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1.
Cell Rep ; 26(5): 1213-1226.e7, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30699350

RESUMO

Pancreatic ß cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in ß cells to regulate Ca2+ influx for insulin secretion. ß cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of ß cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the ß cell plasma membrane, which were markedly decreased in ELKS-KO ß cells. Mechanistically, ELKS directly interacted with the VDCC-ß subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the ß cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.


Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Subunidades Proteicas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Proteínas do Tecido Nervoso/deficiência , Ligação Proteica/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/deficiência
2.
J Cell Biol ; 215(1): 121-138, 2016 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-27697926

RESUMO

The membrane fusion of secretory granules with plasma membranes is crucial for the exocytosis of hormones and enzymes. Secretion disorders can cause various diseases such as diabetes or pancreatitis. Synaptosomal-associated protein 23 (SNAP23), a soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) molecule, is essential for secretory granule fusion in several cell lines. However, the in vivo functions of SNAP23 in endocrine and exocrine tissues remain unclear. In this study, we show opposing roles for SNAP23 in secretion in pancreatic exocrine and endocrine cells. The loss of SNAP23 in the exocrine and endocrine pancreas resulted in decreased and increased fusion of granules to the plasma membrane after stimulation, respectively. Furthermore, we identified a low molecular weight compound, MF286, that binds specifically to SNAP23 and promotes insulin secretion in mice. Our results demonstrate opposing roles for SNAP23 in the secretion mechanisms of the endocrine and exocrine pancreas and reveal that the SNAP23-binding compound MF286 may be a promising drug for diabetes treatment.


Assuntos
Ilhotas Pancreáticas/citologia , Pâncreas Exócrino/citologia , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Células Acinares/metabolismo , Células Acinares/ultraestrutura , Amilases/metabolismo , Animais , Fusão Celular , Exocitose , Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Secreção de Insulina , Camundongos Knockout , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Biológicos , Glândula Parótida/citologia , Transporte Proteico , Proteínas Qb-SNARE/deficiência , Proteínas Qc-SNARE/deficiência , Proteínas SNARE/metabolismo , Vesículas Secretórias/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo
3.
Diabetes ; 65(6): 1648-59, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26953164

RESUMO

VAMP7 is a SNARE protein that mediates specific membrane fusions in intracellular trafficking and was recently reported to regulate autophagosome formation. However, its function in pancreatic ß-cells is largely unknown. To elucidate the physiological role of VAMP7 in ß-cells, we generated pancreatic ß-cell-specific VAMP7 knockout (Vamp7(flox/Y);Cre) mice. VAMP7 deletion impaired glucose-stimulated ATP production and insulin secretion, though VAMP7 was not localized to insulin granules. VAMP7-deficient ß-cells showed defective autophagosome formation and reduced mitochondrial function. p62/SQSTM1, a marker protein for defective autophagy, was selectively accumulated on mitochondria in VAMP7-deficient ß-cells. These findings suggest that accumulation of dysfunctional mitochondria that are degraded by autophagy caused impairment of glucose-stimulated ATP production and insulin secretion in Vamp7(flox/Y);Cre ß-cells. Feeding a high-fat diet to Vamp7(flox/Y);Cre mice exacerbated mitochondrial dysfunction, further decreased ATP production and insulin secretion, and consequently induced glucose intolerance. Moreover, we found upregulated VAMP7 expression in wild-type mice fed a high-fat diet and in db/db mice, a model for diabetes. Thus our data indicate that VAMP7 regulates autophagy to maintain mitochondrial quality and insulin secretion in response to pathological stress in ß-cells.


