetes 20, 257-265. Prentki,M., Crettaz, M., and Jeanrenaud, B. (1980) Biochim. Biophys. Acta 627, 262-269. Le Cam, A., Guillouzou, A., and Freychet, P. (1976) Exp. Cell. Res. 98, 382-395. Gammeltoft, S., Kristensen, L.O., and Sestoft, L. (1978) J. Biol. Chem. 253, 840-8413. Harrison, L.C., and Hin, A. (1980) J. Biol. Chem. 255, 12066-12072. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, 660-672. Van Obberghen, E., De Meyts, P., and Roth, J. (1976) J. Biol. Chem. 6844-6851. 251, Elovson, J. (1980) J. Biol. Chem. 255, 5816-5825. Steer, C.J. and Ashwell, G. (1980) J. Biol. Chem. 255, 3008-3013. Goldstein, J.L., Basu, S.K., Brunschede, G.Y., and Brown, M.S. (1976) Cell 1, 85-95.

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