![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 17, Issue 10, 4249-4256, October 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*Department of Biochemistry, Boston University Medical School, Boston, MA 02118; and Department of Cell Biology, Harvard Medical School, Boston, MA 02115
Submitted April 6, 2006;
Revised June 27, 2006;
Accepted July 18, 2006
Monitoring Editor: Robert Parton
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Watson et al., 2004). Although this process is very well studied, the exact subcellular trafficking pathway(s) and the molecular mechanisms by which insulin recruits GLUT4 to the plasma membrane remain incompletely understood. Kinetic analysis of GLUT4 trafficking first implicated GSV exocytosis as the likely locus of the action of insulin (Satoh et al., 1993; Yeh et al., 1995). More recently, this has been confirmed by data showing that the insulin signaling pathway kinase component Akt2 (for review, see Watson et al., 2004) acts on the GSV exocytosis process, either before the vesicle docking and fusion step (Zeigerer et al., 2004) or directly on this step (Koumanov et al., 2005; van Dam et al., 2005). Evidence from real-time microscopy analysis of GSVs reveals that they can move along cytoskeletal elements (Patki et al., 2001; Semiz et al., 2003) and that the first effect of insulin is to dramatically reduce their mobility, presumably by tethering them to some cellular structure such as the cortical cytoskeleton, followed by a slower fusion with the plasma membrane (Lizunov et al., 2005). Taken together, these data implicate vesicle movement machinery and/or the vesicle fusion apparatus as key endpoint targets of insulin-dependent GLUT4 translocation.
Evidence has been obtained that both the microtubule- (Fletcher et al., 2000; Guilherme et al., 2000; Olson et al., 2001; Liu et al., 2003) and actin-based cytoskeleton participate in the organization of and signaling to GSV exocytosis in fat and muscle cells (Khayat et al., 2000; Omata et al., 2000; Kanzaki and Pessin, 2001; Liu et al., 2003; Brozinick et al., 2004). In both cell types, agents that disrupt actin- or tubulin-based fibers reduce insulin-stimulated glucose uptake and inhibit its underlying mechanism, GLUT4 translocation to the cell surface membrane. Insulin may mediate its affects on cortical actin remodeling via the actin-regulatory protein, neural Wiskott-Aldrich syndrome protein (N-WASP) (Kanzaki et al., 2001; Jiang et al., 2002; Brozinick et al., 2004). In addition, GSV movement has been proposed to require molecular motors moving on microtubules (Emoto et al., 2001) and microfilaments (Bose et al., 2004), but mechanistic links to insulin signaling for these events remain ill defined. The cortical cytoskeleton network has numerous protein components in addition to actin and tubulin (dos Remedios et al., 2003; Winder and Ayscough, 2005), including spectrin family members, which have been suggested to serve membrane-sorting tasks (Beck and Nelson, 1996; Brown and Breton, 2000; Broderick and Winder, 2005). We report here that fodrins, also called nonerythroid spectrins, are abundantly expressed in adipocytes where they serve a role in GLUT4 translocation.
