8/10/2019 J. Biol. Chem.-1993-Yang-4600-3

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Communication

Vol.

268

Xo.

7,

Issue of March

5

p 4600-4603,

1993

0

1993

by

The American Society for Biwhemistry and Mo ku lar Biology Inc.

Printed n U S.A.

T H E J O U R N A LF BIOLOGICALHEMISTRY

Comparison

of

GLUT4 and

GLUT1 Subcellular Trafficking in

Basal and Insulin-stimulated

3T3-Ll Cells*

(Received for publication, November 9, 1992)

Jing Yang and

Geoffrey D.

Holman

From the Department o Biochemistry, University o Bath,

Bath BA2 7AY United Kingdom

The two glucose transporter isoforms GLUT4 and

GLUTl present in 3T3-Ll cells were labeled in the

insulin-stimulated and basal stateswith the imper-

meant bis-mannose photolabel, 2-N-4-(1-azi-2,2,2-tri-

fluoroethyl)benzoyl-1,3-bis- D-mannos-4-yloxy)-2-

propylamine. The redistributions of these labeled

transporters from the plasma membrane to the low

density microsome membrane fraction were followed

while cells were maintained at either insulin-stimu-

lated or basal steady states. In both these steady states

GLUT4nd GLUTlwere continuously recycled.

Analysis of the time courses for tracer-tagged GLUT4

and GLUTl redistribution showed that the endocytosis

rate constants were only ~ 3 0 lower in the insulin-

stimulated (0. 08 and 0.093 min) compared with the

basal (0.1

16

and 0.12 1 min) state. In the insulin-

stimulated state, the rate constants for GLUT4 and

GLUTl exocytosis (0. 086 and 0. 09 6 rnin) were sim-

ilar to those of endocytosis. In contrast, the exocytosis

rate constants of GLUT4 andGLUTl in the basal state

were 0.01 and 0.0 35 min.

W e

therefore conclude that

the main effect of insulin is to increase GLUT4 and

GLUTl exocytosis rate constants by

=9-

and 3-fold,

respectively, and that the unique feature of the GLUT4

isoform is the veryslowrate of exocytosis n the

basal state.

The acute insulin regulation of glucose transport in target

tissues is now known to involve the specialized glucose trans-

porter isoform GLUT4. This isoform was cloned andse-

quenced in 1989 (1-5) and has been shown to be ocalized

only in insulin-responsive tissues. It is expressed at high levels

in white (6) and brown (7) adipose tissue, and in skeletal (8)

and heart(9) muscle. It is the propensity of the GLUT4

isoform to remain localized in the cytoplasmic tubulo-vesic-

ular system in the absence of insulin (7) tha t allows GLUT4-

containing cells to respond acutely to insulin within minutes

and to produce, in response to this stimulation, over 20-fold

increases of glucose transport activity. The GLUT4 isoform

has also been shown

t o

be sequestered to the intracellular-

*

This workwas supported by the Medical Research Council

(United Kingdom) and the British Diabetic Association for financial

support. The costs of publication of this article were defrayed in part

by the payment of page charges. This article must therefore be hereby

marked uduertisement in accordance with 18 U.S.C. Section 1734

solely to indicate this fact.

3

To whom correspondence should be addressed Dept. of Biochem-

istry , University of Bath, Claverton Down, Bath BA2 7AY, United

Kingdom. Tel.: 44-225-826874;Fax: 44-225-826449.

vesicle pool in transfection and expression systems including

3T3-Ll and NIH-3T3 fibroblasts (10, ll ) , oocytes (12), COS

cells (13), and Chinese hamster ovary cells (14,

15).

As

discussed by James and colleagues (7, 15, 16), the se-

questration

of

GLUT4 could be due either to a very rapid

removal of this protein from the plasma membrane

or

to a

very slow rate of exocytosis in the basal state. This is an

important question, as heGLUT4 protein sequence may

contain information that allows a unique cellular processing

by vesicle trafficking and sequestration machinery. If endo-

cytosis is unusual, then the unique processing may occur in

the plasma membrane, whereas if exocytosis is slow, the

targeting recognition events may occur intracellularly. Slot et

al.

