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J Biol Chem, Vol. 274, Issue 35, 24677-24684, August 27, 1999


Identification of Wortmannin-sensitive Targets in 3T3-L1 Adipocytes
DISSOCIATION OF INSULIN-STIMULATED GLUCOSE UPTAKE AND GLUT4 TRANSLOCATION*

Sharon F. HausdorffDagger , Diane C. Fingar§, Kazuko Morioka, Luis A. Garza, Eileen L. Whitemanparallel , Scott A. Summers**, and Morris J. BirnbaumDagger Dagger

From the Howard Hughes Medical Institute, the Cox Institute, and the Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The current studies investigated the contribution of phosphatidylinositol 3-kinase (PI3-kinase) isoforms to insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation. Experiments involving the microinjection of antibodies specific for the p110 catalytic subunit of class I PI3-kinases demonstrated an absolute requirement for this form of the enzyme in GLUT4 translocation. This finding was confirmed by the demonstration that the PI3-kinase antagonist wortmannin inhibits GLUT4 and insulin-responsive aminopeptidase translocation with a dose response identical to that required to inhibit another class I PI3-kinase-dependent event, activation of pp70 S6-kinase. Interestingly, wortmannin inhibited insulin-stimulated glucose uptake at much lower doses, suggesting the existence of a second, higher affinity target of the drug. Subsequent removal of wortmannin from the media shifted this dose-response curve to one resembling that for GLUT4 translocation and pp70 S6-kinase. This is consistent with the lower affinity target being p110, which is irreversibly inhibited by wortmannin. Wortmannin did not reduce glucose uptake in cells stably expressing Myr-Akt, which constitutively induced GLUT4 translocation to the plasma membrane; this demonstrates that wortmannin does not inhibit the transporters directly. In addition to elucidating a second wortmannin-sensitive pathway in 3T3-L1 adipocytes, these studies suggest that the presence of GLUT4 on the plasma membrane is not sufficient for activation of glucose uptake.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The metabolic hormone insulin promotes the disposal of glucose into its peripheral target tissues, adipose and muscle. Insulin-stimulated glucose uptake is mediated primarily by the rapid movement of the fat/muscle-specific glucose transporter GLUT41 from a latent intracellular compartment to the cell surface (1). An insulin-responsive aminopeptidase (IRAP) also resides in this compartment, and this protein also translocates to the plasma membrane after insulin stimulation (2, 3). Insulin-responsive tissues also express GLUT1, a ubiquitous glucose transporter largely responsible for basal uptake (1). Whereas GLUT4 and IRAP reside predominantly in an intracellular compartment in the basal state and are largely excluded from the plasma membrane, GLUT1 localizes significantly to the plasma membrane in addition to the cell interior. Glucose uptake can be regulated by several distinct mechanisms. Acute insulin treatment stimulates the translocation of both GLUT4 and GLUT1 to the cell surface, thus significantly increasing the permeability of the membrane for glucose. Chronic exposure to insulin increases glucose uptake predominantly by up-regulating GLUT1 gene expression through a transcriptional mechanism (4, 5). Additionally, several lines of evidence suggest that the catalytic activity of GLUT4 and GLUT1 on the plasma membrane may be regulated (6).

Recently, much attention has focused on elucidating the signal transduction pathways that regulate insulin-stimulated glucose uptake and GLUT4 translocation (7). A great deal is now known regarding signaling events that occur at the level of the receptor. Insulin binding to and activation of its receptor tyrosine kinase results in the rapid phosphorylation of downstream substrates, such as the insulin receptor substrates, which recruit signaling molecules containing SH2 domains into an active signaling complex. Engagement of these SH2 domain-containing proteins with the tyrosine-phosphorylated motifs on IRS-1 activates many of these molecules, including the phosphatidylinositol 3-kinase (PI 3-kinase), the tyrosine-specific phosphatase SHPTP2, and Grb2/SOS-mediated loading of GTP onto p21ras. Most of these do not mediate insulin-stimulated GLUT4 translocation (8-12).

In recent years, a general consensus has emerged that PI3-kinase is the one IRS-docking protein that is a candidate for an an obligate intermediate in the insulin-signaling pathway leading to accelerated glucose transport. This conclusion derives from two lines of evidence as follows: inhibition of glucose transport by drugs such as wortmannin and LY294002, which inhibit some PI3-kinases (13-15); and antagonism of the response by truncation mutants of the p85 regulatory subunit of PI3-kinase, which block association and activation of the p110alpha and -beta catalytic subunits. However, each protocol suffers significant drawbacks. Questions persist concerning the specificity of inhibitors, and it is already well established that wortmannin inhibits a number of PI3-kinase isoforms with nanomolar efficiency. Similarly, it is difficult to be certain concerning the in vivo specificity of p85 SH2 domain interactions, particularly at the high concentrations achieved by somatic cell microinjection. Thus, we decided to address this problem by two independent strategies as follows: a careful analysis of the effects of a range of concentrations of wortmannin on insulin-stimulated glucose transport, and the utilization of a PI3-kinase isoform-specific neutralizing antisera.

