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1.
Diabetes ; 70(2): 423-435, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33154069

RESUMO

The aim for this study was to elucidate how the hypothalamic hunger-inducing hormone acyl-ghrelin (AG), which is also produced in the pancreas, affects ß-cell function, with particular attention to the role of ATP-sensitive K+ (KATP) channels and the exact site of action of the hormone. AG hyperpolarized the membrane potential and decreased cytoplasmic calcium concentration [Ca2+]c and glucose-stimulated insulin secretion (GSIS). These effects were abolished in ß-cells from SUR1-knockout (KO) mice. AG increased KATP current but only in a configuration with intact metabolism. Unacylated ghrelin counteracted the effects of AG. The influence of AG on membrane potential and GSIS could only be averted in the combined presence of a ghrelin receptor (GHSR1a) antagonist and an inverse agonist. The inhibition of GSIS by AG could be prevented by dibutyryl cyclic-cAMP or 3-isobutyl-1-methylxanthine and the somatostatin (SST) receptor 2-5 antagonist H6056. These data indicate that AG indirectly opens KATP channels probably by interference with the cAMP/cAMP-dependent protein kinase pathway, resulting in a decrease of [Ca2+]c and GSIS. The experiments with SUR1-KO ß-cells point to a direct effect of AG on ß-cells and not, as earlier suggested, to an exclusive effect by AG-induced SST release from δ-cells. Nevertheless, SST receptors may be involved in the effect of AG, possibly by heteromerization of AG and SST receptors.


Assuntos
Grelina/análogos & derivados , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Canais KATP/metabolismo , Animais , Cálcio/metabolismo , Grelina/farmacologia , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Camundongos , Receptores de Somatostatina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Somatostatina/metabolismo
2.
Front Endocrinol (Lausanne) ; 11: 545638, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193079

RESUMO

Objective: Congenital hyperinsulinism (CHI) is a rare disease characterized by persistent hypoglycemia as a result of inappropriate insulin secretion, which can lead to irreversible neurological defects in infants. Poor efficacy and strong adverse effects of the current medications impede successful treatment. The aim of the study was to investigate new approaches to silence ß-cells and thus attenuate insulin secretion. Research Design and Methods: In the scope of our research, we tested substances more selective and more potent than the gold standard diazoxide that also interact with neuroendocrine ATP-sensitive K+ (KATP) channels. Additionally, KATP channel-independent targets as Ca2+-activated K+ channels of intermediate conductance (KCa3.1) and L-type Ca2+ channels were investigated. Experiments were performed using human islet cell clusters isolated from tissue of CHI patients (histologically classified as pathological) and islet cell clusters obtained from C57BL/6N (WT) or SUR1 knockout (SUR1-/-) mice. The cytosolic Ca2+ concentration ([Ca2+]c) was used as a parameter for the pathway regulated by electrical activity and was determined by fura-2 fluorescence. The mitochondrial membrane potential (ΔΨ) was determined by rhodamine 123 fluorescence and single channel currents were measured by the patch-clamp technique. Results: The selective KATP channel opener NN414 (5 µM) diminished [Ca2+]c in isolated human CHI islet cell clusters and WT mouse islet cell clusters stimulated with 10 mM glucose. In islet cell clusters lacking functional KATP channels (SUR1-/-) the drug was without effect. VU0071063 (30 µM), another KATP channel opener considered to be selective, lowered [Ca2+]c in human CHI islet cell clusters. The compound was also effective in islet cell clusters from SUR1-/- mice, showing that [Ca2+]c is influenced by additional effects besides KATP channels. Contrasting to NN414, the drug depolarized ΔΨ in murine islet cell clusters pointing to severe interference with mitochondrial metabolism. An opener of KCa3.1 channels, DCEBIO (100 µM), significantly decreased [Ca2+]c in SUR1-/- and human CHI islet cell clusters. To target L-type Ca2+ channels we tested two already approved drugs, dextromethorphan (DXM) and simvastatin. DXM (100 µM) efficiently diminished [Ca2+]c in stimulated human CHI islet cell clusters as well as in stimulated SUR1-/- islet cell clusters. Similar effects on [Ca2+]c were observed in experiments with simvastatin (7.2 µM). Conclusions: NN414 seems to provide a good alternative to the currently used KATP channel opener diazoxide. Targeting KCa3.1 channels by channel openers or L-type Ca2+ channels by DXM or simvastatin might be valuable approaches for treatment of CHI caused by mutations of KATP channels not sensitive to KATP channel openers.


