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(Received for publication, September 8, 1994; and in revised form, December 13, 1994) From the
GLUT4, the major insulin-responsive glucose transporter isoform
in rat adipocytes, rapidly recycles between the cell surface and an
intracellular pool with two first order rate constants, one for
internalization (k
Insulin plays a key role in glucose homeostasis in humans and
mammals by stimulating glucose transport in muscle and adipose cells (1) . The glucose transport in these cells is catalyzed
primarily by GLUT4(2, 3) , and to a small extent by
GLUT1(4) . GLUT4 is unique in that it is expressed only in
insulin target cells(3, 4) , it is constitutively
sequestered in intracellular organelles, and very little is found at
the plasma membrane(5) . The plasma membrane GLUT4 level
increases greatly after insulin treatment(5, 6) . This
insulin-induced, apparent redistribution of GLUT4 accounts for the
major portion of the acute glucose transport stimulation by insulin in
adipocytes(7) . The insulin effect is reversible; removal of
insulin causes a rapid net movement of GLUT4 from the plasma membrane
back to the storage pool, reestablishing basal levels in both
pools(8) . The GLUT4 storage pool has been isolated by
subcellular fractionation as a subset of intracellular
vesicles(9) . Evidence indicates that GLUT4 co-localizes with
clathrin at the cell surface and in clathrin-coated vesicles in
adipocytes(10) . These findings strongly suggest that GLUT4
redistribution involves vesicle trafficking including exocytotic and
endocytotic pathways(11) . We have demonstrated previously (12) that GLUT4 in rat adipocytes constantly and rapidly
recycles between the plasma membrane and the intracellular storage
pools in a manner describable by two first order rate constants, one
for internalization (k States of protein phosphorylation
regulated by protein kinases and/or protein phosphatases are known to
play a key role in many cellular functions(13) . The importance
of protein phosphorylation in glucose transport regulation has been
suggested by the fact that OKA, ( In an
effort to identify the biochemical basis for the regulation of GLUT4
recycling in adipocytes, we measured in this study how the GLUT4
internalization and externalization rate constants, k
Figure 6:
Time-dependent changes in
[
Figure 7:
Analysis of the steady-state equilibrium
exchange time course of [
Figure 4:
The cell surface-selective incorporation
of [
Figure 5:
[
The
time constant of the process, The measurements of the equilibrium distribution
pool sizes, S Errors in the estimation of
either the time constant, or the equilibrium distribution between the
cytoplasmic and plasma membrane pools, will be reflected in errors in
the computed values of the rate constants. From the relationships
between kinetic coefficients and measured variables given above, it
follows that a given percentage error in the estimation of
Figure 1:
Time courses of
3-OMG equilibrium exchange in adipocytes and the effect of OKA and
insulin. Adipocytes were incubated in the presence of 1 mM 3OMG for 30 min at 37 °C, then further incubated for 20 min
without (squares) and with 1 µM OKA (triangles) or 10 nM insulin (circles),
prior to flux measurement. The time courses were measured using
[
Figure 2:
Effects of OKA on 3OMG equilibrium
exchange in adipocytes as a function of OKA concentration. Each data
point represents a 30 s, 3OMG equilibrium exchange time course measured
in the absence (basal) and in the presence of OKA (stimulated) at a
given concentration, otherwise identical to those illustrated in Fig. 1. The rate of exchange was expressed in terms of the first
order rate constant (per second) calculated as
described(22) .
Figure 3:
Comparison of OKA and insulin effects on
steady-state GLUT4 levels in PM and post-PM fractions measured by
semiquantitative immunoblotting analysis. Adipocytes were
incubated in KRH buffer without (BAS) and with 7 nM insulin (INS) or 1 µM OKA (OKA) for
20 min prior to subcellular fractionation into total membranes (TOT), crude plasma membranes (PM), and microsomes
(post-PM). For each membrane fraction, 50 µg of protein was used
for immunoblotted using PCR3 and
Quantitation of the 50-kDa
[
We have shown previously
that the GLUT4 recycling in basal and insulin-stimulated rat adipocytes
is a kinetically simple, first order equilibrium exchange process in a
closed, two-compartment system. In the steady state, it can be
described by the following
equation,
where S
The observed shift in steady-state GLUT4
distribution from the storage pool to cell surface by OKA would involve
an increase in k A number of diverse agents such as high pH(24) ,
hyperosmolarity (24) , oxidants(25) ,
vanadate(26) , GTPvS(27) , and OKA (16, 17, 18, 19, 20) mimic
the acute metabolic effect of insulin by stimulating glucose transport
activity in adipocytes and muscle cells. Of these insulin mimetics, OKA
is of particular interest as it is a specific inhibitor of
serine-threonine phosphatases 1 and 2A(14) , and also it
induces an insulin-like GLUT4 redistribution(17, 19) .
Our data in the present study ( Fig. 2and Fig. 4and Table 1) clearly demonstrate that OKA-induced glucose transport
stimulation and GLUT4 recruitment are effected at a similar OKA
concentration range (1-100 nM), and to a similar extent
(up to 3.1- and 3.4-fold, respectively), strongly suggesting that OKA,
like insulin, stimulates the transport primarily by GLUT4 recruitment.
Our data here also demonstrate that the extent of the OKA-induced
recruitment is considerably (by about 60%) less than that of
insulin-induced GLUT4 recruitment ( Fig. 3and Table 1),
indicating that OKA mimics insulin action only partially. Our
steady-state kinetic analysis of [ OKA at
concentrations of less than 100 nM was effective in
stimulating glucose transport and in inducing GLUT4 recruitment ( Fig. 2and Fig. 5). This is the same concentration range
where OKA exerts its well known inhibition specific to PP1 and
PP2A(14, 15, 16) and strongly suggests that
a serine-threonine phosphoprotein plays a key role in GLUT4
externalization. It is tempting to propose that insulin and OKA
stimulate the movement of GLUT4 storage vesicle to the cell surface by
invoking a common biochemical event, namely, an increased
phosphorylation state of a serine-threonine phosphoprotein, probably
mediated by an inhibition of PP1 or PP2A. The key phosphoprotein and
the putative phosphatase proposed here are yet to be identified.
Lawrence et al.(17) have shown that OKA increases the
phosphorylation of the GLUT4 and three other proteins in the
intracellular GLUT4 storage vesicles of rat adipocytes. It was also
shown that only a small portion (20%) of GLUT4 in this pool is in a
phosphorylated state, and insulin appears to increase GLUT4
phosphorylation, although slightly, in this
compartment(17, 28) . GLUT4 in the plasma membrane, on
the other hand, is largely (50% or more) in a phosphorylated state in
basal adipocytes, and insulin reduces GLUT4 phosphorylation in this
pool(17) . It is thus not unlikely that GLUT4 is the key
phosphoprotein, whose phosphorylation at the storage vesicles triggers
vesicle movement to the cell surface. Indeed, Not studied in the present investigation is
the effect of OKA on GLUT4 recruitment in insulin-stimulated, rat
adipocytes, where OKA inhibits the insulin
effect(17, 19) . Our preliminary experiments (
Volume 270,
Number 8,
Issue of February 24, 1995 pp. 3938-3943
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
) and the other for
externalization (k
). Insulin decreases k by 2.8-fold and increases k by 3.3-fold, thus increasing the steady-state cell surface GLUT4
level by approximately 8-fold (Jhun, B. H., Rampal, A. L., Liu, H.,
Lachaal, M., and Jung, C.(1992) J. Biol. Chem. 267,
17710-17715). To gain an insight into the biochemical mechanisms
that modulate these rate constants, we studied the effects upon them of
okadaic acid (OKA), a phosphatase inhibitor that exerts a insulin-like
effect on glucose transport in adipocytes. OKA stimulated
3-O-methylglucose transport maximally 3.1-fold and increased
the cell surface GLUT4 level 3.4-fold. When adipocytes were
pulse-labeled with an impermeant, covalently reactive glucose analog,
[
H]1,3-bis-(3-deoxy-D-glucopyranose-3-yloxy)-2-propyl
4-benzoylbenzoate, and the time course of labeled GLUT4 recycling was
followed, the k was found to increase 2.8-fold
upon maximal stimulation by OKA, whereas the k remained unchanged within experimental error. These findings
demonstrate that OKA mimics the insulin effect on only GLUT4
externalization and suggest that insulin stimulates GLUT4
externalization by increasing the phosphorylation state of a
serine/threonine phosphoprotein, probably by inhibiting protein
phosphatase 1 or 2A.