A growing family of PI3-kinase proteins has been identified. Class I PI3-kinases are heterodimers containing an adaptor/regulatory subunit and a tightly associated catalytic subunit. Class IA proteins specifically contain an 85-kDa regulatory subunit, which is activated by tyrosine-phosphorylated proteins, such as IRS-1, upon binding of its SH2 domains to tyrosine-phosphorylated YXXM motifs, and a 110-kDa catalytic subunit (16). Class IB regulatory subunits are unrelated to those in class IA but interact with a largely homologous, although distinct, set of catalytic subunits (16). Class II and III PI3-kinases are also widely expressed (16), and many isoforms retain sensitivity to wortmannin (16, 17). Class III PI3-kinases utilize only PI as a substrate.

Class IA PI3-kinase is thought to mediate insulin's stimulation of GLUT4 translocation, based largely on its recruitment by IRS proteins. As noted above, experiments expressing dominant negative forms of p85 in 3T3-L1 adipocytes support this hypothesis; microinjection of either dominant negative mutants of the p85 regulatory subunit of PI3-kinase or a GST-p85 fusion protein into 3T3-L1 adipocytes blocks GLUT4 translocation (18, 19); similarly, adenovirus-mediated overexpression of an amino-terminal SH2 domain of a p85 domain also blocks insulin-stimulated glucose metabolism (20). However, recent studies indicate that IRS-associated PI3-kinase activity may not be required for insulin-stimulated GLUT4 translocation, and an alternative PI3-kinase-dependent pathway has been proposed (21-23). Therefore, the role of class IA PI3-kinases in insulin-stimulated GLUT4 translocation requires further verification.

The studies described below summarize data accumulated using wortmannin to identify which PI3-kinases regulate its inhibition of insulin-mediated events. At least two different wortmannin targets were identified that regulate its inhibition of glucose uptake; the p110 catalytic subunit of class 1A PI3-kinases was found to be a lower affinity target regulating GLUT4 translocation, and a second, higher affinity target was found which is important for the inhibition of cell-surface glucose transport activity by wortmannin. In addition to demonstrating a role for p110 in insulin-stimulated GLUT4 translocation, these data describe the dissociation of insulin-stimulated glucose uptake and GLUT4 translocation, suggesting the requirement for insulin-induced activation of glucose transporters.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Crystalline porcine insulin was a gift of Lilly. LY294002 was a gift of Dr. Lewis Cantley (Harvard Medical School, Boston). Antibodies against p100alpha and IRAP were kindly donated by Dr. Sara Courtneidge (Harvard Medical School, Boston) (24) and Metabolex, Inc. (Hayward, CA), respectively. Rhodamine-conjugated goat anti-rabbit antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). Bovine serum albumin used in translocation assays was from Calbiochem. Wortmannin was purchased from Sigma and stored as a 10 mM stock in Me2SO. 2-[1,2- 3H]Deoxy-D-glucose (26.2 Ci/mmol) was purchased from NEN Life Science Products. [gamma -32P]ATP (4500 Ci/mmol) and 125I-protein A were purchased from ICN Radiochemicals (Irvine, CA). For the determination of protein concentrations, a bicinchoninic acid protein assay kit was purchased from Pierce. All other chemicals were from Sigma.

Cell Culture-- 3T3-L1 fibroblasts were grown at 37 °C in a humidified atmosphere of 7.5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum (Life Technologies Inc.). Cells were plated onto either 18-mm square 1 coverslips or 12-well plates and differentiated 1 to 2 days post-confluence with dexamethasone (0.4 mg/ml), 1-methyl-3-isobutylxanthine (0.5 mM), and 10% fetal bovine serum as described (25) but without supplemental insulin. Adipocytes were maintained in DMEM containing 10% fetal bovine serum, fed approximately every 4 days, and used at 10-30 days post-differentiation. 3T3-L1/P2 cells were made by retroviral mediated gene transfer of a plasmid encoding an epitope-tagged GLUT4 in which the insulin receptor P2 epitope (RDIYETDYYRKGGKGLLPVR) was inserted in the first extracellular loop.2 3T3-L1 fibroblasts expressing both a myristoylated, constitutively active form of the serine/threonine kinase Akt (Myr-Akt (Delta 4-129)) and a non-myristoylated control mutant (A2-Myr-Akt (Delta 4-129)) were generously provided by Richard Roth, Stanford University, Stanford, CA. The constitutively active Myr-Akt construct includes an amino-terminal myristoylation sequence rendering the molecule constitutively active. The non-myristoylated control has the second glycine in this myristoylation sequence changed to an alanine and is therefore not constitutively active and is regulated normally. The PH domain, amino acids 4-129, was removed from both constructs. The effects of stable expression of these constructs into 3T3-L1s on glucose uptake was described previously (26).

Immunofluorescence of Plasma Membrane Sheets and Intact Cells-- To measure the translocation of GLUT4 and GLUT1, the plasma membrane sheet assay was used (10, 26). Adipocytes were incubated in Leibovitz's L-15 medium (Life Technologies, Inc.) containing 0.2% BSA for 2 h at 37 °C in room air and then treated with Me2SO (vehicle for the drug) or with wortmannin (diluted in Me2SO) for 30 min followed by incubation in the absence or presence of insulin (final concentration 100 nM) for 15 min. Plasma membrane "sheets" were prepared and processed for indirect immunofluorescence using affinity purified antibodies to the carboxyl-terminal portion of GLUT4 or serum-containing antibodies to the carboxyl-terminal portion of GLUT1. Antibodies to GLUT1 were a gift of Miles Pharmaceuticals (West Haven, CT). The amount of glucose transporter on the plasma membrane was quantitated by measuring the fluorescence intensity of at least five fields of sheets for each wortmannin concentration. Digital image processing was performed as described previously (8, 26).

Microinjection of antibody into differentiated 3T3-L1 adipocytes was performed as described previously (12). Anti-PI3-kinase antibody was mixed with a membrane-targeted maltose-binding protein to yield final concentrations of 5 and 1 mg/ml, respectively. Antibody directed against maltose-binding protein was utilized to identify plasma membrane sheets derived from microinjected cells. In all experiments, proteins were injected into the cytoplasm of 50-100 adipocytes. The abundance of GLUT4 on the plasma membrane of microinjected cells was quantitated as described previously (8). Injection of non-immune IgG demonstrated no effect on the distribution of GLUT4 in 3T3-L1 adipocytes (8).

To determine the reversibility of the inhibition of GLUT4 translocation by wortmannin, the drug was removed after the initial 30-min pretreatment by washing the adipocytes three times with L-15 media containing 0.2% BSA and incubating the cells for 30 min in the same media in the absence of wortmannin. Insulin was then added to 100 nM for 15 min, and plasma membrane sheets were prepared as described above.

To measure the accessibility of GLUT4 to the extracellular space, 3T3-L1/P2 adipocytes were treated with wortmannin and insulin as described above for the plasma membrane sheet assay, except indirect immunofluorescence was performed on intact, unpermeabilized cells using affinity purified antiserum generated against the P2 peptide.

IRAP Translocation Assay-- IRAP Translocation was determined using an IRAP biotinylation assay similar to that described previously (27). 3T3-L1 adipocytes in 6-well dishes were washed twice in PBS, once in L-15 media with 0.2% BSA, and left in that media for 2 h at 37 °C. Cells were treated with wortmannin and insulin exactly as for the GLUT4 translocation assay. All subsequent steps were performed at 4 °C. Cells were washed twice in ice-cold KRPH (128 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.25 mM MgSO4, 5 mM NaPO4, 20 mM Hepes, pH 7.4) and treated with 1 ml of 0.5 mg/ml sulfo-NHS-LC-LC-biotin (Pierce) for 30 min. Each plate was then bathed three times for 10 min each in KRPH containing 20 mM glycine, twice with KRPH, and finally lysed in 800 µl of solubilization buffer (1% Triton, 150 mM NaCl, 20 mM Tris-Cl, 5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µM leupeptin, 1 µM pepstatin A, pH 7.4). The lysate was vortexed briefly, incubated for 15 min, and centrifuged at 23,000 × g for 15 min. After filtering the lysates in a 45-µm filter (Millipore), BCA assay (Pierce) was performed to determine protein concentration. 600 µg of protein was diluted to 500 µl with solubilization buffer and immunoprecipitated with 0.6 µl of anti-IRAP sera (Metabolex) overnight, followed by 3-6 h incubation in 30 µl of protein A-Sepharose (Life Technologies, Inc.). These conditions were shown to consistently remove essentially all IRAP from the supernatant. SDS gels of the immunoadsorbates were transferred to PVDF+ membranes (Fisher), blocked in TBS-T with 6% BSA, treated with 1 µg/ml streptavidin-horseradish peroxidase (Pierce) for 2 h, washed in TBS-T, and developed using ECL+ (Amersham Pharmacia Biotech) on a STORM 860 Scanner. The signal intensity of quantitated samples was shown to be within the linear range of detection.

