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1.
Clin Appl Thromb Hemost ; 25: 1076029619853641, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31167567

RESUMO

In nucleated cells, the extrinsic pathway of the programmed cell death (apoptosis) is triggered by interaction of death ligands of the tumor necrosis factor superfamily with the death receptors on external cell surface membrane. In this review, we present evidence that, in contrast to nucleated cells, apoptosis in anucleate platelets can be induced through bypassing the death receptors, using instead specific receptors on the platelet surface mediating platelet activation, aggregation, and blood coagulation. These platelet surface receptors include the protease-activated receptor 1 of thrombin and glycoproteins IIbIIIa and Ibα, receptors of fibrinogen, and von Willebrand factor. The pro-apoptotic BH3 mimetic ABT-737 and calcium ionophore A23187 also trigger platelet apoptosis without using death receptors. These agents induce the intrinsic pathway of platelet apoptosis by direct targeting mitochondrial and extra-mitochondrial apoptotic responses.


Assuntos
Apoptose , Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Coagulação Sanguínea , Plaquetas/citologia , Humanos , Ativação Plaquetária , Agregação Plaquetária , Receptores de Morte Celular/metabolismo
2.
Clin Appl Thromb Hemost ; 24(7): 1009-1013, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29848061

RESUMO

Platelets may selectively execute apoptosis (PL-Apo), activation (PL-Act), and both or no responses when exposed to different chemical agents, shear stresses, and stored under blood banking conditions. Appropriate diagnosis of PL-Apo is an important issue of platelet physiology investigations. However, in diagnosing PL-Apo, there is a risk of a false-negative or false-positive diagnosis. The goal of the current review is to present recommendations that may help to avoid incorrect PL-Apo diagnosis. Analyzing reported studies, we recommend (1) using platelet-rich plasma rather than isolated platelets to minimize artificial stimulation of PL-Apo during platelet isolation, (2) using established optimal conditions for stimulation of PL-Apo and/or PL-Act, (3) using a panel of PL-Apo and PL-Act markers, and (4) appropriate positive and negative controls for quantification of PL-Apo and PL-Act responses.


Assuntos
Plaquetas/metabolismo , Ativação Plaquetária/fisiologia , Apoptose , Reações Falso-Negativas , Reações Falso-Positivas , Humanos
4.
Clin Appl Thromb Hemost ; 23(2): 139-147, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27637909

RESUMO

Availability of universal marker for the diagnosis of platelet apoptosis is an important but currently unresolved goal of platelet physiology investigations. Mitochondrial inner transmembrane potential (▵Ψm) depolarization is frequently used as a marker of apoptosis in nucleated cells and anucleate platelets. Since ▵Ψm depolarization in platelets is also frequently associated with concurrent induction of other apoptotic responses, it may appear that ▵Ψm depolarization is a good universal marker of platelet apoptosis. However, data presented in the current study indicate that this is incorrect. We report here fundamental differences in the effects of potassium ionophore valinomycin and calcium ionophore A23187 on human platelet apoptosis. Although both A23187-triggered and valinomycin-triggered ▵Ψm depolarization are strongly induced, the former is dependent on the opening of mitochondrial permeability transition pore (MPTP) and the latter is MPTP-independent. Furthermore, effects of calcium and potassium ionophores on other apoptotic events are also basically different. A23187 induces caspase-3 activation, proapoptotic Bax and Bak protein expression, phosphatidylserine exposure, and microparticle formation, whereas valinomycin does not induce these apoptotic manifestations. Discovery of targeted ▵Ψm depolarization not associated with apoptosis in valinomycin-treated platelets indicates that this marker should not be used as a single universal marker of platelet apoptosis in unknown experimental and clinical settings as it may lead to a false-positive apoptosis diagnosis.


