Summary

Maintenance of glucose homeostasis requires ‘cross-talk’ between pancreatic insulin secretion and insulin signaling in the peripheral tissues. Both insulin secretion and glucose uptake are regulated exocytotic processes mediated by SNARE protein complexes. SNARE core complexes are heterotrimeric, composed of syntaxin, SNAP-25/23 and VAMP2 proteins in a 1:1:1 ratio. It has become clear that we must thoroughly define the precise mechanisms underlying insulin-stimulated GLUT4 vesicle translocation by skeletal muscle and adipose cells as well as glucose-stimulated insulin secretion by pancreatic beta cells in order to develop therapeutic strategies to better treat and eventually cure diabetic patients. In this chapter, I will examine the molecular interactions responsible for these SNARE dependent events. Similarities and differences in molecular machinery, exocytosis mechanisms as well as the impact of accessory proteins such as Munc18 and Rab GTPases will be discussed. New findings on alterations of particular SNARE proteins upon glucose homeostasis are described in terms of potential for pharmacological targets for intervention.

In 1957 Lacy and colleagues published immunofluorescent and later electron microscopy images of islet beta cells filled with darkened organelles, or insulin granules. These granules were later shown to fuse with the plasma membrane to release insulin.^1-3 Analogously, in 1990 the localization of the insulin-responsive glucose transporter ‘GLUT4’ to intracellular vesicles of adipocytes was revealed, and these GLUT4-storage vesicles were later shown to fuse with the plasma membrane upon stimulation with insulin, to ultimately facilitate glucose disposal.^4,5 However, it was not until 1993 that the molecular basis for vesicle fusion and secretion, termed ‘vesicle exocytosis’, was elucidated by the discovery of SNARE proteins (soluble N-ethylmaleimide sensitive factor attachment protein receptor).^6,7

Vesicle Exocytosis

Vesicle exocytosis entails the pairing of a vesicle associated membrane protein (v)-SNARE (VAMP) with a binary cognate receptor complex at the target membrane composed of SNAP-25/ 23 and Syntaxin proteins (t-SNAREs)^8-14 to form the SNARE core complex (Fig. 1). The v-SNARE VAMP2 (also called synaptobrevin) is an 18 kDa protein oriented with its N-terminus towards the cytoplasm and the carboxyl-terminus spanning the membrane facing the vesicle lumen.^15-17 The t-SNARE Syntaxin is a 35 kDa protein oriented with its amino terminal domain towards the cytoplasm and carboxyl-terminus spanning the plasma membrane facing the extracellular matrix.^8,18 The other t-SNARE, SNAP-23/25 (a 23 or 25 kDa synaptosome associated protein) is tethered to the target membrane via palmitoylated cysteine residues.¹⁹ A combination of electron microscopic, spectroscopic and X-ray crystallographic evidence shows that these SNAREs form a rod shaped complex that is a coiled-coil of four helices: one from VAMP, one from Syntaxin, and the remaining two from SNAP-25.^20-23 Upon forming the core SNARE complex the syntaxin protein increases it helical content whereas VAMP and SNAP-25 convert from unstructured forms into a helices.²⁰ These conformational changes result in the formation of the heterotrimeric complex that is extremely stable, melting only at temperatures greater than 95°C.^6,10,12

Figure 1

Schematic model of SNARE core complexes and Munc18-Syntaxin complexes important in insulin-stimulated GLUT4 translocation and insulin secretion. Syn4 is integrated into the plasma membrane (PM, depicted as a variegated vertical line); SNAP-23/25 is associated (more...)