(7), in theirmmunocytochemistry study on brown adipose

tissue, observed that in the nsulin-stimulated state the pro-

portion of GLUT4 in the plasma membrane was increased to

a level that was ~4 0- fo ld igher than in the basal state. In

the insulin-stimulated state, the proportion of GLUT4 asso-

ciated with coated pits and early endosomes increased 3- and

4-fold, respectively, above basal levels. These authors there-

fore suggested that themain effect of insulin was to increase

exocytosis of GLUT4 from tubulo-vesicular structures to the

plasma membrane and thence into the endosome pathway.

However, such studies on the steady-state level of transporters

do not unequivocally distinguish between an increased exo-

cytosis or decreased endocytosis or to a modification of both

these steps in cycling.

Kinetic, rather han steady-state distribution, studies of

transporter cycling should be able to directly determine and

quantify the site of insulin action on trafficking. Jhun et al.

(17) have recently used a photoreactive probe (B3-GL) to

study insulins effect on GLUT4 translocation kinetics in rat

adipose cells. They have reported tha t insulin has equal effects

on endocytosis and exocytosis, the former being reduced and

the latter ncreased by ~3 -fold. Th etudy by Jhun et

al.

is at

variance with our own investigations of GLUT4 trafficking

kinetics in rat adipose cells and with the hypothesis (15, 16,

19) that themain effect of insulin is to increase exocytosis of

GLUT4. In viewof this controversy, and because

of

the

importance of resolving from kinetic studies the site of insulin

action on GLUT4 trafficking, we have carried out a study

similar to that described by Jhun et al. (17).

GLUT4 and GLUTl are both present t high levels n 3T3-

L1 cells (16,20,21), and o we have been able t o compare the

trafficking of GLUT4 and GLUT1. In contrast to the study

of Jhun

et al.

(17), we have shown that insulin does not

markedly reduce glucose transporter endocytosis but instead

increases exocytosis. This kinetic approach has allowed us to

determine that GLUT4 is unique because of its very low

exocytosis rate in the absence of insulin.

E XPE RI ME NT AL

PROCEDURES

Materials-ATB-[2-3H]BMPAZspecific activity

10

Ci/mmol) was

prepared as described (18).DMEM was romFlow Laboratories.

S.

Satoh, H. Nishimura, A. E. Clark,

I. J.

Kozka,

S. J.

Vannucci,

I.

A. Simpson, M. J. Quon, S.

W .

Cushman, and

G .

D. Holman,

submitted for publication.

The abbreviations used are: ATB-BMPA, 2-N-4-(1-azi-2,2,2-

trifluoroethyl)benzoyl-l,3-bis- D-mannos-4-yloxy~-2-propylamine;

DMEM, Dulbeccos modified Eagles medium; ClzEs, nonoethylene-

glycol dodecyl ether.

4600

8/10/2019 J. Biol. Chem.-1993-Yang-4600-3

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Insulin-stimulated GLUT4 Exocytosis

in 3T3-Ll

Cells 4601

Fetal bovine serum was from Gibco Laboratories. Dexamethasone,

isobutylmethylxanthine, and protein A-Sepharose were from Sigma.

Nonaethyleneglycol dodecyl ether (CIzEg)was from Boehringer.

Monocomponent porcine insulin was a gift from Dr. Ronald Chance,

Eli Lilly Laboratories. Polyclonal antisera were raised against the C-

terminal peptides of GLUT4 CSTELEYGPDEND) and GLU Tl

(CGEELFHPLGADSQV) as described (22, 23).

Cell Culture-3T3-Ll fibroblasts were differentiated to adipocytes

as described (20). Fully differentiated cells were washed with phos-

phate-buffered saline (154 mM NaCl, 12.5 mM sodium phosphate, pH

7.4) and were then incubated for 2 h in serum-free medium containing

25 mM D-glucose. This was followed byhree washes in Krebs-Ringer-

Hepes buffer (KRH buffer, 136 mM NaCl, 4.7 mM KC1,1.25 mM

CaCl,, 1.25 mM MgSO,, 10 mM Hepes, pH 7.4).

A T B - B M P A Photolabeling-Cells in 35-mm dishes were main-

tained a t 37 C either in he absence or the presence of 100 nM insulin

in 1 mlof KRH buffer for 30 min. The buffer was emoved and

replaced by350

p1

of KRH buffer, either with or without insulin

respectively, containing 250 pCi (insulin samples) or 500 pCi (basal

samples) of ATB-[2-3H]BMPA at

18

C. The dishes were placed

between two 2-mm glass plates and were irradiated for 1 min in a

Rayonet photochemical reactor equipped with eight 300-nm bulbs

and eight 350-nm bulbs. The irradiated cells were then either washed

rapidly and immediately homogenized in Tris-EDTA-sucrose (TES

buffer, 10 mM Tris-HC1,0.5 mM EDTA, 255 mM sucrose at

18 c)

r

were maintained a t 37 C in 1ml of KRH buffer containing 10 mM

D-glucose for the times indicated in the figure legends before washing

and homogenization. For each time point he combined material from

two 35-mm dishes was pooled.