Assuntos
Autofagia/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/fisiologia , Proteínas R-SNARE/fisiologia , Trifosfato de Adenosina/biossíntese , Animais , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Intolerância à Glucose/metabolismo , Homeostase , Secreção de Insulina , Masculino , Camundongos , Camundongos Knockout , Proteínas R-SNARE/deficiência
4.
Hum Mutat ; 36(8): 753-7, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25864427

RESUMO

NALCN and its homologues code for the ion channel responsible for half of background Na(+) -leak conductance in vertebrate and invertebrate neurons. Recessive mutations in human NALCN cause intellectual disability (ID) with hypotonia. Here, we report a de novo heterozygous mutation in NALCN affecting a conserved residue (p.R1181Q) in a girl with ID, episodic and persistent ataxia, and arthrogryposis. Interestingly, her episodes of ataxia were abolished by the administration of acetazolamide, similar to the response observed in episodic ataxia associated with other ion channels. Introducing the analogous mutation in the Caenorhabditis elegans homologue nca-1 induced a coiling locomotion phenotype, identical to that obtained with previously characterized C. elegans gain-of-function nca alleles, suggesting that p.R1181Q confers the same property to NALCN. This observation thus suggests that dominant mutations in NALCN can cause a neurodevelopmental phenotype that overlaps with, while being mostly distinct from that associated with recessive mutations in the same gene.


Assuntos
Artrogripose/genética , Ataxia/genética , Deficiência Intelectual/genética , Mutação , Canais de Sódio/genética , Acetazolamida/uso terapêutico , Animais , Artrogripose/metabolismo , Ataxia/tratamento farmacológico , Ataxia/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Pré-Escolar , Feminino , Humanos , Deficiência Intelectual/metabolismo , Canais Iônicos/genética , Proteínas de Membrana , Canais de Sódio/metabolismo
5.
Endocrinology ; 156(2): 444-52, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25426873

RESUMO

The physiological role of serotonin, or 5-hydroxytryptamine (5-HT), in pancreatic ß-cell function was previously elucidated using a pregnant mouse model. During pregnancy, 5-HT increases ß-cell proliferation and glucose-stimulated insulin secretion (GSIS) through the Gαq-coupled 5-HT2b receptor (Htr2b) and the 5-HT3 receptor (Htr3), a ligand-gated cation channel, respectively. However, the role of 5-HT in ß-cell function in an insulin-resistant state has yet to be elucidated. Here, we characterized the metabolic phenotypes of ß-cell-specific Htr2b(-/-) (Htr2b ßKO), Htr3a(-/-) (Htr3a knock-out [KO]), and ß-cell-specific tryptophan hydroxylase 1 (Tph1)(-/-) (Tph1 ßKO) mice on a high-fat diet (HFD). Htr2b ßKO, Htr3a KO, and Tph1 ßKO mice exhibited normal glucose tolerance on a standard chow diet. After 6 weeks on an HFD, beginning at 4 weeks of age, both Htr3a KO and Tph1 ßKO mice developed glucose intolerance, but Htr2b ßKO mice remained normoglycemic. Pancreas perfusion assays revealed defective first-phase insulin secretion in Htr3a KO mice. GSIS was impaired in islets isolated from HFD-fed Htr3a KO and Tph1 ßKO mice, and 5-HT treatment improved insulin secretion from Tph1 ßKO islets but not from Htr3a KO islets. Tph1 and Htr3a gene expression in pancreatic islets was not affected by an HFD, and immunostaining could not detect 5-HT in pancreatic islets from mice fed an HFD. Taken together, these results demonstrate that basal 5-HT levels in ß-cells play a role in GSIS through Htr3, which becomes more evident in a diet-induced insulin-resistant state.


Assuntos
Resistência à Insulina , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Receptores de Serotonina/metabolismo , Serotonina/fisiologia , Animais , Dieta Hiperlipídica , Secreção de Insulina , Masculino , Camundongos Knockout , Receptores de Serotonina/genética , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo
6.
Sci Rep ; 4: 4123, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24535122

RESUMO

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca(2+)] change in the ß-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of ß-cells.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Linhagem Celular , Camundongos
7.
Proc Natl Acad Sci U S A ; 110(48): 19420-5, 2013 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-24218571

RESUMO

In preparation for the metabolic demands of pregnancy, ß cells in the maternal pancreatic islets increase both in number and in glucose-stimulated insulin secretion (GSIS) per cell. Mechanisms have been proposed for the increased ß cell mass, but not for the increased GSIS. Because serotonin production increases dramatically during pregnancy, we tested whether flux through the ionotropic 5-HT3 receptor (Htr3) affects GSIS during pregnancy. Pregnant Htr3a(-/-) mice exhibited impaired glucose tolerance despite normally increased ß cell mass, and their islets lacked the increase in GSIS seen in islets from pregnant wild-type mice. Electrophysiological studies showed that activation of Htr3 decreased the resting membrane potential in ß cells, which increased Ca(2+) uptake and insulin exocytosis in response to glucose. Thus, our data indicate that serotonin, acting in a paracrine/autocrine manner through Htr3, lowers the ß cell threshold for glucose and plays an essential role in the increased GSIS of pregnancy.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Serotonina/farmacologia , Transdução de Sinais/fisiologia , Animais , Feminino , Glucose/metabolismo , Immunoblotting , Imuno-Histoquímica , Secreção de Insulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Gravidez , Receptores 5-HT3 de Serotonina/genética
8.
J Clin Invest ; 123(10): 4513-24, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24051378

RESUMO

Recent genome-wide association studies demonstrated that common variants of solute carrier family 30 member 8 gene (SLC30A8) increase susceptibility to type 2 diabetes. SLC30A8 encodes zinc transporter-8 (ZnT8), which delivers zinc ion from the cytoplasm into insulin granules. Although it is well known that insulin granules contain high amounts of zinc, the physiological role of secreted zinc remains elusive. In this study, we generated mice with ß cell-specific Slc30a8 deficiency (ZnT8KO mice) and demonstrated an unexpected functional linkage between Slc30a8 deletion and hepatic insulin clearance. The ZnT8KO mice had low peripheral blood insulin levels, despite insulin hypersecretion from pancreatic ß cells. We also demonstrated that a substantial amount of the hypersecreted insulin was degraded during its first passage through the liver. Consistent with these findings, ZnT8KO mice and human individuals carrying rs13266634, a major risk allele of SLC30A8, exhibited increased insulin clearance, as assessed by c-peptide/insulin ratio. Furthermore, we demonstrated that zinc secreted in concert with insulin suppressed hepatic insulin clearance by inhibiting clathrin-dependent insulin endocytosis. Our results indicate that SLC30A8 regulates hepatic insulin clearance and that genetic dysregulation of this system may play a role in the pathogenesis of type 2 diabetes.


Assuntos
Proteínas de Transporte de Cátions/genética , Diabetes Mellitus Tipo 2/genética , Insulina/sangue , Fígado/metabolismo , Adulto , Animais , Proteínas de Transporte de Cátions/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Endocitose , Genótipo , Glucagon/metabolismo , Células Hep G2 , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Transporte Proteico , Receptor de Insulina/metabolismo , Análise de Sequência de DNA , Zinco/metabolismo , Transportador 8 de Zinco
9.
Neuron ; 77(6): 1069-82, 2013 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-23522043

RESUMO

A cation channel NCA/UNC-79/UNC-80 affects neuronal activity. We report here the identification of a conserved endoplasmic reticulum protein NLF-1 (NCA localization factor-1) that regulates neuronal excitability and locomotion through the NCA channel. In C. elegans, the loss of either NLF-1 or NCA leads to a reduced sodium leak current, and a hyperpolarized resting membrane potential in premotor interneurons. This results in a decreased premotor interneuron activity that reduces the initiation and sustainability of rhythmic locomotion. NLF-1 promotes axonal localization of all NCA reporters. Its mouse homolog mNLF-1 functionally substitutes for NLF-1 in C. elegans, interacts with the mammalian sodium leak channel NALCN in vitro, and potentiates sodium leak currents in primary cortical neuron cultures. Taken together, an ER protein NLF-1 delivers a sodium leak channel to maintain neuronal excitability and potentiates a premotor interneuron network critical for C. elegans rhythmic locomotion.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Locomoção/fisiologia , Neurônios/metabolismo , Periodicidade , Canais de Sódio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Axônios/metabolismo , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Células Cultivadas , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes/métodos , Canais Iônicos , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Nucleares , Canais de Sódio/genética , Canais de Sódio/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
10.
PLoS One ; 7(10): e47381, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23077605

RESUMO

In glucose-induced insulin secretion from pancreatic ß-cells, a population of insulin granules fuses with the plasma membrane without the typical docking process (newcomer granule fusions), however, its mechanism is unclear. In this study, we investigated the PI3K signaling pathways involved in the upregulation of newcomer granule fusions. Acute treatment with the class IA-selective PI3K inhibitors, PIK-75 and PI-103, enhanced the glucose-induced insulin secretion. Total internal reflection fluorescent microscopy revealed that the PI3K inhibitors increased the fusion events from newcomer granules. We developed a new system for transfection into pancreatic islets and demonstrated the usefulness of this system in order for evaluating the effect of transfected genes on the glucose-induced secretion in primary cultured pancreatic islets. Using this transfection system together with a series of constitutive active mutants, we showed that the PI3K-3-phosphoinositide dependent kinase-1 (PDK1)-Akt pathway mediated the potentiation of insulin secretion. The Akt inhibitor also enhanced the glucose-induced insulin secretion in parallel with the upregulation of newcomer granule fusions, probably via increased motility of intracellular insulin granules. These data suggest that the PI3K-PDK1-Akt pathway plays a significant role in newcomer granule fusions, probably through an alteration of the dynamics of the intracellular insulin granules.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Furanos/farmacologia , Glucose/farmacologia , Hidrazonas/farmacologia , Secreção de Insulina , Camundongos , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Piridinas/farmacologia , Pirimidinas/farmacologia , Piruvato Desidrogenase Quinase de Transferência de Acetil , Vesículas Secretórias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Regulação para Cima/efeitos dos fármacos
11.
Biochem Biophys Res Commun ; 412(4): 556-60, 2011 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-21854759

RESUMO

Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single ß-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2 weeks. Perfusion studies showed that 2 weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters.


Assuntos
Inibidores da Dipeptidil Peptidase IV/farmacologia , Exocitose/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Pirazinas/farmacocinética , Triazóis/farmacocinética , Animais , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Mutantes
12.
Front Biosci (Landmark Ed) ; 16(4): 1197-210, 2011 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-21196227

RESUMO

Insulin, stored in large dense core granules, is biphasically exocytosed by glucose stimulation in pancreatic beta-cells. Several molecules, such as SNARE proteins, and Ca2+ ion are involved in the regulation of insulin exocytosis. Indeed, studies using gene targeting mice revealed critical roles of SNARE proteins and their accessory proteins, which may be associated with diabetes mellitus. In particular, the total internal reflection fluorescent (TIRF) imaging technique shed new light on the molecular mechanism of the insulin exocytotic process. In this review we discuss the mechanism of insulin exocytosis mainly from a point of view of imaging techniques.


Assuntos
Cálcio/metabolismo , Grânulos Citoplasmáticos/metabolismo , Insulina/metabolismo , Proteínas SNARE/fisiologia , Exocitose/fisiologia , Secreção de Insulina , Microscopia de Fluorescência , Fosfatidilinositol 3-Quinase/metabolismo , Vesículas Secretórias/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
14.
PLoS One ; 5(12): e15553, 2010 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-21151568

RESUMO

BACKGROUND: A variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in ß cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from ß cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice. PRINCIPAL FINDINGS: Total internal reflection fluorescence imaging of CDKAL1 KO ß cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K(+)-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca(2+) concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K(+) (K(ATP)) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets. CONCLUSIONS/SIGNIFICANCE: We provide the first report describing the function of CDKAL1 in ß cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in ß cells by facilitating ATP generation, K(ATP) channel responsiveness and the subsequent activity of Ca(2+) channels through pathways other than CDK5-mediated regulation.


Assuntos
Trifosfato de Adenosina/metabolismo , Quinase 5 Dependente de Ciclina/genética , Insulina/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/genética , Animais , Linfócitos B/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Citosol/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Exocitose , Variação Genética , Glucose/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Potássio/química , tRNA Metiltransferases
15.
Biochem J ; 432(2): 375-86, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-20854263

RESUMO

Functional insulin receptor and its downstream effector PI3K (phosphoinositide 3-kinase) have been identified in pancreatic ß-cells, but their involvement in the regulation of insulin secretion from ß-cells remains unclear. In the present study, we investigated the physiological role of insulin and PI3K in glucose-induced biphasic insulin exocytosis in primary cultured ß-cells and insulinoma Min6 cells using total internal reflection fluorescent microscopy. The pretreatment of ß-cells with insulin induced the rapid increase in intracellular Ca2+ levels and accelerated the exocytotic response without affecting the second-phase insulin secretion. The inhibition of PI3K not only abolished the insulin-induced rapid development of the exocytotic response, but also potentiated the second-phase insulin secretion. The rapid development of Ca2+ and accelerated exocytotic response induced by insulin were accompanied by the translocation of the Ca2+-permeable channel TrpV2 (transient receptor potential V2) in a PI3K-dependent manner. Inhibition of TrpV2 by the selective blocker tranilast, or the expression of shRNA (short-hairpin RNA) against TrpV2 suppressed the effect of insulin in the first phase, but the second phase was not affected. Thus our results demonstrate that insulin treatment induced the acceleration of the exocytotic response during the glucose-induced first-phase response by the insertion of TrpV2 into the plasma membrane in a PI3K-dependent manner.


Assuntos
Canais de Cálcio/genética , Células Secretoras de Insulina/fisiologia , Insulina/fisiologia , Canais de Cátion TRPV/genética , Animais , Sequência de Bases , Linhagem Celular , DNA/química , DNA/genética , DNA Complementar/genética , Exocitose , Hormônio do Crescimento/metabolismo , Homeostase , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosfatidilinositol 3-Quinases/metabolismo , Transfecção
16.
Diabetes ; 59(10): 2522-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20622165

RESUMO

OBJECTIVE: Pertussis toxin uncoupling-based studies have shown that Gαi and Gαo can inhibit insulin secretion in pancreatic ß-cells. Yet it is unclear whether Gαi and Gαo operate through identical mechanisms and how these G-protein-mediated signals inhibit insulin secretion in vivo. Our objective is to examine whether/how Gαo regulates islet development and insulin secretion in ß-cells. RESEARCH DESIGN AND METHODS: Immunoassays were used to analyze the Gαo expression in mouse pancreatic cells. Gαo was specifically inactivated in pancreatic progenitor cells by pancreatic cell-specific gene deletion. Hormone expression and insulin secretion in response to different stimuli were assayed in vivo and in vitro. Electron microscope and total internal reflection fluorescence-based assays were used to evaluate how Gαo regulates insulin vesicle docking and secretion in response to glucose stimulation. RESULTS: Islet cells differentiate properly in Gαo(-/-) mutant mice. Gαo inactivation significantly enhances insulin secretion both in vivo and in isolation. Gαo nullizygous ß-cells contain an increased number of insulin granules docked on the cell plasma membrane, although the total number of vesicles per ß-cell remains unchanged. CONCLUSIONS: Gαo is not required for endocrine islet cell differentiation, but it regulates the number of insulin vesicles docked on the ß-cell membrane.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/antagonistas & inibidores , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Diferenciação Celular , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Homeostase , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transativadores/genética
17.
Biochem Biophys Res Commun ; 390(1): 16-20, 2009 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-19766598

RESUMO

To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic beta cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.