Assuntos
Apoptose , Plaquetas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/sangue , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Biomarcadores/análise , Plaquetas/efeitos dos fármacos , Calcimicina/farmacologia , Células Cultivadas , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Valinomicina/farmacologia
6.
Thromb Res ; 133(1): 73-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24268298

RESUMO

BACKGROUND: Depolarization of mitochondrial inner transmembrane potential (ΔΨm) is a key biochemical manifestation of the intrinsic apoptosis pathway in anucleate platelets. Little is known, however, about the relationship between ΔΨm depolarization and downstream morphological manifestations of platelet apoptosis, cell shrinkage and microparticle (MP) formation. OBJECTIVES: To elucidate this relationship in human platelets. MATERIALS AND METHODS: Using flow cytometry, we analyzed ΔΨm depolarization, platelet shrinkage and MP formation in platelets treated with BH3-mimetic ABT-737 and calcium ionophore A23187, well-known inducers of intrinsic platelet apoptosis. RESULTS: We found that at optimal treatment conditions (90min, 37°C) both ABT-737 and A23187 induce ΔΨm depolarization in the majority (88-94%) of platelets and strongly increase intracellular free calcium. In contrast, effects of A23187 and ABT-737 on platelet shrinkage and MP formation are quite different. A23187 strongly stimulates cell shrinkage and MP formation, whereas ABT-737 only weakly induces these events (10-20% of the effect seen with A23187, P<0.0001). CONCLUSIONS: These data indicate that a high level of ΔΨm depolarization and intracellular free calcium does not obligatorily ensure strong platelet shrinkage and MP formation. Since ABT-737 efficiently induces clearance of platelets from the circulation, our results suggest that platelet clearance may occur in the absence of the morphological manifestations of apoptosis.


Assuntos
Compostos de Bifenilo/farmacologia , Plaquetas/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Nitrofenóis/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Materiais Biomiméticos/farmacologia , Plaquetas/citologia , Plaquetas/metabolismo , Calcimicina/farmacologia , Cálcio/sangue , Micropartículas Derivadas de Células/metabolismo , Citometria de Fluxo , Humanos , Membranas Mitocondriais/metabolismo , Piperazinas/farmacologia
7.
Br J Haematol ; 163(3): 377-84, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24033315

RESUMO

The cell plasma membrane is tightly coupled with the vital processes of apoptosis and activation. In the current study, we investigated exposure of the apoptosis marker phosphatidylserine (PS) and activation marker P-selectin (CD62) on the plasma membrane of anucleate platelets. We found that, depending on triggering stimuli, the plasma membrane of human platelets may exist in four states with predominant exposure of (i) PS but not CD62 (75·9 ± 2·8% of total cells), (ii) CD62 but not PS (86·2 ± 1·3%), (iii) both PS and CD62 (89·6 ± 1·0%) or (iv) neither PS nor CD62 (87·9-97·5%), when platelets were treated at optimal conditions with pro-apoptotic BH3 mimetic ABT-737, thrombin, calcium ionophore A23187 or control diluents, respectively. The dynamics of PS exposure induced by ABT-737 is a slow temperature-dependent process requiring 90 min treatment at 37°C rather than at room temperature for obtaining high levels of PS exposure. In contrast, thrombin-induced CD62 exposure and A23187-induced PS and CD62 exposure showed fast temperature-independent dynamics. This model of selective and concurrent stimulation of PS and/or CD62 transition to the platelet surface provides an experimental horizon for elucidating the roles of plasma membrane markers of platelet apoptosis and activation in platelet clearance.


Assuntos
Apoptose/fisiologia , Plaquetas/ultraestrutura , Lipídeos de Membrana/sangue , Proteínas de Membrana/sangue , Selectina-P/sangue , Fosfatidilserinas/sangue , Ativação Plaquetária/fisiologia , Biomarcadores , Compostos de Bifenilo/farmacologia , Plaquetas/química , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Calcimicina/farmacologia , Micropartículas Derivadas de Células , Humanos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Modelos Biológicos , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Sulfonamidas/farmacologia , Temperatura , Trombina/farmacologia
8.
Br J Haematol ; 161(2): 245-54, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23398569

