Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization

Kanda, Hajime; Tamori, Yoshikazu; Shinoda, Hiroaki; Yoshikawa, Mitsuo; Sakaue, Motoyoshi; Udagawa, J.; Otani, Hiroki; Tashiro, Fumi; Miyazaki, Junichi; Kasuga, Masato

doi:10.1172/jci22681

Cited by 120 publications

(104 citation statements)

References 39 publications

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“…These data are consistent with studies of neural Munc18 knockouts and that of lower organisms [61,62]. However another study, found that homozygous Munc18c knockout mice were viable and had increased insulin sensitivity consistent with Munc18c actin as a fusogenic inhibitor [63]. Insulin has also been observed to induce the association of PKC and Munc18c concomitant with a dissociation of the syntaxin4/Munc18c complex [50].…”

Section: Glut4 Vesicle Docking and Plasma Membrane Fusionsupporting

confidence: 86%

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

Hou

Pessin

2007

Current Opinion in Cell Biology

132

116

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SummaryGlucose transporter 4 (GLUT4) is the major insulin regulated glucose transporter expressed mainly in muscle and adipose tissue. GLUT4 is stored in a poorly characterized intracellular vesicular compartment and translocates to the cell surface in response to insulin stimulation resulting in increase glucose uptake. This process is essential for the maintenance of normal glucose homeostasis and involves a complex interplay of trafficking events and intracellular signaling cascades. Recent studies have identified sortilin as an essential element for the formation of GLUT4 storage vesicles during adipogenesis and GGA (Golgi-localized γ-ear-containing Arf-binding protein) as a key coat adaptor for the entry of newly synthesized GLUT4 into the specialized compartment. Insulinstimulated GLUT4 translocation from this compartment to the plasma membrane appears to require the Akt/protein kinase B substrate, termed AS160 (Akt Substrate of 160 kDa). In addition, the VPS9 domain-containing protein Gapex-5 in complex with CIP4 appears to function as a Rab31 guanylnucleotide exchange factor that is necessary for insulin-stimulated GLUT4 translocation. Here we attempt to summaries recent advances in GLUT4 vesicle biogenesis, intracellular trafficking and membrane fusion.

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Section: Glut4 Vesicle Docking and Plasma Membrane Fusionsupporting

confidence: 86%

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

Hou

Pessin

2007

Current Opinion in Cell Biology

132

116

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“…For example, the dissociation of yeast homologs Sly1p (Munc18 homolog) and Sed5p (syntaxin homolog) has been shown to require the small GTPase protein Ypt1p (66). Similarly, in adipocytes, the Munc18c-Syntaxin 4 complex has been proposed to be dissociated upon activation of the Rab4 GTPase (67) or the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation the of Rab GTPase-activating protein AS160 (68,69). In support of this, the crystal structure of Munc18-1 revealed a region that is very similar to the Ypt1p-binding site of Sly1p (70).…”

Section: Discussionmentioning

confidence: 99%

The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis

Thurmond

2006

Journal of Biological Chemistry

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Stimulus-induced tyrosine phosphorylation of Munc18c was investigated as a potential regulatory mechanism by which the Munc18c-Syntaxin 4 complex can be dissociated in response to divergent stimuli in multiple cell types. Use of [ 32 P]orthophosphate incorporation, pervanadate treatment, and phosphotyrosine-specific antibodies demonstrated that Munc18c underwent tyrosine phosphorylation. Phosphorylation was apparent under basal conditions, but levels were significantly increased within 5 min of glucose stimulation in MIN6 beta cells. Tyrosine phosphorylation of Munc18c was also detected in 3T3L1 adipocytes and increased with insulin stimulation, suggesting that this may be a conserved mechanism. Syntaxin 4 binding to Munc18c decreased as Munc18c phosphorylation levels increased in pervanadate-treated cells, suggesting that phosphorylation dissociates the Munc18c-Syntaxin 4 complex. Munc18c phosphorylation was localized to the N-terminal 255 residues. Mutagenesis of one residue in this region, Y219F, significantly increased the affinity of Munc18c for Syntaxin 4, whereas mutation of three other candidate sites was without effect. Moreover, Munc18c-Y219F expression in MIN6 cells functionally inhibited glucose-stimulated SNARE complex formation and insulin granule exocytosis. These data support a novel and conserved mechanism for the dissociation of Munc18c-Syntaxin 4 complexes in a stimulus-dependent manner to facilitate the increase in Syntaxin 4-VAMP2 association and to promote vesicle/granule fusion.In the early 1990s, the discovery of soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) 2 proteins elucidated the molecular basis for synaptic vesicle exocytosis (1, 2). Vesicle exocytosis entails the pairing of a vesicle-associated membrane protein (VAMP) SNARE with a binary cognate receptor complex at the target membrane composed of SNAP-25/23 and syntaxin proteins (target SNAREs) to form the SNARE core complex (3-7). Shortly thereafter, SNARE protein complexes were identified and found to regulate secretory processes in many diverse cell types ranging from yeast and Caenorhabditis elegans to humans, which has led to the general concept that most types of regulated vesicle fusion occur by a common mechanism.Concurrent with the discovery of SNARE proteins was the discovery of the yeast Sec1 secretory protein, which was found to interact directly with the target SNARE syntaxin (8). Homologs in C. elegans (UNC-18), Drosophila melanogaster (ROP), and mammalian cells (Munc18a-c) were also identified (9 -13). Collectively, the Sec1 and Munc18 protein families are now referred to as "SM" proteins for Sec1 and Munc18. Munc18 proteins are ϳ66 -68 kDa in size and are soluble factors with no transmembrane domain (14). They are found localized to the cytosol and also to the plasma membrane through high affinity binding to their cognate syntaxin (15, 16). Munc18a (also known as Munc18-1/neural Sec1/rat brain Sec1) was demonstrated to interact with Syntaxin 1 in a manner mutually exclusive of the other...

