Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration.

BACKGROUND/AIM: In rat pancreatic islets, beta-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. METHODS: 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, beta-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 28-0450. RESULTS: Prolonged culture of rat islets at G5 or G30 instead of G10 triggered beta-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected beta-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to beta-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 beta-cell glucose responsiveness after culture at G5 or G30. CONCLUSIONS: Pharmacological GK activation prevents the alteration of beta-cell survival and function by long-term culture at G5, but mimics glucotoxicity when added to G10. The complex effects of glucose on the beta-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se

Erythropoietin exposure of isolated pancreatic islets accelerates their revascularization after transplantation

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

Early Cytokine-Induced Transient NOX2 Activity Is ER Stress-Dependent and Impacts β-Cell Function and Survival

In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in β-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 β-cells’ function and survival during insulitis, NOX2 might be a potential target for designing therapies against early β-cell dysfunction in the context of T1D onset

Erythropoietin accelerates the revascularization of transplanted pancreatic islets

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

O contributo da articulação entre a família e o jardim de infância para o desenvolvimento da literacia emergente

O presente trabalho enquadra-se na realização do relatório final de Mestrado em Educação Pré-Escolar (EPE) e Ensino do 1.º Ciclo do Ensino Básico (1.º CEB) e tem como tema “O contributo da articulação entre a família e o jardim de infância no desenvolvimento da literacia emergente”. Este relatório final é o reflexo do percurso efetuado em contexto do referido Mestrado. Este relatório encontra-se estruturado em duas partes, sendo que a primeira se inicia com uma reflexão sobre as práticas em contexto de Prática de Ensino Supervisionada (PES) relativas aos semestres da PES II e PES III. Na segunda parte, é apresentado o trabalho de investigação. Com o intuito de perceber o contributo da família e do jardim de infância no desenvolvimento da literacia emergente foi realizada uma investigação de caráter quantitativo, utilizando o questionário direcionado aos educadores e pais/encarregados de educação de um agrupamento de escolas da cidade de Viseu. A análise dos dados recolhidos revelou que as educadoras de infância e os pais/encarregados de educação dizem promover atividades literácitas conducentes à articulação entre ambos os contextos: família e o jardim de infância. Os resultados obtidos permitiram concluir que existe articulação entre a família e as educadoras de infância de um agrupamento de escolas de Viseu, contribuindo para o desenvolvimento da literacia emergente. Assim, a família e o jardim de infância, contextos privilegiados e enriquecedores de literacia, podem contribuir de forma decisiva para o desenvolvimento da literacia emergente na criança.Abstract This work is part of the completion of the final report of Master in Pre-School Education (EPE) and education of the 1st cycle of basic education (1st CEB) and has as its theme "The contribution of the relationship between the family and kindergarten in the development of emergent literacy" This final report is a reflection of the journey effected in the said Master context. This report is structured in two parts, the first of which begins with a reflection on the practice Practice context Supervised Teaching (PES) for the six months of PES PES II and III. In the second part, the research work is presented. In order to realize the contribution of family and kindergarten in the development of emergent literacy one character quantitative research was carried out using a questionnaire directed to educators and parents / guardians of a group of Viseu of city schools. The analysis of the data collected revealed childhood educators and parents / guardians say promote activities leading to the linkage between the two contexts: family and kindergarten. The results showed that there is coordination between the family and the kindergarten teachers of a school group of Viseu, contributing to the development of emergent literacy. So the family and kindergarten, privileged and enriching contexts of literacy, can contribute decisively to the development of emergent literacy in children

Improvement of islet transplantation by the fusion of islet cells with functional blood vessels

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 β-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

Nutrient metabolism, subcellular redox state and oxidative stress in pancreatic islets and β-cells

Insulin-secreting pancreatic β-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) like 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 β-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 β-cell redox changes at the subcellular level. The data indicated that the nutrient regulation of β-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 β-cell subcellular redox state under normal conditions and in the context of T2D, and discuss challenges and opportunities in the field

Glucokinase activation is beneficial or toxic to cultured rat pancreatic islets depending on the prevailing glucose concentration

BACKGROUND/AIM: In rat pancreatic islets, beta-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. METHODS: 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, beta-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 28-0450. RESULTS: Prolonged culture of rat islets at G5 or G30 instead of G10 triggered beta-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected beta-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to beta-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 beta-cell glucose responsiveness after culture at G5 or G30. CONCLUSIONS: Pharmacological GK activation prevents the alteration of beta-cell survival and function by long-term culture at G5, but mimics glucotoxicity when added to G10. The complex effects of glucose on the beta-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se

Prolactin modulates the expression and function of the SNARE proteins, CaMK2 e MAP2 in rat islets

info:eu-repo/semantics/nonPublishe

Prolactin modulates the association and phosphorylation of SNARE and kinesin/MAP-2 proteins in neonatal pancreatic rat islets.

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.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe