BIOCHEMICAL

Vol. 168, No. 3, 1990 May 16, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1089-1094

THE PHORBOL ESTER TPA INDUCES A TRANSLOCATION SENSITIVE Beate

Vogt,

GLUCOSE CARRIER (GLUT4) Joanne

Institut

fUr

Mushack,

Eva Seffer

Diabetesforschung, 40, West

8 MUnchen Received

December

18,

OF THE INSULIN

IN FAT CELLS and H.U.

Kolner Germany

HBring*

Platz

1,

1989

Insulin activates the glucose transport in isolated fat cells through a translocation of the insulin sensitive glucose carrier subtype (GLUT4) and by activation of glucose carriers in the plasma membrane. Protein kinase C stimulating phorbol esters are able to mimick partially the insulin effect on glucose transport. In order to determine whether this phorbol ester effect occurs through a translocation of the insulin sensitive glucose carrier (GLUT41 we used a monoclonal antibody against GLUT4 to determine its distribution in subcellular fractions of rat adipocytes. We found that the phorbol ester TPA is able to increase the amount of GLUT4 in the plasma membrane fraction about two-fold. 0 1990 Rcademic Press, Inc. Insulin It

appears

riers

that

from

underlying direct

stimulates an insulin

membranes

mechanism stimulatory

(3-6).

take

like

(1,2).

kinase like

insulin

C stimulating effect

location

of insulin

to the agents

to the

gested

that

translocation.

protein

membrane kinase

Recently

membrane

plasma to carrier

activity

(7)

sites

from

(3,5).

These

C might

different

play groups

*To whom all correspondence should Abbreviations mABlF8: monoclonal antibody directed table glucose transporter GLUT4 : insulin regulatable glucose 12-0-tetradecanoylphorbol-13-acetate TPA: PBS : phosphat-buffer-saline DTT : dithiothreitol.

is

on glucose by several are

a partial and induce intracellular

observations

a role

a upinsu-

protein insulin a transmemhad sug-

in glucose

identified

the

activity

substances exert

car-

translocation carrier

effect

which

B binding

plasma

to the

can be mimicked

transport

of cytochalasin

branes

of glucose

Among these esters

of adipocytes.

translocation

insulin

effects

(3,5).

uptake

on glucose

overall

phorbol

on glucose

glucose

In addition

effect

Both

acting

the

induced

intracellular

seems to contribute lin

rapidly

several

carrier sub-

be addressed. against

the

insulin

regula-

transporter

0006-291X/90 1089

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Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

BIOCHEMICAL

168, No. 3, 1990 types

of glucose

and their assay rier

is not

(GLUTS)

antibody cose

is

transport

activity

glucose

by a phorbol

carrier

Materials

a monoclonal

car-

antibody

sensitive

glucose

and Pat.

We used

this

of phorbol

ester

on glu-

effect

and on glucose

cytochalasin

ester

different

insulin

the

by the

the

specificity B binding

in muscle

of Pat cells

as determined

be explained

the

whether

between

raised

(13)

expressed

to determine

tribution

tissue

in their

The cytochalasin

to dipferentiate

specifically which

diPPer (a-12).

James et al.

recognizes

rier

RESEARCH COMMUNICATIONS

which

sensitivity

suitable

subtypes.

which

carriers

insulin

AND BIOPHYSICAL

effect

B binding on the

insulin

car-

carrier assay

dismight

sensitive

GLUTS.

and Methods

Materials Porcine insulin was purchased Prom Novo Industrie (Bagsvaerd. Denmark). The monoclonal antibody lF8 was a kind gift of Dr. Pilch, Boston, USA. 1251-goat anti-mouse IgG was obtained from DuPont - New England Nuclear , nitrocellulose from Schleicher & Schuell, electrophoresis chemicals Prom BioRad. All other reagents were of best grade commercially available. Cell

isolation and determination of 3-0-methylglucose Rat adipocytes were prepared as described by HBring et al. Prom male Sprague-Dawley rats (180 - 220 g body wt.). In(14) cubations were carried out at 37OC in the absence (basal) or the presence of insulin or with the phorbol ester TPA as mentioned in the Figure legends. TPA was diluted in pure ethanol, dried with N2, taken up in incubation buffer and sonicated. Glucose transport activity was measured as described by Haring et al. (14).

Western Blotting Rat adipocytes were isolated and subcellular fractions were obtained using a differential centrifugation procedure (3,15,16). The purity of the membrane fractions was assessed by the determination of marker enzymes as described earlier (3). Membranes were subjected to SDS-PAGE on a 7.5 k gel in the presence of 10 mM DTT using the system oP Laemmli (17). Proteins were transfered to nitrocellulose by electroblotting (buPPer: 192 mM glycin, 25 mM Tris, 20 % MeOH, pH 8.3, 4 hrs, 200mA). Following transfer, the filters were blocked with 5 $ non Pat dry milk in PBS for 1 h at 37OC and subsequently incubated with antibody lF8 at a dilution of 3 ug/ml PBS (1 $ dry milk) overni ht at 4OC. Immune complexes were visualized by incubation with l 2fI-goat anti-mouse antisera ( 25.000 cpm/ml) for 1 h at 37OC and 5 hrs at 4OC and autoradiography. The immunolabelled bands of 44 kDa were excised and counted. Background correction was done by counting non labelled areas of nitrocellulose. Results Freshly

prepared

phorbolester. (3-O-MC)

Table uptake.

Pat cells 1 shows

Insulin

were

the

stimulated

stimulation

stimulates

1090

3-O-MG

with of

insulin

or

3-0-Methylglucose

uptake

7-a-fold,

TPA

Vol.

