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1.
Cell ; 169(5): 807-823.e19, 2017 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-28479188

RESUMO

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.


Assuntos
Células-Tronco Hematopoéticas/citologia , Transdução de Sinais , Tretinoína/farmacologia , Vitamina A/administração & dosagem , Animais , Vias Biossintéticas , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular , Dieta , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Poli I-C/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única , Estresse Fisiológico , Vitamina A/farmacologia , Vitaminas/administração & dosagem , Vitaminas/farmacologia
2.
Am J Physiol Endocrinol Metab ; 309(7): E632-9, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26264555

RESUMO

In rat pancreatic islets, ß-cell gene expression, survival, and subsequent acute glucose stimulation of insulin secretion (GSIS) are optimally preserved by prolonged culture at 10 mM glucose (G10) and markedly altered by culture at G5 or G30. Here, we tested whether pharmacological glucokinase (GK) activation prevents these alterations during culture or improves GSIS after culture. Rat pancreatic islets were cultured 1-7 days at G5, G10, or G30 with or without 3 µM of the GK activator Ro 28-0450 (Ro). After culture, ß-cell apoptosis and islet gene mRNA levels were measured, and the acute glucose-induced increase in NAD(P)H autofluorescence, intracellular calcium concentration, and insulin secretion were tested in the absence or presence of Ro. Prolonged culture of rat islets at G5 or G30 instead of G10 triggered ß-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected ß-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to ß-cell survival and function at G5, detrimental at G10, and ineffective at G30. In contrast, acute GK activation with Ro increased the glucose sensitivity of islets cultured at G10 but failed at restoring ß-cell glucose responsiveness after culture at G5 or G30. We conclude that pharmacological GK activation prevents the alteration of ß-cell survival and function by long-term culture at G5 but mimics glucotoxicity when added to G10. The complex effects of glucose on the ß-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se.


Assuntos
Glucoquinase/metabolismo , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Sulfonas/farmacologia , Tiazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Ilhotas Pancreáticas/metabolismo , Masculino , Ratos , Ratos Wistar
3.
Biochem J ; 460(3): 411-23, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24678915

RESUMO

The glucose stimulation of insulin secretion by pancreatic ß-cells depends on increased production of metabolic coupling factors, among which changes in NADPH and ROS (reactive oxygen species) may alter the glutathione redox state (EGSH) and signal through changes in thiol oxidation. However, whether nutrients affect EGSH in ß-cell subcellular compartments is unknown. Using redox-sensitive GFP2 fused to glutaredoxin 1 and its mitochondria-targeted form, we studied the acute nutrient regulation of EGSH in the cytosol/nucleus or the mitochondrial matrix of rat islet cells. These probes were mainly expressed in ß-cells and reacted to low concentrations of exogenous H2O2 and menadione. Under control conditions, cytosolic/nuclear EGSH was close to -300 mV and unaffected by glucose (from 0 to 30 mM). In comparison, mitochondrial EGSH was less negative and rapidly regulated by glucose and other nutrients, ranging from -280 mV in the absence of glucose to -299 mV in 30 mM glucose. These changes were largely independent from changes in intracellular Ca(2+) concentration and in mitochondrial pH. They were unaffected by overexpression of SOD2 (superoxide dismutase 2) and mitochondria-targeted catalase, but were inversely correlated with changes in NAD(P)H autofluorescence, suggesting that they indirectly resulted from increased NADPH availability rather than from changes in ROS concentration. Interestingly, the opposite regulation of mitochondrial EGSH and NAD(P)H autofluorescence by glucose was also observed in human islets isolated from two donors. In conclusion, the present study demonstrates that glucose and other nutrients acutely reduce mitochondrial, but not cytosolic/nuclear, EGSH in pancreatic ß-cells under control conditions.


