Short telomeres compromise β-cell signaling and survival

The genetic factors that underlie the increasing incidence of diabetes with age are poorly understood. We examined whether telomere length, which is inherited and known to shorten with age, plays a role in the age-dependent increased incidence of diabetes. We show that in mice with short telomeres, insulin secretion is impaired and leads to glucose intolerance despite the presence of an intact β-cell mass. In ex vivo studies, short telomeres induced cell-autonomous defects in β-cells including reduced mitochondrial membrane hyperpolarization and Ca(2+) influx which limited insulin release. To examine the mechanism, we looked for evidence of apoptosis but found no baseline increase in β-cells with short telomeres. However, there was evidence of all the hallmarks of senescence including slower proliferation of β-cells and accumulation of p16(INK4a). Specifically, we identified gene expression changes in pathways which are essential for Ca(2+)-mediated exocytosis. We also show that telomere length is additive to the damaging effect of endoplasmic reticulum stress which occurs in the late stages of type 2 diabetes. This additive effect manifests as more severe hyperglycemia in Akita mice with short telomeres which had a profound loss of β-cell mass and increased β-cell apoptosis. Our data indicate that short telomeres can affect β-cell metabolism even in the presence of intact β-cell number, thus identifying a novel mechanism of telomere-mediated disease. They implicate telomere length as a determinant of β-cell function and diabetes pathogenesis.     

Sequential exocytosis of insulin granules is associated with redistribution of SNAP25

We have investigated sequential exocytosis in beta cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in beta cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-beta-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.     

Doc2beta is a novel Munc18c-interacting partner and positive effector of syntaxin 4-mediated exocytosis

The widely expressed Sec/Munc18 (SM) protein Munc18c is required for SNARE-mediated insulin granule exocytosis from islet beta cells and GLUT4 vesicle exocytosis in skeletal muscle and adipocytes. Although Munc18c function is known to involve binding to the t-SNARE Syntaxin 4, a paucity of Munc18c-binding proteins has restricted elucidation of the mechanism by which it facilitates these exocytosis events. Toward this end, we have identified the double C2 domain protein Doc2beta as a new binding partner for Munc18c. Unlike its granule/vesicle localization in neuronal cells, Doc2beta was found principally in the plasma membrane compartment in islet beta cells and adipocytes. Moreover, co-immunoprecipitation and GST interaction assays showed Doc2beta-Munc18c binding to be direct and complexes to be devoid of Syntaxin 4. Supporting the notion of Munc18c binding with Syntaxin 4 and Doc2beta in mutually exclusive complexes, in vitro competition with Syntaxin 4 effectively displaced Munc18c from binding to Doc2beta. The second C2 domain (C2B) of Doc2beta and an N-terminal region of Munc18c were sufficient to confer complex formation. Disruption of endogenous Munc18c-Doc2beta complexes by addition of the Doc2beta binding domain of Munc18c (residues 173-255) was found to selectively inhibit glucose-stimulated insulin release. Moreover, increased expression of Doc2beta enhanced glucose-stimulated insulin secretion by approximately 40%, whereas siRNA-mediated depletion of Doc2beta attenuated insulin release. All changes in secretion correlated with parallel alterations in VAMP2 granule docking with Syntaxin 4. Taken together, these data support a model wherein Munc18c transiently switches from association with Syntaxin 4 to association with Doc2beta at the plasma membrane to facilitate exocytosis.     

Lipid remodelling in neuroendocrine secretion

Neuroendocrine cells secrete hormones and polypeptides through a complex membrane trafficking process that involves the transport of specific organelles, called large dense core secretory granules, from the Golgi apparatus to specialised sites at the plasma membrane where these vesicles are successively exocytosed and recaptured by endocytosis through tightly coupled reactions. The minimal machinery required for exocytosis has been defined as SNARE proteins associated with few accessory proteins. On the other side, clathrin and dynamin constitute major components of some of the most important endocytotic pathways. Although many protein contributors of both exocytosis and endocytosis are now identified, their actual interplay is not well resolved. Furthermore, the necessary tight coupling of exocytosis and compensatory endocytosis to maintain membrane homeostasis in neuroendocrine cells is far from being understood. In this review, we focus on the more recently identified role of lipids in these important processes that are above all membrane remodelling events.     

Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages

Several mechanistically distinct models of nonclassical secretion, including exocytosis of secretory lysosomes, shedding of plasma membrane microvesicles, and direct efflux through plasma membrane transporters, have been proposed to explain the rapid export of caspase-1-processed IL-1 beta from monocytes/macrophages in response to activation of P2X7 receptors (P2X7R) by extracellular ATP. We compared the contribution of these mechanisms to P2X7R-stimulated IL-1 beta secretion in primary bone marrow-derived macrophages isolated from wild-type, P2X7R knockout, or apoptosis-associated speck-like protein containing a C-terminal CARD knockout mice. Our experiments revealed the following: 1) a novel correlation between IL-1 beta secretion and the release of the MHC-II membrane protein, which is a marker of plasma membranes, recycling endosomes, multivesicular bodies, and released exosomes; 2) a common and absolute requirement for inflammasome assembly and active caspase-1 in triggering the cotemporal export of IL-1 beta and MHC-II; and 3) mechanistic dissociation of IL-1 beta export from either secretory lysosome exocytosis or plasma membrane microvesicle shedding on the basis of different requirements for extracellular Ca(2+) and differential sensitivity to pharmacological agents that block activation of caspase-1 inflammasomes. Thus, neither secretory lysosome exocytosis nor microvesicle shedding models constitute the major pathways for nonclassical IL-1 beta secretion from ATP-stimulated murine macrophages. Our findings suggest an alternative model of IL-1 beta release that may involve the P2X7R-induced formation of multivesicular bodies that contain exosomes with entrapped IL-1 beta, caspase-1, and other inflammasome components.     

