Intracellular Insulin-Responsive Glucose Transporter (GLUT4 ...

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Molecular Endocrinology 16(5):1060–1068 Copyright © 2002 by The Endocrine Society

Intracellular Insulin-Responsive Glucose Transporter (GLUT4) Distribution But Not Insulin-Stimulated GLUT4 Exocytosis and Recycling Are Microtubule Dependent SATOSHI SHIGEMATSU, AHMIR H. KHAN, MAKOTO KANZAKI,

AND

JEFFREY E. PESSIN

Department of Physiology & Biophysics, The University of Iowa, Iowa City, Iowa 52242 To investigate the potential role of microtubules in the regulation of insulin-responsive glucose transporter (GLUT4) trafficking in adipocytes, we examined the effects of microtubule depolymerizing and stabilizing agents. In contrast to previous reports, disruption or stabilization of microtubule structures had no significant effect on insulin-stimulated GLUT4 translocation. However, consistent with a more recent study (Molero, J. C., J. P. Whitehead, T. Meerloo, and D. E. James, 2001, J Biol Chem 276:43829–43835) nocodazole did inhibit glucose uptake through a direct interaction with the transporter itself independent of the translocation process. In addition, the initial rate of GLUT4 endocytosis was not significantly affected by microtubule depolymerization. However, these internalized GLUT4 compartments are confined to regions

just beneath the plasma membrane and were not exposed to the extracellular space. Furthermore, they were unable to undergo further sorting steps and trafficking to the perinuclear region. Nevertheless, these apparent early endocytic GLUT4 compartments fully responded to a second insulin stimulation with an identical extent of plasma membrane translocation. Together, these data demonstrate that although microtubular organization may play a role in the trafficking of GLUT4 early endocytic vesicles back to the perinuclear region, they do not have a significant role in insulinstimulated GLUT4 exocytosis, initial endocytosis from the plasma membrane and/or recycling back to the plasma membrane. (Molecular Endocrinology 16: 1060–1068, 2002)

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protein vimentin have been copurified with intracellular GLUT4 containing vesicles (23). Expression of a dominant-interfering vimentin peptide dispersed the perinuclear localized GLUT4 protein and interference with microtubule motor proteins dynein and kinesin attenuated GLUT4 translocation (23, 24). In addition, microtubule depolymerizing agents (nocodazole, colchicine, and vinblastine) disperse the perinuclear localized GLUT4 protein and partially inhibit both insulinstimulated glucose uptake and GLUT4 translocation (24–27). These data support a model in which one component of GLUT4 exocytosis occurs through the insulin-stimulated interaction of GLUT4-containing compartments with microtubules. In contrast, although colchicine was observed to inhibit lipid and glycogen synthesis in primary isolated rat adipocytes, there was no effect on insulin-stimulated glucose uptake (28). More recently, it was reported that disruption of microtubules with nocodazole had no significant effect on GLUT4 translocation (29). However, higher concentrations of nocodazole inhibited glucose transport activity, apparently through a direct interaction with the transporter protein itself independent of microtubule organization. Thus, to further evaluate the role of microtubules in the regulation of GLUT4 trafficking, we have observed that depolymerization of microtubules with nocodazole, colchicine or vinblastine had no significant effect on

ACILITATIVE GLUCOSE UPTAKE is mediated by a family of integral membrane transport proteins that display overlapping but distinct tissue distributions and subcellular compartmentalization (1–4). The major insulin-responsive glucose transporter isoform GLUT4 is primarily expressed in skeletal muscle and adipose tissue (5–9). In the basal state, this transporter undergoes a slow rate of membrane recycling such that 2–5% of the protein is found at the plasma membrane with the bulk localized to multiple intracellular compartments (10–14). Insulin stimulation induces a 10-fold increase in the rate of GLUT4 protein exocytosis concomitant with a smaller 2-fold decrease in the rate of endocytosis (15–18). The combination of these changes in membrane cycling of the GLUT4 protein results in a 10- to 25-fold steady-state increase at the plasma membrane. The insulin-dependent signal transduction pathways regulating this dramatic change in GLUT4 membrane trafficking have been intensively investigated (19–22). Recently, several studies have implicated both the actin and microtubule cell cytoskeleton networks as important structural and regulatory elements in the translocation mechanism. In particular, the microtubular protein ␣-tubulin and intermediate filament

Abbreviations: EGFP, Enhanced green fluorescent protein; GLUT4, insulin-responsive glucose transporter.