There have been a limited number of studies suggesting a link between components of the actin cytoskeleton network and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (for review, see Hong, 2005) that mediate vesicle fusion in the exocytic process (Nakano et al., 2001; Band et al., 2002; Fan and Beck, 2004; Low et al., 2006). GSVs contain VAMP2 as their vesicle SNARE, which interacts with syntaxin 4 (Syn4) at the plasma membrane to mediate vesicle fusion and presents GLUT4 to the extracellular milieu where it can function (for review, see Grusovin and Macaulay, 2003). Interestingly, 
-fodrin has been shown to be a syntaxin 4 binding partner (Nakano et al., 2001). Thus, in the course of a proteomic analysis of adipocyte membrane lipid raft components, we identified - and 
-fodrin as abundant components (-fodrin, 119 nonredundant peptides, 57% coverage; -fodrin, 72 peptides, 35% coverage; our unpublished data) of this membrane fraction. The spectrins/fodrins are widely expressed proteins that form filamentous - heterodimers and bind to actin at both ends, forming a repeating "corral"-like network just beneath the plasma membrane (Bennett and Baines, 2001). This network has a clear role in stabilizing the erythrocyte membrane and has been proposed to serve a variety of functions in other cells, including membrane trafficking/sorting (Beck and Nelson, 1996; Brown and Breton, 2000; Broderick and Winder, 2005). These facts plus the reported association of -fodrin with syntaxin 4 (Nakano et al., 2001) prompted us to examine the behavior of fodrin under conditions of insulin-stimulated GLUT4 translocation. Our data suggest that the cortical actin–fodrin network in rat adipocytes plays a critical role in GSV movement to the plasma membrane (PM) via the interaction of fodrin with Syn4. The insulin-dependent remodeling of fodrin seen by confocal microscopy is remarkably robust, further supporting a role for this protein in GLUT4 translocation to the plasma membrane.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-fodrin (clone AA6, suitable for immunofluorescence [IF]; Molitoris et al., 1996), rabbit anti-syntaxin 4 (catalog no. 110 042), and mouse actin antibodies were purchased from Chemicon International (Temecula, CA), Synaptic Systems (Goettingen, Germany), and Sigma-Aldrich, respectively. We confirmed our results showing insulin-dependent fodrin remodeling with an anti-fodrin antibody (catalog no. 344050; Calbiochem, San Diego, CA) (our unpublished data). Monoclonal antibodies recognizing GLUT4 (1F8) (James et al., 1988) and polyclonal anti-insulin-regulated aminopeptidase (IRAP) have been described previously (Kandror and Pilch, 1998; Zhou et al., 2000). Primary antibodies were detected in Western blots by using secondary antibodies conjugated to horseradish peroxidase (Sigma-Aldrich) diluted 1:3000 and chemiluminescent substrate (PerkinElmer Life and Analytical Sciences, Boston, MA). Cy2 anti-mouse IgG and anti-rabbit Cy5 antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Subcellular Fractionation of Adipocytes
This protocol was performed essentially as originally described previously (Simpson et al., 1983) and as we have previously performed (Kandror and Pilch, 1996). Briefly, epididymal fat pads were removed from male Sprague Dawley rats (150–175 g) and transferred to KRP (12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 0.6 mM Na2HPO4, 0.4 mM NaH2PO4, 2.5 mM glucose, and 2% bovine serum albumin, pH 7.4) at 37°C. Isolated adipocytes were obtained by collagenase (Roche Applied Science, Indianapolis, IN) digestion for 45 min. After recovery from digestion for an additional 45 min, cells were stimulated (or not) with insulin for 15 min. Hormonal action was stopped with 2 mM KCN. Cells were then transferred to HES (20 mM HEPES, 5 mM EDTA, and 250 mM sucrose, pH 7.4) and homogenized with a Teflon-glass tissue grinder. Subcellular fractions (PM, heavy microsomes [HMs], and light microsomes [LMs]) were obtained by differential centrifugation and resuspended in HES. All buffers used in this work contained a mixture of protease inhibitors consisting of 1 µM aprotinin, 10 µM leupeptin, 1 µm pepstatin (American Bioanalytical, Natick, MA), and 5 mM benzamidine (Sigma-Aldrich) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich).
Immunoprecipitation
The PM fraction (50–100 µg of total protein) was suspended in phosphate-buffered saline (PBS) and solubilized with 0.05% Triton-100 overnight at 4°C with constant agitation. Insoluble material was removed by pelleting for 5 min in a microcentrifuge. Monoclonal anti--fodrin antibodies, and nonspecific mouse and rabbit IgG (5 µg), were incubated with the supernatant 2 h at 4°C, and then 20 µl of protein A beads (Santa Cruz Biotechnology, Santa Cruz, CA) was added for 1 h. The supernatant with unbound proteins was collected, and the beads were washed four times with 0.05% Triton-100 in PBS buffer, rinsed once PBS, and eluted in Laemmli sample buffer (Laemmli, 1970) containing 2% SDS, and equal portions were subjected to SDS-PAGE and analyzed by Western blotting.
Confocal Microscopy
Rat adipocytes were washed three times with KRP and fixed with 3% (wt/vol) paraformaldehyde in PBS for 1 h at room temperature. The cells were permeabilized, and nonspecific binding sites were blocked in PBS containing 0.1% saponin, 1% bovine serum albumin, and 3% normal goat serum for 45 min at room temperature. The cells were then incubated with rabbit anti-syntaxin 4 (1:500 dilution) and mouse anti--fodrin antibody (AA2; 1:250) for 2 h at room temperature, and washed three times with PBS containing 0.1% saponin. Next, the cells were incubated with Cy5-conjugated anti-rabbit IgG, Cy2-conjugated anti-mouse IgG (1:200 dilution) and 5 U/ml Alexa Fluor 594-phalloidin for 1 h at room temperature. Finally, the cells were washed with PBS containing 0.1% saponin, mounted in 50% glycerol saturated with n-propylgallate as an antibleaching reagent, and observed with an fluorescence microscope equipped with a laser confocal system (LSM510, Carl Zeiss, Thornwood, NY). Captured images were processed with LSM 5 Image Browser software. The three-dimensional images were reconstructed from serial confocal images taken along the z-axis.
Western Blotting
Proteins were separated by SDS-PAGE, transferred to a 0.2-µm polyvinylidene difluoride membrane and incubated in PBS with 0.1% Tween 20 containing 10% nonfat evaporated milk for 1 h at room temperature. The membranes were then incubated with the primary antibodies described above. Horseradish peroxidase-conjugated secondary antibodies (Sigma-Aldrich), and either an enhanced chemiluminescent substrate kit (PerkinElmer Life and Analytical Sciences) or SuperSignal West Femto Maximum Sensitivity Substrate kit (Pierce Chemical, Rockford, IL) was used for detection.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-fodrin shows a highly punctate signal that overlaps considerably, but not completely, with actin. After insulin exposure, cytoskeletal remodeling occurs, resulting in a more diffuse signal for both proteins, and a lesser degree of overlap (Figures 3 and 9). The punctate nature of the fodrin signal is largely eliminated after insulin exposure in a particularly robust example (Figure 1B) of insulin-dependent cytoskeletal remodeling. Note that there is a strong signal for both fodrin and actin outlining the nucleus. The significance of this is not clear, because the plasma membrane above the nucleus represents <1% of the cell surface and is therefore unlikely to contribute in a major way to insulin-dependent glucose transport/GLUT4 translocation. We next examined the distribution of actin and fodrin in fractionated rat adipocytes by immunoblot analysis as shown in Figure 2. Membrane-associated fodrin and actin are found largely at the plasma membrane, and insulin did not affect the distribution of either protein, whereas having the expected affect on GLUT4 and IRAP translocation (Figure 2), i.e., moving them from the LM to the PM fraction. The results of Figures 1 and 2 suggest insulin stimulation causes remodeling of the cortical fodrin–actin network in rat adipocytes without changing the overall amount of these proteins in the cortical cytoskeleton.
|
|
-fodrin is colocalized with syn4, we reconstructed 3-D images from serial confocal microscopy sections of adipocytes labeled with antibodies to both proteins. As shown in Figure 3A, Syn4 colocalizes with fodrin, and this colocalization is substantially increased after cellular exposure to insulin (more yellow, less green and red). As in Figure 1, the highly punctate fodrin staining is dramatically changed upon insulin exposure whereas the more diffuse Syn4 staining is relatively unchanged. To confirm this possible fodrin-Syn4 interaction, we performed an immunoprecipitation of plasma membrane proteins with anti-fodrin antibody and examined the immunoprecipitate by Western blotting with the indicated antibodies (Figure 3B). The data show that fodrin interacts with Syn4 in the plasma membrane, and the interaction was increased twofold after insulin stimulation (Figure 6). Note that the same result was seen using either Triton X-100 or octyl glucoside to solubilize the membrane, the latter being able to disrupt lipid rafts (Shogomori and Brown, 2003). Thus, these data suggest that there may be a direct link between the plasma membrane actin–fodrin network and the t-SNARE protein involved in GLUT4 translocation.
