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Endocrinology Vol. 144, No. 11 5030-5039
Copyright © 2003 by The Endocrine Society

Insulin Promotes Formation of Polymerized Microtubules by a Phosphatidylinositol 3-Kinase-Independent, Actin-Dependent Pathway in 3T3-L1 Adipocytes

Ann Louise Olson, Craig A. Eyster, Quwanza S. Duggins and John B. Knight

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190

Address all correspondence and requests for reprints to: Dr. Ann Louise Olson, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Room 853-BMSB, Oklahoma City, Oklahoma 73190. E-mail: ann-olson{at}ouhsc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Direct demonstrations implicating the microtubule cytoskeleton in insulin-mediated adipose/muscle-specific glucose transporter (GLUT4) translocation are beginning to emerge, and one role of the microtubule network appears to be the provision of a solid support for GLUT4 vesicle movement. In the current study we show that insulin treatment increases total polymerized {alpha}-tubulin in microtubules in a time- and dose-dependent manner that coincides with established insulin-mediated changes in GLUT4 translocation. Insulin stimulates the growth of microtubules through a pathway that requires tyrosine kinase activity, as indicated by inhibition of the effect after treatment with genistein. Insulin-mediated growth was not inhibited by treatment with the MAPK kinase (MEK) inhibitor, PD98059 or by wortmannin, indicating that the effect does not require activation of extracellular signal-regulated kinase 1/2 or phosphatidylinositide 3-kinase. Depolymerization of the actin cytoskeleton with latrunculin B abrogated the effect of insulin on microtubule polymerization, indicating that an intact actin network is a requirement for insulin-dependent modulation of microtubules. Using methods that measure insulin-dependent GLUT4 translocation in populations of adipocytes as opposed to individual cells, we show a statistically significant reduction in translocation (30% inhibition) in the presence of low concentrations of nocodazole (2 µM). This concentration incompletely depolymerizes the microtubule network, revealing that partial depolymerization of microtubules is sufficient to inhibit GLUT4 translocation. It is likely that stabilization of the microtubule network contributes to insulin stimulation of GLUT4 translocation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A MAJOR PHYSIOLOGICAL action of insulin is to control the uptake and storage of glucose. Insulin exerts a major regulatory influence by signaling an internal vesicular compartment(s) containing the adipose/muscle-specific glucose transporter (GLUT4) to translocate to the plasma membrane (for a recent review, see Ref. 1). Translocation of GLUT4 results in an approximately 5-fold increase in the number of GLUT4 transporters at the plasma membrane, which, in turn, increases glucose transport into the cell (2).

The pool of GLUT4 vesicles that responds to insulin is biochemically distinct from GLUT4 vesicle populations that redistribute to the plasma membrane under other stimuli, such as GTP-{gamma}-S (3) or exercise (4, 5). The precise physical characteristics of the insulin-responsive compartment and the mechanism for intracellular retention have not been established. GLUT4 vesicles were first identified as part of the endosomal compartment by immunolocalization and colocalization with endosomal markers such as the transferrin receptor (6, 7, 8, 9). Newly synthesized GLUT4 is directly targeted to the plasma membrane (10), from which it gains entry into the early endosome. The insulin-responsive pool of GLUT4 vesicles forms as a specialized postendocytic compartment that is separate from the recycling endosome pool (9, 11, 12, 13). Kinetic studies demonstrate that the rate of GLUT4 exocytosis is accelerated by insulin treatment (14, 15, 16, 17). Increased exocytosis may result from increased fission of GLUT4 vesicles from an endosomal precursor, stimulation of the rate of movement of vesicles to the plasma membrane, increased fusion of vesicles with the plasma membrane, or a combination of two or more of these processes. The idea that two or more steps in insulin-dependent GLUT4 translocation may be affected by insulin stimulation is consistent with the observation that insulin-mediated GLUT4 translocation is stimulated by several individual pathways within the insulin signaling cascade (18, 19).

Several laboratories have implicated both actin and microtubule cytoskeletal networks in insulin-dependent GLUT4 translocation. Studies examining the role of the actin cytoskeleton in this process in L6 myotubes, 3T3-L1 adipocytes, and rat adipocytes have shown that disruption of the actin cytoskeleton by either cytochalasin D or latrunculin A inhibits insulin-mediated GLUT4 translocation (20, 21, 22). The actin network may function to transduce the insulin signal to GLUT4 vesicles (23, 24) or to move GLUT4-containing vesicles in the cortical region of the cell (25). The cortical actin network itself undergoes rearrangement when 3T3-L1 cells are treated with insulin, and inhibition of remodeling inhibits GLUT4 translocation, suggesting that the reorganization of actin facilitates GLUT4 translocation (26, 27, 28).

Evidence that the microtubule network plays a role in GLUT4 translocation is accumulating. Several groups have shown, using a variety of pharmacological agents, that either disruption or stabilization of the microtubule network inhibits insulin-mediated GLUT4 translocation (29, 30, 31, 32, 33). Further, depolymerization of the microtubule network using nocodazole, colchicine, or vinblastine causes a redistribution of the perinuclear intracellular GLUT4 storage compartment in 3T3-L1 adipocytes (30, 34), and a disruption of long-distance movement of GLUT4 vesicles (35). Experiments using a dominant interfering light chain mutant of conventional kinesin (KIF5B) to disrupt insulin-dependent GLUT4 translocation, and live cell microscopy to observe tracking of GLUT vesicles along fluorescently labeled microtubules strongly support the involvement of this cytoskeletal network in insulin-mediated GLUT4 vesicle movement (35). Although these studies support one or more roles for the microtubule network in GLUT4 translocation, the role is still being defined and has even been brought into question by some studies (34, 36).

In the current study we demonstrate that the microtubule network itself is an intracellular target of the insulin-signaling cascade and therefore a potentially important point of regulation. We show that insulin promotes the formation of polymerized microtubules, using both a single cell assay and a biochemical analysis measuring polymerized and monomeric tubulin. The effect of insulin on microtubules occurred independently of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and the MAPK pathway, but required tyrosine kinase activity as well as a functional actin cytoskeleton.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
3T3-L1 fibroblasts were obtained from the American Type Tissue Culture repository (Manassas, VA), cultured at 37 C in 5% CO2, and maintained in DMEM containing 25 mM glucose and 10% calf serum. Confluent cultures were induced to differentiate by incubation of the cells with DMEM plus 25 mM glucose, 10% fetal bovine serum, 175 nM insulin, 1 µM dexamethasone, and 0.5 mM isobutyl-1-methylxanthine. After 4 d the medium was changed to DMEM containing 25 mM glucose, 10% fetal bovine serum, and 175 nM insulin, with the incubation period continuing an additional 3 d. More than 95% of the cell population morphologically differentiated into adipocytes under these conditions. Treatment of 3T3-L1 adipocytes with drugs to modify the microtubule cytoskeleton was carried out for the indicated times in serum-free F-12 Ham’s medium.

Plasma membrane sheet assay
Preparation of plasma membrane sheets was carried out as previously described (37). Briefly, cultured adipocytes grown in a six-well cluster dish were treated as described in the figure legends. After experimental treatment, cells were washed with ice-cold PBS and attached to the plate with 0.5% poly-D-lysine. The cells were swollen in hypotonic buffer and then sonicated to release cytoplasmic contents. Pure plasma membrane fragments remaining attached to the plastic dish were scraped into 200 µl solubilization buffer [1% sodium dodecyl sulfate, 20 mM HEPES (pH 7.5), 150 mM NaCl, and 1 mM EDTA]. The protein content of the plasma membrane sheets was measured in a 10-µl aliquot using a spectrofluorometric assay (38).

Extraction of monomeric and polymeric tubulin
Monomeric and polymeric tubulin were differentially extracted from cells plated on 35-mm dishes using a modification of the methods described by Marklund et al. (39). After treatments as indicated, cell monolayers were washed twice with PBS then scraped into 0.4 ml monomeric extraction buffer [20 mM piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES) (pH 6.8), 0.14 M NaCl, 1 mM MgCl2, 1 mM EGTA, 0.5% Nonidet P-40 (NP-40), 0.5 mM phenylmethylsulfonylfluoride, and 4 mM taxol], transferred to an Eppendorf tube, and centrifuged at 13,000 x g for 10 min at room temperature. The NP-40-soluble extract contains monomeric tubulin. The supernatant was transferred to a new tube, and polymeric tubulin was extracted from the remaining insoluble material using 0.4 ml RIPA buffer [10 mM Tris (pH 7.4), 0.15 M NaCl, 1% deoxycholate, 1% NP-40, and 0.1% sodium dodecyl sulfate]. Total protein (bicinchoninic acid assay, Pierce Chemical Co., Rockford, IL) was measured in the monomeric fraction, and the polymeric fraction was determined after solubilization in RIPA buffer according to the manufacturer’s specifications. Equivalent aliquots consisting of 100 ng total protein from polymeric fractions were fractionated by 10% SDS-PAGE and Western-blotted for {alpha}-tubulin. For some experiments equivalent aliquots consisting of 1 µg total protein of the monomeric fraction were also analyzed in the same manner as polymeric tubulin.

Whole cell detergent lysates
One hundred-millimeter plates of treated 3T3-L1 adipocytes were washed twice with ice-cold Tris-buffered saline (TBS), followed by freezing in liquid nitrogen. The plates were thawed on ice and scraped into 1 ml 1% NP-40 lysis buffer [1% NP-40, 20 mM HEPES (pH 7.4), and 2 mM EDTA] containing phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium vanadate, and 1 mM molybdate) and protease inhibitors (10 µM leupeptin, 10 µg/ml aprotinin, 1.5 mM pepstatin A, and 1 mM phenylmethylsulfonylfluoride). The cells were lysed on ice for 20 min, and insoluble material was removed by microcentrifugation for 10 min at 4 C. Protein concentrations of the detergent lysates were determined by a Bradford protein assay (Pierce Chemical Co.) using the manufacturer’s specifications. Gels were loaded with 2 µg total protein/sample.

Electrophoresis and immunoblotting
Samples were fractionated using SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA) in transfer buffer (25 mM Tris and 193 mM glycine, pH 8.5) for 3–4 amp hours at 4 C. Membranes containing tubulin, P42/44 extracellular signal-regulated kinase (ERK), and total protein kinase B (PKB)/akt1 were blocked with 7–10% dried milk and 0.3% Tween 20 in TBS. Antiphosphotyrosine blots were blocked with 2% BSA and 0.1% Tween 20 in TBS. Antiphosphotyrosine blots were carried out using PY99 monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). GLUT4 antiserum was provided by Dr. Gwyn Gould. {alpha}-Tubulin antibody was obtained from Sigma-Aldrich Corp. (T-5168, St. Louis, MO). Phospho-PKB /akt1 antibody specific for Ser473 was obtained from New England Bioloabs (Beverly, MA), and antibody against total PKB/akt 1 was purchased from Transduction Laboratories (Lexington, KY). Immunoblots were visualized using either an enhanced chemiluminescence system (Pierce Chemical Co.) or infrared detection and were quantified using scanning laser densitometry or by infrared spectrophotometry (LiCor, Lincoln, NE).

Immunofluorescence
Cells grown on Lab-Tek culture slides (Nalge-Nunc International, Rochester, NY) were treated as indicated in the figure legends and then fixed in PIPES buffer [0.1 M PIPES (pH 6.8), 5 mM MgCl2, and 50 mM EGTA, pH 7.4] containing 2% paraformaldehyde and 0.1% glutaraldehyde for 10 min at room temperature. Fixed cells were permeabilized in 0.5% Triton X-100, washed, and quenched for 30 min with 66 mM sodium borohydride in 50% ethanol. The cells were incubated with {alpha}-tubulin or {gamma}-tubulin (T-5192, Sigma-Aldrich Corp.), and the antibody was detected using antimouse IgG conjugated with Alexa 488 (Molecular Probes, Eugene, OR) or antirabbit immunoglobulin G conjugated with Alexa 568. The cells were analyzed by confocal microscopy using a Leica TNS confocal microscope (Deerfield, IL). Images displayed were compiled from sections and represent the entire z-axis of the cells (~15 sections of 0.25-µm separation). Image analysis and quantification of {gamma}-tubulin were performed using Leica LCS or Leica LCS Lite software.

Statistical analysis
Statistical analysis was performed using Analyze-It software for Microsoft Excel (Analyze-It Software, Ltd., Leeds, UK).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To begin to examine the function of microtubules in insulin-mediated GLUT4 translocation, we reasoned that the microtubule network itself might be a target of insulin action. To address this question we examined whether cells treated with insulin displayed measurable changes in microtubule dynamics. We first made a morphological examination of microtubules in 3T3-L1 adipocytes treated for various times with a saturating dose of insulin. Insulin treatment promoted an increased intensity of staining of the microtubule network measured by indirect immunofluorescence (Fig. 1Go). Increased staining was prominent throughout cells treated with insulin for 5 min or longer (Fig. 1Go).



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  		FIG. 1. Insulin treatment increases microtubule staining in 3T3-L1 adipocytes. Microtubule immunofluorescence of 3T3-L1 adipocytes treated for 0, 1, 5, 10, 15, or 30 min with 100 nM insulin. Cells were fixed, permeabilized, and stained with anti-{alpha}-tubulin monoclonal antibody. Monoclonal antibody was detected using an antimouse secondary antibody conjugated with Alexa 488. Cells were analyzed by confocal microscopy. Images shown are compiled stacks of the entire z-axis of the cells (15 sections of 0.25 µm) obtained using identical settings on a Leica TCS confocal microscope.

 
Immunofluorescence analysis does not readily provide a quantitative measure of microtubule polymerization in the cell. To quantitate the effect observed by immunofluorescence, we took advantage of the differential solubility of monomeric and polymeric tubulin in nonionic detergents (39, 40). Monomeric tubulin was first extracted in buffer containing 0.5% NP-40. The remaining insoluble material containing polymeric tubulin was then extracted with RIPA buffer. For purposes of quantitation, we established the extreme limits of 100% monomeric tubulin and 100% polymeric tubulin, using nocodazole and taxol treatment, respectively (Fig. 2AGo, lanes 2 and 6) (31). Cells were treated for 0, 1, 5, or 15 min with 100 nM insulin. Western blot analysis revealed that insulin induced an increase in the relative proportion of tubulin found in the SDS-soluble faction (Fig. 2AGo, compare lanes 1, 3, 4, and 5). The insulin-mediated increase in polymeric tubulin, as measured by detergent extraction, was reflected in an insulin-mediated decrease in monomeric {alpha}-tubulin (Fig. 2AGo, compare lanes 1, 3, 4, and 5). In all subsequent studies only sodium dodecyl sulfate-soluble, polymeric tubulin levels were quantitated.



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  		FIG. 2. Insulin treatment increases pool of Triton X-100-insoluble (polymerized) pool of tubulin. Polymerized tubulin and monomeric tubulin were extracted from 3T3-L1 adipocytes treated with 100 nM insulin for 0, 1, 5, and 15 min. Polymerized tubulin was also extracted from cells treated for 1 h with 33 µM nocodazole (Noc) or 10 µM taxol (Tax) to indicate the range from less than 5% polymerized tubulin to greater than 95% polymerized tubulin. Immunoblots from one independent experiment are shown in A. The quantification of polymeric tubulin from five independent experiments is shown in B. The values indicated are the ratio of polymerized tubulin from lanes 1, 3, 4, and 5 of the respective immunoblots, expressed as a percentage of the polymerized tubulin in the taxol-treated sample (the maximum amount of polymerized tubulin). A one-way ANOVA using Dunnett’s pairwise contrasts against the control was performed to compare polymeric tubulin at each time after insulin treatment with the zero time value. *, P < 0.05. C, Quantitation of three independent experiments measuring the dose response of 0, 1, 10, and 100 nM insulin treatment on the formation of polymeric tubulin. A one-way ANOVA using Dunnett’s pairwise contrasts was performed to compare polymeric tubulin at each concentration of insulin with no insulin. *, P < 0.05; **, P < 0.01.

 
Quantitation of the insulin-mediated increase in polymeric tubulin in five independent experiments is shown in Fig. 2BGo. The percentage of polymerized tubulin extracted from cells treated without or with insulin for various times was quantitated by densitometric analysis of Western blots and is presented as the percentage of polymerized tubulin compared with polymerized tubulin extracted from taxol-treated cells (maximum polymerization). This analysis reveals that basal cells have 37 ± 6.2% polymeric tubulin, similar to other cultured cells grown in monolayers (39). Insulin treatment significantly increased polymeric tubulin to 67 ± 4.3% of the maximal value by 5 min of insulin treatment and did not increase above this level after 15 min. The results of the polymeric tubulin extraction are consistent with the increased intensity of staining of microtubules shown in Fig. 1Go.

We next performed a dose-response experiment to determine whether physiological levels of insulin were able to increase polymeric tubulin levels. Cells were incubated for 15 min in the presence of 0, 1, 10, or 100 nM insulin before extraction of polymeric tubulin (Fig. 2CGo). The concentration of insulin required for half-maximal stimulation for tubulin polymerization was between 5–10 nM, a concentration consistent with the half-maximal activation of GLUT4 translocation (41).

To begin characterizing the insulin signaling pathways that lead to tubulin polymerization, we investigated the necessity of tyrosine kinase activation in this process. Cells were pretreated without or with 300 µM genistein for 1 h, followed by treatment with insulin for 0, 1, 5, or 15 min. The efficacy of genistein treatment was tested by Western blotting analysis of total 1% NP-40 cell lysates from cells treated for 0, 1, or 15 min with 100 nM insulin (Fig. 3BGo). Genistein treatment strongly inhibited tyrosine phosphorylation of proteins of approximately 180 and 94 kDa, a size consistent with insulin receptor substrate-1/2 proteins and the ß-subunit of the insulin receptor. The functional significance of inhibition of the insulin receptor tyrosine kinase activity was assessed by measurement of insulin-dependent phosphorylation of PKB/akt1 (Fig. 3CGo). Genistein pretreatment substantially inhibited phosphorylation of PKB/akt1 (Fig. 3CGo, compare lanes 2 and 3 with lanes 5 and 6). Under these conditions, genistein pretreatment also completely inhibited insulin-mediated increases in polymeric tubulin (Fig. 3AGo).



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  		FIG. 3. Insulin-mediated increase in polymerized tubulin requires receptor tyrosine kinase activity. Polymerized tubulin was extracted from 3T3-L1 adipocytes pretreated with genistein and then stimulated with insulin. Cells were treated without or with 300 µM genistein, followed by 100 nM insulin for 0, 1, 5, and 15 min. Polymerized tubulin was also extracted from cells treated for 1 h with 33 µM nocodazole (Noc) or 10 µM taxol (Tax) to indicate the range from less than 5% polymerized tubulin to greater than 95% polymerized tubulin. Immunoblots from one of three independent experiments are shown. Results from the three experiments were quantitated and plotted. Statistical comparison between control and drug-treated samples was carried out using a Pearson correlation test. *, Significant difference (P < 0.05) in polymeric tubulin between control and drug-treated samples at the indicated time points. B, An antiphosphotyrosine blot of whole cell detergent (1.0% NP-40) lysates from cells treated without or with 300 µM genistein and treated for 0, 1, or 15 min with 100 nM insulin. C, An antitotal PKB/akt1 and antiphospho-PKB/akt1 Western blot of the lysates shown in B. The lysates from B and C are from one of three independent and similar experiments.

 
Activated MAPKs have been shown to associate with microtubules during mitosis in somatic cells (42). We tested whether activation of MAPK was required for insulin-mediated microtubule stabilization by inhibiting ERK1/2 activation using PD98059, a specific inhibitor of MAPK kinase (MEK) (43). Pretreatment of cells with 100 µM PD98059 for 1 h before insulin treatment did not inhibit insulin-dependent stabilization of microtubules (Fig. 4AGo). Under these conditions, ERK1/2 activation was prevented, as shown by Western blot analysis using phospho-ERK1/2-specific antibodies (Fig. 4BGo). The inhibition of phospho ERK1/2 was specific for these proteins, as PD98059 treatment did not inhibit phosphorylation of PKB/akt1 (Fig. 4CGo).



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  		FIG. 4. Insulin-mediated increase in polymerized tubulin is not dependent on ERK1/2 activation. Polymerized tubulin was extracted from 3T3-L1 adipocytes treated with 100 nM insulin for 0, 1, 5, and 15 min after pretreatment without or with 100 µM PD98059. Polymerized tubulin was also extracted from cells treated for 1 h with 33 µM nocodazole (Noc) or 10 µM taxol (Tax) to indicate the range from less than 5% polymerized tubulin to greater than 95% polymerized tubulin. Immunoblots from one of five independent experiments are shown. Results from the five experiments were quantitated and plotted in A. Statistical comparison between control and drug-treated samples was carried out using a Pearson correlation test. B, An antitotal ERK1/2 and an antiphospho ERK1/2 Western blot of whole cell detergent (1.0% NP-40) lysates from cells treated without or with 100 µM PD98059 and treated for 0, 1, or 15 min with 100 nM insulin. C, A total PKB/akt1 and phospho-PKB/akt1 Western blot of the lysates shown in B. The lysates from B and C are from one of three independent and similar experiments.

 
Most metabolic actions of insulin are transmitted through activation of PI 3-kinase. To test whether insulin-mediated microtubule stabilization is PI 3-kinase dependent, we pretreated cells with 100 nM wortmannin for 1 h before insulin treatment. Wortmannin treatment had no effect on insulin-mediated microtubule stabilization (Fig. 5AGo). Wortmannin treatment appeared to slightly decrease the level of polymeric tubulin at time zero; however, the insulin-mediated increase was still present, and the rate of this increase was statistically indistinguishable from that in control cells (Fig. 5AGo). To confirm the efficacy of wortmannin treatment, cell lysates were blotted for phospho-ERK1/2 (Fig. 5BGo) and phospho-PKB/akt1 (Fig. 5CGo). Wortmannin efficiently inhibited both of these PI 3-kinase-dependent pathways.



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  		FIG. 5. Insulin-mediated increase in polymerized tubulin is not dependent on PI-3 kinase activation. Polymerized tubulin was extracted from 3T3-L1 adipocytes treated with 100 nM insulin for 0, 1, 5, and 15 min after pretreatment without or with 100 nM wortmannin. Polymerized tubulin was also extracted from cells treated for 1 h with 33 µM nocodazole (Noc) or 10 µM taxol (Tax) to indicate the range from less than 5% polymerized tubulin to greater than 95% polymerized tubulin. Immunoblots from one of three independent experiments are shown. Results from the four experiments were quantitated and plotted. Statistical comparison between control and drug-treated samples was carried out using a Pearson correlation test. No significant difference was detected between control and wortmannin-treated cells. B, An antitotal ERK1/2 and an antiphospho ERK1/2 Western blot of whole cell detergent (1.0% NP-40) lysates from cells treated without or with 100 nM wortmannin and treated for 0, 1, or 15 min with 100 nM insulin. C, A total PKB/akt1 and phospho-PKB/akt1 Western blot of the lysates shown in B. The lysates from B and C are from one of three independent and similar experiments.

 
The morphological analysis shown in Fig. 1Go is consistent with either an increase in the numbers of microtubules or increased polymerization/stabilization of existing microtubules. To determine whether insulin acts to increase the number of growing microtubules (either by increasing the number of nucleation sites or by increasing the total number of microtubules at a given site), we performed a microtubule regrowth assay. In this assay microtubules were completely depolymerized by 33 µM nocodazole treatment. The nocodazole-containing medium was washed out and replaced with fresh medium containing 100 nM insulin or no insulin, then incubated for 1, 3, 10, and 30 min at 37 C. Cells were fixed and incubated with antibodies against {alpha}-tubulin and {gamma}-tubulin, followed by treatment with secondary antibodies conjugated to different fluorochromes. (Fig. 6Go). The efficiency of microtubule nucleation at microtubule-organizing centers (MTOC) was indicated by the intensity of {gamma}-tubulin staining of the MTOC, as described previously by Ploubidou et al. (44). {gamma}-Tubulin staining of MTOC was measured for each time point in 20–22 cells imaged from samples treated without or with insulin, and the resulting intensity values were averaged (Table 1Go). The {gamma}-tubulin-stained MTOC were analyzed using confocal microscopy, and the fluorescence of {gamma}-tubulin was quantitated using Leica LCS software to obtain a measurement of the fluorescence intensity. The degree of {gamma}-tubulin staining of each {gamma}-tubulin center as well as the number of these nucleation centers per cell were indistinguishable in images collected from samples treated without or with insulin (Fig. 6Go and Table 1Go). In contrast, the intensity of polymerized {alpha}-tubulin was increased at all times in cells refed medium that contained insulin compared with cells refed medium that did not contain insulin (Fig. 6Go). This suggests that rather than promoting de novo nucleation of organizing centers or initiation of new microtubules, insulin acts to promote the growth and/or stabilization of preexisting microtubules.



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  		FIG. 6. Insulin does not increase the efficiency of microtubule nucleation. Immunofluorescent microtubules of 3T3-L1 adipocytes were treated with nocodozole and then stimulated with insulin. Cells were exposed to 33 µM nocodazole for 1 h to completely depolymerize microtubules. Nocodazole was washed out and replaced with fresh medium without or with 100 nM insulin. Cells were fixed at 0, 1, 3, 10, or 30 min after removal of nocodazole. The cells were permeabilized, stained with the anti-{alpha}-tubulin antibody and anti-{gamma}-tubulin, and analyzed as described in Fig. 1Go.

 

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  		TABLE 1. Immunofluorescent staining of {gamma}-tubulin in 3T3-L1 adipocytes

 
Insulin has been shown in previous reports to rearrange the cortical actin of 3T3-L1 adipocytes in a PI 3-kinase-independent manner (26, 28). It is possible that a relationship exists between the insulin-dependent rearrangement of cortical actin and the effect we observed on microtubules. To begin investigating this possibility, we examined insulin-dependent effects on microtubules in the presence or absence of an intact actin cytoskeleton. The actin cytoskeleton was depolymerized by 2 h of pretreatment with 60 µM latrunculin B; cells were stimulated with insulin for 1, 5, or 15 min; and polymeric tubulin was then extracted as described above. Depolymerization of actin completely inhibited insulin-mediated polymerization of the microtubule cytoskeleton (Fig. 7AGo). Quantitation of three independent experiments is shown in Fig. 7BGo. Proximal insulin signaling at the plasma membrane was not inhibited by latrunculin B treatment, as shown by antiphosphotyrosine antibody staining of whole detergent lysates obtained from insulin-treated cells (Fig. 7CGo).



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  		FIG. 7. The insulin-mediated increase in polymerized tubulin is dependent on the actin cytoskeleton. Polymerized tubulin was extracted from 3T3-L1 adipocytes treated with 100 nM insulin for 0, 1, 5, and 15 min after pretreatment without or with 60 µM latrunculin B. Polymerized tubulin was also extracted from cells treated for 1 h with 33 µM nocodazole (Noc) or 10 µM taxol (Tax) to indicate the range from less than 5% polymerized tubulin to greater than 95% polymerized tubulin. Immunoblots from one of three independent experiments are shown. Results from the three experiments were quantitated and plotted. Statistical comparison between control and drug-treated samples was carried out using a Pearson correlation test. *, Significant difference (P < 0.05) in polymeric tubulin between control and drug-treated samples at the indicated time points. B, An antiphosphotyrosine Western blot of whole cell detergent (1.0% NP-40) lysates from cells treated without or with 60 µM latrunculin B and treated for 0, 1, or 15 min with 100 nM insulin.