Glucose Transport Assay-- Hexose uptake, as assayed by the accumulation of 0.1 mM 2-deoxy-D-[3H]glucose, was measured as described previously with the following modifications (10, 25). 3T3-L1 adipocytes in 12-well plates were washed twice with KRP buffer (136 mM NaCl, 4.7 mM KCl, 10 mM NaPO4, 0.9 mM CaCl2, 0.9 mM MgSO4, pH 7.4) warmed to 37 °C and containing 0.2% BSA, incubated in Leibovitz's L-15 medium containing 0.2% BSA for 2 h at 37 °C in room air, washed twice again with KRP containing, 0.2% BSA buffer, and incubated in KRP, 0.2% BSA buffer in the absence (Me2SO only) or presence of wortmannin for 30 min at 37 °C in room air. Insulin was then added to a final concentration of 100 nM for 15 min, and the uptake of 2-deoxy-D-[3H]glucose was measured for the last 4 min. Nonspecific uptake, measured in the presence of 10 µM cytochalasin B, was subtracted from all values. Protein concentrations were determined with the Pierce bicinchoninic acid assay. Uptake was measured routinely in triplicate or quadruplicate for each experiment.

To determine the reversibility of the effect of wortmannin on hexose uptake, the drug was removed after the initial 30-min pretreatment by washing the adipocytes three times with KRP containing 0.2% BSA buffer and incubating the cells for 30 min in KRP containing 0.2% BSA buffer in the absence of drug. Insulin was then added to 100 nM, and the uptake of 2-deoxy-D-[3H]glucose was measured for the last 4 min as described above.

pp70-S6 Kinase Assay-- The activity of pp70-S6 kinase was measured by immune complex kinase assay as described previously (10). Briefly, 3T3-L1 adipocytes in 10-cm plates were incubated for 20-24 h in DMEM containing 0.5% BSA and 10 mM Hepes, pH 7.5, prior to an experiment. The cells were then incubated in the absence or presence of wortmannin for 30 min before the addition of 100 nM insulin for 15 min. Cell lysates were prepared as described, except the lysis buffer was supplemented with detergents (0.5% Nonidet P-40 and 0.1% sodium deoxycholate), and Dounce homogenization was not performed. The lysates were immunoprecipitated with polyclonal antisera generated against the amino terminus of pp70-S6 kinase, adsorbed to protein A-Sepharose, washed, and the phosphotransferase activity toward 40 S ribosomes was measured in vitro as described (10).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Wortmannin inhibits insulin-stimulated hexose uptake and GLUT4 and IRAP translocation in multiple tissues (14, 28-32). Interestingly, we observed that in 3T3-L1 adipocytes, wortmannin inhibited insulin-stimulated glucose uptake and GLUT4 and IRAP translocation with distinct dose responses. These cells were treated with increasing concentrations of wortmannin for 30 min, followed by stimulation with 100 nM insulin for 15 min; the uptake of 2-deoxyglucose and the translocation of GLUT4 and IRAP were then measured in parallel. Whereas wortmannin inhibited glucose uptake and GLUT4 and IRAP translocation, their sensitivities to wortmannin were markedly different (Fig. 1A). The half-maximal dose for inhibition of 2-deoxyglucose uptake was approximately 6 nM wortmannin, whereas that for inhibition of GLUT4/IRAP translocation was approximately 80 nM (Fig. 1, A and B). The most striking disparity was at 10 nM wortmannin, where 2-deoxyglucose uptake was inhibited ~77%, and a full complement of GLUT4 and IRAP was detectable on the cell surface. Similarly, at 30 nM wortmannin, 2-deoxyglucose uptake was reduced to basal levels, and GLUT4/IRAP translocation was inhibited only ~12% (Fig. 1, A and B). These results suggest that either 2-deoxyglucose uptake and translocation are mediated by separate targets of wortmannin or require different amounts of PI 3-kinase activity.


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  		Fig. 1.   Effect of wortmannin on insulin-stimulated 2-[3H]deoxyglucose uptake, GLUT4 translocation, and IRAP translocation in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were pretreated with various concentrations of wortmannin for 45 min with insulin (100 nM) present for the last 15 min. The uptake of 2-[3H]deoxyglucose (2-DOG) or the extent of GLUT4 or IRAP translocation was then determined using the plasma membrane sheet assay or surface biotinylation, respectively. Values in the presence of insulin but the absence of wortmannin were set to 100%, and the other values were normalized accordingly. The 2-[3H]deoxyglucose uptake curve represents the mean ± S.E. of seven experiments, and both translocation curves represent the mean ± S.E. of three experiments. Values obtained in the absence of insulin (NO INSULIN) are also shown. B, a composite photomicrograph of a typical plasma membrane sheet experiment depicting the effect of increasing concentrations of wortmannin on the translocation of GLUT4 to the cell surface. Images such as these were quantitated to give the GLUT4 translocation curve in A. C, a typical streptavidin/horseradish peroxidase-stained Western blot of IRAP immunoprecipitates depicting the effect of increasing concentrations of wortmannin (Wort) on the accessibility of IRAP to the membrane-impermeant biotin reagent.

