j. biol. chem.-1993-yang-4600-3
TRANSCRIPT
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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
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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.
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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-
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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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I. and Seino,
S.
(1989) J. Biol. Chem. 264 , 7736-7779
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Shah, N ., Lodish, H. F., and Jarrett, L.
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