RESUMO

Anucleate platelets perform two fundamental processes, activation and apoptosis. We elaborated an approach for selective and concurrent stimulation of platelet apoptosis and/or activation, processes important in haemostasis and platelet clearance. Human platelets were treated with BH3 mimetic ABT-737, thrombin, calcium ionophore A23187 and matched diluents. Apoptosis was determined as mitochondrial inner membrane potential (ΔΨm) depolarization and activation as P-selectin exposure. At optimal treatment conditions (90-180 min, 37°C), ABT-737 predominantly induced apoptosis, when 77-81% platelets undergo only ΔΨm depolarization. The ABT-737 impact on ΔΨm depolarization is strongly time- and temperature-dependent, and much higher at 37°C than at room temperature. In contrast, when platelets were treated with thrombin for 15-90 min at either temperature, activation-only was predominantly (79-85%) induced, whereas A23187 triggers both apoptosis and activation (73-81%) when platelets were treated for 15-60 min at 37°C or 15-90 min at room temperature. These data demonstrate that, depending on the triggering stimulus, platelets predominantly undergo ΔΨm depolarization-only, P-selectin exposure-only, or both responses, indicating that platelet apoptosis and activation are different phenomena driven by different mechanisms. The described model provides a basis for studying differential pharmacological manipulation of platelet apoptosis and activation and their role in haemostasis, thrombosis and platelet clearance.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Plaquetas/metabolismo , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Hemostáticos/farmacologia , Nitrofenóis/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Sulfonamidas/farmacologia , Trombina/farmacologia , Feminino , Temperatura Alta , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Selectina-P/metabolismo , Piperazinas/farmacologia , Fatores de Tempo
9.
Br J Haematol ; 159(5): 565-71, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23025479

RESUMO

Platelet apoptosis and activation have been studied in human platelets treated with BH3-only mimetic ABT-737 and calcium ionophore A23187, agents triggering apoptosis through the intrinsic mitochondrial pathway. Platelet apoptosis was determined as activation of crucial apoptosis-associated caspases, initiator caspase-9 of intrinsic apoptosis pathway, executioner caspase-3 and initiator caspase-8 of extrinsic death receptor pathway, and platelet activation was detected by P-selectin (CD62) exposure on the platelet surface. We found that ABT-737 predominantly induced activation of caspases-9, -3 and -8 rather than CD62 exposure, whereas A23187 induces both caspases activation and CD62 exposure. Caspase-8 activation was stimulated independently of the extrinsic apoptosis pathway via mitochondrial membrane permeabilization and depolarization. These data suggest that (i) caspase-8 activation is triggered in ABT-737- and A23187-treated anucleate platelets through the mitochondria-initiated caspase activation cascade bypassing the death receptors, and (ii) ABT-737-treated platelets are a useful experimental tool for discerning the role of platelet apoptosis in platelet function and survival.


Assuntos
Compostos de Bifenilo/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Caspases/sangue , Nitrofenóis/farmacologia , Receptores de Morte Celular/sangue , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Materiais Biomiméticos/farmacologia , Plaquetas/citologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Isoenzimas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Piperazinas/farmacologia , Ativação Plaquetária/efeitos dos fármacos
10.
J Thromb Thrombolysis ; 33(4): 397-411, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22383127

RESUMO

Apoptosis, or programmed cell death, is a physiological mechanism that serves for controlled deletion of damaged cells. While long attributed exclusively to nucleated cells, over recent years it has been recognized that apoptosis also occurs in anucleate platelets. We describe here experiences of determining markers of apoptosis in human platelets treated in vitro with pro-apoptotic chemical and physical stimuli. These include depolarization of mitochondrial inner membrane, cytochrome c release, expression of pro-apoptotic and anti-apoptotic proteins of Bcl-2 family, activation of apoptosis executioner caspase-3, exposure of phosphatidylserine, platelet shrinkage, fragmentation to microparticles, blebbing and filopod extrusion on the platelet surface. These assays serve to characterize platelet apoptosis in different cellular compartments (mitochondria, cytosol and plasma membrane) and at the whole-cell level. Methods commonly employed in studies of platelet apoptosis markers include flow cytometry, Western blot analysis and electron microscopy. An integrated methodological approach, based on determination of different platelet apoptosis markers, represents a useful tool for examining platelet apoptosis in various physiological and pathological settings.