Immunoprecipitation and Electrophoresis-The 3T3-Ll cells were

vigorously homogenized to ensure all cell material was broken. This

was carried out in

2

ml of TE S buffer, using a tightly fitting Teflon

homogenizer operating at 1500 rpm and with 15 full strokes. Samples

were then centrifuged at 12,500 X gmaxor 20 min. The crude plasma

membrane and post-plasma membrane supernatants were then solu-

bilized in 1.5mlof detergent buffer containing 2% CI2E9, 5 mM

sodium phosphate, pH 7.2, and with the proteinase inhibitors anti-

pain, aprotinin, pepstatin, and leupeptin, each at 1 pg/ml (24). Any

non-solubilized material was then removed by centrifugation at

20,000 X gmaxor 20 min. Antisera (50 p1 of anti-GLUT4 and 100 pl

of anti-GLUT1) were premixed with 30

11

of swollen protein A-

Sepharose, washed in phosphate-buffered saline, and thenmixed with

the solubilized membranes for

2

h at 0-4 C. The immunoprecipitates

were washed four times (insulin) or five times (basal) in 0.1% then

once in 0.01% Cl2E, detergent buffer. The extent of washing of the

immunoprecipitates was more extensive than previously reported (24)

because of the low counts recovered in basal cells. The labeled glucose

transporters were released from the antibody complexes with 10%

SDS,6 M urea,

10%

mercaptoethanol and subjected to electrophoresis

on 10% acrylamide gels. The radioactivity in gel slices was extracted

with hydrogen peroxide as described (24). For the insulin-treated

samples the background was determined by estimating the radioac-

tivity of slices on either side of the transporter peak. The background

radioactivity in basal samples was determined by immunoprecipitat-

ing with preimmune serum and then running this material on the

same gel as the other basal samples. The preimmune serum did not

produce a peak of radioactivity but allowed a correction of background

from slices, which exactly corresponded to the position of the trans-

porter peak.

Estimation

of

Rate Constants-The glucose transporter levels as

determined from electrophoresis (above) were used to calculate the

fractional loss of plasma membrane label. These data were then fi tted

to an integrated rate equation, describing transporter recycling, by

least squares analysis (weighted for relative error) using Fig P soft-

ware (Biosoft).

RESULTS

We have used the im perm eant glucose transporter photo-

label , AT B-B MP A, o racer-tag cel l surface GL UT 4a nd

G L U Tl in 3T 3- Ll cells. We have then followed the removal

of these ransporters from theplasmame mb rane of cells

maintained in ei ther basal or insulin-stimulated sta tes. T he

maintenance of a steadystate of non-labeled ransporter

distribution w as confirmed by comp aring heamounts of

immunode tec table GLUT4 and GLUTl a t the in i t i a labeling

time and following an addit ional 40 min

of

incubation at

bl 40 m8n

D

t

800

0 1

O L

0 2

4 6 8 1 0 1 2

0 2

4

6

8 1 0 1 2

Sl lce

Number

Sl lce

Number

FIG. 1. Equilibration of tracer-tagged

GLUT4

with the in-

tracellular membrane pool in 3 T3-Ll cells. Confluent 3T3-Ll

cells (two 35-mm dishes) were stimulated for 30 min with 100 nM

insulin and then abeled with 250 pCi of ATB-[2-3H]BMPA.Follow-

ing labeling, the dishes were either homogenized immediately (a) or

were maintained at 37 C in the continuous presence of insulin for

another 40 min and then homogenized b ) .GLUT4 was immunopre-

cipitated from either the plasma membrane 0 ) r the post plasma

membrane supernatant (low density microsomes) 0 ) nd then ana-

lyzed by electrophoresis.

37 C 3 Fig.

l a

shows a n SDS-polyacrylamide gel electropho-

resis profile obtained by labeling and mm unopre cipitating

GL UT 4 in insulin- treated 3T3 -Ll cel ls . Th e label recovered

in hepost-plasmamem brane ract ion of cells th at were

homogenized immediately was very low. Thi s suggests tha t

tracer-tagged GLUT4was not significantly tran sfer red to the

low density m icrosomes duri ng the processing period. Our

resultscontrastwi th hose of Jhu n

et al.