Assuntos
Glucose/farmacologia , Glutationa/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/efeitos dos fármacos , Animais , Cálcio/metabolismo , Catalase/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Mitocôndrias/fisiologia , NADP/metabolismo , Oxirredução , Ratos , Espécies Reativas de Oxigênio/metabolismo , Vitamina K 3/metabolismo
5.
Nat Commun ; 15(1): 334, 2024 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-38184650

RESUMO

Pancreatic ß-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. Here we show, in ß-cells from overweight humans without diabetes and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. RNA-seq of sorted ß-cells suggests altered metabolic pathways early following high fat diet, where we find increased basal oxygen consumption and proton leak, but a more reduced cytosolic redox state. Increased ß-cell exocytosis after 2-day high fat diet is dependent on this reduced intracellular redox state and requires the sentrin-specific SUMO-protease-1. Mice with either pancreas- or ß-cell-specific deletion of this fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day high fat diet. Mechanistically, redox-sensing by the SUMO-protease requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained ß-cell exocytosis, and increases enzyme sensitivity to regulation by redox signals.


Assuntos
Dieta Hiperlipídica , Exocitose , Animais , Humanos , Camundongos , Cisteína Endopeptidases/genética , Citosol , Dieta Hiperlipídica/efeitos adversos , Glucose , Peptídeo Hidrolases
6.
Biochem J ; 441(3): 971-8, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22050124

RESUMO

Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic ß-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in ß-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in ß-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat ß-cells.


Assuntos
Glutationa/análise , Proteínas de Fluorescência Verde/análise , Peróxido de Hidrogênio/análise , Células Secretoras de Insulina/química , Medições Luminescentes/métodos , Mitocôndrias/química , Animais , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Cinética , Masculino , Mitocôndrias/metabolismo , Concentração Osmolar , Oxirredução , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sensibilidade e Especificidade
7.
Gen Physiol Biophys ; 31(1): 65-76, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22447832

RESUMO

Chronic administration of glucocorticoids (GC) leads to characteristic features of type 2 diabetes in mammals. The main action of dexamethasone in target cells occurs through modulation of gene expression, although the exact mechanisms are still unknown. We therefore investigated the gene expression profile of pancreatic islets from rats treated with dexamethasone using a cDNA array screening analysis. The expression of selected genes and proteins involved in mitochondrial apoptosis was further analyzed by PCR and immunoblotting. Insulin, triglyceride and free fatty acid plasma levels, as well as glucose-induced insulin secretion, were significantly higher in dexamethasone-treated rats compared with controls. Out of 1176 genes, 60 were up-regulated and 28 were down-regulated by dexamethasone treatment. Some of the modulated genes are involved in apoptosis, stress response, and proliferation pathways. RT-PCR confirmed the cDNA array results for 6 selected genes. Bax α protein expression was increased, while Bcl-2 was decreased. In vivo dexamethasone treatment decreased the mitochondrial production of NAD(P)H, and increased ROS production. Concluding, our data indicate that dexamethasone modulates the expression of genes and proteins involved in several pathways of pancreatic-islet cells, and mitochondria dysfunction might be involved in the deleterious effects after long-term GC treatment.


Assuntos
Dexametasona/farmacologia , Regulação da Expressão Gênica/fisiologia , Ilhotas Pancreáticas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
8.
Diabetes ; 71(8): 1706-1720, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35622000

RESUMO

Hypoxia-induced islet cell death, caused by an insufficient revascularization of the grafts, is a major obstacle for successful pancreatic islet transplantation. Recently, it has been reported that the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is expressed in pancreatic islets and that its loss protects against hypoxia-induced cell death. Therefore, we hypothesized that the inhibition of NLRP3 in islets improves the survival and endocrine function of the grafts. The transplantation of Nlrp3-/- islets or wild-type (WT) islets exposed to the NLRP3 inhibitor CY-09 into mouse dorsal skinfold chambers resulted in an improved revascularization compared with controls. An increased insulin release after NLRP3 inhibition caused the enhanced angiogenic response. Moreover, the inhibition of NLRP3 in hypoxic ß-cells triggered insulin gene expression by inducing the shuttling of MafA and pancreatic and duodenal homeobox-1 into the nucleus. This was mediated by a reduced interaction of NLRP3 with the thioredoxin-interacting protein (TXNIP). Transplantation of Nlrp3-/- islets or WT islets exposed to CY-09 under the kidney capsule of diabetic mice markedly improved the restoration of normoglycemia. These findings indicate that the inhibition of NLRP3 in isolated islets represents a promising therapeutic strategy to improve engraftment and function of the islets.


Assuntos
Diabetes Mellitus Experimental , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Diabetes Mellitus Experimental/metabolismo , Hipóxia/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo
9.
Antioxidants (Basel) ; 10(8)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34439552

RESUMO

In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in ß-cells. Nonetheless, the temporality of the events triggered and the role of different ROS sources remain unclear. Isolated islets from C57BL/6J wild-type (WT), NOX1 KO and NOX2 KO mice were exposed to a PIC combination. We show that cytokines increase O2•- production after 2 h in WT and NOX1 KO but not in NOX2 KO islets. Using transgenic mice constitutively expressing a genetically encoded compartment specific H2O2 sensor, we show, for the first time, a transient increase of cytosolic/nuclear H2O2 in islet cells between 4 and 5 h during cytokine exposure. The H2O2 increase coincides with the intracellular NAD(P)H decrease and is absent in NOX2 KO islets. NOX2 KO confers better glucose tolerance and protects against cytokine-induced islet secretory dysfunction and death. However, NOX2 absence does not counteract the cytokine effects in ER Ca2+ depletion, Store-Operated Calcium Entry (SOCE) increase and ER stress. Instead, the activation of ER stress precedes H2O2 production. As early NOX2-driven ROS production impacts ß-cells' function and survival during insulitis, NOX2 might be a potential target for designing therapies against early ß-cell dysfunction in the context of T1D onset.

10.
Acta Diabetol ; 58(12): 1637-1647, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34254190

RESUMO

AIMS: The exposure of isolated pancreatic islets to pro-angiogenic factors prior to their transplantation represents a promising strategy to accelerate the revascularization of the grafts. It has been shown that erythropoietin (EPO), a glycoprotein regulating erythropoiesis, also induces angiogenesis. Therefore, we hypothesized that EPO exposure of isolated islets improves their posttransplant revascularization. METHODS: Flow cytometric, immunohistochemical and quantitative real-time (qRT)-PCR analyses were performed to study the effect of EPO on the viability, cellular composition and gene expression of isolated islets. Moreover, islets expressing a mitochondrial or cytosolic H2O2 sensor were used to determine reactive oxygen species (ROS) levels. The dorsal skinfold chamber model in combination with intravital fluorescence microscopy was used to analyze the revascularization of transplanted islets. RESULTS: We found that the exposure of isolated islets to EPO (3 units/mL) for 24 h does not affect the viability and the production of ROS when compared to vehicle-treated and freshly isolated islets. However, the exposure of islets to EPO increased the number of CD31-positive cells and enhanced the gene expression of insulin and vascular endothelial growth factor (VEGF)-A. The revascularization of the EPO-cultivated islets was accelerated within the initial phase after transplantation when compared to both controls. CONCLUSION: These findings indicate that the exposure of isolated islets to EPO may be a promising approach to improve clinical islet transplantation.


Assuntos
Eritropoetina , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Eritropoetina/farmacologia , Peróxido de Hidrogênio , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular/genética
11.
EMBO Mol Med ; 13(1): e12616, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33135383

RESUMO

Pancreatic islet transplantation still represents a promising therapeutic strategy for curative treatment of type 1 diabetes mellitus. However, a limited number of organ donors and insufficient vascularization with islet engraftment failure restrict the successful transfer of this approach into clinical practice. To overcome these problems, we herein introduce a novel strategy for the generation of prevascularized islet organoids by the fusion of pancreatic islet cells with functional native microvessels. These insulin-secreting organoids exhibit a significantly higher angiogenic activity compared to freshly isolated islets, cultured islets, and non-prevascularized islet organoids. This is caused by paracrine signaling between the ß-cells and the microvessels, mediated by insulin binding to its corresponding receptor on endothelial cells. In vivo, the prevascularized islet organoids are rapidly blood-perfused after transplantation by the interconnection of their autochthonous microvasculature with surrounding blood vessels. As a consequence, a lower number of islet grafts are required to restore normoglycemia in diabetic mice. Thus, prevascularized islet organoids may be used to improve the success rates of clinical islet transplantation.