) and the other for
externalization (k), and that insulin modulates
both rate constants, reducing k and increasing k to about 3-fold each, thus increasing the
plasma membrane GLUT4 level by 9-fold. Biochemical mechanisms by which
adipocytes regulate these rate constants constitutively and in response
to insulin are currently unknown.
)a well known inhibitor of
protein phosphatases 1 and 2A (PP1 and PP2A,
respectively)(14) , stimulates glucose transport in adipocytes
and
muscle(15, 16, 17, 18, 19) ,
and this stimulation is largely due to GLUT4 redistribution from the
microsomal storage pool to the plasma
membrane(15, 17) . The extent of this OKA effect,
however, is relatively modest (3-6-fold) compared with the
insulin effects (10-20-fold)(12, 19) . OKA has
no effect on insulin receptor tyrosine kinase activity, indicating that
OKA acts at a step distal to insulin receptor(19) . and k, respectively, are affected by OKA.
We pulse-labeled the cell surface pool of glucose transporters in
steady state with an impermeant glucose analog,
[H]B3GL, and measured the time course of the
label mixing between the plasma membrane and the storage pool for basal
and OKA-stimulated steady states. OKA stimulated 3-OMG equilibrium
exchange flux by as much as 3.8-fold and increases the plasma membrane
GLUT4 pool size as much as 3.3-fold. The steady-state kinetic analysis
of the GLUT4 recycling in these maximally stimulated adipocytes
revealed that OKA increases the GLUT4 externalization rate constant
2.8-fold without significantly changing the internalization rate
constant. We suggest that GLUT4 externalization is regulated by the
phosphorylation state of a serine-threonine phosphoprotein and may
involve the inhibition of a specific PP1 or PP2A.
Materials
B3GL and its radioactive tracer
[H]B3GL (15 Ci/mmol) were synthesized as
described(12) . [
C]3OMG was from
Amersham Corp. OKA was purchased from Calbiochem and stored as a 100
mM stock solution in 50% dimethyl sulfoxide until use. Insulin
was a gift from Lilly. Pansorbin was obtained from Calbiochem.
Adipocytes were isolated from epididymal fat pads using male
Sprague-Dawley rats (150-200 g) as described
previously(21) .Photoaffinity Labeling of Adipocytes with
[
The procedures have been described
elsewhere(12) . Isolated adipocytes were incubated
(30-40% cytocrit) in KRH buffer (130 mM NaCl, 4.7 mM KCl, 1.25 mM MgSOH]B3GL
, 2.5 mM NaH
PO, 2.5 mM CaCl,
and 10 mM Hepes, pH 7.4) containing 0.1% BSA and 2 mM glucose or 1 mM pyruvate at 37 °C. OKA or insulin,
when used, was added in a small volume (2.5 µl/ml of cell
suspension) to a given final concentration, and incubated further for
20-30 min. The cells were chilled in water bath (10 °C) for 5
min and mixed with 24 µM of [H]B3GL
(1.7 mCi total) in 1.5 ml of prechilled, BSA-free KRH buffer in a
polypropylene dish kept on ice. The mixture was irradiated on ice for
60 s using a 450-watt mercury arc lamp (Conrad-Hanovia, Newark, NJ) at
a distance of 6.5 cm from silica sleeve and through a layer of water as
a filter. Irradiated cells were immediately washed with 20 volumes of
prechilled (10 °C) incubation buffer to remove unreacted
[H]B3GL.[
The procedures were described elsewhere in
detail (12) . Briefly, labeled adipocytes were resuspended in
preheated (37 °C) KRH buffer containing 0.1% BSA and 2 mM glucose or 1 mM pyruvate and incubated at 37 °C
(chase incubation) for a given duration for up to 60 min. Immediately
after each chase incubation, the cells were rapidly chilled by adding
20 volumes of prechilled KRH buffer containing 1% BSA and 2 mMD-glucose, which reduced the cell suspension temperature
below 10 °C, and washed twice with the same KRH buffer at 10
°C.H]B3GL-labeled GLUT4 Exchange Time
Course MeasurementMembrane Fractionation
Adipocytes were homogenized
and, after removal of the fat cakes, centrifuged at 16,000 
g for 15 min. The resulting pellet was recovered (crude plasma
membrane fraction or PM). The 16,000 g supernatant was
again centrifuged at 212,000 g for 90 min, and the
pellet was recovered (total microsomal fraction or post-PM). When
specified, the 16,000 g supernatant was further
separated into high density and low density microsomal fractions. Total
membrane fraction was obtained by pelleting total homogenate directly
by centrifugation at 212,000 g for 90 min. These
fractionation procedures have been detailed elsewhere(22) .Immunoprecipitation of
[
Membrane fractions
were solubilized in 500 µl of TBS buffer (150 mM NaCl, 10
mM Tris-HCl, pH 7.4) containing 2%
CH]B3GL-labeled GLUT4
E
, and undissolved particulates were removed
by centrifugation (185,000 g for 15 min at 4 °C).
GLUT4 in the particulate-free supernatant was immunoprecipitated at 4
°C as described (12) using PCR3 (affinity-purified,
anti-GLUT4 antibodies obtained by immunizing rabbits against the
synthetic peptide corresponding to rat GLUT4 amino acid sequence
443-483) and Pansorbin (Calbiochem). The precipitates were
solubilized with electrophoresis sample buffer (10% glycerol, 3.0%
sodium dodecyl sulfate, 1.0 mM EDTA, 2.5%
-mercaptoethanol, and 62.5 mM Tris-HCl, pH 7.4) and
separated from Pansorbin by centrifugation (14,000 rpm, 5 min) in an
Eppendorf microcentrifuge.Other Methods
Glucose transport activity was
quantitated by following the 3OMG equilibrium exchange time course
using [C]3OMG as a tracer, and calculating the
first order rate constant for tracer exchange as described
earlier(22) . Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed according to
Laemmli(23) . The gel lanes were cut into 4-mm slices with a
Bio-Rad gel slicer, and gel slice radioactivities were counted in a
liquid scintillation counter (LKB Rackbeta, Pharmacia Biotech Inc.).
Semiquantitative immunoblotting and protein determination were
performed as described elsewhere(12) .Calculation of GLUT4 Recycling Kinetic Coefficients,
k
In the
present study, the experimental measurements are obtained in the form
of the time constant, and k, and Error Estimation
, for relaxation into the steady-state
distribution in [H]B3GL pulse-chase time course
(see Fig. 6and Fig. 7), on the one hand, and an
evaluation of the relative distribution between the two compartments
once steady state has been achieved (S
and S
) (see Table 1), on the other. The
kinetic coefficients are calculated from the relationships
= (k + k)
, and S/S = k/k = K
, the distribution equilibrium constant.
H]B3GL label in PM and post-PM fractions of
basal and OKA-stimulated rat adipocytes. OKA-stimulated (1 µM OKA for 20 min at 37 °C) adipocytes (A) or basal
adipocytes (B) were pulse-labeled with
[H]B3GL in cold as in Fig. 4and subjected
to chase at 37 °C in steady state for an increasing interval as
described under ``Experimental Procedures.'' The PM (left
panels) and post-PM (right panels) fractions were
separated immediately after each chase, and the 50-kDa
[H]B3GL label intensities were quantitated by
SDS-PAGE separation and radioactivity assay as described in the legend
to Fig. 5. Chase intervals studied were 0 min (open
circles), 2 min (solid circles), 5 min (open
triangles), 7.5 min (solid triangles), 10 min (open
squares), and 30 min (solid
squares).
H]B3GL-labeled GLUT4 in
basal and OKA-stimulated adipocytes. Results of labeled GLUT4
pulse-chase time course determinations performed as illustrated in Fig. 6are analyzed for basal (open circles) and
OKA-stimulated (solid circles) adipocytes. Amounts (in
fraction of original) of the labeled GLUT4 in PM yet to be equilibrated
at each chase ((S
,eq - S,
)/(S,eq
- S,0)) were calculated and
plotted as a function of chase time according to as
described in the text, but in linear scale. Solid curves (c for basal; = 2.5 min, and d for
OKA-stimulated; = 3.03 min) are the least squares
nonlinear regression analysis of data points. Also shown in interrupted
curves are the theoretical equilibration time courses where only one of
the recycling rate constants, namely, k (curve a; = 8.3 min) or k (curve b; = 2.15 min) is
affected.
H]B3GL into GLUT4 in rat adipocytes.
Adipocytes were incubated with 1 µM OKA for 20 min at 37
°C, then photolabeled with [H]B3GL (24
µM, 1.7 mCi in 3 ml of suspension) at 10 °C for 60 s
as described under ``Experimental Procedures.'' PM (open
circles) and post-PM (solid circles) fractions were
isolated immediately after labeling, and GLUT4 immune complexes were
precipitated using PCR3 and Pansorbin.
[H]B3GL-labeled proteins in the precipitates
(originated from 500 µg of membrane protein) were separated on
SDS-PAGE, and analyzed by slicing gels, counting each gel for
radioactivity and expressing in disintegrations/gel. The molecular mass
standards were bovine albumin (66 kDa), egg albumin (45 kDa), and
carbonic anhydrase (29 kDa), respectively.
H]B3GL
incorporation into GLUT4 in PM of OKA-stimulated adipocytes as a
function of OKA concentration. Each bar represents a single
experiment similar to that illustrated in Fig. 4, using
adipocytes incubated without (basal) or with a varying concentration of
OKA, where a 50-kDa protein label with [H]B3GL
was evident as in Fig. 4. The intensity of the 50-kDa radiolabel
was quantitated from the total radioactivity of gels 11 to 14 after
correction for the background radioactivity which was arbitrarily
calculated by taking an average of the radioactivities associated with
gels other than 11-14. The incorporated label intensities were
expressed in relative quantities taking that of basal adipocytes as
1.0.
, is a particularly robust
measurement in the sense that it is not compromised by several
potential sources of experimental error. A time lag in the sampling
process, for example, provided it is constant at each sampling, will
have no effect upon the accuracy of time constant determination. This
lag will appear only as a displacement of the time axis; since the time
constant evaluation depends upon differential measurements in the time
domain, it is insensitive to such a constant lag. The apparent time
constant, , is also insensitive to incomplete separation between
the plasma membrane transporter pool, and the cytoplasmic compartment,
again assuming the degree of cross-contamination is constant over the
series of samples. and S (Table 1), on the other hand, is sensitive to
cross-contamination between the two GLUT4 pools in immunoblot
experiments. In the present study, we minimized this problem by
assessing the pool sizes in two separate steps. With insulin-stimulated
adipocytes, we assessed the equilibrium pool sizes by immunoblot after
subcellular fractionation. In the insulin-stimulated state, the
difference between the two pool sizes is much reduced (37 and 63%) in
comparison with the basal state, and thus the error due to
cross-contamination is minimal(12) . We then measured relative
changes in the plasma membrane pool size S between control and insulin-stimulated and OKA-stimulated states
by [H]B3GL labeling, where cross-contamination
introduces little error(12) .
appears as the same fractional error in the values of the rate
constants. Added to this is the percentage error in the measured value
of K, attenuated by the factors K/(1 + K) for k and 1/(1 + K) in the
case of k.
Effect of OKA on Glucose Transport in Rat
Adipocytes
Data in Fig. 1illustrate a typical time
course of 1 mM 3OMG equilibrium exchange in isolated rat
adipocytes and how it is affected by incubation with insulin and OKA.
Insulin (7 nM, incubated for 20 min at 37 °C) stimulated
the exchange flux 8.1 ± 2.2-fold (mean of 12 determinations with
± S.D.). OKA (1 µM, incubated for 20 min at 37
°C) also stimulated the exchange flux, but the stimulation was
significantly less in extent (3.1 ± 0.5-fold, mean of three
determinations ± S.D.) compared with that of insulin. The OKA
effect was fully expressed at 5-min incubation and did not increase
appreciably with further incubation for up to 60 min (not illustrated).
The stimulation was a saturable function of OKA concentration, showing
an apparent half-maximal effect at about 5 nM OKA (Fig. 2). These data with OKA are in accord with the previous
reports by others(16, 17, 19) , where OKA at
submicromolar concentrations was shown to stimulate net uptake of
2-deoxyglucose and 3-O-methylglucose in isolated rat
adipocytes.
C]3OMG as a tracer (see ``Experimental
Procedures'') and analyzed in semilog plots(22) , where C
, C
, and C
represent the cell-associated
radioactivities at the start, after t seconds, and after 15
min (complete tracer equilibrium), respectively. Each data point
represents single determination. Straight lines are drawn by
the least squares linear regression
analysis.
Effect of OKA and Insulin on Steady-state GLUT4
Subcellular Distribution Measured by Immunoblot
Data of
subcellular fractionation and semiquantitative immunoblotting analysis
have shown that OKA increases the cell surface GLUT4 level in rat
adipocytes by recruiting GLUT4 from the storage
pool(17, 19) . We reproduced this OKA effect on GLUT4
recruitment here (Fig. 3). An acute (20 min) incubation of rat
adipocytes with 1 µM OKA did not change total cellular
(total membrane fraction) GLUT4 content, but significantly increased
the cell surface (PM) GLUT4 level with a concomitant reduction in the
microsomal (post-PM) GLUT4 level. The plasma membrane GLUT4 level was
increased 2.8 ± 0.6-fold (mean with S.D., n = 3)
in the presence of 1 µM OKA. The extent of this OKA
effect, however, was considerably less compared with that of the
insulin effect (Fig. 3). The PM GLUT4 pool size of
insulin-stimulated (10 nM, 20 min at 37 °C) adipocytes was
readily detectable by immunoblot (Fig. 3) and amounted to more
than one-half of the storage pool size (Table 1). That the error
due to incomplete separation of the storage pool and plasma membrane in
this assessment is minimal is confirmed by the observed changes in
GLUT4 immunoblot intensity of LDM, the fraction that is little
contaminated with plasma membranes: OKA (1 µM) and insulin
(10 nM, 20 min) reduced LDM GLUT4 pool size to 85.4 ±
9.6 and 67.1 ± 6.1%, respectively (means of five independent
determinations ± S.D.) of that of basal (Table 1).
I-protein A as
described(22) . Similar results were reproduced in two other
experiments.
Effects of OKA and Insulin on the Plasma Membrane GLUT4
Level in Adipocytes
Relative changes in the plasma membrane
GLUT4 level in adipocytes can be quantitated more precisely by the use
of [H]B3GL, a membrane-impermeable, covalently
active, glucose analog, and immunoprecipitation(12) .
OKA-treated adipocytes were pulse-labeled with
[H]B3GL in the presence of OKA at 10 °C (see
``Experimental Procedures'') and immediately subjected to
subcellular fractionation to separate the crude PM and microsomal
(post-PM) fractions. When the PM fraction was immunoprecipitated with
anti-GLUT4 antibody (PCR3), and the immunoprecipitate separated by
SDS-PAGE, a single radiolabeled polypeptide of an apparent molecular
mass of 50 kDa was observed (Fig. 4), reproducing the pattern of
[H]B3GL incorporation to GLUT4 reported
previously(12) . As also shown in (12) , this 50-kDa
radiolabeling was greatly suppressed when adipocytes were pulse-labeled
in the presence of an excess of maltose, a nonpermeable, competitive
inhibitor of glucose transporter (not illustrated), indicating that the
50-kDa label is due to glucose transporter. Little 50-kDa protein
labeling was detectable when the post-PM fraction was subjected to the
same immunoprecipitation and SDS-PAGE separation (Fig. 4),
indicating that the labeling is essentially cell surface
pool-specific(12) .H]B3GL labeling intensity revealed that the
OKA-induced increase in the plasma membrane GLUT4 content is a
saturable function of OKA concentration (Fig. 5). The effect was
half-maximal at around 10 nM, and maximal at 100 nM with no further increase at 1 µM OKA. The GLUT4 level
in PM was 3.36 ± 0.31-fold (mean of three independent
experiments ± S.D.) greater in the OKA (1
µM)-stimulated adipocytes compared with that in untreated
adipocytes (Table 1). Since [H]B3GL
labeling of microsomal GLUT4 was negligible (Fig. 4), this -fold
increase calculation should be free from error due to imperfect
separation of the two pools. In parallel experiments, insulin (10
nM, 30 min) increased the plasma membrane GLUT4 pool size by
6.37 ± 1.5-fold (average of three sets of determination ±
S.D.) compared with basal adipocytes (Table 1).Effects of OKA on the Steady-state GLUT4 Recycling
Kinetics in Rat Adipocytes
Isolated adipocytes were incubated
either in KRH buffer alone or in KRH buffer containing 1 µM OKA for 30 min at 37 °C, then photolabeled with
[H]B3GL for 60 s at 10-15 °C as above (Fig. 4). The labeled cells were quickly resuspended into a
prewarmed (37 °C) KRB buffer and incubated at 37 °C (chase
incubation) for a specified time interval up to 60 min. Where
OKA-stimulated cells were used, the same concentration of OKA was
continuously present throughout the experiment, including chase
incubation, to maintain steady-state conditions. At the end of each
chase incubation, the cells were immediately subjected to
homogenization and membrane fractionation at 10 °C. The resulting
PM and post-PM were immunoprecipitated using PCR3, and the 50-kDa
radiolabel intensities of the immunoprecipitates were quantitated after
SDS-PAGE separation as in Fig. 4. During the 37 °C chase,
the 50-kDa label intensity (labeled GLUT4) decreased in the PM
fraction, with the decrement being stoichiometrically recovered in the
post-PM fraction (Fig. 6). This apparent redistribution of
labeled GLUT4 between PM and post-PM was quite rapid, and virtually
complete within 10 min at 37 °C for both basal and OKA-stimulated
adipocytes. Immunoblot data (not illustrated) indicated that the
amounts of GLUT4 protein in PM and post-PM remained constant throughout
the chase incubation in these experiments. The radiolabel mixing time
courses similar to these have been observed for basal and
insulin-stimulated adipocytes(12) .