Multiple isoforms of each SNARE protein were found to exist and it is hypothesized that by the particular pairing and compartmentalization of these proteins, specificity of vesicle targeting could be achieved.^24-32 On the basis of these studies in neuroendocrine cells, investigators from diabetes-related fields discovered that SNARE protein core complexes were also responsible for regulated secretion of insulin from islet beta cells in response to increased blood glucose,^33-40 as well as facilitating the downstream action of insulin on peripheral glucose disposal via the translocation to and integration of intracellular glucose transporter (GLUT4) vesicles into the plasma membrane of adipocytes and skeletal muscle transverse tubules/sarcolemma.^41-50

Regulation of Exocytosis by Munc18 Proteins

Concurrent with the discovery of SNARE proteins came evidence from yeast genetics suggesting that other proteins also participate in exocytosis and play a role in the regulation of the SNARE complex,⁵¹ leading to the discovery of Sec1 secretory proteins. Yeast Sec1 was found to interact directly with the t-SNARE Syntaxin leading to the quick identification of Sec1 homologues in C. elegans (unc18), D. melanogaster (Rop)^52-61 and mammals.^{53,54,59,62,63} Three homologues were identified in mammalian plasma membranes, and named Munc18 (for Mammalian unc18) proteins. Munc18 proteins are ˜66-68 kDa in size and are soluble factors with no transmembrane domain,⁵⁴ although they are found localized to the plasma membrane through their high-affinity for binding to their cognate Syntaxin.⁶⁴ Munc18-1 (also called Munc18a/n-Sec1/rbSec1) was demonstrated to interact with Syntaxin 1 in a manner mutually exclusive of the other SNARE core complex proteins,^52,54,65 as depicted as a separate binary complex in Figure 1. By 1995 Munc18b and Munc18c were identified but found to be ubiquitously expressed, unlike Munc18a which was expressed only in neurons and islet cells.^56,66-71 In addition, it was shown that Munc18a and Munc18b shared binding preferences for Syntaxin isoforms 1-3, while only Munc18c bound Syntaxin 4 (Syn4) with high affinity.^67,68,70,71 Then it was in 1997 that Syntaxin 4 was demonstrated to be the functional isoform in insulin-stimulated GLUT4 vesicle translocation,^46,72 and in 1998 we and others found that Munc18c over-expression inhibited this process^64,73 (Table 1). Collectively, the Sec1 and Munc18 protein family are now referred to as ‘SM’ proteins (Sec1 and Munc18).

Table 1

Comparison of SNARE protein composition utilized for GLUT4 translocation and insulin exocytosis.

The evidence cited above strongly suggests that Munc18 proteins are essential regulators of SNARE mediated exocytosis. The most recent crystallographic and NMR structural analyses support and extend this concept, showing that Munc18-1 holds Syntaxin 1 in a “closed” conformational state in a 1:1 Munc18:Syntaxin molar ratio and unable to interact with VAMP2. Munc18-1 is proposed to orchestrate the conversion of Syntaxin 1 to the “open” conformational state to facilitate the interaction between Syntaxin 1, VAMP2 and SNAP-25.^54,74-80 Structural studies have led to the general conclusion that Munc18 proteins share a similar overall structure in which a small folded N-terminal domain mediates the interaction with Syntaxins,^81,82 whereas the remainder of the C-terminal domain carries out the poorly understood effector function that appears essential for fusion. Dulubova and colleagues have furthermore speculated that a particular loop2/3 domain of Munc18 proteins may be critical for this effector function,^81,83 which is consistent with studies showing that an inhibitory peptide directed at this region or a single point mutation within it alters Syntaxin 4-Munc18c interaction in 3T3L1 adipocytes.^84,85

One common theme in studying SNARE protein complexes and accessory proteins is that over-expression of one member of the complex can alter that stoichiometry which is optimal for the exocytotic event, resulting in loss/inhibition of function. Throughout the chapter this will become evident, as most initial studies of SNARE and SNARE accessory proteins were conducted using over-expression. Now as more data on SNARE knockout mice and siRNA-depletion evolves we will be able to disseminate the essentiality of these proteins in the various exocytotic processes.