To evaluate further whether either of these effects were dependent upon PI3-kinase, the dose response for wortmannin's inhibition of another PI3-kinase-dependent response, activation of pp70 S6-kinase (15, 33), was also measured. Wortmannin inhibited activation of pp70 S6-kinase in 3T3-L1 adipocytes toward the 40 S ribosome with a dose response resembling that obtained for GLUT4 translocation (Fig. 2A). Although pp70 S6- kinase displayed full activity with 30 nM wortmannin pretreatment, kinase activity fell precipitously upon treatment with 100 nM wortmannin. This result suggests that the target mediating pp70 S6-kinase and GLUT4 translocation are the same and is most likely to be a class I PI3-kinase (33). LY294002, which is chemically unrelated to wortmannin, also inhibited insulin-stimulated hexose uptake and GLUT4 translocation with identical dose responses, suggesting that these responses are blocked due to inhibition of p110 PI3-kinase (Fig. 3). This effect is in marked contrast to that with wortmannin (Fig. 1), suggesting that the site of action of wortmannin responsible for selectively inhibiting hexose uptake is not sensitive to LY294002.


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  		Fig. 2.   Effect of wortmannin on insulin-stimulated activation of pp70-S6 kinase in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were serum-deprived for ~24 h and treated with various concentrations of wortmannin for 45 min in the absence (-) or presence (+) of 100 nM insulin (INS) for the last 15 min. Equal amounts of protein from whole cell lysates were immunoprecipitated with anti-pp70 S6-kinase antisera, and in vitro kinase reactions were performed using 40 S ribosomes as substrate. Ribosomal S6 protein was resolved by SDS-polyacrylamide gel electrophoresis, and the incorporated phosphate was visualized by autoradiography. B, the autoradiograph in A was quantitated on a Molecular Dynamics PhosphorImager. The level of S6 phosphorylation obtained with insulin in the absence of wortmannin was set to 100%, and other values were normalized accordingly. Results are plotted against the dose-response curve for inhibition of GLUT4 translocation from figure (Fig. 1A).


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  		Fig. 3.   Effect of LY294002 on insulin-stimulated GLUT4 translocation and 2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes. 3T3-L1 adipocytes were pretreated with various concentrations of LY294002 for 45 min in the absence (No Insulin) or presence of 100 nM insulin for the last 15 min. The uptake of 2-[3H]deoxyglucose (2-DOG) or the extent of GLUT4 translocation to the cell surface was determined as described, and results are presented as the mean ± S.E. of three experiments. The level of 2-[3H]deoxyglucose uptake or GLUT4 translocation obtained with insulin in the absence of LY294002 was set to 100%, and other values were then normalized accordingly.

When proteins from cells treated with wortmannin are separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with an antibody to wortmannin, a single band of 110 kDa is present, suggesting that wortmannin binds the p110 subunit covalently (34, 35). We therefore hypothesized that if wortmannin were washed away prior to stimulation with insulin, the wortmannin would remain bound to p110 but possibly not to the high affinity target regulating transport. If this were correct, removal of wortmannin would shift the dose-response curve defining the inhibition of transport by wortmannin, and a curve resembling the one for GLUT4 translocation would result. Consistent with this idea, the sensitivity of GLUT4 translocation to inhibition by wortmannin was identical regardless of whether drug was present or had been washed away 30 min previously (Fig. 4A). However, when hexose uptake was measured after removal of wortmannin, there was a rightward shift in the dose-response curve (Fig. 4B). The dose responses of these two events were nearly superimposable (Fig. 4C), suggesting strongly that p110 is responsible for GLUT4 translocation and therefore is the target displaying the lower sensitivity to wortmannin.


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  		Fig. 4.   Reversibility of inhibition by wortmannin of insulin-stimulated GLUT4 translocation and 2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were treated with various concentrations of wortmannin for 45 min in the absence (No Insulin) or presence of 100 nM insulin for the last 15 min. The cells were then either not washed (-wash) or washed (+wash) three times with KRP containing 2% BSA and allowed to recover for 30 min before the addition of insulin (100 nM final) for 15 min. Plasma membrane sheets were prepared, stained for GLUT4, and quantitated. The graph shows the mean ± S.E. of three experiments. B, 3T3-L1 adipocytes were incubated with wortmannin, washed, and stimulated as described above. The uptake of 2-[3H]deoxyglucose (2-DOG) was measured, and results are presented as the mean ± S.E. of three experiments that were performed in parallel with the GLUT4 translocation experiment depicted in A. C, comparison of the curve for GLUT4 translocation (-wash) to the curve for 2-[3H]deoxyglucose uptake (+wash).