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insulin-stimulated GLUT4 translocation to the plasma membrane. In addition, the initial endocytosis of GLUT4 after insulin removal and subsequent reexocytosis after a second round of insulin stimulation were unaffected by nocodazole treatment. However, depolymerization of microtubules did inhibit the intracellular sorting of GLUT4 and resulted in the apparent accumulation of endocytic vesicles beneath the plasma membrane.

RESULTS Microtubule Depolymerization/Stabilization Does Not Affect Insulin-Stimulated GLUT4 Exocytosis Immunofluorescence labeling of fully differentiated 3T3L1 adipocytes with an ␣-tubulin antibody demonstrated the presence of polymerized microtubules in the perinuclear region and around the inner surface of the plasma membrane (Fig. 1A, panel a). Incubation with 3 ␮M nocodazole for 30 min disrupted nearly all the microtubules beneath the plasma membrane and markedly reduced the amount of microtubules emanating from the perinuclear region (Fig. 1A, panel b). The disruption of microtubule structure was reversible as washing the cells to remove nocodazole resulted in a recovery of microtubule organization (Fig. 1A, panel c). As typically ob-

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served, the endogenous GLUT4 protein was localized to the perinuclear region and in small vesicle structures scattered throughout the cell (Fig. 1B, panel a). Nocodazole treatment resulted in a time-dependent dispersion of the perinuclear localized GLUT4 protein that was apparent by 30 min and complete within 120 min (Fig. 1B, panels b and c). Based upon these data, we pretreated 3T3L1 adipocytes with and without 3 ␮M nocodazole for 120 min and examined the insulin-stimulated translocation of GLUT4 using the established plasma membrane sheet assay (Fig. 2). Insulin stimulation resulted in the robust appearance of GLUT4 immunofluorescence in the plasma membrane sheets indicative of GLUT4 translocation (Fig. 2, panels a and c). Despite the disruption of microtubules and dispersion of the perinuclear GLUT4 compartments, nocodazole treatment had no significant effect on insulin-stimulated GLUT4 translocation (Fig. 2, panels b and d). Similarly, insulin was fully capable of inducing GLUT4 translocation in adipocytes pretreated with colchicine and vinblastine under conditions that completely disrupted microtubule organization (data not shown). In addition, cytoplasmic acidification to inhibit dynein motor activity also dispersed the perinuclear GLUT4 protein but had no effect on insulin-stimulated GLUT4 translocation (data not shown). Together, these data demonstrate that depolymerization of microtubules in fully differentiated

Fig. 1. Nocodazole Treatment Results in the Depolymerization of Microtubules and the Dispersion of Perinuclear Localized GLUT4 A, Fully differentiated 3T3L1 adipocytes were incubated in the absence (panel a) or presence (panels b and c) of 3 ␮M nocodazole for 30 min at 37 C. The cells were then washed and incubated for an additional 120 min (panel c) in the absence of nocodazole. The cells were then fixed and subjected to confocal immunofluorescence microscopy using an ␣-tubulin antibody as described in Materials and Methods. B, Fully differentiated 3T3L1 adipocytes were incubated in the absence (panel a) or presence of 3 ␮M nocodazole for 30 (panel b) or 120 (panel c) min at 37 C. The cells were then fixed and subjected to confocal immunofluorescent microscopy using a GLUT4 antibody as described in Materials and Methods. These are representative images obtained from two independent determinations.

