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J. Biol. Chem., Vol. 277, Issue 6, 3809-3812, February 8, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/6/3809    most recent C100645200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nelson, B. A. Articles by Buse, M. G. Search for Related Content PubMed PubMed Citation Articles by Nelson, B. A. Articles by Buse, M. G. Social Bookmarking                      What's this?

ACCELERATED PUBLICATION
Insulin Acutely Regulates Munc18-c Subcellular Trafficking

ALTERED RESPONSE IN INSULIN-RESISTANT 3T3-L1 ADIPOCYTES^*

Bryce A. Nelson^§, Katherine A. Robinson, and Maria G. Buse^¶

From the  Department of Medicine, Division of Endocrinology, Diabetes and Medical Genetics and the ^¶ Department of Biochemistry/Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

Received for publication, November 7, 2001, and in revised form, December 13, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Preincubation of 3T3-L1 adipocytes in high glucose or glucosamine decreases acute insulin (100 nM)-stimulated glucose transport provided that insulin (0.6 nM) is included during preincubation. GLUT4 expression is unchanged (Nelson, B. A., Robinson, K. A., and Buse, M. G. (2000) Diabetes 49, 981-991). Munc18-c, a Syntaxin 4-binding protein, is a proposed regulator of the docking/fusion of GLUT4-containing vesicles with the plasma membrane. We examined the subcellular distribution of Munc18-c in response to acute (15-min) insulin (100 nM) stimulation after preincubation in 5 or 25 mM glucose ± 0.6 nM insulin. Immunoblotting detected Munc18-c mainly in the Triton X-100-soluble plasma membrane (TS-PM) and the Triton X-100-insoluble low density microsomal (TI-LDM) fraction. Under each condition except high glucose + insulin preincubation, acute insulin increased Munc18-c (~50-200%) in TS-PM and decreased Munc18-c (~60%) in TI-LDM. Munc18-c traffic was time-dependent with a lag time of ~3 min compared with GLUT4. Preincubation with high glucose + 0.6 nM insulin significantly impaired acute insulin-stimulated Munc18-c trafficking and decreased basal Munc18-c in the TI-LDM. Preincubation with glucosamine + insulin had similar effects. Total cellular Munc18-c remained unchanged. In conclusion, acute insulin stimulation promotes the translocation of Munc18-c, apparently from a TI-LDM-associated compartment to the TS-PM. Chronically increased glucose flux or exposure to glucosamine disrupts this process, which may negatively impact the fusion of GLUT4-containing vesicles with the plasma membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Insulin lowers plasma glucose in part by accelerating glucose transport into insulin-responsive tissues, i.e. skeletal muscle, heart muscle, and adipocytes, which express the insulin-responsive glucose transporter GLUT4.¹ Under basal conditions, glucose transporters constitutively cycle between the plasma membrane (PM) and intracellular compartments, but GLUT4-containing vesicles (GCVs) are for the most part segregated intracellularly. Insulin accelerates GCV exocytosis much more than endocytosis (recycling), resulting in a net increase in PM-associated GLUT4. Acceleration of glucose transport requires that the translocated GCVs dock at and fuse with the PM (for a review, see Ref. 2). The latter process mimics small synaptic vesicle traffic in neurons (3). SNARE proteins in the vesicle membrane (v-SNARE) bind specifically and with high affinity to SNARE proteins in the target membrane (t-SNARE). Adipocytes express isoforms of both t-SNARE and v-SNARE proteins, and insulin-stimulated GLUT4 trafficking requires SNARE complex interaction (4). Specifically, the interaction of VAMP-2 (5) in GCVs with Syntaxin 4 (6-8) and SNAP-23 (9, 10) at the PM is required for the docking and fusion of GCVs in response to insulin stimulation.

The interaction of VAMP-2 and Syntaxin 4/SNAP-23 is regulated by several proteins (for a review, see Ref. 11). We focused on a syntaxin-binding protein, Munc18-c. Munc proteins share homology with two vesicle-trafficking regulators Sec1 in Saccharomyces cerevisiae and unc-18 in Caenorhabditis elegans. Munc18-a is expressed only in neuronal tissue (12). Munc18-b and -c are expressed in adipocytes, but only the latter regulates GLUT4 trafficking (7, 13, 14). Overexpression of Munc18-c inhibited GLUT4 translocation in response to insulin (15). This inhibitory effect was mitigated by co-expression of Syntaxin 4 (14). In cells that were co-transfected with tagged Munc18-c and Syntaxin 4, a 30-min insulin exposure decreased the localization of transfected Munc18-c to the PM and the association between Munc18-c and Syntaxin 4. Interestingly, microinjection of Munc18-c peptide fragments, which compete with endogenous Munc18-c for binding to Syntaxin 4, resulted, upon insulin stimulation, in GCVs clustered at but not fused with the PM (14). Thus Munc18-c may play a profusogenic role in GCV trafficking.