Since most of the experiments implicating p110 in insulin signaling to glucose transport have relied on potentially problematic dominant inhibition strategies, we set out to confirm the necessity for this isoform by an alternative strategy. We have previously shown that the aforementioned GLUT4 translocation assay can be applied to microinjected cells (8). Microinjection of anti-p110 antibodies drastically inhibited insulin-stimulated GLUT4 translocation without having any effect on unstimulated cells (Fig. 5). This result directly demonstrates the requirement for p110 PI3-kinase in GLUT4 translocation and lends further credence to the assessment that p110 represents the lower affinity target of wortmannin.


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  		Fig. 5.   Effect of microinjection of anti-p110 antibodies on GLUT4 translocation. 3T3-L1 adipocytes were injected with either MBPras (1 mg/ml) and anti-p110 antisera (5 mg/ml) or MBPras alone. Cell-surface GLUT4 levels were ascertained as described under "Experimental Procedures."

Since many studies have suggested that translocation of GLUT4 is the major contributor to stimulation of hexose uptake by insulin, we were intrigued by the observation that 2-deoxyglucose uptake is inhibited even when there is a full complement of GLUT4 on the plasma membrane. This raised the possibility that in the presence of wortmannin, GLUT4-containing vesicles were docked at the membrane but were unable to fuse. A pre-fusion, docked GLUT4 vesicle population has been invoked to explain the lag between increases in plasma membrane GLUT4 and hexose uptake in rat adipocytes (36). To test this hypothesis in wortmannin-treated cells, we measured translocation in 3T3-L1 adipocytes expressing a GLUT4 construct with an epitope tag (derived from insulin receptor peptide P2 (37)) inserted into the first extracellular loop (3T3-L1/P2 cells). GLUT4/P2 protein can be detected on the cell surface of intact, unpermeabilized 3T3-L1/P2 cells only when GLUT4-containing vesicles have fully fused with the plasma membrane, thus exposing the P2 epitope to the extracellular space (Fig. 6). 3T3-L1/P2 adipocytes were treated with wortmannin for 30 min and stimulated with insulin for 15 min, as in the other translocation assays. Fig. 6 shows that the inhibition curve for translocation of GLUT4/P2 in intact cells was identical to that for endogenous GLUT4 (compare with Fig. 1B), indicating that transporter present on the plasma membrane sheets was accessible to the extracellular space.


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  		Fig. 6.   Accessibility of GLUT4 to the extracellular space in the presence of increasing concentrations of wortmannin. 3T3-L1/P2 adipocytes were treated with various concentrations of wortmannin for 45 min in the absence (-insulin) or presence (+insulin) of 100 nM insulin for the last 15 min. The cells were fixed in 3% paraformaldehyde, and the intact cells were processed in the absence of detergent for indirect immunofluorescence with affinity purified anti-P2 peptide antibodies.

Since GLUT1 also moves to the plasma membrane in response to insulin, we next examined whether translocation of GLUT1-containing vesicles was inhibited by wortmannin. Since the "fold" increase in GLUT1 on the plasma membrane in response to insulin is substantially less than that for GLUT4 (38, 39), changes in the former transporter were more difficult to ascertain by the sheet assay. Nonetheless, treatment of 3T3-L1 adipocytes with varying concentrations of wortmannin and LY294002 for 30 min, followed by stimulation with 100 nM insulin for 15 min, significantly inhibited the translocation of GLUT1 to the cell surface (Fig. 7). Inhibition of GLUT1 translocation was quantitated by image processing, and the results show that translocation of GLUT1 is almost fully inhibited by 1 nM wortmannin (Fig. 7). Thus, translocation of GLUT1 was significantly more sensitive to inhibition by wortmannin than GLUT4 translocation in 3T3-L1 adipocytes.


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  		Fig. 7.   Effect of wortmannin on insulin-stimulated GLUT1 translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were pretreated with various concentrations of wortmannin (A) or LY294002 (B) for 45 min in the absence (NO INSULIN) or presence of 100 nM insulin for the last 15 min. The extent of GLUT1 translocation to the cell surface was determined by the plasma membrane sheet assay and was quantitated as described. The abundance of GLUT1 on the plasma membrane sheets in the presence of insulin but without drugs was set to 100%, and all other values were then normalized accordingly. Results are presented as the means ± S.E. of three experiments.