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Fig. 2. Nocodazole Treatment Does Not Inhibit Insulin-Stimulated GLUT4 Translocation Fully differentiated 3T3L1 adipocytes were incubated in the absence (panels a and c) or presence (panels b and d) of 3 ␮M nocodazole for 120 min at 37 C. The cells were then incubated in the absence (panels a and b) or presence (c and d) of 100 nM insulin for 30 min. Plasma membrane sheets were prepared and subjected to confocal fluorescent microscopy using a GLUT4 antibody as described in Materials and Methods. These are representative images obtained from two independent determinations.

3T3L1 adipocytes does not significantly influence insulin-stimulated GLUT4 exocytosis. Microtubule stabilization also inhibits microtubule trafficking functions by preventing protein movement along the growing plus ends (30). Incubation of adipocytes with the microtubule stabilizing agent taxol increased the amount of microtubule labeling both in the perinuclear region and beneath the plasma membrane (Fig. 3A, panels a and b). Because GLUT4 is predominantly localized to the perinuclear region, taxol stabilization of microtubules had no significant effect on the intracellular distribution of GLUT4 (Fig. 3A, panels c and d). The taxol stabilization of microtubules at the inner plasma membrane surface was also readily detectable by examining ␣-tubulin immunofluorescence in isolated plasma membrane sheets (Fig. 3B, panels b, f, d, and h). Importantly, in the same plasma membrane sheets, insulin-stimulated the same extent of GLUT4 translocation in taxol treated cells compared with the untreated adipocytes (Fig. 3B, panels a, c, e, and g). Thus, these data further demonstrate that, in addition to microtubule depolymerization, dynamic microtubule turnover is also not necessary for insulinstimulated GLUT4 translocation. Because insulin-stimulated glucose uptake in adipocytes primarily reflects the GLUT4 protein, we also assessed the effect of nocodazole on glucose transport activity (Fig. 4). As typically observed, insulin stimulation resulted in an approximate 10-fold increase in glucose uptake compared with unstimulated cells (Fig. 4, solid vs. open symbols). Treatment with various


doses of nocodazole before insulin stimulation resulted in a dose-dependent inhibition of glucose uptake (Fig. 4, solid circles). Importantly, in this assay nocodazole was maintained throughout the entire procedure including the wash and transport assay buffers. It should also be noted that there was no significant inhibition of glucose uptake at 3 ␮M nocodazole, a concentration sufficient to depolymerize microtubules (Fig. 1). In these assays, nocodazole was maintained throughout the entire procedure including the wash and transport assay buffers. However, when the cells were pretreated with nocodazole but the nocodazole was left out of the wash and assay buffers, there was only a small dose-dependent inhibition of insulinstimulated glucose uptake (Fig. 4, solid squares). Thus, these data demonstrate that the effect of nocodazole on glucose transport was reversible during the uptake assay, suggesting a direct inhibitory effect of nocodazole on the glucose transporter itself independent of insulin stimulation. To confirm this observation, adipocytes were stimulated with insulin first, then cooled to 4 C and incubated with various doses of nocodazole. Under these conditions, there was an identical inhibition of insulin-stimulated glucose uptake compared with cells continuously maintained in the presence of nocodazole (Fig. 4, solid triangles). These data are fully consistent with nocodazole functioning as an inhibitor of glucose transport activity per se and without necessarily affecting the insulin-stimulated GLUT4 exocytosis process. Microtubule Depolymerization Alters GLUT4 Endocytosis and Intracellular Sorting Several studies have implicated a role for microtubules in plasma membrane endocytosis and intracellular sorting events (31–35). To determine whether the disruption of microtubules affected GLUT4 endocytosis, we next performed a series of studies to directly assess the endocytosis and subsequent exocytosis of the same population of GLUT4. Initially, we examined the time-dependent localization of a GLUT4-enhanced green fluorescent protein (EGFP) fusion protein in the absence of insulin prior and subsequent to the addition of nocodazole (Fig. 5). In the basal state, GLUT4EGFP was predominantly concentrated in the perinuclear region and in small compartments scattered throughout the cell cytoplasm (Fig. 5, panel a). After the addition of nocodazole (time 0), there was a timedependent accumulation of GLUT4-EGFP at the cell surface that was readily apparent by 40–60 min (Fig. 5, panels b–e). At this and subsequent times, GLUT4EGFP was also found to accumulate in smaller punctate compartments just beneath the plasma membrane that remained apparent for the entire time course examined (Fig. 5, panels f–h). We next examined the trafficking patterns of GLUT4-EGFP in living cells (Fig. 6 and supplementary movie, http://mend.endojournals.org/). The data presented in Fig. 6 represent individual images taken from