|
Latrunculin A Disrupts the Actin Network in Rat Adipocytes
LatA binds to actin subunits and prevents their polymerization, thereby disrupting cortical actin structure (Coue et al., 1987). As shown in Figure 4 by confocal microscopy, LatA treatment virtually eliminates the actin signal at the PM, indicating disruption of all actin filaments. In contrast, there was little disruption of the punctate fodrin signal under these conditions, and the insulin-induced remodeling seen in Figures 1A and 3A was blocked by LatA treatment, indicating fodrin-remodeling is dependent on an intact actin network. The Syn4 distribution was unaffected by LatA treatment, and it shows significant colocalization with fodrin as in the prior figures.
|
), and the distribution of Syn4 was unchanged under all conditions. Note that the proportions of cytosol and PM were normalized to one another for this experiment, whereas equal protein was analyzed in Figure 2.
|
|
). To confirm that the results we have obtained in prior figures are due to cortical actin disruption, we performed subcellular fractionation and confocal microscopy on CytoD-treated rat adipocytes. As show in Figure 7A, CytoD treatment causes actin disassociation from PM, albeit to a lesser extent than is caused by LatA, indicating there are cytoD-sensitive and -insensitive actin pools in the PM fraction. In contrast, there was little or no change in fodrin distribution, and the translocation of GLUT4 was not inhibited. Figure 7B shows that there is a small affect of CytoD on the actin signal obtained by confocal microscopy, consistent with the biochemical data, and that CytoD has no effect on the interaction of Syn4 and fodrin as determined by this methodology (our unpublished data).
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Khayat et al., 2000; Kanzaki and Pessin, 2001; Patki et al., 2001; Jiang et al., 2002), cells in which it also enhances glucose transport as a result of GSV translocation to the cell surface. The disruption of the cortical cytoskeleton by latrunculin A or by dominant-negative N-WASP constructs markedly inhibits GLUT4 translocation (Khayat et al., 2000; Kanzaki and Pessin, 2001; Kanzaki et al., 2001; Jiang et al., 2002), and an active role for cortical actin in this process has been proposed based on these data. Similarly, latrunculin A in primary rat adipocytes (Omata et al., 2000) and latrunculin B in isolated rat epitrochlearis muscle (Brozinick et al., 2004) disrupts cortical actin filaments and blocks GLUT4 translocation. However, a biochemical link from the insulin signaling pathway and GSVs to the cortical cytoskeleton has not been firmly established, and insulin-induced-actin remodeling has not been observed in primary fat or muscle cells. Primary adipocytes differ from their cultured counterparts in their large size, completely round shape and a cytoarchitecture consisting of a single lipid (triglyceride) droplet surrounded by a thin cytosolic rim. Cultured adipocytes have multiple lipid droplets (e.g., Hosaka et al., 2005); are round in two dimensions, and like most cultured cells, are thought to represent a less than fully mature cellular phenotype. Here, we reconstructed, from serial confocal sections, 3-D images of primary rats adipocytes to study the spatial organization of the cortical cytoskeleton, its insulin-dependent remodeling, and the relationship of these parameters to GLUT4 translocation.
Our novel findings are that fodrin is a highly expressed component of the cortical cytoskeleton of primary adipocytes that undergoes a robust insulin-dependent remodeling upon insulin action (Figure 1). -Fodrin interacts with Syn4, the t-SNARE required for GLUT4 translocation, and insulin enhances this interaction (Figures 3 and 6). In contrast, disruption of cortical actin by latrunculin A reduces the fodrin–Syn4 interaction, blocks fodrin remodeling, and inhibits GLUT4 translocation (Figures 4
–6). Cytochalasin D treatment has little effect on the cortical cytoskeleton and does not block insulin-mediated GLUT4 translocation (Figure 7). Once insulin-dependent fodrin remodeling has occurred, it becomes insensitive to LatA (Figure 8), indicating that it is only the exocytic step of GLUT4 translocation that requires this aspect of the cortical cytoskeletal function. The data fit a model derived from various recent studies by using different methodology (Zeigerer et al., 2004; Koumanov et al., 2005; Lizunov et al., 2005; van Dam et al., 2005), which together, show that the GSV exoctyic step, at or near the plasma membrane and involving the cortical cytoskeleton, is the major target of insulin signaling to GLUT4 translocation (Figure 10). Our present results complement these studies and describe for the first the involvement of fodrin and its insulin-dependent interaction with Syn4 in GSV trafficking.