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“…To generate N-terminal fluoroprotein-labeled constructs, the following cDNAs were subcloned into the SalI-XbaI sites of pDNR-dual for use with the Cre recombinase-mediated Creator System (Clontech): rat Munc18a (pGex-KG-Munc18a), rat syntaxin 1A (pGEX-syntaxin 1A 11 ), rat Munc18c (pcDNA3-FLAG-Munc18c), human syntaxin 4 (pcDNA4/TO/syntaxin 4-Myc 2 -His), and human SNAP23 (pcDNA3-SNAP23) (gifts from J. Pevsner, R. Scheller, J. Pessin, and T. Weimbs (syntaxin 4 and SNAP23), respectively). The recipient vectors pLoxP-ECFP-C1 and pLoxP-EcYFP-C1 (Q39M mutant of pEYFP-C1; citrine) were generated and mutated to their monomeric forms (A206K) from pLoxP-EGFP-C1 (Clontech) as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

“…Based on overexpression studies, Munc18c was initially proposed to function as a negative regulator of GLUT4 vesicle translocation in 3T3L1 adipocytes (5,6). Furthermore, the failure of the phosphatidylinositol 3-kinase inhibitor wortmannin to suppress GLUT4 externalization in adipocytes derived from Munc18c-null mice suggests that Munc18c inhibits docking and/or fusion under the control of the phosphatidylinositol 3-kinase pathway (11). Although the exact mechanism for this inhibition is not fully understood, Munc18c has been proposed to preclude the binding of syntaxin 4 to its cognate SNAREs, VAMP2 (vesicle-associated membrane protein 2) and SNAP23 (synaptosome-associated protein of 23 kDa), preventing GLUT4 externalization in the absence of insulin (5,6,12).…”

mentioning

confidence: 99%

“…However, genetic studies suggest that the mechanism of Munc18c action may be different from Munc18a. For example, the insulin-induced fusion of GLUT4 vesicles was enhanced in adipocytes prepared from Munc18c-null mice (11), whereas Munc18a deficiency was demonstrated to eliminate both evoked and spontaneous synaptic exocytosis (28). Additionally, insulin (6) as well as N-ethylmaleimide (26), which inhibits NSFmediated SNARE core complex disassembly, have been reported to dissociate Munc18c from syntaxin 4.…”

mentioning

confidence: 99%

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Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

Merrins

Chang

Lam

et al. 2007

Journal of Biological Chemistry

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In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4 LE -SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4 LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4 LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational "opening" of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking.Soluble N-ethylmaleimide-sensitive factor (NSF) 3 attachment protein receptors (SNAREs) are central to vesicle fusion events in all eukaryotes. SNARE proteins anchored to both transport vesicles and their target membranes overcome the forces necessary for fusion by binding with high affinity in a ternary core complex (1). Although formation of SNARE core complexes is sufficient for membrane fusion (2, 3), essential regulatory proteins of the Sec1/Munc18 (SM) family are believed to control SNARE complex formation. Seven vertebrate SM gene family members (Munc18a/b/c, Sly1, Vps45, and Vps33a/b) have been described, which affect membrane trafficking in distinct subcellular pathways, predominantly through their association with specific syntaxin Q-SNAREs (4).Of the varied SM-syntaxin partners, Munc18c-syntaxin 4 interactions are believed to exert a critically important role in the temporal control of insulin-stimulated GLUT4 storage vesicle exocytosis in muscle and adipose tissue (5-8). The delivery of GLUT4 to the cell surface occurs by distinct signaling processes: 1) GLUT4 vesicle recruitment to the plasma membrane, followed by 2) phosphatidylinositol 3-kinase-dependent vesicle docking and fusion mediated by syntaxin 4-containing SNARE complexes (9, 10). Based on overexpression studies, Munc18c was initially proposed to function as a negative regulator of GLUT4 vesicle translocation in 3T3L1 ad...

show abstract

Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization

Cited by 120 publications

References 39 publications

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking

The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis

Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

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