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Fig. 3. Microtubule Stabilization Does Not Alter the Perinuclear Distribution or Insulin-Stimulated GLUT4 Translocation A, Fully differentiated 3T3L1 adipocytes were incubated in the absence (panels a and c) or in the presence (panels b and d) of 100 nM taxol for 120 min at 37 C. The cells were then fixed and subjected to confocal fluorescent microscopy using ␣-tubulin (panels a and b) or GLUT4 (panels c and d) antibodies as described in Materials and Methods. B, Fully differentiated 3T3L1 adipocytes were incubated in the absence (panels a, b, e, and f) or in the presence (panels c, d, g, and h) of 100 nM taxol for 120 min at 37 C. The cells were then incubated in the absence (panels a–d) or presence (e–h) of 100 nM insulin for 30 min. Plasma membrane sheets were prepared and subjected to confocal fluorescent microscopy using ␣-tubulin (panels b, d, f, and h) or GLUT4 (panels a, c, e, and g) antibodies as described in Materials and Methods. These are representative images obtained from two independent determinations.

the beginning and end of the time-lapse video. Insulin stimulation of GLUT4-EGFP-transfected cells resulted in the characteristic appearance of GLUT4-EGFP at the plasma membrane (Fig. 6, A and B). The absence of perinuclear GLUT4 was due to the plane of sectioning so that the internalized GLUT4-EGFP would be readily apparent. In any case, after insulin washout there was a time-dependent decrease in cell surface GLUT4-EGFP concomitant with the appearance in the

interior of the cell (Fig. 6C). In contrast, nocodazole pretreatment completely blocked the intracellular accumulation of GLUT4-EGFP after insulin removal (Fig. 6D). In addition, the plasma membrane GLUT4-EGFP became significantly thicker with the formation of multiple small vesicle compartments beneath the plasma membrane. The time-dependent changes in GLUT4EGFP distribution are dramatically apparent when visualized in a time-lapse movie format (see supple-


mentary material). Thus, disruption of microtubule organization prevented the trafficking of GLUT4 from the plasma membrane to perinuclear compartments. Because GLUT4 remained closely associated with the plasma membrane in the presence of nocodazole,

Fig. 4. Nocodazole Inhibits Glucose Uptake by Directly Interfering with the Glucose Transport Process Itself Fully differentiated 3T3L1 adipocytes were pretreated with various concentrations of nocodazole for 120 min and then stimulated with (solid symbols) or without (open symbols) 100 nM insulin for 15 min at 37 C. The cells were then washed at 4 C in the absence (squares) or presence (circles) of the same nocodazole concentration and 2-deoxyglucose uptake was determined as described in Materials and Methods. In parallel, 3T3L1 adipocytes were first stimulated with 100 nM insulin for 15 min at 37 C then washed and cooled to 4 C. The cells were then incubated with nocodazole for 4 min and the rate of 2-deoxyglucose uptake was then determined. These data represent the averages of three independent experiments each performed in duplicate.