Sustained hyperglycemia causes insulin resistance in humans and reduces the insulin response of glucose transport in experimental animals and cell culture models (for reviews, see Refs. 16 and 17). We found that in 3T3-L1 adipocytes preincubation in high glucose (25 mM) or in low glucose (5 mM) supplemented with GlcN (1-2.5 mM) decreased the subsequent acute insulin response of glucose transport by 40-50% (compared with cells pre-exposed to low glucose) but only if a relatively low dose of insulin (0.6 nM) was included in all media during preincubation. As assessed by the PM lawn assay, high glucose did not reduce insulin-stimulated GLUT4 translocation, while GlcN did. Total GLUT4 and GLUT1 protein expression was not affected by any of the preincubation conditions. IRS-1-associated PI 3-kinase activation in response to acute insulin stimulation was also unaffected (1). However, pre-exposure to high glucose, but not to glucosamine, diminished acute insulin-stimulated Akt activation (18). We hypothesized that high glucose preincubation may have impacted GCV docking and/or fusion (1). In this paper we examined whether acute insulin stimulation regulates the subcellular distribution of Munc18-c and whether this process is altered in insulin-resistant 3T3-L1 adipocytes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- A site-specific polyclonal rabbit antibody against Munc18-c was generously provided by Dr. Jeffrey Pessin (University of Iowa, Iowa City, IA; Ref. 13), and one specific for GLUT4 was provided by Dr. Mike Mueckler (Washington University, St. Louis, MO). A polyclonal antibody against Caveolin-1 was from Santa Cruz Biotechnology (Santa Cruz, CA), horseradish peroxidase-conjugated goat anti-rabbit IgG was from Jackson Immunoresearch Laboratories (West Grove, PA), and enhanced chemiluminescence reagents were from Pierce. Other reagents were purchased from previously identified suppliers (1).

Cell Culture and General Methods-- 3T3-L1 fibroblasts were grown in 100-mm culture dishes and differentiated into adipocytes as described previously (1). Before experiments, cells were preincubated for 18 h in Dulbecco's minimal essential medium containing 1% FBS and sugars at concentrations indicated in the figure legends with or without 0.6 nM insulin. They were re-equilibrated in serum and insulin-free media for 2 h and then stimulated for 15 min with or without 100 nM insulin (1).

Subcellular Fractionation-- Subcellular fractions were generated by differential centrifugation (13, 18). Pellets of the prepared PM and LDM fractions were resuspended in HES buffer containing 1% Triton X-100, solubilized for at least 1 h at 4 °C, and then centrifuged for 75 min at 200,000 × g. The Triton X-100-soluble fractions (TS-PM and TS-LDM) were collected, and the Triton X-100-insoluble pellets (TI-PM and TI-LDM) were resuspended in phosphate-buffered saline containing 1% SDS and heated for 10 min at 95 °C.

SDS-PAGE and Immunoblotting-- Proteins (15 µg) from the appropriate fractions were separated by SDS-PAGE, transferred to nitrocellulose or polyvinylidene difluoride membranes, and immunoblotted at 4 °C overnight with either rabbit anti-Munc18-c (1:2000), rabbit anti-GLUT4 (1:500), or rabbit anti-Caveolin-1 (1:1000). Immunoreactive bands were detected and quantified, and protein concentrations were determined as described previously (1).