 
Complete depolymerization of microtubules was not required for inhibition of insulin-mediated GLUT4 translocation. Treatment of cells with 2 µM nocodazole partly destabilized microtubules, whereas 10 µM nocodazole caused more complete depolymerization (Fig. 8AGo). Despite the differences in microtubule depolymerization, both 2 and 10 µM nocodazole resulted in a 35% inhibition of insulin-dependent GLUT4 translocation (Fig. 8Go, B and C), indicating that complete depolymerization of microtubules is not necessary to inhibit GLUT4 translocation.



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  		FIG. 8. GLUT4 translocation is inhibited with only partial destabilization of microtubules by nocodazole treatment. Immunofluorescent microtubules in cells treated without or with either 2 or 10 µM nocodazole for 30 or 60 min are shown in A. The cells were stained and analyzed as described in Fig. 1Go. B and C, GLUT4 translocation in plasma membrane fragments from cells treated without or with 100 nM insulin for 20 min without or with pretreatment with 2 or 10 µM nocodazole for 1 h before insulin treatment. The blot shows one independent experiment performed in triplicate. The histogram is the quantitation of three independent experiments, each performed in triplicate. A two-tailed t test was performed to compare GLUT4 levels between control and nocodazole-treated samples. *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies from several laboratories, including our own, have implicated the microtubule network in insulin-mediated GLUT4 translocation, but the precise role is unknown (29, 31, 32, 33). There are several possible functions that microtubules may perform in insulin-dependent GLUT4 translocation, including, but not limited to, provision of a supporting structure to specifically retain the insulin-responsive pool of GLUT4 vesicles in the absence of an insulin signal or providing a platform for migration of the vesicles to the plasma membrane upon insulin stimulation. Each of these possibilities is supported by experimental evidence. For example, disruption of the microtubule network by pharmacological agents causes the insulin-responsive, perinuclear pool of GLUT4 vesicles to disperse, suggesting that microtubules may tether GLUT4 vesicles in this intracellular localization (32, 34). Real-time microscopy of insulin-stimulated adipocytes reveals that GLUT4 vesicles migrate along a straight trajectory, and this movement is inhibited by disruption of the microtubule network (29, 33, 35). In a recent report live cell microscopy of the path of GLUT4 vesicle movement coinciding precisely with labeled microtubules, and loss of GLUT4 translocation in the presence of a dominant-interfering kinesin added strong support to a role for microtubules in this process (35). Finally, interfering with the function of kinesin proteins, molecular motors that move from the negative to positive poles of microtubules, inhibits insulin-mediated GLUT4 translocation (32, 35).

In this paper we provide strong evidence that the microtubule network itself is a target of insulin action. Using biochemical and immunohistochemical approaches, we show that physiological doses of insulin increase the polymeric tubulin concentration in 3T3-L1 adipocytes. Increased fluorescent labeling of {alpha}-tubulin was observed at early times after insulin treatment and continued through 30 min of continuous exposure to insulin. In an alternative approach, tubulin was partitioned into detergent-soluble ({alpha}-ß dimers) and detergent-insoluble (polymer) fractions to quantitate the insulin-dependent changes we observed using fluorescent methods. Regulation by insulin is not unexpected, because microtubules change in response to other intracellular signals and are involved in a variety of cellular functions, such as cell growth and chemotaxis (45).

Two lines of evidence indicate that the effect of insulin on microtubules is mediated through the insulin receptor. First, the half-maximal response occurred with insulin levels between 5–10 nM, consistent with the half-maximal dose for GLUT4 translocation in this cell type (41). Second, pretreatment of cells with genistein to inhibit insulin receptor tyrosine kinase activity prevented the effect. Taken together, these data strongly support the hypothesis that insulin signals to microtubules and increases the steady state concentration of polymeric tubulin in the cell.

Pharmacological inhibitors of the PI 3-kinase pathway and the ERK1/2 pathway did not inhibit microtubule polymerization, indicating that these pathways are not necessary for insulin signaling to the microtubule network. Although a majority of the metabolic actions of insulin signal through the PI 3-kinase pathway, a novel PI 3-kinase-independent pathway leading to the activation of the small molecular weight guanosine triphosphatase, TC10, and to the reorganization of cortical actin in 3T3-L1 adipocytes has been reported (26, 46). Reorganization of cortical actin has been shown to be required for insulin-mediated GLUT4 translocation. The similarities between dynamic cortical actin and dynamic microtubules, each stimulated by insulin in a PI 3-kinase independent manner, suggest a possible functional link between the two cytoskeletal systems in insulin-mediated GLUT4 translocation. In fact, destabilization of the actin cytoskeleton by treatment with latrunculin B completely prevented the insulin-dependent changes in the microtubule polymerization. This experiment is consistent with a functional link between these two cytoskeletal networks with respect to intracellular insulin action. Although the specific mechanistic link between the actin and microtubule networks is not clear at this point, interactions between these two networks have been identified in other cellular functions, such as cell motility and cellular kinesis (47, 48). Further work must be conducted to determine whether insulin-dependent changes in cortical actin are dependent on an intact microtubule network.