Insulin Action: GLUT4 Vesicle Translocation

The insulin-responsive glucose transporter GLUT4 is predominantly expressed in striated muscle and adipose tissue and is responsible for the majority of insulin-stimulated glucose uptake.⁸⁶ In the basal noninsulin stimulated state, GLUT4 localizes to tubulovesicular elements and small intracellular vesicles throughout the cell cytoplasm.^87,88 Upon stimulation with insulin these GLUT4-containing compartments undergo a series of regulated steps leading to their eventual fusion with the plasma membrane.^86,89-95 This ultimately results in a large increase in the number of functional glucose transporters on the cell surface (a process termed translocation), which accounts for nearly all of the insulin-stimulated glucose uptake.

The insulin-stimulated translocation of GLUT4-containing vesicles is a complex multi-step process (Fig. 2). Several specific molecular events and signaling molecules have been identified. Initially, insulin binding to the insulin receptor activates the intrinsic protein kinase of the receptor β subunit resulting in its autophosphorylation and tyrosine phosphorylation of several proteins, most notably the IRS family of insulin receptor substrate proteins.⁹⁶ The tyrosine phosphorylation of IRS results in the association, activation and targeting of the phosphatidylinositol kinase (PI 3-kinase).^97-100 The active PI 3-kinase can then generate phosphatidylinositol-3,4,5-trisphosphate, which is necessary for the stimulation of both Protein Kinase B (PKB/Akt) and atypical Protein Kinase C isoforms through activation of the phosphoinositide-dependent protein kinases (PDK1 and PDK2) and/or through engagement of the PKB pleckstrin homology (PH) domain.^101-103 In 2002, the Akt substrate AS160 was identified.¹⁰⁴ AS160, a Rab GTPase-activating protein, becomes phosphorylated in response to insulin and is required for insulin-stimulated GLUT4 translocation.^105-107 Unlike neuronal vesicles, the GLUT4 vesicles are mostly intracellularly localized and physically separated from the plasma membrane, and following insulin receptor signaling, these intracellular GLUT4 storage vesicles traffic towards the plasma membrane, afterwards becoming docked just below the plasma membrane. Docked vesicles undergo priming mediated by binding between syntaxin 4, SNAP-23 and VAMP2 and ultimately the primed vesicles are functionally incorporated into the plasma membrane through the physical mixing of the two lipid bilayers by membrane fusion events, leaving the GLUT4 protein positioned to facilitate the uptake of glucose.^92,94

Figure 2

Schematic model depicting the cascade of events occurring in the process of insulin-stimulated GLUT4 translocation. Insulin binds to the insulin receptor (IR) present on the cell surface of insulin-responsive adipose and skeletal muscle cells, triggering (more...)

Substantial advances have been made in our understanding of the priming and fusion steps of GLUT4 translocation. GLUT4 vesicles copurify with the VAMP2 v-SNARE, and specific proteolytic cleavage of VAMP2, expression of a dominant-negative VAMP2 mutant or inhibitory peptides all impair insulin-stimulated GLUT4 translocation.^{42,43,45,46,48,49} Although VAMP2 (-/-) knockout mice have been generated, no reports on their glucose homeostasis are available at this time as they fail to thrive upon birth.¹⁰⁸ Using dominant-interfering mutants and inhibitory peptides, Syntaxin 4 and SNAP-23 have been identified as the t-SNARE proteins required for insulin-stimulated GLUT4 translocation.^{41,44,46,47,72} Syntaxin 4 (-/+) mice show deficiencies in skeletal muscle GLUT4 translocation and glucose uptake, although adipocyte glucose uptake is normal.¹⁰⁹ While VAMP3 was initially found to be part of this process in 3T3L1 adipocytes, it was later determined to be nonessential since VAMP3 (-/-) knockout mice showed no defects in skeletal muscle or adipocyte insulin-stimulated GLUT4 translocation or alterations in whole-body glucose homeostasis.¹¹⁰ In all, these data provide compelling evidence that VAMP2 functions by directing the association of the GLUT4 containing vesicles with Syntaxin 4 and SNAP-23 at the plasma membrane.