Assuntos
Diabetes Mellitus Experimental , Células Secretoras de Insulina , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Células Endoteliais , Insulina , Camundongos
12.
J Mol Biol ; 432(5): 1461-1493, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31634466

RESUMO

Insulin-secreting pancreatic ß-cells play a critical role in blood glucose homeostasis and the development of type 2 diabetes (T2D) in the context of insulin resistance. Based on data obtained at the whole cell level using poorly specific chemical probes, reactive oxygen species (ROS) such as superoxide and hydrogen peroxide have been proposed to contribute to the stimulation of insulin secretion by nutrients (positive role) and to the alterations of cell survival and secretory function in T2D (negative role). This raised the controversial hypothesis that any attempt to decrease ß-cell oxidative stress and apoptosis in T2D would further impair insulin secretion. Over the last decade, the development of genetically-encoded redox probes that can be targeted to cellular compartments of interest and are specific of redox couples allowed the evaluation of short- and long-term effects of nutrients on ß-cell redox changes at the subcellular level. The data indicated that the nutrient regulation of ß-cell redox signaling and ROS toxicity is far more complex than previously thought and that the subcellular compartmentation of these processes cannot be neglected when evaluating the mechanisms of ROS production or the efficacy of antioxidant enzymes and antioxidant drugs under glucolipotoxic conditions and in T2D. In this review, we present what is currently known about the compartmentation of redox homeostatic systems and tools to investigate it. We then review data about the effects of nutrients on ß-cell subcellular redox state under normal conditions and in the context of T2D and discuss challenges and opportunities in the field.


Assuntos
Compartimento Celular , Células Secretoras de Insulina , Ilhotas Pancreáticas , Estresse Oxidativo , Animais , Antioxidantes/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes , Glucose/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Mitocôndrias/metabolismo , Nutrientes/metabolismo , Nutrientes/toxicidade , Oxirredução , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Superóxido Dismutase/metabolismo
13.
Br J Pharmacol ; 177(7): 1651-1665, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31721150

RESUMO

BACKGROUND AND PURPOSE: Pancreatic islet transplantation is a promising therapeutic approach for Type 1 diabetes. A major prerequisite for the survival of grafted islets is a rapid revascularization after transplantation. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to promote angiogenesis. Therefore, we investigated in this study whether EPO improves the revascularization of transplanted islets. EXPERIMENTAL APPROACH: Islets from FVB/N mice were transplanted into dorsal skinfold chambers of recipient animals, which were daily treated with an intraperitoneal injection of EPO (500 IU·kg-1 ) or vehicle (control) throughout an observation period of 14 days. In a second set of experiments, animals were only pretreated with EPO over a 6-day period prior to islet transplantation. The revascularization of the grafts was assessed by repetitive intravital fluorescence microscopy and immunohistochemistry. In addition, a streptozotocin-induced diabetic mouse model was used to study the effect of EPO-pretreatment on the endocrine function of the grafts. KEY RESULTS: EPO treatment slightly accelerated the revascularization of the islet grafts. This effect was markedly more pronounced in EPO-pretreated animals, resulting in significantly higher numbers of engrafted islets and an improved perfusion of endocrine tissue without affecting systemic haematocrit levels when compared with controls. Moreover, EPO-pretreatment significantly accelerated the recovery of normoglycaemia in diabetic mice after islet transplantation. CONCLUSION AND IMPLICATIONS: These findings demonstrate that, particularly, short-term EPO-pretreatment represents a promising therapeutic approach to improve the outcome of islet transplantation, without an increased risk of thromboembolic events.


Assuntos
Diabetes Mellitus Experimental , Eritropoetina , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Eritropoetina/farmacologia , Camundongos , Neovascularização Fisiológica , Estreptozocina
14.
EMBO Mol Med ; 12(4): e09271, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32187826

RESUMO

The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ-specific manner. Insulin flux across the endothelium to muscle cells is a rate-limiting process influencing insulin-mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non-obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC-specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high-fat diet-induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes.


Assuntos
Células Endoteliais/metabolismo , Resistência à Insulina , Insulina , Músculo Esquelético/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Glucose/metabolismo , Insulina/metabolismo , Camundongos
15.
Mol Cell Endocrinol ; 273(1-2): 32-41, 2007 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-17573185