The RalA GTPase is a central regulator of insulin exocytosis from pancreatic islet beta cells

RalA is a small GTPase that is thought to facilitate exocytosis through its direct interaction with the mammalian exocyst complex. In this study, we report an essential role for RalA in regulated insulin secretion from pancreatic beta cells. We employed lentiviral-mediated delivery of RalA short hairpin RNAs to deplete endogenous RalA protein in mouse pancreatic islets and INS-1 beta cells. Perifusion of mouse islets depleted of RalA protein exhibited inhibition of both first and second phases of glucose-stimulated insulin secretion. Consistently, INS-1 cells depleted of RalA caused a severe inhibition of depolarization-induced insulin exocytosis determined by membrane capacitance, including a reduction in the size of the ready-releasable pool of insulin granules and a reduction in the subsequent mobilization and exocytosis of the reserve pool of granules. Collectively, these data suggest that RalA is a critical component in biphasic insulin release from pancreatic beta cells.     

Insulin-stimulated insulin secretion in single pancreatic beta cells

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca2+ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.     

Palmitoylation-defective asialoglycoprotein receptors are normal in their cellular distribution and ability to bind ligand,but are defective in ligand uptake and degradation

The examination of insulin exocytosis at the single cell level by conventional electrophysiologic and amperometric methods possesses inherent limitations, and may not accurately reflect the morphologic events of exocytosis of the insulin granule. To overcome some of these limitations, we show by epifluorescent microscopy of a fluorescent dye, FM1-43, its incorporation into the plasma membrane and oncoming insulin granules undergoing exocytosis, and their core proteins. Using this method, we tracked exocytosis in real-time in insulinoma INS-1 and single rat islet beta cells in response to KCl and glucose. We observed both single transient and multi-stepwise increases in membrane FM1-43 fluorescence, suggesting single granule exocytosis as well as sequential and compound exocytosis, respectively. Confocal microscopy of nonpermeabilized cells shows that some of the exocytosed insulin granules labeled by the FM1-43 dye could also be labeled with insulin antibodies, suggesting prolonged openings of the fusion pores and slow dissolution of the granule core proteins on the membrane surface.     

A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca²⁺ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn²⁺ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K⁺ (K_ATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the K_ATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca²⁺ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K_ATP channel, inhibition of voltage-gated Na⁺ (TTX) and N-type Ca²⁺ channels (ω-conotoxin), but not L-type Ca²⁺ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca²⁺ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell K_ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Enhanced assay of endothelial exocytosis using extracellular matrix components

Vascular inflammation plays a key role in the pathogenesis of atherosclerosis. The first step in vascular inflammation is endothelial exocytosis, in which endothelial granules fuse with the plasma membrane, releasing prothrombotic and proinflammatory messenger molecules. The development of cell culture models to study endothelial exocytosis has been challenging because the factors that modulate exocytosis in vitro are not well understood. Here we report a method for studying endothelial exocytosis that optimizes extracellular matrix components, cell density, and duration of culture. Human umbilical vein endothelial cells plated on collagen I-coated plates and cultured in the confluent state for 7–12 days in low-serum medium showed robust secretion of von Willebrand factor when stimulated with various agonists. This exocytosis assay is rapid and applicable to high-throughput screening.     

Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1a

Secretory vesicle exocytosis is a highly regulated process involving vesicle targeting, priming, and membrane fusion. Rabs and SNAREs play a central role in executing these processes. We have shown recently that Rab27a and its effector, granuphilin, are involved in the exocytosis of insulin-containing secretory granules through a direct interaction with the plasma membrane syntaxin 1a in pancreatic beta cells. Here, we demonstrate that fluorescence-labeled insulin granules are peripherally accumulated in cells overexpressing granuphilin. The peripheral location of granules is well overlapped with both localizations of granuphilin and syntaxin 1a. The plasma membrane targeting of secretory granules is promoted by wild-type granuphilin but not by granuphilin mutants that are defective in binding to either Rab27a or syntaxin 1a. Granuphilin directly binds to the H3 domain of syntaxin 1a containing its SNARE motif. Moreover, introduction of the H3 domain into beta cells induces a dissociation of the native granuphilin-syntaxin complex and a marked reduction of newly docked granules. These results indicate that granuphilin plays a role in tethering insulin granules to the plasma membrane by an interaction with both Rab27a and syntaxin 1a. The complex formation of these three proteins may contribute to the specificity of the targeting process during the exocytosis of insulin granules.     