Assuntos
Hiperinsulinismo Congênito/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/administração & dosagem , Células Cultivadas , Hiperinsulinismo Congênito/metabolismo , Óxidos S-Cíclicos/administração & dosagem , Dextrometorfano/administração & dosagem , Diazóxido , Humanos , Secreção de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Canais KATP/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nifedipino/administração & dosagem
3.
Endocrine ; 68(3): 526-535, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32146655

RESUMO

Novel agonists of the nuclear liver-X-receptor (LXR) are designed to treat metabolic disorders or cancer. The rationale to develop these new drugs is based on promising results with established LXR agonist like T0901317 and GW3965. LXRα and LXRß are expressed in ß-cells, and expression is increased by T0901317. The aim of the present study was to evaluate whether effects of these drugs on ß-cell function are specific and reliably linked to LXR activation. T0901317 and GW3965, widely used as specific LXR agonists, show rapid, non-genomic effects on stimulus-secretion coupling of mouse pancreatic ß-cells at low µM concentrations. T0901317 lowered the cytosolic Ca2+ concentration, reduced or completely inhibited action potentials, and decreased insulin secretion. GW3965 exerted similar effects on insulin secretion. T0901317 affected the production of reactive oxygen species and ATP. The involvement of the classical nuclear LXRs in T0901317- and GW3965-mediated effects in ß-cells could be ruled out using LXRα, LXRß and double knockout mice. Our results strongly suggest that LXR agonists, that are considered to be specific for this receptor, interfere with mitochondrial metabolism and metabolism-independent processes in ß-cells. Thus, it is indispensable to test novel LXR agonists accompanying to ongoing clinical trials for acute and chronic effects on cell function in cellular systems and/or animal models lacking classical LXRs.


Assuntos
Células Secretoras de Insulina , Receptores Nucleares Órfãos , Animais , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Receptores X do Fígado , Camundongos , Camundongos Knockout
4.
Mol Pharmacol ; 97(3): 202-211, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31911428

RESUMO

Bile acids such as chenodeoxycholic acid (CDC) acutely enhance insulin secretion via the farnesoid X receptor (FXR). Statins, which are frequently prescribed for patients with type 2 diabetes who suffer from dyslipidemia, are known for their diabetogenic risk and are reported to interact with the FXR. Our study investigates whether this interaction is relevant for beta cell signaling and plays a role for negative effects of statins on glycemic control. Experiments were performed with islets and islet cells from C57BL/6N wild-type and FXR-knockout (KO) mice. Culturing islets with atorvastatin (15 µM) for 24 hours decreased glucose-stimulated insulin secretion by approximately 30% without affecting ATP synthesis. Prolonged exposure for 7 days lowered the concentration necessary for impairment of insulin release to 150 nM. After 24-hour culture with atorvastatin, the ability of CDC (500 nM) to elevate [Ca2+]c was diminished and the potentiating effect on insulin secretion was completely lost. Mevalonate largely reduced the negative effect of atorvastatin. Nuclear activity of FXR was reduced by atorvastatin in a mouse FXR reporter assay. The atorvastatin-induced decrease in insulin release was also present in FXR-KO mice. Although not a prerequisite, FXR seems to influence the degree of damage caused by atorvastatin depending on its interaction with CDC: Preparations responding to CDC with an increase in insulin secretion under control conditions were less impaired by atorvastatin than preparations that were nonresponsive to CDC. Extended stimulation of FXR by the synthetic agonist GW4064, which is suggested to induce translocation of FXR from the cytosol into the nucleus, increased the inhibitory effect of atorvastatin. In conclusion, atorvastatin inhibits insulin release and prevents positive effects of bile acids on beta cell function. Both interactions may contribute to progression of type 2 diabetes mellitus. SIGNIFICANCE STATEMENT: This study shows that the diabetogenic risk of statins is coupled to the activity of farnesoid X receptor (FXR)-dependent signaling pathways in beta cells. On the one hand, statins abolish the insulinotropic effects of bile acids and on the other hand, FXR determines the level of impairment of islet function by the statin.


Assuntos
Atorvastatina/metabolismo , Ácidos e Sais Biliares/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Células Secretoras de Insulina/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Atorvastatina/efeitos adversos , Ácidos e Sais Biliares/antagonistas & inibidores , Células Cultivadas , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/prevenção & controle , Relação Dose-Resposta a Droga , Feminino , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Risco
5.
Theranostics ; 10(1): 398-410, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31903128

RESUMO

Non-invasive imaging of ß-cells represents a desirable preclinical and clinical tool to monitor the change of ß-cell mass and the loss of function during pre-diabetic stages. Although it is widely accepted that manganese (Mn) ions are actively gated by voltage-dependent calcium channels (VDCC) in response to glucose metabolism, little is known on its specificity in vivo for quantification of islet ß-cell function using Mn and magnetic resonance imaging (MRI). On the other hand, glucagon-like-peptide-1 receptor (GLP-1R) represents a validated target for the estimation of ß-cell mass using radiolabeled exendin-4 (Ex4) and positron emission tomography (PET). However, a multiparametric imaging workflow revealing ß-cell mass and function quantitatively is still missing. Methods: We developed a simultaneous PET/MRI protocol to comprehensively quantify in vivo changes in ß-cell mass and function by targeting, respectively, GLP-1R and VDCC coupled with insulin secretion. Differences in the spatial distribution of Mn and radiolabeled Ex4 were monitored overtime in native and transgenic pancreata, characterized by spontaneous pancreatic neuroendocrine tumor development. Follow-up with mass spectrometry imaging (MSI) and autoradiography allowed the ex vivo validation of the specificity of Mn and PET tracer uptake and the detection of endogenous biometals, such as calcium and zinc, throughout the endocrine and exocrine pancreas. Results: Our in vivo data based on a volumetric PET/MRI readout for native pancreata and insulinomas connects uptake of Mn measured at early imaging time points to high non-specific binding by the exocrine tissue, while specific retention was only found 24 h post injection. These results are supported by cross-validation of the spatial distribution of exogenous 55Mn and endogenous 44Ca and 64Zn as well with the specific internalization of the radiolabeled peptide targeting GLP-1R. Conclusion: Simultaneous PET/MR imaging of the pancreas enabled the comprehensive in vivo quantification of ß-cell function and mass using Mn and radiolabeled Ex4. Most important, our data revealed that only late time-point measurements reflect the Mn uptake in the islet ß-cells, while early time points detect non-specific accumulation of Mn in the exocrine pancreas.


Assuntos
Células Secretoras de Insulina , Imageamento por Ressonância Magnética , Pâncreas , Neoplasias Pancreáticas/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Animais , Canais de Cálcio/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/ultraestrutura , Manganês/química , Camundongos , Camundongos Transgênicos , Pâncreas/diagnóstico por imagem , Pâncreas/patologia , Compostos Radiofarmacêuticos/química
6.
Exp Clin Endocrinol Diabetes ; 128(10): 644-653, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30986881

RESUMO

Glucose-stimulated insulin secretion (GSIS) is the gold standard for ß-cell function. Both experimental and clinical diabetology, i. e., preceding transplantation of isolated human islets, depend on functional testing. However, multiple factors influence GSIS rendering the comparison of different in vitro tests of glucose responsiveness difficult. This study examined the influence of bovine serum albumin (BSA)-coupled fatty acids on GSIS. Isolated islet preparations of human donors and of 12-months old mice displayed impaired GSIS in the presence of 0.5% FFA-free BSA compared to 0.5% BSA (fraction V, not deprived from fatty acids). In aged INS-1E cells, i. e. at a high passage number, GSIS became highly sensitive to FFA-free BSA. Readdition of 30 µM palmitate or 30 µM oleate to FFA-free BSA did not rescue GSIS, while the addition of 100 µM palmitate and the raise of extracellular Ca2+from 1.3 to 2.6 mM improved glucose responsiveness. A high concentration of palmitate (600 µM), which fully activates FFA1, largely restored insulin secretion. The FFA1-agonist TUG-469 also increased insulin secretion but to a lesser extent than palmitate. Glucose- and TUG-induced Ca2+oscillations were impaired in glucose-unresponsive, i. e., aged INS-1E cells. These results suggest that fatty acid deprivation (FFA-free BSA) impairs GSIS mainly through an effect on Ca2+sensitivity.


Assuntos
Ácidos Graxos não Esterificados/metabolismo , Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Insulinoma , Compostos de Anilina/farmacologia , Animais , Cálcio/metabolismo , Bovinos , Linhagem Celular Tumoral , Humanos , Camundongos , Palmitatos/farmacologia , Fenilpropionatos/farmacologia
7.
Endocrine ; 63(2): 270-283, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30229397

RESUMO

PURPOSE: The role of ATP, which is secreted by pancreatic ß-cells, is still a matter of debate. It has been postulated that extracellular ATP acts as a positive auto- or paracrine signal in ß-cells amplifying insulin secretion. However, there is rising evidence that extracellular ATP may also mediate a negative signal. METHODS: We evaluated whether extracellular ATP interferes with the Ca2+-mediated negative feedback mechanism that regulates oscillatory activity of ß-cells. RESULTS: To experimentally uncover the Ca2+-induced feedback we applied a high extracellular Ca2+ concentration. Under this condition ATP (100 µM) inhibited glucose-evoked oscillations of electrical activity and hyperpolarized the membrane potential. Furthermore, ATP acutely increased the interburst phase of Ca2+ oscillations and reduced the current through L-type Ca2+ channels. Accordingly, ATP (500 µM) decreased glucose-induced insulin secretion. The ATP effect was not mimicked by AMP, ADP, or adenosine. The use of specific agonists and antagonists and mice deficient of large conductance Ca2+-dependent K+ channels revealed that P2X, but not P2Y receptors, and Ca2+-dependent K+ channels are involved in the underlying signaling cascade induced by ATP. The effectiveness of ATP to interfere with parameters of stimulus-secretion coupling is markedly reduced at low extracellular Ca2+ concentration. CONCLUSION: It is suggested that extracellular ATP which is co-secreted with insulin in a pulsatile manner during glucose-stimulated exocytosis provides a negative feedback signal driving ß-cell oscillations in co-operation with Ca2+ and other signals.