represents the amount of
[H]B3GL-labeled GLUT4 in the plasma membrane
pool, is the time constant (= t
/ln 2) for the exchange process, and the
subscripts 0, t, and eq signify values at the start, at
arbitrary time t, and after complete tracer equilibration
(estimated after 60 min) of the chase incubation, respectively. The
plasma membrane GLUT4 label intensities after each chase revealed in Fig. 6were plotted against chase time in Fig. 7on a
semi-log scale corresponding to . It is clear from this
analysis that the GLUT4 recycling in OKA-stimulated adipocytes appears
also to be a simple, first order process of equilibrium exchange in a
closed, two-compartment system in steady state. The values of the
steady-state unidirectional flux 
, time constant, and kinetic
rate coefficients calculated for basal and OKA-stimulated adipocytes
are shown in Table 2.
, a decrease in k, or changes in both. To determine how OKA
affects these two rate constants, we calculated k and k for the basal and maximally
OKA-stimulated steady states. The calculation uses the values for S, S
, and .
Steady-state GLUT4 compartment sizes (S and S) are obtained from the combined data of
immunoblots and [H]B3GL labeling for
insulin-stimulated adipocytes as well as basal and OKA-stimulated
adipocytes (Table 1). Values of calculated k and k are summarized in Table 2. OKA
increased k by 2.8-fold and reduced k by 40%. The uncertainty associated with these
values of the kinetic coefficients reflects both the error in the
measurement of and that introduced through evaluation of the
steady-state distribution, expressed in terms of the distribution
equilibrium constant, K (see ``Experimental
Procedures''). Table 1shows the K values calculated from the measured pool sizes; in each case, the
error is estimated to be about 12%. Table 2indicates the error
associated with the measured time constant values, ranging from 16 to
24%. Since these errors should be statistically independent, they are
combined as the root square sum, yielding the kinetic coefficient error
estimates indicated in Table 2. The observed 40% change in k value in the presence of OKA is not significant
within these experimental errors, whereas the effect of OKA on k observed here is highly significant.
H]B3GL
pulse-labeled GLUT4 chase time courses here ( Fig. 6and Fig. 7and Table 2) demonstrates that the GLUT4 in
OKA-stimulated adipocytes also recycles with two distinct first order
rate constants, k and k,
and that the value of k in OKA-stimulated cells
is 2.8-fold greater than that of basal (control) adipocytes, whereas
that of k is not significantly different between
the basal and OKA-stimulated adipocytes. This is the first clear
demonstration that OKA induces GLUT4 recruitment primarily, if not
exclusively, by modulating its externalization pathway. This is in
contrast to the insulin-induced GLUT4 recruitment in rat adipocytes,
where both k and k are
shown to be affected; insulin increased the former 3.4-fold and reduced
the latter 2.8-fold(12) . OKA thus mimics the insulin effect on
GLUT4 externalization rate constant (k) almost
quantitatively in rat adipocytes, but fails to mimic the insulin effect
on its internalization rate constant (k). This
would in part explain the fact that the maximum extent of GLUT4
recruitment is considerably smaller with OKA than with insulin ( Fig. 3and Table 1)(17, 19) .
P-labeled
GLUT4 in adipocyte LDM was shown to be dephosphorylated in vitro by PP1 and PP2A. GLUT4 translocation by GLUT4 phosphorylation,
however, is yet to be demonstrated(29) . Insulin and OKA also
cause a redistribution of Rab4 from GLUT4 storage vesicles to the
cytosol in rat adipocytes, and the two agents are equipotent in this
Rab4 redistribution(30) , suggesting that phosphorylation of
Rab4 or its associated protein, rather than GLUT4 itself, may trigger
GLUT4 externalization.)(not shown) suggest that this second effect of OKA
requires a slightly higher OKA concentration (0.1-1
µM), where it blocks the insulin-induced reduction of
GLUT4 internalization. This is consistent with the recent proposal that
insulin increases plasma membrane GLUT4 level largely by preventing
GLUT4 internalization as a result of GLUT4 dephosphorylation at a
serine residue(17, 19) .
))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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