we asked the question whether this GLUT4 was exposed to the extracellular space or underwent initial endocytosis into distinct intracellular compartments. To address this issue, an exofacial myc epitope was cloned into the GLUT4-EGFP cDNA to generate a double epitope tagged reporter that can be used for single cell quantitation of GLUT4 translocation (36). Analysis of the quantitative exposure of the myc epitope to the extracellular medium demonstrated an approximate 8-fold insulin stimulation of myc-GLUT4EGFP translocation (Fig. 7A). After insulin removal, there was a time-dependent decrease in the cell surface exposure of the myc epitope that decayed with a t1/2 of approximately 20 min. An identical rate of loss of myc-GLUT4-EGFP exposure to the extracellular space also occurred in the presence of nocodazole. These data demonstrate that the initial steps of GLUT4 endocytosis were not perturbed by microtubule depolymerization. We next determined whether nocodazole would perturb the ability of insulin to stimulate the exocytosis of the newly internalized GLUT4 protein. This was accomplished by first stimulating myc-GLUT4-EGFP expressing cells with insulin and then labeling the cell surface exposed myc epitope with the myc antibody. The cells were then washed to remove insulin and any unbound antibody, subsequently restimulated, and then fixed. The amount of cell surface accessible myc antibody from the previously internalized myc-GLUT4EGFP was then determined by labeling with a Texas Red-conjugated antimouse IgG antibody (Fig. 7B). As previously observed, both the insulin-stimulated translocation and subsequent endocytosis of the mycGLUT4-EGFP fusion protein were unaffected by microtubule depolymerization with nocodazole. More importantly, there was no significant effect of nocoda-

Fig. 5. Nocodazole Induces the Appearance of GLUT4 at the Plasma Membrane and Accumulates GLUT4 Beneath the Plasma Membrane Fully differentiated 3T3L1 adipocytes were transfected with 50 ␮g of the GLUT4-EGFP cDNA as described in Materials and Methods. The cells were allowed to recover for 18 h and placed into a perfusion chamber maintained at 37 C. The cells were continuously imaged by confocal fluorescent microscopy in a single plane before and after the addition of 3 ␮M nocodazole for the times indicated. These are representative images obtained from three independent determinations.


Fig. 6. Nocodazole Inhibits Intracellular Sorting of GLUT4 Fully differentiated 3T3L1 adipocytes were transfected with 50 ␮g of the GLUT4-EGFP cDNA as described in Materials and Methods. The cells were allowed to recover for 18 h and placed into a perfusion chamber maintained at 37 C. The cells were then treated with 100 nM insulin in the absence (panels A and C) or in the presence (panels B and D) of 3 ␮M nocodazole. The cells were then perfused in serum-free medium without (panels A and C) and with (panels B and D) 3 ␮M nocodazole to washout the insulin. The cells were then continuously imaged by confocal fluorescent microscopy in a single plane above the perinuclear region to more clearly visualize the internalize GLUT4-EGFP protein. The initial images are presented in panels A and B and after 2 h subsequent to insulin washout (panels C and D). The full time-lapse images are presented in the accompanying supplementary movie (http://mend.endojournals.org/). These are representative images obtained from three independent determinations.

zole on the time and rate of reappearance of the myc antibody to the extracellular surface after a second insulin stimulation. These data demonstrate that microtubule organization is not required for successive rounds of insulin-stimulated GLUT4 translocation.

DISCUSSION Although microtubules have been generally thought to play critical roles in the long range transport of membrane compartments in neurons (37–40), their role in short-term membrane transport vesicle trafficking has been more difficult to resolve. For example, exocytosis of vesicular stomatitis virus G protein from the transGolgi network and the recycling of the transferrin receptor in some adherent cell lines appear to be independent of microtubules (41, 42). In contrast, kinesin motors and microtubules have been implicated in apical membrane transport in polarized epithelial cells (43), axonal transport in neuronal cells (38), RNA trafficking (44) and chromosomal segregation (45). More