Using astral regrowth experiments, we showed that insulin treatment does not increase the efficiency of microtubule nucleation at centrosomes, as indicated by {gamma}-tubulin staining of these structures. Insulin appears to modify the growing microtubule fibers, either by enhancing plus-end polymerization or by preventing destabilization of the microtubule. Microtubule dynamics are described by four parameters: rate of polymerization, rate of depolymerization, frequency of catastrophe (the conversion from polymerization to depolymerization), and frequency of rescue (the conversion from depolymerization to polymerization). Insulin may act at one or more of these points to alter the steady state of microtubule polymerization. For instance, catastrophe can be prevented by stabilization of a GTP or GDP plus Pi-tubulin cap structure at the plus end of the tubulin polymer (45). Whether insulin promotes the formation of a stabilizing cap structure remains to be determined.

In this study we have demonstrated that low-dose treatment of nocodazole does inhibit the translocation of endogenous GLUT4 in adipocytes. This is in contrast to other laboratories that have shown that translocation of the GLUT4 reporter was not inhibited by low doses of nocodazole (34, 36). The differences in these results may reflect the different assays used to assess GLUT4 translocation. In our work GLUT4 translocation was assessed by Western blot analysis of equivalent lysate aliquots representing an entire plate of cells. In contrast, the other studies used a type of single cell assay in which GLUT4 translocation is assessed on a per cell basis. Therefore, the total pool of cells sampled in the single cell assay is several orders of magnitude lower than that in the type of assay performed in the current study. Given the heterogeneity of GLUT4 expression and the translocation among individual 3T3-L1 adipocytes on a single plate of cells, the method of sampling is critical for detecting 30–40% decreases in insulin-mediated GLUT4 translocation. Thus, the single cell assay may not be sensitive enough to effectively measure small changes in GLUT4 translocation.

The inhibition of GLUT4 translocation using 2 and 10 µM nocodazole was less than that observed using 33 µM nocodazole (31), suggesting that the high dose nocodazole treatment inhibits GLUT4 translocation independently of its effect on microtubules, as reported by Molero et al. (34). Immunofluorescent staining of cells treated with 2 and 10 µM nocodazole show that microtubule depolymerization is not complete under these conditions, with intact microtubules being evident in the perinuclear region of the cell, but not at the cell margins. These data are consistent with model in which the portion of the microtubule fiber that is sensitive to low concentrations of nocodazole (e.g. the growing ends) is responsible for the entire effect of nocodazole on GLUT4 translocation. In this scenario, the growing end of the microtubule could be exerting a force to perform work on another cellular structure (such as the membrane or a vesicle), thus facilitating GLUT4 translocation or other changes in cellular function mediated by insulin. Such a role for the growing ends of microtubules as molecular machines has been described in other systems (49). It is attractive to speculate that changes in microtubule stability along with rearrangement of cortical actin may play a role in facilitating the association of GLUT4 vesicles with the plasma membrane for docking and fusion.

In summary, we have shown that insulin increases the level of polymerized tubulin in a dose-dependent manner that is dependent on tyrosine kinase activity and is independent of PI 3-kinase activity. In light of accumulating evidence demonstrating a role for microtubules in insulin-mediated GLUT4 translocation, the effect of insulin on the microtubule network can be viewed as a potentially important point of regulation in the propagation of insulin action. The insulin-mediated increase in polymeric tubulin depends on an intact actin cytoskeleton and may reflect a functional interaction between actin and microtubules that is important in insulin-dependent GLUT4 translocation. Both the actin cytoskeleton and the microtubule network have been shown to play a role in insulin-mediated GLUT4 translocation. It is possible that the role(s) of microtubules in insulin-dependent GLUT4 translocation may be directly related to the changes in microtubule stability during insulin stimulation.


   Acknowledgments
 
We wish to thank Mr. Jim Henthorn (Flow and Image Cytometry Laboratory, William K. Warren Medical Research Institute, Oklahoma City, OK) for expert assistance with confocal microscopy.


   Footnotes
 
This work was supported by research grants from the NIH (DK-62341) and the Oklahoma Center for Advancement of Science and Technology.

Abbreviations: ERK, Extracellular signal-regulated kinase; GLUT4, adipose/muscle-specific glucose transporter; IRS-1, insulin receptor substrate 1; MTOC, microtubule-organizing centers; NP-40, Nonidet P-40; PI 3-kinase, phosphatidylinositide 3-kinase; PIPES, piperazine-N,N'-bis[2-ethanesulfonic acid]; PKB, protein kinase B; TBS, Tris-buffered saline.

Received May 19, 2003.

Accepted for publication August 5, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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