Since the identity of the higher affinity target for wortmannin is still unknown, it was critical to ascertain whether wortmannin inhibits the transporters directly. To accomplish this, we assayed the the effect of wortmannin on 3T3-L1 adipocytes expressing a constitutively active form of the serine/threonine kinase Akt (Myr-Akt). Akt is positively regulated by insulin via a PI3-kinase-dependent mechanism, and constitutively active forms of Akt stimulate numerous events involving PI3-kinase (40). For example, Myr-Akt increases glucose uptake by both stimulating GLUT4 translocation and increasing expression of GLUT1 (26), although its necessity in insulin-stimulated glucose transport has been questioned (41). A 30-min pretreatment with wortmannin does not inhibit Myr-Akt-stimulated glucose transport (Fig. 8A) or GLUT4 translocation (Fig. 8B).


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  		Fig. 8.   Effect of wortmannin on Myr-Akt-stimulated glucose uptake and GLUT4 translocation. 3T3-L1 adipocytes expressing empty vector, A2-Myr-Akt, or Myr-Akt were pretreated with wortmannin (500 nM) for 45 min in the absence or presence of 100 nM insulin for the last 15 min. A, the uptake of 2-[3H]deoxyglucose was measured as described, and data are presented as means ± S.E. of three independent experiments. B, plasma membrane GLUT4 levels were determined using the plasma membrane "sheet" assay. Sheets were fixed and stained as described under "Experimental Procedures," and the data presented are representative of two independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite years of intensive investigation, the only post-receptor activity generally accepted as required for insulin-stimulated glucose transport is PI3-kinase. Yet, many questions remain not only in regard to other signaling factors but even to the extent of PI3-kinase activation required and the role of each PI3-kinase isoform. The current study provides three novel findings. First, we have shown directly by antibody microinjection a requirement for the p110 isoform of PI3-kinase in the regulation of GLUT4 translocation. Although a number of studies have arrived at a similar conclusion based on the use of inhibitors or dominant-inhibitory proteins, this is the only investigation utilizing isoform-specific immunological reagents. Second, we have demonstrated the existence of a second, high affinity target of wortmannin whose activity is required for stimulation of hexose uptake unrelated to GLUT4 translocation. Finally, we have demonstrated a requirement for maintenance and/or stimulation of GLUT1 or GLUT4 catalytic activity independent of GLUT4 redistribution. Although such a process has been suggested in the past, this is the first clear demonstration that cell-surface, exofacially exposed glucose transporters require additional factors to maintain activity.

While evaluating wortmannin's effects on insulin action, we observed that the drug inhibited insulin-stimulated GLUT4 translocation and glucose uptake with distinct dose dependence. These findings suggest that wortmannin can affect glucose transport in 3T3-L1 adipocytes by two independent mechanisms. The data presented suggest that the lower affinity target is likely to be the p110 catalytic subunit of PI3-kinase and that this subunit is critical for insulin-stimulated GLUT4 translocation. First, wortmannin inhibited another p110 PI3-kinase-dependent event, pp70 S6-kinase activation, with a dose response identical to that for insulin-stimulated GLUT4 translocation (Fig. 2). Second, another inhibitor of PI3-kinases, LY294002, inhibited both glucose transport and GLUT4 translocation with a superimposable dose response (Fig. 3). Third, washing out wortmannin shifted the dose response for glucose transport inhibition to one resembling its inhibition of GLUT4 translocation and pp70 S6-kinase, suggesting that the lower affinity response is due to wortmannin's irreversible effect (35) on p110 PI3-kinase (Fig. 4). Fourth, inhibitory anti-p110 antibodies block the effect of insulin on GLUT4 translocation (Fig. 5). These data confirm the importance of p110 PI3-kinase in GLUT4/IRAP translocation but additionally raise two other questions. 1) What is the "high affinity" target of wortmannin that mediates inhibition of hexose uptake? 2) What is the mechanism that mediates inhibition of deoxyglucose uptake when GLUT4 is on the cell surface? One possible answer to the first question is that wortmannin could be affecting another member of the growing PI3-kinase family. Recently, several of these have been shown to be sensitive to wortmannin at relatively low concentrations. In platelets there appears to be a class II, C2 domain-containing PI3-kinase, which is inhibited by 20 nM wortmannin (42). Moreover, the human homologue of the yeast Vps34p, which phosphorylates PI exclusively, is inhibited by wortmannin with an IC50 of 2.5 nM (43). Nonetheless, whether the high affinity target of wortmannin is either of these proteins, and what the mechanism is by which uptake is inhibited, remains to be determined.

