We have investigated sequential exocytosis in β 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 β 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-β-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.
Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1alpha cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1alpha in pancreatic beta cells. Expression of collectrin was decreased in the islets of HNF-1alpha (-/-) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the beta cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca(2+) influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.
Mutations in the hepatocyte nuclear factor (HNF)-4␣ gene cause a form of maturity-onset diabetes of the young (MODY1) that is characterized by impairment of glucose-stimulated insulin secretion by pancreatic -cells. HNF-4␣, a transcription factor belonging to the nuclear receptor superfamily, is expressed in pancreatic islets as well as in the liver, kidney, and intestine. However, the role of HNF-4␣ in pancreatic -cell is unclear. To clarify the role of HNF-4␣ in -cells, we generated -cell-specific HNF-4␣ knock-out (HNF-4␣KO) mice using the Cre-LoxP system. The HNF-4␣KO mice exhibited impairment of glucose-stimulated insulin secretion, which is a characteristic of MODY1. Pancreatic islet morphology, -cell mass, and insulin content were normal in the HNF-4␣ mutant mice. Insulin secretion by HNF-4␣KO islets and the intracellular calcium response were impaired after stimulation by glucose or sulfonylurea but were normal after stimulation with KCl or arginine. Both NAD(P)H generation and ATP content at high glucose concentrations were normal in the HNF-4␣KO mice. Expression levels of Kir6.2 and SUR1 proteins in the HNF-4␣KO mice were unchanged as compared with control mice. Patch clamp experiments revealed that the current density was significantly increased in HNF-4␣KO mice compared with control mice. These results are suggestive of the dysfunction of K ATP channel activity in the pancreatic -cells of HNF-4␣-deficient mice. Because the K ATP channel is important for proper insulin secretion in -cells, altered K ATP channel activity could be related to the impaired insulin secretion in the HNF-4␣KO mice. Hepatocyte nuclear factor (HNF)2 -4␣, a transcription factor belonging to the nuclear hormone receptor superfamily (NR2A1), is expressed in the liver, kidney, intestine, and pancreas (1). Similar to other nuclear receptors, HNF-4␣ has several functional domains, including the N-terminal transactivation domain (AF-1), a DNAbinding domain, a functionally complex C-terminal region that forms a ligand-binding domain, and a dimerization interface and a transactivation domain (AF-2). In the liver, HNF-4␣ plays an important role in regulating various genes involved in glucose, fatty acid, amino acid, and cholesterol metabolism, as well as blood coagulation and hepatic development and differentiation (2, 3).Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic disorder that accounts for 2-5% of type 2 diabetes. It is characterized by autosomal dominant inheritance and an early age of onset (usually at Ͻ25 years old) (4). We have shown previously that heterozygous mutations of the HNF-4␣ gene can cause a particular form of MODY (MODY1) (5). Functional studies of the mutations in MODY1 patients have shown that the diabetes is caused by loss-offunction mutations (6, 7). Clinical studies have also shown that the primary cause of MODY1 is an impairment of glucose-stimulated insulin secretion by pancreatic -cells rather than liver dysfunction (8, 9), indicating that loss of HNF-4␣ le...
We developed a novel approach allowing intracellular GLUT4 dynamics to be analyzed directly at the single molecule level using Quantum dot to quantitatively establish the behavioral nature of GLUT4. With this approach, we defined the actual steps at which insulin signals directly converge and impact the process of dynamic GLUT4 trafficking events.
When cells release hormones and neurotransmitters through exocytosis, cytosolic Ca(2+) triggers the fusion of secretory vesicles with the plasma membrane. It is well known that this fusion requires assembly of a SNARE protein complex. However, the timing of SNARE assembly relative to vesicle fusion--essential for understanding exocytosis--has not been demonstrated. To investigate this timing, we constructed a probe that detects the assembly of two plasma membrane SNAREs, SNAP25 and syntaxin-1A, through fluorescence resonance energy transfer (FRET). With two-photon imaging, we simultaneously measured FRET signals and insulin exocytosis in beta cells from the pancreatic islet of Langerhans. In some regions of the cell, we found that the SNARE complex was preassembled, which enabled rapid exocytosis. In other regions, SNARE assembly followed Ca(2+) influx, and exocytosis was slower. Thus, SNARE proteins exist in multiple stable preparatory configurations, from which Ca(2+) may trigger exocytosis through distinct mechanisms and with distinct kinetics.
] i and thereby bypassed the ATP-sensitive K + channel-dependent mechanism of glucose sensing, were therefore studied. A high concentration (20 mM) of glucose potentiated CIE within 1 min, and this effect was blocked by inhibitors of PKA. This PKA-dependent action of glucose required glucose metabolism, given that increasing the intracellular concentration of cAMP by treatment with forskolin potentiated CIE only at the high glucose concentration. Finally, PKA appeared to reduce the frequency of 'kiss-and-run' exocytic events and to promote full-fusion events during GIE. These data indicate that a PKA-dependent mechanism of glucose sensing, which is operative even at the basal level of PKA activity, plays an important role specifically in the first phase of GIE, and they suggest that the action of PKA is mediated at the level of the fusion reaction.
We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of ∼7 s even though exocytosis was induced with large increases in cytosolic Ca 2+ concentration by uncaging of a caged-Ca 2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.
Nedachi T, Hatakeyama H, Kono T, Sato M, Kanzaki M. Characterization of contraction-inducible CXC chemokines and their roles in C2C12 myocytes.
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