Impact of Syntaxin4 Binding Proteins Upon GLUT4 Vesicle Translocation

Munc18c

We and others have demonstrated that the Munc18c-Syntaxin 4 complex is functional in insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes. Over-expression of Munc18c resulted in the inhibition of insulin-stimulated glucose uptake and GLUT4 translocation, while having no effect upon GLUT1 translocation or the trafficking of mannose-6-phosphate receptor or transferrin receptor.^64,73,111 Furthermore, competition for the association of endogenous Munc18c-Syntaxin 4 complexes using an interfering peptide of Munc18c demonstrated a specific requirement of this complex for insulin-stimulated GLUT4 vesicle fusion,⁸⁴ but was not necessary for proximal trafficking steps.85 This functional profile is consistent with the role of the homologous Sec1 protein in yeast.^112,113

Post-translational modification of Munc18c or alteration of Munc18c abundance has been related to insulin resistance. For example, glucosamine-induced insulin resistance in adipocytes has recently been correlated with increased O-linked glycosylation of Munc18c and a reduced association of VAMP2 with Syntaxin 4.^114,115 In vivo, localized over-expression of Munc18c in skeletal muscle via adenoviral particle injection markedly impaired insulin-stimulated GLUT4 translocation to sarcolemmal and transverse tubule membranes.¹¹⁶ Moreover, over-expression of a tetracycline-repressible (tet-off) CMV-driven Munc18c transgene in skeletal muscle, adipose tissue and pancreas of mice significantly impaired whole body glucose tolerance, skeletal muscle glucose uptake and GLUT4 translocation.¹¹⁷ Most recently we have shown that Munc18c (-/+) knockout mice are insulin resistant and have severely impaired insulin-stimulated GLUT4 translocation in skeletal muscle.¹¹⁸ This contrasts however with the response of adipocytes differentiated from mouse embryonic fibroblasts (MEF) derived from an independent line of Munc18c (-/-) knockout mice: the MEF-derived adipocytes showed enhanced appearance of GLUT4 at the cell surface in response to insulin.¹¹⁹ These seemingly conflicting data are actually quite consistent with the emerging data from other genetically-altered SNARE mouse models of insulin resistance, showing defects in GLUT4 translocation and glucose uptake in skeletal muscle but not in adipocytes (Table 2).^{109,117,118,120} An alternate explanation for these differences might be that different genes encoding Munc18c were targeted, a concept which is supported by differences in exon-intron structure depicted in the two reports.^117,118

Table 2

Transgenic and knockout strains of mice with genetic alterations in SNARE proteins.

Rabs

In yeast, the SM protein Sly1p interacts with the small GTPase protein Ypt1p.⁸³ However, an analogous Munc18c interacting protein has remained elusive. Members of the small GTPase Rab protein family play crucial regulatory roles in vesicular trafficking. In neurotransmitter release, Rab3A is involved in regulating the efficiency of vesicle priming and membrane fusion.¹²¹ Also, in the yeast trafficking pathway from Golgi to vacuole, the Rab effector protein Vac1 has been shown to interact with the Sec1 family member Vps45.¹²² Vps45 binds to the yeast syntaxin Pep12,¹²³ connecting the functions of a Rab, a soluble Rab effector and the target-membrane-localized Sec1/syntaxin family. To date, there are more than 30 different Rab GTPases, most with distinct cellular localizations and individual roles in secretory and exocytic pathways. Rabs cycle between an active GTP-bound form and the inactive GDP-bound form such that vesicle-associated Rabs are found in the GTP-bound state and following membrane fusion are hydrolyzed to the inactive form.¹²⁴ Rab4 has been shown to be associated with GLUT4-containing vesicles and implicated in the insulin action on glucose transport in rat adipocytes.¹²⁵ Upon insulin stimulation Rab4 is depleted from the GLUT4 containing microsomal fraction of adipocytes and is redistributed to the cytosolic fraction, where it specifically interacts with the GDP-dissociation inhibitor GDI-1.^126-128 In addition, introduction of a carboxyl-terminal peptide, expression of a carboxyl-terminal Rab4 deletion mutant, or increased expression of wild type Rab4 all inhibit insulin-stimulated GLUT4 translocation,^129-131 consistent with a vital functional role for Rab4 in GLUT4 trafficking.