RESUMO

Prolactin induces maturation of insulin secretion in cultured neonatal rat islets. In this study, we investigated whether the improved secretory response to glucose caused by prolactin involves alteration in the expression, association and phosphorylation of several proteins that participate in these processes. Messenger RNA was extracted from neonatal rat islets cultured for 5 days in the presence of prolactin and reverse transcribed. Gene expression was analyzed by semi-quantitative RT-PCR and by Western blotting for proteins. The gene transcription and protein expression of kinesin and MAP-2 were increased in prolactin-treated islets compared to the controls. The association and phosphorylation of proteins was analyzed by immunoprecipitation followed by Western blotting, after acute exposure to prolactin. Prolactin increased the association between SNARE proteins and kinesin/MAP-2 while the association of munc-18/syntaxin 1A was decreased. Serine phosphorylation of SNAP-25, syntaxin 1A, munc-18, MAP-2 was significantly higher whereas kinesin phosphorylation was decreased in prolactin-treated islets. There was an increase in SNARE complex formation in islets stimulated with prolactin, 22 mM glucose, 40 mM K(+), 200 microM carbachol and 1 microM PMA. The prolactin-induced increase in the formation of SNARE complex and syntaxin 1A phosphorylation was inhibited by PD098059 and U0126, inhibitors of the MAPK pathway. These findings indicate that prolactin primes pancreatic beta-cells to release insulin by increasing the expression and phosphorylation/association of proteins implicated in the secretory machinery and the MAPK/PKC pathway is important for this effect.


Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Prolactina/farmacologia , Proteínas SNARE/metabolismo , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/enzimologia , Cinesinas/genética , Potenciais da Membrana/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Fatores de Tempo
16.
Mol Cell Endocrinol ; 439: 354-362, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27664519

RESUMO

High glucose-induced oxidative stress and increased NADPH oxidase-2 (NOX2) activity may contribute to the progressive decline of the functional ß-cell mass in type 2 diabetes. To test that hypothesis, we characterized, in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks at 10 or 30 mmol/l glucose (G10 or G30), the in vitro effects of glucose on cytosolic oxidative stress using probes sensing glutathione oxidation (GRX1-roGFP2), thiol oxidation (roGFP1) or H2O2 (roGFP2-Orp1), on ß-cell stimulus-secretion coupling events and on ß-cell apoptosis. After 1-2 days of culture in G10, the glucose stimulation of insulin secretion (GSIS) was ∼1.7-fold higher in NOX2-KO vs. WT islets at 20-30 mmol/l glucose despite similar rises in NAD(P)H and intracellular calcium concentration ([Ca2+]i) and no differences in cytosolic GRX1-roGFP2 oxidation. After long-term culture at G10, roGFP1 and roGFP2-Orp1 oxidation and ß-cell apoptosis remained low, and the glucose-induced rises in NAD(P)H, [Ca2+]i and GSIS were similarly preserved in both islet types. After prolonged culture at G30, roGFP1 and roGFP2-Orp1 oxidation increased in parallel with ß-cell apoptosis, the glucose sensitivity of the NADPH, [Ca2+]i and insulin secretion responses increased, the maximal [Ca2+]i response decreased, but maximal GSIS was preserved. These responses were almost identical in both islet types. In conclusion, NOX2 is a negative regulator of maximal GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the in vitro glucotoxic induction of cytosolic oxidative stress and alterations of ß-cell survival and function.


Assuntos
Glucose/toxicidade , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , NADPH Oxidase 2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citosol/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/deficiência , Oxirredução , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Compostos de Sulfidrila/metabolismo , Técnicas de Cultura de Tecidos
17.
Sci Signal ; 9(419): rs1, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26980443

RESUMO

Mapping the in vivo distribution of endogenous oxidants in animal tissues is of substantial biomedical interest. Numerous health-related factors, including diet, physical activity, infection, aging, toxins, or pharmacological intervention, may cause redox changes. Tools are needed to pinpoint redox state changes to particular organs, tissues, cell types, and subcellular organelles. We describe a procedure that preserves the in vivo redox state of genetically encoded redox biosensors within histological tissue sections, thus providing "redox maps" for any tissue and comparison of interest. We demonstrate the utility of the technique by visualizing endogenous redox differences and changes in the context of tumor growth, inflammation, embryonic development, and nutrient starvation.