Phosphatidylinositol transfer proteins couple lipid transport to phosphoinositide synthesis

Phosphatidylinositol transfer proteins (PITPs) are lipid binding proteins that can catalyse the transfer of phosphatidylinositol (PI) from membranes enriched in PI to PI-deficient membranes. Three soluble forms of PITP of 35--38 kDa (PITP alpha, PITP beta and rdgB beta) and two larger integral proteins of 160 kDa (rdgB alpha I and II), which contain a PITP domain, are found in mammalian cells. PITPs are intimately associated with the compartmentalised synthesis of different phosphorylated inositol lipids. PI is the primary inositol lipid that is synthesised at the endoplasmic reticulum and is further phosphorylated in distinct membrane compartments by many specific lipid kinases to generate seven phosphorylated inositol lipids which are required for both signalling and for membrane traffic. PITPs play essential roles in both signalling via phospholipase C and phosphoinositide 3-kinases and in multiple aspects of membrane traffic including regulated exocytosis and vesicle biogenesis.     

Biologically active lipids promote trafficking and membrane association of Rac1 in insulin-secreting INS 832/13 cells

Despite emerging evidence to suggest that glucose-stimulated insulin secretion (GSIS) requires membrane targeting of specific small G proteins (e.g., Rac1), very little is known with regard to the precise mechanisms underlying subcellular trafficking of these proteins in the glucose-stimulated islet -cell. We previously reported activation of small G proteins by biologically active lipids via potentiation of relevant GDP/GTP exchange activities within the -cell. Herein, we studied putative regulatory roles for these lipids in the trafficking and membrane association of Rac1 in cell-free preparations derived from INS 832/13 -cells. Incubation of INS 832/13 cell lysates with polyphosphoinositides (e.g., PIP₂), phosphatidic acid, phosphatidylcholine, and phosphatidylserine significantly promoted trafficking of cytosolic Rac1 to the membrane fraction. Lysophosphatidic acid, but not lysophosphatidylcholine or lysophosphatidylserine, also promoted translocation and membrane association of Rac1. Arachidonic acid, diacylglycerol, calcium, and cAMP failed to exert any clear effects on Rac1 translocation to the membrane. Together, our findings provide the first direct evidence in support of our recent hypothesis (Kowluru A, Veluthakal R. Diabetes 54: 3523–3529, 2005), which states that generation of biologically active lipids, known to occur in the glucose-stimulated -cell, may mediate targeting of Rac1 to the membrane for optimal interaction with its putative effector proteins leading to GSIS. pancreatic -cells; GDP dissociation inhibitor; glucose-stimulated insulin secretion     

The release of secretory vesicle in encysting Giardia lamblia

Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. It secretes an extracellular cyst wall using a poorly understood process. An encystation-specific secretory vesicle (ESV) was previously described containing cyst wall proteins. The process of release of these vesicles has been suggested to occur after fragmentation of large ESV in small secretory vesicles, followed by exocytosis, but it was not demonstrated. The release of the ESV was studied by transmission electron microscopy. It was observed: (1) the moment of vesicle release; (2) that a large vesicle is exocytosed and does not fragment into small vesicles; (3) membrane fusion is distinct from traditional exocytosis since it is incomplete; (4) the occurrence of membrane fragmentation and that those membranes reseal to form ghosts; (5) these membrane ghosts may be endocytosed, adhered to flagellar surface or/and form empty vesicles in the extracellular medium.     

SNAP25, but not syntaxin 1A, recycles via an ARF6-regulated pathway in neuroendocrine cells

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins mediate cellular membrane fusion events and provide a level of specificity to donor-acceptor membrane interactions. However, the trafficking pathways by which individual SNARE proteins are targeted to specific membrane compartments are not well understood. In neuroendocrine cells, synaptosome-associated protein of 25 kDa (SNAP25) is localized to the plasma membrane where it functions in regulated secretory vesicle exocytosis, but it is also found on intracellular membranes. We identified a dynamic recycling pathway for SNAP25 in PC12 cells through which plasma membrane SNAP25 recycles in approximately 3 h. Approximately 20% of the SNAP25 resides in a perinuclear recycling endosome-trans-Golgi network (TGN) compartment from which it recycles back to the plasma membrane. SNAP25 internalization occurs by constitutive, dynamin-independent endocytosis that is distinct from the dynamin-dependent endocytosis that retrieves secretory vesicle constituents after exocytosis. Endocytosis of SNAP25 is regulated by ADP-ribosylation factor (ARF)6 (through phosphatidylinositol bisphosphate synthesis) and is dependent upon F-actin. SNAP25 endosomes, which exclude the plasma membrane SNARE syntaxin 1A, merge with those derived from clathrin-dependent endocytosis containing endosomal syntaxin 13. Our results characterize a robust ARF6-dependent internalization mechanism that maintains an intracellular pool of SNAP25, which is compatible with possible intracellular roles for SNAP25 in neuroendocrine cells.     

Voltage-dependent calcium channels at the plasma membrane, but not vesicular channels, couple exocytosis to endocytosis

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.     

The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.