It is not clear from the literature whether the cytoskeletal involvement in GLUT4 translocation impinges on the formation of a signaling complex (Khayat et al., 2000; Eyster et al., 2005) or is required for the movement of GSVs to plasma membrane (Kanzaki et al., 2001; Bose et al., 2002). These cited studies were performed in cultured adipocytes or myocytes (Khayat et al., 2000) where the intracellular GLUT4 pools are perinuclear and must traverse a considerable distance to the cell periphery in response to insulin, whereas the GSVs only need to move short distances in primary fat cells. Moreover, although latrunculin inhibits insulin signaling to Akt in 3T3-L1 adipocytes (Eyster et al., 2005), it does not do so in primary cells (Omata et al., 2000). Thus, we favor the notion that in primary adipocytes, insulin-dependent cytoskeletal involvement in GLUT4 translocation converges more on the vesicular trafficking aspect than on signaling organization, perhaps by means of a tethering protein (or proteins; see below), although we cannot rule out that it has a direct effect on the signal transduction pathway.
So how is fodrin mediating its effects on GLUT4 translocation? As noted previously, there are a few studies suggesting that fodrin can interact with syntaxins (Nakano et al., 2001; Low et al., 2006), including syntaxin 4 as we now show for adipocytes, cells where fodrin has been described only in a single morphological study (Aoki et al., 1997). Fodrins (spectrins) are located just underneath the plasma membrane (Bennett and Baines, 2001), where they can potentially interact with syntaxin 4, and recent studies indicate that insulin halts GSV movement near the cell surface as a result of their tethering to what is likely a cytoskeletal element (Lizunov et al., 2005), which might be fodrin (but see below). In contrast, studies in cultured adipocytes have suggested that GLUT4-containing vesicles are tethered in the basal state (Oatey et al., 1997) and insulin may release the tether, candidate tethering proteins being TUG (tether, containing a UBX domain, for GLUT4 (Bogan et al., 2003) or p115, a bona fide vesicle tethering protein albeit, one with a predominantly Golgi localization that is present in lesser amounts at the cell surface (Hosaka et al., 2005). TUG and p115 interact with GLUT4 and IRAP, respectively, and the former interaction is reduced by insulin. Conversely, we show here that the syntaxin 4–fodrin interaction is increased by insulin by two independent methods, coimmunoprecipitation (Figures 3 and 6) and confocal microscopy (Figure 3). Indeed, when the interaction of -fodrin with syntaxins was studied in vitro with recombinant proteins (Nakano et al., 2001), its affinity was shown to be 
100 nM, and it was specific for this fodrin isoform. In some cell types, syntaxins have also been shown to cluster in cholesterol-rich regions (Lang et al., 2001; Low et al., 2006; Sieber et al., 2006), where their high concentration may allow more facile vesicular fusion. Indeed, it has been reported that a significant proportion (35%) of syntaxin 4 is localized to lipid rafts in cultured adipocytes (Chamberlain and Gould, 2002). We see some evidence for diffuse clusters of syn4, but we see no effects of insulin on this clustering (Figure 3A).
Thus, we envision the following scenario. Insulin signaling targets the cortical cytoskeleton via actin or associated regulatory proteins such as N-WASP, as discussed previously. This results in a dramatic remodeling of fodrin structures such that their signal by IF goes from highly punctate to diffuse (Figure 1), i.e., they undergo a conformational change such that the IF signal is altered, but the PM association is apparently unchanged (Figure 2). We envision the fodrin-based foci to be dispersed by insulin such that access of GSVs to syntaxin 4 is enhanced and GSV fusion with the PM is facilitated (Figure 10). There seems to be little direct interaction of GSVs with fodrin in the basal state, because their IF signals do not significantly overlap in basal, whereas they do overlap to a significant degree in stimulated cells (Figure 9). In summary, our results document the involvement of a new player, fodrin, in GSV translocation and suggest mechanisms by which this may occur. Efforts are underway to identify the biochemical changes that may take place in the cortical cytoskeletal proteins of primary adipocytes as a result of insulin action.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Address correspondence to: Paul F. Pilch (ppilch{at}bu.edu)
Abbreviations used: CytoD, cytochalasin D; GLUT4, glucose transporter 4; HM, heavy microsome; IRAP, insulin-regulated aminopeptidase; LatA, latrunculin A; LM, light microsome; PM, plasma membrane; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Syn4, syntaxin 4.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Band, A. M., Ali, H., Vartiainen, M. K., Welti, S., Lappalainen, P., Olkkonen, V. M., Kuismanen, E. (2002). Endogenous plasma membrane t-SNARE syntaxin 4 is present in rab11 positive endosomal membranes and associates with cortical actin cytoskeleton. FEBS Lett 531, 513–519.[CrossRef][Medline]
Beck, K. A. and Nelson, W. J. (1996). The spectrin-based membrane skeleton as a membrane protein-sorting machine. Am. J. Physiol 270, C1263–C1270.[Medline]
Bennett, V. and Baines, A. J. (2001). Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol. Rev 81, 1353–1392.