Statistical Analysis-- Means ± S.E. are shown. Where error bars are not indicated in the figures, they are too small for graphical representation. The significance of differences between means were evaluated by one, two, or three way analysis of variance (ANOVA) and Tukey's test for unbalanced design using the Statistica software or by two-tailed, unpaired Student's t test using Microsoft Excel 98 software. p < 0.05 was considered significant.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The subcellular distribution of GLUT4 was assessed with or without maximal acute insulin stimulation in cells preincubated in low or high glucose ± 0.6 nM insulin. The purpose of these studies was to compare cell fractions obtained by standard differential centrifugation (Fig. 1) with those obtained with the PM lawn assay (1). As expected, acute insulin stimulation significantly increased PM-associated GLUT4 irrespective of the preincubation conditions. However, after acute insulin stimulation a small (20%) but significant decrease in PM-associated GLUT4 was observed in cells that had been preincubated with high glucose + 0.6 nM insulin. Similarly, in this group, acute insulin stimulation failed to decrease GLUT4 associated with the TS-LDM significantly, while this effect was significant (p < 0.05) in the other three groups. Thus, in contrast to the results obtained with the PM lawn assay, the cell fractionation studies were consistent with impaired GLUT4 translocation associated with insulin-resistant glucose transport in cells pretreated with high glucose + 0.6 nM insulin. The discrepant results obtained with the two experimental methods may be consistent with impaired docking/fusion of GCVs as the primary defect. In this scenario, a fraction of GCVs, which did not fuse with the PM, would remain associated with it in the lawn assay, but the relatively weak link would be disrupted during the more rigorous cell fractionation procedure.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   The effect of preincubation in high glucose + 0.6 nM insulin on GLUT4 trafficking in response to acute (100 nM) insulin stimulation. Cells were preincubated for 18 h in media containing 1% FBS and 5 or 25 mM glucose ± 0.6 nM insulin. They were washed, re-equilibrated for 2 h in serum- and insulin-free media, and then acutely stimulated (or not = basal) with 100 nM insulin for 15 min. After subcellular fractionation, 15 µg of protein from fractions enriched in Triton X-100-soluble plasma membranes (TS-PM in A and C) and low density microsomes (TS-LDM in B and D) were separated by SDS-PAGE and immunoblotted with anti-GLUT4 antibody. Representative immunoblots (A and B) and densitometric quantitation (means ± S.E.) from seven experiments (C and D) are shown. In each experiment, data were normalized to those from cells preincubated in 5 mM glucose  0.6 nM insulin after 15-min acute insulin stimulation (*, p < 0.001 versus basal; **, p < 0.05 versus basal; , p < 0.05 by paired Student's t test compared with acute insulin stimulation after 5 mM glucose + 0.6 nM insulin preincubation). Ins, insulin.

To further assess a putative docking/fusion defect in glucose-induced insulin resistance, we examined the possibility of altered Munc18-c trafficking in response to acute insulin stimulation. Previous studies found Munc18-c primarily localized to the PM (13). As assessed by immunoblot, Munc18-c levels in our study were highest in the TS-PM, TI-LDM (Fig. 2), and TI-PM. The latter did not change in response to the experimental manipulations (data not shown). The Munc18-c signal was too weak to quantify in the TS-LDM and cytosol (data not shown) and may represent contamination by other membrane fractions. Total Munc18-c expression did not change in response to any of the experimental conditions (data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Munc18-c trafficking in response to acute insulin stimulation. Effects of preincubation in high glucose + 0.6 nM insulin. Cells were preincubated and then acutely stimulated with (or without) 100 nM insulin for 15 min as described in Fig. 1. Following subcellular fractionation, 15 µg of protein from the TS-PM-enriched fraction (A and C) and from the Triton X-100-insoluble TI-LDM fraction (B, D, and E) were separated by SDS-PAGE and immunoblotted with an anti-Munc18-c antibody (A-D) or an anti-Caveolin-1 antibody (E). A and B are representative immunoblots. C-E show densitometric quantitation (means ± S.E.) from 10 experiments in C, six in D, and four in E. Data are normalized to cells preincubated in 5 mM glucose without chronic or acute insulin (*, p < 0.05 versus basal; , p < 0.1 versus basal; **, p < 0.05 compared with basal preincubated without insulin). Ins, insulin.

Acute insulin stimulation increased Munc18-c in the TS-PM ~2-fold in cells preincubated without 0.6 nM insulin in low or high glucose (p < 0.05). This effect was abolished after preincubation with 25 mM Glc + 0.6 nM insulin (Fig. 2, A and C). The interaction between glucose concentration and the presence of 0.6 nM insulin during preincubation was significant by two way ANOVA (p < 0.05). In cells preincubated with 0.6 nM insulin in 5 mM glucose, TS-PM-associated Munc18-c was increased in the basal state (p < 0.05), and the further increase induced by acute insulin stimulation was only borderline significant (p < 0.1). However, in a subsequent study (Fig. 3A), acute insulin stimulation significantly (p < 0.05) increased TS-PM-associated Munc18-c under identical conditions. Thus acute insulin failed to increase Munc18-c in the TS-PM only after preincubation in high glucose + 0.6 nM insulin, the condition that induced insulin-resistant glucose transport.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of preincubation with GlcN on acute insulin-stimulated Munc18-c trafficking. Cells were preincubated for 18 h in media containing 5 mM glucose + 0.6 nM insulin or 5 mM glucose + 2.5 mM GlcN + 0.6 nM insulin. Cells were re-equilibrated and then stimulated (or not = basal) for 15 min with 100 nM insulin as in Figs. 1 and 2. After cell fractionation, 15 µg of protein from the TS-PM- (A) and TI-LDM (B)-enriched fractions were separated by SDS-PAGE and immunoblotted with anti-Munc18-c antibody. Means ± S.E. from densitometric analyses of four separate experiments are shown. Data are normalized as in Fig. 2 (*, p < 0.05 compared with basal; **, p < 0.001 compared with basal after preincubation with 5 mM glucose + 0.6 nM insulin by Student's t test).