(17), who have

rep orte d that in rat adipose cells approx imately on e-third of

the labeled GL UT 4 was recovered in the post plasm a m em-

brane fraction in s amples which were processed immediately

after labeling.

Following 40 min of incubation of the cells

at

37

C,

the

label in the plasma membranewas reduced to approxim ately

one-half its initial value. Th e label tha t was lost from the

plasma mem brane was recovered in the low density micro-

some fraction (Fig. lb). We have not routinely analyzed the

mate rial transf erred to the low density microsome fraction

because of th e need to sim ultane ously process multiple im-

munoprecipi ta ted samples obtained for est imating GLU T4

and GLUTl in the p lasma membrane . Previous s tudiesave

demon strated that const i tut ive turnover of t ransporters oc-

curs (23, 24)' an d tha t label which is lost from the plasma

mem brane is ecovered in the ow density microsome fraction.

In basal cells the labeling of GL UT 4 and GL UT l was low

(Fig.

2,

a and b), and following 40 min of incubation of the

labeled cells at 37

C,

the level of tracer-tagged transporters

in the plasma mem brane were reduced tow ard background

levels.

We have used least squaresurve fittin g to irectly estim ate

exocytosis an d endocytosis rate co nstan ts fro m tim e courses

of loss of t race r- tagged GLU T4 and GLU Tl f rom the p lasma

membrane (Fig.

3,

a a n d b ) . For analytical purposes the rate

of loss of label from th e plasm a mem bran e is assum ed to be

dependenton ust two rateconstants; onedescribing the

exocytosis

(kex)

nd one t hendocytosis

(ken).

Th e rate f loss

of labeled tran spo rter s is then iven by Equation 1 where Lp

is the fract ion f the label in the plasma mem brane.

J.

Yang and G. D. Holman, unpublished results.

8/10/2019 J. Biol. Chem.-1993-Yang-4600-3

3/4

4602 Insulin-stimulated GLUT4 Exocytosisn 3T3-Ll Cells

a1

G lu t4

9

b l G lu t1

4

*

0 2 4 5 8 1 0 1 2 2 4 6 8 1 0 1 2

Slice Number Sl iceumber

FIG.

2.

Equilibrationof tracer-tagged GLUT4 and GLUTl

in basal 3T3-Ll cells. Confluent 3T3-Ll cells

( two 35-mm

dishes)

were labeled in the basal state with 500 pCi

of

ATB-[2-3H]BMPA.

Following labeling the dishes

were

either homogenized immediately

0,

A)

or were maintained at 37 C for another 40 min and then

homogenized 0,A ) . GLUT4 a ) and GLUTl b ) were then immu-

noprecipitated

from

the plasma membrane

fractions

and analyzed by

electrophoresis.

10

20 3 4

5

10 20 3 40

T l m s

(minrl T lme rn inrl

GLUT4 and GLUTl in

3T3-Ll cells. Confluent 3T3-Ll

cells

FIG.

3.

Time courses for equilibration of tracer-tagged

were labeled eithe r in the basal 0 ) r insulin-stimulated

A) tates

and were then maintained in these steady

states

for the indicated

time s before homogenization to obtain the plasma membrane frac-

tion. GLUT4 a ) nd GLUTl

b )were

then immunoprecipitated and

analyzed by electrophoresis.The lines through the data were derived

from least squares fitting to Equation 2.

(see

Results ). The results

are

the mean f S.E. from three separate experiments,

except

in the

case

of GLUT4 in basal cells, where the

data

are

from

four experi-

ments.

Integrat ion of Equation

1

with

L p

= 1.0

at

t = 0 gives

Equa t ion 2.

In the experiments escribed here we have used this equa-

tion to analyze the red istribu tion of label und er ste ady- state

condit ions, but Equation is equally applicable to an analys is

of label equilibration under non-steady-state conditions. All

tha t s required is hat he ota l pool of transp orters be

conserved. It is only necessary to consider the concentration

of unlabeled transporters if there are saturation steps in the

flux path way , as would be the cas e if one were analyzing

equilibrium exchange by a carrier protein.