Assuntos
Trifosfato de Adenosina/farmacologia , Comunicação Autócrina/efeitos dos fármacos , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Insulina/farmacologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
8.
Diabetes ; 68(2): 324-336, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30409782

RESUMO

The Takeda-G-protein-receptor-5 (TGR5) mediates physiological actions of bile acids. Since it was shown that TGR5 is expressed in pancreatic tissue, a direct TGR5 activation in ß-cells is currently postulated and discussed. The current study reveals that oleanolic acid (OLA) affects murine ß-cell function by TGR5 activation. Both a Gαs inhibitor and an inhibitor of adenylyl cyclase (AC) prevented stimulating effects of OLA. Accordingly, OLA augmented the intracellular cAMP concentration. OLA and two well-established TGR5 agonists, RG239 and tauroursodeoxycholic acid (TUDCA), acutely promoted stimulus-secretion coupling (SSC). OLA reduced KATP current and elevated current through Ca2+ channels. Accordingly, in mouse and human ß-cells, TGR5 ligands increased the cytosolic Ca2+ concentration by stimulating Ca2+ influx. Higher OLA concentrations evoked a dual reaction, probably due to activation of a counterregulating pathway. Protein kinase A (PKA) was identified as a downstream target of TGR5 activation. In contrast, inhibition of phospholipase C and phosphoinositide 3-kinase did not prevent stimulating effects of OLA. Involvement of exchange protein directly activated by cAMP 2 (Epac2) or farnesoid X receptor (FXR2) was ruled out by experiments with knockout mice. The proposed pathway was not influenced by local glucagon-like peptide 1 (GLP-1) secretion from α-cells, shown by experiments with MIN6 cells, and a GLP-1 receptor antagonist. In summary, these data clearly demonstrate that activation of TGR5 in ß-cells stimulates insulin secretion via an AC/cAMP/PKA-dependent pathway, which is supposed to interfere with SSC by affecting KATP and Ca2+ currents and thus membrane potential.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Secretoras de Insulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Feminino , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Ácido Oleanólico/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ácido Tauroquenodesoxicólico/farmacologia
9.
J Biol Chem ; 294(10): 3707-3719, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30587573

RESUMO

Neuroendocrine-type ATP-sensitive K+ (KATP) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2)4, comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K+-selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward- or outward-facing conformations with low or high dissociation constant, KD , values for the conformation-sensitive channel antagonist [3H]glibenclamide ([3H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's KD for [3H]GBM. This reduction required the Kir N terminus (KNtp), consistent with KNtp occupying a "transport cavity," thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower KD for [3H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a "swapped" helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases KATP channels toward open states, consistent with SUR1 variants with lower KD values causing neonatal diabetes, whereas increased KD values cause congenital hyperinsulinism.


Assuntos
Trifosfato de Adenosina/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Sulfonilureias/química , Receptores de Sulfonilureias/metabolismo , Difosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Cricetinae , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Hidrólise , Ativação do Canal Iônico , Modelos Moleculares , Mutação , Canais de Potássio Corretores do Fluxo de Internalização/química , Ligação Proteica , Conformação Proteica em alfa-Hélice
10.
Pflugers Arch ; 470(3): 537-547, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29218453

RESUMO

We have shown previously that genetic or pharmacological deletion of KATP channels protect against beta cell dysfunction induced by reactive oxygen species (ROS). Since it is assumed that glucolipotoxicity (GLTx) causes ROS production, we aimed to evaluate whether suppression of KATP channel activity can also prevent beta cell damage evoked by GLTx. We used an in vitro model of GLTx and measured distinct parameters of stimulus-secretion coupling. GLTx gradually induced disturbances of Ca2+ oscillations over 3 days. This impairment in Ca2+ dynamics was partially reversed in beta cells without functional KATP channels (SUR1-/-) and by the sulfonylurea gliclazide but not by tolbutamide. By contrast, the GLTx-induced suppression of glucose-induced insulin secretion could not be rescued by decreased KATP channel activity pointing to a direct interaction of GLTx with the secretory capacity. Accordingly, GLTx also suppressed KCl-induced insulin secretion. GLTx was not accompanied by decisively increased ROS production or enhanced apoptosis. Insulin content of beta cells was markedly reduced by GLTx, an effect not prevented by gliclazide. Since GLTx markedly diminished the mitochondrial membrane potential and cellular ATP content, lack of ATP is assumed to decrease insulin biosynthesis. The deleterious effect of GLTx is therefore caused by direct interference with the secretory capacity whereby reduction of insulin content is one important parameter. These findings deepen our understanding how GLTx damages beta cells and reveal that GLTx is disconnected from ROS formation, a notion important for targeting beta cells in the treatment of diabetes. Overall, GLTx-induced energy depletion may be a primary step in the cascade of events leading to loss of beta cell function in type-2 diabetes mellitus.