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recently, several studies have observed that microtubules play an important role in the insulin-stimulated translocation of GLUT4 compartments in adipocytes (23–27). In particular, ␣-tubulin was identified in purified GLUT4 vesicles and inhibition of the microtubulebased motor dynein by brief cytoplasmic acidification dispersed the perinuclear localized GLUT4 and partially inhibited GLUT4 translocation to the cell surface (23). Expression of the microtubule-binding protein hTau40, which impairs kinesin motors, delayed GLUT4 appearance at plasma membrane in response to insulin, and the microtubule depolymerizing drug, nocodazole, partially inhibited insulin-stimulated glucose transport activity and GLUT4 translocation (24). Together, these studies have suggested the importance of microtubule organization and microtubule motors in GLUT4 trafficking events. On the other hand, a more recent study observed that at low concentrations of nocodazole (2 ␮M) that completely dispersed microtubules and perinuclear GLUT4, there was no significant effect on insulin-stimulated GLUT4 translocation or glucose uptake (29). In addition, although higher nocodazole concentrations did not affect insulin-stimulated GLUT4 translocation, there was an inhibition of glucose uptake due to a direct inhibitory effect on the transport protein itself that was independent of microtubule organization. The data presented in this manuscript are fully consistent with this latter study. We have not observed any significant effect on insulin-stimulated GLUT4 translocation to the plasma membrane in fully differentiated 3T3L1 adipocytes treated with several microtubule depolymerizing or stabilizing agents. Identical results were obtained for both the endogenous GLUT4 protein and in cells expressing the GLUT4-EGFP reporter protein. In addition, we have also observed a dose-dependent inhibition of insulin-stimulated glucose uptake by nocodazole. However, this inhibition is observed only if nocodazole is maintained in the transport assay buffers and is reversible after removal of nocodazole. Although our data do not rule out a functional role for microtubule motors in the translocation process, these findings clearly demonstrate that organized microtubule structures in 3T3L1 adipocytes are not necessary for insulin-stimulated GLUT4 exocytosis. In contrast to exocytosis, some endocytotic and intracellular sorting steps can be affected by microtubule depolymerization (30–35). For example, traffic between early endosomes and lysosomes are also inhibited by microtubule depolymerization, whereas transferrin receptor recycling between the plasma membrane and recycling endosomes are not inhibited by microtubule depolymerization (42). Our data demonstrates that microtubule depolymerization resulted in the accumulation of GLUT4 in compartments beneath the plasma membrane. These compartments were distinct from the plasma membrane as the exofacial domains of GLUT4 were no longer exposed to the extracellular space and therefore probably represent initially formed endocytic vesicles and/or early


Fig. 7. Nocodazole-Induced Accumulated GLUT4 Beneath the Plasma Membrane Is Not Exposed to the Extracellular Space and Is Fully Capable of Undergoing a Second Round of Insulin-Stimulated Translocation A, Fully differentiated 3T3L1 adipocytes were transfected with 50 ␮g of the myc-GLUT4-EGFP cDNA as described in Materials and Methods. The cells were allowed to recover for 18 h and treated in the absence or presence of 100 nM insulin for 60 min. Thirty minutes after insulin stimulation, the cells were either untreated (solid circles) or continuously incubated with 33 ␮M nocodazole (solid squares). Insulin was then removed with an isotonic wash buffer containing 10 mM 2-[Nmorpholino]ethanesulfonic acid (pH 6.0), and the cells were allowed to internalize GLUT4 for 15, 30, 60, or 120 min. The cells were then fixed and subjected to confocal fluorescent microscopy. The fluorescent intensity of the surface myc label was normalized for the EGFP signal intensity and was used as a quantitative estimate for the extent of GLUT4 translocation as described in Materials and Methods. These data represent the averages from two independent experiments. B, The myc-GLUT4-EGFP cDNA transfected adipocytes were either nonstimulated or stimulated with 100 nM insulin for 30 min after pretreatment with (solid circles) or without (open circles) 3 ␮M nocodazole for 2 h. Then the cells were chilled and incubated with c-Myc antibody (9E10) for 1 h at 4 C and then washed to remove insulin and excess c-Myc antibody. Some cells were returned to 37 C and incubated for 2 h to allow c-Myc antibody bound myc-GLUT4-EGFP to internalize. The cells were again stimulated with 100 nM insulin for the time indicated. Nocodazole (3 ␮M) was added in the media of nocodazole-pretreated cells throughout the