Assuntos
Exocitose , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Peptídeo 1 Semelhante ao Glucagon/química , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Vesículas Secretórias/metabolismo
18.
Biochem Biophys Res Commun ; 385(3): 291-5, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19426714

RESUMO

We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca2+ concentration ([Ca2+](PM)) with the Ca2+ indicator Fura Red in rat beta cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca2+](PM) caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca2+](PM) induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca2+](PM) rise directly determines the types of fusing granules.


Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Membranas Intracelulares/fisiologia , Fusão de Membrana , Vesículas Secretórias/fisiologia , Animais , Células Cultivadas , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Masculino , Microscopia de Fluorescência , Ratos , Ratos Wistar , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura
19.
Exp Diabetes Res ; 2009: 278762, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20069052

RESUMO

To investigate the different effects between sulfonylurea (SU) and glinide drugs in insulin secretion, pancreatic beta-cells were repeatedly stimulated with SU (glimepiride) or glinide (mitiglinide). Total internal reflection fluorescent (TIRF) microscopy revealed that secondary stimulation with glimepiride, but not glucose and mitiglinide, failed to evoke fusions of insulin granules although primary stimulation with glucose, glimepiride, and mitiglinide induced equivalent numbers of exocytotic responses. Glimepiride, but not glucose and mitiglinide, induced abnormally sustained [Ca(2+)](i) elevations and reductions of docked insulin granules on the plasma membrane. Our data suggest that the effect of glinide on insulin secretory mechanisms is similar to that of glucose.


Assuntos
Carbamatos/farmacologia , Cicloexanos/farmacologia , Exocitose/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fenilalanina/análogos & derivados , Piperidinas/farmacologia , Compostos de Sulfonilureia/farmacologia , Animais , Cálcio/fisiologia , Fusão Celular , Células Cultivadas , Glucose/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Nateglinida , Fenilalanina/farmacologia
20.
Exp Diabetes Res ; 2008: 818341, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18779868

RESUMO

We examined changes in the ultrastructure and localization of major extracellular matrix components, including 5 types of collagen (type I, III, IV, VI, and VIII), laminin, fibronectin, and heparan sulfate proteoglycan in Descemet's membrane of the cornea of diabetic GK rats. In the cornea of diabetic GK rats, more long-spacing collagen fibrils were observed in Descemet's membrane than in the membrane of the nondiabetic Wistar rats. Both GK and Wistar rats showed an age-dependent increase in the density of the long-spacing collagen. Immunoelectron microscopy showed that type VIII collagen was localized in the internodal region of the long-spacing collagen, which was not labelled by any of the other antibodies used. The antidiabetic agents nateglinide and glibenclamide significantly suppressed the formation of the long-spacing collagen in the diabetic rats. Long-spacing collagen would thus be a useful indicator for studying diabetic changes in the cornea and the effect of antidiabetic agents.


Assuntos
Colágeno/metabolismo , Cicloexanos/uso terapêutico , Lâmina Limitante Posterior/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glibureto/uso terapêutico , Fenilalanina/análogos & derivados , Animais , Colágeno/efeitos dos fármacos , Córnea/efeitos dos fármacos , Córnea/patologia , Córnea/ultraestrutura , Lâmina Limitante Posterior/efeitos dos fármacos , Lâmina Limitante Posterior/ultraestrutura , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Retinopatia Diabética/prevenção & controle , Hipoglicemiantes/uso terapêutico , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Nateglinida , Fenilalanina/uso terapêutico , Ratos , Ratos Endogâmicos , Ratos Wistar , Valores de Referência
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