Assuntos
Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Transgenes , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Sondas Moleculares/genética , Oxirredução
18.
Pancreas ; 44(2): 287-95, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25426612

RESUMO

OBJECTIVES: The aim of the study was to evaluate the potential changes induced by fish oil (FO) supplementation on the redox status of pancreatic islets from healthy rats. To test whether these effects were due to eicosapentaenoic acid and docosahexaenoic acid (ω-3), in vitro experiments were performed. METHODS: Rats were supplemented with FO, and pancreatic islets were obtained. Islets were also treated in vitro with palmitate (P) or eicosapentaenoic acid + docosahexaenoic acid (ω-3). Insulin secretion (GSIS), glucose oxidation, protein expression, and superoxide content were analyzed. RESULTS: The FO group showed a reduction in superoxide content. Moreover, FO reduced the expression of NAD(P)H oxidase subunits and increased superoxide dismutase, without altering ß-cell function. Palmitate increased ß-cell reactive oxygen species (ROS) production, apoptosis, and impaired GSIS. Under these conditions, ω-3 triggered a parallel reduction in ROS production and ß-cell apoptosis induced by P and protected against the impairment in GSIS. There was no difference in mitochondrial ROS production. CONCLUSIONS: Our results show that ω-3 protect pancreatic islets from alterations induced by P. In vivo FO supplementation modulates the redox state of pancreatic ß-cell. Considering that in vitro effects do not involve mitochondrial superoxide production, we can speculate that this protection might involve NAD(P)H oxidase activity.


Assuntos
Antioxidantes/administração & dosagem , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Ilhotas Pancreáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Glutationa/metabolismo , Insulina/sangue , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , NADPH Oxidases/metabolismo , Oxirredução , Ácido Palmítico/toxicidade , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Fatores de Tempo , Técnicas de Cultura de Tecidos
19.
PLoS One ; 7(10): e46831, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23056475

RESUMO

AIM/HYPOTHESIS: Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl(2), a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations. METHODS: Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive ß-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing "redox sensitive GFP" targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA. RESULTS: As observed for Mt1a mRNA levels, ß-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50-100 µM ZnCl(2) to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late ß-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions. CONCLUSION: ZnCl(2) reduces mitochondrial oxidative stress and improves rat ß-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Cloretos/farmacologia , Citoproteção/efeitos dos fármacos , Glucose/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Compostos de Zinco/farmacologia , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Técnicas de Cultura de Células , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Metalotioneína/genética , Metalotioneína/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Fatores de Tempo , Transportador 8 de Zinco
20.
Redox Rep ; 16(4): 173-80, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21888768

RESUMO

OBJECTIVES: Reactive oxygen species (ROS) are involved in many physiological and pathological processes. In the present study, we analysed whether the synthetic glucocorticoid dexamethasone induces oxidative stress in cultured pancreatic islets and whether the effects of dexamethasone on insulin secretion, gene expression, and viability can be counteracted by concomitant incubation with N-acetylcysteine (NAC). METHODS: ROS production was measured by dichlorofluorescein (DCFH-DA) assay, insulin secretion by radioimmunoassay, intracellular calcium dynamics by fura-2-based fluorescence, gene expression by real-time polymerase chain reaction analyses and cell viability by the MTS assay. RESULTS: Dexamethasone (Dexa) increased ROS production and decreased glucose-stimulated insulin secretion after 72 hours incubation. Intracellular ROS levels were decreased and the insulin secretion capacity was recovered by concomitant treatment with Dexa+NAC. The total insulin content and intracellular Ca2+ levels were not modulated in either Dexa or Dexa+NAC groups. There was a decrease in the NAD(P)H production, used as an indicator of viability, after dexamethasone treatment. Concomitant incubation with NAC returned viability to control levels. Dexa also decreased synaptotagmin VII (SYT VII) gene expression. In contrast, the Dexa+NAC group demonstrated an increased expression of SYT VII compared to controls. Surprisingly, treatment with NAC decreased the gene expression of the antioxidant enzyme copper zinc superoxide dismutase soluble. DISCUSSION: Our results indicate that dexamethasone increases ROS production, decreases viability, and impairs insulin secretion in pancreatic rat islets. These effects can be counteracted by NAC, which not only decreases ROS levels but also modulates the expression of genes involved in the secretory pathway and those coding for antioxidant enzymes.


Assuntos
Acetilcisteína/farmacologia , Dexametasona/antagonistas & inibidores , Glucocorticoides/antagonistas & inibidores , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dexametasona/toxicidade , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/metabolismo , Glucocorticoides/toxicidade , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Sinaptotagminas/efeitos dos fármacos , Sinaptotagminas/metabolismo
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