Assuntos
Apoptose , Metabolismo Energético , Células Secretoras de Insulina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Gliclazida/farmacologia , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Canais KATP/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Tolbutamida/farmacologia
11.
Diabetes ; 66(11): 2840-2848, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28864549

RESUMO

Atrial natriuretic peptide (ANP) influences glucose homeostasis and possibly acts as a link between the cardiovascular system and metabolism, especially in metabolic disorders like diabetes. The current study evaluated effects of ANP on ß-cell function by the use of a ß-cell-specific knockout of the ANP receptor with guanylate cyclase activity (ßGC-A-KO). ANP augmented insulin secretion at the threshold glucose concentration of 6 mmol/L and decreased KATP single-channel activity in ß-cells of control mice but not of ßGC-A-KO mice. In wild-type ß-cells but not ß-cells lacking functional KATP channels (SUR1-KO), ANP increased electrical activity, suggesting no involvement of other ion channels. At 6 mmol/L glucose, ANP readily elicited Ca2+ influx in control ß-cells. This effect was blunted in ß-cells of ßGC-A-KO mice, and the maximal cytosolic Ca2+ concentration was lower. Experiments with inhibitors of protein kinase G (PKG), protein kinase A (PKA), phosphodiesterase 3B (PDE3B), and a membrane-permeable cyclic guanosine monophosphate (cGMP) analog on KATP channel activity and insulin secretion point to participation of the cGMP/PKG and cAMP/PKA/Epac (exchange protein directly activated by cAMP) directly activated by cAMP Epac pathways in the effects of ANP on ß-cell function; the latter seems to prevail. Moreover, ANP potentiated the effect of glucagon-like peptide 1 (GLP-1) on glucose-induced insulin secretion, which could be caused by a cGMP-mediated inhibition of PDE3B, which in turn reduces cAMP degradation.


Assuntos
Fator Natriurético Atrial/farmacologia , Glucose/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/genética , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Tolbutamida
12.
Endocrinology ; 158(7): 2145-2154, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28449117

RESUMO

The role of liver X receptor (LXR) in pancreatic ß-cell physiology and pathophysiology is still unclear. It has been postulated that chronic LXR activation in ß-cells induces lipotoxicity, a key step in the development of ß-cell dysfunction, which accompanies type 2 diabetes mellitus. In most of these studies, the LXR ligand T0901317 has been administered chronically in the micromolar range to study the significance of LXR activation. In the current study, we have evaluated acute effects of T0901317 on stimulus-secretion coupling of ß-cells. We found that 10 µM T0901317 completely suppressed oscillations of the cytosolic Ca2+ concentration induced by 15 mM glucose. Obviously, this effect was due to inhibition of mitochondrial metabolism. T0901317 markedly depolarized the mitochondrial membrane potential, thus inhibiting adenosine triphosphate (ATP) production and reducing the cytosolic ATP concentration. This led in turn to a huge increase in KATP current and hyperpolarization of the cell membrane potential. Eventually, T0901317 inhibited glucose-induced insulin secretion. These effects were rapid in on-set and not compatible with the activation of a nuclear receptor. In vivo, T0901317 acutely increased the blood glucose concentration after intraperitoneal application. In summary, these data clearly demonstrate that T0901317 exerts acute effects on stimulus-secretion coupling. This observation questions the chronic use of T0901317 and limits the interpretation of results obtained under these experimental conditions.


Assuntos
Hidrocarbonetos Fluorados/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Receptores X do Fígado/agonistas , Mitocôndrias/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Feminino , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Ligantes , Receptores X do Fígado/genética , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
13.
Endocrinology ; 157(12): 4677-4690, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27715254

RESUMO

The angiotensin-converting enzyme 2/angiotensin (Ang)-(1-7)/Mas axis of the renin-angiotensin system often opposes the detrimental effects of the angiotensin-converting enzyme/Ang II/Ang II type 1 receptor axis and has been associated with beneficial effects on glucose homeostasis, whereas underlying mechanisms are mostly unknown. Here we investigate the effects of Ang-(1-7) and its receptor Mas on ß-cell function. Isolated islets from Mas-deficient and wild-type mice were stimulated with Ang-(1-7) or its antagonists and effects on insulin secretion determined. Islets' cytoplasmic calcium and cAMP concentrations, mRNA amounts of Ins1, Ins2, Pdx1, and Mafa and effects of inhibitors of cAMP downstream signaling were determined. Ang-(1-7) was also applied to mice by osmotic pumps for 14 days and effects on glucose tolerance and insulin secretion were assessed. Ang-(1-7) increased insulin secretion from wild-type islets, whereas antagonists and genetic Mas deficiency led to reduced insulin secretion. The Mas-dependent effects of Ang-(1-7) on insulin secretion did not result from changes in insulin gene expression or changes in the excitation-secretion coupling but from increased intracellular cAMP involving exchange protein activated directly by cAMP. Administration of Ang-(1-7) in vivo had only marginal effects on glucose tolerance in wild-type mice but still resulted in improved insulin secretion from islets isolated of these mice. Interestingly, although less pronounced than in wild types, Ang-(1-7) still affected insulin secretion in Mas-deficient islets. The data indicate a significant function of Ang-(1-7) in the regulation of insulin secretion from mouse islets in vitro and in vivo, mainly, but not exclusively, by Mas-dependent signaling, modulating the accessory pathway of insulin secretion via increase in cAMP.