Tomosyn and Synip

In 1998 the Syntaxin 1 binding protein Tomosyn was identified in neuronal cells and found to dissociate Munc18a from Syntaxin 1.¹³² Tomosyn has a VAMP2-like region that is necessary for its interaction with Syntaxin 1 and for complex formation with Syntaxin 1 and SNAP-25.^133,134 Tomosyn has further been shown to exist as multiple isoforms sharing a conserved structure.^134,135 Since tomosyn over-expression results in down-regulated exocytosis,^132,133 and it dissociates the Munc18a-Syntaxin 1 complex known to be important for exocytosis, tomosyn has been characterized as a negative-regulator of neuronal exocytosis. More specifically, it is proposed to inhibit the priming step.¹³⁶ In an analogous manner, the b-tomosyn isoform is found to bind to Syntaxin 4 through a VAMP2-like domain, form a ternary complex with Syntaxin 4 and SNAP-23, and inhibit the priming of GLUT4 vesicles in 3T3L1 adipocytes when over-expressed.¹³⁷ Thus tomosyn and Munc18c appear to function with Syntaxin 4 at the priming step of GLUT4 vesicle fusion, although the details of their roles remain unclear.

The Syntaxin 4 binding protein Synip was isolated and characterized in 1999 in 3T3L1 adipocytes, and the syntaxin 4-synip complex was found to be dissociated upon stimulation with insulin.¹³⁸ Moreover, deletion analyses suggested that the amino terminal domain may provide a regulatory role in modulating the interaction of the carboxyl terminal domain of synip with syntaxin 4.¹³⁸ More recently, synip was shown to contain an unusual dual Akt/PKB consensus phosphorylation motif, where serine 99 is a substrate for Akt2 but not Akt1 or Akt3.¹³⁹ The expression of a synip-ser99 mutant inhibited insulin-stimulated GLUT4 vesicle docking/fusion, concurrent with its inability to undergo insulin-stimulated dissociation from syntaxin 4. It is thus postulated that insulin activation of Akt2 in adipocytes regulates the docking/fusion step of GLUT4 vesicle translocation through regulation of synip phosphorylation.

Exocyst Complex

In the yeast S. cerevisiae a complex of seven proteins were found to function together at sites on the plasma membrane to promote exocytosis, and were so named collectively the Exocyst.¹⁴⁰ Mammalian homologues were isolated shortly thereafter and one Exocyst protein, rSec6, was specifically immunolocalized to sites of granule exocytosis in neurosecretory PC12 cell processes.¹⁴¹ Another exocyst protein, Exo70, has been identified in 3T3L1 adipocytes and found to associate with other exocyst proteins Sec6 and Sec8.¹⁴² Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, which is suggested to function in the insulin signaling cascade as part of a PI-3-kinase independent pathway convergent at stimulation of GLUT4 trafficking.^94,143 Most recently, Sec6 and Sec8 have also been shown to redistribute to the plasma membrane in response to insulin in 3T3L1 adipocytes,¹⁴⁴ altogether supporting a model whereby the exocyst complex functions in insulin-stimulated GLUT4 vesicle targeting to the docking/fusion sites at the plasma membrane.