In the continuous presence of insulin both tracer-tagged

GLUT4 and GLUTl were rapidly removed from the plasma

mem brane. The values of

k,,

a n d

k n

erived from the m ean

results of three separate experimen ts are shown in Table

I .

TAB LE

Kinetic parameters forlucose tran sporter trafficking in 3T 3- Ll cells

Kinetic parameters were calculated from the mean data shown in

Fig. 3. These data were from three separate experiments (Insulin)

and three separate experiments Basal)

except

in the case of GLUT4

in basal cells where the results were f rom four separate experiments.

The data were fitted oEquation 2 using east

squares

analysis

(weighted

for

relative error).

kx

k.

t1 2

min

min

min

Basal

GLUT4

0.010 k 0.001 0.116 f 0.006

5.6

f .3

GLUTl

0.035 f

0.009

0.121 0.020

4.4

f

0.6

GLUT4 0.086 f

0.011

0.080 f

0.007

4.2 f .3

GLUTl 0.096 .023

0.093 f

0.017

3.7 f .6

Insulin

The es t imated ra te co ns tants were s imila r for both GL UT l

and GLUT4. In addit ion, the end points f the equil ibrat ion

of the tracer -tagg ed transp orter s ere not significantly differ-

ent . At

0

min the evels of t racer-tagged GL UT4 and GL UTl

remaining in the plasma mem brane ere 51.1 f 1.7% n 3)

a n d

53

f .2% n= 3), respectively, of the initial values. Th e

maintenance of the nsulin steady-state n which approxi-

mately one-half of the total t ransp orter ool is a t th e urface

(24) th us occurs because th e exocytosis of transp orter s (de-

pendent n ,) matches an equalate of endocytosis

(dependent onken).

In the basal sta te t racer-tagged GLU T4 was more exten-

sively removed from the plasma membrane than in the insu-

l in-st imulated sta te . At 40 m in he fract ion of the tracer-

tagged GLU T4 rem aining in the p lasma mem braneas only

8.4 f 2.0%

n

= 4) of the nitia l value. Bycontrast , he

fract ion of the tracer-tagged GLU Tl rem aining in the plasma

membrane was 20.8 f 1.3% n = 3) of the nit ial value.

Analysis of the ime course

of

tracer-tagged GLUT4and

G LU Tl redis t ribution (Table I) showed tha t the endocytosis

r at e c o ns ta n ts w e re o nly ~ 3 0 %lower in the insulin-st imu-

lated comp ared with the basal sta te . However, the GLU T4

an d G LU Tl exocytosis ra te constants were 8.6 and 3 t imes

higher, respectively, in the insulin -stim ulated compared with

the basa l s ta te (Table ).

Although we have used curve fittin g to directly obta in th e

rate constants

ke,

and ken, he half-tim e or label equilibration

can also be easily calculated from least squares curve fitting

using Equation 2. The half-t ime s only dependent on he

expo nentia l term , as hown by the following equation.

t1/2

=

ln2/ ke. +

k

(Eq. 3)

T h e values for the half-tim es for equilibration of tracer-

tagged transporte r were therefore calculated from sum ming

the ra te cons tants

e,

a n d

kn

s

in Equation 3 and are shown

in Table I . The ha l f - times for GLU T4 and GLU Tl equi l ibra -

tion in basal cells were 5.6 a nd 4.4 m in, respectively. Thes e

values are similar to, but slightly faster than, the half-times

(6.5 min ) for he decrease in glucose transpor t activity n

3T 3- Ll cells following removal of insulin (24). The calcula ted

half-times for lossof tracer-tagged transporters in the contin-

uous presence of insulin were slightly faster than in its ab-

sence because of the greate r contr ibutio nf t he re-exocytosis,

t he

ke,

term in Equation 3, to the equil ibrat ion rate .

DI SCUSSI ON

Use of bis-hexoses with photorea ctive substituents (17,23)'

has shown that GLU T4 const i tut ively recycles between th e

plasma mem brane and the intracel lular vesicle pool of ra t

adipose cells, and we have also demonstrated this phenome-

8/10/2019 J. Biol. Chem.-1993-Yang-4600-3

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Insulin-stimulated

GLUT4

Exocytosis in 3T3 Ll Cells

4603

non in 3T3-Ll cells (Ref. 24 and this tudy). In the ontinuous

presence of insulin, the GLUT4 and GLUTl soforms recycle

at a similar rates and redistribute to the ntracellular pool SO

that at quilibrium only approximately one-half of the labeled

transporters remain in the plasma membrane. This is to be

expected if all the cellular transporters are involved in the

recycling process and is consistent with our previous studies

on the photolabeling of t he cell-surface and otal-cellular

pools of glucose ransporters in 3T3-Ll cells, where we showed

that approximately one-half of the otal cellular pool of

GLUT4 andGLUTl transporters isocated at the ell surface

of insulin-stimulated cells.