Assuntos
Angiotensina I/farmacologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , AMP Cíclico/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/genética , Resistência à Insulina/fisiologia , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Camundongos Knockout , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Receptores Acoplados a Proteínas G/genética , Transativadores/genética , Transativadores/metabolismo
14.
Pflugers Arch ; 467(11): 2389-97, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25893711

RESUMO

Pancreatic beta-cells respond to an unchanging stimulatory glucose concentration with oscillations in membrane potential (Vm), cytosolic Ca(2+) concentration ([Ca(2+)]c), and insulin secretion. The underlying mechanisms are largely ascertained. Some particular details, however, are still in debate. Stimulus-secretion coupling (SSC) of beta-cells comprises glucose-induced Ca(2+) influx into the cytosol and thus into mitochondria. It is suggested that this activates (mitochondrial) dehydrogenases leading to an increase in reduction equivalents and ATP production. According to SSC, a glucose-induced increase in ATP production would thus further augment ATP production, i.e. induce a feed-forward loop that is hardly compatible with oscillations. Consistently, other studies favour a feedback mechanism that drives oscillatory mitochondrial ATP production. If Ca(2+) influx activates dehydrogenases, a change in [Ca(2+)]c should increase the concentration of reduction equivalents. We measured changes in flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) autofluorescence in response to changes in glucose concentration or glucose-independent changes in [Ca(2+)]c. The FAD signal was altered by glucose but not by alterations in [Ca(2+)]c. NAD(P)H was increased by glucose but even decreased by Ca(2+) influx evoked by tolbutamide. The mitochondrial membrane potential ΔΨ was hyperpolarized by 4 mM glucose. As adding tolbutamide then depolarized ΔΨ, we deduce that Ca(2+) does not activate mitochondrial activity but by contrast even inhibits it by reducing the driving force for ATP production. Inhibition of Ca(2+) influx reversed the Ca(2+)-induced changes in ΔΨ and NAD(P)H. The results are consistent with a feedback mechanism which transiently and repeatedly reduces ATP production and explain the oscillatory activity of pancreatic beta-cells at increased glucose concentrations.


Assuntos
Cálcio/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/enzimologia , Oxirredutases/metabolismo , Animais , FMN Redutase/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Glucose/farmacologia , Hipoglicemiantes/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Tolbutamida/farmacologia
15.
Diabetologia ; 58(7): 1532-41, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25874444

RESUMO

AIMS/HYPOTHESIS: Generation of reduction equivalents is a prerequisite for nutrient-stimulated insulin secretion. Mitochondrial succinate dehydrogenase (SDH) fulfils a dual function with respect to mitochondrial energy supply: (1) the enzyme is part of mitochondrial respiratory chains; and (2) it catalyses oxidation of succinate to fumarate in the Krebs cycle. The aim of our study was to elucidate the significance of SDH for beta cell stimulus-secretion coupling (SSC). METHODS: Mitochondrial variables, reactive oxygen species (ROS) and cytosolic Ca(2+) concentration ([Ca(2+)]c) were measured by fluorescence techniques and insulin release by radioimmunoassay in islets or islet cells of C57Bl/6N mice. RESULTS: Inhibition of SDH with 3-nitropropionic acid (3-NPA) or monoethyl fumarate (MEF) reduced glucose-stimulated insulin secretion. Inhibition of the ATP-sensitive K(+) channel (KATP channel) partly prevented this effect, whereas potentiation of antioxidant defence by superoxide dismutase mimetics (TEMPOL and mito-TEMPO) or by nuclear factor erythroid 2-related factor 2 (Nrf-2)-mediated upregulation of antioxidant enzymes (oltipraz, tert-butylhydroxyquinone) did not diminish the inhibitory influence of 3-NPA. Blocking SDH decreased glucose-stimulated increase in intracellular FADH2 concentration without alterations in NAD(P)H. In addition, 3-NPA and MEF drastically reduced glucose-induced hyperpolarisation of mitochondrial membrane potential, indicative of decreased ATP production. As a consequence, the glucose-stimulated rise in [Ca(2+)]c was significantly delayed and reduced. Acute application of 3-NPA interrupted glucose-driven oscillations of [Ca(2+)]c. 3-NPA per se did not elevate intracellular ROS, but instead prevented glucose-induced ROS accumulation. CONCLUSIONS/INTERPRETATION: SDH is an important regulator of insulin secretion and ROS production. Inhibition of SDH interrupts membrane-potential-dependent SSC, pointing to a pivotal role of mitochondrial FAD/FADH2 homeostasis for the maintenance of glycaemic control.


