It is becoming increasingly apparent that beta cell dysfunction resulting in abnormal insulin secretion is the essential element in the progression of patients from a state of impaired glucose tolerance to frank type 2 diabetes (Del Prato, 2003; Del Prato and Tiengo, 2001). Although extensive studies have examined the molecular, cellular and physiologic mechanisms of insulin granule biogenesis, sorting, and exocytosis the precise mechanisms controlling these processes and their dysregulation in the developed of diabetes remains an area of important investigation. We now know that insulin biogenesis initiates with the synthesis of preproinsulin in rough endoplastic reticulum and conversion of preproinsulin to proinsulin. Proinsulin begins to be packaged in the Trans-Golgi Network and is sorting into immature secretory granules. These immature granules become acidic via ATP-dependent proton pump and proinsulin undergoes proteolytic cleavage resulting the formation of insulin and C-peptide. During the granule maturation process, insulin is crystallized with zinc and calcium in the form of dense-core granules and unwanted cargo and membrane proteins undergo selective retrograde trafficking to either the constitutive trafficking pathway for secretion or to degradative pathways. The newly formed mature dense-core insulin granules populate two different intracellular pools, the readily releasable pools (RRP) and the reserved pool. These two distinct populations are thought to be responsible for the biphasic nature of insulin release in which the RRP granules are associated with the plasma membrane and undergo an acute calcium-dependent release accounting for first phase insulin secretion. In contrast, second phase insulin secretion requires the trafficking of the reserved granule pool to the plasma membrane. The initial trigger for insulin granule fusion with the plasma membrane is a rise in intracellular calcium and in the case of glucose stimulation results from increased production of ATP, closure of the ATP-sensitive potassium channel and cellular depolarization. In turn, this opens voltage-dependent calcium channels allowing increased influx of extracellular calcium. Calcium is thought to bind to members of the fusion regulatory proteins synaptogamin that functionally repressors the fusion inhibitory protein complexin. Both complexin and synaptogamin interact as well as several other regulatory proteins interact with the core fusion machinery composed of the Q- or t-SNARE proteins syntaxin 1 and SNAP25 in the plasmamembrane that assembles with the R- or v-SNARE protein VAMP2 in insulin granules. In this chapter we will review the current progress of insulin granule biogenesis, sorting, trafficking, exocytosis and signaling pathways that comprise the molecular basis of glucose-dependent insulin secretion.

Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion. INTRODUCTIONInsulin-stimulated uptake of glucose by fat and muscle and the maintenance of glucose homeostasis are primarily mediated by the Glut4 glucose transporter (Bryant et al., 2002). Glut4 cycles between the plasma membrane and cytoplasmic storage sites, with most of the transporter residing intracellularly in the absence of insulin signaling because the basal rate of endocytosis exceeds the basal rate of exocytosis. Insulin signaling greatly stimulates the rate of exocytosis, leading to recruitment of up to 50% of the transporter to the cell surface where it facilitates glucose uptake. Each of the major elements of this regulated exocytosis process-vesicle mobilization, trafficking to the plasma membrane, docking, and fusion-have been shown to be rate limiting under different circumstances, and the mechanisms that regulate them remain under investigation.Phospholipase D (PLD), a membrane-associated enzyme regulated by agonist stimulation (reviewed in Frohman and Morris, 1999), has been proposed to function at many different steps in vesicle trafficking, including activation of signaling networks (Andresen et al., 2002), budding of vesicles from the trans-Golgi (Chen et al., 1997), and vesicle fusion (reviewed in McDermott et al., 2004). PLD generates the lipid phosphatidic acid (PA), which has been shown to activate phosphatidylinositol 4-phosphate 5-kinase (Honda et al., 1999) and thus increase the levels of phosphatidylinositol 4,5-bisphosphate, a lipid critically required for exocytosis (Di Paolo et al., 2004). PA also has been reported to serve as a membrane anchor for a growing number of protein targets (Manifava et al., 2001), including yeast and mammalian components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex (Wagner and Tamm, 2001;Nakanishi et al., 2004), and it has been proposed to act as a fusogenic lipid in biophysical modeling studies by lowering the activation...

Following biosynthesis, both GLUT1 and VSV-G proteins appear rapidly (2–3 h) at the plasma membrane, whereas GLUT4 is retained in intracellular membrane compartments and does not display any significant insulin responsiveness until 6–9 h. Surprisingly, the acquisition of insulin responsiveness did not require plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) had no effect on the insulin-stimulated GLUT4 translocation. Furthermore, expression of endocytosis-defective GLUT4 mutants or continuous surface labeling with an exofacial specific antibody demonstrated that GLUT4 did not transit the cell surface prior to the acquisition of insulin responsiveness. The expression of a dominant-interfering GGA mutant (VHS-GAT) had no effect on the trafficking of newly synthesized GLUT1 or VSV-G protein to the plasma membrane, but completely blocked the insulin-stimulated translocation of newly synthesized GLUT4. Furthermore, in vitro budding of GLUT4 vesicles but not GLUT1 or the transferrin receptor was inhibited by VHS-GAT. Together, these data demonstrate that following biosynthesis, GLUT4 directly sorts and traffics to the insulin-responsive storage compartment through a specific GGA-sensitive process

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.

TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/ Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (⌬N-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominantinterfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/⌬VCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization. INTRODUCTIONTC10 is a member of the Rho family of GTP-binding proteins and is closely related to Cdc42 (Neudauer et al., 1998). Although TC10 is primarily expressed in adipose and muscle tissue (Neudauer et al., 1998;Imagawa et al., 1999), its function has only been peripherally examined. In vitro binding assays have indicated that active GTP-bound TC10 can bind a number of potential effectors, including mixed lineage kinase 2, myotonic dystrophy-related Cdc42 kinase, p21-activated protein kinases, the Borg family of interacting proteins, the mammalian partition-defective homolog Par6, and the N-WASP isoform of the Wiskott-Aldrich Syndrome Protein (Neudauer et al., 1998;Joberty et al., 1999Joberty et al., , 2000.However, whether TC10 can interact with any of these potential effectors under physiological conditions has yet to be established. Nevertheless, similar to other members of the Rho family, expression of a constitutively active TC10 mutant (TC10/Q75L) in fibroblasts decreased actin stress fibers concomitant with the formation of plasma membrane microspikes (Murphy et al., 1999). However, expression of wildtype TC10 (TC10/WT) was without any effect on fibroblast cell morphology or actin structures. In contrast, expression of wild-type Cdc42 (Cdc42/WT) or a constitutively active Cdc42 mutant (Cdc42/Q61L) in fibroblasts decreased actin stress fibers in parallel with the induction of actin protrusions and lamellipodia (Coghlan et al., 2000). These data indicate that although fibroblasts express the necessary downstream effectors to modulate ac...

Insulin stimulates glucose transport via phosphatidylinositol 3-kinase-dependent and -independent pathways. The phosphatidylinositol 3-kinase-independent pathway involves activation of the G protein TC10. A cDNA encoding the mouse homolog of TC10 was cloned, and its gene was mapped at the distal end of chromosome 17. Additionally, a second gene was discovered with ϳ70% sequence identity to TC10. We refer to this gene as TC10␤. Both isoforms of TC10 were activated by insulin upon transfection in 3T3L1 adipocytes. Cotransfection of cells with TC10␣ or ␤ plus a dominant negative form of the c-cbl-associated protein CAP prevented the activation by insulin, implicating the CAP/Cbl pathway. Interestingly, both forms of TC10 were also localized in lipid raft fractions in transfected adipocytes. However, although overexpression of TC10␣ completely blocked glucose transport, TC10␤ only partially inhibited this process. Furthermore, TC10␣ overexpression disrupted adipocyte cortical actin, whereas TC10␤ had little if any effect. Thus, there are two isoforms of this key signaling intermediate, both of which are activated by insulin, but they may play different roles in initiating downstream effectors that influence glucose transport.

In muscle and adipose cells, the insulin-responsive aminopeptidase (IRAP) is localized to intracellular storage sites and undergoes insulin-dependent redistribution to the cell surface. Following expression, the newly synthesized IRAP protein traffics to the perinuclear insulin-sensitive compartment and acquires insulin sensitivity 6 -9 h following biosynthesis. Knockdown of GGA1 by RNA interference prevented IRAP from entering, but not exiting, the insulin-responsive compart- The secretory pathway of eukaryotic cells is functionally organized into a series of discrete subcellular membrane compartments, each uniquely outfitted with a relatively stable population of resident proteins (1, 2). Against this fairly constant backdrop, many proteins transiently occupy a succession of membrane compartments before reaching their final destinations. In addition, a select few proteins are initially sequestered within subcellular compartments until an appropriate extracellular signal triggers their regulated exocytosis. Regardless of the specific trafficking itinerary, cargo proteins harbor intrinsic targeting information that directs their subcellular localization (3). Protein targeting begins at the level of the endoplasmic reticulum, which represents the entry point into the secretory pathway for both membrane and secreted proteins. Having cleared the quality control system of the endoplasmic reticulum, correctly folded cargo proteins are efficiently incorporated into COPII carrier vesicles prior to anterograde transport to the Golgi apparatus (4, 5). Although the mechanism for intra-Golgi transport is still under debate, it is generally believed that cargo proteins lacking specific endoplasmic reticulum retrieval signals arrive at the Golgi complex and are subsequently transported through the stacked Golgi cisternae (6, 7).The trans-Golgi network (TGN) 2 is the final subcompartment of the Golgi complex and functions as a key sorting station for proteins and lipids. In this capacity, recent work has shown that the TGN is subdivided into functional domains that recruit distinct coat complexes (8, 9). Indeed, a new family of clathrin adaptors, the GGA (Golgi-localized, ␥-ear-containing, ADP-ribosylation factor-binding proteins), have recently been identified as key players in transmembrane cargo selection at the TGN (10 -14). The three mammalian GGA isoforms are modular adaptors, each comprised of an amino-terminal VHS domain, a middle GAT domain, a hinge region, and a carboxylterminal GAE domain. The VHS domain interacts with a consensus DXXLL sorting motif found in the cytosolic tails of a subset of transmembrane cargo proteins, including the cationdependent and independent mannose-6-phosphate receptors, sortilin, and LRP3, among others. The GAT domain binds GTP-loaded ADP-ribosylation factor during GGA-mediated coat recruitment to the TGN, the hinge region interacts with clathrin, and the GAE domain binds several accessory coat proteins (15, 16).Recently, we investigated the sorting of the insulin-responsive glucose tra...

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 ␤-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/ K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of ␤-cell insulin secretion.