Acute insulin decreased Munc18-c associated with the TI-LDM by ~50-70% under most conditions (Fig. 2, B and D, p < 0.01) when compared with basal. Preincubation with 0.6 nM insulin and 25 mM glucose decreased basal Munc18-c in this fraction by ~50% (Fig. 2D, p < 0.05). Acute insulin caused a further 28% decrease, which was not statistically significant. The interaction between glucose concentration and insulin during preincubation was significant by two way ANOVA (p < 0.05).

Caveolin-1 is mainly localized to the TI-LDM and is tyrosine-phosphorylated upon acute insulin stimulation (19). We immunoblotted for Caveolin-1 in TI-LDM to assess the specificity of the acute insulin-induced decrease in Munc18-c in this fraction. The Caveolin-1 concentration in TI-LDM was unaffected by acute insulin stimulation regardless of the preincubation conditions (Fig. 2E) in agreement with previous studies (19, 20).

Because preincubation with GlcN + 0.6 nM insulin also caused insulin resistance in our model (1), the subcellular distribution of Munc18-c was examined. Acute insulin stimulation again increased Munc18-c associated with the TS-PM in cells preincubated in 5 mM glucose with or without 0.6 nM insulin (Fig. 3A, p < 0.05), but this effect was not statistically significant in GlcN-pretreated cells. Munc18-c decreased in the TI-LDM ~50-70% after acute insulin stimulation under all conditions (Fig. 3B, p < 0.05). Significant effects of GlcN (p < 0.001) and the presence of 0.6 nM insulin (p < 0.01) during preincubation were detected by two way ANOVA. Basal Munc18-c associated with the TI-LDM was unchanged in all conditions except in cells that had been preincubated in 5 mM glucose + 2.5 mM GlcN + 0.6 nM insulin. In the latter group, basal TI-LDM-associated Munc18-c was decreased by ~50% as compared with cells preincubated in 5 mM glucose + 0.6 nM insulin. Although not significant by Tukey's post hoc analysis, the difference was significant by Student's unpaired t test (p < 0.001). Acute insulin stimulation further decreased Munc18-c in TI-LDM by ~50% in this group. We could not account for the decreased TI-LDM-associated Munc18-c in the basal state in cells pretreated with 0.6 nM insulin in the presence of high glucose (Fig. 2) or glucosamine (Fig. 3) by a corresponding increase in another fraction. All fractions, including high density microsomes, were examined. However, the marked dilution of the cytosol precluded accurate quantitation in this fraction.

Acute insulin-stimulated translocation of GCVs to the PM is insulin dose- and time-dependent (21-24). Fig. 4 compares the early time course of GLUT4 translocation from the TS-LDM to the TS-PM with the changes in Munc18-c association with the TS-PM and TI-LDM. GLUT4 translocation was time-dependent and detectable by 2 min after insulin stimulation. The increase in PM-associated Munc18-c after insulin stimulation was much smaller (~20%) in these experiments (n = 3) than in previous studies (Fig. 2). However, the insulin-mediated decrease in TI-LDM-associated Munc18-c was ~40% at 10 min and was time-dependent. There appeared to be an ~3-min lag time between the initiation of GLUT4 translocation and the mobilization of Munc18-c from the TI-LDM, suggesting that Munc18-c translocation does not initiate GCV translocation. Assuming that Munc18-c promotes GCV fusion (14), the first, early phase of the insulin response may be supported by Munc18-c associated with the TS-PM in the basal state, while its translocation from the TI-LDM to the TS-PM may facilitate the full, sustained insulin response. Alternatively, Munc18-c may promote dissociation of the SNARE complex after GCV fusion, which after Munc18-c dissociates may render Syntaxin 4 available for docking of the next wave of GCVs.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Time dependence of Munc18-c and GLUT4 trafficking after acute insulin stimulation. Cells were preincubated in medium containing 1% FBS and 25 mM glucose. They were processed as in Figs. 1-3, except that cells were acutely stimulated with 100 nM insulin for 2, 5, or 10 min. Following subcellular fractionation, 15 µg of protein from fractions enriched in TS-PM (A and B), TS-LDM (A), and TI-LDM (B) were separated by SDS-PAGE and immunoblotted with anti-GLUT4 antibody (A) or anti-Munc18-c antibody (B). Means ± S.E. from three separate experiments are shown. Data are normalized to values obtained without insulin stimulation (indicated by 0 on the abscissa).