Bogan, J. S., Hendon, N., McKee, A. E., Tsao, T. S., Lodish, H. F. (2003). Functional cloning of TUG as a regulator of GLUT4 glucose transporter trafficking. Nature 425, 727–733.[CrossRef][Medline]
Bose, A., Guilherme, A., Robida, S. I., Nicoloro, S. M., Zhou, Q. L., Jiang, Z. Y., Pomerleau, D. P., Czech, M. P. (2002). Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c. Nature 420, 821–824.[CrossRef][Medline]
Bose, A., Robida, S., Furcinitti, P. S., Chawla, A., Fogarty, K., Corvera, S., Czech, M. P. (2004). Unconventional myosin Myo1c promotes membrane fusion in a regulated exocytic pathway. Mol. Cell Biol 24, 5447–5458.
Broderick, M. J. and Winder, S. J. (2005). Spectrin, alpha-actinin, and dystrophin. Adv. Protein Chem 70, 203–246.[CrossRef][Medline]
Brown, D. and Breton, S. (2000). Sorting proteins to their target membranes. Kidney Int 57, 816–824.[CrossRef][Medline]
Brozinick, J. T. Jr, Hawkins, E. D., Strawbridge, A. B., Elmendorf, J. S. (2004). Disruption of cortical actin in skeletal muscle demonstrates an essential role of the cytoskeleton in glucose transporter 4 translocation in insulin-sensitive tissues. J. Biol. Chem 279, 40699–40706.
Bryant, N. J., Govers, R., James, D. E. (2002). Regulated transport of the glucose transporter GLUT4. Nat. Rev. Mol. Cell Biol 3, 267–277.[CrossRef][Medline]
Chamberlain, L. H. and Gould, G. W. (2002). The vesicle- and target-SNARE proteins that mediate Glut4 vesicle fusion are localized in detergent-insoluble lipid rafts present on distinct intracellular membranes. J. Biol. Chem 277, 49750–49754.
Coue, M., Brenner, S. L., Spector, I., Korn, E. D. (1987). Inhibition of actin polymerization by latrunculin A. FEBS Lett 213, 316–318.[CrossRef][Medline]
dos Remedios, C. G., Chhabra, D., Kekic, M., Dedova, I. V., Tsubakihara, M., Berry, D. A., Nosworthy, N. J. (2003). Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol. Rev 83, 433–473.
Emoto, M., Langille, S. E., Czech, M. P. (2001). A role for kinesin in insulin-stimulated GLUT4 glucose transporter translocation in 3T3–L1 adipocytes. J. Biol. Chem 276, 10677–10682.
Eyster, C. A., Duggins, Q. S., Olson, A. L. (2005). Expression of constitutively active Akt/protein kinase B signals GLUT4 translocation in the absence of an intact actin cytoskeleton. J. Biol. Chem 280, 17978–17985.
Fan, J. and Beck, K. A. (2004). A role for the spectrin superfamily member Syne-1 and kinesin II in cytokinesis. J. Cell Sci 117, 619–629.
Fletcher, L. M., Welsh, G. I., Oatey, P. B., Tavare, J. M. (2000). Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulin-stimulated glucose uptake. Biochem. J 352, 267–276.[CrossRef][Medline]
Grusovin, J. and Macaulay, S. L. (2003). Snares for GLUT4–mechanisms directing vesicular trafficking of GLUT4. Front. Biosci 8, d620–d641.[Medline]
Guilherme, A., Emoto, M., Buxton, J. M., Bose, S., Sabini, R., Theurkauf, W. E., Leszyk, J., Czech, M. P. (2000). Perinuclear localization and insulin responsiveness of GLUT4 requires cytoskeletal integrity in 3T3–L1 adipocytes. J. Biol. Chem 275, 38151–38159.