Careful time courses have shown that translocation of GLUT4 precedes the increase in hexose uptake (44), and GLUT4 translocation is now thought to be the primary mechanism by which insulin stimulates glucose transport. Yet, as clearly shown in Fig. 1, translocation of both GLUT4 and IRAP, a protein resident in GLUT4 vesicles, can occur in the absence of increased hexose uptake in 3T3-L1 adipocytes. One possibility from this experiment was that the translocation assay utilized was detecting a "docked" state, and thus the translocated transporters were not capable of transporting glucose because they weren't accessible to the extracellular matrix. IRAP translocation, however, as measured by accessibility to a biotinylation reagent, argues against this possibility; at concentrations of wortmannin where GLUT4 might be docked but not fused, IRAP is accessible to biotin. Furthermore, using a mutant of GLUT4 with an epitope inserted in an exofacial domain, we demonstrated that under wortmannin conditions that dissociate GLUT4 translocation and transport, GLUT4 is exposed to the outer surface of the plasma membrane (Fig. 6). These two independent assays strongly exclude the vesicle fusion as the higher affinity site of action for wortmannin. The lack of effect of wortmannin on Myr-Akt-stimulated glucose transport further excludes a direct effect of the drug on the transporter to suppress activity.

A substantial body of circumstantial evidence supports the conclusion that glucose transport can be activated independently of an effect on GLUT4 translocation. Chronic treatment of 3T3-L1 adipocytes with protein synthesis inhibitors significantly increases hexose uptake without corresponding increases in plasma membrane glucose transporters. This would suggest that one or more proteins with rapid turnover rates function in the basal state to suppress the activity of cell-surface glucose transporters (45, 46). Additionally, in rat adipocytes, insulin-stimulated glucose transport activity is inhibited by isoproterenol and augmented by adenosine with no change in the amount of GLUT4 on the cell surface (47, 48). More recently, two studies demonstrated a similar dissociation between cell-surface GLUT4 and the rate of glucose transport into the cell. Inhibitors of p38 mitogen-activated protein kinase prevent insulin-stimulated glucose transport, but not GLUT4 translocation, in both 3T3-L1 adipocytes and L6 myotubes (49). A similar dissociation was observed in skeletal muscle from transgenic mice overexpressing GLUT1 (50).

The simplest explanation for the data presented is that GLUT1 translocation, and not that of GLUT4, mediates insulin-stimulated glucose transport. GLUT1 translocation was inhibited by wortmannin and LY294002 at concentrations comparable with or lower than that required to inhibit transport (Fig. 6), although the precise relationship is difficult to ascertain due to the relatively modest degree of redistribution of GLUT1. However, since the fold stimulation of GLUT1 translocation is only 2-fold, whereas the stimulation in transport is severalfold, something must explain the relatively low basal transport values in the 3T3-L1 adipocyte. A prior group has reported that GLUT1 activity is inhibited substantially (>90%) in 3T3-L1 adipocytes under basal conditions but is then activated in response to insulin (46, 51). Thus, even if GLUT1 translocation is a substantial contributor for increasing glucose influx, an activation step is likely to exist.

An alternative mechanism is that a wortmannin-sensitive accessory protein regulates GLUT4 catalytic activity. Prior studies also suggest that GLUT4 can exist in active and inactive forms. Vannucci et al. (52) utilized the impermeant, exofacial bismannose photolabel (2-N-[4(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4-yloxy)propyl-2-amine) to demonstrate that in the presence of insulin and isoproterenol, cell-surface GLUT4 cannot be recognized by the photolabel. They proposed that the GLUT4-containing vesicles were docked but not functionally fused with the plasma membrane or that the GLUT4-containing vesicles were resident on the plasma membrane but not catalytically active (52). Our data are consistent with the latter explanation, since, under conditions of insulin and low wortmannin, IRAP and GLUT4/P2 are readily detectable in non-permeabilized adipocytes, but there is no increase in hexose uptake. Whether the use of wortmannin mimics the action of adrenergic agents remains to be determined.

    ACKNOWLEDGEMENTS

We thank Sara Courtneidge (Harvard Medical School) and Margaret Chou (University of Pennsylvania) for kindly donating anti-p110alpha and anti-pp70 S6-kinase antibodies, respectively. Metabolex Inc kindly donated anti-IRAP antibodies. Cass Lutz provided assistance in the typing and editing of this manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK39615 (to M. J. B.) and DK09375 (to S. A. S).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: Ariad, 26 Landsdowne St., Cambridge, MA 02139.

§ Current address: Dept. of Cell Biology, Harvard Medical School, Boston, MA 02115.

Current address: Division of Human Systems, Faculty of Letters, Hokkaido University, Sapporo, Japan.

parallel Supported by a Howard Hughes Pre-doctoral Fellowship.

** Current address: Dept. of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523.

Dagger Dagger Member of the Cox Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Pennsylvania Medical School, Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-5001; Fax: 215-573-9138; E-mail: birnbaum@hhmi.upenn.edu.

2 K. Morioka and M. J. Birnbaum, unpublished results.

    ABBREVIATIONS

The abbreviations used are: GLUT, glucose transporter; PI3-kinase, phosphatidylinositol 3-kinase; BSA, bovine serum albumin; IRAP, insulin-responsive aminopeptidase; DMEM, Dulbecco's modified Eagle's medium; IRS, insulin receptor substrate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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