In the basal state a much greater proportion of labeled

GLUT4 transporters are lost from the plasma membrane.

This is the result that would be expected if over 90% of the

transporters were intracellularly localized. However, he half-

time for removal of these labeled transporters in the basal

state is somewhat slower than is observed in the insulin

steady-state. These results contrast with those of Jhun et al.

(17), who have reported that in basal rat adipose cells the

half-time for tracer-tagged GLUT4 quilibration is faster than

that observed in insulin-stimulated cells. This discrepancy

maybe related to the need to rapidly process samples to

determine thefraction of label removed from the plasma

membrane. This is particularly important in the basal state,

where the fraction of the label removed rom the plasma

membrane is more extensive.

Our data show that the calculated endocytosis rate con-

stants (ken)are similar for GLUT4 and GLUTl and thathese

endocytosis rate constants are only -30% slower in the in-

sulin-stimulated compared with the basal state. These find-

ings are consistentwith our previous observations on the rate

of loss of cell-surface transporters from 3T3-Ll cells under

conditions in which insulin was removed by a low pH buffer.

In those experiments we simply estimated the proportions of

transporters remaining at the cell surface by labeling at dif-

ferent time points following insulin removal. We observed

that cell-surface levels of GLUT4 and GLUTl decreased at a

similar rate. We observed that the fractional loss of GLUTl

was slightly less than GLUT4 and suggested that this was

due to a greater re-exocytosis of th is isoform (24).

We have now shown quantitatively that insulin increases

the rate constantkex) or glucose transporter exocytosis. The

exocytosis of t he GLUT4 isoform is 8.6-fold faster in insulin-

stimulated cells compared with basal cells. The basal exocy-

tosis rate constant may be overestimated because of some

plasma-membrane contamination from label, which transfers

to the ntracellular pool. However, he observed level of stim-

ulation of GLUT4 exocytosis is almost sufficient to account

for the -12-15-fold stimulations of cell-surface appearance of

GLUT4 previously observed using ATB-BMPA

(20,

21,

24).

In turn, this level of recruitment of GLUT4 is almost suffi-

cient to account for the increase in glucose transport activity

in these cells (=l& O-fold). Insulin also stimulates GLUTl

exocytosis but only to -3-fold above basal levels. Again, this

level of stimulation of exocytosis accounts for the smaller, 3-

5-fold increases in cell-surface GLUTl accessible to thepho-

tolabel that we have observed in our previous studies.

The major difference between the trafficking of the GLUT4

and GLUTlsoforms is the much slower exocytosis of GLUT4

in the basal state. However, in the presence of insulin, both

isoforms are processed in the same way. Our data therefore

supports the suggestion tha t in basal cells intracellular proc-

essing steps remove GLUT4 from the normal endosome re-

cycling pathway, possibly to a specialized compartment within

the tubulo-vesicular system

(7).

Insulin may then re-commit

or re-target these transporters from the tubulo-vesicular sys-

tem to the plasma membrane and thence to the early endo-

some system. Both intracellular transporter pools would then

be in rapid equilibrium with each other in the insulin-stimu-

lated state, as our photolabel equilibration data shows tha t

the cells entire complement of glucose transporters are in-

volved n the recycling process both in 3T3-Ll cells (this

study) and in rat adipose cells.'

We have consistently observed that the half-time for the

initialstimulation of glucose transporter ranslocation by

insulin is faster than the half-time for steady-state recycling

of transporters and the half-timeor loss of transporters when

insulin is removed (18,24).' We have suggested hat this may

occur because, immediately following insulin addition, trans -

porters are rapidly committed to the plasma membrane at a

vesicle docking and fusion step and that, once transporters

are committed in this way, they are then recycled at a slower

rate within the early endosomes. The kinetic effects expected

from this type of multiple pool-trafficking system have been

analyzed and have been shown to account for the observed

disparity between the half-times for insulin stimulation of the

initial translocation and he steady-state rates of transporter

recycling?

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Shah, N ., Lodish, H. F., and Jarrett, L.

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