168,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Table 1 GlucosetranSpOrt activity as described in the Method section was calculated as percentage of equilibrium reached after 4 s (mean values + SEM of 5 independent cell preparations) determined in duIncrease and decrease of GLUT4 label are expressed as raplicate. tios of Insulin/Basal, TPA/Basal and Basal/Insulin, Basal/TPA respectively (data from Table 2) Glucose transport activity (%I

PM Increase GLUT4 labelling

Basal

5.2 f 2.9

Insulin (1000 jN/ml)

38.4 r 5.2

TPA (10"

23.5 f 5.2

M)

stimulates lar

4 to S-fold.

were

as determined

earlier

(3)

not

PAGE and transferred As previously (GLUTS) sues

described lF8.

and low

density

of the

insulin with

with

TPA.

low

density

lyzing cose plasma

is

the amount membranes

and a 2.5-fold

The purity

enzymes,was Membrane

subcellu-

of these

similar

proteins

frac-

as reported were

sensitive

shows

membranes

separated

also

an immunoblot

of fat

cells

label

by

and a lower a decrease after

increase in the

incubation

on the

a Q.&fold increase

the

nitrocellulose increase

induced

by

1091

mem-

which

were

stimula-

an

increase

membranes

after

in

esters

fat

or TPA.

radioactivity (Table

after

stimulation

insulin

of GLUT4 induced phorbol

tis-

reveals

GLUT4 label

with

transporter and fat

of plasma

in plasma

of GLUT4 by measuring bands

glucose in muscle

The autoradiography

transporter

microsomes

transporter

Fig.1

insulin

There

preparations

immunolabelled

or TPA.

GLUT4 glucose

stimulation

same cell

(13),insulin

can be specifically

ted with

Basal/TPA 1.5 + 0.2 (= 69 + 24.8 % of insulin effect)

to nitrocellulose.

by antibody

branes

TPA/Basal 2.5 f 0.9 (= 70 f 17 % of insulin effect)

prepared.

shown).

Decrease of GLUT4 labelling

Basal/Insulin 1.9 + 0.3

by marker

(data

of

Insulin/Basal 4.4 f I.7

From the

fractions

membrane

tions,

LDM

cell Ana-

of glu2) we found by insulin

(Table

1).

The

in

Vol.

BIOCHEMICAL

168, No. 3, 1990

Ba

Ins TPA

AND BIOPHYSICAL

Ba

Ins

RESEARCH COMMUNICATIONS

TPA

PM LDM Fig. 1 Western blot analysis of insulin regulatable glucose transporter in subcellular membrane fractions of rat adipocytes following stimulation with insulin (1000 ulJ/ml) and TPA (1 nM) for 20 min. Total protein (300 ug) from plasma membranes (PM) and low density microsomes (LDM) were immunoblotted using mAblF8. Immunolabelled bands were visualized by autoradiography after using 1251-labelled sheep anti-mouse IgC.

data

of low

density

GLUT4 following treatment of the sity

microsomes

insulin

show that insulin

the

effect

microsomes

(Table

show a corresponding

and TPA stimulation. phorbol

ester

in plasma

is

membranes

Data able

decrease obtained

to mimick

as well

of by TPA

about

as in low

70 % den-

1).

Table 2 Radioactivity measured in counts per minute (cpm) of immunolabelled bands of 44 kDa using mAblF8 for immunoblotting and 1251labelled goat anti-mouse antisera (25000 cpm/ml) for immunolabelling in different subcellular fractions, PM: plasma membranes, LDM: low density microsomes. Background correction was made by counting non-labelled areas of nitrocellulose (lo-20 %) Experiment

Basal

PM Insulin (1000 pU/ml)

1

171

2

J9M

TPA (1O"M)

Basal

Insulin

1040

480

1518

959

957

200

793

487

1788

813

1017

3

85

546

284

4

329

1022

417

1976

977

1510

5

310

856

404

2168

935

1543

1092

TPA

Vol.

BIOCHEMICAL

168, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Discussion The western blot

data show that phorbol eaters induce an increase

of GLUT4 in plasma

membranes

and a corresponding

decrease in low

density microaomea of fat cells. While insulin induces a 4.4-fold increase of GLUT4 in the plasma membranefraction and a 1.9-fold decrease in 2.5-fold

low density microsomea, phorbol eaters induce a

increase and a 1.5-fold

decrease. Using the cytochalaain

B binding assay we had earlier already reported that phorboleater are able to induce a glucose carrier tranalocation (3,5). With the present method which is more specific glucose carrier

for the insulin

sensitive

a phorbol eater effect on carrier tranalocation These data are in favour of a role of pro-

can be shown as well.

tein kinaae C in the regulation carrier

in fat cells.

Baaed on our earlier

speculated that protein transfer

of the insulin

sensitive

observations

kinase C might be involved

from the insulin

receptor

glucose we had

in the signal

to the glucose transport

ay-

stem. On one hand the present study provides further data which support this hypothesis as it shows that insulin and stimulators of protein kinaae C regulate the same carrier subtype. On the other hand, if one assumesa role of protein kinase C in insulin one might expect that insulin and phorbol eater should aignalling, be equally effective on GLUT4tranalocation. This is not the case. A possible explanation might be that insulin and phorbol eater are differently efficient in activating the protein kinase C. Another explanation, mitting

however, would be that insulin

system which is more efficient

induce carrier

tranalocation.

C is indeed involved still remains open.

uses

a signal trana-

than protein

kinaae C to

Thus the question if protein

in the insulin

kinaae

signal on glucose transport

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