Hong, W. (2005). SNAREs and traffic. Biochim. Biophys. Acta 1744, 493–517.[Medline]
Hosaka, T., Brooks, C. C., Presman, E., Kim, S. K., Zhang, Z., Breen, M., Gross, D. N., Sztul, E., Pilch, P. F. (2005). p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation. Mol. Biol. Cell 16, 2882–2890.
James, D. E., Brown, R., Navarro, J., Pilch, P. F. (1988). Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature 333, 183–185.[CrossRef][Medline]
Jiang, Z. Y., Chawla, A., Bose, A., Way, M., Czech, M. P. (2002). A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling. J. Biol. Chem 277, 509–515.
Kandror, K. V. and Pilch, P. F. (1996). The insulin-like growth factor II/mannose 6-phosphate receptor utilizes the same membrane compartments as GLUT4 for insulin-dependent trafficking to and from the rat adipocyte cell surface. J. Biol. Chem 271, 21703–21708.
Kandror, K. V. and Pilch, P. F. (1998). Multiple endosomal recycling pathways in rat adipose cells. Biochem. J 331, 829–835.[Medline]
Kanzaki, M. and Pessin, J. E. (2001). Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling. J. Biol. Chem 276, 42436–42444.
Kanzaki, M., Watson, R. T., Khan, A. H., Pessin, J. E. (2001). Insulin stimulates actin comet tails on intracellular GLUT4-containing compartments in differentiated 3T3L1 adipocytes. J. Biol. Chem 276, 49331–49336.
Khayat, Z. A., Tong, P., Yaworsky, K., Bloch, R. J., Klip, A. (2000). Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes. J. Cell Sci 113, 279–290.[Abstract]
Kolega, J., Janson, L. W., Taylor, D. L. (1991). The role of solation-contraction coupling in regulating stress fiber dynamics in nonmuscle cells. J. Cell Biol 114, 993–1003.
Koumanov, F., Jin, B., Yang, J., Holman, G. D. (2005). Insulin signaling meets vesicle traffic of GLUT4 at a plasma-membrane-activated fusion step. Cell Metab 2, 179–189.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Lang, T., Bruns, D., Wenzel, D., Riedel, D., Holroyd, P., Thiele, C., Jahn, R. (2001). SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis. EMBO J 20, 2202–2213.[CrossRef][Medline]
Liu, L. B., Omata, W., Kojima, I., Shibata, H. (2003). Insulin recruits GLUT4 from distinct compartments via distinct traffic pathways with differential microtubule dependence in rat adipocytes. J. Biol. Chem 278, 30157–30169.
Lizunov, V. A., Matsumoto, H., Zimmerberg, J., Cushman, S. W., Frolov, V. A. (2005). Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells. J. Cell Biol 169, 481–489.
Low, S. H., Vasanji, A., Nanduri, J., He, M., Sharma, N., Koo, M., Drazba, J., Weimbs, T. (2006). Syntaxins 3 and 4 are concentrated in separate clusters on the plasma membrane before the establishment of cell polarity. Mol. Biol. Cell 17, 977–989.
Martin, S. S., Haruta, T., Morris, A. J., Klippel, A., Williams, L. T., Olefsky, J. M. (1996). Activated phosphatidylinositol 3-kinase is sufficient to mediate actin rearrangement and GLUT4 translocation in 3T3–L1 adipocytes. J. Biol. Chem 271, 17605–17608.