Assuntos
Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Mitocôndrias/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/metabolismo , Animais , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Flavina-Adenina Dinucleotídeo/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Canais KATP/efeitos dos fármacos , Canais KATP/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Nitrocompostos/farmacologia , Propionatos/farmacologia , Succinato Desidrogenase/antagonistas & inibidores , Compostos de Sulfonilureia/farmacologia
16.
Endocrinology ; 156(4): 1263-71, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25599407

RESUMO

We have recently shown that the bile acid (BA) taurochenodeoxycholate (TCDC) acutely stimulates insulin secretion via activation of the farnesoid X receptor (FXR). Aims of the current investigation were to discriminate between nongenomic (≤1 h) and genomic effects (24-48 h) of BAs on ß-cells and to evaluate whether FXR can modulate the adverse effects of a high-fat diet (HFD). TCDC (500 nM) as well as glycine-conjugated and unconjugated CDC (chenodeoxycholate) increased insulin secretion in acute incubations but did not evoke additional effects after 1-2 days of preincubation. The BAs did not stimulate ß-cells of FXR-knockout (KO) mice and activation of the G protein-coupled BA receptor TGR5 was ineffective, suggesting that FXR is the sole BA receptor in ß-cells activated by TCDC and its analogues. As opposed to lean mice, obese FXR-KO mice did not show HFD-induced glucose intolerance and increased fasting glucose. The beneficial impact of FXR-KO on glucose metabolism cannot be explained by an adaptive compensation of insulin secretion or ß-cell mass. Interestingly, in contrast to its effect on islets from lean mice, the FXR agonist GW4064 was ineffective in stimulating insulin secretion of islets from wild type mice fed a HFD or isolated islets kept in a glucolipotoxic medium. Additional feeding of CDC restored the effect of GW4064. CDC prevented HFD-induced impairment of glucose tolerance and in vitro effects of glucolipotoxicity. The data show that the FXR is the most important BA receptor in ß-cells and that FXR signaling in ß-cells is impaired by overnutrition, which alters activatability of the FXR.


Assuntos
Células Secretoras de Insulina/metabolismo , Obesidade/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Ácido Quenodesoxicólico/farmacologia , Feminino , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Isoxazóis/farmacologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Obesidade/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética , Ácido Tauroquenodesoxicólico/farmacologia
17.
Integr Biol (Camb) ; 6(5): 540-4, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24658612

RESUMO

Extracellular recording of the glucose-induced electrical activity of mouse islets of Langerhans on microelectrode arrays (MEAs) is an innovative and powerful tool to address beta-cell (patho-)physiology. In a dual approach we tested whether this technique can detect concentration-dependent drug effects as well as characterize alterations in beta-cell activity during prolonged culture. First we established conditions that allow long-term investigation of beta-cell function by recording electrical activity. The results provide the first measurements of beta-cell membrane potential oscillations of individual murine islets during long-term culture. Oscillations were recorded for up to 34 days after islet isolation. Importantly, the glucose dependence of electrical activity did not change over a period of one month. Thus we can follow electrophysiological changes of individual islets induced by alterations in the beta-cell environment over weeks. Second, we used the MEA technique to assay beta-cell damage induced by oxidative stress and to evaluate appropriate protection mechanisms. Oxidative stress plays a key role in the development of type 2 diabetes mellitus (T2DM). Examination of the acute effects of H2O2 on electrical activity showed that the oxidant reduced the electrical activity in a concentration-dependent manner. The superoxide dismutase mimetic, tempol, protected against the detrimental effects of H2O2. In conclusion, we demonstrated that MEA recordings can be used to address disease-related mechanisms and protective interventions in beta-cells. In the future, this fundamental work should enable the monitoring of the electrical activity of islets of Langerhans under controlled ex vivo conditions including long-term exposure to oxidative stress, glucolipotoxicity, and other diabetes-inducing agents.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Glucose/farmacologia , Ilhotas Pancreáticas/metabolismo , Potenciais da Membrana/fisiologia , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/farmacologia , Óxidos N-Cíclicos/farmacologia , Relação Dose-Resposta a Droga , Eletrofisiologia/métodos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Camundongos Endogâmicos C57BL , Microeletrodos , Marcadores de Spin
18.
Mol Pharmacol ; 83(1): 51-60, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23024191