endosome compartments. Importantly, after this initial internalization, GLUT4 was unable to undergo further sorting as these GLUT4-containing early endosomes were unable to traffic GLUT4 back to the perinuclear compartments. Nevertheless, these early endocytic compartments were fully capable of undergoing a second round of insulin-stimulated translocation to the plasma membrane. Thus, these data further suggest that the insulin responsive GLUT4 compartments are distinct from those located in the perinuclear region and are localized more proximal to the plasma membrane. Alternatively, the insulin-responsive compartments may be perinuclear localized but after microtubule depolymerization redistribute to the periphery of the cell (23). In either case, how can we reconcile the apparent differences reported on the role of microtubule structure on GLUT4 translocation? One possibility is that during the initial biosynthesis or long-term recycling, GLUT4 travels along microtubules and is sorted within the perinuclear region. Disruption of microtubule organization causes these storage compartments to disperse but still allows them to engage the actin cytoskeleton. Similarly, during the endocytosis process microtubule disruption prevents trafficking back to the perinuclear region but still allows for GLUT4 vesicles to functionally interact with actin. Thus, it remains possible that microtubule organization is necessary for the efficient intracellular compartmentalization and trafficking of GLUT4 by providing a pathway to sort onto the actin cytoskeleton. In summary, our data clearly demonstrate that microtubule organization is not necessary for insulinstimulated GLUT4 translocation in fully differentiated 3T3L1 adipocytes. Although microtubules do play a role in the long range membrane transport of GLUT4 from the plasma membrane to the perinuclear region, microtubules are not necessary for the initial steps of plasma membrane endocytosis or the ability of the early endocytic vesicles/early endosomal compartments to undergo successive rounds of insulin-stimulated translocation. In addition, the effect of nocodazole on insulin-stimulated glucose transport activity does not reflect an inhibition of GLUT4 translocation but instead is due to a direct inhibitory action of nocodazole on the transport protein itself. Because microtubule depolymerization results in the formation of short disorganized microtubule tracks, further studies are still needed to clarify the potential role of microtubule motors in the intracellular trafficking and sorting of GLUT4.

experiment. The cells were fixed but not permeabilized. Cell surface c-Myc antibody bound to the myc-GLUT4-EGFP protein was detected with a secondary Texas Red-labeled antibody and total myc-GLUT4-EGFP were detected with EGFP as described in Materials and Methods. Quantitation of plasma membrane localized myc antibody bound to mycGLUT4-EGFP was then determined as described above. This is a representative experiment ⫾ SE from three independent determinations.