Because Munc18-c does not contain protein domains necessary for membrane targeting, its PM localization is mediated predominantly via protein-protein interactions (25). Specifically, Munc18-c co-immunoprecipitates with Syntaxin 4 in 3T3-L1 adipocytes (13). In agreement with this study, we found Munc18-c highly associated with the PM. We modified the subcellular fractionation method in Ref. 13 in that each fraction was further separated into Triton X-100-soluble and -insoluble components. This allowed us to observe for the first time the apparent translocation of endogenous Munc18-c from the TI-LDM to the TS-PM in response to acute insulin stimulation.

The TI-LDM fraction has been implicated in insulin-regulated traffic of docking/signaling proteins such as IRS-1 and PI 3-kinase (26, 27). The fraction is enriched in cytoskeletal proteins (26) and in caveolar microdomains of the PM, which are rich in caveolin and in detergent-insoluble, glycolipid-enriched lipid rafts (DIG) (19, 20, 27-30). The fraction contains numerous docking, scaffolding, and signaling proteins (28-30) and is also involved in a novel insulin-responsive PI 3-kinase-independent signaling pathway of glucose transport regulation (29, 30). Sec1p, the yeast homologue of Munc18-c, has a higher affinity for assembled SNARE complexes than for the syntaxin homologue itself (31). Thus, docking of GCVs may promote the association of Munc18-c with the PM, possibly facilitating fusion. Alternatively, acute insulin stimulation may cause post-translational modification(s) of Munc18-c. Although there are no data relevant to Munc18-c, Munc18-a function can be regulated by protein kinase C and by cyclin-dependent kinase 5-mediated phosphorylation (32, 33). Protein kinase C activation has been implicated in glucose- and in glucosamine-induced insulin resistance in adipocytes (16). Insulin-regulated trafficking of Munc18-c in this fraction may represent a link between the proximal insulin signaling cascade and GCV docking/fusion.

In the model presented, insulin-resistant glucose transport is associated with a disruption of insulin-regulated Munc18-c trafficking. Whether or not the dynamics of insulin-stimulated GCV docking/fusion are impaired in glucose-induced insulin resistance warrants further study.

	ACKNOWLEDGEMENTS

We gratefully acknowledge the generous gifts of antibodies specific to GLUT4 or to Munc18-c from Drs. Mike Mueckler and Jeffrey Pessin, respectively. We thank Lilly Research Laboratories for gifts of recombinant human insulin.

	FOOTNOTES

^* This work was supported in part by Research Grant DK-02001 from the NIDDK, National Institutes of Health (to M. G. B.). This paper was presented in part at the 2001 National Meeting of the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by a Medical Scientist Training Grant GM-08716 from the NIGMS, National Institutes of Health.

To whom correspondence should be addressed: Dept. of Medicine, Endocrinology, Diabetes and Medical Genetics Division, Medical University of South Carolina, 96 Jonathan Lucas St., Ste. 323, Charleston, SC 29425. Tel.: 843-792-2529; Fax: 843-792-4114; E-mail: busemg@musc.edu.

Published, JBC Papers in Press, December 18, 2001, DOI 10.1074/jbc.C100645200

	ABBREVIATIONS

The abbreviations used are: GLUT, glucose transporter; FBS, fetal bovine serum; GCV, GLUT4-containing vesicle; HES buffer, HEPES, EDTA, sucrose buffer with protease and phosphatase inhibitors; IRS, insulin receptor substrate; LDM, low density microsome; PI, phosphatidylinositol; PM, plasma membrane; TI, Triton X-100-insoluble; TS, Triton X-100-soluble; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; v-SNARE, vesicle membrane SNARE; t-SNARE, target membrane SNARE; ANOVA, analysis of variance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/6/3809    most recent C100645200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nelson, B. A. Articles by Buse, M. G. Search for Related Content PubMed PubMed Citation Articles by Nelson, B. A. Articles by Buse, M. G. Social Bookmarking                      What's this?