Insulin Exocytosis in Pancreatic Beta Cells

Greater than 99% of insulin secreted from the pancreatic beta cell proceeds via a regulated secretory pathway.¹⁴⁵ Insulin is contained within dense core granules located inside the pancreatic beta cell. These granules form in the trans-golgi network (TGN), at which time they are loaded with proinsulin. In these immature beta cell granules the proinsulin is cleaved to insulin and C-peptide. Glucose stimulates these granules to move from their intracellular location to the cell surface, and also from more interior pools to the readily releasable pools located just beneath the cell surface. The amount of insulin contained in beta cells is very constant, as the balance between insulin secretion, proinsulin biosynthesis and insulin granule degradation is carefully maintained.^146,147 Based upon the amount of stored insulin in normal beta cells, it is generally accepted that at least for the initial release of insulin, insulin content is not rate-limiting.¹⁴⁸

As depicted in Figure 3, when extracellular glucose rises, the glucose transporters (GLUT2) at the beta cell surface facilitate the uptake of glucose into the cell, resulting in an increase in ATP through metabolism of the glucose. This ATP upsets the basal ATP/ADP ratio causing the K_ATP-channels to close and cell depolarization,^149,150 leading to the opening of Ca²⁺-channels, overall increasing cytoplasmic Ca²⁺ concentration.¹⁵¹ In response to the rise in intracellular Ca²⁺, some docked and primed insulin storage granules fuse and release insulin, while storage granules traffic to join the readily releasable pool at the cell surface (see review, ref. ¹⁵²). Thus, insulin secretion in islets is biphasic,^153,154 with the first phase resulting from the release of insulin granules from an immediate releasable pool preprimed at the plasma membrane.¹⁵⁵ First phase secretion peaks within 5-10 min, and without returning to baseline insulin secretion gradually rises over time after this if glucose is present (representing a second phase of secretion).¹⁵³ The second phase only occurs with nutrient stimulation¹⁵⁶ and is currently thought to represent secretion from those granules which are mobilized to refill the readily and immediate releasable pools of granules.¹⁵⁷

Figure 3

Model depicting the hypothetical pathways of glucose-stimulated insulin release from insulin secretory granules in islet beta cells regulated by Syntaxin 1 (Syn1) and Syntaxin 4 (Syn4) based SNARE complexes. Primed granules are often found colocalized (more...)

Perspectives

Diabetes now strikes 6.2% of the U.S. population, having risen to afflict approximately 1 in every 17 Americans, and is expected to continue to rise. In addition, although it is well-recognized that frank diabetes develops over years of cellular dysfunction in insulin secretion and insulin action, clear markers of this predisposition have remained elusive. Significant progress has been made in defining signaling cascades leading to the secretion of insulin from the islets and uptake of glucose into skeletal muscle and adipose tissues, however each signaling cascade is rate-limited by the distal steps of vesicle exocytosis.^85,148,200 The functional regulation of Syntaxins by the Munc18 proteins is thought to be crucial in these distal steps, although the details remain unclear. There are also numerous animal models of insulin resistance and diabetes which exhibit alterations in SNARE protein abundance with which therapeutic agents are tested.^{165,166,201,202} However, given the evidence showing that Syntaxin 4-based SNARE core complexes and Syntaxin 4-Munc18c complexes participate in both insulin secretion and insulin action, therapies which target or even affect secondarily the SNARE protein abundances will have consequences in both insulin secretion and insulin action. This could be beneficial if the regulation occurred in parallel, but hazardous if the complex regulation were opposing (i.e., upregulation of Syntaxin 4 resulted in decreased glucose uptake but increased insulin secretion). Lack of such knowledge represents an important gap in our understanding of the etiology of insulin resistance and diabetes. Progress in therapeutic treatment of diabetes will need to take into account the mechanism(s) underlying the regulation of insulin secretion in islets cells and glucose uptake in adipocytes by these SNARE complexes and SNARE accessory proteins, and relate these tissue-specific effects to the overall control of glucose homeostasis.

Acknowledgements

I would like to thank Dr. Herbert Gaisano for personal communications regarding his Syntaxin 1 transgenic mice, and Angela Nevins for her critical review of the manuscript. This work was supported by research grants from the National Institute of Health (DK067912) and the American Diabetes Association (1-03-CD-10).

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