MATERIALS AND METHODS Materials The ␣-tubulin and c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and fluorescent secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). The polyclonal antibody against rat GLUT4 (IAO2) was generated as described previously (46). Nocodazole, taxol, colchicine and vinblastine were obtained from Sigma (St. Louis, MO). Exofacial myc-tagged GLUT4 cDNA was kindly provided by Amira Klip, the GLUT4-EGFP/pcDNA3 and exofacial myc-tagged GLUT4-EGFP/pcDNA3 were constructed as previously described (36, 47). Cell Culture and Transient Transfection by Electroporation 3T3L1 adipocytes (American Type Culture Collection, Manassas, VA) were grown and differentiated as described previously (48). Briefly, 9–10 d post differentiation adipocytes were electroporated with GLUT4-myc/pCXN2, GLUT4EGFP/pcDNA3 or myc-GLUT4-EGFP/pcDNA3 using a lowvoltage electroporation technique (49). 2-Deoxyglucose Uptake 3T3L1 adipocytes were placed in DMEM containing 25 mM glucose plus 0.5% BSA for 2 h at 37 C. The cells were washed with KRPH buffer (5 mM Na2HPO4; 20 mM HEPES, pH 7.4; 1 mM MgSO4; 1 mM CaCl2; 136 mM NaCl; 4.7 mM KCl; 1% BSA) and treated with nocodazole and insulin as described in the figure legends. Glucose uptake was determined at 4 C by incubation with 50 ␮M 2-deoxyglucose containing 0.5 ␮Ci of 2-[3H]deoxyglucose. The reaction was stopped after 5 min by washing the cells three times with ice-cold PBS. The cells were then solubilized in 1% Triton X-100 at 4 C for 30 min, and aliquots were subject to scintillation counting and Bradford protein assay. Live Cell Confocal Microscopy Confocal imaging of GLUT4-EGFP in living cells were obtained by using a Carl Zeiss (Jena, Germany) confocal microscope and closed chamber system FCS2 (Bioptechs, Butler, PA). Eighteen hours after electroporation, the cells expressing GLUT4-EGFP were serum starved for 2 h in DMEM containing 25 mM glucose, 0.1% BSA and 25 mM, HEPES (pH 7.4). The coverslips were then placed in a closed chamber system maintained at 37 C. The cells were then imaged every 10 sec starting 15 min before the addition of nocodazole. Cells expressing GLUT4-EGFP were also treated with insulin for 1 h and then washed with serum-free media with or without nocodazole. Images were collected every 30 sec starting 10 min before insulin removal. Immunofluorescence Microscopy Intact cell immunofluorescence was performed by washing the cells once with ice-cold PBS, followed by fixation with 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) and 0.2% Triton X-100 in PBS at room temperature for 10 min. The cells were then blocked with 5% donkey serum. Plasma membrane sheets were prepared by the method of Robinson et al. (50). Briefly, the membranes were fixed in 2% paraformaldehyde at room temperature for 20 min and blocked with 5% donkey serum. The cells or plasma membrane sheets were then incubated with primary antibod-


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ies for 90 min at 37 C and Texas Red- or FITC-conjugated donkey secondary antibody for 2 h at room temperature. The coverslips were mounted in Vectashield (Vector Laboratories, Inc., Burlington, CA) and examined with 40⫻ or 63⫻ oil immersion objectives in a confocal laser scanning microscope. GLUT4 Recycling Assay 3T3L1 adipocytes expressed exofacial myc-tagged GLUT4EGFP were either nonstimulated or insulin stimulated for 30 min after pretreatment with or without 3 ␮M nocodazole for 2 h. Then the cells were chilled and incubated with c-Myc antibody (9E10) for 1 h at 4 C, and then cells were washed to remove insulin and excess c-Myc antibody as described previously (46). Some cells were returned to 37 C and incubated for 2 h to allow c-Myc antibody bound myc-GLUT4EGFP to internalize. Again the cells were stimulated with insulin for the time indicated. Nocodazole (3 ␮M) was added in the media of nocodazole pretreated cells throughout the experiment. Then the cells were washed once with ice-cold PBS and fixed in 4% paraformaldehyde at room temperature for 10 min, but the cells were not permeabilized, and then blocked with 5% donkey serum. The cells were incubated with Texas Red-conjugated donkey antimouse IgG antibody for 2 h at room temperature, mounted in Vectashield, and examined with a confocal laser scanning microscope. The cell surface c-Myc antibody bound myc-GLUT4-EGFP were detected with Texas Red and total myc-GLUT4-EGFP were detected with EGFP. The fluorescent intensities of Texas Red and EGFP were measured in 20 cells for each time points, and the GLUT4 recycling was determined by the ratio between the Texas Red and EGFP intensities after the second insulin stimulation.

Acknowledgments Received November 26, 2001. Accepted January 28, 2002. Address all correspondence and requests for reprints to: Jeffrey E. Pessin, Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242. E-mail: [email protected]. This work was supported by Research Grants DK-33823, DK-59291, and DK-25295 from the NIH.

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