Molitoris, B. A., Dahl, R., Hosford, M. (1996). Cellular ATP depletion induces disruption of the spectrin cytoskeletal network. Am. J. Physiol 271, F790–F798.[Medline]
Nakano, M., Nogami, S., Sato, S., Terano, A., Shirataki, H. (2001). Interaction of syntaxin with alpha-fodrin, a major component of the submembranous cytoskeleton. Biochem. Biophys. Res. Commun 288, 468–475.[CrossRef][Medline]
Oatey, P. B., Van Weering, D. H., Dobson, S. P., Gould, G. W., Tavare, J. M. (1997). GLUT4 vesicle dynamics in living 3T3 L1 adipocytes visualized with green-fluorescent protein. Biochem. J 327, 637–642.[Medline]
Olson, A. L., Trumbly, A. R., Gibson, G. V. (2001). Insulin-mediated GLUT4 translocation is dependent on the microtubule network. J. Biol. Chem 276, 10706–10714.
Omata, W., Shibata, H., Li, L., Takata, K., Kojima, I. (2000). Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes. Biochem. J 346, 321–328.[Medline]
Patki, V., Buxton, J., Chawla, A., Lifshitz, L., Fogarty, K., Carrington, W., Tuft, R., Corvera, S. (2001). Insulin action on GLUT4 traffic visualized in single 3T3–l1 adipocytes by using ultra-fast microscopy. Mol. Biol. Cell 12, 129–141.
Satoh, S., Nishimura, H., Clark, A. E., Kozka, I. J., Vannucci, S. J., Simpson, I. A., Quon, M. J., Cushman, S. W., Holman, G. D. (1993). Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action. J. Biol. Chem 268, 17820–17829.
Semiz, S., Park, J. G., Nicoloro, S. M., Furcinitti, P., Zhang, C., Chawla, A., Leszyk, J., Czech, M. P. (2003). Conventional kinesin KIF5B mediates insulin-stimulated GLUT4 movements on microtubules. EMBO J 22, 2387–2399.[CrossRef][Medline]
Shogomori, H. and Brown, D. A. (2003). Use of detergents to study membrane rafts: the good, the bad, and the ugly. Biol. Chem 384, 1259–1263.[CrossRef][Medline]
Sieber, J. J., Willig, K. I., Heintzmann, R., Hell, S. W., Lang, T. (2006). The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane. Biophys. J 90, 2843–2851.[CrossRef][Medline]
Simpson, I. A., Yver, D. R., Hissin, P. J., Wardzala, L. J., Karnieli, E., Salans, L. B., Cushman, S. W. (1983). Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions. Biochim. Biophys. Acta 763, 393–407.[Medline]
van Dam, E. M., Govers, R., James, D. E. (2005). Akt activation is required at a late stage of insulin-induced GLUT4 translocation to the plasma membrane. Mol. Endocrinol 19, 1067–1077.
Watson, R. T., Kanzaki, M., Pessin, J. E. (2004). Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes. Endocr. Rev 25, 177–204.
Winder, S. J. and Ayscough, K. R. (2005). Actin-binding proteins. J. Cell Sci 118, 651–654.
Yeh, J. I., Verhey, K. J., Birnbaum, M. J. (1995). Kinetic analysis of glucose transporter trafficking in fibroblasts and adipocytes. Biochemistry 34, 15523–15531.[CrossRef][Medline]
Zeigerer, A., McBrayer, M. K., McGraw, T. E. (2004). Insulin stimulation of GLUT4 exocytosis, but not its inhibition of endocytosis, is dependent on RabGAP AS160. Mol. Biol. Cell 15, 4406–4415.
Zhou, M., Vallega, G., Kandror, K. V., Pilch, P. F. (2000). Insulin-mediated translocation of GLUT-4-containing vesicles is preserved in denervated muscles. Am. J. Physiol. Endocrinol. Metab 278, E1019–E1026.
This article has been cited by other articles:
|
|
 |
|
  J. L. Jewell, W. Luo, E. Oh, Z. Wang, and D. C. Thurmond Filamentous Actin Regulates Insulin Exocytosis through Direct Interaction with Syntaxin 4 J. Biol. Chem., April 18, 2008; 283(16): 10716 - 10726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Liu and P. F. Pilch A Critical Role of Cavin (Polymerase I and Transcript Release Factor) in Caveolae Formation and Organization J. Biol. Chem., February 15, 2008; 283(7): 4314 - 4322. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-D. Chen, S. Podvin, E. Gillespie, S. E. Leeman, and C. R. Abraham Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17 PNAS, December 11, 2007; 104(50): 19796 - 19801. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