Assuntos
Apoptose/fisiologia , Plaquetas/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Biomarcadores/metabolismo , Plaquetas/ultraestrutura , Western Blotting/métodos , Tomografia com Microscopia Eletrônica/métodos , Citometria de Fluxo/métodos , Humanos
11.
Diabetes ; 59(2): 448-59, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19903739

RESUMO

OBJECTIVE The inability of pancreatic beta-cells to appropriately respond to glucose and secrete insulin are primary defects associated with beta-cell failure in type 2 diabetes. Mitochondrial dysfunction has been implicated as a key factor in the development of type 2 diabetes; however, a link between mitochondrial dysfunction and defective insulin secretion is unclear. RESEARCH DESIGN AND METHODS We investigated the changes in islet mitochondrial function and morphology during progression from insulin resistance (3 weeks old), immediately before hyperglycemia (5 weeks old), and after diabetes onset (10 weeks old) in transgenic MKR mice compared with controls. The molecular and protein changes at 10 weeks were determined using microarray and iTRAQ proteomic screens. RESULTS At 3 weeks, MKR mice were hyperinsulinemic but normoglycemic and beta-cells showed negligible mitochondrial or morphological changes. At 5 weeks, MKR islets displayed abrogated hyperpolarization of mitochondrial membrane potential (DeltaPsi(m)), reduced mitochondrial Ca(2+) uptake, slightly enlarged mitochondria, and reduced glucose-stimulated insulin secretion. By 10 weeks, MKR mice were hyperglycemic and hyperinsulinemic and beta-cells contained swollen mitochondria with disordered cristae. beta-Cells displayed impaired stimulus-secretion coupling including reduced hyperpolarization of DeltaPsi(m), impaired Ca(2+)-signaling, and reduced glucose-stimulated ATP/ADP and insulin release. Furthermore, decreased cytochrome c oxidase-dependent oxygen consumption and signs of oxidative stress were observed in diabetic islets. Protein profiling of diabetic islets revealed that 36 mitochondrial proteins were differentially expressed, including inner membrane proteins of the electron transport chain. CONCLUSIONS We provide novel evidence for a critical role of defective mitochondrial oxidative phosphorylation and morphology in the pathology of insulin resistance-induced beta-cell failure.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/fisiologia , Mitocôndrias/patologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Glicemia/metabolismo , Cálcio/metabolismo , DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Humanos , Hiperglicemia/complicações , Hiperglicemia/fisiopatologia , Insulina/metabolismo , Resistência à Insulina/fisiologia , Secreção de Insulina , Células Secretoras de Insulina/patologia , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Fosforilação Oxidativa , Estresse Oxidativo/fisiologia , Proteínas/genética , RNA Ribossômico/genética , RNA Ribossômico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
J Biol Chem ; 284(44): 30441-52, 2009 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-19690348

RESUMO

Voltage-gated eag-related gene (Erg) K(+) channels regulate the electrical activity of many cell types. Data regarding Erg channel expression and function in electrically excitable glucagon and insulin producing cells of the pancreas is limited. In the present study Erg1 mRNA and protein were shown to be highly expressed in human and mouse islets and in alpha-TC6 and Min6 cells alpha- and beta-cell lines, respectively. Whole cell patch clamp recordings demonstrated the functional expression of Erg1 in alpha- and beta-cells, with rBeKm1, an Erg1 antagonist, blocking inward tail currents elicited by a double pulse protocol. Additionally, a small interference RNA approach targeting the kcnh2 gene (Erg1) induced a significant decrease of Erg1 inward tail current in Min6 cells. To investigate further the role of Erg channels in mouse and human islets, ratiometric Fura-2 AM Ca(2+)-imaging experiments were performed on isolated alpha- and beta-cells. Blocking Erg channels with rBeKm1 induced a transient cytoplasmic Ca(2+) increase in both alpha- and beta-cells. This resulted in an increased glucose-dependent insulin secretion, but conversely impaired glucagon secretion under low glucose conditions. Together, these data present Erg1 channels as new mediators of alpha- and beta-cell repolarization. However, antagonism of Erg1 has divergent effects in these cells; to augment glucose-dependent insulin secretion and inhibit low glucose stimulated glucagon secretion.


Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Células Secretoras de Glucagon/química , Células Secretoras de Insulina/química , Ilhotas Pancreáticas/citologia , Animais , Cálcio/metabolismo , Glucagon/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Potenciais da Membrana , Camundongos , Técnicas de Patch-Clamp
14.
Diabetes ; 58(9): 2070-83, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19542200

RESUMO

OBJECTIVE: Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS: Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS: ZnT8(-/-) mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8(-/-) islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn(2+) transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS: ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.


