©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Okadaic Acid Stimulates Glucose Transport in Rat Adipocytes by Increasing the Externalization Rate Constant of GLUT4 Recycling (*)

(Received for publication, September 8, 1994; and in revised form, December 13, 1994)

Amrit L. Rampal Byung H. Jhun Sungsoo Kim (§) Hongzhi Liu Michael Manka Mohsen Lachaal (¶) Robert A. Spangler Chan Y. Jung (**)

From the Biophysical Laboratory, Veterans Administration Medical Center and the Department of Biophysical Sciences, State University of New York at Buffalo, Buffalo, New York 14215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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) 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, [^3H]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.


INTRODUCTION

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) 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.

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, (^1)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) .

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 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, [^3H]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.


EXPERIMENTAL PROCEDURES

Materials

B3GL and its radioactive tracer [^3H]B3GL (15 Ci/mmol) were synthesized as described(12) . [^14C]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 [^3H]B3GL

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 MgSO(4), 2.5 mM NaH(2)PO(4), 2.5 mM CaCl(2), 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 [^3H]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 [^3H]B3GL.

[^3H]B3GL-labeled GLUT4 Exchange Time Course Measurement

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.

Membrane Fractionation

Adipocytes were homogenized and, after removal of the fat cakes, centrifuged at 16,000 times g for 15 min. The resulting pellet was recovered (crude plasma membrane fraction or PM). The 16,000 times g supernatant was again centrifuged at 212,000 times g for 90 min, and the pellet was recovered (total microsomal fraction or post-PM). When specified, the 16,000 times 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 times g for 90 min. These fractionation procedures have been detailed elsewhere(22) .

Immunoprecipitation of [^3H]B3GL-labeled GLUT4

Membrane fractions were solubilized in 500 µl of TBS buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4) containing 2% CE(8), and undissolved particulates were removed by centrifugation (185,000 times 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% beta-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 [^14C]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 and k, and Error Estimation

In the present study, the experimental measurements are obtained in the form of the time constant, , for relaxation into the steady-state distribution in [^3H]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(e), the distribution equilibrium constant.


Figure 6: Time-dependent changes in [^3H]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 [^3H]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 [^3H]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).




Figure 7: Analysis of the steady-state equilibrium exchange time course of [^3H]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.






Figure 4: The cell surface-selective incorporation of [^3H]B3GL into GLUT4 in rat adipocytes. Adipocytes were incubated with 1 µM OKA for 20 min at 37 °C, then photolabeled with [^3H]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. [^3H]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.




Figure 5: [^3H]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 [^3H]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.



The time constant of the process, , 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.

The measurements of the equilibrium distribution pool sizes, S 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 [^3H]B3GL labeling, where cross-contamination introduces little error(12) .

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 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(e), attenuated by the factors K(e)/(1 + K(e)) for k and 1/(1 + K(e)) in the case of k.


RESULTS

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.


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 [^14C]3OMG as a tracer (see ``Experimental Procedures'') and analyzed in semilog plots(22) , where C(t), C(o), 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.




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) .



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).


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 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 [^3H]B3GL, a membrane-impermeable, covalently active, glucose analog, and immunoprecipitation(12) . OKA-treated adipocytes were pulse-labeled with [^3H]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 [^3H]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) .

Quantitation of the 50-kDa [^3H]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 [(3)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 [^3H]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) .

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 represents the amount of [^3H]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.



The observed shift in steady-state GLUT4 distribution from the storage pool to cell surface by OKA would involve an increase in k, 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 [^3H]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(e) (see ``Experimental Procedures''). Table 1shows the K(e) 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.


DISCUSSION

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 [^3H]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) .

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, 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 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 (^2)(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) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK13376, the American Heart Association, and the Veterans Administration Medical Center, Buffalo, NY. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Kyung Hee University, College of Medicine, Seoul 130-701, Korea.

Recipient of the American Heart Association Clinical Scientist Award 900443.

**
To whom correspondence and reprint requests should be addressed.

(^1)
The abbreviations used are: OKA, okadaic acid; 3OMG, 3-O-methyl-D-glucose; B3GL, 1,3-bis-(3-deoxy-D-glucopyranose-3-yloxy)-2-propyl 4-benzoylbenzoate; PM, plasma membrane; post-PM, post plasma membrane fraction; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.

(^2)
M. Lachaal, A. L. Rampal, and C. Y. Jung, unpublished results.


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