RESUMO

Earlier studies suggest that glitazones exert beneficial effects in patients with type 2 diabetes by directly affecting insulin secretion of ß-cells, besides improving the effectiveness of insulin in peripheral tissues. The effects of glitazones on stimulus-secretion coupling (SSC) are poorly understood. We tested the influence of troglitazone and pioglitazone on different parameters of SSC, including insulin secretion (radioimmunoassay), cell membrane potential, various ion currents (patch-clamp), mitochondrial membrane potential (ΔΨ), and cytosolic Ca(2+) concentration (fluorescence). Troglitazone exerted stimulatory, inhibitory, or no effects on insulin secretion depending on the drug and glucose concentration. It depolarized the ΔΨ, thus lowering ATP production, which resulted in opening of ATP-dependent K(+) channels (K(ATP) channels) and reduced insulin secretion. However, it also exerted direct inhibitory effects on K(ATP) channels that can explain enhanced insulin secretion. Troglitazone also inhibited the currents through voltage-dependent Ca(2+) and K(+) channels. Pioglitazone was less effective than troglitazone on all parameters tested. The effects of both glitazones were markedly reduced in the presence of bovine serum albumin. Glitazones exert multiple actions on ß-cell SSC that have to be considered as undesired side effects because the influence of these compounds on ß-cells is not controllable. The final effect on insulin secretion depends on many parameters, including the actual glucose and drug concentration, protein binding of the drug, and the drug by itself. Troglitazone and pioglitazone differ in their influence on SSC. It can be assumed that the effects of pioglitazone on ß-cells are negligible under in vivo conditions.


Assuntos
Cromanos/farmacologia , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Tiazolidinedionas/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/fisiologia , Bovinos , Células Cultivadas , Citosol/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Canais KATP/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Pioglitazona , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Radioimunoensaio , Soroalbumina Bovina/farmacologia , Troglitazona
19.
Islets ; 4(5): 333-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23073079

RESUMO

Bile acids (BAs) are important signaling molecules that are involved in the regulation of their own metabolism, lipid metabolism, energy expenditure and glucose homeostasis. The nuclear farnesoid X receptor (FXR) and the G-protein-coupled TGR-5 are the most prominent BA receptors. FXR is highly expressed in liver and activation of liver FXR profoundly affects glucose homeostasis. Strikingly, the effect of FXR activation on glucose metabolism seems to depend on the nutritional status of the organism, i.e., slimness or obesity. Recently, it became evident that FXR is present in pancreatic ß cells and that activation of ß cell FXR contributes to the regulation of glucose homeostasis. Interestingly, FXR activation increases glucose-induced insulin secretion by non-genomic effects on stimulus-secretion coupling. The first chapter of this review shortly introduces the role of liver FXR in glucose metabolism, the second part focuses on the impact of FXR in lean and obese animals, and the third chapter highlights the significance of FXR in ß cells.


Assuntos
Células Secretoras de Insulina/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Glicemia/análise , Humanos , Obesidade/metabolismo
20.
Free Radic Biol Med ; 53(8): 1574-83, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22922339

RESUMO

The cGMP-dependent protein kinase I (cGKI) is a key mediator of cGMP signaling, but the specific functions of its two isoforms, cGKIα and cGKIß, are poorly understood. Recent studies indicated a novel cGMP-independent role for cGKIα in redox sensing. To dissect the effects of oxidative stress on the cGKI isoforms, we used mouse embryonic fibroblasts and vascular smooth muscle cells (VSMCs) expressing both, one, or none of them. In cGKIα-expressing cells, but not in cells expressing only cGKIß, incubation with H2O2 induced the formation of a disulfide bond between the two identical subunits of the dimeric enzyme. Oxidation of cGKIα was associated with increased phosphorylation of its substrate, vasodilator-stimulated phosphoprotein. H2O2 did not stimulate cGMP production, indicating that it activates cGKIα directly via oxidation. Interestingly, there was a mutual influence of H2O2 and cGMP on cGKI activity and disulfide bond formation, respectively; preoxidation of the kinase with H2O2 slightly impaired its activation by cGMP, whereas preactivation of the enzyme with cGMP attenuated its oxidation by H2O2. To evaluate the functional relevance of the noncanonical H2O2-cGKIα pathway, we studied the regulation of the cytosolic Ca²âº concentration ([Ca²âº](i)). H2O2 suppressed norepinephrine-induced Ca²âº transients in cGKIα-expressing VSMCs and, to a lower extent, in VSMCs expressing only cGKIß or none of the isoforms. Thus, H2O2 lowers [Ca²âº](i) mainly via a cGKIα-dependent pathway. These results indicate that oxidative stress selectively targets the cGKIα isoform, which then modulates cellular processes in a cGMP-independent manner. A decrease in [Ca²âº](i) in VSMCs via activation of cGKIα might be a major mechanism of H2O2-induced vasodilation.


Assuntos
Cálcio/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Citosol/metabolismo , Embrião de Mamíferos/enzimologia , Fibroblastos/enzimologia , Peróxido de Hidrogênio/farmacologia , Músculo Liso Vascular/enzimologia , Animais , Western Blotting , Células Cultivadas , GMP Cíclico/metabolismo , Dissulfetos/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Oxidantes/farmacologia , Transdução de Sinais
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