Assuntos
Glicemia/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Grânulos Citoplasmáticos/metabolismo , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Exocitose/fisiologia , Feminino , Expressão Gênica/fisiologia , Células HeLa , Homeostase/fisiologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo Genético , Fatores de Risco , Transportador 8 de Zinco
15.
Lab Invest ; 89(4): 374-84, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19238135

RESUMO

The role of the mitochondrial permeability transition pore (MPTP) in apoptosis of nucleated cells is well documented. In contrast, the role of MPTP in apoptosis of anucleated platelets is largely unknown. The aim of this study was to elucidate the contribution of MPTP in the control of different manifestations of platelet apoptosis by analyzing the effect of cyclosporin A (CsA), a potent inhibitor of MPTP formation. Using flow cytometry, we studied the effect of pretreatment of platelets with CsA on apoptotic responses in human platelets stimulated with calcium ionophore A23187. We found that CsA inhibited A23187-stimulated platelet apoptosis, completely preventing (i) depolarization of mitochondrial inner membrane potential (DeltaPsim), (ii) activation of cytosolic apoptosis executioner caspase-3, (iii) platelet shrinkage, and (iv) fragmentation of platelets to microparticles, but (v) only partially (approximately 25%), inhibiting phosphatidylserine (PS) exposure on the platelet surface. This study shows that MPTP formation is upstream of DeltaPsim depolarization, caspase-3 activation, platelet shrinkage and microparticle formation, and stringently controls these apoptotic events in A23187-stimulated platelets but is less involved in PS externalization. These data also indicate that CsA may rescue platelets from apoptosis, preventing caspase-3 activation and inhibiting the terminal cellular manifestations of platelet apoptosis, such as platelet shrinkage and degradation to microparticles. Furthermore, the results suggest a novel potentially useful application of CsA as an inhibitor of platelet demise through apoptosis in thrombocytopenias associated with enhanced platelet apoptosis.


Assuntos
Apoptose/fisiologia , Plaquetas/fisiologia , Ciclosporina/farmacologia , Potencial da Membrana Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Calcimicina/farmacologia , Caspase 3/metabolismo , Ativação Enzimática , Humanos , Técnicas In Vitro , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial
16.
J Biol Chem ; 283(15): 10184-97, 2008 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-18250168

RESUMO

During insulin secretion, pancreatic alpha-cells are exposed to Zn(2+) released from insulin-containing secretory granules. Although maintenance of Zn(2+) homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn(2+)-transporting pathways and the regulation of Zn(2+) in alpha-cells. To examine the effect of Zn(2+) on glucagon secretion and possible mechanisms controlling the intracellular Zn(2+) level ([Zn(2+)](i)), we employed a glucagon-producing cell line (alpha-TC6) and mouse islets where non-beta-cells were identified using islets expressing green fluorescent protein exclusively in beta-cells. In this study, we first confirmed that Zn(2+) treatment resulted in the inhibition of glucagon secretion in alpha-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of K(ATP) channels by Zn(2+). We then determined that Zn(2+) was transported into alpha-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca(2+) channel blocker Gd(3+) partially inhibited Zn(2+) influx in alpha-TC cells, whereas the L-type voltage-gated Ca(2+) channel inhibitor nitrendipine failed to block Zn(2+) accumulation. To investigate Zn(2+) transport further, we profiled alpha-cells for Zn(2+) transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn(2+) transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn(2+)](i), we examined the effects of oxidizing agents on Zn(2+) mobilization within alpha-cells. 2,2'-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn(2+)](i) in alpha-cells. Together these results demonstrate that Zn(2+) inhibits glucagon secretion, and it is transported into alpha-cells in part through Ca(2+) channels. Zn(2+) transporters and the redox state also modulate [Zn(2+)](i).


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Células Secretoras de Glucagon/metabolismo , Zinco/metabolismo , Animais , Proteínas de Transporte de Cátions/antagonistas & inibidores , Linhagem Celular , Gadolínio/farmacologia , Glucagon/metabolismo , Células Secretoras de Glucagon/citologia , Insulina/metabolismo , Secreção de Insulina , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Camundongos , Camundongos Transgênicos , Oxidantes/farmacologia , Vesículas Secretórias/metabolismo
17.
Diabetes ; 56(11): 2722-31, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17682092

RESUMO

OBJECTIVE: Prolonged elevation of glucose can adversely affect beta-cell function. In vitro studies have linked glucose-induced beta-cell dysfunction to oxidative stress; however, whether oxidative stress plays a role in vivo is unclear. Therefore, our objective was to investigate the role of oxidative stress in an in vivo model of glucose-induced beta-cell dysfunction. RESEARCH DESIGN AND METHODS: Wistar rats were infused intravenously with glucose for 48 h to achieve 20 mmol/l hyperglycemia with/without co-infusion of one of the following antioxidants: taurine (2-amino ethanesulfonic acid) (TAU), an aldehyde scavenger; N-acetylcysteine (NAC), a precursor of glutathione; or tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (TPO), a superoxide dismutase mimetic. This was followed by islet isolation or hyperglycemic clamp. RESULTS: A 48-h glucose infusion decreased glucose-stimulated insulin secretion (GSIS) and elevated reactive oxygen species (ROS), total superoxide, and mitochondrial superoxide in freshly isolated islets. TPO prevented the increase in total and mitochondrial superoxide and the beta-cell dysfunction induced by high glucose. However, TAU and NAC, despite completely normalizing H(2)DCF-DA (dihydro-dichlorofluorescein diacetate)-measured ROS, did not prevent the increase in superoxide and the decrease in beta-cell function induced by high glucose. TPO but not TAU also prevented beta-cell dysfunction induced by less extreme hyperglycemia (15 mmol/l) for a longer period of time (96 h). To further investigate whether TPO is effective in vivo, a hyperglycemic clamp was performed. Similar to the findings in isolated islets, prolonged glucose elevation (20 mmol/l for 48 h) decreased beta-cell function as assessed by the disposition index (insulin secretion adjusted for insulin sensitivity), and co-infusion of TPO with glucose completely restored beta-cell function. CONCLUSIONS: These findings implicate superoxide generation in beta-cell dysfunction induced by prolonged hyperglycemia.


Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Superóxidos/metabolismo , Animais , Glucose/administração & dosagem , Hiperglicemia/metabolismo , Infusões Intravenosas , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo
18.
J Endocrinol ; 192(3): 605-14, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17332528

RESUMO

The mammalian hypothalamus comprises an array of phenotypically distinct cell types that interpret peripheral signals of energy status and, in turn, elicits an appropriate response to maintain energy homeostasis. We used a clonal representative hypothalamic cell model expressing proopiomelanocortin (POMC; N-43/5) to study changes in AMP-activated protein kinase (AMPK) activity and glucose responsiveness. We have demonstrated the presence of cellular machinery responsible for glucose sensing in the cell line, including glucokinase, glucose transporters, and appropriate ion channels. ATP-sensitive potassium channels were functional and responded to glucose. The N-43/5 POMC neurons may therefore be an appropriate cell model to study glucose-sensing mechanisms in the hypothalamus. In N-43/5 POMC neurons, increasing glucose concentrations decreased phospho-AMPK activity. As a relevant downstream effect, we found that POMC transcription increased with 2.8 and 16.7 mM glucose. Upon addition of leptin, with either no glucose or with 5 mM glucose, we found that leptin decreased AMPK activity in N-43/5 POMC neurons, but had no significant effect at 25 mM glucose, whereas insulin decreased AMPK activity at only 5 mM glucose. These results demonstrate that individual hypothalamic neuronal cell types, such as the POMC neuron, can have distinct responses to peripheral signals that relay energy status to the brain, and will therefore be activated uniquely to control neuroendocrine function.


Assuntos
Glucose/farmacologia , Hipotálamo/metabolismo , Leptina/farmacologia , Complexos Multienzimáticos/metabolismo , Neurônios/metabolismo , Pró-Opiomelanocortina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Linhagem Celular Tumoral , Células Clonais , Regulação da Expressão Gênica , Humanos , Insulina/farmacologia , Microscopia de Contraste de Fase , Complexos Multienzimáticos/genética , Canais de Potássio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos
19.
Endocrinology ; 147(10): 4655-63, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16857746

RESUMO

In contrast to mouse, rat islet beta-cell membrane potential is reported not to oscillate in response to elevated glucose despite demonstrated oscillations in calcium and insulin secretion. We aim to clarify the electrical activity of rat islet beta-cells and characterize and compare the electrical activity of both alpha- and beta-cells in rat and mouse islets. We recorded electrical activity from alpha- and beta-cells within intact islets from both mouse and rat using the perforated whole-cell patch clamp technique. Fifty-six percent of both mouse and rat beta-cells exhibited an oscillatory response to 11.1 mm glucose. Responses to both 11.1 mm and 2.8 mm glucose were identical in the two species. Rat beta-cells exhibited incremental depolarization in a glucose concentration-dependent manner. We also demonstrated electrical activity in human islets recorded under the same conditions. In both mouse and rat alpha-cells 11 mm glucose caused hyperpolarization of the membrane potential, whereas 2.8 mm glucose produced action potential firing. No species differences were observed in the response of alpha-cells to glucose. This paper is the first to demonstrate and characterize oscillatory membrane potential fluctuations in the presence of elevated glucose in rat islet beta-cells in comparison with mouse. The findings promote the use of rat islets in future electrophysiological studies, enabling consistency between electrophysiological and insulin secretion studies. An inverse response of alpha-cell membrane potential to glucose furthers our understanding of the mechanisms underlying glucose sensitive glucagon secretion.


Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Animais , Cálcio/metabolismo , Separação Celular , Eletrofisiologia , Células Secretoras de Glucagon/efeitos dos fármacos , Humanos , Insulina/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Especificidade da Espécie
20.
J Biol Chem ; 281(14): 9361-72, 2006 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-16407176

RESUMO

In pancreatic beta-cells Zn(2+) is crucial for insulin biosynthesis and exocytosis. Despite this, little is known about mechanisms of Zn(2+) transport into beta-cells or the regulation and compartmentalization of Zn(2+) within this cell type. Evidence suggests that Zn(2+) in part enters neurons and myocytes through specific voltage-gated calcium channels (VGCC). Using a Zn(2+)-selective fluorescent dye with high affinity and quantum yield, FluoZin-3 AM and the plasma membrane potential dye DiBAC(4)(3) we applied fluorescent microscopy techniques for analysis of Zn(2+)-accumulating pathways in mouse islets, dispersed islet cells, and beta-cell lines (MIN6 and beta-TC6f7 cells). Because the stimulation of insulin secretion is associated with cell depolarization, Zn(2+) (5-10 mum) uptake was analyzed under basal (1 mm glucose) and stimulatory (10-20 mm glucose, tolbutamide, tetraethylammonium, and high K(+)) conditions. Under both basal and depolarized states, beta-cells were capable of Zn(2+) uptake, and switching from basal to depolarizing conditions resulted in a marked increase in the rate of Zn(2+) accumulation. Importantly, L-type VGCC (L-VGCC) blockers (verapamil, nitrendipine, and nifedipine) as well as nonspecific inhibitors of Ca(2+) channels, Gd(3+) and La(3+), inhibited Zn(2+) uptake in beta-cells under stimulatory conditions with little or no change in Zn(2+) accumulation under low glucose conditions. To determine the mechanism of VGCC-independent Zn(2+) uptake the expression of a number of ZIP family Zn(2+) transporter mRNAs in islets and beta-cells was investigated. In conclusion, we demonstrate for the first time that, in part, Zn(2+) transport into beta-cells takes place through the L-VGCC. Our investigation demonstrates direct Zn(2+) accumulation in insulin-secreting cells by two pathways and suggests that the rate of Zn(2+) transport across the plasma membrane is dependent upon the metabolic status of the cell.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Proteínas de Transporte de Cátions/biossíntese , Células Secretoras de Insulina/metabolismo , Zinco/farmacocinética , Membrana Celular , Perfilação da Expressão Gênica , Humanos , Insulinoma/patologia , Íons , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Células Tumorais Cultivadas
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